optimization, characterization and applications of glucose oxidase ...

1

OPTIMIZATION, CHARACTERIZATION AND

APPLICATIONS OF GLUCOSE OXIDASE PRODUCED

FROM ASPERGILLUS AWAMORI MTCC 9645 FOR FOOD

PROCESSING AND PRESERVATION

A THESIS

Submitted by

P. SATHIYA MOORTHI, M.Sc.,

for the award of the degree

of

DOCTOR OF PHILOSOPHY

DEPARTMENT OF INDUSTRIAL BIOTECHNOLOGYDr. M.G.R.

EDUCATIONAL AND RESEARCH INSTITUTEUNIVERSITY

(Declared U/S 3 of the UGC Act 1956)

CHENNAI 600 095

AUGUST 2009

2

Dr. M.G.R.EDUCATIONAL AND RESEARCH INSTITUTE

UNIVERSITY(Declared U/S 3 of the UGC Act 1956)

CHENNAI 600 095

BONAFIDE CERTIFICATE

Certified that this thesis entitled “OPTIMIZATION,

CHARACTERIZATION AND APPLICATIONS OF GLUCOSE

OXIDASE PRODUCED FROM ASPERGILLUS AWAMORI MTCC

9645 FOR FOOD PROCESSING AND PRESERVATION” is the

bonafide work of Mr. P. SATHIYA MOORTHI, who carried out the

research under our supervision. Certified further that to the best of our

knowledge the work reported herein does not form part of any other

thesis or dissertation on the basis of which a degree or award was

conferred on an earlier occasion of this or any other candidate.

SIGNATUREDr. M. DEECARAMAN(CO-SUPERVISOR)Dean,Dept. of Industrial Biotechnology,Dr.M.G.R. Educational and Research Institute,Maduravoyal, Chennai-600 095.

SIGNATUREDr. P.T. KALAICHELVAN(SUPERVISOR)Professor,Centre for Advanced Studies in Botany,University of Madras, Guindy Campus,Chennai-600 025.

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DECLARATION

I declare that the thesis entitled “OPTIMIZATION,

CHARACTERIZATION AND APPLICATIONS OF GLUCOSE

OXIDASE PRODUCED FROM ASPERGILLUS AWAMORI MTCC

9645 FOR FOOD PROCESSING AND PRESERVATION” submitted

by me for the degree of Doctor of Philosophy is the record of work

carried out by me during the period from January 2006 to July 2009

under the supervision of Dr. P.T. Kalaichelvan, Professor, Centre for

Advanced Studies in Botany, University of Madras, Guindy Campus,

Chennai and the co-supervision of Dr. M. Deecaraman, Dean,

Department of Industrial Biotechnology, Dr. M.G.R. Educational and

Research Institute University, Maduravoyal, Chennai and has not formed

the basis for the award of any degree, diploma, associate-ship, fellowship

and titles in this or any other University or other similar institution of

Higher learning.

Signature of the candidate

(P. SATHIYA

MOORTHI)

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ACKNOWLEDGEMENT

I sincerely submit my respectful regards to our Chancellor Thiru

A.C. Chanmugam, BA., B.L., and Pro-Chancellor Er. A.C.S. Arunkumar

Dr.M.G.R. Educational and Research Institute University, Maduravoyal, Chennai

with whose blessing this work has been completed successfully.

I express my gratitude to our Vice-Chancellor Dr. K. Padmanabhan and

Vice-President (Academic) Dr. P.T. Manogaran, Dr.M.G.R. Educational and

Research Institute University, Maduravoyal, Chennai for their valuable advice.

I am extremely thankful and deeply indebted to my research supervisor

Dr. P.T. Kalaichelvan, Professor, Centre for Advanced Studies in Botany,

University of Madras, Chennai for his valuable suggestions, constructive criticism,

unstinted encouragement and care on me throughout my tenure.

I would like to place my deep sense of indebtedness to my co-supervisor

Dr. M. Deecaraman, Dean, Department of Industrial Biotechnology, Dr.M.G.R.

Educational and Research Institute University, Maduravoyal, Chennai for his

invaluable guidelines, suggestions and support through out this work.

I express my sincere gratitude to Dr. Rama vaidyanathan, HoD, Department

of Industrial Biotechnology, Dr.M.G.R. Educational and Research Institute

University, Maduravoyal, Chennai for providing laboratory facilities and her constant

encouragement to motivation of research.

I probably express my sincere thanks, Dr. M. Vijayalakshmi, Deputy HoD,

Department of Industrial Biotechnology, Dr.M.G.R. Educational and Research

Institute University, Maduravoyal, Chennai for her constant advice and motivation

thought my research.

I express my gratitude to Dr. P. Aravindan, Dean Research, Dr.M.G.R.

Educational and Research Institute University, Maduravoyal, Chennai for his constant

support and encouragement throughout the research.

I express my sincere thanks to Dr. S. Senthilvelan, Dean, Engineering and

Technology, Dr.M.G.R. Educational and Research Institute University, Maduravoyal,

Chennai for his valuable suggestions.

I would like to express my sincere thanks to Dr. N. Padmanaban, HoD,

Department of Chemical Engineering, Dr.M.G.R. Educational and Research Institute

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University, Maduravoyal, Chennai for his expert comments, valuable suggestions and

constant support throughout the research.

I would like to express my sincere thanks to Dr. V. Cyril Raj, HoD,

Department of Computer Science and Engineering, Dr.M.G.R. Educational and

Research Institute University, Maduravoyal, Chennai for his motivation and constant

support throughout the research.

I probably express my sincere thanks to Dr. R. Rengasamy, Professor and

Director, Centre for Advanced Studies in Botany, University of Madras, for his expert

commends and valuable suggestions throughout this research.

I express my sincere thanks to Dr. B.P.R. Vital, Professor, Centre for

Advanced Studies in Botany, University of Madras, for helping in fungal

identification and constant support throughout the research.

I would like to thank gratefully Er. Ap. Prabhaker, Managing Director,

Chinnu Exports, Chennai, for his constant support.

I would be failing in my duty if I forget the help rendered by my research lab

friends at Department of Industrial Biotechnology, Dr.M.G.R. Educational and

Research Institute University, Maduravoyal, Chennai.

I heartfelt thank to my research friends at Centre for Advanced Studies in

Botany, University of Madras, Chennai.

I also extend my thanks to the teaching and non-teaching staff of the

Department of Industrial Biotechnology, Dr.M.G.R. Educational and Research

Institute University, Maduravoyal, Chennai, for their kind co-operation.

My heartfelt thanks to all my friends, who have support and encourage me

during the research period.

Words are inadequate to thank my beloved father, mother and sister always

been with me in all my endeavors.

(P. SATHIYA MOORTHI)

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TABLE OF CONTENTS

CHAPTER No. TITLEPAGE

No.

ABSTRACT

LIST OF TABLES

LIST OF FIGURES

LIST OF SYMBOLS, ABBREVIATIONS AND

NOMENCLATURE

vi

viii

x

xiv

1. INTRODUCTION 1–4

2. LITERATURE SURVEY 5–52

2.1 Glucose oxidase production 9

2.2 Media optimization 10

2.3 Natural occurrence of GOx and its applications 10

2.4 Food processing additive 11

2.4.1 Bread making 11

2.4.2 Dry egg powder 12

2.4.3 Antioxidant/preservative (oxygen scavenger) 13

2.4.4 GOx in sea foods 14

2.4.5 Dairy and the lactoperoxidase system (LPS) 14

2.4.6 Reduced alcohol wine 16

2.4.7 Gluconic acid production 17

2.4.8 Glucose sensor/assay 18

2.4.9 Fuel cell 19

2.4.10 Other uses of GOx 20

2.5 Vegetable and food processing 22

2.5.1 Chlorination 22

2.5.2 Chlorine dioxide 22

2.5.3 Biodegradable Packaging 23

2.5.4 Edible coatings and films 24

2.5.4.1 Cellulose 26

7

2.5.4.2 Alginate 26

2.5.4.3 Zein 27

2.5.4.4 Banana powder 27

2.6 Antimicrobial agents in film coating 27

2.6.1 Ethylene diamine tetra acetic acid (EDTA) 28

2.6.2 Bacteriocin 29

2.6.3 Lysozyme 30

2.7 Microbial contaminates in vegetable products 31

2.8 Spices and edible oil in film making 32

2.9 Apple puree 32

2.9.1 Glucose oxidase-catalase system 33

2.9.2 Lactoperoxidase 34

2.10 Minimally processed fruits and vegetables 36

2.11 Reasons for quality changes in minimally processed produce 36

2.12 Methods to improve the shelf life and safety of minimally processed

produce

37

2.13 Microbial spoilage of vegetables and fruits 38

2.14 Pesticides as a source of microbial contamination of salad vegetables 43

2.15 Cut vegetable and fruit preservation techniques in practice 43

2.15.1 Combination preservation 45

2.15.2 Chemical preservative agents 46

2.16 Calcium ions on vegetables and fruits 47

2.16.1 Calcium sources to maintain the shelf-life of fresh vegetables

and fruits

48

2.17 Koruk juice 50

2.18 Effect of blanching 51

2.19 Cold storage 51

2.20 Economic loss 52

8

3. OBJECTIVE OF THE PRESENT WORK 53

4. MATERIALS AND METHODS 54–99

4.1 General 54

4.1.2 Sterilization 54

4.1.3 Chemicals 54

4.2 ISOLATION AND SCREENING OF GLUCOSE OXIDASE

PRODUCING FUNGI AND OPTIMIZATION OF MEDIUM

54

4.2.1 Isolation of GOx producing fungi 54

4.2.2 Screening of GOx producing fungi 55

4.2.3 Identification of fungi 56

4.2.4 Preparation of spore suspension 56

4.3 Analytical methods 57

4.3.1 Assay of GOx activity 57

4.3.2 Estimation of protein 59

4.3.3 Estimation of fungal biomass 60

4.3.4 Analysis of glucose 60

4.3.5 Analysis of gluconic acid 60

4.4 Culture condition for GOx production 61

4.4.1 Selection of suitable medium for GOx production 61

4.5 Media optimization 63

4.5.1 Single factor analysis (SFA) for GOx production 63

4.5.1.1 Effect of carbon source on GOx production 63

4.5.1.2 Effect of nitrogen sources on GOx production 63

4.5.1.3 Effect of Di-ammonium hydrogen phosphate,

potassium di-hydrogen phosphate and magnesium

sulphate on GOx production

63

4.5.1.4 Effect of calcium carbonate supplementation on GOx

production

64

4.5.1.5 Effect of pH and temperature on GOx production 64

4.5.1.6 Effect of fermentation time on GOx production 64

4.5.2 Statistical optimization by Response Surface Methodology 64

4.5.2.1 Experimental design of RSM for optimization of 65

9

media components

4.5.3 Modified composition of production medium GOxM 3 69

4.6 Production of GOx by laboratory batch fermentor 70

4.7 Morphological studies 70

4.7.1 Quantification and qualification of different types of

bioparticles

70

4.7.2 Large pellets (>3 mm diameter) 71

4.7.3 Small bioparticles (< 3 mm diameter) 71

4.7.4 Measurement and calculations 71

4.7.5 Time course study on cell growth, GOx production, substrate

utilization and acid formation during fermentation period

72

4.8 Purification and characterization of GOx 72

4.8.1 Preparation of enzyme for purification 72

4.8.2 Dialysis 72

4.8.3 Lyophilization 73

4.8.4 Ion exchange chromatography 73

4.8.5 Size exclusion chromatography 73

4.9 Polyacrylamide Gel Electrophoresis (PAGE) 74

4.9.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis 74

4.9.2 Native polyacrylamide gel electrophoresis 77

4.9.2.1 Zymogram analysis 79

4.9.3 Confirmation of enzyme activity using plate assay 79

4.10 Characterization of glucose oxidase 80

4.10.1 Kinetic charecterization 80

4.10.2 Effect of temperature and pH on GOx activity 80

4.10.3 Stability testing 80

4.10.4 Stability and inhibitory studies of GOx 81

4.10.4.1 Effect of metal ions on GOx activity 80

4.10.4.2 Effect of calcium ions on GOx activity 80

4.10.5 Preparation of carrier based enzyme 81

4.11. APPLICATION OF GLUCOSE OXIDASE IN FOOD


82

4.11.1 Enhancing the storage stability of vegetable by coating of 82

10

edible film incorporated with glucose oxidase,

lactoperoxidase and lysozyme

4.11.1.1 Microorganisms and culture conditions 82

4.11.1.2 Preparation and analysis of antimicrobial enzymes 82

4.11.1.3 Evaluation of antimicrobial activity of GOx, LPS

and lysozyme with EDTA

85

4.11.1.4 Film making 85

4.11.1.5 Antimicrobial film activity 86

4.11.1.6 Surface sterilization of carrots 86

4.11.1.7 Coating procedure 87

4.11.1.8 Determination of weight loss 87

4.11.1.9 Determination of soluble protein content 88

4.11.1.10 Microbial analysis by viable plate count method 88

4.11.1.11 Sensory analysis 88

4.11.2 Control of browning and enhancing shelf-life of apple

puree by applying glucose oxidase-catalase system with

lactoperoxidase

89

4.11.2.1 Preparation and analysis of antimicrobial enzymes 89

4.11.2.2 Determination antimicrobial activity of GOx, LPS

and catalase

90

4.11.2.3 Preparation of apple puree 90

4.11.2.4 Effect of GOx and ascorbic acid on dissolved

oxygen consumption

91

4.11.2.5 Experimental design 91

4.11.2.6 Evaluation of browning on apple puree 91

4.11.2.7 Examination of microbial populations 92


4.11.3 Studies on the effect of glucose oxidase-catalase with

calcium ions in stabilizing and improving the fruit salad

quality

93

4.11.3.1 Preparation of enzyme 93

4.11.3.2 Collection of fruits 93

4.11.3.3 Preparation of koruk juice 93

11

4.11.3.4 Analysis of antimicrobial activity of GOx, calcium

ions and koruk juice

93

4.11.3.5 Preparation of treatment solution 94

4.11.3.6 General fruit salad preparation procedure 94


4.11.3.8 Experimental design of RSM for optimization of

calcium ions

96

4.11.3.9 Effect of optimized calcium ions on fruit salad

preparation

98

4.11.3.10 Measurement of weight loss 98

4.11.3.11 Microbial analysis of fruit salad by viable plate

count method

98

4.12 Statistical analysis 99

5. RESULTS AND DISCUSSION 100–197

5.1 ISOLATION, MEDIA OPTIMIZATION AND PRODUCTION

OF GLUCOSE OXIDASE

100

5.1.1 Isolation of GOx producing fungi 100

5.2 Optimization of medium for glucose oxidase production 104

5.2.1 Single Factor Analysis (SFA) for GOx production 104

5.2.1.1 Effect of carbon source on GOx production 104

5.2.1.2 Effect of nitrogen sources on GOx production 106

5.2.1.3 Effect of di-ammonium hydrogen phosphate,

potassium di-hydrogen phosphate and magnesium

sulphate on GOx production

106

5.2.1.4 Effect of calcium carbonate on GOx production 109

5.2.1.5 Effect of pH on GOx production 109

5.2.1.6 Effect of temperature on GOx production 111

5.2.1.7 Effect of fermentation time on GOx production 111

5.2.2 Response surface methodology for GOx production 113

5.2.2.1 SET 1: Optimization of glucose, proteose peptone

and calcium carbonate for GOx production

113

5.2.2.2 SET 2: Optimization of di-ammonium hydrogen

phosphate, potassium di-hydrogen phosphate and

116

12

magnesium sulphate for GOx production

5.2.2.3 SET 3: Optimization of pH and temperature 126

5.3 Production of glucose oxidase by laboratory batch fermentor 130

5.3.1 Spore aggregation and pellet formation during the early

cultivation time

130

5.3.2 Morphological studies 133

5.3.3 Time course study on cell growth, substrate utilization, GOx

and gluconic acid production during fermentation period

133

5.4 Purification and characterization of glucose oxidase produced

from Aspergillus awamori MTCC 9645

136

5.4.1 DEAE-Cellulose column chromatography 136

5.4.2 Sephacryl S-200 column chromatography 136

5.4.3 Molecular mass determination of purified GOx by SDS-PAGE 140

5.4.4 Purified GOx activity on native-PAGE 140

5.4.5 Confirmation of enzyme activity using plate assay 140

5.4.6 Kinetic characterization of GOx 143

5.4.7 Effect of temperature and pH on GOx activity 143

5.4.8 Stability testing 143

5.4.9 Stability and inhibitory activity of GOx 148

5.4.9.1 Effect of metal ions on GOx activity 148

5.4.9.2 Effect of calcium ions on GOx activity (1mM) 149

5.4.9.3 Effect of calcium ions on pH stability of GOx 149

13

5.4.9.4 Effect of calcium ions on temperature stability of

GOx (1mM)

149

5.5 APPLICATION OF GLUCOSE OXIDASE IN FOOD


152

5.5.1 Edible films incorporated with glucose oxidase,

lactoperoxidase and lysozyme for carrot preservation

152

5.5.1.1 Assay of antimicrobial enzymes 152

5.5.1.2 Evaluation of antimicrobial activity of GOx, LPS and

lysozyme with EDTA

152

5.5.1.3 Antimicrobial activity of alginate film 154

5.5.1.4 Surface sterilization of carrot 156


5.5.1.6 Measurement of soluble protein content 160

5.5.1.7 Enumeration of bacterial population from treated and

control carrots

160


5.5.2 Control of browning and enhancing the shelf-life of apple

puree by glucose oxidase, catalase and lactoperoxidase

164

5.5.2.1 Assay of antimicrobial enzymes 164

5.5.2.2 Evaluation of antimicrobial activity of GOx, catalase

and LPS

164

5.5.2.3 Effect of GOx and ascorbic acid on removal of

dissolved oxygen from apple puree

166

5.5.2.4 Effect of GOx, catalase, LPS and ascorbic acid on

controlling of browning in apple puree

167

5.5.2.5 Examination of microbial populations 171


14

5.5.3 Studies on the effect of glucose oxidase-catalase with

calcium ions for the improvement of fruit salad quality

173

5.5.3.1 Evaluation of antimicrobial activity of GOx, catalase

calcium ions and koruk juice

173


5.5.3.3 Sensory analysis of GOx-catalase treated fruit salad 176

5.5.3.4 Sensory analysis of calcium chloride treated fruit salad 176

5.5.3.5 Sensory analysis of calcium propionate treated fruit

salad

176

5.5.3.6 Sensory analysis of calcium lactate treated fruit salad 178

5.5.3.7 Sensory analysis of koruk juice treated fruit salad 178

5.5.3.8 Optimization of calcium ions concentrations by

response surface methodology

182

5.5.3.9 Effect of RSM optimized combined of calcium ions,

GOx-catalase and koruk juice on fruit salad

194


5.5.3.11 Enumeration of bacterial population from treated

and control fruit salads

197

6. SUMMARY 199–202

7. CONCLUSIONS AND SCOPE FOR FUTURE WORK 203–204

REFERENCES i–xxviii

LIST OF PUBLICATIONS, PRESENTATIONS AND CONFERENCES xxix–xxxi

CURRICULUM VITAE xxxii

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ABSTRACT

The glucose oxidase (GOx) enzyme has considerable industrial

importance and used in different food products (dried egg, bread,

beverages and vegetables) for processing and preservation. Among the

different fungi isolated from various sugar rich products, Aspergillus

awamori MTCC 9645 was found to be produce high amount of GOx. The

compositions of production medium were optimized by Single Factor

Analysis (SFA) and a statistical tool Response Surface Methodology

(RSM). Glucose and proteose peptone were found to be a good carbon

and nitrogen source for maximum GOx production respectively. Addition

of di-ammonium hydrogen phosphate, potassium di-hydrogen phosphate

and magnesium sulphate supports the GOx production. Remarkably GOx

production was increased in the presence of 35–40 g/l calcium carbonate.

The pH 5.0–6.0 and the temperature between 30 and 35ºC were found to

be optimum for GOx production. The cell free extract was purified and

characterized. Enzyme stability studies were carried out with different

calcium ions in which calcium lactate was found to be effective for

stabilizing GOx activity. Metal ions such as mercuric chloride, copper

sulphate, and silver nitrate were significantly inhibit the activity of GOx.

The enzyme was subjected for the processing and preservation of food

products such as vegetable, apple puree and fruit salad. Alginate film

16

coated carrot with the formulation of GOx and lactoperoxidase (LPS),

lysozyme and EDTA exhibited good shelf-life. Apple puree was

processed with the combination of GOx-catalase with LPS exhibited

good antibrowning and antibacterial efficacy. The combination of GOx-

catalase with calcium ions showed antibacterial and antibrowning activity

in salad that increased the storage stability and also high sensory score.

The results from the present findings were clearly revealed that potential

of GOx would use as processing and preservation of food products.

Key words: Apple puree, Aspergillus awamori (MTCC 9645); Carrot;

Glucose oxidase; Optimization, Preservation; RSM; Salad;

Vegetable process.

17

LIST OF TABLES

TABLE No. TITLE OF TABLE

Table 2.1 Requirements for the commercial manufacture of pre-peeled and/or

sliced, grated or shredded fruit and vegetables

Table 4.1 Different production medium for GOx

Table 4.2 Set 1 Optimization of glucose, proteose peptone and calcium

carbonate for GOx production

Table 4.2.1 Design summary

Table 4.2.2 Experimendal design and of 23 factorial design

Table 4.3 Set 2 Optimization of (NH4)2HPO4, KH2PO4 and MgSO4 for GOx

production



Table 4.4 Set 3 Optimization of pH and temperature for GOx production



Table 4.5 Optimization of calcium ions for salad preparation


Tanle 4.5.2 Experimendal design and results of 23 factorial design

Table 5.1 Glucose oxidase production by different fungi

Table 5.2 Set 1 Optimization of glucose, proteose peptone and calcium

carbonate for the GOx production by CCD of response surface

methodology (23 factorial design)

Table 5.2.1 F-test analysis ( ANOVA for Response Surface Quadratic Model)

Table 5.2.2 Comparition of R2 predicted and estimated

Table 5.2.3 Model coefficient estimated by linear regression

Table 5.3 Set 2 Optimization of (NH4)2HPO4, KH2PO4 and MgSO4 for the GOx

production by CCD of response surface methodology

(23 factorial design)

Table 5.3.1 F-test analysis (ANOVA for Response Surface Quadratic Model)



18

Table 5.4 Set 3 Optimization of pH and temperature for the GOx production by

CCD of response surface methodology (23 factorial design)




Table 5.5 Spore aggregation and pellet formation during the early cultivation

time in the laboratory batch fermentor

Table 5.6 Summary of purification of GOx from A. awamori MTCC 9645

Table 5.7 Bacterial counts (CFU/ml± SD) on surface washing carrot treated

with chlorine dioxide at different concentration and time of exposure

Table 5.8 Soluble protein content (mg/g of dry wt ± SD) in alginate coated

(Formulation I) and uncoated control carrots.

Table 5.9 Optimization of calcium ions for salad preparation by CCD of

response surface methodology (23 factorial design)




Table 5.9.4 Comparison of R2 predicted and estimated


19

LIST OF FIGURESFIGURE No. TITLE OF FIGURES

Figure 2.1 Enzymatic conversion of glucose to gluconic acid by GOx

Figure 2.2 3-Dimentional structure of glucose oxidase

Figure 5.3 Isolation, screening and identification of A. awamori (GOP-3)

Figure 5.4: Isolation, screening and identification of A. awamori (GOP-7) MTCC 9645

Figure 5.5 Effect of different media for GOx production

Figure 5.6 Effect of carbon sources on GOx production

Figure 5.7 Effect of different nitrogen sources for the GOx production

Figure 5.8 Effect of di-ammonium hydrogen phosphate on GOx production

Figure 5.9 Effect of potassium di-hydrogen phosphate and magnesium sulphate of

GOx production

Figure 5.10 Effect calcium carbonate on glucose oxidase production

Figure 5.11 Effect of pH on GOx production

Figure 5.12 Effect of temperature on GOx production

Figure 5.13 Effect of fermentation time on GOx production and cell growth

Figure 5.14 The contour and 3D response surface plot showing the effect of glucose and

calcium carbonate on GOx production

Figure 5.15 The contour and 3D response surface plot showing the effect of glucose and

peptone on GOx production

Figure 5.16 The contour and 3D response surface plot showing the effect of peptone and

calcium carbonate on GOx production

Figure 5.17 The contour and 3D response surface plot showing the effect of

(NH4)2HPO4 and KH2PO4 on GOx production

Figure 5.18 The contour and 3D response surface plot showing the effect of

(NH4)2HPO4 and MgSO4 on GOx production

Figure 5.19 The contour and 3D response surface plot showing the effect of KH2PO4

and MgSO4 on GOx production

Figure 5.20 The contour and 3D response surface plot showing the effect of pH and

temperature on GOx production

Figure 5.21 Production of GOx in A. awamori MTCC 9645 in laboratory bioreactor

Figure 5.22 Time course study on substrate utilization, production of gluconic acid and

biomass during the fermentation

20

Figure 5.23 Time course study on production of GOx and protein during the

fermentation

Figure 5.24 Flow chart for the purification and characterization of extracellular GOx of

A. awamori MTCC 9645

Figure 5.25 Purification of GOx from A. awamori MTCC 9645

Figure 5.25(a) Elution profile on DEAE column chromatography

Figure 5.25 (b) Elution profile on Sephacryl S-200 column chromatography

Figure 5.26 Molecular mass determination on SDS-PAGE (10%) of GOx from A.

awamori MTCC 9645

Figure 5.27(b) Zymogram of GOx (Iso enzyme patterning) on native-PAGE (8%)

developed with horse radishproxidase, o-dianisidine and glucose

Figure 5.27(b) Confirmation of GOx from A. awamori MTCC 9645 by plate assay

Figure 5.28 Kinetic parameters (apparent Km and Vmax) for purified GOx of A. awamori

MTCC 9645 by Lineweaver's Burk plot


MTCC 9645 by Eadie-Hofstee plot


MTCC 9645 by Hanes plot

Figure 5.31 Effect of temperature and pH on GOx activity

Figure 5.32 Effect of temperature stability of purified GOx

Figure 5.33 Effect of metal ions (1mM) on GOx activity

Figure 5.34 Effect of calcium ions (1mM) on GOx activity

Figure 5.35 Effect of calcium ions (1mM) on pH stability of GOx activity

Figure 5.36 Effect of calcium ions (1mM) on temperature stability of GOx

Figure 5.37 Effect of antimicrobial activity of GOx, LPS and lysozyme with EDTA

against E. coli

Figure 5.38 Effect of antimicrobial activity of GOx, LPS and lysozyme with EDTA

against S. aureus

21

Figure 5.39 Effect of antimicrobial alginate films incorporated with partially purified

enzymes of different formulations

Figure 5.40

(a&b)

Effect of GOx, LPS and lysozyme incorporated with alginate film coated

carrots stored at (a) Stored at ~26°C and (b) 6°C

Figure 5.41

(a&b)

Effect of alginate coated (Formulation VII) and uncoated carrots stored on

weight loss (%) during the storage period at (a) 6°C (b) and (b) ~26ºC

Figure 5.42 Showed the microbial population (10-6 dilution) the treated and control

carrots after the storage period (10 d) stored at 6ºC ~26ºC

Figure 5.43

(a&b)

Evaluation of the sensory profile of treated and control carrots after the

storage period (10 d) stored at (a) 6ºC and (b) ~26ºC

Figure 5.44

(a&b)

Effect of antimicrobial activity of GOx, catalase and LPS against (a) E. coli

and (b) S. aureus

Figure 5.45 Effect GOx and ascorbic acid on removal of dissolved oxygen from apple

puree

Figure 5.46 Effect GOx, catalase, LPS and ascorbic acid on controlling of browning in

apple puree

Figure 5.47

(a&b)

Effect GOx and ascorbic acid on controlling of browning in (a) Cut apple

and (b) Apple puree

Figure 5.48 Showed the microbial population of treated and control apple puree

Figure 5.49 Evaluation of the sensory profile of treated and control apple puree

Figure 5.50

(a&b)

Effect of antimicrobial activity of GOx, calcium ions and koruk juice

against (a) E. coli and (b) S. aureus

Figure 5.51

(A–G)

Effect of calcium ions, koruk juice and GOx on controlling browning in cut

(a) apple, (b) pomegranate and (c) guava

Figure 5.52

(a&b)

Evaluation of the sensory profile of different concentration of (a) GOx and

(b) calcium chloride treated and control fruit salad

Figure 5.53

(a&b)

Evaluation of the sensory profile of different concentration of (a) calcium

propionate and (b) calcium lactate treated and control fruit salad

Figure 5.54 Evaluation of the sensory profile of different concentration of koruk juice

treated and control fruit salad

Figure 5.55

(a&b)

Effect of GOx and koruk juice on fruit salad

22

Figure 5.56 The contour and 3D response surface plot showing the weight loss % of

calcium lactate and calcium propionate treatment on fruit salad


calcium propionate and calcium chloride treatment on fruit salad


calcium chloride and calcium lactate treatment on fruit salad

Figure 5.59 The contour and 3D response surface plot showing the overall sensory

acceptability profile of calcium propionate and calcium lactate treatment on

fruit salad


acceptability profile of calcium propionate and calcium chloride treatment

on fruit salad


acceptability profile of calcium lactate and calcium chloride treatment on

fruit salad

Figure 5.62 Effect of RSM optimized calcium ions on fruit salad

Figure 5.63 Evaluation of the sensory profile of combined calcium ions (obtained from

RSM) and GOx-catalase and koruk juice treated and control fruit salad

Figure 5.64

(a,b&c)

Effect of GOx-catalase, calcium ions and koruk juice on fruit salad

Figure 5.65 Effect of GOx, calcium ions and koruk juice for controlling weight loss on

fruit salad

Figure 5.66 Enumeration of microbial population of different treated and control fruit

salad

23

LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE

$ Dollar

% Percentage

° Degree

°C Degree Celsius

°F Degree Fahrenheit

~ Tilde

< Less than

> Greater than

∆ Delta

µ Micro

µl Microlitre

A. awamori Aspergillus awamori

A. niger Aspergillus niger

ANOVA Analysis of variance

B. amyloliquefaciens Bacillus amyloliquefaciens

CBB Coomassie brilliant blue

CCD Central composite design

CFU Colony forming unit

cm Centimeter

CMC Carboxy methylcellulose

CT Cholera toxin

d Day

D Dolton

Df Dilution factor

dl Decilitre

DNA Deoxyribonucleic acid

DO Dissolved oxygen

E. coli Escherichia coli

E. faecium Enterococcus faecium

EAEC Enteroaggregative E. coli

24

EAEC Diffusely adherent E. coli

EAF EPEC Adherence factor

EDTA Ethylene diamine tetra acetic acid

EHEC Enterohemorrhagic E. coli

EIEC Enteroinvasive E. coli

EPEC Enteropathogenic E. coli

ETEC Enterotoxigenic E. coliFAD Flavine adenine dinucleotide

FDA Food and drug administration

g Gram

g Gravitational force

GOx Glucose oxidase

GP Gas permeability

GRAS Generally regarded as safe

h Hour

HOSCN Hypothiocyanous acid

HPMC Hydroxypropyl methylcellulose

IU International unit

kDa Kilo dalton

Kg Kilogram

Km Michaelis constant

L Litre

L. monocytogenes Listeria monocytogenes

LAB Lactic acid bacteria

LB Luria Bertani

LPS Lactoperoxidase

Ltd Limited

Lyz Lysozyme

M Molar

mA Milliamps

MAP Modified atmosphere packaging

mbar Millibar

25

MC Methylcellulose

MEA Malt extract agar

mg Milligram

min Minutes

ml Millilitre

mM Millimolar

mm Millimeter

MPV Minimally processed vegetables

MTCC Microbial type culture collection

N Normal

nm Nano meter

OP Oxygen permeability

OSCN- Hypothiocyante

P Probability

P. amagasakiens Penicillum amagasakiens

P. glaucum Penicillum glaucum

P. notatum Penicillum notatum

P. variabile Penicillum variabile

PAGE Polyacrylamide gel electrophoresis

PBS Phosphate buffer solution

PCR Polymerase chain reaction

PDA Potato dextrose agar

PEF Pulsed electric fields

pH Potential hydrogen ion concentration

POD Peroxidase

ppm Parts per million

psi Pound per square inch

rpm Revolution per minute

RSM Response surface methodology

S. aureus Staphylococcus aureus

S. cerevisiae Saccharomyces cerevisiae

S. enteritidis Salmonella enteritidis

S. typhimurium Salmonella typhimurium

26

SCN− Thiocyanate

SDS Sodium dodecyl sulphate

SFA Single factor analysis

t Time

TDM Total dry mass

TEMED Tetramethyl ethylene diamine

TMB Tetramethylbenzidine

U Unit

UHP Ultra high pressure

US United states

UV Ultra violet

V. vinifera Vitis vinifera

v/v Volume per volume

Vmax Maximal limiting rate velocity

VP Vacuum packaging

W(t) Sample weight at time t

w/v Weight per volume

W0 Initial sample weight

WL Weight loss

WVP Water vapour permeability

X Magnification unit

α Alpha

β Beta

δ Delta

27

CHAPTER-1

INTRODUCTION

Enzymes possess very diverse specificity, reactivity and other

physiochemical, catalytic and biological properties highly desirable for various

industrial and medical applications. The use of enzymes, either alone or as part

of live cells, can be traced back to the dawn of civilization. Now, enzymes are

being rigorously and systematically developed as economically viable and

environment-friendly industrial biocatalysts, along with the fast advancement

and expansion of modern biotechnology (Feng Xu, 2005).

Current industrial enzyme sales are around $2 billion per year with >500

products for >50 major applications (Kirk et al., 2002). Although being a very

small part of the whole speciality chemicals market, the industrial enzyme field

is projected to grow fast, at a rate close to double digits annually for the near

future. Based on application, ~65% of the commercial enzymes are technical

enzyme applications such as detergent, textile and starch, ~25% are food

enzymes and ~10% are feed enzymes.

The majority of the commercial enzymes are hydrolases, while

oxidoreductase account for a miniscule share but it is in contrast to the high

occurrence of oxidoreductase in nature (Kirk et al., 2002; Burk, 2003). The gap

between vast natural oxidoreductase products creates the space and potential

for developing more oxidoreductase based biocatalysts. Currently, the

industrial-technical, food and environmental applications are the only markets

with a significant oxidoreductase commercialization although; it remains very

less in comparison to that of hydrolytic industrial enzymes. Further, improving

the cost competitiveness of existing oxidoreductase products and enhancing the

innovation effort in applying oxidoreductases to new field are vital for the

28

future growth of industrial oxidoreductase biocatalysts (Feng Xu, 2005).

Today, enzymes are used for an increasing range of food applications: bakery,

cheese making, starch processing and production of fruit juices and other

drinks. One such enzyme is glucose oxidase (GOx), which is used on food

industries, as an oxygen and glucose scavenger from foods and beverages

(Sathiya moorthi et al., 2007).

Glucose oxidase (EC 1.1.3.4) belongs to class oxidoreductase and

catalyzes the oxidation of β-D-glucose to gluconic acid, utilizing molecular

oxygen as an electron acceptor with the simultaneous production of hydrogen

peroxide. It is a dimeric protein composed of two identical subunits, each

subunit, or monomer, binds into domain: one domain binds to the substrate

glucose while the other domain binds non-covalently to a cofactor flavin

adenine dinucleotide (FDA).

The success of Aspergillus niger group for industrial production of

biotechnological products is largely due to the metabolic versatility of this

strain. A. niger is well known to produce a variety of enzymes, organic acids,

plant growth regulators, mycotoxins and antibiotics. The industrial importance

of A. niger groups are not limited on its >335 native products but also on

development and commercialization of the new products which are derived by

modern bioprocess and molecular biology techniques.

Glucose oxidase was first isolated from mycelia of A. niger and

Penicillium glaucum by Muller (1928) and later they were detected in different

sources (insects, honey, algae and micro fungi) and recently was obtained from

Penicillum amagasakiense and especially from A. niger. The most common

sources of GOx were A. niger, P. notatum, P. glaucum, P. amagasakiense,

P. purpurogenum, P. variabile and Alternaria alternate (Caridis et al., 1991).

29

Recent researches are focused on factors that regulate the production of

GOx. There are several media constituents like carbon, nitrogen, CaCO3 and

environmental factors such as temperature, pH and agitation involved in the

production of GOx. The above factors have to be optimized for the effective

production of GOx. There are few reports concerned in the optimization of

cultural conditions for the production of GOx from various fungi (Sandip et al.,

2008). Optimization of media composition and other factors by using statistical

method have few advantages over the single factor analysis at a time. It can be

used for easy determination of important parameter from a large number of

factors and the study of the interactions between the variables of the media

constituents.

Glucose oxidase has considerable industrial importance and used for the

removal of trace amounts of oxygen or glucose from different sources such as

dried egg, beer, wine and fruit juices (Reed and Underkoer, 1966). It has been

used to remove residual glucose and oxygen in foods and beverages in order to

prolong their shelf- life. The hydrogen peroxide produced by the enzyme acts

as a good bactericide and can be later removed using catalase which converts

hydrogen peroxide to oxygen and water. GOx can also be used to remove

oxygen from the top of bottled beverages before they are sealed. The GOx was

also found to be antagonistic potential against different food borne pathogens

like Salmonella infantis, Staphylococcus aureus, Clostridium perfringens,

Bacillus cereus, Campylobacter jejuni and Listeria monocytogenes (Tiina and

Sandhlm, 1989). In the glucose detection kits, GOx act as a basis of glucose

sensor (Degani and Heller, 1988).

Minimally processed vegetables (MPV) sold in ready-to-eat (salads) or

ready-to use forms have become a very important area of potential economic

growth for fresh-cut vegetables and fruit industry. Today MPV products have

gained popularity mainly because consumers perceive such products, besides

30

their well-known nutritional qualities, as fresh, healthy, convenient, tasty and

easy to use.

Current technologies for preservation and shelf-life extension of food

include chemical preservatives, heat processing, modified atmosphere

packaging, vacuum packaging or refrigeration. However, these steps do not

eliminate undesirable pathogens from these products or delay microbial

spoilage entirely.

Alternative preservation techniques such as novel non-thermal

technologies and naturally derived antimicrobial ingredients are under

investigation for their application to food products. Alginate-based edible films

and calcium ions have the ability to limit moisture loss and enhancing shelf-life

of vegetables has been studied by the incorporation of various natural

antimicrobial agents.

A number of naturally occurring antimicrobial agents have been

investigated that includes GOx, LPS, lysozyme, lactoferrin, avidin, various

plant extracts such as spices and their essential oils, sulfur and phenolic

compounds (Davidson et al., 2001). Combinations of preservation treatments

allow the required level of protection to be achieved while at the same time

retaining the natural qualities of the product such as, colour, flavour, texture

and nutritional value.

31

CHAPTER-2

LITREATURE SURVEY

Utilization of highly selective oxidizing biocatalysts such as oxygenases

and oxidases is an emerging field of white biotechnology, since, it may lead to

an environment-friendly production of enantiomerically pure compounds

serving as valuable chiral synthons for subsequent chemical syntheses of

biologically active substances (Stottmester et al., 2005).

Glucose oxidase a flavoprotein which catalyses the oxidation of β-D-

glucose to D-glucono-δ-lactone and hydrogen peroxide using molecular

oxygen as the electron acceptor. The reaction can be divided into a reductive

and an oxidative step. In the reductive-half reaction, GOx catalyses the

oxidation of β-D-glucose to D-glucono-δ-lactone which can in certain fungi

such as Aspergillus sp., be enzymatically or spontaneously hydrolyzed to

gluconic acid. Subsequently, the flavine adenine dinucleotide (FAD) ring of

GOx is reduced to FADH2 (Witt et al., 2000). In the oxidative half reaction the

reduced GOx is re-oxidised by oxygen to yield hydrogen peroxide (Figure 2.1).

The kinetics, mechanism of action, properties and molecular structure of

GOx were studied by many researchers (Swoboda and Massey, 1965; Tsuge et

al., 1975; Takegawa et al., 1991; Hecht et al., 1993). GOx from A. niger is a

homodimer with a molecular weight of 150–180 kDa. It contains two tightly

bound FAD molecules (Pazur and Kleppe, 1964). Dissociation of the sub-units

only occurs under denaturation conditions and is accompanied by the loss of

the cofactor FAD (Jones et al., 1982). The amino acid sequence for the 583

residues protein has been derived from the DNA sequence independently by

Kriechbaum et al. (1989) and Frederick et al. (1990).

32

Figure 2.1: Enzymatic conversion of glucose to gluconic acid by GOx

33

The enzyme is highly specific for β-D-glucose with other

monosaccharides, being oxidized at much lower rate (Adams et al., 1969).

GOx from A. niger is a highly glycosylated protein, the carbohydrate content is

ranged from 10% to 24% of its molecular weight (Pazur et al., 1965; Hayashi

and Nakamura, 1981). The glycosylated protein contains 190 mannose and 16

N-acetyl glucosamine residues. Several functions have been proposed for the

carbohydrate moiety of glycoproteins including correct targeting or proteins,

transport through membranes, biological function, immune response and

stabilization of the three dimensional structure (Figure 2.2) of the protein

(Kalisz et al., 1991). In case of GOx, the deglycosylation did not significantly

affect the three dimensional structure of the enzyme. Other properties such as

thermal stability, pH and temperature optimum of GOx activity and substrate

specificity were not affected. Thus, the carbohydrate moiety of GOx, like that

of other glycoproteins, does not appear to contribute significantly to the

biological properties of enzyme (Kalisz et al., 1991). On the other hand, the

carbohydrate-depleted GOx was more rapidly precipitated by the addition of

trichloroacetic acid and ammonium sulfate than the native enzyme. These

results show that the N-linked sugar chains of GOx contribute to the high

solubility of the enzyme in water (Takegawa et al., 1989).

Glucose oxidase was suitable in large-scale technological application

since, 1950’s, which includes the enzymatic determination of glucose with

biosensor technology (Vodopivec et al., 2000) for the production of gluconic

acid and as a food preservative. Implantable glucose sensors may find

significant application for monitoring of glucose in diabetics (Gerritsen, 2001).

Enhancement of the properties of GOx is still receiving more attention,

presumably due to the current and extensive applications of this enzyme.

34

Overall topology of GOx homoenzyme

Subunit structure of GOx showing FAD (red space fill)

Figure 2.2: 3-Dimentional structure of glucose oxidase

35

2.1 Glucose oxidase production

The carbon sources used for GOx production ranged from simple C3

such as glycerol up to a complex carbon source such as starch. Glucose was

mainly used in all cultivation media with a concentration ranging from 40 to 80

g/l. However, in the industrial scale, sucrose or corn steep liquor can be used in

case of bulk production of GOx in non-purified form which is utilized in

gluconic acid production, food preservation and other non-analytical purposes.

Also, different organic and inorganic nitrogen sources were added to the

cultivation medium to enhance production. Peptone or polypeptone with a

concentration ranging from 3 to 20 g/l or yeast extract were the main common

organic nitrogen sources. On the other hand, sodium nitrate was mainly used as

inorganic nitrogen source for GOx production followed by ammonium di-

hydrogen phosphate. The source of phosphate was in the form of either

potassium di-hydrogen phosphate or di-potassium hydrogen phosphate with a

concentration ranging from 0.2 to 1.0 g/l reported by Nakamatsu et al. (1975).

Inorganic salts for supplementation with Mg++, Fe++, Zn++ and K+

cations were also added in low quantity. Moreover, either magnesium

carbonate or calcium carbonate were used in some publications in case of

cultivation in shake flask to neutralize the acidity of the cultivation medium

due to gluconic acid production which was concomitant with GOx production

on using glucose as carbon source. Nakamatsu et al. (1975) studied the effect

of different complex carbon sources as well as different nitrogen sources on

GOx production.

Zetelaki and Vas (1968) investigated the effect of aeration and agitation

on the GOx production by A. niger in a 5 l stirred tank bioreactor. They found

that the maximum enzyme production was recorded at 700 rpm. Further

increases in the agitation speed resulted in neither a higher growth rate nor

higher activity.

36

2.2 Media optimization

There are some advantages of using statistical methods, over the one-

factor-at-a-time classical method for optimization of media. However,

statistical design enables easy selection of important parameters from a large

number of factors and explains the interactions between important variables. A

number of statistical experimental designs were used for optimizing the

fermentation variables. The Plackett–Burman design (Plackett and Burman,

1946) is a well known and widely used statistical technique for screening and

selection of most significant culture variables, while the central composite

design (CCD) provides an important information regarding the optimum level

of each variable along with its interactions with other variables and their

effects on product yield (Pardeep and Satyanarayana, 2006).

2.3 Natural occurrence of GOx and its applications

Glucose oxidase is naturally produced by various fungi and insects. The

main function of GOx is to act as antibacterial and antifungal agent through the

production of hydrogen peroxide. Permanent oxidative stress through the

maintenance of hydrogen peroxide at low concentration by GOx’s continued

catalytic activity was reported by many researchers (Tiina and Sandholm 1989;

Dobbenie et al., 1995) to be very effective against bacterial or fungal growth.

Breakdown of hydrogen peroxide by the microorganism’s intrinsic

catalase may protect it against hydrogen peroxide’s antibacterial or antifungal

effect. Presence of catalase at millimolar level of hydrogen peroxide was

required to inhibit the cell growth. Whereas, in the presence of GOx, the

micromolar level of hydrogen peroxide was constantly maintained by the

catalytic activity of GOx was already sufficient to inhibit the cell growth.

Interestingly, natural functions of GOx include assisting in plant infection,

lignin degradation, lowering pH of the environment, etc., (Chun Ming Wong et

al., 2008).

37

Detailed below are many industrial and commercial applications where

GOx may be used in. The usefulness of GOx in diverse fields had triggered the

search for new sources of GOx from other species of fungi (including A. niger;

Hatzinikolaou et al. 1996) and insects to satisfy the demand for improved

properties such as higher catalytic activity (Fiedurek and Gromada, 1997). The

following discussion will attempt to cover the major applications together with

the respective working principle utilized.

2.4 Food processing additive

Glucose oxidase has Generally Regarded As Safe (GRAS) status under

FDA classification (FDA/CFSAN 2002 a, b) and is available in bulk for use in

food industry as an additive in liquid or powder form. It is often classified with

antioxidant, preservative and stabilizer properties. There are many food

products in which GOx can be used. Some of them are elaborated below, and a

detailed list can be found in the database of Codex Alimentarius Commission

(2007a). The food grade GOx preparation used typically contains a mixture of

GOx and catalase because the two enzymes are found naturally together in the

mycelium cell wall (Witteveen et al., 1992). Separation of GOx from catalase

is costly and not essential in food grade preparations. Furthermore, catalase

assists in the breakdown of hydrogen peroxide produced by GOx, thereby

reducing inhibition and deactivation by hydrogen peroxide (Bao et al., 2001

and Bao et al., 2003).

2.4.1 Bread making

Maturing/oxidizing agents are an essential additive to flour. One of its

purposes is to strengthen gluten, thereby improve the bread’s final texture. This

is achieved through the oxidation of two proteins within flour, gliadin and

glutenen to allow more bonds to form when gluten develops. Gluten forms

when gliadin and glutenen are in contact with water and its maturation is

assisted by the actions of yeast (Corriher, 2001). Only a small amount of

38

maturing agents in the level of parts per million is needed in this process and

traditionally, potassium bromate was used (Figoni, 2003). However, it has

been recognized that bromate is carcinogenic, causing DNA damage invitro

and invivo that may contribute towards cancer (Moore and Chen, 2006). As a

result, most countries have prohibited the use of bromate in food, and an

alternative such as GOx is used in bakery (Enzyme Technical Association,

2001).

Glucose oxidase is an effective oxidant to produce bread with improved

texture and increased loaf volume (Vemulapalli et al., 1998; Rasiah et al.,

2005). The basis of oxidation by GOx has been validated to be a result of the

hydrogen peroxide produced which yields dough that is more elastic and

viscous than the control without GOx (Vemulapalli et al., 1998). In addition,

GOx also causes a drying effect on dough that is attributed to gel formation of

water soluble pentosans (Vemulapalli and Hoseney, 1998). As it is recognized

that potassium bromate does not cause this drying effect, it is postulated that

this effect is induced by GOx (Vemulapalli et al., 1998). Although, the exact

mechanisms by which hydrogen peroxide produced by GOx improves the

dough properties are not completely understood, more work are in progress,

and some theories have been proposed (Rasiah et al., 2005; Franziska Hanft

2006). Nevertheless, GOx is known to cause cross-linking of dough protein

(Rasiah et al., 2005) and exert effects such as reducing the sulfhydryl content

as well as increasing viscosity in the water soluble portion of dough

(Vemulapalli and Hoseney 1998).

2.4.2 Dry egg powder

Maillard non-enzymatic browning is a result of reaction between the

amino group of proteins or amino acid and reducing sugars. In the production

of dried egg powder this reaction causes undesirable browning and formation

of unwanted flavour. Therefore, the glucose present (~4 g/l) in liquid egg is

39

typically removed before spray drying (Sisak et al., 2006). Glucose removal

also has the added benefit of longer shelf-life and enhanced microbial

tolerance. Besides that, hydrogen peroxide produced in the reaction can also

kill or inhibit growth of microorganisms commonly present in liquid egg

(Dobbenie et al., 1995). One of the way to remove glucose from egg is by

adding GOx before spray drying takes place, as allowed under FDA

regulations. This is typically a batch process and contains the enzyme as an

impurity in the product. Through immobilization, continuous process is

possible, and the enzyme can also be retained and recycled instead as being an

impurity in the product. Hence, there were studies into the viability of reactors

for de-sugaring based on immobilized GOx (Sisak et al., 2006). It should be

noted that undesirable browning caused by Maillard reaction is not unique to

eggs. Potato products also suffer the same issue, and GOx can be employed to

reduce glucose content, thereby reducing browning (Low et al., 1989).

2.4.3 Antioxidant/preservative (oxygen scavenger)

The presence of oxygen is a problem in many food products. In high-fat

foods such as mayonnaise and salad dressing, lipid oxidation can cause

deterioration and rancid taste (Isaksen and Adler-Nissen, 1997). The same is

true for beverages such as wine and beer, keeping oxygen out of the drink

helps to maintain taste and flavour (McLeod and Ough, 1970; Labuza and

Breene, 1989). In canned/bottled/packaged food, oxygen also promotes

bacterial growth, hence, it is desirable to remove oxygen from the headspace to

maintain an anaerobic environment (Kirk et al., 2002). In puree processing,

oxygen contributes to maillard non-enzymatic browning, therefore appropriate

controls must be in place (Parpinello et al., 2002).

The overall reaction catalyzed by GOx involves the consumption of two

glucose molecules and one oxygen molecule to produce two gluconic acid

molecules. This reaction consumes oxygen, a trait that allows GOx to be used

40

as an active oxygen scavenger, antioxidant and preservative in various food

applications. Detailed analysis on using GOx as an oxygen scavenger can be

found in Labuza and Breene (1989). Moreover, the catalytic product, gluconic

acid, is safe for human consumption and WHO has not specified a limit on its

acceptable daily intake. This combined with the demand from consumers to

replace chemical antioxidant and oxygen scavenger with natural compounds

which makes GOx an ideal for food preservation.

2.4.4 Glucose oxidase in sea foods

As a preservative, GOx has been demonstrated to be useful to extend the

shelf-life of some sea food. Fillets or whole fish dipped with GOx/glucose

solution before being refrigerated could be stored for up to 21 versus 15 days

for control (Field et al., 1986), and similarly treated shrimp could also be

stored for up to 11 versus 6 days for control (Dondero et al., 1993). This

phenomenon is most likely due to growth inhibition of the spoilage bacteria

such as Pseudomonas fragi, which is commonly present in fish (Yoo and Rand

1995); Pseudomonas fluorescens, which is associated with shrimp (Kantt et al.,

1993); enterotoxic bacteria E. coli and Salmonella derby (Massa et al., 2001).

Other than the food industry, the “natural” advantage of GOx has also triggered

interests from the pharmaceutical company to pursue the use of GOx to replace

traditional antioxidant in their formulations (Uppoor et al., 2001).

2.4.5 Dairy and the lactoperoxidase system (LPS)

One of the most important applications of GOx in food processing

industry is food preservation. The LPS system, when used in conjunction with

GOx, is a very useful antimicrobial agent. LPS is part of the immune system’s

innate defense mechanism against foreign microorganisms and can be found in

mammalian secretions such as milk, tears and saliva. This system consists of

three components like LPS, thiocyanate and hydrogen peroxide. LPS activation

41

occurs only in the presence of thiocyanate and hydrogen peroxide. Catalysis by

LPS generates active intermediates, which has antimicrobial properties and is

completely safe to humans. The presence of GOx and its substrate (glucose)

allows hydrogen peroxide required by LPS to be continuously generated and

replenished (Seifu et al., 2005). Sathiya moorthi et al. (2008) state that, LPS

has been recognized as an effective antimicrobial agent since many years and

used extensively as an antibacteriostatic agent in reducing micro flora in milk

as well as while making cheese.

For the transportation and/or storage of raw milk, the use and the

activation of the LPS is effective against spoilage and it is recommended to be

used when refrigeration is unavailable or as a complement to refrigeration.

Experiments have demonstrated that the shelf-life of milk with active LPS

enzyme almost doubled the comparison to milk with inactivated LPS (Marks et

al., 2001). In addition, activation of the LPS is also suggested as a pre-

treatment for dairy products to enhance bacterial deactivation including

mastitis pathogens (Sandholm et al., 1988) and to allow lower temperature

treatments during pasteurization (Seifu et al., 2005).

The same LPS–GOx system mentioned previously can also be used in

cheese production. The hydrogen peroxide produced by GOx is utilized by the

LPS for cold, i.e. room temperature sterilization, while the gluconic acid

produced is used for direct acidification (Fox and Stepaniak, 1993). It should

be noted that this LPS-GOx antimicrobial system is not limited to food and has

been used in toothpaste (Biotene, 2006; National Library of Medicine, 2007a),

lotions (National Library of Medicine, 2007b), shampoos, cosmetics, meat

processing (Food Standards Australia New Zealand, 2002) and fish farming

(Seifu et al., 2005).

Further more, during incomplete reduction of molecular oxygen, the

super oxide radical, is generated hydrogen peroxide may lead together with the

42

super oxide radical and trace amounts of transition metal ions [eg., Fe(II)] in

the so called Fenton reaction to the formation of the extremely biocidal

hydroxyl radical (Luo et al., 1994). A wide range of both Gram-negative

bacteria (Wray and Mc Laren, 1987; Borch et al., 1989) and Gram-positive

bacteria (Oram and Reiper, 1966; Siragusa and Johnson, 1989) are inhibited by

LPS. However, studies have shown that Gram-negative bacteria were generally

found to be more sensitive to LPS mediated, (food) preservation than Gram-

positive species (Marshal and Reoter, 1980; De Wit and Van Hooydonk,

1996).

2.4.6 Reduced alcohol wine

Sugar is an important ingredient when it comes to alcohol production

through fermentation because it is the primary substrate used by

Saccharomyces cerevisiae to produce alcohol. Hence, reduction of glucose is

necessary to obtain the lower alcohol content. As there are demands for

reduced alcohol wines, partly driven by its lower tax and tariffs, there were

investigations on the feasibility of using various technologies (Gary, 2000).

One of them is to use GOx to reduce the amount of glucose available,

subsequently yielding lower alcohol content.

One of the easiest ways to do this is to add GOx to the must before

fermentation. GOx consumes some of the glucose present making them

unavailable for alcohol fermentation, thereby resulting in wine with reduced

alcohol (Pickering et al., 1998, 1999a, 1999b and 1999c). At the same time,

hydrogen peroxide generated may reduce the activity or growth of the

S. cerevisiae used for alcohol fermentation. Nevertheless, experiments had

shown that the must containing GOx completed fermentation in 10 days,

whereas the control required 12 days (Pickering et al., 1999a). Another

approach examined was to genetically engineer the S. cerevisiae used during

the fermentation process to express GOx. This approach was considered viable

43

but requires more work to be done before it can reach the market. One added

advantage in the use of GOx is that the hydrogen peroxide produced acts as a

bactericide, helping to act as preservative for the wine (Malherbe et al., 2003).

2.4.7 Gluconic acid production

Gluconic acid and its derivative salts are GRAS and can be used in a

wide range of industries (Ramachandran et al., 2006) including textile dying,

metal surface cleaning, food additives, detergents, concrete, cosmetics (Yu and

Scott, 1997) and pharmaceuticals (BACAS, 2004). As a food additive, it can be

used as an acidity regulator, raising agent, colour stabiliser, antioxidant and

chelating agent in bread, feed and beverage (Brookes et al., 2005; Codex

Alimentarius Commission, 2007b). Industrially, gluconic acid is mostly

produced from fermentation (Singh et al., 2005), with an estimated global

production of about 50,000–100,000 ton/year (BACAS, 2004; EuropaBio and

ESAB, 2005). Further information regarding the properties, applications and

microbial production of gluconic acid has been described by Ramachandran et

al. (2006).

As with all fermentation processes, there are some disadvantages.

Cultures require various added nutrients and at least a few days to grow and

perform bioconversion. In addition, culture solutions produce and contain

unwanted by-products, need downstream purifications and consume substrates

prohibiting high conversion efficiency. Hence, the use of enzyme-based

conversion is considered a viable method to reduce production cost and time

(Nakao et al., 1997). For example, during 1997 and 2003, there were patents

filed making claims of GOx based process that is capable of almost 100%

conversion efficiency, require less time than fermentation and do not contain

impurities (Vroemen and Beverini, 1999; Lantero and Shetty, 2004).

Bioreactor using immobilized GOx is one of the preferred setups being

investigated (Godjevargova and Turmanova, 2004). Immobilization allows the

44

enzymes to be recycled, reduces cost as well as permitting relatively easier

design and construction of reactor to produce and remove of the desired

product, gluconic acid continuously. Despite these potential advantages, the

lack of industrial adoption implies that there are major hurdles to overcome

before mass adoption of enzyme-based bioconversion process occurs. This is

evident, as there is patent covering industrial scale production of gluconic acid

from glucose using GOx dating decades old (Bergmeyer and Jaworek, 1976).

Nevertheless, electrodeionisation-based separative bioreactor (a technology

used to make deionised water; Arora et al., 2007) backed by the US

Department of Energy Biomass Program seems promising and may be adopted

by the industry in the near future.

2.4.8 Glucose sensor/assay

Recently estimated the world’s market value of biosensors to be about

$5 billion dollars, and 85% is attributed to glucose biosensors. Many glucose

sensors available in the market are based on immobilized GOx, and more

information about glucose sensors available in the market can be found in the

review article by Newman and Turner (2005).

Glucose oxidase is commonly used to construct amperometric

biosensors for medical (Wilkins and Atanasov, 1996; Newman and Turner,

2005) and food industry (Mello and Kubota, 2002). A constant electric

potential is applied between working and reference electrode, promoting the

catalytic reaction, which drive the current flow that is proportional to the

concentration of the target molecule (Terry et al., 2005). Such amperometric

glucose sensors based on GOx can be divided into three generations according

to its principle of operation and historical development (Wilkins and Atanasov,

1996; Newman and Turner, 2005; Park et al., 2006).

45

In the medical industry use of GOx in glucose sensors is not only

limited to the traditional “fingerpricking” blood glucose measurement devices

but has also been investigated to be used in continuous monitoring of glucose

in vivo (Wilson and Hu, 2000; Klonoff, 2005) such as fluorescent-based

glucose sensing (Pickup et al., 2005, Brown et al., 2006; Brown and McShane,

2006; Yang et al., 2006). Fluorescent-based glucose sensing has some

advantages over “fingerpricking” sensors, for e.g., extreme sensitivity and non-

invasive.

2.4.9 Fuel cell

The use of GOx in fuel cell is not a recent trend. Investigations on GOx

based fuel cells have been on-going since 1960s (Davis and Yarbrough, 1962;

Yahiro et al., 1964). Despite these efforts and ability to produce cells with near

100% current (faradic) efficiency (Weibel and Dodge, 1975), biofuel cells are

still not ready for applications outside the laboratory. As pointed out in many

litreatures (Calabrese Barton et al., 2004; Bullen et al., 2006; Davis and

Higson, 2006), two main hurdles of biofuel cells are the limited lifetime and

limited power output of the cells. Higher enzyme stability is needed to improve

lifetime of the cells from days/months to years, while higher enzyme catalytic

rate is needed to improve power output by several orders of magnitude.

One viable approach to tackle these problems is by using directed

evolution. For example, by using GOx detection assay which measures the

amount of NADPH produced from the downstream catalysis of GOx end-

products at 340 nm, Zhu et al., (2006) obtained GOx mutant E4, which

displays a higher catalytic activity. GOx are typically used in the anode of

biofuel cells to oxidise glucose, i.e., extract electrons and transfer it to the

anode electrode from which the electrons will flow through the load in the

circuit to the cathode at which the electron will be used to reduce molecules,

e.g., oxygen to water (Weibel and Dodge, 1975). At the same time, ions such

46

as proton, i.e. H+ will diffuse from the anode compartment through the

separating semi-permeable membrane to the cathode compartment to complete

the circuit. Typically, this semi-permeable membrane is necessary to prevent

mixing of materials in the two compartments, which may cause interference to

the operation of the cell.

2.4.10 Other uses of Glucose oxidase

Use of GOx is not limited to the applications described above. In the

textile industry, there are considerable interests to replace chemical bleaching

with environmentally friendly bio-bleaching processes. Chemical bleaching

requires around pH 10.5–11 and near boiling temperature, whereas bio-

bleaching can be conducted at lower temperature and around neutral pH. This

means significant cost saving in energy and effluent treatment. Research

conducted so far have shown promising results in using GOx to produce

hydrogen peroxide for bleaching, utilizing the glucose generated from the

upstream desizing and bioscouring processes (Buschle-Diller et al., 2002;

Tzanov et al., 2002). Although it was noted that the cost of the enzyme is too

expensive for textile processing (Hamlyn, 2000), a patent by Novozyme North

America, Inc. (Salmon et al., 2006) can be found, which covers the use of

carbohydrate oxidase, including GOx to bleach textiles. In other words, it

would be reasonable to speculate that the use of GOx in textile industry should

be economically viable in the near future.

While bio-bleaching can be performed in the factories, it is also possible

to do the same in the everyday laundry by adding GOx to laundry detergent

preparations (Pramod, 1999). In the laboratory, GOx also has diverse uses. For

example, GOx can be used in various immunoassays and/or staining

procedures as well as removal of excess glucose (Rathlev, 1983; Porter and

Porter, 1984; Pfreundschuh et al., 1988; Blais and Yamazaki, 1992; Dosch et

al., 1998; Megazyme, 2003). Whereas, in real-time fluorescent microscopy for

47

biological samples, GOx-catalase is often used for oxygen scavenging to

reduce photodamage (Desai et al., 1999). In geochemicalprospecting, heap

leaching, pollution studies, etc., GOx can be used to prepare mineral leaching

solutions as both hydrogen peroxide and gluconic acid produced facilitates

leaching (Clark, 1995; Clark, 1996).

In genetic engineering, expression of GOx in plants can provide

resistance to bacterial infection (Wu et al., 1995). Obviously, there is no

restriction to the number of potential applications that GOx may be employed

in. Other than the known uses mentioned in this article, there seems to be

plenty of room for new novel applications. For instance, when GOx is used as

preservative in packaged food, changes in pH due to glucose hydrolysis can

potentially be monitored using a pH strip visible outside the package. As the

GOx catalyzed reaction is oxygen limited, if the package is broken and air

leaks in, it will provide the necessary oxygen for GOx to hydrolyze glucose

into gluconic acid, causing pH to drop. Alternatively, a specially designed

container could permit some degree of air penetration, providing the necessary

oxygen for GOx to generate and maintain low level of hydrogen peroxide for

microbial inhibition.

Enzymes, including GOx are gaining importance and popularity in the

industry as an environmental friendly alternative to the traditional chemical

treatments, especially when it becomes more cost effective to produce. With

the enzyme market predicted to have annual growth rate at 7.6% per annum

and market value increase from $4.1 to $6 billion by 2011 (The Freedonia

Group, 2007), and they expecting more breakthroughs, investments as well as

innovations in the applications of GOx in the future.

48

2.5 Vegetable and food processing

2.5.1 Chlorination

Disinfection by chlorination has had many applications in the

propagation, production, harvest, post harvest handling, and marketing display

of fresh fruits and vegetables for many decades (Winston et al., 1953;

Endemann, 1969; Anon, 1970; Hough and Kellerman, 1971; Goodin, 1977;

Rabin 1986, Bartz and Lill, 1988). In the past, maintaining wash tank and

flume concentrations of 3,000 mg/ml for tomatoes and 6,000 mg/ml for citrus

were recommended to control decay (Winston et al., 1953). The primary uses

of chlorine have been to inactivate or destroy pathogenic bacteria, fungi,

viruses, cysts, and other propagates of microorganisms associated with seed,

cuttings, irrigation water, farm or horticultural implements and equipment,

contact surfaces, and human contact with fresh produce. Chlorination has been

routinely used to treat post harvest cooling water, in post harvest treatments

(i.e., calcium for firmness enhancement) and during rehydration at shipping

destinations. Chlorine, primarily as sodium or calcium hypochlorite, has been

an important part of a properly managed horticultural sanitation program for

several decades.

2.5.2 Chlorine dioxide

Chlorine dioxide (ClO2) has been recognized as a strong oxidizing agent

with a broad biocidal effectiveness due to the high oxidation capacity of about

2.5 times greater than chlorine. Many studies have demonstrated its

antimicrobial activity. Since its use was allowed in washing fruits and

vegetables by the efficacies of aqueous chlorine dioxide, ozonated water and

thyme essential oil alone or sequentially in killing mixed strains of E. coli

O157:H7 inoculated on alfalfa seeds. They found that the sequential washing

procedure (thyme oil followed by ozonated water and aqueous ClO2 was

significantly more effective in removal of E. coli O157:H7. However, the use

49

of the combination of these techniques may adversely affect the organoleptical

properties.

Chlorine dioxide has received a lot of attention in the last few years

because its effectiveness is less affected by pH and organic matter content than

that of chlorine. Another advantage is its high oxidative action, which has been

observed to be 2.5 times greater than chlorine. However there are some

disadvantages also. These include its poor stability, virus resistance, and its

tendency to explode at high concentrations. Chlorine dioxide decomposes at

temperatures above 30°C (86°F) and when it is exposed to light. Despite these

disadvantages, use of chlorine dioxide has been increasing because of new

technologies that permit shipment to areas of use instead of onsite generation.

Concentrations should not exceed 5 ppm for treating unpeeled fruits and

vegetables. Chlorine dioxide is approved as a wash treatment for uncut

produce, and is being reviewed for approval as a wash treatment for pre-cut

produce (Rabin, 1986).

2.5.3 Biodegradable packaging

The purposes of food packaging are to protect foods from outside

contamination during distribution, transmission and storage, to maintain the

correct moisture, oxygen or carbon dioxide content in a product or maintain a

desired atmosphere in the headspace around a product. Materials, especially

those used in the food and agriculture industries, have rapidly developed in last

few years. The availability in large quantities at low cost and favorable

functional characteristics enabled the broad application of petrochemical based

plastics such as polyolefins, polyesters, polyamides, and etc., However, their

non biodegradable characteristics lead to environmental pollution and

packaging material has been the target of environmental and consumer activist

groups as being a major contributor to the solid waste stream. Biodegradable

packaging materials received a great attention because of their functionality

50

and environmental-friendly attributes. Among them, edible coatings and films

show special advantages in increasing the shelf-life of product.

2.5.4 Edible coatings and films

Edible packaging and coatings must be free of toxic compounds and

should have a high biochemical, physico-chemical and microbiological

stability, before, during and after application (Risch, 2000). They also should

have good sensory qualities and good barrier and mechanical properties. The

main components of edible packaging that provide good film-forming

properties are polysaccharides, proteins and lipids. They are not only

biodegradable products from various food sources, but can also serve as

carriers for certain additives, such as antioxidants, preservatives, flavours, etc.,

(Day, 1998). Edible coatings could also provide a barrier against visible and/or

UV light which can modify the food characteristics via oxidation of lipids and

pigments (Risch, 2000).

Polysaccharide and protein based films have good mechanical properties

and present excellent barriers for gases, aromas, and lipids, but are highly

permeable to moisture (Krochta and Mulder-Johnson, 1997). Wu et al. (2001)

described that starch-alginate based edible films had the ability to limit

moisture loss and lipid oxidation of pre-cooked beef patties but the abilities

differed with the composition of films. Films made from high amylose starch

showed lower water vapour permeability (WVP) and gas permeability (GP)

than regular corn starch films while addition of oil decreased WVP of starch-

based films (Garcia et al., 2000).

Protective coatings based on zein are commercially available for use on

confectionery items, shelled nuts, and pharmaceutical tablets. As the other

protein films, zein films have high WVP. Wu et al. (2001) tested zein and

zein/lipid films and found that the addition of plasticizer and lipid to film

51

matrix lowered the WVP and increased the elasticity of the films. Studies

showed that, contrary to hydrophilic polysaccharide and protein based films,

hydrophobic lipid based films have poor mechanical properties but high

moisture resistance (Yang and Paulson, 2000). Therefore, significant efforts

have been directed towards the development of edible films by incorporation of

both hydrophilic and hydrophobic molecules into the film-forming matrix to

improve film’s physico-chemical properties. Yang and Paulson (2000) showed

that addition of lipids to gellan films significantly improved the WVP, but

lower the mechanical properties and caused the films to become opaque.

Garcia et al. (2000) found that the addition of sunflower oil to starch-based

film decreased the WVP and lowered the crystalline-amorphous ratio

compared to films without additives. The increase of wax content in lactic

acid-casein based edible films could significantly decrease WVP. Sebti et al.

(2002) introduced stearic acid in cellulose films what resulted in decreased

water vapor transmission rate, increased contact angle, decreased tensile

strength, and lowered air permeability of the films. Similarly, Ozdemir and

Floros (2003) reported that increasing the amount of beeswax resulted in

decreased potassium sorbate diffusivity in whey protein films.

As film-forming biopolymers have the ability to act as carriers of small

molecules, various additives have been applied in the films and coatings.

Incorporation of essential oils into the films and coatings may not only

improve the mechanical characteristics of the films, but also enhance their

antimicrobial properties. Combination of naturally occurring antimicrobial

components, chitosan and essential oils, may provide a unique system with

enhanced antimicrobial properties. Incorporation of essential oils into the

chitosan films could reduce loss of active components due to evaporation and

establish possibilities for prolonged antimicrobial action and improved safety

of foods. Furthermore, the hydrophobic compounds of the oils may enhance

barrier and mechanical properties of the films.

52

The usual approach to improve mechanical properties of edible films is

to add a plasticizer such as glycerol, which is a low molecular weight non

volatile substance, into the film to reduce biopolymer chain to chain interaction

resulting in the improvement of film flexibility and stretch ability. However

plasticizers also increase the film permeability (Yang and Paulson, 2000).

2.5.4.1 Cellulose

Cellulose is the most abundant natural polymer on earth and it is an

essentially linear natural polymer of anhydroglucose. As a consequence of its

chemical structure, it is highly crystalline, fibrous and insoluble. Several water-

soluble, composite coatings are made commercially from cellulose, carboxy

methylcellulose (CMC) with sucrose-fatty acid esters. Derivatives of cellulose,

such as methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC),

form strong and flexible water-soluble films (Baldwin et al., 1996).

2.5.4.2 Alginate

Alginates which are extracted from brown seaweeds of the Phaephyceae

class, are the salts of alginic acid, a linear co-polymer of D-mannuronic and L-

guluronic acid monomers. The ability of alginates to react with di and trivalent

cations is being utilized in alginate film formation. Calcium ions which are

more effective than magnesium, manganese, aluminum, ferrous and ferric ions,

have been applied as gelling agents.

Wu et al. (2001) described that starch-alginate-based edible films had

the ability to limit moisture loss and lipid oxidation of pre-cooked beef patties

but the abilities differed with the composition of films. Films made from high

amylose starch showed lower WVP and GP than regular corn starch films

while addition of oil decreased WVP of starch-based films (Garcia et al.,

2000).

53

2.5.4.3 Zein

Corn zein, the prolamin fraction of corn protein has been used

commercially in coating formulations for shelled nuts, candy and

pharmaceutical tablets. Corn zein coating provides a good barrier to oxygen

and its WVP is about 800 times higher than that of a typical shrink-wrap film.

The aqueous alcohol-soluble protein extracted from corn gluten has been

reported to form films with relatively good water barrier properties. Park et al.

(1999) reported that corn zein film had an effect on delaying ripening and

colour change in tomatoes during storage and confirmed that the degree of

colour change which is an indicative of ripening was mainly dependent on the

coating thickness.

2.5.4.4 Banana powder

Banana mainly consists of starch and pectin (Kotecha and Desai, 1995);

these compounds might possibly provide sufficient properties to form

renewable, biodegradable and inexpensive films and packages. Recently

starches isolated from banana, okenia and mango were used to form edible

films (Romero-Bastida et al., 2005). Banana flour obtained from the whole

banana consists mainly of both starch and pectin, which might form films with

good oxygen barrier and mechanical properties. These films would reduce the

need to isolate the starch, making material preparation easier. Therefore,

obtaining films with good oxygen permeability (OP) and desirable film

mechanical properties would be an indication of the possible use of banana

flour as an alternative secondary packaging. Banana films can be used for dried

products and may have a potential to be commercial.

2.6 Antimicrobial agents in film coating

Antimicrobial agents incorporated into edible films or coatings are

released onto the surface of food to control microbial growth. Such coatings

can also serve as a barrier to moisture and oxygen. Edible coatings have

54

become popular in the food industry because they produce less waste, are cost

effective and offer protection after the package has been opened. Hershko and

Nussinovitch (1998) reported on the behavior of hydrocolloid coatings on

vegetative materials. Park reviewed the development of systematic means of

selecting edible coatings to maximize quality and shelf life of fresh fruits and

vegetables.

Fresh foods may contain microorganisms both on their surfaces and

within. These microorganisms, if not destroyed, lead to food spoilage. The

prevention of food spoilage by inhibiting or destroying microorganisms is the

basis of food preservation. Antimicrobial agents incorporated into edible films

or coatings are released onto the surface of food to control microbial growth.

Such coatings can also serve as a barrier to moisture and oxygen.

2.6.1 Ethylene diamine tetra acetic acid (EDTA)

It has been recognized since 1960s that susceptibility of gram-negative

organisms to lysis by Lysozyme can be increased by the use of membrane

disrupting agents such as detergents and chelators. EDTA, a chelator, exhibits

antimicrobial effect by limiting the availability of cations and can act to

destabilize the cell membranes of bacteria by complexion of divalent cations

which act as salt bridges between membrane macromolecules, such as

lipopolysaccharrides (Boziaris and Adams, 1999). Cutter et al. (2001)

reported improved antimicrobial activity of nisin-incorporated PE or PE oxide

blend films by formulation change and addition of food grade chelator-EDTA.

EDTA and other antimicrobial agents such as nisin, lysozyme and GFSE were

mixed and incorporated into Na-alginate and κ-carrageenan for hurdle

effectiveness to Gram-positive and Gram-negative bacteria.

55

2.6.2 Bacteriocin

The bacteriocins produced by lactic acid bacteria (LAB) offer several

desirable properties that make them suitable for food preservations are: (i)

generally recognized as safe substances, (ii) are not active and nontoxic on

eukaryotic cells, (iii) become inactivated by digestive proteases, having little

influence on the gut micro biota, (iv) usually pH and heat-tolerant, (v) have a

relatively broad antimicrobial spectrum, against many food-borne pathogenic

and spoilage bacteria, (vi) they show a bactericidal mode of action, usually

acting on the bacterial cytoplasmic membrane: no cross resistance with

antibiotics and (vii) their genetic determinants are usually plasmid-encoded,

facilitating genetic manipulation. The accumulation of studies carried out in

recent years clearly indicate that the application of bacteriocins in food

preservation can offer several benefits (Thomas et al., 2000) are: (i), an

extended shelf-life of foods, (ii) provide extra protection during temperature

abuse conditions, (iii) decrease the risk for transmission of food borne

pathogens through the food chain, (iv) ameliorate the economic losses due to

food spoilage, (v) reduce the application of chemical preservatives, (vi) permit

the application of less severe heat treatments without compromising food

safety: better preservation of food nutrients and vitamins, as well as

organoleptic properties of foods, (vii), permit the marketing of “novel” foods

(less acidic, with a lower salt content, and with a higher water content), and

(viii) they may serve to satisfy industrial and consumers demands.

In this respect some of the trends of the food industry in Europe, such as

the need to eliminate the use of artificial ingredients and additives, the

demands for minimally-processed and fresher foods, as well as for ready-to-eat

food or the request for functional foods and nutraceuticals (Robertson et al.,

2004) could be satisfied, at least in part, by application of bacteriocins. The

present review will address different aspects related to food preservation by

bacteriocins including factors influencing bacteriocin activity in food systems,

56

hurdle technology, and the impact of recent advances in molecular biology and

the analysis of bacterial genomes on bacteriocin studies and application.

2.6.3 Lysozyme

Lysozyme is a single peptide protein which possesses enzymatic activity

against the β-1-4 glycosidic linkages between N-acetylmuramic acid and N-

acetylglucosamine found in peptidoglycan. Peptidoglycan is a major

component of the cell wall of both Gram-positive and Gram-negative bacteria.

Hydrolysis of the cell wall by lysozyme can damage the structural integrity of

cell wall and result in the lysis of bacterial cells. Lysozyme is of interest for

use in food systems as it is a naturally occurring enzyme that is produced by

humans and many animals, which have activity against cellular structure

specific to bacteria (Gill and Holley, 2000).

Appendini and Hotchkiss, (1997) investigated the feasibility of

incorporating lysozyme into polymers suitable for food contact. Among the

immobilized polymers (polyvinyl alcohol, nylon and cellulose triacetate)

tested, cellulose triacetate yielded the highest activity. The possible

inadequacies of ice preservation of fresh fish for distribution in the expanding

marketplace were noted and the need for its replacement and supplementation

was affirmed. The combination of the antibacterial effect of the enzyme and

acid production could prove to be an effective agent in the preservation of fresh

fish. Field et al. (1986) evaluated the preservative capabilities of GOx on fish

and attempted to design and implement a GOx system for the preservation of

fresh fish. Soares and Hotchkiss (1998) developed cellulose acetate films with

naringinase immobilized to reduce the naringin concentration in grapefruit

juice.

57

2.7 Microbial contaminates in vegetable products

The increasing popularity of salad bars containing freshly cut vegetables

in supermarkets and convenience stores has introduced new environments

which support the growth of food-borne pathogens including

L. monocytogenes. During handling and packaging of these foods, it is

sometimes difficult to maintain cold temperatures which may provide an

opportunity for pathogens to grow in food. In such cases it may be possible to

exploit bacteriocin producing cultures to provide an extra hurdle to these

pathogens. In this respect, Cai et al., (1997) demonstrated that a nisin

producing L. lactis strain HPB 1688, when co-inoculated with

L. monocytogenes on fresh-cut ready-to-eat salad, was able to reduce the

number of L. monocytogenes by approx. 10-fold after storage for 10 days at 7

and 10°C. This group also showed that a bacteriocin-producing E. faecium was

able to reduce the numbers of L. monocytogenes in Caesar salad.

Another problem associated with fresh vegetables is the possibility of

carry-over contamination with coliforms as a result of poor hygienic practices.

This possibility was addressed by Vescovo et al. (1995) who showed that

strains of Lb. casei had a remarkable inhibitory effect, strongly reducing or

eliminating coliforms or enterococci from the third day of refrigerated storage

of ready-to-eat vegetables.

Lactic acid fermentation of cabbage and other vegetables is a common

method for preserving fresh vegetables in the Western World, China and

Korea. In typical sauerkraut fermentation, Lc. mesenteroides initiates growth,

producing carbon dioxide which creates an anaerobic environment, and organic

acids which lower the pH thereby inhibiting the development of undesirable

microorganisms. However, microorganisms such as food spoilage lactobacilli

which can survive in these adverse conditions can lead to spoilage of the

fermented food.

58

2.8 Spices and edible oil in film making

The possibility of edible film or edible coating to carry some food

additives such as antioxidants, antimicrobials, colourants, flavours, fortified

nutrients and spices are being studied (Pena and Torres 1991). The method is

different from direct application, as the incorporation of antimicrobial agents

into edible film or edible coating localizes the functional effect at the food

surface. The antimicrobial agents are slowly released to the food surface, and

therefore, they remain at high concentrations for extended periods of time

(Ouattara et al., 2000a). Antimicrobial agents used in food application include

organic acids, bacteriocins, enzymes, alcohols and fatty acids (Han, 2000). In

addition, spice extracts have been introduced for their ability to control meat

spoilage (Ouattara et al., 2000b). The beneficial effects obtained by using

edible film and coating in terms of physical, mechanical, and biochemical

benefits have been reported in many publications (Krochta and Mulder-

Johnston, 1997). Gennadios and Weller (1990) reported the ability of edible

film in retarding moisture, oxygen, aromas and solute transport.

Spices such as garlic, onion, cinnamon, cloves, thyme and sage have

been investigated for their antimicrobial activity. The antimicrobial compounds

in plant materials are commonly present in the essential oil fraction and it has

more inhibitory effect than the corresponding ground form (Nychas, 1995).

2.9 Apple puree

Browning is due to condensation reactions between phenolic

compounds. The oxidation of o- and p-diphenols give condensation reactions

with the formation of brown polymers, which are more stable than the

monomer forms (Sims and Morris, 1984). Circumstances like normal storage

temperatures (Sommers and Pockock, 1990), light and oxygen dissolution are

favorable for apple browning. The first step of this process is the oxidation of

59

phenols to quinones and it can be a non-enzymatic chemical reaction catalyzed

by metals like copper and iron (Singleton, 1987) or an enzymatic reaction with

the intervention of polyphenol oxidase (Metche, 1986). The non-enzymatic

chemical reaction is predominant in apples is because the polyphenol oxidase

activity, present within itself (Rapp et al., 1977). Besides, the polyphenol

oxidase enzyme is able to catalyze the oxidation of phenols to quinines but

polymerization reactions are non-enzymatic.

The control of purees browning has always been a challenge for the

fruit-processing industry, the use of chemical antioxidant (e.g., ascorbic acid

and sulfites) and high temperature being the most common solutions. In the

recent years, however, there is an increasing interest in the market of foods wit

natural ingredients; in particular, the presence of sulfites in foods has been

related to adverse health effect, whereas ascorbic acid plays also a pro-oxidant

role. Thus, the research for natural antibrowning agents has been stimulated.

Enzymes represent a great potentiality for food processing.

These differences in the mechanism of inhibition may allow the use of

combinations of antibrowning agents that may result in enhancement of

inhibition. Most combinations of antibrowning agents or commercially

available are ascorbic acid-based compositions. Mixtures of ascorbic and

cyclodextrins were reported to be effective in the inhibition of apple juice

browning (Pizzocarno et al., 1993).

2.9.1 GOx-catalase system

The GOx-catalase system is able to scavenge the oxygen and thus

stabilizes foods and beverages against problems related to product oxidation

and browning (Mistry and Min, 1992a). The GOx has recently been used to

control the colour change in grape juice during high-pressure processing

60

(Castellari et al., 2000) and to produce wine with low alcohol content

(Pickering et al., 1998).

2.9.2 Lactoperoxidase

Lactoperoxidase (EC 1.11.1.7) is a member of the peroxidase family, a

group of natural enzymes, widely distributed in nature and found in plants and

animals, including man (Kussendrager and Van Hooijdonk, 2000).The

structure, function and antimicrobial properties of LPS have been reviewed

recently by De Wit and Hooydonk (1996). Next to xanthine oxidase, LPS is

the most abundant enzyme in milk and is found almost exclusively in the whey

after cheese making.

Lactoperoxidase has been identified as an antimicrobial agent in milk,

saliva and tears. LPS is a natural bacterial defence system through the

oxidation of thiocyanate ions by hydrogen peroxide. LPS has proven to be both

bactericidal and bacteriostatic to a wide variety of microorgnisms, while

having no effect on the proteins and enzymes of the organisms producing LPS

(Ekstrand, 1994).

The mechanism of action of the LPS has been explained in detail by

DeWit and Hooydonk (1996). LPS in more active at acidic pH levels (Wever et

al., 1982), but is less stable under acidic conditions showed that the LPS-

catalysed reactions yield short lived intermediary oxidation products of SCN-,

providing antibacterial activity. The major intermediary oxidation product is

hypothiocyante (OSCN-), which is produced in an amount of about 1 mol per

mol of hydrogen peroxide. At the pH optimum of 5.3, the OSCN- is in

equilibrium with HOSCN. The unchanged HOSCN is considered to be more

bactericidal of the two forms (Thomas et al., 1983).

The action of LPS against bacteria is reported to be caused by sulfydryl

(-SH) oxidation (Aune and Thomas, 1978). The oxidation of -SH groups in

61

the bacterial cytoplasmic membrane results in loss of the ability to transport

glucose and also in leaking of potassium ions, amino acids and peptides .

The microbial specificity of LPS has been reviewed by Kurhonen

(1980). Gram-negative, catalase positive organisms are more readily inhibited

by LPS than are Gram-positive, catalase negative bacteria. Gram-negative,

catalase positive organism, (coliforms, Salmonella, etc.,) are not only inhibited,

but are killed if sufficient hydrogen peroxide is provided chemically,

eznymatically or by hydrogen peroxide producing microorganism (Bjorck,

1992). On the other hand, the action of LPS against Gram-positive organisms

is generally bacteriostatic and not lethal.

Gould, (1995) found that there was a critical combination of LPS, GOx

glucose, iodide and thiocyanate to be effective in cosmetics. The treatment

was effective against a range of yeasts, fungi and viruses, as well as bacteria

for periods of up to 4 months. Although the system has been shown to have

potential as a bio-preservative, its potency has so far been extensively

investigated in only dairy and meat products. Its effectiveness against

pathogens in other foods is generally unexplored. Therefore, use of the system

against S. enteritidis in several foods suspected of harboring the pathogen was

investigated.

New preservation techniques are being applied and researched that will

increase the number of inactivating techniques that are available. These include

the addition of bactericidal enzymes such as lysozyme and the LPS, GOx, non-

enzymic proteins such as lactoferrin and lactoferricin, and bacteriocins (Gould,

1995). In this present investigation an attempt is made to control microbial

contaminations and browning during processing and storage of apple puree by

using GOx, LPS and catalase enzyme system.

62

2.10 Minimally processed fruits and vegetables

Minimally processed vegetables sold in ready-to-eat (salads) or ready-to

use forms have become a very important area of potential economic growth for

fresh-cut industry. Today MPV products have gained popularity mainly

because consumers perceive such products, besides their well known

nutritional qualities, as fresh, healthy, convenient, tasty and easy to use (Garret

et al., 2003). For reasons of expense, labour and hygiene, the catering industry

aims to purchase vegetables and fruit that are already peeled and possibly also

sliced, grated or shredded, that is, minimally processed. Consumers are

increasingly demanding convenient, ready-to-use and ready-to-eat fruit and

vegetables with a fresh-like quality, and containing only natural ingredients’.

Minimal processing of raw fruit and vegetables has two purposes. First, it is

important to keep the produce fresh, yet supply it in a convenient form without

losing its nutritional quality. Second, the product should have a shelf-life

sufficient to make its distribution feasible to its intended consumers4. In an

ideal case, minimal processing can be seen as ‘invisible’ processing

(Ahvenainen et al., 1994).

2.11 Reasons for quality changes in minimally processed produce

As a result of peeling, grating and shredding, produce will change from

a relatively stable product with a shelf-life of several weeks or months to a

perishable one that has only a very short shelf-life, even as short as l-3 d at

chill temperatures. Minimally processed produce deteriorates because of

physiological ageing, biochemical changes and microbial spoilage, which may

result in degradation of the colour, texture and flavour of the produce. During

peeling and grating operations, many cells are ruptured, and intracellular

products such as oxidizing enzymes are liberated (Ahvenainen et al., 1994).

63

2.12 Methods to improve the shelf-life and safety of minimally processed

produce

Minimally processed vegetables can be manufactured on the bases of

several different working principles (Table 2.1). If the principle is that products

are prepared today and consumed tomorrow, then very simple processing

methods can be used. Most fruit and vegetables are suitable for this type of

preparation. Such products are suitable for catering but not for retailing

purposes. The greatest advantage of this principle is the low requirement for

investment. If products are required to have a shelf-life of several days up to

one week, or even more in the case of products intended for retailing, then

more advanced processing methods and treatments using the hurdle concept

are needed, as well as the correct choice of raw materials that are suitable for

minimal processing. Preservation is based on combination of several

treatments. As the table shows, not all produce is suitable for this type of

preparation.

2.13 Microbial spoilage of vegetables and fruits

Contamination of vegetable products with food borne pathogens is very

common (Nguyen-The and Carlin, 1994). Effective and feasible means are

needed to remove pathogens and also to prevent food borne diseases associated

with consumption of fresh fruits and vegetables (Roever, 1998; Francis et al.,

1999).

Sanitizers studied for their effectiveness in removing pathogens from

fruits and vegetables include generally chlorine and various acids such as

acetic, ascorbic, citric, and lactic acids (Weissinger et al., 2000; Burnham et

al., 2001; Singh et al., 2002). Natural products may have applications in

controlling pathogens in foods (Bowles and Juneja, 1998), especially in ready-

to-eat foods. There are many studies which have investigated the antimicrobial

activity of different kinds of plant extracts in vitro system (Hsieh et al., 2001;

Cuspinera et al., 2003; Jayaprakasha et al., 2003). On the other hand,

64

Table 2.1. Requirements for the commercial manufacture of pre-peeled and/or sliced, grated or shredded fruit and vegetables

Working principle Demands for processing Customers Shelf-life at5°C (d)

Examples ofsuitable fruit and

vegetables

Preparation todayConsumptionTomorrow

Standard kitchen hygiene and tools.No heavy washing for peeled andshredded produce; potato is anexceptionPackages can be returnable,Containers.

Cateringindustry,restaurants,Schools,industry.

1–2 most fruit andvegetables

Preparation today,theCustomer uses theproduct within 3-4d

DisinfectionWashing of peeled and shreddedproduce with waterPermeable packages; potato is anexception

Cateringindustry,restaurants,schools, industry

3–5carrot, cabbages, Iceberg lettuce ,potato,beetroot, strawberry

Products are alsointended forretailing

Good disinfectionChlorine or acid washing for Peeledand shredded producePermeable packages; potato is anexceptionAdditives

Retail shops inaddition to thecustomers listedabove

5–7

Carrot, Chinesecabbage redcabbage, potato, beetroot, acid fruit,berries.

65

reports on the antimicrobial effects of table type acidulants that are used with

salad vegetables at household applications such as lemon juice (fresh and

commercial) and vinegar are very limited (Sengun and Karapinar, 2004, 2005a,

2005b; Vijayakumar and Wolf-Hall, 2002a, 2002b).

Illnesses caused due to the consumption of foods contaminated with

pathogens such as L. monocytogenes have a wide economic and public health

impact worldwide (Gandhi and Chikindas, 2007). L. monocytogenes can adapt to

survive and grow in a wide range of environmental conditions as well as in a large

variety of raw and processed foods, including milk and dairy products, or fresh

produce. Food spoilage includes physical damage, chemical changes, such as

oxidation, colour changes, or appearance of off-flavours and off-odors resulting

from microbial growth and metabolism in the product (Gram et al., 2002). The

spoilage of refrigerated meat is caused in part by Pseudomonas species which are

responsible for the off-odors, off-flavours, discolouration, gas production and

slime production (Oussalah et al., 2006). In some cases, a change in atmosphere

by vacuum-packing inhibits the aerobic pseudomonads causing a shift in the

microflora to lactic acid bacteria (LAB) and Enterobacteriaceae (Gram et al.,

2002). The pseudomonads are also found in pasteurized milk and are generally

from post-process contamination (Eneroth et al., 2000).

The spoilage microflora associated with fresh vegetables includes

Pseudomonas spp. as well as other Gram-negative bacteria, such as Enterobacteria

(Ragaert et al., 2007). Current technologies for preservation and shelf-life

extension of food include chemical preservatives, heat processing, modified

atmosphere packaging (MAP), vacuum packaging (VP) or refrigeration.

Unfortunately, these steps do not eliminate undesirable pathogens such as

L. monocytogenes from these products or delay microbial spoilage entirely.

Alternative preservation techniques such as novel non-thermal technologies and

66

naturally derived antimicrobial ingredients are under investigation for their

application to food products.

Staphylococcus aureus is highly vulnerable to destruction by heat treatment

and nearly all sanitizing agents. Thus, the presence of this bacterium or its

enterotoxins in processed foods or on food processing equipment is generally an

indication of poor sanitation. S. aureus can cause severe food poisoning. It has

been identified as the causative agent in many food poisoning outbreaks and is

probably responsible for even more cases in individuals and family groups than

the records show. Foods are examined for the presence of S. aureus and/or its

enterotoxins to confirm that S. aureus is the causative agent of food borne illness,

to determine whether a food is a potential source of "staph" food poisoning, and to

demonstrate post-processing contamination, which is generally due to human

contact or contaminated food-contact surfaces. Conclusions regarding the

significance of S. aureus in foods should be made with circumspection. The

presence of a large number of S. aureus organisms in a food may indicate poor

handling or sanitation; however, there is not sufficient evidence to incriminate a

food as the cause of food poisoning. The isolated S. aureus must be shown to

produce enterotoxins. Conversely, small staphylococcal populations at the time of

testing may be remnants of large populations that produced enterotoxins in

sufficient quantity to cause food poisoning. Therefore, the analyst should consider

all possibilities when analyzing a food for S. aureus.

Methods used to detect and enumerate S. aureus depend on the reasons for

testing the food and on the past history of the test material. Processed foods may

contain relatively small numbers of debilitated viable cells, whose presence must

be demonstrated by appropriate means. Analysis of food for S. aureus may lead to

legal action against the party or parties responsible for a contaminated food.

67

Escherichia coli are one of the predominant species of facultative

anaerobes in the human gut and usually harmless to the host; however, a group of

pathogenic E. coli has emerged that causes diarrheal disease in humans. Referred

to as Diarrheagenic E. coli (Nataro and Kaper, 1998) or commonly as pathogenic

E. coli, these groups are classified based on their unique virulence factors and can

only be identified by these traits. Hence, analysis for pathogenic E. coli often

requires that the isolates be first identified as E. coli before testing for virulence

markers. The pathogenic groups includes enterotoxigenic E. coli (ETEC),

enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC),

enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely

adherent E. coli (DAEC) and perhaps others that are not yet well characterized

(Levine, 1987; Nataro and Kaper, 1998). Of these, only the first 4 groups have

been implicated in food or water borne illness.

ETEC is recognized as the causative agent of travelers' diarrhea and illness

is characterized by watery diarrhea with little or no fever. ETEC infections occurs

commonly in under-developed countries but, in the U.S., it has been implicated in

sporadic waterborne outbreaks as well as due to the consumption of soft cheeses,

Mexican-style foods and raw vegetables. Pathogenesis of ETEC is due to the

production of any of several enterotoxins. ETEC may produce a heat-labile

enterotoxin (LT) that is very similar in size (86 kDa), sequence, antigenicity, and

function to the cholera toxin (CT).

EIEC closely resemble Shigella and causes an invasive, dysenteric form of

diarrhea in humans (Dupont et al., 1971). Like Shigella, there are no known

animal reservoirs; hence the primary source for EIEC appears to be infected

humans. Although the infective dose of Shigella is low and in the range of 10 to

few hundred cells, volunteer feeding studies showed that at least 106 EIEC

organisms are required to cause illness in healthy adults. Unlike typical E. coli,

EIEC are non-motile, do not decarboxylate lysine and do not ferment lactose, so

68

they are anaerogenic. Pathogenicity of EIEC is primarily due its ability to invade

and destroy colonic tissue. The invasion phenotype, encoded by a high molecular

weight plasmid, can be detected by invasion assays using HeLa or Hep-2 tissue

culture cells (Mehlman et al., 1982, Dupont et al., 1971) or by PCR and probes

specific for invasion genes.

EPEC causes a profuse watery diarrheal disease and it is a leading cause of

infantile diarrhea in developing countries. EPEC outbreaks have been linked to the

consumption of contaminated drinking water as well as some meat products.

Through volunteer feeding studies the infectious dose of EPEC in healthy adults

has been estimated to be 106 organisms. Pathogenesis of EPEC involves intimin

protein (encoded by eae gene) that causes attachment and effacing lesions (Hicks

et al., 1998); but it also involves a plasmid-encoded protein referred to as EPEC

adherence factor (EAF) that enables localized adherence of bacteria to intestinal

cells (Tobe et al.,1999).

Numerous epidemiologic studies exhibited, that a reduced risk of

degenerative diseases correlates with a high intake of fruits and vegetables

(Steinmetz and Potter, 1996). Ready-to-use salads can suit as useful sources of

minerals and physiologically active substances such as polyphenols, as the

increasing popularity of this convenience product indicates. Due to high bacterial

counts of raw vegetables after harvest up to 106–109 colony forming units per

gram fresh salad (CFU/g), ready-to-use sliced salads are usually contaminated by

microorganisms, too (Jaques and Morris, 1995). Even bacterial pathogens have

been detected in prepackaged salads (Lin et al., 1996) and lettuce (Park and

Sanders, 1992). High initial counts are not substantially reduced during

conventional cold washing.

69

2.14 Pesticides as a source of microbial contamination of salad vegetables

Pesticides are routinely used in the cultivation of vegetables to control

insects, weeds, spoilage bacteria and fungi, and other pests. Pesticides are

commonly grouped into three categories: insecticides, herbicides and fungicides

(Hislop, 1976). These categories have different target populations and active

ingredients, and usually come in a concentrated powder or liquid form.

Reconstitution and dilution of the pesticide with water is required before

application to vegetables, and this is usually done on the farm by the farmer. In

recent years, there has been increased interest in the microbiological quality and

safety of fresh produce such as salad vegetables, as they have been linked to

outbreaks of food borne microbial disease (Beuchat, 1996, 2002; Heard, 1999,

2002; Nguyen-The and Carlin, 2000). While postharvest contamination is often

the source of the implicated microorganisms, there is increasing concern that

preharvest contamination presents significant public health risks. The main

sources of preharvest contamination are fertiliser, irrigation water and soil (Lund,

1992; Beuchat, 1996, 2002). As mentioned already, a diversity of pesticides are

regularly applied to vegetable produce, but they are rarely considered to be a

source of microbial contamination.

2.15 Cut vegetable and fruit preservation techniques in practice

Minimally processed vegetable sold in ready-to-eat or ready-to use form

have become a very important area of potential economic growth for fresh-cut

industry. Today MPV products have gained popularity mainly because consumers

perceive such products, besides their well known nutritional qualities, as fresh,

healthy, convenient, tasty and easy to use (Garret et al., 2003). Nevertheless

potential growth of food-borne pathogens is greater on MPV than on raw produce,

since their characteristics such as high humidity and large number of cut surfaces

that can create ideal conditions for several microorganisms growth (Nguyen-The

and Carlin, 1994; Francis et al., 1999; Alzamora et al., 2000). Although microbial

70

numbers in MPV products are kept low by the combined effect of washing,

modified atmosphere packaging (MAP) and low temperature, several pathogens,

including Campylobacter jejuni, Salmonella spp., E. coli O157:H7, Shigella spp.,

Aeromonas hydrophila, Yersinia enterocolitica and L. monocytogenes can grow

and cause diseases depending on the type of product, storage conditions (time,

temperature and atmosphere composition) and the presence of competitive

microorganisms (Gleeson and O’Beirne, 2005; Mukherjee et al.,2006).

With the objective to improve quality and safety of MPV products, and

reduce preservatives the industry is seeking novel and alternative technologies

with the objective of improving quality and safety of food products. Recently, the

use of lactic acid bacteria (LAB) and/or their natural products for the preservation

of foods (biopreservation) looks as a promising strategy, for reducing growth of

pathogens according to the hurdle technology strategy (Leistner and Gorris, 1995;

Leistner, 2000; Allende et al., 2006; Galvez et al., 2007; Settanni and Corsetti,

2008; Trias et al., 2008).

The preparation of fresh-cut products causes damage to plant tissues,

rendering a more perishable product with shortened shelf-life, compared to intact

fruits and vegetables (Guerzoni et al., 1996; Watada et al., 1996). This problem is

primarily due to a higher respiration rate and the significant damage resulting

from cutting (Pirovani et al., 1997). Fresh-cut processing affects quality factors

such as appearance, flavour, and colour, and product deterioration may proceed

rapidly. The MAP is effective in prolonging the shelf-life of horticultural

commodities by decreasing oxygen (O2) and increasing carbon dioxide (CO2)

concentrations in the package atmosphere achieved via the interaction between

respiratory O2 uptake and CO2 evolution of packaged produce, and Gas transfer

from the package films (Schlimme and Rooney, 1994; Jacxsens et al., 1999;

Makino, 2001).

71

In general, major factors affect the equilibrium gas concentrations of

packaged produce include packaged product weight and its respiration rate,

package film oxygen/carbon dioxide transmission rate and the respiring surface

area (Bell, 1996), and storage temperature. However, for packaged fresh-cut

vegetables in the retail market, package surface area and product fill weight are

often pre-determined to certain degree to achieve a market appeal, and the

respiration rate is also influenced by numerous factors, including storage

temperature, cut size and vegetables types etc. Therefore, selecting package films

with suitable OTRs plays an important role in developing MA packages for

improved quality and shelf-life of fresh-cut produce.

2.15.1 Combination preservation

Food preservation is carried out during food processing in an attempt to

maintain the raw material quality, physico chemical properties and functionality

whilst providing safe products that have a low spoilage potential. This is mainly

achieved through purposely designed processing that varies from one product to

the next. Hence, in preservation processes, which form about a third of the unit

operations used in food processing (Farkas, 1977), the general aim is to employ

combination processes where, for e.g., a mild heat stress and a low concentration

of preservatives are combine in order to fulfil all the above listed objectives.

Additionally, there is currently significant interest in using alternative physical

treatments such as ultra high pressure (UHP) or pulsed electric fields (PEF) to

replace the classical heat treatment. Progress in these areas has been discussed in

this issue by UHP presumably denatures microbial cell wall proteins such that

access to the rest of, e.g., the fungal wall and membrane is greatly facilitated

(Brul, 1999).

Indeed combination preservation treatments are often advocated and where

possible patented (Knorr, 1998). Combinations of preservation treatments allow

72

the required level of protection to be achieved while at the same time retaining the

organoleptic qualities of the product such as, colour, flavour, texture and

nutritional value. The potential use of some of the novel, “natural” preservatives,

discussed previously in this review, in combinations with physical treatments (i.e.,

mildheat, UHP and PEF), has not been extensively evaluated and may lead to the

development of novel mild preservation regimes tailored to the organolyptic

quality needs of individual products.

2.15.2 Chemical preservative agents

The most common classical preservative agents are the weak organic acids,

for example acetic, lactic, benzoic and sorbic acid. These molecules inhibit the

outgrowth of both bacterial and fungal cells and sorbic acid is also reported to

inhibit the germination and outgrowth of bacterial spores (Sofos and Busta, 1981;

Blocher and Busta, 1985).

In solution, weak acid preservatives exist in a pH dependent equilibrium

between the undissociated and dissociated state. Preservatives have optimal

inhibitory activity at low pH because this favors the uncharged, undissociated

state of the molecule which is freely permeable across the plasma membrane and

is thus able to enter the cell. Therefore, the inhibitory action is classically believed

to be due to the compound crossing of plasma membrane in the undissociated

state. Subsequently, upon encountering the higher pH inside the cell, the molecule

will dissociate resulting in the release of charge anions and protons which cannot

cross the plasma membrane.

Microbial resistance to weak organic acid can involve various mechanisms.

For bacteria, significant knowledge exists on their intrinsic, noninducible

resistance mechanisms against these compounds (Russel, 1991). Gram-positive

bacteria do not possess an outer membrane and the exclusion limit of the cell wall

73

of vegetative cells of bacillus megaterium has even been calculated to be as high

as 30,000D (Lambart, 1983). Hence preservatives can easily enter the cells and

their intrinsic resistance is relatively low. In Gram-negative bacteria, resistance

mechanisms are more complicated since these organisms’ posses an inner and

outer membrane.

2.16 Calcium ions on vegetables and fruits

Titchenal and Dobbs (2007) point out some dark green leafy cabbage

family vegetables and turnip greens as good calcium sources, and most leafy

vegetables as potential calcium sources. The major source of calcium in the

United States diet is dairy products, which supply 75% of the intake, and

vegetables, fruits and grains which supply the rest (Allen, 1982). The awareness of

consumers on the benefits of calcium is relatively high. The calcium content in the

diet is critical in most stages of life (Gras et al., 2003). Dietary calcium raises

concern for consumers and health specialists due to the number of processes it is

involved in, the high amount present in the body, and the continuous research

highlighting the benefits of an adequate intake.

Nowadays, an increasing part of the products in the food industry are

fortified, especially dairy products followed by beverages and snacks (Caceres et

al., 2006). A result of evidences linking osteoporosis, hypertension and cancer to

calcium deficiency. While the cause of these diseases is multifactor and poorly

understood, there is some evidence to support the hypothesis that increased

calcium intake might reduce the risk of suffering from these diseases (Appel et al.,

1997; Cumming et al., 1997).

Also, the use of phosphorous-free sources of calcium, such as gluconate,

citrate, lactate, acetate and carbonate calcium salts, can help to obtain a good

balance of calcium and phosphorous in the diet. To give consumers the

74

opportunity to increase their calcium intake without resorting to supplementation,

the industry has been encouraged to fortify food and beverages with calcium

(Cerklewski, 2005). This opens new ways of supplementing calcium intake by

increasing the calcium content in these commodities. For this reason, the use of

natural sources of calcium as preservative with a nutritional fortification effect

presents an advantage for the industry and for the consumer.

2.16.1 Calcium sources to maintain the shelf-life of fresh vegetables and fruits

Different calcium salts have been studied for decay prevention, sanitation

and nutritional enrichment of fresh fruits and vegetables. Calcium carbonate and

calcium citrate are the main calcium salts added to foods in order to enhance the

nutritional value (Brant, 2002). Other forms of calcium used in the food industry

are calcium lactate, calcium chloride, calcium phosphate, calcium propionate and

calcium gluconate, which are used more when the objective is the preservation

and/or the enhancement of the product firmness (Luna-Guzman and Barrett, 2000;

Alzamora et al., 2005; Manganaris et al., 2007).

The selection of the appropriate source depends on several factors:

bioavailability and solubility are the most significant, followed by flavour change

and the interaction with food ingredients. Calcium chloride has been widely used

as preservative and firming agent in the fruits and vegetables industry for whole

and fresh-cut commodities. Sams et al. (1993) and Chardonnet et al. (2003)

studied the effect of calcium chloride on fruit firmness and decay after the harvest

of whole apples.

Saftner et al. (2003) work was also focused on the firming effect of calcium

chloride treatment on fresh-cut honeydew. Luna-Guzman and Barrett (2000)

compared the effect of calcium chloride and calcium lactate dips in fresh-cut

cantaloupe firmness, microbial load, respiration and sensorial evaluation. Other

75

researchers (Garcia-Gimeno and Zurera-Cosano, 1997; Main et al., 1986; Morris

et al., 1985; Rosen and Kader, 1989; Suutarinen et al., 1998) used calcium

chloride as firming agent for processed strawberries.

Wills and Mahendra (1989) examined the effect of calcium chloride on

fresh-cut peaches from a quality point of view, meanwhile Conway and Sams

(1984) evaluated the safety of strawberries treated with calcium chloride. Other

fruits and vegetables, in which the effect of calcium chloride was studied, showing

significant improvement in the quality of the final product, are grape fruit, hot

peppers and diced tomatoes (Mohammed et al., 1991; Floros et al., 1992).

The use of calcium chloride is associated with bitterness and off-flavours

(Bolin and Huxsoll, 1989; Ohlsson, 1994), mainly due to the residual chlorine

remaining on the surface of the product. Calcium lactate, calcium propionate and

calcium gluconate have shown some of the benefits of the use of calcium chloride,

such as product firmness improvement, and avoid some of the disadvantages, such

as bitterness and residual flavour (Yang and Lawsless, 2003). Also, the use of

calcium salts other than calcium chloride could avoid the formation of

carcinogenic compounds (chloramines and trihalomethanes) linked to the use of

chlorine.

Manganaris et al. (2007) compared the effect of calcium lactate, calcium

chloride and calcium propionate dipping in peaches. Calcium increased in tissues

with no dependence on the source used. Calcium incorporation by impregnation

with two calcium sources, calcium lactate and calcium gluconate, was studied in

fresh-cut apple (Anino et al., 2006). Another source of calcium is the calcium-

amino acid chelate formulations which had been patented as nutritionally

functional chelates. Lester and Grusak (1999) showed that the use of calcium

chelate doubled the shelf-life of whole honeydew melon.

76

2.17 Koruk juice

Koruk juice (Unriped grape juice) is commonly used with salad

vegetables as an acidifying and flavouring agent in Turkey and neighboring

countries. It is also consumed as a drink after being sweetened. Currently grape

compounds have attracted increased attention especially in the Welds of nutrition,

health, and medicine (Waterhouse and Walzem, 1998). Phenolic compounds in

grape juice, grape seed and wine have been investigated by many researchers to

show their potent antioxidant, antimutagenic, antibacterial, antiviral, antifungal

and antiulcer activities (Takechi et al., 1985; Liviero et al., 1994; Caccioni et al.,

1998; Saito et al., 1998; Jayaprakasha et al., 2001; Baydar et al., 2004). However,

there is no information about the antimicrobial activity of koruk juice. The

antimicrobial effect of koruk juice varied depending on the culture strains and

products used (P<0.05). There was no significant difference in cell reduction in

samples exposed to koruk juices for 15, 30 and 60 minutes (P>0.05) whereas

reduction obtained at 0 time differed significantly (P<0.05). However, koruk juice

exerted a shock antimicrobial effect on S. typhimurium strains.

Although there is no previous microbiological study related to the

antimicrobial effect of koruk juice, other grape products such as grape seed

extracts, grape juice and wine have been studied by several researchers to

investigate the antimicrobial activity of these products. It has been reported that

grape seed extract reduced the number of S. typhimurium attached on chicken skin

between 1.6 and 1.8 log at 0.1% and 0.5% concentrations (Xiong et al., 1998). In

another study, Baydar et al. (2004) reported that the grape baggase extracts had no

inhibitory effects on the 15 bacteria tested, while the grape seed extracts inhibited

all the bacteria except B. amyloliquefaciens at 20% concentration. Jayaprakasha

et al. (2003) also studied the antimicrobial effect of grape seed extract on three

bacteria and found that, Gram-positive bacteria were inhibited at lower

concentrations of grape seed extracts than Gram-negative bacteria.

77

2.18 Effect of blanching

Generally, the antioxidant potential of vegetables is affected by thermal

processing. (Puupponen-Pimia et al., 2003) found that blanching reduced the

antioxidant capacity by 23% for cauliflower, but increased it by 9% for cabbage.

Wu et al. (2004) found a reduction of 14% in ORAC values for cooked broccoli

and an increase of 41% for cooked red cabbage. The blanching temperature

ranged between 96–98oC. Significant effects of various processing methods on the

reduction of vitamin C by leaching and thermal breakdown have been reported

(Davey et al., 2000). Household and industrial processing might thus affect the

flavanoid, GLS and vitamin contents and in turn affect the health related quality.

2.19 Cold storage

Heat treatments of vegetables are mostly intended to tenderize the

vegetable for consumption or, as a pretreatment for freezing or canning, to

inactivate enzymes and to remove air. The decontamination treatments and low

temperature storage can be combined with modified atmosphere packaging as a

multiapproach stratergy to prolong he shelf-life of MPV. After minimal

processing, a relatively stable agricultural product with a shelf-life of several

weeks or months will become one that has only a very short shelf-life. MPFV

should have storage lives of at least 4–7 days, but preferably longer, up to 21 days,

depending on the market (Ahvenainen, 1996; Barry-Ryan and O’Beirne, 1997).

Cut and damaged surfaces in MPV release nutrients and some intracellular

enzymes, such as polyphenol oxidase, and provide good conditions for some

enzymatic activities and possible microbial spoilage. Enzymatic and microbial

deterioration in MPV continues and shortens their shelf-life even when they under

the recommended chilling conditions (0–8ºC). O’Connor and Skarshshewski

(1992) reported that the shelf-life of some MPV products is less than 5 days at

4oC.

78

2.20 Economic loss

The economic potential is shown by the solid growth of the industry in the

recent past as illustrated by increasing consumption and increasing space devoted

to fresh-cut vegetable products in super markets and on restaurant menus in most

parts of the world (Kaufman et al., 2000).The cost of the chemical preservatives

used for storage of the fruits is higher than the cost of the fruits used for our

consumption or export. This leads to the greater economic loss in most of the food

industries.

79

CHAPTER-3

OBJECTIVE OF THE PRESENT WORK

With this background, in the present study was taken up to address the following

objectives:

· Isolation of glucose oxidase producing fungi from various sugar rich

products.

· Optimization of medium composision and suitable culture condition for the

selected fungus for GOx production.

· Production of extracellular glucose oxidase by laboratory fermentor,

purification and characterization.

· Application of glucose oxidase in food processing and preservation.

80

CHAPTER-4

MATERIALS AND METHODS

4.1 General

The glassware were soaked for overnight in chromic acid solution (10%

potassium dichromate solution in 25% concentrated sulphuric acid) and then

washed thoroughly in running tap water. Finally, they were rinsed in distilled

water and dried in a hot air oven.

4.1.2 Sterilization

The glasswares and medium were sterilized in an autoclave at 121°C at 15

psi for 20 minutes.

4.1.3 Chemicals

The Horse radish peroxidase, o-dianisidine were purchased from Sigma

Chemicals Co. Ltd, USA. Standard protein markers were purchased from Geni

laboratories, Bangalore, India. All other laboratory chemicals were purchased

from SRL and Qualigen, Mumbai, India.

4.2 Isolation and screening of glucose oxidase producing fungi and

optimization of medium

4.2.1 Isolation of GOx producing fungi

Different fungi were isolated from various sugar rich products such as

honey hive collected from Salem, dates, fruits and soil samples from

Maduravoyal, Chennai, Tamil nadu. Isolation was performed by serial dilution

and direct method. In direct method the segments were plated on Potato Dextrose

Agar (PDA) medium amended with streptomycine (30 mg/l). All the plates were

incubated at 28°C for 4–6 days. Single colonies were transferred to fresh plates

and screened their GOx secretion.

81

PDA medium composition

Dextrose - 20 g

Potato extract - Extracted from 200 g of potato

Agar - 20.0 g

The above constituents were made up to 1000 ml by adding distilled water and the

pH was adjusted to 5.8±0.2.

4.2.2 Screening of GOx producing fungi

The isolated fungi were screened for their GOx producing capability

according to the method described by Eun-Ha Park et al. (2000). The fungi were

grown on the media consists (g/l);

Glucose - 80

Peptone - 3.0

(NH4)2HPO4 - 0.388

KH2PO4 - 0.188

MgSO4 - 0.156

Agar - 20.00

The above composition was prepared in sodium acetate buffer (pH 5.5). A

disc of fungal culture was taken from the peripheral zone of the colony and

transferred to the middle of the Petri plate. It was incubated at 35°C for 3 days.

Then the plate was treated with the following mixture;

Glucose - 5 % (w/v)

Glycerol - 2% (v/v)

o-dianisidine - 0.1% (w/v)

Horse radish peroxidase - 60 IU/ml

Agar - 1% (w/v)

82

The above composition was prepared in sodium acetate buffer (pH 5.5) and

overlayed on the fungal culture medium and incubated for one hour. Then the

colour changes were observed. The appearance of brown colour indicates that the

fungi produced the GOx. The following enzymatic reaction will occur giving rise

a brown colour.

The positive colonies were selected and grown on PDA at 28°C.

4.2.3 Identification of fungi

The higher GOx producing fungus was identified as Aspergillus awamori

under the group of A. niger by Prof. B.P.R Vittal, Mycologist, Centre for

Advanced Studies in Botany, University of Madras, Chennai, India. The identified

fungi was submitted to Microbial Type Culture Collection (MTCC), Chandigarh,

India and designated as Aspergillus awamori MTCC 9645. The selective cultures

were maintained on PDA slants at 4°C and sub-cultured at an interval of 30 days.

The fungus A. awamori MTCC 9645 was used for further studies.

4.2.4 Preparation of spore suspension

The Malt Extract Agar (MEA) medium for sporulation of A. awamori

MTCC 9645, that consists of (g/l);

Malt extract - 20.00

Glucose - 20.00

Agar - 20.00

Distilled water - 1000 ml

pH - 5.5±0.2

83

The conidia suspension was prepared from the fungi that grown on the

MEA medium for 3 days at 28°C. After that the fungal colonies were washed with

sterile tween 80 solution (0.1% w/v) to yield a stock spore suspension of 2×107

conidiospores/ml using hemocytometer.

4.3 Analytical methods

4.3.1 Assay of GOx activity (Bergmeyer et al., 1988)

Principle

β-D-glucose + O2 + H2O GOx D-glucano-1,5-lactone+H2O2

H2O2+O-Dianisidine (reduced) POD O-Dianisidine

Where,

GOx- GOx; POD-Peroxidase

Conditions : Temperature =35ºC, pH = 5.1, A500nm, light path =1cm

Method: Continuous spectrophotometric rate determination

Reagents

A. Sodium acetate buffer (50 mM), pH 5.5.

B. o-Dianisidine solution (0.21mM), 50mg of o-Dianisidine

dihydrochloride was dissolved in 7.6 ml of Millipore water, this solution

was diluted to 1.0 ml to 100ml with reagent A.

C. β-D-glucose (10% w/v) substrate solution (this was prepared using β-

D-glucose in 10ml of Millipore water).

D. Reaction cocktail was prepared by o-Dianisidine (0.17 mM) and 1.72%

(w/v) of glucose solution. (The combination of 24 ml of reagent B and 5

ml of reagent C were mixed and adjusted the pH to 5.5. This solution

was prepared immediately before use as fresh solution).

E. Peroxidase enzyme solution (POD)

84

(Freshly prepared solution containing 60 purpurogallin units/ml of

peroxidase, type II was prepared in cold Millipore water).

Procedure

The following reagents were pipette out in to quartz cuvettes:

Reagents Test (ml) Blank (ml)

Reaction cocktail

(reagent D)2.90 2.90

Reagent E (POD)0.10 0.10

Then the mixture was mixed well by inversion and equilibrates to 35ºC.

The colour a change was read in a UV-Visible spectrophotometer at A500nm. After

that following reagents were added,

Reagents Test (ml) Blank (ml)

Reagent F

(enzyme solution)0.10 -

Reagent A (buffer) - 0.10

The above solutions were mixed thoroughly by inversion and recorded the

changes in A500nm for approximately 5 minutes. Maximum linear rate for both test

and blank were obtained at A500nm.

Calculations

(D A500nm / minutes test - D A500nm / minutes blank)(3.1) (df)Units/ml enzyme = (7.5) (0.1)

....... (4.1)

3.1 = volume (in millilitres) of assay

df = dilution factor

85

7.5 = millimolar extinction coefficient of oxidized o-dianisidine at 500 nm.

0.1 = volume (in millilitres) of enzyme used

Units/ml enzymeUnits/mg solid = .......(4.2) mg solid/ml enzyme

Unit definition

One unit of GOx will oxidize 1.0 µmole of β-D-glucose in to D-

gluconolactone and H2O2 per minute in the pH of 5.5 at 35ºC (equivalent to an O2

uptake of 22.4 µl per minute).

4.3.2 Estimation of protein

The protein content of the sample was estimated according to the method of

Bradford (1976).

Reagents

Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 50 ml of

ethanol. Along this 100 ml of 85% (v/v) o-phosphoric acid was added and made

up to one litre with glass distilled water. The concentrations in the reagent were

0.01% (w/v) of CBB G-250, 4.7% (v/v) of ethanol and 8.5% (v/v) of ortho

phosphoric acid.

Procedure

Protein sample (100 µl) was added to 900 µl of sodium acetate buffer (pH

6.5), then, 3 ml of CBB-G250 solution was added and incubated at room

temperature for 5 minutes. Absorbance was read in UV-Visible spectrophotometer

at 595 nm against the blank containing one millilitre of sodium acetate buffer and

3 ml of CBB-G250 solution. The amount of protein was calculated using Bovine

Serum Albumin as a standard protein (Sigma Chemicals Co., USA).

86

4.3.3 Estimation of fungal biomass

The fungal biomass was estimated by by Sandip et al. (2008) method. The

fermented broth sample (50 ml) was acidified to pH 2.5 using 4 M HCl, because

the insoluble calcium carbonate was converted in to soluble calcium chloride and

carbon dioxide. Unless CaCO3 is used, the above procedure was omitted. The

samples were centrifuged at 4,000 g for 15 minutes. The mycelial pellets from the

samples were washed with distilled water and dried (90°C, 36 h) to a constant

mass. The biomass concentrations were expressed in dry weight of biomass per

litre of culture medium and the experiments were done in triplicates.

4.3.4 Analysis of glucose

The glucose content was estimated by dinitro-salicylic acid (DNSA)

method using glucose as the standard (Miller, 1959). The DNSA reagent consisted

of one gram DNSA dissolved in 20 ml of 2N sodium hydroxide and 50 ml

Millipore water. Thirty grams of potassium sodium tartarate tetrahydrate was

added and the volume was brought up to 100 ml with Millipore water. The

glucose was measured as follows: Fifty microlitre of sample was added to 1.95 ml

Millipore water and two millilitre DNSA reagent were boiled for 5 minutes

followed by cooling to room temperature and diluting to 24 ml. Glucose content

was determined by using linear glucose standard curve.

4.3.5 Analysis of gluconic acid

The quantity of gluconic acid was determined by titration of the culture

solution with 0.1 N sodium hydroxide at room temperature. Excess alkali was

added and the solution was maintained at the temperature of 50–55°C. At this

temperature it was found that any glucose present did not react with appreciable

quantities of the sodium hydroxide and that the gluconic acid lactone was

completely hydrolyzed to the acid in from 1 to 2 minutes. After 2 minutes at this

temperature, the solution was titrated with 0.1 N sulfuric acid and the gluconic

87

acid resulting from the hydrolysis of the lactone was added to the first titration. As

a rule the lactone equaled from 5 to 10% of the total quantity of gluconic acid.

The total acid was then calculated in gram of gluconic acid. To check these

results several determinations of gluconic acid, as calcium gluconate, were made

by neutralizing an aliquot portion of the culture liquor with calcium carbonate,

heating to boiling, filtering and precipitating the salt with 3 volumes of 95% (v/v)

ethanol. The mixture was allowed to stand for 2 days to insure complete

precipitation and was then filtered in a weighed crucible. The precipitate was

washed with 60% (v/v) ethanol, after which it was dried to constant weight at

90°C. The agreement between the quantity of acid calculated from the titration

and the quantity actually recovered was satisfactory.

4.4 Culture condition for GOx production

The five millilitre of conidiospore suspension (2×107 spores/ml) was

inoculated in to the Erlenmeyer flask (500 ml) containing 100 ml of the medium.

The cultures were incubated in an orbital shaker (200 rpm) at 32±2ºC for 120 h.

The cell free supernatant was subjected to GOx activity.

4.4.1 Selection of suitable medium for GOx production

Seven different media were chosen to find out a suitable medium for

maximum GOx production (Table 4.1). Among the seven different media, the

production medium GOxM 3 supported maximum GOx production from A.

awamori MTCC 9645 and therefore, it was chosen for further investigation.

88

Table 4.1: Different production medium for GOx

Media No. Media composition (g/l) Reference

GOxM 1

Glucose, 60.0; NH4NO3, 0.3; KH2PO4, 0.25;MgSO4. 7H2O, 0.25; urea, 2.0; CSL, 8 ml; pH,6.0

Li and Chen,1994

GOxM 2

Glucose, 80.0; peptone, 3.0; NaNO3, 5.0; KCl,0.5; KH2PO4, 1.0; FeSO4. 7H2O, 0.01; CaCO3,35.0; pH, 6.0

Petruccioli etal., 1995

GOxM 3

Glucose, 80.0; peptone, 3.0; (NH4)2HPO4,0.388; KH2PO4, 0.188; MgSO4. 7H2O, 0.156;CaCO3, 35.0; pH, 6.0

Fiedurek andSzczodrak,1995

GOxM 4Glucose, 40.0; NH4NO3, 1.0;KH2PO4, 1.0; MgSO4.7H2O, 0.25; pH 6.5

Träger et al.,1992

GOxM 5

Glucose, 40.0; NaNO3, 2.0; KCl, 0.5;KH2PO4,1.0; MgSO4 7H2O, 0.5; FeSO4.7H2O,0.01; yeast extract, 2.0; pH, 6.0

Nakamatsu etal., 1975

GOxM 6

Sucrose, 50.0; Ca(NO3)2, 2.0; citric acid, 7.5;KH2PO4, 0.25; KCl, 0.25; MgSO4. 7H2O, 0.25;FeCl3. 6H2O, 0.01; CSL, 20.0; pH, 6.0

Zetelaki, 1970

GOxM 7

Starch hydrolysate, dextrose basis, 200.0;(NH4)2HPO4, 0.2; CSL, 0.4; KH2PO4, 1, MgSO4.7H2O, 0.1; urea, 0.4; antifoam H-601, 0.5 ; pH,6.5

Shah andKothari, 1993

*Glucose and CaCO3 were sterilized separately and added to the medium beforeinoculation.

89

4.5 Media optimization

The production medium was optimized by classical Single Factor Analysis

(SFA) method and a statistical method of Response Surface Methodology (RSM).

The optimization procedures were carried out by above described shake flask

method.

4.5.1 Single factor analysis for GOx production

The media components were optimized by SFA. The following factors

were analyzed for the optimization of basal production medium components

which includes the effect of different carbon, nitrogen sources, environmental

factors (pH and temperature) and fermentation time.

4.5.1.1 Effect of carbon source on GOx production

The various carbon sources like glucose, sucrose, fructose, maltose,

xylose and rhamnose were analyzed. The carbon sources were checked in different

concentrations such as 20, 40, 60, 80, 100, 120, 140 and 160 g/l. The carbon

sources were separately sterilized and added in to the medium.

4.5.1.2 Effect of nitrogen sources on GOx production

The effect of different nitrogen sources on the production of GOx was

studied. The nitrogen sources such as mycological peptone, proteose peptone,

bacteriological peptone, yeast extract and beef extract were studied in different

concentrations like 1, 2, 3, 4, 5 and 6 g/l.

4.5.1.3 Effect of Di-ammonium hydrogen phosphate, potassium di-hydrogen

phosphate and magnesium sulphate on GOx production

The effect of MgSO4 and (NH4)2HPO4 were evaluated at the concentration

of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 g/l. The KH2PO4 was supplemented in

the production medium at the concentration of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 g/l.

90

4.5.1.4 Effect of calcium carbonate supplementation on GOx production

The effect of the addition of CaCO3 in to the media was analyzed for

biosynthesis of GOx at the concentration of 10, 20, 30, 40, 50 and 60 g/l.

4.5.1.5 Effect of pH and temperature on GOx production

The different pH (3, 4, 5, 6, 7 and 8) was employed for the production of

GOx. To determine the optimum temperature for GOx production the

fermentation medium was incubated at 20, 25, 30, 35, 40 and 45ºC.

4.5.1.6 Effect of fermentation time on GOx production

The fermentation time was determined for the economic production of

GOx. The production profile of GOx and biomass were calculated at every 12

hour interval of 0 to 144 h.

4.5.2 Statistical optimization by Response Surface Methodology (RSM)

To find out the optimum concentration of medium constituents were

analyzed by RSM using central composite design. The experiments were carried

out by using Design-Expert 7.1.6 software package.

RSM is a collection of statistical and mathematical techniques useful for

developing, improving and optimizing the process. RSM defines the effects of the

independent variables, alone or in combination, on the process. In addition to

analyzing the effects of the independent variables, this experimental methodology

generates a mathematical model that accurately describes the overall process. It

has been successfully applied to optimizing conditions in food, chemical and

biological processes.

91

4.5.2.1 Experimental design of RSM for optimization of media components

The optimal levels of eight variables such as glucose, mycological peptone,

CaCO3, MgSO4, KH2PO4, (NH4)2HPO4 and environmental factors (temperature

and pH) were optimized for GOx production. It was carried out by three sets of

experiments. The first set was carried out by the first three variables like glucose,

mycological peptone and CaCO3. After that the second set of variables [MgSO4,

KH2PO4 and (NH4)2HPO4] were done with the optimized concentrations of first

set variables. The third set was optimized by the two variables like temperature

and pH. For that purpose, the response surface approach by using a set of

experimental design (central composite design with five coded levels) was

performed. For the three factors, this design was made up of a full 23 factorial

design with its eight points augmented with three replications of the center points

(all factors at level 0) and the six star points, that is, points having for one factor

an axial distance to the center of ±α, whereas the other two factors are at the level

of 0. The axial distance α was chosen to be 1.68 to make this design orthogonal. A

set of 20 experiments were carried out for the three variables and 13 experiments

for two variables. The central values (0 level) chosen for experimental design

were (g/l):

Set-1: glucose (90.0), mycological peptone (4.0) and CaCO3 (35.0) –Table 4.2.1–4.2.2

Set-2: (NH4)2HPO4 (0.48), KH2PO4 (0.32) and MgSO4 (0.23) –Table 4.3.1–4.3.2

Set-3: pH (6.0) and temperature (35°C) –Table 4.4.1–4.4.2

In developing the regression equation, the test factors were coded according to the

following equation:

....... (4.3)

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Table 4.2: Set 1 Optimization of glucose, proteose peptone and calcium carbonate for GOx production

Table 4.2.1: Design summary

Variables UnitsLevels

-1 -α 0 +1 +-α

Glucose (A) g/l 40 5.910 90 140 174.089Proteosepeptone (B) g/l 1 -1.045 4 7 9.045

CaCO3 (C) g/l 10 -7.044 35 60 77.045

Table 4.2.2: Experimendal design and of 23 factorial design

RunA:

Glucose(g/l)

B:Proteose peptone

(g/l)

C:CaCO3

(g/l)1 90 -1.045 352 40 1 103 90 4 354 40 7 105 5.91 4 356 140 1 607 90 4 358 40 1 609 174 4 3510 90 4 77.0411 90 4 3512 90 4 3513 90 9.045 3514 140 7 6015 140 1 1016 90 4 3517 90 4 3518 140 7 1019 40 7 6020 90 4 -7.044

93

Table 4.3: Set 2 Optimization of (NH4)2HPO4, KH2PO4 and MgSO4 for GOx

production



-1 -α 0 +1 +-α

(NH4)2HPO4 (A) g/l 0.1 -0.13863 0.45 0.8 1.038627

KH2PO4 (B) g/l 0.05 -0.13749 0.325 0.6 0.787493

MgSO4 (C) g/l 0.05 -0.1034 0.275 0.5 0.653403


Run A:(NH4)2HPO4

(g/l)

B:KH2PO4

(g/l)

C:MgSO4

(g/l)1 0.45 0.787 0.2752 0.45 0.325 0.2753 0.45 0.325 0.2754 0.1 0.05 0.55 0.45 0.325 -0.1036 0.1 0.05 0.057 0.1 0.6 0.58 0.45 0.325 0.2759 1.038 0.325 0.27510 0.45 0.325 0.27511 0.8 0.05 0.512 0.1 0.6 0.0513 0.8 0.6 0.514 0.8 0.6 0.0515 0.45 0.325 0.27516 0.45 -0.137 0.27517 -0.138 0.325 0.27518 0.8 0.05 0.0519 0.45 0.325 0.65320 0.45 0.325 0.275

94

Table 4.4: Set 3 Optimization of pH and temperature for GOx production



-1 -α 0 +1 +-α

pH (A) 3.0 1.757 6.0 9 10.242

Temperature (B) °C 20.0 13.786 35.0 50.0 56.213


Run A:pH

B:Temperature (°C)

1 3 502 9 503 1.79 354 6 355 6 13.76 6 56.27 6 358 6 359 6 3510 10.24 3511 3 2012 6 3513 9 20

95

Where as xi is the coded value of the ith independent variable, Xi the natural value

of the ith independent variable, X0 the natural value of the ith independent variable

at the center point, and ∆Xi the step change value of variables. For a three-factor

system, the model equation is:

...... (4.4)

Where: Y-predicted response; b0-intercept; b1, b2, and b3 - linear coefficients; b11,

b22, and b33-squared coefficients; and b12, b13, and b23-interaction coefficients

(Myers and Montgomery, 2002).

4.5.3 Modified composition of production medium GOxM 3

Based on the above observation the following composition was formulated

for the maximum production of GOx medium. The environmental factors are also

proposed for the high production of GOx from A. awamori MTCC 9546. The

modified (GOxM 3) media consists of (g/l);

Glucose - 92.7

Proteose peptone - 3.24

(NH4)2HPO4 - 0.48

KH2PO4 - 0.32

MgSO4 - 0.23

CaCO3 - 36.8

Distilled water - 1000 ml

pH - 5.83

Temperature - 30.7°C

96

4.6 Production of GOx by laboratory batch fermentor

In this study two litre laboratory batch fermentor was used (Lark

Innovation, Chennai, India). After the optimization of medium, the GOx

production was carried out in the fermentor equipped with the instrumentation for

the measurement and control of temperature and pH with working volume of one

litre culture medium. The fermentor vessel containing the optimized production

medium (one litre) was moist heat-sterilized at 121ºC for 20 minutes. The medium

was inoculated with 5% of conidiospore suspensions (2x107 spores/ml) of a 24 h

culture from A. awamori MTCC 9645.

Agitation was performed with a double four bladed impeller at 200-500

rpm. It was carried out at 200 rpm for the initial five hours of the fermentation and

increased up to 500 rpm for the rest of the fermentation period. Aeration was

performed by membrane filtered sterile air passed through the sparger. In the

initial hours the agitation and aeration were decreased to prevent the spore

flotation and adhesion to the walls of the fermentation vessels. The dissolved

oxygen content was maintained at 11 to 12 mg/l during the fermentation. The pH

level was maintained at 5.83±0.2 and it was maintained by the addition of 2N

NaOH or 2N HCl. Fermentation was carried out up to 90 h at 30.7ºC.

4.7 Morphological studies

Fungal morphological characteristics like spore aggregation and pellet

formation were checked during the early stage of fermentation in the bioreactor,

that was checked by slightly modified method of Hesham El-Enshasy (2006).

4.7.1 Quantification and qualification of different types of bioparticles

Any discrete biomass in a culture was considered as `bioparticle`;

bioparticles may consist of a single spore, an aggregation of spores or fungal

pellet.

97

4.7.2 Large pellets (>3 mm diameter)

A sample of the suspension was transferred to a petriplate and the diameter

of pellets was measured using a varnier caliper. Calibration was achieved by

means of a standard millimeter scale. Pellet diameters were measured and the

mean value of the pellet diameter was calculated.

4.7.3 Small bioparticles (< 3 mm diameter)

Small bioparticles (spores, aggregates and small pellets) were obtained

during the cultivation and measured by an ocular micrometry. The maximal

magnification of this unit was 1500X. The average diameter of both spores and

small aggregates were measured manually. The microscopic magnification was set

at 1000X and the bioparticles were observed randomly. In all cases, the data

represented in this study are an average of randomly selected (80–100)

bioparticles.

4.7.4 Measurement and calculations

The average diameter of each bioparticle and length in case of germ tube

was measured. The microscope magnification was set at 50X for pellet

measurement and 450X for spore, germ tube and hyphae measurement. For each

sample, the process was repeated at least 25 times using new positions on the

same and on different bioparticles. The microscopic morphology, that is the

average total hyphal length and the average diameter of the hyphal element, has

been quantified during batch cultivation.

98

4.7.5 Time course study on cell growth, GOx production, substrate utilization

and acid formation during fermentation period

The GOx production, substrate glucose utilization, gluconic acid formation,

protein secretion and cell growth were studied during the fermentation period at

12 h of intervals up to 120 h.

4.8 Purification and characterization of GOx

4.8.1 Preparation of enzyme for purification

After the fermentation, the fermented broth was filtered through Whatman

No.1 filter paper and centrifuge at 10,000 g for 10 minutes. The cell free extract

(600 ml) was mixed with 150 ml of GOx buffer (Sodium acetate buffer 50 mM,

pH 5.5) because ammonium sulphate is known to slightly acidify the extract. The

gradient ammonium sulphate precipitation was carried out [40–85% (w/v)] at 4ºC

with constant stirring. The aggregated proteins were separated by centrifugation at

10,000 g for 15 minutes. All the gradient precipitations were analyzed for protein

and GOx activity. The maximum GOx activity producing precipitates were

dialysised and concentrated by lyophilization.

4.8.2 Dialysis

Dialysis membrane of 110 kDa cut-off range was selected (Hi-Media,

Mumbai, India) and required length was taken. The dialysis membrane consists of

glycerate a plasticizer and some sulphorous compound as stabilizer. It was

activated by immersing the membrane in hot water for 15 minutes at 80°C. The

dialysis tube was then washed with Millipore water. One end was tighten with the

help of dialysis tube closure and the sample was loaded leaving a sufficient head

space the other end was also tightened and then immersed in sodium acetate buffer

(10 mM, pH 5.5). It was kept in a magnetic stirrer and the buffer was changed in

an interval of 4, 12 and 24 h.

99

4.8.3 Lyophilization

The dialyzed protein sample was concentrated by lyophilization. Freeze

drying was performed at the machine working (pump) temperature of -70±5 °C

and 0.01 mbar vacuum. The lyophilized enzyme was stored at -20 °C and it was

used for further purifications.

4.8.4 Ion exchange chromatography

The partially purified enzyme was loaded in to DEAE cellulose anion

exchange column (16 h x 2.4 dia. cm gel bed) pre-equilibrated with 100 ml of 50

mM sodium acetate buffer (pH 5.5). After that the protein sample was loaded and

the bound protein was eluted with linear gradient from 50–400 mM NaCl in

sodium acetate buffer (50 mM, pH 5.5). Elution was performed at 2.0 ml/minute

flow rate and the 5.0 ml fractions were collected separately. The GOx activity and

protein content were analyzed in all the fractions. The high GOx activity

contained fractions were pooled together and dialyzed. The dialyzed suspension

was lyophilized and stored at -20 °C.

4.8.5 Size exclusion chromatography

Sephacryl S-200 (Pharmacia Biotech, Sweden) was allowed to swell

overnight in sterilized distilled water. The floating gel beads were removed and

the gel slurry was packed into a glass column (10 h X 1.6 dia. cm), which

contained sintered filter at bottom. While packing, care was taken to avoid air

bubbles. The packed gel column was equilibrated with 10 mM sodium acetate

buffer pH 5.5.

Concentrated enzyme was loaded on to Sephacryl S-200 column and eluted

with 10 mM sodium acetate buffer (pH 5.5) at the flow rate of 3 ml per 10

minutes. The GOx activity and protein content were analyzed by previously

100

described method and the active fractions were pooled together and concentrated

by lyophilization.

4.9 Polyacrylamide Gel Electrophoresis (PAGE)

The purity of the GOx sample was checked by SDS-PAGE. SDS-PAGE

was performed in 10% polyacrylamide and the proteins were detected with

coomassie brilliant blue R-250 (Laemmli, 1970).

4.9.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-

PAGE)

The SDS-PAGE is a standard technique used for the qualitative analysis of

protein mixtures. Since the method involves the separation of proteins according

to size, it is useful in determining the relative molecular weight of proteins and

monitoring enzyme purification. SDS is an anionic detergent and under defined

experimental conditions the relative electrophoretic mobilities of proteins in the

presence of SDS are related to their relative molecular masses. Samples to be run

on SDS-PAGE are boiled for 5 minutes in the presence of β-mercaptoethanol and

SDS. The β-mercaptoethanol results in the reduction of any tertiary structure

disulphide bonds and the SDS strongly binds the proteins and denatures them.

Each protein in the mixture is fully denatured into a rod-shaped structure

containing a series of negatively charged SDS molecules along the polypeptide

chain.

The SDS-poly acrylamide gel electrophoresis was performed on slab gel

with separating gels (10 and 5% w/v) by the following method of Laemmli

(1970).

101

Reagents

Stock solutions

Solution A 1.5 M Tris-HCl buffer (pH 8.8) with 0.4% (w/v) SDS

Solution B 0.5 M Tris-HCl buffer (pH 6.8) with 0.4% (w/v) SDS

Solution C 30% (w/v) acrylamide with 0.8% bisacrylamide

Solution D 1.4% ammonium persulphate

Solution E 1% SDS

Solution F N,N,N,N’ tetramethyl ethylene diamine (TEMED)

Preparation of gel

Separating gel [10% (w/v)] Stacking gel [5% (w/v)]

Solution A 0.75 ml Solution B 0.38 ml

Solution C 2.0 ml Solution C 0.50 ml

Solution D 0.3 ml Solution D 0.15 ml

Solution E 0.6 ml Solution E 0.3 ml

Millipore water 2.6 ml Millipore water 1.98 ml

Solution F 0.005 ml Solution F 0.005 ml

102

Sample buffer

Glycerol 2.0 ml

β-mercaptoethanol 1.0 ml

10% SDS (w/v) 4.0 ml

Solution B 1.7 ml

Bromophenol blue (aqueous) 0.2 ml

Millipore water 0.6 ml

Tank buffer (pH 8.3)

Tris 3.0 ml

Glycine 14.4 ml

SDS 1.0 ml

Millipore water 1.0 ml

Procedure

The enzyme solution was mixed with an equal volume of sample buffer,

boiled in a water bath for 5 minutes, cooled and added to the wells. Then the

power supply was connected with cathode in the upper tank and anode in the

lower tank. Electrophoresis was carried out at room temperature with at 20mA

current supply until the tracer dye reached 0.5 cm above the lower end.

103

Staining of separated proteins

At the end of electrophoresis, gel was removed and stained with CBB stain

[40% methanol, 0.7% acetic acid, 0.075% Coomassie dye (CBB-R250)] and

destained using 40% methanol, 0.7% acetic acid. The subunit molecular mass of

GOx from A. awamori MTCC 9645 was determined by calibration against a set of

protein standards.

4.9.2 Native polyacrylamide gel electrophoresis

Native-PAGE is also known as non-denaturing gel in which the entire process

does not contain SDS. It helps in studying the character of the protein such as

without denaturing the protein. It is used to study the enzymes. As the protein will

be intact, the protein bonds will be less in native-PAGE than SDS-PAGE. The

sample buffer does not contain SDS and β-mercapto ethanol. The sample is not

heated here. In native the bands are resolved based on molecular weight and net

charge. Native-PAGE was performed as per the method of Davis, (1964).

Reagents

Solution A Solution B

1 N HCl 48.0 ml Acrylamide 30.0 g

Tris 36.6 g N,V’-methyl bisacrylamide 0.8 g

Millipore water 100 ml

Millipore water 100 mlSeparating gel (pH 8.8)

Stacking gel (pH 6.8)

104

Solution C Solution D

Ammonium persulphate 0.14 g N,N,N,N’-tetramethyl ethylene diamine

(TEMED)Millipore water 100 ml

Separating gel [8% (w/v)] Stacking gel [5% (w/v)]

Solution A 0.74 ml Solution A 0.35 ml

Solution B 2.0 ml Solution B 0.50 ml

Solution C 0.3 ml Solution C 0.15 ml

Millipore water 2.89 ml Millipore water 2.0 ml

Solution D 0.005 ml Solution D 0.005 ml

Tank buffer (pH 8.3)

Tris 60.0 g

Glycine 28.8 ml

Millipore water 100 ml

Tank buffer stock solution, was made up to 100 ml with sterile Millipore

water, adjusted to pH of 8.3 and used as tank buffer. Slab gel electrophoresis was

carried out on glass plates of 10.5 x 10.5 cm.

105

Electrophoresis procedure

Polymerization of separating gel was carried out on the glass plates.

Stacking gel was polymerized over the separating gel after inserting a comb. The

known amount of enzyme sample mixed with sample buffer with bromophenol

blue was loaded into the wells and then the power supply was connected with

cathode in the upper tank and anode in the lower tank. Electrophoresis was carried

out at 4°C with constant voltage and 20mA current supply for 2 h until the tracer

dye reached 0.5 cm above the lower end.

4.9.2.1 Zymogram analysis

The native page was developed by overlaying of 1.5% soft agar containing

glucose, o-dianisidine and horse radish peroxidase and kept in dark for 10 minutes

for development of brown colour.

Preparation of soft agar

Agar 1.5% (w/v)

o-dianisidine 1.0 ml [0.21mM(w/v)]

Horse radish peroxidase 0.3 ml (100U)

Glucose 2% (w/v)

4.9.3 Confirmation of enzyme activity using plate assay

Two percentage (w/v) of agar with substrate [5% (w/v) of glucose] was

prepared and poured in a Petri plate. Three wells of 6mm size were made with the

help of cork borer. One kept as control, one well with 50 μl of purified enzyme

and the other well with 50 μl of authentic commercial GOx (Sigma, GOx from A.

niger) enzyme and observed for brown colour formation.

4.10 Characterization of glucose oxidase

4.10.1 Kinetic charecterization

106

100samplecontrolinactivityEnzymesamplein treatedactivityEnzymeactivityrelativeof% ´=

The apparent Km for glucose of the purified GOx was determined by

measuring initial velocities over a range of glucose concentration (100mM to

1000mM) and constant enzyme concentration (20 µg protein). The kinetic

constants for the purified GOx was determined from Lineweaver’s Burk plot,

Hanes-Woolf linear plot and Eadie-Hofstee plot. The Michaelis constant (Km), the

maximal limiting rate velocity (Vmax) were all calculated.

4.10.2 Effect of temperature and pH on GOx activity

The effect of temperature and pH on GOx activity was determined by

Simpson et al. (2006) method. The GOx assay reagents were equilibrated for 10

minutes at the temperatures ranging from 20 to 80°C, before initiating the

reactions with the addition of the GOx at 0.2 U/ml. The pH profile for GOx was

performed in universal buffer containing 50mM potassium dihydrogen

orthophosphate, 33mM citric acid and 50.7 mM boric acid, adjusted the pH values

ranging from 3 to 9 with potassium hydroxide. The assay reagent buffer was

replaced with universal buffer and a GOx concentration of 0.4 U/ml was used to

initiate the reaction. The activity of GOx was expressed as relative activity and it

was calculated by the formula as,

……. (4.5)

4.10.3 Stability testing

The stability of purified GOx was determined based on the method

described by Simpson et al. (2006). It was performed at 25 and 37°C, since these

temperatures correlated to potential applications of GOx, namely implantable

glucose biosensors for human application and food processing (operating at 37°C

and physiological pH of 7.2) and glucose determination test strips (room

temperature operation 25°C). Purified GOx at a concentration of 0.15U/ml was

prepared by dissolution in Millipore water. Volumes of 20 ml were placed in

107

water baths pre-equilibrated to ~25 and 37°C. Samples (0.5 ml) were removed

periodically (10-90 minutes) and analyzed for GOx activity. The GOx activities of

the samples were compared to an initial sample taken at the onset of the

experiment. A lyophilized GOx preparation was stored at -20°C and assayed

monthly for 6 months to provide an indication of shelf-life.

……. (4.6)

4.10.4 Stability and inhibitory studies of GOx

4.10.4.1 Effect of metal ions on GOx activity

The effects of metal ions on enzyme activity were studied at 1 mM

concentration of different metal ions such as lead acetate, cobaltous chloride,

copper acetate, silver sulphate, copper sulphate and mercuric chloride. The 100µl

of enzyme with enzyme activity of 1U/ml was incubated along with 0.9 ml of 1

mM metal ions for 20 minutes. After the incubation period the enzyme activity

was analyzed.

4.10.4.2 Effect of calcium ions on GOx activity

Enzyme (0.1 ml) with the activity of 1 U/ml was incubated with 0.9 ml of 1

mM calcium ions such as calcium carbonate, calcium lactate and calcium

propionate. The enzyme activity was measured after 20 minutes.

4.10.5 Preparation of carrier based enzyme

The enzyme active fractions were mixed with the aqueous stabilizing solution

(Poly ethylene glycol, 1% w/v). The above mixture was freeze dried and stored at

-20°C. The carrier based enzyme was used for further application processes.

4.11. APPLICATION OF GLUCOSE OXIDASE IN FOOD PROCESSING

AND PRESERVATION

100incubationafteractivityEnzymeincubationbeforeactivityEnzymeactivityresidualof% ´=

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4.11.1 Enhancing the storage stability of vegetable by coating of edible film

incorporated with glucose oxidase, lactoperoxidse and lysozyme

This present study was to evaluate the applicability of antimicrobial effect

by purified GOx, partially purified LPS and lysozyme with EDTA for enhancing

the shelf-life of carrot.

4.11.1.1 Microorganisms and culture conditions

The pure culture of E. coli (MTCC 443) and S. aureus (MTCC 96) were

obtained from Microbial Type Culture Collection, Chandigargh, India. The

cultures were frequently subcultured and maintained in Luria Bertani (LB) and

nutrient agar slants at 4ºC respectively. The composition of the LB medium are:

tryptone 10.0 g; yeast extract 5.0 g; NaCl 10.0 g distilled water 1.0 litre and the

nutrient agar medium are: beef extract 1.0g; yeast extract 2.0 g; peptone 5.0 g

NaCl 5.0 g; agar 15.0 g and distilled water one litre.

4.11.1.2 Preparation and analysis of antimicrobial enzymes

Glucose oxidase

The GOx was produced from Aspergillus awamori MTCC 9645. GOx

activity was measured according to Bergmeyer, (1988).

Lactoperoxidase

The partially purified LPS was obtained from whey permeate by Martin

Morrison (1957) method. The initial step in the preparation of whey permeate was

prepared from one litre of milk was taken and heated to 50ºC. Then 10 ml of

acetic acid solution was added to the milk. After that the milk was stirred at room

temperature until coagulation occurred. All subsequent steps were carried out at

4ºC. The coagulated milk was centrifuged for 15 minutes at 3000 g. The pellet and

the solid casein were removed from milk and the supernatant was collected for the

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preparation of partially purified LPS. The collected sample was subsequently

subjected to ammonium sulphate for protein precipitation.

Forty millilitre of wet Amberlite IR-120 (NH4+, form) was added to one

litre of whey permeate and then the suspension was stirred for 3 h and allowed to

stand for 30 minutes to let the resin settle. The resin was then transferred to a

Buchner funnel, and was washed with water and 50 mM sodium acetate (1 litre

and two litres each per litre of whey, respectively). Then resin was transferred to a

column (1.5 X 50 cm) and elution was carried out in two steps using 0.5 M

sodium acetate and 1.0 M sodium acetate at a flow rate of 2-5 ml/minutes. The

ammonium sulphate precipitated protein sample containing LPS was eluted using

0.5M sodium acetate solution and this elution was lyophilized. The partially

purified LPS was stored at -8 ºC.

The enzyme activity of partially purified LPS was measured according to

the method described by Shindler et al., (1976). The oxidation of 3’3, 5,5’-

tetramethylbenzidine (TMB) (Sigma) by LPS was measured

spectrophotometrically at 413 nm using UV-Visible spectrophotometer. The

reaction was conducted in 0.1 M acetate buffer, pH 5.2 at 20°C containing

appropriate concentrations of TMB, HZ02 (Fisher) and LPS in a volume of 1 ml.

Result was expressed in units/ml, where one unit (U) is defined as the amount of

enzyme that oxidizes 1µmol TMB/min.

Lysozyme

The partial purification of lysozyme was carried out by Çifdem Mecitoflu

et al. (2006) method. Lysozyme was prepared from the hen egg. The egg whites

and yolks were separated and the egg white portion was used for the partial

purification. The separated egg white was diluted with two volumes of 0.05M

NaCl solution. The lysozyme was precipitated from the mixture of proteins in the

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egg white by lowering the pH. It was performed by bringing down the pH up to

4.0 by using 1N acetic acid and it was diluted with equal volume of 60% (v/v)

ethanol. It was stored at room temperature for 6 h and the mixture was centrifuged

at 15,000 g for 15minutes at 4°C. Precipitate was removed and the supernatant

containing lysozyme was first dialyzed with cellulose acetate membrane (5 kDa,

Hi-Media, India) at 4°C for 21 h by three changes of 2000ml distilled water. Then

it was concentrated by using lyophilizer at the machine working (pump)

temperature of -70±5°C and 0.01 mbar vacuum. The lyophilized enzyme was

stored at -20 °C and it was used for the film preparation.

The lysozyme enzyme activity was analyzed by spectrophotometrically at

A660nm adopted by Çifdem Mecitoflu et al. (2006). The reaction mixture was

formed by mixing 0.1 ml of enzyme extract or enzyme containing solution can be

prepared by dissolving films in distilled water. Then 2.9 ml of Micrococcus

lysodeikticus cell suspension was prepared (0.26 mg/ml, prepared in 0.05 M Na-

phosphate buffer at pH 7) and incubated at 30°C. The mixture was rapidly

vortexed and immersed into a water bath at 30°C. The absorbance of the reaction

mixture was determined at the end of first minute and the difference between

absorbance value and the initial absorbance value was used for the calculation of

enzyme activity (the absorbance-time curves were linear for 1.5–2 minutes). The

enzyme activity was expressed as units (0.001 absorbance change in one minute).

The average of three measurements was used in all tests.

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4.11.1.3 Evaluation of antimicrobial activity of GOx, LPS and lysozyme with

EDTA

The antimicrobial activity of GOx, LPS and lysozyme with EDTA was

determined against E. coli and S. aureus. The E. coli and S. aureus were taken

from the stored culture and grown in 10 ml of fresh nutrient broth at 37ºC for 24 h.

One millilitre of the culture was centrifuged at 8000 g for 20 minutes. After

centrifugation, the supernatant was discarded and the bacterial pellet was

resuspended with phosphate buffer solution (PBS - pH 7.0). The effect of

antimicrobial enzyme activity was evaluated by the formulations like, (I) GOx,

(II) LPS (III) Lysozyme with EDTA (IV) GOx and LPS, (V) GOx, lysozyme with

EDTA, (VI) LPS and lysozyme with EDTA, (VII) GOx, LPS and lysozyme with

EDTA, (VIII) Control. For every formulation one millilitre of PBS bacterial

suspension was added. The enzyme concentration in the test solution contains

GOx (5 mg/ml), LPS (10 mg/ml) and lysozyme (0.5 mg/ml) with EDTA (0.3

mg/ml). The control treatment contains no antimicrobial enzymes. Triplicates

were incubated at 37ºC for 48 h on rotary platform shaker at 250 rpm. The treated

formulations were analyzed for every 6 h of incubation up to 48 h. The inhibitory

activity of the E. coli and S. aureus was analyzed by serial dilution in PBS by

plating in duplicate on nutrient agar and incubating plates at 37ºC for 48 h. The

results were expressed in colony forming units per millilitre (log CFU/ml).

4.11.1.4 Film making

Film making was carried out by the modified method of Çifdem Mecitoflu

et al. (2006). One gram of sodium alginate was dissolved in 10 ml of distilled

water and glycerol (0.50 ml) was then added to the alginate solution. This mixture

was dissolved thoroughly by using boiling water bath with constant stirring and

boiled for 5 minutes.

112

The hydrocolloid was brought down to room temperature. Then the film

solution was prepared by mixing with GOx, LPS and lysozyme and di-sodium

EDTA.2H2O to get final concentrations of GOx (5 mg/ml), LPS (10 mg/ml) and

lysozyme (0.5 mg/ml) with EDTA (0.3 mg/ml) di-sodium EDTA 2H2O. This bio-

film hydrocolloids was made to stand by for 30 minutes to avoid air bubbles. Then

the film solution was poured on a clean glass plate to form a uniform layer. The

films were dried at room temperature and 6 mm diameter film discs were cut by

using standard stainless steel well cutter. This film discs were used for

antimicrobial test. The above mentioned different formulations of enzymes were

added to the film (alginate) forming solution.

4.11.1.5 Antimicrobial film activity

Antimicrobial activity of the film was performed by using E. coli and

S. aureus as test microorganisms. The over night cultures of E. coli and S. aureus

were prepared in nutrient broth and incubated at 37ºC for 48 h incubation. For

antimicrobial test, discs (six mm in diameter) were prepared from each film by

sterile well cutter under aseptic condition. These discs were placed carefully onto

Petri dishes containing nutrient agar on which 0.1ml culture was spreaded. The

Petri dishes were incubated at 37°C for 24 h and the zone of inhibition (diameter)

was measured with the help of centimeter scale.

4.11.1.6 Surface sterilization of carrots

Fresh carrots (Daucus carota sp.) were purchased in a local supermarket

and stored at 4°C before processing. In this study chlorine dioxide has been

chosen for surface sterilization of carrots. The concentration and soaking time

were optimized for efficient and minimal use of chlorine dioxide. The samples

were sanitized with chlorine dioxide at different concentrations (2, 4, 6, 8 and

10ppm) prepared from the stock solution. Carrots were surface washed with these

different concentrations of chlorine dioxide solution at different time periods (10,

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20 and 30 minutes). After surface sterilization, the sterility was determined by

soaking in sterile distilled water. The microbial population in soaked water were

measured by viable plate method. Number of microbes ware measured as colony

forming units.

4.11.1.7 Coating procedure

Carrots were surface sterilized with chlorine dioxide (6 ppm) for 20

minutes. Surface sterilized whole carrots were taken for alginate coating with

different enzyme formulations. Fresh carrots were dipped completely into the

coating solution for 5 seconds at room temperature and then taken out. This

dipping procedure was repeated thrice and excess coating material was allowed to

drain completely. Then it was immersed in 2% (w/v) of calcium chloride solution

for 5 minutes and the coating was dried with an air blower. Coated and uncoated

carrots were kept in both room temperature (~26°C) and (6°C) before the analysis

of water loss.

4.11.1.8 Determination of weight loss

The shelf-life can be defined as the length of time which the vegetable can

maintain their appearance, safety and nutritional value that appeals to the

consumer. So water loss (weight loss) and soluble protein content were monitored

in carrots stored at both ~26°C and at 6 °C. The coated and uncoated carrots were

weighed and packed in perforated polypropylene covers and kept at both ~26°C

and 6 °C. They were reweighed every 48 h in order to follow the moisture loss as

a function of time over a period of 10 d. A digital electronic balance (Shimadzu,

Japan) was used to determine the product weight. Weight loss percentage

calculated was calculated using the formula,

……. (4.7)

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where: %WL(t) is the percentage weight loss at time t, W0 is the initial sample

weight and W(t) is sample weight at time t.

4.11.1.9 Determination of soluble protein content

Soluble protein content was measured by taking 0.1 g of freeze dried carrot

sample mixed with of two millilitre distilled water and vortexed for 5 minutes at

room temperature. The tubes were centrifuged for 20 minutes at 5000 rpm. The

supernatant was collected in eppendorf tubes and stored at 4°C for 2 h before

analyzing. The soluble protein content of the extracted samples was estimated

according to the method of Bradford (1976). Protein extract was diluted 1:50 in

distilled water and one millilitre of sample was mixed with 5 ml of Coomassie

Brilliant Blue G-250 solution and incubated at room temperature for 20 minutes.

Absorbance value was measured at 595nm in UV-Vissible spectrophotometer.

4.11.1.10 Microbial analysis by viable plate count method

Ten grams of carrot sample was taken and mixed with 90 ml of sterile

distilled water and vortexes for 10 minutes. Then it was serially diluted up to 10-8

dilutions. One millilitre of 10-6 dilution was delivered to Petri dishes containing

nutrient agar medium and spread uniformly. The plates were incubated at 37ºC for

24 h and the results were expressed in colony forming units (CFU/g) of sample.

4.11.1.11 Sensory analysis

The sensory qualities of treated and untreated carrots were performed

during the storage period using five member trained panel with an age of 25–35.

The panel consists of three females and two males who were trained to be familiar

with sensory properties of carrots. The sensory testing method was an acceptance

test in which the sensory parameters were scored on a descriptive scale of 1–5.

The investigated sensory parameters include: (i) taste, (ii) colour, (iii) texture, (iv)

appearance and (v) overall acceptance. Descriptions for each score were as

115

follows: 5-likely very much, 4-likely slightly, 3-neither like nor dislike 2-dislike

slightly and 1-dislike very much. Sensory trials were replicated thrice.

4.11.2 Control of browning and enhancing shelf-life of apple puree by

applying glucose oxidase-catalase system with lactoperoxidase

4.11.2.1 Preparation and analysis of antimicrobial enzymes

GOx: The GOx was produced from Aspergillus awamori MTCC 9645 and the

activity was measured according to Bergmeyer et al. (1988).

Lactoperoxidase: The partially purified LPS was obtained from whey permeate

by Martin Morrison (1957). The enzyme activity of partially purified LPS was

measured according to the method described by Shindler et al., (1976).

Catalase: Enzyme Catalase was brought from M/s Speciality enzymes and

Biochemicals Co, USA. Catalase activity was measured spectrophotometrically by

observing the decrease in light absorption at 525 nm during decomposition of

H2O2 by enzyme. The reaction mixture contained one millilitre of 0.1 M McIl-

Valine buffer pH 6.8, 0.05 ml of suitability diluted enzyme, and 0.03 mg of 100

mM H2O2 solution and was incubated for 30 minutes at 30º C. The reaction was

developed with 0.2 mg o-di-anisidine, 0.06 mg peroxidase (500/mg), and 0.8 ml

glycerol in 2ml of 0.2 M. Tris-phosphate buffer pH 6.8. After 30 minutes

incubation at 30ºC, the enzymatic reaction was stopped by adding 4 ml of 5 N

HCl. The absorbance at 525 nm was measured against a blank, i.e., a control

mixture composed and incubated as described above but deprived of hydrogen

peroxide and peroxidase. One unit (U) of catalase activity was defined as the

amount of enzyme catalyzing the decomposition of 1µmol hydrogen peroxide per

minutes at 30ºC.

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4.11.2.2 Determination antimicrobial activity of GOx, LPS and catalase

The antimicrobial activity of GOx, LPS and Lysozyme with EDTA was

determined against E. coli (MTCC 443) and S. aureus (MTCC 96. The E. coli and

S. aureus were taken from the stored culture and grown in 10 ml of fresh nutrient

broth at 37°C for 24 h. One millilitre of the culture was centrifuged at 8000 g for

20 minutes using eppendorf centrifuge model. After centrifugation, the

supernatant was discarded and the bacterial pellet was resuspended with

phosphate buffer solution (PBS-pH 7.0). The effect of antimicrobial enzyme

activity was evaluated by the formulations (mg or ml /10ml) like, of (I) GOx (100

mg), (II) LPS (40mg), (III) Catalase (0.1ml), (IV) GOx (100mg) +LPS (40mg) (V)

GOx (100mg)+Catalase (0.1ml), (VI) LPS (40mg)+Catalase (0.1ml) and (VII)

GOx (100mg)+Catalase (0.1ml)+LPS (40mg), (VIII) Control.

For every 10 ml of enzyme formulation one millilitre of PBS bacterial

suspension was added. The control treatment contains no antimicrobial enzymes.

Triplicate were incubated at 37°C for 48 h on rotary platform shaker at 250 rpm.

The treated formulations were analyzed for every 6 h of incubation up to 48 h.

The inhibitory activity of the E. coli and S. aureus was enumerated by serial

dilution in PBS by plating in duplicate on nutrient agar and incubating plates at

37°C for 48 h. The results were expressed in colony forming units per millilitre

(CFU/ml) of sample.

4.11.2.3 Preparation of apple puree

Fresh fruits of golden delicious apples were purchased in local super

market, Chennai, India. Apples were culled and apples with fine shiny skins which

were free of physical damage. The apple puree sample was prepared based on the

method described by Parpinello et al. (2002). The apple puree was extracted by

the following process. The selected apples were washed thoroughly, peeled with

stainless steel clean knife and the peeled apples were cut in to small pieces. It was

117

heated at 95ºC for 5 minutes in hot water bath and ground with domestic juice

extractor (Preethi Chepro Plus, India) until puree was obtained. Then it was heated

at 95ºC for 10 minutes, cooled up to 25ºC, homogenized and stored at 4ºC. The

fruit puree was characterized by the following parameters: colour (browning),

dissolved oxygen content, microbial analysis and sensory analysis.

4.11.2.4 Effect of GOx and ascorbic acid on dissolved oxygen consumption

Measurement of dissolved oxygen (DO) was performed in digital DO

Meter Model 801(EI), India. The DO probe was placed in 2% (w/v) of sodium

sulphite solution. The display is allowed to attain equilibrium. The zero knob was

brought to display to read 0.0 by keeping the temperature knob to actual

temperature of the solution. Calibrate knob was then brought to extreme right

position. As given in the instrument manual chart water at 25ºC was taken as the

standard which has an oxygen solubility of 8.2. The above mentioned steps were

repeated with the apple puree samples which were treated with GOx 50, 100 and

150 mg; ascorbic acid 50, 100 and 150 mg/l concentration per litre of apple puree.

4.11.2.5 Experimental design

The effect of browning (colour changes) and antimicrobial activity on apple

purees for combination of GOx-catalase system and LPS. The effect of

antimicrobial enzyme activity was evaluated by the formulations (mg or ml /l of

puree) like, of (I) GOx (100 mg), (II) LPS (40mg), (III) Catalase (0.1ml), (IV)

GOx (100mg) +LPS (40mg) (V) GOx (100mg)+Catalase (0.1ml), (VI) LPS

(40mg)+Catalase (0.1ml) and (VII) GOx (100mg)+Catalase (0.1ml)+LPS (40mg)

and (VIII) Control.

4.11.2.6 Evaluation of browning on apple puree

Right after the extraction of puree and the addition of respective

combination of enzymes and antibrowning agents, the puree was bottled and

118

stored at room temperature in 750 ml glass bottles with cork caps. Browning was

observed during storage by analyzing all the samples at an interval of 5 days up to

30 days. The sample of 20 ml apple puree was taken from each sample and

centrifuged at 10000 g for 15 minutes. The browning was evaluated in the

supernatant by measuring the absorbance at 420 nm. Browning is characterized by

spectro photometric measurements at 420 nm to detect browning pigments

(Toribio and Lozano, 1986; Nagy et al., 1990; Wong and Stanton, 1992).

4.11.2.7 Examination of microbial populations

The prepared samples with various combinations of enzymes and

antibrowning agents were bottled and corked. These corked bottles were stored at

room temperature. These samples were checked for the antimicrobial activity at an

interval of 5 days up to 30 days. The microbial population was enumerated from

the apple puree by viable plate count method as described earlier. One millilitre of

the apple puree sample was uniformly distributed to the nutrient agar medium and

incubated for 24 h at 37°C. The results were expressed in CFU/ml of apple puree.


The sensory qualities of apple puree were performed during the storage

period using 5 member trained panel with an age of 25 to 35 years. The panel

consists of two females and three males who were trained to be familiar with

sensory properties of apple puree. The sensory testing method was an acceptance

test in which the sensory parameters were scored on a descriptive scale of 1-6. The

sensory parameters investigated included the following: (i) taste, (ii) colour, (iii)

flavour, (iv) texture, (v) appearance and (vi) overall acceptance. Descriptions for

each score were as follows: 5–likely very much, 4–likely slightly, 3–neither like

nor dislike 2–dislike slightly and 1–dislike very much. Sensory trials were

replicated thrice.

119

4.11.3 Studies on the effect of glucose oxidase-catalase with calcium ions

in stabilizing and improving the fruit salad quality

4.11.3.1 Preparation of enzyme

The GOx was produced from A. awamori (MTCC 9645) and the activity

was measured according to Bergmeyer et al. (1988). Catalase was brought from

M/s Speciality enzymes and Biochemicals Co, USA and analyzed as described

earlier.

4.11.3.2 Collection of fruits

Fruits like apple, guava, orange, grapes, mango, banana, cherry and lemon

were purchased from local super market, Chennai. Fruits were selected based on

the characters like without physical damage, without microbial spoilage, matured

and equal in size.

4.11.3.3 Preparation of koruk juice

Unripe grapes were purchased from the agriculture farm located at

Dindigul in Tamilnadu. The stalks of the grapes were removed and the grapes

were washed. The grapes were weighed to one kilogram and crushed thoroughly

in grinder. The extracted juice was used for fruit salad preparation.

4.11.3.4 Analysis of antimicrobial activity of GOx, calcium ions and koruk

juice

The antimicrobial activities of calcium ions, GOx and koruk juice were

tested against E. coli (MTCC 443) and S. aureus (MTCC 96). Phosphate buffer

solution was prepared with E. coli and S. aureus (approximately 9 logs CFU/ml).

One ml of PBS solution was treated with 50 µl of following test formulations. The

effect of antimicrobial activity was evaluated by the different treatments like,

120

I) GOx (5mg/ml), II) calcium lactate (2% w/v), III) calcium chloride

(2% w/v), IV) calcium propionate (2% w/v), V) optimized calcium ions

combination (2% w/v), VI) koruk juice (2% v/v), VII) GOx + koruk juice

+calcium ions combination (2% w/v) and VIII) Control.

A control sample consisted of bacterial pellets dissolved in one millilitre of

PBS, which contained no antimicrobial formulation. Six sets of PBS cell

suspension were prepared for each formulation respectively. Triplicate samples

were then incubated at 37°C for 48 h on a rotary platform shaker at 250 rpm. The

E. coli and S. aureus were enumerated by serial dilutions in PBS, plating in

duplicate on nutrient agar and incubated at 37°C for 48 h. The treated samples

were analyzed by every six hours of incubation up to 48 h.

4.11.3.5 Preparation of treatment solution

1. Three different concentrations of GOx like 0.25, 0.5 and 1.0% (w/v) were

dissolved in sterilized distilled water.

2. Calcium lactate, calcium propionate and calcium chloride were prepared at

the concentrations of 0.5, 1.0 and 2% (w/v) in sterile distilled.

3. The extracted koruk juice at the concentration of 0.5, 1.0 and 1.5% (v/v)

mixed in sterilized distilled water.

4. Combined calcium ions (0.5, 1.0, 1.5, 2.0 and 2.5% w/v) with 0.25% (w/v)

of GOx-catalase and Koruk juice (1% v/v) were mixed sterile water.

4.11.3.6 General fruit salad preparation procedure

Fresh fruits like apple, guava, orange, grapes, mango, banana, cherry and

lemon were washed with water and cut it small pieces using sterilized stainless

steel knife. Then it was soaked for 15 minutes in the above treatment solutions.

After that the fruits were taken out from the solutions and used for fruit salad

preparation. Lemon juice (4 ml) was poured to the 1.0 Kg sliced fruits and 150 g

121

of half melted sugar solution added. Then it was transferred to a clean plastic

container and stored at room temperature.


The sensory quality was analyzed after a day of storage such as taste,

colour, texture, appearance and overall acceptance. These qualities were

performed using 5 member trained panel with an age of 25 to 35 years. The panel

consists of three females and two males who were trained to be familiar with

sensory properties of fruit salad. The sensory testing method was an acceptance

test in which the sensory parameters were scored on a descriptive scale of 1-6. The

sensory parameters investigated included the following: (i) taste, (ii) colour, (iii)

texture, (iv) odour and (vi) overall acceptance. Descriptions for each score were as

follows: 5–likely very much, 4–likely slightly, 3–neither like nor dislike 2–dislike

slightly and 1–dislike very much. Sensory trials were replicated thrice.

4.11.3.8 Experimental design of RSM for optimization of calcium ions

The optimization of calcium ions was performed using Design-Expert 7.1.6

software package by Response Surface Methodology (RSM). The optimal levels

of three variables like calcium chloride, calcium lactate, calcium propionate was

done. For that purpose, the response surface approach by using a set of

experimental design (central composite design with five coded levels) was

performed. For the three factors, this design was made up of a full 23 factorial

design with its eight points augmented with three replications of the center points

(all factors at level 0) and the six star points, that is, points having for one factor

an axial distance to the center of ±α, whereas the other two factors are at level 0.

The axial distance α was chosen to be 1.68 to make this design orthogonal. A set

of 20 experiments were carried out for the three variables. The central values (0

level) chosen for experimental design were (% w/v) Calcium lactate-1.50,

Calcium propionate-0.88, Calcium chloride-0.60 (Table 4.5.1–4.5.2).

122

In developing the regression equation, the test factors were coded

according to the following equation:

……… (4.8)

Were xi is the coded value of the ith independent variable, Xi the natural

value of the ith independent variable, X0 the natural value of the ith independent

variable at the center point, and ∆Xi the step change value (∆Xi is 1.50 for calcium

lactate, 0.88 for calcium propionate, 0.60 for calcium chloride). For a three-factor

system, the model equation is:

……… (4.9)

Where: Y, predicted response; b0, intercept; b1, b2, and b3, linear coefficients; b11,

b22, and b33, squared coefficients; and b12, b13, and b23, interaction coefficients

Results were analyzed by the experimental design module of the design expert

7.0. The model permitted evaluation of the effects of linear, quadratic and

interactive terms of the independent variables on the chosen dependent variables.

Three-dimensional surface plots were drawn to illustrate the main and interactive

effects of the independent variables on calcium ions optimization. The optimum

values of the selected variables were obtained by solving the regression equation

and also by analyzing the response surface contour plots (Myers and Montgomery,

2002).

123

Table 4.5: Optimization of calcium ions for salad preparation



-1 -α 0 +1 +αCalcium lactate (A) % (w/v) 0.5 -0.181 1.5 2.5 3.181

Calcium propionate (B) % (w/v) 0.25 -0.176 0.88 1.5 1.926

Calcium chloride (C) % (w/v) 0.2 -0.072 0.60 1 1.272

Tanle 4.5.2: Experimendal design and results of 23 factorial design

RunA

Calcium lactate(%,w/v)

BCalcium

Propionate (%, w/v)

CCalcium chloride

(% ,w/v)

1 1.50 1.93 0.602 1.50 0.88 0.603 1.50 0.88 0.604 0.50 1.50 1.005 0.50 0.25 1.006 2.50 0.25 1.007 1.50 0.88 -0.078 2.50 0.25 0.209 -0.18 0.88 0.6010 1.50 0.88 0.6011 2.50 1.50 0.2012 2.50 1.50 1.0013 1.50 -0.18 0.6014 0.50 0.25 0.2015 1.50 0.88 0.6016 3.18 0.88 0.6017 1.50 0.88 1.2718 1.50 0.88 0.6019 0.50 1.50 0.2020 1.50 0.88 0.60

124

4.11.3.9 Effect of optimized calcium ions on fruit salad preparation

The RSM optimized calcium ions were; calcium lactate- 1.45% (w/v),

calcium propionate- 0.68% (w/v), calcium chloride- 0.55% (w/v). The optimized

combination of calcium ions were analyzed in fruit salad preparation. This

experiment was also done with GOx-catalase and koruk juice.

4.11.3.10 Measurement of weight loss

Weight loss percentage of fruit salad was calculated after 48 h of storage at

room temperature. A digital electronic balance (Shimadzu, Japan) was used to

determine the product weight. Weight loss percentage calculated was calculated

by the formula,

……… (4.10)

where: %WL(t) is the percentage weight loss at time t, W0 is the initial sample

weight and W(t) is sample weight at time t.

4.11.3.11 Microbial analysis of fruit salad by viable plate count method

Thirty gram of sample was taken and mixed with 270 ml of sterile distilled

water. With a sterile pipette, one millilitre of suspension was transferred to the

second tube containing 9.0 ml of sterile water. This was continued for up to 10-8

dilutions. One millilitre of 10-6 to 10-8 dilutions was delivered to Petri dishes

containing nutrient agar medium and spread uniformly. The results were

expressed in colony forming units (CFU/g) of sample.

125

4.12 Statistical analysis

All values are expressed as means ± standard deviation. The results were

analyzed using one-way analysis of variance (ANOVA) and the differences

among the treatments means were analyzed using the Tukey-Kramer multiple

comparison test. P value<0.05 was considered as least significant. The software

GraphPad InStat was employed for the statistical analysis.

126

CHAPTER-5

RESULTS AND DISCUSSION

The present study focused on the screening of potential GOx producing

fungi and their application in the food processing and preservation. The initial part

of the investigation was concentrated on isolation of fungi from various sources

and enhancing the GOx production through the optimization medium

compositions was carried out. Production of GOx was carried out in laboratory

batch fermentor. The cell free extract was purified by various purification

methods. The purified enzyme was characterized by enzyme kinetic constant,

environmental factors, inhibitory activity and stability. The final part of the study

was to applying the GOx in the novel areas such as enhancing shelf-life of

vegetables, preparation in apple puree and fruit salad prepared with calcium ions.

5.1 Isolation, media optimization and production of GOx

5.1.1 Isolation of GOx producing fungi

Glucose oxidase producing fungi were isolated from various sugar rich

products (fruits, dates and honey hive) and also from soil samples. The fungi were

screened based on their GOx producing capability. Screening was carried out from

the 35 isolates of Aspergillus niger group and 17 isolates of Penicillum sp. in

order to identify the highest GOx (extra cellular) activity in the screening medium.

The eleven strains were secreted considerable amount of GOx from the 52 isolates

(Table 5.1). The strains such as GOP3 and GOP7 (Figure 5.3 and 5.4)

(Aspergillus awamori belongs to the group of Aspergillus niger) isolated from

dates and honey hive produced the highest extracellular GOx activity in the basal

production medium as 4.2±0.14 and 5.1±0.22 U/ml respectively. The isolated

higher GOx producing fungus (GOP7) was submitted in Microbial Type Culture

Collection (MTCC), Chandigarh, India and it was designated as Aspergillus

awamori MTCC 9645. The fungus A. awamori MTCC 9645 was produced high

127

amount of extra cellular GOx from the different isolates and this fungus was used

for further optimization and production of GOx.

Table 5.1: Glucose oxidase production by different fungi

Organism

codeName of the fungi Source

GOx activity

(U/ml)

GOP1 Aspergillus awamori Dates 1.25±0.58

GOP2 Aspergillus niger Orange 1.15±0.067

GOP3 Aspergillus awamori Dates 4.2±0.14

GOP4 Aspergillus niger Soil 0.51±0.021

GOP5 Aspergillus niger Soil 2.5±0.092

GOP6 Aspergillus niger Honey hive 3.35±0.11

GOP7 Aspergillus awamori Honey hive 5.1±0.22

GOP8 Penicillium sp Soil 0.75±0.038

GOP9 Penicillium sp Honey hive 1.70±0.67

GOP10 Penicillium sp Sugarcane baggase 2.35±0.14

GOP11 Penicillium variale Dates 4.3±0.18

Values are mean of three replicates (±S.D)

128

Figure 5.3: Isolation, screening and identification of A. awamori (GOP-3)

(D) Spores of A. awamori (GOP-3)

(B) A. awamori (GOP-3) on PDA (C) Browning on screening media

(A) Dates

1000 X450 X

129

(A) Honey hive

(C) Browning on screening media(B) A. awamori (GOP-7) on PDA

(D) Spores of A. awamori MTCC 9645

450 X 1000 X

Figure 5.4: Isolation, screening and identification of A. awamori (GOP-7) MTCC 9645

130

5.2 Optimization of medium for glucose oxidase production

Seven different medium were used for the selection of suitable medium for

GOx production in which GOxM3 supported maximum GOx activity (Figure 5.5).

The optimization of medium was carried out by change the constituents and

concentrations of the cultivation medium through the utilization of various carbon,

nitrogen sources, di-ammonium hydrogen phosphate, potassium di-hydrogen

phosphate, magnesium sulphate, CaCO3 and environmental factors (pH and

temperature). For this investigation two type of optimization were performed one

is classical single factor analysis and another one is statistically by response

surface methodology using central composite design.

5.2.1 Single Factor Analysis (SFA) for GOx production

5.2.1.1 Effect of carbon source on GOx production

A number of carbon sources were analyzed in order to determine their

effect on GOx production in the single factor analysis. The different carbon

sources were supplemented to the basal medium constituents. Carbon source

exerts a great influence on the extracellular GOx production. It was supported

GOx production with different extent and results were expressed in the Figure 5.6.

The highest GOx production was obtained in glucose followed by sucrose. A.

awamori MTCC 9645 was able to grow in all the carbon sources evoluated, while

significant (P<0.05) GOx production was obtained in the glucose and sucrose.

Hatziuikolaou and Macris (1995) described that glucose is a principal inducer for

the transcription of GOx gene.

The GOx has a high specificity to glucose, when compared to other

carbohydrates (Rogalski, 1988; Schomburg and Stephan, 1995). During the

microbial fermentations, the carbon source not only acts as a major constituent for

building of cellular material, while it also used in synthesis of polysaccharide and

131

0 1 2 3 4 5 6

GOxM 1

GOxM 2

GOxM 3

GOxM 4

GOxM 5

GOxM 6

GOxM 7D

iffer

ent p

rodu

ctio

n m

ediu

m

GOx activity (U/ml)

b

b

a

a

c

b

c

0

1

2

3

4

5

6

7

20 40 60 80 100 120 140 160Concentrations (g/l)

GO

x ac

tivity

(U/m

l)

Glucose Fructose Sucrose Maltose Xylose Rhamnose

a

dde

d

a

a

e

ab

e

a

a

a

a

b

c

b

b

a

d

bccb

cc

cc

cd

d

cc

d

d

a

d

a

c

a

d

b

b

a

c

a

a

b

ab

Figure 5.5: Effect of different media for GOx production Legends followed by the same letter are not significantly different (P<0.05).

Values represent the mean of triplicates with standard deviation.

Figure 5.6: Effect of carbon sources on GOx production Legends followed by the same letter are not significantly different (P<0.05) for the same concentration. Values represent the mean of triplicates with standard deviation.

132

as energy source (Stanbury et al., 1997). The present study was also correlated

with Traeger et al. (1991) and Sandip et al. (2008) report such as the glucose and

sucrose were more effective in the production of GOx in the A. niger.

5.2.1.2 Effect of nitrogen sources on GOx production

The various types of nitrogen sources were analyzed for the effective GOx

production. Proteose peptone was found to be effective nitrogen source for GOx

production when compared to other nitrogen sources. The detailed results were

expresed in figure 5.7. The optimum concentration of nitrogen sources for the

production of GOx was found to be 3–4 g/l. Increasing concentration of nitrogen

source produced higher biomass, but reduces the GOx production. A similar result

was obtained by Sandip et al. (2008). Hatziuikolaou and Macris (1995) reported

that peptone was an effective nitrogen source for the production of GOx.

Obviously, the low concentration of peptone has a positive effect on process

economy.

5.2.1.3 Effect of di-ammonium hydrogen phosphate, potassium di-hydrogen

phosphate and magnesium sulphate on GOx production

The enhancement of GOx production was obtained from the addition of

di-ammonium hydrogen phosphate and potassium di-hydrogen phosphate. In the

levels of 0.4 g/l of di-ammonium hydrogen phosphate and 0.2 g/l of potassium di-

hydrogen phosphate were found to be maximum production. Addition of

magnesium sulphate increased the amount of GOx production in the fermentation

medium, while above 0.2 g/l of magnesium sulphate affected the GOx production

(Figure 5.8 and 5.9). Nakamatsu et al. (1975) reported that ammonium di-

hydrogen phosphate, potassium di-hydrogen phosphate and Mg++ were found to be

increase the GOx production.

133

0

1

2

3

4

5

6

7

1 2 3 4 5 6Concentrations (g/l)

GO

x ac

tivity

(U/m

l)

Bacteriological peptoneMycological peptoneProteose peptoneYeast extractBeaf extract

a

aa

a

a

a

a

a

bb

bb

bb

cc

cc

cc

dd

b

b

c

d

dc

bb

Figure 5.7: Effect of different nitrogen sources for the GOx production

Legends followed by the same letter are not significantly different (P<0.05) for the same concentration. Values represent the mean of triplicates with standard deviation.

134

0

1

2

3

4

5

6

7

0 0.1 0.2 0.3 0.4 0.5 0.6Concentration (g/l)

GO

x ac

tivity

(U/m

l)a

c

bc

ab

a

b

a

0

1

2

3

4

5

6

7

0 0.05 0.15 0.2 0.25 0.3 0.35 0.4

Concentration (g/l)

GO

x ac

tivity

(U/m

l)

KH2PO4 MgSO4

a

b

B

bAabab AA

c

bc

A

D

bc

C

B

Figure 5.8: Effect of di-ammonium hydrogen phosphate on GOx production

Figure 5.9: Effect of potassium di-hydrogen phosphate and magnesium sulphate of GOx production

Legends followed by the same letter are not significantly different (P<0.05) for the same concentration. Values represent the mean of triplicates with standard deviation.

135

5.2.1.4 Effect of calcium carbonate on GOx production

The effect of calcium carbonate on the production of GOx is representing

in figure 5.10. Remarkably, GOx production was increased by the

supplementation of 30–40 g/l calcium carbonate. The level of 3.5% CaCO3 has

been reported to be essential for the induction of GOx synthesis in the production

medium by Sandip et al. (2008). Hatzinikolaou and Macris (1995) was also

reported that CaCO3 as a strong inducer for the production of GOx that

accompanied by a metabolic shift from glycolysis to the pentose phosphate

pathway. Liu et al. (1999) and Rogalski et al. (1988) were suggested that the GOx

synthesis was increased by CaCO3, due to a high calcium ion concentration or an

insoluble salt.

Fructose-6-phosphate, which is derived from glucose-6-phosphate

(catalyzed by glucose-6-phosphate isomerase) may enter the Embden–Meyerhof–

Parnas (EMP) or HMP (hexose mono phosphate) pathway. The addition of CaCO3

in to the growth medium made an alteration in the production of GOx by 6-

phosphofructokinase and glucose-6-phosphate dehydrogenase. The 6-

phosphofructokinase is a key regulatory enzyme in the EMP pathway in most

living cells. Cells grown in media without CaCO3 produced high levels of 6-

phosphofructokinase and low amounts of glucose-6-phosphate dehydrogenase and

GOx. The Addition of CaCO3 in to the growth medium was enhanced the

production of GOx and decreased the synthesis of 6-phosphofructokinase. The

induction of CaCO3 was accompanied by a metabolic shift from the glycolytic

pathway (EMP) that induced direct oxidation of glucose by GOx (Liu et al.,

2001).

5.2.1.5 Effect of pH on GOx production

Maximum GOx production was observed at pH between 5 and 6 (Figure

5.11). GOx production was minimal below the pH of 4.0, which could be due to

acidophilic characteristics of the fungi. The pH of the culture medium plays a vital

136

0 1 2 3 4 5 6

10

20

30

40

50

60C

onc.

of c

alci

um c

arbo

nate

(g/l)

GOx activity (g/l)

b

a

c

b

a

d

0

1

2

3

4

5

6

7

3 4 5 6 7 8pH

GO

x ac

tivity

(U/m

l)

0

2

4

6

8

10

12

14

16

Tota

l dry

mas

s (g

/l)

GOx activity TDMa

c

b

c

b

a

C

B

AA

B

C

Figure 5.10: Effect calcium carbonate on glucose oxidase production

Figure 5.11: Effect of pH on GOx production

Legends followed by the same letter are not significantly different (P<0.05). Values represent the mean of triplicates with standard deviation.

137

role for fungal growth and GOx production. The optimum pH range is necessary

for the production of GOx. Rogalski et al. (1988) concluded that calcium

carbonate appeared to be important in preventing the acidification of the broth

culture during cultivation. Petruccioli et al. (1995) stated that the addition of

calcium carbonate to the growth medium in shake flasks and fermenters prevented

pH drop during cultivation.

5.2.1.6 Effect of temperature on GOx production

The optimum temperature was found to be from 30 to 35ºC for the

production of GOx. The increase in temperature affects the GOx production as

well as biomass in the basal medium (Figure 5.12). The optimum temperature for

GOx production was observed at 27.5ºC (Hatzinikolaou and Macris, 1995). Other

researchers reported that the slight increase in temperatures (up to 35°C) enhances

the GOx production (Rogalski et al., 1988; Markwell et al., 1989; Traege et al.,

1991; Caridis et al., 1991).

5.2.1.7 Effect of fermentation time on GOx production

The optimum fermentation time for the GOx production was found to be 84

h and the GOx activity exhibited different patterns in the course of fermentation

(Figure 5.13). Hatzinikolaou and Macris (1995) reported that the optimum

cultivation time for GOx production was 70 h, while Sandip et al. (2008) found at

96 h. In this present investigation after 84 h, the enzyme activity was decreased

drastically and the total dry mass remained stationary.

138

0

1

2

3

4

5

6

7

20 25 30 35 40 45

Temperature (C)

GO

x ac

tivity

(U/m

l)

0

2

4

6

8

10

12

14

Tota

l dry

mas

s (g

/l)

GOx activity Total dry mass

A

C

AB

C

B

A

d

b

a

a

ab

c

0

1

2

3

4

5

6

7

0 12 24 36 48 60 72 84 96 108 120 132 144

Incubation period (h)

GO

x ac

tivity

(U/m

l)

0

2

4

6

8

10

12

14

16

18

20

Tota

l dry

mas

s (g

/l)

GOx activity Total dry mass

d

AAA

BBC

C

D

E

F

H

d

bc

ba

aaa

a

b

c

e

AA

Figure 5.12: Effect of temperature on GOx production

Figure 5.13: Effect of fermentation time on GOx production and cell growth

Legends followed by the same letter are not significantly different (P<0.05). Values represent the mean of triplicates with standard deviation.

139

5.2.2 Response surface methodology for GOx production

5.2.2.1 SET 1: Optimization of glucose, proteose peptone and calcium

carbonate for GOx production

The central composite design (CCD) was employed for the optimization of

glucose, proteose peptone and calcium carbonate on the GOx production and the

results are presented in Table 5.2.

The F-value of 14.30 represent as significant model. There is a chance of

0.01% variation in "model-F value" due to noice (Table 5.2.1). The values of

"Prob > F" and less than 0.05 indicate that model terms are significant. In this

case A, A2, B2 and C2 are significant model terms. Values greater than 0.1

indicate that the model terms are not significant.

The "Lack of Fit F-value" of 14.11 exhibit as significant and there is only

slight chance of 0.57% variation in "Lack of Fit F-value" due to noise. The lack

of fit value is not significant, so the model has to be change to fit as significant.

The "Pred R-Squared" of 0.4706 is not as close to the "Adj R-Squared" of

0.8630 (Table 5.2.2). "Adeq Precision" measures the signal to noise ratio and a

ratio greater than 4 is desirable. The ratio of 11.322 indicates an adequate signal.

This model can be used to navigate the design space. The model coefficient was

estimated by linear regression (Table 5.2.3).

Final equation in terms of coded factors

GOx = 9.346642634+1.238727515 × A +0.065968246 ×B +0.281339953 ×C

+0.0875×A ×B +0.1475 ×A ×C+0.0125 ×B ×C-1.668147103 ×A^2-1.275702839

×B^2-1.189082258 ×C^2

140

Table 5.2: Set 1 Optimization of glucose, proteose peptone and calcium carbonate for the GOx production by CCD of response surface methodology (23 factorial design)

RunA:

Glucose(g/l)

B:Proteosepeptone

(g/l)

C:CaCO3

(g/l)

ResponseGOx activity

(U/ml)

1 90 -1.045 35 5.52

2 40 1 10 4.363 90 4 35 9.56

4 40 7 10 5.16

5 5.91 4 35 1.23

6 140 1 60 5.947 90 4 35 9.75

8 40 1 60 4.72

9 174 4 35 7.21

10 90 4 77.04 6.6411 90 4 35 9.3

12 90 4 35 8.93

13 90 9.045 35 5.14

14 140 7 60 7.1415 140 1 10 6.22

16 90 4 35 9.56

17 90 4 35 9.12

18 140 7 10 6.1419 40 7 60 4.34

20 90 4 -7.044 4.51

141

Table 5.2.1: F-test analysis ( ANOVA for Response Surface Quadratic Model)

SourceSum ofSquares df

MeanSquare

FValue

p-valueProb > F

Model 92.876 9 10.319 14.298 0.0001 A-Glucose 20.955 1 20.955 29.034 0.0003 B-Peptone 0.059 1 0.059 0.082 0.7800 C- CaCO3 1.080 1 1.080 1.497 0.2491 AB 0.061 1 0.061 0.084 0.7768 AC 0.174 1 0.174 0.241 0.6340 BC 0.001 1 0.001 0.002 0.9676 A^2 40.102 1 40.102 55.563 < 0.0001 B^2 23.453 1 23.453 32.495 0.0002 C^2 20.376 1 20.376 28.232 0.0003Residual 7.217 10 0.721Lack of Fit 6.739 5 1.347 14.111 0.0057Pure Error 0.477 5 0.095Cor Total 100.091 19

Table 5.2.2: Comparition of R2 predicted and estimated

Std. Dev. 0.849 R-Squared 0.927

Mean 6.524 Adj R-Squared 0.862

C.V. % 13.021 Pred R-Squared 0.470

PRESS 52.994 Adeq Precision 11.322

Table 5.2.3: Model coefficient estimated by linear regression

FactorCoefficientEstimate df

StandardError

95% CILow

95% CIHigh VIF

Intercept 9.346 1 0.346 8.574 10.118A-Glucose 1.238 1 0.229 0.726 1.750 1B-Peptone 0.065 1 0.229 -0.446 0.578 1C- CaCO3 0.281 1 0.229 -0.230 0.793 1AB 0.087 1 0.301 -0.581 0.756 1AC 0.147 1 0.301 -0.521 0.816 1BC 0.012 1 0.300 -0.656 0.681 1A^2 -1.668 1 0.223 -2.16 -1.169 1.018B^2 -1.275 1 0.223 -1.774 -0.777 1.018C^2 -1.189 1 0.223 -1.687 -0.690 1.018

142

Final equation in terms of actual factors

GOx = -2.763181152+0.138417808×Glucose +1.097614161 ×Peptone

+0.133144144 ×CaCO3+0.000583333 ×Glucose ×Peptone 0.000118 ×Glucose

×CaCO3+0.000166667 ×Peptone ×CaCO3-0.000667259 ×Glucose^2-0.14174476

×Peptone^2-0.001902532 ×CaCO3^2

The contour and three-dimensional response surface curves were plotted

and expressed in Figure 5.14-5.16. Maximum GOx production (9.32 U/ml) was

achieved at 92.7 g/l of glucose, 3.24 g/l of proteose peptone and 36.82 g/l of

calcium carbonate. The higher amount of proteose peptone was enhanced the

biomass, but reduced the GOx production. This result suggested that it may reduce

the fermentation cost.

5.2.2.2 SET 2: Optimization of di-ammonium hydrogen phosphate, potassium

di-hydrogen phosphate and magnesium sulphate for GOx

production

The results of central composite design experiments for studying the effects

of second set of three independent variables such as (NH4)2HPO4, KH2PO4 and

MgSO4 on GOx production are presented in Table 5.3.

The F-value of 2.66 represent as significant model. There is a chance of

7.19% variation in "model-F value" due to noice (Table 5.3.1). The values of

"Prob > F" and less than 0.05 indicate that model terms are significant. In this

case A2, B2 and C2 are significant model terms. Values greater than 0.1 indicate

that the model terms are not significant.


slight chance of 0.01% variation in "Lack of Fit F-value" due to noise.

143

Design-Expert® Software

Glucose oxidaseDesign Points9.75

1.23

X1 = A: GlucoseX2 = C: CaCo3

Actual FactorB: Peptone = 4.00

40.00 65.00 90.00 115.00 140.00

10.00

22.50

35.00

47.50

60.00Glucose oxidase

A: Glucose

C: C

aCo3

5.86409

6.61134

7.35859

8.10583

8.85308

6


Glucose oxidaseDesign points above predicted valueDesign points below predicted value9.75

1.23

X1 = A: GlucoseX2 = C: CaCo3

Actual FactorB: Peptone = 4.00

40.00

65.00

90.00

115.00

140.00 10.00

22.50

35.00

47.50

60.00

5.1

6.275

7.45

8.625

9.8

Glu

cose

oxi

dase

A: Glucose C: CaCo3

Figure 5.14: The contour and 3D response surface plot showing the effect of glucose and calcium carbonate on GOx production

144



1.23

X1 = A: GlucoseX2 = B: Peptone

Actual FactorC: CaCo3 = 35.00

40.00

65.00

90.00

115.00

140.00 1.00

2.50

4.00

5.50

7.00

5.1

6.275

7.45

8.625

9.8

Glu

cose

oxi

dase

A: Glucose B: Peptone



1.23

X1 = A: GlucoseX2 = B: Peptone

Actual FactorC: CaCo3 = 35.00

40.00 65.00 90.00 115.00 140.00

1.00

2.50

4.00

5.50

7.00Glucose oxidase

A: Glucose

B: P

epto

ne

5.88183

5.88183

6.62113

7.360438.09973

8.09973

8.839036

Figure 5.15: The contour and 3D response surface plot showing the effect of glucose and peptone on GOx production

145



1.23

X1 = B: PeptoneX2 = C: CaCo3

Actual FactorA: Glucose = 90.00

1.00 2.50 4.00 5.50 7.00

10.00

22.50

35.00

47.50


B: Peptone

C: C

aCo3

7.01656 7.01656

7.48607 7.48607

7.95559

7.955597.95559

8.4251

8.89461

6



1.23

X1 = B: PeptoneX2 = C: CaCo3

Actual FactorA: Glucose = 90.00

1.00

2.50

4.00

5.50

7.00

10.00

22.50

35.00

47.50

60.00

6.5

7.325

8.15

8.975

9.8

Glu

cose

oxi

dase

B: Peptone C: CaCo3

Figure 5.16: The contour and 3D response surface plot showing the effect of peptone and calcium carbonate on GOx production

146

"Adeq Precision" measures the signal to noise ratio and a ratio greater than

4 is desirable. The ratio of 5.013 indicates an adequate signal (Table 5.3.2). This

model can be used to navigate the design space. The model coefficient was



GOx = 9.68+0.32 ×A+0.025 ×B-0.24 ×C-0.025×A ×B 0.25 ×A ×C -0.60 ×B ×C-

0.62 ×A2-0.69 ×B

2-0.62 ×C2


GOx =+6.09373+4.68728 ×(NH4)2HPO4+8.83236 ×H2PO4+7.42811

×MgSO4-0.25974 ×(NH4)2HPO4 ×KH2PO4+3.17460 ×(NH4)2HPO4 ×MgSO4-

9.69697 ×KH2PO4×MgSO4-5.08266 ×((NH4)2HPO4)2-9.16808 ×KH2PO4-

12.29877 ×(MgSO4)2


and represented in Figure 5.17-5.19. Maximum GOx production (9.71 U/ml) was

achieved at (NH4)2HPO4−0.48 g/l, KH2PO4–0.32 g/l and MgSO4–0.23 g/l.

Higher amount of MgSO4 significantly affected the GOx production followed by

KH2PO4.

147

Table 5.3: Set 2 Optimization of (NH4)2HPO4, KH2PO4 and MgSO4 for the GOx production by CCD of response surface methodology (23

factorial design)

RunA:

(NH4)2HPO4(g/l)

B:KH2PO4

(g/l)

C:MgSO4

(g/l)


(U/ml)

1 0.45 0.787 0.275 8.4

2 0.45 0.325 0.275 9.65

3 0.45 0.325 0.275 9.54

4 0.1 0.05 0.5 6.9

5 0.45 0.325 -0.103 8.1

6 0.1 0.05 0.05 7.8

7 0.1 0.6 0.5 6.2

8 0.45 0.325 0.275 9.72

9 1.038 0.325 0.275 9.9

10 0.45 0.325 0.275 9.6

11 0.8 0.05 0.5 7.8

12 0.1 0.6 0.05 8.6

13 0.8 0.6 0.5 6.1

14 0.8 0.6 0.05 8.4

15 0.45 0.325 0.275 9.7

16 0.45 -0.137 0.275 8.2

17 -0.138 0.325 0.275 7.118 0.8 0.05 0.05 6.8

19 0.45 0.325 0.653 8.9

20 0.45 0.325 0.275 9.65

148


Source Sum ofSquares df Mean

SquareF

Valuep-value

Prob > FModel 20.64 9 2.293 2.658 0.0719A-(NH4)2HPO4 0.008 1 0.008 0.009 0.9239B- KH2PO4 0.775 1 0.775 0.898 0.3654C- MgSO4 0.005 1 0.005 0.005 0.9408AB 0.5 1 0.5 0.579 0.4641AC 2.88 1 2.88 3.337 0.0977BC 5.586 1 5.586 6.474 0.0291 A^2 6.927 1 6.927 8.023 0.0177 B^2 5.586 1 5.586 6.474 0.0291 C^2 8.629 10 0.862Residual 8.607 5 1.724 396.04 < 0.0001Lack of Fit 0.021 5 0.004Pure Error 29.272 19


Std. Dev. 0.928926 R-Squared 0.70522Mean 8.353 Adj R-Squared 0.439918C.V. % 11.12087 Pred R-Squared -1.25073PRESS 65.88533 Adeq Precision 5.012998


Factor CoefficientEstimate df Standard

Error95% CI

Low95% CI

High VIFIntercept 9.67675 1 0.378861 8.832595 10.52091A-(NH4)2HPO4

0.315 1 0.251 -0.244 0.875 1

B- KH2PO4 0.024 1 0.251 -0.535 0.584 1C- MgSO4 -0.238 1 0.251 -0.798 0.321 1AB -0.025 1 0.328 -0.756 0.706 1AC 0.25 1 0.328 -0.481 0.981 1BC -0.6 1 0.328 -1.331 0.131 1A^2 -0.622 1 0.244 -1.167 -0.077 1.018B^2 -0.693 1 0.244 -1.238 -0.148 1.018C^2 -0.622 1 0.244 -1.167 -0.077 1.018

149



6.1

X1 = A: (NH4)HPO4X2 = B: KH2PO4

Actual FactorC: MgSO4 = 0.28

0.10

0.28

0.45

0.63

0.80

0.05

0.19

0.33

0.46

0.60

7.9

8.375

8.85

9.325

9.8

Glu

cose

oxi

dase

A: (NH4)HPO4 B: KH2PO4

Figure 5.17: The contour and 3D response surface plot showing the effect of (NH4)2HPO4 and KH2PO4 on GOx production



6.1

X1 = A: (NH4)HPO4X2 = B: KH2PO4

Actual FactorC: MgSO4 = 0.28

0.10 0.28 0.45 0.63 0.80

0.05

0.19

0.33

0.46

0.60Glucose oxidase

A: (NH4)HPO4

B: K

H2P

O4

8.28249

8.56934

8.56934

8.85619

8.85619

9.14304

9.42989

6

150



6.1

X1 = A: (NH4)HPO4X2 = C: MgSO4

Actual FactorB: KH2PO4 = 0.33

0.10 0.28 0.45 0.63 0.80

0.05

0.16

0.28

0.39

0.50Glucose oxidase

A: (NH4)HPO4

C: M

gSO

4

7.97798

8.32829

8.6786

9.0289

9.02899.37921

6



6.1

X1 = A: (NH4)HPO4X2 = C: MgSO4

Actual FactorB: KH2PO4 = 0.33

0.10

0.28

0.45

0.63

0.80

0.05

0.16

0.28

0.39

0.50

7.6

8.15

8.7

9.25

9.8

Glu

cose

oxi

dase

A: (NH4)HPO4 C: MgSO4

Figure 5.18: The contour and 3D response surface plot showing the effect of (NH4)2HPO4, and MgSO4 on GOx production

151



6.1

X1 = B: KH2PO4X2 = C: MgSO4

Actual FactorA: (NH4)HPO4 = 0.45

0.05 0.19 0.33 0.46 0.60

0.05

0.16

0.28

0.39

0.50Glucose oxidase

B: KH2PO4

C: M

gSO

4

7.90732

8.26754

8.26754

8.62776

8.62776

8.98798

8.98798

9.34819

6



6.1

X1 = B: KH2PO4X2 = C: MgSO4

Actual FactorA: (NH4)HPO4 = 0.45

0.05

0.19

0.33

0.46

0.60

0.05

0.16

0.28

0.39

0.50

7.5

8.075

8.65

9.225

9.8

Glu

cose

oxi

dase

B: KH2PO4 C: MgSO4

Figure 5.19: The contour and 3D response surface plot showing the effect of KH2PO4 and MgSO4 on GOx production

152

5.2.2.3 SET 3: Optimization of pH and temperature


of pH and temperature on GOx production are presented in Table 5.4. The F-value

of 347.29 showed the model is significant. There is a onlychance of 0.01%

variation in "model-F value" due to noice (Table 5.4.1). The values of "Prob > F"

and less than 0.05 indicate that model terms are significant. In this case A2 and

B2 are significant model terms. Values greater than 0.1 indicate that the model

terms are not significant.


slight chance of 0.57% variation in "Lack of Fit F-value" due to noise. The lack

of fit value is not significant, so the model has to be change to fit as significant.

The "Pred R-Squared" of 0.972 is in reasonable agreement with the "Adj R-

Squared" of 0.993. "Adeq Precision" measures the signal to noise ratio and a ratio

greater than 4 is desirable. The ratio of 37.456 indicates an adequate signal (Table

5.4.2). This model can be used to navigate the design space. The model

coefficient was estimated by linear regression (Table 5.4.3).


GOx = 10.10-0.13 A-0.24 ×B-0.075 ×A×B-4.92×A2-4.40 ×B2


GOx = -33.07707+6.57270 ×pH+1.36371 ×Temperature-1.66667E-003 ×pH ×

Temperature-0.54639 ×pH2 -0.019567 ×Temperature2


and presented in the Figure 5.20. Maximum GOx production (10.08 U/ml) was

achieved at the pH 5.83 and the temperature at 30.7°C.

153

Table 5.4: Set 3 Optimization of pH and temperature for the GOx production by CCD of response surface methodology (23 factorial design)

Run A:pH

B:Temperature (°C)


(U/ml)

1 3 50 0.72 9 50 03 1.79 35 0.544 6 35 10.15 6 13.7 2.16 6 56.2 1.17 6 35 9.98 6 35 10.29 6 35 10.110 10.24 35 0.611 3 20 0.812 6 35 10.213 9 20 0.4

154

Table 5.4.1: F-test analysis ( ANOVA for Response Surface Quadratic

Model)


SquareF

Valuep-value

Prob > FModel 268.939 5 53.787 347.287 < 0.0001 A-pH 0.128 1 0.128 0.831 0.392 B-Temperature 0.458 1 0.458 2.957 0.129

AB 0.022 1 0.022 0.145 0.714 A^2 168.221 1 168.221 1086.142 < 0.0001 B^2 134.833 1 134.831 870.555 < 0.0001Residual 1.0841 7 0.154Lack of Fit 1.0241 3 0.341 22.759 0.005Pure Error 0.06 4 0.015Cor Total 270.023 12

Table Table 5.4.2: Comparition of R2 predicted and estimated

Std. Dev. 0.393 R-Squared 0.995


C.V. % 9.016 Pred R-Squared 0.972

PRESS 7.376 Adeq Precision 37.456

Table Table 5.4.3: Model coefficient estimated by linear regression

Factor CoefficientEstimate df Standard

Error95% CI

Low95% CI

High VIF

Intercept 10.1 1 0.176 9.683 10.516

A-pH -0.126 1 0.139 -0.455 0.202 1B-Temperature -0.239 1 0.139 -0.568 0.089 1

AB -0.075 1 0.196 -0.540 0.390 1

A^2 -4.917 1 0.149 -5.270 -4.564 1.017

B^2 -4.402 1 0.149 -4.755 -4.049 1.017

155



0

X1 = A: pHX2 = B: Temperature

3.00

4.50

6.00

7.50

9.00

20.00

27.50

35.00

42.50

50.00

0

2.75

5.5

8.25

11

Glu

cose

oxi

dase

A: pH B: Temperature



0

X1 = A: pHX2 = B: Temperature

3.00 4.50 6.00 7.50 9.00

20.00

27.50

35.00

42.50


A: pH

B: T

empe

ratu

re

3.59365 3.59365

3.593653.59365

5.22106

5.22106

5.22106

6.84846

8.47587

5

Figure 5.20: The contour and 3D response surface plot showing the effect of pH and temperature on GOx production

156

Response surface methodology was reduced the fermentation time (65-70

h) and significantly improved the GOx production, when compared to single

factor analysis. According to Sandip et al. (2008), the changes in the cultivation

time and increase in GOx production was probably due to change in the

concentrations of media constituents obtained by the statistical analysis central

composite design.

5.3 Production of glucose oxidase by laboratory batch fermentor

5.3.1 Spore aggregation and pellet formation during the early cultivation time

Aspergillus awamori MTCC 9645 was cultivated in the two litre of

laboratory batch fermentor (Figure 5.21) at constant pH 5.83. The dissolved

oxygen content and temperature were maintained during the fermentation as 11 to

12 mg/l and 30.7ºC respectively.

The number of spores at the beginning of cultivation time was 1.0×106/ml.

After 5 h, the numbers of free spores were decreased to 2.4X104/ml, when the

spore aggregates began to develop and reached 1.6X103/ml with an average

diameter of about 0.051±0.04 mm. Spore aggregates were varied in their shape

and the number of spores per aggregate could not be easily determined. After 10

h, the number of free spores and aggregates were continually decreased. After

spore germination, the aggregates number was reduce and subsequently the

average diameters of aggregates were increased to 0.18±0.06 mm. After 15 h of

cultivation, no free spores were observed in the cultivation medium followed by

the numbers of aggregates were decreased. The complete pellet structure was

formed after 20 h with a number of 46±0.3 pellets/ml and the average diameter of

1.4±0.3 mm (Table 5.5).

157

Culture conditions

Medium : Modified GOxM3pH : 5.83Temperature : 30.7°CDO : 11-12 mg/lAgitation : 200-500Duration : 84 hVolume : 1000 ml

Figure 5.21: Production of GOx in A. awamori MTCC 9645 in laboratory bioreactor

158

Table 5.5: Spore aggregation and pellet formation during the early

cultivation time in the laboratory batch fermentor

Cultivationtime (h)

No. of freespores/ml

No. ofaggregates/ml

AverageDiameters ofaggregates

(mm)

Total No.ofBioparticles/ml

0 1.0X106 - - 1.0X106

5 2.4X104 1.6X104 0.051±0.04 4.0X104

10 3.5X102 1.2X102 0.18±0.06 4.7X102

15 0.56X102 0.9X102 0.8±0.6 1.46X102

20 - 46.0 1.4±0.3 46±4.0

25 - 46.0 1.4±0.3 46±4.0

30 - 48.0 1.5±0.5 48±6.0

35 - 51.0 1.5±0.2 51±5.0

Values are mean of three replicates (±S.D)

159

5.3.2 Morphological studies

After the complete formation of compact pellet structure, both the pellet

number and diameter were constant at 46-51 pellets per ml and 1.5±0.2mm

respectively during the rest of cultivation time (Table 5.5). Further cell growth

was attributed in the inside of the pellets and also spores germinated in the pellet

core.

The mycelial stage of fungus may grow as dispersed hyphal fragments or

pellets. The growth of the pellet was exponential until the density of the pellet

causes diffusion limitation. In this limitation, the biomass was not increased

because of the insufficient nutrient supply. Further growth was started from the

outer shell of the pellet. New pellet was formed from the fragments of old pellets

(Sandip et al., 2008). The enzyme production rate was decreased after 86 h,

probably because of the depletion of nutrient in the medium (substrate limitation)

(Stanbury et al., 1997).

5.3.3 Time course study on cell growth, substrate utilization, GOx and

gluconic acid production during fermentation period

Based on the previous results, after a lag time of five hour, spores were

germinated giving rise to a short germ tube. The fungus apparently grew

exponentially up to 72 h and reached maximum concentration of 12.5±0.63 g/l cell

dry mass and maintained more or less constant for the rest of cultivation period. A

similar observation was observed by Hesham El-Enshasy, (2006). After this time,

the rate of increase in cell mass was less due to the depletion of glucose, since

glucose is converted to gluconic acid (a less suitable carbon source for cell

growth, Lakshminarayana et al., 1969) by the GOx. The utilization of glucose

started higher after 12 h and complete consumption of glucose was observed at

50–60 h of fermentation. Production of gluconic acid was started at 12 h and

maximum amount was observed at 48 h as 62.3±4.1 g/l. It was slowly decreased

160

in the rest of fermentation period due to the utilization of the fungus as carbon

source (Figure 5.22).

The extracellular GOx secretion was started about 10–12 h and then

reached maximum GOx activity at 72 h as 12±0.63 U/ml. The increase of the GOx

production was mainly through the increase in cell mass. Therefore, increasing

extracellular GOx titer with cultivation time is mainly due to excretion of the

enzyme rather than new production. The initial protein content in the fermentation

medium was high due to the presence of proteose peptone. It was decreased

drastically from 35.5±1.61 to 8.1±0.54 µg/ml at 36 h of fermentation. The level of

the protein content was increased after the secretion of protein by the fungus

(Figure 5.23).

The lower enzyme production and excretion in this culture were due to

morphological and physiological problems. It is known that the growth of fungal

cells in pellet form larger than 46 mm in diameter causes severe mass transfer

limitations between medium and pellet regarding substrate transport into the pellet

as well as product transport from the pellet into the medium (Schügerl et al.,

1983). Moreover, not all cells were biologically active and the biologically active

layer was restricted only in the outer layer of pellet. Also the conversion of

glucose (a good carbon source for both cell growth and enzyme induction) to

gluconic acid (a less suitable carbon source for cell growth and non-GOx inducer)

due to enzyme production resulted in termination of enzyme synthesis. This

problem was also observed in fed batch cultures feeded either with glucose or

yeast extract. The growth morphology was identical in form of dense pellet in all

cultures and the complete transformation of glucose to gluconic acid was observed

after a few hours of enzyme production.

161

0

20

40

60

80

100

120

0 12 24 36 48 60 72 84 96 108 120

Fermentation period (h)

Glu

cose

/Glu

coni

c ac

id (g

/l)

0

2

4

6

8

10

12

14

16

Cel

l dry

mas

s (g

/l)

Gucose Gluconic acid Cell dry mass

0

2

4

6

8

10

12

14

0 12 24 36 48 60 72 84 96 108 120Fermentation period (h)

GO

x ac

tivity

(U/m

l)

0

5

10

15

20

25

30

35

40

Prot

ein

cont

ent (

µg/m

l)

GOx Protein

Figure 5.22: Time course study on substrate utilization, production of gluconic acid and biomass during the fermentation

Figure 5.23: Time course study on production of GOx and protein during the fermentation

Values represent the mean of triplicates with standard deviation.

162

5.4 Purification and characterization of glucose oxidase produced from

A. awamori MTCC 9645

The total protein was taken from the cell free culture filtrate (600 ml) of 72

h old A. awamori MTCC 9645 culture. The protein was precipitated using

ammonium sulphate fractions from 40–85% (w/v). The 60–70% (w/v) of

ammonium sulphate fractions showed higher GOx activity than the rest of the

fractions. The activity was not observed from 40% (w/v) saturation.

The precipitated proteins were used for the purification of GOx. The

precipitate was dialyzed, concentrated by lyophilization and fractionated by

chromatographic techniques. The flow chart of GOx purification is presented in

Figure 5.24.

5.4.1 DEAE-Cellulose column chromatography

The concentrated protein was loaded in the DEAE-Cellulose column.

Eighty fractions of each 4 ml were collected. GOx was eluted from 26–38

fractions (Figure 5.25 a). The enzyme active fractions were pooled, concentrated

by lyophilization, dialyzed against sodium acetate buffer (10 mM, pH 5.5) and

used for the further purification.

5.4.2 Sephacryl S-200 column chromatography

The concentrated protein was loaded on Sephacryl S-200 column and

eluted with sodium acetate buffer (100 mM, pH 5.5). Eighty fractions of 3.0 ml

were collected and the active fractions were observed from 31 to 43 fractions. The

GOx active fractions were pooled dialyzed and freeze dried (Figure 5.25b).

The details of purifications of GOx are summarized in the Table 5.6. The

enzyme activity was purified up to 9.19 folds with a final recovery of 12.98%. The

specific activity of purified GOx was 282.27 U/mg proteins. There were slight

163

Cell free culture filtrate

Salting out with ammonium sulphate up to 70% (w/v) saturation

Centrifuged at 10,000 g for 15 minutes at 4°C

Dialyzed against sodium acetate buffer (10 mM, pH 5.5)

Lyophilized and dialyzed proteins

Loaded the proteins on DEAE-cellulose column chromatography

A. awamori MTCC 9645 grown in optimized medium for 72 h

Harvested the culture by filtration and centrifugation at 10,000 g for 10 minutes

Collected GOx active fractions, dialyzed (10 mM, pH 5.5) and lyophilized

Loaded the proteins on Sephacryl S-200 column chromatography

Collected GOx active fractions, dialyzed and lyophilized

SDS-PAGE,

Native-PAGE

Zymogram

Characterization

Enzyme kinetics

pH optimum and stability

Temperature optimum and stability

Effect of metal ions

Figure 5.24: Flow chart for the purification and characterization of extracellular GOxof A. awamori MTCC 9645

164

0

5

10

15

20

25

30

35

40

45

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76Fraction

GO

x ac

tivity

(U/m

L)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Abs

orba

nce

at 2

80 n

m

Enzyme Protein- 400

- 50

- 200

-100

- 300

- 150

- 250

- 350

Nac

l (m

M)

Nacl

0

5

10

15

20

25

30

35

1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76

Fraction

GO

x ac

tivity

(U/m

L)

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

Abs

orba

nce

at 2

80 n

m

Enzyme Protein

Figure 5.25 (a): Elution profile on DEAE column chromatography

Figure 5.25 (b): Elution profile on Sephacryl S-200 column chromatography Figure 5.25: Purification of GOx from A. awamori MTCC 9645

165

Table 5.6: Summary of purification of GOx from A. awamori MTCC 9645

SampleDescription

TotalProtein

(mg)

Totalactivity(Units)

Specificactivity

(Units/ mgprotein)

Yield(%)

Foldpurification

Initial sample 314.5 9660.0 30.7 100 1

Calcium removal 292.2 9120.0 31.2 94.4 1.01Ammoniumsulphateprecipitation

42.2 5841.0 138.41 60.4 4.50

Dialysis 26.4 3847.0 145.71 39.8 4.74Anion exchangechromatograpy 9.51 2794.0 293.79 28.9 9.56

Dialysis 6.75 2264.0 335.4 23.43 10.92

Lyophilization 5.84 1816.0 310.9 18.8 10.1Size-exclusionchromatograpy 4.91 1669.0 339.9 17.2 11.07

Lyophilization 4.41 1254.0 282.27 12.98 9.19

166

decreases in specific activity during the freeze drying steps since no stabilizers

were added which could potentially interfere with subsequent purification steps.

5.4.3 Molecular mass determination of purified GOx by SDS-PAGE

The purity of GOx was analyzed by sodium dodecyl sulfate polyacrylamide

gel electrophoresis (SDS-PAGE) with 10% of gel. On SDS-PAGE, the purified

GOx showed a single band indicating electrophoretically homogenous (Figure

5.26). The molecular weight of 71.1 kDa by comparing with relative mobility of

the molecular weight of standard protein marker. Simpson et al., (2006) reports

that GOx had a molecular weight of 70 kDa, which was similar to the present

investigation.

5.4.4 Purified GOx activity on native-PAGE

The activity of purified GOx was determined on native-PAGE. It was

developed by overlaying 1.5% of soft agar containing glucose, o-dianisidine and

Horse radish peroxidase. Brown color band was formed in the native gel. Single

zymogram analysis is shown in Figure 5.27a.

5.4.5 Confirmation of enzyme activity using plate assay

Dark brown colour was formed immediately in purified GOx and the

authentic GOx. It was confirmed the activity of the GOx in the final product

(Figure 5.27b). The purified GOx was further subjected to kinetic characterization.

167

kDa

205

97.4

66

43

29

KDa

71.5

L1 L2 L3

L1-Marker

L2-Ion exchange chromatography

L3-Purified GOx (Size exclusion)

Figure 5.26: Molecular mass determination on SDS-PAGE (10%) of

GOx from A. awamori MTCC 9645

168

2

1

3

L1 L2

L1-Purified GOx

L2-Authentic GOx

Figure 5.27 (a): Zymogram of GOx (Iso enzyme patterning) on native-PAGE

(8%) developed with horse radishproxidase, o-dianisidine

and glucose

1. Control (Buffer)

2. Purified GOx

3. Authentic GOx

Figure 5.27(b): Confirmation of GOx from A. awamori MTCC 9645 by

plate assay

169

5.4.6 Kinetic characterization of GOx

The Lineweaver’s Burk plot, Eadie-Hofstee plot and Hans-Woolf linear

plots were used to determine the Vmax and Km values of the GOx from A. awamori

MTCC 1945. The initial velocity was determined by the o-dianisidine-horseradish

peroxidase assay described in materials and method. The enzyme was kinetically

characterized and displayed characteristics with a Vmax of 5.77 (U/20µg) and Km

of 119.44 (mM ) in Lineweaver’s Burk plot, Vmax of 5.73 (U/20µg) and Km of

115.65 (mM ) in Eadie-Hofstee plot, Vmax of 5.71 (U/20µg) and Km of 114.05

(mM ) in Hans-Woolf linear and presented in figure 5.28–5.30.

5.4.7 Effect of temperature and pH on GOx activity

The optimum temperature of GOx was found at 30±2ºC and about 62.3%

of relative activity was observed at the temperature between 20 and 50ºC. More

than 50ºC decreased the GOx activity rapidly. Gouda et al. (2003) reported that

the dissociation of FDA from free holoenzyme in aqueous medium was at 59ºC

and concluded that dissociation of FDA from the holoenzyme was responsible for

the thermal inactivation of GOx. Gul Ozyilmaz et al. (2005) also observed that,

temperature affected the activity of the GOx sharply and maximum activity at

35ºC. At 60ºC the relative activity of GOx were found as 33%. The optimum pH

was observed at 5.5 and showed more than 70% of the maximum relative activity

between pH 4 and 7 (Figure 5.31). A similar observation was reported by Gul

Ozyilmaz et al. (2005) as the optimum pH of 5.5.

5.4.8 Stability testing

The stability of the purified GOx was tested at 25 and 37ºC and the relative

activity not affected over 12 h at 25ºC, while showed a half life of 40 minutes at

37ºC (Figure 5.32). It indicates that the GOx would not be effective at 37ºC

without prior stabilization. Combes and Monsan (1988) reported that GOx

170

Figure 5.28: Kinetic parameters (apparent Km and Vmax) for purified GOx ofA. awamori MTCC 9645 by Lineweaver's Burk plot

The initial velocity was determined by the o-dianisidine-horseradish peroxidase assay described in materials and method

y = 20.7x + 0.1733R2 = 0.983

-0.5

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

-0.015 -0.01 -0.005 0 0.005 0.01 0.0151/[S0]

1/[V

0]

Slope=km/Vmax=20.7Intercept=1/Vmax=0.1733km=20.7/0.1733=119.44Vmax=1/0.1733=5.77

171

y = -115.64x + 5.7329R2 = 0.9435

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8

9

10

-0.1 -0.075 -0.05 -0.025 0 0.025 0.05 0.075 0.1 0.125 0.15

[V0]/[S0]

[V0]

Slope=-km=-115.65km=115.65Intercept=Vmax=5.7317

Figure 5.29: Kinetic parameters (apparent Km and Vmax) for purified GOx ofA. awamori MTCC 9645 by Eadie-Hofstee plot


172

Figure 5.30: Kinetic parameters (apparent Km and Vmax) for purified GOx ofA. awamori MTCC 9645 by Hanes plot


y = 0.175x + 19.96R2 = 0.995

-100

-50

0

50

100

150

200

250

-300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100

[S0]

[S0]

/[V0]

Slope=1/Vmax=0.175Vmax=5.714Intercept=km/Vmax=19.96km=19.96X5.714=114.05

173

0

20

40

60

80

100

120

20 30 40 50 60 70 80

Temperature (C)

Rel

ativ

e G

Ox

activ

ity (%

)3 4 5 6 7 8 9

pH

pH profile Temperature profile

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70 80 90Incubation at 37 ºC (minutes)

Res

idua

l GO

x ac

tivity

(%)

6 12 18 24 30 36 42 48 54 60Incubation at 25 ºC (h)

37 ºC 25 ºC

Figure 5.31: Effect of temperature and pH on GOx activity

Figure 5.32: Effect of temperature stability of purified GOx

174

required effective prior stabilization like immobilization, polyhydric alcohols

(polyethylene glycol). The lyophilized freeze dried GOx powder stored at -20ºC

remained same without loss of activity for 6 months. A similar observation also

reported by Simpson et al. (2006).

5.4.9 Stability and inhibitory activity of GOx

5.4.9.1 Effect of metal ions on GOx activity

The mercuric chloride, copper sulphate, cobaltous chloride and silver

nitrate remarkably inhibited the GOx activity even at 1mM concentration. The

detailed results expressed in figure 5.33. Satoshi Nakamura and Yasuyuki Ogura,

(1986) reported that the reaction catalyzed by the GOx from A niger was markedly

inhibited by Ag+, Hg++ and Cu++. All the metal ions shows various degrees of

inhibition; the inhibition sequence was in the order: copper acetate > silver

sulphate > cobaltous chloride > mercuric chloride > copper sulphate at same

concentration.

The reason for the activation and inhibitions is that lower concentrations of

metal ions can stabilize the conformation of GOx and cause change in

conformation to a more active form, so GOx is activated by metal ions. GOx

having interactions of the FAD molecule at the active site of GOx are 23 potential

hydrogen bonds mostly involving the ribose and pyrophosphate groups. At higher

metal ion concentration, the great number of ions will compete with FAD for the

binding sites on the pyrophosphate or ribose groups, which causes the interactions

of the hydrogen bonds between the FAD molecule and pyrophosphate or ribose

groups to become weakened. They will also compete with the substrate for the

binding sites on the enzyme, so GOx activity was partially inhibited. Divalent

metal ions bind to pyrophosphate and ribose more strongly than monovalent ions,

so the inhibition of divalent ions is stronger than that of monovalent ions.

175

5.4.9.2 Effect of calcium ions on GOx activity (1mM)

Effect of calcium ions on GOx activity was analyzed and it was found that

relative activity of calcium lactate was more i.e., 119.5% when compared to

control. Calcium lactate, calcium carbonate, calcium propionate shows a broad

range of activation. The activation sequence is calcium lactate > calcium

carbonate > calcium propionate (Figure 5.34).

5.4.9.3 Effect of calcium ions on pH stability of GOx

Figure 5.35 shows the effect of calcium ions on pH stability of GOx.

Maximum GOx activity was achieved using calcium lactate and calcium

propionate at pH level 5–6. All these values were compared with control in which

maximum activity was obtained at pH 5–6 while, it was shifted to 7.0 for the

calcium lactate followed by calcium propionate treated GOx. Activity of the

calcium lactate treated GOx was less sensitive to pH changes at acidic and

alkaline pH as compared with that of untreated control enzyme.

5.4.9.4 Effect of calcium ions on temperature stability of GOx (1mM)

The effect of calcium ions on temperature stability of GOx was found to

be effective and it was observed a maximum stability at 50°C in calcium lactate

treated GOx as 86.67% but in control showed only 43.3% of relative activity

(Figure 5.36). It was observed that calcium ions provide temperature stability of

GOx and less sensitive at lower and higher temperature when compared with

control.

176

0

20

40

60

80

100

120

Leadacetate

Cobaltouschloride

Copperacetate

Coppersulphate

Silversulphate

Silvernitrate

Mercuricchloride

Zincchloride

Nickelchloride

Control

Rel

ativ

e ac

tivity

(%)

0

20

40

60

80

100

120

140

Calcium lactate Calcium chloride Calcium propionate Calcium carbonate Control

Rel

ativ

e ac

tivity

(%)

Figure 5.33: Effect of metal ions (1mM) on GOx activity

Figure 5.34: Effect of calcium ions (1mM) on GOx activity

177

0

20

40

60

80

100

120

140

3 4 5 6 7 8 9pH

Rel

ativ

e ac

tivity

(%)

ControlCalcium chlorideCalcium lactateCalcium Propionate

0

20

40

60

80

100

120

140

20 30 40 50 60 70

Temperature (C)

Rel

ativ

e ac

tivity

(%)

Control

Calcium chloride

Calcium lactate

Calcium Propionate

Figure 5.35: Effect of calcium ions (1mM) on pH stability of GOx activity

Figure 5.36: Effect of calcium ions (1mM) on temperature stability of GOx

178

5.5 Application of GOx in food processing and preservation5.5.1 Edible films incorporated with glucose oxidase, lactoperoxidase

and lysozyme for carrot preservation

5.5.1.1 Assay of antimicrobial enzymes

The formulated GOx activity was found to be 5.20 U/mg. This enzyme

works in a pH range 4–6. The total activity of lyophilized LPS (0.5 M sodium

acetate elutes) showed 35 U/mg and the optimum pH between 5.5 and 6.5. The

total activity of lyophilized enzyme was 2400 U/mg.

5.5.1.2 Evaluation of antimicrobial activity of GOx, LPS and lysozyme with

EDTA

After 48 h of incubation, formulation VII treatment showed a significant

activity (P<0.05) against both E. coli and S. aureus by decreasing the cell

population to 0.052 and 0.21 CFU/ml respectively. However, the inhibitory effect

of formulation-VII was found to be more potent when compared with other

formulation. It was followed by a significant activity (P<0.05) obtained from the

formulation-V showing 3.1 CFU/ml with E. coli and 3.65 CFU/ml with S. aureus.

Other formulations showed moderate activity against both the test organisms.

There was no reduction obtained from control (Figure 5.37 and 5.38).

A number of naturally occurring antimicrobials have been investigated,

including GOx, LPS, lactoferrin, lysozyme, avidin, various plant extracts such as

spices and their essential oils, sulfur and phenolic compounds (Davidson et al.,

2001). GOx is one of the antimicrobial enzymes immobilized onto various

substrates. GOx is a typical example of oxidoreductase systems that do not

themselves possess antimicrobial activity. However, the reaction products from

reaction catalyzed by a given antimicrobial oxidoreductase system exhibit

antimicrobial activity (Appending et al., 2002; Suye et al., 1998). In spite of these

known activities, there is still a controversy as to whether the effects of the system

179

0

2

4

6

8

10

12

0 6 12 18 24 30 36 42 48Incubation period (h)

E. c

oli

popu

latio

n (L

og C

FU/m

l)

GOx LPSLysozyme with EDTA GOx and LPSGOx, Lysozyme with EDTA LPS and Lysozyme with EDTAGOx, LPS and Lysozyme with EDTA Control

a

aa

aa

aaa

c

bb

b

bb

bb

b

d

c

d

ecc

c

c

c

e

dd

d

cd

d

e

ee

e

e

f

d

0

2

4

6

8

10

12

0 6 12 18 24 30 36 42 48

Incubation period (h)

S. a

ureu

s p

opul

atio

n (L

og C

FU/m

l)

GOx LPSLysozyme with EDTA GOx and LPSGOx, Lysozyme with EDTA LPS and Lysozyme with EDTAGOx, LPS and Lysozyme with EDTA Control

a a a a a a

a ab bb b

bb

bb

c

cc

c

cc

c c

d

d

d

dd

d

de

e

d

ee

e

Figure 5.37 : Effect of antimicrobial activity of GOx, LPS and lysozyme with EDTA against E. coli

Figure 5.38: Effect of antimicrobial activity of GOx, LPS and lysozyme with EDTA against S. aureus

Legends followed by the same letter are not significantly different (P<0.05) for thesame incubation period.Values represent the mean of triplicates with standarddeviation.

180

on gram-negative and gram-positive bacteria are the same. In addition, its

effectiveness against such pathogens like E. coli O157 and L. monocytogenes in

foods has recently been questioned by a number of investigators. Difference

between reported findings may have resulted from differences in experimental

conditions.

The LPS, when used in conjunction with GOx, is a very useful

antimicrobial agent. LPS is part of the immune system’s innate defense

mechanism against foreign microorganisms and can be found in mammalian

secretions such as milk, tears and saliva. This system consists of three components

like LPS, thiocyanate (SCN-) and hydrogen peroxide. LPS activation occurs only

in the presence of thiocyanate and hydrogen peroxide. Catalysis by LPS generates

active intermediates, which has antimicrobial properties and is completely safe to

humans. The presence of GOx allows hydrogen peroxide required by LPS to be

continuously generated and replenished (Seifu et al., 2005). The hydrogen

peroxide produced by GOx is utilized by LPS for cold, i.e. room temperature

sterilization, while the gluconic acid produced is used for direct acidification (Fox

and Stepaniak, 1993). It should be noted that this LPS-GOx antimicrobial system

is not limited to food and has been used in toothpaste (Biotene 2006; National

Library of Medicine 2007a), lotions (National Library of Medicine 2007b),

shampoos, cosmetics, meat processing (Food Standards Australia New Zealand

2002) and fish farming (Seifu et al., 2005).

5.5.1.3 Antimicrobial activity of alginate film

The discs bored from these films were used for the antimicrobial testing

(Figure 5.39). The film disc with formulation-VII showed significant (P<0.05)

zone of inhibition inferring good antimicrobial activity. There was no clear zone

of clearance found with S. aureus but when compared with E. coli showed lager

partial lysis in all enzyme formulations.

181

0

5

10

15

20

25

I II III IV V VI VII VIIIEnzyme formulations

Zone

of i

nhib

ition

(mm

)

E. coli S. aureus

b

a a

cc

d

e

A

A

A

B

A

C

D

Figure 5.39: Effect of antimicrobial alginate films incorporated with partially purified enzymes of different formulations

I-GOx, II-LPS, III-Lyz with EDTA, IV-GOx and LPS, V-GOx, Lyz with EDTA, VI-LPS and Lyz with EDTA, VII-GOx, LPS and Lyz with EDTA, VIII-Control.

Legends followed by the same letter are not significantly different (P<0.05) for the same microbial treatment. Values represent the mean of triplicates with standard deviation.

182

5.5.1.4 Surface sterilization of carrotA significant inhibitory activity (P<0.05) obtained from above 6 ppm of

chlorine dioxide showed higher inhibitory activity. Six ppm of chlorine dioxide

with soaking of 20 minutes was found to be optimum for surface sterilization of

vegetables. The plate count readings were given in Table 5.7 in terms of CFU/ml.

Chlorine dioxide had received a lot of attention in the past few years because its

effectiveness is less affected by pH and organic matter content than that of

chlorine. Another advantage is its high oxidative action, which has been observed

to be 2.5 times greater than chlorine (Benarde et al., 1967).


The alginate coated films extended the shelf-life of carrots by retarding the

evaporation rate and hence there was no weight loss from the carrot. Carrots

coated with the formulations-VII examined to have lower weight loss than

uncoated ones and retain freshness of the carrots (Figure 5.40). The effect of

lowering the water loss was found to be significant (P<0.05) with the coat

containing formulation-VII. The water loss of the sample increased significantly

during storage. This was expected since fresh vegetables usually lose water after

processing and throughout storage. There was a drastic increase in water loss

reported for the first 2 days then the water loss was stabilized and increased

relatively slow until the tenth day. The difference in weight loss between carrots

stored at 6ºC and ~26°C was minimal, though there was relatively less weight loss

in the carrots stored at 6ºC. The loss of water is a natural process of the catabolism

in fresh-cut vegetables and is attributed to the respiration and other senescence-

related metabolic processes during storage (Watads and Qui 1999). The

percentage weight loss due to water loss until the end of the 10th day for coated

and uncoated carrots is represented in Figure 5.41a and 5.41b.

183

Table 5.7: Bacterial counts (CFU/ml± SD) on surface washing carrot treated

with chlorine dioxide at different concentration and time of exposure

Values are mean of three replicates (± S.D)

Incubationtime inminutes

Bacterial count (CFU/ml ±S.D)Concentration of chlorine dioxide (ClO2)

2ppm 4ppm 6ppm 8ppm 10ppm

10 36±6 14.33±2.52 7.66±1.53 3±1 Absent

20 21.33±4.16 7.33±1.53 Absent Absent Absent

30 11±2 3.33±1.58 Absent Absent Absent

184

I II III IV

V VI VII

(a) Stored at ~26°C

(b) Stored at 6°C

Figure 5.40 (a&b): Effect of GOx, LPS and lysozyme incorporated with

alginate film coated carrots stored at (A) Stored at ~26°C and 6°C (I) Control (II) Sodium alginate (III) GOx (IV) LPS (V) Lysozyme with EDTA

(VI) GOx+LPS (VII) GOx, LPS and Lysozyme with EDTA

I II III IV

V VI VII

185

0

5

10

15

20

25

30

0 2 4 2 8 10Storage period (d)

Wei

ght l

oss

(%)

Alginate film+F-VII Alginate film Control

b

a

a

aa

a

b

b

bb

b

b

c

bcb

0

5

10

15

20

25

30

35

40

45

50

0 2 4 2 8 10Storage period (d)

Wei

ght l

oss

(%)


b

a

a

aa

a

bb

bb

b

b b cbc

c

Figure 5.41 (a&b): Effect of alginate coated (Formulation VII) and uncoated carrots stored on weight loss (%) during the storage period at (a) 6°C and (b) ~26ºC

Legends followed by the same letter are not significantly different (P<0.05) for the same storage period. Values represent the mean of triplicates with standard deviation.

(a): Stored at 6°C

(b): Stored at ~26°C

186

5.5.1.6 Measurement of soluble protein content

There was no significant variation obtained in soluble protein content

during the storage period. The initial soluble protein content of treated carrot

(Formulation-VII) was 0.51±0.0271 mg/g dry weight which dropped significantly

and reached its lowest value of 0.423±0.088 mg/g dry weight. The increment in

soluble protein during the last few days of storage may be attributed to the

formation of some stress proteins, or to the senescence, degradation and enzymatic

activities that soften the texture and lead to the formation of more soluble proteins.

The decline in SPC activities between 2–6 days could be related to the utilization

of soluble proteins in some metabolic activities such as a substrate for respiration,

when the carbohydrate sources become very limited. Similar conclusion was

reported by King et al. (1990), who indicated that more CO2 was released from

asparagus spears over 3–5 days than could be accounted by carbohydrates loss

(Table 5.8).

5.5.1.7 Enumeration of bacterial population from treated and control carrots

The plate count observations were expressed in the Figure 5.42 in terms of

CFU/g. The results showed that treated carrots formulation-VII was less in

microbial population when compared to the control.


The results of sensory analysis of treated and untreated carrots are

expressed in Figure 5.43(a) and 5.43(b). The carrot treated with formulation-VII

[GOx (5 mg/ml), LPS (10 mg/ml) and Lysozyme (0.5 mg/ml) with EDTA (0.3

mg/ml)] were accepted throughout the storage period both 6ºC and ~26°C. There

was no significant difference found in taste of alginate coated and alginate with

formulation-VII but good score was obtained on colour and texture. However,

higher overall acceptances of carrots were received from the treatment of alginate

with formulation-VII with respect to appearance, taste, texture and colour.

187

Table 5.8: Soluble protein content (mg/g of dry wt±SD) in alginate coated (Formulation VII) and uncoated

control carrots

Values are mean of three replicates (S.D). Numbers followed by the same letter are not significantly different (P < 0.05) for the same incubation period.

TreatmentSoluble protein content (mg/g of dry wt ± SD

Storage (days)0 2 4 6 8 10

Formulation VII 0.51±0.0271a 0.532±0.051a 0.513±0.038a 0.47±0.036a 0.453±0.022a 0.423±0.088a

Alginate film 0.524±0.024 a 0.512±0.068 a 0.492±0.048 a 0.462±0.0584a 0.442±0.054 a 0.418±0.087 a

Control 0.520±0.062a 0.512±0.038a 0.470±0.082a 0.43±0.03a 0.411±0.034a 0.378±0.068a

188

0

20

40

60

80

100

120


Mic

robi

al p

opul

atio

n (C

FU/g

)

~26°C 6°C

A

B

C

a

b

c

Figure 5.42: Showed the microbial population (10-6 dilution) the treated and control carrots after the storage period (10 d) stored at 6ºC and ~26ºC

Legends followed by the same letter are not significantly different (P<0.05) for thesame storage temperature. Values represent the mean of triplicates with standarddeviation.

189

0

1

2

3

4

5Appearance

Colour

TextureTaste

Overall acceptance

Formulation VII Alginate film Control

0

1

2

3

4

5Appearance

Colour

TextureTaste

Overallacceptance

Formulation VII Alginate film Control

Figure 5.43 (a&b): Evaluation of the sensory profile of treated and control carrots after the storage period (10 d) stored at (a) 6ºC and (b) ~26ºC. Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much.

Values represent the mean of triplicates.5.5.2 Control of browning and enhancing the shelf-life of apple puree by

glucose oxidase, catalase and lactoperoxidase

(b) Stored at ~26°C

(a) Stored at 6°C

190

5

6

7

8

9

10

log

CFU

/ml

Control

GOx (100 mg)

LPS (40mg)

Catalase (0.1ml)

GOx (100mg) +LPS (40mg)

a

cc

c cc

ab

b bb

bb

b

aa a a a

aa

dd

bc

c

c

cdc

b

cc

bc

cd

dc

cd

5.5.2.1 Assay of antimicrobial enzymes

The formulated GOx activity was found to be 5.2 U/mg and the optimum

pH range between 5and 6. The total activity of lyophilized LPS (0.5 M

sodium acetate elutes) showed 40 U/mg and the optimum pH between 5.5 and 6.5.

The Catalase activity was found to be 2000U/ml and the optimum pH for its

activity was 5–6.

5.5.2.2 Evaluation of antimicrobial activity of GOx, catalase and LPS

The effect of antimicrobial activity of enzyme was evaluated by the

formulations containing; (mg or ml /10ml) like, of (I) GOx (100 mg), (II) LPS

(40mg), (III) Catalase (0.1ml), (IV) GOx (100mg) +LPS (40mg) (V) GOx

(100mg)+Catalase (0.1ml), (VI) LPS (40mg)+Catalase (0.1ml) (VII) GOx

(100mg)+Catalase (0.1ml)+LPS (40mg) and (VIII) Control. The inhibitory

activity of the above enzymes and their combinations were evaluated against

E. coli and S. aureus. After 48 hours of incubation, the formlation-VII

combinationshowed effective control against both E. coli and S. aureus, that

decreasing the cell population to 2.01 and 2.1 log CFU/ml respectively (Figure

5.44a and 5.44b).

Davidson et al. (2001) studied a number of naturally occurring

antimicrobials including GOx, LPS, lactoferrin, lysozyme, avidin, plant extracts

(spices, essential oils, sulfur and phenolic compounds). Seifu et al. (2005) state

that the presence of GOx allows hydrogen peroxide required by LPS to be

continuously generated and replenished. The hydrogen peroxide produced by

GOx is utilized by the LPS for room temperature sterilization, while the gluconic

acid produced is used for direct acidification (Fox and Stepaniak, 1993).

191

0

1

2

3

4

5

6

7

8

9

10


log

CFU

/ml

Control

GOx (100 mg)

LPS (40mg)

Catalase (0.1ml)

GOx (100mg) +LPS (40mg)

GOx (100mg)+Catalase (0.1ml)

LPS (40mg)+Catalase (0.1ml)

GOx (100mg)+Catalase(0.1ml)+LPS (40mg)

a

eef

f

d

ddc

b

c

d

ed

dd d

b

cc

c c

cc

aa

a bb

bb b

a aa

a aa

a

a

ff

b

cdc

c c

b

cc

d

Figure 5.44 (a&b): Effect of antimicrobial activity of GOx, catalase and LPS against (a) E. coli and (b) S. aureus

Legends followed by the same letter are not significantly different (P<0.05) for the same incubation period. Values represent the mean of triplicates with standard deviation.

The LPS, when used in conjunction with GOx, is a very useful

antimicrobial agent. LPS is part of the immune system’s innate defense

mechanism against foreign microorganisms and can be found in mammalian

secretions such as milk, tears and saliva. This system consists of three components

like LPS, thiocyanate and hydrogen peroxide. The LPS activation occurs only in

(b) S .aureus

(a) E. coli

192

the presence of thiocyanate and hydrogen peroxide. Catalysis by LPS generates

active intermediates, which has antimicrobial properties and is completely safe to

humans. The LPS and GOx combination was not only used as a antimicrobial

agent and interestingly they also used in preparation of toothpaste (Biotene 2006;

National Library of Medicine 2007a), lotions (National Library of Medicine

2007b), shampoos, cosmetics and also applied in meat processing (Food Standards

Australia New Zealand 2002) and fish farming (Seifu et al., 2005).

5.5.2.3 Effect of GOx and ascorbic acid on removal of dissolved oxygen from

apple puree

Three concentrations of ascorbic acid (50, 100 and 150 mg/l) were tested on

the apple puree for the curb of oxygen. The complete oxygen disappeared

happened on 100 and 150 mg/l of ascorbic acid at 10 and 8 minutes respectively.

The GOx also applied in three concentrations such as 50, 100 and 150 mg/l and

the complete removal of dissolved oxygen appeared at 8 (50 mg/l), 6 (100 mg/l)

and 5 (mg/l) minutes. Based on the observation, GOx exhibit better response than

the ascorbic acid (Figure 5.45).

Parpinello et al. (2002) reported that the treatment of fruit purees with GOx

removed the 99% of dissolved oxygen within 120 seconds. On the other hand,

fruit purees treated with ascorbic acid required up to 200 minutes to achieve

similar results. These findings are supported to this study for the controlling of

oxidative browning reactions of fruit purees controlled by the treatment of GOx.

193

5.5.2.4 Effect of GOx, catalase, LPS and ascorbic acid on controlling of

browning in apple puree

The formulations containing GOx-catalase and ascorbic acid were showed

effective control of browning when compared with other treatments (Figure 5.46).

The LPS and catalase did not showing significant antibrowning activity. However,

GOx was very effective in the presence of catalase combination (Figure 5.47a and

5.47b). It is clearly exhibiting the oxygen is a key factor in fruit purees browning.

The preparation of apple puree with the combination of GOx-catalase and LPS

was exhibited good antibrowning and antibacterial activity. A similar observation

was reported by Mistry and Min (1992a) that the GOx-catalase system is able to

scavenge the oxygen and thus stabilizes foods and beverages against problems

related to product oxidation and browning.

Sathiya moorthi et al. (2007) state that browning reaction occurs during

storage of fruits as well as purees and juices. In other words, this reaction has a

negative effect on the quality and shelf-life of the product. GOx-catalase system

helps to scavenge the oxygen and it thus helps in preventing oxidation and

browning. Heating has been the unique treatment able to control the enzymatic

browning of fruit purees, whereas GOx showed efficient in reducing to low level

the dissolved oxygen content in apple purees and showed an interesting capability

to control the non-enzymatic browning during apple purees storage.

Enzymic browning starts with the initial enzymic oxidation of phenols to

quinones by the enzyme polyphenol oxidase in the presence of oxygen. Then these

quinones are subjected to further reactions, enzymically catalyzed or not, leading

to the formation of pigments. Cloudy apple juice has increasing market value due

to its sensory and nutritional qualities. Although a typical amber-like hue is

commercially desirable in clarified apple juice, both apple puree and cloudy juice

194

0

2

4

6

8

10

12

14

0 1 2 3 4 5 6 7 8 9 10

Incubation period (minute)

Dis

solv

ed o

xyge

n co

nten

t (pp

m)

Control Ascorbic acid 50mg Ascorbic acid 100mgAscorbic acid 150mg GOx50mg GOx100mgGOx150mg

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Figure 5.45: Effect GOx and ascorbic acid on removal of dissolved oxygen from apple puree Legends followed by the same letter are not significantly different (P<0.05) for the same incubation period. Values represent the mean of triplicates with standard deviation.

Figure 5.46: Effect GOx, catalase, LPS and ascorbic acid on controlling of browning in apple puree Legends followed by the same letter are not significantly different (P<0.05) for the same storage period (d). Values represent the mean of triplicates with standard deviation

195

Control Ascorbic acid GOx

(b) Apple puree

Control Ascorbic acid Formulation-VII

(a) Cut apple

Figure 5.47 (a&b): Effect GOx and ascorbic acid on controlling of browning in (a) Cut apple and (b) Apple puree

Formulation-VII: GOx (100mg) + catalase (0.1ml)+LPS (40mg)

196

are expected to have the yellowish colour which characterizes the fresh product

(Lozano et al., 1994). The food industry has given increasing attention to

minimally processed products. This might be achieved in raw juices by membrane

filtration, ultra high pressure treatment and preservation by freezing.

The control of enzymatic browning has great importance just at the start of

the processes. An approach for the prevention of enzymatic browning of fruit

juices has been the use of antibrowning agents. The most widely used

antibrowning agents are sulfiting agents. Due to adverse health effects, several

studies have been devoted to the non sulfite anti browning agents such as reducing

agents (ascorbic acid and analogs, glutathione, L-cysteine), enzyme inhibitors

(aromatic carboxylic acids, substituted resorcinols, anions, peptides), chelating

agents (phosphates, organic acids), acidulants (citric acid, phosphoric acid),

complexing agents (cyclodextrins) (Labuza et al., 1992; Lambrech, 1995;

Martinez and Whitaker, 1995).

However, ascorbic acid decreased the non-enzymatic browning of fruit

purees to a larger extent. Browning reactions occur during post harvest of fruits

processing, storage of purees and juices that may have a negative effect on the

quality and shelf-life of the products. These differences in the mechanism of

inhibition may allow the use of combinations of antibrowning agents that may

result in enhancement of inhibition. Most combinations of antibrowning agents or

commercially available are ascorbic acid-based compositions (Pizzocarno et al.,

1993).

197

5.5.2.5 Examination of microbial populations

The formulation-VII [(GOx (100mg) + Catalase (0.1ml) + LPS (40mg)]

was found to be effective control of microbial growth, when compared with other

treatments (Figure 5.48). Seifu et al. (2005) state that GOx is one of the most

important enzyme in food processing industry for food preservation. The

combinations of LPS and GOx were very useful antimicrobial agent. Further,

reported that the presence of GOx allows hydrogen peroxide required by LPS to

be continuously generated and replenished. The hydrogen peroxide produced by

GOx is utilized by the LPS for cold, i.e. room temperature sterilization, while the

gluconic acid produced is used for direct acidification (Fox and Stepaniak, 1993).


The sensory analyses of treated and untreated apple puree are expressed in

figure 5.49. The apple puree treated with Formulation-VII contains GOx

(100mg)+Catalase(0.1ml)+LPS(40mg) was accepted throughout the storage

period. The ascorbic acid treated apple puree had a good sensory score on colour

and appearance, while GOx-catalase and LPS treatment produces a similar result

along with good sensory scores with respect to taste, flavour and overall

acceptance.

198

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/ml)

ControlGOx (100 mg)LPS (40mg)Catalase (0.1ml)GOx (100mg) +LPS (40mg)GOx (100mg)+Catalase (0.1ml)LPS (40mg)+Catalase (0.1ml)GOx (100mg)+Catalase (0.1ml)+LPS (40mg)Ascorbic acid (100mg)

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Control GOx+Catalase+LPS Ascarbic acid

Figure 5.48: Showed the microbial population of treated and control apple puree Legends followed by the same letter are not significantly different (P<0.05) for the same storage period. Values represent the mean of triplicates with standard deviation.

Figure 5.49: Evaluation of the sensory profile of treated and control apple puree Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.5.5.3 Studies on the effects of glucose oxidase-catalase with calcium ions

199

for the improvement of fruit salad quality

5.5.3.1 Evaluation of antimicrobial activity of GOx, catalase, calcium ions and

koruk juice

The GOx, calcium propionate, koruk juice and also combined treatment

showed Significant (P<0.05) inhibitory activity against E. coli and S. aureus.

There were no significant inhibitory activities exhibited with calcium chloride and

calcium lactate treatments when compared with other treatments against both the

tested microbes (Figure 5.50a and 5.50b). The hydrogen peroxide produced by the

GOx acts as a good bactericide and can be later removed using catalase which

converts hydrogen peroxide to oxygen and water. The GOx was also found to be

antagonistic potential against different food borne pathogens such as Salmonella

infantis, S. aureus, Clostridium perfringens, Bacillus cereus, Campylobacter jejuni

and L. monocytogenes (Tiina and Sandhlm, 1989). Phenolic compounds in grape

juice, grape seed and wine have been investigated by many researchers to show

their potent antioxidant, antimutagenic, antibacterial, antiviral, antifungal and

antiulcer activities (Takechi et al., 1985; Liviero et al., 1994; Caccioni et al.,

1998; Saito et al., 1998; Baydar et al., 2004; Jayaprakasha et al., 2003). Koruk

juice showed good antimicrobial effect against S. aureus


After 24 h of storage period the sensory analysis scores were expressed in

the radar plots. The effect of antibrowning activity of calcium ions, GOx and

koruk juice on cut apple, pomegranate and guava were shown in figure 5.51

(A-G).

200

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/ml

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Figure 5.50 (a&b): Effect of antimicrobial activity of GOx, calcium ions and koruk juice against (a) E. coli and (b) S. aureus

Legends followed by the same letter are not significantly different (P<0.05) for the same incubation period. Values represent the mean of triplicates with standard deviation

(a) E. coli

(b) S. aureus

201

5.5.3.3 Sensory analysis of GOx-catalase treated fruit salad

The fruit salad prepared with GOx-catalase was found to be no changes in

their appearance, odour and browning in the sensory analysis. The concentration

of 0.25% (w/v) of GOx-catalase produced a maximum sensory score of 4.6

(Figure 5.52a and 5.55a). Mistry and Min (1992b) reported that the GOx-catalase

system scavenge the oxygen and resulted to stabilizes foods and beverages against

problems related to product oxidation and browning. According to Bao et al.

(2001 and 2003) the food grade GOx preparation used typically contains a mixture

of GOx and catalase because the two enzymes are found naturally together in the

mycelium of the cell wall (Witteveen et al., 1992). Catalase assists in the

breakdown of hydrogen peroxide produced by GOx, thereby reducing inhibition

and deactivation by hydrogen peroxide.

5.5.3.4 Sensory analysis of calcium chloride treated fruit salad

Calcium chloride was produced better texture, but browning was appeared

at 0.5% (w/v) concentration. Higher concentration of calcium chloride revealed

bitter taste and off odour. It was found to be optimum with a sensory score of 4.2

at 1% (w/v) (Figure 5.52b). Ohlsson (1994) reported that the use of calcium

chloride is associated with bitterness and off-flavour.

5.5.3.5 Sensory analysis of calcium propionate treated fruit salad

The fruit salad prepared with calcium propionate was found to have a

sensory evaluation of good taste, slightly better appearance and odour but slightly

browning was observed in the 0.5% (w/v) concentration. Higher concentration

exhibited sour taste and lowering the colour of the fruit salad. Satisfactory sensory

qualities were obtained at the concentration of 1.0% (w/v) with the sensory score

of 4.4 (Figure 5.53a).

202

0

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Overalacceptance

0.50% 1.0% 2.0% Control

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5Texture

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TasteOdour

Overal acceptance

0.25% 0.5% 1.0% Control

Figure 5.52 (a&b): Evaluation of the sensory profile of different concentration of (a) GOx and (b) calcium chloride treated and control fruit salad Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.

(b) Calcium chloride

(a) Glucose oxidase

203

5.5.3.6 Sensory analysis of calcium lactate treated fruit salad

The fruit salad prepared with calcium lactate was found to have a sensory

report of good taste, odour, colour but the texture of the fruit salad remained

slightly bad than the other parameters at the concentration of 0.5% (w/v).

However, the other two concentrations 1.0 and 2.0% (w/v) exhibited good score

and 1.0% (w/v) was found to be optimum with a sensory score of 4.6 (Figure

5.53b) Calcium lactate, calcium propionate and calcium gluconate have shown

some of the benefits of the use of calcium chloride, such as product firmness

improvement, and avoid some of the disadvantages, such as bitterness and residual

flavour (Yang and Lawsless, 2003).

5.5.3.7 Sensory analysis of koruk juice treated fruit salad

The fruit salad prepared with koruk juice exhibits no change in taste, very

less odour, little change in appearance and with less browning, when compared the

control fruit salad. It was found to be the optimum concentration at 1.0% (v/v)

with a sensory score of 4.4 (Figure 5.54 and 5.55b). Koruk juice is commonly used

in the preparation of fruit and vegetable salads as an acidifying and flavouring

agent in Turkey and neighboring countries. It is also consumed as a drink after

being sweetened. Currently grape compounds have attracted increased attention

especially in the Welds of nutrition, health and medicine (Waterhouse and

Walzem, 1998).

204

0

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TasteOdour

Overalacceptance

0.50% 1.0% 2.0% Control

0

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5Texture

Colour

TasteOdour

Overalacceptance

0.50% 1.0% 2.0% Control

Figure 5.53 (a&b): Evaluation of the sensory profile of different concentration of (a)calcium propionate and (b) calcium lactate treated and control fruit salad Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.

(b) Calcium lactate

(a) Calcium propionate

205

0

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3

4

5Texture

Colour

TasteOdour

Overal acceptance

0.5% 1.0% 1.5% Control

Figure 5.54: Evaluation of the sensory profile of different concentration of koruk juice treated and control fruit salad Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.

206

(a) GOx

(b) Extract of koruk juice

Figure 5.55 (a&b): Effect of GOx and koruk juice on fruit salad

207

5.5.3.8 Optimization of calcium ions concentrations by response surface

methodology


of set of three independent variables such as calcium lactate, calcium propionate

and calcium chloride on combination are presented in the table 5.9.

The Model F-value of 6.92 implies the model is significant. There is only

0.34% chance that a "Model F-Value" that large could occur due to noise. Values

of Prob > F" less than 0.05 indicate model terms are significant. In that case A, C

were significant model terms. Values greater than 0.1 indicate the model terms are

not significant. If there are many insignificant model terms (not counting those

required to support hierarchy), model reduction may improve the model (Table

5.9.1).The "Lack of Fit F-value" of 9.87 exhibit as significant and there is only

slight chance of 1.02% variation in "Lack of Fit F-value" due to noise.

The "Pred R-Squared" of 0.2886 is in reasonable agreement with the "Adj

R-Squared" of 0.4831. "Adeq Precision" measures the signal to noise ratio and a

ratio greater than 4 is desirable. The ratio of 9.246 indicates an adequate signal.

This model can be used to navigate the design space. The model coefficient was



Weight loss = 27.14-2.56×A-1.44×B-1.65×C


Weight loss = 35.46090-2.55814×Calcium lactate-2.30105×Calcium

propionate-4.11712×Calcium chloride

208

Table 5.9: Optimization of calcium ions for salad preparation by CCD of response surface methodology (23 factorial design)

Run

ACalciumlactate

(%,w/v)

BCalcium

Propionate(%,w/v)

CCalciumchloride(%,w/v)

Response1weight loss

(%,w/v)

Response2overall

acceptence(sensoryscore)

1 1.50 1.93 0.60 21.2 4

2 1.50 0.88 0.60 24.5 4.6

3 1.50 0.88 0.60 25.7 4.8

4 0.50 1.50 1.00 30.5 3.2

5 0.50 0.25 1.00 32.3 2.8

6 2.50 0.25 1.00 24.7 3.6

7 1.50 0.88 -0.07 28.5 3.4

8 2.50 0.25 0.20 29.4 4

9 -0.18 0.88 0.60 31.5 3

10 1.50 0.88 0.60 23.7 4.8

11 2.50 1.50 0.20 29.7 4.2

12 2.50 1.50 1.00 24.7 3.8

13 1.50 -0.18 0.60 30.5 4

14 0.50 0.25 0.20 34 3.8

15 1.50 0.88 0.60 25.5 4.6

16 3.18 0.88 0.60 22.5 4.2

17 1.50 0.88 1.27 22.5 4

18 1.50 0.88 0.60 26.1 4.8

19 0.50 1.50 0.20 31.5 3.8

20 1.50 0.88 0.60 23.8 4.6

209


SourceSum ofSquares

df MeanSquare

FValue

p-valueProb >

Model 154.6567 3 51.55223 6.920266 0.0034

A- Calcium lactate 89.3715 1 89.3715 11.99705 0.0032

B-Calcium propionate 28.24633 1 28.24633 3.79173 0.0693C- Calcium chloride 37.03885 1 37.03885 4.97202 0.0404

Residual 119.1913 16 7.449457

Lack of Fit 113.943 11 10.35845 9.868326 0.0102

Pure Error 5.248333 5 1.049667

Cor Total 273.848 19


STD DEV 2.729369 R-SQUARED 0.564754


C.V. % 10.05663 Pred R-Squared 0.288577PRESS 194.8217 Adeq Precision 9.246421

210

The results of central composite design experiments for studying the effects of

set of three independent variables, calcium lactate, calcium propionate, calcium

chloride on overall acceptance are presented in Table 5.9.

The Model F-value of 7.09 implies the model is significant. There is only a

0.26% chance that a "Model F-Value" that large could occur due to noise.Values

of "Prob > F" less than 0.0500 indicate model terms are significant.

In that case A, A2, B2, C2 were significant model terms. Values greater than

0.100 indicate the model terms are not significant. The "Lack of Fit F-value" of

14.35 implies the Lack of Fit is significant (Table 5.9.3). There is only 0.55%

chance that a "Lack of Fit F-value" that large could occur due to noise.

The "Pred R-Squared" of 0.0211 is not as close to the "Adj R-Squared" of

0.7427 as one might normally expect."Adeq Precision" measures the signal to

noise ratio and a ratio greater than 4 is desirable. The ratio of 7.609 indicates an

adequate signal (Table 5.9.4). This model can be used to navigate the design

space. The model coefficient was estimated by linear regression.

That may indicate a large block effect or a possible problem with the

model and/or data. Things to consider are model reduction, response

tranformation, outliers, etc. "Adeq Precision" measures the signal to noise ratio. A

ratio greater than 4 is desirable (Table 5.9.5). The ratio of 7.609 indicates an

adequate signal. That model can be used to navigate the design space and the

model coefficient estimated by linear regression

211



SquareF

Valuep-value

Prob > FModel 5.879 9 0.653 7.094 0.003 A-Calcium lactate 1.182 1 1.182 12.839 0.005 B-Calcium propionate 0.046 1 0.046 0.508 0.491 C-Calcium chloride 0.141 1 0.1416 1.538 0.243 AB 0 1 0 0 1.000 AC 0.08 1 0.08 0.868 0.373 BC 0.02 1 0.02 0.217 0.651 A^2 2.324 1 2.324 25.240 0.0005 B^2 0.975 1 0.975 10.593 0.008 C^2 1.932 1 1.932 20.991 0.001Residual 0.920 10 0.092Lack of Fit 0.860 5 0.172 14.346 0.005Pure Error 0.06 5 0.012

Table 5.9.4: Comparison of R2 predicted and estimated

STD DEV 0.30344 R-SQUARED 0.864Mean 4 Adj R-Squared 0.742C.V. % 7.586 Pred R-Squared 0.021PRESS 6.656 Adeq Precision 7.608

212


Factor CoefficientEstimate

Df StandardError

95% CILow

95% CIHigh

VIF

Intercept 4.701 1 0.123 4.426 4.977A-Calcium lactate 0.294 1 0.082 0.112 0.477 1B-Calciumpropionate 0.058 1 0.082 -0.124 0.241 1

C-Calcium chloride -0.101 1 0.082 -0.284 0.081 1AB 0 1 0.107 -0.239 0.239 1AC 0.1 1 0.107 -0.139 0.339 1BC 0.05 1 0.107 -0.189 0.289 1A^2 -0.401 1 0.079 -0.579 -0.223 1.018B^2 -0.260 1 0.079 -0.438 -0.082 1.018C^2 -0.366 1 0.079 -0.544 -0.188 1.018


Overal acceptancy =+4.70+0.29×A+0.059×B-0.10×+0.000 ×A ×B+0.10 ×A×C

+0.050 × B × C-0.40×A2-0.26×B

2-0.37×C

2


Overal acceptancy = 2.42389+1.34896×Calcium lactate+1.13924×Calcium

propionate +1.94207 ×Calcium chloride +2.32374E-015×Calcium lactate

×Calcium propionate + 0.25000 ×Calcium lactate ×Calcium

chloride+0.20000×Calcium propionate ×Calcium chloride - 0.40158×Calcium

lactate2-0.66601×Calcium propionate2-2.28891×Calcium chloride2

The contour and three dimensional response surface curves were plotted.

Maximum overall acceptance was achieved at: Calcium lactate-1.45% (w/v),

calcium propionate-0.68% (w/v), calcium chloride-0.55% (w/v). For the optimized

values the weight loss and overall acceptance was observed maximum of 27.2%

and 4.70 respectively. If the values of the calcium ions concentration was changed

the weight loss and overall acceptance were changing in the response (Figure

5.56-5.61).

213Design-Expert® Software

Weight lossDesign Points34

21.2

X1 = A: Calcium lactateX2 = B: Calcium propionate

Actual FactorC: Calcium chloride = 0.60

0.50 1.00 1.50 2.00 2.50

0.25

0.56

0.88

1.19

1.50Weight loss

A: Calcium lactate

B: C

alci

um p

ropi

onat

e

24.4758

25.807927.14

28.4721

29.8042

6


Weight lossDesign points below predicted value34

21.2



0.5

1

1.5

2

2.5

0.25 0.56 0.88 1.19 1.50

23.1

25.125

27.15

29.175

31.2

Wei

ght l

oss

A: Calcium lactate

B: Calcium propionate

Figure 5.56: The contour and 3D response surface plot showing the weight loss % of calcium lactate and calcium propionate treatment on fruit salad



21.2

X1 = B: Calcium propionateX2 = C: Calcium chloride

Actual FactorA: Calcium lactate = 1.50

0.25 0.56 0.88 1.19 1.50

0.20

0.40

0.60

0.80

1.00Weight loss


C: C

alci

um c

hlor

ide

25.0833

26.1117

27.14

28.1683

29.1967

6



21.2



0.25

0.56

0.88

1.19

1.50

0.20 0.40 0.60 0.80 1.00

23.6

25.275

26.95

28.625

30.3

Wei

ght l

oss


C: Calcium chloride

Figure 5.57: The contour and 3D response surface plot showing the weight loss % of calcium propionate and calcium chloride treatment on fruit salad



21.2

X1 = C: Calcium chlorideX2 = A: Calcium lactate

Actual FactorB: Calcium propionate = 0.88

0.20 0.40 0.60 0.80 1.00

0.50

1.00

1.50

2.00

2.50Weight loss

C: Calcium chloride

A: C

alci

um la

ctat

e

24.3367

25.7383

27.14

28.5417

29.9433

6



21.2

X1 = C: Calcium chlorideX2 = A: Calcium lactate


0.20

0.40

0.60

0.80

1.00

0.50 1.00 1.50 2.00 2.50

22.9

25.025

27.15

29.275

31.4

Wei

ght l

oss

C: Calcium chloride

A: Calcium lactate

Figure 5.58: The contour and 3D response surface plot showing the weight loss % of calcium chloride and calcium lactate treatment on fruit salad


Overal acceptancyDesign Points4.8

2.8



0.50 1.00 1.50 2.00 2.50

0.25

0.56

0.88

1.19

1.50Overal acceptancy

A: Calcium lactate

B: C

alci

um p

ropi

onat

e

3.86601

4.04462

4.223234.40184

4.40184

4.58045

6


Overal acceptancyDesign points above predicted valueDesign points below predicted value4.8

2.8



0.50

1.00

1.50

2.00

2.50

0.25 0.56

0.88 1.19

1.50

3.6

3.925

4.25

4.575

4.9

Ove

ral a

ccep

tanc

y

A: Calcium lactate


Figure 5.59: The contour and 3D response surface plot showing the overall sensory acceptability profile of calcium propionate and calcium lactate treatmenton fruit salad

217



2.8

X1 = A: Calcium lactate

0.80


C: C

alci

um c

hlor

ide

3.65818

3.87829

4.31852



2.8



0.25 0.56 0.88 1.19 1.50

0.20

0.40

0.60

0.80



C: C

alci

um c

hlor

ide

4.00624.14728

4.28835 4.28835

4.28835

4.42943

4.429434.5705

6



2.8



0.25

0.56

0.88

1.19

1.50

0.20 0.40

0.60 0.80

1.00

3.86

4.095

4.33

4.565

4.8

Ove

ral a

ccep

tanc

y


C: Calcium chloride

Figure 5.60: The contour and 3D response surface plot showing the overall sensory acceptability profile of calcium propionate and calcium chloride treatment on fruit salad

218



2.8

X1 = A: Calcium lactateX2 = C: Calcium chloride


0.50

1.00

1.50

2.00

2.50

0.20 0.40

0.60 0.80

1.00

3.4

3.775

4.15

4.525

4.9

Ove

ral a

ccep

tanc

y

A: Calcium lactate

C: Calcium chloride

Figure 5.61: The contour and 3D response surface plot showing the overall sensory acceptability profile of calcium lactate and calcium chloride treatment on fruit salad

219

5.5.3.9 Effect of RSM optimized combined of calcium ions, GOx-catalase and

koruk juice on fruit salad

The calcium ions have its own merits and demerits with respect to sensory

qualities. To overcome this problem fruit salad was prepared with the combination

of calcium ions. The fruit salad prepared with the combination of calcium ions was

found to have a good sensory score of better appearance, good taste, odour and

texture with less browning and a sensory score of 4.6 (Figure 5.62 and Figure

5.63). Manganaris et al. (2007) state that the calcium lactate, calcium chloride,

calcium phosphate, calcium propionate and calcium gluconate, which are used

more when the objective is the preservation and/or the enhancement of the product

firmness.

Fruit salad prepared with the combination of optimized calcium ions, GOx-

catalase and koruk juice exhibited good sensory score with respect to colour,

texture, taste, odour and overall acceptance. In this treatment was found to be very

effective in all the parameters analyzed (Figure 5.64a , 5.64b and 5.6c).

Indeed combination of preservation treatments are often advocated (Knorr,

1998). Combinations of preservation treatments allow the required level of

protection to be achieved while at the same time retaining the organoleptic

qualities of the product such as, colour, flavour, texture, taste and nutritional

value. The potential use GOx, calcium ions and koruk juice in combinations in the

preparation of cut fruit and vegetable may lead to preserve the organolyptic quality

needs to individual products.

220

0

1

2

3

4

5Texture

Colour

TasteOdour

Overalacceptance

RSM optimized calcium ions+GOx-catalase+koruk juiceRSM optimized calcium ionsControl

Figure 5.62: Effect of RSM optimized calcium ions on fruit salad

Figure 5.63: Evaluation of the sensory profile of combined calcium ions (obtained from RSM) and GOx-catalase and koruk juice treated and control fruit salad Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.

221

(c) Control

(b) GOx-catalase+combined calcium+koruk juice

ions

(a) GOx-catalase+combined calcium ions

Figure 5.64 (a,b&c): Effect of GOx-catalase, calcium ions and koruk juice on fruit salad

222


The fruit salad treated with all the formulations examined to have lower

weight loss than treated fruit salads. The effect of lowering the water loss was

found with the treatment of (IV) optimized calcium ions combination and GOx-

catalase + calcium ions combination + koruk juice. The water loss of the sample

increased significantly during storage period (after 48 h). This was expected since

fresh-cut fruits and vegetables usually lose water after processing and throughout

storage. The loss of water is a natural process of the catabolism in fresh-cut

vegetables and is attributed to the respiration and other senescence-related

metabolic processes during storage (Watads and Qui 1999). The percentage

weight losses due to water loss of the fruit salad in the different formulations are

represented in Figure 5.65.

5.5.3.11 Enumeration of bacterial population from treated and control fruit salads

The plate count observations were expressed in terms of CFU/g (Figure

5.66). The GOx-catalase, koruk juice, calcium propionate and combined treated

fruit salad with were exhibited less in microbial population when compared with

untreated. Numerous epidemiologic studies exhibited, that a reduced risk of

degenerative diseases correlates with a high intake of fruits and vegetables

(Steinmetz and Potter, 1996). Ready-to-use salads can suit as useful sources of

minerals and physiologically active substances such as polyphenols, as the

increasing popularity of this convenience product indicates. Due to high bacterial

counts of raw vegetables after harvest up to 106–109 colony forming units per

gram fresh salad (CFU/g), ready-to-use sliced salads are usually contaminated by

microorganisms (Jaques and Morris, 1995). Even bacterial pathogens have been

detected in prepackaged salads (Lin et al., 1996) and lettuce (Park and Sanders,

1992). High initial counts are not substantially reduced during conventional cold

washing.

223

0 5 10 15 20 25 30 35 40 45

Calcium chloride (1%)

Calcium lactate (1%)

Calcium Propionate (1%)

Combined calcium ion (1%)

Koruk juice (1%)

Glucose oxidase (0.25%)

GOx+combined calcium ion

GOx+combined calcium ion+koruke juice

Control

Weight loss (%)

a

e

d

c

c

b

b

d

d

0

50

100

150

200

250

Calciumchloride (1%)

Calcium lactate(1%)

Calciumpropionate (1%)

Combinedcalcium ion

(1%)

Koruk juice(1%)

Glucoseoxidase(0.25%)

GOx+combinedcalcium ion

GOx+combinedcalcium ion+koruke juice

Control

Mic

robi

al p

opul

atio

n (C

FU/g

)

bc

bb

c

b

c c

b

a

Figure 5.65: Effect of GOx, calcium ions and koruk juice for controlling weight loss on fruit salad Legends followed by the same letter are not significantly different (P<0.05). Values represent the mean of triplicates with standard deviation.

Figure 5.66: Enumeration of microbial population of different treated and control fruit salad Legends followed by the same letter are not significantly different (P<0.05). Values represent the mean of triplicates with standard deviation.

224

CHAPTER-6

SUMMARY

The current study focused on the screening of GOx producing fungi and

their application in the food processing and preservation. GOx producing fungi

were isolated from various sugar rich products and the strains GOP3 and GOP7

(Aspergillus awamori belongs to the group of niger) isolated from dates and honey

hive produced the highest extra cellular GOx activity in the basal production

medium of 4.2±0.14 and 5.1±0.22 U/ml, respectively. The isolated higher GOx

producing fungus (GOP7) was submitted in Microbial Type Culture Collection

(MTCC), Chandigarh, India and it was designated as Aspergillus awamori MTCC

9645. This fungus was used for further studies.

Seven different medium were used for the selection of suitable medium for

GOx production in which GOxM3 supported maximum GOx activity. Production

medium was optimized by classical single factor analysis and also statistically by

response surface methodology using central composite design.

In single factor analysis, the highest GOx production was obtained in

glucose followed by sucrose at 80–100 g/l. Proteose peptone (3–4 g/l) was a

suitable nitrogen source for GOx production when compared to other nitrogen

sources. In the levels of 0.4 g/l of di-ammonium hydrogen phosphate and 0.2 g/l of

potassium di-hydrogen phosphate were found to be maximum production.

Addition of magnesium sulphate increase of the amount of GOx production in the

fermentation medium, while above 0.2 g/l of magnesium sulphate affects the GOx

production. Remarkably, GOx production was increased by the supplementation

of 30–40 g/l of calcium carbonate. Maximum GOx production was observed at pH

between 5 and 6. The optimum temperature was found to be from 30 to 35ºC for

the production of GOx and the optimum fermentation time for the GOx production

was at 84 h.

225

In Response Surface Methodology, the maximum GOx production (10.08

U/ml) was achieved at 92.7 g/l of glucose, 3.24 g/l of proteose peptone, 36.82 g/l

of calcium carbonate, 0.48 g/l of (NH4)2HPO4, 0.32 g/l of KH2PO4 and 0.23 g/l of

MgSO4. Higher amount of MgSO4 significantly affects the GOx production

followed by KH2PO4. Maximum GOx production was achieved at the pH 5.83 and

the temperature at 30.7°C. Response Surface Methodology was reduced the

fermentation time (65–70h) and significantly improved the GOx production, when

compared to single factor analysis.

Aspergillus awamori MTCC 9645 was cultivated in the two litre laboratory

batch fermentor. The fungal morphological structure was studied. The fungus

apparently grew exponentially up to 72 h and reached maximum concentration of

12.5±0.63 g/l cell dry mass and maintained more or less constant for the rest of

cultivation period. The utilization of glucose started higher after about 12 h and

complete consumption of glucose was observed 50–60 h of fermentation.

Production of gluconic acid was started at 12 h and maximum amount was

observed at 48 h as 62.3±4.1 g/l. The extracellular GOx secretion was started from

10 to 12 h and then reached maximum GOx activity at 72 h as 12±0.63 U/ml.

The concentrated protein obtained from ammonium sulphate precipitation

was purified in the DEAE-Cellulose column followed by Sephacryl S-200 column.

The enzyme activity was purified up to 9.19 folds with a final recovery of 12.98%.

The specific activity of purified GOx was 282.27 U/mg protein. On SDS-PAGE,

the purified GOx showed a single band indicating electrophoretically homogenous

with the molecular weight of 71.1 kDa. A single brown color band was formed in

the native gel. The Lineweaver’s Burk plot, Eadie-Hofstee plot and Hans-Woolf

linear plots were used to determine the Vmax and Km values of the GOx from A.

awamori MTCC 1945.

226

The optimum temperature and pH of GOx activity were found to be 30±2ºC

and pH of 5.5, respectively. The stability of the purified GOx was tested at 25 and

37ºC and the relative activity not affected over 12 h at 25 ºC, while showed a half

life of 40 minutes at 37ºC. All the metal ions showed various degrees of inhibition;

the inhibition sequence was in the order: copper acetate > silver sulphate >

cobaltous chloride > mercuric chloride > copper sulphate at same concentration.

The activity of calcium lactate treated GOx was less sensitive to pH

changes at acidic and alkaline pH as compared with that of untreated control

enzyme. The effect of calcium ions on temperature stability of GOx was found to

be effective and it was observed a maximum stability at 50°C in calcium lactate

treated GOx as 86.67% but in control showed only 43.3% of relative activity.

The combined effect of GOx, LPS and Lysozyme with EDTA were showed

good inhibitory activity than the other formulations against Escherichia coli and

Staphylococcus aureus. The enzymes incorporated into the alginate film (GOx,

LPS, Lysozymes with EDTA) showed good antimicrobial and minimizing the

microbial contamination during storage period. Good sensory score was obtained

in the carrot treated with formulation VII [GOx (5 mg/ml), LPS (10 mg/ml) and

lysozyme (0.5 mg/ml) with EDTA (0.3 mg/ml)] and accepted throughout the

storage period both 6 ºC and ~26°C.

The control of browning and enhancing the shelf-life of apple puree by

GOx, catalase and LPS were studied. The LPS and catalase did not show

significant antibrowning activity. However, GOx was very effective in the

presence of catalase combination. The formulation-VII [(GOx (100mg) + Catalase

(0.1ml) + LPS (40mg)] was found to be effective control of browning and

microbial growth in apple puree. GOx-catalase and LPS treatment produced good

sensory scores.

227

The effects of GOx-catalase with calcium ions for the improvement of fruit

salad quality were evaluated. Fruit salad prepared with the combination of GOx-

catalase, optimized calcium ions exhibited effective antimicrobial, antibrowning

activities. Further, it provided good sensory score with respect to colour, texture,

taste, odour and overall acceptance.

228

CHAPTER 7

CONCLUSIONS AND SCOPE FOR FUTURE WORK

In the present investigation, glucose oxidase producing

Aspergillus awamori MTCC 9645 was isolated and optimum media constituents

were analyzed by single factor analysis and central composite design. The single

factor analysis method indicated glucose, proteose peptone, calcium carbonate and

di-ammonium hydrogen phosphate contributing higher GOx production. Statistical

method for media optimization resulted significant increase in GOx production.

Alginate film coated carrot with the formulation of GOx, LPS and lysozyme with

EDTA exhibited good shelf-life. Apple puree was processed with the combination

of GOx-catalase with LPS exhibited good antibrowning and antibacterial

properties. The combination of GOx-catalase with calcium ions showed

antibacterial and antibrowning activity in salad that increased the storage stability

and high sensory score. The observations from the present findings were clearly

showed that potential of glucose oxidase would use in food processing and

preservation with combined preservation treatments.

Currently, the industrial and food applications of the commercialization of

GOx have an important role. Although, it is remains very less in comparison to

that of hydrolytic enzymes. This enzyme can be used in different formulation for

innovative food processing, preservations. This leads to substantial increase of

utilization in food industry.

Nowadays, consumers are more concerned over the safety of food and so,

the demand for natural foods has spurred the search for biopreservatives. The

antimicrobial packaging is a rapidly developing technology that can be employed

for controlling food borne microbial outbreaks caused mainly by the easily

prepared and minimally processed fresh vegetables and fruits. A greater emphasis

229

on safety features associated with the addition of natural antimicrobial agents

maybe the next area for food processing and preservation. Many of the

antimicrobial compounds studied are not permitted for food application, as they

need to migrate to the food to be effective. Technical challenges exist in

implementing appropriate antimicrobial agents into the process. Researchers have

focused primarily on developing new approaches and testing new methods on

model systems and not quite as much on applications in real food products.

Antimicrobial enzymes and natural compounds must focus on the technical

feasibility, consumer acceptance and food safety aspects of antimicrobial agents in

addition to their chemical, microbiological and physiological effects.

230

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258

LIST OF PUBLICATIONS, PRESENTATIONS AND

CONFERENCES

List of publications in National and International Journals

· Sathiya moorthi P, Rajkumar R and Kalaichelvan PT (2007). Applications of glucoseoxidase enzyme in food Industries. Modern Food Processing 3(1): 74–76.

· Sathiya moorthi P, Periyar selvam S, Deecaraman M and Kalaichelvan PT (2008).

Efficiencies of alternate carrier electives for Rhizobium bio-fertilizer. ICFAI Journal of

Life Sciences 2(2): 41–51.

· Sathiya moorthi P, Deecaraman M and Kalaichelvan PT (2009). Co-immobilization ofBacillus megaterium and Rhizobium leguminoserum for efficient phosphorus andnitrogen fixation on Cicer arietinum. Journal of Plant Disease Sciences 4(1): 41–51.

· Sathiya moorthi P, Jijendrakumar P, Jayakumar and Kalaichelvan PT (2008).Lactoperoxidase enzyme system: The future of preservation. Modern Food Processing3(11): 60–64.

· Sathiya moorthi P, Periyar selvam S, Sasikalaveni A, Murugesan K and KalaichelvanPT (2006). Biological decolorization of textile dyes and their effluents using white rotfungi. African Journal of Biotechnology, 6(4): 425–429.

· Sathiya moorthi P, Periyar selvam S, Deecaraman M, Murugesan K and KalaichelvanPT (2008) Biosorption of textile dyes and effluents by Pleurotus florida, Tameteshirsuta and evaluation of their laccase enzyme activity. Iranian Journal ofBiotechnology, 5(2): 1–5.

· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT (2008). Bioremediation ofauto mobile oil effluent by Pseudomonas sp. Journal of Advanced Biotechnology,6(12): 34–37.

· Sathiya moorthi P, Deecaraman M, Praveen kumar K, Kishan vaidyanat N andKalaichelvan PT (2009). Screening of α- Amylase inhibitors from Natural sources;with particular reference to Stevia rebaudianl. Journal of Advanced Biotechnology,8(10): 33–36.

· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. Enhancing shelf-life ofcarrot by using sodium alginate film incorporated with glucose oxidase,lactoperoxidase and lysozyme. Journal of Food Science (Under Revision).

259

List of paper presentation

· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. “Screening of glucoseoxidase producing fungi and factors regulating production”. National Conferenceon “Resent Trends in Mycological Research” on December 28th and 29th, 2006,Mycological Society of India at J.J. College of Science, Pudukkottai, Tamil Nadu.

· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. “Preparation of ediblecarboxy methyl cellulose film incorporated with glucose oxidase, lactoperoxidaseand lysozyme for prawn preservation” at Young Scientist conference at LoyolaCollege, Chennai 3rd and 4th December 2007.

· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. “Incorporation ofpartially purified antimicrobial enzymes in to sodium alginate films for carrotpreservation”. Safety assessment and consumer production with reference toDairy and Food industry. Dept. of Dairy Science, Madras Veterinary University,Chennai. 28th and 29th November 2007.

· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. “Industrial enzymes forfood processing and preservation”. Safety assessment and consumer productionwith reference to Dairy and Food industry. Dept. of Dairy Science, MadrasVeterinary University, Chennai. 28th and 29th November 2007.

· Sathiya moorthi P, Rekha G, Sasikalaveni A, and Kalaichelvan PT.“Decolorization of Textile dyes by white rot fungi” in the National Conference onCurrent Perspectives in Aquatic Biology, held at University of Madras, Chennai.During 17th and 18th March 2006.

· Praveeven kumar K, Kishan vaidyanat N and Sathiya moorthi P. "Production ofedible films incorporated with anti microbial enzymes for the preservation ofcarrots" in the International conference, Bangalore Bio-2007, during 7th to 9th

June 2007.

260

List of conference and work shop participated

· Training course in “Fruit and Vegetable preservation and nutrition” coducted byGovernment of India, Ministry of Food and Nutrition Board, during the period of9th –13th July 2007.

· Participated in the Annual Conference on “Strategies for food safety and qualityin India. Jointly Organized by TNAU Coimbatore and University of California,Dvis, USA at TNAU, Coimbatore, during 26-28th October 2006.

· Participated in the National workshop on “Recent trends in Biotechnology andanalytical instrumentation” on 5–7th March 2008 conducted by Department ofPlant Biology and Biotechnology, Presidency College and SophisticatedAnalytical Instrument Facility, IIT Chennai.

· Participated in the conference on “Agro Food Processing Technologies” (Qualityand Safety in Fruits and Vegetables Processing), Organized by Confederation ofIndian Industry and USAID during 24th July 2009.

· Participated in the Biotechnology: UK and Indian Perspectives Organized byBritish council and Anna University, durind 17th and 18th Febraury 2008.

· Participated in the seminor on Herbal analysis and applications, Centre for HerbalSciences, University of Madras, Chennai, 29th October 2008.

· Participated in the “Theme workshop on Emerging Trends in EnvironmentalBiotechnology” held during 12th–14th January 2009 at National Institute ofTechnology Karnataka, Surathkal.

· Workshop on “Medical Biotechnology” at Biomedical Research Unit and LabAnimal Centre (BRULAC), Saveetha University, Chennai, during 4th–8th June2007.

· Attended the advanced technical workshop on “Water treatment solutions andwaste water management at Living Waterfine Technologies Pvt Ltd, Chennai,during 11th August 2006.

· Participated in the training programme at CIMAP Hyderabad on“Entrepreneurship development through medicinal and aromatic plantstechnologies” 9th–11th January 2008.

· Participated in the National Conference at University of Madras on “Trends inAlgal Biotechnology” 17th–19th February 2008.

261

Curriculum vitae of P. Sathiya moorthi

He was born on 2nd May 1981 at Salem, Tamilnadu. He have completed

under graduation in B.Sc., Microbiology (2001) at A.V.S. College of Science,

Periyar University and successfully finished his post graduation in M.Sc.,

Industrial Microbiology (2003) at Centre for Advanced Studies in Botany,

University of Madras. After completion of his Master Degree, he joined at Chinnu

Exports Bio-Products Division as Microbiologist and plant in-charge from 2003 to

2005. During this period he was gained the experience in various optimizations of

industrial processes, post harvest technology, value addition of agricultural

product, natural food processing and preservation, drinking water and effluent

treatment.

After that he was joined as a full time Research Scholar in the department

of Industrial Biotechnology, Dr. M.G.R. Educational and Research Institute

University, Maduravoyal, Chennai under the supervision of Prof. P.T.

Kalaichelvan and co-supervision of Prof. M. Deecaraman. He started his research

work on January 2006 in the production of glucose oxidase from fungi and their

applications in food processing and preservation. During the research period he

published several research papers and articles at National, International Journals

and scientific magazines. Further, he was also participated and presented many

research papers in various conferences and seminars. Moreover, he acquired the

following experience during his studies like Microbiological Techniques,

Biochemical analysis, Bioprocess techniques and assesses the research activities to

project students.

***

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