Study of the effectiveness of staphylococcins in biopreservation of Minas fresh (Frescal) cheese with a reduced sodium content.

Reducing salt content in foods such as cheeses, while limiting the growth of spoilage microorganisms and foodborne pathogens, is a difficult challenge. One method that may prove useful is use of staphylococcins, which are bacteriocins produced by staphylococci. Therefore, staphylococcin antimicrobial activity against six strains of S. aureus isolated from cheese was tested aiming at their industrial application in biopreservation of Minas fresh (Frescal) cheese with reduced sodium content. Three staphylococcins were selected for these tests: Pep 5, aureocin A53 and lysostaphin. All three staphylococcins proved to be bacteriolytic against all six strains of S. aureus. The antimicrobial activity of the partially purified staphylococcins was subsequently investigated against strains S. aureus Q1 and QJ3 in cheese matrices (6.0 log CFU/g) with different NaCl contents (control, a 25% reduction, and a 50% reduction), kept under refrigeration at 4 °C, for 21 days. Both strains were shown to be of concern for food industry as they carry the SEA, SEB and SEH enterotoxin genes, and are resistant to ß-lactam drugs and moderate biofilm formers when grown in TSB. When used singly, Pep5, aureocin A53 and lysostaphin reduced approximately 95%, 99% and 99.99% of the viable cell counts, respectively, irrespective of the sodium content of the cheese matrix. The combined action of aureocin A53 and Pep5 resulted in an additional and significant reduction (p < 0.05) of ~1.0 log CFU/g when compared with the reduction caused by the use of either one singly. The combined action of lysostaphin and aureocin A53 or lysostaphin and Pep5 resulted in a reduction similar to or slightly smaller (p > 0.05) than that observed when lysostaphin was employed singly. Lysostaphin also proved to reduce the number of the staphylococcal viable cells to a level (~ 2.0 log CFU/g) at which enterotoxin production should not reach a sufficient quantity to cause food poisoning. Therefore, lysostaphin may have a practical application in the food industry to control staphylococcal contamination of Minas fresh cheese with a sodium content reduced up to 50%, providing consumers with more safe options to reduce their intake of sodium.

The four-component aureocin A70 as a promising agent for food biopreservation.

Aureocin A70 is the only four-component bacteriocin described to date. As it inhibits the growth of a wide range of Gram-positive bacteria, including Listeria monocytogenes strains isolated from food, its potential for improving food safety was investigated in this study. Aureocin A70 (10,240AU/mL) proved to be bactericidal, but not extensively lytic, against listerial strains. The antibacterial activity of aureocin A70 (16AU/mL) was then tested in UHT-treated skimmed milk inoculated with the food-associated L. monocytogenes L12 strain (4-log CFU/mL) during storage at 4°C for one week. Aureocin A70 caused a time-dependent reduction in the listerial viable cell counts (5.51-log units) up to 7days of incubation. Aureocin A70 was neither toxic to the Vero and the L-929 cell lines nor exhibited a hemolytic activity against sheep red blood cells. Aureocin A70 proved to be completely stable for one month at 25°C, 16weeks at 4°C and 20weeks at -20°C. Aureocin A70 exhibited a time-dependent susceptibility to simulated gastric juice and bile salts mimicking gastrointestinal conditions. The entrapment of aureocin A70 in an alginate/gelatin matrix revealed that this bacteriocin can be released from this matrix. Moreover, it remained adsorbed to and active on a low-density polyethylene plastic surface suggesting that aureocin A70 may be employed in bioactive packaging to control the growth of undesirable bacteria. Taken together these results suggest that aureocin A70 is a promising alternative to be used in food applications.

Immunity to the Staphylococcus aureus leaderless four-peptide bacteriocin aureocin A70 is conferred by AurI, an integral membrane protein.

Aureocin A70, which is produced by Staphylococcus aureus A70, is the only four-component bacteriocin described thus far. The genetic determinants responsible for its production are arranged as three transcriptional units encoded by the 7.9-kb plasmid pRJ6. While the transcriptional unit formed by the genes aurABCD encodes the bacteriocin structural peptides, a second divergent gene, aurT, codes for an ABC transporter involved in bacteriocin externalization. The third transcriptional unit is composed of two genes, orfAB, whose functions were hitherto unknown. RT-PCR analysis of orfAB expression revealed that they are arranged as an operon. When orfAB, either with or without the transcriptional terminator found downstream of orfB, was expressed in two different S. aureus strains sensitive to aureocin A70, all strains became immune to this bacteriocin. Cloning of orfB alone, with or without the transcriptional terminator, confirmed orfB participation in immunity, although full immunity was not observed. An increase in immunity was achieved when two copies of orfB were cloned oriented with the exogenous Plac promoter present in the expression vector pT181mcs. orfB (here referred to as aurI) was shown to be responsible for aureocin A70 immunity, but the full immunity phenotype seems to depend on translational coupling involving orfA, which encodes a putative transcriptional regulator, and aurI.