Evaluation of antimicrobial activities of lactic flora isolated from production stages of Maskeh against food indicator bacteria

Lactic acid bacteria (LAB) are a group of gram positive, catalase negative bacteria which possess unique metabolic, physiological and morphological characteristics. In addition, lactic acid bacteria are considered as a valuable source of antimicrobial agents including bacteriocins. Bacteriocins are defined as non-toxic peptides produced by LAB. "Maskeh" as an Iranian traditional butter carries indigenous LAB which secrete and harbor bacteriocins. Our objective in this study was to evaluate the influence of antimicrobial compoundsproduced by isolated LAB from Maskeh, a local traditional butter, against some pathogenic bacteria suchas: staphylococcus aureus, Listeria innocoa and E. coli using culture-based methods. In the following step, influence of some technological parameters including different temperatures, pHs and proteinase K were determined on stability of antimicrobial compounds in bacteriocins.
Three samples of milk, yoghurt and local butter known as Maskeh were collected from different regions in the south ofKhorasanRazavi, Gonabadcity.
Indicator microorganisms and culture condition: In order to activate the indicators, following the cultivation in corresponding and suitable media, they were incubated for 24 hours in suitable temperatures.
Survey of antimicrobial activities: In Agar Spot, Antagonistic activities of isolates were evaluated in solid media. Finally clear zone of inhibition was measured in mm. In Well- Diffusion Assay (WDA), positive isolates from previous step, were selected for this assay. In this method, cell-free supernatant (CFS) of isolates were examined for their antagonistic activities. Finally clear zone of inhibition were evaluated around the wells.
Determination of antimicrobial compound nature: Effect of proteinase K on CFS: In order to evaluate the enzyme sensitivity of bacteriocin like compounds produced by LAB, CFS of isolates were subjected to proteinase K (final concentration 0.5 mg/ml). Mixture of enzyme- CFS was incubated in 37C for four hours. Finally, the remaining inhibitory activity was determined using WDA.
Influence of different temperatures on CFS: Heat sensitivity of bacteriocin like compounds was evaluated with subjecting the CFS of isolates to various temperatures. Again remaining activity of isolates was evaluated with the help of WDA.
Influence of different levels of pH on CFS: CFS of isolates was adjusted at different pHs and their inhibitory effects were determined using WDA.
Discussion & Following the sequencing, 51 isolates were identified from different steps of Maskeh production. Results showed that 44 out of 51 isolates were effective at least against one indicator. It was clear that E.coli, Staph.aureus, Listeria. innocoa, Lb.plantarum, Lb.sake, Lac.lactis ssp. lactis,Lac.lactis ssp. cremoris were inhibited by 33, 7,11,12,7,35 and 7 isolates, respectively. Among pathogenic indicator bacteria, E.coli was inhibited maximally and regarding the non-pathogenic indicators, Lac.lactis ssp. Lactis was inhibited by the most of the isolates. Among the isolates, Ent.faecium B161 and Str.thermophilus B258 presented the highest inhibitory effect against Listeria.innocoa.Interestingly both these strains had been isolated from Maskeh, implying that they originated the same source. Formation of clear inhibition zone in agar spot method is related to colony-associated antimicrobial compounds like H2O2, lactic acid and other organic acids. Also, the strongest clear zone of inhibition was against Listeria.innocoa. Lb.plantarum B120 showed inhibitory effect against 6 out of 7 indicator bacteria. This strain has been isolated from Maskeh, and only did not affect on Lac.lactis ssp. lactis.In the next step, in WDA, CFS obtained from isolates were subjected to inhibition activity evaluation. In this assay, 39 isolates showed inhibitory activity against at least one indicator and 12 isolates had no inhibition against indicators. E.coli was inhibited by the most of the isolates (11), followed by Listeria.innocoa by 8 and Staph.aureus by 2 isolates. But noticeable point is that the strongest influence was seen against Listeria. In the last step, influence of technological parameters such as : temperature, pH and enzyme were determined on antimicrobial and inhibitory activity of CFS. In this survey, only those isolates were chosen which showed inhibitory effect against Listeria innocoa. Regarding the temperature, with increasing of this factor, the diameter of clear zone had decreasing trend. This means that bacteriocin- like compounds are sensitive to heat. Among analyzed isolates, CFS of two isolates namely Aerococcusviridans M156 and M141 possess the highest sensitivity. In contrast, the highest resistance was related to Aerococcusviridans M165. Evaluation of antagonistic activity of CFS of isolates at different pHsexperienced, in pH between 3-5, growing trend of inhibitory activity which is due to produced lactic acid. Maximum of antagonistic activity was seen at pH=3 and along with pH increase toward the alkaline condition, it decreased.Aerococcusviridans M165 and M141 showed clear zone of inhibition equal to 6.5 and 6, respectively at pH=7.Proteniase K as a proteolytic enzyme, exhibited decomposing influence on antimicrobial effects of all isolates, except two of them. This phenomenon implies the proteinaceous nature of antimicrobial compound in CFS, because of decomposition as a result of proteolytic enzyme. But regarding two isolates, the clear zone did mot destroyed even after enzyme treatment.
Some CFS or their producing isolates can be exploited as bio-preservatives; CFS of Aerococcusviridans M 165 can be applied in foods subjected to high heat treatment. In acidic and low pH food, we can use those isolates which theirCFS remain their activity at low pH.