Antibacterial Activity Test of Bacteriocin from Lactobacillus brevis , Lactobacillus casei and Lactobacillus plantarum Against Gram Positive Pathogenic Bacteria

Bacteriocin is a secondary metabolite product of lactic acid bacteria (LAB) which have an antimicrobial and potentially as a natural preservative. LAB isolates used in this study were Lactobacillus brevis, Lactobacillus casei and Lactobacillus plantarum. This study aimed to determine the antibacterial activity of bacteriocin produced by each isolate of LAB including the influence of pH and heating variation against Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis. Antibacterial activity test was done by using disc diffusion method. method. Confirmation test using proteolytic enzyme aimed to analyse that the inhibition zone produced from the activity of bacteriocin. The inhibition zone produced from L. brevis, L. casei and L. plantarum against B. cereus were 15.70, 16.43 and 14.50 mm, against B. subtilis were 13.37, 14.10 and 12.53 mm and against S. epidermidis were 11.37, 14.50 and 12.45 mm. The activity of each bacteriocin decreased with the addition of trypsin and catalase, bacteriocin was active in the pH range of 2-10 and heating temperature of 40-121C. Statistical test showed that the addition of trypsin, catalase and the variation of pH also heating had significant differences (p<0.05) to antibacterial activity produced by bacteriocin from L. brevis, L. casei and L. plantarum. Article history: Received 15/08/2018 Received in revised form 06/11/2018 Accepted 06/11/2018


Introduction
Foodborne disease that commonly called food poisoning is one of the causes of morbidity and mortality in Indonesia.Some of pathogenic bacteria that related to food poisoning are Bacillus cereus, Bacillus subtilis and Staphylococcus epidermidis.According to WHO in 2009 incidence rate due to B. cereus ≥ 100 cases per 1000 population (Arisman, 2009).B. subtilis is the most common bacteria found in 10 out of 24 cutlery samples (41,7%) in Kandou Manado Hospital (Riga et al., 2015).Based on the research (Podkowik et al., 2016), there were 32 S. epidermidis isolates that found from 164 samples of readyto-eat meat products.
Bacteriocin is a secondary metabolite product of lactic acid bacteria (LAB) that has potential as a natural preservative (biopreservative).The advantages of bacteriocin that potentially used as a biopreservative are that it is not a toxic substance, easily degraded by proteolytic enzymes because bacteriocin is a protein compound, does not harm intestinal microflora and stable over wide pH and temperature range (Cleveland et al., 2001).LAB that can produce bacteriocin can be found from fermented food products and processed food products (Palacios et al., 1999).
Es pisang ijo is a Makassar typical drink that is good for health and isolates of LAB that had been identifiedin it was Lactobacillus brevis (Syahputria, 2016).Sotong kering is a preserved food product in West Kalimantan and LAB isolates that had been identified in itwas Lactobacillus casei (Yurinda, 2016).Ce hun tiau is a Chinese Pontianak typical drink that has been proven as a producer of LAB and LAB isolates that has been identified in it was Lactobacillus plantarum (Malik et al., 2010) (Sari et al., 2016).
The aim of this research was to find out that the antibacterial activity of bacteriocin produced from L. brevis, L. casei and L. plantarum include the influence of pH and : 10.22146/jtbb.38138heating temperature variation against B. cereus, B. subtilis and S. epidermidis bacteria and confirmation test using proteolytic enzyme aimed to analyse that the inhibition zone produced from the activity of bacteriocin.

Effect of pH on Bacteriocin Activity
An amount 5 mL of each bacteriocin supernatant from L. brevis, L. casei and L. plantarum were put in different tubes, each tube was adjusted to pH 2, 4, 6, 8, and 10 using NaOH or HCl 0, 1 N.After having incubation for 4 hours at room temperature, then bacteriocin activity was tested using agar diffusion method (Kusmarwati et al., 2014).

Effects of Heating on Bacteriocin Activity
An amount 5 mL of each bacteriocin supernatant from L. brevis, L. casei and L. plantarum were heated at 40, 60, 80, and 100°C for 30 min in waterbath thermostatic and 121°C for 15 min in autoclave (Saad et al., 2015).Then bacteriocin activity was tested using agar diffusion method.

Data Analysis
Inhibition zone diameter data (proteolytic enzyme, pH and heating treatment) were measured using callipers, then each data was analysed using Statistical Program Service Solution (SPSS) using ANOVA test.

Gram Staining Results
Gram staining showed that each isolate of bacteria observed (L.brevis, L. casei and L. plantarum) was Gram positive bacteria characterized by violet colour in bacterial cells.Gram positive bacteria retain the violet colour due to the low lipid content in bacteria when it washed with alcohol, bacterial cell wall more easily hydrated.The hydrated cell wall causes the cell pore size to become small and its permeability decreases therefore the violet colour from crystal violet cannot leave the cell and the cell will remain violet (Assani, 1994).Character morphology was examined are rod shape for those bacteria.This is in accordance with Bergey's Manual of Determinative Bacteriology, the characteristics of the genus Lactobacillus are rod-shaped, Gram-positive and facultative anaerobic (Breed et al., 1957).bacteria filter with a diameter of 0.22 μm which purposed to free the supernatant from the remaining bacterial cells due to bacterial cells the remaining ones could contaminate the resulting supernatant.Therefore, the supernatant that had been produced was containing bacteriocin.
Table 3. Bacteriocin inhibition zone diameter with the addition of proteolytic enzyme against B. subtilis.

Bacteriocin Antibacterial Activity
Bacteriocin antibacterial test activity purposed to determine the inhibitory activity of bacteriocin from those bacteria against B. cereus, B. subtilis and S. epidermidis.
Bacteriocin is mentioned to have antibacterial activity characterized by the formation of inhibitory zones around the disc.The larger the inhibitory zone is, the greater the bacteriocin inhibition activity against pathogenic bacteria.(Bhunia et al., 1991).Bacteriocin is a protein which is easily degraded by proteolytic enzymes.The addition of proteolytic enzymes can eliminate the activity of bacteriocin by decreasing or loss of the inhibitory zone around the disc.Proteolytic enzymes will disturb the bonds of amino acids that arrange the bacteriocin that caused bacteriocin activity will be lost and unstable (Parada et al., 2007).Sensibility to proteolytic enzymes evidences the proteinaceous characteristic of bacteriocins (De Martins et al., 2003).Proteolytic enzymes used were trypsin and catalase enzyme.Bacteriocin also has an α-helical structure with opposite polar and nonpolar sides of bacteriocin, making bacteriocin interact with both water and lipid phases when attached to the surface of a sensitive bacterial cell membrane, the cell destabilizes functionally and the cell dies (Ray et al., 2007).
The existence of α-helical structure which is still good Catalase test were performed to ensure that the antimicrobial activity produced was an activity of bacteriocin and was not activity of hydrogen peroxide.Hydrogen peroxide is one of the metabolites produced by LAB which is also antibacterial.The depletion of the inhibitory zone may J. Trop. Biodiv. Biotech., Vol. 3 (2018), 85-91 89 be due to the breakdown of hydrogen peroxide by the catalase enzyme contained in the supernatant so that the bacteriocin produced by the three isolates still contains another metabolite that was hydrogen peroxide (Sari et al., 2011).Based on this character, the three bacteriocin studied can be used as a food preservative, which the process of that food production involves heating.

Conclusions
Bacteriocin produced from L. brevis was isolated from es pisang ijo, L. casei was isolated from sotong kering and L.

Figure 1 .
Figure 1.Bacteriocin inhibition zone diameter in different pH conditions against B. cereus

Figure 2 .
Figure 2. Bacteriocin inhibition zone diameter in different pH conditions against B. subtilis

Figure 3 .
Figure 3. Bacteriocin inhibition zone diameter in different pH conditions against S. epidermidis although bacteriocin has degraded by trypsin enzyme, causing bacteriocin from L. brevis, L. casei and L. Plantarum still have inhibitory activity against test bacteria.The results similar with the study (Ayuningtyas, 2012) which stated that plantaricin inhibitory activity of four purified L. plantarum strains still exist although it has been degraded by trypsin enzyme.Based on this result, it can be concluded that the antibacterial supernatant produced by the three isolates of LAB was bacteriocin.

Figure 4 .Figure 5 .
Figure 4. Bacteriocin inhibition zone diameter in different heating temperatures against B. cereus

Figure 1 ,
Figure 1, 2 and 3 resulted of the pH effect on bacteriocin activity, each bacteriocin of L. brevis, L. casei and L. plantarum showed antibacterial activity in a wide range of pH (2-10).This could be due to the presence of alkaline amine or carboxylic group of amino acid that arrange the bacteriocin (Balvinder et al., 2011) that caused bacteriocin could retain its antimicrobial activity when there was a shift to acidic or basic range.The results were similar with the study (Gautam, 2009) that reported bacteriocin produced by Lactobacillus brevis

Figure 6 .
Figure 6.Bacteriocin inhibition zone diameter in different heating temperatures against S. epidermidis.

Figure 4 ,
Figure 4,5 and 6 stated that the bacteriocin from L. brevis, L. casei and L. plantarum showed antibacterial activity subjected heating temperature of 40-100ºC for 30 min to 121°C for 15 min.The heat stability of bacteriocin might be due to formation of small globular form, presence of stable cross-linkages, strong hydrophobic regions and the presence of amino acid cysteine which is able to maintain the bacteriocin structure from heating (Barman et al., 2018) (Nugroho et al., 2003).Similar results reported (Ogunbawo, 2003) that bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OG1 still have activity on heating temperature of 100 º C for 10-30 min and 121 ºC for 10-60 min.The results of (Ullah et al., 2017) stated that bacteriocin Lin333 from Lactobacillus casei isolated from Jiangshui Cai (fermented vegetables from China) was stable at 60-121°C.
plantarum was isolated from ce hun tiau had antibacterial activity against B. cereus, B. subtilis and S. epidermidis.The activity of each bacteriocin decreased with the addition of trypsin and catalase.Bacteriocin was active in the pH range of 2-10 and heating temperature of 40-121 o C.

Table 1 .
Bacteriocin inhibition zone diameter will turn turbid due to the release of antimicrobials on the medium.LAB produces the optimum bacteriocin at the incubation time of 24 hours and the maximum incubation time is 48 hours, because at longer incubation times protease or other inactivators can be formed and activated which can reduce the bacteriocin activity.The maximum time in the growth cycle for bacteriocin production depends on the type of bacteria and can occur from the log phase to the initial stationary phase(Hoover et al., 1993).

Table 2 .
Bacteriocin inhibition zone diameter with the addition of proteolytic enzymes against B. cereus one of the metabolites of LAB.The acidic supernatant needed to be conditioned into a neutral state at pH 7 with the addition of 0.1N NaOH which aimed to ensure that the inhibitory zone formed was not derived from organic acids but was purely an activity of bacteriocin(Ogunbawo, 2003).The filtrate that had been neutralized then sterilized with a

Table 4 .
Bacteriocin Inhibition Zone Diameter with the addition of proteolytic enzyme against S. epidermidis.
supernatant has been neutralized with NaOH solution.Gram positive bacteria inhibition mechanism by bacteriocinis caused by bacteriocin that attaches to Gram positive bacteria and forms complexes with lipoteichoic acid found on Gram positive bacterial cell wall it can cause destabilization of the cell wall.Lipoteichoic acid is a specific receptor and is