Antimicrobial agents are used in food and medical industries to control hazardous microorganisms. The risk of exposure to these microorganisms is increasing while their resistance to conventionally used antimicrobials is on the rise. Additionally, consumers are increasingly avoiding foods containing synthetic antimicrobials and prefer products with clean labels. Driven by these urgencies, continuous innovations are needed for discovering new sources of effective natural antimicrobials. Candidates for these natural antimicrobials include bacteriocins, which are ribosomally-synthesized, occasionally post-translationally modified, peptides produced by bacteria. Among bacteriocin-producing bacteria, lactic acid bacteria (LAB) have been used safely for decades to manufacture fermented foods and known by their contributions to improving food safety and human health. Thus, the application of LAB as bioprotective starter/adjunct cultures or their purified/crude bacteriocins possess a high potential to replace synthetic preservatives in food and other products.
The main objective of this study was to search for novel food-isolated LAB, producing effective antimicrobial peptides, possessing desirable safety and probiotic characteristics, and potentially usable in ensuring food safety and promoting human health. To expedite the search process, genomic, phenotypic, and mass spectroscopic analyses were efficiently implemented to characterize the new LAB strains, elucidate the structure of their produced bacteriocins, and assess their safety and probiotic traits. Additionally, new approaches were utilized to produce, purify, and scale-up the production of these LAB bacteriocins, using low-cost media and innovative material separation techniques.
In this current study, we discovered two new bacteriocinogenic LAB strains, Lactobacillus paraplantarum OSY-TC318 and Enterococcus durans OSY-EGY. The crude extract of L. paraplantarum OSY-TC318, a Turkish artisanal cheese isolate, inhibited the growth of various pathogenic and spoilage bacteria such as Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria monocytogenes, Salmonella Typhimurium, and Staphylococcus aureus. Considering its potential usefulness for food and medical applications, genomic analysis was implemented to reveal the novelty of the strain, OSY-TC318, and to predict the biosynthetic pathway and chemical structure of its produced bacteriocin, paraplantaricin TC318. The bacteriocin was purified from L. paraplantarum OSY-TC318 cells using High Performance Liquid Chromatography (HPLC), followed by structural elucidation using Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. This genome-guided MS analysis revealed that the molecular mass of paraplantaricin TC318 is 2263.900 Da, its chemical formula is C106H133N27O22S4 and its primary sequence is: F-K-S-W-S-L-C-T-F-G-C-G-H-T-G-S-F-N-S-F-C-C. This lantibiotic, which differs from mutacin 1140 at amino acid residues 9, 12, 13 and 20, is considered a new member of the epidermin group in class I lantibiotics. The new strain, E. durans OSY-EGY, is an Egyptian artisanal cheese isolate that produces multiple antimicrobial peptides. Replacing microbiological media, it was possible to use the strain to ferment reconstituted skim milk, supplemented with 1% yeast extract; this approach increased the concentration of the antimicrobials by 4-fold. The produced milk-based fermentate inhibited Gram-positive pathogenic and spoilage bacteria such as B. cereus, C. sporogenes, E. faecalis, L. monocytogenes, and S. aureus. Furthermore, a new approach was developed to purify the bacteriocins, from milk fermented using E. durans OSY-EGY, using XAD-16N resin beads. Following this approach, the concentration of the antimicrobials increased by 16-fold, compared to that resulting from microbiological media. The developed purification protocol could be used for recovering high yields of scarcely produced bacteriocins, thus facilitating further studies and applications. The HPLC-purified bacteriocins were structurally elucidated using FT-ICR-MS analysis. The analysis revealed that E. durans OSY-EGY produces two enterocins with molecular mass of 5215.37 and 5203.37 Da, respectively; these showed similarity with the formulated forms of the leaderless enterocins L50A and L50B discovered initially in E. faecium L50. Although the stability, wide spectrum, and effectiveness of these enterocins were well documented, the commercial implementation of such bacteriocins is hampered by the cost of the production media.
In the current study, the processing low-cost waste, cheese acid whey, was efficiently used as a production medium for the natural enterocins produced by E. durans OSY-EGY and E. faecalis OSY-RM6; the latter was isolated in this laboratory from raw milk, and the structure of its potent enterocin has been elucidated in 2013. After optimizing production parameters, enterocins produced by OSY-EGY and OSY-RM6 were semi-purified and concentrated from the fermented whey using resin beads followed by ultrafiltration and freeze drying. The concentrations of OSY-EGY and OSY-RM6 in the final antimicrobial powders were 512,000 and 128,000 AU/g, respectively. To our knowledge, this is the first report of antimicrobial powder derived from cheese acid whey. Considering that E. durans OSY-EGY could be used directly in food fermentations, the bacterium was assessed for its safety and probiotic characteristics using comparative genomics and phenotypic analyses. The comparative genomic analysis revealed that E. durans OSY-EGY is distantly removed from the potentially pathogenic Enterococcus spp. The genome was devoid of genes encoding acquired antibiotic resistance or marker of virulence factors associated with potentially pathogenic enterocooci. The phenotypic analyses showed that the bacterium is sensitive to the antibiotics, vancomycin, ampicillin, tetracycline, chloramphenicol, and aminoglycosides and it does not express hemolytic or gelatinase activity, or cytotoxic effect against Caco-2 cells. Altogether, these findings illustrate the safety characteristics of E. durans OSY-EGY. Mining E. durans OSY-EGY genome for probiotic-related sequences revealed genes associated with acid and bile tolerance, cell adhesion, competitiveness and antioxidant activities, essential amino acids production, and vitamins biosynthesis. Phenotypically, E. durans OSY-EGY strain was tolerant to acidic pH (3.0) and presence of 0.3% bile salts. The bacterium showed adhesion capability to Caco-2 cells of 29 %, cholesterol lowering effect of 44.7 % and free radical scavenging activity of 9 %.
In conclusion, the efficient use of genomic, phenotypic, and mass spectroscopic analyses led to the discovery of novel LAB strains and a new bacteriocin. Additionally, new approaches were implemented successfully to produce, purify, and scale-up the production of LAB bacteriocins. One of the discovered strains possesses desirable antimicrobial and probiotic traits and deemed safe for application in the food and pharmaceutical industries.