Phenotypic and Genotypic Characterization of Bacteriocin-Producing Lactic Acid Bacteria in Laban, a Middle Eastern Fermented Milk

Abstract

The literature is replete with studies on bacteriocins and probiotic properties of lactic acid bacteria (LAB) isolated from various dairy products. However, less is known about laban, a fermented milk product widely consumed in the Middle East. This study, therefore, aims to isolate and characterize laban-borne LAB for their possible bacteriocinogenic and probiotic properties. Twenty-nine laban samples were obtained from several retail stores in the Washington DC metropolitan area and processed for total lactic-acid-bacterial plate count and characterization of the LAB. The LAB count ranged from 4.15 to 8.37 log10 CFU/ml, with a mean of 5.83±1.07. Of the total 238 lactic acid bacteria, twelve isolates exhibiting the highest antimicrobial activity against the standard indicator bacteria used for screening bacteriocins were selected. The indicator bacteria included known pathogens such as Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecium, Pseudomonas aeruginosa, Escherichia coli O157:H:7, Salmonella typhi, Shigella sonnei, Acinetobacter baumannii, Bacillus cereus; and food-spoilage bacteria such as Pseudomonas fluorescens and Micrococcus luteus. The cell-free supernatant (CFS) of the twelve LAB isolates retained a broad-spectrum antimicrobial activity against all the indicator bacteria. A time-lapse study showed a bactericidal activity of the CFS with an average killing time of 26 minutes. Phenotypic characterization and 16s rRNA sequencing identified the twelve isolates as Lactobacillus casei, L. paracasei, L. helveticus, L. plantarum, L. acidophilus, L. delbrueckii subsp. bulgaricus, and L. harbinensis. SDS PAGE analysis of the CSF revealed protein bands with a molecular weight of 7-61 kDa, which is well within the range reported for LAB bacteriocins. PCR analysis detected the abundance of three bacteriocin vi genes, ped, helveticin, and laf. Five of the twelve isolates of the bacteriocinogenic LAB, Lactobacillus casei (HUL5 and HUL12), Lactobacillus helveticus (HUL10), and Lactobacillus delbrueckii subsp. bulgaricus (HUL15), and Lactobacillus harbinensis (HUL20), were selected for whole-genome sequencing (WGS) to confirm the PCR results. Analysis of the WGS confirmed detected helveticin J and two additional genes, lactococcin 972, enterolysin A. Also detected in the isolates, were genes related to multiple probiotic functions, such as host metabolism and stress responses. Antibiotic resistance and virulence genes were not detected. The isolates grew at low pH (1.9-2.25), high salt concentration (>6%), and their cell-free supernatant retained the antimicrobial activity above typical protein denaturing temperatures. Together, these constitute desirable properties for potential probiotic applications of the LAB isolates. Moreover, it was noteworthy to find that the LAB isolates exhibited a broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative pathogenic bacteria, including the spore-formers. Overall, findings from this study reveal that a diverse Lactobacillus spp. with potential application in food preservation and probiosis exist in laban. Further genomic studies of these diverse lactic acid bacteria may reveal their novelty.