- vol. 26(3) May/June 2015 - Published by Tekno Scienze Srl

International Journal of Nutraceuticals Functional Foods and Healthy Ingredients

ISSN 1722-6996 AIHTEIISSN 1722-6996 AIHTEIISSN 1722-6996 AIHTEI

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Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015

Agro FOOD Industry Hi Techvol 26(3) MayJune 2015

Agro FOOD Industry Hi Tech Agro FOOD Industry Hi Tech Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015- vol 26(3) - MayJune 2015- vol 26(3) - MayJune 2015

TEKNO SCIENZE SRLViale Brianza 22 20127 Milano (Italy) Tel +39-02-2680937528381260Fax +39-02-2847226e-mail infoteknoscienzecomweb wwwteknoscienzecom

Editor in Chief Carla Scesa - infoteknoscienzecomEditorial Director Silvana Maini - silvanateknoscienzecomAssociate EditorsGayle De Maria - gayleteknoscienzecomFlorian Weighardt - florianteknoscienzecomMarketing amp SalesGiulio Fezzardini - giulioteknoscienzecomSilvia Baldina - silviateknoscienzecomSimona Rivarollo - simonateknoscienzecomMarketing amp EventsPaola Passadore - paolateknoscienzecomProduction ManagerElisa Novaresi - elisateknoscienzecomProduction Assistants amp IT departmentLuis A Alburqueque Diaz - luisteknoscienzecom Matteo Cattane - matteoteknoscienzecomAdministrative amp Account Alba Aprea - albateknoscienzecom

PRINTING OFFICEArti Grafiche Colombo (Muggiograve - MI Italia)Authorization from Tribunale di Milano n 514 of July 27th 2006 (Autorizzazione del Tribunale di Milano n 514 del 27 Luglio 2006)

SCIENTIFIC ADVISORY BOARDAnton Alldrick (CCFRA)Sybille Buchwald-Werner (Vital Solutions GmbH)Undurti N Das (UND Life Sciences)Inga Koehler (Pfizer Consumer Health Care)Ray A Matulka (Burdock Group)Vicente Micol (University Miguel Hernaacutendez) Arthur Ouwehand (University of Turku)Demosthenes B Panagiotakos (Harokopio University)Denis Poncelet (ONIRIS and Capsulae sarl)Igor Pravst (Nutrition Institute ndash Slovenia)Joe Romano (Waters)Yang Zhu (TNO Quality of Life)Ralf Zink (DMK - Deutsches Milchkontor)

All rights reserved to Tekno Scienze No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopy-ing recording or otherwise without the prior per-mission of the publisher Tekno Scienze Srl

Associated to USPI - Unione della Stampa Periodica Italiana

CONTENTS

1Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015

FROM THE EDITORIAL BOARD2 Expo 2015 feeding the planet energy for life A necessity a priority

S Maini

SENIOR NUTRITION4 Benefits of a Mangifera indica L fruit powder for well-aging

In vitro investigation of effects on master regulators for the aging processS Roumlchter C Ehrhardt S Buchwald-Werner K Berger-Buumlter E Schatton

JOINT HEALTH8 Mangosteen extract for short-term pain management Preclinical approach and pilot clinical investigation on volunteers with soft

tissue pain C Romain J Cases PRODUCT FOCUS14 NEMreg brand Eggshell Membrane - The Natural Choice and Fast

Acting Solution for Joint Health wwwstratumnutritioncomEU

CHOCOLATE16 Rise in ldquohealthierrdquo ldquobetter for yourdquo chocolate M Naprta

FUNCTIONAL FOOD20 Complex foods versus functional foods nutraceuticals and dietary

supplements differential health impact (Part 2) A Fardet FOOD PROCESSING24 The utilization of solid carbon dioxide in the extraction

of extra-virgin olive oil VOOEVOO yield and quality as a function of extraction conditions

adopted A Zinnai F Venturi C Sanmartin I Taglieri G Andrich 28 Optimization of ultrasonic assisted extraction of antioxidants

and phenolic compounds from red basil using response surface methodology

S Ahmadi R A Ghavidel Z Sheikholeslami M Elahi S Elahi

FOOD ANALYSIS34 1H-NMR spectra ndash a highly sensitive tool in the analysis of Grana

Parmigiano-Reggiano type hard cheese M Vezzani F Bellesia L Caglioti A Pinetti C Zucchi G Paacutelyi 38 Flavonoids in mulberry fruit Identification of nonanthocyanin phenolics in some mulberry fruit

species (Morus alba L Morus rubra L and Morus nigra L) V Miljkovic L Nikolic N Radulovic B Arsic G Nikolic D Kostic Z

Bojanic J Zvezdanovic

FOOD SAFETY43 Factors affecting consumersrsquo awareness of food safety O Kilic G A Eryilmaz

SUSTAINABILITY47 Date pits (Phoenix dactylifera (L)) waste to best

K Mahmood M S Alamri A Mohamed S Hussain

51 NEWS

56 EVENTS

International Journal of

Nutraceuticals Functional Foods and Healthy Ingredients


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Agro FOOD Industry Hi Tech is a peer-reviewed journal devoted to functional food and nutraceuticals addressed to a readership belonging to the industry The 2013 impact factor is 0294 according to the Thomson Reuters Journal Citation Reports

2

Several years ago I was in Cairo We had been invited to attend a medical conference It actually turned out to be a very nice experience Everyone gave us a warm and friendly welcome and all the Egyptians we met were so nice to us and the city was amazing so beautiful and full of history Irsquod also like to take the opportunity of this editorial piece to wish Egypt a future of peace and development much deserved by a country whose beauty and kind people strike you at fi rst encounter Having said that as we went around the conference speaking to people about the editorial content of our journals we also had the chance to illustrate our A-FOOD Industry hi tech journal to the organisers of the conference thus described that the journal focuses on health ingredients functional food food supplements as all our readers know very well After letting me talk for a bit one of the people listening to me told me ldquoDear Doctor thank you for your very interesting presentation however our main issue here is not dietary supplements but rather the very food wersquore lackingrdquoMilan Expo 2015 the international exhibition event eagerly awaited by many and for many reasons has just started The main theme is ldquofeeding the planet energy for liferdquo Though we certainly shouldnrsquot need an event like EXPO 2015 to remind us of food availability problems which we are all well aware of it was however well necessary to bring the issues in the spotlight and the international exhibition is the best chance we have to do that With a rapidly growing world population the gap between those who have too much to eat and those who have too little or none at all is still far too broad These issues are well known and have been debated time and time again The risk of endless debating that leads to little or no policy-making is to simply resort to only ldquomissionary-likerdquo actions such as having fundraisers out of churches after Sunday mass or just leave the burden to charities which strive to raise funds also through media campaigns organised for collecting contributions But charity work is simply not enough Famine exists and persists in the world It is not like some ghost lurking in the dark we may be able not to see if wersquore lucky it is a true monster showing clearly every day that kills thousands of innocents every year In the western world we live a paradox which is the opposite of famine-stricken countries a growing number of diseases related to wrong far-too-abundant food unhealthy eating We go on diets to eat less food and research strives to provide solutions to unhealthy eating habits which impact on individuals as well as raising healthcare expenditure This is exactly one of the main topics our journal focuses on This is why I feel involved I feel so challenged by widespread food scarcity Certainly on a personal level I am committed to contributing to the important issue of improving the condition of populations suffering from famine At the same time I also feel encouraged to pursue the mission of our journal with renewed energy The research and development in food industry responds effectively to the needs of the certainly peculiar situation of a part of the world that eats regularly as opposed to famine-stricken countries yet also builds scientifi c knowledge and expertise which benefi t everyone in the end all over the world The study of the human body and its mechanisms the answers wersquore getting today that were unimaginable only a few decades ago understanding the ways the human body reacts and what substances ingredients can help out in many diseases are for the benefi t of all and greatly help to achieve human progress and a better quality of life And the scope of the journal is not limited to that Awareness towards sustainable development is increasingly growing We talk a lot about it in our journals and not because itrsquos a hot topic We donrsquot deal with sustainability just to generally invite readers to remember to switch off lights carefully close the tap or take good care in separating garbage to support waste recycling Simply talking about sustainability nowadays is no longer enough we need to apply it take action to implement it Sustainability is about looking after increasingly scarce resources taking the due care of populations experiencing precarious situations Yet sustainability is also about providing the food sector the technology and innovation that will improve food supply and consumption the practical tools that will teach us a ldquofood culturerdquo that can benefi t everyoneIn these terms EXPO 2015 is an opportunity to exploit It mustnrsquot just be an event where one can stroll around to taste some of the local specialties as we like to do on holiday when we go wine-tasting to some nice vineyards area Indeed having fun and enjoying the event is important yet when you leave the fair pavilions (by the way the Italian pavilion hosts FAB FOOD a wonderful guide through food specialties not to be missed) think back to the day yoursquove spent to try to understand if it has been able to make you more aware of food-related matters and issues Has it triggered you to commit more to improving nutrition a vital aspect for the very existence of man Itrsquos about feeling the urge to meet the needs of people suffering from obesity and cardiovascular disease for instance itrsquos about improving child nutrition or sports nutrition just as it is about providing a glass of drinking water to those who need it the most in some distant sun-scorched or toxic waste-polluted landIf visiting EXPO 2015 will leave us more aware of these issues then the event will have rewarded the efforts and met the expectations of all those who through perseverance hard work and sacrifi ce have provided their time and skills to organise the international exhibition fair in Milan These people deserve our praise encouragement and gratitude

FROM THE EDITORIAL BOARD

Silvana MainiEditorial Director

Agro Food industry Hi Tech TKS Publisher

Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015

Silvana Maini

Expo 2015 feeding the planet energy for lifeA necessity a priority

4 Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015

KEYWORDS Mangifera indica L well-aging SIRT1 AMPK mitochondrial biogenesis SOD

AbstractMango (Mangifera indica L) is not only a delicious fruit but also known for its nutritional value The fruits contain significant levels of bioactive compounds including polyphenols and vitamins A special Mangifera

indica L fruit powder was used to investigate the activation of master regulators of the aging process such as sirtuin 1 and AMP activated protein kinase Furthermore related effects like mitochondrial biogenesis as well as anti-oxidative effects based on superoxide dismutase activity augmentation were investigated As a result the special Mangifera indica L fruit powder showed activation of these master regulators and related modulation underlining potential beneficial effects for well aging

Benefits of a Mangifera indica L fruit powder for well-agingIn vitro investigation of effects on master regulators for the aging process

INTRODUCTION

Aging is associated with several structural and metabolic changes leading to a decrease in physical strength and endurance as well as to a decline in efficiency of body organs As we age the function of mitochondria declines leading to disturbances in energy homeostasis and metabolism and proper cell viability Thus the risk of age related diseases such as cardiovascular diseases metabolic diseases including type 2 diabetes overweight and age related muscle degeneration increases Today there is a difference of at least seven years between the ldquohealthy life expectancyrdquo and total life expectancy at birth (1)More recent scientific studies associate nutrition in particular calorie restriction with healthy aging (2) Energy homeostasis and related metabolic processes are steered by the evolution and conserved metabolic sensors sirtuin 1 (SIRT1) and AMP activated protein kinase (AMPK) During calorie restriction SIRT1 and AMPK are activated and stimulate a cascade of metabolic processes eg increased fatty acid oxidation inhibition of cholesterol fatty acid as well as triglyceride synthesis enhanced muscle glucose uptake and modulation of insulin secretion (3) Beside metabolic processes SIRT1 and AMPK are able to suppress endothelial and vascular smooth muscle cell proliferation or activate the transcription of PCG1alpha a master regulator of mitochondrial biogenesis (4) Additionally SIRT1 beneficially impacts cellular antioxidative stress defence mechanism (2) Studies indicate that natural compounds stimulate SIRT1 and AMPK and thereby may

prevent and reverse the effects of age-related metabolic decline (5) One of the natural activators ndash resveratrol was identified to have crucial impact on energy homeostasis via SIRT1 and AMPK (5) Mango (Mangifera indica L) is one of the most important tropical fruits grown in more than 87 countries throughout the world (6) It has been shown that the variety differ from region to region as well as the traditional use In many countries mango fruits are known as a delicious fruit with nutritional value and in some areas mangos are used as traditional plant like for example to treat diabetes in Aryuvedic medicine (7) Meanwhile scientific studies have been published confirming the beneficial effects on diabetes and inflammation (eg 8) Here we investigated whether a special Mangifera indica L fruit powder from an Indian variety is able to modulate master regulators of the aging process in vitro

MATERIALS AND METHODS

Characterization of Mangifera indica L sample preparationMangifera indica L fruit powder is a 100 pure mango fruit obtained from fruits harvested in India at a special degree of ripeness The fruits are characterized by a higher level of secondary plant ingredients and a lower level of sugar compared to fully ripe fruits The investigated mango fruit powder is commercially available under the brand name Carelesstrade from Vital Solutions Swiss AG Samples for in vitro studies were obtained by treating the fruit powder with water

SENIOR NUTRITION

SIGRID ROumlCHTER1 CHRISTINA EHRHARDT1 SYBILLE BUCHWALD-WERNER2 KARIN BERGER-BUumlTER1 ELENA SCHATTON1

Corresponding author1 Vital Solutions Swiss AG Hauptstr 137C 8274 Taumlgerwilen Switzerland

2 Vital Solutions GmbH Hausingerstrasse 6 40764 Langenfeld Germany

Sigrid Rochter

Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015 5Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015

diabetes Metformin in a concentration of 2 mM (corresponding to a final assay concentration of 285 microgml) and resveratrol in a concentration of 100microM (228 microgml) was used in the assay Cytotoxicity of the test substances and the cell count was performed with Cell Counting Kit-8 from Dojindo Laboratories

Mitochondrial biogenesisMitochondrial biogenesis was investigated in a Human Hepatic cell line (HepG2 DSMZ ACC180) and in a mouse myoblast cell line (C2C12 DSMZ ACC565) Both cell lines were grown to confluence and incubated in growth medium for 72 hours In the experiment with HepG2 cells three different batches of Mangifera indica L sample preparation in the concentration of 200 microgml were investigated In the experiment with C2C12 cells Mangifera indica L batch 3 in the concentrations of 200 microgml and 400 microgml or resveratrol in the concentrations 25 microM (57 microgml) 50 microM (114 microgml) and 100 microM (228 microgml) were analyzed Mitochondria staining after the incubation time was performed with Chromeo Live Cell Mitochondria Staining Kit from Active Motif which is based on a membrane-permeable dye that diffuses into live cells and stain mitochondria The fluorescence intensity is directly proportional to the amount of mitochondria The data are expressed as compared to control values from untreated cells (set to 0) Cytotoxicity of the test substances and the cell count was performed with Cell Counting Kit-8 from Dojindo Laboratories

SOD assaySOD activation was investigated in Human Epidermal Keratinocytes (HEKa from Gibco) The cells were grown to confluence and incubated in growth medium with different concentrations (250 microgml 500 microgml 1000 microgml) of Mangifera indica L sample preparation or Ascorbic acid (10 microM) as a positive control for 24 hours After this incubation time cells were washed twice to remove the Mangifera indica L sample preparation and oxidative stress was induced by adding 200 microM hydrogen peroxide (H2O2) solution in HBSS for 20 minutes SOD enzymatic activity in the cell homogenates was determined using a SOD Assay kit ndash WST (Dojindo) Cytotoxicity of the test substances and the cell count was performed with Cell Counting Kit-8 from Dojindo Laboratories

StatisticsAll the experiments were performed in duplicates or triplicates for each test sample concentration or control Cell experiments were performed using the same cell passage Results are expressed as mean+- standard deviation

RESULTS

Mangifera indica L fruit powder activate SIRT1 and AMPK two key targets for energy homeostasis and cellular metabolismSIRT1 resultsSIRT1 belongs to a class of enzymes (sirtuins) that regulate cellular metabolic functions important for energy homeostasis and cell protection Calorie restriction one of the most effective means to slow down the pace of aging is known to activate SIRT1 the master sensor for energy and nutrient availability (5 10 11) All batches of Mangifera indica L fruit powder showed activation of SIRT1 In pretests the concentration of 500 microgml of Mangifera indica L

(110 raw material water ratio) and filtered through a glass fibre filter (GF6 Diameter 110mm from GE Healthcare Life Sciences Whatman) The strength of the sample preparation is defined with a raw material sample preparation ratio from approx 31 The concentrations of sample preparations used in the different assays and the corresponding amount of Mangifera indica L fruit powder is summarized in table 1 Three to four different Mangifera indica L sample batches obtained from two harvest periods were investigated (table 2) The samples have been produced in the same way and have the same specification

SIRT1 AssayActivation of human recombinant SIRT1 isoenzyme was investigated using two validated commercial available assays (SIRT1 Direct Fluorescent Screening Assay Kit Cayman Chemical and the more sensitive SIRT1-Glo Luminescence Assay Promega with a hrSIRT1 isoenzyme expressed in Sf9 cells from SignalChem) Both assays are based on an acetylated peptide substrate that can be deacetylated by SIRT1 The measured fluorescence or luminescence is directly proportional to the deacetylation activity of the SIRT1 enzyme The samples backgrounds were measured and subtracted from the samples value Mangifera indica L sample preparations out of four different batches in a concentration of 500 microgml were analyzed As a positive control trans-resveratrol (Cayman Chemical) was used in the concentration of 100 microM or corresponding to 228 microgml (9) Data are expressed as compared to control values (set to 0)

AMPK assayActivation of AMPK was investigated in terminally differentiated Human Hepatic Progenitor cells (HepaRG cell line Invitrogen) The cells were plated on a collagen coated cell culture plate and after attachment and spreading incubated for 24 hours with Mangifera indica L sample preparation or control samples in the basal medium without supplements but with 30 mM glucose AMPK activation in the cell lysates was measured with AMPKα [pT172] ELISA kit from Invitrogen Three batches of Mangifera indica L sample preparation in a concentration of 300 microgml were analyzed As a positive control the first-line drug treatment for type 2

Table 1 Concentrations of sample preparation used in the assays and corresponding amount of Mangifera indica L fruit powder

Table 2 Characteristics of the different batches of Mangifera indica L fruit powder


concentration of 200 microgml of Mangifera indica L sample preparation was shown to be the optimal concentration for the assay We furthermore repeated the test in a mouse muscle myoblast cell line (C2C12) and we could show an average activation of 7 by Mangifera indica L sample preparation with the concentration of 200 microgml and 400 microgml A dose dependency could not be observed and further investigations with a range of different concentration of Mangifera indica L sample preparation are necessary to prove these results In contrast resveratrol led to a dose dependent activation between 7 and 17

Mangifera indica L fruit powder augment SOD activity during oxidative stressThe enzyme superoxide dismutase (SOD) catalyzes the breakdown of the superoxide anion into oxygen and hydrogen peroxide and serves as the first line of defense that the body has against superoxide free radicals SOD is present in almost all aerobic cells and extracellular fluids and is considered to be one of the most effective antioxidant As shown in Figure 4 induction of oxidative stress in Human Keratinocytes (HEK cells) with H2O2 led to an increase in SOD activity Interestingly preincubation with Mangifera indica L sample preparation before exposure to oxidative

stress augmented SOD enzyme activity showing an improved defence capacity of the body

DISCUSSION

The results clearly demonstrated that the special Mangifera indica L fruit powder is able to activate master regulators of the aging process such as SIRT1 and AMPK In addition we could show that the investigated special fruit powder stimulated mitochondrial biogenesis Furthermore SOD activity during oxidative stress was augmented by Mangifera indica L fruit powder These targets have been confirmed to be important for energy homeostasis and metabolic processes to prevent the deleterious changes of aging (5) Promoting healthy aging includes maintaining healthy body composition healthy glucose management and lipid metabolism cell protection and energy homeostasis in order to reduce risk factors for age related diseases Furthermore maintaining physical power and muscle mass makes living more comfortable during aging Constituents in the special Mangifera indica L fruit powder which are responsible for the beneficial effects on the master regulators for well-aging are not completely identified Phytochemical analysis revealed that major compounds known to activate SIRT1 and AMPK are resveratrol quercetin and kaempferol We could show that Mangiferin which occurs in the Mangifera indica L fruit powder also activates AMPK However the observation that Mangifera indica L fruit powder

sample preparation was shown to be the optimal concentration for the assay This concentration led to an activation of more than 30 measured with the direct fluorescent screening assay (Figure 1a) Pure resveratrol which was used as positive control activated SIRT1 by approx 50 SIRT1 activation was also measured with a more sensitive luminescence assay with an average activation of 68 after stimulation with Mangifera indica L sample preparation The SIRT1 activation by batch 4 was slightly smaller compared to the other batches but this variation is still in the range for natural products The positive control resveratrol led to an average activation of 96 (Figure 1b)

AMPK resultsAMPK plays a central role in cellular metabolism and energy homeostasis Here we could demonstrate that all three batches of Mangifera indica L fruit powder showed reproducible activation of AMPK by more than 50 (Figure 2) Positive control Metformin led to an average AMPK activation of 130 and pure resveratrol to an average AMPK activation of 82 in the concentration of 100 microM

Mitochondrial biogenesis is stimulated by Mangifera indica L fruit powderEffective mitochondrial biogenesis and turnover is critical for the maintenance of energy production the prevention of oxidative stress and well-aging In a Human Hepatic cell line (HepG2) all batches of Mangifera indica L sample preparation were able to stimulate mitochondrial biogenesis by approx 15-30 (Figure 3) In pretests the

Figure 1 Activation of SIRT1 by Mangifera indica L sample preparation (mean values plusmn standard deviation) Four different batches were investigated Activation in compared to control values (set to 0) Pure resveratrol (100 microM) was used as a positive control (a) Activation of SIRT1 measured with a direct Fluorescent Screening Assay kit (b) Activation of SIRT1 measured with the Luminescence SIRT1-Glo Assay and the sensitive hrSIRT1 isoenzyme expressed in Sf9 cells

Figure 2 Activation of AMPK (phosphorylated AMPK) in HepaRGtrade cells by three different batches of Mangifera indica L sample preparation in a concentration of 300 microgml (mean values plusmn standard deviation) Activation in compared to control values (set to 0) pure resveratrol (100 microM) and Metformin (2 mM) were used as positive control


Based on the in vitro evidence we can assume that this special Mangifera indica L fruit powder may help attenuate the negative metabolic changes associated with aging Product concepts for maintenance of physical energy due to enhanced mitochondrial biogenesis in muscle cells or concepts for healthy body composition and nourished skin due to SIRT1 and AMPK activation could benefit consumers at any age Particularly the aging population may benefit from concepts to reduce the risk to develop metabolic age-related changes as well as from concepts for cell and DNA protection

REFERENCES

1 Euromonitor httpblogeuromonitorcom201401ageing-related-food-and-drink-focus-onlengthening-healthy-life-expectancyhtml21082014

2 Nogueiras R Habegger KM Chaudhary N et al ldquoSirtuin 1 and sirtuin 3 physiological modulators of metabolismrdquo Physiol Rev 92 (3) 1479-1514 (2012)

3 Zhang BB Zhou G Li C ldquoAMPK an emerging drug target for diabetes and the metabolic syndromerdquo Cell Metab 9 (5) 407-16 (2009)

4 Canto C Auwerx A ldquoPGC-1alpha SIRT1

activates AMPK cannot be completely attributed to its Mangiferin content because the activation by the Mangifera indica L fruit powder is greater than that of an equivalent amount of Mangiferin Investigation showed that the Mangifera indica L fruit powder has a 250 fold greater effect than pure Mangiferin of an equivalent dosage Therefore it can be assumed that the efficacy on AMPK of the special Mangifera indica L fruit powder can be ascribed to a synergistic interplay of naturally occuring bioactive ingredients Phytochemical investigations to identify additional potential molecules with anti-aging activity out of Mangifera indica L are ongoing Another mango fruit preparation was already investigated in a mouse model where the authors could show that mango modulates body fat and plasma glucose and lipids in mice fed a high-fat diet (12) These promising results could be partly confirmed in a human study with obese individuals (13) However the properties of mango fruits might be highly dependent on the variety cultivation ripeness and post-harvest handling of the raw material (14) Mango polyphenols are highest during fruit growth and decrease with ripening The Mangifera indica L fruit powder which was investigated in the present in vitro study are harvested at a special degree of ripeness in order to ensure a high concentration of bioactive compounds The results demonstrate a small batch to batch variation eg batch 4 in the SIRT1 luminescence assay showed less activity than the other batches However the variation is still in the range for natural products Whether these promising in vitro results can be transferred to the human system have to be investigated in a randomized placebo controlled nutritional study

Figure 3 Mitochondrial biogenesis in (a) HepG2 cells stimulated with 3 different batches of Mangifera indica L and (b) C2C12 cells after 72 hours treatment with batch 3 of Mangifera indica L sample preparation in the concentrations 200 microgml and 400 microgml and resveratrol (mean values plusmn standard deviation) Activation in compared to control value (set to 0)

Figure 4 SOD activation in Human Keratinocytes (HEK cells) Oxidative stress was induced by hydrogen peroxide (200 microM) after preincubation with different concentration (250 microgml 500 microgml 1000 microgml) of batch 3 of the Mangifera indica L sample preparation or Ascorbic acid (10 microM) as a positive control (mean values plusmn standard deviation) Activation in compared to control value (set to 0)

and AMPK an energy sensing network that controls energy expenditurerdquo Curr Opin Lipidol 20 (2) 98-105 (2009)

5 Hubbard B Sinclair DA ldquoSmall molecule Sirt1 activators for the treatment of aging and age-related diseasesrdquo Trends in Pharmacological Sciences 35 146-154 (2013)

6 Tharanathan RN Yashoda HM Prabha TN ldquoMango (Mangifera indica L) ldquothe king of fruitsrdquo ndash an overviewrdquo Food Rev Int 22 95-123 (2006)

7 Shah KA Patel MB Patel RJ et al ldquoMangifera indica (mango)rdquo Pharmacog Rev 4 (7) 42-8 (2010)

8 Miura T Iwamoto N Kato M Ichiki H Kubo M Komatsu Y Ishida T Okada M Tanigawa K ldquoThe suppressive effect of mangiferin with exercise on blood lipids in type 2 diabetesrdquo Biol Pharm Bull 24 (9) 1091-2 (2001)

9 Borra MT Smith BC Denu JM ldquoMechanism of human SIRT1 activation by resveratrolrdquo J Biol Chem 280 (17) 17187-95 (2005)

10 Sinclair Da Guarente L ldquoSmall molecule allosteric activators of Sirtuinsrdquo Annu Re Pharmacol Toxicol 14 363-380 (2014)

11 Quideau S ldquoPlant polyphenolic small molecules can induce a caloric restriction-mimetic life-span extension by activating sirtuins will polyphenols someday be used as chemotherapeutic drugs in western medicinerdquo Chem Bio Chem 4 427-430 (2004)

12 Lucas EA Li W Peterson SK et al ldquoMango modulates body fat and plasma glucose and lipids in mice fed a high-fat dietrdquo Br J Nutr 106 (10) 1495-505 (2011)

13 Evans SF Meister M Mahmood M et al ldquoMango supplementation improves blood glucose in obese individualsrdquo Nutr Metab Insights 28 (7) 77-84 (2014)

14 Talcot ST Moore JP Lounds-Singleton AJ et al ldquoRipening Associated Phytochemical Changes in Mangos (Mangifera indica) Following Thermal Quarantine and Low-Temperature Storagerdquo J Food Sci 70 (5) C337-41 (2005)


KEYWORDS Mangostin xanthone pain inflammation soft tissue

AbstractSoft tissue pain is a common and debilitating disorder linked to a wide range of unhealthy conditions mainly involving inflammatory processes Although frequently prescribed to manage

inflammatory conditions and attendant pain non-steroidal anti-inflammatory drugs (NSAIDs) are nevertheless associated with side and unwanted effects Accordingly providing safe and natural alternatives is a current challenge In this study an extract of mangosteen rich in α- and γ-mangostins is demonstrated to significantly decrease production of the inflammation marker tumor necrosis factor-α (TNF-α) in a murine model of inflammation and to significantly reduce soft tissue pain in volunteers during a clinical investigation

Mangosteen extract for short-term pain management Preclinical approach and pilot clinical investigation on volunteers with soft tissue pain

INTRODUCTION

Soft tissue pain may arise from a variety of conditions ranging from acute post-traumatic injuries which ordinarily occur during physical exercise to chronic joint complaints such as osteoarthritis or rheumatic disorders The general population may be affected with soft tissue-related injury throughout the course of their life regardless of circumstances and the resulting pain represents a leading clinical cause of physical disability and impaired quality of life Soft tissue-associated pain is the consequence of a cascade of inflammatory events occurring as a physiological response to injured tissue Accordingly inflammatory cells ie monocytes are rapidly mobilized from the circulation to infiltrate in the form of macrophages at site of the damage where they release several mediators of inflammatory response The magnitude of inflammatory reaction is the root of initiated and sustained pain Among the most important mediators involved in inflammation-related pain prostaglandins

particularly prostaglandin E2 (PGE2) is able to directly activate pain-responsive nociceptors (1) PGE2 is generated from a specific polyunsaturated fatty acid (arachidonic acid) released from the lipid membrane of cells mainly through enzymatic activity of cyclooxygenase-2 (COX-2) COX-2 is therefore a valuable therapeutic target for pain management Nevertheless pain mechanisms are complex and other mediators such as cytokines directly and indirectly contribute to nociceptor sensitization and activation (23) As a relevant example numerous studies have highlighted a key role for TNF-α in inflammatory nociception (45)Current pharmacologic treatments NSAIDs are commonly prescribed to patients to treat pain Their pharmacological effects are mostly attributed to the inhibition of COX-2 Although they offer benefic anti-inflammatory and analgesic effects NSAIDs are nonetheless often associated with excessive risks of severe side effects Indeed medium- to long-term use of NSAIDs might be associated with substantial gastrointestinal hepatic renal

JOINT HEALTH

CINDY ROMAIN JULIEN CASES Corresponding author

Fytexia SAS ZAE via Europa 3 rue drsquoAthegravenes 34350 Vendres France

Cindy Romain


Lecco Italy) weighing ~25 g each were randomly divided into three groups of six animals They were housed at 23 plusmn 1degC with relative humidity 60 subjected to a 12 h12 h lightdark cycle and had free access to food and water Animals were handled in compliance with national and international laws and policies The control group was treated with LPS alone (LPS) The supplemented group was

treated with LPS + mangosteen extract (MGS) whereas the standard group received only the

mangosteen extract (STD)

Experimental designMice were orally supplemented with the mangosteen extract at an acute dose of 60 mgkg (MGS

and STD groups) or with tap water as the vehicle (LPS group) thirty

minutes before intraperitoneal injection of LPS (100 microgkg) Ninety

minutes after LPS blood sampling was obtained from the retro-orbital plexus with

animals under ether-induced anesthesia

TNF-α assayConcentrations of TNF-α were determined in

plasma using an ELISA kit from RampD Systems Minneapolis MN USA

Human clinical studySubjectsInclusion criteria incorporated twenty-four volunteers of both sexes suffering with acute (n=15) or chronic (n=9) soft tissue conditions namely osteoarthritic pain inflammation of the tendons and post-traumatic inflammation Volunteers were recruited for the clinical study after they agreed to sign a written informed consent form Exclusion criteria comprised subjects receiving chronic or acute treatment for pain or inflammation

Experimental designA 5-day single-blind randomized and drug reference-controlled clinical trial was conducted Once enrolled subjects were assigned to one of two groups For 5 days one group (n=12 6 males and 6 females) received orally 100 mg of nimesulide daily in two equal doses and the other group (n=12 5 males and 7 females) was supplemented with 600 mg of the mangosteen extract daily in two equal doses Volunteers reported to the research center 2 times during the 5-day intervention period at baseline (D0) and at the end of the study (D5)

Pain assessmentPain rating according to intensity of pain perception by the volunteers was assessed using a visual analogue scale (VAS) at D0 and D5 Volunteers rated the following corresponding scores for pain 0 for no pain 1ndash3 for mild pain (incommodious interfering little with activities of daily living (ADL) 4ndash6 for moderate pain (interfering significantly with ADL) and scores in the range of 7ndash10 for severe pain (disabling unable to perform ADL)

StatisticsStatistical analyses were performed using Statview software version 4511 (Abacus Concepts Berkeley CA USA)

and cardiovascular complications (6) which is why common anti-inflammatory pharmaceuticals are proscribed for chronic pain prevention This clearly increases the importance of natural and safer alternatives for which the majority of patients and practitioners may have a preference especially for preventive action and during long-term supplementationMangosteen (Garcinia mangostana L) is a tropical fruit native to South East Asia and is popularly referred to as ldquothe queen of fruitsrdquo [Note the term from which the phrase is derived was invented in 1903 by David G Fairchild an American plant collector in Miami FL who referred to mangosteen as ldquothe queen of tropical fruitrdquo KJ] (7) Apart from its edible tasty pulp the fruit has been used for centuries in traditional medicine for the treatment of various conditions including dysentery wounds inflammation fever and skin infections (8) Health benefits of mangosteen have been attributed to the presence of specific secondary metabolites naturally occurring in the fruit as xanthones Abundant bioactive xanthones found in the fruit are α- and γ-mangostins (9) for which antioxidant (10) and anti-inflammatory (11) properties have been reported in the literatureIn the present study we assessed a dietary supplement obtained by a traditional extraction process of mangosteen whereby the representative original fingerprint of bioactive xanthones in the fruit are mainly α- and γ-mangostins in a ratio of 5-101 Taking a mechanistic approach an in vivo pre-clinical model of inflammation was used to evaluate supplement efficacy on the release of TNF-α Salutary effects on pain management have been assessed and compared to an NSAID reference in a clinical investigation of volunteers suffering from acute or chronic soft tissue inflammation

MATERIAL AND METHODS

MaterialsMangoSelectreg an extract of Mangosteen prepared by traditional extraction methods was obtained from Fytexia France The supplement is produced by hydro-alcoholic extraction of whole fruit and contains 10 of a 5-101 ratio of α- and γ-mangostinsFor the NSAID studied in the clinical investigation nimesulide was purchased from a local drugstore in FranceIn the preclinical study inflammation was induced using lipopolysaccharides (LPS) from Escherichia coli serotype 055B5 purchased from Sigma (St Louis MO USA)

Pre-clinical model of inflammationAnimals Eighteen male CD-1 mice (Charles River Italia Calco


the case during chronic tissue injuries such as osteoarthritis or rheumatoid arthritis two leading causes of disability and decline in physical function and mobility (12) More generally individuals practicing intensive sport training and those in poor physical condition who start new physical activities are also prone to develop acute soft tissue pain such as tendonitis or sorenessAt baseline (D0) of the present study regardless of the origin of pain all volunteers declared to be affected with severe pain ranging in score from 7 to 9 (data not shown) and registered an average VAS score of 82 As expected 5-days of treatment with the reference drug nimesulide significantly reduced pain perception (-541) Nimesulide is an efficient NSAID analgesic that has been extensively documented in the literature (13ndash15) In the present study nimesulide lowered volunteersrsquo pain scores from severe to mild-moderate levels Nevertheless it is noteworthy that several side-effects linked to prolonged treatment with nimesulide as with other NSAIDs such as diclofenac or ibuprofen and so many others have been reported with increased risks of developing gastrointestinal damage (16)With similar efficacy 5 daysrsquo supplementation with mangosteen extract 600 mg daily was effective in significantly relieving pain (-374) Despite the fact that nimesulide appeared to be a slightly more efficient solution for the management of acute pain in the short period of treatment the mangosteen extract provided the same benefits as the NSAID in the subgroup of volunteers suffering from chronical pain who showed respective averaged pain scores of 38 (n=4) and 425 (n=5) (p=038) (data not shown) This suggests that the extract of mangosteen in the absence of any side or unwanted effects may have the ability to provide a comparatively better risk-benefit balance for the management of chronic pain Analgesic activity of active compounds extracted from mangosteen has previously been demonstrated in a model of pain in mice (17) in which pretreatment with either α- or γ-mangostin significantly decreased pain responses in two validated pain tests the hot-plate and formalin testGiven that soft tissue injury is associated with inflammatory pain the anti-inflammatory effect of the mangosteen extract was examined in an animal model of LPS-induced

The data are expressed as mean plusmn standard deviation (SD) Differences between groups were evaluated using unpaired Studentrsquos t-test

RESULTS

Pre-clinical model of inflammationLPS induces a significant releases of TNF-α in plasma as observed in the LPS group Pre-supplementation with mangosteen extract 30 min before LPS injection significantly lowered LPS-induced TNF-α release (-69 plt005) in the MGS group TNF-α levels remained under the limit of detection by the ELISA assay (lt0031 ngmL) in the STD group (Figure 1) Human clinical studyAt baseline all volunteers were experiencing severe pain with an average VAS score of 82 After 5 days of treatment nimesulide significantly lowered pain scores from severe to mild-moderate resulting in an average score of 38 (plt000001) Pain categories of all the volunteers changed 4 out of 12 reported in the mild category and 8 in the moderate range Volunteers supplemented with the extract of mangosteen reported a significant reduction in pain scores from severe to moderate perception rating an average score of 5 (plt000001) With the exception of 1 individual who remained in the severe category (poor responder) all volunteers showed an improved pain category (Figure 2)

DISCUSSION

The present study provides evidence for the first time that a 5-day supplementation with an extract of mangosteen rich in xanthones (5ndash101 ratio of α- and γ-mangostins) is beneficial for improving inflammation and alleviating pain in patients experiencing chronic or acute soft tissue injury In the general population such painful conditions undoubtedly deteriorate quality of life This is particularly

Figure 2 Perception of intensity of pain at D0 and after 5 days (D5) of nimesulide treatment or mangosteen extract Values are means plusmn SD indicates a significant difference between D0 and D5 (plt0001)

Figure 1 Serum TNF-α concentrations in mice treated with LPS alone (LPS) LPS plus mangosteen extract (MGS) or mangosteen extract only (STD) Values indicate means plusmn SD For each treatment bars with different index letters are significantly different (plt005)


suppression of inhibitor of κB kinase (IKK) activity leading to the inactivation of the COX-2 transcription factor nuclear factor-κB (NF-κB) (23) α- and γ-mangostin have also been described for their ability to modulate functions within mitogen-activated protein kinase (MAPK) signaling pathways involved in the inflammatory response Thus in human macrophage cell models (2425) as well as in primary human adipocyte cells in culture (26) treated with either α- or γ-mangostin phosphorylation of MAPK members ndash ie c-Jun N-terminal kinases (JNK) extracellular signal-regulated kinases (ERKs) and p38 MAPK ndash are significantly decreased Inhibitory effects are mediated through transcription of both NF-κB and activator protein-1 (AP-1) and result in attenuated gene expressions of pro-inflammatory cytokines TNF-α Interleukins 1β (IL-1β) 6 (IL-6) and 8 (IL-8) Finally α- and γ-mangostin are noted to show competitive effects in blocking LPS-induced suppression of peroxisome proliferator-activated receptor-γ (PPAR-γ) (26) a negative modulator of NF-κB signaling (27) Another mechanism of action involved in the anti-inflammatory effect of mangostins is explained by their antioxidant properties (10) namely their capacity to scavenge oxygen reactive species and other free radicals (2829)

Taken together prior mechanistic studies strongly support an anti-inflammatory role for the xanthones α- and γ-mangostin as a result of their capacity to modulate decisive pathways involved in inflammation-associated pain (Figure 3) This is further supported at least in part in the present study with the capacity of α- and γ-mangostin to decrease TNF-α release and by a significant decrease in the level of volunteersrsquo pain from severe to first stages of moderate pain after taking the extract of mangosteen

systemic inflammation Mice pre-treated with the extract 30 minutes before injection with LPS displayed a significantly lower level of TNF-α in plasma (-69) when compared to LPS-treated mice The role of TNF-α in inflammatory pain has been brought to light in studies showing a strong correlation between levels of TNF-α and pain intensity (18) Evidence suggested that a TNF-α-induced mechanism of pain might be elicited through direct and indirect pathways The latter includes activation of COX-2 expression and the subsequent release of the mediator of central pain PGE2 (19) Numerous authors have also highlighted the modulation of pain through a direct sensitization of nociceptors suggesting a key role of TNF-α in pain initiation and sustained pain (20) Accordingly the significant decrease in LPS-induced release of TNF-α measured in the present study would explain at least in part the pain-relieving effect of the extract for which the naturally occurring xanthones α- and γ-mangostin its main bioactive constituents have previously been confirmed to show anti-inflammatory properties in the absence of side effects (8) Mechanisms of action associated with the anti-inflammatory properties of both xanthones are complex and include a modulation of diverse inflammatory pathways In the murine RAW 2647 macrophages model both α- and γ-mangostin were demonstrated to inhibit PGE2 and nitric oxide (NO) production (1121) through the respective inhibition of COX-2 mRNA transcription and modulation of inducible NO synthase (iNOS) It has additionally been established in a C6 rat glioma cell model that γ-mangostin can directly compete with arachidonic acid for binding to the COX-2 active site inhibiting its activation and subsequently the release of PGE2 (22) Such effects were interrelated with the

Figure 3 Possible mechanisms of action linked to anti-inflammatory properties of mangostins


16 Goldstein JL Cryer B ldquoGastrointestinal injury associated with NSAID use a case study and review of risk factors and preventative strategiesrdquo Drug Healthc Patient Saf 7 31ndash41 (2015)

17 Cui J Hu W Cai Z et al ldquoNew medicinal properties of mangostins analgesic activity and pharmacological characterization of active ingredients from the fruit hull of Garcinia mangostana Lrdquo Pharmacol Biochem Behav 95 (2) 166ndash172 (2010)

18 Tak PP Smeets TJ Daha MR et al ldquoAnalysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activityrdquo Arthritis Rheum 40 (2) 217ndash225 (1997)

19 Lin CC Hsiao LD Chien CS et al ldquoTumor necrosis factor-alpha-induced cyclooxygenase-2 expression in human tracheal smooth muscle cells involvement of p42p44 and p38 mitogen-activated protein kinases and nuclear factor-kappaBrdquo Cell Signal 16 (5) 597ndash607 (2004)

20 Zhang XC Kainz V Burstein R et al ldquoTumor necrosis factor-α induces sensitization of meningeal nociceptors mediated via local COX and p38 MAP kinase actionsrdquo Pain 152 (1) 140ndash149 (2011)

21 Tewtrakul S Wattanapiromsakul C Mahabusarakam W ldquoEffects of compounds from Garcinia mangostana on inflammatory mediators in RAW2647 macrophage cellsrdquo J Ethnopharmacol 121 (3) 379ndash382 (2009)

22 Nakatani K Nakahata N Arakawa T et al ldquoInhibition of cyclooxygenase and prostaglandin E2 synthesis by gamma-mangostin a xanthone derivative in mangosteen in C6 rat glioma cellsrdquo Biochem Pharmacol 63 (1) 73ndash79 (2002)

23 Nakatani K Yamakuni T Kondo N et al ldquogamma-Mangostin inhibits inhibitor-kappaB kinase activity and decreases lipopolysaccharide-induced cyclooxygenase-2 gene expression in C6 rat glioma cellsrdquo Mol Pharmacol 66 (3) 667ndash674 (2004)

24 Bumrungpert A Kalpravidh RW Chuang CC et al ldquoXanthones from mangosteen inhibit inflammation in human macrophages and in human adipocytes exposed to macrophage-conditioned mediardquo J Nutr 140 (4) 842ndash847 (2010)

25 Liu SH Lee LT Hu NY et al ldquoEffects of alpha-mangostin on the expression of anti-inflammatory genes in U937 cellsrdquo Chin Med 7 (1) 19 (2012)

26 Bumrungpert A Kalpravidh RW Chitchumroonchokchai C et al ldquoXanthones from mangosteen prevent lipopolysaccharide-mediated inflammation and insulin resistance in primary cultures of human adipocytesrdquo J Nutr 139 (6) 1185ndash1191 (2009)

27 Chinetti G Fruchart JC Staels B ldquoPeroxisome proliferator-activated receptors (PPARs) nuclear receptors at the crossroads between lipid metabolism and inflammationrdquo Inflamm Res 49 (10) 497ndash505 (2000)

28 Martiacutenez A Galano A Vargas R ldquoFree radical scavenger properties of α-mangostin thermodynamics and kinetics of HAT and RAF mechanismsrdquo J Phys Chem B 115 (43) 12591ndash12598 (2011)

29 Chang HF Wu CH Yang LL ldquoAntitumour and free radical scavenging effects of γ-mangostin isolated from Garcinia mangostana pericarps against hepatocellular carcinoma cellrdquo J Pharm Pharmacol 65 (9) 1419ndash1428 (2013)

Nevertheless further investigations would be necessary to evaluate the effects of medium- to long-term supplementation with the extract gain a better understanding of the mechanisms of action involved and to confirm its propensity to extend a change in pain category toward mild pain or even to completely mitigate pain in the absence of any side and unwanted effects

REFERENCES AND NOTES

1 Chen L Yang G Grosser T ldquoProstanoids and inflammatory painrdquo Prostaglandin Other Lipid Mediat 104-105 58ndash66 (2013)

2 Shubayev VI Kato K Myers R Cytokines in Pain Chapter 8 in Translational Pain Research From Mouse to Man In Kruger L Light AR editors (2010)

3 Verri WA Cunha TM Parada CA et al ldquoHypernociceptive role of cytokines and chemokines Targets for analgesic drug developmentrdquo Pharmacol Ther 112 116ndash138 (2006)

4 Cunha TM Verri WA Silva JS et al ldquoA cascade of cytokines mediates mechanical inflammatory hypernociception in micerdquo Proc Natl Acad 102 (5) 1755ndash1760 (2005)

5 Junger H Sorkin LS ldquoNociceptive and inflammatory effects of subcutaneous TNFalphardquo Pain 85 (1-2) 145ndash151 (2000)

6 Patrignani P Tacconelli S Bruno A et al ldquoManaging the adverse effects of nonsteroidal anti-inflammatory drugsrdquo Expert Rev Clin Pharmacol 4 (5) 605ndash621 (2011)

7 Chin YW Kinghorn AD ldquoStructural Characterization Biological Effects and Synthetic Studies on Xanthones from Mangosteen (Garcinia mangostana) a Popular Botanical Dietary Supplementrdquo Mini Rev Org Chem 5 (4) 355ndash64 (2008)

8 Pedraza-Chaverri J Caacuterdenas-Rodriacuteguez N Orozco-Ibarra M et al ldquoMedicinal properties of mangosteen (Garcinia mangostana)rdquo Food Chem Tox 46 (10) 3227ndash3239 (2008)

9 Walker EB ldquoHPLC analysis of selected xanthones in mangosteen fruitrdquo J Sep Sci 30 1229ndash1234 (2007)

10 Jung HA Su BN Keller WJ et al ldquoAntioxidant xanthones from the pericarp of Garcinia mangostana (Mangosteen)rdquo J Agric Food Chem 54 2077ndash2082 (2006)

11 Chen LG Yang LL Wang CC ldquoAnti-inflammatory activity of mangostins from Garcinia mangostanardquo Food Chem Tox 46 688ndash693 (2008)

12 Lee DM Weinblatt ME ldquoRheumatoid arthritisrdquo Lancet 358 (9285) 903ndash911 (2001)

13 Binning A ldquoNimesulide in the treatment of postoperative pain a double-blind comparative study in patients undergoing arthroscopic knee surgeryrdquo Clin J Pain 23 (7) 565ndash570 (2007)

14 Fioravanti A Storri L Di Martino S et al ldquoA randomized double-blind multicenter trial of nimesulide-beta-cyclodextrin versus naproxen in patients with osteoarthritisrdquo Clin Ther 24 (4) 504ndash519 (2002)

15 Bianchi M Broggini M ldquoA randomised double-blind clinical trial comparing the efficacy of nimesulide celecoxib and rofecoxib in osteoarthritis of the kneerdquo Drugs 63 (S1) 37ndash46 (2003)


500 mg dose per day Signifi cant results were observed for both pain and stiffness in as early as 7 days after beginning supplementation

Following the positive results from the pilot studies a randomized double-blind placebo-controlled multicenter study was conducted to investigate NEMregrsquos effects on 67 individuals with

mild to moderate osteoarthritis of the knee This study clearly confi rmed the results from the pilot trials Once again a single 500 mg daily dose resulted in statistically positive results in the treatment group compared to the placebo group from the fi rst assessment at day 10 The positive trend continued to the end of the 60-day trial No adverse events associated with the use of NEMreg were reported

An independent open label clinical trial involving six centers was later conducted in Germany This study reaffi rmed the results from the prior trials and substantiated once again the safety and effi cacy of NEMreg Though this study was not placebo-controlled the consistency of results at six different facilities was impressive This study was also signifi cant in that it revealed the benefi ts from NEMreg supplementation on more than one affected joint The knowledge of NEMreg was expanded further through one in vitro trial followed by an animal study These two studies investigated NEMregrsquos observed anti-infl ammatory activity and its effects in ameliorating infl ammation through inhibition of the cytokines that contribute to unresolved infl ammation The in vitro trial also documented the effects of the human digestive process on NEMreg functioning The data showed that the inhibition of certain infl ammatory markers was

Maintaining strong joint function throughout life is a global health goal In Europe a recent survey reported that 38 of individuals over 18 are concerned about the health of their joints making it the number one health concern Wear and tear on aging joints along with the joint stress caused by obesity or injury all contribute to the increasing risk of developing joint dysfunction suffi cient enough to impact daily activities

Over-the-counter pharmaceutical pain relievers and anti-infl ammatories are the most common treatments for joint pain and stiffness but these are not ideal for long-term management and are only effective after symptoms appear They can also damage the gastrointestinal lining increasing the risk for GI bleeding As a result a growing number of individuals have opted for a proactive and more natural approach to joint care through lifestyle changes and nutritional supplementation Most individuals who seek out a joint supplement want it to contain ingredients that are safe backed by published research and convenient to use ideally one that can be taken only once a day Within this large group of ingredients NEMreg stands out as a joint support ingredient that naturally possesses attributes that meet all of these criteria

NEMreg is a food-sourced joint support ingredient composed of the thin inner membrane of a chicken eggshell This membrane has a unique composition of proteins and peptides including several types of collagen as well as other components naturally found in our own joint tissue such as hyaluronic acid and chondroitin sulfate

The published research supporting NEMregacutes benefi ts to joint tissue is impressive and continues to expand The initial research included two small pilot studies They were both conducted in healthy individuals with discomfort and stiffness in a variety of joints including knees hips ankles neck and elbows The information gained from these open label studies provided the incentive for further research as they showed a clear indication of safety and effi cacy in decreasing discomfort and increasing fl exibility of the joints with one

PRODUCT FOCUS Agro FOOD Industry Hi Tech customers publication

NEMreg brand Eggshell MembraneThe Natural Choice and Fast Acting Solution for Joint Health


acknowledged NEMregrsquos benefi ts where it has been granted authorized claims for the treatment of osteoarthritis

NEMreg is protected by the issuance of multiple patents

For the perfect joint support ingredient formulators need look no further than NEMreg

Study references available upon request

even enhanced after the in vitro digest Both studies yielded favorable results with the animal model study supporting the results gained from the in vitro trial

Even though the safety of NEMreg can be assumed because of its source from a common conventional food it has nonetheless been the subject of extensive safety testing and self-affi rmed GRAS determination With the exception of allergenicity to eggs there are no known side effects from NEMreg consumption even at excessively high dosage levels The production of NEMreg is fully traceable from the egg source and throughout production in a US GMP facility Therefore safety issues arising from adulteration or contamination are not areas of concern NEMreg does not interact or interfere with the metabolism of other ingredients and is therefore the perfect joint support ingredient in proprietary formulations

The impressive effi cacy research on NEMreg and its exceptional safety record have expanded beyond its use in the US The stringent regulatory bodies in Canada have even

Edifi ci CEPID Tecnoparc Avingunda Cambra del Comerccedil 42

43204 Reus SPAIN

Email NEMstratumnutritioncomwwwstratumnutritioncomEU


KEYWORDS Chocolate health claims marketing

AbstractThis paper discusses the rise in appearance of ldquohealthierrdquo ldquobetter for yourdquo chocolates not only in health food stores but in mainstream supermarkets It looks into the most popular claims being made on new

product launches and provides several relevant examples for each group of claims

Rise in ldquohealthierrdquo ldquobetter for yourdquo chocolate

MARKET OVERVIEW

ldquoHealthierrdquo chocolate claimsThe overall number of new product launches (NPLs) within the chocolate confectionery category has been somewhat volatile over the last five years (1) (Mintel 2015) This can to some extent be explained by a mature market volatility in cocoa prices and other economical factors At the same time the percentage of chocolate products that are launched with some sort of a health-related marketing claim has grown from 15 in 2010 to 175 in 2014 (1) (Mintel 2015) This trend is most likely a result of industryrsquos response to consumersrsquo increasing health concerns and a demand for healthier treats in light of worrying obesity figures particularly in Europe and North America Furthermore consumers are increasingly looking for smart solutions in their food In their multi-tasking lives they need their food to multi-task too and chocolate seems like a suitable platform for ldquoadded goodnessrdquo So the question is can chocolate remain a treat while becoming ldquohealthierrdquo and ldquobetter for usrdquo (Figure 1)

Looking at the types of claims being made on the NPLs within the chocolate confectionery category (Figure 2) it is evident that the most popular claim is ldquono additivespreservativesrdquo Consuming products without additives or preservatives has become increasingly important to the consumers over the last decade or so and with chocolate being no exception the industry is responding accordingly Some examples of this claim include

bull Recently re-branded Ferrerorsquos Kinder Joy Chocolate Eggs for Boys and Kinder Joy Chocolate Eggs for Girls marketed as being ldquofree from artificial colours or preservativesrdquo

bull Krogerrsquos Simple Truth Organic Dark Chocolate with Quinoa which contains no preservatives and is also USDA organic and Fair Trade certified

Organic and all natural claims come second highest on the list of claims These kinds of chocolates are popping up especially in organicnatural supermarkets such as Planet Organic and Whole Foods in UK and similar concepts worldwide but they are also demanding ever increasing shelf space within the regular supermarkets with some

CHOCOLATE

MIA NAPRTALeatherhead Food Research

Randalls Way (off Randalls Road) Leatherhead Surrey KT22 7RY United Kingdom

Mia Naprta

Figure 1 ldquoHealthierrdquo chocolate as a percentage of all chocolate New Product Launches (NPLs)

supermarketsrsquo own labels also making organic and all natural chocolates Recent product launches bearing this claim include

bull Pana Chocolatersquos Coconut + Goji Chocolate Tablet ndash a raw organic handmade chocolate naturally sweetened with agave nectar Furthermore the product is described as being suitable for vegans having low glycaemic content and being free from dairy soy gluten and refined sugar

bull DMrsquos natural line Alnatura launched Lime amp Poppy Seed White Chocolate described as ldquoa fine fruity organic specialityrdquo The product retails in a 50g pack (note the trend for smaller size chocolates) and features the BIO7 Initiative (2) logo

There are more and more allergen free and in particular gluten free chocolates around ndash as a response to an increasing number of consumers focusing on either diagnosed or self-diagnosed allergies and intolerances These claims also appeal to those consumers who have no perceived health problems but chose ldquofree fromrdquo foods as a matter of preference Several examples of chocolates bearing allergen free and gluten free claims include

bull For Christmas 2014 Dutch supermarket Albert Heijn has launched Santa Claus Chocolate Figures made with real Belgian milk chocolate and containing no gluten

bull Dammenbergrsquos Milk-Free Raspberry Chocolates are free from gluten but also nuts egg and milk

In these times when obesity is a continuous problem it comes as no surprise that an increasing number of manufacturers have been reformulating their products in order to reduce or remove fat (especially transfats) andor sugar

bull Sugarless Bliss Chocolitersquos Sugar Free Mint Flavoured Dark Chocolates claim to contain ldquo20 less calories than standard chocolatesrdquo and are sweetened with Splenda making them suitable for diabetics and dieters They are fortified with zinc magnesium and chromium and are free of cholesterol and trans fat helping to improve insulin and glucose utilisation

bull Loackerrsquos Milk Chocolate with Milk Cream Filling and

Figure 2 Number of claims for chocolate confectionery NPLs launched from 2010-2014



and GMO organic contains UTZ-certified cocoa and is suitable for vegans

A couple of other trends that have been popping up especially in the last year or two include ldquo100 cocoardquo and ldquoraw chocolaterdquo

bull Madagascan bean-to-bar chocolate company Madeacutecasse has extended its range of ldquoExquisitely Africanrdquo handcrafted bars with the addition of a 100 cocoa variant made using three of the countryrsquos rare heirloom cocoa varieties Pure Ancient Criollo Amelonado and Trinitario cocoa It is available in a 75g format

bull Moulin des Moinesrsquos Pure Organic Cocoa Mass Chocolate has been made from 100 cocoa using traditional French methods This Fairtrade and organic-certified product retails in a 100g pack

bull Happy Day Raw Foods Raw Chocolate has been sweetened with low glycaemic index sustainable coconut sugar The bite-sized pieces of chocolate are said to be made with the finest raw cacao available from suppliers who care about fair trade and sustainability

bull SuroVitalrsquos Almond Cappuccino amp Mulberry Raw Chocolate Tablet is handmade from unroasted Criollo cocoa beans This organic chocolate claims to be ldquoa good source of magnesium calcium and ironrdquo and retails in a 50g pack

In terms of number of ldquohealthierrdquo ldquobetter for yourdquo type of claims being made on new products within the chocolate category the USA leads the way with 2738 claims made over the five year period from 2010-2014 (3) It is followed by the United Kingdom with 1645 claims Canada with 1032 Germany with 804 and Brazil 727 while other markets are also catching onto this trend (1)

From the marketrsquos point of view it is likely that this trend will continue to grow in the coming years and that chocolate will become even more multi-functional bearing some of the claims described above and possibly some new ones At the same time the industry is likely to get increasingly challenged by the medical profession demanding a proof that soft marketing claims are backed by scientific tests and evidence


1 Mintel (2015) Global New Products Database (GNPD) Accessed wwwgnpdcom 26 March 2015

2 The Alnatura Bio 7 Initiative stands for ldquoorganic food based on a holistic philosophy which encompasses the values and production methods of organic farmingrdquo

3 Note that number of claims can be larger than number of new product launches in some instances due to the fact that some product will bear more than just one claim

Crispy Wafer is marketed as being low in hydrogenated fat The product retails in an 87g pack reinforcing the trend for reduced bar sizes from the usual 100g

While ingredients such as sugar and fat are being taken out many perceived ldquobetter for yourdquo ingredients are often being added to chocolate Examples include added antioxidants super fruits high fibre vitamins and minerals etc

bull Aronia Original Organicrsquos Aronia Berries in Dark Chocolate is described as a ldquofibre rich product consisting of berries that have been carefully dried to retain all the important nutrients then covered with organic Belgian chocolaterdquo This vegetarian snack is free from gluten lactose added flavours colouring preservatives and GMO

bull Jrsquos Raw Cacao amp Black Sesame Chocolate Bar is described as ldquohandmade from 100 raw plant-based stone-ground and natural ingredientsrdquo This dairy- gluten- and soy-free product claims to be rich in fibre iron magnesium phosphorus as well as omega 6 oil It contains 300mg calcium per bar It retails in an 85g pack featuring the Great Taste 2014 logo

bull Xin Weirsquos Black Tea Flavoured Oatmeal Chocolate contains grains and whole nuts According to the manufacturer ldquoblack tea helps the body to fight against cold symptoms and aids in digestion while oatmeal is rich in carbohydrate and dietary fibre which provide energy to the bodyrdquo

bull Nestleacute Milorsquos Nuggets Fun Set Chocolate Flavoured Confectionery has been repackaged in an 80g pack containing two 40g sachets and a cup The product comes with Actigen-E formula which is a combination of vitamins and minerals to help optimise the release of energy and is aimed at children

Functional claims including improving brain power aiding slimming etc although comparatively small are also on the rise

bull Vosges Haut Chocolat Super Dark Reishi Mushroom amp Walnut Super Foods amp Dark Chocolate contains antioxidants omega 3 fatty acids polyphenols and polysaccharides and is loaded with potassium calcium and magnesium This chocolate claims to bring health benefits such as ldquostress reduction heart health enhanced brain function and general wellnessrdquo This Womenrsquos Business Enterprise National Council (WBENC)-certified product is all natural suitable for vegans gluten free and contains 72 cacao

bull Scho-Ka-Kolarsquos Whole Milk Chocolate contains coffee and cola nut This UTZ-certified product claims to ldquogive mental energy for more power and concentrationrdquo and retails in a 100g pack suggesting 26 portions

bull NibMor Extreme Dark Chocolate with Cacao Nibs contains methylxanthines such as caffeine and theobromine ndash ldquoplant components that facilitate focusrdquo This kosher-certified product is free from gluten


KEYWORDS complex foods functional foods nutraceuticals food structure synergy human studies health potential

AbstractWhat differentiates natural complex foods from nutraceuticals and dietary supplements is food structure this latter involving nutrient interaction and synergism and a complex mixture at nutritional doses Scientific

evidence showed that functional foods nutraceuticals and dietary supplements (FND) have failed stopping chronic diseases epidemics most conclusions of recent meta-analyses and systematic reviews are lack of significant health effect and needs for further studies Why such disappointing results Probably because FND results from a curative and reductionist nutritional approach while complex foods participates in a preventive and holistic approach Indeed reductionism has led to fractionate foods isolating compounds from them for use at supra-nutritional doses in FND Holism considers foods as complex systems in which the whole is more than sum of the parts leading to more sustainable health effects and technological treatments more respectful of food structure

Complex foods versus functional foods nutraceuticals and dietary supplements differential health impact (Part 2)

An emphasis on synergismSynergy means that 1 + 1 is not equal to 2 but is higher than 2 eg as eloquently shown in vitro with a mix of rutin p-coumaric acid abscisic acid (1) In other words the whole is more than the sum of the parts For example the strength of a cable is higher than the sum of the strengths of each steel rope constitutive of the cable and taken separately Examples could be multiplied indefi nitely In addition there are more and more papers showing convincing results about this issue

Thus Rayalam et al have studied synergism between resveratrol and other phytochemicals and the implications for obesity and osteoporosis They concluded that ldquocombining resveratrol with other phytochemicals may provide an extraordinary potential for preventing obesity and osteoporosis by not only decreasing the dose of each compound thereby avoiding potential toxic side effects but also by targeting multiple signaling pathways affecting adipogenesis apoptosis lipolysis and osteogenesis simultaneouslyrdquo and added that ldquothese phytochemical synergies may make possible novel safe potent and effi cacious therapiesrdquo(2) Further Wang et al concluded that ldquocombining foods across food categories was more likely to create an antioxidant synergismrdquo (3)

In another study authors have investigated the effect of botanical diversity on antioxidant status in healthy women The reduction in urinary isoprostanes is higher following consumption of eighteen fruits and vegetable species than with only fi ve botanical species Authors concluded that ldquobotanical diversity plays a role in determining the bioactivity of high-vegetable and fruit diets and that smaller amounts of many phytochemicals may have greater benefi cial effects than larger amounts of fewer phytochemicalsrdquo (4) The second study is an observational study in which authors have tested the effect of fruit and vegetable variety versus quantity consumed the consumption of a high variety of fruits and vegetables but not quantity was associated with signifi cant better cognitive test scores (5)

These examples well illustrate that increasing botanical varieties within diets increases the number and diversity of bioactive compounds rather than their amount leading to benefi cial synergetic effects which are less predominant when increasing the quantity of only a few food compounds This may have important implications for nutritional policies For example it is not suffi cient recommending increasing fruit and vegetable consumption - such as in the French Programme National Nutrition Santeacute guidelines marks - one must also recommend to increase botanical diversity

FUNCTIONAL FOOD

ANTHONY FARDETINRA UMR 1019 UNH CRNH Auvergne

F-63000 CLERMONT-FERRAND amp Clermont Universiteacute Universiteacute dAuvergne Uniteacute de Nutrition Humaine

BP 10448 F-63000 CLERMONT-FERRAND France

Anthony Fardet


and supplements do not lead to the same kinetics of micronutrient release within organism but for which effects Indeed the differential health effects of different kinetics of micro- and phyto-nutrients release are not known on a long term Therefore I think this is the most important and urgent research issue to address However in some specific nutritional situations this is undoubtedly useful to increase micronutrient bioavailability as for example through vitamin A supplementation in some developing countries where severe deficiencies can occur Indeed in population that mainly consumes vegetable products there may be problem with micronutrient bioavailability to organism

So according to food matrix characteristics nutrients are not equally bioavailable and released at the same kinetic within digestive tract This is true for starch but also for lipids and proteins The same is also true for fiber according to their physico-chemical characteristics such as solubility degree of polymerization water-holding capacity porosity viscosity etc they are not fermented equally thus soluble fiber are rapidly fermented releasing a high amount of short-chain fatty acids at a given colonic site while insoluble fibre are partially and more progressively fermented

In addition to macronutrients and fiber there are also free and bound micronutrients For example free and bound ferulic acid have not the same nutritional effect due to different kinetics of release free ferulic acid (1-5) is released in the upper digestive tract where it may play a role in cell signaling while bound ferulic acid (95-99) is more progressively released and would play a major antioxidant role at colonic level

TOWARD NEW TECHNOLOGICAL PROCESSES

However between supplements and natural food products maybe there is a golden mean to find Indeed technological processes may well play the role to modify nutrient bioavailability provided it is for the best (15) For example gelatinization of starch increases its digestibility One can also beneficially search to increase bioavailability of vitamins and polyphenols For example degrading phytates and increasing mineral absorption may be very interesting in developing countries where there are mineral deficiencies Thus pre-fermentation soaking and germination are very used in developing countries to improve nutritional density of grain products and to increase micronutrient bioavailability (13) Here regarding fermentation and germination of rye results clearly show that fermentation plus germination is the most efficient in increasing amount of bioactive compounds (some bound compounds being initially physiologically inactive) (16)

This leads to the concept of lsquominimal processingrsquo that is a good compromise between highly refined processed foods and natural foods sometimes not edible as such (17) I think that the main issue of processing is to preserve both food structure and nutrient density but also palatability (because it has been shown that a highly nutritious food will not be consumed if its organoleptic properties are low) Finally processing may help to reach the following objective to be a lsquofriendrsquo of natural foods

The role of food structureAfter synergy effect the beneficial roles played by structure of complex foods must be emphasized The number of human studies illustrating the role played by food structure is many (6) The first having emphasized this point was led in 1977 intact apple apple pureacutee and apple juice were tested in healthy subjects for their effect on post-prandial glycaemia insulinaemia and satiety Results showed that the more the original apple is destructured or refined the less it is satiating and the higher the insulinemic response while no significant effect on glycaemia (7) There is also another interesting study by Granfeldt et al in 1991 about durum wheat and the influence of processing on food structure pasta and bread made of the same ingredients ie durum wheat exhibited different glycaemic and insulinaemic responses due to their different food structure (8) In addition to food form food cohesiveness or density may also play an important role on food health effect For example breads with different crumb density have not the same glycaemic and satiety responses Thus in the studies by Burton et al and by Saulnier et al a significant correlation between bread crumb density and glycaemic response has been found (9-10)

Besides the role of food structure characteristics on physiology there is also the role played by nutrient interaction within the complex food matrix they can be either deleterious as for antinutrients or beneficials as for other kinds of interactions eg with fiber

AntinutrientsAntinutrients are well known to chelate minerals and to limit their bioavailability (11) They are polyphenols phytic acid and other phytochemicals such as lectins and some saponins They are generally accumulated in the plant under stress conditions or pro-oxidative conditions eg to fight against insects So plant-based micronutrients in their natural form are generally only partially bioavailable For example iron in cereal products is no more than 20 bioavailable in humans magnesium no more than 30 and zinc no more than 20 (12) Therefore processing is an important means 1) to reduce anti-nutrients levels eg legume and cereal pre-fermentation and pre-germination as used in developing countries but also 2) to release bound form of some micronutrients into more bioavailable free form (13)

The fiber co-passengersIn addition to interaction with phytic acid there are also interactions with the fiber fraction The concept of lsquofiber co-passengersrsquo has been developed recently apparently first by Vitaglione et al in 2008 fiber co-passengers are all the non-energy nutrients bound to fiber and that arrive at colonic level where they exert their physiological effect eg phenolic antioxidants bound to fiber (14) According to Vitaglione et al antioxidants bound to fiber would be protected by them within food structure to reach the colon where they can exert their full potential ie the trapping of free radicals released by bacteria metabolism (protection against colon cancer) and the progressive release of antioxidants that pass through the intestinal barrier to reach bloodstream where they can protect from LDL oxidation then from cardiovascular disease risk such as atherosclerosis (14)

Food structure and bioavailabilityIn a nutritional supplement micronutrients are generally almost 100 bioavailable Therefore complex foods


REFERENCES

1 Parker T L Miller S A Myers L E et al ldquoEvaluation of Synergistic Antioxidant Potential of Complex Mixtures Using Oxygen Radical Absorbance Capacity (Orac) and Electron Paramagnetic Resonance (Epr)rdquo Journal of Agricultural and Food Chemistry 58 209-17 (2010)

2 Rayalam S Della-Fera M A Baile C A ldquoSynergism between Resveratrol and Other Phytochemicals Implications for Obesity and Osteoporosisrdquo Molecular Nutrition and Food Research 55 1177-85 (2011)

3 Wang S Meckling K A Marcone M F et al ldquoSynergistic Additive and Antagonistic Effects of Food Mixtures on Total Antioxidant Capacitiesrdquo Journal of Agricultural and Food Chemistry 59 960ndash8 (2011)

4 Thompson H J Heimendinger J Diker A et al ldquoDietary Botanical Diversity Affects the Reduction of Oxidative Biomarkers in Women Due to High Vegetable and Fruit Intakerdquo Journal of Nutrition 136 2207-12 (2006)

5 Ye X Bhupathiraju S N Tucker K L ldquoVariety in Fruit and Vegetable Intake and Cognitive Function in Middle-Aged and Older Puerto Rican Adultsrdquo British Journal of Nutrition 109 503-10 (2013)

6 Structure Des Aliments Et Effets Nutritionnels Edited by Fardet A Souchon I Dupont D Ed Quae Versailles France (2013)

7 Haber G B Heaton K W Murphy D et al ldquoDepletion and Disruption of Dietary Fibre Effects on Satiety Plasma-Glucose and Serum-Insulinrdquo Lancet 2 679-82 (1977)

8 Granfeldt Y Bjorck I Hagander B ldquoOn the Importance of Processing Conditions Product Thickness and Egg Addition for the Glycaemic and Hormonal Responses to Pasta A Comparison with Bread Made from lsquoPasta Ingredientsrsquordquo European Journal of Clinical Nutrition 45 489-99 (1991)

9 Burton P Lightowler H J ldquoInfluence of Bread Volume on Glycaemic Response and Satietyrdquo British Journal of Nutrition 96 877-82 (2006)

10 Saulnier L Micard V ldquoImpact De La Structure De Lrsquoaliment Sur Les Proprieacuteteacutes Nutritionnelles Et Lrsquoacceptabiliteacute Du Pain Et Des Pacirctesrdquo Innovations Agronomiques 19 63-74 (2012)

11 Lopez H W Leenhardt F Coudray C et al ldquoMinerals and Phytic Acid Interactions Is It a Real Problem for Human Nutritionrdquo International Journal of Food Science and Technology 37 727-39 (2002)

12 Fardet A ldquoNew Hypotheses for the Health-Protective Mechanisms of Whole-Grain Cereals What Is Beyond Fibrerdquo Nutr Res Rev 23 65-134 (2010)

13 Fardet A ldquoA Shift toward a New Holistic Paradigm Will Help to Preserve and Better Process Grain Product Food Structure for Improving Their Health Effectsrdquo Food amp Function DOI 101039C4FO00477A (2014)

14 Vitaglione P Napolitano A Fogliano V ldquoCereal Dietary Fibre A Natural Functional Ingredient to Deliver Phenolic Compounds into the Gutrdquo Trends in Food Science and Technology 19 451-63 (2008)

15 Fardet A ldquoEditorial - Are Technological Processes the Best Friends of Food Health Potentialrdquo Advances in Nutrition and Food Technology 1 103 (2014)

16 Katina K Liukkonen K-H Kaukovirta-Norja A et al ldquoFermentation-Induced Changes in the Nutritional Value of Native or Germinated Ryerdquo Journal of Cereal Science 46 348-55 (2007)

17 Imran P Farhan S Sultan M T et al ldquoRecent Developments in Minimal Processing A Tool to Retain Nutritional Quality of Foodrdquo Critical Reviews in Food Science and Nutrition 54 340-51 (2014)

18 Fardet A Rock E ldquoToward a New Philosophy of Preventive Nutrition From a Reductionist to a Holistic Paradigm to Improve Nutritional Recommendationsrdquo Advances in Nutrition 5 430-46 (2014)

19 Visioli F ldquoCan Experimental Pharmacology Be Always Applied to Human Nutritionrdquo International Journal of Food Sciences and Nutrition 63 10-3 (2012)

and help them to give the best For example cereal micronutrient density can be preserved via less refined flours and less drastic thermal treatments

CONCLUSIONS AND PERSPECTIVES

Functional foods nutraceuticals and dietary supplements do not really succeed in stopping obesity and diabetes epidemics and in preventing from cancers and cardiovascular diseases in developed and emerging countries but they remain very useful in some circumstances Besides there is today a body of evidence showing that the whole is best than the sum of the parts because of synergism interactions with digestive environment and satiety effect Therefore it is now time to develop new minimal processes because processing is probably the most significant lever to rapidly improve food nutritional quality (eg more rapid than genetic selection and more efficient on a long term than dietary supplements)

Otherwise while the last decades were characterized by a reductionist and pharmacologic approach applied to human nutrition the future should include a more holistic approach applied to foods and diets and complementary to the reductionist approach (6) Focus should be put more on preventive nutrition and complex foods A holistic view of food will help preserve more its complex and natural matrix Consequently this will help improving nutritional recommendations (6)

Concerning the reductionist approach it is well characteristic from Western societies (18) It has led to major scientific discoveries Reductionism aims at isolating components of a system to better understand how it works Therefore its major drawback is to see the system through a reduced view with the risk to conclude that this partial view explain the whole Thus during these last twenty years of research one has mainly focused on the physiological and health effect of food nutrient taken one by one which has led to associate one compound with one physiological mechanism This has led to think that only one compound may prevent the organism from chronic diseases as for isolated antioxidants Obviously this is not true foods are complex systems and not drugs and human organism is complex as well so there is today a real need for more holistic approaches historically originating from Asia For this we need more generalist holistic and transversal researchers to work together with highly specialized and reductionist researchers (18)

This questioning is not really new as we can read in the paper of Visioli in 2011 ldquoIn summary the development of new experimental paradigms to appropriately study the effects of food items on human health should be actively sought after and public health authorities should reconsider their approach to regulations and guidelinesrdquo (19)

In the end foods being not drugs it is now urgent to shift from a reductionist and pharmacological approach to a holistic and integrative approach in nutrition research


KEYWORDS Olive oil Cryogen Solid CO2 Extractability Extraction conditions Mass transfer

AbstractThe impact of the addition of solid CO2 on olive oil (EVOOVOO) production in terms of extraction yield and chemical quality was studied at pilot scale in an industrial oil-press in two different crop seasons The

addition of CO2s to the fruits induces the intracellular water freezing and the consequent laceration of cellular membranes could induce the diffusion of many cellular compounds In the experimental conditions adopted the addition of the cryogen to the olives during pre-milling phase greatly increased the extraction yield (ranging from cong 1 to cong 21 ) with respect to the related control Furthermore the use of solid CO2 had no negative effects on chemical composition of the olive oil maintaining the highest quality according to EU legal standards showed by control Therefore the utilization of solid CO2 as cryogen could be an appropriate technology to improve olive oil yield and quality and increase its shelf-life

The utilization of solid carbon dioxide in the extraction of extra-virgin olive oilVOOEVOO yield and quality as a function of extraction conditions adopted

INTRODUCTION

In recent years the Mediterranean diet has become increasingly popular gaining widespread attention among the nutrition and research communities (1-2) and their unreplaceable element in this dietary style is extra virgin olive oil (EVOO) For these reasons extra-virgin olive oil (EVOO) is characterized as one of the highest economic values for vegetable oils being the main dietary fat in the Mediterranean countries (3-6)One of the most important industrial handicaps of VOO and EVOO production is the low efficiency of current extraction techniques (7) Nowadays several studies have pointed out the importance of the different virgin olive oil processing stages on the extraction yield as well as the minor composition found in the final product and the most used solution in oil mil for improve extraction is increase malaxation time andor temperature (37-8) Although the important research effort devoted to find innovative mild techniques to enhance VOO and EVOO production (79-11) at industrial level the entire virgin olive oil (VOO) process has changed very little over the last twenty years (3) The addition of a cryogen is a physical technology that has been proposed for improving mass transfer processes in food industry mainly in wine-making technology (12) The method is based on the evidence that the volume occupied by the same amount of water in the solid state is greater than that in the liquid phase The addition of a cryogen to the fruits induces the intracellular water

freezing a corresponding increase of endocellular volume occurs and the consequent laceration of cellular membranes (cellular break) induces the immediate diffusion in the liquid phase of many cellular compoundsWith the aim to contribute to advance in VOOEVOO technology at DAFE of University of Pisa an innovative VOOEVOO extraction technology (Patent ndeg ITRM20100617) involving the addition of a cryogen to the olives (13) were developed in order to increase the extraction yield of an high quality EVOO As a function of the temperature adopted at atmospheric pressure carbon dioxide (CO2) can be present only in a solid (CO2S = carbonic snow T lt -785degC) andor in a gaseous state (T gt -785degC) (12) Because of the direct contact between the cryogen (carbonic snow) and the olives the pre-milling addition of solid carbon dioxide CO2s (carbonic snow) to the olives causes a partial solidification of cellular water In this way it could be possible to obtain extra virgin olive oils characterized by a greater amount of metabolic compounds deriving from the olives even because the relevant amount of gas (gaseous CO2G) which evolves during the direct contact between olives and cryogen decreases the amount of atmospheric oxygen diffused in the liquid phase and then inhibits the possible oxidation of the phenolic substancesThe main objective of the present study was to demonstrate at pilot scale in an industrial oil-press mill the potential benefit of the addition of cryogen (CO2s) directly to olives during pre-milling phase on the yield and quality of the oil extracted in order to obtain a preliminary

FOOD PROCESSING

ANGELA ZINNAI FRANCESCA VENTURI CHIARA SANMARTIN ISABELLA TAGLIERI GIANPAOLO ANDRICHCorresponding author

University of Pisa Department of Agriculture Food and Environment (DAFE) Via del Borghetto 80 56124 Pisa Italy

Angela Zinnai


In all the experimental run the ratio ldquomass of cryogenrdquoldquomass of fruitsrdquo was maintained constant close to 02With the aim to avoid that the differences in the composition of olives (ie cultivar andor ripening stage as well as water content) utilised in different experimental runs can influence determination of the oil extraction yield this parameter has been expressed as ldquoextractabilityrdquo according to the following equation

Extractability index (E) = (kg of extracted oil)(kg of milled olives)x of oil inside the olives Eq 1

Chemical analysis of oil qualityGeneral chemical parameters free acidity ( of oleic acid) peroxide value (meq O2Kg) K270 and K232 were determined according the analytical methods described in the Regulation 25681991 of the European Union Commission and later modifications

RESULTS AND DISCUSSION

In this paper we take in account the olives harvested in two different crop seasons (2010 and 2014) characterised by a very different weather trend that significantly influenced the phytosanitary status of the fruits (14)While the 2010 crop season in Tuscany was characterised by a general good weather trend that allowed to obtain an increase in the production of oil (+150) if compared with the previous year as well as a general high quality of oil produced the 2014 has been defined as ldquoThe Black Year of Italian olive oilrdquo Also in Tuscany there are multiple causes for the disastrous fall in 2014 when the trees were turning flowers to fruit in the spring freezing weather suddenly turned scorching causing the trees to drop olives summer was hot and humid leading to all sorts of problems then in mid-September there was a major hail storm knocking much of the fruit that remained onto the ground Finally compounding the problems with the weather was a troublesome infestation of a fruit fly spreading a disease known as ldquoolive tree leprosyrdquo In Tuscany these very hard weather conditions during 2014 season caused both a reduction of oil production ranging from 50 to 70 than the last year and the oil produced is characterised by a quality level lower than that generally showed by Tuscan EVOO As a consequence of the worst weather trend during 2014 season the extractability index appear significantly lower than that obtained in the same experimental conditions during 2010 crop season

Impact of cryogen addition on oil extraction yieldIn Table 2 are reported the different indexes of oil extraction calculated for each experimental run according to eq 1 with

evaluation of the suitability of the new proposed methodology for VOOEVOO production

Materials and methods The study were conducted on samples of olive oil produced from monovarietal and polyvarietal (mix) olives collected in Tuscany during two different crop season (Table 1) characterised by a very different weather trend during the ripening and harvesting period for olives In each experimental run olive oil samples were obtained from homogeneous batches of olives with or without the addition of carbon dioxide in the solid state (ldquocarbonic snowrdquo) and main chemical parameters were determined so to compare the oil yield as well as the general quality obtained as a function of different process conditions adopted

Determination of oil extraction yield The extraction runs were carried out utilising a micro olive-press (Oliomio Babyreg produced by ldquoToscana Enologica Morirdquo Tavarnelle Val di Pesa - Italy) which is able to mill 20divide30 kg of olives and it was suitably modified to allow the addition of carbonic snow directly to olive fruits andor to their paste The main process steps followed by this micro olive-press can be so schematically presented olives properly cleaned and washed are poured into the receiving hopper where a screw feeds the crusher equipped with a hollow knife impeller The produced paste falls into the lower mixer and a helicoidally shaped stirrer promotes its malaxation during the malaxation phase the temperature reached by the paste is maintained in the wished range by a thermal regulation system (temperature sensor put inside the olive paste connected with a double jacket heating system) The wished flow of the olive paste is then sent to a biphasic decanter by a pump equipped with a speed change gear The decanter (4200 rpm) promotes the separation of oil from the solid parts of olive mixed with vegetable water plus the fraction possibly added to allow an efficient separation of these two phases In each experimental run control olive oil production was also obtained to make comparison In this case the extraction process was realised avoiding any contact between olives and cryogen In order to allow a suitable comparison between the results obtained in the presence or in the absence of solid CO2 during the oil extraction process the olive fruits utilised in each experimental run were suitably mixed and then the homogeneous sample (~ 60 kg) was divided into two aliquots (30 Kg of each) Both olives batches were processed in the same day with (EC) or without (ET) addition of solid CO2 in order to minimize the effect of physiological degradation of the whole fruits during storage on the extraction yield andor quality of the oil obtained as a function of extraction conditions adopted (cryo-extraction process vs conventional one)

Table 1 Experimental runs origin and cultivar of the raw matter

Table 2 Oil extractability yield EC = extractability yield by innovative method (with cryogen) ET = extractability yield by traditional method (without cryogen)


(such as phenols and aromatic compounds) diffused from the solid parts of the olives into the lipid phase during the different experimental runs could be very helpful in order to individuate among several combinations of working conditions the optimal one Moreover since the impact of the addition of cryogen to the olives appear positive also when the oil extractability decreases the application of this innovative technology can be very useful especially when the production of the olive oil shows an uncertain trend as a consequence of bad weather conditions during ripening of fruits

REFERENCES

1 Huang CL Sumpio BE ldquoOlive oil the mediterranean diet and cardiovascular healthrdquo J Am Coll Surg 207 407ndash416 (2008)

2 Dairi S Galeano-Diacuteaz T Acedo-Valenzuela MI et al ldquoMonitoring oxidative stability and phenolic compounds composition of myrtle-enriched extra virgin olive during heating treatment by flame oven and microwave using reversed phase dispersive liquidndashliquid microextraction (RP-DLLME)-HPLC-DAD-FLD methodrdquo Ind Crops and Prod 65 303ndash314 (2015)

3 Clodoveo M L ldquoNew advances in the development of innovative virgin olive oil extraction plants Looking back to see the futurerdquo Food Res Int 54 726ndash729 (2013)

4 Inarejos-Garciacutea AM Goacutemez-Alonso S Fregapane G et al ldquoEvaluation of minor components sensory characteristics and quality of virgin olive oil by near infrared (NIR) spectroscopyrdquo Food Res Int 50 250ndash258 (2013)

5 Scheidel A Krausmann F ldquoDiet trade and land use A socio-ecological analysis of the transformation of the olive oil systemrdquo Land Use Policy 28 47ndash56 (2011)

6 Bengana M Bakhouche A Lozano-Saacutenchez J et al ldquoInfluence of olive ripeness on chemical properties and phenolic composition of Chemlal extra-virgin olive oilrdquo Food Res Int 54 1868ndash1875 (2013)

7 Pueacutertolas E Martiacutenez de Marantildeoacuten I ldquoOlive oil pilot-production assisted by pulsed electric field Impact on extraction yield chemical parameters and sensory propertiesrdquo Food Chem 167 497ndash502 (2015)

8 Fregapane G Salvador MD ldquoProduction of superior quality extra virgin olive oil modulating the content and profile of its minor componentsrdquo Food Res Int 54 1907ndash1914 (2013)

9 Helms M ldquoFood sustainability food security and the environmentrdquo British Food J 106(5) 380ndash387 (2004)

10 Hadj-Taieb N Grati N Ayadi M et al ldquoOptimization of olive oil extraction and minor compounds content of Tunisian olive oil using enzymatic formulations during malaxationrdquo Biochem Eng J 62 79ndash85 (2012)

11 Clodoveo ML Hbaieb RH ldquoBeyond the traditional virgin olive oil extraction systems Searching innovative and sustainable plant engineering solutionsrdquo Food Res Int 54 1926ndash1933 (2013)

12 Zinnai A Venturi F Andrich G ldquoTime evolution of phenol extractions from Sangiovese grapes with and without the addition of solid carbon dioxiderdquo Agrochimica LV(4) 1-10 (2011)

13 Andrich G Venturi F Zinnai A ldquoInnovative technology for the production of olive oil characterized by organoleptic and nutritional high qualityrdquo Original Patent of Universitagrave di Pisa (Patent ndeg RM2010A000617) (2010)

14 Mraicha F Ksantini M Zouch O et al ldquoEffect of olive fruit fly infestation on the quality of olive oil from Chemlali cultivar during ripeningrdquo Food and Chem Tox 48(11) 3235-3241 (2010)

(EC) or without (ET) the addition of cryogen during pre-milling phase In order to evaluate the effect induced by the addition of cryogen on the oil extraction yield it was determined the Extractability Index Variation (EIV) as the percentage of the variation of oil extractability using CO2s compared with the same parameter obtained by a conventional extraction process

EIV = (EC-ET)ET 100 Eq 2

The experimental values (Table 2) showed as the direct addition of cryogen to the olives during pre-milling phase could induce a general increase in the oil extractability ranging from cong 1 to cong 21

Impact of cryogen addition on general chemical parametersTable 3 shows the main chemical parameters of oils produced in all the experimental runs Regardless the extraction method adopted (conventional or cryo) the values related to all the general chemical parameters remained below the maximum limit for extra virgin olive oil according to EU legislation (Regulation EC 19892003) The impact of addition of a cryogen to the olives in pre-milling phase did not seem have an influence on the general quality of the produced oil

CONCLUSIONS

Even if number of experimental runs carried out until now could appear quite reduced the data obtained gave a positive indication about the suitability of the new proposed method for the oil extraction from olives so that it will be used again during the next crop season Furthermore in order to individuate the best working conditions that could allow to obtain the best results in olive oil production by applying the innovative extraction process in the next future it will be necessary to adopt several combination of the main working parameters (ie amount of cryogenamount of olives fruit ripening stage etc) during olive oil production In this context also the measure of the concentrations of high quality components

In relation to each couple of runs (A1divideG7) for each parameter values not sharing the same letter have significantly different mean

Table 3 Oil general chemical parameters EC = extraction with cryogen (runs labeled with number from 1 to 7) ET = extraction without cryogen (runs labeled with letter from A to G)

∆


KEYWORDS Anthocyanin antioxidant extraction red basil sonication

AbstractToday the use of oils in the food industry has greatly expanded Therefore preventing oxidation of food is critical to increase shelf life and food quality Antioxidants are compounds that delay or slow the lipid

peroxidation and free radical generation in cells Antioxidant activity in red basil (Ocimum tenuiflorum) is one of major causes of its pharmacological actions Phenolic compounds in red basil extracts have been proved as good antioxidants In this the effect of time (10 20 and 30 min) and temperature (40 50 and 60degC) on optimization of anthocyanin extraction was evaluated using surface response method The study was aimed to determine maximum extraction of anthocyanin from red basil using ultrasound method The optimum conditions for basil extraction were 40 min at 6282degC Anthocyanin concentration extraction efficiency ferric reducing ability total phenolic compounds and EC50 were registered 9647407 7804989 5829404 3922485 and 3851161 respectively

Optimization of ultrasonic assisted extraction of antioxidants and phenolic compounds from red basil using response surface methodology

INTRODUCTION

Lipid peroxidation is one of the most important biologically relevant free radical chain reactions that contributes mainly to the damage or non-integrity of lipid containing tissues during food processing at high temperature Increase in oil and fats oxidative stability is an efficient approach to solve this problem (1) Lipid peroxidation during food processing not only lowers the nutritive value of food but also provokes free radical generation The biochemical role of free radicals in the development of many diseases including atherosclerosis cancer and premature aging is clearly known (2) Antioxidants delay lipid peroxidation and reduce free radical production Antioxidants are divided into two groups natural and synthetic antioxidants Natural antioxidants consist of several compounds such as phenolic compounds (flavonoids tocopherols and phenolic acids) nitrogen compounds (alkaloids Chlorophyll derivatives amino acids and amines) and carotenoids On the other hand synthetic antioxidants are mainly phenolic for instance butylated hydroxytoluene (BHT) butylated hydroxyanisole (BHA) tert-butyl hydroquinone (TBHQ) and propyl gallates (PG) which are used widely in many greasy foods The adverse effects of these compounds on human health are reported by Mahdavi et al (3) Moreover it has been reported that synthetic antioxidants should be used in limited quantity because they exhibit toxicity and physiological disorders in human body (4) Due to limited sources of natural antioxidants and low efficiency of

these compounds such as tocopherols identify other important sources is necessary Basil (Ocimum tenuiflorum family Lamiaceae) is a popular herb in the Mediterranean diets an annual herb commonly used in many kinds of food preparations in Mediterranean diets Red basil is cultivated widely in Iran and its antioxidative properties are clearly known Antioxidant activity of components in red basil is one of major causes of its pharmacological actions Phenolic compounds in red basil extracts including eugenol cirsilineol isothymusin isothymonin rosmarinic acid orientin and vicenin have been proved as good antioxidant compounds and zinc an antioxidant mineral has been found to be significantly high in

FOOD PROCESSING

SAEED AHMADI1 REIHANEH AHMADZADEH GHAVIDEL2 ZAHRA SHEIKHOLESLAMI3 MOLOUD ELAHI4 SHADI ELAHI5

Corresponding author1 Young Researchers and Elite Club Quchan Branch Islamic Azad University Quchan Iran

2 Department of Food Science and Technology Quchan Branch Islamic Azad University Quchan Iran3 Technical and Engineering Research Section Agricultural and Natural Resources

Research Center of Khorasan Razavi Iran4 Young Researchers and Elite Club Quchan Branch Islamic Azad University Quchan Iran

5 Department of Chemical Engineering Biotechnology Trends Quchan Branch Islamic Azad University Quchan Iran Reihaneh Ahmadzadeh

Ghavidel

Figure 1 Calibration curve of phenolic compounds at 765 nm


investigations to identify medicinal plants and build up a socio-economic profile of herbal medicines The current study was aimed to determine maximum extraction of anthocyanin from red basil using ultrasound waves and surface response method In other words different extraction time and temperature and their effects on antioxidative properties and phenolic compounds of red basil were investigated


Mature and middle-sized red basil (Ocimum basilicum) plants were purchased form local field in Sabzevar Iran The genius and species of the plants were confirmed by Tehran National Botanic Garden The green and red leaves were separated and covered with plastic bags and stored in freezer at -18degC All chemicals were purchased from Merck Chemical Company (Germany)

Ultrasonic extractionSamples were weighted and mixed with ethanol-hydrogen chloride solvent (1585) The samples were placed in ultrasonic bath (BANDELIN SONOREX digitec model DT 510 H Germany) for different times (10 20 and 30 min) and temperatures (40 50 and 60degC) the device was set on 35 KHZ The extracts were filtered using Whatman filter paper (NO 5) and vacuum pomp Solvent was removed using rotary (Laborota 40024003 control Germany) at 40-50degC The materials were transferred onto plates and dried at 45-50degC Plates were covered with aluminum sheets to prevent light degradation The samples were kept in freezer at -18degC until further analysis (9 10)

Anthocyanin assay by pH differential method Anthocyanin was measured according to Fuleki and Fransis method (11) Absorbance was recorded at 700 nm and pH 1 and 45 The anthocyanin content was calculated using following formula Cyanidin-3 5-glucoside was dominant anthocyanin with maximum lambda 520 nm

∆a= [(A 520- A700) pH = 1] ndash [(A 520- A700) pH =45] C=∆a times M times D Ɛ times L

Where C anthocyanin intensity M dominant anthocyanin molecular weight D diluting factor L cuvette length Ɛ molar absorption

Phenolic compound assay For this purpose 5 ml extract was dissolved in methanol and then 25 ml folin-ciocalteus reagent was added After 3 min 5

red basil However there is a great deal of diversity in the composition of red basil cultivated in different locations The high economic value of basil oil is due to the presence of phenyl propanoids like eugenol chavicol and their derivatives or terpenoids like monoterpen alchole linalool methyl cinnamate and limonene The antioxidant activity of phenolic compounds is mainly due to their redox properties which can be play an important role in absorbing and neutralizing free radicals quenching singlet and triplet oxygen or decomposing peroxides (5)Extraction of antioxidants compounds from herbs and spices can be performed by using many methods including i) steam distillation ii) hydrocarbon extraction iii) chlorinated solvent extraction iv) enzymatic treatment and fermentation v) super critical carbon dioxide extraction (6) Conventional techniques for the extraction are time and solvent consuming thermally unsafe and the analysis of plant constituents is also limited by some extraction steps These extraction techniques are mostly based on the use of solvents and heat or agitation to increase the solubility of the desired compounds and to improve the mass transfer (7) In recent years the use of alternative ways for the extraction of plant constituents has shown interest and potentialities both in research and industrial level The ultrasound assisted extraction is relatively new extraction techniques that use ultrasound treatments with traditional solvent extraction This systems has been used for the extraction of natural compounds (eg polyphenolics anthocyanins aromatic compounds polysaccharides and functional compounds) from different plant species by varying the main parameters that may influence the extraction efficiency namely the nature and volume of solvent extraction time ultra waves power matrix characteristics and temperature (8) In recent years much attention has been paid to applying herbal medicines on account of harmful effects of synthetic drugs Therefore it is necessary to carry out comprehensive studies and

Figure 2 Response surface and optimization of red basil extract as a function of time and temperature

Figure 3 Comparison between optimum treatments and control treatment (without ultrasound) for each test in red basil


relationships between explanatory variables and response variables using Expert 700 software Independent variables consist of time (three levels) and temperature (three levels) The number of experiments would decrease using response surface methodology (12)


Suitable model selection In order to assess the validity of the fitted models lack of fit test coefficient of variation R-squared and Adj R-Squared were determined The most important parameter in statistical analysis table is lack of fit parameter Statistically the suitable model is the model that its lack of fit test is not significant This parameter represents suitability of the model (13) R-Squared and Adj R-Square for a model with good fit should ideally be more than 1 Response values for each test under different time and temperature are given in Table 1 Although quadratic model for extracted anthocyanin ferric reducing ability total phenolic compounds and DPPH was significant (plt001) lack of fit test was not significant (pgt005) This result represents fitted model propriety Significant terms for model consist time (plt001 A) and temperature (plt001 B) The results shown in Table 2 indicate suitable accordance between calculated model and observed values and model accuracy

Optimization Optimum condition for red basil extraction by ultrasound method was performed using numerical optimization technique (Figure 2) This technique was used for anthocyanin concentration extraction efficiency total phenolic compounds free radical scavenging activity and ferric reducing ability Values for optimization of each response are shown in Table 3 Finally obtained results for red basil extraction optimization are presented din Table 4 Tiwari et al (14) studied the effect of time on anthocyanin extraction of black berry using ultrasound method They have

ml sodium carbonate (75) was added into aquatic phase and after 1 min made up to 50 ml with distilled water After 24 h the absorbance was recorded at 765 nm and phenolic compounds were calculated according to following formula and standard curve (10) (Figure 1)P= Y W times 1000X= absorbance absorbance - control absorbance Y= 10776 x2 + 02644 x + 00099Where P phenolic compounds (mg ml-1) W sample weight

Ferric reducing ability measurement Briefly fresh FRAP solution was prepared by mixing acetate buffer TPTZ reagent and 20 mM iron chloride hexahydrate (10 1 1) and then mixture was kept in a dark place TPTZ was prepared by mixing 234 mg TPTZ reagent with 75 ml 40 mM HCL Acetate buffer (03 M pH 36) was made from a mixture of acetic acid (16 ml) and sodium acetate trihydrate (31 g) made up to 1000 ml with distilled water Depend on radical scavenging activity of sample 100 ml of sample was dissolved in 10 ml methanol and then 30 microl of the solution was added into 900 microl FRAP reagent solved into 90 microl distilled water The samples were heated using benmary bath after being vortexed The samples were cooled to 37degC and then absorbance was recorded at 595 nm Ferric reducing ability was calculated using following formula Y= 1782 x ndash 9211X = sample absorbance ndash control absorbance Where Y micromol Fe II per liter X absorbance at 595 nm

Free radical scavenging activity assay (DPPH)The DPPH solution (0006) was prepared by dissolving DPPH in methanol The solution was added into test tubes containing plant samples After mixing well absorbance was recorded at 512 nm Free radical scavenging activity was calculated using following formula A () = AC ndash AS AC times 100Where A Free radical scavenging activity () AC control absorbance AS sample absorbanceEC50 was defined as the concentration of antioxidants which caused 50 inhibition of free radicals (1)

Extraction efficiency Solvent was removed from the extracts by benmary bath The dry matter was weighted and extract efficient was expressed as mg g-1 dry weight (6)

Statistical analysisThe experimental design was a completely randomized design with three replications Analysis of variance (ANOVA) was carried out and response surface methodology was used to optimize extraction condition and understand

Table 1 Response values during extraction process

Table 2 Statistical analysis and fitted quadratic model for differnet red basil parameters


The best temperature was 30degCAccording to the results anthocyanin concentration was 9647407 at optimum point while the anthocyanin concentration in control treatment was 18575 Extraction efficiency at optimum point and control treatment was 7804989 and 37283 respectively (Figure 3) Ferric reducing ability at optimum point and control treatment was

5829404 and 363227 respectively Total phenolic compounds at optimum point and control treatment was 3922485 and 168662 respectively The EC50 at optimum point and control treatment was 3851161 and 10998 respectively

CONCLUSION

The current experiment was aimed to determine free radical scavenging activity anthocyanin content phenolic compounds ferric reducing ability and extraction efficiency in red basil using ethanol-HCl solvent and ultrasonic waves In addition the effect of time and temperature were studied on these parameters According to the results the best condition for red basil extraction was 30 min extraction at 5503degC At this point anthocyanin concentration and extraction efficiency were found 964 and 780 respectively Moreover ferric reducing ability was recorded 58294 and phenolic compounds and EC50 were 392248 and 385 respectively Finally results suggest that red basil can be introduced as good source of antioxidants on account of its phenolic compounds

REFERENCES

1 Shaddel R laquoOptimization of Extraction Bioactive Substances in Pistacia Atlantica Using Response Surface Method and Study on Its Antioxidant Effect Master thesisraquo Ferdowsi University of Mashhad 2011

2 Ahmadi F Kadivar M Shahedi M laquoAntioxidant Activity of Kelussia Odoratissima Moza In Model and Food Systemsraquo Food Chemistry 105 57-64 (2007)

3 Mahdavi DL Deshpande SS Salunkhe DK Food Antioxidant 1st edn New York Marcel Dekker Inc USA 1995

4 Sharif A Farhoosh R Khodaparast MHH Antioxidant Activity of Bene Hull Oil Compared with Sesame and Rice Bran Oils during the Frying Process of Sunflower Oil Journal of Food lipids 16 (3) 394-406 (2009)

5 Asami DK Hong Y Barrett D M et al ldquoComparison of the total phenolic and ascorbic acid content of freeze-dried and air-dried marionberry strawberry and corn grown using conventional organic and sustainable agricultural practicesrdquo J Agric Food Chem 51(5)1237- 1241 (2003)

6 Peter K V ldquoIntroduction in Handbook of Herbs and Spicesrdquo Woodhead Pubblishing Limited In K V Peter (Ed) vol 1 (2001)

7 Mandal V Y Mohan Hemalatha S ldquoMicrowave Assisted Extraction ndash An Innovative and Promising Extraction Tool for Medicinal Plant Researchrdquo Pharmacognosy Reviews 1(1) 7-18 (2007)

8 Delazar A Nahar L Hamedeyazdan S et al ldquoMicrowave-assisted extraction in natural products isolationrdquo Methods in Moleulcar Biology 86489-115 (2012)

reported that anthocyanin content increased from 20 to 40 min extraction and then remained stable until 100 min extraction They have stated that this is because of the balance between solvent and extracted substances The highest anthocyanin (5614 mg g-1) was obtained after 40 minutes Similar results were obtained by Huang et al (15) who studied anthocyanin content in sweet potato Increase in anthocyanin content with increasing extraction time has also been reported by Borges et al (16) in Euterpe edulis and Yang et al (17) in purple corn Similarly Fan et al (18) found a linear relationship between extraction time and anthocyanin content in sweet potato The effect of time and temperature on extraction efficiency by ultrasound method in grape was studied by Lieu and Li (19) They concluded that extraction efficiency increased with increasing time and temperature In addition optimum temperature and time were 74degC and 13 min respectively Similar results were achieved by Sheng et al (20) who studied the effect of time and temperature on extraction efficiency of Forsythia suspense In their study the optimum temperature and time were registered as 6681degC and 8543 min respectively In another study Shad del et al (1) have reported that increase in extraction time and temperature increases ferric reducing ability and phenolic compounds content in Pistacia atlantica The effect of temperature (110 and 160degC) on reducing ability of canola meal extract was evaluated by Hassas-Roudsari et al (21) They concluded that there is no significant difference between two temperatures as to reducing ability At higher temperature extraction would decrease due to oxidation (22) With increasing sample to solvent ratio in short time contact phenolic compounds slightly increased whereas in long time contact phenolic compounds slightly decreased This might be due to extraction of impure compounds by solvent (23) Increase in phenolic compound has been reported by Wang et al (24) who studied wheat bran and indicated that 10-30 min ultrasonic increases phenolic compound Similar results have been found by Rajaei et al (25) in pistachio They reported that increase in temperature up to 65degC increases extraction efficiency however there was no significant change at higher temperatures (65-85degC) Increase in free radical scavenging activity has been reported by Kishk et al (26) in ginger up to 30 min and after 30 min this activity remained stable In addition free radical scavenging activity increased when temperature increased from 20 to 54degC Increase and then decrease in free radical scavenging activity was observed in common bean by Lai et al (27)

Table 3 Applied values for red basil optimization and their characteristics

Table 4 Optimization of red basil extraction affected by time and temperature

Treatment in Juice Processing Ultrasonics sonochemistry 17 273-279 (2009)

20 Sheng Z Li J Li Y Optimization of Ultrasonic-Assisted Extraction of Phillyrin from Forsythia Suspensa Using Response Surface Methodology Journal of Medicinal Plants Research 6 1633-1644 (2012)

21 Hassas-Roudsari M Chang PR Pegg RB et al ldquoAntioxidant Capacity of Bioactives Extracted from Canola Meal by Subcritical Water Ethanolic and Hot Water Extractionrdquo Food Chemistry 114 717-726 (2009)

22 Rostagno MA Palma M Barroso CG Ultrasound-Assisted Extraction of Soy Isoflavones Journal of Chromatography A 1012 119-128 (2003)

23 Chan SW Lee CY Yap CF et al ldquoOptimization of Extraction Conditions for Phenolic Compounds from Limau Purut (Citrus Hystrix) Peelsrdquo International Food Research 16 203-213 (2009)

24 Wang J Sun B Cao Y et al Optimization of Ultrasound-Assisted Extraction of Phenolic Compounds from Wheat Bran Food Chemistry 106 804-810 (2008)

25 Rajaei A Barzegar M Hamidi Z Optimization of Extraction Conditions of Phenolic Compounds from Pistachio (Pistachia Vera) Green Hull through Response Surface Methodrdquo Journal of Agricultural Science and Technology 12 605-615 (2010)

26 Kishk YFM El Sheshetawy HE ldquoOptimization of Ginger (Zingiber officinale) phenolics extraction conditions and its antioxidant and radical scavenging activities using Response Surface Methodologyrdquo World Journal of Dairy amp Food Sciences 5 188-196 (2010)

27 Lai J Xin C Zhao Y et al Optimization of ultrasonic assisted extraction of antioxidants from black soybean (Glycine max var) Sprouts Using Response Surface Methodology Molecules 2013 18 1101-1110

9 Pedramnia A Sharifi A Tavakoli pour H ldquoOptimization of Extraction of Barberry by Ultrasound Wavesrdquo Food Science and Technology 12 45-52 (2010)

10 Bahman Abadi J Optimization of Extraction of Barberry Using Ultrasonic and Response Surface Methods Master thesisi Islamic Azad University Ghuchan Ranch 2011

11 Fuleki T Francis FJ ldquoQuantitative Methods for Anthocyanins 1 Extraction and Determination of Total Anthocyanin in Cranberriesrdquo Journal of Food Science 33 72-78 (1968)

12 Li J Ding S Ding X ldquoOptimization of the Ultrasonically Assisted Extraction of Polysaccharides from Zizyphus Jujube Cv Jinsixiaozaordquo Journal of Food Engineering 80 176-183 (2007)

13 Atkinson AC Donev AN Optimum Experimental Designs Oxford University Press 1992

14 Tiwari BK OrsquoDonnell CP Cullen PJ ldquoEffect of Sonication on Retention of Anthocyanins in Blackberry Juicerdquo Journal of Food Engineering 93 166ndash171 (2009)

15 Huang CL Liao WC Chan CF et al ldquoOptimization for the Anthocyanin Extraction from Purple Sweet Potato Roots Using Response Surface Methodologyrdquo Journal of Taiwan Agricultural Research 59 143-150 (2010)

16 Borges GDSC Vieira FGK Copetti C Optimization of the Extraction of Favanols and Anthocyanins from the Fruit Pulp of Euterpe Edulis Using the Response Surface Methodologyrdquo Food Research International l44 708ndash715 (2011)

17 Yang Z Zhai W ldquoOptimization of Microwave-Assisted Extraction of Anthocyanins from Purple Corn (Zea mays L) Cob and Identification with HPLC ndash MSrdquo Innovative Food Science and Emerging Technologies 11 470ndash476 (2010)

18 Fan G Han Y Gu Z et al ldquoOptimizing Conditions for Anthocyanins Extraction from Purple Sweet Potato Using Response Surface Methodology (RSM)rdquo LWT 41 155ndash160 (2007)

19 Lieu LN Le VVM Application of Ultrasound in Grape Mash

NEWS NEWS NEWS NEWS NEWS NEWS NEWS NEWS

Company ContactUL Information amp Insights

Whitney BlackGlobal Marketing Manager

7930 Santa Fe Ste 300Overland Park KS 66204

USATel +1 913 953 8928

whitneyblackulcom

SOLVING THE NEXT RampD CHALLENGE WITH PROSPECTORreg

UL (Underwriters Laboratories) demonstrated the companyrsquos comprehensive ingredients database at Vitafoods Europe 2015

At the Prospectorreg booth during Vitafoods Europe visitors experienced fi rst hand how ULrsquos innovative search engine simplifi es product development Listing some 70000 ingredients the global database is a valuable time-saving tool when it comes to the formulation of food beverages and dietary supplements

Researching new ingredients can be an arduous task tying up resources and holding up the formulation process ldquoThis can be very challenging for formulatorsrdquo says Jill Frank Certifi ed Food Scientist and Food Industry Expert at UL ldquoespecially with new product developments that go beyond the normal scope of the jobrdquo At Vitafoods UL experts demonstrated just how helpful their platform can be when it comes to searching for ingredients contacting suppliers and requesting samples and starter formulations Containing details of more than 70000 ingredients from hundreds of suppliers ULProspectorcom is incredibly easy quick and free to use Designed to speed up the search for ingredients and accelerate the RampD process the database is maintained and kept up to date by ULrsquos experts so users can be sure that the information is always currentWith 11000 functional food and nutrition ingredients Prospectorreg is a game-changing application for the nutraceutical sector ldquoOwing to the comprehensive breadth of information itrsquos easy to fi nd the right ingredients for specifi c health positionings and claims Many of our visitors had a lightbulb moment when they realized the opportunities that our database offersrdquo added FrankFor more information please visit wwwULprospectorcom

34 Agro FOOD Industry Hi Tech - vol 26(2) - MarchApril 2015

KEYWORDS Parmigiano-Reggiano 1H-NMR of suspensions of hard cheese concentration gradients in Parmigiano-Reggiano instrumental control of analytical parameters in Parmigiano-Reggiano sample size from Parmigiano-Reggiano

AbstractSuspensionssolutions of grated Grana Parmigiano-Reggiano type hard cheese give well-resolved 1H-NMR spectra These spectra show very specific patterns which are also sensitive to inner inhomogeneities of

these hard cheeses This enables to use the 1H-NMR spectra obtained from Grana Parmigiano-Reggiano as indicators of subtle changes in the concentration gradients of its parts This possibility was used to analyze the phenomenon of crumbling of this cheese yielding pieces of a few centimetre size The results indicate that sample size should be preferably gt5 g before homogenization by grinding or grating

1H-NMR spectra ndash a highly sensitive tool in the analysis of Grana Parmigiano-Reggiano type hard cheese

INTRODUCTION

Cheeses are complex mixtures of organic and of some inorganic compounds In these systems also minor components are of importance sensitively influencing nutritive properties taste flavour and therefore also the commercial value of these popular productsThe complex character of the analytical problem required that a broad range of instrumental analytical methods were adapted to cheeses such as GC-MS (1) for volatile components in combination with PampT (23) and SPME (4) (or both (2)) type sampling variations of HPLC (56) high-pressure TLC (7) electrophoretic techniques (8) occasionally in combination with biochemical methods (8 9) as well as spectroscopic analyses (UV-VIS (10) IR (11)) to name only the most efficient ones Several excellent reviews cover the field too We cite here only the last one (12) Interestingly the use of NMR spectroscopy one of the most popular tools in the organic analysis was reported only in a few communications (13-15) In the present paper we describe an application of 1H-NMR spectra for some problems of Grana Parmigiano-Reggiano hard cheesesThe Parmigiano-Reggiano cheese is one of the most valuable types of the so-called ldquoparmesanrdquo cheese family This registered trade mark is given only to products of a consortium of cooperatives and small factories joined into the Consorzio del Formaggio ldquoParmigiano-Reggianordquo which was founded 70 years ago with the goal of

standardizing the quality and coordinating marketing efforts (1617) The production rules are based on easily measurable parameters (eg density pH temperatureetc) We are involved in a program aiming at the extension of the control parameters towards methods enabling a molecular-level analysis of the composition of the Grana Parmigiano-Reggiano cheeses (1 3 18-22)It is an interesting feature of the Grana Parmigiano-Reggiano type cheeses that upon action of mechanical fracture strain applied for example by the tip of traditional knife-like cheese splitting devices (ldquocoltello a mandorlardquo or ldquocoltello a gocciardquo) millimetre ndash to centimetre ndash size splinters (ldquoscaglierdquo) are easily separated (Figure 1) The formation of these splinters is due most probably to internal inhomogeneities in the bulk of the cheese We studied these splinters with 1H-NMR spectroscopy as described in this paper

FOOD ANALYSIS

MARCELLO VEZZANI1 FRANCO BELLESIA2 LUCIANO CAGLIOTI3 ADRIANO PINETTI1 CLAUDIA ZUCCHI1 GYULA PAacuteLYI1

Corresponding author 1 Department of Life Sciences University of Modena and Reggio Emilia Via G Campi 183 I-41125 Modena Italy

2 Department of Chemical and Geological Sciences University of Modena and Reggio Emilia Via G Campi 183 I-41125 Modena Italy

3 Department of Chemistry and Technology of Natural Substances University ldquoLa Sapienzardquo Roma Piazzale A Moro 5 I-00185 Roma Italy

Claudia Zucchi

Figure 1 Splinters (ldquoscaglierdquo) of Grana Parmigiano Reggiano hard cheese

Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015Agro FOOD Industry Hi Tech - vol 26(2) - MarchApril 2015 35

Spectroscopic measurementsNMR spectra were obtained by a Bruker AMX-400 and a Bruker Avance 400 instruments 1H resonances were measured at 40013 MHz with both instruments 1D spectra were acquired with a presaturation sequence of the water peak (54 dB for decoupled power level for presaturation and 2s of presaturation during relaxation delay) and integrated with respect to dichloroacetic acid added to samples in known quantity as internal standard 1H-1H COSY spectra were obtained with presaturation during relaxation delay of the water peak (54 dB for decoupled power level for presaturation and 1s of presaturation during relaxation delay) a spectral width of 4800 Hz in both dimensions 512 increments 8 scans and 2k data points 1H-1H TOCSY spectra were acquired with presaturation during relaxation delay of the water peak (54 dB for decoupled power level for presaturation and 1s of presaturation during relaxation delay) a spectral width of 4800 Hz in both dimensions 512 increments 16 scans and 2k data points


The basis of the analysis of parmesan cheese with liquid phase NMR is the fact that finely grated cheese samples are giving liquid phases if are shaken for a few hours with water (or D2O) These non transparent (milky) liquid phases are visibly of colloidal nature we are investigating this aspect currently in our Laboratories From phenomenological viewpoint the important consequence of this observation is that these liquid phases are stable enough to ldquosurviverdquo at least till the end of the registration of NMR spectra Control experiments have shown that these samples remained unaltered even after a few hoursThe 1H-NMR spectra obtained from these samples are of excellent quality comparable to spectra obtained from (transparent) homogeneous solutions The NMR spectra are very rich of bands which are an advantageous feature for ldquodactyloscopicrdquo identification


Sample preparationTwo kinds of samples were prepared(a) Splinters of Parmigiano-Reggiano cheese (24 months

aged) were split off with the traditional hard cheese knife These splinters were of about 10x10x20 mm size Four of these samples were sliced with a very sharp (razor) knife into 12-15 mm slices as shown in Figure 2

(b) Another set of samples was prepared by cutting out from the splinters small 1-15 mm3 portions (Figure 3)

Samples prepared as described above were finely pulverized in a mortar and then the samples were shaken (mechanically) for 25 h in D2O (075 mL of D2O per 006divide015 g sample) A milky non-transparent liquid was obtained which was filtered and then used immediately (within 2 h) for NMR spectroscopic measurementsWe found that the pH of these apparently colloidal solutions was stable until the measurement was performed We did not stabilize the pH by buffer solutions to avoid salting-out effects

Figure 2 Slicing of a splinter

Figure 3 Concentration gradients in a Parmigiano Reggiano cheese splinter Internal quantitative standard Cl2CHCOOH Measured amino acid (a) serine (b) tyrosine (c) lysine


application complements earlier efforts at individual characterization of Parmesan cheese samples (24)

ACKNOWLEDGMENT

Help in collection the Parmigiano Reggiano cheese samples is gratefully acknowledged to the Consorzio del formaggio ldquoParmigiano-Reggianordquo (Modena)


Marcello Vezzani last year student of the University of Modena and Reggio Emilia passed away June 5 2003 This paper is based in part on his Diploma Thesis which remained unfinished This paper is dedicated to His memory

1 Thomsen M Gourrat K Thomas-Danguin T GuichardE ldquoMultivariate approach to reveal relationships between sensory perception of cheeses and aroma profile obtained with different extraction methodsrdquo Food Res Internat 62 561-671 (2014)

2 Bellesia F Pinetti A Pagnoni UM Rinaldi R Zucchi C Caglioti L Paacutelyi G ldquoVolatile components of Grana Parmigiano-Reggiano type hard cheeserdquo Food Chem 83 55-61 (2003)

3 Bellesia F Bianchi A Pinetti A Trillini B ldquoThe volatile organic compounds of Tuber uncinatum from middle Italyrdquo J Essential Oil Res 10 483-487 (1998)

4 Innocente N Munari M Biasutti M ldquoCharacterization by solid-phase microextraction-gas chromatography of the volatile profile of protected designation of origin Montasio cheese during ripeningrdquo J Dairy Sci 96 26-32 (2013)

5 Sforza S Galaverna G Neviani E Pinelli C Dossena A Marchelli R ldquoStudy of the oligopeptide fraction in Grana Padano and Parmigiano Reggiano cheeses by liquid chromatography ndash electrospray ionization mass spectrometryrdquo Eur J Mass Spectrom 10 421-427 (2004)

6 Bertolino M Dolci P Giordano M Rolle L Zeppa G ldquoEvolution of chemico-physical characteristics during manufacture and ripening of Castelmagno PDO cheese in wintertimerdquo Food Chem 129 1001-1011 (2011)

7 Kovaacutecs Aacute Simon-Sarkadi L Mincsovics E ldquoDetermination of biogenic amines by overpressured thin-layer chromatographyrdquo Eacutelelmiszervizsg Koumlzl 43 104-111 (1997)

purposes but it makes somewhat difficult the assignment of each peak At this point the 2D correlations were indispensable and we obtained essentially the same assignments as described in ref 14 We shall come back to certain aspects of the assignment process in a separate publication NMR signals corresponding to natural amino acids could be confidentially assigned The quantitative evaluation of such signals was combined by the use of an internal (quantitative) standard this was dichloroacetic acid Cl2CHCOOH which gives a sharp singlet signal at δ = 612 ppm low field from the middle of the spectrum in a range which was completely empty in all the proton resonance spectra obtained from cheese samples without this additive This quantitative method was tested for 3 amino acids (Tyr Ser Lys) which were of lower and medium abundance (respectively) in our independent HPLC study (22) The results of this analysis are shown in Figure 3 These results indicate that the splinters of the Parmigiano-Reggiano cheese have still their own microstructure which was studied also with another approach as described later in the present paper In another approach to the analysis of the splinter samples (Figures 1 and 2) of Parmigiano-Reggiano cheese these were finely cut into slices and the slices were pulverized subsequently treated with D2O and then analysed by 1H-NMR In an attempt at avoiding assignment uncertainties we treated quantitatively these spectra in a ldquoblack boxrdquo manner we did not assign bands at all but we calculated intensities of small ranges of spectra (reasonably spaced 005-02 ppm segments were used) and plotted the intensities of these ranges against the chemical shift scale of the spectra Very characteristic diagrams were obtained (Figure 4) These plots indicate sensitively the concentration gradients in the inner bulk of the splinters across the slices (Figure 5) These diagrams can be transformed into concentration vs slice number diagrams which make the comparison of the single slices even more specific

CONCLUSIONS

We believe that this work has resulted the following main consequences(i) 1H-NMR qualitative and quantitative analysis can be used

for ldquodelicaterdquo problems of cheese chemistry as the detailed study of the inner texture of cheeses

(ii) 1H-NMR quantitative analysis can be applied in cheese chemistry for the study of concentration gradient problems without detailed assignment of the signals

(iii) The sharp concentration gradients (and their irregular changes) in the bulk of Parmigiano-Reggiano cheeses warn at caution in the sampling process (in any kind of analysis) according to the results obtained in course of the present work we suggest that the size of a reliable representative sample should be between 5divide10 g

(iv) The very individual shapes of the diagrams shown in Figures 5 and 6 offer a new possibility for obtaining ldquofingerprintrdquo-like individual patterns which are characteristic for almost each sample This potential

Figure 4 Integrated areas of spectra against chemical shift ranges (integrated intensity of the Cl2CHCOOH peak at 6121 ppm was taken as equiv 1)


8 Andrighetto C Marcazzan G Lombardi A ldquoUse of RAPD-PCR and TTGE for the evaluation of biodiversity of whey cultures for Grana Padano cheeserdquo Lett Appl Microbiol 38 400-405 (2004)

9 Gaiaschi A Beretta B Poiesi C Conti A Giuffrida MG Galli CL Restani P ldquoProteolysis of beta-casein as a marker of Grana Padano cheese ripeningrdquo J Dairy Sci 84 60-65 (2001)

Figure 5 Concentration gradients in the inner bulk of the splinters across the slices ((a) (b) (c) (d) are the indication of the different samples A B C D indicate consecutive slices)

10 Wallace J M Fox PF ldquoRapid spectrophotometric and fluorimetric methods for monitoring nitrogenous (proteinaceous) compounds in cheese and cheese fractions A reviewrdquo Food Chem 62 217-224 (1998)

11 Gori A Maggio R M Cerretani L Nocetti M Caboni M F ldquoDiscrimination of grated cheeses by Fourier transform infrared spectroscopy coupled with chemometric techniquesrdquo Internat Dairy J 23 115-120 (2012)

12 Jaster H Pries de Campos A Leonelli A Bach L ldquoQuality evaluation of parmesan-type cheese A chemometric approachrdquo Food Sci Technol 34 181-188 (2014)

13 Zucchi C Bellesia F Pinetti A Simon-Sarkadi L Weimer B Caglioti L Paacutelyi G ldquoFeaturing Compounds of the Greasy Base of the Parmesan Cheese Aromardquo 3rd Italian-French Meeting on Organic Chemistry Organic Chemistry towards Interfaces (Pisa) Abstr P60 (2002)

14 Shintu L Caldarelli S ldquoHigh-resolution MAS NMR and chemometrics Characterization of the ripening of Parmigiano Reggiano cheeserdquo J Agricult Food Chem 53 4026-4031 (2005)

15 Bordoni A Picone G Babini E Vignali M Danesi F Vali V Di Nunzio M ldquoNMR composition of in vitro digestion of Parmigiano Reggiano cheese aged 15 and 30 monthsrdquo Magn Resonance Chem 49 (S1) S61-S70 (2011)

16 Eg A review on measurements executed between 1865 and 1932 (c) Mameli E Cultrera Rrdquo The composition of Parmesan cheeserdquo Ind Ital Conserve Aliment 7 301-311 (1932)

17 Pecorari M Gambini G Riverberi P Caroli A ldquoInfluence of technological factors on glycolysis in Parmesan cheeserdquo Sci Tec Latt-Casearia 54 287-299 (2003)

18 Bellesia F Pinetti A Pagnoni UM Rinaldi R Zucchi C Caglioti L Paacutelyi G ldquoVolatile compounds in food aroma Biosynthesis and biotransformationsrdquo in Progress in Biological Chirality (G Paacutelyi C Zucchi L Caglioti Eds) edited by Elsevier Amsterdam-Oxford 253-256 (2004)

19 Bellesia F Pinetti A Pagnoni UM Zucchi C Caglioti L Paacutelyi G ldquoA key process of aroma evolution Fatty acid beta-oxidation in Parmesan cheese ripeningrdquo Agro-Food Ind Hi-Tech 16 (1) 52-56 (2005)

20 Diploma Thesis of Sabrina De Paola University of Modena and Reggio Emilia (Identificazione di Composti Organici Volatili nellrsquoAroma del Formaggio Grana Parmigiano Reggiano Correlazione con Dati di Analisi Sensoriale) (AA 200203)

21 Diploma Thesis of Gian Luca Garuti University of Modena and Reggio Emilia (Formazione di Aromi nel Parmigiano Reggiano e nel Burro Influenza di Nuove Tecnologie) (AA 200304)

22 Bellesia F Pinetti A Simon-Sarkadi L Zucchi C Csapoacute J Weimer B Caglioti L Paacutelyi G Internat J Food Eng accepted for publication

23 Plutowska B Wardencki W ldquoAromagrams ndash Aromatic profiles in the appreciation of food qualityrdquo Food Chem 101 845-872 (2007)


KEYWORDS Mulberry mass spectrometry NMR techniques Flavonoids

AbstractMulberry fruits were shown to possess good antioxidant properties Flavonoids were extracted from three mulberry species (Morus alba L Morus rubra L and Morus nigra L) and characterized using mass

spectrometry The methanol extract was subjected to 1D and 2D NMR analysis in DMSO-d6 using TMS as an internal standard Mass spectrometry in combination with NMR spectra of crude mixture (methanol extract) was shown as a reliable tool for the characterization of flavonoids with already published NMR spectra in DMSO-d6 However in case of compounds not very well established it does not show relevance so separation of the crude mixture into individual components and their 1D and 2D NMR characterization is necessary However the results of this article would be useful in mulberry drink quality control and mulberry functional food research

Flavonoids in mulberry fruitIdentification of nonanthocyanin phenolics in some mulberry fruit species (Morus alba L Morus rubra L and Morus nigra L)

INTRODUCTION

Mulberry (Morus Moraceae) is known as food and mulberry leaves have been used as feed for silkworms In general there are 10-16 species of mulberry generally accepted by the majority of botanical authorities (1) Morus nigra L is a rustic plant growing wildly cultivated in gardens or commonly used in sericulture It is hard to commercialize products made from mulberry because of fragile structure and low stability on storage Morus alba and Morus nigra have their origins in western Asia and the red mulberry is a native of North America (2) It was shown that dried mulberry fruits possess anti-oxidant anti-microbial and anti-infl ammatory properties (3 4) Mulberry plants contain various bioactive components such as alkaloids and fl avonoids (1 2 5 6) Qualitative analysis of the nonanthocyanin phenolics from 2 mulberry cultivars was performed using HPLC-DAD-ESI-MSMS method As a result of the analysis six nonanthocyanin phenolics were identifi ed (procatechuic acid chlorogenic acid 4-caffeoylquinic acid taxifolin rutin quercetin) and three others (35-diCQA taxifolin-hexoside kaempferol-hexoside) were tentatively identifi ed (7) Similarly the fruits of black and white mulberries were studied and fi ve compounds from the methanol extract were identifi ed using HPLCPDAESI-MS (8) Mulberries are anthocyanins-rich fruits known as traditional Chinese medicine used for dizziness and blurred vision (9) Flavonoids are present in mulberry fruits (at least four of them including rutin) (8) They show anti-infl ammatory anti-oxidant

anti-allergic hepatoprotective anti-thrombotic antiviral and anti-carcinogenic activities Anthocyanins represent natural phenolic compounds responsible for the colouring of plant leaves fl owers and fruits Twenty different anthocyanins were identifi ed in plants six of them can be used as food additives (2) Two methods were used for the analysis and quantifi cation of anthocyanins in mulberry-pH-differential method (UV-VIS spectrophotometry) and high performance liquid chromatography (HPLC) (8-11) Anthocyanins are sensitive to pH oxygen light temperature and the presence of chemicals (12) Resveratrol (trans-3 4rsquo 5-trihydroxystilbene) and oxyresveratrol (trans-23rsquo45rsquo-tetrahydroxystilbene) are hydroxystilbenes found in numerous plant species such as grapes peanuts and mulberries (13) Resveratrol has potential as a neuroprotectant (14 15) and it shows cardioprotective effects (16) The objective of the research was the identifi cation of fl avonoids in some mulberry species (Morus alba L Morus rubra L and Morus nigra L)

EXPERIMENTAL

Sample preparationPreparation of mulberry extracts (black red and white) was performed on the following way Mulberry fruits picked in June 2013 were kept in the

FOOD ANALYSIS

VOJKAN MILJKOVIC1 LJUBISA NIKOLIC1 NIKO RADULOVIC2 BILJANA ARSIC2 GORAN NIKOLIC1DANIJELA KOSTIC2 ZORAN BOJANIC3 JELENA ZVEZDANOVIC1

Corresponding author1 Faculty of Technology University of Nis Bulevar Oslobodjenja 124 Leskovac Serbia

2 Faculty of Sciences and Mathematics University of Nis Visegradska 33 Nis Serbia3 Faculty of Medicine University of Nis Bulevar Dr Zorana Djindjica 81 Nis Serbia


Avance III 400 MHz NMR spectrometer (1H at 400 MHz 13C at 101 MHz) equipped with a 5-mm dual 13C1H probe head The 1H spectra were recorded with 16 scans 1 s relaxation delay 4 s acquisition time 0125 Hz digital FID resolution 51 280 FID size with 6410 Hz spectral width and an overall data point resolution of 00003 ppm The 13C spectra were recorded with Waltz 161H broadband decoupling 12 000 scans 05 s relaxation delay 1 s acquisition time 05 Hz digital FID resolution 65 536 FID size 31 850 Hz spectral width and an overall data point resolution of 0005 ppm1Hndash1H gDQCOSY and NOESY spectra were recorded at spectral widths of 5 kHz in both F2 and F1 domains 1 K times 512 data points were acquired with 32 scans per increment and the relaxation delays of 20 s The mixing time in NOESY experiments was 1 s Data processing was performed on a 1K times 1K data matrix Inverse-detected 2D heteronuclear correlated spectra were measured over 512 complex points in F2 and 256 increments in F1 collecting 128 (gHMQC) or 256 (1Hndash13C gHMBC) scans per increment with a relaxation delay of 10 s The spectral widths were 5 and 27 kHz in F2 and F1 dimensions respectively The gHMQC experiments were optimized for CndashH couplings of 165 Hz the 1Hndash13C gHMBC experiments were optimized for long-range CndashH couplings of 10 Hz Fourier transforms were performed on a 512 times 512 data matrix π2 Shifted sine-squared window functions were used along F1 and F2 axes for all 2D spectra


The mulberry extracts show the presence of the same components quinic acid gallic acid apigenin naringenin catechin quercetin myricetin chlorogenic acid cyanidin-3-O-arabinoside peonidin-3-O-arabinoside delphinidin-3-O-arabinoside cyanidin-3-O-galactoside or cyanidin-3-O-glucoside peonidin-3-O-glucoside or peonidin-3-O-galactoside or malvidin-3-O-arabinoside delphinidin-3-O-glucoside or delphinidin-3-O-galactoside petunidin-3-O-glucoside or petunidin-3-O-galactoside or petunidin-3-O-arabinoside malvidin-3-O-glucoside or malvidin-3-O-galactoside rutin luteoline perhaps 4-hydroxypanduratin A and 10-formyl-78-dihydrofolic acid (10-FDHFA) They were found using mass spectrometry and proved in NMR spectra (1D and 2D) of mixture obtained

refrigerator (-17degC) until the beginning of the extraction process Prior to the extraction 150 g of each mulberry type were blended Extraction was performed three times using 500 ml pa methanol (Macron Fine chemicals Norway) on a magnetic stirrer at room temperature for 1 h Afterwards the first extract was separated and 250 ml of methanol were added to the rest and the extraction was repeated once again The second extract was separated later (filtrate) using Buchner funnel This extract was merged with the first extract and the residue was for the third time extracted with 250 ml of methanol The third extraction lasted 30 min and it was performed under the same conditions as before The third extract was merged with two previous extracts overall it was 1000 ml of methanol mulberry extract (black red and white) 200 ml of the extract were taken for evaporation on a vacuum evaporator until slurry residue was obtained ready for further analysis

Analytical methodsMass spectrometryUltra high performance chromatography (UHPLC) analysis was carried out using a Dionex Ultimate 3000 UHPLC system equipped with a diode array (DAD) detector set on four wavelengths (520 nm 360 nm 320 nm and 280 nm) and also connected with LCQ Fleet Ion Trap Mass Spectrometer (Thermo Fisher Scientific Germany) The separations were performed on a Hypersil gold C18 column (50x21 mm 19 microm) (Thermo Fisher Scientific Germany) at 25degCDuration of PLC-MS analysis was 15 min In case of ESI-MS the temperature of the capillary was 275degC voltage of drop in the source 45 kV sheet gas flow 32 arb units and auxiliary gas flow 8 arb units

NMRAll NMR spectra were recorded at 25degC in CD3SOCD3 with TMS as an internal standard Chemical shifts are reported in ppm (δ) and referenced to TMS (δH = 0 ppm) in 1H NMR spectra andor to 13CD3SOCD3 (δC = 3952 ppm) in heteronuclear 2D spectra Scalar couplings are reported in Hertz 10 mg of a sample were dissolved in 1 ml of CD3SOCD3 and 07 ml of the solution were transferred into a 5 mm Wilmad 528-TR-7 NMR tubeThe 1H and 13C NMR spectra were recorded on a Bruker

Figure 1 Full mass spectra of mulberry methanol extracts of A) Morus alba L and B) Morus nigra L


(19) for pure quinic acid in DMSO-d6 (broad singlet at δH=447 ppm and peaks with chemical shifts δH=325 ppm δH=380 ppm and δH=383 ppm) and peaks in 13C NMR spectrum (Figure 3B) (three peaks in the region from 65-80 ppm and one in the region of the spectrum 175-180 ppm)

by the evaporation of the methanol extract of Morus rubra L using well-known published spectra of pure compounds in DMSO-d6 (TMS as an internal standard) We detected more compounds than in similar works published before (7 8) DMSO-d6 was used as a solvent for recording of the mixture because it yields well resolved spectra while in pure D2O the methylene carbons are isochronous quaternary carbons are of very low intensity requiring extensive instrument time (limited solubility long relaxation times) (17) However it is not recommended as a routine NMR solvent for flavonoids for several reasons 1) the spectra show an increased temperature sensitivity 2) the occurrence of additional OH signals potentially troubles the analysis of overlapping essential CH resonances and gives more complex coupling pattern without any gain in essential information 3) due to enhanced temperature sensitivity of the methylene protons the spectra are harder to reproduce At least one more solvent especially for new derivatives should be used in order to establish the structure (17) Full mass spectra of methanol extracts of Morus alba L and Morus nigra L show very similar fragmentation pattern (Figure 1) In both of them it is noticeable the presence of very strong peak at mz=381 It can be a sign of the presence of quinic acid derivative ([M-H]-(mz)=381 fragments at 191 and 110) (17) which can be true for the extract of Morus nigra L or the presence of the hexose in the structure (MSMS fragments 201 and 219) (18) which is presumably correct for Morus alba L and Morus nigra LThe analysis of mz peak at 381 from the methanol extract of Morus rubra L proves the presence of both quinic acid (fragment mz=192) and a hexose (mz=219) (Figure 2)The presence of quinic acid can be detected by protons in 1H NMR spectrum (Figure 3A) of the mixture using literature data

Figure 2 The analysis of mz peak at 381 from the methanol extract of Morus rubra L

Figure 3 NMR spectra of metahanol extract of Morus rubra L A) 1H NMR spectrum B) 13C NMR spectrum


(some peaks are absent like δC=1564 ppm and δC=1566 ppm) The same can be said for luteoline because of the absence of the 1H peak characteristic for the compound in 1H spectrum of the mixture (δH=69 ppm (1H H-5rsquo)) and 13C peaks (eg δC=1822 ppm (C-4) δC=1647 ppm (C-2)) (21) NMR spectra of compounds 4-hydroxypanduratin A (24) and 10-formyl-78-dihydrofolic acid (10-FDHFA) (25) were not recorded in DMSO-d6 as pure compounds so in order to be sure that these compounds with presented mass spectra are really compounds as we suggested we must separate components of the methanol extract mixture and record 1D and 2D NMR spectra of pure components Presence of anthocyanins (cyanidin-3-O-arabinoside

peonidin-3-O-arabinoside delphinidin-3-O-arabinoside cyanidin-3-O-galactoside or glucoside peonidin-3-O-glucoside or galactoside or malvidin-3-O-arabinoside delphinidin-3-O-glucoside or galactoside petunidin-3-O-glucoside or galactoside malvidin-3-O-glucoside or galactoside) was detected by mass spectrometry but the exact determination of their type is not possible without separation of the methanol extract into components and their NMR characterization We gave qualitative analyses of flavonoids in three Morus species (Morus alba L Morus rubra L and Morus nigra L) and proof of existence of some of them using NMR spectra (methanol extract of Morus rubra L) The presence of particular anthocyanin peak in MS spectra still needs more proof Determination of major and minor components in crude extract iequantitative analyses will be our further study

CONCLUSION

Three mulberry species (Morus alba L Morus rubra L and Morus nigra L) were subjected to analyses on the presence of flavonoids Mass spectrometry revealed presence of numerous flavonoids which can be detected in the mixture of compounds without previous separations In case of Morus rubra L the presence of anthocyanins was proven using 1D and 2D NMR techniques However the presence of particular anthocyanin peak in MS spectra still needs more proof This study might be interesting as a rapid method of characterization of nonanthocyanin phenolics in crude extracts of mulberry species Results of this article can be useful in control the quality of mulberry drinks and food products

The rest of 1D and 2D spectra of the methanol extract of Morus rubra L is available in Supplementary Material Similar concept was used for the determination of other components in the methanol extract Fragmentation patterns of some compounds are represented in Figure 4Gallic acid is present in the mixture in extremely small quantities compared to other compounds detected using mass spectrometry (20) The same conclusion on the content of apigenin can be brought as well The peaks correspond to apigenin (21) in 1H and 13C NMR spectra are absent Naringenin detected using mass spectrometry was present in small amounts due to absence of peaks in 1H and 13C NMR spectra characteristic for this compound eg δH=588 δH=731 ppm δC=19691 ppm δC=1642 ppm δC=1673 ppm and δC=1586 ppm (22) Catechin was present as a minor component in the mixture (again the peaks correspond to this compound (23) were absent in recorded 1H and 13C NMR spectra of the methanol extract) Quercetin is also not present as a major component or in a pure form the absence of characteristic peaks like δH=769 ppm (1H d H-2rsquo) δH=690 ppm (1H d H-5rsquo) δH=755 ppm (dd H-6rsquo) in 1H NMR spectrum of the mixture or δC=14679 (C-2) ppm in 13C NMR spectrum leads to this conclusion (20) Myricetin is present in traces-the absence of peaks corresponded to this compound (eg δC=1464 ppm (C-2) δC=1357 ppm (C-3) δC=1613 ppm (C-5) δC=1759 (C-4) in 13C NMR spectrum of the mixture (20) Chlorogenic acid is present we believe as a derivative because of the presence of some of the peaks (eg δC=735 ppm δC=682 ppm) and absence of some of them (eg δC=175 ppm δC=1658 ppm) in 13C NMR spectrum of the mixture (8) Rutin is present as it was deduced by mass spectrometry but in small quantities or as its derivative

Figure 4 Fragmentation patterns for somecompounds isolated from the methanolic extract of Morus rubra L A) gallic acid (mz 170) B) apigenin (mz 270) C) naringenin (mz 271) D) catechin (mz 286)

powder produced by spray dryingrdquo J Food Eng 88 411-418 (2008)13 Golkar L Ding X Z Ujiki M B et al ldquoResveratrol inhibits

pancreatic cancer cell proliferation through transcriptional induction of macrophage inhibitory cytokine-11rdquo J Surg Res 138 163-169 (2007)

14 Wang Q Xu J Rottinghaus G E et al ldquoResveratrol protects against global cerebral ischemic injury in gerbilsrdquo Brain Res 958 439-447 (2002)

15 Bastianetto S Zheng W H Quirion R ldquoNeuroprotective abilities of resveratrol and other red wine constituents against nitric oxide related toxicity in cultured hippocampal neuronsrdquo Br J Pharmacol 131 711-720 (2000)

16 Bradamante S Barenghi L Piccinini F et al ldquoResveratrol provides late-phase cardiprotection by means of a NO and adenosine mediated mechanismrdquo Eur J Pharmacol 465 115-123 (2003)

17 Pauli G F Kuczkowiak U Nahrstedt A ldquoSolvent effects in the structure dereplication of caffeoyl quinic acidsrdquo Magn Reson Chem 37 (11) 827-836 (1999)

18 Simirgiotis M J ldquoAntioxidant capacity and HPLC-DAD-MS profiling of Chilean peumo (Cryptocarya alba) fruits and comparison with German peumo (Crataegus monogyna) from Southern Chilerdquo Molecules 18 (2) 2061-2080 (2013)

19 wwwsigmaaldrichcomspectrafnmrFNMR00112PDF accessed 25 June 2014

20 Liu J-X Di D-L Shi Y-P ldquoDiversity of chemical constituents from Saxifraga Montana Hrdquo J Chinese Chem Soc 55 (4) 863-870 (2008)

21 Moussaoui F Zellagui A Segueni N et al ldquoFlavonoid constituents from Algerian Launaea resedifolia (OK) and their antimicrobial activityrdquo Rec Nat Prod 4 (1) 91-95 (2010)

22 Kyriakou E Primikyri A Charisiadis P et al ldquoUnexpected enzyme-catalyzed regioselective acylation of flavonoid aglyconesrdquo Organic amp Biomolecular Chemistry 10 (9) 1739-1742 (2012)

23 Hye M A Taher M A Ali M Y et al ldquoIsolation of (+)-catechin from Acacia catechu (cutch tree) by a convenient methodrdquo J Sci Res 1 (2) 300-305 (2009)

24 Trakoontivakorn G Nakahara K Shinmoto H et al ldquoStructural analysis of a novel antimutagenic compound 4-hydroxypanduratin A and the antimutagenic activity of flavonoids in a Thai spice fingerroot (Boesenbergia pandurata Schult) against mutagenic heterocyclic aminesrdquo J Agric Food Chem 49 (6) 3046-3050 (2001)

25 Holcapek M Jandera P ldquoInterpretation of electrospray and atmospheric pressure chemical ionization mass spectra of 10-formyl-78-dihydrofolic acid and 5-formyl-5678-tetrahydropteroic acidrdquo Rapid Commun Mass Spectrom 13 (14) 1423-1426 (1999)

ACKNOWLEDGEMENTS

Authors want to thank for the financial support to Ministry of Science Education and Technological Development of Republic of Serbia (Project No TR 34012 174007)

REFERENCES

1 Dimitrijevic D S Kostic D A Stojanovic G S at al ldquoPolyphenol contents and antioxidant activity of five fresh fruit Morus spp (Moraceae) extractsrdquo Agro Food Ind Hi Tech 24(5) 34-37 (2013)

2 Hassimotto N M A Genovese M I Lajolo F M ldquoIdentification and characterisation of anthocyanins from wild mulberry (Morus nigra L) growing in Brazilrdquo Food Sci Technol Int 13 17-25 (2007)

3 Sadiq Butt M Nazir A Tauseef Sultan M et al ldquoMorus alba L naturersquos functional tonicrdquo Trends Food Sci Tech 19 505-512 (2008)

4 Bunea A Rugina D Sconta Z et al ldquoAnthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic propertiesrdquo Phytochemistry 95 436-444 (2013)

5 Nitra N Kornkanok I Wiroje K et al ldquoQuantitative determination of 1-deoxynojirimycin in mulberry leaves using liquid chromatography-tandem mass spectrometryrdquo J Pharm Biomed Anal 44 853-858 (2007)

6 Albishi T John J A Al-Khalifa A S et al ldquoAntioxidant anti-inflammatory and DNA scission inhibitory activities of phenolic compounds in selected onion and potato varietiesrdquo J Funct Foods 5 930-939 (2013)

7 Zhang W Han F Duan C ldquoHPLC-DAD-ESI-MSMS analysis and antioxidant activities of nonanthocyanin phenolics in mulberry (Morus alba L)rdquo J Food Sci 73 (6) C512-C518 (2008)

8 Pawlowska A M Oleszek W Braca A ldquoQuali-quantitative analyses of flavonoids of Morus nigra L and Morus alba L (Moraceae) fruitsrdquo J Agric Food Chem 56 3377-3380 (2008)

9 Du Q Zheng J Xu Y ldquoComposition of anthocyanins in mulberry and their antioxidant activityrdquo J Food Compos Anal 21 390-395 (2008)

10 Ozgen M Serce S Kaya C ldquoPhytochemical and antioxidant properties of anthocyanin-rich Morus nigra and Morus rubra fruitsrdquo Sci Hort 119 275-279 (2009)

11 Lee J H Kang N S Shin S-O et al ldquoCharacterization of anthocyanins in the black soybean (Glycine max L) by HPLC-DAD-ESIMS analysisrdquo Food Chem 112 226-231 (2009)

12 Tonon R V Brabet C Hubinger M D ldquoInfluence of process conditions on the physicochemical properties of acai (Euterpe olraceae Mart)

Industry perspective

NOME COGNOMECorresponding authorindirizzo 1indirizzo 2

SEZIONE


KEYWORDS Food safety awareness level consumer Turkey

AbstractThe objective of this research is to determine the factors affecting the food safety awareness of consumers in the urban areas of Samsun Province in Turkey In this research food safety awareness level was divided

into three classes low medium and high Survey data obtained from 384 consumers was analyzed with the Ordered Probit Model The results indicated that socio-economic variables (gender education and household income) and behavioral variables (consumption of organic food and consumption of low priced genetically modified food) had considerable effects on consumersrsquo awareness of food safety The results also showed that if the education level of consumers has increased the probability of their awareness level would be increased Furthermore it was found that women were more sensitive to food safety issues than men Therefore firms must develop marketing strategies for woman consumers and public institutions should make much more effort to increase the awareness level of man consumers

Factors affecting consumersrsquo awareness of food safety

INTRODUCTION

Human health depends on adequate and balanced nutrition It improves individual cognitive developments and work efficiency as well as increasing average life span by reducing health risks As individuals get mature cognitive development takes place too Consumers gradually develop a sense of balanced consumption and they begin to investigate the effects of various foods on human health (1) In order to feed the continually increasing population in the world two fundamental problems must be solved The first one is food security which means accessing sufficient amount of food by consumers and the other one is food safety representing the reliability of the accessed food in terms of human nutrition and health Although the food security issue has been largely solved by many developed countries it still remains as a fundamental issue for both developing and undeveloped countries especially in many African and Asian countries Almost all countries in the world are faced by factors such as nutrition based diseases genetically modified foods fat contents or other similar factors Food consumption is very important for human life due to supplying their physiological needs Today the safety of consumed food in terms of taste quality and health has become important particularly in countries that have solved more basic food safety problems Attainment of adequate food is the first step towards a sustainable nutrition Healthy and a sustainable life is possible only with the simultaneous

improvement of food security and safety The emergence of diseases originated by food in the recent years such as avian flu has put food safety into question At the same time socio-economic developments have increased consumersrsquo interest in consuming safe foods Sensitive consumers besides the productsrsquo ability to satisfy their needs consider many other features of a product There is an increase of consumersrsquo interest in environmental friendly products The changing consumersrsquo expectations combined with unfavorable health incidence have increased the importance of studies concerning food safetyThere have been studies about consumersrsquo awareness perceptions attitudes and behavior about food safety In a review study by Wilcock et al (2) different attitudes of consumers do not necessarily lead to behaviors that increase the safety of food consumed Another review study (3) states that knowledge attitudes intentions and self-reported practices do not correspond to observed behaviors suggesting that observational studies provide a more realistic indication of the food hygiene actions that are used in domestic food preparation It concludes that food safety education influences the behavior of consumers Field studies indicate that consumers are willing to pay higher prices for certified products (4) organic food (5) and food labeling but the perception on quality may sometimes differ across consumers (6) Some studies were extensively focused on the consumersrsquo behavior on low-carbon agri-food purchase (7) their perception on the health aspect of organic food (8) and

FOOD SAFETY

OSMAN KILIC GAMZE AYDIN ERYILMAZCorresponding AuthorDepartment of Agricultural Economics Faculty of Agriculture Ondokuz Mayis University 55139 Samsun Turkey

Gamze Aydin EryilmazOsman Kilic


The Ordered Probit Model was used to estimate the effect of consumersrsquo socio-economic and behavioral characteristics on their levels of food safety awareness The qualitative and dependent variable that had more than two values were ordered according to their magnitude The Ordered Probit Model was explicitly explained in different literatures (15-19) and one of the recommended consecutive response models of data sets with these types of variables


According to the survey results 64 of the consumers were women Forty-one percent of consumers had less than 9 years of education and 19 of consumers had more than 13 years of education Housewives who earned no income constituted 34 of the consumers surveyed Forty-eight percent of families had more than three individuals Twenty five percent of families had children aged 6 or less Fifty-seven percent of the consumers were aware of at least one certificate relevant to food safety quality or environmental management (ISO 9001 ISO 22000 ISO 14001 HACCP and GLOBALGAP) In the research only 8 of consumers purchased organic foods However 27 of consumers stated that they preferred genetically modified (GM) food if its price is lower than the standard priceThe dependent variable of the model was consumersrsquo food safety awareness level and this variable was classified as low awareness level (0) medium awareness level (1) or high awareness level (2) Socio-economic characteristics of consumers (age and education level household income household size and number of children) and the consumersrsquo consumption behaviours (awareness of at least one certificate about food safety organic food consumption purchasing GM food at low prices) constituted the independent variables of

factors affecting consumersrsquo consumption of packed and unpacked fluid milk (9) Several studies have examined the level of food safety awareness among people in Turkey and in other parts of the world (10-13)The objective of this research was to determine the factors affecting consumersrsquo level of food safety awareness in the urban areas of Samsun province The results of this research not only primarily serve as a guide for consumers but also can be used by people and institutions related to food production storage and transportation The results may also shed light on the policies of public institutions responsible for food inspection


The main material of the research was the questionnaire data collected from consumers in the Atakum Canik and Ilkadim districts of Samsun province The consumers were surveyed in January and February 2011 The sample size was determined by ungrouped one stage random likelihood sampling method based on households (14) using the following formula

Where n is the sample size Zα2 is the significance level (assumed to be 95) p is the probability of the examined situation occurring (p=05 is used to the absence of preliminary information concerning consumersrsquo food safety awareness level) q is the probability of situation not occurring (q=1-p) and d is the margin of error (assumed to be 5) Estimated sample size was calculated as 384 and distributed proportionally according to the population resident in the various districts Consumers were classified into three groups according to their food safety awareness levels To determine their levels consumers were asked twenty questions in order to assess their behaviours concerning examining label information purchasing storing and consuming food To these questions the consumers were limited to five answers (never 1 rarely 2 sometimes 3 generally 4 always 5) and the total number of points was considered to represent the consumerrsquos level of food safety awareness Consumers having a standard deviation point lower and higher than the average number of points were labeled as low and high level awareness respectively Consumers having a standard deviation point between low and high level were regarded as consumers within a medium level awareness At the end of calculations 73 respondents were assigned to the low level 248 respondents to the medium and 63 respondents to the high level of awareness group The consumersrsquo awareness level in this research about food safety has been executed considering all food staffs together

Table 1 Descriptive statistics of explanatory variables


increase in the likelihood of ldquolowrdquo awareness level and a decrease in ldquomediumrdquo and ldquohighrdquo frequently food safety awareness level Both the ldquoCHILDrdquo and ldquoCERTIFICATErdquo variables in the model positively affected consumersrsquo food safety awareness level However these variables were not significant statistically Consumersrsquo organic food consumption coefficient had a significant positive correlation with food safety awareness level Consumersrsquo consumption of organic food decreased the probability of being at low food safety awareness level by 16 while it increased the probability of being at medium and high food safety awareness level by 3 and 13 respectively Based on the statistically significant coefficients consumersrsquo consumption of genetically modified food negatively affected their food safety awareness levels When the consumers consumed these foods the probability of being in low awareness level increased by 6 and the probability of being in medium and high awareness level decreased by 1 and 4 respectively

the model The dependent and independent variables used in the model are defined in Table 1The results of the Ordered Probit Model for variables affecting the consumersrsquo level of food safety awareness are given in Table 2 The threshold (μ1) value in the model was found to be positive and statistically significant at the 001 level This means that the consumer characteristics included in the model significantly affected food safety awareness levelThe marginal effects of consumersrsquo socio-economic and behavioural characteristics on food safety awareness level are given in Table 3 Coefficients in Table 2 and marginal effects in Table 3 were evaluated together in order to pay attention to marginal effects Based on the results coefficients related to consumersrsquo gender were positively correlated and statistically significant (plt001) If a given consumer was a woman the probability of low level of awareness decreased by 7 whereas the possibility of medium and high level of awareness increased by 2 and 6 respectivelyVariables related to the ages of consumers (AGE2 and AGE3) negatively affected consumersrsquo food safety awareness level However ldquoAGE2rdquo and ldquoAGE3rdquo variables were not found to be statistically significant It was also determined that the education levels of consumers positively affected food safety awareness level The variables (EDU2 and EDU3) relating to education level were found to be statistically significant As consumersrsquo education increased probability of food safety awareness level also increased Estimated coefficients in ldquoEDU2rdquo and ldquoEDU3rdquo increased with education level According to the marginal effects while the probability of being in low awareness level decreased for the consumers in level ldquoEDU2rdquo and ldquoEDU3 the probability of being in medium and high awareness levels increased Household income had a statistically positive effect on consumersrsquo food safety awareness level Variables regarding to household income (INC2 INC3 and INC4) gave significant positive correlations with food safety awareness level The positive sign of the variablesrsquo coefficient indicated that when the household income was more than 450 TL per month an increment in the ldquoINC2rdquo ldquoINC3rdquo and ldquoINC4rdquo variables increased consumersrsquo food safety awareness level The probabilities increased by 64 and 112 respectively as the household income category changed from the lowest (INC2) to the highest income (INC4) categoryThe marginal effects of household size ldquoHSIZErdquo indicate an

Table 2 Estimates of the ordered probit model for food safety awareness level

Table 3 The Marginal effects of factors on the probability of relative frequencies for food safety awareness level


and other mass communication tools Finally training facilities in schools may make a significant contribution to create a sensible public opinion for food safety

REFERENCES

1 Topuzoglu A Hidiroglu S Ay P et al ldquoConsumersrsquo Knowledge Related to Food Products and Their Attitudes to Health Risksrdquo Bulletin of TSK Preventive Medicine 6 (4) 253-258 (2007)

2 Willcock A Pun M Khanona J et al ldquoConsumer Attitudes Knowledge and Behavior a Review of Food Safety Issuesrdquo Trends in Food Science and Technology 15 (2) 56-66 (2004)

3 Redmond E C Griffith C J ldquoConsumer Food Handling in the Home Areview of Food Safety Studiesrdquo Journal of Food Protection 66 (1) 130-161 (2003)

4 Wang Z Mao Y Gale F ldquoChinese Consumer Demand for Food Safety Attributes in Milk Productsrdquo Food Policy 33 (1) 27-36 (2008)

5 Krystallis A Chryssohoidis G ldquoConsumersrsquo Willingness to Pay for Organic Food Factors That Affect It and Variation Per Organic Product Typerdquo British Food Journal 107 (5) 320-343 (2005)

6 McCluskey J J Loureiro M L ldquoConsumer Preferences and Willingness to Pay for Food Labeling A Discussion of Empirical Studiesrdquo Journal of Food Distribution Research 34 (3) 95-102 (2003)

7 Chuanmin S Xiaomin Y Yukun Z et al ldquoConsumer Behavior on Low-carbon Agri-food Purchase A Carbon Labelling Experimental Study in Chinardquo Agric Econ- Czech 60 (3) 133-146 (2014)

8 Kutnohorska O Tomsik P ldquoConsumersrsquo Perception of the Health Aspect of Organic Foodrdquo Agric Econ- Czech 59 (7) 293-299 (2013)

9 Kilic O Akbay C Tiryaki G Y ldquoFactors Affecting Packed and Unpacked Fluid Milk Consumptionrdquo Agric Econ- Czech 55 (11) 557-563 (2009)

10 Jevsnik M Hlebec V Raspor P ldquoConsumersrsquo Awareness of Food Safety from Shopping to Eatingrdquo Food Control 19 (8) 737-745 (2008)

11 Uzunoz M Buyukbay O E Bal G S ldquoConscious Levels of Rural Women in the Subject of Food Safety (Case of Tokat Province)rdquo Journal of Agricultural Faculty of Uludag University 22 (2) 35-46 (2008)

12 Gozener B and Buyukbay E O ldquoInvestigating Knowledge Level of Students About The Term of Food Safetyrdquo Gaziosmanpasa University Journal of Agriculture Faculty 26 (2) 45-53 (2009)

13 Sanlier N ldquoThe Knowledge and Practice of Food Safety By Young and Adult Consumersrdquo Food Control 20 (6) 538-542 (2009)

14 Collins M Sampling Consumer Market Research Handbook (1986)

15 Maddala G S Limited-Dependent and Qualitative Variables in Econometrics New York Cambridge University Press (1983)

16 Greene WH Econometric Analysis Prentice-Hall International Inc 1000 p (1997)

17 Chen K Ali M Veeman M et al ldquoRelative Importance Rankings for Pork Attributes by Asian-Origin Consumers in California Applying an Ordered Probit Model to a Choice-Based Samplerdquo Journal of Agricultural and Applied Economics 34 (1) 67-79 (2002)

18 Mutlu S Yurdakul O ldquoInvestigating Consumers Payment Wilingness for Food Safety with Ordered Probit Model (A Case Study on Red Meat Consumption in Urban Areas of Adana)rdquo VII Agricultural Economics Congress Antalya Congress Report Book 632-641 (2006)

19 Akbay C Tiryaki G Gul A ldquoConsumer Characteristics Influencing Fast Food Consumption in Turkeyrdquo Food Control 18 (8) 904-913 (2007)

CONCLUSIONS In recent years the appearance of food-borne diseases affecting human health has made consumers more interested in safe food products This interest has caused researchers to ask which factors might contribute to food safety from the production process to the time of consumption There are various people and institutions serving different roles in regulating food consumption The most active elements among them are the consumers demanding the products For this reason consumersrsquo food safety awareness level must be determined Assessing the factors affecting safe food consumption and presenting consumersrsquo relevant socio-economic factors and behavioral characteristics are of great importance for manufacturers in the food sector The Ordered Probit Model results used in this research had shown that gender education level household income organic food consumption and low-priced GM food consumption had significant effects on consumersrsquo awareness of food safety While the low-priced GM food consumption negatively affected awareness level it had been positively affected by the gender education of consumers household income and organic food consumption In other words consumersrsquo consumption of low-priced GM food decreases their probability of being at the high awareness level while the other variables increase this probability In the research 16 of surveyed consumers were at the high awareness level while the remaining consumers were at the medium or low awareness levels Because of the importance of food safety in terms of human health this percentage must be increased The food safety awareness level of the consumers varied according to gender Woman consumers were more aware of food safety than men Therefore new products developed by food companies and studies concerning existing productsrsquo reliability must be devoted to gaining the confidence of woman The changes in Turkeyrsquos socio-cultural structure in recent years have increased womenrsquos role in purchasing food In addition works targeting the awareness level of men should be increased due to the awareness level of men which is generally lower than that of womanIt was determined that only 8 of consumers consumed organic food When importance of organic food is considered for human health studies concerning organic food consumption should be increased Therefore unit costs should be decreased with increasing organic food production Thus organic foods may be sold to consumers at lower prices On the other side GM foods are of great importance among the subjects remaining on the food safety agenda and also among the much debated questions in terms of health In this research 27 of consumers had a tendency to prefer GM foods if they were cheap Consumers are not yet aware of the adverse effects of GM foods on human health Therefore consumers must be informed about these foods prior to their consumptionSafe food consumption is an individual behavior which is gained in early ages Therefore it is one of the priority tasks of parents to teach their children what kind of foods they must consume However the government must take initiative and play a major role in food safety by providing information for consumers especially through newspaper television internet



SEZIONE


KEYWORDS Date pits fibre functional foods

AbstractPhoenix dactylifera (L) fruit is considered one of the oldest and nutritionally richest fruits on earth Its ever-growing production and consumption yield pits about 10-15 of the total fruit production that threatens its

recycling Several researchers have evaluated its chemical composition and phytochemicals profile that have shown bioactivities in vitro In milieu to that various functional foods and beverages have also been tried This mini review summarizes the potential usefulness of date pit as a viable healthy ingredient in some processed foods

Date pits (Phoenix dactylifera (L)) waste to best

INTRODUCTION AND NUTRITIONAL COMPOSITION

Date stone the core of phoenix dactylifera (L) fruit is the main collateral stream obtained concomitantly with many date products ie date paste date syrup and confectionery etc Normally date stone accounts 10-13 or sometimes even higher of the fruitrsquos total weight on a dry mass basis depending upon the cultivar and cultural conditions The ever-growing production of date fruit till today with nearly 8 million tons in 2010 (1) has been generating a handful of unwanted waste in the form of date stones threatening its recycling even to the developed countries like USA However wise utilization of this waste date-pit might be helpful in date cultivation and finally in income enhancement of this sector Morphologically date pit ranges 20 to 25 cm in length with 6 to 8 mm thickness The stone colour varies from light brown to dark brown based on varietal variations without an obvious flavour and scent but with a slightly bitter sensation Chemically date seed has been evaluated extensively indicating the significant amount of crude oil (90g100g) protein (51g100g) and a major proportion of the dietary fibres approximately 73 (2) these fractions were found comparably higher than fruit flesh Along with a minor fraction of soluble fibres cellulose hemicellulose and lignin make up the main insoluble portion of the seed In case of minerals many of the varieties presented higher concentrations of potassium (05 ) other minerals like calcium iron selenium boron cobalt copper magnesium manganese zinc sodium phosphorus and fluorine were also observed in fair amounts Moreover some cultivar seeds also presented traces of aluminium lead cadmium chloride and sulphur (2 3 4) Fairly large amounts of phenolics and

antioxidants have also been reported with 3942 mg100g as Gallic acid equivalents (GAE) and 80400 micromol100g as trolox equivalent respectively (2) Besbes et al (5) evaluated the total sterols that were approximately 3000 mgkg for various cultivars where β-sitosterol accounted 83 of the total Oestrone one of the naturally occurring oestrogens has also been reported Some water soluble galactomannan (6) and alkali-soluble heteroxylan type of polysaccharide fractions (7) have been isolated and analysed as well However some anti-nutritional factors like tannins and phytates have been reported recently (32)The date seeds yield greenish yellow non-drying moisturizing oil This oil has been credited to contain significant amounts of essential fatty acids main fractions are oleic (4747 ) linoleic (1023 ) lauric (1024 ) palmitic (1241 ) stearic (336 ) and myristic acid (1452 ) along with traces of linolenic (012 ) and arachidic acid (034 ) (8) Table 1 summarizes some recent studies on date seed and oil physico-chemical components evaluated experimentally Date seed oil is believed to protect against UV exposures which has made it suitable for use in cosmetic and soap-like products Occasionally date seed powder and oil have been evaluated as an alternatively cheaper source of many functional ingredients in human diet Formulations similar to coffee jam high fibre bread and seed oil mayonnaise are some valorised food applications of date stones In the meat sector from cattle to fish it has proved an authenticated substrate for feed base although sometimes additional nitrogen sources are admixed exogenously Chemically up to 65 oxalic acid can be obtained by date seed processing The production of activated carbon through chemical or

SUSTAINABILITY

KAISER MAHMOOD MOHAMMED S ALAMRI ABDELLATIF MOHAMED SHAHZAD HUSSAINCorresponding AuthorDepartment of Food Science and Nutrition College of Food and Agricultural Sciences King Saud University PO Box 2460 Riyadh 11451 Saudi Arabia

Kaiser Mahmood


given by the FDA and other health organizations

Date seed extract as preservativeBeing enriched with phenols and antioxidants date seed extract itself has been reported as a preservative for food commodities (22) Amany et al (11) evaluated the seed extract of lsquoKhalasrsquo dates on lipid oxidation and integrity of ground beef during storage The total phenolic contents and antioxidant capacities of the extract were determined through Folin-Ciocalteu reagent and ferric reducing antioxidant power assay (FRAP) For quality assurance of the meat thiobarbituric acid assay (TBA) was conducted at the start of the experiment and also after a storage of 10 days The

stored meat was found to be intact in quality with reduced hydroperoxides concentrations even after one and half week storage at 0degC Oumlzcan and Al-Juhaimi (23) determined the oxidative stability of olive oil at 60degC after adding date seed extract at 05 10 and 15 where butylated hydroxyanisole (BHA) was compared as a control All the tested concentrations indicated better antioxidative characters compared to BHA suggesting the extract a potent oxidation inhibitor against many reactive species

Healthy bread formulationConsidering the high fibre proportions of date pits Almana and Mahmoud (24) firstly attempted addition of coarse and fine powder at 5 10 15 in dough for preparation of high fibre lsquoMafroodrsquo bread - a Saudi flat bread marketed all over the Arabian Peninsula The bread made with date seed powder was found richer in fat but lower in protein while a substantial increase in soluble and total dietary fibres were also noticed A fourfold increase of total dietary fibres was seen in bread containing coarse fractions at 10 addition without any tentative adversity in bread quality The odour colour flavour chewiness and overall acceptability were not much affected Similarly Bouaziz et al (25) prepared defatted date seed fine and coarse fibre concentrates from two cultivars namely lsquoAllig and Deglet Nourrsquo and added in the bread the resulting performance of the final bread was tested after addition at 1 to 3 level The bread volume was observed to be decreased after the addition of the fibre concentrates The bread colour was also changed and the firmness was increased Alternatively reduced specific volume and firmness were observed for coarse particles Overall acceptable bread quality was obtained with a substantially higher fibre dose Similarly another effort of bread making was made using pits of lsquoBehrirsquo cultivar (26)

physical strategies has been extensively utilized in controlling the environmental pollution The date pits would be a worthy by-product of date fruit that could be manipulated as a potent source of bioactive fractions in the human diet

FUNCTIONAL FOODS AND BEVERAGES APPLICATIONS

Besides the use of fresh dates in the production of date syrup date juice date paste and other bakery items date seeds are being used as a functional ingredient in many food recipes Products like coffee and cordial jam mayonnaise and bread have been reported to be formulated by different researchers Higher consumption of dietary fibre delivered through functional foods containing date pits could be claimed for fulfilment of recommended daily intakes (RDI)

Table 1 Some recent studies on date seed chemical composition

Figure 1 Date palm seeds with extracted oil


gums It was claimed that the pudding provides 072 g soluble fibre fractions per serving Finer particles (149- microm sieve) of date pits were added to avoid granular texture of the pudding Yeast was also added in one of the pudding recipes for which the sensory ranking was observed closer to commercial one Other than soluble fibres a significant amount of the insoluble portion was also claimed for this healthy pudding

Date pits as coffee alternativeAbdillah and Andriani (17) introduced a healthier and cheaper alternative drink - a non-caffeinated coffee - with coffee like flavour The roasted date seeds were processed by drying grinding and sieving to get a particular granule size The various types of drink combinations were prepared by the direct mixing of date seed with warm water or ginger water The date powder could directly be boiled in water or milk and honey or sucrose could be used to make it sweet and tasty As a second choice date powder could be used in making lsquoCappuccino Choco Floatrsquo where it is mixed with chocolate milk powder creamer granulated sucrose shaved ice and finally garnished with chocolate ice cream Due to the fair concentrations of minerals and antioxidants this cheaper drink can contribute in satisfying daily recommended dietary requirements Cordial preparation A study was conducted for the preparation of functional cordial drinks from date kernel powder (9) Five different types of drinks with varying concentrations of water sucrose and citric acid were formulated and evaluated for aroma colour taste and aftertaste and also for overall acceptability rating on a 5-point hedonic scale The highest liking (43) was observed for formulation containing 150 ml water with 30 g and 4 g of sucrose and citric acid respectively It was further suggested that process optimization for this cordial at commercial scale might get fame as an appealing and tasty drink However under the umbrella of nanotechnology date seed extract has been encapsulated by whey proteins that are expected to be easily utilized in some beverages and liquid foods with minimal sensorial changes in the product quality in addition to health claims (31)

CONCLUSIONS

In the context of sustainable use of agricultural produce their waste by-products utilization has a cardinal role Being an in expensive and nutritionally rich source of dietary fibres and antioxidants numerous studies explored date-pit powder in various food recipes However to be a functional ingredient of commercial food products it still needs a multivariate evaluation in terms of process optimization Additionally to better estimate the pure effect of each group of functional components they should be tested separately

ACKNOWLEDGEMENT

The authors extend their appreciation to the Deanship of Scientific Research College of Food and Agriculture Sciences King Saud University Saudi Arabia for funding current work

In bread formulation the date seed powder replacement was made at 4 8 and 12 of wheat flour It was further claimed that highest level of replacement could provide 133 g of soluble fibres per serving of 30 g and may help in meeting the FDA requirements The bread loaves with reduced volume higher staling and darker colour were observed The pan bread fortification was done at 5 10 and 15 date pits powder The bread with 15 date pits obtained the highest sensory acceptability compared to control and other substitution levels When the bread was fed to Albino rats with induced hyperglycemia a control on diabetic conditions affirmed its suitability in decreasing the risk of disease (27)

Date seed oil and mayonnaise preparation Along the value addition to agricultural by-products date seed oil has also been considered as an alternative source of vegetable oil and a renewable source of energy Besbes et al (28) found high medium-triglycerides of 32-42 carbon chain making about 27 of lsquoDeglet Nour and Alligrsquo seed oils The main unsaturated fatty acid detected through gas liquid chromatography (GLC) was oleic acid (3917ndash4213 ) while lauric acid was the main saturated fatty acid with 2434 Moreover it was observed that date seed oil can easily be stored till 40 days even at temperature as high as 60degC In another study heating stability of oil from two cultivars namely lsquoDeglet Nour and Alligrsquo was estimated (21) The Rancimat model system was adopted to assess the oxidative stability of oil The melting enthalpies of the oils were estimated through differential scanning calorimetry (DSC) The observed data indicated stability for a longer period of heating (30-40 h) nominating date seed oil as a substantial alternative to other frying and cooking oils and could easily afford refining temperature Other than the direct culinary use of date pit oil for cooking a successful manipulation has been made by mayonnaise formulation (a kind of French sauce) using lsquoKhalasrsquo seed oil egg yolk and vinegar with few optional herbs or spices (12) A superior sensory scoring was shown by date seed oil mayonnaise compared to one prepared from corn oil signifying its product development potentials Furthermore it can also be used in greasing of baking pan preparing vegetable butter and other commodities similar to other vegetable oils

Edible jam preparationLike other high-pectin fruit jam date seed jam was also prepared to consider its functionally important constituents (29) The date-seed powder was supplemented in jam formulation to improve the fibre contents without compromising the product quality taste-wise The main ingredients of the jam formulation were sucrose pectin and gum arabic with citric and ascorbic acid as preservatives and taste enhancers According to the hedonic scale of 4 points the average overall acceptability score was 298 as the product was liked by nearly 60 of panellists

High fibre pudding formulationPudding is usually a dessert and typically it is a milk protein based semisolid food Alamri et al (30) developed a high fibre pudding Its ingredients were date pit powder corn starch sucrose whole milk powder flaxseed and xanthan

49

Conference on Economics Business and Management (ICEBM) Indonesia pp 80-87 (2012)

18 M Akbari R Razavizadeh et al African Journal of Biotechnology 11 pp 12088-12093 (2012)

19 M Biglar M Khanavi Iran J Pharm Res 11 pp 873-878 (2012)20 MS Rahman S Kasapis et al J Food Eng 80 pp1ndash10 (2007)21 S Besbes C Blecker et al Food Chem 91 pp 469ndash476 (2005)22 MES Mirghani MA Mikail et al Date seed extract as

preservative Chapter 16 in Current Research and Development in Biotechnology Engineering (volume III) Edited by MD Alam AT Jameel A Amid IIUM Press Kuala Lumpur pp 139-145 (2011)

23 MM Oumlzcan F Al-Juhaimi J Food Sci Tech Mys pp 1-5 (2013)24 HA Almana RM Mahmoud Ecol Food Nutr 32 pp 261-270

(1994) 25 MA Bouaziz WB Amara et al J Texture Stud 41 pp 511-531

(2010) 26 MS Alamri A Mohamed et al Ital J Food Sci 26 pp 1-11

(2014)27 MS Halaby MH Farag et al Int J Food Sci Nutr 3 pp 49-59

(2014)28 S Besbes C Blecker et al J Food Lipids 11 pp 251ndash265 (2004)29 MES Mirghani A Al-Mamun et al Aust J Basic Appl Sci 6 pp

22-29 (2012)30 MS Alamri A Mohamed et al Eur Food Res Technol 239 pp

755-763 (2014)31 L Bagheri A Madadlou et al Food Res Int 5 pp 866ndash871

(2013)32 EAA Al-Tamim J Am Sci 10 pp 154-159 (2014)


1 ZX Tang Lu-E Shi et al J Sci Food Agr 93 pp 2351-2361 (2013)2 MA Al-Farsi CY Lee Crit Rev Food Sci 48 pp 877minus887 (2008)3 M Al-Farsi C Alasalvar et al J Agr Food Chem 53 pp

7592minus7599 (2005)4 AY Ali Mohamed AS Khamis J Agr Food Chem 52 pp

6522minus6525 (2004)5 S Besbes C Blecker et al Food Sci Technol Int 10 pp 333ndash338

(2004) 6 O Ishrud M Zahid et al Carbohyd Res 335 pp 297-301 (2001)7 O Ishurd Y Ali et al Carbohyd Res 338 pp 1609-1612 (2003)8 HM Habib H Kamal et al Ind Crops Prod 42 pp 567- 572

(2013)9 MES Mirghani Adv Nat Appl Sci 6 pp 575-582 (2012)10 H Najib YM Al-Yousef Int J Poult Sci 11 pp 624-629 (2012)11 MMB Amany MA Shaker et al International Food Research

Journal 19 pp 223-227 (2012)12 AMM Basuny MA Al-Marzooq Food and Nutrition Sciences 2

pp 938-943 (2011)13 MRS Ardekani M Khanavi et al Iran J Pharm Res 9 pp 141-146

(2010)14 EB Saafi M Trigui et al Int J Food Sci Tech 43 pp 2033ndash2037

(2008)15 N Chaira A Ferchichi et al Pakistan journal of Biological

Sciences 10 pp 2202-2207 (2007)16 M Al-Farsi C Alasalvar et al Food Chem 104 pp 943ndash947

(2007) 17 LA Abdillah M Andriani Proceeding of International



A cutting-edge research project comprising research institutes and communication experts across Europe has set out to study the role of health-related claims and symbols in consumer behaviour One of the goals of CLYMBOL is to understand how claims and symbols appear on food and drink products in their context and how this information can help guide consumer behaviour In addition a toolbox will be produced to support guidance in measuring the impact of claims and symbols on understanding purchase and consumption behaviour Findings will be used to design better communication activities and draw implications for policy makers and the European food industry

There are 14 partners working on the CLYMBOL project which is more than half way through its planned four years It is split into six main work areas and receives partial funding from the European Unionrsquos Seventh Framework Programme for research technological development and demonstration

The projectrsquos fi rst work area focusses on the history of health-related claim and symbol use across EU member states their current prevalence on food packaging and in which context the claims and symbols appear Among other things 53 European key representatives from national food authorities representatives of the food industry and consumer organisations were interviewed Results showed that there are vast differences in Europe when it comes to how health claims and symbols have been regulated before 2006 and how their use was monitored The stakeholder groups did express a strong interest in evaluating the impact of health claims and symbols in particular (1) the role of health claims and symbols in consumer behaviour (2) their impact on public health and (3) economic effects Building on these fi ndings researchers have sampled more than 2000 food and drink products in fi ve EU member states and are currently analysing the packaging for product- and health-related information They are reviewing which claims and symbols are found how they are classifi ed and the nutritional composition of foods carrying those claims and symbols

Consumer needs and wants with regards to health claims and symbols are important to identify in CLYMBOL In a second work area of the project the researchers are looking at consumer models of health (eg their beliefs and inferences related to specifi c health outcomes) and how they use these models to interpret nutrition and health claims Furthermore an online study of European

consumers in 10 countries was undertaken to assess how motivated and able food shoppers were in processing health claims and symbols on food products and whether there are country-specifi c or segment-specifi c differences such as social demographics In general European consumersrsquo motivation and ability to process health claims differed little between claims and symbols How motivated people were to process health-related claims depended strongly on their lsquoneed for informationrsquo while lsquosubjective knowledge about the healthiness of foodrsquo correlated strongly with how able people were to process claims

A methodological toolbox will be developed as part of the third work area to enable researchers and other stakeholders such as public authorities industry and consumer organisations to measure the impact of health claims and symbols on consumer understanding purchase and consumption behaviour The toolbox will cover a range of tested and validated methods explaining how to apply each technique which research questions are most suitable and how to undertake the analysis and interpretation The toolbox will be made

publicly available at the end of the project

In work area four a wide range of European studies will be undertaken to empirically investigate the effects of health claims and symbols on consumer understanding purchase and consumption behaviour This will take the form of in-store and experimental studies in selected supermarkets Methods that will be applied include eye-tracking of consumers household panel data surveys and actual food and nutrient intake during consumption This work will be completed by early 2016

The implications of fi ndings from work areas one to four for different stakeholders (consumers industry retailers non-governmental organisations policy makers and others) including actionable recommendations for communication and education around health claims and symbols will be analysed in the fi fth work package Furthermore researchers will study the effect on consumer awareness perception and understanding of a social media-based health logo campaign which ran in the Netherlands in 2014

The project also has a separate work area which focusses on the dissemination and communication of CLYMBOL All project information can be accessed through the project website at wwwclymboleu wwweufi corg

The CLYMBOL project how health-related claims and symbols impact consumer behaviour

NAROGENreg ENERGY COLLAGEN MASKNarogenreg Energy Collagen Mask is a natural water-soluble extract which moisturizes rejuvenates and renews your skin cells Because Narogenreg Energy Collagen Mask is rich in amino acids minerals and antioxidants it plays an important role in metabolism Therefore Narogenreg Energy Collagen Mask facilitates the production of collagen and the function of repairing damaged skin cells By sloughing off dead skin cells and impurities your skin tone becomes lighten and fi rm Collagen and elastin are natural skin moisturizer which can absorb up to thirty times of water therefore they can improve the moisture of your skin help skin cell repair and smoothen your skin Apply Narogenreg Energy Collagen Mask regularly to help prevent wrinkleswwwfebicocom

OPENING OF CHOCOLATE ACADEMYtrade CENTER IN MOSCOWThe Barry Callebaut Group has announced the opening of its new state-of ndashthe-art CHOCOLATE ACADEMYtrade centre in Russiarsquos capital Moscow In 2008 Barry Callebaut opened its fi rst training centre for chocolate professionals on the premises of its chocolate factory in Chekhov 60 km south of Moscow Relocated now to the city centre of Moscow the new CHOCOLATE ACADEMYtrade will remain a unique training centre in Russia It is the only such centre to offer a diverse range of seminars demonstrations theoretical courses and practical workshops designed to inspire artisans and culinary professionals including chocolatiers pastry chefs bakery and other chocolate experts as well as culinary professionals working in hotels restaurants and the catering business The centre will also serve as a meeting place for chocolate professionals encouraging the exchange of technical expertise among each other The new CHOCOLATE ACADEMYtrade centre will showcase the knowledge accumulated over more than 150 years by Barry Callebautrsquos global Gourmet chocolate brands Callebautreg (Belgium artisanal chocolate) Cacao Barryreg (French artisanal chocolate) and Carmareg (Swiss artisanal chocolate) The well-known French chef Wielfried Hauwell will share his experience in the art of chocolate and act as the Head of the new CHOCOLATE ACADEMYtrade center in Moscow ldquoWith our relocated modernized training centre we aim to empower all chocolate professionals in the region to create and express themselves with all the chocolates we are offering ndash way beyond the technical barriers of this wonderful working materialrdquo comments Hauwell ldquoChefs and confectioners hardly fi nd an occasion or a central place in Russia where they can further develop their skills learn from experienced expert teachers and exchange new ideas among each other Our new training centre for chocolate now centrally located in Russiarsquos capital Moscow will serve as such a spot It is a unique place where we are cultivating an atmosphere of curiosity creativity and inspiration for both professionals and chocolate loversrdquo he addswwwbarry-callebautcom

On March 20 2015 Villa San Martino in Barasso (Italy) hosted the fi rst edition of NUTRA DAY a 360ordm seminar on nutraceuticals organized by Cum Grano Salis LtdThe different topics of high scientifi c relevance were articulated in several speeches on generic key nutraceutical issuesThe scientifi c curator and moderator of the day Prof Martina of Pavia University made an opening recommendation to be rigorous in suggesting nutraceuticals and then works started with the analysis of ldquoThe Pharmacy as a marketrdquo by Dr Brambilla of IMS Health in which the OTC market in pharmacies emerged as in constant expansion and supporting fi gures showed nutraceuticals are a signifi cant chunk of the Italian OTC pieThe very actual subject ldquoNutraceuticals and self-care Medical Devices formulation and legal product innovationrsquos opportunitiesrdquo saw Dr Di Fulvio explaining that ECJ (European Court of Justice) has already recognised that the capacity to bring back to functional levels fi x and modify physiological functions is not exclusive to medicines and that dose-effect relationship characterisation and safety are fundamentals to identify new opportunities also when it comes to Nutraceutical Medical DevicesDr Mogna from Probiotical with ldquoInnovative technologies to support probiotics effectivenessrdquo analysed probiotics considering their fundamental role within the nutraceutical world both for practices and pharmacies and he underlined how modern selection criteria of probiotics go hand-in-hand with new needs

such as biocompatibilityProf Eandi from AIFA in ldquoEconomic-health value of nutraceuticals for patients national health system and Italian societyrdquo spoke of nutraceuticals under the different perspective of ldquonutri-economyrdquo based on experiences of pharma-economy studies and having developed the concept of ldquocomplex systemsrdquo He explained how scientifi c evidence on the role of nutrients within diseasesrsquo prevention and progression has pushed health decision makers to promote the importance of natural supplements as a health tool Prof Daglia of Pavia University spoke about ldquoNutraceutical active products present situation evolution and developmentsrdquo on the long awaited evolution of nutraceutical supplements highlighting how claims must be based on widely accepted scientifi c evidence understandable by the average consumer Dr Benatti from Sigma-Tau showed us the steps of a nutraceutical supplement from the inception till its market launch in ldquoDeveloping the ideal nutraceuticalrdquoDr Francolini from Filarete Foundation took us into the hi-tech research world applied to nutraceuticals with ldquoThe role of in vitro models in screening and creating new nutraceuticalsrdquo which taught us how in vitro tests open new screening and evaluation opportunities of nutraceuticals

The next edition of NUTRA DAY is set to take place in Milan Italy during March 2016wwwcgseucom

NUTRA DAY 2015


52 Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015

Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015 53Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015

NEWS NEWS NEWS NEWS NEWS NEWS NEWS

Chocolate has many health benefi ts mdash it can potentially lower blood pressure and cholesterol and reduce stroke risk But just as connoisseurs thought it couldnrsquot get any better therersquos this tasty new tidbit Researchers have found a way to make the treat even more nutritious ndashndash and sweeterOn March 24th 2015 they described their research at the 249th National Meeting amp Exposition of the American Chemical Society (ACS) the worldrsquos largest scientifi c society The meeting featured nearly 11000 reports on new advances in science and other topics Cocoa undergoes several steps before it takes shape as a candy bar Workers cut down pods from cocoa trees then split open the pods to remove the white or purple cocoa beans They are fermented in banana-lined baskets for a few days and then set out to dry in the sun Roasting the next step brings out the fl avor But some of the healthful polyphenols (antioxidants) are lost during the roasting process so the researchers wanted to fi gure out a way to retain as much of the polyphenols and good fl avors as possibleldquoWe decided to add a pod-storage step before the beans were even fermented to see whether that would have an effect on the polyphenol contentrdquo said Emmanuel Ohene Afoakwa PhD who is at the University of Ghana ldquoThis is not traditionally done and this is what makes our research fundamentally different Itrsquos also not known how roasting affects polyphenol contentrdquoAfoakwarsquos team divided 300 pods into four groups that were either not stored at all or stored for three seven or 10 days before processing This technique is called ldquopulp preconditioningrdquo After each storage period passed fermentation and drying were done as usual He reported that the seven-day storage resulted in the highest antioxidant activity after roasting

To assess the effects of roasting the researchers took samples from each of the storage groups and roasted them at the same temperature for different times The current process is to roast the beans for 10-20 minutes at 248-266 degrees Fahrenheit he explains Afoakwarsquos team adjusted this to 45 minutes at 242 degrees Fahrenheit and discovered that this slower roasting at a lower temperature increased the antioxidant activity compared to beans roasted with the conventional method In addition the beans that were stored and then roasted for 45 minutes had more polyphenols and higher antioxidant activity than beans whose pods were not stored prior to fermentation said Afoakwa He explained that pulp preconditioning likely allowed the sweet pulp surrounding the beans inside the pod to alter the biochemical and physical constituents of the beans before the fermentation ldquoThis aided the fermentation processes and enhanced antioxidant capacity of the beans as well as the flavorrdquo he said He added that the new technique would be particularly useful for countries in Southeast Asia and Latin America where cocoa beans produce a chocolate with a less intense chocolate flavor and have reduced antioxidant activityLooking to the future he said the team will be studying in more detail the effects of roasting on the flavor of freshly picked compared to stored cocoa beans They will be testing different temperatures and roasting and storing times to determine if even higher amounts of antioxidants can be retained through the processThe researchers acknowledge funding from the Belgium Government under the VLIR TEAM Cocoa Project between Ghent University Ghent Belgium and the University of Ghana Accra Ghanawwwacsorg

More flavorful healthful chocolate could be on its way

NEW NOVOZYMESrsquo ENZYME SAVES CASH FOR MALTOSE SYRUP PRODUCERSNovozymes has announced the launch of a new enzyme targeting the production of maltose syrups The new product known as Secura helps to optimize operations and reduce processing costs The starch industry is one of the longest-standing markets for enzymes and produces a wide range of sweeteners and ingredients used within the food industry Secura is a thermostable low-pH beta amylase enzyme for maltose syrup production Secura has a higher product activity than plant-sourced beta amylase products ndash and this activity level remains stable during storage This stability results in simpler more consistent dosing and processing that does not need constant monitoring As Secura is microbial-based it is Chometz-free Kosher and Halal-certifi ed which is becoming increasingly important in the sweetener market ldquoThe fact that Secura tolerates higher temperatures offers an important benefi t to starch producersrdquo says Frederik Mejlby Marketing Director for Novozymes Grain Processing ldquoMaltose syrups

are typically produced at lower temperatures 55deg-60deg C and have a relatively low osmotic pressure This means that there is a risk that unwanted microorganisms can grow during saccharifi cation Keeping saccharifi cation at higher temperature minimizes the risk of bacterial infections - and Secura works well at temperatures as high as 70degCrdquowwwnovozymescom

NEWS


Broccoli sprout extract protects against oral cancer in mice and proved tolerable in a small group of healthy human volunteers the University of Pittsburgh Cancer Institute (UPCI) partner with UPMC Cancer Center has announced at the American Association for Cancer Research (AACR) Annual Meeting in Philadelphia The promising results will be further explored in a human clinical trial which will recruit participants at high risk for head and neck cancer recurrence later this year This research is funded through Pitts Specialized Program of Research Excellence grant in head and neck cancer from the National Cancer Institute People who are cured of head and neck cancer are still at very high risk for a second cancer in their mouth or throat and unfortunately these second cancers are commonly fatal said lead author Julie Bauman MD MPH co-director of the UPMC Head and Neck Cancer Center of Excellence So were developing a safe natural molecule found in cruciferous vegetables to protect the oral lining where these cancers formPrevious studies including large-scale trials in China have shown that cruciferous vegetables that have a high concentration of sulforaphane - such as broccoli cabbage and garden cress - help mitigate the effects of environmental carcinogens Dr Bauman collaborated with Daniel E Johnson PhD professor of medicine at Pitt and a senior scientist in the UPCI Head and Neck Cancer Program to test sulforaphane in the laboratory For several months Dr Johnson and his team

gave sulforaphane to mice predisposed to oral cancer and found that it signifi cantly reduced the incidence and number of tumors The clear benefi t of sulforaphane in preventing oral cancer in mice raises hope that this well-tolerated compound also may act to prevent oral cancer in humans who face chronic exposure to environmental pollutants and carcinogens said Dr Johnson Dr Bauman treated 10 healthy volunteers with fruit juice mixed with sulforaphane-rich broccoli sprout extract The volunteers had no ill-effects from the extract and protective changes were detectable in the lining of their mouths meaning it was absorbed and directed to at-risk tissue These fi ndings were enough to prompt a clinical trial that will recruit 40 volunteers who have been curatively treated for head and neck cancer The participants will regularly take capsules containing broccoli seed powder to determine if they can tolerate the regimen and whether it has enough of an impact on their oral lining to prevent cancer From there larger clinical trials could be warrantedWe call this green chemoprevention where simple seed preparations or plant extracts are used to prevent disease said Dr Bauman also an associate professor in Pitts School of Medicine Green chemoprevention requires less money and fewer resources than a traditional pharmaceutical study and could be more easily disseminated in developing countries where head and neck cancer is a signifi cant problemwwwupmccommedia

Broccoli sprout extract promising for head and neck cancer prevention

SPORTS NUTRITION FOOTBALLERS CAN BENEFIT FROM CURCUMINIndenarsquos involvement in the growing sports nutrition market is confi rmed by the companyrsquos participation in the Football Medicine Strategies for Player Care Conference The Queen Elizabeth II Conference Centre on April 11th and 12th For over two decades this conference has been the meeting point of the international Football Medicine Community On this occasion Indena presented a study on Merivareg its proprietary lecithin-based and diet inspired delivery system of curcumin confi rming that its specifi c curcumin formulation may aid in sports nutrition by attenuating Delayed Onset Muscle Soreness (DOMS) due to eccentric muscle activitywwwindenacom

OMNIACTIVE PRESENTS POSTERS ON THE EFFECTS AND SAFETY OF HIGHLY SOLUBLE CURCUMINVijaya Juturu PhD FACN Manager of Global Scientifi c and Clinical Affairs with OmniActive Health Technologies presented two posters on emerging science surrounding highly soluble curcumin (CurcuWINtrade) during this yearrsquos Experimental Biology (EB) meeting March 28th - April 1st at the Boston Convention and Exhibit Centre Boston Massachusetts (US) The curcumin posters were based on in vitro and in vivo model studies conducted by OmniActive in collaboration with researchers from Firat University (Turkey) The fi rst study-Curcumin (CurcuWIN) Improves Antioxidant Capacity and Reduces Infl ammation Following Downhill Running-Induced Muscle Damage-investigated the effects of curcumin supplementation on changes in serum parameters and antioxidant status of muscle in rats after exhaustive exercise on a motor-driven rodent treadmill A signifi cant decrease in LDL-cholesterol and triglycerides was also observed in CurcuWIN treated groups These results suggest CurcuWIN is a potential ingredient for preventing muscle damage as well as improving exercise performance and increase in time of exhaustion wwwomniactivescom

NEWS

Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 201554 Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015

NEWS NEWS NEWS NEWS NEWS NEWS NEWS NEWSNEWSNEWSNEWS NEWS NEWS NEWS NEWS NEWS NEWS NEWS NEWS NEWS NEWS NEWS NEWS


drugs than is possible with current cocoa-butter-based systemsCollaborating on the research were Siela Maximova senior scientist and professor of horticulture and Yufan Zhang who received a doctoral degree in plant biology in January of 2015In a previous study a stearoyl-acyl carrier protein desaturase (SAD) gene family was fi rst described in the chocolate tree by Guiltinans laboratory and collaborators from the International Cocoa Genome Consortium which sequenced the entire genome of the plantIn this follow-up research described online in Frontiers in Plant Science (Plant Genetics and Genomics Section) the SAD gene family is examined in detail and the expression patterns of each SAD gene in various cacao tissues is explored along with functional analysis to study how the enzyme worksResearchers discovered that a single gene -- TcSAD1 -- is primarily involved in the synthesis of cocoa butter and is responsible for its melting pointWe used state-of-the-art plant science techniques to gain evidence for the role of the SAD1 gene in cocoa butter biosynthesis Guiltinan said The other SAD genes appear to play other roles in the growth of the chocolate tree such as fl ower and leaf development where these fatty acids play important roles as key components of various membrane systems This information can be used to develop biomarkers for screening and breeding of new cacao varieties with novel fatty acid compositions of cocoa butterNotably cocoa butter consists of almost equal amounts of palmitic acid stearic acid and oleic acid Its exact composition determines its melting temperature which is very close to human body temperature thus providing the smoothness and feel of chocolate in the mouth as well as the creamy texture of cosmetics on skinDuring cacao seed development large amounts of fatty acids are synthesized hat are rich in saturated and monounsaturated lipids Guiltinan said An important part of this process is the activity of an enzyme produced by the SAD1 gene that creates a special double bond critical in determining the melting point of chocolate that is very close to the human body temperature and makes cocoa butter quite uniqueThe research was supported by Penn State the Universitys College of Agricultural Sciences The Huck Institutes of Life Sciences and the American Research Institute Penn State Endowed Program in the Molecular Biology of CacaoPenn States College of Agricultural Sciences

The discovery of a gene involved in determining the melting point of cocoa butter -- a critical attribute of the substance widely used in foods and pharmaceuticals -- will likely lead to new and improved products according to researchers in Penn States College of Agricultural SciencesThe fi nding by plant geneticists also should lead to new varieties of the cocoa plant that could extend the climate and soil-nutrient range for growing the crop and increase the value of its yield they said providing a boost to farmers incomes in the cocoa-growing regions of the worldCacao Theobroma cacao L is an understory tropical tree domesticated in the Amazon basin and today widely cultivated in West Africa Central and South America and Southeast Asia Around the world more than fi ve million cocoa farmers -- and more than 40 million people total -- depend on cocoa for their livelihood according to the World Cocoa Foundation which puts annual cocoa production worldwide at 38 million tons valued at $118 billionCacao pods each containing around 40 seeds are harvested approximately 20 weeks after pollination The seeds contain about 50 percent total lipids (cocoa butter) which provides a main raw ingredient for chocolate manufacturing as well as ingredients for pharmaceutical and cosmetic productsCocoa butter with altered melting points may fi nd new uses in specialty chocolates cosmetics and pharmaceuticals said lead researcher Mark Guiltinan professor of plant molecular biology who has been conducting research on the cacao tree for three decades For example a chocolate with a higher or lower melting point would be useful for production of chocolate with specifi c textures and specialty applicationsCacao seeds after harvest A mixture of lipids called cocoa butter makes up about half of each seed The natural melting point of cocoa butter is close to human body temperature This trait gives chocolate its melt-in-your-mouth texture and provides a creamy texture to lotions applied to the skinThe snap and melt of chocolate are two very important textural features that determine the appeal of chocolate to consumers and having new varieties of the cocoa plant that produce butter with different melting points would be a valuable resource to control those characteristics Guiltinan explained Medical applications could include production of drug-delivery products with slower release of

Discovery of gene that determines cocoa butter melting point to have far-reaching effects

IMCD AND DSM ANNOUNCE EXTENSION OF FOOD ENZYME PARTNERSHIP IN EUROPEIMCD NV and DSM have announced they have further expanded their successful partnership for food enzymes The new multi-territory cooperation includes the Benelux France Germany Italy Poland South Africa SEE Nordics Turkey and UK amp Ireland and represents the conclusion of successful business transitions in all territories The main food enzyme market sectors include bakery brewing confectionery dairy and fruit amp vegetable processing Gokhan Oran IMCD Business Group Director Food amp Nutrition comments IMCD is delighted to strengthen its partnership with DSM in Europe As a leading supplier in the food enzymes market the DSM range will further enhance our product and technical offering to our customers We hope to build on our successes to date and extend this cooperation with DSM to include more regions and to expand into other areas of the business in the future wwwimcdgroupcom



RAPID AND EFFICIENT DNA CHIP TECHNOLOGY FOR TESTING 14 MAJOR TYPES OF FOOD BORNE PATHOGENSToshiba Ltd and Kawasaki City Institute for Public Health collaborate in the development of a rapid and effi cient DNA chip technology for testing 14 major types of food borne pathogens for applications in hygiene management in food manufacture pharmaceuticals and cosmetics The researchers are affi liated with the Kawasaki INnovation Gateway at SKYFRONT Japan Conventional methods for testing food-borne pathogens is based on the cultivation of pathogens a process that is complicated and time consuming So there is demand for alternative methods to test for food-borne pathogens that are simpler quick and applicable to a wide range of potential applications Now Toshiba Ltd and Kawasaki City Institute for Public Health have collaborated in the development of a rapid and effi cient automatic abbreviated DNA detection technology that can test for 14 major types of food borne pathogens The so called lsquoDNA chip cardrsquo employs electrochemical DNA chips and overcomes the complicated procedures associated with genetic testing of conventional methods The lsquoDNA chip cardrsquo is expected to fi nd applications in hygiene management in food manufacture pharmaceuticals and cosmeticswwwanalytica-worldcom

TATE amp LYLE EXPANDS KRYSTARreg CRYSTALLINE FRUCTOSE PRODUCTION AT FACILITY IN INDIANA USTate amp Lyle announces plans for a further expansion of its operations in Lafayette Indiana US investing $65 million The expansion is expected to be operational by late 2016 Tate amp Lyle will install new equipment at its Lafayette South facility signifi cantly expanding production of its KRYSTARreg Crystalline Fructose sweetener to meet growing demand in the Americas and Asia In addition to the expansion Tate amp Lyle will also phase in environmental effi ciencies as part of the investment This announcement marks Tate amp Lylersquos second Indiana expansion within the last year following an investment of more than $90 million at our Lafayette Sagamore facility for a number of projects including an increase of its specialty food starch production capacity ldquoThis expansion not only allows us to stay ahead of the global demand for crystalline fructose but also gives us the opportunity and fl exibility to continue to grow within the North American and emerging marketsrdquo said Joan Braca President of Specialty Food Ingredients at Tate amp Lyle ldquoWersquod like to thank both the state of Indiana and the city of Lafayette for their ongoing support of projects like these and look forward to our continued relationship with themrdquowwwtateandlylecom

EVENTS EVENTS EVENTS EVENTS EVENTSEVENTS

Vitafoods Asia continues to grow as Asiarsquos only dedicated event for professionals involved in the nutraceutical functional food and beverages and dietary supplements industries Entering its fi fth year it is taking place on 2-3 September 2015 at AsiaWorld-Expo Hong KongThe event delivers over 170 international exhibiting companies alongside the most focused audience in the region with over 3000 visitors from across Asia-Pacifi c who attend to discover the latest trends and innovations meet new and existing suppliers and place orders Vitafoods Asia provides exhibitors with access to the key buyers and distributors in the expanding and lucrative Asian nutraceutical industryVitafoods Asia continues to demonstrate why it is the most important meeting place in Asia for industry professionals with the addition of new features an expanded educational programme and exhibitors from across the globe Confi rmed exhibitors include Arnet Pharmaceutical Aroma New Zealand Axialys Innovations Biosearch Life Chr Hansen K-W Pfannenschmidt Novastell Pharmanager Group Pharmatech Asia Group Probiotical Healthcare and Vidya Herbs amongst many othersThe New Products Zone allows visitors to fi nd the latest functional food and ingredients on the market The Finished Products Pavilion includes the Tasting Bar where visitors can see touch taste and sample the latest functional food and beverages Free-to-attend visitor attractions include the Exhibitor Presentation Theatre offering a programme of presentations and seminars by leading industry professionals as well as the Market Insights Hub which offers short presentations by given by specialists The Vitafoods Asia Conference which launched in 2014 takes place alongside the exhibition and features the most relevant topics across the research product development trends and regulation in the nutraceutical and functional food industries in Asia Find out more and register for free at wwwvitafoodsasiacom


Agro FOOD Industry Hi Techvol 26(3) MayJune 2015

Agro FOOD Industry Hi Tech Agro FOOD Industry Hi Tech Agro FOOD Industry Hi Tech - vol 26(3) - MayJune 2015- vol 26(3) - MayJune 2015- vol 26(3) - MayJune 2015

TEKNO SCIENZE SRLViale Brianza 22 20127 Milano (Italy) Tel +39-02-2680937528381260Fax +39-02-2847226e-mail infoteknoscienzecomweb wwwteknoscienzecom

Editor in Chief Carla Scesa - infoteknoscienzecomEditorial Director Silvana Maini - silvanateknoscienzecomAssociate EditorsGayle De Maria - gayleteknoscienzecomFlorian Weighardt - florianteknoscienzecomMarketing amp SalesGiulio Fezzardini - giulioteknoscienzecomSilvia Baldina - silviateknoscienzecomSimona Rivarollo - simonateknoscienzecomMarketing amp EventsPaola Passadore - paolateknoscienzecomProduction ManagerElisa Novaresi - elisateknoscienzecomProduction Assistants amp IT departmentLuis A Alburqueque Diaz - luisteknoscienzecom Matteo Cattane - matteoteknoscienzecomAdministrative amp Account Alba Aprea - albateknoscienzecom

PRINTING OFFICEArti Grafiche Colombo (Muggiograve - MI Italia)Authorization from Tribunale di Milano n 514 of July 27th 2006 (Autorizzazione del Tribunale di Milano n 514 del 27 Luglio 2006)

SCIENTIFIC ADVISORY BOARDAnton Alldrick (CCFRA)Sybille Buchwald-Werner (Vital Solutions GmbH)Undurti N Das (UND Life Sciences)Inga Koehler (Pfizer Consumer Health Care)Ray A Matulka (Burdock Group)Vicente Micol (University Miguel Hernaacutendez) Arthur Ouwehand (University of Turku)Demosthenes B Panagiotakos (Harokopio University)Denis Poncelet (ONIRIS and Capsulae sarl)Igor Pravst (Nutrition Institute ndash Slovenia)Joe Romano (Waters)Yang Zhu (TNO Quality of Life)Ralf Zink (DMK - Deutsches Milchkontor)

All rights reserved to Tekno Scienze No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means electronic mechanical photocopy-ing recording or otherwise without the prior per-mission of the publisher Tekno Scienze Srl

Associated to USPI - Unione della Stampa Periodica Italiana

CONTENTS


FROM THE EDITORIAL BOARD2 Expo 2015 feeding the planet energy for life A necessity a priority

S Maini

SENIOR NUTRITION4 Benefits of a Mangifera indica L fruit powder for well-aging

In vitro investigation of effects on master regulators for the aging processS Roumlchter C Ehrhardt S Buchwald-Werner K Berger-Buumlter E Schatton

JOINT HEALTH8 Mangosteen extract for short-term pain management Preclinical approach and pilot clinical investigation on volunteers with soft

tissue pain C Romain J Cases PRODUCT FOCUS14 NEMreg brand Eggshell Membrane - The Natural Choice and Fast

Acting Solution for Joint Health wwwstratumnutritioncomEU

CHOCOLATE16 Rise in ldquohealthierrdquo ldquobetter for yourdquo chocolate M Naprta

FUNCTIONAL FOOD20 Complex foods versus functional foods nutraceuticals and dietary

supplements differential health impact (Part 2) A Fardet FOOD PROCESSING24 The utilization of solid carbon dioxide in the extraction

of extra-virgin olive oil VOOEVOO yield and quality as a function of extraction conditions

adopted A Zinnai F Venturi C Sanmartin I Taglieri G Andrich 28 Optimization of ultrasonic assisted extraction of antioxidants

and phenolic compounds from red basil using response surface methodology

S Ahmadi R A Ghavidel Z Sheikholeslami M Elahi S Elahi

FOOD ANALYSIS34 1H-NMR spectra ndash a highly sensitive tool in the analysis of Grana

Parmigiano-Reggiano type hard cheese M Vezzani F Bellesia L Caglioti A Pinetti C Zucchi G Paacutelyi 38 Flavonoids in mulberry fruit Identification of nonanthocyanin phenolics in some mulberry fruit

species (Morus alba L Morus rubra L and Morus nigra L) V Miljkovic L Nikolic N Radulovic B Arsic G Nikolic D Kostic Z

Bojanic J Zvezdanovic

FOOD SAFETY43 Factors affecting consumersrsquo awareness of food safety O Kilic G A Eryilmaz

SUSTAINABILITY47 Date pits (Phoenix dactylifera (L)) waste to best

K Mahmood M S Alamri A Mohamed S Hussain

51 NEWS

56 EVENTS

International Journal of

Nutraceuticals Functional Foods and Healthy Ingredients


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Agro FOOD Industry Hi Tech is a peer-reviewed journal devoted to functional food and nutraceuticals addressed to a readership belonging to the industry The 2013 impact factor is 0294 according to the Thomson Reuters Journal Citation Reports

2






Silvana Maini







INTRODUCTION





SENIOR NUTRITION




Sigrid Rochter






RESULTS











DISCUSSION









REFERENCES
























INTRODUCTION



JOINT HEALTH



Cindy Romain























RESULTS


DISCUSSION





























































43204 Reus SPAIN







MARKET OVERVIEW






CHOCOLATE



Mia Naprta













































FUNCTIONAL FOOD




Anthony Fardet














REFERENCES
































INTRODUCTION



FOOD PROCESSING



Angela Zinnai















REFERENCES



















CONCLUSIONS




∆






INTRODUCTION



FOOD PROCESSING






Ghavidel





























CONCLUSION


REFERENCES




































USATel +1 913 953 8928

whitneyblackulcom










INTRODUCTION



FOOD ANALYSIS





Claudia Zucchi















ACKNOWLEDGMENT












CONCLUSIONS






























INTRODUCTION



EXPERIMENTAL


FOOD ANALYSIS




















CONCLUSION


















ACKNOWLEDGEMENTS


REFERENCES















SEZIONE






INTRODUCTION



FOOD SAFETY



















REFERENCES























SEZIONE









SUSTAINABILITY


Kaiser Mahmood














CONCLUSIONS


ACKNOWLEDGEMENT






49











































NUTRA DAY 2015










NEWS







NEWS














2






Silvana Maini







INTRODUCTION





SENIOR NUTRITION




Sigrid Rochter






RESULTS











DISCUSSION









REFERENCES
























INTRODUCTION



JOINT HEALTH



Cindy Romain























RESULTS


DISCUSSION





























































43204 Reus SPAIN







MARKET OVERVIEW






CHOCOLATE



Mia Naprta













































FUNCTIONAL FOOD




Anthony Fardet














REFERENCES
































INTRODUCTION



FOOD PROCESSING



Angela Zinnai















REFERENCES



















CONCLUSIONS




∆






INTRODUCTION



FOOD PROCESSING






Ghavidel





























CONCLUSION


REFERENCES




































USATel +1 913 953 8928

whitneyblackulcom










INTRODUCTION



FOOD ANALYSIS





Claudia Zucchi















ACKNOWLEDGMENT












CONCLUSIONS






























INTRODUCTION



EXPERIMENTAL


FOOD ANALYSIS




















CONCLUSION


















ACKNOWLEDGEMENTS


REFERENCES















SEZIONE






INTRODUCTION



FOOD SAFETY



















REFERENCES























SEZIONE









SUSTAINABILITY


Kaiser Mahmood














CONCLUSIONS


ACKNOWLEDGEMENT






49











































NUTRA DAY 2015










NEWS







NEWS



















INTRODUCTION





SENIOR NUTRITION




Sigrid Rochter






RESULTS











DISCUSSION









REFERENCES
























INTRODUCTION



JOINT HEALTH



Cindy Romain























RESULTS


DISCUSSION





























































43204 Reus SPAIN







MARKET OVERVIEW






CHOCOLATE



Mia Naprta













































FUNCTIONAL FOOD




Anthony Fardet














REFERENCES
































INTRODUCTION



FOOD PROCESSING



Angela Zinnai















REFERENCES



















CONCLUSIONS




∆






INTRODUCTION



FOOD PROCESSING






Ghavidel





























CONCLUSION


REFERENCES




































USATel +1 913 953 8928

whitneyblackulcom










INTRODUCTION



FOOD ANALYSIS





Claudia Zucchi















ACKNOWLEDGMENT












CONCLUSIONS






























INTRODUCTION



EXPERIMENTAL


FOOD ANALYSIS




















CONCLUSION


















ACKNOWLEDGEMENTS


REFERENCES















SEZIONE






INTRODUCTION



FOOD SAFETY



















REFERENCES























SEZIONE









SUSTAINABILITY


Kaiser Mahmood














CONCLUSIONS


ACKNOWLEDGEMENT






49











































NUTRA DAY 2015










NEWS







NEWS



















RESULTS











DISCUSSION









REFERENCES
























INTRODUCTION



JOINT HEALTH



Cindy Romain























RESULTS


DISCUSSION





























































43204 Reus SPAIN







MARKET OVERVIEW






CHOCOLATE



Mia Naprta













































FUNCTIONAL FOOD




Anthony Fardet














REFERENCES
































INTRODUCTION



FOOD PROCESSING



Angela Zinnai















REFERENCES



















CONCLUSIONS




∆






INTRODUCTION



FOOD PROCESSING






Ghavidel





























CONCLUSION


REFERENCES




































USATel +1 913 953 8928

whitneyblackulcom










INTRODUCTION



FOOD ANALYSIS





Claudia Zucchi















ACKNOWLEDGMENT












CONCLUSIONS






























INTRODUCTION



EXPERIMENTAL


FOOD ANALYSIS




















CONCLUSION


















ACKNOWLEDGEMENTS


REFERENCES















SEZIONE






INTRODUCTION



FOOD SAFETY



















REFERENCES























SEZIONE









SUSTAINABILITY


Kaiser Mahmood














CONCLUSIONS


ACKNOWLEDGEMENT






49











































NUTRA DAY 2015










NEWS







NEWS


















DISCUSSION









REFERENCES
























INTRODUCTION



JOINT HEALTH



Cindy Romain























RESULTS


DISCUSSION





























































43204 Reus SPAIN







MARKET OVERVIEW






CHOCOLATE



Mia Naprta













































FUNCTIONAL FOOD




Anthony Fardet














REFERENCES
































INTRODUCTION



FOOD PROCESSING



Angela Zinnai















REFERENCES



















CONCLUSIONS




∆






INTRODUCTION



FOOD PROCESSING






Ghavidel





























CONCLUSION


REFERENCES




































USATel +1 913 953 8928

whitneyblackulcom










INTRODUCTION



FOOD ANALYSIS





Claudia Zucchi















ACKNOWLEDGMENT












CONCLUSIONS






























INTRODUCTION



EXPERIMENTAL


FOOD ANALYSIS




















CONCLUSION


















ACKNOWLEDGEMENTS


REFERENCES















SEZIONE






INTRODUCTION



FOOD SAFETY



















REFERENCES























SEZIONE









SUSTAINABILITY


Kaiser Mahmood














CONCLUSIONS


ACKNOWLEDGEMENT






49











































NUTRA DAY 2015










NEWS







NEWS
















REFERENCES
























INTRODUCTION



JOINT HEALTH



Cindy Romain























RESULTS


DISCUSSION





























































43204 Reus SPAIN







MARKET OVERVIEW






CHOCOLATE



Mia Naprta













































FUNCTIONAL FOOD




Anthony Fardet














REFERENCES
































INTRODUCTION



FOOD PROCESSING



Angela Zinnai















REFERENCES



















CONCLUSIONS




∆






INTRODUCTION



FOOD PROCESSING






Ghavidel





























CONCLUSION


REFERENCES




































USATel +1 913 953 8928

whitneyblackulcom










INTRODUCTION



FOOD ANALYSIS





Claudia Zucchi















ACKNOWLEDGMENT












CONCLUSIONS






























INTRODUCTION



EXPERIMENTAL


FOOD ANALYSIS




















CONCLUSION


















ACKNOWLEDGEMENTS


REFERENCES















SEZIONE






INTRODUCTION



FOOD SAFETY



















REFERENCES























SEZIONE









SUSTAINABILITY


Kaiser Mahmood














CONCLUSIONS


ACKNOWLEDGEMENT






49











































NUTRA DAY 2015










NEWS







NEWS



















INTRODUCTION



JOINT HEALTH



Cindy Romain























RESULTS


DISCUSSION





























































43204 Reus SPAIN







MARKET OVERVIEW






CHOCOLATE



Mia Naprta













































FUNCTIONAL FOOD




Anthony Fardet














REFERENCES
































INTRODUCTION



FOOD PROCESSING



Angela Zinnai















REFERENCES



















CONCLUSIONS




∆






INTRODUCTION



FOOD PROCESSING






Ghavidel





























CONCLUSION


REFERENCES




































USATel +1 913 953 8928

whitneyblackulcom










INTRODUCTION



FOOD ANALYSIS





Claudia Zucchi















ACKNOWLEDGMENT












CONCLUSIONS






























INTRODUCTION



EXPERIMENTAL


FOOD ANALYSIS




















CONCLUSION


















ACKNOWLEDGEMENTS


REFERENCES















SEZIONE






INTRODUCTION



FOOD SAFETY



















REFERENCES























SEZIONE









SUSTAINABILITY


Kaiser Mahmood














CONCLUSIONS


ACKNOWLEDGEMENT






49











































NUTRA DAY 2015










NEWS







NEWS




































RESULTS


DISCUSSION





























































43204 Reus SPAIN







MARKET OVERVIEW






CHOCOLATE



Mia Naprta













































FUNCTIONAL FOOD




Anthony Fardet














REFERENCES
































INTRODUCTION



FOOD PROCESSING



Angela Zinnai















REFERENCES



















CONCLUSIONS




∆






INTRODUCTION



FOOD PROCESSING






Ghavidel





























CONCLUSION


REFERENCES




































USATel +1 913 953 8928

whitneyblackulcom










INTRODUCTION



FOOD ANALYSIS





Claudia Zucchi















ACKNOWLEDGMENT












CONCLUSIONS






























INTRODUCTION



EXPERIMENTAL


FOOD ANALYSIS




















CONCLUSION


















ACKNOWLEDGEMENTS


REFERENCES















SEZIONE






INTRODUCTION



FOOD SAFETY



















REFERENCES























SEZIONE









SUSTAINABILITY


Kaiser Mahmood














CONCLUSIONS


ACKNOWLEDGEMENT






49











































NUTRA DAY 2015










NEWS







NEWS














- vol. 26(3) May/June 2015 - Published by Tekno Scienze Srl

Documents

Transcript of - vol. 26(3) May/June 2015 - Published by Tekno Scienze Srl