Characterization of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based on Phenotypic, Genotypic, and Cell Wall Protein Analyses

Abstract

Non-starter lactic acid bacteria (NSLAB) were isolated from 12 Italian ewe cheeses representing six different types of cheese, which in several cases were produced by different manufacturers. A total of 400 presumptive Lactobacillus isolates were obtained, and 123 isolates and 10 type strains were subjected to phenotypic, genetic, and cell wall protein characterization analyses. Phenotypically, the cheese isolates included 32% Lactobacillus plantarum isolates, 15% L. brevis isolates, 12% L. paracasei subsp. paracasei isolates, 9% L. curvatus isolates, 6% L. fermentum isolates, 6% L. casei subsp. casei isolates, 5% L. pentosus isolates, 3% L. casei subsp. pseudoplantarum isolates, and 1% L. rhamnosus isolates. Eleven percent of the isolates were not phenotypically identified. Although a randomly amplified polymorphic DNA (RAPD) analysis based on three primers and clustering by the unweighted pair group method with arithmetic average (UPGMA) was useful for partially differentiating the 10 type strains, it did not provide a species-specific DNA band or a combination of bands which permitted complete separation of all the species considered. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis cell wall protein profiles clustered by UPGMA were species specific and resolved the NSLAB. The only exceptions were isolates phenotypically identified as L. plantarum and L. pentosus or as L. casei subsp. casei and L. paracasei subsp. paracasei, which were grouped together. Based on protein profiles, Italian ewe cheeses frequently contained four different species and 3 to 16 strains. In general, the cheeses produced from raw ewe milk contained a larger number of more diverse strains than the cheeses produced from pasteurized milk. The same cheese produced in different factories contained different species, as well as strains that belonged to the same species but grouped in different RAPD clusters.Non-starter lactic acid bacteria (NSLAB) usually increase from a low number in fresh curd to dominate the microflora of mature cheese (36). In contrast to starters, NSLAB tolerate the hostile environment of cheese during ripening; this environment typically is characterized by 32 to 39% moisture, 4 to 6% salt in moisture, pH 4.9 to 5.3, 5 to 13°C, and a deficiency of nutrients (15,48). Mesophilic lactobacilli predominate in the NSLAB community, although pediococci and micrococci may also be found (6,11,15). Lactobacillus casei subsp. casei, L. casei subsp. pseudoplantarum, Lactobacillus paracasei subsp. paracasei, and Lactobacillus plantarum are the most frequently isolated taxa, but other facultatively and obligately heterofermentative species of lactobacilli are also found (18, 28). Adventitious mesophilic lactobacilli are usually present because of postpasteurization contamination but may also constitute part of the raw milk microflora and survive pasteurization (48).The role of NSLAB in ripening has not been resolved satisfactorily yet, although inclusion of adjunct cultures of some strains of NSLAB or use of ra...

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