THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY MA THI NGOC DIEP Topic title UTILIZATION OF YEAST AND ENZYME PRODUCTS FOR ADDED VALUE OF THAI HONEY NEO-FOOD FOR AGING SOCIETY BACHELOR THESIS Study course : Full-time Major : Food Technology Faculty : Biotechnology and Food Technology Period : 2014 – 2018 Thai Nguyen, 06/2018 h THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY MA THI NGOC DIEP Topic title: UILIZATION OF YEAST AND ENZYME PRODUCTS FOR ADDED VALUE OF THAI HONEY NEO-FOOD FOR AGING SOCIETY BACHELOR THESIS Study course : Full-time Major : Food Technology Faculty : Biotechnology and Food Technology Period : 2014 – 2018 Supervisor : Assoc. Nitnipa Soontorngun Msc. Dinh Thi Kim Hoa Thai Nguyen, 06 /2018 h i DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Food Technology Student name Ma Thi Ngoc Diep Student ID DTN1453170074 Utilization of yeast and enzyme products for added-value Thesis title of Thai honey neo-food for aging society 1. Nitnipa Soontorngun Supervisors 2.
Dinh Thi Kim Hoa There are many factors that affect the normal activity of the cell, cell damage, cell death, because of aging and diseases such as alcohol, smoking, mental stress, UV rays, fast food, pollution [39]. Humans protect themselves from these damaging compounds, in part, by absorbing antioxidants from high-antioxidant foods [40]. So antioxidants can play important roles in treatment of several dieases [24]. Many kinds of food have antioxidants which have benefits for health, for example fruits, vegetables, drink, fish, chocolate [33].
Honey have high proportion of antioxidants (Tualang 18.511mgRE/100 g Honey; Gelam 32.886 mgRE/100g Honey; Acacia 30. People can produce honey in many products in life that is good for health and can apply in cosmetics, food, and pharmaceuticals. It is easy to see many famous products in the market such as: Pharmacy Honey Potion Warming Face Mask (America) [36], Honeyed whiskey wine , (America) and Royal bee (Thailand), Wildflower honey, Manuka honey, Acacia Honey (Canada) [25], Manukaguard Medical Grade Manuka Honey 12+ Dietary Supplement, 8. In addition, with social development my research focuses on antioxidant content of trigonal.
honey collected from Thailand. Purpose of this research is to create new product from h ii honey helpful for society, specially which is rich in antioxidant contents with great value such as anti-aging, anti-cancer, anti-inflammatory. My research focuses on utilization of yeast and enzyme products for added-value of Thai honey neo-food for aging society. Firstly, we determine the total flavonoid content to estimate the antioxidant ability of honey.
After that, we use yeast strains S. cerevisiae without two genes called the Sod1, Cta1 for checking the role of these genes in the survival of yeast strains S. If honey has an activity of two strains it means that honey can remove activity of reactive oxygen species (ROS) and can protect cell [2]. The activity of Sod1 and Cta1 is important because these enzymes can remove free radicals which can damage cells.
The activities of superoxide dismutase (Sod1), catalase (Cta1) constitute a first line antioxidant defense system which plays a key and fundamental role in protection cells from oxidative stress [23]. My research also focuses on characterization of superoxide dismutase (Sod1), Catalase (Cta1) role in antioxidance. Sod1 catalyzes the dismutation of superoxide anion (O2−) to H2O2 and O2 [13], Sod1 plays important role of enzyme activity to decrease the free radicals. Cta1 is a common enzyme that catalyzes the decomposition of hydrogen peroxide to water and oxygen [9].
Key words Antioxidant, Reactive oxygen species, Flavonoid, S. cerevisiae, Superoxide dismutase, Catalase Number of pages 28. h iii ACKNOWLEDGEMENT This thesis was completed by the support and assistance of a number of people whom I would like to personally thank. First and foremost, I would like to express my gratitude to my supervisor Assoc.
Nitnipa Soontorngun the school of Bioresources and Technology, King Mongkut’s University of Technology Thonburi (KMUTT), Thailand. I would like to thank Mrs. Dinh Thi Kim Hoa from the Faculty of Biotechnology and Food Technology, Thai Nguyen University of Agriculture and Forestry (TUAF). Thier expertises and understanding guided me through my internship, providing useful advice for the improvement of this work.
Some special thanks also to my lab facilitator, Mrs. Siriporn Thongmee for sharing her knowledge and her time, for making amazing experiments. Big thanks also go to every people on Gene Technology Lab Program at KMUTT for helping me fit in and feel welcome from the moment and for the unlimited patience to explain me every doubt I had during my internship. I would also like to acknowledge my teachers at TUAF, MSc.
Dinh Thi Kim Hoa, MSc.Trinh Thi Chung that contributed to make this work and had an enjoyable and fulfilling experience. Thai Nguyen, 10th, June, 2018 Many thanks and best regards, Student Ma Thi Ngoc Diep h iv CONTENTS DOCUMENTATION PAGE WITH ABSTRACT. iv LIST OF ABBREVIATION. vi LIST OF TABLE.
vii LIST OF FIGURES. viii PART 1: INTRODUCTION. Honey and the situation of honey production in the world. Activity of gene superoxide dismutase and catalase.
Overall goal of the research. Scientific significance and practical meaning. 13 PART 2: MATERIALS AND METHODS. Equipment and Materials .2 The method for determining the total flavonoid content .3 Function of Sod1 and Cta1 under hydrogen peroxide assay .2 Function of Sod1 and Cta1 under hydrogen peroxide assay .4 Activity Sod1 of honey in saccharomyces cerevisiae under hydrogen peroxide assay .2 Activity Sod1 of honey in Saccharomyces cerevisiae under hydrogen peroxide assay.
18 PART 3: RESULTS AND DISCUSSIONS .2 Total flavonoid content .3 Function of Sod1 under hydrogen peroxide assay………………………….4 Activity Sod1 of honey in saccharomyces cerevisiae under hydrogen peroxide assay….25 PART 4: CONCLUSIONS AND SUGGESTIONS. 28REFERENCES 29 h vi LIST OF ABBREVIATION Cta1 Catalase Sod1 Superoxide dismutase Nm Nanometer Mg Milligram Ml Milliliter µl Microliter mg/ml Milligram per milliliter 0 C Degree Celsius WT Wild type % Percent MeOH Methanol CH3COOH Acid acetic Rpm Revolutions per minute M Molar Mm Millimolar Ccs1p Copper Chaperone for SOD1 Deletion S. cerevisiae Saccharomyces cerevisiae h vii LIST OF TABLE Table 2.1: Materials and formula for make YPD broth and YPD agar…………………….2: Materials and formula for make YPD broth and YPD agar…………………….Error! Bookmark not defined.2: Absorbance of rutin ……………………………………………………….3: Total flavonoid content of 2 honey fractions…………………………………….4: Compare total flavonoid content in honey fractions from Thailand and Malaysia…23 h viii LIST OF FIGURES Figure 1.1: Types of flavonoids and sources……………………………………………….2: Basis structure of flavonoids………………………………………………….3: Causes of oxidative stress…………………………………………………….4: Free radical effects to heathy atoms…………………………………………….5: Benefits of antioxidant molecules…………………………………………….6: Antioxidant activity of enzyme superoxide dismutase………………………….7: Process catalase catalyzes the converted of hydrogen peroxide to oxygen and water…………………………………………………………………………………….1: Standard curve of rutin (mg/ml)………………………………………………. The chart of Total flavonoid content (mg/ml) of Honey fraction 1 and Honey fraction 2…………………………………………………………………………………………22 Figure 3.3: Cell survival of WT, Sod1 and Cta1 strains……………………………….4: Cell survival of Sod1 strain under different conditions……………………….
Honey and the situation of honey production in the world Honey is a sweet and natural product made from flower nectar, combined with an enzyme secreted by honey bees, then concentrated by reducing moisture in the honeycomb cells. Honey has many nutrients so honey has been used humans since ancient times, nearly 5500 years ago, most ancient population, including the Greeks, Thailand, Chinese, Egyptians, Romans, Mayans, and Babylonians with purpose consume honey both for nutrition aims and for its medicinal properties [31]. Honey is a food rich in nutrients and good for health, such as antioxidants, anti- aging, anti-inflammatory, honey help increase the health of people and increase the resistance to health [31]. Honey is food has great potential, nutritional food, functional foods, cosmetics, and medicine.
So many countries invest in honey production [1]. Honey has producing largest countries in the world such as (2018): China, Turkey, United States of America, Iran, Russian Federation, and India. Besides, there are countries in honey productions: Thailand, Laos, Cambodia, Indonesia, and Vietnam. Honey is natural product from bee consisting of a highly concentrated solution with element main is the complex mixture of sugar and little quantities of other constituents, such as minerals, proteins, vitamins, organic acids, flavonoids, phenolic acids, enzymes, and volatile compounds [34].
The quantity of these different compounds varies greatly depending on the floral and geographical origin of the honey [34]. Special, Honey components reported to be responsible for its antioxidant effects are flavonoids, phenolic acids, ascorbic acid, catalase, carotenoids. Numerous studies have reported that most chronic disease such as cancer, coronary, heart disease, neurological and many other diseases originated by oxidative damage. Studies are also proven that the therapeutic potential and increase health of honey h 2 with people is always associated with antioxidant capacity against reactive oxygen species [11].
Therefore, in recent years apply for studies have been focused on the composition special of kinds of honey is their biological properties such as antioxidant, anti-inflammatory, anti-aging, anticancer and antimicrobial activities in wound healing, as well as in the treatment of skin ulcers and gastrointestinal disorders [11]. Research has contributed to increase the value of using honey and many honey product, in today's life, honey is a very popular product. Honey has many antioxidant ingredients. Especially in honey contains flavonoid ingredients should have ability good antioxidant.
Flavonoids are polyphenolic compounds that contain a C15 flavone skeleton (diphenylpropane) and are collectively known as vitamin P [20]. Currently, Honey have many studies research about total flavonoid content, currently, there are many research studies that show high levels of flavonoids: (Tualang 18.511mgRE/100 g Honey; Gelam 32.886mg RE/100 g Honey; Acacia 30.74mgRE/100g Honey) from Malaysia [5], so honey has good antioxidant properties. Flavonoids have important in the protection of health such as: anti-inflammatory (Yamamoto & Gaynor, 2001), anti-microbial (Tim Cushnie & Lamb, 2005), antioxidant (Shahidi & Wanasundara, 1992), anti- cancer (Wei, Tye, Bresnick, & Birt, 1990) activity as well as the prevention of osteoporosis (Migliaccio & Anderson, 2003) [10]. Honey has many benefits in life that promote health and prevent disease.
My research was to determine total flavonoid content in two honey samples in Thailand Trigona spp. stingless honey bee from Bee park (Collected 6/12/2017) and Trigona spp. stingless bee honey from the resort (Collected 4/12/2017) and check activity of gene Sod1, Cta1, in the honey sample, to determine antioxidant content in honey. Flavonoid Introduction flavonoid Flavonoids are a large family of substances (more than 4000, of which several hundred are found in edible plants) flavonoid are antioxidant compounds found in plants, fruits and some animals [27].
Moreover, Honey have a lot of flavonoid compounds it good for health human can protect body attack of reactive oxygen species, protect the cell. Flavonoids are a ubiquitous group of naturally occurring polyphenolic compounds characterized by the flavan nucleus and is one of the most prevalent classes of compounds in fruits and insect, vegetables and plant-derived beverages. In addition, honey contains flavonoids characteristic of antioxidants. The flavonoids structure have been identified and many of which are responsible for the attractive colors of flowers, fruits and leaves function create color.
Flavonoids in plants can protect plants from the harmful effects of insects [28]. Flavonoids a class of polyphenolic compounds widely distributed in plants. Quercetin and rutin are among the most largely found flavonoids in a great variety of fruits and vegetables, insect, including tea, coffee, and other grains. Special, rutin found into insect have [42] Flavonoids have the potential to neutralize free radicals, giving an electron an antioxidant molecule that stabilizes free radicals, free radicals that contain unstable molecules in the electron-donning state of normal molecules [18].
The antioxidant will give one electron for free radical help free radical become normal molecular The antioxidant properties of flavonoids are based on an ability the activity of enzymes involved in the remove of free radicals and in the elimination of active free radicals. Flavonoid is compound remove activity of reactive oxygen species. In addition, flavonoids have the function to prevent oxidation of vitamin C and other detrimental reactions caused by active oxygen.