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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 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.Prof.Dr Nitnipa Soontorngun Msc Dinh Thi Kim Hoa Thai Nguyen, 06 /2018 i DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Major Food Technology Student name Ma Thi Ngoc Diep Student ID DTN1453170074 Thesis title Supervisors Utilization of yeast and enzyme products for added-value of Thai honey neo-food for aging society Assoc.Prof.Dr Nitnipa Soontorngun Msc 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 30.741 mgRE/100g Honey) [11] 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, 8.8 Ounce (New Zealand) [37] In addition, with social development my research focuses on antioxidant content of trigonal.ssp honey collected from Thailand Purpose of this research is to create new product from 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 cerevisiae [26] 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, cerevisiae, Superoxide dismutase, Catalase Number of pages 28 Flavonoid, S 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.Prof.Dr 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 iv CONTENTS DOCUMENTATION PAGE WITH ABSTRACT i ACKNOWLEDGEMENT iii CONTENTS iv LIST OF ABBREVIATION vi LIST OF TABLE vii LIST OF FIGURES viii PART 1: INTRODUCTION 1.1 Background 1.1.1 Honey and the situation of honey production in the world 1.1.2 Flavonoid 1.1.3 Free radical 1.1.3 Antioxidant 1.2 Saccharomyces cerevisiae 1.3 Activity of gene superoxide dismutase and catalase 1.3.1 Superoxide Dismutase 10 1.3.2 Catalase 12 1.4 Objectives 13 1.4 Overall goal of the research 13 1.4.2 Detail goal 13 1.5 Scientific significance and practical meaning 13 1.5.1 Scientific significance 13 1.5.2 Practical meaning 13 PART 2: MATERIALS AND METHODS 14 2.1 Equipment and Materials 14 2.1.1 Materials 14 2.1.2 Chemicals 14 2.1.3 Equipment 14 2.2 Methods 15 v 2.2.1 Honey extraction 15 2.2.2 The method for determining the total flavonoid content 16 2.2.3 Function of Sod1 and Cta1 under hydrogen peroxide assay 16 2.2.3.1 Prepare media 16 2.1.2.2 Function of Sod1 and Cta1 under hydrogen peroxide assay 17 2.2.4 Activity Sod1 of honey in saccharomyces cerevisiae under hydrogen peroxide assay 17 2.2.4.1 Prepare media 17 2.2.4.2 Activity Sod1 of honey in Saccharomyces cerevisiae under hydrogen peroxide assay 18 PART 3: RESULTS AND DISCUSSIONS 20 3.1 Honey extraction 20 3.2 Total flavonoid content 20 3.3 Function of Sod1 under hydrogen peroxide assay………………………… 23 3.4 Activity Sod1 of honey in saccharomyces cerevisiae under hydrogen peroxide assay… 25 PART 4: CONCLUSIONS AND SUGGESTIONS 27 4.1 CONCLUSIONS 27 4.2 SUGGESTIONS 28REFERENCES 29 vi LIST OF ABBREVIATION Cta1 Catalase Sod1 Superoxide dismutase Nm Nanometer Mg Milligram Ml Milliliter µl Microliter mg/ml Milligram per milliliter Degree Celsius C 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 vii LIST OF TABLE Table 2.1: Materials and formula for make YPD broth and YPD agar…………………… 17 Table 2.2: Materials and formula for make YPD broth and YPD agar…………………… Error! Bookmark not defined Table 3.1: Honey extraction…………………………………………………………… 20 Table 3.2: Absorbance of rutin ……………………………………………………… 20 Table 3.3: Total flavonoid content of honey fractions…………………………………… 21 Table 3.4: Compare total flavonoid content in honey fractions from Thailand and Malaysia…23 viii LIST OF FIGURES Figure 1.1: Types of flavonoids and sources……………………………………………….4 Figure 1.2: Basis structure of flavonoids………………………………………………… Figure 1.3: Causes of oxidative stress…………………………………………………… Figure 1.4: Free radical effects to heathy atoms…………………………………………….5 Figure 1.5: Benefits of antioxidant molecules…………………………………………… Figure 1.6: Antioxidant activity of enzyme superoxide dismutase………………………… 10 Figure 1.7: Process catalase catalyzes the converted of hydrogen peroxide to oxygen and water…………………………………………………………………………………… 12 Figure 3.1: Standard curve of rutin (mg/ml)……………………………………………… 21 Figure 3.2 The chart of Total flavonoid content (mg/ml) of Honey fraction and Honey fraction 2…………………………………………………………………………………………22 Figure 3.3: Cell survival of WT, Sod1 and Cta1 strains……………………………… 23 Figure 3.4: Cell survival of  Sod1 strain under different conditions……………………… 25 19 Step 12: Put 500µl of cell with 500µl of DI water and measure OD=600nm, formula make start cell 0.01 in volume 100µl Step 13: Put 2mM of H2O2 and H2O (2µl) with cell and YPD broth or 50% methanol or honey 1mg/ml into well plate Step 14: Incubation hour at 30 degree Celsius (use biosan thermos machine) Step 15: After that make serial dilution solution 10% (4 time) Step 16: Use 2µl of solution for spot (dark room) Step 17: Spread use 50µl on plate (dark room) Step 18: Incubation days after calculate cells survival 20 PART RESULTS AND DISCUSSIONS 3.1 Honey extraction Table 3.1: Honey extraction Sample Honey crude Honey extraction Sample 40 ml 22.615 mg Sample 40 ml 14.7 08 mg Raw honey were separated by adding ethyl acetate to remove sugar and water The result from table 3.1 showed that sample (Honey from bee park of MMUTTThailand) had higher extraction yield than that of sample (Honey from resort of KMUTT- Thailand), with amount of 22.615 mg and 14.708 mg, correspondingly It can be inferred that with the same volume of raw honey (40ml) the difference of amount between two extracted samples can be explained due to various ingredients in honey For instant, Sample may have higher ratio of sugar than that of sample Honey fraction dissolved in 50% MeOH at ratio (mg/ml): Sample were 22.615 mg of honey fraction was dissolved in 50% MeOH (22.615 mg), Sample were 14.708 mg of honey fraction was dissolved in 50% MeOH (14.708 mg) to keep them pure and carry out next experiments 3.2 Total flavonoid content Table 3.2: Absorbance of rutin (mg/ml) Concentration of rutin (mg/ml) 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 Ads Abs Abs Abs Abs Average 0.225 0.230 0.238 0.602 0.609 0.862 1.108 1.156 1.174 1.488 0.215 0.266 0.336 0.843 0.849 0.880 1.106 1.243 1.394 1.440 0.195 0.267 0.280 0.569 0.700 0.851 0.952 1.115 1.273 1.360 0.319 0.402 0.636 0.926 1.108 1.188 1.420 1.780 1.968 2.280 0.341 0.662 0.692 0.759 0.861 0.993 1.243 1.300 1.642 2.002 0.259 0.365 0.436 0.739 0.825 0.955 1.166 1.319 1.490 1.714 21 Absorbance of rutin (mg/ml) increate with concentration of rutin (mg/ml), absorbance at 415nm Absorbance of rutin high more at 0.1 (mg/ml) rutin and low more at 0.01 (mg/ml) rutin This table build standard curve of rutin (mg/ml)  Standard curve of rutin (mg/ml) Figure 3.1: Standard curve of rutin (mg/ml) From this fig can see standard curve of rutin (mg/ml) at absorbance 415nm Spend on formula Y= 16,24x + 0,0336 with R2 = 0.9905 can calculate total flavonoid of honey (mg/ml) Table 3.3: Total flavonoid content of honey fractions Concentration of honey fraction (mg/ml) Honey fraction 1(mg/ml) Honey fraction 2(mg/ml) mg/1 ml 0.143 0.131 0.5 mg /1 ml 0.069 0.567 0.25 mg/1 ml 0.060 0.049 0.12 mg/1 ml 0.056 0.036 0.06 mg/1ml 0.022 0.033 Table 3.3 show total flavonoid content of honey fractions at different concentration of honey fraction (mg/ml) Total flavonoid of honey fraction at 22 1mg/1ml honey fraction has 0.143 and honey fraction has 0.131 we can see honey fraction has high concentration more than honey fraction Total flavonoid content at lower concentration see that honey fraction has high concentration more than honey fraction  Total flavonoid content (mg/ml) of Honey fraction and Honey fraction with concentration of mg/ml Figure 3.2 The chart of Total flavonoid content (mg/ml) of Honey fraction and Honey fraction It can be seen from the table 3.5 and bar chart 3.2 that flavonoid content of two honey samples was different Honey Bee Park (sample 1) had total flavonoid content at 0.144 mg of rutin/mg, and Honey Resort (sample 2) had total flavonoid content at lower level with 0.131 mg of rutin/mg The difference between these total flavonoid contents may be because of their floral source and its collection region This is also reported in the publication of Lee Suan Chua in 2013 [5] Comparing the total flavonoid contents of two honey samples in this research with honey samples from Malaysia, the result was shown in table 3.6 23 Table 3.4: Compare total flavonoid content in honey fraction from Thailand and Malaysia Honey from Malaysia Honey from Thailand Honey Gelam 0.00032 mg Honey Bee Park 0.143 mg RE/mg of RE/mg of honey fraction honey fraction Honey Acacia 0.00030mg Honey Resort 0.131 mg RE/mg of RE/mg of honey fraction honey fraction With the same method to determine the total flavonoid content, it was inferred that the honey from Thailand has much higher total flavonoid content than that of Malaysia Honey Therefore, we can suggest that activity of antioxidant in honey from Thailand may be stronger than that in Honey from Malaysia 3.3 Function of Sod1 under hydrogen peroxide assay  Cell survival of WT, Sod1 and Cta1 strains WT 0mM H2O2 Sod1 Cta1 WT  Sod1 3mM H2O2 Cta1 WT Sod1 Cta1 5mM H2O2 Figure 3.3: Cell survival of WT, Sod1 and Cta1 strain 24 - First: Under condition 0mM hydrogen peroxide stress - Second: Under condition 3mM hydrogen peroxide was toxic for growing cell - Third: Under condition 5mM hydrogen peroxide was too toxic for growing cell Condition: 0mM hydrogen peroxide, 3mM hydrogen peroxide and 5mM hydrogen peroxide  0mM hydrogen peroxide - The First: Yeast WT strain media containing 0mM hydrogen peroxide grew better than  Sod1 and Cta1 - The Second: Cta1 strain in media containing 0mM hydrogen peroxide grew better than sod1 - The Third: Sod1 strain in media containing 0mM hydrogen peroxide had the number of survival cell smallest  3mM hydrogen peroxide - The First: Yeast WT strain in media containing 3mM hydrogen peroxide grew better than sod1 and Cta1 - The Second: Cta1 strain in media containing 3mM hydrogen peroxide grew better than Sod1 - The Third: Sod1 strain in media containing 3mM, almost cells died although there are still a few survival cells  5mM hydrogen peroxide - The First: Yeast WT strain in media containing 5mM hydrogen peroxide grew better than Sod1 and Cta1 - Second: Cta1 strain in media containing 5mM hydrogen peroxide grew better than yeast Sod1 - Third: Sod1 didn’t have any survival cell So it is clear that all Sod1 strains go to die under condition 5mM hydrogen 25 Discuss about the function of Sod1 and Cta1 The growth of WT will be better than that of Cta1 because WT has gene Cta1 which will convert hydrogen peroxide to oxygen and water but strain with Cta1 can’t convert hydrogen peroxide to water and oxygen, in some cases it can convert hydrogen peroxide to water less than WT so we can observer some survival cells The growth of WT was better than Sod1 because WT has gene Sod1 will convert superoxide radical to hydrogen peroxide and water but Sod1 which lost gene Sod1 still convert superoxide radical to hydrogen peroxide and oxygen and sometimes convert superoxide radical to hydrogen peroxide and water less than WT The growth of Cta1 was better than Sod1 because Cta1 still can convert hydrogen peroxide to water and oxygen and Sod1 can convert superoxide radical to hydrogen peroxide and oxygen In the cell if there is a lot of radical superoxide, it is dangerous for cell growth The growth of Sod1 can’t be seen under condition 5mM hydrogen peroxide stress because hydrogen peroxide is a toxic for Sod1 3.4 Activity Sod1 of honey in saccharomyces cerevisiae under hydrogen peroxide assay Sod1 YPD broth + H20 YPD broth + H202 50% Methanol + H20 50% Methanol + H202 2mM Hydrogen peroxide 0.1 mg Honey fraction + H20 0.1 mg Honey fraction + H202 Figure 3.4: Cell survival of  Sod1 strain under different conditions 26  Survival of cell in  Sod1 strain under different conditions It is clear that in the YPD broth with water condition and YPD broth with hydrogen peroxide condition the cell grew, under YPD broth with water condition the cell grew better than under YPD broth with hydrogen peroxide condition Under the condition 2: 50% methanol with water and 50% methanol with hydrogen peroxide the yeast cell also grew, under condition 50% methanol with water the cell grew better than under condition 50% methanol with hydrogen peroxide Under the condition 3: 0.1mg honey fraction with water and 0.1mg honey fraction with hydrogen peroxide we can see survival cells, under condition 0.1mg honey fraction with hydrogen peroxide the growth of cells was better than 0.1mg honey fraction with water Discuss about Function of Sod1 in cell Under Condition 1: Under condition YPD broth with water had cell growing better than under condition YPD broth with hydrogen peroxide, under condition YPD broth with water the activity of Sod1 was better than under condition YPD broth with hydrogen peroxide Under Condition 2: Under condition 50% methanol with water we can see the cell survived better than under condition 50% methanol with hydrogen peroxide, under condition 50% methanol with water the activity of Sod1 was better than condition 50% methanol with hydrogen peroxide Under Condition 3: 0.1 mg honey fraction with water and 0.1mg honey fraction with hydrogen peroxide had cell growing well In honey, the activity of gene Sod1 converting superoxide radical to hydrogen peroxide and oxygen help cell survive well 27 PART CONCLUSIONS AND SUGGESTIONS 4.1 CONCLUSIONS  Experiment 1: Honey extraction - Sample contained 22.615 mg of honey fraction 1, dissolved in 50% MeOH (22.615 mg), Sample contained 14.708 mg of honey fraction 2, dissolved in 50% MeOH (14.708 mg) - Honey sample had higher concentration of flavonoid than that of sample  Experiment : Total flavonoid content - Honey Bee Park had 0.144 mg RE/mg of honey fraction and honey Resort had 0.131 mg RE/mg of honey fraction - These flavonoid contents may prevent free radicals and protect cells with many health benefits  Experiment : Function of Sod1 under hydrogen peroxide assay - Under condition hydrogen peroxide stress, growth of WT was better than than that of Sod1 and Cta1 strains - Under condition hydrogen peroxide stress, growth of cta1 grew better than that of Sod1 because Cta1 with deleted gene Hydrogen peroxide is less toxic than superoxide radical The Sod1 with deleted SOD1 gene could not convert from superoxide radical to hydrogen peroxide, therefore very sensitive to hydrogen peroxide  Experiment 4: Activity Sod1 of honey in Saccharomyces cerevisiae under hydrogen peroxide assay - Cell survival (Sod1 strain) under condition YPD broth with water and hydrogen peroxide was grow It means the cell lacked activity of Sod1 to convert superoxide radical to hydrogen peroxide and water and YPD broth give nutrients for help cells grow - Cell survival (Sod1 strain) under condition 50% methanol with hydrogen peroxide and water was grow It means the cell lacked activity of Sod1 to convert 28 superoxide radical to hydrogen peroxide and water plus 50% methanol solution was too toxic to the cells - Cell survival (Sod1 strain) under condition 0.1 mg of honey with hydrogen peroxide and water, cell could grow better It means the cell has compensated for activity of Sod1 The honey may have activity Sod1 help cell survive  Experiment 3: Yeast Sod1 strain in honey fraction cell can grow, the cell grew better It means the cell has compensated for activity of Sod1 The honey may have activity Sod1 help cell survive  Experiment 1: Honey Bee Park has (0.144 mg RE/mg of honey fraction 1) and honey resort (0.131 mg RE/mg of honey fraction 2) had high concentration of antioxidant compounds 4.2 SUGGESTIONS Perform total vitamin C assay for determining total vitamin C in honey bee park (KMUTT- Thailand) Perform experiment in which enzyme is added to honey for increasing value of honey bee park (KMUTT- Thailand) Create new food products from honey for the elderly 29 REFERENCES [1] Ahmed S, Sulaiman SA, Baig AA, (2017) " Honey as a Potential Natural Antioxidant Medicine: An Insight into Its 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