Isolation and identification of cultivable lactic acid bacteria in traditional yak milk products of Gansu Province in China

Various traditional fermented yak milk and raw milk foods could be considered as an abundant resource for obtaining novel lactic acid bacteria (LAB) with unique properties. Eighty-eight samples of yak milk products were collected from Gansu Province in China. Three hundred and nineteen strains of LAB isolated from these samples were identified by phenotypic methods, 16S rRNA gene sequence analysis and PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technology. Among the isolates, one hundred and sixty-four isolates (51.41% of the total) were classified under Lactobacilli, and one hundred and fifty-five (48.59%) belonged to cocci. All the isolates were classified to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Weissella) and twenty-one species. Lactobacillus helveticus (87 strains), Leuconostoc mesenteroides subsp. mesenteroides (49 strains), Streptococcus thermophilus (39 strains), Lactobacillus casei (31 strains) and Lactococcus lactis subsp. lactis (19 strains) were considered as the predominant populations in the yak milk products. The results showed that there were abundant genus and species LAB existing in yak milk products in Gansu Province in China. The obtained LAB pure cultures may be a valuable source for further starter selection.     

Dominant lactic acid bacteria and their technological properties isolated from the Himalayan ethnic fermented milk products

Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China consume a variety of indigenous fermented milk products made from cows milk as well as yaks milk. These lesser-known ethnic fermented foods are dahi, mohi, chhurpi, somar, philu and shyow. The population of lactic acid bacteria (LAB) ranged from 10(7) to 10(8) cfu/g in these Himalayan milk products. A total of 128 isolates of LAB were isolated from 58 samples of ethnic fermented milk products collected from different places of India, Nepal and Bhutan. Based on phenotypic characterization including API sugar test, the dominant lactic acid bacteria were identified as Lactobacillus bifermentans, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus kefir, Lactobacillus hilgardii, Lactobacillus alimentarius, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Enterococcus faecium. LAB produced a wide spectrum of enzymes and showed high galactosidase, leucine-arylamidase and phosphatase activities. They showed antagonistic properties against selected Gram-negative bacteria. None of the strains produced bacteriocin and biogenic amines under the test conditions used. Most strains of LAB coagulated skim milk with a moderate drop in pH. Some strains of LAB showed a high degree of hydrophobicity, suggesting these strains may have useful adhesive potential. This paper is the first report on functional lactic acid bacterial composition in some lesser-known ethnic fermented milk products of the Himalayas.     

内蒙古呼伦贝尔地区传统发酵乳中乳酸菌的多样性分析

【目的】对内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌资源的生物多样性进行研究。【方法】采用纯培养和16S rRNA基因序列分析法对内蒙古呼伦贝尔地区传统发酵乳中的乳酸菌进行多样性分析。【结果】从8份传统发酵乳制品(6份酸牛奶和2份酸马奶)样品中分离到24株乳酸菌,通过16S rRNA基因序列分析和系统进化关系分析将24株乳酸菌鉴定为2株Lactobacillus kefiranofaciens、2株Lactobacillus kefiri、5株Lactobacillus paracasei、3株Lactobacillus plantarum、1株Lactobacillus rhamnosus、6株Lactococcus lactis subsp.lactis、2株Leuconostoc mesenteroides subsp.dextranicum、2株Streptococcus thermophilus和1株Enterococcus faecium。【结论】Lactococcus lactis subsp.lactis为内蒙古呼伦贝尔地区传统发酵乳制品的优势菌种,占总分离株的25%,其次为Lactobacillus paracasei,占总分离株的20.83%。     

Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.     

Phenotypic and phylogenetic analysis of lactic acid bacteria isolated from forage crops and grasses in the Tibetan Plateau

A total of 140 lactic acid bacteria (LAB) strains were isolated from corn, alfalfa, clover, sainfoin, and Indian goosegrass in the Tibetan Plateau. According to phenotypic and chemotaxonomic characteristics, 16S rDNA sequence, and recA gene PCR amplification, these LAB isolates were identified as belonging to five genera and nine species. Corn contained more LAB species than other forage crops. Leuconostoc pseudomesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus brevis, and Weissella paramesenteroides were dominant members of the LAB population on alfalfa, clover, sainfoin, and Indian goosegrass, respectively. The comprehensive 16S rDNA and recA-based approach effectively described the LAB community structure of the relatively abundant LAB species distributed on different forage crops. This is the first report describing the diversity and natural populations of LAB associated with Tibetan forage crops, and most isolates grow well at or below 10°C. The results will be valuable for the future design of appropriate inoculants for silage fermentation in this very cold area.     

Characterization of lactic acid bacteria isolated from the one humped camel milk produced in Morocco

One hundred and twenty (120) strains of lactic acid bacteria (LAB) were enumerated and isolated from raw dromedary milk in Morocco using various cultured media. Strains isolated were characterized by phenotypic, physiological and biochemical properties. Results showed that high counts of LAB were found. Presumptive lactobacilli counts ranged from 2.5x10(2) to 6x10(7)cfu/ml, presumptive lactococci levels varied from 5x10(2) to 6x10(7)cfu/ml, presumptive streptococci counts varied from 4.2x10(2) to 8x10(7)cfu/ml, presumptive leuconostoc levels ranged from 5.4x10(2) to 5.4x10(7)cfu/ml. Results showed also that Lactobacillus and Lactococcus were the predominant genera with 37.5% and 25.8%, respectively. The dominated species found were Lactococcus lactis subsp. lactis (17.5%), Lactobacillus helveticus (10%), Streptococcus salivarius subsp. thermophilus (9.20%), Lactobacillus casei subsp. casei (5.80%) and Lactobacillus plantarum (5%). This is the first report on the characterization of LAB strains isolated from the one humped camel milk produced in Morocco.     

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.     

Redox potential to discriminate among species of lactic acid bacteria

AIMS: To verify to what degree reducing capacity is a characterizing parameter of a species, and of the strains themselves within a given species, of lactic acid bacteria. METHODS AND RESULTS: Eighty-eight strains belonging to 10 species of lactic acid bacteria (LAB) isolated from traditional Italian cheeses were studied for their reduction activity: Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Streptococcus thermophilus, Lactococcus lactis ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus and Pediococcus pentosaceus. It was observed that the lactococci reached minimum redox potential before the lactobacilli. The reduction rate of Enterococcus spp. and L. lactis ssp. lactis was higher than that of the streptococci and Lactobacillus spp. All the P. pentosaceus strains had poor reduction activity compared with the other species. CONCLUSIONS: The evolution of the redox potential in milk over a time span of 24 h has been found to be a parameter that characterizes a species: the different courses corresponding to the species in question are clearly evident, and interesting differences can also be noted within the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: The reduction aptitude of strains might be used to select and adapt appropriate strains for use as starters for dairy products.     

Resident lactic acid bacteria in raw milk Canestrato Pugliese cheese

AIMS: Investigation of the autochthonous lactic acid bacteria (LAB) population of the raw milk protected designation of origin Canestrato Pugliese cheese using phenotypic and genotypic methodologies. METHODS AND RESULTS: Thirty phenotypic assays and three molecular techniques (restriction fragment length polymorphism, partial sequencing of the 16S rRNA gene and recA multiplex PCR assay) were applied to the identification of 304 isolates from raw milk Canestrato Pugliese cheese. As a result, 168 of 207 isolates identified were ascribed to genus Enterococcus, 25 to Lactobacillus, 13 to Lactococcus and one to Leuconostoc. More in details among the lactobacilli, the species Lactobacillus brevis and Lactobacillus plantarum were predominant, including 13 and 10 isolates respectively, whereas among the lactococci, Lactococcus lactis subsp.cremoris [corrected] was the species more frequently detected (seven isolates). CONCLUSIONS: Except for the enterococci, phenotypic tests were not reliable enough for the identification of the isolates, if not combined to the genotype-based molecular techniques. The polyphasic approach utilized allowed 10 different LAB species to be detected; thus suggesting the appreciable LAB diversity of the autochthonous microbial population of the Canestrato Pugliese cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: A comprehensive study of the resident raw milk Canestrato Pugliese cheese microbial population has been undertaken.     

Biodiversity of lactic acid bacteria in Moroccan soft white cheese (Jben)

The bacterial diversity occurring in traditional Moroccan soft white cheese, produced in eight different regions in Morocco, was studied. A total of 164 lactic acid bacteria were isolated, purified and identified by whole-cell protein fingerprinting and rep-PCR genomic fingerprinting. The majority of the strains belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Enterococcus. Sixteen species were identified: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, Lactococcus garvieae, Lactococcus raffinolactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc citreum, Eterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus saccharominimus and Streptococcus sp.     

Microbial Diversity in Ngari,Hentak and Tungtap,Fermented Fish Products of North-East India

Ngari, hentak and tungtap are traditional fermented fish products of North-East India. Eighteen samples of ngari, hentak and tungtap were collected and were analysed for microbial load. Lactic acid bacteria, endospore-forming rods, yeasts and aerobic mesophilic counts ranged from 4.0 to 7.2, 3.3–4.6, <1–3.5 and 4.3–7.3 log c.f.u./g, respectively. Lactic acid bacteria were identified as Lactococcus lactis subsp. cremoris, Lactococcus plantarum, Enterococcus faecium, Lactobacillus fructosus, Lactobacillus amylophilus, Lactobacillus coryniformis subsp. torquens and Lactobacillus plantarum. Endospore-forming rods were identified as Bacillus subtilis and Bacillus pumilus, aerobic coccal strains were identified as Micrococcus. Yeasts were identified as species of Candida and Saccharomycopsis. Pathogenic contaminants were detected in all samples, however, none of the sample contained more than 10² c.f.u./g of Bacillus cereus, 10³ c.f.u./g of Staphylococcus aureus and enterobacteriaceae population, respectively. Enzymatic and antimicrobial activities of the isolates were tested. None of the strains produced biogenic amines in the method applied. Most strains of LAB had a high degree of hydrophobicity, indicating their ‘probiotic’ characters. This study has demonstrated the microbial diversity within the species of lactic acid bacteria, Bacillus and yeasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

The clam Meretrix lamarckii (Bivalvia: Veneridae) is a rich repository of marine lactic acid bacterial strains

Lactic acid bacteria (LAB) were isolated from the intestinal tract of the wild clam Meretrix lamarckii caught from the coastal waters of Kashima, Ibaraki, Japan. As many as 415 isolates were obtained using the culture method, of which 70 were considered presumptive LAB strains based on phenotypic tests. Phylogenetic analysis of these presumptive isolates of LAB based on the sequence of the 16S rRNA gene demonstrated that the species belonged to several genera of Lactobacillus, Lactococcus and Pediococcus. Interestingly, however, the species composition was different between the samples in July and October 2010. Further analyses based on the fermentation profiles revealed that the LAB from the clam caught in July 2010 were identified to be Lactobacillus curvatus, Lactobacillus plantarum, Lactococcus lactis subsp. cremoris and Pediococcus pentosaceus, whereas those in October 2010 were identified to be Lactobacillus plantarum, Lactococcus lactis subsp. lactis and P. pentosaceus. The diversity of LAB in the intestinal tract of the clam suggests that the filter feeder bivalves such as M. lamarckii are a rich repository of marine isolates of LAB.     

Phenotypic and genotypic characteristics of lactic acid bacteria isolated from sour congee in Inner Mongolia of China

Sour congee is a popular food in the western regions of Inner Mongolia in China. It has a complex microbial population, which contributes to its unique flavor and functional properties. In this study, we chose 28 sour congee samples that were collected from different areas in Inner Mongolia for analysis of the microbial community of lactic acid bacteria (LAB) by classical biochemical tests, 16S rRNA gene sequencing, multiplex PCR assay of recA gene and restriction fragment length polymorphism (RFLP) analysis of the tuf gene (encoding elongation factor Tu). The results revealed that all the isolates were identified as Lactobacillus (L.) paracasei (38 strains), L. fermentum (28 strains), L. plantarum (7 strains), L. brevis (4 strains), L. reuteri (2 strains), L. amylolyticus (1 strain), Enterococcus (E.) faecalis (3 strains), E. italicus (2 strains) or Lactococcus lactis subsp. lactis (1 strain). The predominant LAB were L. casei and L. fermentum in sour congee samples. The diversity of LAB derived from sour congee could offer useful information for further research on sour congee, and the results demonstrated that the combination of tuf gene and RFLP patterns can be considered as a useful tool for differentiation of the L. casei group.     

Monitoring the bacterial community of maize silage stored in a bunker silo inoculated with Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri

Aims: To monitor variations in the bacterial community and fermentation products of maize silage within and between bunker silos. Methods and Results: Silage samples were collected in 2008 and 2009 from three dairy farms, wherein the farmers arranged for a contractor to produce maize silage using bunker silos. Silage was prepared using a lactic acid bacteria (LAB) inoculant consisting of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri. Eight samples were collected from each bunker silo; 4 ‘outer’ and 4 ‘inner’ samples were collected from near the top and the bottom of the silo. The dry matter, lactic acid, acetic acid, ethanol, 1‐propanol and 1,2‐propanediol contents differed between bunker silos in both sampling years. Higher acetic acid, 1‐propanol and 1,2‐propanediol contents were found in the bottom than the top layers in the 2008 samples, and higher lactic acid content was found in the top than the bottom layers in the 2009 samples. The bacterial community varied more between bunker silos than within a bunker silo in the 2008 samples, whereas differences between the top and the bottom layers were seen across bunker silos in the 2009 samples. The inoculated LAB were uniformly distributed, while several nonconventional silage bacteria were also detected. Lactobacillus acetotolerans, Lactobacillus panis and Acetobacter pasteurianus were detected in both years. Stenotrophomonas maltophilia was detected in the 2008 samples, and Lactobacillus reuteri, Acinetobacter sp. and Rahnella sp. were detected in the 2009 samples. Conclusions: Although differences were seen within and between bunker silos, the bacterial community may indicate a different relationship between bunker silos and sampling locations within a bunker silo from that indicated by the fermentation products. Significance and Impact of the Study: Analysis of bacterial community can help understand how diverse non‐LAB and LAB species are involved in the ensiling process of bunker‐made maize silage.     

Isolation and identification of lactic acid bacteria from Tarag in Eastern Inner Mongolia of China by 16S rRNA sequences and DGGE analysis

Tarag is a characteristic fermented dairy product with rich microflora (especially lactic acid bacteria), developed by the people of Mongolian nationality in Inner Mongolia of China and Mongolia throughout history. One hundred and ninety-eight samples of Tarag were collected from scattered households in Eastern Inner Mongolia, and total of 790 isolates of lactic acid bacteria (LAB) were isolated by traditional pure culture method. To identify these isolates and analyze their biodiversity, 16S rRNA gene sequences analysis and PCR-DGGE were performed respectively. The results showed that 790 isolates could be classified as 31 species and subspecies. Among these isolates, Lactobacillus helveticus (153 strains, about 19.4%), Lactococcus lactis subsp. lactis (132 strains, about 16.7%) and Lactobacillus casei (106 strains, about 11.0%) were considered as the predominated species in the traditional fermented dairy products (Tarag) in Eastern Inner Mongolia. It was shown that the biodiversity of LAB in Tarag in Inner Mongolia was very abundant, and this traditional fermented dairy product could be considered as valuable resources for LAB isolation and probiotic selection.     

Microbiological profile in Serra ewes' cheese during ripening

The microflora of Serra cheese was monitored during a 35 d ripening period at three different periods within the ewe's lactation season. After 7 d ripening, the numbers of micro-organisms reached their maximum, and lactic acid bacteria (LAB) and coliforms were the predominant groups. Pseudomonads were not detected after 1 week of ripening. At all stages of ripening, cheeses manufactured in spring exhibited the lowest numbers of LAB and yeasts, whereas cheeses manufactured in winter showed the lowest numbers of coliforms and staphylococci.
Leuconostoc lactis was the most abundant LAB found in Serra cheese whereas Enterococcus faecium and Lactococcus lactis spp. lactis exhibited the highest decrease in percentage composition. Numbers of both Leuc. mesenteroides and Lactobacillus paracasei tended to increase throughout ripening. The most abundant coliform was Hafnia alvei. Klebsiella oxytoca was found in curd but declined in number during ripening. Staphylococcal flora of curd was mainly composed of Staphylococcus xylosus, Staph. aureus and Staph. epidermidis. Staphylococcus xylosus was the major species found at the end of ripening. Pseudomonas fluorescens , was the only Pseudomonas species isolated from the curd. Although a broad spectrum of yeasts were found in Serra cheese, Sporobolomyces roseus was the most abundant yeast isolated.     

Use of real time PCR to determine population profiles of individual species of lactic acid bacteria in alfalfa silage and stored corn stover

Real-time polymerase chain reaction (RT-PCR) was used to quantify seven species of lactic acid bacteria (LAB) in alfalfa silage prepared in the presence or absence of four commercial inoculants and in uninoculated corn stover harvested and stored under a variety of field conditions. Species-specific PCR primers were designed based on recA gene sequences. Commercial inoculants improved the quality of alfalfa silage, but species corresponding to those in the inoculants displayed variations in persistence over the next 96 h. Lactobacillus brevis was the most abundant LAB (12 to 32% of total sample DNA) in all of the alfalfa silages by 96 h. Modest populations (up to 10%) of Lactobacillus plantarum were also observed in inoculated silages. Pediococcus pentosaceus populations increased over time but did not exceed 2% of the total. Small populations (0.1 to 1%) of Lactobacillus buchneri and Lactococcus lactis were observed in all silages, while Lactobacillus pentosus and Enterococcus faecium were near or below detection limits. Corn stover generally displayed higher populations of L. plantarum and L. brevis and lower populations of other LAB species. The data illustrate the utility of RT-PCR for quantifying individual species of LAB in conserved forages prepared under a wide variety of conditions.Disclaimer: Mention of products is for informational purposes only and does not imply a recommendation or warranty by USDA over other products that may also be suitable     

Isolation,identification and characterization of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> LB44 and <Emphasis Type="Italic">Weissella confusa</Emphasis> LM85 for the presence of bacteriocin-like inhibitory substances (BLIS)

Lactic acid bacteria (LAB) were isolated from different soil samples such as dairy, garden, rhizospheric soil and sediments of lake. Thirty nine strains of bacteria were isolated using direct spreading method but only one showed the characteristics of LAB. Whereas, using accumulation with incubation method, fifty seven strains were found to be LAB out of sixty eight strains of bacteria isolated. Therefore, accumulation with incubation method was found to be more effective and faster. On the basis of higher growth and antimicrobial activity, two potential isolates LB44 and LM85 were selected for the further characterization. Strain LB44 and LM85 were identified using 16S rDNA amplification and sequencing as Pediococcus pentosaceus LB44 and Weissella confusa LM85, respectively. Cell free supernatant (CFS) of these strains showed antimicrobial activity, stability at higher temperatures, acidic pH and in the presence of organic solvents, detergents and surfactants. Antimicrobial activity was not affected by treatment with catalase and lipase but reduced after treatment with proteinase K reveals the presence of bacteriocin-like inhibitory substance in CFS. CFS inhibited the growth of other Gram-positive and Gram-negative bacteria such as Micrococcus luteus, Lactobacillus delbrueckii, L. curvatus, L. plantarum, L. acidophilus, Enterobacter cloaceae, Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Bacillus subtilis, B. licheniformis, Salmonella typhi and Escherichia coli.     

Study of the ecology of fresh sausages and characterization of populations of lactic acid bacteria by molecular methods

In this study, a polyphasic approach was used to study the ecology of fresh sausages and to characterize populations of lactic acid bacteria (LAB). The microbial profile of fresh sausages was monitored from the production day to the 10th day of storage at 4 degrees C. Samples were collected on days 0, 3, 6, and 10, and culture-dependent and -independent methods of detection and identification were applied. Traditional plating and isolation of LAB strains, which were subsequently identified by molecular methods, and the application of PCR-denaturing gradient gel electrophoresis (DGGE) to DNA and RNA extracted directly from the fresh sausage samples allowed the study in detail of the changes in the bacterial and yeast populations during storage. Brochothrix thermosphacta and Lactobacillus sakei were the main populations present. In particular, B. thermosphacta was present throughout the process, as determined by both DNA and RNA analysis. Other bacterial species, mainly Staphylococcus xylosus, Leuconostoc mesenteroides, and L. curvatus, were detected by DGGE. Moreover, an uncultured bacterium and an uncultured Staphylococcus sp. were present, too. LAB strains isolated at day 0 were identified as Lactococcus lactis subsp. lactis, L. casei, and Enterococcus casseliflavus, and on day 3 a strain of Leuconostoc mesenteroides was identified. The remaining strains isolated belonged to L. sakei. Concerning the yeast ecology, only Debaryomyces hansenii was established in the fresh sausages. Capronia mansonii was initially present, but it was not detected after the first 3 days. At last, L. sakei isolates were characterized by randomly amplified polymorphic DNA PCR and repetitive DNA element PCR. The results obtained underlined how different populations took over at different steps of the process. This is believed to be the result of the selection of the particular population, possibly due to the low storage temperature employed.