Enterococcus populations in artisanal Manchego cheese: biodiversity, technological and safety aspects.

Enterococci represent a considerable proportion of the microbiota in Manchego cheeses. In this study, a total of 132 enterococci isolated from good quality Manchego cheeses from two dairies at different ripening times were genotypically characterized and identified using molecular techniques. Representative isolates from the clusters obtained after genotyping were assayed for some enzymatic activities considered to have a potential role in cheese ripening, and for 2,3-butanedione and acetoin production, evaluation of odor intensity and appearance in milk and safety evaluation. Enterococcus faecalis was the predominant specie, accounting for 81.8% of the total isolates, while Enterococcus faecium, Enterococcus hirae and Enterococcus avium were present in low proportions. The number of genotypes involved at each ripening time varied both between dairies and with the ripening times; genotype E. faecalis Q1 being present in almost all the samples from both dairies. Eight isolates showed a higher proteolytic activity and 3 isolates produced high quantities of acetoin-diacetyl, for which reason they are interesting from a technological standpoint. A low antibiotic resistance was found and almost all the strains were susceptible to clinically important antibiotics. On the contrary, only four isolates (E. faecalis C4W1 and N0W5, and E. faecium N32W1 and C16W2) did not harbor some of the virulence genes assayed.

Changes in the ovalbumin proteolysis profile by high pressure and its effect on IgG and IgE binding.

Egg proteins are responsible for one of the most common forms of food allergy, especially in children, and one of the major allergens is ovalbumin (OVA). With the aim to examine the potential of high pressure to enhance the enzymatic hydrolysis of OVA and modify its immunoreactivity, the protein was proteolyzed with pepsin under high-pressure conditions (400 MPa). Characterization of the hydrolysates and peptide identification was performed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS). The antigenicity (binding to IgG) and binding to IgE, using the sera of patients with specific IgE to OVA, were also assessed. The results showed that, upon treatment with pepsin at 400 MPa, all of the intact protein was removed in minutes, leading to the production of hydrolysates with lower antigenicity than those produced in hours at atmospheric pressure. However, the exposure of new target residues only partially facilitated the removal of allergenic epitopes, because the hydrolysates retained residual IgG- and IgE-binding properties as a result of the accumulation of large and hydrophobic peptides during the initial stages of hydrolysis. These peptides disappeared at later stages of proteolysis, although reactivity toward IgG and IgE was not completely abolished. Some fragments identified in the hydrolysates (such as Leu124-Phe134, Ile178-Ala187, Leu242-Leu252, Gly251-Ile259, Lys322-Gly343, Phe358-Phe366, and Phe378-Pro385) carried previously identified IgE-binding epitopes. Because some of the peptides found, such as Phe358-Phe366, probably contain only one binding site for IgE, the possibility to use high pressure to tailor hydrolysates that contain mostly peptides with only one IgE-binding site, which may help the immune system to tolerate egg proteins, is suggested.

Unfolding and refolding of beta-lactoglobulin subjected to high hydrostatic pressure at different pH values and temperatures and its influence on proteolysis.

The unfolding of beta-lactoglobulin during high-pressure treatment and its refolding after decompression were studied by 1H NMR and 2H/1H exchange at pH 6.8 and 2.5 and at 37 and 25 degrees C. The extent of unfolding increased with the pressure level. The structure of beta-lactoglobulin required higher pressures to unfold at pH 2.5 than at pH 6.8. More flexibility was achieved at 37 degrees C than at 25 degrees C. Results indicated that the structural region formed by strands F, G, and H was more resistant to unfold under acidic and neutral conditions. The exposure of Trp19 at an earlier time, as compared to other protein regions, supports the formation of a swollen structural state at pH 2.5. Refolding was achieved faster when beta-lactoglobulin was subjected to 200 MPa than to 400 MPa, to 37 degrees C than to 25 degrees C, and to acidic than to neutral pH. After treatment at 400 MPa for 20 min at neutral pH, the protein native structure was not recovered. All samples at acidic pH showed that the protein quickly regained its structure. Hydrolysis of beta-lactoglobulin by pepsin and chymotrypsin could be related to pressure-induced changes in the structure of the protein. Compared to the behavior of the protein at atmospheric pressure, no increased proteolysis was found in samples with no increased flexibility (100 MPa, 37 degrees C, pH 2.5). Slightly flexible structures were associated with significantly increased proteolysis (100 MPa, 37 degrees C, pH 6.8; 200 MPa, 37 degrees C, pH 2.5). Highly flexible structures were associated with very fast proteolysis (>or=200 MPa, 37 degrees C, pH 6.8; >or=300 MPa, 37 degrees C, pH 2.5). Proteolysis of prepressurized samples improved only when the protein was significantly changed after the pressure treatment (400 MPa, 25 degrees C, 20 min, pH 6.8).

Changes in chymotrypsin hydrolysis of beta-lactoglobulin A induced by high hydrostatic pressure.

Beta-lactoglobulin (beta-Lg) was subjected to high pressures up to 400 MPa and proteolysis with chymotrypsin. The hydrolysates were analyzed by SDS-PAGE and RP-HPLC, and the fragments obtained were identified by ESI-MS/MS. The results obtained showed that beta-Lg was hydrolyzed by chymotrypsin in a "progressive proteolysis" manner at either atmospheric or high pressure. Proteolysis during or after high-pressure treatment showed longer and more hydrophobic peptides than proteolysis at atmospheric pressure. Chymotrypsin showed a behavior similar to that of trypsin, with some differences, probably related to the orientation of the target residues specific for each enzyme. The similarities between proteolytic fragments produced by the two enzymes support that proteolysis enhancement under high pressure depends on the substrate changes rather than the enzyme. High pressure seemed to accelerate the first steps of proteolysis, probably through dimer dissociation, while leaving portions of the molecule more resistant to the enzyme.

Influence of high hydrostatic pressure on the proteolysis of beta-lactoglobulin A by trypsin.

This work describes the effect of the hydrolysis time and pressure (0.1-400 MPa) on the proteolysis of beta-lactoglobulin A (beta-lg A) with trypsin, either conducting hydrolysis of beta-lg under pressure or hydrolysing beta-lg that was previously pressure treated. Pressurisation, before or during enzyme treatments, enhanced tryptic hydrolysis of beta-lg. Trypsin degraded pressure-modified beta-lg and pressure-induced beta-lg aggregates, favouring proteolysis to the intermediate degradation products: (Val(15)-Arg(40)), (Val(41)-Lys(69))S-S(Leu(149)-Ile(162)) and (Val(41)-Lys(70))S-S(Leu(149)-Ile(162)). These were further cleaved at the later stages of proteolysis to yield: (Val(15)-Tyr(20)), (Ser(21)-Arg(40)), (Val(41)-Tyr(60)), (Trp(61)-Lys(69))S-S(Leu(149)-Ile(162)) and (Trp(61)-Lys(70))S-S(Leu(149)-Ile(162)). Particularly, in the tryptic hydrolysates of pre-pressurized beta-lg, two other fragments linked by disulphide bonds: (Lys(101)-Arg(124))S-S(Leu(149)-Ile(162)) and (Tyr(102)-Arg(124))S-S(Leu(149)-Ile(162)), were found. These corresponded to rearrangement products induced by SH/SS exchange between the free thiol group of Cys(121) and Cys(160), that normally forms the disulphide bond Cys(66)-Cys(160). In the light of these results, structural modifications of beta-lg under high pressure are discussed.