A Novel Approach for Gut Ecosystem Resilience: Evaluating Lacti-plantibacillus plantarum-12INH as a Promising Natural Antibacterial Agent

Abstract

A healthy colon aids in the body’s absorption of nutrients from food and the development of the immune system, which in turn helps the neurological system and hormones to function normally. The presence of natural antibacterial agents in the digestive tract can activate the human immune system. Antibiotic resistance can develop in the body, and probiotic bacteria in the digestive system can decline as a result of incorrect antibiotic use, which can also limit the creation of lactic acid and bacteriocins. Our research’s aim was to identify local Indonesian probiotic bacteria isolated from breast milk and to evaluate the efficacy of the bacteriocin generated. The findings revealed that the isolated probiotic bacterium was Lactiplantibacillus plantarum-12INH, which differed from species listed in the NCBI in terms of its Gram-positive cell size, resistance to high temperatures (30, 37, and 45 °C), low pH tolerance (2, 3, and 4), and tolerance to 0.3% bile salts. In addition, it is capable of producing bacteriocins with an inhibition zone against E. coli ATCC 25922 bacterial pathogens of 12.48 mm similar to amoxicillin and tetracycline antibiotics, pH 2–10, and stability at high temperatures (40, 60, 80, 100, and 121 °C). Future applications for L. plantarum-12INH in processed food products include balancing colonic microbiota, repairing the colon wall, and promoting immune system development.

1. Introduction

Breast milk contains the complete nutrition needed by newborns because it contains vitamins (A, B1, B12, D, and K), iodine, iron [1], and diverse microbiota [2] such as probiotic bacteria [3]. It is a source of probiotic bacteria which is essential for maintaining the microflora of the digestive tract in infants, because the probiotics in breast milk can boost the immune system and can provide protection against pathogenic bacteria in the digestive system [4]; therefore, probiotic bacterial isolates from human habitats are anticipated to be able to adapt easily in the digestive system when applied to ready-to-eat food products [2]. In order to obtain pure isolates of indigenous probiotic bacteria with well-defined species and comprehensive physical, chemical, and biochemical characteristics, the determination of probiotic bacteria must be continued by evaluating their capacity to produce bacteriocin bioactive compounds, through biochemical and molecular testing.

Frequent causes of gastrointestinal tract infections [3] are foods and beverages that contain pathogenic microbes. Antibiotics are frequently employed to eliminate microorganisms in the digestive tract. Antibiotic resistance can be caused by inappropriate or excessive use of antibiotics, which is presently a global health concern [4]. Natural antibacterial agents that are generated by probiotic bacteria are the best way to maintain healthy intestinal conditions, allowing the nervous system and hormones to function properly and stimulating the development of the immune system, one of whose functions is to protect against the COVID-19 virus.

It has been clinically demonstrated that Lactiplantibacillus plantarum 299v does not produce antibiotic resistance, aids in the absorption of iron, and can be used to treat irritable bowel syndrome (IBS) and other digestive system infections [5]. The harmful bacteria Escherichia coli ATCC 30105, Bacillus subtilis ATCC 6633, and Micrococcus luteus ATCC 4698 cannot develop because Lactiplantibacillus plantarum, which is found in the feces of Bamei pigs, is capable of producing antibiotics from organic acid [6]. L. plantarum has been analyzed from various types of food in several regions. Currently, L. plantarum has been isolated from Bulgarian yoghurt [6], Korean radish water kimchi [7], Bamei pig feces in China [6], and Fufu Nigerian fermented food [7]. The native Indonesian strain found in this study that was isolated from breast milk has a good possibility of colonizing the human intestine. Therefore, more research is needed, especially regarding the attributes that characterize probiotic capabilities.

2. Materials and Methods

2.1. Probiotic Characterization

The probiotic bacteria isolated from breast milk were characterized on cell morphology [8] by microscopy with a scanning electron microscope (JEOL JSM-6360LA), a catalase test, as well as bile salt [9], high temperature resistance (30, 37, and 45 °C) (Hayakawa, 1992), and low pH resistance test (2, 3, and 4) [10], followed by a biochemical test with a Vitex 2.0 compact Biomereux using the card type: ANC testing instrument 00001658F4A9 (12903), lot number 2441139403 bionumber 33377110304.

2.2. Molecular Identification and Bioinformatic Analysis

Species-level identification was performed using the PCR primers 27F and 1492R, 16S rRNA sequencing, and DNA barcoding (1400 bp). For DNA purification, we used genomic DNA extraction with a Quick-DNA Fungal/Bacterial Miniprep Kit D6005 (Zymo Research Company, Irvine, CA, USA). The pure DNA samples were processed by PCR amplification with My Taq HS Red Mix (Bioline, BIO-25048), and 1400 gene pairs of 16S rDNA gene were amplified using bidirectional sequencing. The PCR protocol stages consisted of the following: denaturation at 95 °C for 2 min, 35 denaturation cycles at 95 °C for 45 s, temperature hardening at 54 °C for 1 min, elongating at 72 °C for 1 min, and extension at 72 °C for 5 min. Then, 5 µL of each amplified mixture was added for electrophoresis in a 1.5% solution consisting of 1.5 g agarose powder in 100 mL TEA buffer. Electrophoresis lasted for 45 min at a voltage of 90 V, to obtain mRNA. The PCR product was carried out by Sanger sequencing. Prior to online BLASTN using the NCBI database, the DNA sequences obtained were trimmed on all edges to remove invalid DNA sequences using Chromash (http://technelysium.com.au/wp/chromas/, accessed on 20 January 2023). The results of combining DNA sequences in the forward and reverse positions were carried out using an online open-source system through Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 20 January 2023). The DNA sequnce in the reverse position had been previously reversed through online (https://www.bioinformatics.org/sms/rev_comp.html, accessed on 20 January 2023). After obtaining a DNA sequence that had good quality, then, online BLASTN was performed on the nucleotide sequence (https://blast.ncbi.nlm.nih.gov/, accessed on 20 January 2023). After validation of the species name, Mega X was used to compile the phylogenetic tree by simultaneously entering several sequences obtained from the NCBI database as comparison data [11]. Species identification was performed molecularly with 16S rRNA sequencing, followed by a phylogenetic analysis.

2.3. Bacteriocin Production and Antimicrobial Activity Test

Eight Erlenmeyer flasks were prepared containing 25 mL of MRS broth (Oxoid) each added with 2% suspension and vortexed until homogeneous, followed by incubation for 0–16 h anaerobically at 37 °C. Every 2 h, observations were made of OD₅₈₀. Each solution was centrifuged at a speed of 8000 rpm, at 4 °C, for 15 min. The supernatant was neutralized with 0.1 N NaOH solution to pH 6. It was filtered with 0.22 µm millipore paper. The result of filtration was a crude bacteriocin extract which was used to soak sterile paper disks. Sterile MHA media were prepared, and then poured on sterile petri dishes and allowed to solidify. The Escherichia coli bacterial suspension was swabbed on the agar surface. The paper disk was placed on the agar surface and incubated at 37 °C for 24 h. The larger the diameter of the clear zone produced was an indicator of bacteriocin production [9,12]. Amoxicillin and tetracycline 30 mg were chosen because they are antibiotics that have the ability to kill Gram-negative bacteria.

2.4. Bacteriocin Activity Test: pH Resistance

Crude bacteriocins were conditioned at different pH values (2, 4, 6, 8, and 10) by adding 1 N HCL and 1 N NaOH. Then, each sample was incubated for 4 h at 30 °C. For temperature resistance, five test tubes containing crude bacteriocin were heated at different temperatures (40, 60, 80, 100, and 121 °C) for 15 min. All samples were tested for antimicrobial activity [13,14].

2.5. Bacteriocin Purification and Protein Molecular Weight Analysis

For bacteriocin purification and the protein molecular weight analysis, (NH₄)₂SO₄ was added to crude bacteriocin until the solution concentration was 80% at 4 °C, homogenized using a stirrer, incubated for 24 h, and centrifuged at 10,000 rpm for 20 min at 4 °C. The precipitate was diluted with 0.2 M phosphate buffer, pH 7 [15].

3. Results

3.1. Characterization Probiotic

According to our findings, the physical characteristics of the colonies growing on MRS media were as follows: oval, white, convex elevation, unbroken colony margins, and colony growth located in the center. Bacteria with purple bacilliwere observed for examination under a microscope at a magnification of 100 × 40 (see Figure 1).

The results showed that the probiotic bacterial candidate isolates did not produce bubbles after being dripped with hydrogen peroxide (H₂O₂), indicating that the bacterial isolate was catalase negative, could grow in 0.3% bile salt conditions (Figure 2a), could live at 30 and 37 °C for 4 days and grew slowly at 45 °C (Figure 2b), and could survive well at pH 2, 3, and 4 for 90 min (Figure 2c).

The biochemical characterization test attempts to determine the species of indigenous probiotic bacteria using the Vitek 2.0 compact Biomerieux (2012) instrument based on the capacity to decrease carbohydrate and amino acid sugars. According to the results of the Vitek testing, the selected Lactobacillus plantarum organisms were produced with 98% probability, bionumber 33377110304 biochemical characteristics. It can be concluded that indigenous bacterial isolates are species of L. plantarum bacteria with the ability to ferment the following: D-galactose, D-cellobiose, saccharose/sucrose, beta-galactopyranosidase indoxyl, maltotriose, leucin arylamidase, tyrosine arylamidase, arbutin, esculin hydrolysis, N-acetyl-D-glucosamine, D-ribose 2, phenylalanine arylamidase, D-glucose, 5-bromo-4-chloro-3-indoxyl-beta-glucoside, L-proline arylamidase, D-mannose, and D-maltose.

3.2. DNA Molecular Identification and Phylogenic Trees

The isolate of bacteria LPX 14_12INH that was molecularly characterized is determined to be L. plantarum based on the similarity of the sequence DNA on the 16S rRNA gene region (Figure 3). This is demonstrated by the fact that the sequence’s similarity to the reference CP055123 is one hundred percent. At this point, in the phylogenetic tree reconstruction, L. plantarum species established a distinct clade, distinct from L. argentoratensi (OM370998) and L. pentosus (MW714761 and OM618135). However, the genetic difference between these species remains quite small. The L. plantarum strain LPX 14_12INH shared the most similarities with the same species strain 2964 (MT611900) from China.

Table 1. BLAST NCBI results of the 16S rRNA sequencing analysis.

Description	Results
Species	Lactiplantibacillus plantarum strain NCIMB 8826
Maximum score	8851
Total score	1770
Query coverage	100%
e-Value	0.0
Percent identity	100%
Accession number	CP037961.1

shows the BLAST NCBI results of the 16S rRNA sequencing analysis.

The next step was the phylogenetic test to determine clumps of species that were similar to indigenous bacterial isolates. The results obtained are shown in Figure 4.

3.3. Bacteriocin Production and Antimicrobial Activity

Further investigation was conducted by examining the timing and amount of bacteriocin generated by indigenous L. plantarum. The incubation period was 16 h, with measurements of OD580 and the ensuing clear zone in suppressing the growth of pathogenic bacteria E. Coli performed every 2 h. The wider the diameter of the clear zone generated, the more bacteriocin is produced. The antibiotic tetracycline was utilized as a positive control to compare antibiotic inhibition with the resultant bacteriocins. Tetracycline was chosen as an antibiotic because it can kill Gram-negative bacteria (Figure 5).

3.4. Bacteriocin Activity against pH Resistance and Temperature Resistance

The findings demonstrated that L. plantarum-12INH-produced bacteriocin was capable of inhibiting the development of the pathogenic bacterium E. coli under pH conditions as low as 10 and as high as 12, with the largest resistance area of 12.48 mm. The bacteriocin was deemed to have a modest ratio since it was able to inhibit E. coli growth when heated between 40 and 121 degrees Celsius with a resistance zone between 9 and 87 mm. In Figure 6, a more thorough explanation is provided.

3.5. Bacteriocin Molecular Weight

The results of SDS-PAGE electrophoresis with 12% Tris-glycine SDS-PAGE gel, voltage 150 V, time of 120 min, and sample loading 20 µL, obtained a molecular weight of bacteriocin from L. plantarum-12INH of 13.59 kDa (Figure 7) based on the equation y = −1.1085x + 2.0917 with a value of R² = 0.9937.

4. Discussion

The formation of bacterial colonies in the middle of the media indicates that the bacteria that grow are anaerobic, or can grow in the absence of oxygen (Figure 1). The purple bacteria under the microscope indicate that they are Gram-positive bacteria, because the thick peptidoglycan on the bacterial cell wall absorbs the crystal violet color, allowing it to adhere to the cell wall and not to be dissolved by ethanol [11]. The cell diameters of the indigenous bacterial isolate ranged from 1.26 to 2.34 m.

The cell morphology and bacterial colony indicates that the bacteria found have probiotic potential. The catalase test is the next characteristic. The catalase reaction produces positive results when air bubbles develop that indicate the presence of O₂, and negative results when no gas bubbles occur [16]. Because indigenous bacterial isolates are catalase negative, they can be studied further as potential probiotic microorganisms. Because H₂O₂ compounds can be generated during aerobic metabolism, the catalase test can also be used to identify bacterial groupings based on oxygen demand because only bacteria that can grow in aerobic settings can breakdown these compounds.

According to Figure 2, indigenous bacterial isolates have potential qualities as probiotic bacteria due to their strong development in acid and bile salt environments, allowing them to survive the digestive tract. The human digestive tract begins in the mouth with a pH condition determined by food intake, travels through the stomach at pH 2, then in the small intestine at pH 7–8, and finally in the colon at pH 5–8 [17]. LAB can be employed as a probiotic agent if it can live in the digestive tract; is resistant to heat, acids, and bile salts; and can correctly develop and colonize the intestines [16]. This enhances the case for indigenous bacteria as probiotic bacterium candidates.

According to the findings in Figure 2b, indigenous bacterial isolates can survive at high temperatures. The third day of incubation at 45 °C in the exponential phase resulted in a 360.45% increase in OD. This supports the notion that isolated indigenous bacteria are potential probiotic bacteria. Lactiplantibacillus pentosus, a probiotic bacteria, may grow optimally at 45 °C after 24 h of fermentation, yielding 20.53 mM gamma-aminobutyric acid [18]. As a result, additional screening or selection testing is required, such as sensitivity to low pH, gastric fluids, bile salts, pancreas, and preventing the growth of other harmful microorganisms.

Next, the bacteria are evaluated for their capacity to grow at low pH levels, strengthening the prediction outcome. The purpose of this test is to confirm that the discovered bacteria are capable of surviving in an acidic environment. Figure 2c demonstrates the ability of native bacterial isolates to endure at pH 2, 3, and 4. The isolated bacterial isolates are possibilities for probiotic bacteria if they can survive at pH 2 for 100 min. Due to its capacity to keep an extracellular pH that is more alkaline than its cytoplasmic pH, LAB has a high level of tolerance. A significant proton gradient would not emerge in LAB since the intracellular pH changes dynamically along with the drop in external pH. When a large proton gradient forms, poor conditions result because it may lead to proton translocation, which consumes a lot of energy, as well as the buildup of anions and organic acids in the cytoplasm, which are toxic to these cells [19].

D-Cellobiose, saccharose/sucrose, arbutin, esculin hydrolysis, phenylalanine arylamidase, D-glucose, 5-bromo-4-chloro-3-indoxyl-beta-glucoside, D-mannose, and L-pyrrolidonyl arylamidase can all be assimilated by native bacterial strains. Native bacterial isolates had a 98% chance of L. plantarum based on these biochemical traits. The outcome of the sequence 1166 gaps was used to corroborate this identification (Figure 3). Based on the similarities and differences in its genetic traits compared to other existing species, a phylogenetic tree was constructed to ascertain the evolutionary relationship of the native L. plantarum. Indigenous L. plantarum isolates are genetically related to other L. plantarum species that have been previously identified, but Figure 4 demonstrates that they have different characteristics, confirming that indigenous L. plantarum is a different species from Indonesian bacteria species, which can then be registered internationally.

In addition to physical and biochemical traits, a molecular analysis of the 16S RNA gene area was also conducted in this work to confirm the species. Based on the morphological, biochemical, and molecular traits, the L. plantarum species was legitimate. The species L. plantarum forms a clade and is distinguished from other species in the same genus in the phylogenetic tree reconstruction (Figure 4). Although there is no genetic difference between the L. plantarum species in this gene area, there is a genetic difference between L. plantarum and the strains of L. pentosus 19234 (0.00070) and L. xiangfangensis HBUAS59038 (0.009888). Nevertheless, since 30 June 2020, the species L. plantarum’s legal name has been changed. The official name has been changed from L. plantarum to Lactiplantibacillus plantarum [5]. However, more research on the DNA traits of the L. plantarum strain LPX 14_12 INH from several area genes is required.

The growth of putrefactive bacteria such as E. coli can be inhibited by probiotic bacteria, which are LAB capable of creating secondary metabolites in the form of organic acids, hydrogen peroxide, diacetyl, and peptides known as bacteriocins. While some other studies have taken longer than 10 h to produce bacteriocin (

Table 2. The abilities of Lactiplantibacillus spp. related to temperature resistance, pH resistance, zone inhibition against E. coli, and time required to produce bacteriocins.

Bacteria	Time Produce Bacteriocin (h)	Zone Inhibition against E. coli (mm)	pH Resistance	Temperature Resistance (°C)	Originated	References
L. plantarum-12INH	10	9.25–12.48	2–10	37–121	Breast milk native Indonesia	Our research
L. plantarum IIA-1A5	24	8.94	6.35–6.48	3–5	-	[20]
L. plantarum DU10	16	5–10	6.5	37	Raw camel milk	[21]
L. fermentum	24	10	6.5	37	Human milk	[22]
L. plantarum	24	6–9	4–8	30–90	Cheese	[23]
L. plantarum H1	18–24	4	-	37	Pickle	[24]
L. plantarum V1	18–24	18	-	37	Fermented durian flesh	[25]
L. plantarum QP19	24	12–15	5–5.5	37	Pig feces	[6]

), L. plantarum-12INH produced optimal bacteriocin crude extract at the 10th h, which was characterized by an increase in the OD580 value and a decrease in pH as an indicator of organic acid formation as well as the ability to inhibit the growth of pathogenic bacteria E. coli (Figure 5).

Probiotic bacteria can compete with pathogenic bacteria, preventing them from adhering to the surface of the intestinal epithelium and suppressing them. The efficiency of each probiotic bacteria’s secondary metabolites can be seen by viewing the clear zone of the anti-microbial test [16]. Bacteriocins are generated during the exponential phase of cell growth, in the same way as proteins are. This system is controlled by extrachromosomal plasmid DNA and is impacted by a number of important variables, including pH. Bacteriocins are typically produced as peptides via the ribosomal route and subsequently modified. The optimal circumstances of the associated LAB and the medium where the optimal LAB growth is the facultative anaerobic conditions will impact LAB growth.

In the present study (Table 2), L. plantarum-12INH produces bacteriocins faster, is stable up to pH 2, and can be heated to 121 °C. This demonstrates that the bacteriocin produced by L. plantarum-12INH works well in human digestive tract conditions, has the ability to inhibit the growth of pathogenic bacteria equivalent to amoxicillin and tetracycline antibiotics (Figure 6), and can be used as a natural preservative and antibiotic candidate in food products.

Bacteriocins produced by Gram-positive bacteria can be classified into four classes: Class I bacteriocins are antibiotics with a molecular weight of 5 kDa, Class II bacteriocins are non-antibiotics with a molecular weight of 10 kDa, Class III bacteriocins are heat-labile lytic, and Class IV bacteriocins are antibiotics with lipids or carbohydrate parts [26]. Glycine (a Class II bacteriocin) is a heat-resistant amino acid with a pl value of 8–11, is hydrophobic, amphiphilic, and has a molecular weight of less than 10 kDa [27]. The findings of this work provide a new reference indicating Gram-positive bacteria can create bacteriocins with molecular weights greater than 10 kDa (13.59 kDa) that are durable at high temperatures up to 121 °C and pH 2–10.

5. Conclusions

According to our results, native Lactiplantacillus plantarum-12INH from Indonesia has the following characteristics:

• Rod-shaped, catalase negative, measuring 1.26–2.34 m, able to survive at low pH (2, 3, and 4), 3% bile salts, and temperatures of 30, 37, and 45 °C;
• Can produce bacteriocin secondary metabolites with a molecular weight of 13.59 kDa which have the ability to be an anti-microbial against bacterial growth E. coli under conditions of pH 2, 4, 6, 8, and 10 and temperatures 40 °C, 60, 80, 100, and 120 °C similar to amoxicillin and tetracycline antibiotics;
• Has the potential to be used as a functional food and as an alternative to standard natural antibacterial agents for assisting digestion while maintaining a healthy gut microbiome.