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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
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 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
1.1. 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


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

0

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 2 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…………………………………………………... 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

1

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, antiaging, 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


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), anticancer (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.

3

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].

4

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


5

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: )
1.1.3 Free radical

Figure 1.4: Free radical effects to healthy atoms
(Source: )
Figure 1.4: Free radical effects to heathy atoms
Free radicals can be defined as any molecule that has the ability to survive

independently containing an unpaired electron in an atomic orbit and they have
unstable characteristics. Free radicals is most unstable and highly reactive free
radicals can take an electron from a normal molecule that is harmful to the cell. Free
radicals can donate an electron or accept an electron from other molecules, thus


6

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


7

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 darkcolored 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,
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.


8

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.


9

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


10

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


11

(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].


12

1.3.2. Catalase

Figure 1.7: Process catalase catalyzes the converted of hydrogen peroxide to
oxygen and water
Catalase is the most common enzyme found in nearly all living organisms that
are exposed to oxygen (such as bacteria, plants, and animals). It catalyzes the
decomposition of hydrogen peroxide to water and oxygen [9]. It is a very important
enzyme in protecting the cell from affect oxidative damage by reactive oxygen
species (ROS). Catalase catalyzes the convert from hydrogen peroxide to oxygen
and water. Likewise, catalase has the fastest catalyst in all enzymes; a catalase
molecule can convert millions of hydrogen peroxide molecules into water and
oxygen per second. So catalase is a biological catalyst [16].
Yeast is the enzyme, called catalase that acts as a catalyst for the reaction that

breaks down hydrogen peroxide into oxygen and water. Cta1 encodes catalase A
[1], hydrogen peroxide detoxification in the peroxisomal and mitochondrial to water
and oxygen and detoxify the cell [38]. In mitochondria, hydrogen peroxide is
produced by the superoxide dismutase Sod1p and Sod2p in defending against
reactive oxygen species (ROS) [40]. In peroxisomes, the primary source of
hydrogen peroxide is that produced during fatty acid beta-oxidation [19].
Catalase plays an important role in eliminating the activity of hydrogen
peroxide in the cell and protects the cell from the effect of hydrogen peroxide. Cta 1
activity is also increased during caloric restriction and during oxidative stress
caused by the processes of aging, acid stress adaptation, and thermotolerance. Cta1p


13

activity is important during oxidative stress response and in protecting proteins
against oxidative inactivation [35].
Transcription of Cta1 is regulated in response to oleic acid, glucose, heme and
oxygen [16]. This transcriptional response is mediated by the transcriptional
activators Adr1p, Oaf1p, Rtg1p, and Rtg2p via binding to overlapping ORE and
UAS1 elements in the Cta1 promoter [10].

1.4. Objectives
1.4. Overall goal of the research
1. Utilization of yeast and enzyme products to add value of Thai honey as neofood for aging society
2. Research to create new products from honey for the elderly
1.4.2. Detail goal
1. Study the activity of antioxidant enzymes in honey fraction (Collect from
Bee Park and Resort of KMUTT-Thailand)
2. Study determine antioxidant content in honey fraction (Collect from Bee
Park and Resort of KMUTT-Thailand)

1.5. Scientific significance and practical meaning
1.5.1. Scientific significance
Determine activity of antioxidant and concentration antioxidant in honey
(Collect from Bee Park and Resort of KMUTT-Thailand)
1.5.2. Practical meaning
The result of the study could indicate the antioxidant content of local honey
and determine whether honey has high or low levels of antioxidants to be further
use as new food product for elderly.


14

PART 2
MATERIALS AND METHODS
2.1. Equipment and Materials
2.1.1. Materials
- Honey from bee park of MMUTT- Thailand (Collect 06/12/2017)
- Honey from resort of KMUTT- Thailand (Collect 04/12/2017)
2.1.2 Chemicals
- Ruin hydrate, R514350G, SIGMA, CHINA
- Methanol (CH3OH), M209712500, QREC, New Zealand
- Aluminum chloride hydrated, 879500 G, HAZARDOUS, New Zealand
- Ethyl acetate, E/0900/21, LE11 5RG, UK
- Formic acid 85%, F503332501, QREC, New Zealand
- Yeast extract powder, RM027-500G, HIMEDIA, INDIA
- Peptone Bacteriological, GM001-500G, HIMEDIA, INDIA
- D-(+)-Glucose anhydrous, GRM077-500G, HIMEDIA, INDIA
- Agar Powder Bacteriological, GRM026-500G, HIMEDIA, INDIA
- Hydrogen peroxide 30%, Thailand
- DI water, Biotechnology KMUTT

- Yeast strain without superoxide dismutase, Saccharomyces cerevisiae
- Yeast strain without catalase, Saccharomyces cerevisiae
- Ethanol 70%, 180207, Thailand
2.1.3 Equipment
 MICROFUGE Centrifuge, MBB10B010, Beckman coulter, GEMANY
 Laboratory fume hood, E0200204, HSIANGTAIFHBT 41908, ProLAB,
Thailand
 Automatic autoclave, LS-2D, REYALL, TAIWAN.
 UV-1200 Spectrophotometer, UEC 1305007, MAPADA INSTRUMENTS
 Vortex, G-560E, Scientific Industries, INC, U.S.A
 BJPX-ElM Incubator,P11136, GIBTHAI CO.lio , LTD


15

 Incubator shaker, ZWY-2102C, 60E0200203, Lio Lab international
CO.,LTD
 Biohazard Safety cabinet, BHA72, FF-TECH, ITALY
 Class II Biological safety cabinet, ESCO, life Sciences (Thailand) CO.,
LTD
 SOXHLET EXTRACTOR, PP 25-1287, Catalogue, UKAS
 Water bath, NTT-2400, EYELA, Japan
 100m Erlenmeyer flasks, APPROX. VOL, Germany
 96-Well Polystyrene, Thermo Scientific™ Nunc™,USA
 Cuvette, 759170, Brandtech, Macro
 Falcon® Conical Tubes, 50 mL
 Test Tubes, extra-large, 20 x 150 mm
 MicroPette, Scilogex 71312118, Plus Single-Channel Fixed Volume
Pipettor
 SCILOGEX, MS-H280-Pro,Circular-top LED Digital Hotplate Stirrer

 Eppendorf™, 0030119401, Fisher Scientific, England
 Duran bottle, Z305189, SIGMA-ALDRICH, Caps capacity 250 ml,
Thailand
 Glass Graduated Cylinder, 43237-2, Ronyes Lifescience, Singapore
 Analytical balance, KERN ABS 80-4N,scales Maesuring instruments
 Drying oven, J-300S, J-300M, Jisico, CO.,TLD
2.2. Methods
2.2.1 Honey extraction
- Step 1: Use formic acid 0.1% (10ml) mixed 10 ml of honey crude
- Step 2: Vortex 30 minute
- Step 3: Add ethyl acetate and shake few minute (3 times)
- Step 4: Separate honey and remove sugar