One gram of naniura from Medan restaurant, North Sumatera, was diluted in 100 mL de Man, Rogosa, and Sharpe (MRS) broth (Himedia, India) and incubated at 37°C for 24 h. The bacteria suspension was collected, the supernatant was pipetted at approximately 1 ml and then put into 9 ml sterile physiological NaCl solution (10^-1). Subsequently, the dilution was performed up to 10^-5, and after completion, 0.1 ml was pipetted from each dilution tube to MRS agar using a spreader, and then the Petri dishes were incubated for 48 h at 37°C. The growing bacteria were observed over the next 2 days.

Isolation of LAB was performed using MRS agar (Millipore, Merck, Germany), prepared by dissolving in distilled water, then sterilized in an autoclave, along with Petri dishes and other glassware. The sterile MRS agar was prepared in a Petri. The bacteria that grew were counted for the number of colonies. They were then purified by separating into several colonies and regrowing on MRS agar before reincubation. This purification was carried out until they became pure. Bacteria with the same colony shape and color in 1 Petri were considered single. Finally, the purified bacteria were stored in 25% (v/v₀) glycerol at -20°C.

Gram staining was performed on about 9 isolates, and each strain was tested for gram color using a violet, iodine, 96% alcohol, and safranin solution. The test started by fixing the collected bacterial isolate with an ose needle and scratching it on a slide while being heated over a fire until the colonies dried up. The slide was dripped on the isolate with violet solution, left for 1 min, and rinsed with distilled water. Furthermore, it was dripped with iodine solution on the isolate, left for 1 min, and rinsed with distilled water. It was then rinsed with 96% alcohol until the color of the iodine and violet solution did not flow. The glass was again dripped with safranin solution on the isolate, left for 45 sec, then rinsed with distilled water. It was dried on paper towels, then observed under a microscope with a 100x lens. The gram color of the isolate is positive when it appears purple. However, when it is pink, then the gram color of the isolate is negative. Therefore, to classify a cell as bacilli, it should be rod-shaped, while a round cell is called cocci.

The catalase assay was conducted on all strains, the slide was prepared, and then some bacteria were scratched using an ose needle. H₂O₂ solution was dripped on the isolate and the bubbles formed were observed. The catalase assay was declared positive when bubbles were formed, implying that the strain can produce catalase enzyme. Meanwhile, it is declared negative when there is no formation of bubbles, which indicates that the strain cannot produce catalase enzyme.

This test was conducted twice to test the ability of bacteria strains to inhibit the growth of pathogenic isolates, namely Escherisia coli and Staphylococcus aureus. At this stage, nine isolates were used because they had negative results in the catalase test. This test used chloramphenicol (30 µl) and MRS broth as positive and negative control, respectively. The test was performed using the diffusion method, starting with growing each strain and pathogen in MRS and Nutrient agar (NA) (Merck, Germany) in a test tube for 24 h. Each strain was collected using a needle ose part of the colony and then put into a physiological solution. Furthermore, the absorbance of the suspensions was calculated using UV-Vis spectrophotometry at a wavelength of 600 nm with an OD of 0.1. After observing that the number of cells followed OD 0.1, the suspense was ready for the assay. Each suspension of pathogens was collected with a sterile cotton swab and then streaked evenly onto the NA plate. After the pathogens were scratched onto the media, each assay strain was obtained by dipping sterile disk paper, draining, and placing on NA, which the pathogens had scratched. The media was incubated at 37°C for 24 h, and after 1 day, the inhibition zone was observed to be clear, after which the diameter was measured.

For the preparation of the stomach and intestines, the strains need to be identified to know their availability for each in acid and bile salt conditions because the strains were included as LAB from the previous assay. One of the strains with higher availability was taken for the next identification. MRS broth was adjusted to pH 3 with 1 M HCl. Bile solution was prepared by adding 0.3% bile acid factors (Jarrow Formula, Los Angeles) into MRS broth. Cell harvested by nine strains at an exponential growth phase (18 h) were inoculated in MRS broth, acid, and bile solution and incubated at 37°C for 4 h. The inoculates were adjusted to OD₆₀₀ using spectrophotometry UV-Vis (Shimatzu). The following formula was used to measure the percentage of cells that survived in an acid solution [7]:

(1)

(2)

After incubation for 24 h in MRS broth, cultured BAMA 4 was harvested by centrifugation, and cells were disrupted using a homogenizer (Bactosoni, Bandelin, Germany). The disrupted cells were centrifuged at 4500 rpm for 15 min, and the supernatant was used for an inhibition assay, while acarbose was employed as a positive control. The determination of α-glucosidase was based on inhibiting crude glucosidase formation from the substrate sucrose [8]. This α-glucosidase inhibitory activity (%) was determined according to the following equation [9]:

(3)

Where: %test activity = Abs. total test sample/Abs. control x 100%. Total test sample absorbance = test sample absorbance + sucrose + crude enzyme. Absorbance A1= Absorbance of the test sample + sucrose.

The isolation of DNA from the strain was conducted using the Quick-DNA^TM Bacterial Miniprep kit, and the procedure can be completed in 15 minutes. The strain was added directly to a ZR BashingBead^TM Lysis Tube (0.1 and 0.5 mm). Furthermore, it was rapidly and efficiently lysed by bead beating without using organic denaturants or proteinases. The DNA was then isolated and purified using Zymo-Spin^TM Technology, which was ideal for downstream molecular-based applications, including PCR and array. The Genomic lysis buffer was added to a final dilution of beta-mercaptoethanol of 0.5% (v/v). Subsequently, 50-100 mg of bacterial cells resuspended in up to 200 μl of water or isotonic buffer (PBS) were placed in a ZR Bashing Bead™ lysis tube (0.1 mm and 0.5 mm). A total of 750 µl of BashingBead™ buffer was added to the tube. The ZR BashingBead^TM lysis tube (0.1 mm and 0.5 mm) was centrifuged in a microcentrifuge at 10,000xg for 1 min. Approximately 400 µl of the supernatant was transferred to a Zymo-Spin^TM III-F filter. A total of 1200 µl of genomic lysis buffer was mixed with the filtrate in the collection tube from the previous step. The mixture at 800 µl was transferred to a Zymo-Spin^TM IICR Column in a Collection tube and centrifuged at 10,000xg for 1 min. The flow-through was discarded from the Collection tube, and the precious step was repeated. A 200 µl DNA pre-wash buffer was added to the Zymo-Spin^TM IICR Column in a new Collection tube and centrifuged at 10,000x g for 1 min. g-DNA wash buffer at 500 µl was mixed with the Zymo-Spin^TM IICR Column and centrifuged at 10000x g for 1 min. The Zymo-Spin^TM was transferred to a clean 1.5 ml microcentrifuge tube, and 100 µl DNA elution buffer was directly mixed with the column matrix. The mixture was centrifuged at 10000x g for 30 sec to elute the DNA. A strain designated primarily as LAB was identified in its 16S rRNA with 27F/1492R PCR primer. PCR amplification of genes was used (2x) My Taq HS Red Mix (Bioline, BIO-25048). Its products at 1 µl were assessed by electrophoresis with 0.8% TBE agarose at non-template control (NTC). The nucleotides were sequenced with Bi-directional. Meanwhile, the sequences were compared with those in the NCBI Genbank database (http://www.ncbi.nlm .nih.gov/BLAST/) using BLAST (NCBI, Bethesda, MD, USA).

The total number of bacteria isolates obtained from this study was 9, with isolates codes as BAMA 1, 2, 3, 4, 5, 6, 7, 8, and 9. The characteristics of the isolates include convex shape, brownish-white color, small, and rounded edges. Gram staining and catalase test were carried out on isolates, and the following results are listed in Table 1.

Based on the test results above, the nine isolates were negative in the catalase test. LAB is a bacterial that produces negative results in the catalase test. From the characteristics of LAB, which was negative from the catalase test, the nine isolates were considered LAB.

The results of the measurement of the diameter of the inhibition zone or the clear zone are in Table 2 below, also, the results of the average inhibition zone measurement include the size of the paper disc. This test was carried out twice, the diameter of the inhibition zone was measured vertically and horizontally. The positive control used in this assay was chloramphenicol 30 µg and the negative control was sterile MRS broth. The following are the results of the calculation of the average diameter of the antibacterial test from 9 isolates listed in Table 2 and shown in Fig. (1).

Fig. (1). Bar graph of the diameter of the inhibition zone of antibacterial test results from BAMA 1 – 9 isolates against E.coli and S.aureus. Meanwhile, for the test isolates, the more optimal diameter of the inhibition zone was BAMA 4 isolate. BAMA 4 isolate was able to inhibit S. aureus which was almost equal in value to the diameter of BAMA 7, 8, and 9 isolates and higher than the diameter of the inhibition zone of BAMA 4 isolates. BAMA isolates 1, 2, 3, 5, and 6. Meanwhile, in inhibiting E.coli, the diameter was higher than the diameter of the inhibition zone of BAMA isolates 7, 8, and 9.

Fig. (2). Percentage cell growth of BAMA 1 – 9 strains on acid and bile.

This compares the absorbance of cell growth in acid with normal media and the absorbance 600nm of cell growth in bile with normal media, with an incubation time of 4 hours.

The α-glucosidase inhibition activity assay of the BAMA 4 strain was 65%, while that of acarbose was 70%. This indicates that the supernatant of isolate BAMA 4 grown in MRS broth media contained compounds that had the ability to inhibit the hydrolysis of crude glucosidase enzymes to sucrose.

Based on the results of molecular identification, BAMA 4 strain was of Pediococcus acidilactici species, in the form of lactic acid bacteria, gram-positive and has cocci cells. Genomic DNA of P. acidilactici BAMA 4 (Fig. 2) was observed on Nanodrop methods and the absorbance can be seen in Table 3 (Fig. 3), while the sequences shown in Table 4 and results of electrophoresis PCR product (Fig. 4). The hit BLAST results against the NCBI database, excluding uncultured sample sequences and P. acidilactici and the phylogenetic tree can be seen in Fig. (5).

Fig. (3). BAMA 4 isolate in MRS Agar medium plate.

Fig. (4). Gel photo of PCR product of 16S rRNA P. acidilactici BAMA 4. Results of electrophoresis PCR product have been known with 1465 bp.