Identification and characterization of Leuconostoc carnosum, associated with production and spoilage of vacuum-packaged, sliced, cooked ham

Abstract

Leuconostoc carnosum was shown to be the specific spoilage organism in vacuum-packaged, sliced, cooked ham showing spoilage during 3 weeks of shelf life. Identification of the specific spoilage organism was done by use of phenotypic data and ClaI, EcoRI, and HindIII reference strain ribopatterns. One hundred L. carnosum isolates associated with the production and spoilage of the ham were further characterized by pulsed-field gel electrophoresis (PFGE), together with some meat-associated Leuconostoc species: L. citreum, L. gelidum, L. mesenteroides subsp. dextranicum, and L. mesenteroides subsp. mesenteroides. ApaI and SmaI digests divided the industrial L. carnosum strains into 25 different PFGE types, ApaI and SmaI types being consistent. Only one specific PFGE type was associated with the spoiled packages. This type also was detected in air and raw-meat mass samples. The spoilage strain did not produce bacteriocins. Only seven isolates belonging to three different PFGE types produced bacteriocins. Similarity analysis of the industrial L. carnosum strains revealed a homogeneous cluster which could be divided into eight subclusters consisting of strains having at most three-fragment differences. The L. carnosum cluster was clearly distinguished from the other meat-associated leuconostoc clusters, with the exception of the L. carnosum type strain. Ribotyping can be very helpful in the identification of L. carnosum, but its discriminatory power is too weak for strain characterization. PFGE provides good discrimination for studies dealing with the properties of homogeneous L. carnosum strains.

FIG. 1

ClaI ribopatterns. Lanes 4 and 11, phage lambda DNA cleaved with HindIII as a fragment size marker; lane 1, Weissella viridescens ATCC 12706^T; lane 2, Weissella halotolerans ATCC 35410^T; lane 3, Weissella paramesenteroides DSM 20288^T; lane 5, Leuconostoc mesenteroides subsp. mesenteroides DSM 20343^T; lane 6, Leuconostoc mesenteroides subsp. cremoris CCUG 21965^T; lane 7, Leuconostoc mesenteroides subsp. dextranicum DSM 20484^T; lane 8, Leuconostoc pseudomesenteroides DSM 20193^T; lane 9, Leuconostoc carnosum NCFB 2776^T; lane 10, Leuconostoc gelidum NCFB 2775^T; lane 12, Leuconostoc lactis CCUG 30064^T; lane 13, Leuconostoc fallax CCUG 30061^T; lane 14, Leuconostoc citreum (Leuconostoc amelibiosum) D1.

FIG. 2

EcoRI ribopatterns. Lanes 4 and 11, phage lambda DNA cleaved with HindIII as a fragment size marker; lane 1, Weissella viridescens ATCC 12706^T; lane 2, Weissella halotolerans ATCC 35410^T; lane 3, Weissella paramesenteroides DSM 20288^T; lane 5, Leuconostoc mesenteroides subsp. mesenteroides DSM 20343^T; lane 6, Leuconostoc mesenteroides subsp. cremoris CCUG 21965^T; lane 7, Leuconostoc mesenteroides subsp. dextranicum DSM 20484^T; lane 8, Leuconostoc pseudomesenteroides DSM 20193^T; lane 9, Leuconostoc carnosum NCFB 2776^T; lane 10, Leuconostoc gelidum NCFB 2775^T; lane 12, Leuconostoc lactis CCUG 30064^T; lane 13, Leuconostoc fallax CCUG 30061^T; lane 14, Leuconostoc citreum (Leuconostoc amelibiosum) D1.

FIG. 3

HindIII ribopatterns. Lanes 4 and 11, phage lambda DNA cleaved with HindIII as a fragment size marker; lane 1, Weissella viridescens ATCC 12706^T; lane 2, Weissella halotolerans ATCC 35410^T; lane 3, Weissella paramesenteroides DSM 20288^T; lane 5, Leuconostoc mesenteroides subsp. mesenteroides DSM 20343^T; lane 6, Leuconostoc mesenteroides subsp. cremoris CCUG 21965^T; lane 7, Leuconostoc mesenteroides subsp. dextranicum DSM 20484^T; lane 8, Leuconostoc pseudomesenteroides DSM 20193^T; lane 9, Leuconostoc carnosum NCFB 2776^T; lane 10, Leuconostoc gelidum NCFB 2775^T; lane 12, Leuconostoc lactis CCUG 30064^T; lane 13, Leuconostoc fallax CCUG 30061^T; lane 14, Leuconostoc citreum (Leuconostoc amelibiosum) D1.

FIG. 4

SmaI (lanes 1 to 9) and ApaI (lanes 10 to 18) ribopatterns. Lanes 1, 9, 10, and 18, Leuconostoc carnosum NCFB 2776^T; lanes 2 and 11, Leuconostoc mesenteroides subsp. mesenteroides DSM 20343^T; lanes 3 and 12, Leuconostoc mesenteroides subsp. dextranicum DSM 20484^T; lanes 4 and 13, Leuconostoc pseudomesenteroides DSM 20193^T; lanes 5 and 14, Weissella paramesenteroides DSM 20288^T; lanes 6 and 15, Leuconostoc gelidum NCFB 2775^T; lanes 7 and 16, Leuconostoc citreum (Leuconostoc amelibiosum) D1; lanes 8 and 17, Leuconostoc carnosum V-8a.

FIG. 5

Dendrogram based on SmaI ribopatterns. The similarity between all pairs was expressed by Dice coefficient correlation, and the unweighted pair-group method with arithmetic averages was used for the construction of the dendrogram.