International Journal of Research and Review Vol.7; Issue: 1; January 2020 Website: www.ijrrjournal.com Research Paper E-ISSN: 2349-9788; P-ISSN: 2454-2237

Antibacterial Activity Test of plantarum Bacteriocin Isolated from Dadiah

Sura Aisyah Tampubolon1, Netti Suharti2, Edison3, Andani Eka Putra2

1Master of Biomedical Science Program, Faculty of Medicine, Universitas Andalas, Padang City, 2Department of Microbiology, Faculty of Medicine, Universitas Andalas, Padang City, Indonesia 3Department of Public Health and Community Medicine, Faculty of Medicine, Universitas Andalas, Padang City, Indonesia

Corresponding Author: Sura Aisyah Tampubolon

ABSTRACT traditional food that is served at weddings, inaugurations of marriage and other Background: Dadiah is a traditional food in traditional processions. Prizes are obtained , Indonesia which is obtained from from the results of the buffalo the buffalo milk fermentation process. The aim fermentation process which is inserted into a of this study to determine antibacterial activity tube and covered with banana test of bacteriosin insulated from dadiah. leaves for one to two days. This Methods: This research was an experimental of fermentation process will produce dadiah completely randomized design with determined which is milky white with a slippery texture, [1] minimum inhibitory concentration and and has a sour taste and distinctive aroma. minimum bactericidal concentration which Dadiah contains (LAB) determined by using dilution method in some which is the group of bacteria that most concentrations of 100%, 80%, 60%, 40%, 20%, produce compounds called bacteriocin. [2] and 0%. Identification of bacteriocin from Bacteriocin compounds from LAB obtained Lactobacillus plantarum isolates was carried out from dadiah is known to have antibacterial using the sodium dodecyl sulfate activity so that it has the potential to be a polyacrylamide gel electrophoresis (SDS- natural alternative antibiotic to protect PAGE) method. Results: The results of the study known that humans from the effects of resistant microbes that cause disease without causing percentage of Bacteriocin of minimum [3] inhibitory concentration from Lactobacillus health problems. plantarum against P. aeruginosa was 98.57% Bacteriocin is an antimicrobial (p<0.001). Meanwhile, the results of minimum peptide produced by LAB during the bactericidal concentration cannot be identified. exponential growth phase. The large amount Our result showed that the bacteriocin protein of bacteriocin produced can kill or inhibit band was in the range of 17-28 kDa. other bacteria that compete in the same Conclusion: This analysis confirmed that the ecology. [4] Bacteriocin can inhibit the bacteriocin compound from Lactobacillus growth of pathogenic microbes and has the plantarum had a bacteriostatic effect on P. potential to be used as an antimicrobial in aeruginosa. the food industry. As an synthesis agent, Keywords: Dadiah, Lactobacillus plantarum, bacteriocin in biopreservatives also Bacteriocin, Pseudomonas aeruginosa contributes greatly to the health sector as a natural antibiotic agent and a [5] INTRODUCTION food. Dadiah is a traditional food from Previous study known cell-free West Sumatra buffalo milk which is a filtrate from LAB indigenous dadiah isolates (L. plantarum D-0 and L. Lactis D-

International Journal of Research and Review (ijrrjournal.com) 159 Vol.7; Issue: 1; January 2020 Sura Aisyah Tampubolon et al. Antibacterial activity test of lactobacillus plantarum bacteriocin isolated from dadiah

01) are proven to produce antimicrobial concentration which determined by using substrates that can inhibit the growth of dilution method in some concentrations of indicator pathogenic bacteria such as S. 100%, 80%, 60%, 40%, 20%, and 0%. enteritidis ser. Typhimurium ATCC 14028, Identification of bacteriocin from E. coli ATCC 25922 and S. aureus ATCC Lactobacillus plantarum isolates was carried 25923 indicated by the clear zone of out using the sodium dodecyl sulfate inhibition in the well diffusion polyacrylamide gel electrophoresis (SDS- method.[6] Antoher study it was explained PAGE) method. Bacteriocin filtrate was put that bacteriocin from Lactobacillus sp. 1223 into each test tube containing a SCG isolated from fresh cow's milk can predetermined volume of nutrient broth and inhibit the growth of pathogenic bacteria 0.1 ml bacterial suspension. Each treatment such as Salmonella thypimurium and E. combination amounts to 2.1 ml for 100% of coli.[7] Other research showed that L. casei the mixture. The lowest concentration that has antimicrobial activity (positively can inhibit more than 90% growth of inhibits Escherichia coli, Staphylococcus pathogenic bacterial colonies after 24-hour aureus, and Enterococcus faecalis). L. casei incubation is determined as the MIC value. which is a local isolate has characteristics While the minimum bactericidal that have the potential to be used as concentration value is determined by probiotic microbes. [8] assessing the lowest concentration of the Based on studies that have been filtrate which shows no growth of done, it is not yet known about the pathogenic bacteria. The quantitative antibacterial activity of bacteriocin from variables were recorded as mean and Lactobacillus plantarum obtained from standard deviation. Hypothesis test used dadiah against P. aeruginosa so that in this one-way anova. A two-tailed P-value of study testing the antibacterial activity of <0.05 was considered statistically Lactobacillus plantarum bacteriocin was significant. Data were analyzed using the isolated from dadiah against Pseudomonas SPSS version 21.0. aeruginosa. RESULT MATERIALS & METHODS In the results of this study showed This research was an experimental bacteriocin molecular weights ranged from of completely randomized design with 17 to 28 kDa (indicated by a red circle given determined minimum inhibitory an arrow). concentration and minimum bactericidal

Figure 1: Bacteriocin molecular weights

International Journal of Research and Review (ijrrjournal.com) 160 Vol.7; Issue: 1; January 2020 Sura Aisyah Tampubolon et al. Antibacterial activity test of lactobacillus plantarum bacteriocin isolated from dadiah

The effect of bacteriocin filtrate culture on The tape produced in the acrylamide gel via Pseudomonas aeruginosa can be seen in SDS-PAGE is a single protein. [9,10] Table 1. In this study, testing the effect of L. plantarum bacteriocin filtrate culture on P. Table 1: Antibacterial activity of bacteriocin against some P. aeruginosa. Bacteriocin L. Plantarum has aeruginosa bacteria Sample Concentration Average of P. aeruginosa p value an influence on the growth of P. aeruginosa (%) (log 10 cfu/ml) bacteria. This is because bacteriocin has an 100 5.041 80 5.848 antibacterial role. The results of 60 6.207 <0.001* antibacterial test from bacteriocin showed 40 8.153 20 9.633 that the smaller the bacteriocin Control (0) 10.021 concentration, the lower the ability of *p<0.001, significant bacteriocin to inhibit bacterial growth. The results of this study showed that L. The results showed the average number of plantarum bacteriocin filtrate isolated from P. aeruginosa bacteria at a concentration of dadiah did not have a bactericidal effect on 100% ie found 1.1 × 105 CFU/ ml, 80% 5 P. aeruginosa bacteria. This is because the concentration found 7.05 × 10 CFU/ ml, growth of P. aeruginosa bacteria is still 60% concentration found 1.61 × 106 CFU/ 8 found. ml, 40% concentration found 1.422 × 10 Bacteriocin is a metabolite CFU/ ml, 20% concentration found 4.3 × (extracellular) in the form of a protein that is 109 CFU/ ml and in the control there was 9 synthesized directly in the ribosome of a 1.05 × 10 CFU/ ml. In this study the bacterium and has a broad spectrum in minimum inhibitory concentration (MIC) inhibiting the growth of pathogenic bacteria. was determined from a decrease in the Bacteriocin can work on target bacteria in number of bacterial colonies to 90% of two stages, firstly through absorption on the controls. From the results of this study surface of bacterial cells, and secondly, found a decrease in the number of bacterial lethal effects through: (i) bacteriostatic colonies of more than 90% at a effect; (ii) bactericidal effect without cell concentration of 60% to 100% compared to lysis or (iii) with cell lysis. The mechanism controls. Conversely, at a concentration of of action of bacteriocin compounds against 20% to 40% a decrease in the number of bacteria is generally known through the bacteria is less than 90%. In the results of bond between bacteriocin and bacterial cell this study it was found that L. plantarum wall or membrane which can cause bacteriocin did not have a minimum instability in the cell membrane. This results bactericidal concentration against P. in the formation of holes or pores in aeruginosa. The MIC of bacteriocin L. bacterial cell membranes. [11] The formation plantarum against P. aeruginosa was of these pores will increase membrane determined at a concentration of 40%. permeability, disrupt the function of proton motive force (PMF) and cause the loss of DISCUSSION intracellular material from bacteria. The Determination of bacteriocin disruption of PMF will cause disruption in molecular weight is done through the SDS- the formation of energy in bacterial cells, PAGE method using acrylamide gel. The resulting in stunted cell growth and lead to function of determining bacteriocin bacterial cell death. [12] molecular weight with SDS-PAGE is to Most of the bacteriocins produced separate the purity of a protein. The protein by LAB are cationic (because they contain obtained is likely to be contaminated by excess lysil and arginil residues) composed molecular weights originating from the of 12-45 amino acids. The electrostatic same molecule, so the SDS-PAGE method interaction of positively charged and must be used to test the purity of a product. hydrophobic bacteriocin with a negatively

International Journal of Research and Review (ijrrjournal.com) 161 Vol.7; Issue: 1; January 2020 Sura Aisyah Tampubolon et al. Antibacterial activity test of lactobacillus plantarum bacteriocin isolated from dadiah charged phosphate group on the target cell 5. Sunaryanto R, Marwoto B. Isolasi, membrane results in the binding of Identifikasi, dan Karakterisasi Bakteri bacteriocin to the target membrane and this Asam Laktat dari Dadiah Susu Kerbau. is the initial stage of bacteriocin to initiate Jurnal Sains dan Teknologi Indonesia. cell wall damage. After bonding with the Balai Pengkajian Bioteknologi BPP target membrane absorption of helical Teknologi. 2012;14(3):228-233. structures from bacteriocin to the cell 6. Aslam M, Shahid M, Rehman FU, membrane through its hydrophobic part, Naveed NH, Batool AL, Sharif S, Asia while the hydrophilic part is exposed to A. Purification and characterization of solvents. When the bacteriocin bacteriosin isolated from Streptococcus concentration threshold is reached, the thermophilus. African Journal. 2011; peptide will unite and form a trans 5(18):2642-2648. membrane membrane, with the hydrophobic 7. Sankar NR, Priyanka VD, Reddy PS, part facing outward, and the hydrophilic Rajanikanth P, Kumar VK,Indira M, et part facing inward, thus forming a pore on al. Purification and characterization of the membrane. Pore formation causes the bacteriocin produced by Lactobacillus loss of intracellular molecules and the entry plantarum Isolated from cow milk. Int J of extracellular molecules, this causes Microbiol Res 2012;3(2):133-137. changes in cellular pH. These events affect 8. Barbosa MS, Todorov SD, Ivanova IV, the growth of pathogenic bacterial cell et al. Characterization of a two-peptide growth and can cause the death of bacterial plantaricin produced by Lactobacillus cells that are sensitive to bacteriocin. [13,14] plantarum MBSa4 isolated from Brazilian salami. Food Control. 2016; CONCLUSION 60:103–12. The conclusion of this study confirmed that 9. Hernandes D, Cardell E, Zarate V. the bacteriocin compound from Antimicrobial activity of lactid acid Lactobacillus plantarum had a bacteriostatic bacteria isolated from Tenerife cheese: effect on P. aeruginosa. initial characterization of plantaricin TF711, a bacteriocin-like substance REFERENCES produced by Lactobacillus plantarum 1. Neetles CG, Barefoot SF. Biochemical TF711. Journal of Applied and genetic characteristic of bacteriocins Microbiology. 2005;99:77 – 84 of food-associated lactic acid bacteria. J 10. Gong HS, Meng XC, Wang H. Mode of Food Prot. 1993; 56:338-356 action of plantaricin MG, a bacteriosin 2. Savadogo A, Ouattara CAT, Basssole active against Salmonella typhimurium. IHN, Traoer SA. Bacteriocins and lactic Journal of Basic Microbiology. 2010; acid bacteria- a minireview. Afr J 50:37– 45. Biotechnol. 2006; 5:678–683. 11. Maunatin A, Khanifa. Uji potensi 3. Vasiljevic T, Shah NP. -From probiotik Lactobacillus plantarum secara Metchnikoff to bioactives. Int Dairy in-vitro. Alchemy. 2012;2(1):26-3 Journal. 2008;18:714-728. 12. Cleveland J, Montville TJ, Nes IF, et al. 4. Martins IM, Corte JCG, Munoz J, Bacteriocins: safe, natural antimicrobials Moreno B, Ramos M, Clemente JA, for food preservation. Intern J Food Duran A, Ribas JC. Differential Microbiol. 2007;71:1–20. Activities Of Three Families Of Specific 13. Arief IS, Budiman C, Jenie BS. β (1,3) Glucan Synthase Inhibitors in Plantaricin IIA-1A5 from Lactobacillus Wild-type and Resistant Strains of plantarum IIA-1A5 displays bactericidal Fission Yeast. The Journal of Biological activity against Staphylococcus aureus. Chemistry. 2011; 286(5): 3484-3496 Beneficial Microbes. 2015;6(4):603–13.

International Journal of Research and Review (ijrrjournal.com) 162 Vol.7; Issue: 1; January 2020 Sura Aisyah Tampubolon et al. Antibacterial activity test of lactobacillus plantarum bacteriocin isolated from dadiah

14. Elayraja S, Ennamalai N, Mayavu P, Balasubramanian T. Production, How to cite this article: Tampubolon SA, purification, and characterization of Suharti N, Edison, Putra AE. Antibacterial bacteriocin from Lactobacillus murinus activity test of lactobacillus plantarum AU06 and its broad antibacterial bacteriocin isolated from dadiah. International spectrum. Asian Pac J Trop Biomed Journal of Research and Review. 2020; 7(1): 159-163. 2014; 4(Suppl 1):305-311.

******

International Journal of Research and Review (ijrrjournal.com) 163 Vol.7; Issue: 1; January 2020