Biotechnological use of dairy by-products for the production and microencapsulation of the food preservative enterocin CRL35.

Bacteriocins from Gram-positive bacteria have been proposed as natural food preservative and there is a need for large-scale production for commercial purposes. The aim of the present work is to evaluate whey, a cheese industrial by-product, for the production and microencapsulation of enterocin CRL35. Whey proved to be a promising basal medium for bacterial growth although the bacteriocin production was quite low. However, it could be much favored with the addition of yeast extract at concentrations as low as 0.5%. Besides improving bacteriocin production, this peptide was successfully microencapsulated by spray drying using whey protein concentrate and a chitosan derivative as wall materials. Microcapsules averaging 10 ± 5 µm diameter were obtained, with good structural integrity and high antimicrobial activity with a stability of at least 12 weeks at 4°C. In summary, sustainable bacteriocin production and microencapsulation was achieved recycling whey or its derivatives. In addition, the formulation owns high antimicrobial activity with a long shelf life. The development of a food preservative may represent a green solution for handling whey.

Antimicrobial activity of MccJ25(G12Y) against gram-negative foodborne pathogens in vitro and in food models.

The use of bacteriocins is a promising alternative to improve food security through the biocontrol of food pathogens and spoilage microorganisms. Gram-negative produced microcin J25(G12Y), known as (MccJ25(G12Y)) is a variant of the well-studied and characterized antimicrobial peptide, microcin J25 (MccJ25). In the present work, we explored the activity of this microcin against Gram-negative bacteria linked to foodborne diseases. We evaluated the in vitro antimicrobial activity of MccJ25(G12Y) in solid medium against a collection of pathogenic and food-altering strains and studied its activity and stability in meat and dairy food systems. We show that MccJ25(G12Y) exhibited the same in vitro antimicrobial spectrum as its parental microcin (MccJ25) against different Gram-negative foodborne pathogens and spoilage strains. We highlight that low concentrations of MccJ25(G12Y) between 0.45 and 29.4 µM were able to inhibit a substantial number of pathogens, including Salmonella, Escherichia, Shigella and Enterobacter genus. We also demonstrate the antimicrobial effectiveness of the peptide against Escherichia coli O157:H7 NCTC 12900, Enterobacter cloacae CECT 194, and Salmonella enterica CECT 4396 in fish and beef burgers and yogurt. MccJ25(G12Y) was added or not to food matrices inoculated with the foodborne pathogens at 105 CFU/g or mL. Afterward, food products were stored at 4 °C and selective media for the specific enumeration were used to determine the antimicrobial susceptibility of each pathogen to MccJ25(G12Y). The viability of the three pathogens was significantly reduced in the different food biological environments. In yogurt, the peptide decreased E. coli numbers on day 5 by about 4 log 10 CFU/mL as compared to non-treated samples. For S. enterica and E. cloacae no viable cells were detected at the end of the treatment. Adding MccJ25(G12Y) to fish burgers decreased E. cloacae numbers during storage 2 log10 CFU/g on the first day, reaching a difference of about 5 log 10 CFU/g after 10 days compared to non-treated control. Finally, the peptide decreased E. coli O157:H7 numbers on the beef burgers samples during storage on day 10 by about 3 log 10 CFU/g as compared to non-treated samples. The stability analysis demonstrated that MccJ25(G12Y) is capable of remaining active in these food matrices for a considerable time during the storage at refrigeration temperatures. These results reinforce the studies on the potential applicability of this microcin as a biopreservative in the food industry.

Physiological and proteomic response of Escherichia coli O157:H7 to a bioprotective lactic acid bacterium in a meat environment.

The enterohemorrhagic Escherichia (E.) coli (EHEC) is a pathogen of great concern for public health and the meat industry all over the world. The high economic losses in meat industry and the high costs of the illness highlight the necessity of additional efforts to control this pathogen. Previous studies have demonstrated the inhibitory activity of Enterococcus mundtii CRL35 towards EHEC, showing a specific proteomic response during the co-culture. In the present work, additional studies of the EHEC-Ent. mundtii interaction were carried out: i) differential protein expression of E. coli O157:H7 NCTC12900 growing in co-culture with Ent. mundtii in a meat environment, ii) the reciprocal influence between these two microorganisms in the adhesion to extracellular matrix (ECM) proteins and iii) the possible induction of the phage W933, coding for Shiga toxin (Stx1), by Ent. mundtii CRL35. Proteomic analysis showed a significant repression of a number of E. coli NCTC12900 proteins in co-culture respect to its single culture, these mostly related to the metabolism and transport of amino acids and nucleotides. On the other hand, statistically significant overexpression of EHEC proteins involved in stress, energy production, amino acid metabolism and transcription was observed at 30 h respect to 6 h when EHEC grew in co-culture. Data are available via ProteomeXchange with identifier PXD014588. Besides, EHEC showed a decreased adhesion capacity to ECM proteins in the presence of the bioprotective strain. Finally, Ent. mundtii CRL35 did not induce the lytic cycle of W933 bacteriophage, thus indicating its potential safe use for eliminating this pathogen. Overall, this study expands the knowledge of EHEC- Ent. mundtii CRL35 interaction in a meat environment, which will certainly contribute to find out effective biological strategies to eliminate this pathogen.

PrfA activation in Listeria monocytogenes increases the sensitivity to class IIa bacteriocins despite impaired expression of the bacteriocin receptor.

BACKGROUND: The scope of the present work was to characterize the activity of class IIa bacteriocins in Listeria (L.) monocytogenes cells that constitutively express an activated form of PrfA, the virulence master regulator, since bacteriocin sensitivity was only characterized in saprophytic cells so far. The mannose phosphotransferase system (Man-PTS) has been shown to be the class IIa bacteriocin receptor in Listeria; hence, special attention was paid to its expression in virulent bacteria. METHODS: L. monocytogenes FBprfA* cells were obtained by transconjugation. Bacterial growth was studied in TSB and glucose containing-minimal medium. Sensitivity to antimicrobial peptides was assessed by killing curves. Membranes of L. monocytogenes FBprfA* cells were characterized using proteomic and lipidomic approaches. RESULTS: The mannose phosphotransferase system (Man-PTS) was downregulated upon expression of PrfA*, and these cells turned out to be more sensitive to enterocin CRL35 and pediocin PA-1, while not to nisin. Proteomic and lipidomic analysis showed differences between wild type (WT) and PrfA* strains. For instance, phosphatidic acid was only detected in PrfA* cells, whereas, there was a significant decline of plasmalogen-phosphatidylglycerol in the same strain. CONCLUSIONS: Our results support a model in which Man-PTS acts just as a docking molecule that brings class IIa bacteriocins to the plasma membrane. Furthermore, our results suggest that lipids play a crucial role in the mechanism of action of bacteriocins. GENERAL SIGNIFICANCE: This is the first demonstration of the link between L. monocytogenes virulence and the bacterial sensitivity toward pediocin-like peptides.

Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes.

BACKGROUND: Enterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain. METHODS: Listeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques. RESULTS: The growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes. CONCLUSIONS: These results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains. GENERAL SIGNIFICANCE: Highly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.

28-mer Fragment Derived from Enterocin CRL35 Displays an Unexpected Bactericidal Effect on Listeria Cells.

Two shorter peptides derived from enterocin CRL35, a 43-mer bacteriocin, were synthesized i.e. the N-terminal fragment spanning from residues 1 to 15, and a 28-mer fragment that represents the C-terminal of enterocin CRL35, the residues 16 to 43. The separate peptides showed no activity when combined. On one hand, the 28-mer peptide displayed an unpredicted antimicrobial activity. On the other, 15- mer peptide had no consistent anti-Listeria effect. The dissociation constants calculated from experimental data indicated that all peptides could bind at similar extent to the sensitive cells. However, transmembrane electrical potential was not dissipated to the same level by the different peptides; whereas the full-length and the C-terminal 28-mer fragment induced almost full dissipation, 15-mer fragment produced only a slow and incomplete effect. Furthermore, a different interaction of each peptide with membranes was demonstrated based on studies carried out with liposomes, which led us to conclude that activity was related to structure rather than to net positive charges. These results open up the possibility of designing new peptides based on the 28-mer fragment with enhanced activity, which would represent a promising approach for combating Listeria and other pathogens.