ABSTRACT:

Food deterioration caused by microorganisms during the different stages of food handling has a great influence on food safety, shelf life and quality. The use of natural antimicrobials in place of classical antibiotics and chemical preservatives is gaining interest due to the challenge of resistance of microorganisms to antibiotics and the deleterious effects associated with the use of chemical preservatives. Lactic acid bacteria (LAB) produce bacteriocins which are of interest because they are active at nanomolar quantities, are nontoxic, and are readily available in fermented foods. Because of the high prevalence of foodborne infections in humans and the financial losses associated with food deterioration, the goal of this research was to isolate and identify LAB and test their bioactive qualities against food-borne bacteria.

Fermented food samples (Fufu, Garri, Kunu, Nono and Ogi), which were used for the research were purchased from local vendors in Benin City, Edo State and in Oshodi, Lagos State. LAB were isolated from these food samples using De Mann, Rogosa and Sharpe (MRS) agar. Cell-free supernatants (CFS) obtained from 18 - 24 hour LAB cultures grown on MRS broth were subjected to agar well diffusion assay against potential foodborne pathogenic and spoilage bacteria. The test organisms were Bacillus cereus, Salmonella typhimurium, Staphylococcus aureus, Leuconostoc mesenteroides, Klebsiella pneumoniae, Bacillus tequilensis, Achromobacter xylosoxidans and Achromobacter insolitus. The effect of organic acid was removed by changing the pH of the supernatants to 7.0 with 1M NaOH, and the effect of hydrogen peroxide was removed by using catalase enzyme. The supernatant was then filter-sterilized using a membrane filtration device with a 0.2 μm pore size millipore filter and tested against foodborne antibiotic-resistant pathogenic bacteria isolated from meat pie and spoilage bacteria isolated from abacha, using an agar well diffusion assay. The antibacterial effectiveness of the bacteriocin was compared with that of green synthesized Zinc Oxide nanoparticles ZnONPs. The selected strain of LABwas subjected to different culture conditions to determine the optimum conditions for growth and bacteriocin production in MRS broth. The molecular weight of purified bacteriocin was estimated using Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to characterize the bacteriocins. All the isolates obtained in this study were identified using phenotypic and genotypic characterization. The sequences of each strain's 16S rRNA gene were compared, and the strains with the highest similarity were used to construct the phylogenetic tree.

A total number of 162 LAB isolates were isolated from the fermented food samples. The antibacterial sensitivity test of the LAB against Staphylococcus aureus ATCC 25923, yielded positive result for 45 LAB isolates while the growth of Escherichia coli ATCC 25922 was suppressed by 52 LAB isolates.The selected pure LAB culture showed optimal growth with optimal bactericidal protein production in MRS broth at pH 5.0 and 2.5 % NaCl when cultures were incubated at 40 °C.The CFS obtained from these LAB isolates gave a zone of inhibition (ZOI) ranging from 9.67 ± 0.88 mm to 21.33 ± 1.76 mm against potential pathogenic strains of Leuconostoc mesenteroides, Klebsiella pneumonia, Salmonella typhimurium and Bacillus cereus isolated from meat pie. Similarly, the effect of the LABbacteriocin on spoilage bacteria associated with Abacha wasevaluated using agar well diffusion assay. The bacteriocinogenic LAB identified as Limosilactobacillus pontis strain EOINONO, Limosilactobacillus pontis strain OGENONO, Limosilactobacillus pontis strain SEOGARI, Lactiplantibacillus plantarum strain MJIKUNU and Limosilactobacillus pontis strain EEIKUNU were observed to impede the growth of the food spoilage bacteria. The spoilage organisms were identified as Bacillus tequilensis strain SEOABACHA, Bacillus tequilensis strain EEIABACHA, Achromobacter xylosoxidans strain IMABACHA and Achromobacter insolitus strain MJIABACHA. There was no significant difference in the zone of inhibition produced by bacteriocin against the test isolates compared to the zone of inhibition produced by ZnONPs. Therefore, it portends that the bacteriocins produced by the LAB isolated from these food products could act as probiotics for effective inhibition of the growth of antibiotic-resistant foodborne pathogenic and spoilage organisms.