Phenotypic and PCR-based characterization of the microflora in Präst cheese during ripening

Microbiological sampling of Präst cheese from three cheese factories was done during ripening. The evolution of total bacterial counts, lactococci, lactobacilli, enterococci presumptive leuconostoc and pediococci was investigated after 30, 90, 180 and 270 days of ripening. Isolates (140) of non-starter lactic acid bacteria (NSLAB) from 12 Präst cheeses after 90, 180 and 270 days of ripening were examined. The isolates were tested by physiological and biochemical assays, species-specific PCR and 16S rDNA sequencing. The predominant NSLAB species was Lactobacillus paracasei. The development and evolution of the NSLAB microflora in Präst varied according to dairy plant and ripening time.     

Microbial diversity and succession during the manufacture and ripening of traditional, Spanish, blue-veined Cabrales cheese, as determined by PCR-DGGE

The diversity and dynamics of the dominant microbial communities arising during the manufacture and ripening of four batches of naturally fermented Cabrales cheese were investigated by the PCR-DGGE culture-independent technique. Total microbial DNA was extracted from cheese milk, curd and cheese samples and used as template material in PCR experiments to amplify the V3 region of the bacterial 16S rRNA gene, plus the D1 region of the eukaryotic 26S rRNA gene. These regions were then analysed using DGGE. Eukaryotic and bacterial bands were identified by isolation, reamplification and sequencing. The results were compared to those obtained in a previous microbial characterization of the same four batches using classical culturing methods. Great variability was recorded between batches by the PCR-DGGE technique. This was also shown by culturing, and underlines the uniqueness of artisanal products. Lactocococcus lactis subsp. lactis was dominant from the cheese milk stage until the end of ripening, whereas populations of certain Lactobacillus species appeared during ripening. Populations of species never isolated by culturing were found to be numerous by the PCR-DGGE method, in particular Lactococcus garvieae and Lactococcus raffinolactis. Other, completely unknown lactococci were also detected. The dominant eukaryotic populations from day 15 onwards were those of Penicillium roqueforti and Geotrichum candidum.     

Phenotypic and PCR-based characterization of the microflora in Norvegia cheese during ripening

Microbiological sampling of Norvegia cheese from three cheese factories was done during ripening. The evolution of aerobic mesophilic bacteria, lactococci, lactobacilli, enterococci, presumptive leuconostoc and pediococci was investigated after 30, 90, 180 and 270 days of ripening. Isolates (135) of non-starter lactic acid bacteria (NSLAB) from nine Norvegia cheeses after 90, 180 and 270 days of ripening were examined. The isolates were tested by physiological and biochemical assays, species-specific PCR and 16S rDNA sequencing. After 90 days of ripening Leuconostoc spp., most probably from the starter, and the NSLAB specie Lactobacillus paracasei dominated among the isolates, however, after longer ripening Lb. paracasei dominated. The development and evolution of the microflora in Norvegia varied according to dairy and ripening time.     

Artisanal and experimental Pecorino Siciliano cheese: microbial dynamics during manufacture assessed by culturing and PCR-DGGE analyses

Traditional artisanal Pecorino Siciliano (PS) cheeses, and two experimental PS cheeses were manufactured using either raw or pasteurised ewes' milk with the addition of starter cultures. The bacterial diversity and dynamics of the different cheese types were evaluated both by culturing and characterisation of isolates, and a culture-independent approach based on the 16S ribosomal RNA (rRNA) gene. Following cultivation, artisanal and experimental cheese types showed similar microbial counts, and isolates belonging to Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecalis and Leuconostoc mesenteroides were identified by phenotypic characterisation and comparison of the restriction fragment length polymorphism (RFLP) of the 16S rRNA gene to that of reference species. The culture-independent fingerprinting technique PCR and denaturing gradient gel electrophoresis (DGGE) of V6 to V8 regions of the 16S rRNA gene of samples taken during artisanal PS cheese manufacture, from raw milk to the ripened cheese, indicated relevant shifts in the microbial community structure. The dominance of Streptococcus bovis and Lactococcus lactis species in the traditional artisanal PS was revealed by 16S rRNA gene sequencing. Comparison of DGGE profiles of samples from milk to ripened cheese, derived from artisanal procedure and the two experimental PS cheeses during production showed similar trends with the presence of intense bands in common. Nevertheless, the profiles of several artisanal cheeses from different farms appeared more diverse, and these additional species are probably responsible for the generally superior flavour and aroma development of traditional PS cheese.     

Hot topic: Geographical distribution and strain diversity of Lactobacillus wasatchensis isolated from cheese with unwanted gas formation

Lactobacillus wasatchensis, an obligate heterofermentative nonstarter lactic acid bacteria (NSLAB) implicated in causing gas defects in aged cheeses, was originally isolated from an aged Cheddar produced in Logan, Utah. To determine the geographical distribution of this organism, we isolated slow-growing NSLAB from cheeses collected in different regions of the United States, Australia, New Zealand, and Ireland. Seven of the cheeses showed significant gas defects and 12 did not. Nonstarter lactic acid bacteria were isolated from these cheeses on de Man, Rogosa, and Sharpe medium supplemented with ribose, a preferred substrate for Lb. wasatchensis. Identification was confirmed with 16S rRNA gene sequencing and the API50CH (bioMérieux, Marcy l'Etoile, France) carbohydrate panel. Isolates were also compared with one another by using repetitive element sequence-based PCR (rep-PCR). Lactobacillus wasatchensis was isolated only from cheeses demonstrating late-gas development and was found in samples from 6 of the 7 cheeses. This supports laboratory evidence that this organism is a causative agent of late gas production defects. The rep-PCR analysis produced distinct genetic fingerprints for isolates from each cheese, indicating that Lb. wasatchensis is found in several regions across the United States and is not a local phenomenon.     

Isolation, characterization, and influence of native, nonstarter lactic acid bacteria on Cheddar cheese quality

To determine whether adventitious nonstarter lactic acid bacteria (NSLAB) might affect cheese flavor and quality, we studied a population of NSLAB present in 30 premium quality Cheddar cheeses (3-mo ripened) produced at a commercial facility in the United States. DNA fingerprinting analysis with a sensitive strategy for arbitrary priming polymerase chain reaction showed that 75 isolates corresponded to at least 18 distinct nonstarter organisms. According to ribotype database comparisons of representatives from the 18 groups, 9 matched Lactobacillus (closest to paracasei species), 8 matched Streptococcus thermophilus, and 1 matched to a Lactococcus species. This finding indicated that among the 75 NSLAB isolates, Lactobacillus made up 64%, S. thermophilus 32%, and Lactococcus 4%. Isolates representing 11 NSLAB groups were characterized for protease, peptidase, and diacetyl production. Based on this phenotypic analysis, two Lactobacillus isolates were evaluated as adjuncts in Cheddar cheese. All of the NSLAB identified from the adjunct cheese at 3 mo by DNA fingerprinting consisted of the adjunct lactobacilli, showing that the adjunct strains predominated throughout the early stages of ripening. The impact of adjunct lactobacilli was evident after 6 mo when free amino acids significantly increased and sensory scores improved in adjunct cheese as compared with a control cheese. The largest impact was found in adjunct cheese containing a blend of both lactobacilli strains. These results show that certain adventitious NSLAB positively contribute to flavor development.     

Evolution of microbial populations during traditional Feta cheese manufacture and ripening

In three different dairies (A, B and C) located in Peloponess region (Southern Greece), traditional Feta cheese trials took place February to March using mixtures of sheep's and goat's milk. Only small variations in the evolution of microbial groups were observed during the whole ripening period. The main groups, such as thermophilic cocci, mesophilic lactococci, thermophilic lactobacilli, nonstarter lactic acid bacteria (NSLAB), presumptive Leuconostoc, enterococci and micrococci, reached their highest levels during the first 16 days, and then declined approximately 1-2 log units until the end of ripening. The remaining groups investigated, comprising yeasts, coliforms and Escherichia coli, were highest at day 4. The yeasts remained constant, while coliforms and E. coli decreased sharply and were not detectable after 120 days of ripening. A number of 146 isolates (dairy A) taken from all stages of the manufacturing and ripening process were purified and studied. Lactobacillus plantarum (58/146) and isolates of related species Lactobacillus pentosus and Lactobacillus paraplantarum (16/146) were the most common microorganisms found during cheese ripening. Streptococcus thermophilus (23/146) and Lactobacillus delbrueckii subsp. bulgaricus (20/146) were detected in high levels up to 20 days, and then gradually reduced. Enterococcus faecium (29/146) was found in all manufacturing and ripening stages.     

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.     

Biofilm formation and contamination of cheese by nonstarter lactic acid bacteria in the dairy environment

Defects in cheese, such as undesirable flavors, gas formation, or white surface haze from calcium lactate crystals, can result from growth of nonstarter lactic acid bacteria (NSLAB). The potential for biofilm formation by NSLAB during cheese manufacturing, the effect of cleaning and sanitizing on the biofilm, and bacterial growth and formation of defects during ripening of the contaminated cheese were studied. Stirred-curd Cheddar cheese was made in the presence of stainless steel chips containing biofilms of either of two strains of erythromycin-resistant NSLAB (Lactobacillus curvatus strain JBL2126 or Lactobacillus fermentum strain AWL4001). During ripening, the cheese was assayed for total lactic acid bacteria, numbers of NSLAB, and percentage of lactic acid isomers. Biofilms of L. curvatus formed during cheese making survived the cleaning process and persisted in a subsequent batch of cheese. The starter culture also survived the cleaning process. Additionally, L. curvatus biofilms present in the vat dislodged, grew to high numbers, and caused a calcium lactate white haze defect in cheese during ripening. On the other hand, biofilms of L. fermentum sloughed off during cheese making but could not compete with other NSLAB present in cheese during ripening. Pulsed-field gel electrophoresis results verified the presence of the two biofilm strains during cheese making and in the ripening cheese. Probable contamination sites in the plant for other NSLAB isolated in the cheese were identified, thus supporting the hypothesis that resident NSLAB biofilms are a viable source of contamination in the dairy environment.     

Microbiological characterization of artisanal Raschera PDO cheese: analysis of its indigenous lactic acid bacteria

The aim of this research was to study the bacterial populations involved in the production of artisanal Raschera PDO cheese (Italian Maritime Alps, northwest Italy) in order to collect preliminary knowledge on indigenous lactic acid bacteria (LAB). A total of 21 samples of Raschera PDO cheese, collected from six dairy farms located in the production area, were submitted to microbiological analysis. LAB were randomly isolated from M17 agar, MRS agar and KAA plates and identified by combining PCR 16S-23S rRNA gene spacer analysis, species-specific primers and 16S rRNA gene sequencing. Biodiversity of Lactococcus lactis subsp. lactis isolates was investigated by RAPD-PCR. LAB microflora showed the highest count values among all microbial groups targeted. They reached counts of 10(9) colony forming unit (cfu)/g in cheese samples after 3 days of salting and 15 days of ripening. Yeast population also showed considerable count values, while enterococci and coagulase-negative cocci (CNC) did not overcome 10(7)cfu/g. L. lactis subsp. lactis was the species most frequently isolated from Raschera PDO samples at all different production stages while in aged cheeses Lactobacillus paracasei was frequently isolated. RAPD-PCR highlighted that isolates of L. lactis subsp. lactis isolated from Raschera PDO were highly homogeneous.     

非发酵剂乳酸菌A-3和D-3对半硬质山羊奶干酪成熟的影响

目的获得加速半硬质山羊奶干酪成熟的非发酵剂乳酸菌菌株(non-starterlacticacidbacteria,NSLAB)。方法以前期分离自地中海地区山羊奶干酪中的2株优良NSLAB菌株为研究对象,测定其对干酪成熟过程中组成成分、微生物菌群、蛋白质水解和质构的影响。结果添加NSLAB菌株对干酪组成成分没有显著影响, NSLAB菌株没有影响乳球菌生长,在干酪成熟期间pH 4.6-SN和12%TCA-SN逐渐增加,且添加NSLAB的干酪在成熟30 d后显著增加了pH 4.6-SN和12%TCA-SN含量, 5%PTASN/TN的增加主要是由于乳酸菌中肽酶作用的结果, SDS-PAGE电泳结果说明添加NSLAB菌株的干酪中小分子多肽含量明显比对照干酪多,RP-HPLC分析得出干酪水溶性中肽的数量随着成熟时间增加。添加NSLAB菌株A-3没有改变干酪的硬度,使干酪的弹性增加。结论添加菌株A-3作为NSLAB的干酪样品中微生物自溶率高,蛋白水解程度强,质构性能良好,具有加速干酪成熟的潜力,是山羊奶干酪工业化生产的优良NSLAB。     

Diversity and dynamic of lactic acid bacteria strains during aging of a long ripened hard cheese produced from raw milk and undefined natural starter

The aim of this study was to explore diversity and dynamic of indigenous LAB strains associated with a long ripened hard cheese produced from raw milk and undefined natural starter such as PDO Grana Padano cheese. Samples of milk, curd, natural whey culture and cheeses (2nd, 6th, 9th and 13th months of ripening) were collected from 6 cheese factories in northern Italy. DNA was extracted from each sample and from 194 LAB isolates. tRNA^Ala-23S rDNA-RFLP was applied to identify isolates. Strain diversity was assessed by (GTG)5 rep-PCR and RAPD(P1)-PCR. Finally, culture-independent LH-PCR (V1–V2 16S-rDNA), was considered to explore structure and dynamic of the microbiota. Grana Padano LAB were represented mainly by Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus helveticus and Pediococcus acidilactici, while the structure and dynamic of microbiota at different localities was specific. The strength of this work is to have focused the study on isolates coming from more than one cheese factories rather than a high number of isolates from one unique production. We provided a valuable insight into inter and intraspecies diversity of typical LAB strains during ripening of traditional PDO Grana Padano, contributing to the understanding of specific microbial ecosystem of this cheese.     

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase‐positive cocci (GCC) population in sucuk, a traditional Turkish dry‐fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA‐PCR (RAPD‐PCR) and repetitive extragenic palindrome‐PCR (rep‐PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real‐time PCR DNA melting curve analysis and high‐resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species‐specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep‐PCR and/or PCR‐RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.     

Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system

Isolates of Non-Starter Lactic Acid Bacteria (NSLAB) from six ripened Danbo cheeses of different ages and of different brands were examined. Special emphasis was on the genus Lactobacillus with the aim of investigating their role in cheese maturation. Thirty-three isolates were typed by the PCR-based method, Randomly Amplified Polymorphic DNA (RAPD). Ten RAPD types were found and 70% of the isolates were of RAPD types found in more than one cheese. The different RAPD types were identified to species level by Temporal Temperature Gradient Gel Electrophoresis (TTGE). Most of the isolates were identified as Lactobacillus paracasei (76%), but also Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus rhamnosus and some taxa originating from the starter culture were detected. In one cheese, no lactobacilli were found.One strain of the most frequent Lactobacillus RAPD type from each of the five cheeses with a Lactobacillus flora was used as adjunct cultures in a cheese model system. Four of the five adjuncts were re-isolated during ripening. Two adjunct containing model cheeses received higher flavour scores than the control while two other were associated with off-flavours. The two model cheeses with off-flavour had a similar microflora and both were after 13 weeks of ripening dominated by a strain identified as L. plantarum.     

The microbial ecology of a typical Italian salami during its natural fermentation

We have investigated the bacteria and yeast ecology of the typical Italian Ciauscolo salami that is produced in Central Italy using a polyphasic approach based on culture-dependent and -independent methods. The physico-chemical analyses showed a progressive drop in pH and water activity (aw) during ripening. The viable counts revealed a dominance of lactic acid bacteria (LAB) over coagulase negative cocci (CNC) and yeasts. From the molecular identification of the isolates, the prevalence of Lactobacillus curvatus, Lb. plantarum and Staphylococcus xylosus was shown among the bacteria, while Debaryomyces hansenii was the prevalent species among the yeasts, and it was isolated throughout the whole ripening process. Minority species, namely Rhodotorula mucillaginosa and Trichosporon brassicae, were also recovered from the meat batter. The total microbial community was profiled without cultivation by analyzing the DNA that was directly extracted from the salami samples. Moreover, the cultivable community was profiled by analyzing the DNA recovered from bulk cells that were obtained by harvesting the colonies from serial-dilution agar plates. The 16S rRNA gene V1 and V3 regions were used as targets in the denaturing gradient gel electrophoresis (DGGE) profiling of the LAB and CNC communities, respectively, while the diversity and dynamics of the yeast population were assessed by analyzing a portion of the 28S rRNA gene. Our findings suggest that the microbial diversity of fermented meat products can be successfully investigated by this polyphasic approach that is based on the assessment of both the total and the cultivable community diversity.     

Microbial dynamics of Castelmagno PDO, a traditional Italian cheese, with a focus on lactic acid bacteria ecology

The dynamics of dominant microflora throughout the manufacture and ripening processes were evaluated in three batches of traditional Castelmagno PDO cheese. Milk, curd and cheese samples, at different stages during cheesemaking, were collected and subjected to culture-dependent and -independent analysis. Traditional plating and genetic identification of lactic acid bacteria (LAB) isolates, and PCR-DGGE analysis of V1 region of 16S rRNA gene were carried out. The collected samples were also monitored by HPLC for the presence of organic acids, sugars and ketones. LAB resulted to be the prevailing microflora in all production stages although enterococci, coagulase-negative cocci and yeasts also showed considerable viable counts probably related to the presence, in the dairy samples analysed, of free short-chain fatty acids detected by HPLC. Lactococcus lactis subsp. lactis was the species most frequently isolated during Castelmagno PDO manufacture, while Lactobacillus plantarum and Lactobacillus paracasei were isolated with the highest frequencies from ripened Castelmagno PDO cheese samples. Occasionally strains of Lactobacillus delbrueckii subsp. lactis, Lactobacillus coryniformis subsp. torquens and Lactobacillus casei were isolated. The results obtained on Castelmagno PDO microflora underlines a partial correspondence between culture-dependent method and DGGE analysis. Thus, in this study, it is highlighted once more the importance to combine molecular culture-independent approaches with classical microbiological methods for the study of complex environmental communities occurring in food matrices.     

Biodiversity of the microbial community in a Spanish farmhouse cheese as revealed by culture-dependent and culture-independent methods

The microbial diversity within Alberquilla cheese, made from a spontaneously fermented mixture of raw goats' and sheep's milk in the Alpujarra mountains (Granada, south-east Spain), has been studied by the classical culturing method and also by molecular analysis of community DNA. A collection of 206 isolates was obtained from the cheese on different selective/differential media, which were then re-grouped to 52 after randomly amplified polymorphic DNA (RAPD)-PCR analyses. Isolates on Man-Rogosa and Sharpe-agar (MRS), M17-glucose agar and Kenner Fecal (KF)-agar medium were identified by specific PCR or 16S rRNA gene sequencing and belonged mainly to the lactic-acid bacteria group. The predominant genus was Lactobacillus, which accounted for more than 50% of the isolates, the most abundant species being Lactobacillus paracasei, followed by considerably less quantities of Lb. plantarum and Lb. brevis. Other lactic-acid bacteria identified were Pediococcus urinaequi, Leuconostoc pseudomesenteroides, Leuc. mesenteroides, Lactococcus lactis and even the enterococci Enterococcus faecium and E. devriesei. Cluster analyses of RAPD-PCR patterns revealed a high degree of diversity among the lactobacilli. The Gram-negative bacterial strains belonged mainly to Hafnia alvei species. The microbes occurring in Alberquilla cheese were also studied by PCR temporal temperature-gradient gel electrophoresis (TTGE) of the 16S rRNA V3 region and partial 16S rRNA sequencing of the TTGE bands. The results showed a major presence of lactic-acid bacteria closely related to Lc. lactis, Lb. paracasei, Lb. plantarum, Lb. brevis, Lb. acidophilus and Enterococcus sp. The non-lactic-acid bacterium detected was identified as Escherichia coli. All the Enterococcus strains showed great susceptibility to the most clinically relevant antibiotics, harbouring only the virulence gene efaAfm. On the basis of their antimicrobial activity against Listeria monocytogenes we chose two strains of Ln. mesenteroides that produced mesenterocin B105 and mesenterocin Y105, as revealed by PCR techniques.     

Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model

Tyramine and histamine are the biogenic amines (BAs) most commonly found in cheese, in which they appear as a result of the microbial enzymatic decarboxylation of tyrosine and histidine respectively. Given their toxic effects, their presence in high concentrations in foods should be avoided. In this work, samples of three cheeses (Zamorano, Cabrales and Emmental) with long ripening periods, and that often have high BA concentrations, were screened for the presence of BA-degrading lactic acid bacteria (LAB). Seventeen isolates were found that were able to degrade tyramine and histamine in broth culture. All 17 isolates were identified by 16S rRNA sequencing as belonging to Lactobacillus casei. They were typed by plasmid S1-PFGE and genomic macrorestriction-PFGE analysis. Two strains (L. casei 4a and 5b) associated with high degradation rates for both BAs were selected to test how this ability might affect histamine and tyramine accumulation in a Cabrales-like mini-cheese manufacturing model. The quantification of BAs and the monitoring of the strains' growth over ripening were undertaken by RP-HPLC and qPCR respectively. Both strains were found to reduce histamine and tyramine accumulation. These two strains might be suitable for use as adjunct cultures for reducing the presence of BAs in cheese.     

Rapid differentiation of lactic acid bacteria from autochthonous fermentation of Iberian dry-fermented sausages

The populations of lactic acid bacteria (LAB) in different types of Iberian dry-fermented sausages from central-west Spain were identified. A simple and rapid electrophoretic method of whole-cell protein profiles was evaluated, correlating it with 16S rRNA gene sequence analysis and biochemical identification by API 50 CHL. A total of 96 isolates were identified by SDS-PAGE showing stable profiles corresponding to 30-45 polypeptides in the range 95-8kDa that were clearly different for the different species and were grouped with those of the 9 reference strains used in this study. The SDS-PAGE method showed that the predominant species were Pediococcus acidilactici (48%) followed by Lactobacillus plantarum (23%) and Lactobacillus brevis (18%). The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database. However, biochemical identifications by API 50 CHL showed different errors at the genus and species level. In sum, the SDS-PAGE analysis showed itself to be a rapid and accurate differentiation method for the most commonly encountered LAB isolates in dry-fermented sausages.     

An assessment of factors characterising the microbiology of Grana Trentino cheese,a Grana‐type cheese

In this study, microbiological aspects of Grana Trentino, a variant of Grana Padano cheese, were defined by plate counts, random amplified polymorphic DNA (RAPD) PCR genotying, 16S rRNA gene sequencing of bacterial isolates and PCR–denaturing gradient gel electrophoresis (PCR–DGGE). Results showed variability in monthly fluctuations of whey culture counts, differences in the diffusion of bacterial genotypes among producers and dairy plant‐specific microbial associations. Moreover, the presence of bacteria not previously reported in this cheese type was highlighted, including coagulase‐negative staphylococci and Lactobacillus sanfranciscensis‐like micro‐organisms.