Research Journal of Recent Sciences _________________________________________________ ISSN 2277-2502 Vol. 4(ISC-2014), 173-176 (2015) Res. J. Recent. Sci. International Science Congress Association 173 Antagonistic Effect of Lactobacillus Isolates from cow Milk on selected Pathogenic Bacteria Chundawat Hemant and Joshi Harshada* Microbiology Laboratory, Department of Biotechnology, Vigyanbhawan, Block B, Mohanlalsukhadia University, Udaipur, Rajasthan, INDIAAvailable online at: www.isca.in, www.isca.me Received 26th November 2014, revised 7th February 2015, accepted 25th February 2015 AbstractThe viable microbial food supplements which beneficially influence the health of humans are known as probiotics. Antibacterial activity is one of the important probiotic properties for a strain to be a functional probiotic.The three Lactobacillus strains namely Lactobacillus plantarum CM1, Lactobacillus fermentum CM4 and Lactobacillus casei subsp. casei CM6 were isolated from cow milk samples collected from Udaipur city. Their antagonistic activity was tested against five pathogenic bacteria such as Serratia marcescens NCDC 108, Enterobactor aerogens NCDC 106, Proteus vulgaris NCDC 73, Pseudomonas aeruginosa NCIM 5029, and Micrococcus luteus NCDC 131 using agar well assay method. The results showed that the Lactobacillus strains namely Lactobacillus plantarum CM1, Lactobacillus fermentum CM4 and Lactobacillus casei subsp. casei CM6 were able to inhibit the growth of all the pathogenic bacteria in varying degrees. All the three Lactobacillus isolates were found to be the most sensitive against Micrococcusluteus than the rest of the pathogenic bacteria used in the study. The inhibition observed in the case of all the isolates that have antagonistic effect may be due to the production of organic acids as well as other compounds such as bacteriocins, hydrogen peroxide etc.Keywords: Probiotics, Lactobacillus isolates, antagonistic activity, pathogenic bacteria. Introduction Recent concerns on the rampant and indiscriminate use of antibiotics for disease treatments and growth promotion of livestock and the development of antibiotic resistant pathogens have led to increased interest in the application of probiotics and their antimicrobial metabolites as alternative antimicrobial strategies for treatment and prevention of infections. Hence, antimicrobial activity against pathogens is a desirable property of a potential probiotic strain. Probiotics are defined as ‘Live microorganisms, which when administered in adequate amounts confer a health benefits to the host. Lactobacilli are known as probiotic bacteria due to beneficial health effects exerted by them. Lactobacilli have antimicrobial activity because of the production of organic acids (lactic and acetic acid), diacetyl, hydrogen peroxide, and bacteriocins e.g. acidocin, acidophilin, lacticin and nicin. Many researchers have been determined the antagonistic activity of lactobacilli against gram-positive and gram-negative pathogenic bacteria3,4. The present study was conducted to determine the antagonistic activity of Lactobacillus isolates against some pathogenic bacteria. Material and Methods The materials and methods used in the present study were as follows: Maintenance and preservation of cultures: Lactobacillus strains: Lactobacillus isolates used in this study were isolated from cow milk samples, collected from various street venders and local dairies in Udaipur city. The isolates were stored in litmus milk at 10ºC. Cultures were activated in MRS broth with incubation at 37ºC for 24 h. Pathogenic cultures: The pathogenic cultures used in this study were Serratia marcescens NCDC 108, Enterobactor aerogens NCDC 106, Proteus vulgaris NCDC 73, Pseudomonas aeruginosa NCIM 5029, and Micrococcus luteus NCDC 131All the strains were obtained from National Dairy Research Institute, Karnal and NCIM, Chandigarh. The cultures were maintained on nutrient agar slant and activated in nutrient broth at 24h interval. Detection of antagonistic activity: The isolates were screened for the antibacterial substance by inoculating active culture into MRS broth and incubating overnight at 37C. The stationary cells were centrifuged at 3000 rpm for 20 min. The culture supernatants neutralized with and without 1N NaOH were tested for antibacterial activities against different pathogenic bacteria. For determining the antibacterial activity of the isolates against the potential pathogenic bacteria agar well assay was used. Finally the diameter of inhibition zone extending laterally around the well was measured with scale. The inhibition zone of more than 1 mm diameter was considered as positive inhibition.Results and Discussion Result: The antibacterial activity was determined in the two fractions of the cell free supernatants: normal cell supernatant Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 4(ISC-2014), 173-176 (2015) Res. J. Recent. Sci. International Science Congress Association 174 and cell supernatant neutralized with 1N NaOH. The normal cell supernatants of the three isolates showed higher antibacterial activities than the supernatants neutralized with NaOH. The bacterial supernatants without NaOH of all the three Lactobacillus isolates Lactobacillus plantarum CM1, Lactobacillus fermentum CM4 and Lactobacillus casei subsp. casei CM6 showed highest inhibition zones against gram-positive indicator organisms (table 1 and figure 1). The sizes of inhibition zones obtained for Lactobacillus plantarum CM1, Lactobacillus fermentum CM4 and Lactobacillus casei subsp. casei CM6 against Micrococcus luteus were 19.5, 26.0 and 24.00 mm respectively. For Lactobacillus plantarum CM1 the sizes of inhibition zones against rest of the indicator organisms were ranged from 14.0 to 16.0 mm. For Lactobacillus fermentum CM4 and Lactobacillus casei subsp. casei CM6 the sizes of inhibition zones against rest of the indicator organisms were ranged from 9.5 to 18.0 mm and 11.0 to 20 mm respectively. Table-1 Antibacterial activities of Lactobacillus isolates against different pathogenic bacteria (Without NaOH) S. No. Name of organism Diameter of clear zone (mm) including well diameter Serratia marcescensEnterobacter aerogenesProteus vulgarisPseudomonas aerouginosaMicrococcus luteusMean 1. Lactobacillus plantarum CM1 16.0 14.0 15.0 16.0 19.5 16.1 2. Lactobacillus fermentum CM4 10.5 14.0 09.5 18.0 26.0 15.6 3. Lactobacillus casei subsp. casei CM6 13.0 15.0 11.0 20.0 24.0 16.6 4. Mean 13.167 14.333 11.833 18.0 23.167 16.1 CRD ANOVA for Antibacterial activities of Lactobacillus isolates against different pathogenic bacteria (Without NaOH) S. No. Source DF SS MS F SE(m) CD(5) CD(1) 1. A 2 0.0499909 0.0249954 2.777 0.03 0.09043 0.1251 2. B 4 5.00866 1.25217 139.130** 0.03873 0.1167 0.1615 3. A x B 8 1.19334 0.149167 16.574** 0.06708 0.2022 0.2797 4. Error 15 0.134999 0.00899994 CV = 5.8924, A = Lactobacillus isolates, B = Indicator bacteria, *, ** Significant at 5 and 1 percent level of significance Figure-1 Antibacterial Activity of Lactobacillus Isolates against Different Pathogenic Bacteria (Without NaOH) Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 4(ISC-2014), 173-176 (2015) Res. J. Recent. Sci. International Science Congress Association 175 The bacterial supernatants with NaOH of all the three Lactobacillus isolates Lactobacillus plantarum CM1, Lactobacillus fermentum CM4 and Lactobacillus casei subsp. casei CM6 showed highest inhibition zones against Micrococcus luteus (table 2 and figure 2). The sizes of inhibition zones obtained for Lactobacillus plantarum CM1, Lactobacillus fermentum CM4 and Lactobacillus casei subsp. casei CM6 against Micrococcus luteus were 15.5, 21.0 and 14.0 mm respectively. For Lactobacillus plantarum CM1 the sizes of inhibition zones against rest of the indicator organisms were ranged from 10.0 to 12.0 mm. For Lactobacillus fermentum CM4 and Lactobacillus casei subsp. casei CM6 the sizes of inhibition zones against rest of the indicator organisms were ranged from 11.0 to 13.5 mm and 9.0 to 12.5 mm respectively. Table-2 Antibacterial activities of Lactobacillus isolates against different pathogenic bacteria (With NaOH) S. No. Name of organism Diameter of clear zone (mm) including well diameter Serratia marcescensEnterobacter aerogenesProteus vulgarisPseudomonas aerouginosaMicrococcus luteusMean 1. Lactobacillus plantarum CM1 10.5 11.5 10.0 12.0 15.5 11.9 2. Lactobacillus fermentum CM4 12.0 11.0 13.5 11.0 21.0 13.7 3. Lactobacillus casei subsp. caseiCM6 11.5 10.5 09.0 12.5 14.0 11.5 4. Mean 11.333 11.000 10.833 11.833 16.833 12.37 CRD ANOVA for Antibacterial activities of Lactobacillus isolates against different pathogenic bacteria (With NaOH) S. No. Source DF SS MS F SE(m) CD(5) CD(1) 1. A 2 0.274669 0.137335 27.466** 0.0224 0.0674 0.09325 2. B 4 1.53134 0.382834 76.565** 0.02887 0.08702 0.1204 3. A x B 8 0.548664 0.0685831 13.716** 0.05 0.1507 0.2085 4. Error 15 0.0750017 0.00500011 CV = 5.7179, A = Lactobacillus isolates, B = Indicator bacteria, *, ** Significant at 5 and 1 percent level of significance Figure-2 Antibacterial Activity of Lactobacillus Isolates against Different Pathogenic Bacteria (With NaOH) Research Journal of Recent Sciences ______________________________________________________________ ISSN 2277-2502Vol. 4(ISC-2014), 173-176 (2015) Res. J. Recent. Sci. International Science Congress Association 176 Discussion: Antibacterial activity is one of the important properties for a strain to be a functional probiotic. The production of antimicrobial compounds by a probiotic organism provides a competitive edge to their survival and proliferation in GI tract and can also help in eradication of pathogenic bacteria. A variety of antimicrobial compounds such as organic acids, hydrogen peroxide, ethanol, acetaldehyde, acetoin, fatty acids, low molecular mass compounds and bacteriocins6 are produced by lactobacilli. In the present study both the bacterial supernatants normal supernatant and supernatant neutralized with NaOH of all the three Lactobacillus isolates showed the demonstrable antibacterial activities against both gram-positive and gram-negative pathogenic bacteria (table3.1 to 3.2). This observation showed stronger agreement with the report which reveals that Lactobacillus strains showed antibacterial activities against gram-positive as well as negative bacteria. All the three Lactobacillus isolates showed broad inhibitory spectrum against both gram-positive and gram-negative bacteria8,9. The bacterial supernatant without NaOH of all the three strains showed stronger activity as compare to that of neutralized supernatant. Similar findings were also reported byresearcher who proposed that the weak antibacterial activities in neutralized supernatant may be due to the other factors except acid and may be due to the production of bacteriocins that are active against pathogenic organisms at the optimum pH10. This may be the possible reason for antibacterial activities in neutralized supernatant in the present study. Conclusion As the antagonistic activity is one of the crucial property for a strain to be a probiotic. All the three Lactobacillus isolates showed demonstrable antibacterial activity. These Lactobacillus isolates could be used as potent probiotics. Further research related to the evaluation of these isolates for antibacterial compounds will be conducted. 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