Isolasi Dan Identifikasi Bakteri Asam Laktat Penghasil Bakteriosin Dari Urutan, Sosis Fermentasi Tradisional Bali Isolation And Identification Of Bacteriocin Producing Lactic Acid Bacteria From Urutan, Balinese Traditional Fermented Sausage

Lactic acid bacteria (LAB) are the main bacterial group in the urutan which play an important role in the fermentation process and inhibit the growth of pathogenic bacteria. Urutan is a Balinese sausage, that is traditionally produced and naturally fermented. The objective of this study was to isolate and identify LAB that can produce bacteriocins from the urutans. The urutans used in this study were purchased from several traders in the Baturiti and Mengwi region, Bali. A total of 14 bacterial isolates that inhibited indicator bacterial growth on overlay agar procedure, Metamorfosa:Journal of Biological Sciences 8(1): 81-88 (Maret 2021) eISSN: 2655-8122 82 were isolated and confirmed as LAB by Gram-positive bacteria and negative results on the catalase test. Bacteriocin confirmation test was carried out by a disk diffusion method using Escherichia coli ATCC8739, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC29212 and Lactobacillus plantarum E12.1 as indicator bacteria. Crude bacteriocin from Lactobacillus sp. J2 and J6 inhibited the growth of L. plantarum E12.1 and E. coli ATCC8739. Crude bacteriocin of Lactobacillus sp. J6 also inhibited the growth of E. faecalis ATCC29212. Analysis of 16S rRNA gene sequences showed that Lactobacillus sp. J2 and J6 were identified as Lactobacillus plantarum with the similarity of the Lactobacillus sp. J2 sequence is 99.39% and the similarity of the Lactobacillus sp. J6 sequence is 99.35%. The conclusion of this study is L. plantarum J2 and L. plantarum J6 were LAB which produced bacteriocin in the urutan. Keyword: Lactic Acid Bacteria, Bacteriocin, Lactobacillus plantarum, Urutan


INTRODUCTION
Lactic acid bacteria (LAB) is an important group of microorganisms involved in the production of various fermented food. LAB could produce various compounds with antagonistic activity against other microbes (Perez et al., 2014). The ability to inhibit the growth of spoilage microorganisms and pathogenic bacteria contribute to the maintenance of hygienic and quality of the product or host health (Kormin et al.,2001). Those compounds include organic acids, H2O2, CO2, diacetyl, acetaldehyde, and bacteriocins (Yang et al.,2012).
Bacteriocins are proteins or complex proteins biologically active with antimicrobial action against other bacteria, principally closely related species (Deraz et al (2005) in Parada et al (2007). The bacteriocins produced by LAB are classified as Generally Recognized as Safe (GRAS) by the Food and Drug Administration (FDA). The bacteriocins also have superior properties such as tolerance to high temperatures, relatively wide antimicrobial spectrum but most at closed related bacteria, activity over a wide pH range, colorless, odorless, and tasteless (Perez et al., 2014).
Due to the advantages, bacteriocins have caught the attention of food researcher and industries as natural food bio preservatives to replace the use of chemical-based food preservatives that according to Bali et al., (2011) could cause undesirable side effects such as alteration in the constituents,nutritional and organoleptic properties of the food and toxic effects on human health.
Bacteriocin as a biopreservative agent is not widely available and relatively expensive. On the other hand, Indonesia has various types of fermented food which are natural sources of LAB. One of the naturally fermented foods that has not been much studied is urutan, a traditional fermented sausage from Bali.
Antara, et al. (2002)  Aryanta (2013) also isolated Pediococcus cerevisiae from urutans that were allowed to ferment naturally. These studies showed that several LAB species grew during the fermentation process of urutan, and they are possibly bacteriocin-producing LAB. However, the capability of LAB isolates to produce bacteriocins were not tested in these studies. Therefore, in this study authors aim to obtain bacteriocin-producing LAB isolates from urutans and to identify the species on the basis of their 16S rDNA sequences compared with those deposited at the GeneBank.

MATERIALS AND METHODS Bacterial Strains and Culture Media
Escherichia coli ATCC8739, Staphylococcus aureus ATCC25923, and Enterococcus faecalis ATCC29212 were obtained from the Microbiology Laboratory of the Faculty of Medicine, Udayana University, while Lactobacillus plantarum E12.1 was obtained from the author's collection, were used as indicator bacteria in the assays. Blood agar plate supplemented with 5%-defibrinated sheep blood was used to culture Escherichia coli ATCC8739, Staphylococcus aureus ATCC25923, and Enterococcus faecalis ATCC29212. On the other hands, Lactobacillus plantarum E12.1 was cultured by using MRS (De Man, Rogosa, and Sharpe) agar.

Isolation and Screening for Antibacterial-Producing LAB
Screening for antibacterial-producing LAB was carried out by the overlay method, as specified in Subagiyo et al. (2016). A quantity of 10 g urutans were homogenized in 90 mL of sterile saline solution (0.85% NaCl) and diluted in a series of multiples of ten. From the 10 -5 and 10 -6 dilution rate, each as much 0.1 mL were spread over two different MRS agar plates and incubated at 37 0 C for 48 hours under anaerobic conditions. After 48 hours, the bacterial colonies that grew on the first plate was overlayered with 10 mL MRS soft agar which added with 100 μL suspension of 0.5 McFarland E. faecalis ATCC29212, while the second plate was overlayered with 10 mL MRS soft agar added with 100 μL suspension of 0.5 McFarland L. plantarum E12.1. The plates were then incubated at 37 0 C for 48 hours under anaerobic conditions to produce inhibition zones. Bacterial colonies with clear zones were collected and examined with the catalase test and Gram stain test to ensure that the isolates were LAB.

Confirmation Test for Bacteriocin-Producing LAB
Confirmation tests were carried out using the disk diffusion method. The indicator bacteria were S. aureus ATCC25923, E. coli ATCC8739, E. faecalis ATCC29212, and L. plantarum E12.1. Production of crude bacteriocins and inhibition test were conducted according to Sari et al. (2016). Antibacterialproducing LAB colonies were cultivated in MRS broth and incubated at 37 0 C for 18-20 hours under anaerobic conditions. This was followed by centrifugation at 8,000 rpm for 15 minutes to separate the cells from the supernatant. The supernatant was neutralized by adding 1M NaOH until the pH reached 6-7, then filtered with a 0.22 milipore membrane to obtain the neutral cell-free supernatant (crude bacteriocin). A total of 20 μL of crude bacteriocin were dropped on a blank disc until completely absorbed. The disc was placed on Nutrient Agar (NA) which had been rubbed with 0.5 McFarland indicator bacterial suspension. After incubation at 37 o C for 24 hours, the diameter of the inhibition zone (clear zone as shown in figure 1) was measured. The test was carried out in triplicates. The average diameter of the inhibition zones was interpreted according to Pan et al. (2009) as follows: 0-3 mm is weak, 3-6 mm is moderate, and > 6 mm is strong.

Identification of Bacteriocin-Producing LAB Isolates using 16S rRNA gene sequencing
The identification was carried out by amplification of the 16S rRNA gene using the PCR method, followed by sequencing. DNAs were isolated using the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, IN, USA) according to the manufacturer's instruction. The primer design and PCR procedure were conducted according to Arief et al. (2015). The universal primers 9F (5'-GAG TTT GAT CCT GGC TCA G-3') and 1541R (5'-AAG GAG GTG ATC CAG CC-3') were used with slight modification on the PCR condition. The PCR formulation consisted of 15 μL of GoTaq® Green Master Mix, 2,4 μL of 10 μM primers, 3 μL of template DNA, and H2O until the total volume of 30 μL was reached.
DNAs were amplified using Thermal Cycler (Biometra) under the following conditions: pre-denaturation at 95 o C for 5 minutes followed by 30 cycles of denaturation at 95 o C for 1 minute, annealing at 55 o C for 1 minute, extension at 72 o C for 1 minute, and elongation at 72 o C for 7 minutes. PCR products were electrophoresed on 1% agarose gel containing 0,003% GelRed® (Biotium) and visualized on UV-transilluminator. The expected DNA was then sequenced to identify the nucleotide base sequence (First Base, Malaysia).
The DNA sequence was edited with the Biological Sequence Alignment Editor (BioEdit) then presented in the form of Fasta files and aligned with the nucleotides of known bacteria strains on the Basic Alignment Search Tool (BLAST) available on the website of https://blast.ncbi.nlm.nih.gov/Blast.cgi.

RESULTS AND DISCUSSION Isolation for Bacteriocin-Producing LAB
LAB was isolated from five samples of indigenous fermented urutans. A total of 14 colonies with clear zones that inhibited the growth of indicator bacteria were isolated in this study. Those 14 isolates confirmed as LAB based on Gram staining result that showed as Gram-positive bacilli or cocci and negative catalase test result. These results were in line with those reported by Todar (2012) with LAB isolate characteristics of Gram-positive cocci, coccobacilli, or bacilli, non-forming spores, generally anaerobic, and lack of catalase enzymes.
LAB could produce various compounds with antagonistic activity against other microbes. Those compounds include organic acids, H2O2, CO2, diacetyl, acetaldehyde, and bacteriocins (Yang et al., 2012). To confirm that the inhibition zones were due to bacteriocins affect, the confirmation test was carried out using the neutralized supernatant (crude bacteriocins). Crude bacteriocins of 14 isolates were tested, only two isolates named J2 and J6 showed inhibition zones against indicator bacteria ( Table 1).
Neutralization of culture supernatants resulted in the loss of organic acid activity. According to Daeschel (1989), organic acids are relatively more abundantly produced than other antimicrobial compounds (Artha, 2016). Hu et al. (2019) also stated that the key to strong antimicrobial activity in three strains of L. plantarum against S.aureus, E.coli, and Salmonella is the organic acid. Therefore, after the supernatant was neutralized, LAB isolates with no ability to produce bacteriocins lost their activity. Note: Ø = diameter, Int = Interpretation, (-) = no inhibition zone formed, (+) = weak, (++) = moderate, (+++) = strong Bacteriocins are classified into several types. Classical type bacteriocins are active only against homologous species, while the second type can show inhibitory activity against a broad range of Gram-positive microorganisms (Parada et al., 2007). In this study, the bacteriocins produced by Lactobacillus sp. J2 and J6 showed moderate (5,07 mm) to strong (10,27 mm) inhibition zones against L. plantarum E12.1 and weak (2,7 mm) inhibition zones against E. faecalis ATCC29212 by Lactobacillus sp. J6. Similar results are shown by Romadhon and Margino (2012), where they found that the bacteriocins produced by two LAB isolates from the tiger shrimp intestine effectively inhibited the growth of Pediococcus accidilactici LB 42. Both isolates were identified as Pediococcus accidilactici. Kormin et al. (2001) found that L. plantarum from tempeh inhibited the growth of Enterococcus faecalis N-I-103. Bacteriocins produced by Lactobacillus sp. J2 and J6 did not show inhibitory activity against the Gram-positive S. aureus, but showed weak to moderate inhibitory activity against the Gram-negative E. coli. Similar results were also reported by Sari et al. (2016), in which the bacteriocins produced by L. plantarum isolated from the Ce Hun Tiau drink did not show an inhibition zone against S. aureus but they inhibited the growth of E. coli. The target of bacteriocins is the cytoplasmic membrane. Lipopolysaccharide (LPS) outside the membrane layer of Gramnegative bacteria protects the bacteria from attack by bacteriocins. Therefore, bacteriocins are generally only active against Gram-positive bacterial cells (Parada et al., 2007 andBromberg et al., 2004). However, mutations or exposure to stress such as heating, freezing, and thawing could damage the outer membrane, which allows access of the bacteriocin to the cytoplasmic membrane thereby increasing sensitivity to bacteriocins (Bromberg et al., 2004). Different mechanisms of action from bacteriocins were also described, such as changing enzymatic activity, inhibition of spore germination, and inactivation of carrier anions through the formation of selective and nonselective pores (Abee, 1995;Martinez andDe Mantis, 2006 in Parada et al., 2007).
Bacteriocin's inhibitory activity against Gram-negative bacteria was also shown in several other studies. Bromberg (2004) found that from 128 bacteriocin-producing LAB strains, 47.6% inhibited the growth of Gramnegative bacteria Pseudomonas sp CTC 32, 38.3% inhibited E. coli ATCC25422, and 37.5% inhibited Salmonella typhimurium ATCC14028. Lade et al. (2006) in Parada et al., (2007) found that Lactobacillus plantarum and Lactobacillus lactis isolated from vegetable wastes produced bacteriocins which inhibited the growth of E. coli. Bacteriocin can be bactericidal or bacteriostatic. Bacteriocin's antibacterial activity is greatly influenced by factors such as environmental conditions and strain of indicator bacterial species used, concentration and purity of the bacteriocin, nutrient content in the medium (Romadhon and Margino, 2012;Sari, et al., 2018), and experimental environment (Cintas et a.l, 2001). Stern et al. (2006) in their study on Lactobacillus salivarius showed that the activity of purified bacteriocin was 89.8% higher than the supernatant from bacterial cultures (Artha, 2016).

Identification of Bacteriocin-Producing LAB Isolate
Identification of Lactobacillus sp. J2 and J6 isolates were carried out by amplification of the 16S gene using 9F and 1541R primers. The PCR products were ± 1500 bp ( Figure 2) then sequenced, and the results were aligned with the database at the Gene Bank. The size of the Lactobacillus sp. J2 and J6 16S rRNA gene sequences was 1481 bp and 1494 bp, respectively. Aligned of the Lactobacillus sp. J2 sequence on BLAST showed 99.39% similarity with Lactobacillus plantarum strain 33X, while the Lactobacillus sp. J6 sequence showed 99,35% similarity with Lactobacillus plantarum strain 7232 (multiple sequences alignment/MSA showed in figure 3). Therefore, both Lactobacillus sp. J2 and J6 isolates belong to species of Lactobacillus plantarum. Lactobacillus plantarum is known as a bacteriocin-producing LAB species. According to Baltasar (2015), bacteriocin-producing L. plantarum strains have been isolated from various vegetable and animal products such as cereals, wines, meats, and dairy products. Bacteriocins produced by L. plantarum are generally indicated as plantaricin which are classified into two classes, namely bacteriocin class I (lantibiotic) and bacteriocin class II (non-lantibiotic). These two strains were in the same clade as the J2 isolate, which indicated that the three strains of bacteria came from the same bacteria. As a comparison, J2 and J6 isolates were also aligned with several strains of L. plantarum, Lactobacillus species, and other LAB genera ( Phylogenetic tree analysis showed that the J6 isolate was closely related to L. plantarum E 12.1.