INTRODUCTION
Background
Multi-drug resistant bacteria pose significant clinical challenges globally, with increased resistance among pathogens linked to the widespread use of antibiotics Infections caused by these resistant microorganisms lead to higher healthcare costs and increased morbidity and mortality rates, particularly in developing countries Pseudomonas aeruginosa, an opportunistic gram-negative bacterium, is a major contributor to nosocomial infections, accounting for nearly 10% of hospital-acquired infections in surgical sites, the respiratory tract, and the urinary tract This bacterium exhibits high genotypic diversity, particularly in cystic fibrosis patients, and is commonly associated with otitis media and burn wound infections P aeruginosa demonstrates inherent resistance to many antibiotics, including aminoglycosides and fluoroquinolones In contrast, Staphylococcus aureus, a gram-positive cocci, is often found in the anterior nares of humans, creating favorable conditions for infection.
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 naturally occurring in the human body and have been shown to be effective in treating various bacterial infections through oral administration Research indicates that their beneficial effects may stem from their ability to inhibit pathogen growth by secreting antibacterial substances such as lactic acid and hydrogen peroxide This study specifically aims to investigate whether Lactobacillus can inhibit the growth of Staphylococcus aureus and Pseudomonas aeruginosa.
Objectives
This in vitro experiment aims to identify bacterial strains to analyze their antibacterial activity and evaluate the effectiveness of probiotic bacteria under various biological and abiotic conditions The findings could lead to the development of disinfectant agents that replace harmful chemical disinfectants, leveraging the antibacterial properties of the identified strains Ultimately, this study seeks to promote environmental protection by proposing probiotic-based cleaning agents that not only eliminate pathogenic bacteria but also serve as eco-friendly alternatives to traditional chemical cleaners.
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.
LITERATURE REVIEW
Definition of Probiotics
The term "probiotic," derived from Greek meaning "for life," has evolved alongside growing interest in viable bacterial supplements and their mechanisms of action Initially, it referred to substances produced by one microorganism that stimulated the growth of others, later encompassing tissue extracts and animal feed supplements that positively influenced intestinal flora balance Fuller’s definition in 1989 described probiotics as live microbial feed supplements that enhance the host animal's microbial balance Currently, the Food and Agriculture Organization and World Health Organization define probiotics as live microorganisms that, when administered in adequate amounts, confer health benefits to the host, particularly when consumed as part of food.
The potential uses of probiotics in food and agricultural products are gaining attention, with a focus on selecting new strains of beneficial bacteria and developing innovative applications The agricultural sector is seeing a rise in probiotic applications for animal, fish, and plant production However, uncertainties still exist concerning the technological, microbiological, and regulatory factors involved in these developments.
Biofertilizers
Bio-fertilizers promote natural growth stimulants for plants, eliminating the need for harmful chemical compounds that can jeopardize user health They enhance soil fertility and productivity by increasing humus through microorganisms that decompose organic residues The application of bio-fertilizers boosts the nutrient cycle and creates a "biological buffer" to mitigate extreme cultivation conditions Additionally, beneficial microorganisms in bio-fertilizers stimulate the plant's immune system, offering protection against pests and significantly reducing the reliance on pesticides.
Biofertilizer technology is an eco-friendly alternative to chemical fertilizers, utilizing renewable energy resources and minimizing environmental pollution This low-cost solution is particularly beneficial for developing nations with affordable labor and limited access to chemical nutrient inputs for crops The production of biofertilizers involves various microorganisms that form symbiotic relationships with plant roots, enhancing nutrient uptake, especially nitrogen and phosphorus, which are crucial for plant growth and metabolism Future advancements in biofertilizers may incorporate a combination of nitrogen-fixing and phosphate-mobilizing microorganisms, along with research focused on transferring nitrogen-fixing genes from bacteria to plants, paving the way for reduced reliance on chemical fertilizers.
MATERIAL AND METHOD
Equipment and materials
No Name of device Type Country
6 Dry bath incubator MD-02N Taiwan
9 Orbital shaker incubator LM-570RD Singapore
14 Thermo scientific MY SPIN 6 HSF 51902 Taiwan
16 Laminar airflow cabinet JW.4N Taiwan
Under the guidance of my supervisor, Mr Douglas JH Shyu, two groups of beneficial bacteria were identified to promote health and support joint function This study evolved from an initial project focused on utilizing probiotics to improve the health of animals and plants, ultimately branching into initiatives aimed at environmental protection.
Figure 2.1 The powder of bacteria in this study
MRS broth and American Bacteriological Agar were utilized as nutrient media for the cultivation of plant growth bacteria in this study, with concentrations of 52.25 g/L for MRS broth and 16 g/L for American Bacteriological Agar, adjusted to a pH of 7.3 The media were sterilized by autoclaving at 121ºC for one hour and subsequently stored at 4ºC.
Figure 2.2 MRS broth medium and American Bacteriological Agar
To extract DNA from gram-positive bacteria, a Lysozyme buffer was prepared, consisting of 20 µg/ml lysozyme, 20 nM Tris-HCl, 2 nM EDTA, and 1% Triton X-100, adjusted to a pH of 8.0 The buffer was sterilized by autoclaving at 121°C for 20 minutes and stored at 4ºC for future use.
Figure 2.3 Lysozyme Buffer for DNA extraction
The Escherichia coli strain DH5α, illustrated in Figure 2.4, was utilized for cloning purposes and was generously provided by the Functional Genomics Laboratory at the Department of Biology, National Pingtung University of Science and Technology.
Figure 2.4 The cell of Escherichia coli (DH5α) in this study.
Method
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)
1 Firstly, prepared plate agar and liquid medium to culturing bacteria
15.675 MRS Broth + 300ml of H2O (mix well) and add 3ml liquid on tubes Add 3ml tubes
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
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
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 200àl Lysozyme Buffer and vortex ( keep in room temperature 10 min and shake it by every 3 min)
3 Add 200àl GB Buffer ( vortex 10 times to mix sample)
4 Incubate 70°C for 10 minutes until the sample lysate is clear, invert the tube every 3 minutes At this time, preheat required Elution Buffer at 70°C DNA binding
5 Add 200àl 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 to the GD Column
8 Close the cap and centrifuged at 13,000rpm for 2 minutes Discard the flow-through and return the GD column to the 2ml collection tube
9 Add 400àl W1 Buffer to the GD Column Centrifuge at 13,000rpm for 1min then discard the flow-through and return the GD Column to the 2ml Collection Tube
10 Add 600àl 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
12 Transfer dried GD Column into a clean 1.5ml microcentrifuge tube
13 Add 50àl 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
DNA electrophoresis was performed using 1.2% agarose gel to validate the gene extracted from DNA samples To prepare the gel, approximately 0.3 grams of agarose was mixed with 30 ml of 0.5X TAE buffer, which was derived from a 50X TAE buffer solution The mixture was heated in a microwave for 3 minutes to ensure complete dissolution, then cooled and poured into a casting tray to solidify for about 30 minutes Once solidified, the gel was placed in an electrophoresis tank and submerged in 1X TAE buffer DNA samples were combined with a 6X loading dye and loaded into the wells, with the first well containing a 1 kb DNA marker for size reference After loading, the electrophoresis apparatus was powered on, and the gel was run at 100V for 30 minutes, monitoring the migration of the marker until it reached the penultimate line The gel was subsequently stained with Vison DNA for visualization.
* DNA electrophoresis was conducted in 0,5X TAE buffer
PCR reactions were conducted with a final volume of 10 µL, utilizing a Takara PCR Thermal Cycler Dice® Gradient (Code TP600) The protocol included an initial denaturation at 94ºC for 5 minutes, followed by 25 cycles of denaturation at 94ºC for 30 seconds and annealing at 53ºC for 60 seconds, culminating in a final extension at 72ºC for 10 minutes This procedure was developed at the Biotechnology Laboratory, National Pingtung University of Science and Technology.
Table 2.5 The component of PCR reaction amplification gen 16S rRNA
Table 2.6 The sequences of primer used for PCR reaction to identify
Characterization of rhizobacteria in sesame
16S-F3R3 TAC GGG AGG CAG CAG
TAC CTT GTT ACG ACT TCA
The temperature cycles for PCR reaction
3.2.6 DNA purification from Agarose gel
PCR products often contain impurities such as primers, buffers, and nucleotides, which can interfere with the separation process To ensure accurate results, it is essential to purify the PCR product Following confirmation of the PCR product through DNA electrophoresis, the target fragment is carefully excised under UV light with a clean scalpel and transferred to a microcentrifuge tube DNA gel purification is then performed using the FavorPrep™ Gel/PCR purification kit (Favorgen Biotech Corp., Taiwan), following the established protocol.
Figure 2.6 The process of DNA purification from Agarose gel
1 Excise the agarose gel with a clean scalpel
• Remove the extra agarose gel to minimize the size of the gel slice
2 Transfer up to 200 mg of the gel slice into a microcentrifuge tube
3 Add 500 àl of FADF Buffer to the sample and mix by vortexing
4 Incubate at 60 °C for 10 minutes and vortex the tube every 2 ~ 3 minutes until the gel slice dissolved completely
5 During incubation, interval vortexing can accelerate the gel dissolved
6 Make sure that the gel slice has been dissolved completely before proceed the next step
7 Cooldown the sample mixture to room temperature And place a FADF Column into a Collection Tube
8 Transfer all of the sample mixtures to the FADF Column Centrifuge at 13,000 x g for 1 minute, then discard the flow-through
9 Add 750 àl of Wash Buffer (ethanol added) to the FADF Column Centrifuge at 13,000 x g for 30 1 minute, then discard the flow-through
• Make sure that ethanol (96-100 %) has been added into Wash Buffer when first use
10 Centrifuge again at full speed (~ 18,000 x g) for an additional 3 minutes to dry the column matrix
• Important step! The residual liquid should be removed thoroughly on this step
11 Place the FADF Column to a new microcentrifuge tube
12 Add 40àl of Elution Buffer to the membrane center of the FADF Column Stand the FADF Column for 1 min
• Important step! For effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely
• Important: Do not elute the DNA using less than the suggested volume (10àl) It will lower the final yield
13 Centrifuge at 13,000 x g for 1 min to elute DNA Keep the sample at -20 o C
3.2.7 Cloning of Screened Gene into yT&A-Vector following Transformed into DH5α 3.2.7.1 Ligation
The PCR product was cloned into the yT&A-Vector following the RBC Rapid Ligation Kit protocol from RBCBioscience, Taiwan A ligation mixture was prepared on ice in a microcentrifuge tube, comprising 1.5 µl of the PCR product, 1X reaction buffer, 0.5 µl of the yT&A cloning vector, and 0.5 µl of T4 DNA ligase, with distilled water added to achieve a final volume of 10 µl The mixture was gently pipetted to ensure proper mixing and then incubated at 4°C for 18 hours.
Table 2.7 The component of the ligation reaction
The mixture of reaction ligation was transformed into competent cell E coli
DH5 α by heat shock (Foger and Hall, 2007) The process includes the following basic steps (Figure 2.8)
Figure 2.7 The process of transformation reaction
1 Take 20ul DH5a to a centrifuge tube
2 Add 200ul CaCl2(0.1M) into centrifuge tube
3 Add 10ul ligation products keep 40min of the mixture in the ice and shake it every 10 min
4 Move centrifuge tube to an incubator for 42oC in the only 90secound
5 Then keep centrifuge tube 5mins more in ice
6 Add to mixture 600ul of LB
7 Culture bacteria in 1hour at 37oC
8 Centrifuge 8,000rpm for 1min and discard 600ul of the liquid and mix well
9 Spread 100ul of a mixture to LB plate(add AP) and culture at 37oC for 12 hours
Culture bacteria at 37 °C over 12 hours
Bacteria cultivated in liquid LB medium with 100 µg/ml of Ampicillin were incubated at 37°C in a shaking incubator for 6 to 12 hours Plasmid extraction was performed using the Favor Prep Plasmid Extraction Mini Kit (Favorgen) following the standard protocol.
1 Transfer 1 ml of well-grown bacterial culture into a centrifuge tube
2 Centrifuge the tube at 13,000 x g for 1 minute to pellet the cells and discard the supernatant completely
3 Add 200 àl of FAPD1 Buffer (RNase A added) to the cell pellet and resuspend the cells completely by pipetting
• No cell pellet should be visible after resuspension of the cells
4 Add 200 àl of FAPD2 Buffer and gently invert the tube 5 ~ 10 times Incubate the sample mixture at room temperature for 2 ~ 5 minutes to lyse the cells
• Do not vortex, a vortex may shear genomic DNA If necessary, continue inverting the tube until the lysate becomes clear
• Do not proceed with the incubation over 5 minutes
5 Add 300 àl of FAPD3 Buffer and invert the tube 5 ~ 10 times immediately to neutralize the lysate
• Invert immediately after adding FAPD3 Buffer will avoid asymmetric precipitation
6 Centrifuge at 13,000 x g for 10 min to clarify the lysate During centrifugation, place a FAPD Column in a Collection Tube
7 Transfer the supernatant carefully to the FAPD Column and centrifuge at 13,000 x g for 5 minutes Discard the flow-through and place the column back to the Collection Tube
• Do not transfer any white pellets into the column
8 Add 400 àl of W1 Buffer to the FAPD Column and centrifuge at 13,000 x g for 1 minute Discard the flow-through and place the column back to the Collection Tube
9 Add 600 àl of Wash Buffer to the FAPD Column and centrifuge at 13,000 x g for 1 minute Discard the flow-through and place the column back to the Collection Tube
• Make sure that ethanol (96-100 %) has been added into Wash Buffer when first use
10 Centrifuge at full speed (~ 18,000 x g) for an additional 3 minutes to dry the FAPD Column
• Important step! The residual liquid should be removed thoroughly on this step
11 Place the FAPD Column to a new 1.5 ml microcentrifuge tube
12 Add 35àl of Elution Buffer to the membrane center of the FAPD Column Stand the column for 1 minute
• Important step! For effective elution, make sure that the elution solution is dispensed on the membrane center and is absorbed completely
13 Centrifuge at 13,000 x g for 5 minutes to elute plasmid DNA and store the DNA at -20 °C
Figure 2.8 The process of Plasmid DNA extraction
The screened gene was validated through restriction enzyme digestion using EcoRI and HindIII The eluted DNA plasmid was combined with 10 µL of CytSmart and the restriction enzymes, then incubated at 37°C for 2 hours To analyze the digestion, approximately 3 µL of the mixture with dye was loaded onto a 1.2% agarose gel Gel electrophoresis was conducted for 30 minutes at 100V, followed by staining with ethidium bromide (EtBr) and imaging to confirm successful digestion.
Table 2.8 The component of restriction enzyme digestion reaction
Pouch hold method (A formula has been shown by my professor to test antibacterial activity)
Each strain of bacteria will be tested antibacterial activity assay with 2 pathogenic bacteria are staphylococcus aureus and pseudomonas aeruginosa about
To evaluate the effectiveness of probiotics, gradually increase the concentration of biological bacteria from 100 µl to 200 µl and then to 300 µl over a period of 10 to 12 hours This method will help determine if a higher concentration of probiotics leads to stronger results.
Figure 2.9 The image illustrates how to prepare work on the MRS agar plate
A mixture of randomly selected strains will be created in a 1:1 ratio, totaling 200ml This combined solution will undergo an antibacterial activity assay against two pathogenic bacteria, with testing conducted over a duration of 10 to 12 hours.
RESULTS
The result of the culture of bacteria
The bacteria strains were grown in MRS Broth medium at 37ºC in 48 hours in the incubator After two days, the bacteria culture appeared singles colony (Figure 3.1)
Figure 3.1 The two groups of bacteria isolated growth on media
The result of gram staining
Gram staining is an experimental method that distinguishes bacterial species into two groups (Gram-positive and Gram-negative) based on the physical and chemical properties of the cell wall
Observe the slide under a microscope
Gram-positive: dark blue or purple
Gram-negative: yellow red or burgundy
Figure 3.2: The shape and arrangements of bacteria observed under the microscope
The image illustrates the color and shape of Gram-positive bacteria, which appear purple after Gram staining These bacteria are characterized by their rod-shaped structure and typically form straight rods that can arrange themselves in chains of varying lengths.
The results of the DNA extraction of six strains in the study
Bacteria samples were collected and processed using a DNA extraction kit in the laboratory The extracted DNA was analyzed through 1.2% agarose gel electrophoresis and stained with Clear Vision DNA stain The results of the total DNA extraction are presented in Figure 3.2.
Figure 3.3 The results of DNA extraction
The successful extraction of DNA from the cell envelope structure demonstrates its viability for molecular biology research and applications The total DNA obtained was intact, with no degradation observed, and the resulting bands were bright and clear, confirming its suitability for subsequent experimental use.
The results of PCR amplification
Figure 3.4 The results of PCR amplification with primer pair 16S-F3R3
After optimizing the priming temperature, DNA samples were cloned using PCR with specific research primers The successful PCR amplification products were analyzed through electrophoresis on a 1.2% agarose gel for 30 minutes The results showed a single, clear band corresponding to approximately 1100 bp using the primer pair 16S-F3R3, indicating successful amplification These PCR products can now be utilized for further vector splitting.
The results of gene cloning
4.5.1 Results of transforming plasmid DNA into variable cells of E coli DH5α
Colony Ht1 Colony Ht4 Colony Ht6
Figure 3.5 The result of transforming the recombinant vector into competent cells
The transformation of the recombinant vector into variable cells yielded positive results, as demonstrated in Figure 3.5, with the culture plate displaying a sufficient number of white colonies for easy selection These colonies were subsequently cultured in 3 ml of LB liquid medium supplemented with 1 ml/l of ampicillin and shaken at 200 rpm overnight to facilitate DNA plasmid extraction.
4.5.2 The results of DNA Plasmid Extraction by Restriction Enzyme digestion
After extracting the plasmids, they should be digested with EcoRI and Hind III restriction enzymes to verify the presence of the target gene segment The expected outcome of this recombinant enzyme digestion is the separation of the plasmid into two components: i) the lower portion contains the transformed DNA fragment, and ii) the upper portion consists of the residual plasmid after the DNA fragment has been removed.
Healthy colony group Figure 3.6 The results of electrophoresis of enzyme-cut products by EcoRI and
HindIII enzyme of the 16S-F3R3 primer
The plasmid separation results are divided into two parts, including size DNA bands of 600 bp and 1100 bp respectively that exactly as originally planned.
Identify and analyze the nucleotide sequence of the DNA markers
After successful cloning, the DNA segments will be sent to Kiron Meek Genomics, a leading sequencing company in Taiwan The sequencing will be performed using an automatic sequence reader based on Sanger's principle Following this, the sequences will be processed with Bioedit software and compared to data from GenBank (NCBI) for analysis, utilizing a specific primer for further study.
Figure 3.7 The software to compare sequences on gene banks on the NCBI website
Results of identification and sequence analysis of 16S-F3R3 gene
The J2-5; J3-1; J6-7; Ht1-6; Ht4-4 and Ht6-10 strains were sequenced with 16S-F3R3 gene sequences with the following results:
The analytical gene sequence measures approximately 1100 bp, revealing significant differences valuable for classification The J2-5 strain exhibits a 99.3% homology with the DNA sequence of Lactobacillus pentosus or Lactobacillus plantarum, while the J6-7 strain shows a 98.6% homology with Lactobacillus plantarum The Ht4-4 strain demonstrates a 99.7% homology to Lactobacillus rhamnosus, and Ht6-10 has a 97.9% similarity to Lactobacillus paracasei Additionally, the J3-1 and Ht1-6 strains remain unclassified, with sequencing results indicating "vector." These four identified strains are beneficial bacteria documented in various countries within the NCBI database.
Figure 3.8 Phylogenetic tree gene and homologous rate of J2-16S-F3R3 strain
Figure 3.9 Phylogenetic tree gene and homologous rate of J6-16S-F3R3 strain
Figure 3.10 Phylogenetic tree gene and homologous rate of Ht4-16S-F3R3 strain
Figure 3.11 Phylogenetic tree gene and homologous rate of Ht6-16S-F3R3 strain
Table 3.1 Identify 6 strains in the study on gene bank
No Strains Primer Size (bp) Similar
J2: Lactobacillus pentosus or Lactobacillus plantarum subsp plantarum
The results of the antibacterial activity assay
4.7.1 Antibacterial of mix strains of bacterial
Table 3.2 The antibacterial level of mix strains is measured in units of millimeters
Figure3.12 The level of anti-Staphylococcus aureus
In the study of bacterial resistance against Staphylococcus aureus, the J2-J6 and J6-Ht4 strains demonstrated the highest antibacterial activity, with inhibition zones measuring 3 mm Following closely is the J6-Ht6 strain, which exhibited a 2.5 mm antibacterial circle The other tested groups had inhibition zones not exceeding 2 mm, while J2-Ht4 showed the least antibacterial effect, with a mere 0.5 mm zone.
Figure3.13 The level of anti- Pseudomonas aeruginosa
Experiments with Pseudomonas aeruginosa revealed that the J6-Ht4 and J6-Ht6 groups achieved the best results, with a notable inhibition zone of 3mm In contrast, the J2-Ht6 group underperformed, likely due to an insufficient bacterial concentration to effectively combat pathogenic bacteria compared to the other groups.
In short, all bacterial groups have the ability to fight off pathogenic bacteria Among them, the three groups J2-J6, J6-Ht4 and J6-Ht6 are the best about the antibacterial ability
Table 3.3 The anti-Staphylococcus aureus level
Figure3.3 The anti- Staphylococcus aureus level of J2, J6, HT4, and HT6 strain
Table 3.5 reveals that bacteria J2 and J6 exhibit comparable anti-Staphylococcus aureus activity, as evidenced by their similar inhibition zones In contrast, Ht4 and Ht6 demonstrate significant bacterial invasion across the entire agar plate Consequently, J2 and J6 are determined to possess superior antibacterial properties compared to Ht4 and Ht6.
Table 3.4 The anti-Pseudomonas aeruginosa
Figure 3.15 The anti-Pseudomonas aeruginosa level of J2, J6, HT4, and HT6 strain
Table 3.4 clearly illustrates the relationship between bacterial concentration and the inhibition of pathogenic bacteria, demonstrating that higher concentrations result in stronger antibacterial effects Notably, all four bacterial strains show anti-Pseudomonas aeruginosa properties, with similar sizes of antibacterial zones at equivalent concentrations This indicates that these strains possess nearly identical antibacterial efficacy.
CONCLUSIONS
Conclusion
The study identified a total of six bacterial strains within the Joint and Health bacteria groups, following experiments and sequencing of samples Out of these, four distinct strains were successfully identified.
J2: Lactobacillus pentosus or Lactobacillus Plantarum subsp plantarum J6: Lactobacillus Plantarum
The primary objective of this experiment is to evaluate antibacterial activities, revealing that strains J2 and J6 exhibit superior antibacterial properties compared to strains Ht4 and Ht6 against pathogenic bacteria Notably, increased bacterial concentration correlates with enhanced inhibition Additionally, experiments involving mixed bacterial strains demonstrate effective collaboration in suppressing pathogenic bacteria.
The results indicate varying levels of bacterial inhibition among random mixed strains and four specific Lactobacillus strains: Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus pentosus, and Lactobacillus plantarum subsp plantarum.
In this experiment, it was found that all tested bacteria possess the capability to inhibit pathogenic bacteria The effectiveness of this antibacterial action, indicated by the width of the inhibition zone surrounding the hole, is directly related to the concentration of the bacteria used.
Table 3.4 presents the antibacterial cycle sizes, revealing that the J6 and Ht4 mixture effectively eliminated both Staphylococcus aureus and Pseudomonas aeruginosa, achieving an elimination cycle width of approximately 3mm In contrast, the J2 and Ht4 group exhibited the smallest antibacterial effect against Staphylococcus aureus, measuring only 0.5mm Notably, the J2-Ht6 group demonstrated no antibacterial activity against Pseudomonas aeruginosa, while the antibacterial cycle sizes of the other groups did not exceed 3mm.
In conclusion, four lactobacillus strains have ability resistant to the two types of pathogenic bacteria, which is actually potential bacteria for producing friendly cleaning agents.
Recommendations
- Based on the antibacterial properties of probiotic bacteria, which are beneficial bacteria, it is necessary to continue research and development
- Testing of this product to purify the environment from this probiotic bacterium in the future
- Experimental survey and compare the superiority of probiotics compared with conventional detergents
- It is therefore suggested the idea of the bacterial strain or a group of bacteria strains that have the best cleaning as an environmental cleaner
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