Neda Nayyeri; Mohammad Reza Edalatian Dovom; Mohammad Bagher Habibi Najafi; Masoumeh Bahreini
Abstract
Introduction : Nowadays, attention to foods free from chemical preservatives is on the rise. Recently, consumers have concerned about foods containing these preservatives in their formulation .Therefore,the use of antimicrobial peptides produced by Lactic acid bacteria (LAB) are strongly highlighted. ...
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Introduction : Nowadays, attention to foods free from chemical preservatives is on the rise. Recently, consumers have concerned about foods containing these preservatives in their formulation .Therefore,the use of antimicrobial peptides produced by Lactic acid bacteria (LAB) are strongly highlighted. Ribosomally-synthesized peptides, which possess antimicrobial properties, are produced by a vast range of organisms including prokaryotes and Eukaryotes.Yeasts are the most important microorganisms which are responsible for fruit juice and soft drink spoilage. In case of growth and production of by-products like CO2, acid and other contaminants, the spoilage will be visible.Lactic acid bacteria isolated from natural, local sources such as dairy products have presented potentially antifungal features against food spoilage fungi. Among these, Rodotorula and Penicillium are regarded as the most critical genera in fruit juice spoilage.The main objective ofthis study was the evaluation of anti-yeast activity of Lactobacillus plantarum isolates from different stages of Lighvan production against Rodotorulamucilaginosa as fruit juice spoilage indicator. In the next step, technological parameters effects were analyzed on antimicrobial potential of cell-free supernatant of these isolates. Finally, we aimed at finding the most effective isolate on aforementioned yeast.Materials and methods: NineteenLb.plantarumisolates, which were identified previously, were subjected to antifungal assay. For this purpose, RodotorulamucilaginosaPTCC 5257 was selected. Agar spot and Well Diffusion Assay (WDA) were applied for antifungal assay in solid and liquid media, respectively.Determination of yeast colony: Following the cultivation of yeasts in Potato Dextrose Broth, it was determined using Spectrophotometer.Preparation of Lb.plantarum cell-free supernatant (CFS) was carried out. In agar spot method, clear zones of inhibition around the spotted colonies were evaluated after 24-48h incubation. In WDA, CFS of Lb.plantarum isolates were poured in wells and clear zones were evaluated around each well after 24-48 h incubation.Technological properties: The influence of different levels of pH (2, 3, 4, 5, 6, and 7) was analyzed on CFS of Lb.plantarum isolates. This assay was done according to Wang et al., 2011. Finally, results were reported using the measurement of clear zone diameter in mm. All experiments were performed in duplicates.The effects of various temperatures were applied on CFS and remaining the antifungal activity was evaluated according to Rouse et al., 2008. Finally, all CFS of Lb.plantarum isolates were subjected to Proteniase K and antifungal properties of CFS were assayed by WDA according to Ghrairi et al., 2005.Results and discussion:Agar spot results showed the highest and lowest clear zone of inhibition related to C28 and LF49 with 16 and 6 mm diameter, respectively. In this method, 11 out of 19 isolates (60%) presented anti-yeast activity with clear zone formation. In comparison between two incubation temperatures (25 and 30◦C), all isolates stronger clear zone in 30◦C than in 25◦C. This was due to enhancement of yeast growth in 25C rather than in 30C. Also, with respect to the mesophilic nature of Lb.plantarum isolates, the possibility of metabolite production are more likely in 30C. It was reported that antifungal activity of LAB is mostly due to synergistic effect of lactic acid and acetic acid. In agar spot, some colony-associated antimicrobial compounds are responsible for antifungal activity. In WDA, 8 out of 19 isolates (42%) were positive for their inhibitory effects. The highest anti-yeast activity was seen at pH=2. It seems that antimicrobial compounds are likely more stable at acidic conditions than at alkaline ones. Among isolates, C28 presented the highest stability at alkaline conditions. With pH increase, the antifungal activity decreased so that no anti-yeast activity was seen at pH=7. Regarding the different temperatures, we should mention that thermal resistance of isolates' CFSwitnessed declining trend with increasing of temperature. This fact implies the presence of low- molecular weight compounds in CFS. Finally, all isolates' CFS was subjected to proteinase K. All isolates have lost their anti-yeast activity after enzyme treatment showing their proteinaceous nature.Conclusion: In WDA, the number of positive isolates showing anti-yeast activity declined in comparison with agar spot. Since some isolates retain their inhibitory activity toward food spoilage yeast at low pH, their CFS can be applied in acidic foods like fruit juice. Also, some isolates showed their antifungal activity at high temperatures (80C and 100C) which are applied for fruit juice pasteurization, so they can be applied in fruit juices as a bio-preservative.
Mohammad Reza Edalatian Dovom; Masoud Yavarmanesh; Fariba Ghiamati Yazdi; Morteza Khomeiri; Neda Nayyeri
Abstract
Introduction : 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 ...
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Introduction : 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.Materials and methods: 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 & Results: 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.Conclusion: 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.
