Study on the evaluation of probiotics as environmental cleaning agents

THAI NGUYEN UNIVERSITYUNIVERSITY OF AGRICULTURE AND FORESTRY TRINH THI MY DUYEN Topic title: STUDY ON THE EVALUATION OF PROBIOTICS AS ENVIRONMENTAL CLEANING AGENTS BACHELOR THESIS Study

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

TRINH THI MY DUYEN

Topic title:

STUDY ON THE EVALUATION OF PROBIOTICS

AS ENVIRONMENTAL CLEANING AGENTS

BACHELOR THESIS

Study Mode : Full-time

Thai Nguyen, 10 / 2019

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

TRINH THI MY DUYEN

Topic title:

STUDY ON THE EVALUATION OF PROBIOTICS

AS ENVIRONMENTAL CLEANING AGENTS

BACHELOR THESIS

Assoc Prof Tran Van Dien

Thai Nguyen, 10 / 2019

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Major Environmental Science and Management

Student name Trinh Thi My Duyen

Student ID DTN155429007

Thesis Title Study on the evaluation of probiotics as environmental cleaning

agentsSupervisors 1 Assoc Prof Douglas J H Shyu2 Assoc Prof Tran Van Dien

Abstract: This is an innovative and innovative project developed from aproject

to use probiotics to enhance the health of animals and plants into projects onenvironmental protection This is a study of probiotic bacteria About theeffectiveness

of probiotic bacteria in different biological and abiotic conditions From there, it ispossible to create disinfectant reagents to replace the use of chemicals from theantibacterial activities of each strain of bacteria

Health group bacteria and joint group bacteria have been used in thisexperiment Health bacteria contain three strains of biological bacteria Its functionis

to maintain health Joint bacteria contain three strains of biological bacteria Thefunction of the general group is to strengthen the health of the joint By sequencingthe 16S rRNA and DNA genes amplified through polymerase chain reaction using16S-F3R3 primers The sequence of bacteria was sequenced after that the result wascompared with the gene bank on the NCBI website Genetic analysis showed that the

3

Then test the antibacterial activity of each strain with pathogenic bacteria arestaphylococcus aureus and pseudomonas aeruginosa.

This study was created to evaluate the environmental cleanup feature ofprobiotic bacteria

Keywords Antibacterial, 16S rRNA, 16S-F3R3, NCBI,

Number of pages 48

Date of Submission 10 /2019

4

From the bottom of my heart, I would like to express my deepestappreciation

to all those who provided me the opportunity to complete this research

First and foremost, I would like to express my sincere gratitude and deepregard to my supervisors: Dr Douglas J H Shyu of National Pingtung University

of Science and Technology Who kindly was very patient with my knowledgegaps

and in guiding me wholeheartedly when I implemented this research

I also want to express my thanks to Dr Tran Van Dien of Thai NguyenUniversity of Agriculture and Forestry, the second supervisor, for his supervision,encouragement, advice, and guidance in writing this thesis

In addition, formal thanks should be offered to the dean and AssociateProfessor of office of international affairs of National Pingtung University ofScience and Technology, Dr Pomin Li for granting my internship acceptance

I would also like to acknowledge with much appreciation to the Douglas J

H Shyu, PhD Associate Professor Functional Genomics Laboratory Department

of Biological Science and Technology National Pingtung University of Scienceand Technology, who gave the permission to use all required equipment and thenecessary materials to conduct my research in Laboratory of Department ofBiological Science and Technology

I wish to thank the technicians who work in the Department of BiologicalScience and Technology

My supervisor Dr Douglas J H Shyu who provided me probiotic bacteriafor free Without them, this research could not be accomplished on time.

Of course, I would like to thank my Taiwan friends - Yu Wen, Liyu, Wily,Ito, Chau, and others for their invaluable support and encouragement when Istayed

in Pingtung

Finally, special thanks to my family, my friends for their love and moralsupport throughout my studies

Thankyou very much and best regards.

Thai Nguyen, September 25

Student

Trinh Thi My Duyen

TABLE OF CONTENT

3.2.1 3.2.7 Cloning of Screened Gene into yT&A-Vector following Transformed intoDH5a 25

3.2.7.1 3.2.7.2

3.2.7.3LIST OF FIGURES

3.2.7.4 3.2.7.5 3.2.7.6Figure 3.8 Phylogenetic tree gene and homologous rate of J2-16S-F3R3strain 38

3.2.7.7Figure 3.9 Phylogenetic tree gene and homologous rate of J6-16S-F3R3strain 38

3.2.7.8Figure 3.10 Phylogenetic tree gene and homologous rate of Ht4-16S-F3R3strain 38

3.2.7.9Figure 3.11 Phylogenetic tree gene and homologous rate of Ht6-16S-F3R3strain 39

3.2.7.10 Figure3.12 The level of anti-Staphylococcus aureus 40

10

3.2.7.11 Figure3.13 The level of anti- Pseudomonas aeruginosa 403.2.7.12 Figure3.14 The anti- Staphylococcus aureus level of J2, J6, HT4, and HT6 strain 41

3.2.7.13 Figure3.15 The anti-Pseudomonas aeruginosa level of J2, J6, HT4, and HT6 strain

3.2.7.14 42

11

3.2.7.15 LIST OF TABLES

3.2.7.16

3.2.7.17

3.2.7.66 PART I INTRODUCTION1.1 Background

3.2.7.67 Multi-drug resistant bacteria are the cause of numerous

throughout the world Increased resistance among pathogens causing nosocomialand community-acquired infections is known to be related to the widespreadutilization of antibiotics (Pfaller et al., 1997) Infectious diseases caused byresistant microorganisms are accountable for increased health costs as well ashigh

morbidity and mortality, especially in developing countries Pseudomonasaeruginosa is an opportunistic gram-negative bacterium which is a major cause ofnosocomial infections, usually occurring in the context of serious underlyingdiseases and accounting for nearly 10% of all hospital-acquired infections ofsurgical sites, the respiratory tract and the urinary tract (Jarvis et al., 1992) Highgenotypic diversity of Pseudomonas aeruginosa strains isolated from patients withcystic fibrosis in the Czech Republic Res in Microbiol 2007 It is alsoprevalently

related to otitis media and nasal infections and represents a leading cause ofmorbidity due to burn wound infection (Brook, 1994; Lyczak et al., 2000) P.Aeruginosa has an inherent resistance to most available antibiotics, includingaminoglycosides, anti-pseudomonal penicillins, newer cephalosporins, imipenem,and íluoroquinolones as treatment options for systemic infections (Burgess, 2005;Paul et al., 2004; Patzer et al., 2007) Similarly, Staphylococcus aureus (S aureus)belongs to a gram-positive group with a cocci shape It was found in grape-like

clusters Frequently, S aureus is mostly found in the anterior nares in humanswhich are the favorable conditions for maintenance and infection Especially,

3.2.7.68 S.aureus has a characteristic of biofilm formation When S aureus

circulatory system, it avoids the detection by the immune system, binds to aspeciíic surface including infection area and forms a bioíilm to survive in the host(Lowy et al., 1998) Moreover, its bioíilm can be created on both biotic andabiotic

surface; so, S aureus shows resistance to antibiotics that becomes a problem intreatment (Fedtke et al., 2004; Greenber et al., 1989; Herrmann, 2002; D Joh etal., 1999; PW Park et al., 1996; Patti et al., 1994) Some infectious diseasesrelated

to the S aureus’ bioíilm formation were arthritis, endocarditis, and cystic fibrosis(Costa et al., 1999; Lancet, 1998; Rajan, 2002) Bacteria could havecharacteristics

to form a biofilm The extracellular polymeric matrix is made from thecombination of exopolysaccharides, proteins, teichoic acids, enzymes, andextracellular DNA (Melchior et al., 2006; Parra-Ruiz et al., 2012) Regarding toprevious studies, the matrix’s structure is changed from strains to strains due tothe

environment and the conditions (Rohde et al., 2001; Landini, 2009) By living in

a community, biofilms have various benefits and advantages from their parts andone of them is resistant to the immune system and antibiotics Recent reports havedocumented the role of exogenous Lactobacilli in the prevention and treatment ofsome infections Lactobacillus acidophilus is gram-positive bacteria naturallyliving in the human and animal digestive system Lactobacillus is also used indairy

products including milk, yogurt in combination with other microbes.Lactobacillus has the potential to be used as an antibiotic medicine and a drugdeliver ( TTV Doan et al., 2013) Recent reports have documented the role ofexogenous Lactobacilli in the prevention and treatment of someinfections.Lactobacillus strains are commensal in the human body Oral

Lactobacillus strains has been found to be useful in various bacterial infections(Gordon et al., 1957; Vandenbergh, 1993; Carson, Riley, 2003) Its beneíicialeffect may be associated to its ability to inhibit the growth of pathogens,apparently

by the secretion of antibacterial substances including lactic acid, hydrogenperoxide and etc (Gibbs, 1987) As the above statement, the aim of the study ofLactobacillus was to know whether Lactobacillus inhibits Staphylococcus aureusand Pseudomonas aeruginosa or not

effectiveness of probiotic bacteria in different biological and abiotic conditions.From there, it is as a premise possible to create disinfectant reagents to replace theuse of chemicals from the antibacterial activities of each strain of bacteriaidentified The main aim of this study is to contribute to protecting theenvironment Propose the cleaning agent from probiotic to substitute chemicalscleaning agent, which does not eliminate pathogenic bacteria only but also be anenvironmentally friendly agent.

1.3 Scope of study

- The provided bacteria cultured in MRS broth environment

- The bacteria DNA extraction The ampliíication genes by PCR reactionusing 16S rRNA primers

- Ligation and transformation reactions to joining of two nucleic acidfragments through the action of an enzyme performed using T4 DNAligase

- Sequencing of bacterial gene and compare to with gene bank in the NCBI.

- Use DNA star software to identify selected strains of bacteria

- Test and evaluate antibacterial activity by the pouch hold method withidentified bacteria strains

3.2.7.70 PART II LITERATURE REVIEW2.1 Definition of Probiotics

3.2.7.71 Etymologically the term probiotic is derived from the Greeklanguage

meaning “for life” but the deíinition of probiotics has evolved over timesimultaneously with the increasing interest in the use of viable bacterialsupplements and in relation to the progress made in understanding theirmechanisms of action The term was originally used to describe substancesproduced by one microorganism that stimulated the growth of others and was laterused to describe tissue extracts that stimulated microbial growth and animal feedsupplements exerting a beneficial effect on animals by contributing to theirintestinal flora balance (Fuller, 1999.) Until recently the most widely useddeíinition which contributed to the development of the probiotic concept inseveral

ways was that of Fuller: “probiotics are live microbial feed supplements whichbeneíicially affect the host animal by improving microbial balance” (Fuller,1989)

The definition used at present was given by the Food and AgricultureOrganization

of the United Nations World Health Organization, according to which probioticsare redefined as “live microorganisms which when administered in adequateamounts confer a health beneíit on the host.” In relation to food the definition can

be adjusted by emphasizing that the beneficial effect is exerted by themicroorganisms “when consumed in adequate amounts as part of food”

(FAO/WHO, 2001).

3.2.7.72 The potential applications of probiotics in unrecognized foodand

agricultural products have not been formally recognized Recently, there hasbeenincreasing interest in food and agricultural applications of probiotics, theselection

of new strains of biobacteria and the development of new applications havegained

a lot importance Agricultural applications of probiotics related to animal, fishand

plant production have increased Agricultural applications of probiotics related toanimal, fish and plant production have increased Nevertheless, there remain someuncertainties regarding the technological, microbiological and regulatory aspects(Krốckel L, 2006)

fertilizers increases the nutrient cycle in the soil and forms a "biological buffer" toimprove the extreme conditions / stress of cultivation It is the useful

microorganisms that are added to the soil when fertilizing bio-fertilizers to helpstimulate the host immune system and protect plants from pests (Vikas Ghumare

et al., 2014) Therefore, bio-fertilizers can help reduce the use of large amounts ofpesticides

3.2.7.74 Unlike chemical fertilizers, biofertilizer technology does not

environmental pollution and it is based on renewable resources of energy.Biofertilizer technology is a low-cost technology It is most suitable for growingnations where labour is inexpensive and nutrient input for crop cultivation throughchemical sources is meagre Various micro-organisms and associations withplantswhich are involved in biofertilizer production like their usage on the farmare

reviewed in here In addition to nitrogen, phosphorus also plays an important role

in plant growth and metabolismThe microorganisms responsible for dissolving thephosphate occur in intimate symbiotic relationships with the roots and thefunction

is the liaison between the tree and the surrounding soilIn the future, biofertilizerswill probably include a blend of nitrogen-fixing and phosphate-mobilizingmicroorganisms Research aimed at growing these and at transferring nitrogen-fixing genes from bacteria into plants will result in a new biotechnology to reduceour dependence on chemical fertilizers (Rao, N S S., 1982)

3.2.7.75 PART III MATERIAL AND METHOD

3.1 Equipment and materials

3.2.7.84 Switzerland3.2.7.85

2 3.2.7.86 Deep freezer 3.2.7.87CF-U23V M 3.2.7.88n Japa3.2.7.89

3 3.2.7.90 Drying Oven 3.2.7.91 3.2.7.92 Taiw

an3.2.7.93

5 3.2.7.98 Vortex shaker 3.2.7.99M2000 V 3.2.7.100an Taiw3.2.7.101

6

3.2.7.102 Dry bath

incubator

3.2.7.103 MD-02N

3.2.7.104 Taiw

an3.2.7.105

8 3.2.7.110machine Centrifuge 3.2.7.11137520 D- 3.2.7.112many Ger3.2.7.113

9

3.2.7.114 Orbital shaker

incubator

3.2.7.115 LM-570RD

3.2.7.116 Singapore

12 3.2.7.126 Microwave

3.2.7.127 TMO-202

3.2.7.128 Taiw

an3.2.7.129

13 3.2.7.130 Nichipet EX 3.2.7.131 3.2.7.132 Japa

n3.2.7.133

16 3.2.7.142cabinet Laminar airflow 3.2.7.1434N JW. 3.2.7.144an Taiw3.2.7.145

18 3.2.7.150balance Analytical

3.2.7.151 AB54-S

3.2.7.152 Switzerland3.2.7.153

3.2.7.156 Figure 2.1 The powder of bacteria in this study.

3.2.7.157

3.I.2.2 Preparing Bacteria growth media

3.2.7.158 MRS broth and American Bacteriological Agar (Figure 2.2)

nutrients medium for the growth of plant growth bacteria used in this study withthe rate of the former was 52.25g/l and the latter was 16g/l (pH 7.3) Sterilized itby

autoclaving at 121°C for 1 hour and keep it at 4°C

3.2.7.160 3.1.2.3 Lysozyme Buffer

3.2.7.161 Preparing Lysozyme buffer (Figure 2.3) was used to DNA

gram-positive bacteria included 20pg/ml lysozyme; 20nM Tris- HCl; 2nM EDTA; 1%Triton X-100 and pH 8.0 Sterilized it by autoclaving at 121°C for 20 min andkeep at

3.2.7.162 4°C

Figure 2.2 MRS broth medium and American Bacteriological Agar

3.2.7.163 Figure 2.3 Lysozyme Buffer for DNA

extraction

3.2.7.164 3.1.2.4 Escherichia coli (DH5a)

3.2.7.165 Escherichia coli strain (DH5a) (Figure 2.4) was used for

3.2.7.171 Table 2.2 Formular in g/l (Medium to facilitate de growth of

2 3.2.7.179 Dipotassium Phosphate 3.2.7.180 2.003.2.7.181

3 3.2.7.182 Bacteriological Peptone 3.2.7.183 10.003.2.7.184

4 3.2.7.185 Ammonium Citrate 3.2.7.186 2.003.2.7.187

5 3.2.7.188 BeeíExtract 3.2.7.189 8.003.2.7.190

6

3.2.7.191 Tween 3.2.7.192 1.00

3.2.7.193

7 3.2.7.194 Sodium Acetate 3.2.7.195 5.003.2.7.196

8 3.2.7.197 Magnesium Sulfate 3.2.7.198 0.203.2.7.199

9

3.2.7.200 Yeast Extract 3.2.7.201 4.00

3.2.7.202

10 3.2.7.203 Manganase Sulfate 3.2.7.204 0.053.2.7.205

1 Firstly, prepared plate agar and liquid medium to culturing bacteria

2 The bacteria were removed from the original storage tube and then diluted

it to cultured spread on the surface of the medium plate according to the

by the culture rods that heated on the fire of alcohol

3 Then it was incubated in the incubator at 37°C for 48 hours

4 The result would be the appearance of single colonies

5 Picked up each one single colony in a plate put in one liquid culture tube,then the sample shook and incubated in the incubator for 37°C overnight tofacilitate DNA extraction in the next process

3.2.2 Gram staining

1 Add about 1 drop of crystal violet stain over the fixed culture Let standfor 60 seconds

2 Pour off the stain and gently rinse the excess stain with a stream of H2O

3 Add about 1 drop of the iodine solution on the smear, enough to coverthe fixed culture Let stand for 30 seconds

4 Pour off the iodine solution and rinse the slides with running water

Shake off excess water from the surface

5 Add a few drops of alcohol so the solution trickles down the slide Rinse

it off with water after 5 seconds Stop when the solvent is no longer

colored as it

flows over the slide

6 Counterstain with 5 drops of the Safranin solution for 20 seconds

7 Wash off the red SaEranin solution with water Blot with bibulous paper toremove any excess water Alternatively, the slide may be shaken to remove

8 Examine the finished slide under a microscope

3.2.7.209 Genomic DNA extraction kit which was used for DNA extraction in my

study The process includes the following basic steps (Figure 2.5)

3.2.7.210 Centriíuge of bacteria culture tube for 1 min at

13,000 rpm and discard the supermatant

3.2.7.211 Add 200 pl Lysozyme Buíter and mix gently.

Incubate in vert the tube every 3 mins3.2.7.212 ị

3.2.7.213 Add 200 |_il GB Buíter to the sample, vontex íor 5

3.2.7.218 Apply all the mixture to the GD column and

centriíuge at 13,000 rpm for 1 min

3.2.7.219 _- I

3.2.7.220 Discard the flow-through and add 400 pl of W1

Buffer (ethanol added)3.2.7.221 ị

3.2.7.222 Discard the flow-through and add 600 pl of Wash

Buííer (ethanol added) Centrituge 13,000 rpm for

1 min.

3.2.7.223 ị

3.2.7.224 Go on discard and centriíuge more 3 mins.

Transter GD column into new mirocentrituge

tube.

3.2.7.225 ị

3.2.7.226 Add 50 pl Elution Butíer and centriíuge at 13,000

rpm for 1 min and keep at -20°C

3.2.7.227 Figure 2.5 The process of DNA extraction bacteria

1 Add 1ml sample to microtube, centrifuge at 13000rpm/1min then removesupernatant

2 Add 200pl Lysozyme Buffer and vortex ( keep in room temperature 10

3.2.3 DNA

extraction

32

min and shake it by every 3 min).

3 Add 200pl GB Buffer ( vortex 10 times to mix sample)

3.2.7.228is clear, invert theIncubate 70°C for 10 minutes until the sample lysate

3.2.3 DNA

extraction

33

3.2.7.229 tube every 3 minutes At this time, preheat required Elution Buffer

binding

4 Add 200pl of Ethanol 95% to the sample lysate and vortex immediatelyfor 10 seconds to mix samples If precipitate appears, break up bypipetting

5 Place a GD Column on a 2 ml Collection Tube

6 Apply all the mixture (including any precipitate) from the previous step

9 Add 600pl of Wash Buffer to the GD Column Centrifuge at 13000rpmfor 1min

10 Discard the flow-through and return the GD Column the 2ml CollectionTube Centrifuge for an additional 3 minutes to dry the column

3.2.7.230 DNA Elution

11 Transfer dried GD Column into a clean 1.5ml microcentrifuge tube

12 Add 50pl of preheated Elution Buffer to the center of the columnmatrix

13 Allow standing for 2 minutes until the Elution Buffer is absorbed bythe matrix

34

14 Centrifuge at 13000rpm for 1min to elute puriíied DNA.

35

3.2.7.231 3.2.4 DNA Electrophoresis.

3.2.7.232 DNA electrophoresis was conducted using agarose gel to

obtained from the result of DNA extraction To make 1.2% agarose gel (Table2.4),

approximately 0.3 grams of agarose to 30 ml of 0,5X TAE buffer (dilution from50X TAE buffer (Tab 2.3) and warp the upper part of the flask Some holes on thewarp were needed to decrease the pressure when heating Then the flask wasplaced in the microwave for 3 minutes to dissolve the particles completely Afterthe agarose was dissolved, the solution was cooled and poured into the cast until itbecame solid around 30 minutes Place the gel in the electrophoresis tank andsubmerged in 1X TAE buffer The DNA samples were mixed with a 6X loadingdye and loaded in the wells The first well was treated with 1 kb (Meru et al.,2013)

DNA marker in order to observe the size of DNA samples After the samples werecompletely loaded, the electrophoretic device was energized and colloid wasobserved after 30 minutes at 100V The gel was stopped until the maker run to thepenultimate mark line The gel was stained before running by Vison DNA

3.2.7.233 Table 2.3 50X TAE buffer composition.

3.2.7.243 * DNA electrophoresis was conducted in 0,5X TAE buffer.

3.2.7.244 Table 2.4 1,2 % Agarose gel composition

3.2.7.254 Table 2.5 The component of PCR reaction amplification gen

3.2.7.267 Total 3.2.7.268 10

3.2.7.269

38

3.2.7.270 Table 2.6 The sequences of primer used for PCR reaction

ACT TCA 3.2.7.28518 3.2.7.2863.2.7.287

3.2.7.288 The temperature cycles for PCR reaction

3.2.7.289 16S-F3R3: 25 Cycles

3.2.7.290

3.2.7.291

3.2.7.292 3.2.6 DNA purification from Agarose gel.

3.2.7.293 PCR products obtained after gene amplification have

primers, PCR buffers, nucleotides and sometimes may contain by-products thatwill affect the results of the separation process Therefore, it was necessary topurify the PCR product After the PCR product was coníirmed by DNAelectrophoresis, the target fragment was excised on the UV lightbox using a cleanscalpel then transferred the gel slice into a microcentrifuge tube DNA gelpurification was conducted with FavorPrepTM Gel/PCR purification kit(Favorgen Biotech Corp., Taiwan) according to the basic steps (Figure2.6)

39