In-vitro and In-silico approach for characterization of antimicrobial-peptide from probiotics against Staphylococcus aureus and Escherichia coli


 The present aim was to determine the characteristic feature and stability of antimicrobial compound (AMC) produced by probiotic strains of Enterococcus durans MCC4243, Lactiplantibacillus plantarum (Basanym: Lactobacillus plantarum MCC4246) and Limosilactobacillus fermentum (Basonym: Lactobacillus fermentum MCC4233) against Staphylococcus aureus MTCC96 and Escherichia coli MTCC118. Growth kinetic assay revealed 24h of incubation to be optimum for bacteriocin production. Ammonium sulphate precipitation-dialysis was found to be favorable method for extraction of AMC compared to other methods employed. The partially purified compound after ion-exchange chromatography was found to be thermo-resistant upto 90°C and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, α-amylase and lipase. The apparent molecular weight of AMC from MCC4243 and MCC4246 was found to be 3.5KDa. PCR confirmed the presence of plantaricinA gene in MCC4246. Translated partial amino acid sequence of plnA gene of MCC4246 displayed 48 amino acid sequence which had 100% similarity with plantaricinA of Lactobacillus plantarum (WP 0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns and functions on cytoplasm with 10.82 isoelectric point and 48.6% hydrophobicity. From the study, the amino acid sequence “KSSAYSLQMGATAIKQVKKLFKKWGW” of peptide was predicted to be responsible for antimicrobial activity.


Introduction
In recent years, people are being attracted towards consumption of food, positively associated with health and nutrition. The attitude towards un-acceptance of food products containing some of the arti cial preservatives has increased due to growing awareness of its ill-effect on human and animal health (Anand and Sati 2013). Hence it is a major challenge in food sector to satisfy consumers demand for products free of chemical preservatives and also to ensure natural means of food safety. Further, the chemically synthesized antibiotics and their non judicious usage has resulted in emerging multidrug resistant pathogens and their devastating damage thereafter. Consequently, the need for new strategies and methodologies to control infectious pathogens is increasing. The health bene ts executed by some of the traditional foods have attracted researchers to dig the mechanism and apply the knowledge for betterment of life. In this regard, antimicrobial compounds (AMC) or the bacteriocins from food grade lactic acid bacteria (LAB) have engrossed because of its safety and broad range of antibacterial spectrum.
Bacteriocins are ribosomally synthesized proteinaceous compound produced by bacteria to protect themselves as well as to competitively colonize in the gut through elimination of undesirable organisms.
Bacterial ability to inhibit spoilage organisms has been exploited for its application as natural preservatives in various food formulations. LAB being the natural micro ora of human intestine has added advantage for its application in food due to their GRAS (Generally recognized as Safe) status.
Bacteriocins from LAB are of low molecular peptides which vary in their size, post-translation modi cations, stability and mode of action (Amortegui et al. 2014). According to Drider et al (2006) and Nes and Holo (2000) bacteriocins, have been classi ed into 3 groups based on presence or absence of post-translational modi cation: Class I-Lantibiotics containing lanthionine or ß-methyl-lanthionine; Class II-heat stable unmodi ed bacteriocins; Class III-large heat liable bacteriocin. Class II bacteriocins are further subdivided into Class IIa (one peptide), Class IIb (two peptide), Class IIc (circular), Class IId (unmodi ed, linear, non-pediocin like) and Class IIe (Microcin like). Class IIa bacteriocins are <10 kDa in size with 37-48 amino acids that inhibit growth of food spoilage organisms such as Listeria monocytogenes, Bacillus cereus, Clostridium perfringens, Staphylococcus aureus and E. coli (Rodrigues et al. 2005).
Bacteriocin produced by Lactococcus lactis subsp. lactis ie., Nisin is the only bacteriocin approved by US-FDA for commercial application in food products. However the application of nisin is limited in neutral and alkaline pH. Long-time preservation of foods by biopreservative process and inhibition of pathogenic microbes through natural means is therefore universal need in the present scenario. Considering this, the present work was carried out to optimize the method for e cient extraction of antimicrobial compound (AMC) from LAB isolated from fermented foods. These LAB cultures including Enterococcus durans DB-1aa (MCC4231), Enterococcus faecium DB-b2-15b (MCC4263), Lactiplantibacillus plantarum (Basanym: Lactobacillus plantarum Cu2-PM7 (MCC4246), Limosilactobacillus fermentum (Basonym: Lactobacillus fermentum Cu3-PM8 (MCC4233) and Lactobacillus fermentum IB-PM15 have been previously characterized for their potential probiotic properties (Bindu and Lakshmidevi 2020). In the present study, we report the effect of physical and chemical parameter on the activity of partially puri ed AMC and PCR approach has been applied for characterization of antimicrobial peptide.

Bacterial cultures and growth condition
The selected probiotic cultures Enterococcus durans DB-1aa (MCC4231), Enterococcus faecium DB-b2-15b (MCC4263), Lactobacillus plantarum Cu2-PM7 (MCC4246), Lactobacillus fermentum Cu3-PM8 (MCC4233) and Lactobacillus fermentum IB-PM15 were isolated from fermented food products, identi ed and deposited at Microbial Culture Collection Center, Pune (Bindu and Lakshimidevi 2020). activity was further quanti ed by the two-fold dilution method. Brie y, cell suspension of pathogenic culture grown in BHI broth was adjusted to turbidity of 0.5 McFarland which is equivalent to 1-2 x 10 5 CFU/mL. Antibacterial activity was quanti ed by testing the two fold serially diluted sample of AMC by agar well diffusion assay and the results were expressed as AU/ml. Arbitrary unit (AU) is de ned as the reciprocal value of the highest dilution at which the zone of inhibition is observed.
Minimum Inhibitory Concentration (MIC) MIC was estimated by using different concentration of the sample (10-100 µg) in a 96 well ELISA plate.
Freshly, grown pathogenic cultures at a nal concentration of 10 5 CFU/mL was inoculated into each well and the plate was incubated at 37°C for 24 h. Chloramphenicol (5-50 µg) was used as a positive control and sample with un-inoculated broth as negative control. After incubation absorbance was measured at 600nm. MIC was de ned as the lowest protein concentration (in mg/mL) which inhibited the growth of pathogenic culture completely. All assays were performed in triplicate.

Determination of proteinacious nature of AMC
The proteinacious nature of AMC was checked agar well diffusion assay by treating the crude AMC with proteinase K at a nal concentration of 2 mg/ml for 30 min. The untreated CFS and the enzyme alone in buffer (pH 7.0) served as controls.
Probiotic growth and AMC production Growth kinetics and AMC production was studied for a period of 5 days. The selected cultures were inoculated (1% v/v) in 250 ml MRS broth and incubated at 37°C without agitation. An aliquot of sample was drawn after every 12 h during growth and checked for bacterial growth and antimicrobial activity.
Bacterial growth was determined by measuring cell density at 600nm. The antimicrobial activity was evaluated after lter sterilizing the neutralized CFS by well diffusion assay.

Extraction of the AMC
Optimization of the extraction procedure The CFS of the selected probiotic cultures was subjected to various extraction procedures to determine a suitable protocol for maximum recovery of AMC. Protocol 1: CFS was mixed with ice-cold ethanol (1:1 v/v) and allowed for precipitation overnight at 4°C. The precipitate was air-dried, resuspended in phosphate buffer (pH 7.0) and checked for inhibitory activity. Protocol 2: CFS was blended with equal volumes of chloroform on a magnetic stirrer for 1 h. The mixture was left at room temperature undisturbed for phase separation. AMC in the interphase was collected carefully and the residual chloroform was evaporated under nitrogen and monitored for the inhibitory activity. Protocol 3: Fine powder of ammonium sulfate was added slowly to the CFS until a saturation level of 80% under constant stirring. The procedure was followed at 4°C under aseptic condition. The precipitate obtained was dialyzed against phosphate buffer (pH 7.0) with three changes of buffer and then analyzed for inhibitory activity. Protocol 4: Butanol (1: 0.5 v/v) was added to CFS and vigorously mixed in a separating funnel. The mixture was left at room temperature for phase separation. The butanol layer was carefully collected and evaporated to dryness using rotary evaporator and checked for the activity.

Ion exchange chromatography
The dialyzed sample (60 mg protein) of each culture was loaded separately onto a DEAE cellulose column (18 X 3 cm) pre-equilibrated with sodium phosphate buffer (0.2 mM; pH 7.0). A linear gradient of 0.0-1.0M NaCl in the same buffer was used to elute the protein fractions at a ow rate of 1 ml/min. Fractions (2 ml) was collected and checked for absorbance at 280 nm. The fractions showing the highest protein content were pooled, lyophilized and examined for antimicrobial activity.
Stability of partially puri ed AMC

Effect of Temperature and pH
The effect of temperature was evaluated by incubating the partially puri ed AMC for 30 min at different temperatures (30,40,50, 60, 70, 80, 90°C). The residual activity was checked by agar well diffusion assay after cooling to room temperature. The effect of pH was studied by adjusting the AMC to different pH (3-9) using 1M NaOH or 1M HCl. The sample was incubated at 37°C for 30 min. The inhibitory activity was later checked after neutralization of AMC.

Effect of enzymes
The effect of enzymes such as trypsin, amylase and lipase on inhibitory activity was evaluated by treating the partially puri ed AMC with each enzyme at a nal concentration of 1mg/ml. Samples were incubated at 37°C for 30 min. After incubation, samples were heat treated at 90°C for 5 min to inactivate the enzymes and then the residual activity was determined as described earlier.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and overlay assay to detect AMC Crude sample, dialyzed sample as well as column puri ed fractions of bacteriocin were subjected to SDS-PAGE (Laemmli 1970) along with low range protein marker ranging from 3.5-43 KDa. The antimicrobial activity in the partially puri ed sample was determined by overlay assay (Barboza-Corona et al. 2007).
The sample containing 80 µg of protein was mixed with equal volume of non-reducing loading buffer and directly loaded in the SDS-PAGE gel without boiling. Following electrophoresis, the gel was cut vertically and immersed in xing solution (25% isopropanol, 10% acetic acid) for 1 h and washed with doubledistilled water with three changes after every 30 min. Later, the gel was sterilized by exposing to UV for 30 min and then aseptically placed in a sterile petriplate and overlaid with BHI agar pre-inoculated with 1% (equivalent to 10 8 CFU/ml) overnight grown test organism (Staphylococcus aureus or E. coli). The petriplate was later incubated at 37°C for 24 h and observed for the presence of inhibition zone.
Detecting bacteriocin genes by polymerase chain reaction (PCR) The presence of bacteriocin gene was identi ed in L. plantarum Cu2-PM7 using plantaricin A (F: GTA CAG TAC TAA TGG GAG; R: CTT ACG CCA ATC TAT ACG) and P1/P2 primers (F: AAAATATCTAACTAATACTTG; R: TAAAAAGATATTTGACCAAAA). The gene was ampli ed in 25 µl reaction mixture containing 2 µl of 1X PCR buffer, 1.5 µl of MgCl 2 , 5 µl of dNTPs, 0.5 µl of each (Forward and reverse) primer, 2.5 U of Taq polymerase and 100 ng of genomic DNA. PCR conditions include denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 50°C for pln primer and 52°C for P1/P2 primer, for 45 sec and extension at 72°C for 1 min and nal extension of 72°C for 10 min. The ampli ed PCR product was sequenced at GENESPY Research Services, Mysore. Sequence was matched with similar sequence from NCBI data base.

Bacteriocin peptide analysis
Bacteriocin peptide structure for the putative amino acid was predicted by using Geneious Prime® 2019.2.1 Software (Kearse et al. 2012). Using the software, secondary structure, antigenic regions, signal cleavage sites, transmembrane regions and protein domains prediction plugins was determined. Further the amino acid sequence data was analyzed. The peptide showing activity was later analyzed by using protein predict server (https://open.predictprotein.org/) followed by PDB database (Armstrong et al. 2020).

Statistical analysis
The experiments were conducted in triplicates and the data analysis was done using STATISTICA version 6 software (Statsoft Inc., Tulsa, USA). The signi cant different between the samples were analyzed by using ANOVA (analysis of variance), and the mean separation was accomplished by Duncan's multiple range test.

Minimum inhibitory concentration of AMC
According to the results obtained, MIC was in the range of 2200 to 2400 µg against S. aureus and E. coli. Higher activity against S. aureus (228.6 AU/mg) and E. coli (114.3 AU/mg) was noticed in AMC of L. plantarum Cu2-PM7, followed by L. fermentum Cu3-PM8 (139.1 and 69.6 AU/mg against S. aureus and E. coli respectively). Enterococcus faecium DB-b2-15b displayed lowest activity (38.1 and 38.1 AU/mg against S. aureus and E. coli respectively). Based on the data, three cultures viz., Enterococcus durans DB-1aa, L. plantarum Cu2-PM7 and L. fermentum CU3-PM8, showing higher activities were selected for further study.
Proteinaceous nature of AMC from probiotic culture Proteinaceous nature of the neutralized CFS was con rmed by treating with proteinase K. According to the results obtained, the activity was not lost after neutralization but was signi cantly reduced on treating with proteinase K. This con rms that the inhibitory activity was not due to acidity but was due to proteinaceous compound.

Growth kinetics and bacteriocin production
The selected cultures entered the late exponential phase in 24 h and remained constant in stationary phase upto 5 days. The OD (optical density) at 600nm is presented in Table 1. Inhibition zone in the range of 12-16 mm dia was observed upto 3 days against S. aureus. On day 4 th and 5 th , no activity was observed. MIC against S. aureus was 2400 µg for all the tested AMC after 24 h of growth (Table 1).
However on the 5 th day MIC was 5200, 5000 and 3800 µg for AMC from DB-1aa, Cu2-PM7 and Cu3-PM8 respectively. Activity was highest at 24 th h, later the activity gradually decreased. According to the data obtained, maximum activity (266.02 AU/mg) was noticed in L. plantarum Cu2-PM7. The AMC compound of Cu2-PM7 and Cu3-PM8 showed inhibitory activity against E. coli upto 5 days, but DB-1aa displayed zone of inhibition upto 3 days (Table 1). With increase in incubation days, a gradual reduction in the size of inhibition zone was observed. The MIC was 2400 µg on the 1 st day but displayed 5600, 5200 and 3800 µg on the 5 th day. The activity was maximum (133.01 AU/mg) with L. plantarum Cu2-PM7 after 24 h of incubation.
Extraction of AMC from selected cultures AMC from selected cultures was extraction by four different techniques to determine the best method for extraction of AMC. Ethanol precipitation did not show any inhibitory activity against S. aureus, however against E. coli lesser zone of inhibition (10 mm dia) was observed (Fig. S1). Comparatively, sample after ammonium sulphate precipitation-dialysis displayed maximum zone of inhibition than chloroform extract and butanol extract ( Table 2). MIC against S. aureus was in the range of 1900 -2200 µg and 2000 -2300 µg against E. coli. According to the data obtained, ammonium sulphate precipitation-dialysis was found to be the best method for extraction which showed an activity of 155.27, 212.72 and 179.80 AU/mg with E. durans DB-1aa, L. plantarum Cu2-PM7 and L. fermentum Cu3-PM8 respectively. Hence, further step of puri cation was carried out after ammonium sulphate precipitation and dialysis.
Puri cation of bacteriocin by ion exchange chromatography lyophilization. The antimicrobial activity assay con rmed the zone of inhibition in fraction 1 and 2 of all the three culture against S. aureus and E. coli (Fig. 1). The data obtained revealed highest inhibition zone of 20 and 18 mm against S. aureus and E. coli by fraction-1 of L. plantarum Cu2-PM7 with MIC of 1200 and 1300 µg respectively. The details of puri cation data are summarized in Table 3. The nal yield of bacteriocin obtained after ion-exchange chromatography was 0.4, 0.5 and 0.4% for DB-1aa, Cu2-PM7 and Cu3-PM8 respectively against S. aureus. Correspondingly, a nal yield of 0.35, 0.76 and 0.68% was found against E. coli.
Effect of temperature and pH on bacteriocin activity According to the results obtained, the bacteriocins from all the tested cultures were active in all the temperature tested (Table 4). However, bacteriocin from Cu3-PM8 showed slight reduction in the activity at higher temperature. Further, the pH stability of bacteriocin puri ed from selected probiotic culture was analyzed at various pH ranging from pH 3-10 ( Table 5). The data revealed that the inhibitory activity was more active at acidic pH (inhibition zone ranging from 14-20 mm dia). As the pH approached neutral, the activity reduced and was completely inhibited at alkaline pH.

Effect of enzymes on bacteriocin activity
According to the data obtained, the inhibitory activity was retained in all the three bacteriocin after treating with a-amylase and lipase. However, in presence of trypsin 55, 34 and 43% activity was retained.

SDS-PAGE analysis of antimicrobial compound
Molecular weight of the antimicrobial compound from selected probiotic culture (Cu2-PM7 and Cu3-PM8) was determined by SDS-PAGE (Fig. 2). Accordingly, apparent molecular weight of both AMC was found to be 3.5 kDa. Inhibition zone corresponding to their respective protein bands was developed on performing activity assay by overlaying indicator strain (Fig. 2).
Detecting plantaricin gene by PCR PCR analysis of plantaricin gene was carried out in Cu2-PM7 using plnA and P1/P4 primer. A good ampli cation of 450 bp was observed with plnA F/R primer. On the other hand, no ampli cation was detected with P1/P4 primer. The control culture, E. durans DB-1aa did not show any ampli cation in presence of any of the tested primer. Further, the PCR product was sequenced and the obtained sequence was subjected to BLASTn search in NCBI database and aligned with hit sequences. The results indicated 100% homology with plantaricin plnA sequence of Lactobacillus plantarum strain EG.LP 18.7 (MN172266.1) (Fig. 3).
The nucleotide sequence was translated to aminoacid sequence and checked for homology with BLAST search. Translated partial amino acid sequence of pln A gene of Cu2-PM7 displayed 48 amino acid sequence which had 100% similarity with plantaricin A of L. plantarum (WP 0036419). Fig. 4 represents the similarity of plnA-PM7 amino acid sequence with related sequence from NCBI data base.

Bacteriocin peptide analysis
The pln A gene sequence was translated and the 138 amino acid sequence thus obtained was studied for its structural and functional properties. It was observed that the partial sequence of Cu2-PM7 possessed 10.82 isoelectric point and 48.6% hydrophobicity. The sequence possessed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns and functions on cytoplasm. One topological domain, which encodes the bacteriocin-like signal sequence (MKQLSNKEMQKIVGG) was noticed with a GG cleavage motif at their N-terminal region. Further, four antigenic regions were detected in the acquired sequence which were located at sites 6-26, 28-46, 65-77 and 122-135 with sequence read LSILRCAVNVILSSKYVQSNQ, LFKIVTLTFIKYLIALEFH, THYPKSEVIIMKI and WLIYITVFQVIFRIDGV, respectively. A 3D structure for the bacteriocin-like peptide was inferred from Phyre2 and indicated alpha and beta sheet structure fragments (Fig. 5). The protein-predict database showed maximum homology towards bacteriocin plantaricin A of Lactobacillus plantarum WCFS1, with the ID P80214. Hence, it can be perceived that the amino acid sequence stretch of our native isolate Cu2-PM7 possessed "KSSAYSLQMGATAIKQVKKLFKKWGW" peptide showing a molecular weight of 3 kDa.

Discussion
The increasing awareness among consumers about the side effect caused by some of the chemically synthesized arti cial 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 con rm 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 nulli ed 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. 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 rst step of puri cation, 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 rst step of bacteriocin puri cation 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 puri cation of bacteriocin. In the present investigation, therefore ion exchange chromatography has been applied for puri cation of AMC. According to the data obtained, increase in the puri cation 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, puri cation fold of 2. Stability study of partially puri ed 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 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 puri ed 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 signi cantly 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 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 con rms that the bacteriocin produced by Lactobacillus spp have molecular weight lower than 10 kDa In an attempt to determine the plantaricin gene in the selected probiotic strain L. plantarum Cu2-PM7, PCR was carried out using speci c primers. As expected, an ampli ed product of 450 bp was found with plnA F/R primer in L. plantarum Cu2-PM7. However no ampli cation was observed with P1/P4 primer. E. durans DB-1aa genomic DNA was used as control where no ampli cation 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. 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 ve 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 modi cation (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 con rmed 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.

Conclusions
The data support the potential inhibitory activity of bacteriocin against Staphylococcus aureus and E. coli. On the basis of two important observations, i.e. proteinaceous nature of antagonistic substances and their heat stability, they can be considered as bacteriocins. The peptide analysis support the plantaricin A type bacteriocin present in the native probiotic culture L. plantarum Cu2-PM7. Overall the study highlights the characteristic features of bacteriocins produced from probiotic bacteria isolated from fermented foods which could be employed in food as preservative agent. The data obtained enhances our knowledge on the biocontrol of undesirable bacteria that could be used as an alternative therapy for treating infectious disease.  Values are average of two experiments performed individually (Mean ± SD). '-' indicate no activity.   Values are average of two experiments performed individually (Mean ± SD).  Plantaricin peptide analysis (A) Amino acid sequence similarity between plnA-PM7 and related sequence from NCBI data base (B) Amino acid sequence aligned (dark shade represents maximum homology) Figure 5