Khảo sát quy trình công nghệ sản xuất thức ăn hỗn hợp dạng viên cho lợn sữa tại tập đoàn BMG

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].. 5 Figure 1.4: Free radical effects to heathy a

Study course : Full-time

Faculty : Biotechnology and Food Technology

Thai Nguyen, 06/2018

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

Faculty : Biotechnology and Food Technology Period : 2014 – 2018

Supervisor : Assoc.Prof.Dr Nitnipa Soontorngun Msc Dinh Thi Kim Hoa

Thai Nguyen, 06 /2018

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Student name Ma Thi Ngoc Diep

Thesis title Utilization of yeast and enzyme products for added-value

of Thai honey neo-food for aging society

Supervisors 1 Assoc.Prof.Dr Nitnipa Soontorngun

2 Msc Dinh Thi Kim Hoa

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

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, Flavonoid, S

cerevisiae, Superoxide dismutase, Catalase

Number of pages 28

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, 10 th , June, 2018

Many thanks and best regards,

Student

Ma Thi Ngoc Diep

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

1.1.Background 1

1.1.1.Honey and the situation of honey production in the world 1

1.1.2 Flavonoid 3

1.1.3 Free radical 5

1.1.3 Antioxidant 6

1.2 Saccharomyces cerevisiae 7

1.3 Activity of gene superoxide dismutase and catalase 9

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

2.2.1Honey extraction 15

2.2.2 The method for determining the total flavonoid content 16

2.2.3Function of Sod1 and Cta1 under hydrogen peroxide assay 16

2.2.3.1 Prepare media 16

2.1.2.2Function 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

3.1Honey 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

LIST OF ABBREVIATION

Cta1

Sod1

Catalase Superoxide dismutase

MeOH

Methanol Acid acetic

Rpm Revolutions per minute

Copper Chaperone for SOD1 Deletion

S cerevisiae Saccharomyces cerevisiae

LIST OF TABLE Table 2.1: Materials and formula for make YPD broth and YPD agar……… 17

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 2 honey fractions……… 21 Table 3.4: Compare total flavonoid content in honey fractions from Thailand and Malaysia…23

LIST OF FIGURES

Figure 1.1: Types of flavonoids and sources……… 4

Figure 1.2: Basis structure of flavonoids……… 4

Figure 1.3: Causes of oxidative stress……… 5

Figure 1.4: Free radical effects to heathy atoms……….5

Figure 1.5: Benefits of antioxidant molecules……… 6

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 1 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

PART 1 INTRODUCTION 1.1 Background

1.1.1 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, 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

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

1.1.2 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 Flavonoid is compound have the main function protect the cell against activity reactive oxygen radical [18]

Figure 1.1: Types of flavonoids and sources

Figure 1.2: Basis structure of flavonoids

Causes of oxidative stress

In life, there are many effects on the normal functioning of molecules, harmful

cell, cell will death Exogenous effect free radicals: the environment that acts as radiation is ultraviolet radiation, X-rays, gamma rays, smoking, chemicals that promote superoxide formation are herbicides, stress, alcohol, fast foods, pollutants, infection Exogenous effects to the cell and which damage activity of cell Endogenous effects to the cell: Free radicals form in the body due to natural metabolism such as respiratory cell metabolism, free radicals that can damage

cellular organs such as proteins, DNA and membrane by stealing their electrons [39] The effects of exogenous causes affect the natural metabolism that results in

the activity of free radicals

Figure 1.3: Causes of oxidative stress

(Source: http://www.beyondlimits.net.au) 1.1.3 Free radical

Figure 1.4: Free radical effects to healthy atoms

acting as an oxidizer or reducing agent Most highly reactive radicals can take an electron from a normal molecule that is harmful to the cell [29]

Free radicals are hydroxyl radicals, superoxide anionic radicals, hydrogen peroxide, oxygen singlet, hypochlorite, nitric oxide and peroxiynitrite, which cause many illnesses in the human body These are highly reactive species that have the ability in the nucleus and in the cell membrane to damage biologically relevant molecules such as DNA, proteins, carbohydrates and lipids [29] Free radicals molecules lead to cellular injury and cell death In particular, the nucleic acids, fats and proteins are the main targets of free radicals Leading to cell death and is the cause of many dangerous diseases [29]

1.1.3 Antioxidant

Figure 1.5: Benefits of antioxidant molecules

Antioxidants help your body repair cells damaged by free radicals Antioxidants are beta-carotene, lycopene and vitamins A, C, E and flavonoid Antioxidant increased health of people and antioxidants can also be found in fruits, vegetables, and teas Antioxidants from food is the most healthful way to obtain them but they may also be taken as supplements [21]

These reactive oxygen species include the superoxide anion, O2-, the hydroperoxyl radical, HO2-·, hydrogen peroxide, H2O2 and the hydroxyl radical, ·OH, all intermediates in the reduction of O2 to H2O and the reactive oxygen species can cause oxidation of proteins, RNAs, and DNAs and peroxidation of membrane lipid DNA breaking of strand link, mutation of bases, Lipids increase membrane fluidity and

permeability and breaking of lipid chains and make modified of amino acids and breaking of the peptide chain [44]

The honey contains high levels of antioxidants, flavonoids Flavonoids are the antioxidants found in honey, so they play the role of antioxidants in honey Flavonoids almost found in yellow color, some color are white, red, blue and purple (Kerry bone and Simon mills, 2013)

In other words, an antioxidant is defined as “any substance that, when present

at low concentration compared with that of an oxidizable substrate, significantly delays

or prevents oxidation of that substrate” (Halliwell and Gutteridge, 1999)

1.2 Saccharomyces cerevisiae

S cerevisiae (commonly known as baker’s yeast) is a single-celled eukaryote that

is frequently used in scientific research S cerevisiae is an attractive model organism

due to the fact that its genome has been sequenced, its genetics are easily manipulated and it is easy to maintain in the lab Yeast proteins are similar in sequence and function

to those found in other organisms, studies performed in yeast can help us to determine how a particular gene or protein functions in higher eukaryotes (including humans) [12]

S cerevisiae is believed to have been originally isolated from the skin of grapes

(one can see the yeast as a component of the thin white film on the skins of some colored fruits such as plums; it exists among the waxes of the cuticle) It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology,

dark-much like Escherichia coli as the model bacterium It is the microorganism behind the most common type of fermentation Structure of S cerevisiae cells are round to ovoid,

5–10 μm in diameter It reproduces by a division process known as budding Many proteins important in human biology were first discovered by studying their homologs

in yeast S cerevisiae these proteins include cell cycle proteins, signaling proteins, and

protein-processing enzymes

S cerevisiae is currently the only yeast cell known to have Berkeley bodies

present, which are involved in particular secretory pathways Antibodies against S

cerevisiae are found in 60–70% of patients with Crohn's disease and 10–15% of

patients with ulcerative colitis (and 8% of healthy controls) [43] As a single-cell

organism, S cerevisiae is small with a short generation time (doubling time 1.25–2

hours [15] at 30 °C or 86 °F) and can be easily cultured [5].

S cerevisiae can grow aerobically on glucose, maltose, and trehalose and fail to

grow on lactose and cellobiose However, growth on other sugars is variable Galactose and fructose are shown to be two of the best fermenting sugars The ability of yeasts to use different sugars can differ depending on whether they are grown aerobically or anaerobically Some strains cannot grow anaerobically on sucrose and trehalose [22] Strains can use ammonia and urea as the sole nitrogen source, but cannot use nitrate, since they lack the ability to reduce them to ammonium ions They can also use most amino acids, small peptides, and nitrogen bases as nitrogen sources Histidine,

glycine, cystine, and lysine are, however, not readily used S cerevisiae does not

excrete proteases, so extracellular protein cannot be metabolized Yeasts also have a requirement for phosphorus, which is assimilated as a dihydrogen phosphate ion, and sulfur, which can be assimilated as a sulfate ion or as organic sulfur compounds such as the amino acids methionine and cysteine Some metals, like magnesium, iron, calcium, and zinc, are also required for good growth of the yeast [22]

Honey has many yeast strain such as Zygosaccharomyces mellis acidi,

Zygosaccharomyces mellis and the other Torula mellis [32], S cerevisiae var bayanus

[36] The research use yeast strain S cerevisiae as a model know activity of genes in yeast strain S cerevisiae from the activity of yeast strain honey can know the activity

of genes in the body S cerevisiae was the first eukaryotic genome to be completely

sequenced [4] The genome sequence was released to the public domain on April 24,

1996 Since then, regular updates have been maintained at the Saccharomyces Genome

Database This database is a highly annotated and cross-referenced database for yeast

researchers Another, S cerevisiae database is maintained by the Munich Information Center for Protein Sequences (MIPS) S cerevisiae genome is composed of about

12,156,677 base pairs and 6,275 genes, compactly organized on 16 chromosomes

Only about 5,800 of these genes are believed to be functional It is estimated at least 31% of yeast genes have homologs in the human genome [6]

1.3 Activity of gene superoxide dismutase and catalase

These molecules collectively act against free radicals to against free radicals and protect cells from damage The effectiveness of the antioxidants which basically

include superoxide dismutase (Sod1) and catalase (Cta1) is important and

indispensable in the entire defense strategy of antioxidants, special honey has the

activity of gene Sod1, Cta1 role antioxidant removes free radical damage cell [23]

There are 2 enzymes can remove free radical They are very fast in neutralizing any molecule with the potential of developing into a free radical or any free radical with the ability to induce the production of other radicals Three key enzymes: first

enzyme superoxide dismutase remove superoxide anion, enzyme catalase, and

glutathione peroxidase are top on the list Two enzymes effect to protect the cell by removing harmful of reactive oxygen species and breakdown of hydrogen peroxide to harmless molecules (H2O2/alcohol and O2) Superoxide dismutase has role important into against reactive oxygen species (ROS), superoxide dismutase is first enzyme catalysis superoxide radical become oxygen and hydrogen peroxide Catalase will catalysis hydrogen peroxide become water and oxygen [23]

These free radicals is neutralized or scavenged by antioxidants In this process, molecules become free radicals themselves, but of lesser damaging effects These ‘new radicals’ are easy to neutralized and make completely harmless by other antioxidants in this groups Special catalase remove hydrogen peroxide radical, convert hydrogen peroxide to water and oxygen, especially, it also can scavenge active free radicals, protect the cell and help cell to survive strongly [29]

The antioxidants response against oxygen radicals by endogenous or exogenous sources

Superoxide dismutase, catalase, and glutathione peroxidase are antioxidant

enzymes which do not only play a fundamental but indispensable role in the

antioxidant protective capacity of biological systems against free radical attack First

superoxide dismutase help cell remove superoxide radicals by enzyme Sod1 The

superoxide radical (O2-) or singlet oxygen radical generated in tissues through metabolism or reactions in cells is catalytically converted to hydrogen peroxide (H2O2) and molecular oxygen (O2) by superoxide dismutase (Sod1) [23] Catalase (Cta1) removes active antioxidant radicals Catalase is the second enzyme to remove

hydrogen peroxide

1.3.1 Superoxide Dismutase

Superoxide dismutase (Sod1) is an enzyme that catalyzes the dismutation of the

superoxide (O2-) radical into either ordinary molecular oxygen (O2) and hydrogen peroxide (H2O2) [17] Superoxide is a byproduct of oxygen metabolism in the cell, which, if not catalyzed by the enzymes required, can damage the cell, causing many types of cell damage

Figure 1.6: Antioxidant activity of enzyme superoxide dismutase

Hydrogen peroxide is also damaged and is degraded by other enzymes such as

catalase Thus, Sod is an important antioxidant defense in nearly all living cells

exposed to oxygen In other cases, such as Lactobacillus plantarum and lactobacilli,

another mechanism has been used to prevent cell damage due to the reaction [17]

Sod1 is Cu-Zn superoxide dismutase encoding Cu-Zn superoxide dismutase in

the cytoplasm that plays a role in oxygen detoxification and in copper ion buffers

[4] Cu-Zn superoxide dismutase (Sod1) is a rich enzyme, Cu-Zn superoxide

dismutase activity into cytosol capable of scavenging superoxide anion [41] Sods

catalyze the breakdown of the superoxide radical, O2-, to an oxygen molecule

(dioxygen) and hydrogen peroxide, Sod with hydrolysis role The active form of

Sod1p is a homodimer, with each 32kD subunit containing one catalytic copper ion and one zinc ion Two conserved cysteine residues of each monomer are joined together in a disulfide bond and this bond is critical for enzymatic activity The

specific copper chaperone Ccs1p delivers the copper ion to Sod1p and also

facilitates formation of the intramolecular disulfide bond [14]

The Cu-Zn-Sod1p is cytosolic; however, a fraction of both Sod1p and its metallochaperone, Ccs1p, localize to the intermembrane space (IMS) of mitochondria where Sod1p performs a physiological role in scavenging

mitochondrial reactive oxygen species Accumulation of Sod1p within mitochondria

is dependent on the presence of the mitochondrial form of Ccs1p, which enhances retention of the immature Sod1p within the IMS [40]

Sod1 is among the first genes to be implicated in the chronological aging of yeast [8] In case of deletion or mutation Sod1 significantly reduces the yeast's lifetime and cloning [30], Sod1 is beneficial for yeast activity in cases of overexpression of both

Sod1 prolonged survival but no effect on metabolic rate [13]

Superoxide dismutase (Sods) are abundant enzymes present in prokaryotes and

eukaryotes Prokaryotes have two forms, one contains iron (Fe) and another contains manganese (Mn) The Cu-Zn form is found in few distantly related bacterial species Eukaryotes have a Mn-containing form in the mitochondrion and

a Cu-Zn containing form in the cytoplasm The Mn and Fe proteins are related to each other, while the Cu-Zn protein is unrelated to either [4]

Sod1 enzyme is necessary for the cell to help cellular protect against free

radicals, effect on oxygen radicals Free radicals will be harmful agents which leads

to promote aging and cell death that makes damage to cell and heath In the human,

levels of Sods will decline with age, in other words, free radical formation increases harmful for the cell Some studies have shown that proper daily Sod1

supplementation can boost the immune system and dramatically reduces the risk of diseases and ultimately slows the aging process of healthy cells [4]