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This study characterized the microflora present in four Queso Fresco‐style cheeses made from raw milk (RM) and two cheeses made from pasteurized milk (PM) obtained directly from six different producers in Mexico. Microbial isolates were initially analyzed on 8 different selective media and further analyzed by 16S rRNA gene sequence analysis. Sequence analysis identified Lactococcus lactis ssp. lactis as an isolate growing on M17 agar in four cheese samples. L. lactis ssp. lactis was the only lactic acid bacterium identified which is traditionally used as a cheese starter culture. Sequence analysis of isolates from MRS medium supplemented with vancomycin identified Leuconostoc mesenteroides in all six cheese samples. A high number of coliforms, enterococci and coagulase‐positive staphylococci were identified in all RM cheeses. The numbers of total coliform species were lower in both PM samples (by 2 log10), but the numbers of staphylococci were lower in only one sample (by 3 log10). The numbers of enterococci were high in both PM samples, and sequence analysis identified Enterococcus faecium as the only enterococcal species isolated from all six cheeses. These results suggest that L. lactis ssp. lactis, L. mesenteroides and E. faecium are species that may contribute to the organoleptic qualities of Queso Fresco‐style cheeses analyzed in this study. PRACTICAL APPLICATIONS This work was performed to define the natural microflora present in traditional‐style Queso Fresco in order to identify bacterial species that may contribute to the organoleptic qualities of the cheese. Identification and enumeration of the “generally regarded as safe” (GRAS) lactic acid bacteria may allow for the development of new culture blends for the production of Queso Fresco using pasteurized milk, while maintaining the desired qualities of the raw milk varieties. In addition, bacteria recovered from the raw milk cheeses may be novel strains offering advantages over commercially available starter and adjunct LAB currently being used to make Queso Fresco. The work also identified potential pathogens and spoilage bacteria associated with traditional‐style Queso Fresco, stressing the importance in developing a desirable pasteurized milk cheese.

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Enterococci are often identified as constituents of the indigenous microflora from raw milk artisanal cheeses and are believed to contribute to the unique organoleptic qualities of these products. Many strains of enterococci are also known to produce antimicrobial peptides, enterocins, which may prevent the growth of certain food-born pathogens. In this study 33 enterococcal isolates from Hispanic-style cheeses were screened for the production of bacteriocins. Of the 33 isolates, 5 Enterococcus faecium and 1 Enterococcus durans isolates inhibited the growth of Listeria spp. The antilisterial activity was lost after treatment with pepsin, trypsin, pronase, proteinase K and alpha-chymotrypsin suggesting the active component was a protein or peptide. The active compounds were heat stable and had molecular weights between 4 and 8 kDa, which is characteristic of Class II enterocins. A PCR screen showed that four E. faecium isolates contained nucleic acid sequences for multiple enterocins. Isolate H41K contained entA and entP; and isolates H51Ca, H51Cb and H41B contained entA, entP and entL50AB, with H41B also containing entB. All PCR tests performed were negative for E. faecium isolate H41D, suggesting the production of a novel enterocin. The isolates were also screened for susceptibility to antibiotics, with only two showing low-level resistance to vancomycin (8 microg ml(-l)). However, three isolates were highly resistant to both tetracycline and kanamycin, with two of the isolates also showing high resistance to erythromycin.

A screen of thermophilic lactic acid bacteria identified Streptococcus thermophilus strain ST110 as the putative producer of a bacteriocin with high level of activity against pediococci. Thermophilin 110 was isolated from culture supernatant after 16 h of growth and partially purified by a chloroform extraction procedure. The bacteriocin inhibited the growth of several lactic acid bacteria and in the case of Pediococcus acidilactici, it induced cell lysis with the concomitant release of OD260-absorbing material and intracellular enzymes. SDS-PAGE analysis revealed two components with estimated sizes between 4.0 kDa and 4.5 kDa, respectively, with possible involvement in bacteriocin activity as indicated by agar overlay assays with P. acidilactici as the target organism. Thermophilin 110 was inactivated by several proteolytic enzymes and also by alpha-amylase, which indicated the putative requirement of a glycosidic component for activity. The bacteriocin produced by S. thermophilus may be especially useful in the food processing industries to control spoilage caused by pediococci.

A pH-dependent adsorption/desorption technique was used to screen Streptococcus thermophilus strains for the production of bacteriocins. Agar-diffusion tests with S. thermophilus strains as targets identified 13 out of 41 strains as producers of antibacterial activity. Thermophilin A, the bacteriocin-like substance present in the culture supernatant of S. thermophilus ST134 was purified to homogeneity by ammonium sulfate precipitation and ion-exchange chromatography, followed by ultrafiltration. Thermophilin A is a relatively heat-stable and apparently glycosylated bacteriocin with a bactericidal mode of action against sensitive cells.

The enzymatic breakdown of milk proteins releases bioactive peptides. Two such peptides are the 11-residue antimicrobial peptide from bovine lactoferrin (BL-11) and the 12-residue hypotensive peptide from alpha(s1)-casein (C-12). These two peptides have now been cloned in Streptococcus thermophilus to develop strains that enhance the functionality and nutritional value of dairy food products. Nucleic acid sequences encoding the peptides were generated by overlapping PCR and were subsequently cloned into a new expression vector under control of the ST(2201) promoter. S. thermophilus transformants were successfully identified using GFP as a selectable marker. The presence of the synthetic gene constructs in S. thermophilus was confirmed by PCR.