Isolation of some yeast strains with high proliferation ability from natural sources in thai nguyen

NGUYEN THI HOATITLE: ISOLATION OF SOME YEAST STRAINS WITH HIGH PROLIFERATION ABILITY FROM NATURAL SOURCES IN THAI NGUYEN BACHELOR THESIS Study Mode : Full – time Major : Food Technolog

NGUYEN THI HOA

TITLE: ISOLATION OF SOME YEAST STRAINS WITH HIGH PROLIFERATION ABILITY FROM NATURAL SOURCES IN THAI NGUYEN

BACHELOR THESIS

Study Mode : Full – time Major : Food Technology Faculty : Advance Education Program Batch : 2016 - 2020

Thai Nguyen, November 11th 2020

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY

NGUYEN THI HOA

TITLE: ISOLATION OF SOME YEAST STRAINS WITH HIGH PROLIFERATION ABILITY FROM NATURAL SOURCES IN THAI NGUYEN

BACHELOR THESIS

Study Mode : Full – time Major : Food Technology Faculty : Advance Education Program Batch : 2016 - 2020

Thai Nguyen, November 11th 2020

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University Of Agriculture And Forestry

Degree program Bachelor of Food Technology

Student name Nguyen Thi Hoa

Student ID DTN1653170035

Thesis title Isolation of some yeast strains with high

proliferation ability from natural sources in Thai Nguyen

Supervisor Msc Vi Dai Lam

Supervisor’s signature

Abstract : The reasearch purpose to isolate the yeast strains from fruit that can

be used in food production A total 10 samples including locally availible source such as bananas, organes, sugarcane juice, grapes, guava are collected from other area of the Thai Nguyen province After culture proliferates, 12 yeast strains were isolated using selective medium Yeast Extract Peptone Dextrose Agar From the differential tests which included morphological and microscopic observation and physiological and biochemical characterization such as tolerant ethanol, 5 strains were selected as possible Saccharomyces strain The result sequencing show S1( from surgarcane juice) strain is

Clavispora lusitaniae

Keywords: Isolation, Yeast, fruits Number of page: 36

Date of submission:

ACKNOWLEGEMENT

First of all, I am grateful to the Thai Nguyen University of Agriculture and Forestry, the Advance Education Program, the Faculty of Biotechnology and Food Technology, who have taught and imparted necessary knowledge and valuable experience to me during the time I studied and researched at Thai Nguyen University of Agriculture and Forestry

I am extremely thankfull to my supervisor Mr.Vi Dai Lam, who supported me for the completion of this thesis I also acknowledge with a deep sense of reverence with all members of my family and friends who are always to help and encourage me on the completion of this thesis

However, due to the limited knowledge and the lack of practical experience, the content of the thesis cannot avoid short comings, I would like to receive further suggestions and advice to make my this thesis is more complete

Best regards

Thai Nguyen,November 11 th ,2020

Nguyen Thi Hoa

List of Figures……….vi

List of Tables ……… vii

List of Abbreviations………viii

PART I INTRODUCTION……….1

1.1 Research rationale……… 1

1.2 Research’s objectives……… 2

1.3 Meaning of the reseach ……….……….2

1.3.1 The scientific meaning of the topic……… 2

1.3.2 The practical meaning of the topic……….2

PART II LITERATURE REVIEW……….4

2.1 Yeast ……… ……….4

2.1.1 Yeast biology properties………4

2.1.2 Metabolism in Yeast……… 7

2.1.3 Yeast habitat……… 7

2.1.4 Application of yeast………8

2.2 Overview of research in the field of the topic……….14

2.2.1 In the world……… 14

2.2.2 In the country………15

PART III METHODS ……… 17

3.1 Objective of study………17

3.1.1 Objective of study…….……….17

3.1.2 Chemicals, Instruments And Machines……….17

3.2 Methods … ………19

3.2.1 Isolation of yeast from samples……….19

3.2.2 Identification of yeast……….24

PART IV RESULTS……….26

4.1 Isolation of yeast from samples……… 26

4.2 Identifiacation of yeast ……… 28

PART V CONCLUSION AND PEPTITION……… 31

5.1 Conclusion……… ……….31

5.2 Peptition ……… 31

REFERENCES ……… 32 APENDENCES

LIST OF FIGURES

Figure 1: Yeast Saccharomyces cerevisiae

Figure 2: Ten-fold serial dilution

Figure 3: Colonies of yeast isolated from fruits Figure 4: Sequencing result

Figure 5: Sequencing result of S1 strain

LIST OF TABLES

Table 1: Nutural yeast habitats

Table 2: Morphological characteristic of B1, S1 and O1 strains Table 3: Growth ability of isolated strains

Table 4: Ethanol tolerance test

LIST OF ABBREVIATION

1 Yeast Extract Peptone Agar( YEPD)

2 Saccharomyces cerevisiae ( S cerevisiae)

3 Carbon dioxide ( CO2)

4 Energy( ATP)

5 Deoxyribonucleic acid( DNA)

6 Polypropylene( PP)

7 Low density polyethylene( LDPE)

8 Biaxially Oriented Polypropylene (BOPP)

9 Gram( g)

10 Miligram(mg)

11 Mililit(ml)

PART 1: INTRODUCTION

1.1 Reaseach rationale

Yeast is a common model in a lot of Biotechnology and Food technology researches Depend on experiments, yeasts can be applied as a gene transfering vector, a fermentation factor, the cell type to increase biomass Yeasts cells have contributed important roles in many food products, such as: Bread, wine, sake and beer These products has many benefits for humans According to many studies, the daily use of bread, a glass of beer or a cup of wine after meals can helps to support the digestive system, helps keep bones strong and creates healthy red blood cells

The production of fermentation foods depends on many factors, yeast is an indispensable ingredient to create delicious taste The yeasts used in bread production

named Saccharomyces cerevisiae [25] This yeast strain uses glucose to create CO2,

ethanol, esters, aldehydes, and amino acids that supply to the development of flavor and aroma of the products The carbon dioxide works to increase the volume of dough and increase the taste Additionally, vitamins and amino acids are also trustworthy for the health and nutritional benefits achieved from bread [9], [12]

In 1970, Burrows listed four functions of yeast in bread making: Firstly, to increase doug volume by evolution of CO2 during fermentation of the available carbohydrates in the flour Secondly, to develop structure and texture in the dough by the stretching due to expansion of gas bubbles Thirdly to improve flavor Finally to add

some nutritive values of bread The time to yeast cells can work effectively should not be

so long because it can cause the spoilage of other ingredients This mean, the number of yeast cells must be enough to ferment in a short time Therefore, the yeast strains which can proliferate quickly is valuable in food production

Depending on the value and the roles of yeast strains in research and application,

the topic “Isolation of some yeast strains from natural sources” is conducted

1.2 Research’s objectives

1.2.1 General objective

Isolation of yeast which can proliferate quickly

1.2.2 Specific objective

Isolation of 01 indigenous yeast strains with good proliferation capacity

1.3 Meaning of the reseach

1.3.1 Scientific significance

The theme encourages students to participate in scientific research in the field of food processing

- Understand and grasp the knowledge learned

- Supplement knowledge through practical research activities, cultivate knowledge, accumulate practical experience, and approach scientific research for research and later work

self Helping students consolidate and systematize learned knowledge into scientific research

PART 2: LITERATURE REVIEW 2.1 Yeast

2.1.1 Yeast biology properties

Yeasts are unicellular eukaryotic fungi with a particle size of 5-10µm[11] Most yeast species belong to Ascomycotina, a few are Basidiomycetes Bakers' yeast and the

yeasts used in brewing, winemaking, and distilling are strains of Saccharomyces cerevisiae , belonging to the family Saccharomycetaceae in Ascomycotina Yeast has an

spherical,egg or ovoid form Its main nutritional source is sugars such as glucose, sucrose, maltose and fructose for energy production Yeast can grow in a pH range of 3 to 5.5 and the optimum pH value is pH 4.5-5.0 The suitable temperature for yeast growth is 25-30oC [4], [14], [20]

Figure 1: Yeast Saccharomyces cerevisiae ( Power & Syred, 2014)

The composition of the yeast cell includes organelles likes nucleus, endoplasmic reticulum, mitochondrion, golgi apparatus, vacuole.[18]

Cell wall: Yeast cell walls have 250 nm thick and acount for nearly 25% of the dry

weight of the cell [15] Most yeasts have cell walls composed mainly of glucan and mannan Other yeasts have cell walls that contain chitin and mannan

Plasma membrane: is composed mainly of lipoprotein and may contain additional

sterols The plasma membrane is a difficulties of solutes, catalyzes metabolic reactions and is a place for a daughter cell to be created

Cytoplasm: is that portion of the cell enclosed by the plasma membrane and excluding

other membrane-bound organelles It is an aqueous colloidal liquid containing a multitude of metabolites [7]

.Mitochondrion: is an organelle where aerobic respiration occurs Mitochondria are the

energy-producing organs of cells

Nucleus: stores the cell DNA and chromosome Nucleus is round, membrane nucleus

inside is the nuclear fluid containing the nucleus,

Vacuole: is a membrane-bound structure that stores nutrients likes acid amin, sugars,

mineral and is also where the cell breaks down proteins

Endoplasmic reticulum: where the ribosome is attacched,is a network of membranes to

exchange information between the cell’s outer surface and internal structures[27]

Microbodies: are mainly made up by glycogen bodies and lipid granules [6]

Yeast cells reproduce both as asexual and sexual The accepted kind of yeast is asexual reproduction by budding, where a small bud is formed on the parent cell New bud develops from the mother cell when the conditions are right and then, when the bud reaches the size of an adult, it separates from the mother cell Some yeasts, including Schizosaccharomyces pombe, reproduce by fission instead of budding, and thereby creating two identically sized daughter cells The transition between haploid and diploid phases of the life cycle is accomplished through either sexual reproduction where two haploid cells of opposite mating type (α and α’ ) communicate with each other using proteins call pheromones and mate to a diploid zygote, or meiosis where one diploid cell undergoes premeiotic S-phase and two divisions, resulting in four haploid cells enclosed

in ascspore walls.[17]

The yeast growth process includes the phases: lag phase, log phase, stationary phase and death phase The frist phase of growth is lag phase The lag phase occurs during the cells’ adjustment to new environment (culture conditions) During this stage, cells have not cleaved yet, but the volume and volume increase markedly due to the increase in new cell components [22] The lag phase usually lasts from minutes to several hours The length of the lag phase can be controlled to some extent due to its dependence on the type

of medium and the initial inoculum size The second growth phase is the log phase This stage is characterized by a period of fastest possible growth if the right culture and culture conditions are encountered During exponential growth, physical healthy cells repeat at a uniform rate, so the rate of cell growth in the culture medium is related to the

number of cells present at any particular moment The yeast population copys regularly until the maximum number of cells is reached The duration of the log period varies according to species and environmental composition The generation time of the species

is calculated from the log phase (linear part) of the growth curve At the stationary phase, the number of cells under going separation identical the number of cells dying There is no further increase in cell number and the population is keep at its maximum level for a period of time The basic factors responsible for this phase are depletion of some necessary metabolites and increase of toxic end products in the medium The final phase of the growth curve is the death phase, which is characterised by a net loss of culturable cells This results from continuing depletion of nutrients and build-up of metabolic wastes Usually, the cells die at a rapid and uniform rate This decrease in population closely parallels the increase in population during the log phase.[17]

2.1.2 Metabolism in Yeast

Process of metabolizing nutrients from the environment to building new cells and for growth

Metabolism includes 3 step

- Cleaves macromolecules outside the cell so they can cross the membrane into the intracellular space

- Break down of small molecules for intermediate metabolites(pyruvate,acetyl) and energy(ATP)

- Thoroughly construe intermediate metabolites into CO2 and H2O

Metabolism in yeast includes anabolism and catabolism

Catabolism: the process of breaking down macromolecular nutrients so that they cross

the membrane into the intracellular space, then further break down compounds with small molecules into intermediate compounds and absorb energy Continue to thoroughly break organic compounds into the final products CO2 and H2O.[2]

Anabolism: use of metabolic intermediate compounds that are catabolized or readily

available in the environment to perform cell structural biosynthesis, such as polysaccharide, proteins, nucleic acids, lipids [2]

Animal Yeast is present in the stomach and on the skin of mammals For

example The yeast Malassezia, found in dogs, causes dermatitis

or fungal skin diseases in dogs Another type of yeast is the candida yeast that exists in the human body, mainly appearing on the skin, in the mouth and intestinal tract, causing diseases such

as dermatitis, thrush, thrush, and digestive disorders

Atsmosphere There are several types of yeasts in the air Rhodotorula and

Sporobolomyces are dispersed by air from layer above soil surfaces

Built

Enviroment

Yeasts are literary ubiquit ous in building,e.g : S cerevisiae is readily isolated from surfaces in wineries

Plants The interface between the degradable nutrients of the plant and

the world of decay is what yeast is all about; Yeast is found in roots, stems, leaves and fruits

Soil Yeast are global in farm and are found only in higher Lipomyces

and schwanniomyces are insulated absolutely from soil Water Yeast dominate in surface layers of fresh and salt waters, but are

not present in great numbers The spceies Debaryomyces hansemi

is a halotolerant yeast that can grow in nearly saturated brine solutions

2.1.4 Application of yeasts

Yeast is widely used in the field of biotechnology and food technology Many types of yeast are use in food production such as baker yeast, brewer's yeast in beer fermentation, and yeast in wine fermentation and for xylitol production Nowadays, yeast is regarded

as a versatile tool for biotechnological purposes [31] [32]

2.1.4.1 Alcoholic beverage

Beer and wine are popular products which are really familiar in Vietnam Beer is a delicious and refreshing drink, with an alcohol content of 4-5% For the most part, all beers include a particular range of ingredients: water, grain, yeast and hops Yeasts and rice is also important in wine production During the wine making process, yeast is responsible for converting sugar (glucose and fructose) in fruit juice or rice into wine Different yeast strains can give different quality levels of products [31]

2.1.4.2 Animal feed

In addition, we see a very important application of yeast in animal production This is because the yeast cells contain substances essential for life in high concentrations To reduce the amount of waste In 2019, reseachers from Hong Kong, China, have been studying food waste that can be used as fish feed, and the addition of baking yeast and enzymes will enhance immunity of fish [19]

2.1.4.3 Bread production

2.1.4.3.1 Bread production process [10]

Step 0 Ingredient

- Raw material: Flour, yeast, water, sugar, salt,additive

- Machine: Oven sanaky

Step 1 Weigh material + Mixing

- Weight material : depending on the type of cake

- Mixing : assures all ingredients are spread out oer the bread evenly

Step 2: Bulk Fermentation

This phase consists of leaving the dough mix just like that for about 30 minutes During this resting period the flour hydrates, more specifically the starch and gluten of the flour are hydrated by water Of great important for the development of bread flavor is the production of organic acid during fermentation Another result of dough fermentation is the production of ,carbon dioxide , this is a gas and causes the dough to expand Often a dough is a lot softer and more flexible after resting It tends to make it easier to knead in the next step

Step 3: Kneading

The role of kneading is to uniformly distribute the ingredients into a homogeneous dough mass Forms elastic gluten networks Prepare the dough block for the next step Kneading by hand : Folding, stretching

Step 5: Final Fermentation

The major goal of the final fermentation is to raise the bread to the desired degree

Step 6: Scoring

Before when the bread is put into the oven, we must scoring the bread The purpose of the cake scoring is to help the cake bloom better and look better

Step 7: Baking

Turn on oven before 15 minutes

Depending on type of bread that set temperature and time

Step 8: Cooling and Eating

Step 9: Packaging and Shelf life [25]

1.Packaging

The ideal bread packaging material must: be attractive, maintain adequate shelf-life, run

on automatic machinery, be strong, be inexpensive, be an adequate moisture barrier, protect the shape of the product

Some examples of food contact materials used in bakery: Polypropylene (PP),

Low density polyethylene (LDPE), Biaxially Oriented Polypropylene (BOPP),

Paper / Foil

2.Shelf life

The shelf life of bakery products is divided into:

a Fresh products - have 24 h shelf life

b Long shelf life products – from a few days, to several months and even years

2.1.3.4.2 Role of Yeast in bread making

Yeast three major functions in the dough: leavening, maturing, and flavor development.[16]

Leavening

Yeast converts the sugars found in flour into alcohol and CO2 CO2 is the agent that increases the volume of dough, the CO2 is stored in the gluten networks in the dough Yeast has to change to the mainly anaerobic environment in the dough as well as to the fermentation of maltose after exhaustion of available free sugars Yeast also has to accept

to sure degree of osmotic pressure exerted when added salt, sugars

Maturation

Yeast action plays an important role in the texture development of the dough, this

is called maturation The mixing results in a network of gluten and air bubbles allowing the CO2 to diffuse in Thanks to the gluten network, the air bubbles formed by CO2 during fermentation are retained

Taste and Flavor

The characteristic and appealing bread aroma would not develop without yeast During the bulk and final fermentation, biochemical reactions produce products such as alcohols, esters, and amino acids In addition to the fermentation process, the aroma of the bread is also determined by the baking process, nearly 70 flavor-causing substances