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.

FIG. 1

Dendrogram, obtained from combined RAPD patterns with three primers, of Lactobacillus isolates from ewe cheeses and type strains. A cluster analysis was conducted with similarity estimates by using UPGMA.

FIG. 2

RAPD patterns obtained with primers P1 (a), P4 (b), and P7 (c), showing the representative fingerprints of the 11 clusters. Lane 1, DNA molecular size standards (123-bp ladder DNA; Gibco BRL, Life Technologies); lanes 2 to 12, clusters 1 to 11, respectively.

FIG. 3

SDS-PAGE patterns of cell wall proteins from Lactobacillus isolates. Lane 1, standard proteins (see Materials and Methods); lane 2, L. fermentum 65H; lane 3, L. brevis 68E; lane 4, L. curvatus 1HD; lane 5, L. casei subsp. casei M16; lane 6, L. paracasei subsp. paracasei 4H3; lane 7, L. casei subsp. pseudoplantarum 115K; lane 8, L. plantarum 144; lane 9, L. pentosus 2HG; lane 10, L. rhamnosus 15H3.

FIG. 4

Dendrogram, obtained from SDS-PAGE patterns of cell wall proteins, of Lactobacillus isolates from ewe cheeses and type strains. A cluster analysis was conducted with similarity estimates by using UPGMA.