High-power Ultrasound to Control of Honey Crystallisationeffect on flavour, HMF) and remove the need...

High-power Ultrasound to Control of Honey

Crystallisation

by Bruce R. D’Arcy

October 2007

RIRDC Publication No 07/145 RIRDC Project No UQ-101A

ii

© 2007 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 541 6 ISSN 1440-6845 High-power Ultrasound to Control of Honey Crystallisation Publication No. 07/145 Project No. UQ-101A The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances.

While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication.

The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors.

The Commonwealth of Australia does not necessarily endorse the views in this publication.

This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165.

Researcher Contact Details Dr Bruce D’Arcy School of Land, Crop and Food Sciences The University of Queensland Brisbane QUEENSLAND 4072 Phone: 07 3346 9190 Fax: 07 3365 1177 Email: [email protected] Website: http://www.uq.edu.au/~gabdarcy/

In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: 02 6271 4100 Fax: 02 6271 4199 Email: [email protected]. Web: http://www.rirdc.gov.au Electronically published in October 2007

iii

Foreword This report details the successful application of ultrasound technology to the liquefaction of candied honey through the development of a new process. But why is such new technology needed by the honey industry? In the honey industry, a crystallisation or candying problem can occur in some floral types of honey, and there is concern about the heating process presently used to control this problem. The potential for overheating or ‘cooking’ honey by beekeepers and honey packers that reduces honey quality is ever present. Therefore, the honey industry requires alternate technologies for liquefying candied honey, such as ultrasound. Using ultrasound to input energy of 70000 joules from a 40 millimetre sonotrode operated at a wavelength amplitude of 12 micrometers (µm) is sufficient to liquefy candied Salvation Jane honey (~250 grams) without compromising honey quality. For example, this ultrasound treatment produces a lower concentration of hydroxymethylfurfural (HMF) formed from honey sugars, no decrease in diastase and invertase (enzyme) activities relative to that produced by a similar heating regime used by the industry. The specific energy input needed to completely liquefy candied Salvation Jane honey is 0.126 kilowatt-hours (kWh) per kilogram (kg). Therefore, ten kg of candied honey will require 1.26 kWh, while 300 kg will require 37.9 kWh. Further, this report shows that ultrasound treatment will not only liquefy candied honey without long exposure to high temperatures, but may make the liquid honey more stable to subsequent crystallisation on storage, relative to liquid honey produced by the present industrial heating regime. The Dyce method for production of creamed honey was evaluated to see if ultrasound treatment could improve the quality and consistency of creamed honey. Little change was evaluated. Untreated canola/red gum creamed honeys had a similar crystal content to ultrasound-treated canola/red gum creamed honeys and untreated alfalfa/blue gum creamed honeys had a similar crystal content to ultrasound-treated alfalfa/blue gum creamed honeys. The importance of this report is that it provides data that can be used by beekeepers and honey packing companies to develop a pilot-scale ultrasound processing system in conjunction with an ultrasound equipment manufacturer. The implications for the honey industry, if this ultrasound technology is adopted and applied on an industrial scale, would be the production of better quality honey (e.g. less effect on flavour, HMF) and remove the need for hot rooms, thus reducing energy costs. This project was funded from industry revenue, matched by funds from the Federal Government. This report, an addition to RIRDC’s diverse range of over 1600 research publications, forms part of our Honeybee R&D program, which aims to improve the productivity and profitability of the Australian beekeeping industry. Most of our publications are available for viewing, downloading or purchasing online through our website: downloads at www.rirdc.gov.au/reports/index.htm purchases at www.rirdc.gov.au/eshop

Peter 0’Brien Managing Director Rural Industries Research and Development Corporation

iv

About the Author Dr Bruce D’Arcy is a food scientist, analytical chemist, and Senior Lecturer in food chemistry in the School of Land, Crop and Food Sciences at The University of Queensland. He is Director of the Australian Honey Research Unit at the University of Queensland, has extensive knowledge of the physicochemical properties of honey, and has been researching various aspects of the chemistry, physical properties and product development of species-specific floral types of Australian honey for the past 14 years.

Acknowledgments The following members of the Australian Honey Research Unit at the University of Queensland made varying levels of contributions to the material and data contained in this report: Researcher Contribution Associate Professor Bhesh Bhandari (Academic at UQ)

Academic input

Mr Tikiri Rajapakse (PhD student at UQ 2003-2007)

Experimental data as part of PhD research project

Ms Cécile Fuches [visiting research student from ISTAB (Institut des Sciences et Techniques des Aliments de Bordeaux), France in 2005]

Experimental data on the effect of ultrasound on the creamed honey process

Mr Renee Linssen (visiting research student from Wageningen University Research, Wageningen, The Netherlands in 2004)

Experimental data on the effect of ultrasound on the creamed honey process

Ryan Becker (4th year BFoodTech student in 2002)

Preliminary ultrasound study

The author gratefully acknowledges Capilano Honey Ltd. for their advice and kind donation of the honey samples used in this research.

v

Glossary Two terms are used in the honey industry and honey research with respect to honey crystallisation: • candied honey • creamed honey

The term ‘candied honey’ will be used throughout this report to mean ‘naturally granulated honey’, or ‘naturally crystallised honey’ which is a solid honey that forms due to crystallisation of D-glucose monohydrate in cold weather, often between extraction from the combs and its arrival in 200 litre drum at the packing factory. Here, different floral types of honey are known to crystallise at different rates and produce crystals of different sizes (termed fine grained and coarse grained naturally crystallised honeys). Candied honey is not preferred by Australian consumers, so heating is used by honey packers to liquefy honey prior to sale. In addition, this liquefaction process must stabilise the honey (remove any nuclei) so that crystallisation of the liquefied honey does not occur in honey jars after packing. The second term ‘creamed’, or ‘soft set’ or ‘whipped’ honey, which is marketed as ‘creamed honey’ in Australia, refers to ‘induced’ crystallised solid honey (finely grained) with a creamy and spreadable texture produced in Australia and overseas using specialised processes generally based on the Dyce method. This involves the use of blends of a number of rapidly crystallising floral types of honey and a floral honey that is strongly flavoured. DSC differential scanning calorimetry G° Shade units HMF hydroxymethylfurfural content J Joules kHz Kilohertz KJ Kilojoules kWh Kilowatts L litres US United States UV Ultra violate

vi

Contents Foreword ................................................................................................................................................ iii About the Author.................................................................................................................................... iv Acknowledgments .................................................................................................................................. iv Glossary................................................................................................................................................... v Executive Summary ............................................................................................................................. xiii 1. Introduction...................................................................................................................................... 1

1.1 What is Honey?......................................................................................................................... 1 1.2 Chemical Composition of Honey ............................................................................................. 1

1.2.1 Carbohydrates in honey.............................................................................................. 2 1.2.2 Moisture content of honey ......................................................................................... 4 1.2.3 pH and acidity of honey ............................................................................................. 5 1.2.4 Ash and mineral content of honey.............................................................................. 5 1.2.5 Hydroxymethylfurfural content of honey .................................................................. 6 1.2.6 Enzymes in honey ...................................................................................................... 7 1.2.7 Colour of honey.......................................................................................................... 8 1.2.8 Flavour and aroma of honey ...................................................................................... 9 1.2.9 Composition of Australian honey ............................................................................ 11

1.3 Physical Characteristics of Honey .......................................................................................... 12 1.3.1 Viscosity of honey.................................................................................................... 12 1.3.2 Specific heat of honey.............................................................................................. 13 1.3.3 Hygroscopicity of honey .......................................................................................... 13 1.3.4 Crystallisation of honey ........................................................................................... 14 1.3.5 Electrical conductivity of honey .............................................................................. 14 1.3.6 Fermentation in honey.............................................................................................. 15

1.4 Processing and Storage of Honey ........................................................................................... 15 1.4.1 Uncapping of the combs........................................................................................... 16 1.4.2 Extraction of honey from the combs ........................................................................ 16 1.4.3 Straining and clarifying of honey............................................................................. 16 1.4.4 Heating of honey during processing......................................................................... 17 1.4.5 Effect of heat on honey composition........................................................................ 18 1.4.6 Storage of honey ...................................................................................................... 19 1.4.7 Control of Crystallisation......................................................................................... 20 1.4.8 Production of creamed honey................................................................................... 20

1.5 Quality, Methods of Analysis and International Regulatory Standards.................................. 21 1.5.1 Moisture content of honey ....................................................................................... 21 1.5.2 Apparent reducing sugar content in honey............................................................... 23 1.5.3 Apparent sucrose content in honey .......................................................................... 23 1.5.4 Water insoluble solids/matter content in honey ....................................................... 24 1.5.5 Ash content of honey .............................................................................................. 25 1.5.6 Electrical conductivity of honey .............................................................................. 25 1.5.7 pH and acidity of honey ........................................................................................... 25 1.5.8 Diastase activity of honey ........................................................................................ 26 1.5.9 Invertase activity of honey ....................................................................................... 26 1.5.10 Hydroxymethylfurfural (HMF) content of honey ................................................... 26 1.5.11 Proline content ........................................................................................................ 27 1.5.12 Specific rotation ...................................................................................................... 27

vii

1.6 Ultrasound in Food Processing ............................................................................................... 28 1.6.1 Equipment for the generation of high-power ultrasound ......................................... 28 1.6.2 Design of ultrasonic systems.................................................................................... 29 1.6.3 Mechanism of ultrasound action .............................................................................. 30 1.6.4 Applications of ultrasound in food processing......................................................... 30 1.6.5 Effect of ultrasound on the liquefaction of candied honey ...................................... 35

1.7 Conclusion .............................................................................................................................. 38 2. Aims and Objectives ...................................................................................................................... 39

2.1 Overall Project Aims .............................................................................................................. 39 2.2 Project Objectives................................................................................................................... 39

3. Methodology .................................................................................................................................. 40

3.1 Effect of Ultrasound on the Cavitation of Sugar Solutions .................................................... 40 3.1.1 Experiment 1 Measurement of the size of the cavitation bubbles generated by

ultrasound using the Malvern Mastersizer/E............................................................ 40 3.1.2 Experiment 2: Measurement of the ultrasound cavitation by analysis of

decomposition of an aqueous iodine solution .......................................................... 42 3.2 Effect of Ultrasound Treatment on the Creamed Honey Production Process......................... 43

3.2.1 Assessment of creamed honeys based on their crystalline D-glucose monohydrate contents determined using differential scanning calorimetry (DSC) ....................... 43

3.2.2 Effect of ultrasound treatment on the crystals and maximum temperature of creamed honey, and the maximum temperature of liquid honey............................................ 46

3.2.3 Effect of ultrasound treatment on the experimental creamed honey process........... 50 3.3 Effect of Ultrasound Treatment on the Liquefaction of Candied honey and for Controlling

Honey Crystallisation ............................................................................................................. 53 3.3.1 Experiment 1: Ultrasound treatment of candied honey with interruption of input

energy, for selection of sonotrode and treatment conditions.................................... 53 3.3.2 Experiment 2: Effect of different input energy levels of continuous ultrasound

treatment (40 mm diameter sonotrode at 12 µm) for liquefying candied honey, and its effect on enzyme activity and the hydroxymethylfurfural concentration............ 56

3.3.3 Experiment 3: A comparative crystallisation study of candied honey liquefied by ultrasound treatment or by heat treatment................................................................ 60

4. Results and Discussion................................................................................................................... 63

4.1 Effect of Ultrasound on the Cavitation of Sugar Solutions .................................................... 63 4.1.1 Introduction .............................................................................................................. 63 4.1.2 Experiment 1 Measurement of the size of the cavitation bubbles generated by

ultrasound using the Malvern Mastersizer/E............................................................ 63 4.1.3 Experiment 2: Measurement of the ultrasound cavitation by analysis of

decomposition of an aqueous iodine solution .......................................................... 64 4.1.4 Summary, conclusions and implications.................................................................. 65

4.2 Effect of Ultrasound Treatment on the Creamed Honey Production Process......................... 66 4.2.1 Assessment of creamed honey blends based on their crystalline D-glucose

monohydrate contents determined using differential scanning calorimetry (DSC) . 66 4.2.2 Effect of ultrasound treatment on the crystals and maximum temperature of creamed

honey, and the maximum temperature of liquid honey............................................ 68 4.2.3 Effect of ultrasound treatment on the experimental creamed honey process........... 69 4.2.4 Summary, conclusions and implications.................................................................. 72

viii

4.3 Effect of Ultrasound Treatment on the Liquefaction of Candied Honey and for Controlling Honey Crystallisation ............................................................................................................. 74 4.3.1 Experiment 1: Ultrasound treatment of candied honey with interruption of input

energy for selection of sonotrode and treatment conditions..................................... 74 4.3.2 Experiment 2: Effect of different input energy levels of continuous ultrasound

treatment (40 mm diameter sonotrode at 12 µm) for liquefying candied honey, and its effect on enzyme activity and the hydroxymethylfurfural concentration............ 91

4.3.3 Experiment 3: A comparative crystallisation study of candied honey liquefied by ultrasound treatment and heat treatment .................................................................. 97

4.3.4 Summary, conclusions and implications................................................................ 104 4.4 Energy Requirement for an Industrial Scale-Up Design of a Commercial Ultrasound

Processor............................................................................................................................... 107 4.4.1 Calculation of the energy requirement for ultrasound liquefaction of candied honey

................................................................................................................................ 107 4.4.2 Design considerations for commercial scale ultrasound liquefaction of candied

honey...................................................................................................................... 109 5. Implications.................................................................................................................................. 115 6. Recommendations........................................................................................................................ 115 7. References Cited .......................................................................................................................... 116

ix

List of Tables Table 1.1 Mean amounts (% of total honey) of constituents of honey……………… 2 Table 1.2 Carbohydrate contents (and/or range) of honeys in different countries…... 3 Table 1.3 Yields of the principal sugars in the oligosaccharide fraction (3.65%) of honey……………………………………………………………………

5

Table 1.4 Mineral elements detected in honey………………………………………. 6 Table 1.5 Enzymes in honey…………………………………………………………. 7 Table 1.6 Invertase activity (g sucrose hydrolysed per 100 g h-1)………………….. 8 Table 1.7 Comparison of diastase activity before and after storage for 15 months…. 8 Table 1.8 Colour designations of extracted honey…………………………………... 9 Table 1.9 Floral varieties and flavours………………………………………………. 10 Table 1.10 Flavour attributes of honey………………………………………………... 10 Table 1.11 Aroma attributes of honey………………………………………………… 11 Table 1.12 Summary of analytical data of Australian honeys………………………… 12 Table 1.13 Specific heat of honey…………………………………………………….. 13 Table 1.14 Approximate equilibrium between relative humidity of air and the water content of clover honey......................................................................

14

Table 1.15 Effect of moisture and yeast counts on fermentation in honey…………… 15 Table 1.16 HMF increases resulting from processing………………………………… 18 Table 1.17 HMF content of honey during processing and packaging………………… 18 Table 1.18 The effect of heating on invertase activity and HMF of various Greek honeys………………………………………………………………

19

Table 1.19 Honey quality standards of Codex Alimentarius and European Directive.. 22 Table 1.20 Proposed new international standards for honey…………………………. 23 Table 1.21 Relationship of water content of honey to refractive index………………. 24 Table 1.22 Current and potential applications for ultrasound in the food industry…… 31 Table 1.23 Some technical parameters of honey liquefied by ultrasonic waves of 23 kHz………………………………………………………………..……

36

Table 1.24 The effect of ultrasonic waves (23 kHz) and heat treatment (60 °C for 30 min) on the HMF levels of honey…………….…...……………………

36

Table 1.25 Crystallisation of honeys that were liquefied by ultrasonic waves and heat treatment……………………………………………………………...

37

Table 1.26 Effect on honey of microwave treatment at different power levels………. 38 Table 3.1 Technical details of ultrasound sonotrodes……………………………….. 47 Table 3.2 Details of treatments in the study of the ultrasound effect on the creamed honey process…….........................................................................

52

Table 3.3 Ultrasound treatment conditions for a combination of sonotrode and amplitude......................................................................................................

55

Table 3.4 Experimental design of the ultrasound treatment conditions……………... 55 Table 3.5 Six ultrasound input energy levels used to treat candied honey………….. 58 Table 3.6 Experimental design of six ultrasound treatment conditions used to treat candied honey………………………………………………………...

58

Table 3.7 Experimental design for the heat and ultrasound treatments……………… 61 Table 4.1 Crystalline D-glucose monohydrate contents of six creamed honey

samples......................................................................................................... 66

Table 4.2 Crystalline D-glucose monohydrate contents of four creamed honey samples.........................................................................................................

67

Table 4.3 Mean content of crystalline D-glucose monohydrate in canola/red gum (70:30) creamed honey…………………………………………………….

70

Table 4.4 Mean content of crystalline D-glucose monohydrate in alfalfa/blue gum (70:30) creamed honey…………………………………………………….

71

x

List of Tables (Continued) Table 4.5 Input energy, maximum net power, treatment time and maximum

temperature of honey treated with the 40 mm diameter sonotrode at the 12 µm amplitude setting …………………………………………………..

75

Table 4.6 Input energy, maximum net power, treatment time and maximum temperature of honey treated with the 40 mm diameter sonotrode at

the 9 µm amplitude setting...........................................................................

75


the 6 µm amplitude setting……………………………………………...…

76


the 3 µm amplitude setting……………………………………………...…

76

Table 4.9 Input energy, maximum net power, treatment time and maximum temperature of honey treated with the 22 mm diameter sonotrode at the 100 µm amplitude setting……………………………………….................

79


the 75 µm amplitude setting…………………………………………..…...

79


the 50 µm amplitude setting……………………..……………...................

80


the 25 µm amplitude setting………………..…………………………...…

80

Table 4.13 Maximum net power, input energy, treatment time and maximum temperature of honey treated with the 7 mm diameter sonotrode at the 175 µm amplitude setting…………………………………………………

83

Table 4.14 Maximum net power, input energy, treatment time and maximum temperature of honey treated with the 7 mm diameter sonotrode at the 131.25 µm amplitude setting ……………………………………………...

83

Table 4.15 Maximum net power, input energy, treatment time and maximum temperature of honey treated with the 7 mm diameter sonotrode at the 87.5 µm amplitude setting ………...............................................................

84

Table 4.16 Maximum net power, input energy, treatment time and maximum temperature of honey treated with the 7 mm diameter sonotrode at the 43.75 µm amplitude setting………………………………………………..

84

Table 4.17 Mean maximum net power achieved over the six ~10000 J energy inputs for the 12 ultrasound treatment combinations……………………..

87

Table 4.18 Mean cumulative treatment times for the six ~10000 J energy inputs for each of the 12 ultrasound treatment combinations ………………………..

89

Table 4.19 Mean maximum temperatures reached after a ~60000 J treatment for each of the 12 ultrasound treatment combinations……………..……….…

90

Table 4.20 Mean actual input energy and instrumentally set input energy for treatment with the 40 mm sonotrode ultrasonic sonotrode operated

at the 12 µm amplitude and 100% cycle (continuous)…………..………...

92

Table 4.21 Mean maximum temperature of Salvation Jane honey, total net power and overall treatment time for treatment by the 40 mm sonotrode ultrasonic sonotrode operated at the 12 µm amplitude and 100% cycle (continuous)..

93

xi

List of Tables (Continued) Table 4.22 Mean hydroxymethylfurfural concentration of ultrasound (40 mm

sonotrode, 12 µm amplitude)-treated Salvation Jane honey and heat-treated Salvation Jane honey………………………………………………

93

Table 4.23 Mean diastase activity of ultrasound (40 mm sonotrode, 12 µm amplitude)-treated Salvation Jane honey and heat-treated Salvation Jane honey………………………………………………………………………

94

Table 4.24 Mean invertase number of ultrasound (40 mm sonotrode, 12 µm amplitude)-treated Salvation Jane honey and heat-treated Salvation Jane honey……………………………………………………………………....

96

Table 4.25 Mean values of input energy, treatment time and maximum temperature of honey treated with ultrasound ……………….……………

98

Table 4.26 Crystallisation of heat-treated honey samples (up to 112 days)…………... 99 Table 4.27 Crystallisation of ultrasound-treated honey samples (up to 112 days)……. 101 Table 4.28 Visual observations of heat-treated and ultrasound-treated honey

containers after 203 days (29 wks)………………………………………... 104

Table 4.29 Ultrasound maximum bulk power, treatment time and maximum energy for an input energy of ~70000 J required to liquefy ~250 g

candied honey……………………………………………………………...

108

xii

List of Figures Figure 3.1 Instrument setup of the Malvern Mastersizer/E and ultrasound processor…...… 41 Figure 3.2 Differential scanning calorimeter…………………………………………......... 45 Figure 3.3 Ultrasound equipment setup………………………………………..…………… 47 Figure 3.4 Image analyser………………………………………………………………….. 49 Figure 4.1 Effect of controlled ultrasound on the crystal shape in crystallised reworked

honey …………………………………………………………………………… 68

Figure 4.2 Mean maximum net power versus each ~10000 J of input energy for honey treated with the 40 mm diameter sonotrode at four amplitude settings…………

77

Figure 4.3 Mean cumulative treatment time versus each ~10000 J of input energy for honey treated with the 40 mm diameter sonotrode at four amplitude settings….

78

Figure 4.4 Mean maximum temperature versus each ~10000 J of input energy for honey treated with the 40 mm diameter sonotrode at four amplitude settings…………

78

Figure 4.5 Mean maximum net power versus each ~10000 J of input energy for honey treated with the 22 mm diameter sonotrode at four amplitude settings…………

81

Figure 4.6 Mean cumulative treatment time versus each ~10000 J of input energy for honey treated with the 22 mm diameter sonotrode at four amplitude settings.....

82

Figure 4.7 Mean maximum temperature versus each ~10000 J of input energy for honey treated with the 22 mm diameter sonotrode at four amplitude settings…………

82

Figure 4.8 Mean maximum net power versus each ~10000 J of input energy for honey treated with the 7 mm diameter sonotrode at four amplitude settings…………..

85

Figure 4.9 Mean cumulative treatment time versus each ~10000 J of input energy for honey treated with the 7 mm diameter sonotrode at four amplitude settings…...

86

Figure 4.10 Mean maximum temperature versus each ~10000 J of input energy for honey treated with the 7 mm diameter sonotrode at four amplitude settings…………..

86

Figure 4.11 Individual value plot of the maximum net power achieved during six ~10000 J energy inputs versus sonotrode diameter and amplitude (95%

confidence interval for the mean)………………………………………….……

88

Figure 4.12 Individual value plot of the cumulative treatment time over six ~10000 J energy inputs versus sonotrode diameter and amplitude (95% confidence interval for the mean)……………………………………………………………

89

Figure 4.13 Individual value plot of the maximum temperature reached after six ~10000 J energy inputs versus sonotrode diameter and amplitude (95%

confidence interval for the mean)……………………….………………………

90

Figure 4.14 Individual value plot of hydroxymethylfurfural concentration versus ultrasound treatment (95% confidence interval for the mean)………..…

94

Figure 4.15 Individual value plot of diastase activity versus ultrasound treatment (95% confidence interval for the mean)….……………………………………………

95

Figure 4.16 Individual value plot of invertase activity versus ultrasound treatment (95% confidence interval for the mean)……………………..………...........................

96

Figure 4.17 Growth of needle crystals with time in heat-treated honey (identical view on one slide of one replicate over time)…………………………………...

99

Figure 4.18 Plate crystal formation within needle crystal masses (selected images from different heat-treated samples from 8 different slides for different replicates)....

100

Figure 4.19 Growth of needle crystals in ultrasound-treated honey (identical view on one slide of one replicate over time)…………………………………………

102

Figure 4.20 Growth of needle crystals on a plate crystal in ultrasound-treated honey (identical view on one slide of one replicate over time)…………………...……

102

Figure 4.21 Growth of plate crystals from the blunt ends in ultrasound-treated honey (identical view on one slide of one replicate over time……………………..…..

103

Figure 4.22 Technical data of the Dr Hielscher GmbH UIP 1000 ultrasonic processor…….. 111 Figure 4.23 Technical data of the Dr Hielscher GmbH UIP 4000 ultrasonic processor…….. 112 Figure 4.24 Factors affecting the temperature during ultrasound treatment……………... 113

xiii

Executive Summary What the report is about? This report details a successful development of a process for liquefying candied honey based on the use of ultrasound technology. This research is important due to the concern within the honey industry that the present heating regime used to liquefy candied honey is reducing the quality of honey, particularly its flavour. Therefore, it was necessary to undertake this research project to determine if it was feasible and cost effective to replace the present heat treatment used by the honey industry for the liquefaction of naturally crystallised or candied honey, with an ultrasound treatment. The experiments undertaken used a laboratory scale ultrasound processor to gather critical data that could be used to support industrial scale-up trials within a honey packing company, or by beekeepers. In addition, the Dyce creamed honey process was examined with respect to the factors that affect the crystallisation of D-glucose monohydrate and thus the quality of creamed honey, and whether ultrasound treatment could improve the quality of creamed honey. Who is the report targeted at? Beekeepers and honey packing companies. Background It is a common problem within the honey industry for heat-treated liquefied honey to crystallise during storage, particularly during cold weather. Since liquid honey is preferred by Australian consumers, and by food companies (for ease of handling), then an alternate method to expensive and time-consuming heating is required to retard the crystallisation process in honey. Creamed honey production is a difficult process to control, particularly related to crystallisation, the size of the D-glucose monohydrate crystals, and thus the quality of the final product related to hardness and spreadability. The process used by the honey industry to produce creamed honey is based on the Dyce method (Dyce, 1931a,b; Dyce, 1976). Ultrasound treatment has the potential to control this crystallisation process. However, before any effects of ultrasound can be determined, it is necessary to be able to produce high quality creamed honey in the laboratory using the Dyce method, and to be able to determine the amount of crystalline D-glucose monohydrate present in creamed honey. Ultrasound treatment was examined to determine if it can improve the creamed honey process and the stability of the final product. Aims and Objectives Aims: (1) To reduce the amount of expensive heating and loss in quality during liquefaction of candied

honey by developing an alternate, cost-effective ultrasound based method for the partial or complete liquefaction of candied honey, with a view to ultrasound having direct application for beekeeper control of honey crystallisation, or for liquefying candied honey prior to decanting in a honey packing plant.

(2) To better control the texture of creamed honey spread by developing an ultrasound based method that enhances the nucleation rate and produces uniform crystal growth in a creamed honey system, with a view to it being used by beekeepers and honey processors for producing consistent and high quality creamed honey.

Objectives: (1) To investigate the effect of ultrasound treatment on candied honey, including individual glucose

monohydrate crystals (2) To determine the ultrasound conditions for liquefying candied honey and for controlling

crystallisation in honey (3) To investigate the effect of ultrasound treatment on the creamed honey production process

xiv

Methods Used 1. Effect of Ultrasound Treatment on the Liquefaction of Candied Honey The main study of the effect of ultrasound on candied honey was divided into three experiments. In the first ultrasound liquefaction experiment, it was necessary to determine which laboratory scale ultrasound sonotrode, out of the available 7 mm, 12 mm or 40 mm diameter sonotrodes, better liquefied candied honey. Candied honey (~250 g) was treated with ultrasound energy interrupted after each of six 10,000 J intervals, for predetermined input energy levels, using three ultrasonic sonotrodes, with the temperature profile in the honey being monitored during each interruption. Input energy, treatment time and power measurements were also recorded from the ultrasonic processor. The optimum sonotrode (40 mm diameter) and amplitude (12 µm) were selected in Experiment 1. The first aim of Experiment 2 was to determine the minimum input energy required for complete liquefaction. The use of too high an input energy not only wastes energy but will unnecessarily increase the temperature and treatment time. The second aim was to determine if the ultrasound treatment adversely affected the quality of the honey. The third aim was to determine the specific energy input (kWh) required to liquefy one kilogram of candied honey, in order for the developed novel ultrasound liquefaction method to be useful for the honey industry. In the second ultrasound liquefaction experiment, candied Salvation Jane honey samples were treated with six different ultrasound input energy levels using this optimum sonotrode and amplitude. These liquefied honey samples were analysed for their hydroxymethylfurfural (HMF) concentrations, and diastase and invertase activities, since these three quality parameters are normally used by the honey industry and regulators to gauge the heating history and quality of honey. To complete the ultrasound liquefaction study, a third experiment was carried out to determine the effect of ultrasound treatment, relative to heat treatment, on the stability of liquid honey with respect to subsequent crystallisation on storage. In the third ultrasound liquefaction experiment, candied reworked mixed honey (~200 g) was completely liquefied by ultrasound treatment. Reworked mixed honey was selected for this trial as it is a very fast crystallising honey that produces large crystals. This permitted a crystallisation study to be completed in a short time-frame. Crystallisation of ultrasound-treated honey under optimum conditions of 14 °C was monitored (using a microscope as part of an image analyser) and compared with crystallisation in honey samples initially treated with a standard heat treatment. 2. Evaluation of the Effect of Ultrasound Treatment on the Creamed Honey Production Process A study was carried out to optimise a method to determine the amount of crystalline D-glucose monohydrate present in creamed honey, and to produce a laboratory creamed honey with a similar level of D-glucose monohydrate crystals to that of the commercial Capilano Honey Ltd. creamed honey. Various honey blends were used to produce creamed honey using the Dyce method, and the amount of crystalline D-glucose monohydrate present in these laboratory creamed honeys was determined using a differential scanning calorimeter (DSC). Results/Key findings 1. Effect of Ultrasound Treatment on the Liquefaction of Candied Honey The main finding from the first experiment was that the 40 mm diameter sonotrode operated at the 12 µm amplitude was optimum for completely liquefying candied honey. While it has a lower maximum net power for any 1 s period during treatment than does the 22 mm diameter sonotrode, the maximum net power for the 40 mm diameter sonotrode increased steadily after each of the six interrupted 10000 J energy inputs as the honey liquefied, while the maximum net power for the 22 mm diameter sonotrode initially increased, but decreased markedly from the fourth 10000 J energy treatment onwards. As the candied honey liquefies, the power output from the sonotrode increases until the honey is liquid, at which point there is little increase in maximum net power. The decrease in net power after an initial increase observed for the 22 mm diameter sonotrode indicates that there was poor efficiency in the emission of energy from the 22 mm diameter sonotrode into the candied honey.

xv

The 7 mm diameter sonotrode produces a lower maximum net power than the other two sonotrodes, again indicating poor output efficiency of energy from this sonotrode into the candied honey. Cumulative treatment times were lower for the 40 mm sonotrode (324 s to 383.3 s; lowest for the 12 µm amplitude) relative to those for the 7 mm (588.3 s to 681.3 s) and 22 diameter (394.7 s to 871.0 s) sonotrodes. In addition, the variation in treatment times among replications was also lower for the 40 mm diameter sonotrode. The more efficient the emission of energy from the sonotrode to the honey, the shorter the treatment times. Finally, the maximum temperature reached after the sixth interrupted 10000 J of energy input was significantly (P<0.05) lower for the 40 mm diameter (66.2 °C to 67.8 °C) sonotrode relative to the 7 mm (78.2 °C to 84.4 °C) and 22 mm (76.4 °C to 82.8 °C) sonotrodes. This reflects the shorter treatment time for the 40 mm diameter sonotrode, which was possibly due to the high maximum net power produced by it. Key Finding: Since treatment times need to be as short as possible and temperatures as low as possible, then the 40 mm diameter sonotrodes operated at an amplitude of 12 µm is the optimum condition for complete liquefaction of honey on a laboratory scale. In the next experiment, a preliminary trial showed that a range of input energies from 50000 J to 70000 J would produce a range of liquefaction efficiencies from partially liquefied to completely liquefied. During a replicated trial involving six input energies between 50000 J and 70000 J, only an input energy of 70000 J completely liquefied candied Salvation Jane honey. The other energy inputs only partially liquefied the candied honey. In addition, the time needed to emit each of the fixed energies from the sonotrode increased from 304 s for 50000 J of input energy to 434.0 s for 70000 J of input energy, since it takes longer for a sonotrode to emit more energy. However, there was no significant (P>0.05) difference in the maximum temperature (which ranged 69 °C to 77.3 °C) after each of the six fixed energy treatments. Further, the maximum net power recorded at any 1 s interval during each energy level treatment was not different from each other. Key Finding: A 70000 J ultrasound energy treatment can be used to completely liquefy candied honey in a relatively short time of 434 s, without it adversely affecting the maximum temperature generated in the honey relative to lower energy treatments. There was no significant (P>0.05) difference in the HMF concentration in honeys treated with between 50000 J and 62500 J of input energy and honeys that were heat-treated. However, the HMF concentrations in the honeys treated with 65000 J and 70000 J of input energy were significantly (P<0.05) lower than the HMF concentration in the heat-treated honeys. This is primarily due to the honey being at the maximum temperature reached of 77.3 °C for a much shorter time (434.0 s) than for a heating regime (55 °C for 16 h and 72 °C for 2 min) which is similar to that presently used by the honey industry. The effect of the energy treatments on enzyme activity was negligible since there were no significant (P>0.05) differences in the diastase activity between honeys treated with any of the six energy inputs and those that were heat-treated, while the invertase activity of most of the ultrasound treated honey was higher than the heat-treated honeys, with this difference not always being significant. Key Findings: Use of an ultrasound input energy of 70000 J from a 40 mm sonotrode operated at an amplitude of 12 µm is sufficient to liquefy candied Salvation Jane honey (~250 g) without compromising honey quality. For example, this ultrasound treatment results in the production of a lower concentration of HMF from honey sugars , and no decrease in diastase and invertase activities, relative to a heating regime (55 °C for 16 h and 72 °C for 2 min) similar to that used by industry. The specific energy input needed to completely liquefy candied Salvation Jane honey is 0.126 kWh/kg. Therefore, 10 kg of candied honey will require 1.26 kWh, while 300 kg will require 37.9 kWh. The first finding from the third experiment was that the D-glucose monohydrate crystallised differently in each type of treated honey. In the heat-treated honey samples, the initial plate crystals that formed at between 14 and 28 days were long thin, spiral-shaped plate crystals. In contrast, in the

xvi

ultrasound-treated honey samples, most of the initial crystals that formed at between 14 and 49 days were large pentagon-shaped plate crystals. In addition, more needle crystal masses were formed in the heat-treated honeys than were produced in the ultrasound-treated honeys at the end of the monitoring period of 112 days (16 weeks). Moreover, in heat-treated honeys, plate crystals grew underneath these needle crystal masses in the later stages of crystallisation. In contrast, in ultrasound-treated honeys, plates formed after the needles, with subsequent needles growing on these initial plates. Key Finding: Ultrasound treatment delays D-glucose monohydrate crystallisation more than does a heat treatment similar to that used by the honey industry. This occurs at both the microscopic level (in a drop of honey) and in bulk samples. In addition, there is a difference in the crystal formation process at the microscopic level in ultrasound-treated honey relative to that in heat-treated honey. The reason for this is not clear, and requires further study. Thus, ultrasound treatment will not only liquefy candied honey without the need for long exposure to high temperatures, but may make the liquefied honey more stable to subsequent crystallisation on storage. 2. Evaluation of the Effect of Ultrasound Treatment on the Creamed Honey Production Process Samples of commercial Capilano Honey Ltd. creamed honey were initially analysed and found to have an average crystalline D-glucose monohydrate content of 39.6-40.1 g/100 g honey. In addition, two blends consisting of 70% alfalfa honey/30% blue gum honey and 70% canola honey/30% red gum honey were chosen for a subsequent replicated study, since these produced laboratory creamed honeys that were soft and spreadable, with crystalline D-glucose monohydrate contents of 21.6 g/100 g honey (after eight days storage at 14 °C) and 21.9 g/100 g honey (after 12 days storage at 14 °C) respectively. Alfalfa and canola honeys are fast crystallising, fine-grained honeys, while blue gum and red gum honeys have strong flavours. But the level of crystals was not high enough. A study was then carried out to increase the level of D-glucose monohydrate crystals in the laboratory-creamed honey produced using the above honey blends. One experimental factor that was changed was the length of storage time at 14 °C, since commercial processes use 42 days of storage to produce the maximum possible level of D-glucose monohydrate crystals. When 39 days of storage were used, a laboratory-creamed honey with a crystalline D-glucose monohydrate content of 47.1 g/100 g honey was produced, which is higher than that found in commercial Capilano Honey Ltd. creamed honey. Thus, the honey blend and creamed honey process had been optimised in the laboratory. To further study the creamed honey process, ultrasound treatment was investigated as a means of reducing the crystal size, and thus improving the spreadability of the product. Prior to the start of this project it was thought that ultrasound treatment of the D-glucose monohydrate crystals in honey would shatter them, thereby reducing their size. However, the use of an image analyser has shown that rather than reducing the crystal size through break up of the crystals, the crystal size is reduced through partial melting or dissolution of the D-glucose monohydrate crystals. The clean, sharp crystal structures (plates) are replaced by irregular shaped plates, indicating that some glucose molecules on the edge of the crystal structure dissolve into the surrounding liquid, producing plate crystals that have melted edges and surfaces. Finally, ultrasound treatment was then applied to the seed honey prior to it being added to the honey blend. The hypothesis is that if the size of the crystals in the seed honey can be reduced by ultrasound treatment, then the creamed honey process could be enhanced (producing a higher level of crystals) and the final honey would have smaller crystals. This was not the case, and the level of crystals was not significantly (P>0.05) different from the control creamed honey produced with seed honey that was not ultrasound treated. Included in this experiment was the use of ultrasound treatment of the seeded honey blends at one day and two days after the addition of the seed honey. Again, such treatments were hypothesised to reduce the crystal size and enhance subsequent crystallisation during storage at 14 °C. However, there was no significant (P>0.05) difference in the crystalline D-glucose monohydrate content relative to the control untreated creamed honey. None of the ultrasound treatments enhanced the level of D-glucose monohydrate crystals relative to the untreated creamed honey.

xvii

Key Finding: The untreated canola/red gum creamed honeys (47.1 g/100 g honey) had similar crystal contents to the ultrasound-treated canola/red gum creamed honeys (44.5 g/100 g honey to 47.1 g/100 g honey), while the untreated alfalfa/blue gum creamed honeys (33.1 g/100 g honey) had similar crystal contents to ultrasound-treated alfalfa/blue gum creamed honeys (32.2 g/100 g honey to 33.1 g/100 g honey). Finally, conditioning of the creamed honey product was investigated. The reason for this is that some of the creamed honey samples produced were creamy in texture and some were semi-solid in texture. There did not seem to be any particular treatment that led to one type of product over the other. In fact, replicates of the same treatment often had both types of texture. As part of the Dyce process (Dyce, 1931a,b; Dyce, 1976), a conditioning step is used prior to the creamed honey being sent to supermarkets for sale. Conditioning is where the creamed honey is stored at 30 °C for a number of days. This study found that such conditioning did produce consistency in texture with all product having a creamy texture. The storage at 30 °C for 14 days produced a small reduction in the crystalline D-glucose monohydrate content, with the final content being similar to that found for commercial Capilano Honey Ltd. creamed honey. Key Finding: The conditioning process dissolves some of the D glucose monohydrate crystals leading to an increase in the amount of liquid honey, and an overall softening of the creamed honey, with a consequent improvement in spreadability. While ultrasound treatment did not produce a product that was different to untreated creamed honey, conditioning the final product before sale is very important for producing a consistent product from one production run to another. Implications for relevant stakeholders for industry Once the data from an industrial trial have been obtained, an ultrasound equipment manufacturing company such as Dr Hielscher GmbH can then provide specifications for liquefying larger amounts of honey such as 300 kg in 200 L drums. However, while the time required to liquefy10 kg or 300 kg of candied honey will depend on the input power of the ultrasound processor and the capacity of the sonotrode/sonotrodes, it will be less than the time now used to liquefy candied honey in hot rooms. The newer, large plastic drums, which have a completely removable lid, would be ideal for use with the proposed ultrasound processing system. However, the one industrial scale problem likely to be encountered relates to the design of the commonly used 200 L galvabond drums. These drums have only small openings which, while permitting limited insertion of the sonotrode, would not permit the moving of the drum up and down and sideways in a predetermined pattern so as to expose all the candied honey to the ultrasonic waves. However, as part of the industrial trials and the subsequent design of the processor system by an ultrasound equipments manufacturer such as Dr Hielscher GmbH, such a limitation in the galvabond drums may be able to be overcome. It is recommended that part of the Australian honey industry such as major honey packing companies should undertake these types of industrial trials to ensure technology transfer from this project to industry. The results of this study of the Dyce creamed honey process will aid beekeepers and honey packing companies to better understand their creamed honey process and improve the quality and consistency of their product from batch to batch. Recommendations Honey packers must make use of this collected data, by taking up the challenge (and rental costs) of participating in a scaled-up industry trial involving an industrial ultrasound processor (much more powerful than used in this project) for liquefying 10 kg candied honey in commonly used plastic containers (e.g. diameter of 270 mm and height of 240 mm). This would be done in consultation with an ultrasound equipment manufacturer such as Dr Hielscher GmbH and the project’s research team. As part of the recommended system design, the ultrasound sonotrodes will have to be inserted into the drum to a particular depth in order to liquefy the honey down to the bottom of the drum. Initially, the sonotrode will be in contact with the hard candied honey near the top of the container. As liquefaction of the surface candied honey proceeds, the honey container will need to be moved upwards and

xviii

sideways in a predetermined pattern, so that the sonotrode is brought in contact with as much of the candied honey as possible, so as to minimise the treatment time required. In addition, or alternatively, a stirrer could be inserted in the semi-melted honey to mix the liquid honey with the remaining candied honey, thereby creating a flow in the container past the treatment region around the sonotrode. Ultrasound waves dissipate quickly at a short distance from the sonotrode, so some mixing is required.

1

1. Introduction This is a review of literature related to composition, physical characteristics, and storage and processing of honey, and the application of ultrasound in food processing operations. Research work on beekeeping and hive management was not investigated in this project. 1.1 What is Honey? Honey is defined as the natural sweet substance produced by honeybees from nectar of plants, from secretions of living plants or excretions of plant sucking insects on the living parts of plants, which honeybees collect and transform by combining with specific substances of their own. These are then deposited, dehydrated, stored left in the honey comb to ripen and mature (Codex Alimentarius, 1998). The biological definition is as follows: Honey is a substance produced by honeybees and some other social insects from nectar or honeydew collected from living plants. They transform the nectar or honeydew by evaporating water and by the action of enzymes they secrete. As a rule, honeybees seal the finished honey in cells of their comb (Crane, 1990). Bee honey is broadly categorised as blossom honey and honeydew honey. Blossom honey is formed from the nectar of plants. Honeydew honey is produced from honeydew which consists of excretions of plant sucking insects (Codex Alimentarius, 1998; Clemson, 1985; Doner, 1977). 1.2 Chemical Composition of Honey The composition of honey depends on the varieties of flowering plants that nectar is collected from and environmental conditions. Therefore, the composition of honey types produced in the world is variable. Clemson (1985) reported that soil moisture, humidity, temperature, sunlight and other atmospheric conditions have an effect on nectar production. Further, different plants require different environmental requirements though they have the same genes. As an example, white box (Eucalyptus albens) and spotted gum (Eucalyptus maculate) trees require frosty nights while yellow box (Eucalyptus melliodora) trees need hot nights and still conditions for better nectar production. Honey is a supersaturated sugar solution, with sugars and water making up 99% of most honeys. The remaining 1% consists of enzymes, aroma constituents, organic acids, minerals and other substances, that are responsible for a number of the characteristics of honey (Crane, 1990). The mean amounts (% of total honey) of constituents of honey produced in Russia, U.S., Rumania and Australia are shown in Table 1.1. The amounts for the minor constituents in U.S. honey samples are also listed. The sugar content of honey is four and a half times the water content of honey. Thus, the high concentration of sugar in honey is responsible for its keeping quality and high viscosity (Crane, 1990).

2

Table 1.1 Mean amounts (% of total honey) of constituents of honey

Constituent Mean Russia U.S. Rumania Australia Australia* Range Fructose 39.3 37.4 38.2 38.4 43.3 37.5 21.7-53.9 Glucose 32.9 35.9 31.3 34.0 30.2 28.1 20.4-44.4 Sucrose 2.3 2.1 1.3 3.1 2.5 1.1 0.0-7.6 Water 17.0 18.6 17.2 16.5 15.6 18.5 13.4-26.6

Disaccharides (as maltose) 7.3 1.1 2.7-16.0 Higher sugars 1.5 0.6 0.1-8.5

Total acids (as gluconic) 0.57 0.17-1.17 Minerals 0.17 0.2 0.02-1.03

Nitrogen (in amino acids and proteins) 0.04 0.00-0.13

Adapted from Crane, 1990; except for Australian honey data by *Mossel (2002). 1.2.1 Carbohydrates in honey Honey consists of a mixture of carbohydrates. White & Hoban (1959), Siddiqui & Furgala (1967, 1968), and Low & Sporns (1988) identified the presence of 23 carbohydrates. Glucose and fructose are monosaccharides that make up 65-75% of the total soluble solids and 85-95% of the honey carbohydrates. Researchers have reported the presence of several disaccharides, trisaccharides and oligosaccharides. Table 1.2 summarises data on carbohydrate contents of honeys from different countries. 1.2.1.1 Monosaccharides in honey Fructose and glucose are monosaccharides that account for about 85-95% of honey carbohydrates (Crane, 1990). Honeybees add the enzyme invertase to the nectar or honeydew, which produces fructose and glucose by hydrolysis of the sucrose present in the nectar. The fructose to glucose ratio is a characteristic of some honeys. Most honey types including robina, salvia, tupelo and sweet chestnut honeys are richer in fructose than glucose. However, canola, dandelion and blue curls honeys are unusually richer in glucose (Amor, 1978). White (1976a) reported the glucose and fructose content of honeys from throughout the world, with for example the average fructose and glucose contents of U.S. honeys being 38.19% and 31.28% respectively (Table 1.2). When the data on Australian honeys reported by White (1976a) and Mossel (2002) are compared, it can be seen that comparable contents of fructose and glucose were obtained for honey samples sourced 26 years apart (Table 1.2). 1.2.1.2 Disaccharides in honey Sucrose is a disaccharide that comprises 1 – 3% of the composition of honey, and consists of fructose and glucose units linked together by a glycosidic bond. White & Hoban (1959) isolated and identified isomaltose, maltulose, turanose and nigerose from honey. They also confirmed the presence of sucrose and maltose in honey. Siddiqui & Furgala (1967) isolated and characterised gentiobiose and laminaribiose, and confirmed the presence of maltose, kojibiose, isomaltose, nigerose and neotrehalose in honey. Low & Spons (1988) reported the presence of sucrose, trehalose, neotrehalose, cellobiose, laminaribiose, nigerose, turanose, maltulose, maltose, kojibiose, gentiobiose, palatinose and isomaltose in honey. Spanish honeys contain sucrose, maltose, and kojibiose (Mateo & Bosch-Reig, 1997). Da Costa Leite et al. (2000) reported that Brazilian honey contains sucrose, maltose, turanose, nigerose, melibiose and isomaltose. Mossel (2002) reported concentrations of sucrose (Table 1.2), maltose (Table 1.2) and turanose (0.37 – 1.44 g/100 g honey) in 126 samples of honeys from 15 different species-specific types of Australian honey.

3

Table 1.2 Carbohydrate contents (and/or range) of honeys in different countries

Country Total Glucose Fructose Sucrose Maltose

reducing

sugars % % % % % (Range) (Range) (Range) (Range) (Range)

EUROPE Britain - 34.6 39.8 - -

30.0-36.8 36.1-44.4 Bulgaria 71.7 - - 1.6 -

Italy - - - - - 58.9-84.8 18.0-43.5 30.2-49.2 0.0-10.1 0.1-4.3

Netherlands - 30.5 41.5 - 4.6 - 20.4-39.5 38.1-53.9 - 1.8-7.5

Portugal - 32.2 - - - - 27.2-34.6 31.0-38.2 0.7-1.2 5.1-8.9

Rumania 75.6 34.0 38.4 3.1 - - - - - -

Spain 68.8 30.8 38.1 1.85 - 55.7-77.9 24.2-38.2 31.6-42.9 0.5-3.7 -

Yugoslavia 73.0 35.2 37.3 1.91 - 65.5-79.1 26.1-43.2 30.1-44.9 0.15-4.70 -

ASIA India - 35.7 39.3 0.6 -

- 34.2-39.2 36.8-40.5 0.3-1.0 - Japan 69.2 32.6 36.0 2.83 -

60.5-76.1 22.2-38.6 30.0-48.5 1.0-5.8 - Pakistan - 40.4 28.26 2.12 -

- 39.0-53.8 27.7-34.2 1.90-2.75 - Korea - 23.6 30.9 1.63 2.68

- 24.0-29.9 29.6-33.4 0.87-3.57 1.97-3.80 Nepal - 41.95 45.9 1.96 3.26

- 36.3-46.3 42.3-50.4 0.0-7.80 1.61-4.13 Taiwan - 28.7 23.5 - -

- 17.1-36.2 20.7-46.8 - -

AFRICA Angola - 33.9 36.4 0.86 6.48

- 32.0-35.0 34.2-38.5 0.15-1.50 4.56-7.79 Mozambique - 32.0 36.2 1.1 6.51

- 28.6-35.3 33.6-38.2 0.65-1.60 5.1-7.58 Portuguese Guinea - 31.2 38.2 1.06 6.36

- 28.5-34.4 35.6-40.8 0.45-2.20 3.55-8.80 Sao Tome+Principe - 31.0 34.8 0.61 5.97

- 28.5-32.4 33.9-36.2 0.10-1.00 5.13-7.34 South Africa - 31.5 35.5 0.54 5.4

- 22.3-39.4 22.3-40.1 0.0-6.24 2.1-10.0

4

Table 1.2 (Continued)

AMERICAS Argentina - 34.3 40.9 - -

- 29.4-37.2 37.7-44.9 - - Canada - 34.6 37.6 0.6 1.6

- 30.4-39.3 35.6-40.2 0.00-6.50 0.63-3.30

Chile 79.2 32.7 44.1 2.5 73.7-80.7 30.1-35.0 39.1-47.0 0.90-4.40 -

Uruguay 67.3 - - 4.9 -

U.S. - 31.28 38.19 4.9 7.31

- 22.0-40.7 37.2-44.2 1.31-7.75 2.74-15.98

OCEANIA -

Australia 73.5 30.2 43.3 2.5 - Australia* 62.58-72.67 25.24-30.58 32.60-41.72 0.49-2.45 0.53-3.27

New Zealand - 36.2 40.0 2.8 -

- 32.4-40.2 38.4-42.0 1.5-4.8 - All data was adapted from White (1976a) except for one set of Australian honey data from *Mossel (2002). Single values are averages; others are ranges; and, - indicates data were not included in the publication.

1.2.1.3 Trisaccharides and oligosaccharides in honey Trisaccharides consist of three simple sugar subunits and oligosaccharides contain more than three sugar units. These are formed from mono and disaccharides. The presence of erlose was first reported by White & Maher (1954). Siddiqui & Furgala (1967) isolated and identified melezitose, 3-α-isomaltosylglucose, maltotriose, 1-kestose, panose, isomaltotriose, erlose, theanderose, isopanose, isomaltotetraose and isomaltopentaose from honey. Low & Sporns (1988) reported the presence of 1-kestose, isopanose, erlose, theanderose, maltotriose, panose and isomaltotriose in honey. Mateo & Bosch-Reig (1997) showed that Spanish honey contains maltulose, raffinose, erlose and melezitose. Da Costa Leite et al. (2000) analysed 70 Brazilian honeys of different floral types and confirmed the presence of maltotriose, panose, melezitose and raffinose. Siddiqui & Furgala (1967, 1968) (cited by Doner, 1977) showed that oligosaccharides make up 3.65% of honey solids (Table 1.3). 1.2.2 Moisture content of honey The moisture content plays an important role in the stability of honey in relation to fermentation and granulation during storage. Normally, the moisture content of ripened honey is below 18.6% (White, 1978). The analysis of U.S. honeys by White et al. (1964) found that the average moisture content of honey is 17% with a range of 13.4 – 22.9%. A recent study of Australian floral honeys showed that the moisture level was 17.22 – 19.60% (Mossel, 2002). Singh & Bath (1997) reported moisture content of 21.8% for Basic jounce honey and 19.4% for Eucalyptus lanceolatus honeys in their analysis of Indian honeys. Junzheng & Changying (1998) analysed 46 varieties of Chinese honeys and reported 29% moisture content for acacia honey and a range of 19.8-27.5% for other honeys. These values are higher relative to the moisture contents of ripened honey. White (1976b) quoted the work by Lochhead (1933) who reported that honeys with a moisture content below 17.1% will not

5

ferment and above 20% are liable to ferment. However, honey with a moisture content between 17.1-20% will ferment depending on the yeast counts in the honey. It also seems that honeybees are unable to reduce the water content of honey to a safe level in regions that have high atmospheric humidity. As an example, the honey from rubber (Hevea brasikiensis) in Sri Lanka may contain 25% water content in sealed honey (Crane, 1990). In addition, honey from certain plant sources characteristically have high moisture content for specific reasons. As an example, heather (Calluna vulgaris) honey has a moisture content between 19.2 and 26% (Crane & Walker, 1984).

Table 1.3 Yields of the principal sugars in the oligosaccharide fraction (3.65%) of honey Disaccharides % Trisaccharides % Higher

oligosaccharides %

Maltose 29.4 Erlose 4.5 Isomaltotetraose 0.33 Kojibiose 8.2 Theanderose 2.7 Isomaltopentaose 0.16 Turanose 4.7 Panose 2.5 Isomaltose 4.4 Maltotriose 1.9 Sucrose 3.9 1-Ketose 0.9 Maltulose (and 2 unidentified ketoses)

3.1 Isomaltotriose 0.6

Nigerose 1.7 Melezitose 0.6 α β-Trehalose 1.1 Isopanose 0.24 Gentiobiose 0.4 Centose 0.05 Laminaribiose 0.09 3-α-isomaltose-glucose trace Adapted from Doner (1977) 1.2.3 pH and acidity of honey The average pH of American honey is 3.9 with a range of 3.4 to 6.1 (Anon, 2003a; White, 1976a). The comparative values for Australian honey is 3.6-6.6 (Langridge (1971), 3.8 - 5.4 (Chandler et al., 1974) and 4.0-4.4 (Mossel, 2002). Honey contains 0.17 - 1.17% organic acids and 0.05 - 0.15% amino acids. The organic acids reported to be present in honey are acetic, butyric, citric, formic, gluconic, lactic malic, pyroglutamic and succinic acid. The dominant organic acid is gluconic acid which forms by glucose oxidase acting on glucose (Stinson et al., 1960). White (1978) reported the probability of the presence of glycolic, α-ketoglutaric and pyruvic, tartaric and 2- or 3-phosphoglyceric acid, α - or β-glycerophosphate and glucose-6-phosphate in honey. Further, a higher acidity level of honey is reported to increase the stability of honey against microbiological actions (Stinson et al., 1960). Finally, there are 18 amino acids present in honey and proline is most dominant (White, 1978). 1.2.4 Ash and mineral content of honey Minerals are found in the ash component of honey. White (1978) reported an average ash content of 0.17%, with a range of 0.02-1.03% in honey. The minerals present in honey are potassium, sodium, calcium, magnesium, iron, copper, manganese, chlorine, phosphorus, sulphur and silica. Potassium is the major element, which on average makes up 33-35% of honey ash, while iron, copper and manganese are present in honey in small amounts. The mineral elements of honey published by White (1976a) are given in Table 1.4. Amor (1978) quoted the work by Schuette & co-workers on the ash content and colour of honey, who reported that light coloured honeys have an average ash content of 0.065% and dark honeys have an average ash content of 0.173%. Australian honeys have ash contents of 0.10-0.37% (Mossel, 2002).

6

1.2.5 Hydroxymethylfurfural content of honey Hydroxymethylfurfural (HMF) is produced by acid catalysed dehydration of hexoses. In honey, glucose and fructose decompose in the presence of gluconic acid to form HMF (Kuster, 1990). This process is reported to be enhanced by heat or storage under elevated temperatures. Bogdanov et al. (1997) reported that practically there in no HMF in fresh honeys, but the level increases during storage depending on the pH of honey and the storage temperature. However, a small amount of HMF is present in fresh honeys, particularly Australian honeys and the amount increases according to the temperature and duration if honey is heated. Honey is heated at different stages of its processing to reduce viscosity, destroy yeast, and dissolve crystals. However, such heat treatments increase the HMF content of honey. Therefore, HMF content can be used as an indicator to detect the heat damage and adulteration of honey. Further, it has been reported that the HMF content of honey increases during storage in the warm climates of tropical and subtropical countries. The latest Codex standards for the HMF content of honey is set as less than 40 mg/kg after processing and/or blending of honey. However, a standard for HMF of less than 80 mg/kg has been set for the honeys produced in countries or regions with tropical ambient temperatures and in blends of these honeys (Codex Alimentarius, 2001. Revised codex standard for honey). The HMF content in Australian honey is 4.09-33.65 mg/kg (Mossel, 2002).

Table 1.4 Mineral elements detected in honey As percentage of ash Mineral Element Honey Colour Range Average Potassium Light

dark 23.0-70.8 2.0-61.6

35.30 33.00

Sodium Light dark

0.96-9.26 0.2-11.20

3.59 4.68

Calcium Light dark

3.54-13.00 0.46-7.30

8.77 3.57

Calcium as lime (CaO) Light dark

4.95-18.19 0.64-10.21

12.27 5.00

Magnesium Light dark

1.00-9.24 0.66-11.47

3.42 2.77

Chlorine Light dark

4.52-13.21 2.26-14.46

10.20 9.67

Phosphorus Light dark

1.03-9.55 0.84-6.67

6.37 3.67

Sulphur Light dark

5.77-16.24 2.67-14.36

11.49 7.98

Silica Light dark

0.58-2.23 0.17-1.79

1.60 1.00

Adapted from White (1976a)

7

1.2.6 Enzymes in honey Enzymes are another important constituent of honey because they play an important role in honey production from the nectar of the plant. Further, enzymes are heat sensitive and extra low levels indicate that honey has been overheated. Further, their activities are decreased during storage and used as indicators of the freshness of honey. Table 1.5 lists the enzymes in honey (Crane, 1990).

Table 1.5 Enzymes in honey

Enzyme Characteristics From worker honeybees Invertase Glucose oxidase

Hydrolyses (inverts) sucrose to glucose and fructose; more heat sensitive than amylase. Oxidises glucose to gluconic acid and hydrogen peroxide in the presence of water.

From plants (nectar/honey dew) Catalase Acid phosphatase α- and β-Amylase (diastase)

Regulates activity of glucose oxidase by controlling hydrogen peroxide equilibrium. Occurs in pollen, and in nectar and honey, but very little in honey stored by honeybees fed sucrose. A small portion in some honey is from plants.

Adapted from Crane (1990) Honeybees add invertase (α-glucosidase) to nectar during the process of harvesting and ripening of honey. The hydrolytic activity of invertase on sucrose finally produces glucose and fructose. Some invertase may be present in extracted honey, which will result in the sucrose inversion taking place during storage (Crane, 1990). Recently, Bonvehi et al. (2000) studied the invertase activity of Spanish monofloral honeys from different floral origins and reported that invertase activity is 6 - 46.2 SN (Table 1.6). Invertase activity is expressed as g sucrose hydrolysed per 100 gh-1 (SN). Chestnut honey (Castenea sativa) had the highest activity of 19.2 to 32.63 SN among the Spanish honey studied. Glucose oxidase is active only in unripe or dilute honey, and most active when the sugar concentration is between 25% and 30%. This enzyme oxidises glucose and produces gluconic acid and hydrogen peroxide. The activity of glucose oxidase decreases with an increase in reducing sugar concentration during honey production, and disappears when nectar is fully converted to reducing sugars. Schade et al. (1958) analysed ten American honey samples for diastase activity before and after storage at 20 °C for 15 months (Table 1.7). The diastase activity decreased slightly in most of the samples. α- and β-Amylase (diastase) break down starch and do not seem to be involved in any chemical reaction in honey. However, it is very heat sensitive and may be used as an indicator to identify overheated honey during processing (Crane, 1990). The data listed in Tables 1.6 and 1.7 show that invertase activity and diastase activity have a wide variability among different floral honeys. Australian honeys have diastase activities of 9.77-18.84 Shade units (G°) (Mossel, 2002).

8

Table 1.6 Invertase activity (g sucrose hydrolysed per 100 g h-1)

Honey floral source X SD Vmax Vmin Castanea sativa 25.61 5.84 32.63 19.20 Rosmarins officinalis 14.43 5.73 24.00 6.00 Dorycnium pentaphyllum 10.09 2.98 13.24 6.69 Erica arborea 13.57 4.98 18.70 5.25 Erica cinerea 18.30 5.59 29.00 14.00 Erica vagans 8.36 2.18 10.34 6.85 Citrus spp 9.15 2.02 12.25 6.97 Eucalyptus spp 10.65 4.89 15.97 6.34 Hedysarum coronarium 4.84 2.26 8.40 0.85 Quercus spp 17.20 4.41 22.80 11.90 Robina psedoacacia 4.04 0.58 4.70 3.70 Lavadula stoechas 14.42 2.33 17.10 11.40 Polyfloral 21.38 9.69 46.20 10.57

X – Mean invertase activity; SD – standard deviation; V max – maximum value detected; Vmin – minimum value detected Adapted from Bonvehi et al. (2000)

Table 1.7 Comparison of diastase activity before and after storage for 15 months

Sample number Diastase Shade Units (G°)

Before After 1 12.0 11.5 4 27.5 26.1 5 5.8 5.2 6 14.8 14.0 7 9.9 7.4 8 4.0 - 9 20.0 16.6

10 22.4 21.6 Adapted from Schade et al. (1958) Catalase, an enzyme from plants, breaks down hydrogen peroxide responsible for antimicrobial activity. Bilberry and heather honeys contain much catalase and show least antimicrobial activity. White clover and Scots pine honeys do not contain catalase. Thus, they have high antimicrobial activity due to high hydrogen peroxide levels. Further, acid phosphatase is in nectar, but the activity of this enzyme in honey is not known. 1.2.7 Colour of honey The colour of honey depends on the floral source and its mineral content (Anon, 2003b). Perez-Arquillue et al. (1994) reported that honey with a higher mineral content is darker in colour. Rogers (1976) reported that the colour of honey sourced from the same plants also depends on the climatic factors and the honey ripening temperature in the hive. Published work reported that honey

9

colour is a temperature sensitive parameter, and honey can become darker as a result of different storage conditions (Anon, 2003b). U.S. Department of Agriculture has classified honey into seven categories (Table 1.8). They are water white, extra white, white, extra light amber, light amber, amber and dark amber. The Pfund colour grader is used by the honey industry for grading honeys. Australian honey studied by Mossel (2002) had Pfund colour readings of 32-78 mm.

Table 1.8 Colour designations of extracted honey USDA Colour Standards Designations

Colour range USDA Colour Standards

Colour Range Pfund Scales in mm

Water White Honey that is a water white or lighter in colour.

8 or less.

Extra White Honey that is darker than water white, but not darker than extra white in colour.

Over 8 to and including 17.

White Honey that is darker than extra white, but not darker than white in colour.


Extra Light Amber Honey that is darker than white, but not darker than extra light amber in colour.


Light Amber Honey that is darker than extra light amber, but not darker than light amber in colour.


Amber Honey that is darker than light amber, but not darker than amber in colour.


Dark Amber Honey that is darker than amber in colour.

Over 114.

Source: U.S. Standards for grades of extracted honey, U.S. Department of Agriculture (Anon, 1985) 1.2.8 Flavour and aroma of honey Honey flavour and aroma are defined as the degree of taste excellence and fragrance or odour of honey (Anon, 1985). Crane (1990) indicated that the substances that give honey its aroma are derived from specific plants. These substances are volatile and evaporate more rapidly, while heating honey above 30 – 35 °C will degrade the flavour and aroma. White (1978) reported that flavour is a characteristic that can be used to identify floral types. Further, flavours range from most delicate to harsh, with lighter coloured honeys having milder flavours. The flavours in relation to floral varieties, and flavour and aroma attributes of honey published by National Honey Board, U.S. are presented in Tables 1.9 - 1.11 respectively (Anon, 2003c). Volatile compounds responsible for flavour and aroma in Australian honeys have been reported by Rowland et al. (1995), and D’Arcy et al. (1997, 2001a).

10

Table 1.9 Floral varieties and flavours

Floral Variety Characteristics Avocado honey Molasses prune and metallic flavour. Clover honey Light colour, spicy cinnamon aroma and spicy cinnamon flavour Blueberry honey Low viscosity, flowery perfume, green and lemon aroma and fruity

flavour. Eucalyptus honey Sweet aroma, clover nectar aroma, sweet flavour, clover nectar flavour,

lingering flavour, sweet aftertaste. Buckwheat honey Dark colour, sharp and chemical and medicinal aroma, barny, medicinal,

green and bitter flavour, pungent aromatic, lingering molasses prune aftertaste and flavour.

Orange blossom honey Sweet, flowery perfume, clover nectar and waxy aroma, sweet, flowery perfume, clover nectar and waxy flavour, sweet and flowery perfume aftertaste.

Tupelo honey Sweet, flowery perfume and spicy cinnamon aroma, flowery perfume and spicy cinnamon flavour, flowery perfume and fruity aftertaste.

Sage honey Thick / viscous with clover nectar flavour. Sourwood honey Anise aroma and flavour, astringent, relatively low on sweet flavour.

Source: Sensory attributes of honey, National Honey Board, U.S. (Anon, 2003c)

Table 1.10 Flavour attributes of honey Flavour Description Sweet Intensity of sweet flavour, a sweet typical of honey. Clover Nectar Intensity of flavour like clover nectar. Green Intensity of green flavour, this can include any green growing thing and

green un-ripened fruit. Spicy Cinnamon Intensity of spicy cinnamon like flavour, tastes like red hots. Waxy Intensity of waxy flavour like beeswax. Flowery Perfume Intensity of flowery or perfumery flavour including white lilies or citrus

blossom. Anise Intensity of anise flavour like black licorice or anise candy. Molasses Prune Intensity of flavour like molasses, brown sugar, a caramelised flavour,

horehound candy, prunes or raisins. Medicinal Intensity of medicine like or medicinal flavour may be like varnish, outer

part of the mango. Fruity Intensity of fruity flavour like apricot, peach, lemon, orange. Barny Intensity of a barn-like flavour characteristic of some dark honey. Metallic Intensity of metallic flavour or iron flavour. Bitter Intensity of bitter flavour.

Source: Sensory attributes of honey, National Honey Board, U.S. (Anon, 2003c)

11

Table 1.11 Aroma attributes of honey Aroma Description Sweet Intensity of sweet aroma, sweet as typically comes from honey. Clover Nectar Intensity of aroma like clover nectar, a sweet flowery nectar but quite

delicate. Green Intensity of green aroma, this can include any green plant and green un-

ripened fruit. Spicy Cinnamon Intensity of spicy cinnamon like aroma, smells like red hots. Waxy Intensity of waxy aroma like beeswax. Flowery Perfume Intensity of flowery or perfumery aroma including white lilies or citrus

blossom. Anise Intensity of anise aroma like anise candy or black licorice. Molasses Prune Intensity of aroma like molasses, sorghum molasses, prunes, raisins,

Horehound candy or brown sugar. Sharp Intensity of a strong, sharp, barny aroma. Lemon Intensity of lemon-like aroma. Chemical / Medicinal Intensity of chemical or medicinal aroma may be like varnish.

Source: Sensory attributes of honey, National Honey Board, U.S. (Anon, 2003c) 1.2.9 Composition of Australian honey Australian honey is produced from many varieties of flowering plants in scattered areas of the country. Thus, the variation in its composition is large. Honey produced in Australia by eucalypt trees represents 70-80% of the total Australian honey production. Further, Australia produces a large number of unifloral honey types through the use of specific hive locations and season of production. The hives are carefully located near one species of plants during the flowering season, which enables controlled foraging of honeybees to those plants. This enables the production of species-specific honeys. Chandler et al. (1974) have analysed the composition of Australian honey samples from 60 eucalypt flora, 18 non-eucalypt flora and 21 exotic flora, while Mossel (2002) analysed the composition of 126 Australian honey samples from ten eucalypt flora and five non-eucalypt flora (Table 1.12). The samples in both studies were collected from all honey producing areas in Australia. Eucalypt honeys show uniformity in chemical composition and consist of three colour grades; white, extra light amber and light. However, non-eucalypt honeys are dark in colour, pale to medium amber, and are lower in fructose and higher in sucrose content than eucalypt honeys. Moisture contents of eucalypt honeys are 13.2-17.4% (Chandler et al., 1974) and 15.40-20.6% (Mossel, 2002). Further, glucose contents of 20.2-38.3% (Chandler et al., 1974) and 17.60-40.85% (Mossel, 2002), and fructose contents of 40.2-49.9% (Chandler et al., 1974) and 28.27-44.34 (Mossel, 2002) have been reported for eucalypt honeys. The pH of eucalypt honeys are 3.54-4.9 (Chandler et al., 1974) and 3.82-4.61 (Mossel, 2002). Analysis of non-eucalypt honeys have shown moisture contents of 14.0-20.2% (Chandler et al., 1974) and 15.20-23.60% (Mossel, 2002), glucose contents of 22.2-34.8% (Chandler et al., 1974) and 16.96-33.91% (Mossel, 2002) and fructose contents 32.0-46.2% (Chandler et al., 1974) and 31.24-44.40% (Mossel, 2002). Australian non-eucalypt honeys have a pH values of 3.54-4.9 (Chandler et al., 1974) and 3.57-4.91 (Mossel, 2002). In conclusion, it is interesting to note these similarities and differences between the Australian honey composition data produced by Chandler et al. (1974) and recently by Mossel (2002), where there is a 28 year difference in when the honey was produced. Differences probably reflect the different floral types of honey analysed in each study.

12

Table 1.12 Summary of analytical data of Australian honeys Compositional Criteria Range* Range+ Colour (Pfund value in mm) 0 – 114 32 – 78 Moisture (%) 14.5 – 17.5 17.22 – 19.60 Ash (%) 0.20 – 0.50 0.10 – 0.35 pH 3.8 – 5.4 4.02 – 4.43 Free acid (mequiv/kg) 6.0 – 40.0 11.41 – 24.28 Total acid (mequiv/kg) 7.0 – 50.0 13.13 – 31.86 Apparent sucrose (%) 0.0 – 19.3 - Glucose (%) 20.1 – 40.0 25.24 – 30.58 Apparent fructose (%) 32.0 – 53.9 32.60 – 41.72 Diastase number (G°) 9 - 44 9.77 – 18.84 HMF (mg/kg) 0 - 40 4.09 – 33.65 *Adapted from Chandler et al. (1974) +Adapted from Mossel (2002) 1.3 Physical Characteristics of Honey Honey is a viscous liquid mainly consisting of glucose and fructose. However, its physical properties are different from an invert sugar solution with the same moisture content. Thus, the physical characteristics of honey are largely determined by the types and concentrations of sugars and other compounds in honey. 1.3.1 Viscosity of honey Viscosity is an important property in the handling and processing of honey. The flow properties depend on the composition, moisture content, and temperature of honey. Generally, honey samples with high moisture contents have a low viscosity. Further, the composition of individual sugars, and the amount and size of the colloids in honey are reported to influence honey viscosity (Bhandari et al., 1999a). In most published work, honey is reported to be a Newtonian fluid (White, 1978; Junzheng & Changying, 1998; Mossel, 2002). The viscosity of Newtonian fluids is independent of the shear rate and previous shear history, and depends only on composition and temperature. Further, honey is ‘thixotropic’ if the viscosity decreases with an increase in time for constant temperature and shear rate. In addition, if the viscosity of a fluid increases with an increase in shear rate at constant temperature, it is called as ‘dilatant’. Munroe (1943) reported that New Zealand manuka honey and Indian karvi honey behave as thixotropic due to protein in those honeys. In addition, Mossel (2002) showed that Australian jellybush honey is ‘thixotropic’. These honeys will not flow without agitation making it difficult for beekeepers to extract honey from the combs. Dilatancy is reported in opuntia honey from Nigeria, which contains dextran. Recently, there have been a number of studies of the rheological behaviour (e.g. viscosity) of Australian honeys (Bhandari et al., 1999b; Mossel et al., 2000; D’Arcy et al., 2001b; Mossel, 2002; Mossel et al., 2003; Sopade et al., 2003), including at sub-zero temperatures (Sopade et al., 2004a). In addition, a number of studies examined models for predicting the rheological behaviour (e.g. viscosity) of Australian honeys (Mossel et al., 2000; Sopade et al., 2003). Studies of the glass transition temperature of Australian honeys (Sopade et al., 2001) and the effect of moisture levels on the vitrification (glass formation ability) of Australian honey (Sopade et al., 2002) produced important data on the glass forming characteristics of Australian honey. Finally, the flow characteristics, including rheological behaviour and friction factors, of Australian liquid honey pumped in straight

13

pipes provided some important data for the pumping of liquid honey for use by industry (D’Arcy et al., 2001b; Sopade et al., 2004b). 1.3.2 Specific heat of honey Specific heat is determined as a heat related property of honey, and has received very little attention in the past. White (1976b) quoted the work of Helvey (1954) who reported that the specific heat is 0.54 at 20 °C for honey containing 17.4% moisture but that it varies depending on the moisture content (Table 1.13). He also presented the results obtained by MacNaughton for a temperature range of 29 °C to 48 °C, where the specific heat ranged between 0.56 - 0.73. A recent study by Sopade et al. (2006) examined the specific heat capacity of Australian honeys at temperatures from 35 °C to 165 °C using differential scanning calorimetry, and related it to the composition of these honeys.

Table 1.13 Specific heat of honey

Moisture content% Specific heat 20.4 0.60 19.8 0.62 18.8 0.64 17.6 0.62 15.8 0.60 14.5 0.56

coarsely granulated 0.64 finely granulated 0.73

Adapted from White (1976b) 1.3.3 Hygroscopicity of honey Honey is a hygroscopic product that absorbs moisture from the air, making it susceptible to fermentation. Most honeys contain more fructose than glucose, and fructose is a hygroscopic carbohydrate. The gain or loss of moisture in honey, when exposed to the air, depends on temperature, moisture content and relative humidity of the air (White, 1976b). Honey in its normal moisture range of 16.8-18.3% is in equilibrium with air at 55-60% relative humidity (Table 1.14). Further, it was reported that each honey has an equilibrium relative humidity where moisture is absorbed or passed onto another product (White, 1978). The moisture absorbed at the surface disperses slowly throughout honey due to its high viscosity. White (1978) quoted the work by Martin (1958) who reported that honey with 22.5% moisture exposed to 86% relative humidity had a 26% moisture content at the surface and no change at 2cm below after seven days. Water sorption properties of Australian unifloral honeys were recently reported by Yao et al. (2002, 2003)

14

Table 1.14 Approximate equilibrium between relative humidity of air and the

water content of clover honey

Relative humidity (%) Water content (%) 50 15.9 55 16.8 60 18.3 65 20.9 70 24.2 75 28.3 80 33.1

Adapted from White (1978); interpolated from the data of Martin (1958) 1.3.4 Crystallisation of honey Crystallisation is a natural phenomenon in honey, which is a supersaturated sugar solution. The supersaturated state occurs because honey contains more than 70% sugars and less than 20% water. Amor (1978) quoted work by Jackson & Silsbee (1924) on honey granulation who used model solutions of glucose, fructose and water at 30 °C. They found that the solubility of glucose was 55% without fructose and in the presence of 39% fructose, the solubility decreased to 33%. Further, he described the work of Lothrop (1943), who studied the solubility of glucose in fructose solutions at concentrations similar to honey. A sudden increase in glucose solubility was observed at fructose concentrations of 150 g/100 g of water. He also found that the solid phase was crystalline D-glucose monohydrate for glucose solubilities of 85-90 g/100 g water. Further, sucrose solubility does not show any sudden increase in solubility due to an increase in fructose concentration. Amor (1978) also quoted the work of Kelly (1954) who suggested that the presence of fructose reduced the transition temperature of crystalline D-glucose monohydrate from 50 °C to 30 °C. In addition, he reported that glucose does not crystallise from honey until the temperature is below 30 °C. As honey is a supersaturated sugar solution containing glucose and fructose, glucose loses water and crystallises as D-glucose monohydrate at room temperature. These monohydrate crystals serve as seeds for the crystallisation process. In addition, other small particles such as dust, pollen and air bubbles serve as nuclei for crystallisation. The water released by glucose during crystallisation increases the moisture content of honey, making it more susceptible to fermentation. The tendency of honey to crystallise depends on its composition and moisture content. Honey with a glucose content less than 30% rarely crystallises and those with 35% glucose are naturally crystalline (Assil et al., 1991). Doner (1977) reported that honey with glucose/water ratios less than 1.7 are related to non-granulating honey while honeys with ratios greater than 2.1 are rapidly granulating. Bhandari et al. (1999a) reviewed the crystallisation kinetics of honey. Crystallisation of glucose monohydrate in Australian unifloral honeys is also affected by the sorption properties of honey (Yao et al., 2002, 2003). 1.3.5 Electrical conductivity of honey The electrical conductivity of honey depends on the mineral content, organic acids, proteins and some other substances. There is very little information available on the electrical conductivity of honey. White (1976b) quoted the work by Vorwohl (1964) who reported that electrical conductivity of a 20% honey solution of single species honeys ranged from 0.85 to 8.47 x 10-4 ohm.cm. Further, heather honey had electrical conductivity of 7.7x10-4 ohm.cm and floral honeys are in the range of

15

0.6-1.46 x 10-4 ohm.cm. The electrical conductivity of honeydew honey is 6.3–16.4x10-4 ohm.cm. Further, it was found that conductivity values increase with an increase in ash content (White, 1976b). Recently, Mossel (2002) found that the electrical conductivity of Australian honeys varied from 0.24 mS.cm-1 to 0.89 mS.cm-1. 1.3.6 Fermentation in honey Honey contains sugar tolerant yeast in different amounts, and fermentation will take place under favourable conditions, such as at higher moisture content and temperatures. Further, there is a possibility of fermentation after the crystallisation of honey, because crystallisation of D-glucose monohydrate will create a high-moisture liquid phase that is susceptible to fermentation. Except for the production of alcoholic drinks by fermentation of honey (e.g. mead), fermentation spoils honey. All honeys contain sugar tolerant yeasts which multiply if the water content and temperature is favourable, leading to activation of the fermentation process (Crane, 1990). Thus, the moisture content of honey plays an important role in fermentation. Amor (1978) quoted the work by Lockheed (1933) on the effect of moisture content and yeast counts on fermentation (Table 1.15).

Table 1.15 Effect of moisture and yeast counts on fermentation in honey

Moisture content (%) Liability to ferment Below 17.1 safe regardless of yeast count 17.1 - 18.0 safe if yeast count < 1000/g 18.1 - 19.0 safe if yeast count < 10/g 19.1 - 20.0 safe if yeast count < 1/g Over 20.0 Always in danger

Lockheed (1933) cited by Amor (1978) Temperature is another factor that has an effect on the fermentation of honey. Amor (1978) quoted work by Wilson & Marvin (1931) who reported that honey could be stored below 11 °C to prevent fermentation. In addition, they indicated that fermentation is rapid between 13 °C and 21 °C. Above 27 °C, fermentation develops slowly in unripe honeys but probably not at all in well ripened honey. Honey is heated between 62.8 °C to 65.5 °C during processing to destroy yeast to prevent fermentation. 1.4 Processing and Storage of Honey Honey processing includes all the handling of honey during which its physical and chemical properties are changed in order to facilitate handling or to improve certain qualities. The standard system of honey processing consists of the following stages (Crane, 1990). 1. Clearing honeybees from the honey supers or framed combs to be harvested and taking the

combs to the honey house. 2. Warming the combs to 32-35 °C. 3. Uncapping the combs and dealing with the cappings. 4. Extracting the honey from the combs in a centrifuge. 5. Clarifying the honey by passing it through a strainer and/or baffle tank. 6. In large processing plants, flash-heating and pressure-filtering. 7. Storing the honey in bulk containers.

16

At lower temperatures, the rate of flow of honey is very slow. At higher temperatures, the quality of honey may be damaged. Therefore, the temperature of honey will be held at below 55 °C for all processing operations including flow through pipes, pumps and strainers. In temperate countries, sealed honeycombs are warmed to 32 °C to 35 °C before extraction. The supers are held in thermostatically controlled warm rooms in large extracting plants until they reach the required temperature. Warm air is blown through the supers in these rooms to permit effective circulation of air (Crane, 1990). 1.4.1 Uncapping of the combs Uncapping is the first step in extracting honey from the combs. Hand operated, steam heated and electrically heated knifes are used for uncapping. Most electric and steam heated knives contain a thermostat to control the knife temperature. Power uncappers used by beekeepers consist of a pair of heated knives. These knives are mounted horizontally and saw through the each side of the frame as it passes between the knives. Another type of uncappers are designed to remove the wax cappings by placing combs on a stainless steel wire conveyor. Frames on the conveyor are fed through two stainless steel flails located one above and one beneath the comb. The uncapper is adjusted to lightly strike the wax surface of the combs by the tip ends of two flails (Tew, 1992). Cappings are included in honey produced by all these methods. Small scale beekeepers separate the honey and cappings using gravity. The spin/float honey/wax separator is a centrifugal machine designed to separate honey from cappings. The honey and cappings mixture is fed into a perforated revolving drum, which is fitted inside a large stationary drum. Separated honey is directed to the settling tank, and cappings are dropped to a container beneath the capping spinner. Capping melters are also used to separate honey and wax, and cappings are dropped onto a hot surface to melt the wax. The wax melts at 62°-65°C. Liquid wax settles on top of the honey in a tank below the hot surface. Honey and wax have separate outlets in different levels in the tank. Honey from melters may be darkened and caramelised, and should not be mixed with extracted honey. The efficient uncappers have either wax melters or capping spinners underneath them, which eliminate the extra movement of cappings to other devices ( Townsend, 1976; Crane, 1990; Tew, 1992). 1.4.2 Extraction of honey from the combs Extracting honey from the combs is carried out using a low speed centrifuge. It consists of a cylindrical container with a framework inside that spins about the vertical axis. Manual and electrical powered extractors are available with different capacities. Tangential and radial extractors are used for small scale and large scale extraction respectively. The speed of rotation of the centrifuge depends on the viscosity of honey, temperature and mode of extraction. The speed of 380-400 rpm for tangential extractors and 300 – 350 rpm for radial extractors have been found to be suitable. The framework of the extractor holds the frames in vertical position and leaves a depth of 35 to 50 cm at the bottom for the collection of extracted honey. The honey flow from the extractor is normally sent to a sump tank. Honey is cooled due to the rotation of the machine and through evaporation during centrifugation ( Townsend 1976; Crane, 1990; Tew, 1992). 1.4.3 Straining and clarifying of honey The temperature of honey may reduce during extraction due to evaporative cooling. Thus, honey must be warmed up to 35 °C to reduce viscosity leading to a speeding up of the process of straining. Firstly, the refuse materials including small pieces of cappings are removed by sending honey through settling

17

or baffling tanks. In small scale straining, honey passes through a coarse sieve and a fine straining cloth, supported by a screen with 1.6 meshes per cm. The straining cloth is suspended in a container at a height allowing honey to be directed to a storage tank at the bottom. In large-scale continuous flow systems, honey is clarified by passing through a baffle tank with the jacket of the tank able to be warmed by a suitable method. There are a series of five or six baffle plates at right angles to the honey flow with a 2.5 cm gap between alternative plates at the top and bottom. Honey is passed through the baffles and small particles settle at the upper surface of the baffles. This method is useful in clarifying honey before straining in a continuous flow process. Some strainers consist of four cylindrical screens fitted one inside the other, with the finest in the outside position. The screens consist of 5, 12, 20 and 32 meshes per cm and mesh diameters of 18, 23, 28 and 33 cm respectively. Honey is fed at the centre and passes through the screens and flows out at the outer wall. The tank and each screen contain a drainage tap and the output of the strainer is located 10 cm below the top of the tank (Townsend, 1976; Crane, 1990). Tubular pressure strainers are also used for straining clarified honey after a long storage or contamination by unclean equipment. Honey is pumped through two tubular pressure strainers connected parallel in the pipeline in this process. The nylon strainers of these strainers need to be cleaned regularly which is done by opening the strainers at the inlet. Centrifugal strainers are used in large processing plants for coarse pre-strained honey. Honey is piped into the centre of the centrifuge, which has a speed of 2000 to 4000 rpm, and is then forced through fine nylon cloth and piped out of the centrifuge (Crane, 1990). 1.4.4 Heating of honey during processing Heat treatment is found to be an essential component at different stages of honey processing. Honey is heated for fast handling, to destroy yeast, to dissolve the large granules, and to increase keeping quality. It is important to control the period of heating and the temperature to prevent over heating and loss of freshness in honey. Further, heating and cooling must be undertaken rapidly in order to minimise heat damage to the honey. The first heating is undertaken at the honey house by storing honey in a 32-35 °C temperature warm room for a day before uncapping. Then, honey is heated at the extractor as it cools during the extraction process. Heat can be applied within the extractor or in the sump. Extracted honey is passed over the warm water heated coils in the extractor or the bottom of the extractor is heated with hot water. The sump tank is heated by jacketing and surrounding it with warm water to maintain its temperature at 46°C. Heating cables are also used to heat honey pipelines to sumps, settling tanks and filters. Heating cables have a temperature of 48.9 °C on the band and increases the temperature of honey inside the pipe to 37.8 °C depending on the moisture content. Heat exchanger units have been designed to heat honey under controlled conditions. This heating will assist in the straining and filtering of honey. For honey that arrives at the packing plant in a crystallised or candied form in 200 L drums, it needs to be heated in a hot room at a temperature of at least 55 °C overnight prior to decanting and pumping. In addition, honey is heated to a temperature of 60 °C or above in the packing plant to kill the yeast, and thus inhibit fermentation. This process is called pasteurisation. Honey fermentation depends not only on the yeast content but also on the water content, natural granulation behaviour, and the stages of processing. Heating also dissolves any crystals in honey and helps the filtering process, which aids in keeping the honey in a liquid form for a longer duration for those honeys that granulate quickly. Flash heating honey up to 70 °C or even up to 77 °C for 5 min is the commercial practice of pasteurisation (Townsend, 1976; Crane, 1990).

18

1.4.5 Effect of heat on honey composition Heating is an important operation in honey processing. However, heating has been shown to have an effect on honey quality parameters such as HMF, diastase activity, invertase activity and colour. The effect of heating honey can be recognized by an increase in HMF content. HMF is a product produced by the breakdown of sugars, particularly glucose and fructose. Very small amounts of HMF are contained in fresh honeys and rarely exceed 10 mg/kg. White (1994) studied the increase in HMF during commercial processing and packaging of honey in the U.S. Twelve samples intended for bulk sale and 12 samples intended for retail sale were used for the analysis. These samples were collected from commercial packers before and after normal processing practices. An increase of HMF was found for all the samples, and the values were between 1.1 and 20.4 mg/kg. The data of 12 honey samples are shown in Table 1.16 and the average increase in HMF content was 12.7 mg/kg.

Table 1.16 HMF increases resulting from processing

Packer Number of samples

HMF content (mg/kg)

Before After Difference 1 1 17.1 27.9 10.8 2 3 29.9 43.2 13.3 3 3 3.7 24.1 20.4 4 3 12.1 13.2 1.1 5 1 2.8 17.5 14.7 6 2 8.0 24.0 16.0

Adapted from White (1994) White (1994) presented the work of the Sioux Honey Association on the effect of six processing steps practiced by U.S. honey packers and indicated that the average increase in the HMF content from processing after nine days in storage was 8.5 mg/kg. Table 1.17 shows the contribution of various steps of processing to the increase in the HMF content in three samples.

Table 1.17 HMF content of honey during processing and packaging

Process HMF (mg/kg) Sampled from 55 gallon drum 1 2 3

After melting in hot oven 4.2 3.5 4.5 After 15 hr in settling tank 4.7 6.3 5.4 Immediately after bottling 6.0 9.1 7.0

Cased, stacked, stored 9 days 5.8 9.4 8.4 After 1 year storage 11.8 13.0 12.8

Overall increase from processing 27.7 34.1 34.3 Adapted from White (1994). Karabournioti & Zervalaki (2001) studied the effect of heat treatment on five Greek honeys with different botanical origins at temperatures of 35 °C, 45 °C, 55 °C, 65 °C and 75 °C for 24 h. These heat-treated honey samples were analysed for their HMF content and invertase activity (Table 1.18),

19

and an increase in HMF content and decrease in invertase activity were observed after heating. It was found that the heat resistance is different according to the botanical origin of honey, with the pine honey sample being the most resistant. Further, a decrease in invertase activity started at a temperature of 35 °C, and the HMF content was less than the international standard of 40 mg/kg even at 55 °C.

Table 1.18 The effect of heating on invertase activity and HMF of various Greek honeys

Pine Orange Helianthus Cotton Thymus Temp HMF Invertase HMF Invertase HMF Invertase HMF Invertase HMF Invertase

Unheated 1.20 200.30 2.25 23.85 26.8 93.0 9.70 104.10 8.78 70.64 35 1.95 179.30 3.45 18.90 29.20 90.10 9.90 96.50 10.78 65.64 45 2.25 174.50 3.75 12.70 32.60 72.50 11.40 74.20 13.17 53.56 55 4.80 121.30 4.35 10.80 39.00 28.90 16.50 32.40 23.95 20.66 65 12.40 10.65 19.00 3.50 87.60 2.55 52.70 4.0 48.20 6.35 75 43.40 4.90 63.30 0 226.35 0 173.4 0 191.35 1.11

Adapted from Karabournioti & Zervalaki (2001) Karabournioti & Zervalaki (2001) concluded that HMF is the most reliable criterion to detect the effect of heating on honey as it is not present or is present in low levels in fresh honey. On the other hand, invertase started decreasing at 35 °C depending on the origin of honey, and shows a great variation during an increase in temperature. Thus, the combination of HMF content and invertase activity seems to be a better criterion to detect the effect of heat treatment, since the HMF content gives information about overheating, while the invertase activity can detect gentle heating. A study of heat effects on Australian honeys examined the effect of accelerated storage conditions (55°C for 44 days) on honey colour, acidity and total nitrogen content (Wootton et al., 1976a), on the sugar and free amino acid contents of honey (Wootton et al., 1976b), and on volatile honey components (Wootton et al., 1978). 1.4.6 Storage of honey Honey is subjected to fermentation, granulation (crystallisation), discolouration, flavour damage, and destruction of enzymes, and production of hydroxymethylfurfural during storage. The recommended storage temperature for processed honey is 18-24 °C, and unprocessed honey is below 10 °C (Anon, 2003d; Marvin, 1930). The major problem during storage is the fermentation of honey. All honeys with more than 20% moisture are liable to ferment. Honey with less than 17.1% moisture is not liable to ferment as microorganisms (e.g. yeast) are unable to grow due to the low water activity. However, honey containing a moisture content between 17.1 and 20% is fermentable, subject to the amount of yeast present. Honey is safe, if the yeast content is less than 1000 per g for a moisture content of 17.1%-18%. Honeys with a moisture content of 18.1% to 19% and 19.1% to 20% are not fermentable if the yeast count does not exceed 10 per g and 1 per g respectively. Further, storing honey below 11 °C is reported to prevent fermentation and granulation. However, if the moisture content of honey is in the range for which fermentation will take place, the yeast must be destroyed before storage. The recommended pasteurisation method includes flash heating to 77 °C for few minutes or heating at 63 °C for 30 min (Anon, 2003d; Townsend, 1976; White et al., 1964).

20

The other factor that affects honey during storage is the storage temperature. Studies on the storage temperature of honey has shown that a reduction of 5-9 °C in temperature results in a reduction in the rate of HMF production to 1/3 and maximum enzyme loss to 1/5 (White et al., 1964). Honeys first stored at 0 °C for at least 5 weeks and then at 14 °C, have been shown not to crystallise for 2 years. However, honeys stored only at 14 °C have crystallised within 5 weeks (Townsend, 1976). Studies on heating honey at 60 °C for half an hour, followed by storage at room temperature (7-30 °C), 5 °C and 40 °C for periods of 0, 2, 4 and 6 months, have shown that honey stored at 40 °C resulted in deterioration of colour and no granulation. Further, there was no difference in the colour between honey stored at room temperature and 5 °C. However, the results of granulation studies have indicated that, for honeys where granulation started after 30 days, the honey could be prevented from granulating for another two months by heat treatment and storage at 5 °C (Gupta et al., 1992). 1.4.7 Control of Crystallisation Honey is a supersaturated sugar solution consisting of mainly glucose and fructose. In extracted and stored honey, the excess glucose precipitates in the form of crystalline D-glucose monohydrate and releases water. The rate of crystallisation depends on the origin of honey and the method of processing. Natural crystallisation, before heating, usually produces fine grained crystals because of the presence of fine seed crystals and initiators such as dust, pollen and fine air bubbles. These seed crystals or other small particles are not present in heated or filtered honey, since filtering removes the potential crystallisation nuclei such as fine crystals, pollen, dust and other particles, and heating dissolves small crystals. Thus, crystallisation of filtered or heated honey produces coarse grained crystals and the process is slow. D-Glucose monohydrate crystals form a lattice within honey, which immobilises the other components into a suspension. Thus, the moisture in the liquid phase of honey, due to its high water activity, encourages yeast growth and finally initiates fermentation. Flash heating and pasteurisation delays the crystallisation by dissolving any crystals present, and kills yeast, thereby preventing fermentation (Assil et al., 1991; Crane, 1990; White, 1978). However, crystallisation is a natural phenomenon in most honeys and takes place at some time during storage. Further, each honey crystallises differently. Honeys with less than 30% glucose such as Tupelo and Robina honeys never crystallise. Further, some honeys crystallise rapidly such as rape honeys, and some crystallise slowly. In addition, some honeys have finely granulated crystals and others have coarsely granulated crystals (Assil et al., 1991; Bhandari et al., 1999a; Crane & Walker, 1984; Tarbouret et al., 1992). Therefore, naturally crystallised or candied honey needs to be heated to dissolve the D-glucose monohydrate crystals prior to the handling and packing processes. Commercial honey processing plants have hot rooms that are kept at a temperature high (e.g. 55 °C) enough to liquefy the crystals in the drums. The drums of candied honeys can be placed on their sides on a sloping rack in the hot room, which is heated by hot air to 60 °C – 70 °C. These drums have to be heated for long periods (e.g. 24 h) to convert candied honey to a pumpable state for further processing activities. 1.4.8 Production of creamed honey Creamed honey is a very fine grain semisolid product produced by controlled crystallisation. The texture of creamed honey is a three-dimensional interlaced network of crystals in a liquid phase, and depends on the moisture content, glucose content and the quality of seed material. Dyce (1931a,b) developed the process for producing creamed honey. Firstly, honey is heated to 49 °C and strained to remove small particles. Honey is again heated to 66 °C, cooled rapidly to 24 °C, and 5-10% seed honey is added with vigorous mixing. Crystallised seed honey is prepared by grinding candied honey and keeping it for 5-7 days at a temperature of 14 °C. The temperature of the honey at the point of adding seeds should be no warmer than 24 °C and not as low as 14 °C, otherwise, the warm honey may dissolve small crystals in the seed honey. Next, the thoroughly mixed honey is filled into

21

containers, sealed and kept at 14 °C, after which a fine textured creamed honey results in 4 to 6 days. A critical granulation temperature has been identified as 14 °C, at which supersaturation and viscosity is balanced and resulting crystallisation (granulation) proceeds rapidly. Finally, the finely granulated honey needs to be conditioned at 30 °C until a soft texture results, making the creamed honey acceptable to consumers. After such softening, creamed honey will not return to its original hardness even if stirred. Honey for creaming should have a moisture content of 17.5-18%. Creamed honey produced from honeys with this moisture range is found to be neither hard nor soft. Further, the smoothest texture was achieved by ensuring a large number of very fine crystals and fragments are present in the seed material (Amor, 1978; Dyce, 1976; White, 1978) . 1.5 Quality, Methods of Analysis and International Regulatory

Standards The honey quality standards and methods presented below are formulated and published by the International Honey Commission. The draft honey standards of Codex Alimentarius and European Directive are detailed in Table 1.19. Pressed honey is defined as honey obtained by pressing broodless combs with or without the application of moderate heat. These standards apply to all honeys produced by honeybees. Further, it also covers honey packed in containers with the intention of repacking for retail. It is stated that any food ingredient foreign to honey, including food additives, should not be added. Further, any flavour and aroma should not be absorbed during the processing and storage of honey. Honey should not be heated to a level that changes the composition, and any chemical or biological treatment should not be undertaken to control crystallisation (Bogdanov et al., 1999a,b). Further, new honey standards for sugar content and electrical conductivity are also being proposed (Table 1.20). The methods used to determine the standard parameters indicated in Tables 1.19 and 1.20 are detailed in the Harmonised Methods of the International Honey Commission (Bogdanov et al., 1997; Bogdanov, 2002). 1.5.1 Moisture content of honey Moisture content is an important criterion and determines the capability of honey to remain stable in storage without fermentation. Generally, a maximum moisture content of 21 g/100 g honey is suggested. The analysis of 30,000 honey samples conducted by the International Honey Commission in 1989-97 found that 91-95% of all honeys had a water content of less than 20 g/100 g honey. In Switzerland, a maximum value of 20 g/100 g honey is accepted, while Germany, Belgium, Austria, Italy and Spain accept maximum moisture contents of 17.5 to 18.5 g/100 g honey for special honeys (Bogdanov et al., 1999a,b).

22

Table 1.19 Honey quality standards of Codex Alimentarius and European Directive

Quality Criteria Codex Draft EU Draft

Moisture Content general heather, clover Industrial or bake honey

≤ 21 g/100 g ≤ 23 g/100 g ≤ 25 g/100 g

≤ 21 g/100 g ≤ 23 g/100 g ≤ 25 g/100 g

Apparent Reducing Sugars Content honeys not listed below honeydew honey or blends of honeydew honey and blossom honey Xanthorrhoea pr.

≥ 65 g/100 g ≥ 45 g/100 g ≥ 53 g/100 g

≥ 65 g/100 g ≥ 60 g/100 g ≥ 53 g/100 g

Apparent Sucrose Content honeys not listed below Robina, Lavandula, Hedysarum, trifolium, Citrus, Medicago, Eucalyptus cam., Eucryphia luc., Banksia enz., Rosemarinus Calothamnus san., Eucalyptus scab., Banksia gr., Xanthorrhoea pr., honeydew honey and blends of blossom with honeydew honey

≤ 5 g/100 g ≤ 10 g/100 g ≤ 15 g/100 g

≤ 5 g/100 g ≤ 10 g/100 g -

Water-Insoluble Solids Content general pressed honey

≤ 0.1 g/100 g ≤ 0.5 g/100 g

≤ 0.1 g/100 g ≤ 0.5 g/100 g

Mineral Content (ash) general honeydew or blends of honeydew and blossom honey or chestnut honey

≤ 0.6 g/100 g ≤ 1.2 g/100 g

≤ 0.6 g/100 g ≤ 1.2 g/100 g

Acidity ≤ 50 meq/kg ≤ 40 meq/kg Diastase Activity (Diastase number in Schade scale) after processing and blending (Codex) general for all retail honey (EU) honeys with natural low enzyme content

≥ 8 ≥ 3

≥ 8 ≥ 3

Hydroxymethylfurfural Content after processing and/or blending (Codex) general for all retail honey (EU)

≤ 60 g/100 g

≤ 40 g/100 g

Adapted from Bogdanov et al. (1999a,b). 1.5.1.1 Determination of the moisture content of honey Moisture content of honey is determined by a refractive index method. The moisture content is obtained from a standard table consisting of respective refractive index values versus moisture values. An Abbe or digital refractometer is used for measuring the refractive index of honey. Water content of honey and refractive index at a temperature of 20 °C are given in Table 1.21. This table was created by using the following formula developed by Wedmore (1955) using the data of Chataway (1932).

002243.0)1.log(73190.1 −−

=IRW

Where, W is water content in g / 100 g honey, and RI is refractive index.

23

The refractive index method is reproducible, simple, and is used satisfactorily in honey quality control around the world (Bogdanov et al., 1997; Bogdanov, 2002). The water content for temperatures higher or lower than 20 °C is determined by adding or subtracting 0.00023 per degree (°C).

Table 1.20 Proposed new international standards for honey

Quality Criteria Proposed value Sugar Content sum of fructose and glucose blossom honeys honeydew honey or blends of honeydew honey and blossom honey Sucrose honeys not listed below Banksia, Citrus, Hedysarum, Medicago, Robinia, Rosmarinus, Lavandula

≥ 60 g/100 g ≥ 45 g/100 g ≤ 5 g/100 g ≤ 10 g/100 g ≤ 15 g/100 g

Electrical Conductivity Blossom honeys excepted the honeys listed below and blends with them; blends of honeydew and blossom honey Honeydew and chestnut honey, excepted the honeys listed below and blends with those Exceptions: Arbutus, Banksia, Erica, Eucalyptus, Eucryphia, Leptospermum, Melaleuca, Tilia

≤ 0.8 mS/cm ≤ 0.8 mS/cm

Adapted from Bogdanov et al. (1999a,b). 1.5.2 Apparent reducing sugar content in honey Apparent reducing sugar content corresponds to the majority of honey sugars. However, honeydew honeys contain high amounts of non-reducing sugars such as melezitose, maltotriose and raffinose. Thus, Codex as well as the EU draft have set the standard for apparent reducing sugar content of blossom honeys as a minimum of 65 g/100 g honey. Standards for honeydew honey and blends are set at a minimum of 45 g/100 g honey and 60 g/100 g honey by Codex and EU draft respectively. Apparent reducing sugars are defined as the sugars, that reduce a Fehling’s solution under specified conditions. Reducing sugar content is determined by titration of a modified Fehling’s solution at its boiling point against a solution of honey. Methylene blue is used as the internal indicator. 1.5.3 Apparent sucrose content in honey The general standard for apparent sucrose content was set at a minimum of 5 g/100 g honey, excluding some unifloral honeys like banksia, citrus, hedysarum, medicago and robina honeys with up to 10 g/100 g honey, and honeys with up to 15 g/100 g of sucrose. The apparent sucrose content is calculated by multiplying difference of percent invert sugar values by 0.95 and expressed as grams, apparent sucrose per 100 g honey (Bogdanov et al., 1997; Bogdanov, 2002).

24

Table 1.21 Relationship of water content of honey to refractive index

Water Content

(g/100 g) Refractive Index

(at 20 °C) Water Content

(g/100 g) Refractive Index

(at 20 °C) 13.0 1.5044 19.0 1.4890 13.2 1.5038 19.2 1.4885 13.4 1.5033 19.4 1.4880 13.6 1.5028 19.6 1.4875 13.8 1.5023 19.8 1.4870 14.0 1.5018 20.0 1.4865 14.2 1.5012 20.2 1.4860 14.4 1.5007 20.4 1.4855 14.6 1.5002 20.6 1.4850 14.8 1.4997 20.8 1.4845 15.0 1.4992 21.0 1.4840 15.2 1.4987 21.2 1.4835 15.4 1.4982 21.4 1.4830 15.6 1.4976 21.6 1.4825 15.8 1.4971 21.8 1.4820 16.0 1.4966 22.0 1.4815 16.2 1.4961 22.2 1.4810 16.4 1.4956 22.4 1.4805 16.6 1.4951 22.6 1.4800 16.8 1.4946 22.8 1.4795 17.0 1.4940 23.0 1.4790 17.2 1.4935 23.2 1.4785 17.4 1.4930 23.4 1.4780 17.6 1.4925 23.6 1.4775 17.8 1.4920 23.8 1.4770 18.0 1.4915 24.0 1.4765 18.2 1.4910 24.2 1.4760 18.4 1.4905 24.4 1.4755 18.6 1.4900 24.6 1.4750 18.8 1.4895 24.8 1.4745

25.0 1.4740 Adapted from Bogdanov et al. (1999a,b). 1.5.4 Water insoluble solids/matter content in honey Pollen, honeycomb debris, honeybee and filth particles are considered as insoluble matter, which is a criteria for cleanliness. The insoluble solids content is helpful in detecting impurities in relation to the permitted maximum of 0.1 g/100 g honey set by the Codex and EU standards (Bogdanov et al., 1999a,b). Water insoluble matter is defined as the material found by analysis to be insoluble in water and expressed as g per 100 g (Bogdanov et al., 1997; Bogdanov, 2002).

25

1.5.5 Ash content of honey The ash content of honey is used as criterion for botanical origin. Blossom honeys are lower in ash content than honeydew honey. The standard set for maximum ash content is 6% (Bogdanov et al., 1999a,b). 1.5.6 Electrical conductivity of honey Electrical conductivity is used to distinguish between blossom honey and honeydew honey, and is expressed in units of milli Siemens cm-1 . It depends on the ash and acid content of honey. It has been found that, ash content (A) has a linear relationship with electrical conductivity (C):

C = 0.14 + 1.74 A Where, C = electrical conductivity (in milli Siemens cm-1) A = ash content (in g/100 g) Based on published data, an electrical conductivity of less than 0.8mS/cm applies for blossom honeys and mixtures of blossom and honeydew honeys, while an electrical conductivity of more than 0.8 mS/cm applies for honeydew and chestnut honeys (Bogdanov et al., 1999a,b). Electrical conductivity is defined as that of a 20% w/v solution in water at 20°C. The 20% refers to honey dry matter. The electrical conductivity is determined by measuring the electrical resistance of the solution using a conductivity meter. The following additional criteria, such as invertase activity, proline content and specific rotation are presented to be used for determination of honey quality outside the international honey regulations. 1.5.7 pH and acidity of honey pH is measure of activity of hydrogen ions in a solution and is defined as the negative logarithm of the concentration of active hydrogen ions. A honey sample of 10 g is dissolved in 75 mL of carbon dioxide free water, and the pH is measured using a pH meter. Acidity is a quality criterion and fermentation of honey increases the acidity. Thus, a maximum value for acidity is useful. The maximum value of 50 meq/kg is set in the Codex draft, as some honeys have a high acidity. Free acidity is the content of all free acids in honey. The honey sample, that is used to determine pH, is titrated with 0.1M sodium hydroxide solution to pH 8.30 to determine free acidity. Free acidity is expressed as milliequivalents per kilogram of honey, and is equivalent to the volume of 0.1M sodium hydroxide required for titration in mL multiplied by 10. Free acidity = volume of 0.1M NaOH (mL) x 10 Further, lactone acidity is defined as the combined acidity, which is not titratable and expressed as milliequivalents per kilogram. Total acidity is the sum of free acidity and lactone acidity. The lactone acidity is obtained by adding an excess of sodium hydroxide and determination of pH at the equivalence point.

26

1.5.8 Diastase activity of honey Diastase activity is an indicator of freshness and overheating of honey. There is a large natural variation in diastase activity. The standard for diastase activity is set as a Diastase Number (DN) of eight. Codex and EU directive are the same for the minimum requirement of diastase activity. However, Codex standard refers to processed and blended honey, while the EU standard refers to retail honey. The diastase activity is likely to reduce during storage. The Gothe units (or Schade Units) is used to measure the diastase activity. It is defined as the amount of enzyme which will convert 0.01 g starch to a prescribed end-point in 1 h at 40 °C. There are two methods, the Schade method and the Phadebas method, used for the analysis of diastase activity in honey. The diastase activity is determined using the Schade method, and is calculated as diastase number (DN) (Bogdanov et al., 1997; Bogdanov, 2002): DN = 300 / tx

Where, tx = reaction time (min) 1.5.9 Invertase activity of honey Invertase activity is also used as a freshness indicator, and invertase is susceptible to heat and storage damage. It is given as Invertase Number and standards are set at 10 for fresh and unheated honeys, and 4 for honeys with low enzymatic activity. Invertase activity also has a natural variation in honeys. Invertase (sucrase, glucosidase, transglucosidase) transforms sucrose to glucose and fructose. Invertase Number is defined as the amount of sucrose per g hydrolysed in 1 hour by the enzymes contained in 100 g of honey and expressed in units. One unit is defined as the number of micromoles of substrate destroyed per minute, and expressed per kilogram of honey (Bogdanov et al., 1997; Bogdanov, 2002). 1.5.10 Hydroxymethylfurfural (HMF) content of honey The hydroxymethylfurfural (HMF) content is the main quality indicator of the freshness and heating history of honey. Practically, there is no HMF in fresh honey. However, it tends to increase during storage, and depends on the pH of honey and the storage temperature. A maximum value of 40 mg/kg of HMF is accepted in the international honey market. Codex proposes a maximum of 60 mg/kg considering honey storage in warm climatic countries. The European Union (EU) standard proposes 40 mg/kg, as it has validity in European countries. However, the Codex standard refers to processed and blended honeys, while the EU standard is set for all retail honeys. (Bogdanov et al., 1999a,b). HMF content is the concentration of 5-(hydroxymethyl)-furan-2-carbaldehyde and expressed as milligrams per kilogram. HMF is determined in filtered, aqueous honey solution by reverse phase High Performance Liquid Chromatograph equipped with UV detection. Another method based on the work of White (1979) determines the UV absorbance of HMF at 284 nm. The interference of other components at this wavelength is avoided by determination of the difference between the absorbance of a clear aqueous honey solution and the same solution after addition of sodium bisulphite. HMF content is calculated by subtraction of the background absorbance at 336 nm. The absorbance of the sample solution and reference solution at 284 nm and 336 nm is determined in this method. If the

27

absorbance at 284 nm exceeds a value of about 0.6, the sample solution is diluted with water and the reference solution with sodium bisulphite solution.

Dilution, 10

solution sample of volumeFinal=D

The HMF content (in mg/kg) is calculated using the following formula (Bogdanov et al., 1997; Bogdanov, 2002):

HMF content = (A284 - A336) x 149.7 x 5 x D/W

Where, A284 = absorbance at 284 nm A336 = absorbance at 336 nm D = dilution factor, if dilution necessary W = Mass of honey sample (g) 1.5.11 Proline content Proline content is used to determine honey ripeness and adulteration, and a minimum value of 180 mg/kg is set as the standard. Further, there is a considerable natural variation in different honey types. Proline content is defined as the colour developed with ninhydrin in comparison with standard proline, and expressed as a proportion of the mass of honey in mg/kg. 1.5.12 Specific rotation Specific rotation is the overall value of optical rotation created by all the sugars in honey. In Greece, Italy and United Kingdom, the specific rotation measurement is used to differentiate between blossom and honeydew honeys. In Italy, optical rotation of blossom honeys and honeydew honeys have shown negative values and positive values respectively. Specific rotation is defined as the angle of rotation of polarised light at the wavelength of the sodium D line at 20°C. An aqueous solution of one decimetre depth containing 1 g/mL of substance is used for analysis. Specific rotation is measured using a polarimeter, and the value is related to the carbohydrate composition. Specific angular rotation is calculated by the following formula (Bogdanov et al., 1997; Bogdanov, 2002).

Specific angular rotation [α]20D 100

p x lxα

=

Where, α = angular rotation l = length of polarimeter tube in dm p = mass of honey taken for analysis

28

1.6 Ultrasound in Food Processing Ultrasound is defined as sound waves with a frequency greater than that of human hearing range. Human hearing is in the frequency range of 0.016 to 18 kHz, and the power intensity of normal conversation is about 1 Wcm-2. Ultrasound is broadly divided into two categories. Low power high frequency ultrasound consists of very high frequencies between 2 to 20 MHz and power intensity between 100mWcm-2 and 1 Wcm-2. This type of ultrasound is used in medical scanning and chemical analysis of food properties. In addition, these ultrasonic waves pass through the materials without causing any physical or chemical changes in its properties. Ultrasound waves with a frequency range of 20 kHz to 100 kHz are called high-power ultrasound, when it produces the power levels of between 10 to 1000 Wcm-2. This low frequency high-power ultrasound causes physical disruption of the applied material and promotes certain chemical reactions. Further, low frequency high-power ultrasound with power intensities of more than 10 Wcm-2 will generate cavitation effects. Cavitation is known to disrupt some physical properties and modify many chemical reactions (Leadley & Williams, 2002; Mason, 1998; McClements, 1995). Ultrasound waves with frequencies more than 18 kHz are generated by the application of a vibration force to the surface of a material. When the vibration force is applied to the surface of a material, it is transmitted through the bonds within molecules. Further, each of the molecules transmits the motion to an adjoining molecule before returning to approximately its original position in this process. If ultrasound is applied perpendicular to the surface of the material, then a compression wave is generated within the material. Similarly, a shear wave is generated by the application of ultrasound parallel to surface. The ultrasound waves cause the layers in the material to oscillate in their original positions at the same frequency as the ultrasound waves. Thus, displacement of a fixed position in the material varies sinusoidal with time, and the time difference between two maximum positions is the period of oscillation (T). The wavelength (λ) is the distance between successive maximum positions, and amplitude (A) is the displacement of the layers from its equilibrium position at a given time. An ultrasonic wave is characterized by its amplitude (A) and the frequency (f); and ultrasonic velocity (C) is given by the formula, C = f λ (McClements, 1995). 1.6.1 Equipment for the generation of high-power ultrasound The first step in the use of high-power ultrasound is to derive a method for generating acoustic energy. A transducer is the device used to convert mechanical or electrical energy into sound energy. Three main types of transducers namely, liquid driven transducers, magnetostrictive transducers, and piezoelectric transducers are available. Liquid driven transducers consist of a liquid whistle, where a liquid is forced across a thin metal blade causing it to vibrate at ultrasound frequencies (Leadley & Williams, 2002; Mason, 1998; Mason & Lorimer, 1988). The rapidly alternating pressure and the effect of cavitation generates a high degree of mixing in the liquids. As it involves pumping a liquid, processing applications are limited to mixing and homogenisation. Magnetostrictive transducers are devices that use the magnetostriction effect of some ferromagnetic materials such as iron or nickel. Magnetostriction is the change of dimension of the materials on the application of magnetic field. A magnetostrictive transducer is in the form of a rod acting as the magnetic core within a solenoid. The core is an assembly of layers of thin nickel plates forming a closed square loop and coils wound around two opposite sides of it. The application of current to the coil results in a reduction of the dimensions of the core, and a reduction in the dimensions of the transducer thereafter. The maximum frequency generated by these transducers is limited to 100 kHz, while the energy efficiency is about 60%. However, these transducers can withstand long exposure to high temperatures. Piezoelectric transducers are the most commonly used transducers for the generation of ultrasound. These transducers utilize ceramics containing piezoelectric materials such as barium titanate or lead metaniobate. The piezoceramic element is produced in the form of a disk with a central hole. Ceramic transducers are normally clamped

29

between metal blocks to protect the delicate material and prevent it from overheating. Piezoelectric transducers usually consist of two ceramic elements and are 95% electrically efficient, thus resulting in their possible use over the whole ultrasonic frequency range. However, they are unable to withstand long exposure to temperatures of more than 85 °C (Leadley & Williams, 2002; Mason, 1998; Mason & Lorimer, 1988). 1.6.2 Design of ultrasonic systems Most devices used for the generation of ultrasound are based on electroacoustic systems. Piezoelectric transducers are mostly used in these systems. The equipment required to convey ultrasound energy to a liquid system consists of following three parts: (1) a generator to convert mains electricity into high frequency alternating current to drive the transducer assembly; (ii) a transducer element that converts the high frequency alternating current into mechanical vibrations; (iii) a delivery system that conveys the vibration to the liquid. The shape and dimensions of a piezoelectric transducer is dependent on its working frequency. A 20 kHz transducer has twice the length of 40 kHz transducer. The transducer is attached to the upper fixed horn to connect it to the delivery system. Further, the tip of the horn can become eroded, with the overall horn length being reduced after prolonged use. Hence, replaceable screw-threaded tips are made to fix at the end of the horn. The availability of power through a transducer is inversely proportional to the square of frequency. Therefore, lower frequencies are selected for high power applications (Leadley & Williams, 2002; Mason, 1998). The ultrasonic systems are broadly categorised as ultrasonic bath and sonotrode systems. In an ultrasonic bath system, the transducers are fixed to the underside of the tank, mostly operated at 40 kHz frequency, and the ultrasonic energy is directly delivered to the liquid in the tank. Further, these are low power systems because the volume of liquid in the tank is large. Due to the presence of standing waves produced by the reflection of sound waves at the liquid-air interface separated by the distances equivalent to the half wave lengths, high intensities are achieved at fixed levels in the bath. The sonotrode system consists of an ultrasonic horn attached to the transducer, and the length of the horn is a half wavelength (or multiple). The sonotrode systems produce high power as the emitter surface is directly in contact with the solution. This method also provides an amplification of acoustic energy. The amplitude gain depends on the shape and difference in diameter of the horn between the driven face and the emitting face. If the horn has the same diameter along its length, no gain in amplitude will occur, but the horn transfers the acoustic energy to the medium. Gain is defined as a specific ratio of the diameter of the driven face (D) and the emitting face (d). The ratio is D/d for an exponential or linear tapered horn, and (D/d)2 for a stepped horn. Standing waves are created only if the sound waves meet the reflecting surface of the liquid / air interface at a distance of a half wave length from the source. Further, very high activity zones are generated in the nodes of the standing wave. The highest energy conveyable depends on the type of the material used to make the transducer and the horn, and the area of the emitting surface. Hence, highly flexible materials like titanium or aluminium alloys are selected for fabrication to achieve better transmission of ultrasound. Aluminium is vulnerable to chemical reactivity with liquids. Thus, most of the ultrasonic horns are made of titanium. Ultrasound horns with a small emitting surface produce efficient ultrasound transmission (Leadley & Williams, 2002; Mason, 1998). Ultrasonic equipment transmits about 75% of input energy into the liquid. The mean efficiencies of the horn and ultrasonic generator are 89% and 84% respectively (Loning et al., 2002).

30

1.6.3 Mechanism of ultrasound action The application of ultrasound to a liquid creates alternate rarefactions and compressions. Thus, sound waves with a sufficiently high amplitude produce bubbles or cavities, and this incident is called ‘cavitation’. These cavitation bubbles have a limited lifetime and break up into smaller bubbles or completely disappear. There are two types of cavitation; stable or transient. Stable cavitation occurs due to the oscillation created by ultrasound waves, which forms small bubbles in the liquid. It takes so many oscillatory cycles for the bubbles to increase their size in a stable cavitation. As the ultrasound waves pass through the liquid, they vibrate these bubbles and strong current is produced in the surrounding liquid. Further, it attracts the other small bubbles into the sonic field and microcurrents are created in the liquid. This effect is called microstreaming, which provide a substantial force causing the cells to shear and breakdown without the collapse of bubbles. The shear force created by this process is one of the actions that lead to disruption of cells. In transient cavitation, the bubble size changes in a few oscillatory cycles and it collapses with different intensities. The larger bubbles eventually collapsed producing high pressures of up to 100 MPa and high temperatures up to 5000 °K instantly. The pressure produced during bubble collapse is also sufficient to disrupt cell walls and eventually lead to cell disruption. Application of ultrasound to a liquid also leads to the formation of free radicals by sonolysis of water due to these high pressures and temperatures (Leadley & Williams, 2002; Sala et al., 1995; Scherba et al., 1991; Suslick, 1988). 1.6.3.1 Factors affecting cavitation during ultrasound treatment Cavitation depends on the frequency and amplitude of the ultrasound, the viscosity of the treatment medium, and the temperature. The intensity of cavitation depends on the amplitude of the ultrasound, with large amplitudes producing high intensity cavitation. The frequency of ultrasound determines the size of the bubbles formed during cavitation. Bigger bubbles are created at lower frequencies of 20 kHz and cavitation does not take place above 2.5 MHz frequency. Further, transient cavitation dominates for ultrasound frequencies lower than 100 kHz. In addition, the intensity of bubble collapse depends on the temperature and viscosity of the treatment medium, and the frequency of the ultrasound. The bubbles develop more rapidly as the temperature increases, but the intensity of collapse is reduced. This effect may be due to an increase in vapour pressure and decrease in tensile strength. As a result, the increase in temperature makes cavitation less effective (Alliger, 1975; Arakeri & Chakraborty, 1990; Leadley & Williams, 2002; Suslick, 1988). 1.6.4 Applications of ultrasound in food processing Power ultrasound has been used in food processing for mixing, emulsification and tenderisation. The potential areas of application in the processing of food are crystallisation control, enzyme inhibition, inactivation of microorganisms, and improved heat and mass transfer (Leadley & Williams, 2002). Current and potential applications of high-power ultrasound in the food industry are listed in Table 1.22. 1.6.4.1 Effect of the food system on ultrasound effectiveness The effectiveness of ultrasound depends on the characteristics of the food. Generally, the killing effect of ultrasound treatment is reduced in foods containing high fat content. Differences in the effects may be due to inherent effects of the environment on cavitation, or to changes in ultrasound penetration and distribution in the material. Ultrasound waves will passes easily through low viscosity fluids causing cavitation. However, to achieve the same level of penetration in highly viscous solutions, ultrasound waves with higher intensity have to be applied. Thus, low frequency high-power ultrasound will be better in penetrating viscous products such as honey than high frequency ultrasound, since high frequency ultrasound will be dispersed in the solution causing a reduction in the overall intensity of the input energy (Earnshaw, 1998; Leadley & Williams, 2002; Loning et al., 2002).

31

Table 1.22 Current and potential applications for ultrasound in the food industry

Application Comments Crystallisation of fats and sugars Enhances the rate and uniformity of seeding. Degassing Carbon dioxide removal from fermentation liquors. Foam breaking Foam control in pumped liquids and during container filling. Extraction of solutes Acceleration of extraction rate and efficacy; research on coffee,

tea, brewing. Ultrasonically aided drying Increased drying efficiency when applied in warm air resulting in

lower drying temperatures, lower air velocities or increased product throughput.

Mixing and emulsification On-line commercial use often using a liquid whistle. Can also be used to break emulsions.

Spirit maturation and oxidation processes

Including rapid oxidation in alcoholic drinks. 1 MHz ultrasound has possible applications for accelerating whisky maturation through the barrel wall.

Meat tenderisation Alternative to pounding or massaging; evidence for enhanced myofibrillar protein extraction and binding in reformed and cured meats.

Humidifying and fogging Ultrasonic nebulisers for humidifying air with precision and control; possible application in disinfectant fogging.

Cleaning and surface decontamination

On-line commercial use for cleaning poultry processing equipment; possible pipe fouling and fresh produce cleaning applications.

Cutting Commercial units available capable of cutting difficult products with less wastage, more hygienically, and at high speeds.

Effluent treatment Potential to break down pesticide residues. Precipitation of airborne powders Potential for wall transducers to help precipitate dust in the

atmosphere. Inhibiting enzyme activity Can inhibit sucrose inversion, and pepsin activity; generally

oxidases are inactivated by sonication but catalases are only affected at low concentrations, while reductases and amylases appear to be highly resistant to sonication.

Stimulating living cells Low power sonication can be used to enhance the efficiency of whole cells without cell wall disruption.

Ultrasonically assisted freezing Control of crystal size and reduced freezing time through the zone of ice crystal formation.

Ultrasonically aided filtration Rate of flow through a filter medium can be increased substantially.

Enhanced preservation (thermal and chemical)

Sonication in combination with heat and pressure has the potential to enhance microbial inactivation; this could result in reduced process times and/or temperatures to achieve similar lethality.

Adapted from Leadley & Williams (2002)

32

1.6.4.2 Effect of ultrasound on the oxidation process Cavitational collapse of the bubbles in transient cavitation generates extremely high local pressures and temperatures. Thus, during the sonication of water, the violent collapse of the cavities form reactive hydrogen atoms and hydroxy radicals. These radicals joined together to produce hydrogen peroxide responsible for oxidation reactions (Weissler, 1959). Further, the sonication of aqueous potassium iodide solution has been studied extensively, and it is reported that H • , HO • and H • O2 radicals are formed. These radicals produce iodine and hydrogen peroxide by reacting with the liquid, or they combine together (Naidu et al., 1994) . Further, carbon tetrachloride is used as a catalyst in the oxidation of aqueous potassium iodide solutions (Contamine et al., 1995; Feng et al., 2002). High frequency, low-power ultrasound has been used for ageing fermented products, while it induces rapid oxidation in alcoholic drinks for flavour development and early maturation. Ultrasound of 1 MHz frequency has altered the alcohol/ester balance and possibly accelerates maturation of whisky (Leadley & Williams, 2002; Roberts, 1993). 1.6.4.3 Effect of ultrasound on the stimulation of living cells Low-power ultrasound is reported enhance the efficiency of cells without cell disruption. Ultrasound seems to affect membrane/seed permeability and increases the transport of nutrients. Research work on the use of ultrasound in yoghurt manufacture as a processing aid has shown that production time can be reduced by up to 40%. Ultrasound is also used in lactose-hydrolysed fermented milk production. Sonication depleted the initial lactose by 71-74% in comparison with 39-51% lactose hydrolysis that is achieved in non-sonicated milk. Sonication also improves the consistency and texture of the products from these two processes. The application of 20 kHz frequency ultrasound to an aqueous environment improves the germination of lotus seeds by 30%. Further, 20 kHz frequency ultrasound has been used for activation of immobilised enzymes by increasing the transport of substrate to the enzyme immobilised in a support gel. However, attention was given to the power intensity, as enzymes will be denatured by high power intensities. Further, high frequency ultrasound of 7 MHz increases amylase activity, with the effect possibly being due to microstreaming of reagents to the immobilised enzyme (Leadley & Williams, 2002; Toba et al., 1990). 1.6.4.4 Effect of ultrasound on emulsification Power ultrasound is commonly used in emulsification of food products by using liquid driven transducers. The resultant shock wave created by the collapse of cavitation bubbles near the phase boundary of two immiscible liquids provides efficient mixing of layers. Tests have shown that the size of the fat globules is reduced by up to 80% after ultrasonication, and emulsions are more stable than those produced by conventional methods. In addition, better particle distribution was achieved by ultrasonication at temperatures of 70 °C and 75°C. Ultrasound emulsification is used to manufacture food products like salad cream, tomato ketchup and peanut butter (Leadley & Williams, 2002; Mason, 1998; Mason et al., 1996). 1.6.4.5 Effect of ultrasound on extraction The mechanical effects of ultrasound increases the extraction processes through greater penetration of solvent into cellular materials and by improving mass transfer. Further, it disrupts biological cell walls to facilitate the release of cell contents (Mason, 1998). As a result of these effects, power ultrasound improves the extraction of sugar from sugar beet (Chendke & Fogler, 1975). An efficient process has been developed for protein extraction from defatted soya beans by using 20 KHz frequency ultrasound. A continuous process involving ultrasound treatment using a sonotrode with 550 Watts of power applied to a slurry has resulted in better extraction than the previously available methods (Wang, 1975, 1981). The extraction of tea solids from leaves is an important step in the

33

production of instant tea (instant tea is produced by removing water from a pure tea infusion by spray drying). An experiment on the use of ultrasound has increased extraction by about 20% at 60°C. The ultrasonic extraction was greater than thermal extraction, and required less time for the process (Mason & Zhao, 1994). 1.6.4.6 Effect of ultrasound on filtration Filtration is a common process in most sectors of the food industry. The process involves the separation of suspensions of solids from a liquid. Further, it may be used for the production of a solid free liquid or the isolation of solid from its mother liquors. Ultrasonication has two specific effects that can improve the filtration process. Sonication will increase filtration by agglomeration of fine particles and supply sufficient vibration energy to the filter to keep particles suspended above the filtering medium to prevent clogging. Ultrasound filtration of fruit extracts and drinks increases the amount of fruit juice extracted from pulp. The extraction is further enhanced by applying an electrical potential across the pulp. The filter itself is the cathode, and the anode functions as a source of attraction of negatively charged particles in this process (Leadley & Williams, 2002; Mason & Lorimer, 1988). 1.6.4.7 Effect of ultrasound on drying Ultrasonically enhanced drying reduces the probability of oxidation and dehydration in the material, as it can be carried out at lower temperatures than conventional methods. The application of ultrasound to particles in a warm air convection drier has led to increased drying efficiency, and resulted in lower drying temperatures, lower air velocities, and an increase in throughput of the product. Sonication may reduce the pressure above the particles and promote water loss into the warm air passing over the bed of the drier. Experiments on hot air drying of carrots with sonication have revealed a reduction in treatment times and the achievement of final moisture contents of 1% (Leadley & Williams, 2002). The use of acoustic drying of rice, gelatine beads, and orange crystals in a rotary dryer with an ultrasound frequency of 10.9 kHz increases throughput (Mason et al., 1996). Sonication increases the drying rate of shelled corn, and whole and crushed wheat during the drying process, with the effect being prominent at low temperatures. An increase of 130% in the drying rate was produced at 21 °C in the presence of ultrasound. Further, at 63 °C and 79.5 °C, drying rate increases were achieved (Huxsoll & Hall, 1970). 1.6.4.8 Effect of ultrasound on enzyme inhibition Several hypotheses have been developed regarding the factors affecting the ultrasound inactivation of enzymes. This inactivation may be due to local intense pressure, high temperature, and shear forces generated by ultrasound, which denature the protein (Leadley & Williams, 2002). The suggested mechanisms are: (a) Acoustic cavitation – where the cavitation bubble/liquid interface causes acoustic streaming

which creates severe shear stress promoting enzyme denaturation. (b) Cavitational collapse in transient cavitation produces very high pressures and temperatures

which will inactivate the enzymes in hot spots. (c) Ultrasound promotes chemical reactions by decomposition of water and hydrogen, with

hydroxyl free radicals being formed in the process. These free radicals induce the formation of peroxy radicals, which in the presence of oxygen leads to protein fragmentation.

The amplitude of the ultrasound is also reported to influence the inactivation of enzymes. Studies on the enzymes, peroxidase and lipoxygenase, has shown that inactivation decreased linearly with

34

increasing amplitude (Leadley & Williams, 2002). Further, exposure to high intensity ultrasound for a long duration reduces the catalytic activity of enzymes in foods (Roberts, 1993). This effect may be due to generation of high pressures, temperatures, and shear forces by the ultrasound, which denature protein. The effect of ultrasound on enzymes is generalised by suggesting that oxidases are inactivated by sonication and catalases are affected only at low temperatures. However, amylases seem to be highly resistant to sonication (Mason, 1998). 1.6.4.9 Effect of ultrasound on the destruction of microorganisms Several hypotheses have been developed regarding the factors affecting the ultrasound inactivation of microorganisms. The microbial inactivation by power ultrasound is considered to occur due to cavitation, localised heating and free radical formation. During transient cavitation, the bubble size increases quickly and these bubbles collapse producing temperatures up to 5000 °K and pressure up to 100 MPa. Such a pressure is sufficient to disrupt the cell wall structures leading to cell disruption. The localized high temperature also leads to denaturation of proteins and enzymes. However, these temperatures occur instantly, and the immediate vicinity of the cells is likely to be affected. In addition, microstreaming created by stable cavitation produces a shear force, which rubs against the surface cells causing the microbial cells to shear and breakdown. In addition, free radicals are formed during the application of ultrasound to liquids due to sonolysis of water, and these free radicals have a bactericidal effect (Leadley & Williams, 2002). It appears that ultrasound is more effective when used with other decontamination methods like heating, chlorination, and extremes of pH (Lillard, 1994). 1.6.4.10 Effect of ultrasound on the processing of meat Power ultrasound has been used as an alternative to pounding and tumbling in meat processing. Ultrasound treatment enhances myofibrillar protein extraction and improves binding in reformed and cured meat after ultrasonication. The evaluation of the effect of salt tumbling, sonication in aqueous liquor, or both, has shown that ultrasound-treated meat has better binding strength, water holding capacity, product colour and yield (Vimini et al., 1983). In addition, ultrasound used in combination with tumbling in brine improves these qualities of meat (Roberts, 1991). 1.6.4.11 Effect of ultrasound on manothermosonication The process of application of high energy ultrasound and heat under moderate pressure is called manothermosonication. Manothermosonication inhibits the activity of enzymes and microorganisms more rapidly than heat treatment alone under the same temperatures. Further, manothermosonication increases the inactivation efficiency, and has a negative effect on the quality parameters of foods (Lopez et al., 1994; Vercet et al., 2002). Bacillus subtilis when suspended in distilled water, glycerol or milk, and simultaneously treated with 20 kHz ultrasound and heat treatment shows decimal reduction times that were reduced by 63% for glycerol and 79% for milk. Further, spores that were suspended in water and treated at a 70-95 °C temperature range, show a reduced heat resistance of between 99.9% and 70%. The effect of manothermosonication is at a minimum at the boiling point of water (Garcia et al., 1989). Finally, the effect of simultaneous application of 20 KHz ultrasound and heat treatment on the survival of a strain of Staphylococcus aureus in ultra-heat-treated milk involved a decrease in the decimal reduction time of 43% (Ordonez et al., 1987). 1.6.4.12 Effect of ultrasound on crystallisation High-power ultrasound improves crystallisation in aqueous solutions and sugar solutions. Three possible mechanisms have been suggested for the process. Firstly, it is possible to break up existing crystals and nuclei due to the stresses produced during cavitation. Further, the fragments produced by

35

this action will act as nucleation centres. As a result, large numbers of small crystals are produced in the final product. Experiments on concentrated sucrose solutions subjected to ultrasound have revealed that the number of small crystals can be increased. The crystals produced by normal crystallisation are large and uneven in size. The size of these crystals can be reduced by sonication later (Mason et al., 1996). Application of ultrasound in saturated mediums in the pharmaceutical industry initiates seeding and controls subsequent crystal growth. The cause of this effect is due to ultrasound disintegration of the crystals in the medium, and cavitation bubbles produced in the process acting as nuclei. Therefore, selection of the correct sonication conditions makes it possible to produce uniform crystals with designated sizes (Price, 1997). Power ultrasound has been used to cavitate the medium during the salting-out crystallisation process. The results have shown that size and size distribution of crystals can be controlled by ultrasonication. Thus, crystallisation of supersaturated solutions can be controllable by the application of ultrasound (Li et al., 2003). In addition, sonication assists in the crystallisation of D-fructose and sorbitol, which are disinclined to crystallise (VanHook & Frulla, 1952). 1.6.5 Effect of ultrasound on the liquefaction of candied honey Kaloyereas (1955) undertook the first experiment on the ultrasound treatment of honey. Honey was subjected to 9 kHz frequency ultrasound for 30 min and stored at various temperatures between +39 °C to -40°C. Crystals were examined after storage periods of 1 to 4 weeks, with no crystals being found in treated samples. Further, analysis of yeast cells revealed that the number of yeast cells in honey is reduced after ultrasound treatment. Liebel (1978) confirmed these findings by analysis of honey exposed to 18 kHz ultrasound for 5 min. In addition, the use of ultrasound treatment to inhibit granulation has the advantage of being a process that occurs at the normal extracting and bottling temperature of about 33°C. Treatment at these temperatures also facilitate the flow of honey for processing operations and should not effect the flavour and aroma, although this has not been reported. Thrasyvoulou et al. (1994) studied the liquefaction of ten candied honey samples by two methods: 23 kHz frequency ultrasound and heat treatment for 30 min at 60 °C. Untreated honey samples were used as the control. Ultrasound treatment of 300 g honey samples showed an increase in the honey temperature to 76 to 82°C, with the time required for liquefying honey samples being between 18 and 25 min (Table 1.23). Thrasyvoulou et al. (1994) reported that such difference in time may be due to the difficulty in immersing the tip of the sonotrode to the optimum depth due to the granulated condition of the samples. Honey samples were also analysed for hydroxymethylfurfural (HMF) content, sealed and stored at a room temperature of 25±4°C. The HMF content of samples increased after ultrasound treatment, as well as after heat treatment, in comparison with untreated samples. However, the average increase in HMF content due to ultrasound treatment and heat treatment were 85.7% and 128.7% respectively (Thrasyvoulou et al., 1994) (Table 1.24). Thus, the increase in the HMF content due to ultrasonication is less than that due to the conventional heating method. However, such a change in HMF on ultrasound treatment may have been due to the increase in temperature that occurs during ultrasound treatment, rather than actually being due to the ultrasound waves, particularly when the ultrasound treatment time averaged 22.3 min. This is a large treatment time for 300 g of candied honey. The ultrasound treatment as well as heat treatment also affected the diastase activity of honey. The average decrease in diastase activity for ultrasonic treatment and heat treatment were 16.2% and 23.1% respectively, again probably due to the long treatment time. In addition, Thrasyvoulou et al. (1994) reported no significant (p>0.05) effect on the moisture content, electrical conductivity and pH of honey from the ultrasound treatment. Further, analysis of crystallisation showed that honey samples treated by ultrasound remained in the liquid state for a much longer period than did the heat-treated samples, e.g. samples 2, 3 and 4 in Table 1.25. This is an important result and needs further studies involving Australian honeys.

36

Table 1.23 Some technical parameters of honey liquefied by ultrasonic waves of 23 kHz

Sample Number

Time (min)

Temperature (°C)

Energy (kWh)

1 24 79 0.1408 2 21 76 0.1232 3 22 76 0.1290 4 18 76 0.1056 5 24 79 0.1408 6 25 82 0.1466 7 21 80 0.1232 8 21 76 0.1232 9 24 80 0.1408

10 23 78 0.1348 Mean 22.3 78.2 0.1308 SD 2.1 2.2 0.0123

SD: standard deviation. Adapted from Thrasyvoulou et al. (1994) Table 1.24 The effect of ultrasonic waves (23 kHz) and heat treatment (60 °C for 30 min) on

the HMF levels of honey

Sample number Control (ppm) Ultrasonic Treatment Heat Treatment ppm Increase (%) ppm Increase

(%) 1 8.0 16.1 101.2 22.6 182.5 2 12.2 15.0 22.9 16.7 36.8 3 5.8 6.9 18.9 13.2 127.5 4 8.7 15.0 72.4 17.3 98.8 5 4.3 9.3 166.2 8.2 90.6 6 5.1 10.4 103.9 14.7 188.2 7 2.8 6.9 146.4 8.2 192.8 8 4.6 8 73.9 12.4 169.5 9 7.0 12.0 71.4 11.4 62.8

10 6.4 14.7 129.6 15.2 137.5 Mean 6.5 11.4 85.7 14.0 128.7

SD 42.2 55.2 SD: standard deviation. Adapted from Thrasyvoulou et al. (1994)

37

Table 1.25 Crystallisation of honeys that were liquefied by ultrasonic waves and heat treatment

Sample Number Days after Treatment

Ultrasound Heat 1 177 177 2 452 288 3 465 288 4 183 190 5 460 288 6 265 202 7 450 450 8 276 234 9 259 253

10 450 450 Mean 344.3

SE 38.8 SE: standard error Adapted from Thrasyvoulou et al. (1994) Thrasyvoulou et al. (1994) concluded that there is a negative effect of ultrasound treatment on the honey quality parameters of HMF and diastase activity. However, the effect was less in comparison with the heat treatment method. In addition, a positive effect of an increase in duration of the liquid state after ultrasound treatment was observed in comparison with heat treatment alone. Thus, Thrasyvoulou et al. (1994) suggested undertaking experiments on evaluating the effects of different ultrasound frequencies, and the development of a mechanism for reducing the temperature increase during treatment. Hebber et al. (2003) studied the effect of microwave heating to prevent deterioration of honey by fermentation during storage at ambient temperatures in tropical countries. The microwave oven used for the experiment has frequency of 2450 MHz and maximum power of 850 W. The raw honey was strained, heated to 40°C, filtered and stored at 4 °C prior to the experiment. Honey samples were drawn from storage and allowed to reach ambient temperature before treatment. Here, a 50 g honey sample in a 250 mL glass beaker was used for all the tests. Experiments were carried out at different power levels of 10 to 100 (175-850 W) and for different heating periods of 15 to 90 seconds. The temperature of honey after each treatment was measured and recorded at peak temperature. Then, samples were cooled to room temperature rapidly and analysed for HMF content, yeast content, diastase number and moisture content. HMF content increased marginally at lower heating durations and increased rapidly at higher power levels (Table 1.26). However, these values are much lower than the Codex Alimentarius standard of 60 mg/kg. Further, diastase activity, expressed as diastase number, decreased in all the heating conditions. However, most of the values are above the diastase number standard of eight. Hebber et al. (2003) concluded that though it is possible to achieve an acceptable level of yeast reduction through a combination of time and power level of microwave heating, consideration should be given to controlling the increase in the honey temperature.

38

Table 1.26 Effect on honey of microwave treatment at different power levels Sample Number

Power Level (W)

Power Intensity

(W/g)

Duration (s)

Peak Temperature

(°C)

Moisture (%)

Yeast Count

(cfu/mL) (x103)

HMF (mg/kg)

Diastase Number

Control 20 21.8 7.00 2.0 16.6 1 10 3.5 15 28 21.8 2.90 2.6 15.8 2 10 3.5 30 32 21.8 1.50 3.0 15.2 3 10 3.5 45 40 21.2 0.80 3.6 14.2 4 10 3.5 60 51 21.2 0.50 4.2 13.6 5 10 3.5 90 66 20.8 0.30 4.7 12.5 6 30 6.3 15 36 21.8 2.30 2.8 14.8 7 30 6.3 30 45 21.2 0.80 3.4 13.4 8 30 6.3 45 62 20.8 0.50 4.2 11.3 9 30 6.3 60 73 20.8 0.40 4.5 10.3

10 30 6.3 90 106 20.2 0.20 4.9 9 11 50 9.1 15 45 21.2 2.10 3.4 13.1 12 50 9.1 30 56 21.2 0.60 3.9 11.9 13 50 9.1 45 84 20.8 0.40 4.4 10.0 14 50 9.1 60 96 19.8 0.20 4.7 8.6 15 50 9.1 90 106 19.8 0.15 5.5 8 16 70 11.9 15 50 21.2 1.60 3.6 12.3 17 70 11.9 30 66 20.8 0.50 4.2 10.8 18 70 11.9 45 96 20.2 0.30 4.8 9.0 19 70 11.9 60 101 19.8 0.20 5.3 8.0 20 100 16.0 15 54 21.2 0.45 3.8 12.0 21 100 16.0 30 89 20.8 0.25 4.6 9.4 22 100 16.0 45 105 19.8 0.15 5.4 8.1 23 100 16.0 60 110 19.2 0.10 7.2 7.0

Adapted from Hebber et al. (2003) 1.7 Conclusion This review of the literature has shown there has been little study of the effect of high-power ultrasound on crystallised honey, and the effect of ultrasound on honey quality parameters. However, there are many studies that have reported detrimental effects of heat on honey quality. While heating is the standard method for liquefying candied honey throughout the world, there is an increasing interest from the honey industry to improve the quality, particularly, the flavour quality, of its honey as consumers become more demanding. There is also a continuing problem with crystallisation or candying of honey between liquefaction (by heating) during honey packing and purchase by food industry customers and consumers. In Australia, honey is expected to be sold in supermarkets in a liquid form, and crystallised honey is considered inferior. In addition, the food industry, which uses sizeable amount of honey, prefers a liquid product to make handling of honey easier since it can be pumped when in a liquid form. Thus, it is concluded that an alternate honey liquefaction technology to heating needs to be studied. Based on this review of the literature, high-power ultrasound has the potential to be such a alternate technology for liquefying candied honey, as long as it does not affect quality parameters such as HMF concentration, and enzyme activities such as for diastase and invertase. However, little has been studied on ultrasound liquefaction of candied honey with only two studies in 1955 and 1994 having been carried out. Thus, there is a gap in our understanding of this type of technology as it is applied to honey liquefaction, and a detailed study of ultrasound liquefaction of candied honey is urgently required.

39

2. Aims and Objectives 2.1 Overall Project Aims • To reduce the amount of expensive heating and loss in quality during liquefaction of candied

honey by developing an alternate, cost-effective ultrasound based method for the partial or complete liquefaction of candied honey by 2006, with a view to ultrasound having direct application for beekeeper control of honey crystallisation, or for liquefying candied honey prior to decanting in a honey packing plant.

• To better control the texture of creamed honey spread by developing an ultrasound based method

that enhances the nucleation rate and produces uniform crystal growth in a creamed honey system by 2006, with a view to it being used by beekeepers and honey processors for producing consistent and high quality creamed honey.

2.2 Project Objectives • To investigate the effect of ultrasound treatment on the cavitation of sugar solutions. • To investigate the effect of ultrasound treatment on the creamed honey production process. • To investigate the effect of ultrasound treatment on candied honey, including individual glucose

monohydrate crystals. • To determine the ultrasound conditions for liquefying candied honey and for controlling

crystallisation in honey.

40

3. Methodology The project is divided into three main sections based on the objectives:

• Effect of ultrasound treatment on the cavitation of sugar solutions.

• Effect of ultrasound treatment on the creamed honey production process.

• Effect of ultrasound treatment on candied honey, including individual glucose monohydrate crystals, and the determination of the ultrasound conditions for liquefying candied honey and for controlling crystallisation in honey.

3.1 Effect of Ultrasound on the Cavitation of Sugar Solutions 3.1.1 Experiment 1 Measurement of the size of the cavitation bubbles

generated by ultrasound using the Malvern Mastersizer/E The objective of this experiment is to develop a method for measuring cavitation in pure water and sugar solutions by determination of cavitation bubble sizes using a laser diffraction instrument. 3.1.1.1 Preliminary trial 1: Effect of ultrasound cavitation on pure water using a 22

mm diameter sonotrode A preliminary trial was undertaken to explore the possibility of measuring the size of the cavitation bubbles generated using ultrasound, by setting up the ultrasound processor within the Malvern Mastersizer/E. A rectangular glass vessel of 50 x 50 x 150 mm3 size was fabricated and placed in the space between the laser beam generator and detectors of the Malvern Mastersizer/E (Figure 3.1). A Dr Hielscher UP400S ultrasound processor, with an operational frequency of 24 kHz and fitted with a 22 mm diameter sonotrode, was mounted on a test rig designed and fabricated to hold the instrument. The sonotrode was inserted into the glass vessel containing distilled water. The ultrasound sonotrode, the glass vessel, and laser beam of the Malvern Mastersizer/E were set up to capture the bubbles generated closest to the tip of the sonotrode. The Malvern Mastersizer/E was fitted with a lens of 300 mm focal length. The ultrasound processor and the Malvern Mastersizer/E were operated by Cronolog UPCCTRL version 2.3 Win software and the Malvern Mastersizer/E SB.0B computer software. The ultrasonication time was set for the minimum setting of one second, as any increase in time generated larger bubbles inside the glass vessel due to secondary cavitation. The test parameters of amplitude, cycle and ultrasonication time were set in the control view of the ultrasound processor.

41

Figure 3.1 Instrument setup of the Malvern Mastersizer/E and ultrasound processor The optical measuring unit of the Malvern Mastersizer/E was aligned by horizontal and vertical adjustments knobs, and by rotating the glass vessel to obtain satisfactory intensity. The detectors in the Malvern Mastersizer/E measure the laser beams diffracted by the cavitation bubbles in the vessel. The Malvern Mastersizer/E was set in master mode and command syntax written to capture the bubble field ten times, one measurement after the other, and to save data in a file. The ultrasound processor and Malvern Mastersizer/E were started at the same time by pressing the START keys of the respective computer key boards. Ten measurements were taken by the Malvern Mastersizer/E corresponding to 3000 sweeps of the bubble field. These measurements were analysed using the Mastersizer/E SB.0B computer software to obtain particle size versus percentage frequency by volume plots, and particle size versus percentage undersize by volume plots. The results table shows the percentage frequency by volume between each of the two particle size diameters, and percentage undersize by volume for respective particle size diameters. These plots and results were used to examine the bubble size distribution patterns generated by ultrasound. A number of tests were conducted for the ultrasound processor with the maximum amplitude setting of 100 µm (100% amplitude) and the 100 % cycle (continuous) with an ultrasonic treatment time of one second. Distilled water was used as the test liquid and the bubble field was captured by the Malvern Mastersizer/E.

Ultrasound processor

Glass vessel

Sonotrode

Test rig

Malvern Mastersizer/E

Detector

Laser beam generator

42

3.1.1.2 Preliminary trial 2: Effect of cavitation on pure water and fructose solutions

using a 7 mm diameter sonotrode In this preliminary trial, the 7 mm diameter sonotrode was used instead of the 22 mm diameter sonotrode, with the ultrasound processor operated for 5 s to examine the size of the cavitation bubbles. The Dr Hielscher UP400S ultrasound processor with a 7 mm diameter sonotrode was mounted on a test rig and the sonotrode was inserted into the 50 x 50 x 150 mm3 size glass vessel containing distilled water. The glass vessel was placed between the laser beam generator and the detector of the Malvern Mastersizer/E. The ultrasound processor was operated for 5 s with 100 % emission cycle (continuous) and different amplitude settings of 43.75 µm, 87.5 µm, 131.25 µm and 175 µm (i.e. 25 %, 50 %, 75 % and 100 % amplitude) using distilled water as the test liquid. The Malvern Mastersizer/E was programmed to capture 10 measurements of the bubble field for all these tests. These records were analysed to obtain the particle size versus percentage frequency by volume plots and results tables, and percentage undersize by volume plots and results tables. Similarly, another set of tests was conducted to investigate the cavitation in fructose solutions with concentrations of 20 %, 30 %, 40 %, 60 % and 80 %. The bubble fields of the fructose solutions were captured 10 times for 20 s of operation of the ultrasound processor. 3.1.2 Experiment 2: Measurement of the ultrasound cavitation by analysis of

decomposition of an aqueous iodine solution A preliminary trial was conducted to develop a calibration curve of iodine concentration versus absorbance using standard iodine solutions. This was necessary in order to determine the amount of iodine liberated after ultrasonication in relation to the absorbance of UV-Visible spectrophotometer. Standard iodine solutions with concentrations of 0.3 – 1.3 x 10 –3 mg/mL were prepared by dissolving the corresponding amount of resublimed iodine in 0.1 M potassium iodide solution. The absorbance of these iodine solutions was measured at 354 nm using a Pharmacia LKB Ultraspec III spectrophotometer. Potassium iodide solution (0.1 M) was used as the solvent blank. In addition, other iodine solutions of 0.094 x 10 –3 mg/mL and 0.19 x 10 –3 mg/mL were prepared, and the absorbance was measured at 354 nm using a Pharmacia LKB Ultraspec III spectrophotometer, in order to extend the quantification calibration curve between 0.1 x 10 –3 mg/mL and 0.3 x 10 –3 mg/mL.

43

3.2 Effect of Ultrasound Treatment on the Creamed Honey

Production Process 3.2.1 Assessment of creamed honeys based on their crystalline D-glucose

monohydrate contents determined using differential scanning calorimetry (DSC)

3.2.1.1 Honey samples The following five honey samples were supplied by Capilano Honey Ltd. for use in creamed honey production:

• Salvation Jane honey • red gum honey • iron bark honey • clover honey • canola honey.

3.2.1.2 Preparation of honey samples All the honey samples were heated at 55 °C for 16 h in a water bath to dissolve the crystals and then permitted to cool to room temperature. The liquid honey samples were homogenized by mixing before sampling. Then, these well mixed honeys were kept in a temperature controlled incubator (SANYO Cooled Incubator Model: MIR-253) at 14 °C for two days before being used in the production of creamed honey. 3.2.1.3 Method for the preparation of experimental creamed honey Creamed honey preparation was based on the Dyce (1931a,b, 1976) method, which is the basis of most creamed honey production processes used in industry throughout the world. Commercial Capilano Honey Ltd. creamed honey used as seed honey in the experiment was kept at 14 °C for two days before use. Here, commercial Capilano Honey Ltd. creamed honey (15 g) as seed honey was added to 85 g of each honey blend in a 250 mL plastic container, and the final blend was well mixed with a small glass stirring rod. The containers containing the honey blend and the glass stirring rod were then sealed with a screw plastic top (to maintain the moisture content), and stored at 14 °C in a temperature controlled incubator (SANYO Cooled Incubator Model: MIR-253). The honey samples were then mixed for 1 min every 3 h (for a total of four times a day), for two days. In commercial production of Capilano Honey Ltd. creamed honey, the honey blend and seed honey are kept at 18 °C and maintained at this temperature after mixing and while being continually agitated for two days (Personal correspondence with Ralph Williams, Capilano Honey Ltd.). Commercial creamed honey is produced in about three days, and is ready for sale in six days.

44

3.2.1.4 Differential scanning calorimetry (DSC) instrumentation Crystal content in creamed honey was determined as crystalline D-glucose monohydrate using differential scanning calorimetry. A Perkin Elmer Pyris 1 differential scanning calorimeter (DSC) with Pyris software for Windows version 3.51 and Pyris thermal analysis system version 3.51 were used to determine the amount of crystalline D-glucose monohydrate content in the creamed honeys (Figure 3.2). The DSC operates on the principle of thermal transition. There are two aluminium pans, one is the reference pan and the other is the sample pan, and each pan sits on a heater. Heat flow was calibrated using Indium standard at the scan rate of 10 °C per min. The reference pan was kept empty and about 8-10 mg of creamed honey was weighed into a second aluminium sample pan and sealed with a lid. These two pans were heated at the same rate according to a given method: 0 °C to 90 °C at the rate of 30 °C / min. The DSC produces a graphic output of temperature versus heat flow. When the crystals in the creamed honey dissolve, heat is absorbed. After the temperature of sample pan reaches the dissolution temperature of the crystals, the temperature will not increase until all the crystals are dissolved. Therefore, the heater under the sample pan has to heat the sample to dissolve the crystals, as well as increase the temperature at the same rate as the reference pan. This extra heat flow during dissolution of the crystals is shown as a peak in the DSC plot. The analysis of the plot by Pyris thermal analysis system version 3.51 software determined the onset of dissolution, dissolution temperature, end of dissolution, and the area under the curve of the peak. Enthalpy change (ΔH) for the honey sample was calculated by dividing the area under the curve by the mass of the sample. The enthalpy change is equal to the energy required to dissolve D-glucose monohydrate crystals in honey. 3.2.1.5 Determination of the enthalpy change of crystalline D-glucose monohydrate

using DSC Pure samples of crystalline D-glucose monohydrate were analysed in triplicate using DSC, and the average enthalpy change (J) per g was calculated as described in 3.2.1.4. This value was used to determine the amount of crystalline D-glucose monohydrate in creamed honey (Section 3.2.1.6). 3.2.1.6 .Determination of the crystalline D-glucose monohydrate content in commercial

Capilano Honey Ltd. creamed honey using DSC For the Capilano Honey Ltd. creamed honey, three subsamples were analysed using the DSC (a triplicate analysis) to obtain the enthalpy change from the recorded thermograms. The crystalline D-glucose monohydrate content of commercial Capilano Honey Ltd. creamed honey was calculated using following equation:

Enthalpy change of creamed honey D-glucose monohydrate content (g/100 g) = x 100 Enthalpy change of crystalline D-glucose monohydrate

45

Figure 3.2 Differential scanning calorimeter 3.2.1.7 Determination of the crystalline D-glucose monohydrate content in

experimental creamed honeys using DSC 3.2.1.7.1 Preliminary creamed honey production experiment No 1 In the first preliminary experiment, six creamed honeys were prepared for DSC analysis using the following honey blends at ratios of 70:30 and the methodology in Section 3.2.1.3:

• Salvation Jane and red gum honeys • Salvation Jane and iron bark honeys • clover and red gum honeys • clover and iron bark honeys • canola and red gum honeys • canola and iron bark honeys.

For each creamed honey, three subsamples were analysed using the DSC (a triplicate analysis) at 1, 4, 5, 6 and 12 days after adding the seed honey to the blend (after production). The enthalpy changes were obtained from the thermograms, while the crystalline D-glucose monohydrate contents were calculated as described in 3.2.1.6. 3.2.1.7.2 Preliminary creamed honey production experiment No 2 In the second preliminary experiment, four creamed honeys were prepared for DSC analysis using the following honey blends and the methodology in Section 3.2.1.3:

• clover and canola honeys at a ratio of 70:30 • clover and canola honeys at a ratio of 30:70 • alfalfa and iron bark honeys at a ratio of 70:30 • alfalfa and blue gum honeys at a ratio of 70:30

46

The clover and canola honeys are both fast crystallising and fine grained honeys. For each creamed honey, three subsamples were analysed using the DSC (a triplicate analysis) at 2, 5 and 8 days after adding the seed honey to the blend (after production). The enthalpy changes were obtained from the thermograms, while the crystalline D-glucose monohydrate contents were calculated as described in 3.2.1.6. 3.2.1.8 Comparison of commercial Capilano Honey Ltd. creamed honey with the

experimental creamed honeys, based on the crystalline D-glucose monohydrate contents

The content of crystalline D-glucose monohydrate in the experimental creamed honeys was compared with the content in commercial Capilano Honey Ltd. creamed honey. 3.2.2 Effect of ultrasound treatment on the crystals and maximum temperature

of creamed honey, and the maximum temperature of liquid honey Preliminary experiments were carried out to determine the effect of ultrasound treatment on the crystal size and maximum temperature of creamed honey. Image analysis was used to examine the crystals in the creamed honey. In addition, a preliminary experiment on the effect of ultrasound treatment on the maximum temperature of liquid honey was carried. 3.2.2.1 Ultrasound processor A Dr Hielscher UP400S ultrasound processor used for this study (Figure 3.3) has an operational frequency of 24 kHz, and is designed for both batch and continuous application to process fluid or solid media on a laboratory scale. The ultrasound waves produced from the system are designed for the purpose of disintegration, such as cell disruption, emulsifying and homogenizing in laboratory vessels. The processor has an effective output power of 400 W and 300 W in liquid media. The efficiency of the processor is more than 85%. Ultrasonic waves generated by the processor are transmitted to the media by sonotrodes that emit from the front face. Sonotrodes are composed of titanium alloy and screwed to the electroacoustic transducer of the processor. There were three sonotrodes with diameters of 7 mm, 22 mm and 40 mm available for testing. These sonotrodes have different maximum amplitudes, maximum immerse depths and maximum sonic power densities (Table 3.1).

47

Figure 3.3 Ultrasound equipment setup 3.2.2.2 Operation and control of the ultrasound processor The processor was controlled by Cronolog UPCCTRL version 2.3 Win software. Amplitude, pulse interval, ultrasonic time or input energy parameters were entered numerically into the dialog boxes of the computer control display. Amplitudes and pulse intervals were entered as percentages, with ultrasound waves reaching the maximum amplitude at the 100% setting (Table 3.1). The amplitude setting can be adjusted between 25% and 100%, and the pulse interval within the range of 20% to 100%. At a 100% pulse interval, ultrasound is emitted continuously; so this was used for all experiments in this project. The ultrasonic treatment time depends on the input energy setting and vice versa. Setting one parameter, such as ultrasonic time determines the input energy into the medium. The ultrasonic processor was activated by the START function of the computer software, and the treatment was terminated by setting the ultrasonic time or input energy for the experiment.

Table 3.1 Technical details of ultrasound sonotrodes

Sonotrode Diameter

(mm)

Maximum Amplitude

(µm)

Maximum Immerse Depth (mm)

Maximum Sonic Power Density

(W/cm2) 7 175 90 300

22 100 45 85 40 12 20 12

Sonotrode

48

3.2.2.3 Calibration of the ultrasound processor Sonotrodes were fitted to the processor one by one and calibrated in air using the calibrate function of the software. These sonotrode files were saved for re-loading before each operation of the processor with the respective sonotrodes during the ultrasound experiments. 3.2.2.4 Ultrasound treatment methodology The processor was mounted vertically and a 250 mL plastic vessel with dimensions of 70 mm (diameter) x 79 mm (height) was placed on an adjustable stand for the treatment (Figure 3.3). The height of the adjustable platform was raised until the sonotrode just touched the top layer of honey inside the container. The specific sonotrode file was loaded for the fitted sonotrode, and amplitude and time settings for the treatment were set by entering the values. The treatment was started by the computer as described in 3.2.2.2. The sample vessel was rotated around the sonotrode so as to cover all the surface area of the top honey layer, and moved vertically using the adjustable stand. These horizontal and vertical movement are critical, otherwise, only the area close to the sonotrode will be treated since the high viscosity of honey prevents penetration of the ultrasonic waves into the honey. The time taken for one rotation was 15 – 20 s. One vertical movement of the platform was approximately 4 - 5 mm. 3.2.2.5 Image analysis of honey crystals 3.2.2.5.1 Image analyser instrument The image analyser consisted of a Prism Optical microscope fitted with a QImaging Micropublisher 3.3 RTV digital colour camera connected to a personal computer (Figure 3.4). 3.2.2.5.2 Analysis of a honey drop using the image analyser instrument The microscope slides (well type) were prepared by placing a drop of honey in the well of the microscope glass slide and covering it with a cover slip, followed by sealing through the application of “Depex” mounting medium. Each honey drop was then visualised using a light microscope connected to a computer monitor. Images were manually focussed to obtain clear images. Images displayed on the monitor were captured as several fields of view and saved by QCapture software. ImageJ software was used for image processing and mathematical measurements. 3.2.2.6 Measurement of the temperature of the honey The temperature profile of the creamed honey and liquid honey during ultrasound treatments was recorded using a temperature sensor and a data logger. 3.2.2.7 Determination of the effect of ultrasound treatment conditions on crystals and

the maximum temperature of commercial Capilano Honey Ltd. creamed honey Three preliminary experiments were carried out to determine the effect of different ultrasound treatments on the crystal size (as measured by an image analyser) and the maximum temperature of commercial Capilano Honey Ltd. creamed honey.

49

Figure 3.4 Image analyser

3.2.2.7.1 Preliminary ultrasound experiment No 1 Preliminary experiments were conducted in order to select a sonotrode for treatment of commercial Capilano Honey Ltd. creamed honey (100 g). Samples of commercial Capilano Honey Ltd. creamed honey were treated by the 22 mm diameter sonotrode (at amplitudes of 25 µm, 50 µm, 75 µm, 100 µm) and 40 mm diameter sonotrode (at amplitudes of 3 µm, 6 µm, 9 µm, 12 µm) at the 100% pulse interval for 40 s. The ultrasound pulse interval of 100% was used for all the tests as it transmits ultrasound waves continuously to the honey. Drops of treated creamed honey were analysed using the image analyser to observe the incorporation of air bubbles and to determine the size of the crystals after treatment. 3.2.2.7.2 Preliminary ultrasound experiment No 2 Further tests were undertaken using the 40 mm diameter sonotrode with different amplitude settings (3 µm, 6 µm, 9 µm, 12 µm) and a 100% pulse interval to treat of commercial Capilano Honey Ltd. creamed honey (100 g) for 60 s in order to select suitable treatment conditions. The objective of these tests was to melt large crystals into small ones and incorporate air bubbles which will act as nuclei during crystallisation. The ultrasound treatment pulse interval of 100% was used for all the tests as it transmits ultrasound waves continuously to honey. The temperature profile of the honey during treatment was recorded as in Section 3.2.2.6. Drops of honey samples were observed under the microscope equipped with a camera, and images were captured and analysed by the image analyser software (Figure 3.4).

50

3.2.2.7.3 Preliminary ultrasound experiment No 3 Next, creamed honey samples (100 g) were treated with the 40 mm diameter sonotrode at amplitude settings of 3 µm and 6 µm for 40 s, and the temperature profile of the honey during treatment was recorded as in Section 3.2.2.6. 3.2.2.8 Determination of the effect of ultrasound treatment conditions on the

maximum temperature of liquid honey Tests were also conducted to select which ultrasound conditions for the treatment of liquid honey (100 g) raised the honey temperature the least. Thus, liquid honey (100 g) was treated with the 40 mm diameter sonotrode operated at different amplitude settings of 3 µm, 6 µm, 9 µm and 12 µm, and an ultrasonic treatment time of 30 s. The temperature profile of the honey during treatment was recorded as in Section 3.2.2.6. 3.2.3 Effect of ultrasound treatment on the experimental creamed honey

process The objective of this experiment was to evaluate the effect of ultrasound treatment on creamed honey production by treating seed honey, as well as the honey blends mixed with seed honey. A replicated experiment was undertaken, and the crystalline D-glucose monohydrate contents (determined using DSC analysis) in the final experimental creamed honey samples were used as the determining factor to statistically evaluate the quality of creamed honey. 3.2.3.1 Selection of honey samples for blends Alfalfa and blue gum, and canola and red gum honeys were selected for use in blends for this study, based on results of the two preliminary experiments (Section 3.2.1.7). 3.2.3.2 Preparation of honey blends A 70:30 honey blend (1600 g) was made by mixing alfalfa honey (1120 g) and blue gum (480 g). Similarly, a canola and red gum 70:30 honey blend was prepared. The covered containers of honey blends (1600 g) were heated at 55 °C for 16 h in a water bath so as to dissolve all the crystals. For each of the two warm honey blends, 16 subsamples of honey (85 g) were then transferred into 250 mL plastic vessels, and allowed to cool to room temperature. Then, these 32 vessels were kept in an incubator at 14 °C for two days before production of the creamed honey. These honey samples were stirred every 3 h for 1 min, four times a day, while in the incubator (2 days). 3.2.3.3 Preparation of seed honey Commercial Capilano Honey Ltd. creamed honey was kept in the incubator at 14 °C for two days before being used as seed honey. In addition, ‘ultrasound-treated’ creamed honey was prepared by treating commercial Capilano Honey Ltd. creamed honey under the following ultrasound conditions, with a Dr Hielscher UP400S

51

ultrasound processor with an operational frequency of 24 kHz using the methodology detailed in Section 3.2.2.4:

• 40 mm diameter sonotrode • 6 µm amplitude • 100% pulse interval • 30 s treatment time

This ultrasound treatment combination was the one that produced the lowest maximum temperature for liquid honey (Section 3.2.2.8) and some dissolution of crystals to smaller ones. This ‘ultrasound-treated’ Capilano Honey Ltd. creamed honey was kept in a temperature controlled incubator (SANYO Cooled Incubator Model: MIR-253) at 14 °C for one day before being used as ‘ultrasound-treated’ seed honey. 3.2.3.4 Preparation of experimental creamed honey from the honey blends and the

seed honey Capilano Honey Ltd. creamed honey (15 g), as seed honey, was added to 85 g of each honey blend prepared as in Section 3.2.3.2, and the final blend was well mixed with a small glass stirring rod. The containers containing the honey blend and the glass stirring rod were then sealed with a screw plastic top (to maintain the moisture content), and stored at 14 °C in a temperature controlled incubator (SANYO Cooled Incubator Model: MIR-253). The honey samples were then mixed for 1 min every 3 h (for a total of four times a day), for three days. After this period, the creamed honey samples were stored at 14 °C. 3.2.3.5 Experimental design Four replicates of four different treatments (Table 3.2) of two honey blends were used in this replicated experiment, resulting in a total of 32 experimental creamed honey samples being produced. Using the ultrasound methodology detailed in Section 3.2.2.4, the following ultrasound conditions were used to prepare treatments T3 and T4:

• 40 mm diameter sonotrode • 6 µm amplitude • 100% pulse interval • 30 s treatment time.

3.2.3.6 Determination of crystalline glucose content in creamed honeys The 32 experimental creamed honey samples were analysed by DSC in a completely randomised manner. For each creamed honey sample, four subsamples were taken for DSC analysis, and the crystalline D-glucose monohydrate content was determined as described in Section 3.2.1.6. The first DSC analysis of the creamed honey samples occurred at 5 days after first addition of seed honey to the blends. The next DSC analyses for the alfalfa/blue gum creamed honey samples were carried out after 13 days, while the canola/red gum creamed honey samples were analysed again after 18 days.

52

Table 3.2 Details of treatments in the study of the ultrasound effect on the creamed honey process

Treatment Description

T1 15 g of seed honey is added to 85 g of honey blend, mixed and kept in the incubator at 14 °C.

T2 15 g of ‘ultrasound-treated’ seed honey is added to 85 g of honey blend, mixed and kept in the incubator at 14 °C.

T3 15 g of seed honey is added to 85 g of honey blend, mixed and kept in the incubator at 14 °C. Honey mixture was treated by ultrasound one day after the addition of the seed honey.

T4 15 g of seed honey is added to 85 g of honey blend, mixed and kept in the incubator at 14 °C. Honey mixture was treated by ultrasound two days after the addition of the seed honey.

Next, DSC analysis of canola/red gum creamed honey samples were undertaken after 39 days and alfalfa/blue gum creamed honey samples after 47 days. Because only 8 sub samples (two sample containers) could be analysed using the DSC in a day, the difference in 39 and 47 days is purely due to the analytical capacity of the DSC. 3.2.3.7 Statistical analysis of data for the crystalline glucose contents Statistical analysis of the data for the crystalline D-glucose monohydrate content of the experimental creamed honey samples was carried out using Minitab for Windows Release 14 according to a completely randomised experimental design of “2 honey blends x four treatments x four replicates”. Analysis of variance was calculated using the general linear model to obtain the variability between the treatments at the 95% significant level. 3.2.3.8 Conditioning of creamed honey Conditioning of creamed honey is done to soften the honey, since creamed honey is generally hard and difficult to spread immediately after production. In this study, it was observed that replicates of the same treatment had different final consistencies, with some replicates having the required creamy consistency, while other replicates had a semi-solid hard consistency. Thus, an investigation into the effect of conditioning on the consistency and content of crystalline D-glucose monohydrate was carried out. 3.2.3.8.1 Canola/red gum creamed honey Two different replicate samples of canola/red gum creamed honey produced using treatment T1 were selected for conditioning. One of these replicate creamed honey samples had a creamy consistency, while the other had a semi-solid consistency. These creamed honeys were stored at room temperature (25 °C) for 14 days (conditioning). Three subsamples from each of these two conditioned creamed honey samples were analysed by DSC for their the crystalline D-glucose monohydrate content as described in Section 3.2.1.6.

53

Next, one replicate canola/red gum creamed honey sample, produced using treatment T2, was stored at room temperature (25 °C) for 3 days, while another canola/red gum creamed honey sample, also produced using treatment T2, was stored in a water bath at 30 °C for 3 days in order to evaluate the effect of a conditioning heat treatment of 30 °C. Three subsamples from each of these two conditioned creamed honey samples were analysed by DSC for their the crystalline D-glucose monohydrate content as described in Section 3.2.1.6. 3.2.3.8.2 Alfalfa/blue gum creamed honey Two alfalfa/blue gum creamed honey samples with different physical consistencies (creamy and semi-solid), produced using treatment T2, were also stored at room temperature (25 °C) for 14 days (conditioning). Three subsamples from each of these two conditioned creamed honey samples were analysed by DSC for their the crystalline D-glucose monohydrate content as described in Section 3.2.1.6. 3.3 Effect of Ultrasound Treatment on the Liquefaction of Candied

honey and for Controlling Honey Crystallisation 3.3.1 Experiment 1: Ultrasound treatment of candied honey with interruption

of input energy, for selection of sonotrode and treatment conditions The objective of this experiment was to identify the most effective sonotrode and ultrasound treatment condition for liquefying candied honey. A Dr Hielscher UP400S processor with three sonotrodes of diameter 7 mm, 22 mm and 40 mm were used for the experiment along with four amplitude settings. The effective amplitude (µm) of the 25%, 50%, 75% and 100% amplitude settings depends on the maximum amplitude of each sonotrode. The maximum amplitudes of the three sonotrodes were 175 µm, 100 µm and 12 µm for the 7 mm, 22 mm and 40 mm diameter sonotrodes respectively. The ultrasound input energy was interrupted at ~10000 J intervals to monitor the temperature profile within honey after each ~10000 J treatment, and to evaluate the effect of different sonotrodes at different amplitude levels. In general, sonotrodes with a smaller front face produce lower sonic power. However, the actual emitted sonic power depends on the amplitude and sonic power density of the sonotrode. The 7 mm diameter sonotrode has the highest sonic power density of 300 W/Cm2 while the 22 mm and 40 mm diameter sonotrodes have 85 and 12 W/Cm2 respectively. Any future scale of a sonotrode for commercial use in the honey industry will require knowledge of how the diameter of the front face of a sonotrode affects the dissolution of crystals in honey. 3.3.1.1 Honey samples Candied (fully crystallised) Salvation Jane honey was used for the experiment. This honey was supplied by Capilano Honey Ltd.. The candied honey was kept at room temperature until required for the experiment.

54

3.3.1.2 Preparation of honey samples About 250 g of candied Salvation Jane honey was weighed as a sample into a 250 mL plastic container, 70 mm in diameter and 79 mm in height. 3.3.1.3 Ultrasound treatment conditions Honey samples of ~250 g in plastic containers were treated by ultrasound as described in Section 3.2.2.4. A Dr Hielscher UP400S ultrasound processor used for this study (Figure 3.3) has an operational frequency of 24 kHz, and is designed for both batch and continuous application to process fluid or solid media on a laboratory scale. The following ultrasound conditions were entered in the numerical fields of computer control dialog boxes: input energy of ~10000 J; amplitudes as different percentages between 25 - 100% depending on the treatment condition; and pulse control at 100% cycle (continuous). The tests were activated by the START function of the Cronolog UPCCTRL version 2.3 Win software. The termination criterion of each treatment was an input energy value of ~10000 J. These ultrasonic sonotrodes have different maximum amplitudes which is achieved at the 100% amplitude setting in the computer software of ultrasound processor. As an example, the 7 mm diameter sonotrode has a maximum (100%) amplitude of 175 µm, thus the 75% amplitude for the 7 mm sonotrode refers to an ultrasonic wave of 131.25 µm amplitude. 3.3.1.4 Experiment 1 - Preliminary trial A preliminary trial was carried out to determine the ultrasonic energy required to liquefy~250 g of candied Salvation Jane honey with interruptions after each input energy of ~10000 J. A Dr Hielscher UP 400S ultrasound processor with a 40 mm diameter sonotrode, four amplitude settings of 12, 9, 6 and 3 µm, and the 100% cycle setting (continuous) were used for the trial. The operation and equipment set up is detailed in Section 3.2.2.4. 3.3.1.5 Experiment 1 - Replicated trial A replicated experiment was conducted in order to identify the most effective ultrasound sonotrode and treatment condition for liquefying candied honey, and to investigate the distribution and increase of temperature in honey during treatments. There was a total of 12 treatments consisting of 3 diameters of sonotrode x 4 amplitudes. The 100% cycle was used for each sonotrode and six ~10000 J energy inputs, with interruptions after each ~10000 J, were utilised. A total input energy of approximately 60000 J was thus supplied to each honey sample. Full details of the ultrasound treatment conditions used in the experiment are given in Table 3.3. The data file, which consists of ultrasonic time, amplitude (as a percentage), pulse or cycle (as a percentage always 100%), bulk power, net power and input energy produced by the Cronolog UPCCTRL version 2.3 Win software, was saved after each interruption during the treatments and exported to Microsoft Excel for further analysis. Each of the 12 treatments was replicated three times. Thus, 36 honey samples of ~250 g each were used for this replicated experiment. In addition, six ultrasound tests were carried out in any one day. Therefore, the experiment consisted of 36 honey samples that was carried out over six days using an incomplete randomised block design (Table 3.4).

55

Table 3.3 Ultrasound treatment conditions for a combination of sonotrode and amplitude

Treatment No. Treatment Conditions

T1 40 mm diameter sonotrode, 100% amplitude (12 µm), 100% cycle



T4 40mm diameter sonotrode, 25% amplitude (3 µm), 100% cycle






T10 7 mm diameter sonotrode, 75% amplitude (131.5 µm), 100% cycle

T11 7 mm diameter sonotrode, 50% amplitude (87.5 µm), 100% cycle

T12 7 mm diameter sonotrode, 25% amplitude (43.75 µm), 100% cycle Energy input of 6 x ~10000 J was used, with an interruption between each ~10000 J input in order to measure the bulk power, net power, ultrasonic time and temperature after each ~10000 J of energy input.

Table 3.4 Experimental design of the ultrasound treatment conditions

Day Treatment Conditions 1 T11R2 T8R2 T9R2 T10R2 T1R2 T5R2 2 T2R2 T6R2 T7R2 T3R2 T4R2 T12R2 3 T1R3 T3R3 T5R3 T8R3 T4R3 T12R3 4 T9R3 T6R3 T10R3 T2R3 T7R3 T11R3 5 T7R1 T2R1 T5R1 T3R1 T10R1 T11R1 6 T8R1 T9R1 T4R1 T12R1 T1R1 T6R1

T1 to T12 and R1 to R3 are denoted as 12 ultrasound treatment conditions and 3 replicates respectively. 3.3.1.6 Determination of temperature profile The temperature profile was monitored inside the honey sample by inserting a K-type thermocouple from the top to the bottom and back to the top within approximately one minute. The thermocouple was connected to a Center 309 data logger thermometer and temperature data were recorded for each one second interval. These data were loaded to a personal computer through Testlink SE309 – RS232 software for analysis of the temperature profile in honey and to determine the maximum temperature after each ~10000 J energy treatment.

56

The temperature of all the honey samples was measured at room temperature before the treatment. It was not possible to use the data logger thermometer during the ultrasound treatment due to interference created by the ultrasound waves. Consequently, the temperature was measured using the data logger, as described above, during the whole of the~60 s interruption period between each ultrasound treatment, and for 60 s after the sixth ultrasound treatment. The temperature files recorded by the data logger thermometer were analysed in order to determine the temperature profile and maximum temperature in honey after each interruption and after a total of six individual ~10000 J of energy treatment. 3.3.1.7 Determination of bulk power, net power, ultrasonic time and temperature The data files of experimental data of each ~10000 J ultrasound treatments captured by the Cronolog UPCCTRL software were analysed in order to determine:

• time required to supply the fixed amount of input energy to the honey • maximum and minimum bulk power • net power of the ultrasound processor during the treatment (including the maximum net power

for any 1 s period) • actual input energy emitted to the honey.

The net power of the ultrasound processor is an important parameter in the design of industrial type ultrasound processors for liquefying candied honey. The relationship of bulk power to net power provides the efficiency of the ultrasound processor during the treatment of honey (Personal correspondence with staff from the ultrasound equipment manufacturer, Dr Hielscher GmbH) 3.3.1.8 Statistical analysis Statistical analysis of maximum net power, treatment time and maximum temperature measured after each treatment was carried out using Minitab for Windows release 14 and SAS Statistical software version 8.0 according to an incomplete randomised block design with twelve treatments, six blocks and three replicates. Analysis of variance was calculated using the general linear model to obtain the variability between the treatments at the 95% significant level. 3.3.2 Experiment 2: Effect of different input energy levels of continuous

ultrasound treatment (40 mm diameter sonotrode at 12 µm) for liquefying candied honey, and its effect on enzyme activity and the hydroxymethylfurfural concentration

The first and main objective of this experiment was to determine the input energy requirement for completely liquefying an amount of candied honey by a continuous ultrasound treatment. The second aim was to determine the effect of ultrasound treatment on the hydroxymethylfurfural concentration, and on the diastase and invertase activities of honey in comparison with the heat treatment. The actual maximum net power during the treatment is a design criterion for the scale-up of the ultrasound processor for industrial use. The specific input energy (kWh/kg) for liquefying candied honey is a function of the maximum net power, treatment time, and the mass of honey used.

57

3.3.2.1 Honey samples Candied Salvation Jane honey was used for the experiment. These honey samples were supplied by Capilano Honey Ltd. 3.3.2.2 Preparation of honey samples About 250 g of candied Salvation Jane honey was weighed as a sample into a 250 mL plastic container, 70 mm in diameter and 79 mm in height. 3.3.2.3 Ultrasound treatment of honey at different input energy levels The Dr Hielscher UP400S ultrasound processor used for this study (Figure 3.3) has an operational frequency of 24 kHz, and is designed for both batch and continuous application to process fluid or solid media on a laboratory scale. The ultrasonic sonotrode of 40 mm diameter was selected with 100% amplitude (12 µm) and 100% cycle (continuous) for this experiment, as it is the most effective treatment condition found in Experiment 1. Honey containers were treated by different ultrasound input energies using the method detailed in Section 3.2.2.4. First, the sonotrode file for the 40 mm diameter sonotrode was loaded. Then, the test parameters of amplitude and cycle for all the tests were entered as 100% to obtain maximum amplitude (12 µm) and continuous emission of ultrasound. The required input energy values were entered for each test and treatment was activated by the START function of the UPCCTRL Cronolog 2.3 Win software. The ultrasound treatment was terminated by the computer software when the input value of the ultrasonic input energy was reached. 3.3.2.4 Experiment 2 - Preliminary trial A preliminary trial was carried out to determine the range of input energy required for liquefying ~250 g of candied Salvation Jane honey by continuous ultrasound treatment with the 40 mm diameter sonotrode operated at the maximum amplitude (12 µm)and 100% cycle (continuous) settings. 3.3.2.5 Experiment 2 - Replicated trial 3.3.2.5.1 Preparation of ultrasound-treated honey A replicated trial was carried out to liquefy ~250g of a candied honey sample in a plastic container using six different ultrasound input energy levels within the range of ~50000 – ~70000 J (Table 3.5). This experiment with six treatments and three replicates (total of 18 samples) was carried out using a randomised complete block design (Table 3.6). One replicate of each of the six treatments were randomly selected and undertaken each day, with the replicated trial being conducted over three days (Table 3.6). The 18 ultrasound-treated honey samples were placed in an ice bath immediately after treatment and the temperature of honey was monitored by a Center 309 data logger thermometer. As soon as the honey reached room temperature (25 °C), the sample container was sealed with a screw cap and transferred to freezer prior to the analysis of the hydroxymethylfurfural (HMF) concentration, and the diastase and invertase activities.

58

Table 3.5 Six ultrasound input energy levels used to treat candied honey

Treatment Input energy (J)

T1 50000 T2 55000 T3 60000 T4 62500 T5 65000 T6 70000

Table 3.6 Experimental design of six ultrasound treatment conditions used to treat candied honey

Day Treatment Conditions 1 T2R1 T5R1 T3R1 T6R1 T4R1 T1R1 2 T6R2 T2R2 T3R2 T1R2 T4R2 T5R2 3 T1R3 T2R3 T6R3 T5R3 T3R3 T4R3

Where, T1 - T6 : Ultrasound input energy levels; R1 - R3 : Replicates 3.3.2.5.2 Preparation of heat-treated honey samples A candied Salvation Jane honey sample (~250 g) was liquefied by heating in a water bath at 55 °C for 16 h followed by heating at 72 °C for 2 min. This is similar to the processing conditions used in the Australian honey industry to liquefy candied honey prior to packaging. One heat-treated honey sample was prepared each day for three days (total of three samples). The three heat-treated samples were cooled rapidly to room temperature (25 °C) in an ice bath, after which the containers were sealed with a screw cap and transferred to a freezer prior to analysis of the hydroxymethylfurfural (HMF) concentration, and the diastase and invertase activities. 3.3.2.6 Determination of the temperature profile and monitoring the temperature in

honey The temperature profile of each honey sample was measured and recorded using a Center 309 data logger thermometer immediately after each treatment. In addition, the temperature of the ultrasound and heat-treated honey samples were continuously monitored until the temperature reached room temperature (25 °C). It was not possible to use the data logger thermometer during the ultrasound treatment due to interference created by the ultrasound waves. 3.3.2.7 Image analysis and visual observation of honey samples after treatment Samples obtained from each of the ultrasound-treated honeys were examined under an optical microscope as described earlier to observe the dissolution of crystals and the incorporation of air bubbles. In addition, visual observations on the status of honey in the containers were carried out after each treatment condition.

59

Samples of heat-treated honey were also observed under the microscope for the presence of crystals, and visual observations of the honey in the containers were recorded. 3.3.2.8 Preparation of honey samples for hydroxymethylfurfural, diastase and

invertase analysis Honey samples were removed from the freezer and kept at room temperature (25 °C) for 16 h in order to prepare honey solutions for chemical analysis. The 21 honey samples (18 ultrasound-treated and 3 heat-treated honey samples) were analysed in duplicate, with each of the 42 sub-samples being selected in a completely randomised order for analysis. 3.3.2.9 Determination of the hydroxymethylfurfural concentration of honey The hydroxymethylfurfural concentration of the honey samples was determined using Method 5.2 of the Harmonised Methods of the International Honey Commission (Bogdanov et al., 1997; Bogdanov, 2002). This method was developed by White (1979) and determines the concentration of hydroxymethylfurfural or 5-(hydroxymethyl-) furan-2-carbaldehyde as mg/kg. A Pharmacia LKB Ultraspec III spectrophotometer was used for analysis of the honey solutions. 3.3.2.10 Determination of the diastase activity of honey The diastase activity of honey samples was determined using Method 6.1 of the Harmonised Methods of the International Honey Commission (Bogdanov et al., 1997; Bogdanov, 2002) and a Pharmacia LKB Ultraspec III spectrophotometer. The original method was developed by Schade et al. (1958) and modified by White & Pairent (1959) and Hadorn & Zurcher (1972). Diastase activity is defined as the amount of enzyme required to convert 0.01 g of starch to the prescribed end point in 1 h at 40 °C. The diminution, by diastase in honey, of the blue colour of a standard starch solution mixed with iodine is measured at intervals. The time (tx) required to reach an absorbance of 0.235 at a wavelength of 660 nm was determined by a regression equation of the trend line of the time versus absorbance plot. Diastase activity was given as Diastase Number (DN) expressed in Gothe units (or Schade units) and was calculated as 300 divided by tx . 3.3.2.11 Determination of the invertase activity of honey The invertase activity of honey was determined as Invertase Number (IN) by Method 9 of the Harmonised Methods of the International Honey Commission (Bogdanov et al., 1997; Bogdanov, 2002) and a Pharmacia LKB Ultraspec III spectrophotometer. p-Nitrophenyl-α-D-glucopyranoside, which was used as the substrate, is converted into glucose and p-nitrophenol by invertase (α-glucosidase) in the honey. The adjustment of the pH to 9.5 stops the enzymatic reaction and transforms p-nitrophenol into the p-nitrophenolate anion. The amount of converted substrate corresponds to amount of produced p-nitrophenolate anion, which was determined using a spectrophotometer at 400 nm. Invertase activity is expressed as Invertase Number and determined by the following equation developed by Hadron et al. (1966):

IN = 21.64 x ΔA400 nm Where, ΔA400 nm is the absorbance of the sample solution subtracted by blank.

60

3.3.2.12 Determination of the deviation from the mean The results of the HMF concentration, and the diastase and invertase activities were calculated using two determinations obtained by the same method on two identical sub-samples (determined in a random order) from the same honey sample, under the same conditions (same operator, same apparatus). For all the 21 honey samples (18 ultrasound-treated and 3 heat-treated) analysed in this experiment, the analysis was repeated until deviation from the mean between two sub-samples was within the precision levels given in the Harmonised Methods of the International Honey Commission (Bogdanov et al., 1997; Bogdanov, 2002) for the particular analytical method. 3.3.2.13 Statistical analysis Statistical analysis of the HMF concentration, and the diastase and invertase activities of the honey samples treated with different ultrasound input energy levels or a heat treatment was carried out using Minitab for Windows release 14 according to a completely randomised block design. Heat-treated honey samples were used as control. Analysis of variance was calculated using the General Linear Model to obtain the variability between the treatments at the 95% significant level, and to determine the effect of ultrasound treatment on each variable in comparison with the heat treatment. 3.3.3 Experiment 3: A comparative crystallisation study of candied honey

liquefied by ultrasound treatment or by heat treatment 3.3.3.1 Preliminary trial Preliminary tests were undertaken to determine the input energy required to liquefy a ~200 g sample of candied reworked mixed honey. The estimated input energy required for liquefying ~200 g of candied honey is 55000 J. As such, the input energy parameter was fixed at 55,000 J with the 40 mm diameter sonotrode operated at the 12 µm amplitude and 100 % cycle setting. The ultrasound processor was operated until the honey sample was completely liquefied. 3.3.3.2 Replicated trial of liquefaction of candied honey by ultrasound or heat

treatment 3.3.3.2.1 Experimental design A replicated trial was carried out to evaluate the effect of ultrasound treatment and heat treatment on crystallisation and crystal growth under the optimum crystallisation temperature of 14 °C. The experiment consisted of two treatments (T1 and T2) and six replicates (R1 - R6) in a completely randomised design (Table 3.7).

61

The two treatments were:

• T1: heat treatment at 55 °C for 16 h and 72 °C for 2 min

• T2: ultrasound treatment of 55000 J of input energy supplied by the 40 mm diameter sonotrode at the 12 µm amplitude and 100 % cycle setting until the honey was liquefied

The conditions of the heat treatment (T1) were chosen to approximate the heating regime used by industry for liquefying candied honey. Three replicates for each of the two treatments were undertaken on each of the two days of the experiment as part of a completely randomised design (Table 3.7).

Table 3.7 Experimental design for the heat and ultrasound treatments

Day Treatment Conditions 1 T1R1 T1R3 T2R2 T2R1 T2R3 T1R2 2 T2R4 T1R6 T1R5 T2R5 T2R6 T1R4

Where, T1 : Heat treatment and T2: Ultrasound treatment R1-R6 : Replicates 3.3.3.2.2 Honey samples Candied reworked mixed honey was used for the experiment. A honey sample of ~200g was used for this experiment. Reworked mixed honey for this trial was supplied by Capilano Honey Ltd. 3.3.3.2.3 Preparation of honey samples About 200 g of reworked mixed honey was weighed as a sample into a 250 mL plastic container, 70 mm in diameter and 79 mm in height. Twelve honey samples of ~200 g each were prepared for the experiment. 3.3.3.2.4 Ultrasound treatment conditions The Dr Hielscher UP400S ultrasound processor used for this study (Figure 3.3) has an operational frequency of 24 kHz, and is designed for both batch and continuous application to process fluid or solid media on a laboratory scale. The 40 mm diameter ultrasonic sonotrode was selected and operated at an amplitude of 12 µm and a 100 % cycle (continuous) for this experiment as it was the most effective sonotrode, amplitude and cycle setting found in Experiment 2 for the liquefaction of honey. Honey containers were treated with an instrumentally set input energy of 55000 J using the method detailed in Section 3.2.2. These samples were allowed to cool naturally to 55 °C by storing them at room temperature (25 °C), and the time taken for this was recorded. The honey samples were then immediately transferred to an incubator at 14 °C.

62

3.3.3.2.5 Heat treatment of honey samples Candied reworked mixed honey samples of ~200 g each were heat treated by placing honey containers in a water bath at 55 °C for 16 h followed by 72 °C for 2 min so as to dissolve the crystals. These samples were allowed to cool naturally to 55 °C by keeping them at room temperature (25 °C). The honey samples were then immediately transferred to an incubator at 14 °C. 3.3.3.2.6 Measuring temperature profile The temperature profile before and immediately after the treatments was monitored in all the honey samples using a Center 309 data logger thermometer. The temperature of honey was monitored continuously after each treatment until it reached 55 °C; the time required for each honey sample to reach 55 °C was recorded. 3.3.3.2.7 Image analysis of slides and visual observations of honey containers Three microscope slides (well type) each were prepared immediately after each replicated test of heat treatment and ultrasound treatment. The slides were prepared by placing a drop of honey in the well of a microscope glass slide and covering it with a cover slip, followed by sealing through the application of “Depex” mounting medium. The prepared slide was then placed inside a plastic test tube which was closed with a screw cap. The honey drop was collected after homogenising the sample after each treatment. These slides were kept at 14 °C inside an incubator, and examined every seven days using an optical microscope at 10 X magnification in order to observe the formation and growth of glucose monohydrate crystals in the honey drop. Microscopic observations every seven days continued up to 8 weeks, after which microscopic examination occurred every 4 weeks until 16 weeks. Here, the monitoring interval of slides was increased from one week to four weeks, as there was no significant change in formation of crystal or growth in crystals after seven days. The twelve honey containers of this replicated trial after heat treatment or ultrasound treatment were initially allowed to cool to 55 °C by storing them at room temperature. These samples were stored in an incubator at 14 °C and visually observed once every seven days for crystal formation in the honey. The following gradings were used:

• No crystals or granules in the top layer 0 • A few crystals and/or granules or clumps in the top layer * • Some crystals and/or granules or clumps in the top layer ** • More crystals and/or granules or clumps in the top layer *** • Top layer is completely crystallised. ****

63

4. Results and Discussion 4.1 Effect of Ultrasound on the Cavitation of Sugar Solutions 4.1.1 Introduction The objective of this experiment was to investigate the effect of water activity on cavitation generated by ultrasound treatment in sugar solutions. The mechanical disintegration of crystals during ultrasound treatment is due to cavitation. In order to study the cavitation in different concentrations of sugar solutions, a method for measuring cavitation was studied. Firstly, laboratory work was undertaken on the development of a method to measure cavitation by way of measuring the size of the cavitation bubbles. Burdin et al. (1999) have reported that the size of cavitation bubbles generated by a 13 mm diameter ultrasound sonotrode with a 20 kHz ultrasound processor inside a 150 x 100 x 270 mm3 glass vessel can be measured by a laser diffraction instrument, the Malvern Mastersizer 2600. The tests were conducted with a 100 mm focal length lens and measurement was captured by a 1000 sweeps setting. Thus, a laboratory trial was undertaken to explore the possibility of using the techniques and measuring instruments described by Burdin et al. (1999) by setting up the ultrasound processor within the Malvern Mastersizer/E to measure the size of cavitation bubbles. Research work by Ciaravino et al. (1983), Shirgaonkar et al. (1997), Naidu et al. (1994), Gogate et al. (2001, 2002) and Senthil Kumar et al. (2000) reported that the decomposition of potassium iodide by ultrasonication produces iodine. Further, the cavitational yield can be determined by measuring the amount of iodine liberated by ultrasonication. The amount of iodine released during sonication is very small and a UV-Visible spectrophotometer was used for such analysis. A preliminary trial was conducted to explore the possibility of determining cavitation by measuring the amount of iodine released during the ultrasound treatment of a potassium iodide solution. 4.1.2 Experiment 1 Measurement of the size of the cavitation bubbles

generated by ultrasound using the Malvern Mastersizer/E 4.1.2.1 Preliminary Trial 1: Effect of ultrasound cavitation on pure water using a 22

mm diameter sonotrode The preliminary trial of ultrasound cavitation of water found that there was turbulence inside the glass vessel for the 1 s operation of the ultrasound processor with the 22 mm diameter sonotrode, and the data capturing speed of the Malvern Mastersizer/E was not fast enough to capture the cavitation area. Therefore, it was difficult to obtain correct measurements of bubble sizes for the 1 s operation. Even though, the ultrasound processor was operated for 1 s, measurement by the Malvern Mastersizer/E takes 14 - 15 s, corresponding to 10 measurements with 3000 sweeps of the bubble field. The data captured by the Malvern Mastersizer/E were analysed, and the particle size versus percentage frequency by volume plots, and particle size versus percentage undersize by volume plots of the

64

records indicated that the plots were incomplete and the measurements were limited at a maximum of 600 µm. The results indicate that values for bubble diameters are in the range of 0 – 600 μm. To minimise the effect of turbulence generated by the 22 mm diameter sonotrode, a 7 mm diameter sonotrode was selected for the subsequent tests, and the ultrasound processor was set for an operation time of 20 s, since the Malvern Mastersizer/E captures the bubble field at the rate of 10 times within each 15 s. 4.1.2.2 Preliminary Trial 2: Effect of cavitation on pure water and fructose solutions

using a 7 mm diameter sonotrode Analysis of the Malvern Mastersizer measurements of bubble fields produced in pure water by ultrasound treatments with the 7 mm diameter sonotrode operated at different amplitudes showed that bubble sizes were in a range of 0 to 600 µm. The plot of particle size versus percentage frequency by volume, and the plot of particle size versus percentage undersize by volume were also incomplete. The tests for the 20 %, 30 % and 40 % fructose solutions were undertaken satisfactorily. However, it was not possible to obtain satisfactory laser intensity data for the 60 % and 80 % fructose solutions using the Malvern Mastersizer/E. The analysis of the results for the 20 % to 40 % fructose solutions showed similar results indicating large bubble sizes (up to 600 µm) and incomplete plots. Therefore, it was assumed that the bubbles attached to the sides of glass vessel had scattered light, and these bubbles may be responsible for the larger bubble diameters. A larger rectangular glass vessel (100 x 100 x 200 mm3) was fabricated in order to avoid the multiple scattering of light. However, there were difficulties in locating this glass vessel, filled with distilled water or fructose solution, in the Malvern Mastersizer/E and aligning the optical measuring unit to obtain the required laser intensity. Therefore, the ultrasound tests were not conducted using this glass vessel as the Malvern Mastersizer/E was unable to capture the bubble field with sufficient laser intensity. 4.1.3 Experiment 2: Measurement of the ultrasound cavitation by analysis of

decomposition of an aqueous iodine solution The chemical effect of ultrasound in liquids is related to the formation of free radicals. According to Ciaravino et al. (1983), Shirgaonkar et al. (1997), Naidu et al. (1994), and Gogate et al. (2001, 2002), the oxidation of a potassium iodide solution can be used as a model reaction for studying the effect of ultrasound waves. Further, if the oxidation of potassium iodide is done by hydrogen peroxide produced by ultrasound, then equivalent amounts of free iodine and hydroxyl ions should be formed as per the following equation.

H2O2 + 2KI -----> I2 + 2KOH Therefore, the cavitational yield can be determined by the fact that iodine ions in aqueous potassium iodide solution can be transformed into iodine by ultrasound treatment. Thus, the amount of iodine liberated can be used to measure the cavitational yield.

65

Iodine liberated by ultrasonication of a potassium iodide solution is very small. Therefore, a UV-Visible spectrophotometer was selected for analysing iodine, and the absorbance at 354 nm was monitored (Naidu et al. (1994). The analysis of absorbance data for iodine concentrations of 0.3 – 1.3 x 10 –3 mg/mL showed a linear regression with a correlation coefficient of 0.99. The linear regression equation between iodine concentration and absorbance was Y = 119.05 X + 0.013. However, a Pharmacia LKB Ultraspec III spectrophotometer was not sensitive enough to measure the absorbance at 354 nm for iodine concentrations of 0.1 – 0.2 x 10 –3 mg/mL, which are the concentrations produced by the ultrasound treatment of potassium iodide. Thus, it was concluded that this method was not able to be used to measure cavitation yield, and this cavitation study was abandoned. 4.1.4 Summary, conclusions and implications Finally, to better understand the effect of ultrasound on honey, a study of cavitation (bubble formation) produced by ultrasound in pure water and sugar solutions was carried out. Here, the results of this ultrasound cavitation study showed that the size of the cavitation bubbles as measured by the Malvern Mastersizer/E in water and fructose solutions using the 7 mm and 22 mm diameter sonotrodes exceeds 600 µm. However, the maximum particle size measurement of the Malvern Mastersizer/E is limited to 600 µm. Even when a larger containment vessel was used there were still difficulties encountered. Here, it was difficult to set up and align the optical measuring unit to obtain the required laser intensity for a larger 100 x 100 x 200 mm3 sized glass vessel and for high concentrations of fructose solutions. It is concluded that this experimental set up of the Malvern Mastersizer/E cannot be used for measuring the size of bubbles with diameters >600 μm that are generated by ultrasound in pure water and fructose solutions. Next, another method for measuring cavitation in sugar solutions was examined for applicability. This involved measuring the cavitation yield through measurement of the amount of iodine produced by ultrasound treatment of an aqueous solution of potassium iodide. The sensitivity of the spectrophotometer used was too low to measure the very low concentrations of iodine likely to be produced by ultrasound treatment. In conclusion, while it was one of the aims of this project to measure the cavitation produced in sugar solutions as a means of understanding the effect of ultrasound treatment on honey, an optimised method to measure such cavitation was not found, and this part of the project was not carried further.

66

4.2 Effect of Ultrasound Treatment on the Creamed Honey Production Process

4.2.1 Assessment of creamed honey blends based on their crystalline D-

glucose monohydrate contents determined using differential scanning calorimetry (DSC)

4.2.1.1 Determination of the enthalpy change of crystalline D-glucose monohydrate

using DSC The enthalpy change of crystalline D-glucose monohydrate is required to calculate the amount of crystalline D-glucose monohydrate in honey. Using pure crystalline D-glucose monohydrate as reference, analysis by differential scanning calorimetry showed the average enthalpy change of crystalline D-glucose monohydrate is 83.35 J per g. 4.2.1.2 .Determination of the crystalline D-glucose monohydrate content in commercial

Capilano Honey Ltd. creamed honey using DSC On analysis by DSC, commercial Capilano Honey Ltd. creamed honey samples had an average crystalline D-glucose monohydrate content of 39.6 – 40.1 g/100 g honey. 4.2.1.3 Determination of the crystalline D-glucose monohydrate content in

experimental creamed honeys using DSC 4.2.1.3.1 Preliminary creamed honey production experiment No 1 Differential scanning calorimeter analysis of creamed honey samples prepared from Salvation Jane/red gum, Salvation Jane/iron bark, clover/red gum, clover/iron bark, canola/red gum, and canola/iron bark honey blends, 12 days after the production, showed that the canola/red gum blend produce creamed honey with the highest amount of crystalline D-glucose monohydrate of 21.9 g/100 g honey (Table 4.1). As a result, the canola/red gum blend was selected for a further replicated experiment on creamed honey (Section 4.2.3.1.1).

Table 4.1 Crystalline D-glucose monohydrate contents of six creamed honey samples

Honey blend Amount of Crystalline D-glucose

Monohydrate (g/100 g) at 12 Days After Production

Salvation Jane (70%) and red gum (30%) 13.7 Salvation Jane (70%) and iron bark (30%) 15.2 clover (70%) and red gum (30%) 14.8 clover (70%) and iron bark (30%) 11.6 canola (70%) and red gum (30%) 21.9 canola (70%) and iron bark (30%) 14.5

67

4.2.1.3.2 Preliminary creamed honey production experiment No 2 Creamed honey samples prepared from clover/canola, alfalfa/blue gum, and alfalfa/iron bark honey blends were analysed by DSC eight days after production. Clover (70%)/canola (30%), and canola (70%)/clover (30%) creamed honeys showed higher crystalline D-glucose monohydrate contents of 26.6 and 29.5 g/100 g honey respectively (Table 4.2). However, these two creamed honey samples involving clover/canola honey blends were completely solidified and not able to be spread. This results from the fact that both these honeys are fast crystallising and fine grained honeys. Normally, for the honey blends used in commercial creamed honey productions, the honey in the blend in the highest amount must be fast crystallising and produce fine grained crystals, while the other honey(s) in the blend in lower amounts must be strongly flavoured so as to impart flavour to the final creamed honey. Thus, it is not ideal to have both honeys in the blend as fast crystallising as was the case with clover and canola. In comparison, the alfalfa/blue gum, and alfalfa/ironbark experimental creamed honeys were soft and spreadable, thus possessing ideal physical properties. Of these two experimental creamed honeys, the one prepared from the alfalfa and blue gum blend had the higher crystalline D-glucose monohydrate content of 21.6 g/100 g honey (Table 4.2). Thus, the alfalfa and blue gum blend was selected for further study (Section 4.2.3.1.2) since it was soft and spreadable after production and had a high crystal content.

Table 4.2 Crystalline D-glucose monohydrate contents of four creamed honey samples

Honey blend Amount of Crystalline D-glucose

Monohydrate (g/100 g) at 8 Days After Production

clover (70%) and canola (30%) 26.5 canola (70%) and clover (30%) 29.5 alfalfa (70%) and blue gum (30%) 21.6 alfalfa (70%) and iron bark (30%) 12.9

4.2.1.4 Comparison of commercial Capilano Honey Ltd. creamed honey with the

experimental creamed honeys based on the crystalline D-glucose monohydrate contents

The amount of D-glucose monohydrate crystals was lower for the experimental creamed honey blends (Tables 4.1 and 4.2) than for the commercial Capilano Honey Ltd. creamed honey (39.6 – 40.1 g/100 g honey). This is probably due to the length of time (12 and 8 days) after production when the DSC analysis was carried out. Up to 42 days are normally used in commercial production In addition, the exact formulation and honey blends used by Capilano Honey Ltd. was probably different to that used in this study (due to company confidentiality), thus further contributing to the lower crystal content. Thus, further work was undertaken to increase the amount of D-glucose monohydrate crystals in the experimental process; namely, the use of ultrasound treatment and increasing the length of the storage period at 14 °C after initial production, before the DSC analysis was carried out.

68

4.2.2 Effect of ultrasound treatment on the crystals and maximum temperature

of creamed honey, and the maximum temperature of liquid honey 4.2.2.1 Determination of the effect of ultrasound treatment conditions on the crystals

and maximum temperature of commercial Capilano Honey Ltd. creamed honey 4.2.2.1.1 Preliminary ultrasound experiment No 1 During this preliminary study, treatment of the commercial Capilano Honey Ltd. creamed honey samples with the 22 mm diameter sonotrode at four amplitudes resulted in complete dissolution of all the crystals during the short time treatment. However, it was found that the 40 mm diameter sonotrode was most effective for the short time (60 s) ultrasound treatment due to even application of ultrasound throughout the honey and the fact that there were some melted crystals of reduced size remaining after the treatment. 4.2.2.1.2 Preliminary ultrasound experiment No 2 To further study the creamed honey process, ultrasound treatment was investigated as a means of reducing the crystal size, and thus improving the spreadability of the product. Prior to the start of this project it was thought that ultrasound treatment of D-glucose monohydrate crystals in honey would shatter them, thereby reducing their size. However, the use of an image analyser showed that rather than reducing the crystal size through break up of the crystals, the crystal size was reduced through partial melting or dissolution of the D-glucose monohydrate crystals. The clean sharp crystal structures (plates) were replaced by irregular shaped plates indicating that some glucose molecules on the edge of the crystal structure were dissolving into the surrounding liquid, producing crystals that appeared to have melted edges and surfaces (Figure 4.1). Before Ultrasound Treatment After Ultrasound Treatment

Figure 4.1 Effect of controlled ultrasound on the crystal shape in crystallised reworked honey

Melted edges as crystals dissolve

69

On ultrasound treatment of commercial Capilano Honey Ltd. creamed honey samples (100 g) using the 40 mm diameter sonotrode at all the amplitude settings (3 µm, 6 µm, 9 µm, 12 µm), and with the 100% pulse interval and a 60 s treatment time, it was found that most of the crystals were dissolved and the creamed honey was liquefied. 4.2.2.1.3 Preliminary ultrasound experiment No 3 The maximum temperatures reached by commercial Capilano Honey Ltd. creamed honey when treated by the 40 mm sonotrode at amplitudes of 3 µm and 6 µm and a treatment time of 40 s, were 30 °C and 40 °C respectively. Microscopic observations showed there was incorporation of a large number of air bubbles, and that melted crystals (now of a smaller size) were still present when the amplitude setting of 6 µm was used. The 6 µm amplitude and 100% pulse interval for a 40 s treatment was selected from this experiment as it increased temperature of honey to the lowest maximum of 40 °C but still resulted in some dissolution of the crystals to smaller sizes. The maximum temperature was taken as the selection criteria since temperatures higher than 55 °C will dissolve all crystals in honey. 4.2.2.2 Determination of the effect of ultrasound treatment conditions on the

maximum temperature of liquid honey Application of continuous ultrasound treatment using the 40 mm sonotrode at amplitudes of 3 µm, 6 µm, 9 µm and 12 µm showed that the treatment involving the 6 µm amplitude at a 100% pulse interval for a 30 s treatment increased the temperature of liquid honey to the lowest maximum of 42°C. Thus, this treatment condition was selected for the other studies below. 4.2.3 Effect of ultrasound treatment on the experimental creamed honey

process 4.2.3.1 Determination of the crystalline glucose content in experimental creamed

honeys Based on the results and conclusions in Section 4.2.1.3, the two honey blends selected for this replicated study of the effect of ultrasound treatment on the creamed honey process were:

• canola and red gum honeys at a ratio of 70:30 • alfalfa and blue gum honeys at a ratio of 70:30.

4.2.3.1.1 Analysis of the crystal content of canola/red gum creamed honey Table 4.3 lists the amount of D-glucose monohydrate crystals (as determined by DSC) after postproduction storage at 14 °C for 18 days and 39 days storage for the experimental canola/red gum creamed honey samples.

70

Table 4.3 Mean content of crystalline D-glucose monohydrate in canola/red gum (70:30) creamed honey

Treatment Mean Amount of Glucose

Monohydrate (g/100 g) 18 Days After Production*

Mean Amount of Crystalline D-glucose Monohydrate (g/100 g)

39 Days After Production* T1 37.1 47.1 T2 33.6 44.5 T3 37.8 47.1 T4 33.7 46.5

*Means of four replicates T1: 15 g of seed honey was added to 85 g of honey blend, mixed and kept in the incubator at 14 °C. T2: 15 g of ‘ultrasound-treated’ seed honey was added to 85 g of honey blend, mixed and kept in the

incubator at 14 °C. T3: 15 g of seed honey was added to 85 g of honey blend, mixed and kept in the incubator at 14 °C.

The honey mixture was treated with ultrasound one day after the addition of the seed honey. T4: 15 g of seed honey was added to 85 g of honey blend, mixed and kept in the incubator at 14 °C.

The honey mixture was treated with ultrasound two days after the addition of the seed honey. The results indicate that the amount of crystalline D-glucose monohydrate in experimental canola/red gum creamed honeys increased with time during the storage period of 18 to 39 days after mixing the seed honey with the honey blend, particularly when compared to the crystal contents (21.9 g/100 g honey) observed for 12 days storage at 14 °C (Table 4.1). Thus, the length of the storage period, after mixing the seed honey with the honey blend, is critical in maximising the crystal content of creamed honey. Statistical analysis of the crystalline D-glucose monohydrate content in the canola/red gum creamed honey samples indicated there was no significant difference (P>0.05) between the different ultrasound-treated creamed honey samples (T2, T3, and T4) and the control creamed honey sample (T1). In addition, there was no significant difference (P>0.05) in the crystalline D-glucose monohydrate content between creamed honey samples prepared with ultrasound-treated seed honey (T2), and those prepared with untreated seed honey but treated with ultrasound 1 day (T3) and 2 days (T4) after mixing of the seed honey with the honey blend. In conclusion, there appears to be no benefit in terms of the amount of crystals formed during the creamed honey process from the use of ultrasound at different stages in the production process. 4.2.3.1.2 Analysis of the crystal content of alfalfa/blue gum creamed honey Table 4.4 lists the amount of D-glucose monohydrate crystals (as determined by DSC) after postproduction storage at 14 °C for 13 days and 47 days storage for the experimental alfalfa/blue gum creamed honey samples.

71

Table 4.4 Mean content of crystalline D-glucose monohydrate in alfalfa/blue gum

(70:30) creamed honey

Treatment Mean Content of Crystalline D-glucose Monohydrate (g/100 g)

13 Days After Production*

Mean Content of Crystalline D-glucose Monohydrate (g/100 g)

47 Days After Production* T1 17.5 33.1 T2 21.9 32.2 T3 21.5 32.8 T4 22.6 31.6

*Means of four replicates T1: 15 g of seed honey was added to 85 g of honey blend, mixed and kept in the incubator at 14 °C. T2: 15 g of ‘ultrasound-treated’ seed honey was added to 85 g of honey blend, mixed and kept in the

incubator at 14 °C. T3: 15 g of seed honey was added to 85 g of honey blend, mixed and kept in the incubator at 14 °C.

The honey mixture was treated with ultrasound one day after the addition of the seed honey. T4: 15 g of seed honey is added to 85 g of honey blend, mixed and kept in the incubator at 14 °C. The

honey mixture was treated with ultrasound two days after the addition of the seed honey. The results indicate that the amount of crystalline D-glucose monohydrate in experimental alfalfa/blue gum creamed honeys increased with time during the storage period of 13 to 47 days after mixing the seed honey with the honey blend. Thus, the storage period after mixing the seed honey with the honey blend is critical in maximising the crystal content of creamed honey. In addition, the crystal content of the alfalfa/blue gum creamed honey after 47 days of storage at 14 °C was lower than the canola/red gum creamed honey after 39 days of storage at 14 °C. Dyce (1976) suggested that storage for 42 days at 14 °C is required to produce a firm creamed honey prior to conditioning. Statistical analysis of the crystalline D-glucose monohydrate content in the alfalfa/blue gum creamed honey samples indicated there was no significant difference (P>0.05) between the different ultrasound-treated creamed honey samples (T2, T3, and T4) and the control creamed honey sample (T1). In addition, there was no significant difference (P>0.05) in the crystalline D-glucose monohydrate content between creamed honey samples prepared with ultrasound-treated seed honey (T2), and those prepared with untreated seed honey but treated with ultrasound 1 day (T3) and 2 days (T4) after mixing of the seed honey with the honey blend. In conclusion, there appears to be no benefit in terms of the amount of crystals formed during the creamed honey process from the use of ultrasound at different stages in the production process. 4.2.3.2 Conditioning of creamed honey High quality creamed honey is firm and creamy at room temperature. Dyce (1931a,b) recommended that if creamed honey is hard and difficult to spread, then it should be stored at 30 °C until it is spreadable. McDonald (1964) found that the honeys conditioned at 32 °C, five days after production did not encountered any difficulty with spreadability. 4.2.3.2.1 Canola/red gum creamed honey Two of the four replicate samples of the canola/red gum creamed honey, produced using the control treatment T1 (no ultrasound treatment), had different final physical consistencies before being subjected to a conditioning process of storage at room temperature (25 °C) for 14 days. After conditioning, the crystalline D-glucose monohydrate content increased from 48.8 g/100 g honey to 51.4 g/100 g honey for the replicate sample that had a creamy consistency but decreased from

72

45.0 g/100 g honey to 39.5 g/100 g honey for the replicate sample with a semi-solid consistency. However, these changes were not measured statistically to see if they were significant. When two replicates samples of the canola/red gum creamed honey, produced using treatment T2, were conditioned at temperatures of 25 °C and 30 °C, the crystal content decreased from 46.1 to 39.1 g/100 g honey (for a creamy creamed honey) and 41.6 to 38.8 g/100 g honey (for a semi-solid creamed honey) respectively. However, these changes were not measured statistically to see if they were significant. 4.2.3.2.2 Alfalfa/blue gum creamed honey The crystalline D-glucose monohydrate contents of two replicate samples (one with a creamy consistency and one with a semi-solid consistency) of the alfalfa/blue gum creamed honey, produced using treatment T2, increased from 34.0 to 39.4 g/100 g honey (original creamy consistency) and 32.5 to 38.6 g/100 g honey (original semi-solid consistency) after conditioning for 14 days in room temperature (25 °C). However, these changes were not measured statistically to see if they were significant. In conclusion, creamed honey samples conditioned at 25 °C and 30 °C reached comparable levels of D-glucose monohydrate crystals to that in commercial Capilano Honey Ltd. creamed honey (39.6 – 40.1 g/100 g). Thus, while conditioning of the experimental creamed honeys led to products that were spreadable with an ideal creamy consistency, the content of crystalline D-glucose monohydrate varied little (2.6-7.0 g/100 g honey) during conditioning. This modification of the physical consistency is probably due to the conditioning process at 25 °C or 30 °C leading to an slight increase in the level of liquid relative to crystal content (as some crystalline D-glucose monohydrate dissolves) making the creamed honey more creamy and spreadable. Dyce (1976) suggests that once creamed honey has become soft due to conditioning, it will not return to its original hardness even if stored at 14 °C and/or stirred. 4.2.4 Summary, conclusions and implications In another study, the creamed honey process was examined to determine if ultrasound could be used to improve it, particularly related to the crystallisation process, the size of the D-glucose monohydrate crystals, and the quality of the final product related to hardness and spreadability. The process used by the honey industry to produce creamed honey is based on the Dyce method (Dyce, 1931a,b; Dyce, 1976). So before any effects of ultrasound could be determined, it was necessary to be able to produce high quality creamed honey in the laboratory using the Dyce method, and to be able to determine the amount of crystalline D-glucose monohydrate present in creamed honey. This was done successfully, with a differential scanning calorimeter (DSC) used to measure the crystal content in the experimental creamed honeys. Samples of commercial Capilano Honey Ltd. creamed honey were initially analysed and found to have an average crystalline D-glucose monohydrate content of 39.6-40.1 g/100 g honey. It was the aim of the preliminary trials to produce a laboratory creamed honey with a similar level of D-glucose monohydrate crystals to that of the commercial Capilano Honey Ltd. creamed honey. Various honey blends were evaluated based on information and floral honey samples supplied by Capilano Honey Ltd. Finally two blends consisting of 70% alfalfa honey/30% blue gum honey and 70% canola honey/30% red gum honey were chosen for a subsequent replicated study, since these produced laboratory creamed honeys that were soft and spreadable, with crystalline D-glucose monohydrate contents of 21.6 g/100 g honey (after 8 days storage at 14 °C) and 21.9 g/100 g honey (after 12 days storage at 14 °C) respectively. Alfalfa and canola honeys are fast crystallising, fine grained honeys, while blue gum and red gum honeys have strong flavours. However, the level of crystals was not high enough.

73

So next a study was carried out to increase the level of D-glucose monohydrate crystals in the laboratory creamed honey produced using the above honey blends. One experimental factor that was changed was the length of storage time at 14 °C, since commercial processes use 42 days of storage to produce the maximum possible level of D-glucose monohydrate crystals. When 39 days of storage were used, a laboratory creamed honey with a crystalline D-glucose monohydrate content of 47.1 g/100 g honey was produced, which is higher than that found in commercial Capilano Honey Ltd. creamed honey. Thus, the honey blend and creamed honey process have been optimised in the laboratory. To further study the creamed honey process, ultrasound treatment was investigated as a means of reducing the crystal size, and thus improving the spreadability of the product. Prior to the start of this project is was thought that ultrasound treatment of D-glucose monohydrate crystals in honey would shatter them, thereby reducing their size. However, the use of an image analyser has shown that rather than reducing the crystal size through break up of the crystals, the crystal size is reduced through partial melting or dissolution of the D-glucose monohydrate crystals. The clean sharp crystal structures (plates) are replaced by irregular shaped plates indicating that some glucose molecules on the edge of the crystal structure dissolve into the surrounding liquid producing plate crystals that have melted edges and surfaces. Thus, ultrasound treatment was then applied to the seed honey prior to it being added to the honey blend. The hypothesis was that if the size of the crystals in the seed honey could be reduced by ultrasound treatment, then the creamed honey process could be enhanced (producing a higher level of crystals) and the final honey would have smaller crystals. This was not the case, and the level of crystals was not significantly (P>0.05) different from the control creamed honey produced with seed honey that was not ultrasound treated. Included in this experiment were ultrasound treatments of the creamed honey mixtures 1 day and 2 days after the addition of the seed honey. Again, such treatments were hypothesised to reduce the crystal size and enhance subsequent crystallisation during storage at 14 °C. However, there was no significant (P>0.05) difference in the crystalline D-glucose monohydrate content relative to the control untreated creamed honey. None of the ultrasound treatments enhanced the level of D-glucose monohydrate crystals relative to that for the untreated creamed honey. For example, the untreated canola/red gum creamed honeys (47.1 g/100 g honey) had similar crystal contents to ultrasound-treated canola/red gum creamed honeys (44.5 g/100 g honey to 47.1 g/100 g honey ), while untreated alfalfa/blue gum creamed honeys (33.1 g/100 g honey) had similar crystal contents to ultrasound-treated alfalfa/blue gum creamed honeys (32.2 g/100 g honey to 33.1 g/100 g honey). Finally, conditioning of the creamed honey product was investigated. The reason for this was that some of the produced creamed honey samples were creamy in texture and some were semi-solid in texture. There did not seem to be any particular treatment that led to one type of product over the other. In fact, replicates of the same treatment often had both types of texture. As part of the Dyce process (Dyce, 1931a,b; Dyce, 1976), a conditioning step is used prior to the creamed honey being sent to supermarkets for sale. Conditioning is where the creamed honey is stored at 30 °C for a number of days. This study found that such conditioning did produce consistency in texture with all product having a creamy texture. Storage at 30 °C for 14 days produced a small change in the crystalline D-glucose monohydrate content with the final content being similar to that found for commercial Capilano Honey Ltd. creamed honey. Thus, it appears that the conditioning process dissolves some of the D glucose monohydrate crystals leading to an increase in the amount of liquid honey, and an overall softening of the creamed honey, with a consequent improvement in spreadability. In conclusion, while ultrasound treatment did not produce a product that was different to untreated creamed honey, conditioning the final product before sale is very important for producing a consistent product from one production run to another. The results of this study of the Dyce creamed honey process will aid beekeepers and honey packing companies to better understand their creamed honey process and improve the quality and consistency of their product from batch to batch.

74

4.3 Effect of Ultrasound Treatment on the Liquefaction of Candied

Honey and for Controlling Honey Crystallisation Three experiments were undertaken to evaluate the effectiveness of using ultrasound treatment to liquefy naturally crystallised or candied honey. Firstly, it was necessary to determine which ultrasound sonotrode of the available 7 mm, 12 mm or 40 mm diameter sonotrodes better liquefies candied honey. Here, ~250 g of candied honey was treated with ultrasound input energy interrupted at 10,000 J intervals, for predetermined input energy levels, using three ultrasonic sonotrodes, with the temperature profile in honey being monitored during each interruption. Input energy, treatment time and power measurements were also recorded from the data file produced by the ultrasonic processor. The most effective ultrasound sonotrode for the treatment conditions was determined during this experiment. Secondly, candied honey samples were treated with six different ultrasound input energy levels using the 40 mm diameter sonotrode operated at an amplitude of 12 µm to determine the optimum input energy required for liquefaction. The effects of ultrasound on the hydroxymethylfurfural concentration, and diastase and invertase activities in honey were determined by chemical analysis and compared with that for standard heat-treated honey samples. Thirdly, ~200 g of candied reworked mixed honey was completely liquefied by ultrasound treatment. Reworked mixed honey was selected for this trial as it was found to be a very fast crystallising honey that produces large crystals. The crystallisation of ultrasound-treated honey was monitored with time under optimum crystallisation conditions of 14 °C, in comparison with honey samples initially treated with a standard heat treatment. 4.3.1 Experiment 1: Ultrasound treatment of candied honey with interruption

of input energy for selection of sonotrode and treatment conditions The results of the preliminary trial showed that the input energy for the 40 mm diameter sonotrode required to liquefy~250 g of candied Salvation Jane honey is a total of six ~10000 J treatments, when interruptions were carried out at every ~10000 J of energy input. In principle, a specific amount of candied honey requires a specific ultrasound input energy for liquefaction. Therefore, a total ultrasonic input energy of 60000 J, with interruption after each ~10000 J treatment, was used for the subsequent replicated trial for liquefying ~250 g of the same Salvation Jane honey. The input energy values of this study showed that the actual input energy emitted to the honey sample was not exactly the same as the instrument set ~10000 J. Even though the termination criteria for the ultrasound processor was fixed at ~10000 J of input energy, the final values emitted to the honey depends on the input power of the last one-second interval and the cumulative input energy up to the last one second of the treatment. The net power is the actual input power transmitted through the sonotrode to the honey sample during the treatment (i.e. input energy per second). The maximum immerse depth of the sonotrodes inside the honey containers after completion of the treatments were 70, 65 and 55 mm for the 40, 22 and 7 mm diameter sonotrodes respectively. However, the manufacturer recommended maximum immersion depths for the 40, 22 and 7 mm diameter sonotrodes are 20, 45 and 90 mm (Table 3.1). Thus, the maximum manufacturer recommended immersion depth was exceeded during the treatments involving the 40 and 22 diameter sonotrodes.

75

4.3.1.1 Effect of ultrasound treatment of candied Salvation Jane honey by a 40 mm

diameter sonotrode The mean values of input energy, maximum net power (for any one second period), treatment time, and maximum temperature for the six interrupted treatments of ~10000 J of energy input by the 40 mm diameter sonotrode are given in Tables 4.5-4.8 for amplitude settings of 12, 9, 6 and 3 µm. Table 4.5 Input energy, maximum net power, treatment time and maximum temperature of

honey treated with the 40 mm diameter sonotrode at the 12 µm amplitude setting

Input Energy (J)

Maximum Net Power (W)

Treatment Time (s)

Maximum Temperature (°C)

Mean* Standard Deviation




10065.7 59.8 183.9 6.6 66.3 1.5 55.8 2.8 10070.1 77.4 182.5 12.7 61.7 4.0 59.2 2.8 10052.9 17.5 209.1 9.2 52.7 3.2 61.1 2.4 10156.6 92.6 231.9 13.8 47.7 4.0 62.5 2.6 10065.0 42.1 241.4 14.3 44.3 2.5 63.9 2.2 10049.6 33.2 230.6 5.7 51.3 2.9 67.8 1.4

*Mean of 3 replications Table 4.6 Input energy, maximum net power, treatment time and maximum

temperature of honey treated with the 40 mm diameter sonotrode at the 9 µm amplitude setting

Input Energy

(J) Maximum Net Power

(W) Treatment Time

(s) Maximum Temperature

(°C) Mean* Standard

Deviation Mean* Standard



Deviation 10035.6 15.8 151.3 7.9 84.0 1.0 54.3 2.6 10070.3 33.7 162.6 2.8 73.0 1.0 58.5 1.5 10072.2 60.2 186.2 10.4 61.0 3.6 61.5 1.6 10122.4 26.9 219.2 8.3 51.7 0.6 62.5 1.3 10110.7 88.0 215.9 3.0 50.3 1.5 63.4 1.7 10113.6 34.0 210.6 7.1 57.3 2.1 66.2 1.0 *Mean of 3 replications

76

Table 4.7 Input energy, maximum net power, treatment time and maximum


Input Energy


(W) Treatment Time






Deviation 10061.1 30.6 151.3 11.2 83.0 5.6 59.5 2.0 10066.3 48.8 150.0 2.8 75.7 2.1 59.1 2.9 10057.6 60.9 180.8 2.1 65.0 1.0 62.1 0.9 10060.0 66.1 219.7 5.6 52.7 0.6 63.5 1.1 10076.8 96.4 221.7 6.8 49.3 1.5 64.8 0.7 10152.7 6.0 216.8 6.5 57.7 0.6 66.8 0.5

*Mean of 3 replications Table 4.8 Input energy, maximum net power, treatment time and maximum


Input Energy


(W) Treatment Time






Deviation 10059.5 12.8 155.0 16.2 81.7 5.8 61.6 4.6 10079.5 39.1 158.7 6.8 74.7 1.5 59.9 1.8 10022.3 13.0 180.7 1.1 63.0 4.2 62.2 0.5 10116.4 87.0 217.4 12.0 54.7 2.1 64.1 1.3 10115.2 71.3 220.8 11.3 50.7 1.5 64.3 0.7 10058.4 49.3 214.5 5.2 55.3 2.1 67.0 0.8

*Mean of 3 replications The maximum net power supplied by the 40 mm sonotrode increased from the first ~10000 J to the fourth ~10000 J of input energy for all the amplitudes and decreased from the fourth ~10000 J to the sixth ~10000 J of input energy for the 9, 6 and 3 µm amplitudes (Figure 4.2). The maximum net power achieved for the 12 µm amplitude setting for the 40 mm diameter sonotrode increased from the second ~10000 J to the fifth ~10000 J of input energy and decreased from the fifth ~10000 J to the sixth ~10000 J of energy input. The maximum net power achievable increases with each consecutive ~10000 J of input of energy due to the progressive dissolution of glucose crystals and a decrease in honey viscosity making it easier for emission of energy from the sonotrode to the honey. This suggests that a consistent dissolution of the glucose crystals occurred with the 40 mm diameter sonotrode when operated at the 12 µm amplitude setting.

77

80

100

120

140

160

180

200

220

240

260

0 1 2 3 4 5 6 7

Cumulative input energy (10 kJ)

Max

imum

net

pow

er (W

)

12 µm 9 µm 6 µm 3 µm

Figure 4.2 Mean maximum net power versus each ~10000 J of input energy for honey

treated with the 40 mm diameter sonotrode at four amplitude settings The individual treatment time required to deliver each ~10000 J of energy input showed a decreasing trend up to the fifth ~10000 J of energy input for all the amplitude settings (Tables 4.5-4.8). Then, the individual treatment time for the sixth ~10000 J of energy input increased in comparison with the preceding individual treatment times for each ~10000 J of energy input for all the amplitudes. When each treatment time is added in a cumulative manner over the six ~10000 J energy inputs, an increasing trend was observed for the cumulative input energy for all the amplitude settings as would be expected (Figure 4.3). However, the cumulative treatment time was always lower for the 12 µm amplitude setting at all ~10000 J energy inputs relative to the other amplitude settings. The reason for this is that the higher net power (Figure 4.2) achievable at each ~10000 J of energy input with the 12 µm amplitude means a shorter time is required to achieve each ~10000 J of input of energy. The maximum temperature measured during the interruption after each ~10000 J of energy input increased up to the sixth ~10000 J of energy input for all the amplitudes, except for the decrease in temperature for the 6 and 3 µm amplitudes from the first ~10000 J to the second ~10000 J of energy input (Figure 4.4). In addition, there was little difference in the temperatures achieved after each ~10000 of energy input between the four amplitudes. In conclusion, the data for the 40 mm sonotrode during this interruption study suggest that the 12 µm amplitude is superior to the other three amplitudes for liquefying candied honey. The reason for this is that it generates higher net power for each ~10000 J of energy input resulting in less treatment time being required to deliver the ~10000 J of energy from the sonotrode to the honey, without there being any difference in the maximum temperatures resulting for each amplitude.

78

Figure 4.3 Mean cumulative treatment time versus each ~10000 J of input energy for honey treated with the 40 mm diameter sonotrode at four amplitude settings

50

52

54

56

58

60

62

64

66

68

70

0 1 2 3 4 5 6 7

Cumulative input energy (10kJ)

Max

imum

tem

pera

ture

(°C

)


Figure 4.4 Mean maximum temperature versus each ~10000 J of input energy for honey

treated with the 40 mm diameter sonotrode at four amplitude settings

50

100

150

200

250

300

350

400

450

0 1 2 3 4 5 6 7


Cum

ulat

ive

treat

men

t tim

e (S

ec)


79


diameter sonotrode The mean values of input energy, maximum net power (for any one second period), treatment time, and maximum temperature for the six interrupted treatments of ~10000 J of energy input by the 22 mm diameter sonotrode are listed in Tables 4.9-4.12 for the amplitude settings of 100, 75, 50 and 25 µm respectively. Table 4.9 Input energy, maximum net power, treatment time and maximum temperature

of honey treated with the 22 mm diameter sonotrode at the 100 µm amplitude setting

Input Energy


(W) Treatment Time






Deviation 10200.8 144.8 358.1 66.3 139.0 115.1 53.6 4.7 10305.2 81.5 392.5 21.8 32.3 4.2 68.7 10.2 10165.1 128.3 373.4 18.8 31.7 4.2 75.1 2.4 10136.7 19.2 299.7 86.3 42.0 12.5 75.3 3.0 10088.9 94.8 208.5 102.1 137.0 135.2 74.2 2.7 10070.2 47.4 194.9 60.4 105.3 39.5 76.9 1.9

*Mean of 3 replications Table 4.10 Input energy, maximum net power, treatment time and maximum temperature


Input Energy


(W) Treatment Time






Deviation 10149.0 79.6 318.6 34.5 77.0 25.5 58.7 5.9 10149.7 128.8 340.4 19.4 34.7 1.5 69.7 2.6 10198.6 165.9 385.5 4.2 29.7 1.5 75.7 2.4 10086.5 6.2 316.0 20.2 37.0 2.0 75.9 0.5 10132.7 37.2 233.0 37.6 60.3 17.6 73.2 2.2 10038.2 53.2 198.6 38.2 156.0 55.0 72.6 3.1

*Mean of 3 replications

80

Table 4.11 Input energy, maximum net power, treatment time and maximum temperature


Input Energy


(W) Treatment Time






Deviation 10187.7 61.5 300.2 38.2 72.7 24.8 58.0 11.9 10189.2 58.9 339.7 25.7 34.3 4.5 72.8 4.7 10206.3 86.3 374.9 0.9 31.3 2.5 74.9 4.5 10118.1 66.8 288.1 39.1 42.3 8.0 76.6 1.5 10092.2 66.5 169.5 75.5 62.3 32.0 73.1 2.3 10029.4 35.9 161.9 20.1 157.7 70.0 77.7 3.5

*Mean of 3 replications Table 4.12 Input energy, maximum net power, treatment time and maximum temperature


Input Energy


(W) Treatment Time






Deviation 10185.7 33.8 360.9 14.3 150.7 73.9 61.3 4.8 10313.9 42.3 359.3 28.8 35.3 5.0 68.9 4.1 10140.2 135.8 253.4 166.5 144.7 191.7 69.1 4.1 10080.6 72.5 144.4 64.5 173.3 139.3 71.1 2.5 10053.3 25.5 114.4 29.7 231.7 93.4 75.4 5.7 10090.1 52.9 160.7 45.6 135.0 3.6 82.9 6.8

*Mean of 3 replications The maximum net power supplied by the 22 mm diameter sonotrode increased from the first ~10000 J to the third ~10000 J of energy input, and decreased from the third ~10000 J to the sixth ~10000 J of energy input for the 75 and 50 µm amplitudes (Figure 4.5). The maximum net power for the 25 µm amplitude setting decreased from the first ~10000 to the fifth ~10000 J of input energy and increased to the sixth ~10000 J of energy input. For the 100 µm amplitude setting, there was an initial increase in the maximum net power between the first and second ~10000 J of energy input, after which there was a decline in the maximum net power to the sixth ~10000 of energy input. Finally, between the third and fifth ~10000 J of energy input, the 25 µm amplitude setting showed a lower maximum net power relative to that for the other amplitudes, suggesting this amplitude was liquefying the honey and decreasing the viscosity less quickly probably due to the emission of energy from the 22 mm sonotrode being less rapid at the 25 µm amplitude setting. The other three amplitudes showed similar maximum net power between these energy inputs.

81

80

130

180

230

280

330

380

430

0 1 2 3 4 5 6 7


Max

imum

net

pow

er (W

)

100 µm 75 µm 50 µm 25 µm

Figure 4.5 Mean maximum net power versus each ~10000 J of input energy for

honey treated with the 22 mm diameter sonotrode at four amplitude settings

The individual treatment time for each ~10000 J of energy input showed a decreasing trend up to the fifth ~10000 J of energy input, but a large increase for the sixth ~10000 J of energy input (Tables 4.9-4.12). However, the cumulative treatment time showed an increasing trend with an increase in the cumulative input energy (Figure 4.6). However, the cumulative treatment time for the 25 µm amplitude setting was numerically higher (Figure 4.6) and showed a more rapid increase from the second ~10000 J to the sixth ~10000 J of input energy relative to that observed for the other three amplitude settings. The reason for this observation is that there was difficulty in the emission of ultrasound energy from the 22 mm diameter sonotrode when using the 25 µm amplitude setting as evidenced by the lower net power achievable (discussed above), resulting in a longer treatment time required to dissipate each ~10000 to the honey. The maximum temperature increased in a similar trend up to the fourth ~10000 J of energy input for the 22 mm sonotrode at amplitudes of 100, 75, and 50 µm (Figure 4.7), but with the major increase occurring between the first and second ~10000 J of energy input. The small increase in the maximum temperature between the second and fifth ~10000 J of energy input is probably due to the decrease in maximum net power that occurred between these two energy input treatments (Figure 4.5). This suggests emission of energy from the 22 mm diameter sonotrode to the honey is not very efficient due to the physical nature of the honey.

82

50

150

250

350

450

550

650

750

850

950

0 1 2 3 4 5 6 7


Cum

ulat

ive

treat

men

t tim

e (S

ec)

100 µm 75 µm 50 µm 25 µm

Figure 4.6 Mean cumulative treatment time versus each ~10000 J of input energy

for honey treated with the 22 mm diameter sonotrode at four amplitude settings

50

55

60

65

70

75

80

85

0 1 2 3 4 5 6 7


Max

imum

tem

pera

ture

(°C)

100 µm 75 µm 50 µm 25 µm

Figure 4.7 Mean maximum temperature versus each ~10000 J of input energy for

honey treated with the 22 mm diameter sonotrode at four amplitude settings

83


diameter sonotrode The mean values of input energy, maximum net power (for any one second period), treatment time, and maximum temperature for the six interrupted treatments of ~10000 J of energy input by the 7 mm diameter sonotrode are given in Tables 4.13-4.16 for the amplitude settings of 175 µm, 131.25 µm, 87.5 µm and 43.75 µm.

Table 4.13 Maximum net power, input energy, treatment time and maximum temperature of honey treated with the 7 mm diameter sonotrode at the 175 µm amplitude setting

Input Energy


(W) Treatment Time






Deviation 10041.3 27.0 97.7 13.2 216.3 17.6 56.6 6.7 10028.4 22.0 129.1 16.4 95.3 10.3 61.0 4.2 10082.0 25.6 141.5 0.4 75.3 6.4 66.8 1.7 10079.4 69.0 163.4 4.9 67.7 2.5 69.0 0.3 10068.9 69.4 180.8 17.9 63.0 7.0 71.3 0.6 10066.4 46.1 161.5 8.7 70.7 2.1 78.3 1.7

*Means of 3 replications

Table 4.14 Maximum net power, input energy, treatment time and maximum temperature of honey treated with the 7 mm diameter sonotrode at the 131.25 µm amplitude setting

Input Energy


(W) Treatment Time






Deviation 10046.2 6.9 84.8 10.4 213.7 37.7 57.1 1.6 10072.5 6.3 108.0 7.6 115.7 3.1 64.4 2.0 10044.6 59.2 120.6 5.4 94.0 7.8 68.2 3.6 10073.7 13.0 138.1 5.0 86.3 3.1 70.1 2.7 10039.6 35.9 139.0 12.7 76.7 6.8 74.5 0.8 10043.2 30.6 131.4 5.7 91.3 3.8 80.3 4.2


84


Input Energy


(W) Treatment Time






Deviation 10028.4 10.7 87.8 5.9 225.0 13.9 60.8 4.2 10066.8 24.4 112.1 7.6 111.7 2.9 63.8 0.7 10039.3 34.9 125.3 10.1 90.3 5.9 67.6 0.8 10059.5 32.6 129.3 10.6 85.0 4.4 70.0 0.5 10062.8 19.8 139.1 9.9 73.3 20.4 74.8 0.9 10070.7 8.7 122.0 1.8 96.0 3.0 84.4 1.8



Input Energy


(W) Treatment Time






Deviation 10048.6 36.4 87.2 5.1 220.0 16.4 56.7 5.1 10042.2 32.3 105.7 4.8 111.0 6.1 65.6 4.8 10068.6 54.2 120.3 11.0 93.0 7.5 66.1 11.0 10068.8 51.1 151.0 12.1 77.3 4.9 71.0 12.1 10067.0 53.8 157.9 18.7 74.7 11.0 72.8 18.7 10072.6 28.7 142.9 9.7 82.3 9.0 78.2 9.7

*Means of 3 replications The maximum net power supplied by the 7 µm diameter sonotrode increased from the first ~10000 J to the fifth ~10000 J of input energy and decreased from the fifth ~10000 J to the sixth ~10000 J of input energy for all the amplitudes (Figure 4.8). This was a similar trend to that observed for the 40 mm diameter sonotrode. The increase is due to the honey becoming more liquefied and less viscous as each ~10000 J energy treatment occurs making energy emission from the sonotrode into the honey easier, the more energy that is emitted. However, the maximum net power achieved by the 7 mm diameter sonotrode at the 175 µm amplitude setting was always higher than that achieved at the other three amplitudes indicating that this amplitude is superior in the emission of energy into the honey. Thus, it can be concluded that the 40 mm diameter sonotrode operated at the 12 µm amplitude (Figure 4.2) and the 7 mm diameter operated at the 175 µm (Figure 4.8) are the superior combinations for the emission of energy from the sonotrode to the honey, since the maximum net power increases with each ~10000 J energy treatment, and these amplitudes produce the highest net power for their respective sonotrodes. The 22 mm diameter sonotrode (Figure 4.5) produced the highest maximum net power during the first three ~10000 J energy treatments but the net power levels decreased rapidly over the subsequent three ~10000 J energy treatments suggesting it was difficult for energy emission from the 22 mm diameter sonotrode to the honey.

85

80

100

120

140

160

180

200

0 1 2 3 4 5 6 7


Max

imum

net

pow

er (W

)

175 µm 131.25 µm 87.5 µm 43.75 µm

Figure 4.8 Mean maximum net power versus each ~10000 J of input energy for honey treated with the 7 mm diameter sonotrode at four amplitude settings

The treatment time for each individual ~10000 J of input energy decreased up to the fifth energy input but there was an increase between the fifth and sixth ~10000 J treatments (Tables 4.13-4.16). The cumulative treatment time increased with an increase in cumulative energy input, with the changes for the 131.25 µm, 87.5 µm and 43.75 µm amplitudes being nearly identical (Figure 4.9). In addition, the lower treatment times for the 175 µm amplitude reflect the higher maximum net power achievable at this amplitude (Figure 4.8), since less time is needed for emission of each ~10000 J of energy from the 7 mm diameter sonotrode to the honey. The maximum temperature in the honey increased with an increase in the cumulative input energy for all the amplitudes (Figure 4.10), with little difference between amplitudes. In summary, treatments by the 7 mm sonotrode showed that the effective area of treatment is comparatively smaller due to the size of the face of the sonotrode, since additional horizontal and vertical movements of the honey container were needed to liquefy each layer of candied honey. This indicates that increasing the surface area of the sonotrode should be the highest priority for any scale-up to larger sonotrodes for liquefying large volumes of honey in a commercial honey packing factory.

86

200

300

400

500

600

700

800

0 1 2 3 4 5 6 7


Cum

ulat

ive

treat

men

t tim

e (S

ec)

175 µm 131.25 µm 87.5 µm 43.75 µm

Figure 4.9 Mean cumulative treatment time versus each ~10000 J of input energy for honey treated with the 7 mm diameter sonotrode at four amplitude settings

50

55

60

65

70

75

80

85

90

0 1 2 3 4 5 6 7


Max

imum

tem

pera

ture

(°C)

175 µm 131.25 µm 87.5 µm 43.75 µm

Figure 4.10 Mean maximum temperature versus each ~10000 J of input energy for honey treated with the 7 mm diameter sonotrode at four amplitude settings

87

4.3.1.4 Comparison of the performance of the ultrasonic sonotrodes for liquefying

candied Salvation Jane honey The variability of the net power of the sonotrodes observed during each individual ~10000 J of input energy was not reflected in the maximum net power generated during any 1 s period over the total six interrupted ~10000 J treatments by the 12 sonotrodes/amplitude combinations (Table 4.17 and Figure 4.11). The variation of maximum net power for each sonotrodes/amplitude combination, as well as variation between replicates, are indicated in Figure 4.11. Treatments with the 40 mm diameter sonotrode were steady and had consistent net power throughout for each individual ~10000 J energy treatment (Table 4.17 and Figure 4.11), while the maximum net power generated increased as dissolution of the glucose crystals proceeded with each ~10000 J energy treatment. The maximum net power produced by the 22 mm sonotrode was higher than for the 40 mm sonotrode, which was higher than for the 7 mm sonotrode. However, while the 22 mm sonotrode showed steady and high maximum net power over the initial three ~10000 J energy treatments (Figure 4.5), the maximum net power reduced markedly over the final three ~10000 J energy treatments suggesting difficulties with the efficiency of emission of energy from the sonotrode to the partially liquefied honey. In conclusion, the 40 mm diameter sonotrode performs best in the emission of energy from the sonotrode to the honey as dissolution of the glucose proceeds with each ~10000 J of energy input.

Table 4.17 Mean maximum net power achieved over the six ~10000 J energy inputs for the 12 ultrasound treatment combinations

Sonotrode

Diameter (mm) Amplitude

(µm) Maximum Net Power (W)*

Mean** Standard Deviation

%CV+

40 12 241.4 14.3 5.9 40 9 222.2 5.6 2.5 40 6 225.6 2.1 0.9 40 3 227.6 1.8 0.8 22 100 400.8 15.0 3.8 22 75 385.5 4.2 1.1 22 50 374.9 0.9 0.2 22 25 374.8 6.9 1.9 7 175 180.8 18.0 9.9 7 131.25 144.8 2.7 1.9 7 87.5 142.0 2.9 3.5 7 43.75 159.1 10.7 11.7

*Maximum net power achieved for each of the 12 treatment combinations over the 6 interrupted ~10000 J energy inputs. **Means of 3 replications for each of the 12 treatment combinations +%Coefficient of variance = (standard deviation / mean) x 100

88

Max

imum

net

pow

er (W

)

Amplitude (µm)

Sonotrode dia (mm)

175.00

131.2

510

0. 00

87.50

75.00

50.00

43. 75

25.00

12.009.0

06.0

03.00

402274022740227402274022740227402274022740227402274022740227

450

400

350

300

250

200

150

100

Amplitude

9.0012.0025.00

(µm)

43.7550.0075.0087.50

100.00131.25175.00

3.006.00

Figure 4.11 Individual value plot of the maximum net power achieved during six ~10000 J energy inputs versus sonotrode diameter and amplitude (95% confidence interval for the mean)

The individual value plot of cumulative treatment time versus sonotrode diameters and amplitude combinations shows that the 40 mm diameter sonotrode was less variable (lower %CV) and the cumulative treatment time was lower than those for the other two sonotrodes (Table 4.18 and Figure 4.12). A high level of variation in the cumulative treatment time for the 22 mm diameter sonotrode occurs specifically for the 25 and 100 µm amplitudes (Table 4.18 and Figure 4.12). In conclusion, since treatment time is critical in any commercial use of ultrasound liquefaction, then the 40 mm diameter sonotrode is preferred due to its lower cumulative treatment time over the whole six ~10000 J energy inputs. The means of the maximum temperatures of honey after complete treatment of six ~10000 J of input energy were 66.2 - 67.8, 76.4 – 82.9 and 78.2 – 84.4 for the 40mm, 22mm and 7 mm diameter sonotrodes respectively (Table 4.19). The maximum temperature data of the twelve ultrasound treatments by the three sonotrodes were analysed statistically in order to determine the effect of the diameter of the sonotrode. Significantly lower (P<0.05) maximum honey temperatures were found for treatment by the 40 mm diameter sonotrode than by the other two sonotrodes (Figure 4.13 and Table 4.19). Further, the coefficient of variation of the 40 mm diameter sonotrode was lower than those for the other two sonotrodes, indicating a high level of consistency for the 40 mm sonotrode in relation to the maximum temperature (Table 4.19). However, there was a high level of variation in the maximum honey temperature for treatment by the 22 and 7 mm diameter sonotrodes (Table 4.19 and Figure 4.13). Thus, this suggests the 40 mm diameter sonotrode is the best since the maximum temperature is required to be as low as possible to prevent heat damage of the honey.

89

Table 4.18 Mean cumulative treatment times for the six ~10000 J energy inputs for each of

the 12 ultrasound treatment combinations

Sonotrode Diameter (mm)

Amplitude (µm)

Cumulative Treatment Time (s)**

Mean Standard Deviation

%CV+

40 12 324.0 14.4 4.5 40 9 377.3 7.6 2.0 40 6 383.3 5.9 1.5 40 3 380.0 3.6 1.0 22 100 487.0 279.0 57.3 22 75 394.7 31.5 8.0 22 50 400.7 64.6 16.1 22 25 871.0 353.0 40.6 7 175 588.3 28.5 4.9 7 131.25 677.7 47.6 7.0 7 87.5 681.3 24.9 3.7 7 43.75 658.3 29.8 4.5

*Means of 3 replications +%Coefficient of variance = (standard deviation / mean) x 100 **Cumulative treatment times do not include the five 60 s interruption periods for each of the 12 treatments

Cum

ulat

ive

treat

men

t tim

e (s

)

Amplitude (µm)

Sonotrode dia (mm)

175.00

131.2

510

0. 00

87.50

75.00

50.00

43. 75

25.00

12.009.0

06.0

03.00

402274022740227402274022740227402274022740227402274022740227

2000

1500

1000

500

0

Amplitude

9.0012.0025.00

(µm)

43.7550.0075.0087.50

100.00131.25175.00

3.006.00

Figure 4.12 Individual value plot of the cumulative treatment time over six ~10000 J energy inputs versus sonotrode diameter and amplitude (95% confidence interval for the mean)

90

In conclusion, with respect to the 40 mm sonotrode, an analysis of the maximum net power and total ultrasonic treatment times for the different amplitudes suggests that the 12 µm amplitude has the highest maximum net power of 241.4 W and lowest cumulative treatment time of 324 seconds, out of the four amplitude settings of 3 µm, 6 µm, 9 µm and 12 µm (Tables 4.17 and 4.18).

Table 4.19 Mean maximum temperatures reached after a ~60000 J treatment for each of the 12 ultrasound treatment combinations

Sonotrode

Diameter (mm) Amplitude

(µm) Maximum Temperature (°C)


%CV+

40 12 67.8 1.4 2.0 40 9 66.2 1.0 1.6 40 6 66.8 0.5 0.7 40 3 67.2 0.4 0.6 22 100 77.9 1.7 2.2 22 75 76.4 1.4 1.8 22 50 78.1 2.9 3.8 22 25 82.9 6.8 8.2 7 175 78.3 1.7 2.2 7 131.25 80.3 4.2 5.3 7 87.5 84.4 1.8 2.1 7 43.75 78.2 5.0 6.3

*Means of 3 replications +%Coefficient of variance = (standard deviation / mean) x 100

Max

imum

tem

pera

ture

(°C

)

Amplitude (µm)

Sonotrode dia (mm)

175.00

131.2

510

0. 00

87.50

75.00

50.00

43. 75

25.00

12.009.0

06.0

03.00

402274022740227402274022740227402274022740227402274022740227

100

90

80

70

60

Amplitude

9.0012.0025.00

(µm)

43.7550.0075.0087.50

100.00131.25175.00

3.006.00

Figure 4.13 Individual value plot of the maximum temperature reached after six ~10000 J energy inputs versus sonotrode diameter and amplitude (95% confidence interval for the mean)

91

4.3.1.5 Image analysis of ultrasound-treated Salvation Jane honey samples Visual observations of the honey containers after treatment found that some samples treated with the 22 mm diameter sonotrode were not completely liquefied and some crystals were observed at the bottom of the container. Visual observations of all the other containers containing honey samples treated with ultrasound indicated the honey was completely liquefied after six interrupted energy treatments of ~10000 J. However, microscopic observation of the honey samples immediately after the sixth 10000 J of input energy showed that there were small crystals or partly melted crystals in all the samples. Samples treated with the 7 mm diameter sonotrode had a comparatively large number of small air bubbles incorporated in honey. 4.3.1.6 Conclusions from Experiment 1 The 40 mm diameter sonotrode produced a significantly lower (P<0.5) maximum temperature than the 22 and 7 mm sonotrodes in relation to the maximum temperature after the six ~10000 J of input energy. Further, use of the 12 µm amplitude with the 40 mm sonotrode produced the highest maximum net power and lowest cumulative treatment time out of the four available 40 mm sonotrode amplitudes. Hence, the 12 µm amplitude was selected from the four amplitude settings for the 40 mm sonotrode, so that the honey is treated in the shortest possible time with the lowest maximum temperature, thus minimising heat effects on the honey. Therefore, the 40 mm diameter sonotrode operated at the 12 µm amplitude and 100% cycle (continuous) setting was used for the subsequent continuous ultrasound experiment (Experiment 2). 4.3.2 Experiment 2: Effect of different input energy levels of continuous

ultrasound treatment (40 mm diameter sonotrode at 12 µm) for liquefying candied honey, and its effect on enzyme activity and the hydroxymethylfurfural concentration

The aim of this experiment was to determine the optimum input energy required to just completely liquefy candied Salvation Jane honey. The results of Experiment 1 above showed that the 40 mm diameter sonotrode operated at the 12 µm amplitude and 100% cycle (continuous) settings was the best ultrasonic conditions to use for this experiment. 4.3.2.1 Input energy requirement for liquefying candied Salvation Jane honey In a preliminary trial, it was found that ~250 g of candied Salvation Jane honey was completely liquefied by a continuous input of energy in the range of ~65000 – ~70000 J from the 40 mm diameter sonotrode operated at the 12 µm amplitude and 100% cycle (continuous) settings. In a replicated trial using the above sonotrode operating conditions, continuous ultrasound treatment of candied Salvation Jane honey was carried out with instrumentally set input energies of ~50000 to ~70000 J (Table 4.20). The treated honeys were visually examined for the presence of crystals. Firstly, it was observed that the actual input energy was not exactly the same as the instrumentally set input energy values. The experimental protocol terminated the ultrasound treatment at an input energy that was closest to the instrumentally set input energy value (Table 4.20). For an actual recorded input energy of 70058.11 J, complete liquefaction of ~250 g of candied honey for all three replications was observed to occur on visually examining the honey in the containers. For

92

an instrumentally set input energy of ~65000 J, only one of the three replicates (65121.76 J of actual input energy) led to the complete liquefaction of the candied honey. Instrumentally set input energies lower than ~65000 J did not dissolve all the crystals in ~250 g of candied honey when it was observed visually in a container. However, image analysis of a drop of ultrasound-treated honey using the optical microscope showed that there were a few small crystals in almost all the samples. Table 4.20 Mean actual input energy and instrumentally set input energy for treatment with

the 40 mm sonotrode ultrasonic sonotrode operated at the 12 µm amplitude and 100% cycle (continuous)

Treatment Instrumentally Set Input Energy (J)

Actual Input Energy (J)*

Mean Standard. Deviation

%CV+

T1 50000 50053 44.7 0.1 T2 55000 55067 77.4 0.1 T3 60000 60080 59.4 0.1 T4 62500 62566 66.0 0.1 T5 65000 65081 47.5 0.1 T6 70000 70058 55.1 0.1

*Means of 3 replications +%Coefficient of variance = (standard deviation / mean) x 100 The actual energy supplied for the three replications of all the treatments (T1-T6) was consistent as indicated by the low coefficient of variation (Table 4.20). 4.3.2.2 Effect of ultrasound treatments using the 40 mm sonotrode (amplitude 12 µm)

on the maximum temperature of Salvation Jane honey, total net power and overall treatment time

The maximum temperature of the Salvation Jane honey samples immediately after the continuous ultrasound treatments was highest (77.3°C) in honey samples treated with ~70000 J input energy (Table 4.21). Maximum temperatures in the other samples were between 69.4 to 73.2°C. However, these differences in maximum temperature between the six input energy levels were not significant (P>0.05). The maximum net power recorded for any one second period during the treatment time was similar for each input energy between 50053 J and 70058 J, indicating the energy was being emitted at the same rate from the 40 mm diameter sonotrode operated at 12 µm, but the time required for emission of energy into the honey increased as the amount of energy increased.

93

Table 4.21 Mean maximum temperature of Salvation Jane honey, total net power and overall treatment time for treatment by the 40 mm sonotrode ultrasonic sonotrode operated at the 12 µm amplitude and 100% cycle (continuous)

Treatment Actual Input Energy

(J)*

Overall Treatment

Time (s)*

Maximum Net Power in any 1 s

Period (W)*

Maximum Temperature Immediately After Treatment

(°C)*


%CV+

T1 50053 304.7 216.3 69.4 0.6 1.0

T2 55067 345.7 211.3 71.4 0.7 1.0

T3 60080 368.3 216.5 73.2 0.8 1.0

T4 62566 393.0 226.4 73.1 1.9 2.5

T5 65081 383.7 222.3 72.9 0.6 0.8

T6 70058 434.0 222.7 77.3 1.7 2.1

*Means of 3 replications +%Coefficient of variance = (standard deviation / mean) x 100 4.3.2.3 Effect of ultrasound treatments using the 40 mm sonotrode (amplitude 12 µm)

on the hydroxymethylfurfural concentrations of Salvation Jane honey The mean values of the hydroxymethylfurfural (HMF) concentrations of the ultrasound-treated Salvation Jane honey samples were between 4.2 to 5.0 mg/kg honey (Table 4.22 and Figure 4.14). Heat-treated Salvation Jane honey samples had a mean HMF concentration of 5.7 mg/kg honey. The HMF concentrations of the ultrasound-treated samples were not significantly different (P>0.05) between each treatment of different input energy. Only the ultrasound-treated samples involving treatments T5 (65081 J of energy input) and T6 (70058 J of energy input) had HMF levels that were significantly lower (P<0.05) than the heat-treated honey (Figure 4.14). Thus, in can be concluded that any effect of an ultrasound treatment needed to liquefy candied honey (T6 with 70058 J of energy input) is less than that produced by a heating regime typically used by the honey industry. Table 4.22 Mean hydroxymethylfurfural concentration of ultrasound (40 mm sonotrode,

12 µm amplitude)-treated Salvation Jane honey and heat-treated Salvation Jane honey

Treatment Mean Input Energy

(J)* HMF (mg/kg)

Mean* Standard. Deviation

%CV+

Ultrasound: T1 50053 4.3 0.6 14.0 Ultrasound: T2 55067 4.5 0.4 8.9 Ultrasound: T3 60080 4.4 0.3 6.8 Ultrasound: T4 62566 5.0 0.1 2.0 Ultrasound: T5 65081 4.5 0.2 4.4 Ultrasound: T6 70058 4.2 0.3 7.1

Heat: T7 5.7 0.2 3.5 *Means of 3 replications +%Coefficient of variance = (standard deviation / mean) x 100

94

Treatment

HM

F co

nten

t (m

g/kg

)

7654321

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

Treatment 1: 50053 J; Treatment 2: 55067 J; Treatment 3: 60080 J; Treatment 4: 62566 J; Treatment 5: 65081 J; Treatment 6: 70058; Treatment 7: heat treatment. 95% CI for means

Figure 4.14 Individual value plot of hydroxymethylfurfural concentration versus

ultrasound treatment (95% confidence interval for the mean) 4.3.2.4 Effect of ultrasound treatments using the 40 mm sonotrode (amplitude 12 µm)

on the diastase activity of Salvation Jane honey The heat treatment used in this study is similar to the standard industry processing used to liquefy candied honeys. Even after this heat treatment, the honey samples had a mean diastase activity of 24.1 Schade units (G°) which is very high for a honey (Codex minimum is 8 G°) (Table 4.23). Thus, it is a good honey to use to measure the effect of ultrasound treatment. Here, the diastase activity in the ultrasound-treated samples was between 24.6 and 26.4 G° Schade units (G°) (Table 4.23).

Table 4.23 Mean diastase activity of ultrasound (40 mm sonotrode, 12 µm amplitude)-treated Salvation Jane honey and heat-treated Salvation Jane honey

Treatment Mean Input Energy

(J)* Diastase Activity Schade Units (G°)


%CV+


Heat: T7 24.1 0.3 1.2 *Means of 3 replications +%Coefficient of variance = (standard deviation / mean) x 100

95

However, there was no significant (P>0.05) differences in the diastase activities between each of the six different ultrasound-treated Salvation Jane honey samples and the heat-treated Salvation Jane honey samples, while each ultrasound treatment level produced honeys with diastase activities that were not significantly (P>0.05) different from each other (Figure 4.15). Thus, it can be concluded that there is no detrimental effect on the diastase activity in honey from any ultrasound treatment.

Treatment

Dia

stas

e ac

tivity

(Sch

ade)

7654321

30

28

26

24

22

20

Treatment 1: 50053 J; Treatment 2: 55067 J; Treatment 3: 60080 J; Treatment 4: 62566 J; Treatment 5: 65081 J; Treatment 6: 70058; Treatment 7: heat treatment.; 95% CI for means

Figure 4.15 Individual value plot of diastase activity versus ultrasound

treatment (95% confidence interval for the mean) 4.3.2.5 Effect of ultrasound treatments using the 40 mm sonotrode (amplitude 12 µm)

on the invertase activity of Salvation Jane honey The mean invertase activity of the heat-treated sample was 0.6 IN which is lower than that found by Mossel (2002) for similarly processed Australian honeys (8.07 IN). However, ultrasound-treated Salvation Jane honey samples had invertase activities of between 2.0 – 4.6 IN (Table 4.24 and Figure 4.16). These results show that the invertase levels in ultrasound-treated samples are higher than the heat-treated samples, which is a favourable result. In particular, there was a significantly higher (P<0.05) invertase activity for only the ultrasound-treated samples produced by the 50053 J (T1), 60080 J (T2) and 65081 J (T5) input energies relative to that for the heat-treated samples. However, there was no significant (P>0.05) difference in the invertase activity between the Salvation Jane honey samples treated with the six different levels of ultrasound input energy. Treatment T6 (70058 J of input energy), which was the only ultrasound treatment that completely dissolved the honey crystals, produced honeys with invertase activities that were not significantly (P>0.05) different from those of the heat-treated samples. Thus, it can be concluded that there is no detrimental effect on the invertase activity from an ultrasound treatment that completely liquefies candied Salvation Jane honey.

96

Table 4.24 Mean invertase number of ultrasound (40 mm sonotrode, 12 µm amplitude)-

treated Salvation Jane honey and heat-treated Salvation Jane honey

Treatment Mean Input Energy (J)*

Invertase Activity (IN)


%CV+


Heat: T7 0.6 0.4 66.7 *Means of 3 replications +%Coefficient of variance = (standard deviation / mean) x 100; values for the %CV are variable and high for some samples.

Treatment

Inve

rtase

act

ivity

7654321

7

6

5

4

3

2

1

0

95% CI for the MeanIndividual Value Plot of Invertase activity vs Treatment

Treatment 1: 50053 J; Treatment 2: 55067 J; Treatment 3: 60080 J; Treatment 4: 62566 J; Treatment 5: 65081 J; Treatment 6: 70058; Treatment 7: heat treatment. 95% CI for means

Figure 4.16 Individual value plot of invertase activity versus ultrasound treatment (95%

confidence interval for the mean)

97

4.3.2.6 Conclusions from Experiment 2 The actual specific energy input requirement for liquefying 1 kg of candied honey as found during this experiment will be useful in the future process of designing an industrial level ultrasound processor for liquefying large amounts of candied honey (e.g. in 200 L drums). In addition to the specific input energy (J) requirements, the capacity of the sonotrode, the dimension of the container and specific treatment time will be the other main parameters needed to be considered in any future design process. For the HMF concentrations, and the diastase and invertase activities, no detrimental effects were found from the ultrasound treatments relative to those observed from a normal heat treatment of honey. 4.3.3 Experiment 3: A comparative crystallisation study of candied honey

liquefied by ultrasound treatment and heat treatment The main objective of this experiment was to determine and compare the rate of the natural crystallisation process and crystal growth in honey that had been liquefied by either ultrasound or heat treatment. A ‘reworked mixed honey’ was supplied by Capilano Honey Ltd. for this study which investigated crystal formation and growth under the optimum crystallisation condition of 14 °C. This honey is a fast crystallising honey that produces large pentagon shaped plate crystals. Thus, it was ideal to use, since the experiment could be completed in a reasonably short time-frame. Honey samples were ultrasound treated and heat treated to dissolve the crystals completely. 4.3.3.1 Preliminary trial Candied reworked mixed honey (~200 g) was completely liquefied at an instrumentally set input energy of 55000 J. Visual observations of the honey container showed that honey was completely liquefied and a transparent liquid honey produced. 4.3.3.2 Replicated trial of liquefaction of candied honey by ultrasound and heat

treatment The candied reworked honey samples of the replicated trial were completely liquefied by ultrasound treatment with 55000 J of input energy, with cavitation (bubble formation) of the liquid honey being observed during the later part of the treatment period when the honey was less viscous. Visual observation of the containers containing honey subjected to ultrasound and heat treatment indicated that the crystals were completely dissolved, and the honey was a clear transparent liquid. The actual mean input energy of 55048.3 J was sufficient to liquefy~200 g of candied honey in an overall mean time of 442.5 s (7.4 min), while the liquid honey reached an overall mean maximum temperature of 80.7 °C (Table 4.25). However, on storage at room temperature (25 °C), the temperature of the liquid honey reached 55 °C after an average 36.5 min. This temperature was chosen as it is a temperature that is similar to that used by industry for liquefying candied honey in hot rooms overnight. Thus, the total time that the ~200 g of honey was at a temperature greater that 55 °C during and after ultrasound treatment was ~44 min (7.4 + 36.5 min), which is much less than 16 h at 55 °C, a treatment that is similar to that used by the honey industry to liquefy candied honey prior to pumping and bottling. Of course, when the ultrasound treatment is scaled up to liquefy 300 kg of

98

candied honey, the time will increase to a level that will depend on the input energy used. However, it likely to be much less than 16 h. Table 4.25 Mean values of input energy, treatment time and maximum temperature of honey

treated with ultrasound

Replicate Input Energy

(J)

Treatment Time

(s)

Maximum Temperature

Reached After Treatment

(°C)

Time Taken to Reach 55 °C after Ultrasound Treatment

Ceased (min)

1 55039.5 456 81.3 35.1 2 55045.0 445 81.1 36.9 3 55094.2 443 81.3 39.8 4 55013.7 468 80.7 34.5 5 55023.3 463 81.2 38.3 6 55074.2 380 78.5 34.5

Mean 55048.3 442.5 80.7 36.5 Standard Deviation 30.7 32.1 1.1 2.2

4.3.3.2.1 Image analysis of slides for crystal formation and growth: heat-treated honey The image analysis of heat-treated honey samples showed that there were melted crystals, small crystals and air bubbles in the honey after the treatment (Figure 4.17). The ultrasound-treated honey samples also showed some melted crystals and small crystals. However, there were more smaller air bubbles in ultrasound-treated samples in comparison with the heat-treated samples. Image analysis of 18 slides of heat-treated samples indicated that first plate crystals were formed in 7 slides at 14 days after treatment (Table 4.26). All these crystals were spiral shaped, long thin plate crystals. Most of these plate crystals did not show any increase in size or growth during the monitoring period up to 112 days. Needles were produced in 12 slides within 49 days (Figure 4.17). These needle crystals tended to grow outwards from the middle of the crystal mass, and large crystal masses were produced with time. Figure 4.17 shows views of the same crystal mass over time and growth of the crystals is clearly visible. In addition, plates were observed to form within the needle crystal masses in 8 slides at between 49 – 84 days after treatment (Figure 4.18). In some instances, needles grow on the existing plates and subsequently completely covered them with needle masses. In the final microscopic observations at 112 days after heat treatment, there were 10 slides with mostly large needle crystal masses and the formation of plates underneath. In addition, three slides had only needles, while there was no crystal formation in two of the slides. Only plate crystals were observed in the other three slides.

99

Table 4.26 Crystallisation of heat-treated honey samples (up to 112 days)

Formation of Initial Crystals Replicate Slide

Number Plates Needles Status of Crystals After 112 days

1 1 14 days No needles. Only initial plate crystals. 1 2 14 days 35 days Needle crystal masses.

1 3 14 days 35 days Needle crystal masses with plates formed underneath.

2 1 No plates. 35 days Needle crystal masses on the plates. 2 2 14 days No needles. Only initial plate crystals. 2 3 No plates. 49 days Needle crystal masses. 3 1 No plates. 49 days Needle crystal masses.


3 3 No plates. No needles. No crystallisation. 4 1 No plates. 14 days Needles grow on the plates.


4 3 No plates. 21 days Needle crystal masses with plates formed underneath.

5 1 28 days No needles. Two long plate crystals. 5 2 No plates. No needles. No crystallisation. 5 3 No plates. No needles. No crystallisation. 6 1 14 days 49 days Needle crystal masses. 6 2 No plates. 49 days Needle crystal masses. 6 3 No plates. No needles. No crystallisation.

Day 49Day 1

Day 112Day 84

181 µm

331 µm

376 µm

Figure 4.17 Growth of needle crystals with time in heat-treated honey (identical view on one slide of one replicate over time)

Air Bubbles

100

356 µm

Day 49

Day 56

290 µm

Day 84

460

µm

445 µm

Day 84

Figure 4.18 Plate crystal formation within needle crystal masses (selected images from different heat-treated samples from 8 different slides for different replicates)

4.3.3.2.2 Image analysis of slides for crystal formation and growth: ultrasound-treated honey Crystallisation of ultrasound-treated samples was initiated by formation of plate crystals in 5 out of 18 slides at 14 days after treatment (Table 4.27). Another six slides produced initial plates at between 21-49 days. Most of these initial crystals were pentagon shaped, large plate crystals. Needle formation was observed in 13 slides at between 21-56 days after the ultrasound treatment. However, there was no plate formation at between 49 - 84 days as occurred in the heat-treated honey samples. The needles produced in the 21 – 56 day period after ultrasound treatment grew and produced large dark crystal masses of needles and some plates underneath in these 13 slides after 112 days (Figure 4.19). In two slides, there was no growth of crystals or any other crystal formation throughout the 112 day monitoring period. For the ultrasound-treated honeys, it was also observed that needle crystal masses grew on the plate crystals produced at initial crystallisation, and large dark needle masses resulted (Figure 4.20).

101

Table 4.27 Crystallisation of ultrasound-treated honey samples (up to 112 days)

Formation of Initial Crystals Replicate Slide Number Plates Needles

Status of Crystals After 112 days


1 2 No plates. 56 days Needle crystal mass with plates formed underneath

1 3 28 days 84 days Needle crystal masses on the plates.

2 1 No plates. No needles. No crystallisation. 2 2 14 days No needles. Only initial plate crystals. 2 3 14 days No needles. Only initial plate crystals.





4 2 No plates. No needles. No crystallisation.


5 1 No plates. No needles. No crystallisation.




6 2 14 days 21 days Plate crystal masses with some needles.

6 3 49 49 Plate crystal masses with some needles.

102

Day 1Day 56

Day 84

Day 112

166

µm

359 µm256

µm

Figure 4.19 Growth of needle crystals in ultrasound-treated honey (identical view on one slide of one replicate over time)

Day 56

Day 84 Day 112

317

µm

650

µm

509

µm

Figure 4.20 Growth of needle crystals on a plate crystal in ultrasound-treated honey (identical view on one slide of one replicate over time)

103

On another slide containing ultrasound-treated honey, formation of plate crystals on larger plate crystals and their subsequent growth were observed. However, no needle crystals grew on these plates (Figure 4.21). These plates have pointed edges and growth was observed from the blunt end.

Day 28 Day 49

Day 84

644 µm

596 µm

488 µm

Day 112

Figure 4.21 Growth of plate crystals from the blunt ends in ultrasound-treated honey (identical view on one slide of one replicate over time)

4.3.3.2.3 Visual observations of honey in the containers The heat-treated and ultrasound-treated honey containers were visually observed every seven days during which a few clumps of crystals were found at the bottom of the containers in all the ultrasound-treated honey containers at three weeks (21 days) after treatment. A few clumps of crystals were also observed at the bottom in one heat-treated container after four weeks. Further, there were a few crystals at the bottom of all heat-treated containers by ten weeks. The formation of small crystal structures in the liquid honey was observed after ten weeks in two containers, one containing a heat-treated honey sample and one containing an ultrasound-treated honey sample. Some of these small crystal structures tended to grow upwards from the bottom or from the middle of the liquid honey to the top layer. A large number of air bubbles appeared inside the heat-treated liquid honey samples after 21 weeks and the liquid became non-transparent. However, the ultrasound-treated honey samples had comparatively less air bubbles and four out of six containers contained clear transparent liquid honey. There was no significant increase in the amount of the initial clumps of crystals that appeared at the bottom of the containers after 29 weeks.

104

After 24 weeks, in all the heat-treated honey samples, the formation of a layer of crystals and granules with air bubbles was observed in the top layer of the container. However, after 24 weeks, for all the ultrasound-treated honeys, there were only a few clumps of granules and a comparatively small amount of air bubbles in the top layer of the containers; much less than in the heat-treated honey samples The thickness of these layers could not be measured at 29 weeks as the thickness was very small at this stage of crystallisation. The amount of crystals and/or granules in the top layer was used as a measuring tool for crystallisation, since the quantity of small crystal structures formed in the rest of the container was comparatively small (Table 4.28). A rating was developed to evaluate the visual observations of honey crystallisation in containers (Table 4.28). The visual observations of honey containers up to 29 weeks indicate that heat-treated honey samples have crystallised faster than ultrasound-treated honey. In addition, the amount of crystals and/or granules formed as a layer at the top was comparatively low in ultrasound-treated samples after 29 weeks relative to that in heat-treated honey samples.

Table 4.28 Visual observations of heat-treated and ultrasound-treated honey containers after 203 days (29 wks)

Treatment Replicate Visual Observations Rating Heat 1 Some crystals and granules in the top layer of honey. ** Heat 2 Some crystals and granules in the top layer of honey. ** Heat 3 Few crystals and granules in the top layer of honey. * Heat 4 More crystals and granules in the top layer of honey. *** Heat 5 Few crystals and granules in the top layer. * Heat 6 Few crystals and granules in the top layer. *

Ultrasound 1 Few clumps of granules in the top layer. * Ultrasound 2 No crystals or granules in the top layer. 0 Ultrasound 3 No crystals or granules in the top layer. 0 Ultrasound 4 Few clumps of granules in the top layer. * Ultrasound 5 Few clumps of granules in the top layer. * Ultrasound 6 Few clumps of granules in the top layer. *

1. No crystals or granules in the top layer. 0 2. A few crystals and/or granules or clumps in the top layer. * 3. Some crystals and/or granules or clumps in the top layer. ** 4. More crystals and/or granules or clumps in the top layer. *** 5. Top layer is completely crystallised. ****

4.3.4 Summary, conclusions and implications The main study of the effect of ultrasound on candied honey was divided into three experiments. In the first ultrasound liquefaction experiment, it was necessary to determine which ultrasound sonotrode, out of the available 7 mm, 12 mm or 40 mm diameter sonotrodes, better liquefies candied honey. Here, ~250 g of candied honey was treated with ultrasound input energy interrupted after each of the six 10,000 J intervals, for predetermined input energy levels, using three ultrasonic sonotrodes, with the temperature profile in the honey being monitored during each interruption. Input energy, treatment

105

time and power measurements were also recorded from the data file produced by the ultrasonic processor. The main finding was that the 40 mm diameter sonotrode operated at the 12 µm amplitude was optimum for completely liquefying candied honey. While it has a lower maximum net power for any 1 s period during treatment than did the 22 mm diameter sonotrode, the maximum net power for the 40 mm diameter sonotrode increased steadily after each of the six interrupted 10000 J energy inputs as the honey liquefied, while the maximum net power for the 22 mm diameter sonotrode initially increased but decreased markedly from the fourth 10000 J energy treatment onwards. As the candied honey liquefies, the power output from the sonotrode increases until the honey is liquid at which point there is little increase in maximum net power. The decrease in net power after an initial increase observed for the 22 mm diameter sonotrode indicates that there was poor efficiency in the emission of energy from the 22 mm diameter sonotrode into the candied honey. The 7 mm diameter sonotrode produced a lower maximum net power than the other two sonotrodes again indicating poor output efficiency of energy from the sonotrode into the candied honey. Cumulative treatment times were lower for the 40 mm sonotrode (324 s to 383.3 s; lowest for the 12 µm amplitude) relative to those for the 7 mm (588.3 s to 681.3 s) and 22 diameter (394.7 s to 871.0 s) sonotrodes. In addition, the variation in treatment times among replications was lower for the 40 mm diameter sonotrode. The more efficient is the emission of energy from the sonotrode to the honey, the shorter the treatment times. Finally, the maximum temperature reached after the sixth interrupted 10000 J of energy input was significantly (P<0.05) lower for the 40 mm diameter (66.2 °C to 67.8 °C) sonotrode relative to those for the 7 mm (78.2 °C to 84.4 °C) and 22 mm (76.4 °C to 82.8 °C) diameter sonotrodes. This reflects the shorter treatment time for this sonotrode, which was possibly due to the high maximum net power produced. Since treatment times need to be as short as possible, and temperatures as low as possible, then the 40 mm diameter sonotrodes operated at an amplitude of 12 µm is the optimum condition for complete liquefaction of honey on a laboratory scale. Once the optimum sonotrode (40 mm diameter) and amplitude (12 µm) were selected in Experiment 1, the first aim of Experiment 2 was to determine the minimum input energy required for complete liquefaction. The use of too higher an input energy not only wastes energy but will unnecessarily increase the temperature and treatment time. The second aim was to determine if the ultrasound treatment adversely affects the quality of the honey. The third aim was to determine the specific energy input (kWh) required to liquefy one kilogram of candied honey, in order for the developed novel ultrasound liquefaction method to be useful for the honey industry. Thus, in the second ultrasound liquefaction experiment, candied Salvation Jane honey samples were treated with six different ultrasound input energy levels using this optimum sonotrode and amplitude. A preliminary trial showed that a range of input energies from 50000 J to 70000 J would produce a range of liquefaction efficiencies from partially liquefied to completely liquefied. During a replicated trial involving six input energies between 50000 J and 70000 J, only an input energy of 70000 J completely liquefied candied Salvation Jane honey. The other energy inputs only partially liquefied the candied honey. In addition, the time needed to emit each of the fixed energies from the sonotrode increased from 304 s for 50000 J of input energy to 434.0 s for 70000 J of input energy, since it takes longer for a sonotrode to emit more energy. However, there was no significant (P>0.05) difference in the maximum temperature (which ranged 69 °C to 77.3 °C) after each of the six fixed energy treatments. Further, the maximum net power recorded at any 1 s interval during each energy level treatment was not different from each other. This suggests that the highest energy treatment (70000 J) can be used to completely liquefy candied honey in a relatively short time of 434 s without it adversely affecting the maximum temperature generated in the honey relative to lower energy treatments. However, such a finding means little if the ultrasound treatment adversely affects the quality of the honey. Therefore, the liquefied honey samples produced in Experiment 2 by ultrasound treatment with different input energies were analysed for their hydroxymethylfurfural (HMF) concentrations, and diastase and invertase activities, since these three quality parameters are normally used by the

106

honey industry and regulators to gauge the heating history of honey. The effect of ultrasound on the HMF concentration showed that there was no significant (P>0.05) difference in the HMF concentration in honeys treated with between 50000 J and 62500 J of input energy and honeys that were heat-treated. However, the HMF concentrations in the honeys treated with 65000 J and 70000 J of input energy were significantly (P<0.05) lower than the HMF concentrations in the heat-treated honeys. This is primarily due to the honey being at the maximum temperature reached of 77.3 °C for a much shorter time (434.0 s) than a heating regime (55 °C for 16 h and 72 °C for 2 min) which is similar to that presently used by the honey industry. The effect of the energy treatments on enzyme activity was negligible since there were no significant (P>0.05) differences in the diastase activity between honeys treated with any of the six energy inputs and those that were heat-treated, while the invertase activity of most of the ultrasound treated honey was higher than the heat-treated honeys, with this difference not always being significant. In conclusion, use of an ultrasound input energy of 70000 J from a 40 mm sonotrode operated at an amplitude of 12 µm is sufficient to liquefy candied Salvation Jane honey (~250 g) without compromising honey quality. For example, this ultrasound treatment results in the production of a lower concentration of HMF from honey sugars , and no decrease in diastase and invertase activities, relative to a heating regime (55 °C for 16 h and 72 °C for 2 min) similar to that used by industry. The specific energy input needed to completely liquefy candied Salvation Jane honey is 0.126 kWh/kg. Therefore, 10 kg of candied honey will require 1.26 kWh, while 300 kg will require 37.9 kWh. To complete the ultrasound liquefaction study, it was necessary to determine the effect of ultrasound treatment, relative to heat treatment, on the stability of liquid honey with respect to subsequent crystallisation. It is a common problem within the honey industry for heat-treated liquefied honey to crystallise on storage, particularly during cold weather. Since liquid honey is preferred by Australian consumers, and by food companies (for ease of handling), then a method to retard the crystallisation process in honey is required. In the third ultrasound liquefaction experiment, candied reworked mixed honey (~200 g) was completely liquefied by ultrasound treatment. Reworked mixed honey was selected for this trial as it is a very fast crystallising honey that produces large crystals. This permitted a crystallisation study to be completed in a short time-frame. Crystallisation of ultrasound-treated honey under optimum conditions of 14 °C was monitored (using a microscope as part of an image analyser) and compared with crystallisation in honey samples initially treated with a standard heat treatment. The first finding was that the D-glucose monohydrate crystallised differently in each type of treated honey. In the heat-treated honey samples, the initial plate crystals that formed at between 14 and 28 days were long thin, spiral shaped plate crystals. In contrast, in the ultrasound-treated honey samples, most of the initial crystals that formed at between 14 and 49 days were large pentagon shaped plate crystals. In addition, more needle crystal masses were formed in the heat-treated honeys than were produced in the ultrasound-treated honeys at the end of the monitoring period of 112 days (16 weeks). Moreover, in heat-treated honeys, plate crystals grew underneath these needle crystals masses in the later stages of crystallisation. In contrast, in ultrasound-treated honeys, plates formed after the needles, with subsequently needles growing on the initial plates. In conclusion, the findings of this crystallisation study indicate that ultrasound treatment delays D-glucose monohydrate crystallisation more than does a heat treatment similar to that used by the honey industry. This occurs at both the microscopic level (in a drop of honey) and in bulk samples. In addition, there is a difference in the crystal formation process at the microscopic level in ultrasound-treated honey relative to that in heat-treated honey. The reason for this is not clear, and requires further study. Thus, ultrasound treatment will not only liquefy candied honey without the need for long exposure to high temperatures, but may make the liquefied honey more stable to subsequent crystallisation on storage.

107

4.4 Energy Requirement for an Industrial Scale-Up Design of a

Commercial Ultrasound Processor 4.4.1 Calculation of the energy requirement for ultrasound liquefaction of

candied honey As per the personnel correspondence of Dr Ing Gerhard Hielscher of the ultrasound equipment manufacturer, Dr. Hielscher GmbH of Germany, determination of specific energy input required for an ultrasound treatment is described below. In this method, the power requirement during the ultrasound treatments were measured by a watt meter and the energy input (W) was calculated by ‘power multiplied by sonication time’. The average power value was used for calculation. The specific energy input (Wspec) in kWh/kg for a specific mass (kg) of a sample is calculated as,

Specific Energy Input (Wspec) = Error! Objects cannot be created from editing field codes.

As industrial devices have the same functional principles as laboratory devices, the above specific energy input can be used for industrial scale calculation (Personnel correspondence with Dr. Hielscher GmbH). It was assumed that the maximum bulk power recorded during the treatment can be used as the power needed to be generated by the ultrasound processor to supply the specific input energy in a specified time. Maximum energy requirement for liquefying 250 g of crystallized honey (W) = maximum bulk power x ultrasonic time (This is similar to the calculation of energy (W) in the Dr. Hielscher’s procedure) Maximum Energy (kWh)

= [maximum bulk power (W) x ultrasonic time (s) / (60 x 60 x 1000))] Specific energy input (Wspec) (in kWh/kg)

= [ maximum bulk power (W) x ultrasonic time (s) / 60 x 60 x 1000 x mass (kg) ] The data for the 3 replicates obtained from Table 4.21 (treatment T6) are given in Table 4.29 for the ultrasound conditions for an instrumentally set energy input of ~70000 J supplied to completely liquefy ~250 g candied honey using the Dr Hielscher UP400S ultrasound processor fitted with the 40 mm diameter sonotrode operated at the 12 µm amplitude and 100% cycle (continuous operation).

108

The measurements of data during the ultrasound treatment includes the bulk power, net power, ultrasonic time and input energy at one second intervals. These data provide the information about the variation of bulk power and net power supplied by the ultrasound processor during the treatment period. Table 4.29 Ultrasound maximum bulk power, treatment time and maximum energy

for an input energy of ~70000 J required to liquefy ~250 g candied honey Replicate Maximum Bulk Power

(W) Ultrasonic Time

(s) Maximum Energy

(Ws) R1 266.1 398 105907.8 R2 254.0 468 118872.0 R3 266.0 436 115976.0

Mean 262.0 434 113585.3 Std. Dev. 7.0 35.0 6804.7

The mean maximum bulk power and mean ultrasonic treatment time were used to calculate the energy requirement to completely liquefy 250 g of candied honey: Energy requirement to completely liquefy 250 g of candied honey = 262 (W) x 434 (s) = 13708.0 Ws

Therefore, the specific energy input = 25.010006060

434262xxx

x = 0.126 kWh/kg

This value can be used to calculate the specific energy requirement for liquefying a specific mass of honey on an industrial scale. Honey is stored in 200 L drums of diameter 580 mm and height 880 mm before it is placed in a hot room. These drums contain 300 kg of honey and have openings of 50 mm and 20 mm diameter. Therefore, the specific energy input requirement for liquefying 300 kg of honey = 300 x 0.126 = 37.9 kWh The time required to liquefy 300 kg of candied honey depends on the input power of the ultrasound processor and the capacity of the sonotrode(s). In addition, the ultrasound sonotrodes have to be inserted into the drum to a particular depth in order to liquefy the honey down to the bottom of the drum. A mechanical platform device would have to be designed in order to rotate the drum around the ultrasonic sonotrode(s) (depending on the diameter of the emitting face of the sonotrode) and move the drum vertically after each layer of honey has been liquefied. A number of beekeepers have expressed interest in being able to liquefy smaller quantities of candied honey such as 10 kg (or smaller) in plastic containers. Therefore, the specific energy input requirement for liquefying 10 kg of honey = 10 x 0.126 = 1.26 kWh

109

4.4.2 Design considerations for commercial scale ultrasound liquefaction of

candied honey It was not possible to scale up the ultrasound processing conditions as part of this study as the necessary industrial processors were not available to the research team. However, the input energy calculation described in Section 4.4.1 was for a laboratory scale processor, but it provides a good starting point for an industrial trial by a honey packing company. Discussions with beekeepers and honey packers have suggested that there is a need to liquefy candied honey quantities of 5-10 kg in plastic containers, and 300 kg in 200 L drums. To initiate the transfer of the results of this research project to the honey industry, preliminary communication with a company that manufactures ultrasound processors was undertaken. The laboratory data collected for liquefaction of candied honey using a laboratory size ultrasound processor has been sent to ultrasound equipment manufacturer Dr Hielscher GmbH, Germany in order to commence the development of the ultrasound processing system for medium and large scale industrial processing. 4.4.2.1 Recommendations for industrial testing of the Dr Hielscher GmbH UIP

ultrasound processors for the ultrasound liquefaction of up to 10 kg of candied honey

The following recommendations are for candied honey in containers where the complete lid can be removed exposing the whole of the top layer of the honey. There is a need for a platform under the honey container that can be moved up and down and sideways so that the sonotrode can be moved throughout the candied honey. Initially, the sonotrode will be in contact with the hard candied honey near the top of the container. As dissolution (or liquefaction) of the surface honey proceeds, the honey container will need to be moved upwards and sideways in a predetermined pattern (similar to that employed in the laboratory scale experiments detailed earlier), so that the sonotrode is brought in contact with as much of the candied honey as possible to minimise the treatment time required. In addition, or alternatively, a stirrer could be inserted in the semi-melted honey to mix the liquid honey with the remaining candied honey creating a flow in the container past the treatment region around the sonotrode. The ultrasound waves dissipate quickly at a short distance from the sonotrode, so some mixing is required. 4.4.2.1.1 Ultrasound processor details for industrial trial on 10 kg candied honey The company, Dr Hielscher GmbH has indicated that UIP 1000 industrial ultrasound processor (Figure 4.22) combined with a 22 mm diameter sonotrode (Model: BS2d22) or a 34 mm diameter sonotrode (Model: BS2d34) should be tested for liquefying candied honey in 10 kg containers in order to collect data in an industrial environment. The UIP 1000 industrial ultrasound processor generates 1000 W of power with an operation frequency of 20 kHz. The BS-1.2 and BS-1.4 boosters can be attached to BS2d22 and BS2d34 sonotrodes to increase the amplitude ratio of the sonotrode to 1:1.2 and 1:1.4 respectively. The amplitude of the ultrasound waves can be adjusted between 50 – 100 %. The power output from this ultrasound processor should be sufficient to liquefy up to 10 kg of candied honey. Generally, 10 kg plastic containers have dimensions of diameter 270 mm and height 240 mm. The BS type sonotrodes are block sonotrodes which are heavy sonotrodes made of titanium alloy and used for homogenising, dispersing etc. The technical details of the UIP 1000 ultrasonic processor are given in Figure 4.22.

110

In addition, the UIP 4000 industrial ultrasound processor is available with 4000 W of power and a cascade sonotrode (Model: KS20d65l6) with a diameter of 60 mm and a length of 630 mm (Figure 4.23). Cascade sonotrodes are heavy sonotrodes made of titanium for use with UIP 2000 and UIP 4000 industrial ultrasonic processors. Such a processor would be used for candied honey quantities greater than 10 kg but less than 300 kg (in 200 L drums). Both these type of sonotrodes (Figures 4.22 and 4.23) may be able to be incorporated into a multiple sonotrode processor reducing the need for significant movement of the honey container and shortening the treatment time. 4.4.2.1.2 Recommended procedure for industrial testing of the Dr Hielscher GmbH UIP 1000

ultrasound processor for the ultrasound liquefaction of up to 10 kg of candied honey The company, Dr Hielscher GmbH has provided the following procedure to be adopted in an industrial setting for testing the UIP 1000 industrial ultrasound processor for the liquefaction of up to 10 kg of candied honey. Such initial testing by interested honey packers and beekeeper is required so that the specific energy input (Wspec) in kWh/kg for the ultrasound liquefaction of candied honey by an industrial ultrasonic processor can be determined. For the industrial trials, the following will need to be supplied from the company, Dr Hielscher GmbH (via rental):

UIP1000 industrial processor Stand ST2 Power Meter Sonotrodes BS2d34 and BS2d22 Booster B2-1.4 Temperature probes (the more, the better)

Considering the height of the container (diameter of 270 mm and height of 240 mm), standard sonotrodes should be used. Initially, the trial should start with the BS2d34 sonotrode (immersed 40 mm into the container). In order to obtain sufficient data, the ultrasound treatment tests should be performed a number of times with the same amount of candied honey and using the same immersion depth. These tests should follow the following 4 steps: Step 1: Power supply to the ultrasound processor to be supplied through a wattmeter in order

to measure the power requirement. Step 2: Measure the absorbed power (no load power) of the ultrasound processor for different

amplitude settings (i.e. between 50 – 100%) by operating the processor in air. Step 3: Measure the power input at each amplitude setting (with the wattmeter) required to

completely liquefy the candied honey, and record the treatment time. Step 4: Tests on candied honey should be repeated with/without a booster and at different

amplitude settings (50-100%, front panel). Temperature probes should record the temperature increase over time (e.g. 0.5, 1, 2, 4, 8, 16 min) at certain distances (e.g. 25, 50, 75, 100 mm) from the sonotrode (horizontal and vertical distance). Figure 4.24 details the factors affect temperature during ultrasound treatment. In addition the actual power should be recorded.

111

Source: Highly intensive ultrasound for laboratories and industries, Dr Hielscher GmbH

Figure 4.22 Technical data of the Dr Hielscher GmbH UIP 1000 ultrasonic processor

112

Source: Highly intensive ultrasound for laboratories and industries, Dr Hielscher GmbH

Figure 4.23 Technical data of the Dr Hielscher GmbH UIP 4000 ultrasonic processor

113

The actual power requirement is calculated by deducting no load power from the absorbed power. The energy requirement is determined by multiplying the actual power by the sonication time. Further, the specific energy input in kWh/kg units for each test needs to be calculated as detailed in Section 4.4.1. Conclusion: The data collected from such an industrial trial on 10 kg of candied honey will help to determine the energy, power, treatment time and setup required for the liquefaction of 300 kg of honey in 200 L drums.

Figure 4.24 Factors affecting the temperature during ultrasound treatment (Source: the company, Dr Hielscher GmbH)

4.4.2.1.3 Ultrasound liquefaction of 300 kg of candied honey The above collected data need to be given to the company, Dr Hielscher GmbH for designing an industrial scale ultrasound processing system, including for liquefying 300 kg of candied honey in 200 L drums. The newer type large plastic drums which have a completely removable lid would be ideal for use with the above processor system. Design Problem: The one industrial scale problem likely to be encountered relates to the design of the commonly used 200 L galvabond drums. These drums have only small openings which while permitting insertion of the sonotrode, will not permit the moving of the drum up and down and sideways in a predetermined pattern so as to expose all the candied honey to the ultrasound waves. However, as part of the industrial trials and the subsequent design of the processor system by Dr Hielscher GmbH, such a limitation in the galvabond drums may be able to be overcome.

114

4.4.2.1.4 Costs of ultrasound processors for industrial trials The UIP 1000 industrial ultrasound processor and sonotrodes with boosters can be rented for $2,486.00 + GST + freight for one month from the local agent of the company, Dr Hielscher GmbH equipments, DKSH Australia Pty Ltd.. In addition, the company, Dr Hielscher GmbH offer a complete UIP 1000 (1000W) 20 kHz industrial ultrasound processing system, including an ultrasonic processor, the two sonotrodes BS2d22 and Bd2d40, the BS-1.2 and BS-1.4 boosters, a height adjustable stand and a power meter for a cost of $21,915. The power meter displays the current power, cumulated energy and cumulated operating time 4.4.2.1.5 Contact details for ultrasound manufacturer Hielscher Ultrasonics GmbH DKSH Australia Pty. Ltd. Am Dobelbach 19 14-17 Dansu Court, D-70184 Stuttgart Hallam Germany Victoria 3803 Tel.: +49 711 234 8 199 Tel : +61 3 9554 6666 Fax: +49 711 234 8 198 Fax: +61 3 9554 6688 Email: [email protected] Email: [email protected] 4.4.2.2.5 Follow up assistance for industrial trials of ultrasound liquefaction of candied honey During the initial trials by honey packers and beekeepers, follow up assistance on the ultrasound treatment of honey can be obtained from the project team members listed below: Dr Bruce D’Arcy Associate Professor Bhesh Bhandari School of Land, Crop and Food Sciences School of Land, Crop and Food Sciences The University of Queensland The University of Queensland Brisbane Brisbane Queensland 4072 Queensland 4072 Phone: 07 3346 9190 Phone: 07 3346 9192 Mobile: 0421 058 160 Fax: 07 3365 1177 Fax: 07 3365 1177 Email: [email protected] Email: [email protected]

115

5. Implications This project has produced data that can be used by beekeepers and honey packing companies to develop a pilot scale ultrasound processing system in conjunction with an ultrasound equipment manufacturer. This will need to not only involve ultrasound treatment of, for example, 10 kg of honey in plastic containers with removable lids, but 300 kg candied honey in large plastic containers with removable lids. Where problems will arise will be in the treatment of honey in 200 L galvabond drums with their two small hole outlets. Insertion of a ultrasound sonotrode will be difficult with such a designed drum, and an alternate sonotrode design to a cylinder type sonotrode will be required. The implications for the honey industry, if this ultrasound technology was applied on an industrial scale, would be to produce better quality honey (less effect on flavour, HMF etc.), and remove the need for hot rooms (and thus reduce energy costs).

6. Recommendations It is recommended that honey packing companies in Australia with engineering expertise should undertake industrial scale trials in conjunction with this project’s research team and a manufacturer of ultrasound equipment. An initial industrial trial has already been designed in conjunction with an ultrasound equipment manufacturer; it is detailed in Section 4.4.2.1 and is based on the experimental data obtained from this project using a small laboratory scale ultrasound processor. It is envisaged that the ultrasound treatment of five or ten kg of candied honey in plastic containers could be automated as part of a processing line. In addition, the treatment of candied honey in larger (>100 L) plastic drums (where the complete lid can be removed) could be automated, involving a much larger and more powerful ultrasound processing system, prior to decanting and pumping. However, engineering difficulties will arise for treatment of honey in a 200 L galvabond drum since there are only two small holes in the lid making insertion of the sonotrode very difficult. A possible alternate sonotrode system could involve a wrap-around ring, containing a series of sonotrodes, that is strapped to the outside of the drum, and a rod type stirrer that is inserted in through the larger of the two openings in the top of the drum. It is important to stir the partially candied honey so as to bring the crystallised honey close to the ultrasound sonotrodes wrapped around the outside of the drum. Here, the depth of penetration of the ultrasound waves into the viscous honey will need to be considered (the waves do not penetrate well into honey), as will the temperature of the honey close to the drum wall. Stirring will help prevent areas of honey where the temperature becomes too high. But again, these are challenges that need to overcome by working directly with an ultrasound manufacturer. Technology transfer of this project’s findings will require further expenditure of money and time by those honey packers and beekeepers interested in reducing or removing their need to heat honey for extended periods in hot rooms prior to pumping and bottling. Those honey producers conscious of improving the quality of their product, particularly the flavour quality, are most likely to take up this scale-up challenge, and ensure this new technology for liquefying candied honey becomes common place within the Australian honey industry. It is recommended that this should occur in the next twelve months.

116

7. References Cited Alliger, H. 1975, Ultrasonic disruption. American Laboratory, 7(10), 75-85.

Amor, D. M. 1978, Composition, properties and uses of honey - A literature survey. The British Food Manufacturing Industries Research Association, Scientific and Technical Surveys No. 108.

Anon. 1985, United States standards for grades of extracted honey, United States Department of Agriculture.

Anon. 2003a, pH and acids in honey, National Honey Board, U.S.

Anon. 2003b, Honey Colour, National Honey Board, U.S.

Anon. 2003c, Carbohydrates and the sweetness of honey, National Honey Board, U.S.

Anon. 2003d, Shelf life and stability of honey, National Honey Board, U.S.

Arakeri, V. H., & Chakraborty, S. 1990, Studies towards potential use of ultrasonics in hydrodynamic cavitation. Current Science, 59(24), 1326-1333.

Assil, H. I., Sterling, R., & Sporns, P. 1991, Crystal control in processed liquid honey. Journal of Food Science, 56(4), 1034-1041.

Bhandari, B., D’Arcy, B., & Chow, S. 1999b, Rheology of selected Australian honeys. Journal of Food Engineering, 41, 65-68.

Bhandari, B., D’Arcy, B., & Kelly, C. 1999a, Rheology and crystallization kinetics of honey: present status. International Journal of Food Properties, 2(3), 217-226.

Bogdanov, S. 2002, Harmonised methods of the international honey commission, International Honey Commission. http://www.alp.admin.ch/themen/00502/00555/00564/index.html?lang=en

Bogdanov, S., Lüllmann, C., Martin, P., von der Ohe, W., Russmann, H., Vorwohl, G., Oddo, L., Sabatini, A., Marcazzan, G., Piro, R., Flamini, C., Morlot, M., Lhéritier, J., Borneck, R., Marioleas, P. T., A., Kerkvliet, J., Ortiz, A., Ivanov, T., D’Arcy, B. & Mossel, B. 1999a, Honey quality, methods of analysis and international regulatory standards: Review of the work of the International Honey Commission. Mitteilungen aus dem Gebiete der Lebensmitteluntersuchung und Hygiene, 90(1), 108-125.

Bogdanov, S., Lüllmann, C., Martin, P., von der Ohe, W., Russmann, H., Vorwohl, G., Oddo, L., Sabatini, A., Marcazzan, G., Piro, R., Flamini, C., Morlot, M., Lhéritier, J., Borneck, R., Marioleas, P. T., A., Kerkvliet, J., Ortiz, A., Ivanov, T., D’Arcy, B., Mossel, B. & Vit, P. 1999b, Honey quality and international regulatory standards: review by the International Honey Commission. Bee World., 80(2), 61-69.

Bogdanov, S., Martin, P. & Lüllmann, C. 1997, Harmonized methods of the European Honey Commission. Apidologie, Extra Issue: 1-59.

Bonvehí, J. S., Torrento, M. S. and Raich, J. M. 2000, Invertase activity in fresh and processed honeys. J. Sci. Food Agric., 80, 507-512.

Burdin, F., Tsochatzidis, N. A., Guiraud, P., Wilhelm, A. M., & Delmas, H. 1999, Characterisation of the acoustic cavitation cloud by two laser techniques. Ultrasonics Sonochemistry, 6, 43-51.

Chandler, B. V., Fenwick, D., Orlova, T., & Reynolds, T. 1974, Composition of Australian honeys. Division of Food Research, CSIRO.

Chataway, H. D. 1932, Determination of moisture in honey. Canadian Journal of Research, 6, 532-547.

Chendke, P. K., & Fogler, H. S. 1975, Macrosonics in industry. Ultrasonics, 31, 31-37.

117

Ciaravino, V., & Miller, M. W. 1983, A comparison of two techniques for measured iodine release as an indicator of acoustic cavitation. Journal of Acoustical Society of America, 74(6), 1813-1816.

Clemson, A. 1985, Honey and Pollen Flora. Department of Agriculture, New South Wales.

Codex Alimentarius. 1998, Draft revised for honey at step 6 of the Codex Procedure.CX5/10.2, CL1998/12-S 1998.

Codex Alimentarius. 2001, Revised codex standard for honey. (No. CODEX STAN 12-1981, Rev.1 (1987), Rev.2 (2001)).

Contamine, F., Faid, F., Wilhelm, A. M., Berlan, J., & Delmas, H. 1995, Chemical reactions under ultrasound: discrimination of chemical and physical effects. Chemical Engineering Science, 49(24B), 5865-5873.

Crane, E. 1990, Bees and Beekeeping. Science, Practice and World Resources. New York: Cornell University Press.

Crane, E., & Walker, P. 1984, Composition of honey from some important honey sources. Bee World, 65(4), 167-174.

Da Costa Leite, J. M., Trugo, L. C., Costa, L. S. M., Quinteiro, L. M. C., Barth, O. M., Dutra, V. M. L., De Maria, C.A.B. 2000, Determination of oligosaccharides in Brazilian honeys of different botanical origin. Food Chemistry, 70, 93-98.

D’Arcy, B., Bhandari, B., Caffin, N., Halley, P., Sopade, P., Mossel, B., D’Arcy, M., Yao, L., & Doebler, C. 2001b, Improving the movement and use of liquid Australian honey within manufacturing processes. Rural Industries Research and Development Corporation. 209 + xxi pp.

D’Arcy, B., Rintoul, G., Krebbers, B., Sancho, T., Jung, D., & Fedorow, M. 2001a, Flavour quality assurance of Australian floral honeys by chemical fingerprinting. Rural Industries Research and Development Corporation. 312+ xxvii pp.

D’Arcy, B., Rintoul, G. B., Rowland, C. Y., & Blackman, A. J. 1997, Composition of Australian honey extractives. 1. Norisoprenoids, monoterpenes, and other natural volatiles from Blue Gum (Eucalyptus leucoxylon) and Yellow Box (Eucalyptus melliodora) honeys. Journal of Agricultural and Food Chemistry, 45, 1834-1843.

Doner, L. W. 1977, The sugars of honey. Journal of the Science of Food and Agriculture, 28, 443-456.

Dyce, E. J. 1931a, Crystallization of honey. Journal of Economic Entomology, 24, 597-602.

Dyce, E. J. 1931b, Fermentation and crystallisation of honey. Cornell University Agricultural Experiment Station Bulletin, 528, 1-76.

Dyce, E. J. 1976, Producing finely granulated or creamed honey. Chapter 10 in Honey : A Comprehensive Survey. E. Crane (Ed.), p. 293-306. London: William Heinemann Ltd.

Earnshaw, R. G. 1998, Ultrasound : a new opportunity for food preservation. In Ultrasound in Food Processing. M. J. W. Povey & T. J. Mason (Eds.), p. 183-192.

Feng, R., Zhao, Y., Zhu, C., & Mason, T. J. 2002, Enhancement of ultrasonic cavitation yield by multi-frequency sonication. Ultrasonics Sonochemistry, 9, 231-236.

Garcia, M. L., Burgos, J., Sanz, B., & Ordonez, J. A. 1989, Effect of heat and ultrasonic waves on the survival of two strains of Bacillus subtilis. Journal of Applied Bacteriology, 67, 619-628.

Gogate, P. R., Shrigaonkar, I. Z., Sivakumar, M., Senthilkumar, P., Vichare, N. P., & Pandit, A. B. 2001, Cavitation reactors: efficiency assessment using model reaction. AIChE Journal, 47(11), 2526-2538.

Gogate, P. R., Tatake, P. A., Kanthale, P. M., & Pandit, A. B. 2002, Mapping of sonochemical

118

reactors: review, analysis, and experimental verification. AIChE Journal, 48(7), 1542-1560.

Gupta, J. K., Kaushuik, R., & Joshi, V. K. 1992, Influence of different treatments, storage temperatures and period on some physico-chemical characteristics and sensory qualities of Indian honey. Journal of Food Science and Technology, 29(2), 84-87.

Hadorn, H. & Zürcher, K. 1972, Eine einfache kinetische methode zur Bestimmung der Diastasezhal in honig. Dtsch Lebensm Rundsch, 68: 209-216.

Hebbar, H. U., Nandani, K. E., Lakshmi, M. C., & Subramanian, R. 2003, Microwave and infrared heat processing of honey and its quality. Food Science and Technology Research, 9(1), 49-53.

Helvey, T. C. 1954, Study on some physical properties of honey. Food Research, 19(3), 282-292.

Huxsoll, C. C., & Hall, C. W. 1970, Effects of sonic irradiation on drying rates of wheat and shelled corn. Transactions of ASAE, 13, 21-24.

Jackson, R. F., Silsbee, C.G. 1924, Saturation relations in mixtures of sucrose, dextrose and levulose. Technological papers of the Bureau of Standards, 18, 278-304.

Junzheng, P., & Changying, J. 1998, General rheological model for natural honeys in China. Journal of Food Engineering, 36, 165-168.

Kaloyereas, S. A. 1955, Preliminary report on the effect of ultrasonic waves on the crystallisation of honey. Science, 121, 339-340.

Karabournioti, S., & Zervalaki, P. 2001, The effect of heating on honey HMF and invertase. Apiacta, 36(4), 177-181.

Kelly, F. H. C. 1954, Phase equilibria in sugar solutions IV. Ternary system of water-glucose-fructose. Journal of Applied Chemistry, 4, 409-411.

Kuster, B. F. M. 1990, 5-Hyroxymethylfurfural (HMF). A review focussing on its manufacture. Starch/Starke, 42(1), 314-321.

Langridge, D. F. 1971, Australian honey in relation to European standards. Food Tech. Aust., March, 116-118.

Leadley, C., & Williams, A. 2002, Power ultrasound - current and potential applications for food processing. UK: Campden & Chorleywood Food Research Association Group.

Li, H., Wang, J., Bao, Y., Guo, Z., & Zhang, M. 2003, Rapid sonocrystallization in the salting-out process. Journal of Crystal Growth, 247, 192-198.

Liebl, D. E. 1978, Ultrasound and granulation of honey. American Bee Journal, 2, 107.

Lillard, H. S. 1994, Decontamination of poultry skin by sonication. Food Technology, 48, 72-73.

Lochhead, A. G. 1933, Factors concerned with the fermentation of honey. Ottawa, Canada: Division of bacteriology, Dominion Experimental Farms.

Loning, J., Horst, C., & Hoffmann, U. 2002, Investigations on the energy conversion in sonochemical processes. Ultrasonics Sonochemistry, 9(3), 169-179.

Lopez, P., Sala, F. J., de la Fuente, J. L., Condon, S., Raso, J., & Burgos, J. 1994, Inactivation of peroxide, lipoxygenase and polyphenol oxidase by manothermosonication. Journal of Agricultural and Food Chemistry, 42, 252-256.

Lothrop, R.E. 1943, ‘Saturation relations in aqueous solutions of some sugar mixtures with special reference to higher concentrations’, Thesis, George Washington University.

Low, N. H., & Sporns, P. 1988, Analysis and quantitation of minor di- and trisaccharides in honey, using capillary gas chromatography. Journal of Food Science, 53(2), 558-561.

Martin, E. C. 1958, Some aspects of hygroscopic properties and fermentation of honey. Bee World, 39(7), 165-178.

119

Marvin, G. E. 1930, Further observations on the deterioration and spoilage of honey in storage. Journal of Economic Entomology, 23, 431-438.

Mason, T. J. 1998, Power ultrasound in food processing - the way forward. In M. J. W. Povey & T. J. Mason (Eds.), Ultrasound in food processing (pp. 105-126).

Mason, T. J., & Lorimer, J. P. 1988, Sonochemistry: theory, application and uses of ultrasound in chemistry.

Mason, T. J., Paniwnyk, L., & Lorimer, J. P. 1996, The uses of ultrasound in food technology. Ultrasonics Sonochemistry, 3, S253-S260.

Mason, T. J., & Zhao, Y. 1994, Enhanced extraction of tea solids using ultrasound. Ultrasonics, 32(5), 375-377.

Mateo, R., & Bosch-Reig, F. 1997, Sugar profiles of Spanish unifloral honeys. Food Chemistry, 60(1), 33-41.

McClements, D. J. 1995, Advances in the application of ultrasound in food analysis and processing. Trends in Food Science and Technology, 6, 293-299.

McDonald, J. L. 1964, Finely crystallised honey. Gleanings in Bee Culture, 92, 614-615, 689, 700.

Munro, J. A. 1943, The viscosity and thixotropy of honey. Journal of Economic Entomology, 36(5), 769-777.

Mossel, B. L. 2002, ‘Antimicrobial and quality parameters of Australian unifloral honey’, Ph.D. Thesis, University of Queensland, Brisbane, Australia.

Mossel, B., Bhandari, B., D’Arcy, B., & Caffin, N. 2000, Use of an Arrhenius model to predict rheological behaviour in some Australian honeys. Lebensmittel-Wissenschaft und Technologie, 33(8), 542-552.

Mossel, B., Bhandari, B.R., D’Arcy, B., & Caffin, N. 2003, Determination of the viscosity of some Australian honeys based on composition. International Journal of Food Properties, 6(1), 87-97.

Naidu, D. V. P., Rajan, R., Kumar, R., Gandhi, K. S., Arakeri, V. H., & Chandrasekeran, S. 1994, Modelling of a batch sonochemical reactor. Chemical Engineering Science, 49(6), 877-888.

Ordonez, J. A., Aguilera, M. A., Garcia, M. L., & Sanz, B. 1987, Effect of combined ultrasonic and heat treatment (thermoultrasonication) on the survival of a strain of Staphylococcus aureus. Journal of Dairy Research, 54, 61-67.

Perez-Arquillue, C., Conchello, P., Arino, A., Juan, T., & Herrera, A. 1994, Quality evaluation of Spanish rosemary (Rosmarinus officinalis) honey. Food Chemistry, 51, 207-210.

Price, C. 1997, Ultrasound-the key to better crystals for pharmaceutical industry. Pharmaceutical Technology Europe(October), 78-86.

Roberts, R. T. 1991, Sound for processing food. Nutrition and Food Science, 130, 17-18.

Roberts, R. T. 1993, High intensity ultrasonics in food processing. Chemistry and Industry (Feb), 119-122.

Rodgers, P. E. W. 1976, Honey quality control. In Honey: A Comprehensive Survey. E. Crane (Ed.), p. 314-325. London: William Heinemann Ltd.

Rowland, C.Y., Blackman, A.J., D’Arcy, B.R., & Rintoul, G.B. 1995, A comparison of organic extractives found in leatherwood (Eucryphia lucida) honey and leatherwood flowers and leaves. Journal of Agricultural and Food Chemistry. 43, 753-763, 1995.

Sala, F. J., Burgos, J., Condon, S., Lopez, P., & Raso, J. 1995, Effect of heat and ultrasound on microorganisms and enzymes. In New methods of food preservation. G. W. Gould (Ed.), pp. 117-204. Glasgow: Blackie Academic and Professional.

120

Schade, J. E., Marsh, G. L., & Eckert, J. E. 1958, Diastase activity and hydroxy-methyl-furfural in honey and their usefulness in detecting heat alteration. Food Research, 23, 446-463.

Scherba, G., Weigel, R. M., & O'Brien, W. D. 1991, Quantitative assessment of the germicidal efficacy of ultrasonic energy. Applied and Environmental Microbiology, 57(7), 2079-2084.

Senthil Kumar, P., Siva Kumar, M., & Pandit, A. B. 2000, Experimental quantification of chemical effects of hydrodynamic cavitation. Chemical Engineering Science, 55, 1633-1639.

Shrigaonkar, I. Z., & Pandit, A. B. 1997, Degradation of aqueous solution of potassium iodide and sodium cyanide in the presence of carbon tetrachloride. Ultrasonics Sonochemistry, 4, 245-253.

Siddiqui, I. R., & Furgala, B. 1967, Isolation and characterization of oligosaccharides from honey. Part I. Disaccharides. Journal of Apicultural Research, 6(3), 139 -145.

Siddiqui, I. R., & Furgala, B. 1968, Isolation and characterization of oligosaccharides from honey. Part II. Trisaccharides. Journal of Apicultural Research, 7(1), 51-59.

Singh, N., & Bath, P. K. 1997, Quality evaluation of different types of Indian honey. Food Chemistry, 58(1-2), 129-133.

Sopade, P.A., Bhandari, B., D’Arcy, B., Halley, P., & Caffin, N. 2002, A study of the vitrification of Australian honey at different moisture contents. In Progress in Amorphous Food and Pharmaceutical Systems. Royal Society of Chemistry. 281, 169-186.

Sopade, P. A., Bhandari, B., Halley, P., D’Arcy, B., & Caffin, N. 2001, Glass transition in Australian honeys. Food Australia, 53(9), 399-404.

Sopade, P. A., Halley, P., Bhandari, B., D’Arcy, B., Doebler, C., & Caffin, N. 2003, Application of the Williams - Landel - Ferry model to the viscosity - temperature relationship of Australian honeys. Journal of Food Engineering, 56, 67-75.

Sopade P. A., Halley P.J., & D’Arcy B.R. 2006, Specific heat capacity of Australian honeys from 35 to 165C as a function of composition using differential scanning calorimetry. Journal of Food Processing and Preservation, 30, 99-.

Sopade, P. A., Halley, P.J., D’Arcy, B.R., Bhandari, B.R., & Caffin, N.A. 2004a, Dynamic and steady-state rheology of Australian honeys at sub-zero temperatures. Journal of Food Process Engineering, 27(4), 284-309.

Sopade, P. A., Halley, P J., D’Arcy, B.R., Bhandari, B. & Caffin, N. 2004b, Friction factors and rheological behaviour of Australian honey in a straight pipe. International Journal of Food Properties, 7(3), 393-405.

Stinson, E. E., Subers, M. H., Petty, J., & White, J. W. 1960, The composition of honey. V. Separation and identification of the organic acids. Archives of Biochemistry and Biophysics, 89, 6-12.

Suslick, K. S. 1988, Ultrasound: its chemical, physical and biological effects. New York: VCH Publishers.

Tarbouret, T., Berdague, J. L., & Lheritier, J. 1992, Tendency of honey to crystallize - a renewed attempt at a statistical approach. Apidologie, 23(5), 415-429.

Tew, J. E. 1992, Honey and Wax - A consideration of production, processing and packaging techniques. In The Hive and the Honey Bee. J. M. Graham (Ed.), p. 657-704. Illinois: Dadant & Sons.

Thrasyvoulou, A., Manikis, J., & Tselios, D. 1994, Liquefying crystallized honey with ultrasonic waves. Apidologie, 25, 297-302.

Toba, T., Hayasaka, I., Taguchi, S., & Adachi, S. 1990, A new method for manufacture of lactose-hydrolysed fermented milk. Journal of the Science of Food and Agriculture, 52, 403-407.

121

Townsend, G. F. 1976, Processing and storing liquid honey. In Honey : A comprehensive survey. E. Crane (Ed.), p. 269-292. London: William Heinemann Ltd.

VanHook, A., & Frulla, F. 1952, Nucleation in sucrose solutions. Industrial and Engineering Chemistry, 44(6), 1305-1308.

Vercet, A., Sanchez, C., Burgos, J., Montanes, L., & Buesa, L. P. 2002, The effects of manthermosonication on tomato pectic enzymes and tomato paste rheological properties. Journal of Food Engineering, 53, 273-278.

Vimini, R. J., Kemp, J. D., & Fox, J. D. 1983, Effects of low frequency ultrasound on properties of restructured beef rolls. Journal of Food Science, 48, 1572-1573.

Vorwohl, G. 1964, Die beziehungen zwischen der elektrischen leitfahigkeit der honige und ihrer trachtmassigen herkunft. Ann. Abeille, 7(4), 301-309.

Wang, L. C. 1975, Ultrasonic extraction of proteins from autoclaved soybean flakes. Journal of Food Science, 40, 549-551.

Wang, L. C. 1981, Soybean protein agglomeration: promotion by ultrasonic treatment. Journal of Agricultural and Food Chemistry, 29, 177-180.

Wedmore, E. B. 1955, The accurate determination of the water content of honeys: Part I. Introduction and results. Bee World, 36(11), 197-206.

Weissler, A. 1959, Formation of hydrogen peroxide by ultrasonic waves: free radicals. Journal of American Chemical Society, 81, 1077-1081.

White, J. W. 1976a, Composition of honey. In Honey: A Comprehensive Survey. E. Crane (Ed.), p. 157-206. London: William Heinemann Ltd.

White, J. W. 1976b, Physical characteristics of honey. In Honey: A Comprehensive Survey. E. Crane (Ed.), p. 207-239. London: William Heinemann Ltd.

White, J. W. 1978, Honey. Advances in Food Research, 24, 288-375.

White, J. W. 1979, Spectrophotometric method for hydroxymethylfurfural in honey. Journal of Association of Official Analytical Chemists, 62(3), 509-514.

White, J. W. 1994, The role of HMF and diastase assay in honey quality evaluation. Bee World, 75(3), 104-117.

White, J. W., & Hoban, N. 1959, Composition of honey. IV. Identification of the disaccharides. Archives of Biochemistry and Biophysics, 80, 386-392.

White, J. W., Kushnir, I., & Subers, M. H. 1964, Effect of storage and processing temperatures on honey quality. Food Technology, 18, 153-156.

White, J. W., & Maher, J. 1954, Sugar analysis of honey by a selective adsorption method. Association of Official Agricultural Chemists, 37(2), 478-486.

White, J. W. & Pairent, F. W. 1959, Report on the analysis of honey. J Assoc Off Anal Chem, 42: 341-348.

Wilson, H. F., & Marvin, G. E. 1931, The effect of temperature on honey in storage. Journal of Economic Entomology, 24, 589-596.

Wootton, M., Edwards, R. A., Faraji-Haremi, R., & Johnson, A.T. 1976a, Effect of accelerated storage conditions on the chemical composition and properties of Australian honeys. 2. Colour, acidity and total nitrogen content. Journal of Apicultural Research, 15(1), 23-28.

Wootton, M., Edwards, R. A., & Faraji-Haremi, R., 1976b, Effect of accelerated storage conditions on the chemical composition and properties of Australian honeys. 2. Changes in sugars and free amino acid contents. Journal of Apicultural Research, 15(1), 29-34.

122

Wootton, M., Edwards, R. A., & Faraji-Haremi, R. 1978, Effect of accelerated storage conditions on the chemical composition and properties of Australian honeys. 3. Changes in volatile components. Journal of Apicultural Research, 17(3), 167-172.

Yao, L., Bhandari, B., D’Arcy, B., & Caffin, N. 2002, Honey business: two common properties of honey. Food Technology International, 133-134, 136-137.

Yao, L., Bhandari, B., Datta, N., Singanusong, R., & D’Arcy, B. 2003, Crystallisation and moisture sorption properties of selected Australian unifloral honeys. Journal of the Science of Food and Agriculture, 83, 884-888.

High-power Ultrasound to Control of Honey Crystallisationeffect on flavour, HMF) and remove the need...

Documents

Transcript of High-power Ultrasound to Control of Honey Crystallisationeffect on flavour, HMF) and remove the need...