The increasing awareness among consumers about the side effect caused by some of the chemically synthesized artificial preservatives motivated the study to characterize bacteriocins from probiotic culture that can positively enhance the characteristic taste, wellness and safety of the product. The present probiotic cultures were isolated from fermented food and were shown to exhibit potential probiotic properties (Bindu and Lakshmidevi 2020). The selected cultures showed acid and bile tolerance, gastrointestinal survival, good adhesion property and also possible functional properties including antioxidant activity and industrially important enzyme production. The selected probiotic cultures displayed large spectrum of inhibitory activity against pathogenic and spoilage bacteria including Micrococcus luteus, Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis, Klebsiella sp., E. coli, Pseudomonas aeroginosa, Yersinia enterocolitica, Salmonella sp, and Enterobacter aeroginosa. In the view of their probiotic candidacy, the AMC from these cultures were characterized in the present investigation.
Each indigenous strain exhibited a variable titre and pattern of inhibition against selected pathogens. On the context of inhibitory activity of all the selected probiotic culture, studies were continued with Staphylococcus aureus and E. coli as indicator organisms because of their know pathogenecity (Croxen et al. 2013; Kadariya et al. 2014). According to the results obtained, inhibitory activity against S. aureus and E. coli was in the following increasing order: DB-b2-15b < DB-1aa < IB-PM15 < Cu3-PM8 < Cu2-PM7. Further studies were therefore continued with DB-1aa, Cu2-PM7 and Cu3-PM8.
Generally, bacteriocins are proteinaceous compound produced by certain bacteria to hinder the growth of similar or closely related strains. Hence, to confirm the proteinaceous nature of the AMC, the cell free supernatant (CFS) of the probiotic culture was treated with proteinase K. The enzyme “proteinase K” is a serine protease responsible for degradation of protein. Accordingly, the CFS treated with the enzyme was unable to inhibit the pathogen indicating the denaturation of proteinaceous compound present in CFS which is responsible for antimicrobial activity. As these probiotic cultures are lactic acid bacteria, the action of lactic acid was initially nullified by adjusting the pH of CFS to7.0 using 1N NaOH.
Growth kinetic studies reveal that the selected probiotic cultures enter late exponential phase within 24 h and in stationary phase for the next 5 consecutive days tested. With respect to S. aureus, inhibitory activity was noticed upto 3 days, later reduction or no activity was seen. In case of E. coli, inhibitory activity was observed upto 5 day. Maximum activity was determined in Cu2-PM7 after 24 h against S. aureus (266.02 AU/mg) and E. coli (133.01 AU/mg). Bello et al. (2018) have shown bacteriocins from L. plantarum Z1116 with 500 AU/ml at the 9th hour. The inhibitory activity gradually increased and reached 12,000 AU/ml after 18th hour of growth in MRS broth at 30°C where high cell density (6.8 OD600 nm) was recorded. In the same study, Enterococcus faecium AU02 and Leuconostoc lactis PKT0003 showed highest level of bacteriocins activity (3200 AU/ml and 1600 AU/ml) at 21st and 24th h of growth where highest (2.8 and 2.75 OD600 nm) cell density was recorded. Milioni et al. (2015) compared bacteriocin production of Lactobacillus plantarum LpU4 grown in MRS broth and MRS broth buffered with citrate at 25°C. In normal MRS broth, the antimicrobial activity was initiated at the beginning of the exponential phase reaching a maximum (1,600 AU/ml) at late exponential phase (24 h) and remained constant during the stationary phase upto 48 h. In buffered MRS broth, the activity increased from 100 to 3,200 AU/ml during the exponential growth phase and was stable until 48 h. According to Barbosa et al. (2013), the optimum condition for bacteriocins production (1600 AU/ml) was 25°C and 20 h of incubation time.
As a first step of purification, the AMC was subjected to various extraction techniques for optimum yield. Accordingly, ammonium sulphate precipitation-dialysis technique was found to be more favorable compared to chloroform extraction, ethanol precipitation or butanol extraction. Likewise, Song et al. (2014) used ammonium sulphate precipitation as first step of bacteriocin purification from Lactobacillus plantarum ZJ5 and reporter an activity of 317.14 AU/mg. Earlier studies have reported most of the bacteriocin to contain positively charged amino acid residues with hydrophobic features (Barbosa et al. 2013). Hence, ion exchange chromatography is the commonly applied strategy which has been successfully used for purification of bacteriocin. In the present investigation, therefore ion exchange chromatography has been applied for purification of AMC. According to the data obtained, increase in the purification fold of 2.75, 1.87 and 1.92 was noticed with respect to DB-1aa, Cu2-PM7 and Cu3-PM8 against S. aureus. With regard to E. coli, purification fold of 2.4, 2.84 and 3.26 was perceived. Song et al. (2014) obtained 1.7% yield after RP-HPLC purification of plantaricin from Lactobacillus plantarum ZJ5. In another study, RP-HPLC purified bacteriocin from Lactobacillus sakei showed 74949.6 AU/mg activity with 32% yield and 40.05 fold increase in the activity as tested against Enterococcus faecalis J2-2 (Barbosa et al. 2013).
Stability study of partially purified AMC at different temperature revealed that the compound is resistant upto 90°C. Earlier literature also support the data that the AMC from Lactobacillus plantarum are heat stable (Todorov 2009). Earlier studies have shown bacteriocins stable upto 60°C (Nowroozi et al. 2004; Sowani and Thorat 2012; Martinez et al. 2013), however at higher temperature above 80°C, a significant reduction in the antimicrobial activity has been reported. In the present study, the tested bacteriocin was stable even at 90°C. Similar results of temperature stability have also been reported for bacteriocins LPBM10, bacST202Ch, bacST216Ch, enterocin AS-48, and plantaricin OL15 (Mourad et al. 2005; Zapata et al. 2009; Todorov et al. 2010). Barbosa et al. (2013) have reported a anti-listerial bacteriocin from Lactobacillus sakei MBSa1 which is stable even at 121°C for 15 min. Lactocin NK24 from Lactococcus lactis display 87% reduction in the inhibitory activity at 100°C and gets completely inactivated after steam sterilization (Lee and Paik 2001). Ferchichi et al. (2001) reports almost 25 and 8.3% reduction in the antimicrobial activity of lactocin MMFII, from Lactococcus lactis at 80 and 110°C respectively. Bacteriocin tolerance to higher temperature would be an essential property for their application in thermally processed food. In this regards, the three bacteriocins purified from the selected probiotic culture indicate their possible application as biopreservative agents.
The data on pH stability showed that the bacteriocins from the selected probiotic cultures are active at acidic pH. The activity, however significantly reduced at neutral pH and alkaline pH Similarly, previous studies have reported that bacteriocins are highly stable at acidic pH but get inactivated at alkaline pH(Todorov et al. 2010; Hernandez et al. 2005). The bacteriocin with anti-listerial activity from Lactobacillus sakei MBSa1 was found stable at pH 2 to 6, but lost part of the activity at pH 8 and 10 (Barbosa et al. 2013). Todorov and Dicks (2005) reports almost 50% reduction in antimicrobial activity of bacteriocins ST28MS and ST26MS from L. plantarum at pH values lower than 4.0. Zapata et al. (2009) observed a marked reduction in the inhibitory activity of bacteriocin from L. plantarum LPBM10 with increase in pH value higher than 5. However, several studies have shown bacteriocins (ST23LD, ST341LD, bacST202Ch, bacST216Ch, and ST71KS) from Lactobacillus plantarum, which are stable between pH 2.0 and 12.0 (Todorov and Dicks 2005; Todorov et al. 2010; Martinez et al. 2013).
The effect of digestive enzyme such as trypsin, lipase and a-amylase on the bacteriocin activity was analyzed and the residual activity was determined. According to the data obtained, bacteriocin of the all the three selected cultures were stable in presence of lipase and a-amylase indicating the lack of carbohydrate or lipid moiety. However in presence of trypsin partial inactivation was observed.
SDS-PAGE analysis and over-lay assay revealed a molecular weight of bacteriocin to be 3.5 kDa in both Cu2-PM7 and Cu3-PM8. Earlier reports confirms that the bacteriocin produced by Lactobacillus spp have molecular weight lower than 10 kDa (Cintas et al. 2001; Cotter et al. 2013) as well as morethan 14 kDa (Todorov et al. 2004; Todorov and Dicks 2006). In the similar line, bacteriocin-producing lactic acid bacteria (LAB) were isolated from fermented Parkia biglobosa seeds and were identified as Lactobacillus plantarum Z1116, Enterococcus faecium AU02 and Leuconostoc lactis PKT0003. They produced bacteriocins of size 3.2 kDa, 10 kDa and 10 kDa, respectively (Bello et al. 2018). Tome et al. (2009) partially purified bacteriocins from nine LAB isolated from vacuum-packaged cold-smoked salmon (CSS) which was active against Listeria monocytogenes, E. faecalis, E. faecium, and Staphylococcus aureus. The molecular size of bacteriocins ranged from 2.8 to 4.5 kDa. Messi et al. (2001) isolated LAB from italian sausages that produced plantaricin with a molecular weight of 4.5 kDa which showed broad spectrum activity against food pathogens including S. aureus, L. monocytogenes and A. hydrophila. Lactobacillus plantarum SA6 isolated from fermented sausage produced a plantaricin peptide with molecular mass of 3.4 kDa. Amortegui et al. (2014) isolated Lactobacillus plantarum from ensiled corn and purified the bacteriocin by ammonium sulphate precipitation (70%) and dialysis. They reported 5 and 10 kDa protein with antagonistic activity against Listeria innocua, Listeria monocytogenes, and Enterococcus faecalis. Milioni et al.  characterized 4.8 kDa plantaricin from Lactobacillus plantarum which was isolated from sheep-milk cheese. Plantaricin IIA-1A5 active against Staphylococcus aureus was purified which showed single band on SDS-PAGE with apparent molecular weight of 6.4 kDa (Arief et al. 2015).
In an attempt to determine the plantaricin gene in the selected probiotic strain L. plantarum Cu2-PM7, PCR was carried out using specific primers. As expected, an amplified product of 450 bp was found with plnA F/R primer in L. plantarum Cu2-PM7. However no amplification was observed with P1/P4 primer. E. durans DB-1aa genomic DNA was used as control where no amplification was observed. The results indicate that the present strain L plantarum Cu2-PM7 harbors the plantaricin A gene. BLASTn analysis of the sequence showed highest similarity with Lactobacillus plantarum strain EG.LP 18.7 (MN172266.1). Similarly, Remiger et al. (1996) reported the binding position of primer plnA5p and S7, outside the structural gene of plantaricin A, which allowed the amplification of 450 bp DNA fragment in L. plantarum strains. Earlier studies on comparative genome analysis have reported significant variation in the plantaricin operon in the gene cluster of Lactobacillus plantarum strains (Maldonado et al. 2004; Rojo-Bezares et al. 2008). Ben Omar et al. (2008) isolated Lactobacillus strains from poto-poto, a congolese fermented maize product and observed significant variation in the number of genes between the strains. The operon of Pln locus of plantaricin encoding genes is either simple or complex (Diep et al. 2009). Devi and Halami (2019) reported different plantaricin types based on the presence or absence of pln genes.
The translated protein sequence of plnA gene of L. plantarum Cu2-PM7 showed 100% homology with plantaricin A of L. plantarum (WP 0036419). As described by Diep et al. (1996), planatricin system in L. plantarum is organized into five operon. (1) plnABCD: The regulatory operon encoding bacteriocin –like peptide (plnA), a histidine protein kinase (plnB) and two cytoplasmic response regulators (pln C and pln D). (2) pln GHSTUV: operon associated with transport (3) plnJKLR (4) plnMNOP (5) plnEF1 : related to plantaricin production and immunity. Plantaricin A is a single peptide bacteriocin without post translational modification (Diep et al. 2009). They are included in subclass IIc. L. plantarum CTC305 originally isolated from fermented sausage and L. plantarum Cll isolated from cucumber fermentation were shown to share the plantaricin A encoding gene plnA (Diep et al. 1994).
In the current analysis, bioinformatic tools were used to characterize the peptide nature. Geneious Prime software predicted the protein domain MKQLSNKEMQKIVGG which codes for bacteriocin like protein. On further analysis of this motif, it was observed that this motif is responsible for the entry of the bacteriocin peptide, with a GG cleavage motif at their N-terminal region. The sequence also confirmed the presence of four antigenic regions at sites 6-26, 28-46, 65-77 and 122-135.
It is well known that bacteriocin encoding genes are found along with immunity proteins and other accessory proteins which are arranged in an operon cluster (Noda et al. 2018). In continuation, the bacteriocin-like signal sequence was targeted to predict the peptide which shows activity. The bacteriocin sequence showed maximum homology towards plantaricin A of Lactobacillus plantarum WCFS1, with the ID P80214. Further, the peptide with bacteriocin plantaricin-A had a PBD ID of IYTR, this ID possessed a sequence of 26 amino acids with 2.99 molecular weight (Fig. 5) (Kristiansen et al. 2005). Hence, it can be perceived that the amino acid sequence stretch of our native isolate Cu2-PM7 possessed “KSSAYSLQMGATAIKQVKKLFKKWGW” peptide which is responsible for antimicrobial activity.