1 Development of a Fermented Pumpkin Water Kefir

1 Development of a Fermented Pumpkin Water Kefir (PWK) Beverage with Anti-hyperglycemic Potential Koh Wee Yin Food Technology Division, School of Industrial technology, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia FOR FURTHER INFORMATION Koh Wee Yin Email: wyin. kelly@gmail. com Tel: +60179196280 DR. Uthumporn Utra@Sapina Abdullah Email: sapina@usm. my Tel: +60125528141 Food Technology Division

2 Introduction/ Literature review • Pumpkin (Cucurbita pepo) • Known as “Nan gua (南瓜)”, “labu manis (Malay)” • Monoecious, flowering, tendril-bearing vine belonging to Cucurbitaceae family • Widely cultivated in frost-free tropical and subtropical lands, warm season crop (18 -30 °C is best for its growth) • Good source of carotenoids, dietary fibers and minerals • Biological effects: Ø Antidiabetic, hepatoprotective, hypotensive, antioxidative, antimicrobial • Pumpkin fruit applications: Ø Culinary Food Technology Division

3 Pumpkin Nutrient-dense and (Elhardallou, 2014). treatment of diabetets Very low in calorie (17 kcal/100 g flesh) (Adams et al. , 2011). Anti-diabetic compounds: trigonellin acid, nicotinic acid (Yoshinari et al. , 2009). Protein-bound polysaccharides favoured by probiotic for growth & adhesion (Du et al. , 2011). Fermented pumpkin fruit possessed antihyperglycemic effects, improved probiotics viability and survival in gastrointestinal tract (Zhao et al. , 2015; Genevois et al. , 2016; Semjonovs et al. , 2013). Rich in beta-carotene: a beautiful natural colorant and or nutritional precursor of Vitamin A (Zhao et al. , 2015). Polysaccharides Food Technology Division

4 Water Kefir ‒ Natural Probiotics Source • Non-dairy fermented beverage (fermented by Water Kefir grains) • Substrates ‒ fruit juices, coconut water, sugar water, fruit figs, cocoa pulp, fruit and vegetable pulp • Mother culture ‒ Water Kefir grains are combination of bacteria and yeasts in a bio-matrix structure of soluble gel-polysaccharide, called Kefiran. • Mostly consist of Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, acetic acid bacteria, and yeast (FAO/WHO, 2001; Farnworth, 2006) Food Technology Division

5 Benefits of Water Kefir Probiotics Improved protein digestibility, micronutrient bioavailability, and biological enrichment (Elizabeth, 2006) Anti-inflammatory, anti-ulcerogenic and angiogenic activities (Rodrigues et al. , 2016; Prado et al. , 2016) DNA protection, antioxidative (Fiorda et al. , 2016) Prevent Helicobacter pylori infection, diarrhea, constipation and colon cancer (Bekar et al. , 2011; Alsayadi et al. , 2014; Farnworth, 2006) Antihyperglycemic activities (Hadisaputro et al. , 2012; Alsayadi et al. , 2014) Water Kefir Grains Anti-microbial and healing activity (Rodrigues et al. , 2005; Huseini et al. , 2012) Delay the progression of diabetic complications (Punaro et al. , 2014) Food Technology Division

6 International Codex Alimentarius Standard ‒ Description of Kefir Definition (CODEX STAN 243 -2003) Starter culture prepared from kefir grains, Lactobacillus kefiri, and species of the genera Lactobacillus, Leuconostoc, Lactococcus and Acetobacter growing in a strong specific relationship. Kefir grains constitute both lactose-fermenting yeasts (Kluyveromyces marxianus) and non-lactosefermenting yeasts (Saccharomyces unisporus, Saccharomyces cerevisiae and Saccharomyces exiguus). (FAO/WHO, 2001) Composition: ØSum of specific microorganisms constituting the starter culture (cfu/g, in total): min. 107 ØYeasts (cfu/g): min. 104 Food Technology Division

7 Water Kefir Dairy free Yeast and bacteria strains (probiotics) and kefiran have hypoglycemic effects (Zheng et al. , 2013; Wang et al. , 2015). Fermented by water kefir grains (lactic acid bacteria (LAB), yeasts, and acetic acid bacteria (AAB) (Alsayadi et al. , 2014). Kefiran Lactic acid bacteria Yeast Acetic acid bacteria Species of microbiota in grains change significantly from kefir grains → kefir → second batch of kefir (Simova et al. , 2002). Water Kefir Grains Use of starter culture allow the production of uniform quality products (Beshkova et al. , 2002). Food Technology Division

8 Main Problem Statement Health benefits of Pumpkin with short shelf life Lactose intolerance and vegetarian consumer Approximately 5 million people die each year as a consequence of diabetes (IDF, 2017). Antidiabetic and probiotic properties Health conscious society Food Technology Division

9 Main objective • The main goal of this work is to produce a naturally sourced alternative of functional anti-hyperglycemic beverage. Food Technology Division

10 Novelty Pumpkin Water Kefir (PWK) 1. Utilization of Agricultural product (Pumpkin fruit) • New products • Pumpkin is low in fat and calories and rich in disease-fighting nutrients such as: § Alpha-carotene § Beta-carotene § Fiber § Vitamins C and E § Potassium and Magnesium Food Technology Division

11 Novelty Pumpkin Water Kefir (PWK) 2. Value added/functional drinks with new taste and flavour • High in antioxidant, vitamins and minerals • High amount of fiber • Unique flavor, authentic taste + Food Technology Division

12 Novelty Pumpkin Water Kefir (PWK) 3. Convenient and easy-to-consume • Ready-to-drink beverage offer convenience to today’s busy consumers 4. Good for digestion and great for heath • Supply gut-friendly good bacteria • Ease of digestibility • Excellent nutritional qualities • Unique functionality: help reduce after-meal blood glucose rise and maintain healthy level of postprandial blood glucose Food Technology Division

13 Novelty Pumpkin Water Kefir (PWK) 5. Probiotic and Antidiabetic properties Food Technology Division

14 Proximate Contents Proximate Analysis Moisture content 89. 23 Ash content 2. 81 Protein content 3. 32 Fiber content 2. 73 Fat content 0. 00 Carbohydrate content Minerals Experimental Value (%) 4. 47 PWK beverage (mg/L) Potassium 363. 01 ± 0. 00 Magnesium 143. 80 ± 0. 01 Calcium 120. 82 ± 0. 01 Sodium 69. 63 ± 0. 00 Phosphorous 11. 83 ± 0. 02 Copper 3. 91 ± 0. 02 Iron 0. 91 ± 0. 00 Manganese 0. 50 ± 0. 00 Selenium 0. 49 ± 0. 00 Zinc 0. 16 ± 0. 00 Food Technology Division

15 Comparison With Competitors Pumpkin Water kefir Orange Juice Apple Juice Mango Juice NUTRITIONAL INFORMATION Size per cup Calories Total carbohydrate Protein Fat Fibre Per 240 ml 60 kcal 10. 7 g 8. 0 g 0. 0 g 6. 5 g Calories Total carbohydrate Protein Fat Fibre Per 240 ml 110 kcal 25. 0 g 2. 0 g 0. 3 g 0. 5 g Calories Total carbohydrate Protein Fat Fibre Per 240 ml 140 kcal 34. 0 g 1. 0 g 0. 5 g 3. 0 g Food Technology Division

16 Comparing with Spritzer Fibre Ingredients: Water, Sugar, Oligofructose-Inulin mixture, Passion Fruit flavour, Citric Acid, Sodium Citrate, Sodium Benzoate, Contain permitted preservative, flavouring and food conditioners ANTIDIABETIC PROPERTIES SIGNIFICANT AMOUNT OF MINERALS Food Technology Division

17 Technological properties of PWK Beverage 12 • PWK beverage maintained 109 CFU/ml viable Lactobacillus mali K 8 strain throughout 28 days refrigerated (4 ◦C) storage. a 10 c d b e 8 6 b a e d c week 0 week 1 week 2 week 3 week 4 a b c e d Lactic acid Ethanol a a c c b c 4 b c d e 2 Figure: p. H value, lactic acid bacteria viability, residual sugar, lactic acid and ethanol formation, in the PWK beverage inoculated with Lactobacillus mali K 8 strain during storage for 4 weeks at 4◦C. Error bars represent standard deviation of all trials. Different superscripts have mean ranks that are significantly different among storage time (weeks). 0 p. H Viability Residual sugar -2 Food Technology Division

18 PWK Beverage: An Effective Inhibitors of α-Glucosidase and α-Amylase PWK beverage α-Amylase inhibitory activity IC 50 (mg/m. L) α-Glucosidase inhibitory activity IC 50 (mg/m. L) Acarbose (standard drug) 22. 22 ± 0. 00 19. 63 ± 0. 07 5. 14 ± 0. 00 0. 048 ± 0. 00 Values are given as means ± standard deviation (n = 3). IC 50, the concentration of pumpkin juice required to cause a 50% decrease in the enzymes’ activity. Food Technology Division

19 α-Glucosidase Inhibitors Mechanism Without enzyme inhibitors With enzyme inhibitors Carbohydrates Jejunu m Ileum Carbohydrate absorption Jejunum Ileum Food Technology Division

20 Effect of PWK on postprandial blood glucose level in vivo Normal rat blood glucose (mmol/L) 11 PWK Control 10 9 B Acarbose Aa Ab Ac 8 7 Bb 6 Bb 5 Ba Bb Ba Cc 4 0 30 60 Blood collection timepoints (minutes) 120 Diabetic rat blood glucose (mmol/L) A PWK Control 25 Acarbose Aa Ac 23 Ab Ba 21 Bb 19 Bc Ca Cb 17 Cc 15 0 30 60 Blood collection timepoints (minutes) 120 Figure: The glycemic response curve in blood glucose levels (mmol/L) after the administration of sample (PWK or standard drug (acarbose) in normal rat (A) and diabetic rat (B) Three treatment groups i. e. acarbose (10 mg/kg); PWK, fermented pumpkin water kefir beverage (109 CFU/m. L/rat); and control (distilled water), were orally co-administered with starch (2 g/kg). Each value is expressed as the mean ± standard deviation of eight rat per group (n=48). Food Technology Division

21 Sensory Properties of PWK Consumer perception on PWK– Acceptable in all attributes and comparable with commercial product Appearance 9 8 7 6 5 4 3 2 1 0 Overall acceptibility Texture Aroma Commercial product PWK Taste Food Technology Division

22 Overall Sensory Acceptance of PWK Neither like nor dislike 13% 2% Like moderately 48% 37% Like very much Like extremely Food Technology Division

23 Comparability to Commercialized Product 11% 89% No Yes Food Technology Division

24 Future Purchase 7% 93% will buy will not buy Food Technology Division

25 Future Planning 2018 ESTABLISHMEN T, PROMOTION, CERTIFICATION 2019 DISTRBUTION (PENINSULAR MSIA), ISO AND HACCP 2020 2021 2022 MAINTAIN DISTRIBUTION, PROMOTION (TV, MAGAZINE) IMPROVISE AND INCREASE THE PUBLICITY OVERSEA MARKETS RESEARCH AND DEVELOPMENT CENTER CONTINUOUS INNOVATION AND CREATIVENESS Food Technology Division

26 Acknowledgements • The Ministry of Science and Technology of China, the People’s Government of Guangdong Province and the Chinese Academy of Sciences • USM Vice-Chancellor Award Scholarship • Research University Grant (RU) ‒ 1001/PTEKIND/811339 (RM 100, 000) (1/3/2016 – 28/2/2019) • ASEAN University Network (AUN) and Korea Association of Southeast Asian Studies (KASEAS) ‒ ASEAN-Korea Exchange Fellowship Program 2016/2017 Food Technology Division

27 Publications • Optimization of a fermented pumpkin-based beverage to improve Lactobacillus mali survival and αglucosidase inhibitory activity: A response surface methodology approach – In Press – Online (Food Science and Human Wellness) • Development of a novel fermented pumpkin-based beverage inoculated with water kefir grains: a response surface methodology approach – Accepted (Food Science and Biotechnology) • Isolation and Characterization of the Probiotic and Anti-hyperglycemic Potential of a Novel Lactobacillus Strain Isolated from Water Kefir Grains – Accepted (Food Science and Biotechnology) • Fermented pumpkin-based beverage extenuates postprandial hyperglycemia in Type-2 diabetic rats by inhibiting carbohydrate digestive key enzymes – Under review (Bioscience, Biotechnology and Biochemistry) Food Technology Division

28 THANK YOU VERY MUCH Food Technology Division