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Growth optimization of Bacillus subtilis 11A isolated from Indonesian native chicken (Gallus domesticus) for bacteriocin production

This study aimed to obtain the optimal growth of Bacillus subtilis 11A isolated from Indonesian native chicken (Gallus domesticus). The growth optimization included pH medium conditions (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5) and different incubation temperatures (31, 33, 35, 37, 39, 41°C). Isolate was incubated for 12 hours using Luria Bertani broth. Optical density was observed using spectrophotometer (λ600) at 0 and 12 hours of incubations. Bacteriocin activity against pathogens Staphylococcus aureus, Salmonella typhimurium and Escherichia coli was determined using a well-diffused method. Data was statistically analyzed using a one-way analysis of variance (ANOVA) followed with Duncan’s New Multiple Range Test (DMRT) to distinguish the treatment means. The results showed that the optimum pH for bacterial growth was at pH 6.5. Optimum temperature for bacterial growth was at 39°C. The highest bacteriocin activity of B. subtilis 11A against indicator pathogens was resulted at stationary phase (1906.14, 2179.79, 2343.07 AU/ml). It can be concluded that B. subtilis 11A isolated from Indonesian native chicken grew optimally for bacteriocin production at pH 6.5 and temperature 39°C. The highest bacteriocin production and activity produced by Bacillus subtilis 11A was at stationary phase.


Introduction
Antibiotics in the field of animal health are used as prevention and treatment of diseases, as well as a growth promoter in chickens. The administration of antibiotics in cattle may cause residues in the products. The residues of tetracycline, sulfonamides, fluoroquinolones and enrofloxacin respectively as much as 1,046.3-1,158.9; 20-800; 37.6-164.7; and 18.32 µg/kg in chicken meat in Nigeria, Pakistan, Portugal, and Iran [1][2][3][4]. Due to antibiotic residues as the negative effects of antibiotics addition in feed, many researches have been conducted to invent natural antibiotics which may be produced by plants (herbs) and microorganism (probiotics). One of natural antibiotic produced by bacteria was bacteriocin, in exampleis bacteriocin produced by genus Bacillus. Bacteriocins produced by Bacillus bacteria actively inhibit the growth of pathogenic bacteria [5]. Bacillus bacteria play a role in maintaining the balance of intestinal microbiota, enhance the natural defense mechanisms of the host, so it will positively affect on the performance and the health of chickens [6]. Growth of Bacillus bacteria related to pH medium and temperature of incubation. The production of bacteriocin is also affected by the growth of bacterial cells [7]. So that, optimum pH medium and incubation temperature were needed to optimize the growth and bacteriocin production by Bacillus bacteria isolated from native chickens (Gallus domesticus).

Bacteriocin activity assay.
Antimicrobial activity assay of bacteriocins against pathogens every phase of growth was performed by growing the isolate Bacillus subtilis 11A on the medium based on the optimal pH and incubation temperature. Samples of medium were were taken every two hours and stored at temperature 4°C for furtherly used in bacteriocins extraction. Extraction of bacteriocin refers to [7], by centrifuging the medium solution at a speed of 10,000×g (4°C) for 15 minutes. Then, a cellfree supernatant was collected for testing against pathogens (Staphylococcus aureus, Salmonella typhimurium and E. coli) as follows: as much as 50 µl of sample is placed in well 5 mm well [8] at Tryptic Soya Agar (TSA, Oxoid, UK) medium and incubated at 37°C for 24 hours. The clear zone was then measured using a caliper and the value of the bacteriocin activity (AU/ml) is determined.

Stastical analysis.
The data of bacterial growth were analyzed using a one-way analysis of variance (ANOVA) and continued by Duncan's New Multiple Range Test [9]. Data of bacterial growth and bacteriocin activity were analyzed descriptively.

Results and discussion
Incubations of medium at different pH and temperature were conducted to obtain optimum pH and the optimum temperature for bacterial growth. Results showed that the optimum temperature for B. subtilis 11A were 39 and 41°C. Incubation at temperature 39°C had a higher absorbance (0.471) compared to the incubation temperature at 41°C (0.460) and the lowest was at 31°C (P<0.05). The effect of different level temperature on the growth of B. subtilis 11A in this study was summarized in Table 1.  3 This study showed that the optimum pH for the growth of B. subtilis 11A was at pH 6.5 and the lowest growth was at pH 5, 5.5 and 8.5 (P<0.05). The effect of different pH medium on the growth of B. subtilis 11A is presented in Table 2. Growth kinetics of B. subtilis 11A at different incubation temperature is presented in Figure 1. Salman [10] and Sumardi [11] reported that the growth of B. subtilis RM16 and Bacillus sp. isolated from native chicken had optimum incubation temperature at 40°C, while Nguyen [12] reported that optimum temperature for the growth of Bacillus subtilis CH16 was 42°C.
Growth kinetics of B. subtilis 11A at different medium pH is presented in Figure 2. Previous research showed that the optimum pH for the growth of B. subtilis was at pH 7 [13] and for B. licheniformis at pH 6.5-7.5.  This study showed that B. subtilis 11A produced bacteriocin at the end of the log phase (10 th hour) and its activity reached the peak at the beginning of the stationary phase (12 th hour). The bacteriocin activity of B. subtilis 11A decreased in line with the decreased in bacterial growth . Antimicrobial activity of bacteriocin produced by Bacillus subtilis 11A is presented in Figure 3. Bacteriocin of B. subtilis 11A had the highest inhibitory activity to E. coli and S. typhimurium and the lowest activity to S. aureus. The inhibitory activity of bacteriocins against S. aureus, S. typhimurium and E. coli were 1,906.14; 2,179.79; and 2,343.07 AU/ml, respectively. This study showed the inhibitory activity of bacteriocin of B. subtilis 11A was more sensitive to Gram negative bacteria than to Gram positive bacteria.

Conclusion
This study showed the optimum conditions for the growth of B subtilis 11A isolated from the digestive tract of native chickens were at pH 6.5 and temperature 39°C. The highest bacteriocin activity was produced at the beginning of the stationary phase and was more sensitive to Gram negative pathogenic bacteria (E. coli and S. typhimurium) than Gram positive pathogenic bacteria (S. aureus).