Diversity and dynamics of communities of coagulase-negative staphylococci in traditional fermented sausages

AIMS: Evaluation of composition and evolution of the coagulase-negative staphylococci (CNS) communities in two traditionally fermented sausages (salsiccia and soppressata lucana) produced in Basilicata, southern Italy. METHODS AND RESULTS: A culture-dependent approach based on isolation on selective media and identification with phenotypic and molecular methods was used. Phenotypic data of 471 strains were analysed by multivariate statistical methods by using 28 strains from culture collections and 48 strains identified by molecular methods (such as 16S rDNA sequencing, species-specific PCR assays, intergenic spacer region-PCR and PCR-denaturing gradient gel electrophoresis) as a reference. The CNS microflora of the sausages was found to be dominated by different biotypes of Staphylococcus xylosus (51.2%), followed by S. pulvereri/vitulus, S. equorum and S. saprophyticus (13.4, 10.2 and 10%, respectively). Other species (S. succinus, S. pasteuri, S. epidermidis, S. warneri and Macrococcus caseolyticus) were also present at lower levels. Identification of 25% of the isolates was impossible. CONCLUSIONS: The composition of CNS communities varied significantly with sausage type, plant and ripening time and clear differences were found among communities of salsiccia and soppressata at the end of ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic characterization, supported by molecular and statistical analyses, can be considered a useful approach for typing a large number of isolates and for monitoring the evolution of staphylococcal communities during sausage fermentation but does not always provide a satisfactory identification of the isolates.     

Effect of mixed starter cultures fermentation on the characteristics of silver carp sausages

To improve the quality and functionality and increase the utilization of silver carp (Hypophthalmichthys molitrix) muscle, three groups of silver carp sausages inoculated with the combinations of Staphylococcus xylosus-12 with Lactobacillus plantarum-15, Pediococcus pentosaceus-ATCC33316, and Lactobacillus casei subsp. casei-1.001, and a batch without any starter (control) were prepared. During the 48 h fermentation at 30°C, silver carp sausages inoculated with mixed starter cultures resulted in a rapid pH decrease, suppression in the growth of Enterobacteriaceae, Pseudomonas, yeasts and molds, and exhibited higher texture profiles (hardness, gumminess, springiness, and chewiness) and whiteness than the control (P < 0.05). The changes in non-protein nitrogen (NPN), free amino acid and SDS-PAGE indicated severe hydrolysis of muscle protein occurred during fermentation. Polyunsaturated fatty acids were higher in quantity in sausages with cultures compare to the control. No significant differences for taste, texture, and appearance were found among batches with mixed starters. The sausage inoculated with the combination of Lactobacillus plantarum-15, S. xylosus-12, and Lactobacillus casei subsp. casei-1.001 (S-PXC) gained highest scores for flavor and overall acceptability. There was an apparent positive correlation (r = 0.87) between the NPN and the overall acceptability in sausages, whereas pH value showed a significantly negative correlations (r = –0.89 to −0.99, P < 0.05) with taste, flavor, texture, appearance, and overall acceptability.     

Microbial ecology of the soppressata of Vallo di Diano, a traditional dry fermented sausage from southern Italy, and in vitro and in situ selection of autochthonous starter cultures

The microbial ecology of "soppressata of Vallo di Diano," a traditional dry fermented sausage from southern Italy, was studied by using both culture-dependent and culture-independent approaches. The ripened fermented sausages were characterized by high microbial loads of both staphylococci and lactobacilli. Using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the variable V3 and V1 regions of the 16S rRNA gene and direct DNA sequencing, it was possible to identify Staphylococcus xylosus, S. succinus, and S. equorum among the staphylococci and Lactobacillus sakei and L. curvatus within the lactobacilli. Moreover, Debaryomyces hansenii was the main yeast species found by targeting the yeast 26S rRNA gene by PCR-DGGE. Selected strains of S. xylosus, L. sakei, and L. curvatus were characterized for their technological properties in the ripening conditions of the fermented sausages so as to select an autochthonous starter formulation. The selection included the determination of nitrate reductase, lipolytic, and antioxidant activity and proteolysis with myofibrillar and sarcoplasmic protein fractions. Such properties were evaluated in both in vitro and in situ assays; the latter were performed by using each strain as a starter in the laboratory-scale manufacture of soppressata of Vallo di Diano and by monitoring the microbiological and chemical changes at the end of ripening. The results show differences between the in vitro and in situ selection results and indicate that in situ evaluation of the technological performance of specific strains is better suited to selecting autochthonous starter cultures for fermented-meat products than in vitro evaluation.     

Phenotypic diversity of lactic acid bacteria isolated from fermented sausages produced in Basilicata (Southern Italy)

AIMS: to evaluate the evolution of lactic acid bacteria (LAB) populations in traditional fermented sausages (salsiccia and soppressata) produced in artisanal and industrial plants in Basilicata (Southern Italy). METHODS AND RESULTS: Four hundred and fourteen lactic acid bacteria (LAB) cultures were isolated from samples of sausages at different stages of ripening. A phenotypic characterization of the isolates was carried out using a set of 28 tests, and 34 clusters were identified at the 80% similarity level using hierarchical cluster analysis. Of the isolates 50% were identified as Lactobacillus sakei (with several biotypes), 22% as Pediococcus spp. (mainly Ped. pentosaceus), 7% as Leuconostoc (Leuc. carnosum, Leuc. gelidum, Leuc. pseudomesenteroides), 6% as Lact. plantarum, 1% as Lact. curvatus. Other lactobacilli, including unidentified species, were present in lower numbers. CONCLUSION: The phenotypic diversity and composition of the LAB flora varied as a function of the production plant, product type and ripening time. SIGNIFICANCE AND IMPACT OT THE STUDY: A new procedure based on bootstrapping and Multidimensional Scaling was successfully used to obtain a graphical representation of the evolution of the LAB populations.     

The influence of starter cultures on the formation of volatile compounds in dry smoked sausages

The differences between the composition of volatile substances in two specimens of dry smoked sausages produced using a standard and experimental (a mixture of propionic acid bacteria and bifidobacteria) cultures were studied by capillary gas chromatography. It was found that the experimental starter culture intensified the flavor-formation processes as compared with the standard culture. The experimental specimen had richer qualitative and quantitative compositions and displayed more intensive aroma and flavor. The contents of lactones and volatile terpenoids in the experimental specimen were much higher than in the control. The organoleptic characteristics of experimental dry smoked sausage specimen were considerably better.     

Ecology and dynamics of coagulase-negative cocci isolated from naturally fermented Italian sausages

Coagulase-negative cocci (CNC) ecology in naturally fermented sausages from Friuli Venezia Giulia region, in the North East of Italy, was investigated. A total of 617 CNC strains, isolated from three different plants during the fermentation process, were identified by traditional methods (biochemical tests) and molecular methods based on species specific PCR, PCR-Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing of the V3 region of the 16S rRNA gene. The identification, by using biochemical tests, was not successful for 130 strains. Moreover, incongruent results were observed comparing the traditional with the molecular identifications. The same species of CNC were found in all three processing plants, but their contribution to the fermentations was different. In two plants Staphylococcus xylosus was the main species involved in fermentation process, while in the third the maturation was carried out equally by three species: S. xylosus, Staphylococcus warneri and Staphylococcus pasteuri.     

Culture-dependent and -independent methods to investigate the microbial ecology of Italian fermented sausages

In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (> 100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.     

Multiphasic approach to study the bacterial ecology of fermented sausages inoculated with a commercial starter culture

In this paper, the ability of a commercial starter culture to perform a sausage fermentation is evaluated. Molecular analysis revealed the presence of several strains of the same species contained in the starter culture with different behavior during the fermentation, and the contribution of Lactobacillus curvatus, which was only marginally isolated during the transformation.     

PCR-DGGE analysis for the identification of microbial populations from Argentinean dry fermented sausages

Different PCR-DGGE protocols were evaluated to monitor fermentation process and to investigate bacterial communities developed in two artisanal Argentinean fermented sausages. Bacterial universal primers frequently used in PCR-denaturing gradient gel electrophoresis (DGGE) were evaluated. Lactic acid bacteria (LAB) and staphylococci species isolated from Tucumán sausages were used to determine the experimental conditions for PCR amplification and DGGE differentiation. Total microbial DNA extracted directly from both fermented sausages was subjected to DGGE analysis. PCR-DGGE results were different for each set of primers used. Primers Bact-0124f(GC)-Uni-0515r and V1f(GC)-V1r showed to be efficient to differentiate LAB and Staphylococcus cultures while the set V3f(GC)-Uni-0515r allowed to demonstrate the succession of different Lactobacillus and Staphylococcus species during ripening process. An intense band corresponding to Lactobacillus sakei was observed to be present in both samples. Staphylococcus saprophyticus was only observed in Tucumán sausage while a band identified as Brochothrix thermophacta was detected in Córdoba sausage. PCR-DGGE analysis of different 16S rDNA amplicons was able to discriminate between LAB and Gram-positive, coagulase-negative cocci, resulting an effective tool to establish the microbiota developed in artisanal dry sausages.     

Flavour profile of idli batter prepared from defined microbial starter cultures

Idli is a traditional cereal/legume-based naturally fermented steamed product with a soft and spongy texture which is highly popular and widely consumed as a snack food item in India. The predominant fermentation microflora comprises lactic acid bacteria and yeasts and causes an improvement in the nutritional, textural and flavour characteristics of the final product. The flavour profile of idli batter prepared with initial levels of 2 × 10⁴ c.f.u. g⁻¹ of Candida versatilis CFR 505 and 2 × 10¹ c.f.u. g⁻¹ of Pediococcus pentosaceus CFR 2123 in 500 g idli batter, packed in polyester polylaminate pouches and stored at 30 ± 2 °C was periodically analysed by GC-MS. The desirable flavour compounds such as ketones, diols and acids were found to be present upto 8 days of storage, whereas undesirable flavours like sulphurous and oxazolidone compounds, ethanone and thiazole appeared in the batter subsequent to 6 days of storage. The sensory attributes of idlis (final product) prepared from the stored batter related well to the determined flavour profile. The present study appeared to indicate that the flavour profile of traditional fermented foods can be a reliable qualitative and quantitative parameter for objective assessment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Functional implications of the microbial community structure of undefined mesophilic starter cultures

This review describes the recent advances made in the studies of the microbial community of complex and undefined cheese starter cultures. We report on work related to the composition of the cultures at the level of genetic lineages, on the presence and activity of bacteriophages and on the population dynamics during cheese making and during starter culture propagation. Furthermore, the link between starter composition and starter functionality will be discussed. Finally, recent advances in predictive metabolic modelling of the multi-strain cultures will be discussed in the context of microbe-microbe interactions.     

Functional implications of the microbial community structure of undefined mesophilic starter cultures

This review describes the recent advances made in the studies of the microbial community of complex and undefined cheese starter cultures. We report on work related to the composition of the cultures at the level of genetic lineages, on the presence and activity of bacteriophages and on the population dynamics during cheese making and during starter culture propagation. Furthermore, the link between starter composition and starter functionality will be discussed. Finally, recent advances in predictive metabolic modelling of the multi-strain cultures will be discussed in the context of microbe-microbe interactions.

     

Identification and tracing of Enterococcus spp. by RAPD-PCR in traditional fermented sausages and meat environment

Aims:  Four local small-scale factories were studied to determine the sources of enterococci in traditional fermented sausages.
Methods and Results:  Different points during the production of a traditional fermented sausage type ( fuet ) were evaluated. Randomly amplified polymorphic DNA (RAPD)-PCR was used to type 596 Enterococcus isolates from the final products, the initial meat batter, the casing, the workers' hands and the equipment. Species-specific PCR-multiplex and the partial sequencing of atpA gene and 16S rRNA gene sequencing allowed the identification of the isolates: Enterococcus faecalis (31·4%), Enterococcus faecium (30·7%), Enterococcus sanguinicola (14·9%), Enterococcus devriesei (9·7%), Enterococcus malodoratus (7·2%), Enterococcus gilvus (1·0%), Enterococcus gallinarum (1·3%), Enterococcus casseliflavus (3·4%), Enterococcus hermanniensis (0·2%), and Enterococcus durans (0·2%) . A total of 92 different RAPD-PCR profiles were distributed among the different factories and samples evaluated. Most of the genotypes found in fuet samples were traced back to their source.
Conclusions:  The major sources of enterococci in the traditional fermented sausages studied were mainly the equipment followed by the raw ingredients, although a low proportion was traced back to human origin.
Significance and Impact of the Study:  This work contributes to determine the source of enterococcal contamination in fermented sausages and also to the knowledge of the meat environment.     

Effects of initial moisture content of Korean traditional wheat-based fermentation starter nuruk on microbial abundance and diversity

The brewing of makgeolli, one of Korea’s most popular alcoholic beverages that is gaining popularity globally, is facilitated by nuruk, a traditional Korean cereal starter. The nuruk microbiome greatly influences the fermentation process as well as the nutritional, hygienic, and aromatic qualities of the product. This study is a continuation of our efforts to examine nuruk biodiversity at a depth previously unattainable. In this study, microfloral dynamics in wheat-based nuruk C, composed of traditional ingredients such as barley, green gram, and wheat and fermented under various internal moisture contents of 20% (C20), 26% (C26), and 30% (C30), was evaluated using 454 pyrosequencing during the 30-day fermentation process. Rarefaction analysis and alpha diversity parameters indicated adequate sampling. C20 showed the greatest fungal richness and diversity, C20 and C26 exhibited similar bacterial richness and diversity, while C30 had low fungal and bacterial richness. Fungal taxonomic assignments revealed that the initial moisture content caused selective enrichment of Aspergillus candidus with a decreasing trend during fermentation, whereas Saccharomycetales sp. exhibited increasing relative abundance with increasing moisture content from day 6 of the fermentation process. Depending on initial moisture level, changes in bacterial communities were also observed in the genera Streptomyces, Bacillus, and Staphylococcus, with decreasing trends whereas Saccharopolyspora exhibited a sigmoidal trend with the highest abundance in C26. These findings demonstrate the possible impact of initial moisture content of nuruk on microfloral richness, diversity, and dynamics; this study is thus a step toward our ultimate goal of enhancing the quality of nuruk.

     

传统发酵肉制品中微生物菌群对风味形成的研究进展

我国传统发酵肉制品种类丰富、风味独特。本文主要介绍参与肉制品发酵的微生物,从蛋白质、脂质和碳水化合物代谢途径的角度概述微生物对发酵肉制品呈香物质形成的作用机制,以及发酵过程中微生物的演替与风味变化的关系。