Study on the evaluation of probiotics as environmental cleaning agents

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY TRINH THI MY DUYEN Topic title: STUDY ON THE EVALUATION OF PROBIOTICS AS ENVIRONMENTAL CLEANING AGENTS... UNIVERSITY OF A

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY

TRINH THI MY DUYEN

Topic title:

STUDY ON THE EVALUATION OF PROBIOTICS

AS ENVIRONMENTAL CLEANING AGENTS

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

Major : Environmental Science and Management

Faculty : Advanced Education Program Office

Supervisors : Assoc Prof Douglas J H Shyu

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Major Environmental Science and Management

Student name Trinh Thi My Duyen

Thesis Title Study on the evaluation of probiotics as environmental cleaning

agents

Supervisors 1 Assoc Prof Douglas J H Shyu

2 Assoc Prof Tran Van Dien Abstract: This is an innovative and innovative project developed from a project

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

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

Health group bacteria and joint group bacteria have been used in this experiment Health bacteria contain three strains of biological bacteria Its function is

to maintain health Joint bacteria contain three strains of biological bacteria The function of the general group is to strengthen the health of the joint By sequencing the 16S rRNA and DNA genes amplified through polymerase chain reaction using 16S-F3R3 primers The sequence of bacteria was sequenced after that the result was compared with the gene bank on the NCBI website Genetic analysis showed that the strains identified were Lactobacillus rhamnosus, Lactobacillus paracasei Lactobacillus pentosus or Lactobacillus Plantarum subsp, Lactobacillus Plantarum

Then test the antibacterial activity of each strain with pathogenic bacteria are staphylococcus aureus and pseudomonas aeruginosa

This study was created to evaluate the environmental cleanup feature of probiotic bacteria

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

Number of pages 48

Date of Submission 10 / 2019

ACKNOWLEDGMENT

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

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

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

of Science and Technology Who kindly was very patient with my knowledge gaps and in guiding me wholeheartedly when I implemented this research

I also want to express my thanks to Dr Tran Van Dien of Thai Nguyen University 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 Associate Professor of office of international affairs of National Pingtung University of Science 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 Science and Technology, who gave the permission to use all required equipment and the necessary materials to conduct my research in Laboratory of Department of Biological Science and Technology

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

My supervisor Dr Douglas J H Shyu who provided me probiotic bacteria for 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 I stayed

in Pingtung

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

Thank you very much and best regards

Thai Nguyen, September 25

Student

Trinh Thi My Duyen

TABLE OF CONTENT

DOCUMENTATION PAGE WITH ABSTRACT i

ACKNOWLEDGMENT iii

TABLE OF CONTENT v

LIST OF FIGURES 1

LIST OF TABLES 3

LIST OF ABBREVIATIONS 4

PART I INTRODUCTION 5

1.1 Background 5

1.2 Objectives 7

1.3 Scope of study 7

PART II LITERATURE REVIEW 9

2.1 Definition of Probiotics 9

2.2 Biofertilizers 10

PART III MATERIAL AND METHOD 12

3.1 Equipment and materials 12

3.1.1 Equipment 12

3.1.2 Materials 13

3.2 Method…… …15

3.2.1 Culture of bacteria 15

3.2.2 Gram staining 17

3.2.3 DNA extraction 18

3.2.4 DNA Electrophoresis 20

3.2.5 PCR amplification 21 3.2.7 Cloning of Screened Gene into yT&A-Vector following Transformed into DH5α 25

3.2.7.1 Ligation 25

3.2.8 Antibacterial activity assay 30

PART IV RESULTS 32

4.1 The result of the culture of bacteria 32

4.2 The result of gram staining 32

4.3 The results of the DNA extraction of six strains in the study 33

4.4 The results of PCR amplification 34

4.5 The results of gene cloning 35

4.5.1 Results of transforming plasmid DNA into variable cells of E coli DH5α 35

4.5.2 The results of DNA Plasmid Extraction by Restriction Enzyme digestion 36

4.6 Identify and analyze the nucleotide sequence of the DNA markers 37

4.7 The results of the antibacterial activity assay 39

4.7.1 Antibacterial of mix strains of bacterial 39

4.7.2 Antibacterial of each strain 41

PART V CONCLUSIONS 43

5.1 Conclusion 43

5.2 Recommendations 44

REFERENCES 45

LIST OF FIGURES

Figure 2.1 The powder of bacteria in this study 13

Figure 2.2 MRS broth medium and American Bacteriological Agar 14

Figure 2.3 Lysozyme Buffer for DNA extraction 14

Figure 2.4 The cell of Escherichia coli (DH5α) in this study 15

Figure 2.5 The process of DNA extraction bacteria 18

Figure 2.6 The process of DNA purification from Agarose gel 23

Figure 2.7 The process of transformation reaction 26

Figure 2.8 The process of Plasmid DNA extraction 29

Figure 2.9 The image illustrates how to prepare work on the MRS agar plate 31

Figure 3.1 The two groups of bacteria isolated growth on media 32

Figure 3.2: The shape and arrangements of bacteria observed under microscope 33

Figure 3.3 The results of DNA extraction 33

Figure 3.4 The results of PCR amplification with primer pair 16S-F3R3 34

Figure 3.5 The result of transforming the recombinant vector into competent cells 35

Figure 3.6 The results of electrophoresis of enzyme-cut products by EcoRI and HindIII enzyme of the 16S-F3R3 primer 36

Figure 3.7 The software to compare sequences on gene banks on the NCBI website 37

Figure 3.8 Phylogenetic tree gene and homologous rate of J2-16S-F3R3 strain 38 Figure 3.9 Phylogenetic tree gene and homologous rate of J6-16S-F3R3 strain 38 Figure 3.10 Phylogenetic tree gene and homologous rate of Ht4-16S-F3R3 strain 38

Figure 3.11 Phylogenetic tree gene and homologous rate of Ht6-16S-F3R3 strain 39

Figure3.12 The level of anti-Staphylococcus aureus 40

Figure3.13 The level of anti- Pseudomonas aeruginosa 40Figure3.14 The anti- Staphylococcus aureus level of J2, J6, HT4, and HT6 strain 41Figure3.15 The anti-Pseudomonas aeruginosa level of J2, J6, HT4,and HT6 strain 42

LIST OF TABLES

Table 2.1 Equipments of studies 12

Table 2.2 Formular in g/l (Medium to facilitate de growth of lactobacilli) 16

Table 2.3 50X TAE buffer composition 20

Table 2.4 1,2 % Agarose gel composition 21

Table 2.5 The component of PCR reaction amplification gen 16S rRNA 21

Table 2.6 The sequences of primer used for PCR reaction to identify Characterization of rhizobacteria in sesame 22

Table 2.7 The component of the ligation reaction 25

Table 2.8 The component of restriction enzyme digestion reaction 30

Table 3.1 Identify 6 strains in the study on gene bank 39

Table 3.2 The antibacterial level of mix strains is measured in units of millimeters 39

Table 3.3 The anti-Staphylococcus aureus level 41

Table 3.4 The anti-Pseudomonas aeruginosa 42

NCBI National Center for Biotechnology Information

EDTA Ethylenediaminetetraacetic acid

LB broth Luria Bertani Broth

Rpm Rotation per minute

MRS Broth De MAN, ROGOSA, and SHARPE broth

1.1 Background

Multi-drug resistant bacteria are the cause of numerous clinical problems throughout the world Increased resistance among pathogens causing nosocomial and community-acquired infections is known to be related to the widespread utilization of antibiotics (Pfaller et al., 1997) Infectious diseases caused by resistant microorganisms are accountable for increased health costs as well as high morbidity and mortality, especially in developing countries Pseudomonas aeruginosa is an opportunistic gram-negative bacterium which is a major cause of nosocomial infections, usually occurring in the context of serious underlying diseases and accounting for nearly 10% of all hospital-acquired infections of surgical sites, the respiratory tract and the urinary tract (Jarvis et al., 1992) High genotypic diversity of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis in the Czech Republic Res in Microbiol 2007 It is also prevalently related to otitis media and nasal infections and represents a leading cause of morbidity due to burn wound infection (Brook, 1994; Lyczak et al., 2000) P Aeruginosa has an inherent resistance to most available antibiotics, including aminoglycosides, anti-pseudomonal penicillins, newer cephalosporins, imipenem, and fluoroquinolones 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 humans which are the favorable conditions for maintenance and infection Especially,

S.aureus has a characteristic of biofilm formation When S aureus enters into the circulatory system, it avoids the detection by the immune system, binds to a specific surface including infection area and forms a biofilm to survive in the host (Lowy et al., 1998) Moreover, its biofilm can be created on both biotic and abiotic surface; so, S aureus shows resistance to antibiotics that becomes a problem in treatment (Fedtke et al., 2004; Greenber et al., 1989; Herrmann, 2002; D Joh et al., 1999; PW Park et al., 1996; Patti et al., 1994) Some infectious diseases related

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

to form a biofilm The extracellular polymeric matrix is made from the combination of exopolysaccharides, proteins, teichoic acids, enzymes, and extracellular DNA (Melchior et al., 2006; Parra-Ruiz et al., 2012) Regarding to previous studies, the matrix’s structure is changed from strains to strains due to the environment and the conditions (Rohde et al., 2001; Landini, 2009) By living in

a community, biofilms have various benefits and advantages from their parts and one of them is resistant to the immune system and antibiotics Recent reports have documented the role of exogenous Lactobacilli in the prevention and treatment of some infections Lactobacillus acidophilus is gram-positive bacteria naturally living in the human and animal digestive system Lactobacillus is also used in dairy products including milk, yogurt… in combination with other microbes Lactobacillus has the potential to be used as an antibiotic medicine and a drug deliver ( TTV Doan et al., 2013) Recent reports have documented the role of exogenous Lactobacilli in the prevention and treatment of some infections

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

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

1.2 Objectives

This in vitro experiment was designed to identify strains of bacteria is a base to analysis the antibacterial activity and to evaluate the antibacterial effectiveness of probiotic bacteria in different biological and abiotic conditions From there, it is as a premise possible to create disinfectant reagents to replace the use of chemicals from the antibacterial activities of each strain of bacteria identified The main aim of this study is to contribute to protecting the environment Propose the cleaning agent from probiotic to substitute chemicals cleaning agent, which does not eliminate pathogenic bacteria only but also be an environmentally friendly agent

1.3 Scope of study

- The provided bacteria cultured in MRS broth environment

- The bacteria DNA extraction The amplification genes by PCR reaction using 16S rRNA primers

- Ligation and transformation reactions to joining of two nucleic acid fragments through the action of an enzyme performed using T4 DNA ligase

- 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 with identified bacteria strains

PART II LITERATURE REVIEW 2.1 Definition of Probiotics

Etymologically the term probiotic is derived from the Greek language meaning “for life” but the definition of probiotics has evolved over time simultaneously with the increasing interest in the use of viable bacterial supplements and in relation to the progress made in understanding their mechanisms of action The term was originally used to describe substances produced by one microorganism that stimulated the growth of others and was later used to describe tissue extracts that stimulated microbial growth and animal feed supplements exerting a beneficial effect on animals by contributing to their intestinal flora balance (Fuller, 1999.) Until recently the most widely used definition which contributed to the development of the probiotic concept in several ways was that of Fuller: “probiotics are live microbial feed supplements which beneficially affect the host animal by improving microbial balance” (Fuller, 1989) The definition used at present was given by the Food and Agriculture Organization

of the United Nations World Health Organization, according to which probiotics are redefined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host.” In relation to food the definition can

be adjusted by emphasizing that the beneficial effect is exerted by the microorganisms “when consumed in adequate amounts as part of food” (FAO/WHO, 2001)

The potential applications of probiotics in unrecognized food and agricultural products have not been formally recognized Recently, there has been

increasing interest in food and agricultural applications of probiotics, the selection

of new strains of biobacteria and the development of new applications have gained

a lot importance Agricultural applications of probiotics related to animal, fish and plant production have increased Agricultural applications of probiotics related to animal, fish and plant production have increased Nevertheless, there remain some uncertainties regarding the technological, microbiological and regulatory aspects (Kröckel L, 2006)

2.2 Biofertilizers

Bio-fertilizer also has a mechanism to form natural growth stimulants for plants that farmers do not need chemical compounds to stimulate plant growth and endanger the health of users Bio-fertilizer also helps to balance soil fertility, increase soil productivity thanks to the amount of humus accumulated by microorganisms, decomposing the source of organic residues in soil Applying bio-fertilizers increases the nutrient cycle in the soil and forms a "biological buffer" to improve the extreme conditions / stress of cultivation It is the useful microorganisms that are added to the soil when fertilizing bio-fertilizers to help stimulate 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 of pesticides

Unlike chemical fertilizers, biofertilizer technology does not contribute to environmental pollution and it is based on renewable resources of energy Biofertilizer technology is a low-cost technology It is most suitable for growing nations where labour is inexpensive and nutrient input for crop cultivation through

which are involved in biofertilizer production like their usage on the farm are reviewed in here In addition to nitrogen, phosphorus also plays an important role

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

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

PART III MATERIAL AND METHOD 3.1 Equipment and materials

3.1.1 Equipment

Table 2.1 Equipments of studies

3.1.2.Materials

3.1.2.1 Bacteria

Two groups of bacteria that function to support health and joint support were provided by my supervisor, Mr Douglas JH Shyu This study project developed from a project to use probiotics to enhance the health of animals and plants into projects on environmental protection

Figure 2.1 The powder of bacteria in this study

3.1.2.2 Preparing Bacteria growth media

MRS broth and American Bacteriological Agar (Figure 2.2) were used as nutrients medium for the growth of plant growth bacteria used in this study with the rate of the former was 52.25g/l and the latter was 16g/l (pH 7.3) Sterilized it by autoclaving at 121ºC for 1 hour and keep it at 4ºC

3.1.2.4 Escherichia coli (DH5α)

Escherichia coli strain (DH5α) (Figure 2.4) was used for cloning purposes provided by Functional Genomics Laboratory in the Department of Biology Science and Technology - National Pingtung University of Science and Technology

Figure 2.4 The cell of Escherichia coli (DH5α) in this study

3.2 Method

3.2.1 Culture of bacteria

In order to culture well-grown bacteria, a specific type of medium was required In this study, I used MRS Broth contains the best nutrition for bacteria to grow well

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

15.675 MRS Broth + 6g Agar American + 300ml H2O (mix well)

Sterilize them all in an autoclave for 1 hour And then spill liquid to the plate Store both liquid and the solid medium in 4° cabinets

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 ‘‘Z’’ shape

by the culture rods that heated on the fire of alcohol

3 Then it was incubated in the incubator at 37ºC 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 37ºC overnight to facilitate 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 stand for 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 cover the 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 Safranin solution with water Blot with bibulous paper to remove any excess water Alternatively, the slide may be shaken to remove most

8 Examine the finished slide under a microscope

3.2.3 DNA extraction

Genomic DNA extraction kit which was used for DNA extraction in my study The process includes the following basic steps (Figure 2.5)

Figure 2.5 The process of DNA extraction bacteria

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

2 Add 200µl Lysozyme Buffer and vortex ( keep in room temperature 10 min and shake it by every 3 min)

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

tube every 3 minutes At this time, preheat required Elution Buffer at 70°C DNA binding

5 Add 200µl of Ethanol 95% to the sample lysate and vortex immediately for 10 seconds to mix samples If precipitate appears, break up by pipetting

6 Place a GD Column on a 2 ml Collection Tube

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

10 Add 600µl of Wash Buffer to the GD Column Centrifuge at 13000rpm for 1min

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

DNA Elution

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

13 Add 50µl of preheated Elution Buffer to the center of the column matrix

14 Allow standing for 2 minutes until the Elution Buffer is absorbed by the matrix

15 Centrifuge at 13000rpm for 1min to elute purified DNA

3.2.4 DNA Electrophoresis

DNA electrophoresis was conducted using agarose gel to confirm the gene obtained from the result of DNA extraction To make 1.2% agarose gel (Table 2.4), approximately 0.3 grams of agarose to 30 ml of 0,5X TAE buffer (dilution from 50X TAE buffer (Tab 2.3) and warp the upper part of the flask Some holes on the warp were needed to decrease the pressure when heating Then the flask was placed in the microwave for 3 minutes to dissolve the particles completely After the agarose was dissolved, the solution was cooled and poured into the cast until it became solid around 30 minutes Place the gel in the electrophoresis tank and submerged in 1X TAE buffer The DNA samples were mixed with a 6X loading dye 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 were completely loaded, the electrophoretic device was energized and colloid was observed after 30 minutes at 100V The gel was stopped until the maker run to the penultimate mark line The gel was stained before running by Vison DNA

Table 2.3 50X TAE buffer composition