Isolation and Characterization of Lactiplantibacillus plantarum Bacteriocin from Fruit-based Fermented Products and its Comparison with Commercial Nisin

Introduction Lactic acid bacteria (LAB) and their bacteriocins are widely used as natural and safe preservatives in food products, to control both pathogenic and spoilage microorganisms. This study aimed to isolate and identify LAB from several traditionally produced fermented fruits and vegetables from different parts of Iran, screening their potentials for producing bacteriocin-like substances production and evaluate their antimicrobial activities against various pathogens. The effect of heat treatment and different pH values on the stability of bacteriocins were also assessed and compared with commercial nisin for their possible application in the food industry as an alternative to chemical preservatives. Materials and Methods Lactic acid bacteria were isolated from several fermented products like hawthorn, mixed fruit pickles containing quince and apple, mango, and medlar. pickle, and tThe isolates were identified using phenotypic (physiological and biochemical) and genotypic (16S rDNA gene sequencing) methods followed by drawing phylogenetic tree based on the neighbor-joining method. The bacteriocins were prepared from the neutralized and cell-free supernatant (CFS). To precipitate the bacteriocins, ammonium sulfate (75%), potassium phosphate buffer, and methanol-chloroform were used, and extraction was completed with a high-speed centrifugecentrifugation. After freeze-drying, the precipitate was kept as crude bacteriocin. The bacteriocin activity was measured by the critical method, and the effect of heat, storage time and pH on the stability of bacteriocins was evaluated. The minimum inhibitory concentration (MIC) and the minimum inhibitory bactericidal concentration (MBC) of the examined bacteriocins were determined on against the pathogenic strains of Escherichia coli and Staphylococcus aureus and compared with commercial nisin. Results and Discussion In this research, from 162 isolated strains of LAB, four isolates (10A, S6, Sa, and Ab) were selected based on the highest amount of antimicrobial compounds and diameter of the inhibitory zone against pathogenic strains. then the isolates were identified as different strains of Lactiplantibacillus plantarum (previously classified as Lactobacillus plantarum). The phylogenetic position of the isolates was determined by drawing a phylogenetic tree. The drawn tree consists of two clusters and the first cluster consist of two sub-clusters, with two different strains of L. plantarum in each of them. In the next step, bacteriocin of the isolates was extracted using saturated ammonium sulfate and high-speed (23000g) centrifugingcentrifugation. Partially purified bacteriocins from different species showed high inhibitory effects on tested indicators, which were estimated, for L. plantarum 3360 (10A) and L. plantarum lb51 (Ab), 64000 AU/ml against Staphylococcus aureus and Escherichia coli. All selected bacteriocins indicated a stable effect at different temperatures of 60 and 121°C for 20 min and 4 and -20°C for 6 months, this effect was the examined bacteriocins were also stable against at acidic and alkaline pHs too. Also, the inhibitory property decreased under very acidic (pH < 3) and very alkaline (pH > 8) conditions, but this reduction was not significant at the 95% confidence level. Bacteriocins with 64000 AU/mL activity had higher antimicrobial properties against the pathogens compared to an equal amount of commercial NiseenNisin-S (680 AU/mL). The results of MIC and MBC showed that isolates 10A and Ab have the highest inhibitory properties compared to other extracted bacteriocins and/or nisin. Since heat and chemical preservatives are used in food preparation, the stability of bacteriocins against heat and different pH is important, therefore, after extraction and purification, the extracted bacteriocins can be used as a biological preservative in the production of various food products in the range of acidic and alkaline pH, including juices, meat products, and sauces. Encapsulation of these peptides and their application in food products needs further investigation.