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The antioxidantwill give one electron for free radical help free radical become normal molecularThe antioxidant properties of flavonoids are based on an ability the activity ofenzymes in

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

MA THI NGOC DIEP

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

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

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 damagingcompounds, in part, by absorbing antioxidants from high-antioxidant foods [40] Soantioxidants can play important roles in treatment of several dieases [24]

Many kinds of food have antioxidants which have benefits for health, forexample fruits, vegetables, drink, fish, chocolate [33] Honey have high proportion

of antioxidants (Tualang 18.511mgRE/100 g Honey; Gelam 32.886 mgRE/100gHoney; Acacia 30.741 mgRE/100g Honey) [11] People can produce honey in manyproducts in life that is good for health and can apply in cosmetics, food, andpharmaceuticals It is easy to see many famous products in the market such as:Pharmacy Honey Potion Warming Face Mask (America) [36], Honeyed whiskeywine , (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

Big thanks also go to every people on Gene Technology Lab Program atKMUTT for helping me fit in and feel welcome from the moment and for theunlimited patience to explain me every doubt I had during my internship.

I would also like to acknowledge my teachers at TUAF, MSc Dinh Thi KimHoa, MSc.Trinh Thi Chung that contributed to make this work and had an enjoyableand 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

6

Sod1

LIST OF ABBREVIATION

Catalase Superoxide dismutase

S cerevisiae Saccharomyces cerevisiae

1

1.1 Background

PART 1 INTRODUCTION

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 thehoneycomb cells Honey has many nutrients so honey has been used humans sinceancient times, nearly 5500 years ago, most ancient population, including the Greeks,Thailand, Chinese, Egyptians, Romans, Mayans, and Babylonians with purposeconsume 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 theresistance to health [31] Honey is food has great potential, nutritional food,functional foods, cosmetics, and medicine So many countries invest in honeyproduction [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

anti-Honey is natural product from bee consisting of a highly concentrated solutionwith element main is the complex mixture of sugar and little quantities of otherconstituents, such as minerals, proteins, vitamins, organic acids, flavonoids,phenolic acids, enzymes, and volatile compounds [34] The quantity of thesedifferent compounds varies greatly depending on the floral and geographical origin

of the honey [34]

Special, Honey components reported to be responsible for its antioxidanteffects 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 oxygenspecies [11] Therefore, in recent years apply for studies have been focused on thecomposition special of kinds of honey is their biological properties such asantioxidant, anti-inflammatory, anti-aging, anticancer and antimicrobial activities inwound healing, as well as in the treatment of skin ulcers and gastrointestinaldisorders [11] Research has contributed to increase the value of using honey andmany honey product, in today's life, honey is a very popular product.

Honey has many antioxidant ingredients Especially in honey containsflavonoid ingredients should have ability good antioxidant Flavonoids arepolyphenolic compounds that contain a C15 flavone skeleton (diphenylpropane) andare collectively known as vitamin P [20] Currently, Honey have many studiesresearch about total flavonoid content, currently, there are many research studiesthat show high levels of flavonoids: (Tualang 18.511mgRE/100 g Honey; Gelam32.886mg RE/100 g Honey; Acacia 30.74mgRE/100g Honey) from Malaysia [5], sohoney 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 ofosteoporosis (Migliaccio & Anderson, 2003) [10] Honey has many benefits in lifethat 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 severalhundred are found in edible plants) flavonoid are antioxidant compounds found inplants, fruits and some animals [27] Moreover, Honey have a lot of flavonoidcompounds it good for health human can protect body attack of reactive oxygenspecies, protect the cell Flavonoids are a ubiquitous group of naturally occurringpolyphenolic compounds characterized by the flavan nucleus and is one of the mostprevalent classes of compounds in fruits and insect, vegetables and plant-derivedbeverages In addition, honey contains flavonoids characteristic of antioxidants Theflavonoids structure have been identified and many of which are responsible for theattractive colors of flowers, fruits and leaves function create color Flavonoids inplants can protect plants from the harmful effects of insects [28] Flavonoids a class

of polyphenolic compounds widely distributed in plants Quercetin and rutin areamong 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 anantioxidant molecule that stabilizes free radicals, free radicals that contain unstablemolecules in the electron-donning state of normal molecules [18] The antioxidantwill give one electron for free radical help free radical become normal molecularThe antioxidant properties of flavonoids are based on an ability the activity ofenzymes involved in the remove of free radicals and in the elimination of active freeradicals Flavonoid is compound remove activity of reactive oxygen species Inaddition, flavonoids have the function to prevent oxidation of vitamin C and otherdetrimental reactions caused by active oxygen Flavonoid is compound have themain 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, harmfulcell, cell will death Exogenous effect free radicals: the environment that acts asradiation is ultraviolet radiation, X-rays, gamma rays, smoking, chemicals thatpromote 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 naturalmetabolism 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 1.4: Free radical effects to healthy ato

(Source: ht t p://w w w.b e yondlimits net.a u)

1.1.3 Free radical

Figure 1.3: Causes of oxidative stress

(Source: ht t p://w w w.b e yondlimits net.a u)

ms

Figure 1.4: Free radical effects to heathy atoms

Free radicals can be defined as any molecule that has the ability to surviveindependently containing an unpaired electron in an atomic orbit and they haveunstable characteristics Free radicals is most unstable and highly reactive freeradicals can take an electron from a normal molecule that is harmful to the cell Freeradicals can donate an electron or accept an electron from other molecules, thus

-

-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, hydrogenperoxide, oxygen singlet, hypochlorite, nitric oxide and peroxiynitrite, which causemany illnesses in the human body These are highly reactive species that have theability in the nucleus and in the cell membrane to damage biologically relevantmolecules such as DNA, proteins, carbohydrates and lipids [29] Free radicalsmolecules lead to cellular injury and cell death In particular, the nucleic acids, fatsand proteins are the main targets of free radicals Leading to cell death and is thecause 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 Antioxidantsare beta-carotene, lycopene and vitamins A, C, E and flavonoid Antioxidantincreased 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 theymay also be taken as supplements [21]

These reactive oxygen species include the superoxide anion, O2 , thehydroperoxyl radical, HO2 ·, hydrogen peroxide, H2O2 and the hydroxylradical, ·OH, all intermediates in the reduction of O2 to H2O and the reactiveoxygen species can cause oxidation of proteins, RNAs, and DNAs and peroxidation

of membrane lipid DNA breaking of strand link, mutation of bases, Lipidsincrease membrane fluidity and

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

The honey contains high levels of antioxidants, flavonoids Flavonoids are theantioxidants 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, whenpresent at low concentration compared with that of an oxidizable substrate,significantly delays or prevents oxidation of that substrate” (Halliwell andGutteridge, 1999)

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 somedark- colored fruits such as plums; it exists among the waxes of the cuticle) It isone of the most intensively studied eukaryotic model organisms in molecular and

cell biology, 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 Manyproteins 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 grownaerobically or anaerobically Some strains cannot grow anaerobically on sucrose andtrehalose [22]

Strains can use ammonia and urea as the sole nitrogen source, but cannot usenitrate, since they lack the ability to reduce them to ammonium ions They can alsouse 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 Yeastsalso have a requirement for phosphorus, which is assimilated as a dihydrogenphosphate ion, and sulfur, which can be assimilated as a sulfate ion or as organicsulfur compounds such as the amino acids methionine and cysteine Some metals,like magnesium, iron, calcium, and zinc, are also required for good growth of theyeast [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 16chromosomes.

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 radicalsand 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 neutralizingany molecule with the potential of developing into a free radical or any free radicalwith 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 byremoving harmful of reactive oxygen species and breakdown of hydrogen peroxide

to harmless molecules (H2O2/alcohol and O2) Superoxide dismutase has roleimportant into against reactive oxygen species (ROS), superoxide dismutase is firstenzyme 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 otherantioxidants in this groups Special catalase remove hydrogen peroxide radical,convert hydrogen peroxide to water and oxygen, especially, it also can scavengeactive 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 (O -) or singlet oxygen radical generated in tissues throughmetabolism 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 hydrogenperoxide (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 manytypes 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 ionand one zinc ion Two conserved cysteine residues of each monomer are joinedtogether 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 anothercontains manganese (Mn) The Cu-Zn form is found in few distantly relatedbacterial 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 toeach 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 ofdiseases and ultimately slows the aging process of healthy cells [4]