Agriculture, Food and Nutrition for Health and Wellness. Proceedings

18th World Congress on Clinical Nutrition (WCCN 2014) การประชุ ม วิ ช าการระดั บ โลก ครั ้ ง ที ่ ๑๘: โภชนาการคลิ น ิ ค (๒๕๕๗) Agriculture, Food and N...
4 downloads 2 Views 24MB Size
18th World Congress on Clinical Nutrition (WCCN 2014) การประชุ ม วิ ช าการระดั บ โลก ครั ้ ง ที ่ ๑๘: โภชนาการคลิ น ิ ค (๒๕๕๗) Agriculture, Food and Nutrition for Health and Wellness

Proceedings December 1-3, 2014 Sunee Grand Hotel & Convention Center Ubon Ratchathani, Thailand

Ubon Ratchathani University International College of Nutrition Faculty of Medicine, Thammasat University Center of Excellence in Applied Thai Traditional Medicine Research

  18  WCCN 2014  WORLD CONGRESS ON CLINICAL NUTRITION  TH

‘Agriculture, Food and Nutrition for Health and Wellness’  December 1‐3, 2014, Ubon Ratchathani, Thailand  Organised by   

UBON RATCHATHANI UNIVERSITY  Tel: +66 (0) 4535 3000‐3, www.ubu.ac.th   FACULTY OF AGRICULTURE (Key Organiser)  Tel: +66 (0) 4535 3500, www.agri.ubu.ac.th  85 Sathonlamark Road, Warin Chamrap, Ubon Ratchathani  34190, Thailand 

 

FACULTY OF MEDICINE, THAMMASAT UNIVERSITY  95 Paholyotin Road Klongluang, Pathumthani 12120 Thailand  Tel: +66 (0) 2926 9669  www.med.tu.ac.th 

 

INTERNATIONAL COLLEGE OF NUTRITION (ICN)  www.icnhealthfoods.com   

 

 

 

 

CENTER OF EXCELLENCE IN APPLIED THAI TRADITIONAL  MEDICINE RESEARCH (CEATMR)  Faculty of Medicine, Thammasat University, Thailand  Tel: +66 (0) 2926 9734, 2926 9749  www.med.tu.ac.th  INDIGENOUS FOOD RESEARCH AND INDUSTRIAL  DEVELOPMENT UNIT (IFRIDU)  Department of Agro‐Industry (Food Technology),   Faculty of Agriculture, Ubon Ratchathani University, Thailand   Tel: +66 (0) 4535 3500 Ext.3599  www.agri.ubu.ac.th 

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

SPONSORS OFFICE OF THE HIGHER EDUCATION COMMISSION (OHEC) 328 Si Ayutthaya Road, Ratchathewi, Bangkok 10400 Thailand Tel: +66 (0) 2610 5391-99, 2610 5401-03 www.mua.go.th THE THAILAND RESEARCH FUND (TRF) 14th Floor, SM Tower 979/17-21 Phaholyothin Road, Samsaen-nai, Phayathai, Bangkok 10400 Thailand Tel: +66 (0) 2278 8200 www.trf.or.th CHAROEN POKPHAND FOODS PUBLIC COMPANY LIMITED (CPF) C.P. Tower, 313 Silom Road, Bangrak, Bangkok 10500 Thailand Tel: +66 (0) 2625 8000 www.cpfworldwide.com BETAGRO PUBLIC COMPANY LIMITED 323 Vibhavadi Rangsit Road, Laksi, Bangkok 10210 Thailand Tel: +66 (0) 2833 8000 www.betagro.com OVO FOODTECH COMPANY LIMITED 9 Soi Ramintra 19 Yak 3, Ramintra Road, Anusawaree, Bangkhen, Bangkok 10220 Thaland Tel: +66 (0) 2900 4075-6, 2522 6175-6 www.ovofoodtech.com CHO THAVEE DOLLASIEN PUBLIC COMPANY LIMITED CHO THAVEE THERMOTECH COMPANY LIMITED 265 Moo 4 Klangmuang Road, Muangkao, Muangkhonkaen, Khonkaen 40000 Thailand Tel: +66 (0) 4334 1412-18 Ext. 108 www.ctvdoll.co.th AGRICULTURAL RESEARCH DEVELOPMENT (PUBLIC ORGANIZATION) (ARDA) 2003/61 Phaholyothin Road, Chatuchak, Bangkok 10900 Thailand Tel: +66 (0) 2579 7435 www.arda.or.th

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

DUTCH MILL COMPANY LIMITED 137/6 Moo 1 Buddha Monthon 8th Road., Kunkaew, Nakhon Pathom 73120 Thailand Tel: +66 (0) 3433 9020-4 www.dutchmill.co.th THE RICH HOTEL (UBON RATCHATHANI) Jangsanit Road, Ubon Ratchathani 34000 Thailand Tel: +66 (0)8 8378 1166, +66 (0) 4531 4881 Email: [email protected] BANK FOR AGRICULTURE AND AGRICULTURAL COOPERATIVES (BAAC) 2346 Phahon Yothin Road, Sena Nikhom, Chatuchak, Bangkok 10900 Thailand Tel: +66 (0) 2558 6555, 2555 0555 (call center) www.baac.or.th FOOD INDUSTRY DEVELOPMENT INSTITUTE (NFI) 2008, Soi Arun Amarin 36, Bangyeekhan, Bang Phlad, Bangkok 10700 Thailand Tel: +66 (0) 2886 8088 www.nfi.or.th BECTHAI BANGKOK EQUIPMENT & CHEMICAL COMPANY LIMITED 300 Phaholyothin Road, Phayathai Bangkok 10400 Thailand Tel: +66 (0) 2615 2929 www.becthai.com

ADDITIONAL SPONSORS: 1. COMMITTEE FOR ENTREPRENEURIAL PROMOTION OF UBON RATCHATHANI UNIVERSITY 2. ASSOC.PROF.DR. ADUN APINAN 3. CHATCHAWAL ORCHID COMPANY LIMITED (www.qualitygreen.com)

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

[email protected] by: Ubon Ratchathani University 85 Sathonlamark Road, Warin Chamrap, Ubon Ratchathani 34190, THAILAND Tel: +66 (0) 4535 3000-3, Fax: +66 (0) 4528 8048 www.ubu.ac.th

Published by: Faculty of Agriculture, Ubon Ratchathani University 85 Sathonlamark Road, Warin Chamrap, Ubon Ratchathani 34190, THAILAND Tel: +66 (0) 4535 3500, Fax: +66 (0) 4528 8373 www.agri.ubu.ac.th

All right reserved. No part of this book may be reproduced, stored in retrieval system or transmitted in any form or by any mean: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without permission from the publishers.

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

ABOUT THE CONFERENCE The conference will provide opportunities to meet and share experiences with representatives from the international pool of experts in agriculture, food, nutrition and healthcare. It will also provide an excellent occasion for forging links with government officials, academics and business communities. Plenary sessions: Invited speakers will deliver lectures on the followingtopics 1. Impact of Nutritional Quality of Bioactive Compounds and Functional Food on LongTerm Health 2. Advances in Delivery of Bioactive Nutrients in Food Matrix 3. Industrial Perspectives on Future Developments in Agriculture, Food and Nutrition for Health and Wellbeing 4. Current Trend in Natural Health 5. Role of Biotechnology in Food and Nutrition: Risks and Benefits 6. Traditional and Alternative Healthcare in Modern World Symposia: Abstracts dealing with any of the following topics may be submitted 1. Role of indigenous products in health and wellness This symposium covers researches on indigenous products which deal with nutritional properties, possible medicinal value, and safety issues which affect health and wellness of the consumers. 2. Advances in processing techniques for bioactive compounds This symposium covers researches on new processing techniques that would increase the efficiency in preserving and/or increasing the quality and efficacy of bioactive compounds from animal or plant materials, e.g. dehydration, size reduction, extraction, encapsulation, etc. 3. Efficacy of bioactive compounds and functional foods This symposium covers researches on nutritional and/or medicinal value of bioactive compounds or functional foods that contain these compounds and how well they affect consumers’ or patients’ health and wellness. 4. Bioactive ingredients from plant and animal sources This symposium covers researches on bioactive ingredients derived from animal or plant materials that can be used as medicinal or nutritional ingredients in traditional medicine, nutraceutical and functional food products. 5. Role of prebiotics and probiotics in health This symposium covers researches on prebiotics and probiotics and how they affect consumers’ gastrointestinal health. 6. Nutrition, risk factors and novel biomarkers This symposium covers researches on nutritional value and possible health risks of bioactive compounds from plant and animal sources, and biomarkers that may be used to identify and quantify these compounds and determine their efficacy in health-related products. i

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

7. Emerging trends in dietary management Most consumers utilize natural products as their primary sources of defenses against diseases. This symposium will cover researches on chemistry, formulation and efficacy of agricultural materials and food derived from natural products in relationships to consumers’ wellbeing. 8. Role of traditional and alternative medicine in healthcare This symposium covers researches on the use of traditional and alternative medicines in mitigating diseases and promoting good health. 9. Food technology and health

ii

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

 

 

STEERING COMMITTEE 

ORGANIZING COMMITTEE 

Prof.Dr. Chomchin Chantaraskul  Assoc.Prof.Dr. Nongnit Teerawatanasuk   Prof.Dr. Buncha Ooraikul  Assoc.Prof.Dr. Visith Chavasit  Mrs. Darunee Edwards  Asst.Prof.Dr. Tanaboon Sajjaanantakul  Dr. Thaweesak Koanantakool   Dr. Krissada Ruangarreerat  Assoc.Prof. Paiboon Thummaratwasik   Assoc.Prof.Dr. Tipvanna Ngarmsak   Mr. Pravit Anantavarasil  Mr. Aumnart Pakarat  Prof.Dr. Metha Wannapat  Assoc.Prof.Dr. Jowaman Khajarern  Assoc.Prof.Dr. Preecha Wanichsetakul                                                           

Dean of Faculty of Agriculture  Vice President for Research and Social  Engagement  Assistance to the President for Human  Resource Management  Vice President for Administration and  International Relations  Vice President for Planning and University  Council Affairs  Vice President for Academic Affairs  Vice President for Student Development  Vice President for Educational Quality  Assurance and Information  Assoc.Prof.Dr. Arunporn Itharat  Charoen Pokphand Foods Public Company  Limited  Betagro Public Company Limited  Kaona Kan Kaset Company  Dutch Mill Company Limited  Dr. Pisuth Lertvilai  Dean of Faculty of Science  Dean of College of Medicine and Public Health  Dean of Faculty of Pharmaceutical Sciences  Dean of Faculty of Nursing  Assoc.Prof.Dr. Watcharapong Wattanakul   Head of Department of Agro‐Industry  Head of Office of Secretary  Mrs. Suchit Uttarmart                   

iii   

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

 

 

SCIENTIFIC COMMITTEE 

WORKING COMMITTEE 

Prof.Dr. Buncha Ooraikul (Canada)   Prof.Dr. Tapan K. Basu (Canada)   Prof.Dr. Lech Ozimek (Canada)   Prof.Dr. Ram B. Singh (India)  Prof.Dr. Sukhinder Kaur (Canada)   Prof.Dr. Daniel Pella (Slovakia)                         Prof.Dr. Asim Dutta Roy (Norway)                Prof.Dr. Lekh Juneja (Japan)                          Prof.Dr. Fabien De Meeter (Belgium)       Prof.Dr. Agnieska Wilcznska (Poland)   Prof.Dr. N. S. Dhalla (Canada)   Prof. Dr. A. K. De (India)   Prof.Dr. D. W. Wilson (United Kingdom)  Prof.Dr. Toru Takahashi (Japan)   Prof.Dr. Hediki Mori (Japan)   Prof.Dr. Mahmood Moshiri (Canada)   Prof.Dr. Harpal S. Buttar (Canada)  Prof.Dr. Toru Takahashi (Japan)   Prof.Dr. Krasimira Hristova (Bulgaria)   Prof.Dr. Svetoslav Handjiev (Bulgaria)   Prof.Dr. Paul Kolodziejczyk (Canada)  Prof.Dr. Michael Gänzle (Canada)  Dr. Le Hoang Lam (Vietnam)  Assoc.Prof. Paiboon Thammaruwasik  (Thailand)  Assoc.Prof.Dr. Tipvanna Ngarmsak (Thailand)  Assoc.Prof.Dr. Wirote Youravong (Thailand)  Assoc.Prof.Dr. Jirawat Yongsawatkul  (Thailand)  Assoc.Prof.Dr. Pairat Sophanodora (Thailand)  Assoc.Prof.Dr. Bung‐orn Sripanidkulchai  (Thailand)  Asst.Prof.Dr. Suwayd Ningsanond (Thailand)  Asst.Prof.Dr. Ekasit Onsaard  (Thailand)  Asst.Prof.Dr. Jittra Singthong (Thailand)  Asst.Prof.Dr. Wiriya Phomkong (Thailand)  Asst.Prof.Dr. Kasem Nantachai  (Thailand)  Asst.Prof.Dr. Weerawet Utto (Thailand)  Asst.Prof.Dr. Ratchadaporn Oonsivilai  (Thailand)  Dr. Chalat Santivarangkna (Thailand)  Dr. Santad Wichienchote (Thailand)  Dr. Sirirat Kiatpathomchai (Thailand)  Dr. Krittaya Utto (Thailand) 

Dean of Faculty of Agriculture  Head of Department of Agro‐Industry  Vice Dean for Administration  Vice Dean for Research  Vice Dean for Academic Affairs  Vice Dean for Student Affairs  Head of Department of Fisheries  Head of Department of Agronomy  Head of Department of Horticulture  Head of Department of Animal Sciences  Head of Office of Laboratory and Farming  Dr. Narintorn Boonbrahm  Asst.Prof.Dr. Nittaya Wanikorn  Asst.Prof.Dr. Udom Tipparach  Asst.Prof.Dr. Thaweesak Juengwatanatrakul  Asst.Prof.Dr. Paowana Panomket   Miss Jaruwan Chubpawa   Asst.Prof.Dr. Ekasit Onsaard  Asst.Prof.Dr. Jittra Singthong  Asst.Prof.Dr. Weerawate Utto  Asst.Prof.Dr. Wiriya Phomkong  Dr. Apinya Ekpong  Dr. Prayong Udomworaparnt  Dr. Jindamanee Sangkarnjanawanich  Dr. Kritsna Siripon  Dr. Wachirapan Boonyaputthipong  Dr. Pan Promchot  Dr. Thidarat Juthong  Dr. Chutima Thongkaew  Dr. Metinee Maweang  Dr. Chatchaya Onumpai  Mrs. Kanlaya Theerapongthanakorn  Miss Sirianong Saengkrajang 

iv   

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

  TABLE OF CONTENTS 

     

Page          i      iii       v 

Mutagenicity and/or Anti‐mutagenicity of Noni and Noni Products by Ames Test 

1

About the conference   Committee       Table of contents   

     

     

     

     

     

     

     

  Tanyong Phatthanawiboon and Nattaya Konsue 

Effect of Blanching and Drying Processes on Antioxidant Activity of Mangosteen Rind 

7

Peamsuk Suvarnakuta, Sarunya Haklin, and Ajcharaporn Srisuriyo 

Process Improvement for Granulated‐non Centrifugal Sugar and its Properties  

13

Monthana Weerawatanakorn, Kanyaphat Meerod, Jiraphat Namnoy, Duangmanee Naktuen,  Kitiya Khamkeanklang, Sasivimon Chittrakorn, Khanitta Ruttarattanamongkol, and Sukeewan  Detyothin 

Inhibition of α‐Amylase and α‐Glucosidase Related to Anti‐hyperglycemic Activity     by Thai Plant Extracts 

19

Parichat Phalanisong, Kanit Vichitphan, Jaehong Han, and SukandaVichitphan

Effect of Emulsifiers on Properties and Stability of Water‐In‐Rice Bran Oil‐In‐Water  (W/O/W) Emulsions 

24

Nattapong Prichapan and Utai Klinkesorn 

Roselle Antioxidant Extracted by Microwave and Its Stability in Sherbet Ice Cream 

29

Pawiboon Supattanakul, Jantima Duangsilakij, and Sirinda Kusump 

Protective Effects of Selected Phenolic Compounds on Oxidative Stress 

37

Tarnrat Pattanawong, Pravate Tuitemwong, Pin‐Dur Duh, and Shih‐Ying Chen 

Antioxidant Activities of Protein Hydrolysates from Red Tilapia                   (Oreochromis niloticus) Fillet  Nur ‘Aliah Daud, Abdul Salam Babji, and Salma Mohamad Yusop 

43

Palm Sugars Improve Starch Digestibility of Bakery Products 

48

Rumpai Thongta and Khongsak Srikaeo 

Comparison of Antioxidant Properties of Hydrolyzed Skipjack Tuna             (Katsuwonus pelamis) Dark Muscle as a Function of Degree of Hydrolysis 

53

Worrapanit Chansuwan and Pavinee Chinachoti 

Nutritional Values, Antioxidant Activities and Physicochemical Properties of Drinking  Pomegranate (Punica granatum L.) Prepared from Pressing and Grinding Methods  Thikumporn Kongsabai, Sophawan Bousopha, and Sitthipong Nalinanon 

v   

60

December 1-3, 2014: Ubon Ratchathani, THAILAND 18th World Congress on Clinical Nutrition (WCCN) "Agriculture, Food and Nutrition for Health and Wellness"

Page Development of Yentafo Sauce from Red Pigment and High Antioxidant Plants 

67

Manatchaya Sungsri‐ina 

Isolation and Identification of Gamma‐Aminobutyric Acid‐producing Lactic Acid  Bacteria from Fermented Food  Watchareeya Wonghan, Kanit Vichitphan, and SukandaVichitphan 

72

Development of Kai Algae (Cladophora sp.)‐enriched Layered Instant Fried Noodles 

77

Suteera Vatthanakul, Bootsrapa Leelawat, Irin Rangsipaiboon, and Suchanat Ratthasomboon 

The Potency of Antioxidant in Wine, which Used Fruit Peel and Fruit Axis as  Substrates 

82

Pornpan Phuapaiboon 

The VARK Learning Style of Nursing Students  

87

Puntipa Kaewmataya,  Apiradee Charoennukul, Kulthida Kulprateepunya,  Sangduan Ginggaw,  and Nusara Prasertsri 

Knowledge Needs Related to Food and Nutrition Resources of a Community in  Thailand: A Case Study of Ubon Ratchathani Province  Jaroonsree Meenongwah, Puntipa Kaewmataya, Yaowaret Prapasanon, Udomwan  Wansri, Waiyaporn Promwong, Lukana Chopsiang, and Kunthida Kulprateepanya

                                 

vi   

92

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Mutagenicity and/or Anti-mutagenicity of Noni and Noni Products by Ames Test Tanyong Phatthanawiboona and Nattaya Konsuea* a Food Technology Program, School of Agro-Industry, Mae Fah Luang University, Chiang Rai 57100, Thailand * Corresponding email: [email protected]

ABSTRACT Noni (Morinda citrifolia L., Rubiaceae) has been used as a folk medicine for decades. Despite being consumed mostly in Thailand as a fermented juice there is no evidence showing that fermentation can enhance therapeutic effect of Noni when compare to fresh juice. In this study, fresh and fermented noni juice were prepared in our laboratory and analyzed for their total phenolic content. Consequently, the mutagenicity and/or antimutagenicity effects were evaluated by Ames test, employing bacteria Salmonella typhimurium TA 98 and TA 100 strains, with and without S9 activation system. Preliminary study showed that both bacteria strains TA98 and TA100 demonstrated suitable characteristics for Ames test as determined by histidine requirement, rfa mutation and Rfactor. However, only TA98 was chosen for further study due to its superior viability. Study of mutagenicity and/or anti-mutagenicity of samples were conducted using 4-nitro-ophenylenediamine (NPD) and 2-aminoanthracene (2AA) as positive controls in the absence and presence of S9 respectively. No toxicity was evident in both fresh and fermented noni juice as indicated by comparable number of histidine revertants per plate being 70, 75 and 72 histidine revertants per plate in control, fresh and fermented juice respectively. The second study revealed that fresh and fermented noni juice inhibited the histidine revertants per plate in a dose dependent manner (with and without S9) when compare with positive controls. This can be concluded that fresh noni juice and fermented noni juice have similar mutagenicity inhibition effect. It is believed to be due to an equal value of total phenolic content presented in both samples being 46.83 and 45.05 mg GAE/ml for fresh and fermented juice respectively. Nevertheless, actual bioactive compounds that responsible for these findings should be further studied. Keywords: Ames test, Mutagenicity, Noni, Fermentation

INTRODUCTION Morinda citrifolia, commonly known as noni, is a widely distributed tropical tree. It grows on the islands of the South Pacific, Southeast Asia, Central America, Indian subcontinent, and in the Caribbean. The fruit and leafs of this tree have a history of using for food and health promotion (West et al., 2011). Noni products are marketed as botanical dietary supplements for health benefits. Noni contains many phytochemicals including anthraquinones, flavonoid, polysaccharide, glycoside, iridoids, lignans, and triterpenoids. Compounds such as scopoletin, rutin, ursolic acid, β-sitosterol, asperuloside, and 1

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

damnacanthal are considered as key compounds of noni fruits (Chan-Blanco et al., 2006). Previously, in vitro and in vivo studies reported that fresh noni juice, extracts, or isolated biological compounds rendered health benefits in terms of scavenging of free radicals, antimutagenicity, anti-carcinoma activity, anti-clastogenic activity, inhibition of low-densitylipoprotein oxidation, anti-inflammatory activity, blood purification, stimulation of the immune system, regulation of cell function, and regulation of cholesterols (Yang et al., 2007). Indeed, a decade ago, the European Union has given noni juice the status of „novel food‟ (SCF, 2002). Noni juice is now used in Europe as a nutraceutical to treat chronic conditions such as arthritis, cancer, cardiovascular disease and diabetes. However, despite being consumed mostly in Thailand as a fermented juice there is no evidence showing that fermentation can enhance therapeutic effect of noni compared to the fresh juice. Mutation is an important factor for carcinogenesis. Preventing the biotransformation of procarcinogens to form ultimate carcinogen occurring in the present of S9 metabolic activation can decrease the rate of mutation. Ames test is one of the model methods developed to investigate mutagenicity modulation. Using of the Ames test is based on the assumption that any substance being mutagenic for the bacteria, Salmonella typhimurium, may also turn out to be a carcinogen. The Salmonella is specifically induced mutagenesis to be unable to synthesize amino acid and, therefore, limit its growth. However, the gene will gain its function in the presence of pro-carcinogen, thus a number of revertant colonies per plate is enormously increased. Besides, anti-mutagenicity of test compounds can be described if those numbers of colonies is decreased by using of carcinogen as a positive control (Mortelmans and Zeiger, 2000). The S9 fraction contains both cytochrome P450 and phase II enzymes. This has been used for Ames test to determine the chemical substances required for metabolic activation. The objectives of this study were to determine total phenolic content as well as the mutagenicity and/or anti-mutagenicity of noni products by Ames test. 4-nitro-ophenylenediamine (NPD) and 2-aminoanthracene (2AA) were used as positive controls in the absence and presence of S9 metabolic activation, respectively.

MATERIALS AND METHODS Chemicals Folin–Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). Nicotinamide adenine dinucleotide phosphate (NADP+) was purchased from Tokyo chemical industry (Japan). Glucose-6-phosphate was obtained from Calbiochem (Canada). 2-aminoanthacene and 4-nitro-o-phenylenediamine were obtained from Sigma Aldrich and phenobarbitalinduced S9 was a gift from Prof. Costas Ioannides, University of Surry, UK. Preparation of sample Ripen noni fruits were collected from Chiang Rai, Thailand. Fermented juice was produced by a local producer in Chiang Rai. In brief, fully ripe fruits were washed, chopped and mixed with brown sugar (10%). The mixture was transferred to a cleaned container and left at room temperature for 12 months fermentation. Fresh juice was prepared by employing hydraulic

2

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

press extraction prior to filtration. Both fresh and fermented noni juices were pasteurized at 80ºC for 3 min before future analysis. Determination of total phenolic content The total phenolic content in each sample was determined by spectrophotometer according to Folin-Ciocalteu procedure (Vu et al., 2012). The total phenolic content of sample was expressed as mg gallic acid equivalent (GAE) per ml of sample. Ames test Mutagenicity testing was carried out according to the method described by Maron and Ames (1983). To a 2 ml top agar containing histidine and biotin, 100 µl of histidine dependent bacteria, 100 µl of noni juice and 500 µl of S9 activation system or phosphate buffer saline (PBS) were added. The mixture was then gently mixed and poured on glucose minimal (GM) agar plate (Bottom agar). Once the top agar was solidified, the plates were incubated in a 37ºC incubator for 48 hr at which the time of the histidine revertants colonies were counted. Anti-mutagenicity testing was similarly conducted but both positive control and noni juice were added on a top agar. In the presence of S9 activation system, 2-aminoanthacene (2AA) was used as positive control, whereas 4-nitro-o-phenylenediamine (NPD) was employed in the absence of S9.

RESULTS AND DISCUSSION Determination of total phenolic content Total phenolic content of decreased significantly after pasteurization process for both fresh and fermented noni juice (Table 1). The result agreed with George and colleagues (2011) who reported that total phenolic content in tomato juice majorly declined after pasteurization. However, fermentation and non-fermentation process did not significantly affect total phenolic content. It can be found hat pasteurized fresh noni juice contained 23.80 mg GAE/ml, whereas pasteurized fermented noni juice was 19.85 mg GAE/ml. The result contradicted with previous report by Lai et al. (2013) whom found that phenolic content in soymilk was decreased offer fermentation process. It was believed that β-glucosidase generated from starter culture was responsible for increasing of phenolic content during fermentation. Nonetheless, fermentation of noni juice is majorly involved with Lactobacillus casei whereas soy bean fermentation was a simultaneous action of Streptococcus thermophiles and Bifidobacterium infantis. Table 1 Total phenolic content in noni samples Sample Fresh noni juice Pasteurized noni juice Fermented noni juice Pasteurized fermented noni juice

Total phenolic content (mg GAE*/ml) 46.83 ± 24.88a 23.80 ± 0.11b 45.05 ± 1.26a 19.85 ± 1.01b

Means with different lowercase superscripts indicate significant difference (P < 0.05) * Gallic acid equivalent

3

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Mutagenicity and anti-mutagenicity test The strain of S. typhimurium, TA98 (frame shift mutagen) was chosen from preliminary study to employ in Ames test. According to mutagenicity testing, samples were tested for their ability to increase histidine revertants per plate compared to spontaneous reversion rate and positive controls. S9 metabolic activation system was used to describe a pro-carcinogenic characteristic of sample whereas the number of colonies increased without S9, thus carcinogenic characteristic would be evident. It was found that pasteurized fresh noni juice and pasteurized fermented noni juice had no mutagenicity effect up to a high concentration of 100 µl per plate for both the presence and absence of S9 activation system. In the presence of S9 fraction, a lower number of histidine revertants per plate comparing with control (2Aminoanthracene) was found to be 72±4 and 85±10 colonies for fermented- and nonfermented noni juice, respectively (Table 2). Similar figure was observed in the absence of S9. However, the previous study reported that noni juice provided mutagenicity effect when tested with S. typhimurium TA1537 strain in the presence of metabolic activation (S9). Nonetheless, the author discussed that flavonoids in red grape juice, which is a constituent of noni juice, might have caused this effect (Westendorfet et al., 2007). Moreover, further analysis foregene mutations in mammalian cells presented negative results (SCF, 2002). Therefore, this can be inferred that fermented and non-fermented noni juice have no adverse effect on consumer health. Table 2 also shows anti-mutagenicity testing results of fresh and fermented noni juice. In the presence of positive control, 2AA and S9, the number of histidine revertants per plate was increased. However, pasteurized noni juice or fermented noni juice was incorporated, decreasing of number of colonies was observed. Comparing between two types of noni product, no significant effect was shown. Moreover, in the absence of S9, similar results were observed and indicated that both fermented- and non-fermented noni juices have an ability to inhibit mutagenicity in a similar extent of the revertants per plate at the same concentration. This results conformed the previous study which reported that noni juice exhibited antitumorigenic activity in human colorectal cancer cell line (Nualsanit et al., 2012) and inhibited the proliferation of human lung and colon cancer cells (Lv et al., 2011). Moreover, reduction of genotoxic effect of lesions induced by mitomycin C (MMC) and doxorrubicin (DXR) in Drosophila melanogaster was reported when commercial noni juice (TNJ) was employed (Franchi et al., 2013). However, it is crucially addressd that this commercial noni juice was composed of fresh noni juice extract and mixed fruit juice. The present work revealed that pure noni juice of both fresh and fermented types could rise anti-mutagenicity and could be a promising candidate for further study on its actual bioactive compound that responsible for anti-mutagenicity ability.

4

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Table 2 The mutagenicity of pasteurized noni juice and pasteurized fermented noni juice Samples

Spontaneous reversion rate ** Pasteurized noni juice (µl) 25 50 100 Pasteurized fermented noni juice (µl) 25 50 100 4-nitro-o-phenylenediamine 2-Aminoanthracene

Mutagenicity of noni juice (No. of histidine revertants/plate) -S9* +S9* b 78 ± 15 70 ± 10b

Anti-mutagenicity of noni juice (No. of histidine revertants/plate) -S9* +S9*

75 ± 70b 89 ± 30b 97 ± 11b

75 ± 12b 72 ± 16b 85 ± 10b

289 ± 43b 262 ± 16b 119 ± 25c

389 ± 43b 363 ± 17b 217 ± 28c

116 ± 33b 140 ± 27b 136 ± 80b 406 ± 36a

72 ± 10b 67 ± 12b 72 ± 40b

329 ± 18b 216 ± 16b 180 ± 18c 406 ± 36a

430 ± 18b 337 ± 23bc 209 ± 96c

589 ± 152a

589 ± 152a

Results are expressed as mean ± SD of triplicates. Means with different lowercase superscripts in column indicate significant difference (P < 0.05) * Phenobarbital induced-S9 ** The number of colonies obtaining from gene reversion without test compound

CONCLUSION Pasteurized fermented and non-fermented noni juice had no toxicity effect and, moreover, rendered anti-mutagenic properties in Ames test. One of the mechanisms of anti-mutagenicity was revealed by using the selected tester strain (TA98). This implies that noni juice prevents tumor formation in a frameshift mutation inhibition manner and the effect for both carcinogen and pro-carcinogen occurring. However, actual bioactive compounds that responsible for these findings and another characteristic of the anti-mutagenicity should be further studied.

ACKNOWLEDGEMENTS This research was financial supported by National Research Council of Thailand, Ministry of Science and Technology, Thailand. The author also would like to thanks Mae Fah Luang University for technical assistant.

REFERENCES Chan-Blanco, Y., Vaillant, F., Mercedes, P.A., Reynes, M., and Brillouet, J-M., and Brat, P. (2006), “The noni fruit (Morinda citrifolia L.): A review of agricultural research, nutritional and therapeutic properties”, Journal of Food Composition and Analysis, Vol. 19, pp. 645-654.

5

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Franchi, L.P., Guimarães, N.N., De Andrade, L.R., De Andrade, H.H., Lehmann, M., Dihl, R.R., and Cunha, K.S. (2013), “Antimutagenic and antirecombinagenic activities of noni fruit juice in somatic cells of Drosophila melanogaster”, An Acad Bras Cienc, Vol. 85 No. 2, pp. 585-594. Lai, L.R., Hsieh, S.C., Huang, H.Y, and Chou. C.C. (2013), “Effect of lactic fermentation on the total phenolic, saponin and phytic acid contents as well as anti-colon cancer cell proliferation activity of soymilk”, Journal of Bioscience and Bioengineering, Vol. 115 No. 5, pp. 552-556. Lv, L., Chen, H., Ho, C.T., and Sang, S. (2011), “Chemical components of the roots of noni (Morinda citrifolia) and their cytotoxic effects”, Fitoterapia, Vol. 82 No. 4, pp. 704708. Maron, D.M. and Ames, B.N. (1983), “Revised methods for the Salmonella mutagenicity test”, Mutat Res, Vol. 113, pp. 173-215. Mortelmans, K. and Zeiger, E. (2000), “The Ames Salmonella microsome mutagenicity assay”, Mutation Research, Vol. 455, pp. 29-60. Nualsanit, T., Rojanapanthu, P., Gritsanapan, W., Lee, S.H., Lawson, D., and Baek, S.J. (2012), “Damnacanthal, a noni component, exhibits antitumorigenic activity in human colorectal cancer cells”, The Journal of Nutritional Biochemistry, Vol. 23 No. 8, pp. 915-923. SCF (Scientific Committee on Food) (2002), “Opinion of the Scientific Committee on Food on Tahitian Noni® juice”, Opinion expressed on 4 December 2002. Vu, K.D., Carlettini, H., Bouvet, J., Côté, J., Doyon, G., Sylvain, J.F., and Lacroix, M. (2012), “Effect of different cranberry extracts and juices during cranberry juice processing on the antiproliferative activity against two colon cancer cell lines”, Food Chemistry, Vol. 132 No. 2, pp. 959-967. West, B.J., Deng, S., and Jensen, C.J. (2011), “Nutrient and phytochemical analyses of processed Noni puree”, Food Research International, Vol. 44 No. 7, pp. 2295-2301. Westendorf, J., Effenberger, K., Iznaguen, H., and Basar, S. (2007), “Toxicological and analytical investigations of noni (Morinda citrifolia) fruit juice”, J. Agric. Food Chem, Vol. 55 No. 2, pp. 529–537. Yang, J., Paulino, R., Janke-Stedronsky, S., and Abawi, F. (2007), “Free-radical-scavenging activity and total phenols of noni (Morinda citrifolia L.) juice and powder in processing and storage”, Food Chemistry, Vol. 102 No.1, pp. 302-308.

6

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Effect of Blanching and Drying Processes on Antioxidant Activity of Mangosteen Rind Peamsuk Suvarnakuta*, Sarunya Haklin, and Ajcharaporn Srisuriyo Department of Food Science and Technology, Faculty of Science and Technology Thammasat University (Rangsit Center), Pathum-thani 12120 * Corresponding email: [email protected]

ABSTRACT This research aimed to investigate the antioxidant activity kinetics of mangosteen rind during drying and to explore the appropriate drying conditions to preserve the antioxidant activity of dried mangosteen rind. Since it was recently found that antioxidant activity in mangosteen rind is inhibited by polyphenol oxidase activity, the blanching process which is the simple method to inhibit polyphenol oxidase enzyme, needs to be perfermed before drying. The effect of the blanching process on the antioxidant activity was therefore also investigated. The antioxidant activity of the ethanolic rind extract was expressed in terms of the inhibition capacity and inhibitory concentration 50% (IC50). The result showed that the antioxidant activity of mangosteen rind could be preserved by blanching process with boiling water at 100°C within >1 minute before drying. The antioxidant activity however decreased with drying time and moisture content. The superheated steam drying (SSD) methods at high temperature conditions (i.e. 160 and 180°C) had shorter drying time than that of all hot-air drying conditions. However, the hot-air drying preserved antioxidant activity more than the superheated steam drying at low temperature as well as blanching process could preserve the antioxidant activity in mangosteen rind. Keywords: Antioxidant activity, DPPH, Polyphenol oxidase, Superheated steam drying

INTRODUCTION Mangosteen rind is known as one of the best natural sources of xanthones. Xanthones belong to a class of polyphenolic compounds, commonly found in higher plant families (Peres et al., 2000). Xanthones and xanthone derivatives have been reported to have high antioxidant activity (Tachakittirungrod et al., 2007). In general, mangosteen rind must be dried prior to extraction of active compounds or even storage to extend its shelf life. However, it was recently found that antioxidant activity is inhibited by polyphenol oxidase activity (Suvarnakuta et al., 2011; Kim et al., 2013).The blanching process which is a simple method to inhibit polyphenol oxidase enzyme was to be performed before drying. Hence, the objective of this study was to investigate the effect of the blanching process on the antioxidant activity of dried mangosteen rind and the kinetics of antioxidant activity during conventional drying and superheated steam drying (SSD) methods.

7

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

MATERIALS AND METHODS Drying of mangosteen rind Mangosteen (Garcinia mangostana Linn.) fruit was purchased from a local market in Pathumthani. Mangosteen rind was separated from the fruit flesh and stored at -18°C until further use. To perform a drying experiment frozen mangosteen rind was thawed and then threated by trimming outer and inner skin off. The rind was then chopped by chopper (National, MK-5080N, Japan) to obtain a particle size of about 1 mm before drying. Then 100 g of prepared mangosteen rind was blanched in 7 liters of water at 80, 90, 100°C with water-bath accuracy ±1°C. For blanching treatments, chopped rind was immersed in a waterbath (MEMMERT, WNB7, Germany) and collected after reaching inactivation time when no activity of peroxidase in mangosteen rind could be detected. Blanched mangosteen rind was then dried at temperatures of 140, 160 and 180°C by using hot-air dryer (MEMMERT, UM 500, Germany) and superheated steam dryer (SHARP, AX-1500X, Japan). Mangosteen rind was dried until reaching the final moisture content in 0.08-0.10 kg/kg (d.b.). All experiments were performed in duplicate. Peroxidase activity analysis A 10 g each of raw and blanched mangosteen rind samples (blanching for 1, 2,..., 10 minutes) was weighed into 100 ml of 1 M sodium chloride solution. The samples were homogenized in a blender for 2 minutes. The slurry was mixed with guaiacol and H2O2 as substrates. The absorbance at 470 nm was recorded using an UV/VIS spectrophotometer (Unicam, UV2-100, England). The steady reading in absorbance of 1.0 per minute and per ml of extract sample defined the inactivation time of peroxidase under the assay conditions. Extraction of mangosteen rind Ethanolic rind extract was performed by using the methods of Suvarnakuta et al. (2011) with some modifications. Mangosteen rind powder (0.2 g) was mixed with 3 ml of 95% (v/v) ethanol. The mixture was then centrifuged at 3000 rpm for 5 min. A supernatant was collected and transferred to a 10 ml volumetric flask. The extraction was repeated both extracted solutions were combined in one 10 ml volumetric flask. The ethanolic extract was then filled up to the final volume of 10 ml with 95% (v/v) ethanol. Antioxidant activity evaluation PPH (2,2-diphenyl-2-picrylhydrazyl) radical scavenging activity of the ethanolic extract was evaluated by using the method of Okonogi et al. (2007) with some modification. A 3.9 ml of 100 μM DPPH radical in ethanol was added to a test tube with 0.2 ml of the ethanolic extract. The mixture was mixed using a vortex mixer for 10 sec. and left to stand in dark at room temperature for 60 min. The absorbance was measured at 540 nm using a UV-visible spectrophotometer (Unicam, UV2-100, England). Pure ethanol was used to calibrate the spectrophotometer. Inhibition capacity of fresh and dried mangosteen rind was calculated from the following equation (Huang, 2005). Inhibition capacity 

% Inhibition Weight of sample (dry basis)

8

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

The percentage inhibition of the DPPH radicals was calculated from the following equation (50% inhibition, IC50): ODblank  ODsample % Inhibition   100 ODblank Where ODblank and ODsample are the optical density of 95% ethanol and of the ethanolic extract, respectively.

RESULTS AND DISCUSSION Peroxidase inactivation Table 1 presents the peroxidase inactivity in mangosteen rind as a function of time for the different tested temperatures of blanching treatment. Peroxidase inhibition is usually used to investigate the causes of oxidative enzyme in fruit and vegetable. The remained polyphenol oxidase is the main effect of enzymatic oxidations. The result showed that the enzyme inactivation was significantly affected by the blanching temperature. The peroxidase residual activity at 90°C decreased to 37.5% of inactivation time. No activity of peroxidase was detected after 1 min. of blanching treatment at 100°C of water. Table 1 Peroxidase inactivation time of blanching treatment Blanching Temperature (oC) 80 90 100

Inactivationtime (min) 8 5 1

Evolutions of antioxidants of mangosteen rind during drying Total antioxidant activity (%inhibition) of the extract of fresh and blanched mangosteen rind which was assessed by DPPH radical scavenging capacity assay were approximately 95.26±3.96%, and 91.41±4.69%, respectively. As shown in Figures 1 and 2, the antioxidant activity of the extract of mangosteen rind during hot-air drying and superheated steam drying decreased when compared with that of fresh mangosteen rind. The drying process would normally affect natural antioxidants in fresh plant materials. For enzymatic degradation, although thermal treatment could inactivate degradative enzyme such as polyphenol oxidases, which is normally found in plant materials, some polyphenolic could be degraded due to initial activity of degradative enzyme prior to enzyme inactivation (Lim and Murtijaya, 2007). The result showed that the retentions of antioxidant activity in both mangosteen rind treatment markedly decreased via hot-air drying and superheated steam drying (Figure 1 and 2) while Figure 1 showed that loss of antioxidant activity after drying may be caused by thermal and enzymatic degradation.

9

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

1600 1400

Inhibition capacity

1200 1000 800

SSD 140 °C SSD 160 °C

600

SSD 180 °C

400

CON 140 °C CON 160 °C

200

CON 180 °C

0 0

0.5

1 Moiture content (d.b.)

1.5

2

Figure 1 Inhibition capacity of blanched mangosteen rind during conventional drying (CON) and superheated steam drying (SSD) 2400 2200 2000 1800

Inhibition capacity

1600 1400 1200

SSD 140 °C

1000 800

SSD 160 °C SSD 180 °C

600

CON 140 °C

400

CON 160 °C

200

CON 180 °C

0 0

0.5

1

1.5

2 2.5 3 Moiture content (d.b.)

3.5

4

4.5

5

Figure 2 Inhibition capacity of unblanched mangosteen rind during conventional drying (CON) and superheated steam drying (SSD) 10

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Although the drying period at low product temperature was expected to have much more ability to preserve antioxidant than drying at high temperature, the antioxidant inhibition capacity per dry weight of mangosteen rind when dried at operating temperature of 140°C undergone by both drying methods, had significantly higher inhibition capacity than that of 160 and 180°C. This showed that drying of mangosteen rind at 140°C required longer drying time than at 160 and 180°C. Extended drying time may result in a loss of bioactive compounds (Chantaro et al., 2008) and it was noticed that mangosteen rind temperatures at operating temperture 140°C, were lower than 180°C. Since polyphenol oxidase is heat labile and is inhibited at 90°C, enzymatic degradation may be the main mechanism causing loss of xanthones during initial period of drying rind at 140°C. Table 2 Antioxidant activity of dried rind underdryingtemperature of 160oC Pre-treatment Unblanching Blanching

IC50 (mg/ml) SSD 0.228 ± 0.071 0.173 ± 0.048

CON 0.176 ± 0.68 0.151 ± 0.013

Average IC50 0.202 ± 0.065b 0.162 ± 0.032a

For each variable means followed by different letters are significantly different at p ≤ 0.05% (Duncan's test)

Similar to the results of inhibition capacity, total antioxidant activity (IC50) of the extract of mangosteen rind during the hot-air drying, was higher than that of superheated steam drying (shown in Table 2). It may be caused by the longer drying time of superheated steam drying. In addition, the blanching process also caused significant decreases in the antioxidant activity of biomaterial (Figure 1-2) because the blanching process decreased phenolic compound and soluble vitamins. However, Table 2 showed that blanching pre-treatment before dryings caused significant increasing in antioxidant activity of dried mangosteen rind under both hotair drying and superheated steam drying. The blanching treatment may have degraded enzyme prior to dryings resulting in the higher phenolic content of dried sample. Table 3 also indicates that the drying with high temperature could have some influences on the stability of antioxidant activity in mangosteen rind. Therefore, the loss of antioxidant activity in the mangosteen rind is related to the influence of enzymatic degradation and thermal degradation. Table 3 Antioxidant activity of dried mangosteen rind underwent blanching pretreatments Temperature (oC) 140 160 180 Average IC50

IC50 SSD 0.167 ± 0.036 0.173 ± 0.049 0.257 ± 0.059 0.198 ± 0.058b

Average IC50 CON 0.156 ± 0.019 0.151 ± 0.013 0.177 ± 0.030 0.161 ± 0.021a

0.161 ± 0.024A 0.162 ± 0.032A 0.217 ± 0.060B

For each variable means followed by different letters are significantly different at p ≤ 0.05% (Duncan's test)

11

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

CONCLUSION The results of this study showed that the antioxidant activity of mangosteen rind could be preserved by blanching process with boiling water in 1 minute before drying to inactivate peroxidase. The antioxidant mangosteen rind significantly decreased during conventional drying and superheated steam drying due to enzymatic degradation and thermal degradation. Hot-air drying at 160°C was the best condition to preserve antioxidant in mangosteen rind because it provided the proper drying time and temperature which could inactivate oxidative enzyme and also halt the thermal degradation. Similarly, the hot air drying could preserve antioxidant activity more than the superheated steam drying at low temperature as well as blanching process could preserve the antioxidant activity in mangosteen rind.

REFERENCES Chantaro, P., Devahastin, S., and Chiewchan, N. (2008), “Production of antioxidant high dietary fiber powder from carrot peels”, LWT - Food Science and Technology, Vol. 41 No.10, pp. 1987-1994. Huang, D., Boxin, O.U., and Prior, R.L. (2005), “The chemistry behind antioxidant capacity assays”, Journal of Agricultural and Food Chemistry, Vol. 53 No. 6, pp. 1841-1856. Kim, M.H., Kim, J.M. and Yoon, K.Y. (2013), “Effects of blanching on antioxidant activity and total phenolic content according to type of medicinal plants”, Food Science and Biotechnology, Vol. 22 No.3, pp. 817-823. Lim, Y.Y. and Murtijaya, J. (2007), “Antioxidant properties of Phyllanthus amarus extracts as affected by different drying methods”, LWT - Food Science and Technology, Vol. 40 No. 9, pp. 1664-1669. Okonogi, S., Duangrat, C., Anuchpreeda, S., Tachakittirungrod, S., and Chowwanapoonpohn, S. (2007), “Comparison of antioxidant capacities and cytotoxicities of certain fruit peels”, Food Chemistry, Vol. 103 No. 3, pp. 839-846. Peres, V., Nagem, T.J., and Oliveira, F.F. (2000), “Tetraoxygenated naturally occurring xanthones”, Phytochemistry, Vol. 55 No. 7, pp. 683-710. Suvarnakuta, P., Chaweerungrat, C., and Devahastin, S. (2011), “Effects of drying methods on assay and antioxidant activity of xanthones in mangosteen rind”, Food Chemistry, Vol. 125 No. 1, pp. 240-247. Tachakittirungrod, S., Okonogi, S., and Chowwanapoonpohn, S. (2007), “Study on antioxidant activity of certain plants in Thailand: Mechanism of antioxidant action of guava leaf extract”, Food Chemistry, Vol. 103 No. 2, pp. 381- 388.

12

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Process Improvement for Granulated-non Centrifugal Sugar and its Properties Monthana Weerawatanakorn*, Kanyaphat Meerod, Jiraphat Namnoy, Duangmanee Naktuen, Kitiya Khamkeanklang, Sasivimon Chittrakorn, Khanitta Ruttarattanamongkol, and Sukeewan Detyothin Department of Agro-Industry, Faculty of Agriculture Natural Resources and Environment Naresuan University, Phitsanulok 65000, Thailand * Corresponding email: [email protected]

ABSTRACT Sugarcane (Saccharum officinarum L) is the leading economic crop of Thailand and is a major source of refined sugar production. Compared with refined sugar, non-centrifugal sugar (NCS), Naam Taan Oi, is high in nutritional value and beneficial to health. NCS has been mostly produced in hardened block form which is useful for storage stability but not for convenient use compared with the granular form. Based on a traditional process, a practical technique to produce Thai NCS in the granular form was developed using a drying method at 80ºC and baking powder to improve the sugar crystallization process. The physico-chemical properties were investigated. The optimum drying periods were 5 and 6 hours for 6 and 4 kg sucrose added respectively. The optimum amount of baking powder was 1.5%. The antioxidant inhibition by DPPH method ranged from 76.14±0.23% to 80.61±0.37%. Total phenolic compounds ranged from 10.02±0.35 to 13.17±0.46 mg gallic acid equivalent/g sample (dw). The total flavonoid content ranged from 1.22±0.03 to 1.90±0.04 mg Rutin equivalent/g sample (dw). Calcium content was 16.25±0.39 to 22.62±0.46 mg/100 g sample. Keywords: Naan Taan Oi, Flavonoid, Antioxidant, Drying

INTRODUCTION Non-centrifugal sugar (NCS) is a food which used to be the dominant form of cane sugar consumption before large-scale production of refined sugar (Jaffe, 2012). It has been consumed in many regions of the world and is known by different names, for example, Kokuto in Japan, Panela in South America, Jaggery in South Asia and Africa, and Naam Taan Oi in Thailand. Whole cane sugar and unrefined brown or black sugar have sometimes been referred to as NCS (Asikin et al., 2014). Unlike refined sugar, the unique production process of NCS includes dehydration and crystallization through the evaporation of whole sugarcane (Saccharum officinarum L.) juice, resulting in a final product packed with natural nutrients and phytochemicals. Scientific data confirmed that NCS has multiple beneficial health effects (Jaffe, 2012). The phenolic compositions of sugarcane and cane products are mainly phenylpropanoids and flavonoids, major representatives of the latter being derivatives of naringenin, tricin, apigenin, and luteolin (Paton, 1992). In Thailand, Naam Taan Oi has mostly been produced in a hardened block form due to the ease of the operation. Compared with the granulated form, the block form is not convenient for application. Based on the

13

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

traditional brown sugar process by artisan farmers, to produce granules from NCS is difficult as it depends on optimum sugar crystallization and degree of saturation which involves many factors, such as boiling time and temperature, acidity during concentration, and sugarcane varieties. The objectives of this study were to develop Thai NCS (NCS) into granular form (GNCS) using a drying process and baking powder based on a traditional process. The physicchemical properties, antioxidant, total phenolic compounds, and flavonoid content were also evaluated.

MATERIALS AND METHODS Development of Thai NCS into granular form Cane juice was evaporated by artisan farmers based on a traditional method using an open pan boiling technique. During evaporation, white sugar (sucrose) was added at 172 and 258 g/L of cane juice to obtain the samples. Then they were poured into the drying tray and dried in a conventional oven at 80ºC for 5, 6, and 7 h. The second part of the study studied the effects of the addition of baking soda. Thick sugar cane syrup (77.3±0.5 ºBrix) obtained by the evaporation of cane juice was used as a raw material. Then the thick cane syrup was heated for 10 min with 20% sucrose adding in the last 2 min. The mixture was poured into a tray containing baking powder (Best Food, Thailand) at 1, 1.5 and 2.0%, manually mixed, left at room temperature for 45 min, and dried at 80ºC for 3, 4, and 5 h in a conventional oven. The dried products were blended in a blender machine for 10 s after cooling at room temperature. The granulated NCS (GNCS) were thoroughly homogenized by a machine for physico-chemical properties analysis. A sample was prepared following Duarte-Almeida (2011) for antioxidant activity and total polyphenol and total flavonoid contents. Physicochemical properties were investigated as follows: Moisture content and aw analyses (AOAC, 2005) Moisture content was evaluated based on drying at 105ºC and Aw was measured with water activity analyzer. Bulk density and color analysis (ICUMSA, 2003) The bulk density was calculated by dividing the mass of the powder by the volume occupied in the cylinder. Color was determined by the standard ICUMSA (International Commission for Uniform Methods of Sugar Analysis). Solubility test The method was modified based on Eastman and Moore (1995). Titratable acidity (TA) Titratable acidity was determined by titration and the result was expressed as % citric acid.

14

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Sugar composition andmineral content (Ca) (AOAC, 2005) Fructose, glucose and sucrose were determined by high-performance liquid chromatography (HPLC) with a Refractive Index (RI) detector. Calcium was characterized and quantified by the atomic absorption spectrophotometry method. 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging (Medini et al., 2014) Total phenol content (Medini et al., 2014) The content was expressed as milligrams of Gallic acid equivalents per gram dry weight. Flavonoids content (Meda et al., 2005) The result was expressed as mg Rutin equivalent (QE)/g sample dry weight.

RESULTS AND DISCUSSION NCS is an unrefined product that has opportunities to supply sugar to a growing health food market. The study found that the sucrose addition at 172 (B) and 258 (C) g/L of cane juice had no significant effect on solubility, water activity, and moisture content but not for color, and bulk density and compared with the control (A), the sucrose adding significantly lowered water activity, moisture contents, color extent of GHCS (data not shown). Bulk density of the control could not be measured due to high water activity and moisture content. The major sugar of GNCS was sucrose (Table 2). The study found that the appropriate level sucrose addition and the optimum drying duration in terms of water activity, color value, and solubility were B and C as indicated in Table 1 and 2. The second part of the study, an assessment of the impact of baking powder on GNCS development, indicated that baking powder level and drying duration had no significant impact on water activity and solubility (data not shown). Moreover, longer drying periods caused increases of color value but increased addition of baking powder decreased color value. Surprisingly, baking powder dramatically decreased water activity of GNCS in the range of between 0.190 and 0.170, leading to easy and rapid grinding. The appropriate level based on color value of baking powder addition and drying period was 1.5% and 3 h (D) respectively, as shown in Table 1. Table 1 Physical properties of granulated non-centrifugal sugar (GNCS) at different levels of sugar and baking powder addition and drying times GNCS

A B C D

Drying time (hours) 0 6 5 3

Color (IU)

% Acidity

Properties Aw

0.54 ± 0.01 0.42 ± 0.01 0.65 ± 0.02 0.47 ± 0.02

0.35 ± 0.00 0.19 ± 0.00 0.27 ± 0.01 0.14 ± 0.02

0.46 ± 0.03 0.42 ± 0.01 0.39 ± 0.00 0.18 ± 0.01

% Solubility Bulk 30ºC density 30.43 ± 0.73 ND 30.86 ± 0.82 4.38 ± 0.49 31.47 ± 0.41 6.07 ± 0.86 33.14 ± 1.00 4.00 ± 0.03

A, without sucrose addition and drying (control); B, sucrose addition of 0.172 Kg/L cane juice; C, sucrose addition of 0.258 Kg/L cane juice; D, addition of sucrose and baking powder at 0.2 Kg and 0.015 Kg/ Kg thick sugar cane syrup, respectively

15

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Table 2 Sugar and calcium contents of granulated non-centrifugal sugar (GNCS) at different levels of sugar and baking powder addition and drying times GNCS

A B C D

Drying time (hours) 0 6 5 3

Fructose (g/100g)

Glucose (g/100g)

Sucrose (g/100g)

Calcium (mg/100g )

9.68 ± 0.13 6.50 ± 0.13 7.03 ± 0.06 10.43 ± 0.28

9.50 ± 0.25 6.63 ± 0.20 4.90 ± 0.42 12.55 ± 0.18

67.27 ± 0.13 74.68 ± 0.13 75.87 ± 0.03 67.35 ± 1.45

22.62 ± 0.46 20.37 ± 0.50 16.25 ± 0.39 18.19 ± 0.38

A, without sucrose addition and drying (control); B, sucrose addition of 0.172 Kg/L cane juice; C, sucrose addition of 0.258 Kg/L cane juice; D, addition of sucrose and baking powder at 0.2 Kg and 0.015 Kg/ Kg thick sugar cane syrup, respectively

As indicated in Table 3, the addition of sucrose slightly lowered the calcium content but the drying time had no impact on it (Table 3). Drying for 5 h (B) significantly increased total phenolic content from 13.17 to 15.13 (mg GE/g dw) due to drying process generated maillard reaction (browning reaction) inducing the formation of antioxidants. Maillard reaction related antioxidants have been postulated: radical scavenging antioxidants (Coghe et al., 2003). the other hand, baking powder (C) caused a significant decrease in the total phenolic compound. There was no significant difference between NCS as OTOP-GNCS from Phayao (D).The study found that total phenolic and flavonoid contents of developed GNCS were higher than GNCS from palm (E). Sucrose addition and drying for 5 h (B) had no effect on the total flavonoid content while baking powder addition and drying for 3 (C) significantly lowered the level of total flavonoids. The total flavonoid content of GNCS from Phayao (D) was the highest which may be due to the differences in the cane varieties used. Antioxidant activity by DPPH slightly decreased when baking powder was added. Table 3 Total phenolic, flavonoids, and antioxidant activity of granulated non-centrifugal sugar (GNCS) Thai NCS

A B C D E

Total phenol contents compounds (mg GE/g dw) 13.17 ± 0.46c 15.13 ± 0.08d 10.02 ± 0.35b 13.95 ± 0.44c 3.07 ± 0.61a

Total flavonoid content (mg RU/g dw)

% DPPH inhibition

1.89 ± 0.72c 1.90 ± 0.04c 1.22 ± 0.03b 2.09 ± 0.01d 0.57 ± 0.02a

79.37 ± 0.34c 78.77 ± 0.17c 76.14 ± 0.23b 71.08 ± 0.52a 79.17 ± 0.26c

Values followed by different letters in the same column are significantly different at p< 0.05. A, without sucrose addition and drying; B, addition of 0.258 Kg/L cane juice with drying for 5 h; C, addition of baking soda 0.015 Kg/Kg thick sugar cane syrup with drying for 3 h; D, the one town one product from Phayao; E, commercial palm sugar powder.

16

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

CONCLUSION Based on the traditional production process of Nam Tann Oi, sucrose and baking powder addition together with conventional drying can be used to develop non-centrifugal sugar (NCS) into granular form (GNCS). The developed product, GNCS, is a valuable nutritional product in terms of calcium content, total phenolic content, flavonoid content, and antioxidant activity.

ACKNOWLEDGEMENTS This project was supported by the National Research Council of Thailand, project No. R2557B070 and the Office of the Higher Education Commission, project NoR2557AH006. Appreciation is expressed to the Office of International Relations at Ubon Ratchathani University for assistance with English.

REFERENCES AOAC. (2005), Official methods of analysis, 18th ed. Association of Official Analytical Chemists., Gaithersburg, MD. Asikin, Y., Kamiya, A.,Mizu, M., Takara K, Tamaki, H., and Wada, K. (2014), “Changes in the physicochemical characteristics, including flavor components and maillard reaction products, of non-centrifugal cane brown sugar during storage”, Food Chemistry, Vol. 149, pp. 170–177. Coghe, S., Vanderhaegen, B., Pelgrims, B., Basteyns, A.-V., and Delvaux, F. (2003), “Characterization of dark specialty malts: New in-sights in colour evaluation and proand antioxidative activity”, Journal of the American Society of Brewing Chemists, Vol. 61, pp. 125–132. Duarte-Almeida J.M., Salatino A. Genovese M.I, and Lajolo F.M. (2011), “Phenolic composition and antioxidant activity of culms and sugarcane (Saccharum officinarum L.) products”, Food Chemistry, Vol. 125, pp. 660–664. Eastman, J.E. and Moore, C.O. (1995), “Cold water soluble granular starch for gelled food composition”, U.S. Patent 4465702. Jaffe, W.R. (2012), “Health effects of non-centrifugal sugar (NCS): A review”, Sugar Tech, Apr-June. Meda, A., Lamien, C.E., and Romito, M. (2005), “Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity”, Food Chemistry, Vol. 91, pp. 571-577.

17

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Medini, F., Fellah, H., Ksouri, R., andAbdelly, C. (2014), “Total phenolic, flavonoid and tannin contents and antioxidant and antimicrobial activities of organic extracts of shoots of the plant Limonium delicatulum”, Journal of Taibah University for Science, Vol. 8, pp. 216–224. Paton, N.H. (1992), “Sugar cane phenolics and first expressed juice colour–Part I”, International Sugar Journal, Vol. 94, pp. 99–108.

18

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Inhibition of α-Amylase and α-Glucosidase Related to Anti-hyperglycemic Activity by Thai Plant Extracts Parichat Phalanisonga,b, Kanit Vichitphana,b*, Jaehong Hanc, and SukandaVichitphana,b a Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, 40002, Thailand b Fermentation Research Center for Value Added Agricultural Products (FerVAAP), Khon Kaen University, Khon Kaen, 40002, Thailand c Department of Systems Biotechnology, Chung-Ang University, South Korea * Corresponding email: [email protected]

ABSTRACT Several Thai plants have been consumed as health foods and traditional medicines for the treatment and prevention of diseases. Hyperglycemia or high blood sugar (glucose) is a common problem for people with diabetes, sometimes leading to serious health conditions involving eyes disorders, kidney failure, and heart diseases. In this study, fifteen Thai plants from North-Eastern Thailand were evaluated for their potential as anti-hyperglycemic agents. The powder of dried samples were extracted using maceration with 95% (v/v) ethanol, dried under reduced pressure, and then dissolved in dimethyl sulfoxide (DMSO). The inhibitory effects against two enzymes (α-amylase and α-glucosidase) related to carbohydrate digestion were used to determine anti-hyperglycemic activity. The results showed that 0.25 mg.mL-1 of all fifteen ethanolic extracts inhibited α-glucosidase activities with percent inhibition in the range of 2.4 to 99% inhibition. Only four extracts, Phyllanthus amarus (Luktaibai), Bauhinia strychnifolia (Yanangdang), Caesalpinia sappan (Fangdang), and Caesalpinia sappan (Fangsom), exhibited a strong inhibitory effect (higher than 80%) on α-glucosidase activities with the half maximal inhibitory concentration (IC50) of 30, 60, 90, and 100 µg.mL-1 respectively. In addition, 15 mg.mL-1 of the extract of C. sappan showed a high inhibitory effect on α-amylase activities with 61-84% inhibition. The inhibitory effects of C. sappan on these two enzymes related to the digestion of carbohydrates suggest that C. sappan can possibly be used as an anti-hyperglycemic agent to control glucose levels in the bloodstream. Keywords: Thai plant, Hyperglycemia, Anti-hyperglycemic agent, Diabetes

INTRODUCTION Diabetes or diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia or high blood glucose levels, resulting from defects in insulin secretion, insulin action, or both (American Diabetes Association, 2014). There are four major types of diabetes, insulindependent diabetes (type 1), non-insulin-dependent diabetes (type 2), gestational diabetes, and diabetes secondary or other conditions that have been classified by the World Health Organization (WHO, 2014). Type 2 diabetes accounts for 90-95% of all diabetes cases and is a major public health concern with high morbidity, mortality, and health-care costs (Bloom et

19

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

al., 2011). The main treatments for people with type 2 diabetes are to control blood glucose levels and to prevent the risk of some chronic diseases related to diabetes, such as stroke, hypertension, and obesity. Recently, nutraceuticals and supplements from plants have played an important role in the management of diabetes and other metabolic disorders. Studies reported that some plant species, for example, Momordica charantia (bitter melon), Allium sativum (garlic), Cinnamonum cassia (cinnamon), Thymus vulgaris (thyme), Trigonella foenum-graecum (fenugreek), Panax ginseng (ginseng), and Gymnema sylvestre (gymnema), could be used to reduce blood glucose (Najm, 2012; Cazzola and Cestaro, 2014). Thailand is located in the tropics and has a high biodiversity of plants, several of which are consumed as health foods and traditional medicines. This study evaluated the inhibitory effects on key enzymes related to carbohydrate digestion and control of glucose levels in the bloodstream of fifteen local Thai plants used in infusion drinks for the reduction of blood sugar levels.

MATERIALS AND METHODS Plant samples and preparation of plant extracts Plant samples used in this study were selected from North-Eastern Thailand. The dried powder samples were extracted using maceration with 95% (v/v) ethanol for 24 h. The filtrate was dried under reduced pressure using a rotary evaporator (EYELA N1100, USA). The crude extracts were kept at -20ºC and then all samples were dissolved in dimethyl sulfoxide (DMSO) to give the final concentration of 250 mg.mL-1. Determination of α-glucosidase inhibition The effects of the extracts on α-glucosidase activity were determined using p-nitrophenyl-αD-glucopyranoside (pNPG) as a substrate. Briefly, 50 µl of the extract, 100 µl of αglucosidase solution (0.2 U.mL-1), and 160 µL of 0.01 M phosphate buffer pH 6.8 were mixed and incubated at 37ºC for 10 minutes. The reaction was started by adding 200 µL of 2.5 mM pNPG. After 10 minutes, the reaction was terminated by adding 700 µL of 0.2 M sodium carbonate solution. The yellow color of p-nitrophenol was determined at 405 nm by a spectrophotometer. The inhibitory effects of the extract on α- glucosidase were determined as the percentage of inhibition and the half maximal inhibitory concentration (IC50) which referred to the extract concentration to inhibit 50% of α-glucosidase activity. Determination of α-amylase inhibition The effects of the extracts on α-amylase activity were determined using soluble starch solution as a substrate. Briefly, 100 µL of the extract, 100 µL of α-amylase solution, and 200 µL of 0.01 M phosphate buffer pH 6.2 were mixed and incubated at 40ºC for 30 minutes. The reaction was started by adding 200 µL of 1% soluble starch. After 30 minutes, the reaction was terminated by adding 200 µL of 3, 5-dinitrosalicylic acid (DNS) reagent and boiling at 100ºC for 5 minutes. The reaction mixture was cooled and 10 mL of deionized water was added. The absorbance of the solution was then measured at 540 nm by spectrophotometer. The inhibitory effects of the extract on α-amylase activity were determined as the percentage of inhibition and IC50.

20

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

RESULTS AND DISCUSSION The percentage of extracting yield was calculated by the weight of the crude extract divided by the weight of the dried sample and multiplied by 100. The percentage of extraction yields were obtained in a range of 2-12% as shown in Table 1. The inhibitory effects on two enzymes, α-amylase and α-glucosidase, related to carbohydrate digestion were determined. The extracts‟concentrations at 15 mg.mL-1 were tested to evaluate the inhibitory effect on α-amylase, while α-glucosidase was tested with extracts at the concentration of 0.25 mg.mL-1. The results showed that α-glucosidase was more sensitive to all tested extracts than α-amylase (Figure 1). Four extracts, Phyllanthus amarus (luktaibai), Bauhinia strychnifolia (yanangdang), Caesalpinia sappan (fangdang), and Caesalpinia sappan (fangsom), exhibited a strong inhibitory effect at higher than 80% on α-glucosidase activities with IC50 30, 60, 90, and 100 µg.mL-1, respectively. The extract of C. sappan (fangsom) also showed a strong inhibitory effect on α-amylase activities with IC50 9 mg.mL-1. The inhibitory effect of C. sappan on both α-glucosidase and α-amylase related to the digestion of carbohydrates is possible delayed glucose absorption and suppressed postprandial hyperglycemia (Tahrani et al., 2010; Tundiset al., 2010). The inhibitory effect of C. sappan (fangsom) extract on the two enzymes displayed promising potential to be selected for further study on bioactive compounds and application for use as an anti-hyperglycemic agent to control blood glucose levels in type 2 diabetes. -1

) -1

)

Figure 1 Percent inhibition of fifteen plant extracts on α-amylase and α-glucosidase activity 21

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Table 1 Plant materials used in this study and the percentage of extraction yields Family Poaceae Zingiberaceae

Fabaceae

Iridaceae Menispermaceae Euphorbiaceae Lamiaceae Rhaminaceae

Scientific name Poganatherum crinitum Oryza sativa (R3, germinated rice berry) Curcuma aromatica Curcuma „Wan En-Leung‟ Curcuma aeruginosa Curcuma zedoaria Kaemferia galanga Bauhinia strychnifolia Caesalpinia sappan (FD, Fangdang) Caesalpinia sappan (FS, Fangsom) Eleutherine americana Coscinium fennestratum Phyllanthus amarus Orthosiphon aristatus Ventilago calycalata

Part used Leaf Seed Rhizome Rhizome Rhizome Rhizome Rhizome Stem Stem Stem Bulb Stem Aerial part Aerial part Rhizome

Yield (%) 3.39 2.48 6.32 12.34 5.89 7.04 4.90 5.02 5.20 7.99 3.60 5.19 3.58 5.64 3.18

CONCLUSION The results showed that the extract of Caesalpinia sappan (fangsom) had a substantial inhibitory effect on α-glucosidase and α-amylase in a concentration-dependent manner. This plant extract may contain bioactive compounds and can possibly be used as an antihyperglycemic agent to control glucose levels in the bloodstream.

ACKNOWLEDGEMENTS This research was financially supported by the Research Center for Value Added Agricultural Products (FerVAAP) and Graduate School, KhonKaen University.

REFERENCES American Diabetes Association (2014), “Diagnosis and classification of diabetes mellitus”, Diabetes Care, Vol. 37 No. 1, pp. 581-589. Bloom, D.E., Cafiero, E.T., Jané-Llopis, E., Abrahams-Gessel, S., Bloom, L.R., Fathima, S., Feigl, A.B., Gaziano, T., Mowafi, M., Pandya, A., Prettner, K., Rosenberg, L., Seligman, B., Stein, A.Z., and Weinstein, C. (2011). “The global economic burden of noncommunicable diseases”, World Economic Forum, Geneva, pp. 1-10. Cazzola, R. and Cestaro, B. (2014). “Antioxidant spices and herbs used in diabetes”, Diabetes: Oxidative Stress and Dietary Antioxidants, Available at: http://dx.doi.org/10.1016/B978-0-12-405885-9.00009-7 (accessed 10 November 2014).

22

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Najm, W.I. (2012), “An overview on nutraceuticals and herbal supplements for diabetes and metabolic syndrome”, Nutritional and Therapeutic Interventions for Diabetes and Metabolic Syndrome, Elsevier, London, pp. 355-362. Tahrani A.A., Piya, M.K., Kennedy, A., and Barnett A.H. (2010), “Glycaemic control in type 2 diabetes: Targets and new therapies”, Pharmacology and Therapeutics, Vol. 125 No. 2, pp. 328–361. Tundis R., Loizzo, M.R., and Menichini, F. (2010), “Natural products as α-amylase and αglucosidase inhibitors and their hypoglycemic potential in the treatment of diabetes: An update”, Medical Chemistry, Vol. 10 No. 4, pp. 315-331. WHO: World Health Organization (2014), “Diabetes”, available http://www.who.int/mediacentre/factsheets/fs312/en/ (accessed 3 October 2014).

23

at:

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Effect of Emulsifiers on Properties and Stability of Water-In-Rice Bran Oil-In-Water (W/O/W) Emulsions Nattapong Prichapana and Utai Klinkesorna* Department of Food Science and Technology, Faculty of Agro-Industry Kasetsart University, 50 Ngamwongwan Rd., Chatuchak, Bangkok 10900 * Corresponding email: [email protected] a

ABSTRACT Water-in-oil-in-water (W/O/W) emulsions can be used as an encapsulation and delivery system of bioactive compounds. Encapsulation of bioactive compounds in W/O/W emulsions can help to protect them from environmental stress, isolate them from other ingredients that they may interact with, and also mask their undesirable flavors or tastes. However, this system is thermodynamically unstable. Selection of proper emulsifier type and concentration is important for production of stable W/O/W emulsions. In the present work, we investigated the effect of emulsifiers on the stability of either water-in-oil (W/O) or W/O/W emulsions. Rice bran oil was used as the oil phase due to its health benefits and high oxidative stability. The effect of whey protein concentrate (WPC; 10, 15, or 20%) as co-emulsifier with polyglycerolpolyricinoleate (PGPR; 6, 8, or 10%) for preparation of W/O emulsions was investigated by measuring droplet size diameter (Z-average) and microstructure. The results showed that emulsions prepared from 6% PGPR in the oil phase and 10% WPC in the water phase had the smallest size of 306.7±7.9 nm. The effect of a hydrophilic emulsifier, sodium caseinate (NaCN), concentration (1-3%) in outer water phase on stability of W/O/W emulsions was also evaluated. We found that stable emulsions stabilized by 2% NaCN showed the smallest droplet size (1902.0±114.0 nm) with bimodal dispersion. For effect of WPC in the inner water phase on the stability of W/O/W emulsions, we found that adding WPC in the inner water phase improved stability of W/O/W emulsions. These emulsions had smaller droplet size (1226.5±17.7 nm) and droplet size was not significantly changed after 7 days storage (P>0.05). The knowledge from this work can be applied to produce stable W/O/W emulsions from rice bran oil for use as a delivery system for bioactive compounds and other nutritional substances. Keywords: Water-in-oil-in-water emulsions, Encapsulation, Rice bran oil

INTRODUCTION Direct fortification of bioactive compounds into food is unstable for long-term storage and may reduce food quality such as taste, flavor, and visual appeal color changes. Encapsulation of these compounds in W/O/W emulsions before introduction into the food can help to solve these problems. W/O/W emulsions can be used to encapsulate water-soluble substances, such as minerals and water-soluble vitamins, for fortification in water-based food. However, using of emulsions have been limited by their thermodynamic instability such as droplet flocculation, phase separation, and leakage of bioactive compounds (McClements, 2005;

24

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Fang and Bhandari, 2010; Dickinson, 2011). Selection of proper emulsifier type and concentration is important for production of stable emulsions. The present work aimed to study the effect of emulsifiers on properties and stability of W/O/W emulsions. In this work, PGPR, WPC and NaCN are selected due to their economic availability and they are generally used as emulsifiers in the food industry. Rice bran oil, a healthy oil, was used as the oil phase because it contains high amounts of natural antioxidants and bioactive compounds (Charoen et al., 2011).

MATERIALS AND METHODS Materials Rice bran oil (Thai Edible Oil Co., Ltd., Bangkok, Thailand) was purchased from a local supermarket. Polyglycerolpolyricinoleate ester (PGPR) was donated by Sakamoto Yakuhin Kogyo Co., (Osaka, Japan). Whey protein concentrate (WPC; protein content not less than 80%, fat content not more than 8.5%, ash not more than 4.5%, moisture not more than 6%) and sodium caseinate (NaCN; protein content not less than 90%, fat content not more than 2%, ash not more than 6%, moisture is not more than 6%) were donated by Vicchi Enterprise Co., Ltd. (Bangkok, Thailand). All other chemicals were reagent grade or better. Methods W/O Emulsion Preparation W/O emulsions were prepared from 80 wt % oil phase and 20 wt % water phase. Water phases were prepared by dispersing 0, 5, 10, or 15 wt % WPC in 100 mM phosphate buffer pH 7 containing 0.02 wt % sodium azide with storage over night to make WPC completely hydrated. Oil phases were prepared by dispersing 6, 8, or 10 wt % PGPR in rice bran oil, and then heated to 40oC, before mixed with water phase for 3 min. The mixture was then homogenized using a handheld homogenizer (IKA-ULTRA-TURRAX® T 25 basic, KIKA®WERKE GMBH & CO.KG, Germany) at 13,500 rpm for 2 min, before passing through a two-stage high-pressure valve homogenizer (15MR-8TA, APV Gaulin Inc., Wilmington, MA, USA) at 3,500 psi, and cooled with cooling water. W/O emulsions were kept at 25oC overnight before analysis. W/O/W Emulsions Preparation W/O/W emulsions were prepared from 80 wt % outer water phase and 20 wt % W/O emulsions. Outer water phases were prepared by dispersing WPC or NaCN in phosphate buffer solution and storing overnight at 25oC. Freshly prepared W/O emulsions were mixed with the outer water phase for 3 min. The mixture was then homogenized using a hand held homogenizer at 12,000 rpm for 2 min, before being passed through a two-stage high-pressure valve homogenizer at 2,000 psi, and cooled with cooling water. The prepared W/O/W emulsions were then kept at 25oC overnight before analysis. Droplet Size Distributio Emulsions were diluted 100 times with mineral oil for W/O emulsions, or with phosphate buffer solution for the W/O/W emulsions to avoid multiple scattering effect, before measurement using a dynamic light scattering instrument (Zetasizer Nano series-Zen 3600, Malvern Instruments, Worcestershire, UK).

25

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

-potential W/O/W emulsions were diluted 100 times with phosphate buffer solution before filling into capillary cells, and analysis using a particle electrophoresis instrument (Zetasizer Nano series-Zen 3600, Malvern Instruments, Worcestershire, UK) at 25oC. Microstructure of Emulsion Emulsions were softly agitated, then samples were dropped on a microscope slide and observed with 40× magnification objective lens using an optical microscope (Axiolab®, Carl Zeiss Ple Ltd., Germany). W/O/W emulsions were diluted 10 times with phosphate buffer solution before being observed. Statistics All experiments were carried out at least two times. The data were subject to the analysis of variance (ANOVA) using SPSS software package. The Duncan’s Multiple Range Test comparisons at p value < 0.05 were made to determine significant differences.

RESULTS AND DISCUSSION Effect of PGPR and WPC concentration on stability of W/O emulsions For the effect of PGPR concentration on droplet size of W/O emulsion without WPC, the results showed that the emulsion stabilized with 8% PGPR had droplet size smaller than emulsion with 6% PGPR (Table 1). This may explain that higher emulsifier concentration lead to stronger interface stabilized against droplet coalescence. This result corresponds to the results of Marquez et al. (2010). However, there was no significant difference in droplet size of emulsion stabilized with 8 and 10% PGPR. For the systems containing 6% PGPR and WPC at different concentrations (Table 1) we found that emulsion containing 10 and 15% WPC had droplet size smaller than emulsion without WPC. It is possible that WPC, which is an amphiphilic molecule, may form an internal layer around water droplets, or form complex interfacial layer that can better stabilize the emulsion (Su et al., 2006; Dickinson, 2011). However, emulsion with 20% WPC had droplet size larger than emulsion with 10 and 15% WPC. Moreover at 8 and 10% PGPR, emulsion containing WPC had droplet size higher than emulsion without WPC. Too much emulsifier concentration may lead to high viscosity of the system resulting in difficulty to breaking in the emulsion into small droplet (Tang et al., 2013). However, no significant differences in physical appearance and microstructure were found between emulsions (data not shown). Table 1 Mean droplet size diameter (nm) of W/O emulsions Concentration of PGPR in oil phase (wt %) 6 8 10

Concentration of WPC in water phase (wt %) 0 487.9 ± 27.4ef 273.2 ± 13.2a 312.4 ± 4.1ab

10 306.7 ± 7.9ab 457.9 ± 0.4ef 377.0 ± 42.3cd

15 321.2 ± 31.7abc 429.1 ± 2.3de 362.7 ± 34.3bc

Means with same letters are not significantly different (P > 0.05)

26

20 510.5 ± 22.3f 487.8 ± 47.9ef 487.2 ± 24.7ef

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Effect of emulsifier type and concentration on stability of W/O/W emulsions The W/O emulsion stabilized with 6% PGPR was used to produce W/O/W emulsion using 2% NaCN or WPC as hydrophilic emulsifier. The results showed that mean droplet size of W/O/W emulsion stabilized with NaCN (1902±114 nm) was smaller than that of WPC (2509±186 nm). This is probably due to the droplets in emulsion stabilized with NaCN have electrical charge or -potential (-20.17±1.59 mV) higher than droplets stabilized by WPC (-11.90±0.46 mV). The higher -potential led to the higher electrostatic repulsion retarding droplet flocculation and coalescence, resulting in smaller emulsion droplets (McClements, 2005). For the effect of NaCN concentration (1, 2, and 3%), the results showed that emulsion stabilized with 1% NaCN had droplet size about 2946±234 nm which is bigger than droplet size of emulsion stabilized with 2 and 3% NaCN (1902±114 and 1931±70 nm, respectively). This may be due to higher NaCN concentration producing a thicker and stronger interface (Mc Clements, 2005). However, there was no significant difference in droplet size between emulsion stabilized with 2 and 3% NaCN. For production of W/O/W emulsions, the results showed that droplet size of W/O/W emulsion prepared from W/O emulsion stabilized with 6% PGPR in the presence of 10% WPC (1226.5±17.7 nm) was smaller than emulsion stabilized by PGPR alone at 6 and 8% (2355±41.0 and 2415.5±9.2 nm, respectively; Figure 1). After 7 days storage, droplet size of W/O/W emulsion prepared from 6 and 8 % PGPR stabilized W/O emulsion decreased to 1537.2±42.1 and 2191.8±37.1 nm, respectively. This may due to water in inner water phase diffusing to outer water phase (Dickinson, 2011). However, droplet size of W/O/W emulsion prepared from W/O emulsion stabilized with 6% PGPR containing 10% WPC in the inner phase did not significantly change (P > 0.05). A possible explanation is that WPC in the inner water phase may form the extra layer as mentioned that can help to retard the diffusion of water molecules.

A

B

C

Figure 1 Optical microscope image of W/O/W emulsions: 6% PGPR and 0% WPC (A), 8% PGPR and 0% WPC (B), and 6% PGPR and 10% WPC (C)

27

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

CONCLUSION W/O/W emulsions prepared from NaCN had -potential higher than that of WPC, so it had more repulsive force between droplets preventing droplet flocculation. The lowest NaCN concentration that provided the smallest droplet size was 2 wt %. The addition of 10 wt % WPC in the inner water phase can reduce the droplet size of W/O emulsion and retard the diffusion of water molecules from inner water phase to outer water phase. This information can be used to produce W/O/W emulsions from rice bran oil for use in delivery systems of bioactive compounds and other nutritional substances.

ACKNOWLEDGEMENTS This research was supported by the Thailand Research Fund under The Royal Golden Jubilee Ph.D. Program (PHD/0001/2555: 2.F.KU/55/I.1.R.03).

REFERENCES Charoen, R., Jangchud, A., Jangchud, K., Harnsilawat, T., Naivikul, O., and McClements, D.J. (2011), “Influence of biopolymer emulsifier type on formation and stability of rice bran oil-in-water emulsions: Whey protein, gum arabic, and modified starch”, Journal of Food Science, Vol. 76, pp. E165-E172 Dickinson, E. (2011), “Double emulsions stabilized by food biopolymers”, Food Biophysics, Vol. 6, pp. 1-11. Fang, Z. and Bhandari, B. (2010), “Encapsulation of polyphenol”, Trends in Food Science & Technology, Vol. 21, pp. 510-523. Marquez, A.L., Medrano, A., Panizzolo, L.A., and Wagner, J.R. (2010), “Effect of calcium salts and surfactant concentration on the stability of water-in-oil (w/o) emulsions prepared with polyglycerolpolyricinoleate”, Journal of Colloid and Interface Science, Vol. 341, pp. 101-108. McClements, D.J. (2005), Food Emulsion: Principles, Practice, and Techniques, CRC Press, Boca Raton. Su, J., Flanagan, J., Hemar, Y., and Singh, H. (2006), “Synergistic effects of polyglycerol ester of polyricinoleic acid and sodium caseinate on the stabilisation of water–oil– water emulsions”, Food Hydrocolloids, Vol. 20, pp. 261-268. Tang, S. Y., Sivakumar, M., and Nashiru, B. (2013), “Impact of osmotic pressure and gelling in the generation of highly stable single core water-in-oil-in-water (W/O/W) nano multiple emulsions of aspirin assisted by two-stage ultrasonic cavitational emulsification”, Colloids and Surfaces B: Biointerfaces, Vol. 102, pp. 653-658.

28

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Roselle Antioxidant Extracted by Microwave and Its Stability in Sherbet Ice Cream Pawiboon Supattanakul*, Jantima Duangsilakij, and Sirinda Kusump Department of Food Science and Technology, Faculty of Science and Technology Thammasat University, Rungsit Campus, Thailand 12110 * Corresponding email: [email protected]

ABSTRACT The conventional extraction method used to isolate antioxidant from plant cells consumes large amounts of time and solvent. The microwave assisted technique was introduced to cause cell disruption within a few minutes, therefore shortening the extraction process. This study aimed to investigate 1) extraction time for separating antioxidant from roselle by microwaving and 2) antioxidant stability of roselle extract in sherbet ice cream. Microwave power of 460 watt was used to extract antioxidant from dry roselle with extraction time ranging from 0.5 to 8 minutes. Roselle extract was tray-dried at 85oC for 15 hours and the roselle extract powder (1 and 2% w/w) was added to sherbet ice cream. The ice cream was kept at -18oC for 9 weeks. Microwave extraction times of 1 and 5 minutes yielded roselle extract powder with the highest antioxidant activity and total anthocyanin content (p < 0.05). After one week of storage, 2% roselle ice cream had higher antioxidant activity, total phenolic compound and total anthocyanin contents than 1% roselle ice cream (p < 0.05). After nine weeks, the antioxidant activity of the 1% roselle ice cream showed a greater reduction than that of 2% roselle ice cream. Over the nine week period, the 1% roselle showed a greater reduction in phenolic compounds than 2% roselle ice cream, whereas the reduction in total anthocyanin compounds was greater in the 2% roselle than the 1%. This study suggests that the microwave-assisted technique could be effective in the extraction of antioxidants from roselle. Keywords: Roselle, Antioxidant, Microwave extraction, Anthocynin, Ice cream

INTRODUCTION Antioxidant activity expressed in plant comes from many phytochemicals including anthocyanins and other phenolic compounds. To release antioxidants from plant cell, solidliquid extraction combined with heat is conventionally used. However, this method is time and solvent consuming. In addition, many research found that some phytochemicals were not heat stable and were able to degrade when exposed to high heat for a long time, which in turn, reduced antioxidant activity (Jiratanan and Liu, 2004; Xu et al., 2007; Lin and Chou, 2009). Microwave is a technique that can heat up solvent rapidly and induces cell rupture in a short time, increase migration rate of phytochemicals from the plant cell, therefore, facilitates the extraction (Wang and Weller, 2006). Extract from microwave extraction contained higher quantities of anthocyanins and phenolic compounds compared to that from conventional method (Garofulić et al., 2013). 29

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Roselle (Hibiscus sabdariffa Linn.) is a good source for anthocyanins and phenolic compounds that can exhibit potent antioxidant activity (Ramírez-Rodrigues et al., 2012). Hot water extraction is a typical method used for isolating anthocyanins from roselle. Nevertheless, anthocyanin content and antioxidant activity of the extract reduced with increasing extraction temperature and increasing extraction time (Chumsri et al., 2008). Furthermore, antioxidant activity of anthocyanin extract was found high in acid food (Ruenroengklin et al., 2008). Therefore, sherbet ice cream, a frozen dessert with pH of lower than 4 may be a good carrier for delivering antioxidants from roselle to consumers. However, there is little information concerning extraction of roselle antioxidant by microwave and addition of roselle extract from microwave in sherbet ice cream. The objectives of this study were (1) to compare antioxidant activity of roselle extract from microwave extraction and that from conventional method and (2) to investigate antioxidant activity during storage of sherbet ice cream added with roselle extract from microwave extraction.

MATERIALS AND METHODS Rolselle extracts preparation Fresh roselles (Hibiscus sabdariffa Linn.) were bought in December, 2013 from a local market in Pathumthani province, Thailand. They were cut, removed the seeds, washed and dried in tray dryer (P.K. Tech Engineering, Thailand) at 75oC for 15 h. Dried roselles were ground and sieved by sieve shaker (Endecotts AS 200, England). Dried powder sized 20 to 100 mesh was mixed with distilled water (powder : water, 1:40 w/v). The mixture was heated with microwave power of 460 watt for 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 min. For the conventional method, the mixture was heated at 90oC for 16 min. After filtered the mixture through Whatman No.1 filter paper, the filtrate was tray dried at 85oC for 15 h. The roselle extract powder was kept in a vacuum sealed laminated bag. Determination of antioxidant activity of roselle extract powder Antioxidant activity was determined by using DPPH radical scavenging assay and reported as efficient concentration value (EC50). The method of determination was slightly modified from Brand-Williams et al. (1995). A DPPH solution was prepared by dissolving 0.01 g of DPPH in 25 ml of methanol. The DPPH solution had an absorbance of 0.60±0.05 units at 515 nm. The roselle extract powder (0.01g) was dissolved in 7 ml of distilled water. One milliliter of roselle extract solution was drawn to mix with 1 ml of methanol to prepare a mother solution. Different concentrations of a sample solution were prepared by using different volumes of the mother solution and final volume was made up with methanol to 1.9 ml. After added 100 µl of the DPPH solution, the sample solution was kept in a dark place for 4 h prior to measurement the absorbance at 515 nm. Roselle extract powder with the highest antioxidant activity was selected into sherbet ice cream. Determination of moisture content of roselle extract powder Moisture content was determined by drying 5 g of roselle extract powder at 105C until constant weight was reached (AOAC, 2000).

30

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Roselle sherbet ice cream preparation Ice cream was formulated from 60.9% of water, 4.28% of cream (38% fat), 3.42% of Skim milk powder (F.A. Food Co.,LTD., Thailand) 24% of sugar, 7.9% of glucose syrup, 0.25% of xanthan gum (Deosen Biochemical Ltd., China) and 0.05% of di-mono glycerol. Ice cream mix was pasteurized at 72oC for 15 min and homogenized for 1 min by a hand-held homogenizer (YstralX10, Ystral GmbH, Germany). The mixture was cooled and refrigerated at 4oC for 16 h before added 0.01%of citric acid (50% w/v) and 1 or 2% of roselle extract powder. The mixture was gently stirred before semi-frozen by ice cream freezer (Homemade®, China). The semi-frozen ice cream was packed in a container and hardened at -18oC for 24 h. The ice cream was stored at -18oC. For the control (0% roselle extract powder), a few drops of red color (Best odour Co., Ltd., Thailand) was used for coloring. Ice cream extract preparation The method of ice cream extract preparation was modified from Hwang et al. (2009). In brief, 5 ml of 500 ml/l methanol was added to 10 g melted ice cream sample. The solution was mixed and kept in the dark at room temperature for 12 h. before filtered with 0.45 µm syringe filter. The filtrate was kept in a 1.5 ml Eppendorf tube and placed in the dark at 4oC until used. Determination of antioxidant activity of roselle sherbet ice cream One hundred and fifty microliter of ice cream extract was added with 800 µl of distilled water and 550 µl of 70% methanol solution to make an ice cream mother solution. Antioxidant activity of the ice cream extract was determined using a DPPH radical scavenging method (Brand-Williams et. al, 1995) as described above. ABTS radical scavenging method (Binsan et al., 2008) was used and reported as efficient concentration value (EC50). An ABTS solution was prepared by mixing 7.4 mM ABTS solution with 2.6 mM potassium persulphate solution in equal volume. The solution was stored in the dark at room temperature for at least 12 h prior to dilution with buffer solution (pH 7.4) to get an absorbance of 1.10±0.05 units at 734 nm. One hundred fifty milliliter of ice cream extract was added with 1350 µl of distilled water to obtain an ice cream solution. Different volume of the ice cream solution was added with buffer solution and the final volume was made up to 0.5 ml. After added with 1.5 ml of the ABTS solution, the mixture was kept in the dark for 3 h prior to measurement the absorbance at 734 nm. Determination of total phenolic compound (TPC) Total phenolic compound was measured by using Folin-Ciocalteu (Julkunen-Titto, 1985). A mother solution was prepared by adding 1350 µl of distilled water to 150 µl of ice cream extract. The reaction mixture consisted of 600 µl of mother solution, 1.4 ml of distilled water and 200 µl of Folin–Ciocalteu was stored in the dark for 3 min then 1 ml of 20% Na2CO3 was added. The mixture was kept in the dark for 40 min and measured the absorbance at 750 nm. The result was calculated and reported as mg gallate equivalent per ml of ice cream extract. Determination of total anthocyanin content The roselle extract powder (0.01 g) was dissolved in 2 ml of distilled water and mixed with 2 ml of methanol (containing 100 g kg-1of HCl) and centrifuged at 3000 x g, 4 oC for 10 min (Picinelliet al.,1994). The supernatant (100 µl) was mixed with 900 µl of 1 molequiv/l HCl. 31

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Absorbance at 520 nm (A520) of the supernatant was read. Anthocyanin content (mg/l) was calculated as A520 x 101 x 18.89. For total anthocyanin content of ice cream, 2 ml of melted ice cream sample was mixed with 2 ml of methanol. Statistical analysis Treatment was performed in three replicates, which were independent batches of control and roselle sherbet. The experiment was divided into extraction and storage. Completely Randomized Design (CRD) was applied for all experiments. Analysis of variance (ANOVA) was applied for the determination of the main effect. Ducan’s new multiple range test was used to separate mean of main effect when significant differences (p  0.05) were observed.

RESULTS AND DISCUSSION Roselle extract powder obtained from microwave and conventional extraction had no significant difference for moisture content and water activity (p > 0.05) which were in the range of 4.11–4.54% and 0.364–0.451, respectively (Table 1). The low water activity indicated that less chemical reaction occurred (Labuza, 1970) and changes to of antioxidant activity of roselle extract powder during its storage should be low. Roselle extract powder using microwave extraction showed lower EC50 and higher total anthocyanin content (Table 2) than that of the control (p < 0.05) with the highest activity at extraction time of 1 and 5 min., indicating the efficiency of the microwave assisted technique. The result was in accordance with previous research (Garofulić et al., 2013) who reported that the microwaveassisted extraction provided the greater amount of phenolic acid and total anthocyanin contents than non-microwave assisted method. However, antioxidant activity and anthocyanin content of the roselle extract powder from microwave were significantly low (p 0.05)

32

Water activity ns 0.384 ± 0.003 0.3875 ± 0.013 0.4065 ± 0.030 0.401 ± 0.020 0.4135 ± 0.013 0.386 ± 0.011 0.402 ± 0.054 0.4355 ± 0.022 0.3785 ± 0.018 0.385 ± 0.008

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Table 2 Antioxidant activity and total anthocyanin contents of roselle extract powder Extraction time (min) 0.5 1 2 3 4 5 6 7 8 Conventional method

EC50 (g sample/mol DPPH) 1.444 ± 0.010a 0.925 ± 0.035f 1.037 ± 0.009de 1.058 ± 0.035d 1.170 ± 0.047c 0.969 ± 0.020ef 1.294 ± 0.059b 1.303 ± 0.040b 1.503 ± 0.013a 1.518 ± 0.057a

Total anthocyanin content (mg/l) 455.986 ±119.025a 539.933 ± 102.192a 508.135 ± 72.709a 474.429 ± 31.944a 478.244 ± 85.416a 527.850 ± 103.736a 142.456 ± 34.990b 181.250 ± 29.985b 139.912 ± 48.014b -

Means within same column followed by different letters are significantly different (p < 0.05)

Figure 1 Antioxidant activity by DPPH method (A) and ABTS method (B), TPC (C) and total anthocyanin (D) of roselle ice cream ( 2%, 1% and control)

33

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

For the first week of storage stability, antioxidant activity of 2% roselle ice cream (RIC) was significantly higher (EC50 of 0.21 g ice cream/mol DPPH and 0.03 g ice cream/mol ABTS) than that of 1% RIC (EC50 of 0.30 g ice cream/mol DPPH and 0.04 g ice cream/mol ABTS) (Figure 1). These results were compatible with of TPC and total anthocyanin content (Figure 1). Over 9-week storage, antioxidant activity, TPC and total anthocyanin decreased with increasing storage time (Figure 1). This may be due to the oxidation reaction between the ice cream ingredients and oxygen present in the ice cream structure. Phenolic compound and anthocyanins could counterbalance with occurring free radicals, leading to the reduction of antioxidant activity. Even though, antioxidant activity, TPC and total anthocyanins of roselle sherbet ice cream reduced, they were higher than that of the initial of the control. Linear regression coefficient for storage time (Table 3) showed that the reduction of antioxidant activity with DPPH method and phenolic compound of 1% RIC was higher than that of 2% RIC. On the other hand, the reduction of anthocyanins using 2% RIC was higher than that of 1% RIC. These results indicated that anthocyanins may not be the only phytochemicals responsible for antioxidant activity in roselle sherbet ice cream. Some phytochemicals including anthocyanins exhibited pro-oxidant activity when present in high concentrations (Yamanaka et al.,1997; Sugihara et al., 1999). Initially, there was higher anthocyanin content of 2% RIC than that of 1% RIC. After storage, anthocyanins may possess pro-oxidant activity and bind with the other compounds, resulting of higher reduction of anthocyanins for 2% RIC. Table 3 Linear regression coefficient on storage time Control 1% 2%

DPPH method 0.0576 0.0392 0.0127

ABTS method 0.0407 0.0036 0.0027

TPC -0.0378 -0.1893 -0.1767

Total anthocyanins -17.379 -14.303 -39.256

CONCLUSION Rapid and efficient extraction of roselle antioxidant can be exploited by using microwave assisted method. Antioxidant stability of the roselle ice cream was higher in ice cream containing higher roselle extract powder quantity, however, anthocyanins may not be the main component contributing to the antioxidant activity of the ice cream.

ACKNOWLEDGEMENTS This work was funded by the Center of Excellence, Thammasat University and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission.

34

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

REFERENCES AOAC International (2000), Official Methods of Analysis 17th ed. AOAC Int., Gaithersbersburg, MD. Binsan, W., Benjakul, S., Visessanguan, W., Roytrakul, S., Tanaka, M., and Kishimura, H. (2008), “Antioxidative activity of Mungoong, an extract paste, from the cephalothorax of white shrimp (Litopenaeus vannamei)”, Food Chemistry, Vol. 106 No. 1, pp. 185– 193. Brand-Williams, W., Cuvelier, M.E., and Berset, C. (1995), "Use of free radical method to evaluate antioxidant activity", Journal of Food Science and Technology, Vol. 28 No. 1, pp. 25-30. Chumsri, P., Sirichote, A., and Itharat, A. (2008), "Studies on the optimum conditions for the extraction and concentration of roselle (Hibiscus sabdariffa Linn.) extract", Songklanakarin Journal of Science and Technology, Vol. 30 (Suppl.1), pp. 133-139. Garofulić, I.E., Dragović-Uzelac, V., Jambrak, A.R., and Jukić, M. (2013), "The effect of microwave assisted extraction on the isolation of anthocyanins and phenolic acids from sour cherry Marasca (Prunus cerasus var. Marasca)", Journal of Food Engineering, Vol. 117 No. 4, pp. 437–442. Hwang, J. Y., Shyu, Y.S., and Hsu, C.K. (2009), "Grape wine lees improves the rheological and adds antioxidant properties to ice cream", Journal of Food Science and Technology, Vol. 42, pp. 312–318. Jiratanan, T. and Liu, R.H. (2004), "Antioxidant activity of processed table beets (Beta vulgaris var.conditiva) and green beans (Phaseolus vulgaris L.)" Journal of Agricultural and Food Chemistry, Vol. 52 No. 9, pp. 2659–2670. Julkunen-Titto, R. (1985), "Phenolic constituents in the leaves of northern willows: Methods for the analysis of certain phenolics", Journal of Agricultural and Food Chemistry, Vol. 33, pp. 213–217. Labuza, T.P. (1970), “Properties of water as related to the keeping quality of foods”, Proceedings of the Third International Congress of Food Science & Technology, Washington, DC., pp. 618-635. Lin, Y.C. and Chou, C.C. (2009), "Effect of heat treatment on total phenolic and anthocyanin contents as well as antioxidant activity of the extract from Aspergillus awamorifermented black soybeans, a healthy food ingredient", Journal of Food and Nutrition Research, Vol. 60 No. 7, pp. 627-636. Picinelli, A., Bakker, J., and Bridle, P. (1994), "Model wine solutions: Effect of sulphur dioxide on colour and composition during ageing", Journal of Grapevine Research, Vol. 33, pp. 31-35.

35

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Ramírez-Rodrigues, M.M., Plaza, M.L., Azeredo, A., Balaban, M.O., and Marshall, M.R. (2012), “Phytochemical, sensory attributes and aroma stability of dense phase carbon dioxide processed Hibiscus sabdariffa beverage during storage”, Food Chemistry Vol. 134, pp. 1425–1431. Ruenroengklin N., Zhong, J., Duan, X., Yang, B., Li, J., and Jiang, Y. (2008), "Effects of various temperatures and pH values on the extraction yield of phenolics from litchi fruit pericarp tissue and the antioxidant activity of the extracted anthocyanins", International Journal of Molecular Sciences, Vol. 9, pp. 1333-1341. Sugihara, N., Arakawa, T., Ohnishi, M., and Furuno, K. (1999), “Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with alpha-linolenic acid”, Journal of Free Radicals in Biology & Medicine Vol. 27, pp. 1313–1323. Wang, L.J. and Weller, C.L. (2006), "Recent advances in extraction of natural products from plants", Trends in Food Science and Technology, Vol. 17, pp. 300–312.

36

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

 

Protective Effects of Selected Phenolic Compounds on Oxidative Stress Tarnrat Pattanawonga,b, Pravate Tuitemwonga, Pin-Dur Duhb, and Shih-Ying Chenb a Food Safety Center, ISTRS, KMUTT, Bangkok 10140, Thailand b R&D Office, Department of Health and Nutrition, Chia Nan University of Pharmacy and Science, 60, sec.1,Erh-jen Road, Jen-te Dist. Tainan, Taiwan 71710

ABSTRACT The aim of this study was to evaluate the effects of selected phenolic compounds on oxidative stress. The phenolic compounds in the range of 50-500 µg/ml showed no cytoxicity in the growth of HepG2 cells except Chrysin, Flavones, Galangin, and Baicalein. Flavonols, such as Quercetin, Morin, Myricetin, Rutin, Fisetin, and Kaemferol, and some phenolic acids such as Caffeic acid and Chlorogenic, showed greater protection of the growth of HepG2 cells induced by 0.1 mM t-BHP than flavones, such as Chrysin, Baicalein, Apigenin, Nobilelin, and Tangeretin. For ROS generation, Quercetin, Morin, Myricetin, Rutin, Fisetin, Kaemferol, and Caffeic acid showed significant protection of these models. For mitochondrial membrane potential tests, Quercetin, Morin, Myricetin, Fisetin, Kaemferol, Caffeic acid and Chlorogenic acid showed significant protection mitochondrial membrane potential of these models. For the acellular tests, Quercetin, Morin, Myricetin, Rutin, Fisetin, Kaemferol, Caffeic acid, and Chlorogenic acid increased SOD activity and the inhibition of DPPH and NO. In cellular and acellular model systems, Quercetin, Morin, Myricetin, Rutin, Fisetin, Kaemferol, Caffeic, and Chlorogenic demonstrated a significant inhibitory effect on oxidative stress due to their remarkable antioxidant activity. Keywords: HepG2 cells, Oxidative stress, t-BHP, Antioxidant

INTRODUCTION Stress plays a significant role in the development of many diseases. A stressful condition leads to the excessive production of free radicals, resulting in oxidative stress and imbalance in the oxidant per antioxidant system. The natural phenolic compounds have received increasing interest recently. Since a great amount of them can be found in plants, the consumption of vegetables and beverages may reduce the risk of the development of several diseases due to their antioxidant power, among other factors. Phenolic compounds are constituted in one of the biggest and most widely distributed groups of secondary metabolites in plants. The antioxidant activity of food phenolic compounds is of nutritional interest, since it has been associated with benefits to human health through the prevention of several diseases1. The objectives of this study were first to evaluate the activity of a wide range of 19 phenolic compounds belonging to different classes and subclasses in a cellular and cellular model systems on oxidative stress. Second, results were used to identify possible mechanisms of action based on structure−activity relationships and molecular docking. The researchers

37   

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

 

believe these new insights into the structure-function relationships may be useful in the prevention of several diseases.

MATERIALS AND METHODS Measurement of HepG2 cells viability HepG2 cells were cultured in MEM medium at 37oC. After 24 h, cells were cultured with phenolic compounds at 50, 200, and 500 μg/ml for 30 min. Then, HepG2 cells were treated by 0.1 mM t-BHP for 24 h. Cell viability was determined by colorimetric measurement (MTT)2. Measurement of intracellular reactive oxygen species (ROS) HepG2 cells were treated by 0.1 mM t-BHP for 18 h, then added with 10 μl of ROS reagent and incubated for 2 h. Reactive oxygen species produced from cells were determined using ELISA reader and emission wavelengths of 485 and 535nm3. Evaluation of the mitochondrial membrane potential (ΨΔm) HepG2 cells were treated by 0.1 mM t-BHP for 18 h, then added with 10 μl of JC-1 dye staining solution and incubated for 30 min. The fluorescence was detected using ELISA reader and emission wavelengths of 560 and 595 nm3. Caspase-3 activity determination HepG2 cells were treated by 0.1 mM t-BHP for 18 h, then, 300 μl of medium was removed and 25 μl of caspase-3 kit was added. The caspase-3 activity was determined by a fluorometric assay kit. Fluorescence was detected using a microplate fluorescence reader with excitation and emission wavelengths of 485 and 530 nm3. Western bolt HepG2 cells were treated by 0.1 mM t-BHP for 18 h. Then, cells were collected and lysed in the ice-cold lysis buffer and kept on ice for 30 min, then centrifuged at 7000 xg for 10 min at 4oC. The supernatants were collected and the protein contents were determined by using the BCA protein assay kit. Each sample, which contained 100 µg proteins, was separated on 4% SDS–polyacrylamide gels. After electrophoresis, gels were transferred to nitrocellulose paper, then immunoblotted with primary antibody at 4oC for 1 h, and then with secondary antibody for 1 h, and visualized using an enhanced chemiluminescence (ECL) kit. The expression levels of Bcl-2 and Bax and ß-action proteins were determined by densitometry and analyzed3.

38   

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

 

RESULTS AND DISCUSSION

120 50 ug/ml 200 ug/ml 500 ug/ml

100 80

*

*

* *

60

*

*

* *

40

* ###

*

20

*

*

*

* *

**

* *

*

*

*

*

* *

*

*

*

*

*

* *

*

*

* **

* *

ro t -B l Qu H er P ce ti M n o r M y r in ic e C h tin r Ka ys e m in F l fe r o av l on Fi es s G a e t in la ng in Ru B a tin ic a A p l e in ig He e n s p in T a e r id n g in er e C a tin G a ffe l lic i c C ac Hy d r o u m id ox ar yb ic Fe en ru zo C h l ic ic lo a c i ro d ge ni c

0

Co

nt

C e ll v ia b ility (% o f c o n tr o l)

Effect of HepG2 cells viability The phenolic compounds in the range of 50-500 µg/ml showed no cytoxicity in the growth of HepG2 cells except Chrysin, Flavones, Galangin, and Baicalein after incubation for 24 hours (data no shown). In Figure 1, 0.1 Mm t-BHP significantly decreased the viability of the cells. However, the treatment of HepG2 cells with t-BHP and various concentrations of selected phenolic compound. The phenolic compounds at 500 µg/mL showed higher protection than those of lower concentrations. Therefore, the 500 µg/mL concentration of phenolic compounds was used in the following tests.

Phenolic Compounds Figure 1 Cell viability of the selected phenolic compounds on HepG2 cells induced by t-BHP Measurement of intracellular reactive oxygen species (ROS) In Figure 2, ROS generation increased significantly when the cells were treated with 0.1 mM t-BHP, indicating that the t-BHP had a strong effect on ROS generation because of its oxidizing nature. When the cells were treated with a phenolic compound and t-BHP, ROS generation was significantly decreased.

39   

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

250

# 500 ug/ml

200

150

100

*

* *

*

*

*

*

50

ac c

ic

ge

ni

ffe Ch

lo

ro

Ca

Ka

id

tin

ac

Ru

id

tin

l

se Fi

fe

et

em

ic yr M

Q

ro

in

in or M

et ue

rc

t -B

ro nt Co

in

HP

0 l

R O S P r o d u c tio n (% o f C o n tr o l)

 

Phenolic Compounds

Figure 2 Effect of the selected phenolic compounds on ROS production in HepG2 cells induced by t-BHP

120 500 ug/ml

*

100 80

*

*

*

60

*

*

*

#

40 20

ti n

l

se Fi

ro fe em

Ka

c ni

Ch

lo

ro

Ca

ge

ffe

yr

ic

ac

ac

id

id

t in

et ic

Ru

in

in or M

rc Q

ue

M

et

in

HP t- B

nt

ro

l

0 Co

M itochondrial m em brane potential

Evaluation of the mitochondrial membrane potential (ΔΨm) The t-BHP decreased the mitochondrial membrane potential (ΔΨm). This meant t-BHP induced mitochondrial damage. When the HepG2 cells were treated with a phenolic compound and 0.1 mM t-BHP, the ΔΨm was increased. This suggested selected phenolic compounds decreased the tendency of the cells to undergo mitochondrial dysfunction.

Phenolic Compounds

Figure 3 Effect of the selected phenolic compounds on mitochondrial membrane potential in

HepG2 cells induced by t-BHP

40   

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

 

#

180 160 140

500 ug/ml

120 100

*

80

*

*

*

*

*

*

*

60 40 20

ru

lic

tin Fe

c ge

ni

Ru

ac

ac

id

id

l ic ff e

Ch

lo

ro

Ca

em Ka

ic yr

fe

et

ro

in

in or M

ce er

M

tin

HP Qu

Co

t -B

ro

l

0

nt

C a s p a s e -3 a c tiv ity (% o f c o n tr o l)

Cytosolic caspase-3 activity determination In Figure 4, the caspase-3 activity increased significantly when the cells were treated with 0.1mM t-BHP, indicating the t-BHP significantly affected the apoptotic process. When the cells were treated with a phenolic compound and t-BHP, the caspase-3 activity was significantly decreased.

Phenolic Compounds

Figure 4 Effect of the selected phenolic compounds on Caspase-3 activity in HepG2 cells induced by t-BHP Evaluation of Bcl-2/Bax ratio The phenolic compounds, except Fisetin, showed increased Bcl-2 expression and a decreased Bax expression in HepG2 cells with t-BHP, indicating that phenolic compounds caused an up-regulation of Bcl-2 protein and a down-regulation of Bax in t-BHP-induced HepG2 cells, leading to an increased ratio of Bcl-2/Bax.

41   

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

 

2.5

B c l-2 /B a x ra tio

*

500 ug/ml

2.0

*

*

*

*

1.5

*

#

1.0

.5

id

ti n Ch

lo

ro

Ca

ge

ffe

ni

c

ic

ac

Ru

ac

id

t in

l

se

ro fe

et ic

em Ka

yr

Fi

in

in or M

rc ue Q

M

et

in

HP t-B

Co

nt

ro

l

0.0

Phenolic Compounds

Figure 5 Effect of the selected phenolic compounds on Bcl-2/Bax ratio in HepG2 cells induced by t-BHP

CONCLUSION The results showed selected phenolic compounds, flavonoids and some phenolic acid showed high protection of the growth of HepG2 cells induced by 0.1mM t-BHP. For protein expression of HepG2 cells induced by 0.1mM t-BHP, Quercetin, Morin, Myricetin, Kaemferol, Caffeic acid, and Chlorogenic acid showed an increased Bcl-2/Bax ratio. In addition, Quercetin, Morin, Myricetin, Rutin, Fisetin, Kaemferol, Caffeic acid, and Chlorogenic acid showed significant protection in ROS generation and mitochondrial membrane potential tests. From this model systems, Quercetin, Morin, Myricetin, Rutin, Fisetin, Kaemferol, Caffeic, and Chlorogenic demonstrated significant inhibitory effects on oxidative stress due to the hydroxyl groups related to the antioxidative stress in each compounds.

REFERENCES Reis Giada Md. L. (2013), Food Phenolic Compounds: Main Classes, Sources and Their Antioxidant Power. Chen, Z.T., Chu, H.L., Chyau, C.C., Chu, C.C, and Duh, P.D. (2012), “Protective effects of sweet orange (Citrus sinensis) peel and their bioactive compounds on oxidative stress’’, Food Chem, Vol. 135, pp. 2119-2127. Lee, C.P., Chen, Z.T., Yu, P.Y., Wang, Y.C., and Duh, P.D. (2012), “Identification of bioactive compounds and comparison of apoptosis induction of three varieties of sugarcane leaves”, Journal of Functional Foods, vol. 4, pp. 391-397.  42   

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Antioxidant Activities of Protein Hydrolysates from Red Tilapia (Oreochromis niloticus) Fillet Nur ‘Aliah Daud, Abdul Salam Babji*, and Salma Mohamad Yusop School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia * Corresponding email: [email protected]

ABSTRACT Antioxidant activities of protein hydrolysates from O. niloticus fillet were evaluated with different antioxidant assays. Hydrolysis was conducted with thermolysin and alcalase enzymes from 0-4 hours at 37oC, pH 7.4. Freeze dried protein hydrolysate was tested for degree of hydrolysis and peptide content. Both enzymatic hydrolysates obtained by the reaction of thermolysin and alcalase were tested for anti-oxidant activity. Hydrolysates after 2 hours incubation with thermolysin and alcalase had degree of hydrolysis of 76.29% and 63.49%, respectively. Both enzymes yielded higher anti-oxidative activities after 1 hour incubation. Studies of low level of molecular weight cut-off hydrolysates showed that, 10 kDa molecular weight cut-off have stronger antioxidant activities than 3 kDa molecular weight cut-off and much higher than hydrolysates without ultrafiltration. Hydrolysates from Red Tilapia may contribute as a health promoting ingredient in functional foods to reduce oxidation stress caused by accumulated free radicals. Keywords: Oxidative stress, Antioxidant peptide, Functional food, Red Tilapia protein

INTRODUCTION Concern regarding health safety has motivated the consumer and food industry to seek natural alternatives for antioxidants. Researches have been conducted through various methods to seek potential natural antioxidant peptides. In recent years, a considerable amount of research has focused on extraction of bioactive peptides which are encrypted within food proteins, with a view to utilizing such peptides as functional food ingredients aimed at health maintenance (Erdmann et al., 2008). Fish, in particular, have attracted interest for extraction of antioxidant peptides. Fish protein hydrolysates such as skin gelatin hydrolysate (See, 2011) and meat protein hydrolysates (You et al., 2010) have been reported to exhibit antioxidant activity. Antioxidative peptides from fish proteins have been considered to be safe and healthy molecules with low molecular weight, easy absorption, low cost, and high antioxidant activity (Sarmadi and Ismail, 2010). Selection of Red Tilapia is based on the growing demand for live fish and tilapia fillet in the domestic market. Thus, Red Tilapia fish protein sources will be more readily available than other freshwater fish. This study focused on evaluation of antioxidant activities of protein hydrolysates, obtained from Red Tilapia (O. niloticus) fillet, hydrolysed by enzyme thermolysin and alcalase.

43

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

MATERIALS AND METHODS Chemicals and reagents All enzymes and chemicals of analytical grade were purchased from Sigma (Sigma–Aldrich Chemical Co., St Louis, MO, USA). Protein hydrolysate, DH, and peptide content Red tilapia fillet was mixed with distilled water ( 2:100 w/v), adjusted to suitable pH and temperature. Hydrolysis was performed for 4 hours, after which the reaction was inactivated at 90°C for 10 minutes. Samples were centrifuged at 3000 x g for 20 minutes and the hydrolysates obtained were freeze-dried. DH was obtained using method as described by Hoyle and Merritt (1994). Peptide content were measured by method of Church et al. (1983). Antioxidant activity tests DPPH and ABTS radical-scavenging activities were determined as described by Binsan et al. (2008). Reducing power of the hydrolysates was measured by the method of Oyaizu (1988). Ultrafiltration Hydrolysate solution was separated into high and low molecular weight fractions by ultrafiltration at 4oC using 10 kDa molecular weight cut-off (MWCO) membrane (Vivaflow 200, Sartorius, Germany) and 3 kDa MWCO membrane (Vivaflow 50), separately. The eluent was then lyophilised and kept for use in further experiment. Statistical analysis All data collected was analyzed using analysis of variance (ANOVA) and Duncan’s multiple range tests. Significant differences in means between samples were determined at 5% confidence level (P < 0.05).

RESULTS AND DISCUSSION Degree of hydrolysis and peptide content of hydrolysates Table 1 showed that thermolysin resulted in higher amount of protein hydrolysate derived from red tilapia fillet than alcalase. The high level of DH by thermolysin treatment suggested that thermolysin has higher affinity and is more efficient than alcalase in the production of hydrolysates of interest. Quantification of peptide from hydrolysate showed that the peptide obtained is directly proportional to DH. With decreased rate of proteolysis, the peptide produced decreased slightly at 3-4 hours incubation. Differences observed in the hydrolysis rate and pattern might be due to the differences in the properties of enzyme cutting sites as well as the accessibility of peptide bonds to each protease (Bordbar et al., 2013).

44

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Table 1 Effect of hydrolysis time and different enzymes on degree of hydrolysis and peptide content of O. niloticus protein hydrolysate (n=2) Hydrolysis time (h) 0.5 1 2 3 4

DH (%) 27.41 47.30 76.29 88.01 92.58

Thermolysin Peptide content (mg/ml) 0.18 ± 0.17d 0.29 ± 0.15c 0.46 ± 0.03b 0.53 ± 0.25a 0.56 ± 0.28a

DH (%) 26.85 34.19 63.49 80.66 85.30

Alcalase Peptide content (mg/ml) 0.14 ± 0.11d 0.21 ± 0.07c 0.38 ± 0.23b 0.49 ± 0.13a 0.51 ± 0.27a

DPPH radical scavenging activity Table 2 shows that antioxidant activity was higher at 1 hour than 2 hours for thermolysin and alcalase, at 1 mg/ml concentration of both samples, with 38.98% and 38.89%, respectively. Size interval of hydrolysates at 10 kDa and below showed a higher antioxidant activities for the hydrolysates compared with 3 kDa and below. Extensive enzymatic hydrolysis of proteins resulted in decreased antioxidant activity. This might be due to free amino acids, where protein hydrolysates (peptides) had more potential antioxidants than free amino acids (Elias et al., 2008). ABTS radical scavenging activity Hydrolysates obtained by thermolysin give higher antioxidant activity than alcalase with 1 hour and 2 hours of incubation, at 1 mg/ml concentration of both sample, as shown in Table 2. Studies at low molecular weight cut-off (10 kDa and 3kDa) showed that hydrolysates by thermolysin have higher ABTS scavenging activity than by alcalase for both MWCO intervals. Several studies also have looked at the contribution of molecular size of peptide mixtures which showed that low molecular weight fractions contained more potent antioxidative peptides (Sampath et al., 2011). Table 2 Effect of hydrolysis time and low molecular weight with different enzymes on antioxidant activity of O. niloticus protein hydrolysate with DPPH and ABTS assay Samples Red tilapia protein Hydrolysates After hydrolysis (chosen time interval, hour) After ultrafiltration (diff. membrane cut-off, MWCO) ab

Size interval -

DPPH activity (%) Thermolysin Alcalase 12.31 ± 2.03d

ABTS activity (%) Thermolysin Alcalase 14.81 ± 0.69 d

1 2

38.98 ± 0.70b 34.70 ± 0.52c

38.89 ± 0.71b 31.09 ± 0.66c

44.78 ± 0.06b 37.82 ± 0.09c

40.41 ± 0.16b 34.64 ± 0.13c

10 kDa 3kDa

42.51 ± 0.67a 14.70 ± 0.01d

44.30 ± 0.01a 11.50 ± 0.06e

54.00 ± 0.01a 20.00 ± 0.07d

44.60 ± 0.04a 13.60 ± 0.03e

Different lower case letters indicate significant differences (P < 0.05) between samples (n=3)

45

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Reducing power capacity Hydrolysate obtained by thermolysin showed slightly higher reducing power capacity compared to that of alcalase at 2 mg/ml. At size 3 kda and below, results obtained showed significant difference between alcalase and thermolysin where hydrolysate by thermolysin resulted in significantly high reducing power than alcalase. The high activity of antioxidant at low molecular weight indicates the ability to bind with iron and chelate pro-oxidative iron resulting in decreased oxidation (Zhu et al., 2006).

(a)

(b)

Figure 1 Effect of hydrolysis time and different enzymes (Thermolysin and Alcalase) on anti-oxidant activity of O. niloticus protein hydrolysate with reducing power assay (a) and anti-oxidant activity on low molecular weight of O. niloticus protein hydrolysate with reducing power assay (b) *A1 is alcalase hydrolysate after 1 hour hydrolysis; A2 is alcalase hydrolysate after 2 hours; T1 is thermolysin hydrolysate after 1 hour; T2 is thermolysin hydrolysate after 2 hours; A10 is alcalase hydrolysate with 10 kDa molecular weight (MW) cut-off; A3 is alcalase hydrolysate with 3kDa MW cut-off; T10 is thermolysin hydrolysate with 10kDa MW cut-off; T3 is thermolysin hydrolysate with 3 kDa MW cut-off ab Different lowercase letters indicate significant differences (P < 0.05) between samples, (n=3)

CONCLUSION Protein hydrolysates of Red Tilapia (O. niloticus) hydrolysed by thermolysin and alcalase have demonstrated some potential antioxidant activities, with the use of thermolysin being slightly more efficient than alcalase. It is recommended that potent anti-oxidant peptides extracted from Red Tilapia O. niloticus be further studied to characterize the anti-oxidant peptides as a health promoting ingredient in functional foods.

ACKNOWLEDGEMENTS This study was financially supported from the project 05-01-02 SF 1007 provided by the Ministry of Agriculture and Agro-Based Industry (MOA) of Malaysia.

46

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

REFERENCES Binsan, W., Benjakul, S., Visessanguan, W., Roytrakul, S., Tanaka, M., and Kishimura, H. (2008), “Antioxidative activity of Mungoong, an extract paste, from the cephalothorax of white shrimp (Litopenaeus vannamei)”, Food Chem, Vol. 106 No. 1, pp.185–193. Bordbar, S., Ebrahimpour, A., Azizah, A. H., Mohd Yazid, A.M., Anwar, F., and Saari, N. (2013). “The improvement of the endogenous antioxidant property of Stone Fish (Actinopyga lecanora) tissue using enzymatic proteolysis”, ISRN Biomed Res Int. Church, F.C., Swaisgood, H.E., Porter, D.H., and Catignani, G.L. (1983), “Spectrophotometric assay using o-phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins”, Journal Dairy Sci, Vol. 66, pp. 1219–1227. Elias, R.J., Kellerby, S.S., and Decker, E.A. (2008), “Antioxidant activity of proteins and peptides”, Critical Reviews in Food Sci Nutr, Vol. 48, pp. 430–441. Erdmann, K., Cheung, B.W.Y., and Schröder, H. (2008), “The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease”, J Nutr Biochem, Vol. 19, pp. 643–654. Hoyle, N.T. and Merritt, J.H. (1994), “Quality of fish protein hydrolysate from Herring (Clupea harengus)”, J Food Sci, Vol. 59, pp. 76–79. Oyaizu, M. (1988). “Antioxidative activities of browning products of glucosamine fractionated by organic solvent and thin-layer chromatography”, Nippon Shokuhin Kogyo Gakkaishi, Vol. 35, pp. 771-775. Sampath, N.S., Nazeer, R.A., and Jaiganesh, R. (2011), “Purification and biochemical characterization of antioxidant peptide from horse mackerel (Magalaspis cordyla) viscera protein”, Peptides, Vol. 32, pp. 149–156. Sarmadi, B.H. and Ismail, A. (2010), “Antioxidative peptides from food proteins”, Peptides, Vol.31, pp. 1949–1956. See, S. F. (2011), “The antioxidant activities of gelatin hydrolysates obtained from african catfish (Clarias gariepinus) and salmon (Salmo salar) skin as influenced by degree of hydrolysis”, The 12th Asean Food Conference, Bangna, Bangkok. You, L., Zhao, M., Regenstein, J.M., and Ren, J. (2010), “Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion”, Food Chem, Vol. 120, No. 3, pp. 810–816. Zhu, K., Zhou, H., and Qian, H., (2006), “Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with alcalase”, Process Biochem, Vol. 41, pp. 1296–1302.

47

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Palm Sugars Improve Starch Digestibility of Bakery Products Rumpai Thongta* and Khongsak Srikaeo Food Science and Technology Program, Faculty of Food and Agricultural Technology Pibulsongkram Rajabhat University, Muang, Phitsanulok 65000 Thailand * Corresponding email: [email protected]

ABSTRACT Palm sugar has been used as a traditional sweetener for thousands of years in Asia. It is now gaining popularity globally because of its natural, occurrence, minimal processing requirement and health giving properties. This paper examins starch digestibility, physical and sensory characteristics of bakery products (breads and cookies) produced by using palm sugars from Borassus flabellifer (palm sugar) and Cocos nucifera (coconut sugar) in comparison with cane sugar (sucrose) and sorbitol. It was found that sorbitol provided the slowest starch digestion rate and consequently the lowest estimated glycemic index (GI) values. Palm and coconut sugars provided better starch digestion rate and lower GI values than those of cane sugar, indicating their nutritional quality over the cane sugar. However, sweeteners affected the physical and sensory characteristics of the products as evidenced by appearance, color, texture, aw and sensory scores. This research suggests that palm sugars might be used to replace cane sugar in bakery products for lowering GI purposes but care must be taken as they induced changes in the properties of the products. Keywords: Palm sugar, Coconut sugar, Bakery product

INTRODUCTION Palm sugar has been used as a traditional sweetener for thousands of years in Asia. It is now gaining popularity globally because of its natural, minimal processed and healthy. One of the major health claims is its glycemic index (GI). Low GI foods play an important role in the dietary management of diabetes, weight reduction, peak sport performance and the reduction of risks associated with heart disease and hypertension (Jenkins et al., 2002). This paper examined the physicochemical properties and starch digestibility of bakery products (breads and cookies) produced by using palm sugars from Borassus flabellifer (palm sugar) and Cocos nucifera (coconut sugar) in comparison with cane sugar (sucrose).

MATERIALS AND METHODS Wheat flour was analyzed for starch composition including total starch (TS), resistant starch (RS) and non-resistant starch (Non-RS) following the approved AACC Method 32-40. It was then mixed with four types of sugars (cane sugar, palm, coconut and sorbitol) at the same sweetness level. The sweetness level for cane sugar, palm and coconut sugars is 1.0 while the sorbitol is 0.6 (Whelan et al., 2008). The mixtures were analyzed for starch digestibility and

48

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

estimated GI using the rapid in vitro digestibility assay (Sopade and Gidley, 2009). The flour was then used to produce breads and cookies using general recipes. The cane sugar was substituted with palm sugar, coconut sugar and sorbitol at the same sweetness level. Physical properties of the products were evaluated including color (L* a* b* system), aw, texture by a texture analyzer and sensory characteristics using 9-point hedonic scaling method.

RESULTS AND DISCUSSION RS, Non-RS and TS of the wheat flour sample were found to be 3.91±0.06, 91.99±0.93 and 96.80±0.62 g/100 g dry sample, respectively. The digestograms are shown in Figure 1.

Figure 1 Starch digestogram of the wheat flour and sweetener mixtures The flours were used to produce the breads and cookies. The appearances of the breads are shown in Figure 2. The physical properties and sensory characteristics of the breads are shown in Tables 1-2 respectively.

49

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Figure 2 Appearances of the breads produced from a) cane sugar (control), b) coconut sugar, c) palm sugar and d) sorbitol Table 1 Physical properties and average estimated glycemic index (GI) of the breads produced using different sweeteners Breads Cane sugar Coconut Palm Sorbitol

L* 65.40 ± 0.71a 61.60 ± 0.42c 60.65 ± 0.07c 62.45 ± 0.64b

Color a* 3.15 ± 0.07b 3.60 ± 0.14a 3.70 ± 0.00a 3.50 ± 0.00a

aw b* 19.55 ± 0.21b 20.55 ± 007a 20.40 ± 0.00a 19.55 ± 0.07a

0.94 ± 0.01 a 0.92 ± 0.00b 0.89 ± 0.01c 0.87 ± 0.00d

Firmness (g) 857.71 ± 1.03b 8 12.66 ± 8.07d 884.88 ± 7.87a 833.18 ± 5.55c

GIAVG 81.34 ± 0.96a 65.67 ± 0.12b 63.92 ± 1.27b 55.78 ± 0.14c

Values with the same letters in column are not significantly different (P > 0.05).

Table 2 Sensory characteristics of the breads produced using different sweeteners Breads Cane sugar Coconut Palm Sorbitol

Color

Odor

Sweetness

Texture

7.67 ± 0.71a 7.40 ± 0.77a 7.60 ± 0.86a 7.77 ± 0.77a

6.67 ± 0.96ab 6.80 ± 0.89ab 6.93 ± 0.69a 6.37 ± 0.96ab

6.80 ± 0.92a 6.80 ± 0.96a 6.67 ± 0.99a 4.23 ± 1.07b

7.20 ± 1.03a 7.03 ± 0.89b 7.03 ± 1.07b 4.47 ± 1.22c

Overall acceptability 7.40 ± 0.89a 7.13 ± 0.90a 7.00 ± 1.08a 4.60 ± 1.30b

Values with the same letters in column are not significantly different (P > 0.05).

Considering the density of the black/white pixels in Figure 2, the appearances as determined by image analysis techniques (Srikaeo et al., 2011), gave acceptable results. The porosity as indicated by percentages of the white pixels in the images was found to be 57.14±0.91%, 57.41±1.23%, 56.03±2.21% and 56.92±1.61% for the breads made with cane sugar, coconut, palm and sorbitol respectively. The color and texture including water activity changed in according to the sugars used. In terms of cookies, the appearances of the cookies are shown in Figure 3. The physical properties and sensory characteristics are shown in Tables 3-4 respectively. Unlike breads, the appearances of cookies made using different types of sugars gave different appearances. Sorbitol gave the lowest sensory scores when compared to those from the other sugars.

50

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Figure 3 Appearances of the cookiesproduced from a) cane sugar (control), b) coconut sugar, c) palm sugar and d) sorbitol Table 3 Physical properties and average estimated glycemic index (GI) of the cookies produced using different sweeteners Cookies Cane sugar Coconut Palm Sorbitol

Color

aw

L*

a*

b*

66.43 ± 0.21b 61.20 ± 0.35d 62.10 ± 0.00c 67.40 ± 0.10a

5.33 ± 0.06c 8.83 ± 0.25b 9.47 ± 0.06a 5.33 ± 0.12c

29.47 ± 0.50c 31.07 ± 0.57b 32.40 ± 0.00a 29.63 ± 0.38c

0.33 ± 0.01c 0.36 ± 0.01b 0.38 ± 0.01b 0.40 ± 0.00a

Expansion ratio (mm) 0.78 ± 0.03b 0.81 ± 0.05b 0.82 ± 0.04b 0.90 ± 0.00a

Break strength (g) 764.21 ± 3.29a 768.85 ± 0.80a 764.34 ± 1.23a 664.23 ± 4.31b

GIAVG 86.14 ± 1.73a 49.39 ± 0.08b 48.14 ± 0.87b 28.31 ± 0.18c

Values with the same letters in column are not significantly different (P > 0.05)

Table 4 Sensory characteristics of the cookies produced using different sweeteners Breads Cane sugar Coconut Palm Sorbitol

Color

Odor

Sweetness

Texture

7.33 ± 0.88a 7.53 ± 0.73a 7.37 ± 0.81a 7.50 ± 0.82a

6.80 ± 1.03b 6.80 ± 1.12b 6.47 ± 1.04b 7.13 ± 0.89a

6.47 ± 1.20a 6.77 ± 1.25a 6.47 ± 1.11a 4.70 ± 0.70b

6.93 ± 0.20a 6.93 ± 0.26a 6.93 ± 0.11a 6.10 ± 0.76b

Overall acceptability 7.23 ± 0.85a 7.37 ± 0.78a 7.23 ± 0.86a 6.57 ± 1.01b

Values with the same letters in column are not significantly different (P > 0.05)

This research highlights the findings from coconut and palm sugars. As mentioned earlier, very few published works have proved that palm sugars were low in GI values and suitable for low GI foods. Only one recent published work has reported that the GI of coconut sap sugar was in low category (Trinidad et al., 2010). The estimated GI values of the breads and cookies made using coconut or palm sugars were found to be significantly lower than those of cane sugar. The major components of palm sugars are sucrose˜70-80%) with glucose (˜3-9%) and fructose (˜3-9%) (Purnomo, 1992). Although, the major sugar component in palm sugars are sucrose, similar to cane sugar, palm sugars are minimally processed and their natural forms are complex and contain other ingredients rather than sugars. Palm sugars were reported to contain significant amounts of dietary fiber, especially inulin (Trinidad et al., 2010; Vayalil, 2012). These could play an important role in lowering the GI values.

51

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

CONCLUSION Sweeteners influenced the starch digestibility, physical and sensory characteristics of the wheat based food products breads and cookies in this study. This paper highlights palm and coconut sugars as alternative healthy sweeteners as they provided benefits in terms of GI values. In this study, palm and coconut sugars can be used to produce breads and cookies with lower GI properties than those made from cane sugar. Although palm and coconut sugars could influence the physical and sensory properties of the products they have milder effects than those of sorbitol.

ACKNOWLEDGEMENTS This research was financially supported by the Thailand Research Fund (TRF)–Grant No. MSD56I0142. The cooperation from the industry partner is greatly acknowledged.

REFERENCES Jenkins, D.J.A, Kendall, C.W.C., Augustin, L.S.A, Franceschi, S., Hamidi, M., Marchie, A., Jenkins, A.L., and Axelsen, M. (2002), “Glycemic index: Overview of implications in health and disease”, The American Journal of Clinical Nutrition, Vol. 76, pp. 266S273S. Purnomo, H. (1992), “Sugar components of coconut sugar in Indonesia”, ASEAN Food Journal, Vol. 7, pp. 200-201. Sopade, P.A. and Gidley, M.J. (2009), “A rapid in-vitro digestibility assay based on glucometry for investigating kinetics of starch digestion”, Starch-Starke, Vol. 61, pp. 245-255. Srikaeo, K., Khamphu, S., and Weerakul, K. (2011), “Peeling of gingers as evaluated by image analysis techniques: A study for pickled ginger process”, International Food Research Journal, Vol. 18, pp. 1387-1392. Trinidad, T.P., Mallillin, A.C., Sagum, R.S., and Encabo, R.R. (2010), “Glycemic index of commonly consumed carbohydrate foods in the Philippines”, Journal of Functional Foods, Vol. 2, pp. 271-274. Vayalil, P.K. (2012). “Date fruits (Phoenix dactylifera Linn): An emerging medicinal food”, Critical Reviews in Food Science and Nutrition, Vol. 52, pp. 249-271. Whelan, A.P., Vega, C., Kerry, J.P., and Goff, H.D. (2008), “Physicochemical and sensory optimisation of a low glycemic index ice cream formulation”, International Journal of Food Science & Technology, Vol. 43, pp. 1520-1527.

52

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Comparison of Antioxidant Properties of Hydrolyzed Skipjack Tuna (Katsuwonus pelamis) Dark Muscle as a Function of Degree of Hydrolysis Worrapanit Chansuwana and Pavinee Chinachotia* a Nutraceutical and Functional Food Research and Development Center, Learning Resources Center Prince of Songkla University, 15 Karnjanavanich Road, Hat Yai, Songkhla 90110 * Corresponding email: [email protected]

ABSTRACT Dark muscle from Skipjack tuna (Katsuwonus pelamis) was hydrolyzed by Alcalase and Flavourzyme at various enzyme concentrations and times. Hydrolysates treated with both enzymes with the same degrees of hydrolysis (DH) (23%, 36%, 45%, and 56%) were prepared and 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity and ferric reducing antioxidant power (FRAP) were analyzed. Hydrolysis profiles of hydrolysates demonstrated that DH and nitrogen recovery increased curvilinearly with incubation time and enzyme concentration. Peptides produced were found to increase in free radical scavenging and reducing power properties with increasing DH. Alcalase hydrolyzed samples were significantly more antioxidative than Flavourzyme hydrolyzed samples. DPPH IC50 decreased with increasing DH from 2.73 mg/ml (0% DH) to 0.6 mg/ml (56% DH). Peptides smaller than 3 kDa showed the highest DPPH radical scavenging capacity by between 10 and 39% from the control (initial or pre-hydrolysis). FRAP reducing power also increased with increasing DH with Alcalase being more effective than Flavouzyme. Hydrolysates from tuna dark muscle were found the most antioxidative (radical scavenging capacity and reducing power) at 56% DH. Keywords: Antioxidant, Hydrolysis, Dark muscle, Skipjack tuna, Protein hydrolysate

INTRODUCTION Tuna processing plants in Songkhla province, Thailand mainly involve Skipjack tuna (Katsuwonus pelamis). Tuna dark muscle is mainly used in pet food products. To find more value-added use, protease hydrolysis can be applied to convert this by-product into functional fish hydrolysate with improved biological functional activities. Antioxidative peptides were reported to improve wound healing, recovery from fatigue, and prevention of diseases related to stress (Young et al.,2013). Dark muscle from several tuna species exhibits antioxidative properties, such as Thunnus obesus (Je et al., 2008) and Thunnus tonggol (Hsu, 2010). The objective of this study was to investigate hydrolyzed peptides from Skipjack tuna dark muscle by enzymatic hydrolysis and the effect of degree of hydrolysis (DH) on antioxidant activities using Alcalase and Flavourzyme.

53

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

MATERIALS AND METHODS Sample and dark muscle hydrolysate preparation Dark muscle from Skipjack tuna (Katsuwonus pelamis) was kindly donated by Songkhla Canning Public Co., Ltd. (Songkhla, Thailand). Dark muscle was collected after steaming of whole tuna meat (100C for 30 min). Dark muscle hydrolysates were prepared following the method as previously reported (Chansuwan and Chinachoti, 2014). Briefly, one hundred gram of sample was continually mixed with deionized water at a ratio of 1:2 (W/V) and homogenized at a speed of 13,000 rpm for 1 min. The pH of homogenates was adjusted to 8.5 for Alcalase and 7.0 for Flavourzyme reaction using 6 N NaOH. The mixtures were then incubated at 55C (Alcalase) and 50C (Flavourzyme) for 20 min prior to enzymatic hydrolysis. The enzymatic hydrolysis was started with the addition of different amounts of enzymes at 0.5, 1, 2, and 4% w/w (protein basis). Hydrolysis was carried out for 0, 30, 60, 90, 120, 180, and 240 min and the reaction was stopped by heating at 95C for 15 min in a water bath with occasional agitation. All samples were cooled immediately in ice and the pHs of the samples were subsequently adjusted to 7.0 using 1 M HCl (if needed). These were then filtered through muslin cloth 3 times and the supernatants were collected. Hydrolysates with specific DH at 23%, 36%, 45%, and 56% were selected for studying the effect of DH on antioxidative activities. Fractionation by Ultrafiltration (UF) membranes Hydrolysates were fractionated using Amicon concentrator equipped with UF membrane (Millipore, USA) with molecular weight cut off (MWCO) of 30, 10, and 3 kDa to isolate four fractions (30 kDa). The process for fractionation was centrifugation samples (10 mg/ml) at 3,000xg at 4C for 30 min. Degree of hydrolysis (DH) DH was estimated from the amount of free amino acid groups which was analyzed by a reaction with Trinitobenzene sulfonic acid (TNBS) (Adler-Nissen, 1979). Nitrogen Recovery (NR) NR was used to describe the hydrolysis yield. Total nitrogen in the soluble fraction and total nitrogen in substrate (non-hydrolyzed sample) were determined using the Kjeldahl method (AOAC, 2000). Protein solubility Protein solubility of dark muscle hydrolysates was determined using the method of Tsumura et al. (2005) with slight modification. DPPH radical scavenging capacity The scavenging effect of hydrolysates against DPPH radicals was determined according to the method described by Brand-Williams et al. (1995) with slight modification. Trolox, ascorbic acid, and -tocopherol were used as positive controls. IC50 value, denoting the concentration of the sample required to scavenge 50% of the DPPH free radicals, was calculated from scavenging capacity (%) versus hydrolysate concentration curves.

54

18th World Congress on Clinical Nutrition (WCCN) “Agriculture, Food and Nutrition for Health and Wellness” Ubon Ratchathani, Thailand, December 1-3, 2014

Ferric reducing antioxidant power (FRAP) FRAP assay was determined according to Benzie and Strain (1996). Trolox was used as a positive control and the activity was expressed as μmol Trolox equivalents (TE)/g sample. Statistical Analysis Data were subjected to Analysis of Variance (ANOVA) and Duncan‟s Multiple Range Test. Paired comparisons were done using t-test (p