Sourdough bacterial dynamics revealed by metagenomic analysis in Brazil.

This study dealt with the influence of the temperature on the bacterial dynamics of two spontaneously fermented wheat sourdoughs, propagated at 21 ±â€¯1 °C (SD1) and 30 ±â€¯1 °C (SD2), during nine backslopping steps (BS1 to BS9). Proteobacteria was the only phylum found in flour. Escherichia hermannii was predominant, followed by Kosakonia cowanii, besides species belonging to the genera Pantoea and Pseudomonas. After one step of propagation, Clostridium and Bacillus cereus group became predominant. Lactobacillus curvatus was found at low relative abundance. For the second backslopping step, Clostridium was flanked by L. curvatus and Lactobacillus farciminis. From BS4 (6th day) onward, lactic acid bacteria (LAB) became predominant. L. farciminis overcame L. curvatus and remained dominant until the end of propagations for both sourdoughs. At 21 °C, Bacillus, Clostridium, Pseudomonas, and Enterobacteriaceae were gradually inhibited. At the end of propagation, SD1 harbored only LAB. Otherwise, the temperature of 30 °C favored the persistence of atypical bacteria in SD2, as Pseudomonas and Enterobacteriaceae. Therefore, the temperature of 21 °C was more suitable for sourdough propagation in Brazil. This study enhanced the knowledge of temperature's influence on microbial assembly and contributed to the elucidation of sourdough microbial communities in Brazil.

Lysozyme affects the microbial catabolism of free arginine in raw-milk hard cheeses.

Lysozyme (LZ) is used in several cheese varieties to prevent late blowing which results from fermentation of lactate by Clostridium tyrobutyricum. Side effects of LZ on lactic acid bacteria population and free amino acid pattern were studied in 16 raw-milk hard cheeses produced in eight parallel cheese makings conducted at four different dairies using the same milk with (LZ+) or without (LZ-) addition of LZ. The LZ-cheeses were characterized by higher numbers of cultivable microbial population and lower amount of DNA arising from lysed bacterial cells with respect to LZ + cheeses. At both 9 and 16 months of ripening, Lactobacillus delbrueckii and Lactobacillus fermentum proved to be the species mostly affected by LZ. The total content of free amino acids indicated the proteolysis extent to be characteristic of the dairy, regardless to the presence of LZ. In contrast, the relative patterns showed the microbial degradation of arginine to be promoted in LZ + cheeses. The data demonstrated that the arginine-deiminase pathway was only partially adopted since citrulline represented the main product and only trace levels of ornithine were found. Differences in arginine degradation were considered for starter and non-starter lactic acid bacteria, at different cheese ripening stages.

Nonstarter lactic acid bacteria volatilomes produced using cheese components.

In long-ripened cheese, flavor formation occurs during ripening. The metabolism of lactic acid bacteria (LAB) leads to the production of different compounds that contribute to the flavor of cheese. The contribution of LAB to the formation of cheese flavor has previously been studied. However, the specific nonstarter LAB (NSLAB) metabolic reactions in ripened cheese that lead to the formation of flavor compounds remain unclear. In ripened cheese, the nutrient sources available include small peptides or amino acids, citrate, lactate, free fatty acids, and starter LAB cell lysis products. Thus, the aim of this study was to evaluate the ability of NSLAB to produce volatile flavor compounds by using an in vitro system that used only the nutrients available in ripened cheese as the energy source. Moreover, the potential contribution of the NSLAB volatilome on total cheese flavor is discussed. For this purpose, the production of volatile compounds on cheese-based medium (CBM) and on starter LAB lysed cell medium (LCM) by 2 Lactobacillus casei and 2 Lactobacillus rhamnosus strains, previously isolated from ripened Parmigiano Reggiano cheese, was investigated. The generated volatile compounds were analyzed with head-space gas chromatography mass spectrometry. Overall, ketones, aldehydes, alcohols, and acids were the most abundant compounds produced. Differences in volatilome production were found between NSLAB grown in LCM and CBM. The catabolic metabolism of amino acids and fatty acids were required for NSLAB growth on LCM. Conversely, pyruvate metabolism was the main catabolic pathway that supported growth of NSLAB in CBM. This study can be considered a first step toward a better understanding of how microbiota involved in the long ripening of cheese may contribute to the development of cheese flavor.

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains.

Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk.

cDNA-amplified fragment length polymorphism to study the transcriptional responses of Lactobacillus rhamnosus growing in cheese-like medium.

AIMS: Lactobacillus rhamnosus is a dominant species during Parmigiano Reggiano cheese ripening and exhibits a great adaptability to unfavourable growth conditions. Gene expression of a Lact. rhamnosus, isolated from Parmigiano Reggiano cheese, grown in a rich medium (MRS) and in a cheese-like medium (CB) has been compared by a novel cDNA-amplified fragment length polymorphism (cDNA-AFLP) protocol. METHODS AND RESULTS: Two techniques, capillary and gel electrophoresis cDNA-AFLP, were applied to generate unique transcript tags from reverse-transcribed messenger RNA using the immobilization of biotinylated 3'-terminal cDNA fragments on streptavidin-coated Dynabeads. The use of three pairs of primers allowed detecting 64 genes expressed in MRS and 96 in CB. Different transcripts were observed when Lact. rhamnosus was cultured on CB and MRS. CONCLUSIONS: The cDNA-AFLP approach proved to be able to show that Lact. rhamnosus modifies the expression of a large part of genes when cultivated in CB compared with growth under optimal conditions (MRS). In particular, the profiles of the strain grown in CB were more complex probably because the cells activate different metabolic pathways to generate energy and to respond to the environmental changes. SIGNIFICANCE AND IMPACT OF STUDY: This is the first research on Lact. rhamnosus isolated from cheese and represents one of the few concerning bacterial transcriptomic analysis towards cDNA-AFLP approaches.

Comparison of phenotypic (Biolog System) and genotypic (random amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) methods for typing Lactobacillus plantarum isolates from raw vegetables and fruits.

The diversity of 72 isolates of Lactobacillus plantarum, previously identified from different raw vegetables and fruits, was studied based on phenotypic (Biolog System) and genotypic (randomly amplified polymorphic DNA-polymerase chain reaction, RAPD-PCR, and amplified fragment length polymorphism, AFLP) approaches. A marked phenotypic and genotypic variability was found. Eight clusters were formed at the similarity level of 92% based on Biolog System analysis. The most numerous clusters grouped isolates apart from the original habitat. Almost all isolates fermented maltose, D,L-lactic acid, N-acetyl-D-mannosamine and dextrin, and other typical carbon sources which are prevalent in raw vegetables and fruits. None of the isolates fermented lactose and free amino acids. At high values of linkage distance, two main clusters were obtained from both UPGMA (unweighted pair group with arithmetic average) dendrograms of RAPD-PCR and AFLP analyses. The two clusters mainly separated isolates from tomatoes and carrots from those isolated from pineapples. At 2.5 linkage distance, a high polymorphism was found and several sub-clusters were formed with both analyses. In particular, AFLP allowed the differentiation of 55 of the 72 isolates of L. plantarum. The discriminatory power of each technique used was calculated through the Simpson's index of diversity (D). The values of the D index were 0.65, 0.92 and 0.99 for Biolog System, RAPD-PCR and AFLP analyses, respectively.

Natural whey starter for Parmigiano Reggiano: culture-independent approach.

AIMS: The aim of this work was to obtain a deeper insight into the knowledge of microbial composition of Parmigiano Reggiano natural whey starters through different culture-independent methods. METHODS AND RESULTS: Eighteen different Parmigiano Reggiano natural whey starters sampled from three different provinces of this cheese production area and the nonacidified wheys from which they arose have been studied by length heterogeneity polymerase chain reaction (LH-PCR) and fluorescent in situ hybridization (FISH). A high microbial composition variability between different samples has been observed. CONCLUSIONS: Revealing different images of the same community, LH-PCR and FISH have given a more accurate view of the not well-known Parmigiano Reggiano whey starter ecosystem. SIGNIFICANCE AND IMPACT OF THE STUDY: New lights have been shed on Parmigiano Reggiano natural whey starters microbial composition, highlighting how culture-independent approach could be used and improved to study this and other food ecosystems.

Transcriptional analysis of the gdhA gene in Streptococcus thermophilus.

AIMS: To study the transcriptional analysis of glutamate dehydrogenase gene, involved in the amino acid conversion to aroma compound in Streptococcus thermophilus. METHODS AND RESULTS: Analysis of the gdhA gene nucleotide sequence of S. thermophilus CNRZ1066 revealed that the coding region is 1353 nucleotides long. The deduced amino acids sequence exhibits the putative GDH active site and some conserved domains characteristic of family I of hexameric GDHs. Phylogenetic analysis revealed that the gdh gene of S. thermophilus clustered with the orthologues of other streptococci such as Streptococcus mutans, Streptococcus agalactiae and Streptococcus infantarius. Studying the structural organization of the gdhA locus the amino acid similarity of GDHs was higher than 87%, but the locus organization was not conserved. A dominant transcript of approximately 1.4 kbp was revealed by Northern blot hybridization, suggesting that gdhA mRNA is monocystronic. Primer extension showed that transcription start point of gdhA was localized 43 bp upstream of the potential start codon (ATG). CONCLUSIONS: The gdhA represents a monocistronic operon highly conserved in phylogenetic-related bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: A deeper knowledge of gdh transcriptional mechanisms could lead to develop S. thermophilus industrial starter cultures with optimized aromatic properties.

Application of molecular approaches to study lactic acid bacteria in artisanal cheeses.

The present review focuses on the application of the most common molecular approaches for the investigation of the diversity of LAB in the artisanal cheese environment, and for their tracking within this complex ecosystem. Historically the detection and identification of lactic acid bacteria (LAB) has largely been hampered by the incomplete knowledge of their optimal in vitro culture conditions. Recent years have seen an explosion in the application of molecular tools for elucidating both qualitatively and quantitatively on the abundance of LAB species and how their presence interacts with the environment. Many of these approaches, predominantly rooted in the use of ribosomal RNA (rRNA) and its encoding genes, are successfully applied to describe the relationship between LAB and their individual identity. In the present work emphasis is given to the methodologies to determine the complex communities as well as to the detection of individual groups of LAB of several artisanal cheeses. Moreover, analyses of viability of LAB during the manufacture and ripening, through culture-independent approaches is also discussed. The potential of novel advances in molecular technologies such as metagenomic for analysis of the cheese ecosystem is also provided.

A model to assess lactic acid bacteria aminopeptidase activities in Parmigiano Reggiano cheese during ripening.

The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid beta-naphthylamide (betaNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an amino-peptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity amino-peptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.

Whey starter for Grana Padano cheese: effect of technological parameters on viability and composition of the microbial community.

This work aimed to investigate the effects of thermal treatments and yeast extract addition on the composition of the microbial community of natural whey starters for Grana Padano cheese. Different natural whey starter samples were held at 4 degrees C for 24 h (cooling treatment), or at -20 degrees C for 24 h (freezing treatment) to evaluate the possibility of conservation, or at 54 degrees C for 1 h (heat treatment) to evaluate the effect of the temperature commonly used during curd cooking. Separately, another set of samples was enriched with 0.3, 0.5, and 1.0% (wt/vol) of yeast extract to study its effect on the growth of lactic acid bacteria (LAB) in the starter. The new approach in this study is the use of 2 culture-independent methods: length heterogeneity (LH)-reverse transcription (RT)-PCR and fluorescence microscopy. These techniques allowed us to easily, quickly, and reproducibly assess metabolically active LAB in the control and treated samples. The LH-RT-PCR technique distinguished microorganisms based on natural variations in the length of 16S rRNA amplified by RT-PCR, as analyzed by using an automatic gene sequencer. Fluorescence microscopy counts were performed by using a Live/Dead BacLight bacterial viability kit. The repeatability of LH-RT-PCR showed that this technique has great potential to reveal changes in the microbial community of natural whey starters for Grana Padano cheese. All species showed low sensitivity to cold (4 degrees C). However, after the freezing (-20 degrees C) and heating (54 degrees C) treatments, different behaviors of the species were reported, with significant changes in their viability and relative composition. Heating treatment during curd cooking profoundly affected the viability and composition of the community that remained in the cheese and that consequently modified the microbial population. At the same time, this treatment produced the selection of LAB in whey and could be considered as the first step in natural whey starter production. Addition of yeast extract stimulated the growth of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. lactis to the detriment of Lactobacillus helveticus species. Because the yeast extract altered the microflora balance, whey starter conservation at -20 degrees C and yeast extract addition cannot be suggested as technological innovations.

A new validated HPLC-FLD method for detecting ochratoxin A in dry-cured meat and in blue cheese.

In the present study, a fast and sensitive method for the quantification of ochratoxin A in two lipidicproteic food matrices has been developed. In particular, the sample preparation procedure has been optimized for dry-cured meat products and blue cheeses and tested for several validation parameters (LOD, LOQ, recovery, repeatability and within-laboratory precision). The procedure has been then applied to several dry-cured meat products and blue cheeses from the market.Ochratoxin A has been occasionally found in dry-cured and smoked ham from the market and the contamination occurred both in the outer and in the inner part of the products. Concerning the blue cheese, the occurrence of ochratoxin A is reported for the first time: OTA was occasionally found at low levels (0.1-3 µg/kg) in commercial samples of Roquefort from France and Gorgonzola from Italy, opening a new issue for risk assessment and quality control.

Fluorescence microscopy for studying the viability of micro-organisms in natural whey starters.

AIMS: The aim of this work was to study the viability and cultivability of microbial populations of different natural whey starters and to evaluate their resistance to thermal treatments (such as exposure to high or low temperatures). METHODS AND RESULTS: Twenty-three natural whey starters for Grana Padano cheese were investigated and subsequently pH measurement, plate count agar using Man-Rogasa-Sharpe (MRS) pH 5.4 agar and whey agar medium (WAM) were performed using these samples. LIVE/DEAD BacLight bacterial viability kit was used. Total count and viability of all the 23 samples were high and similar to each other (CV 20%). However, the cultivable population was lower in terms of cfu ml(-1) and number of cells per millilitre than the viable fraction and highly variable, although its count value was higher in WAM than in MRS pH 5.4. The heating (60 degrees C for 5 min and 54 degrees C for 1 h) and freezing (-20 and -80 degrees C) treatments affected the cultivability and viability of the microbial population. CONCLUSIONS: This study demonstrated the effectiveness of LIVE/DEAD BacLight bacterial viability kit, which has already been used to evaluate bacterial populations, in investigating microbial viability in a complex ecosystem such as a natural whey starter. SIGNIFICANCE AND IMPACT OF THE STUDY: The aim of this study was to quantify the presence of damaged nonviable bacterial cells in natural whey starters. The Thoma Glass is a useful method to obtain fluorescence microscopy counts to evaluate the technological performance of natural whey starters.

High-pressure processing of Gorgonzola cheese: influence on Listeria monocytogenes inactivation and on sensory characteristics.

The presence of Listeria monocytogenes on the rind of Gorgonzola cheese is difficult to avoid. This contamination can easily occur as a consequence of handling during ripening. The aims of this study were to determine the efficiency of high-pressure processing (HPP) for inactivation of L. monocytogenes on cheese rind and to evaluate the influence of HPP treatments on sensory characteristics. Gorgonzola cheese rinds, after removal, were inoculated (about 7.0 log CFU/g) with L. monocytogenes strains previously isolated from other Gorgonzola cheeses. The inoculated cheese rinds were processed with an HPP apparatus under conditions of pressure and time ranging from 400 to 700 MPa for 1 to 15 min. Pressures higher than 600 MPa for 10 min or 700 MPa for 5 min reduced L. monocytogenes more than 99%. A reduction higher than 99.999% was achieved pressurizing cheese rinds at 700 MPa for 15 min. Lower pressure or time treatments were less effective and varied in effectiveness with the cheese sample. Changes in sensory properties possibly induced by the HPP were evaluated on four different Gorgonzola cheeses. A panel of 18 members judged the treated and untreated cheeses in a triangle test. Only one of the four pressurized cheeses was evaluated as different from the untreated sample. HPP was effective in the reduction of L. monocytogenes on Gorgonzola cheese rinds without significantly changing its sensory properties. High-pressure technology is a useful tool to improve the safety of this type of cheese.

A fluorescence-based method for the detection of adhesive properties of lactic acid bacteria to Caco-2 cells.

AIMS: The ability of probiotic micro-organisms to adhere to the intestinal surface is regarded as a substantial advantage in terms of bacteria persistence in the gastrointestinal tract. The aim of the present study was the development of a method based on fluorescent staining of bacteria and subsequent spectrofluorimetric detection to quantify the adhesion of several strains of Lactobacillus and Bifidobacterium to Caco-2 cells. METHODS AND RESULTS: Lactic acid bacteria strains were subjected to fluorescent staining using the viable probe carboxyfluorescein diacetate and subsequently incubated on Caco-2 monolayers. The adhesion of the micro-organisms was determined by spectrofluorimetry following the lysis of the attached bacterial cells and expressed as adhesion percentage. The values obtained for the micro-organisms tested ranged from 4% for Bifidobacterium infantis Bi1 to 10% for a Bifidobacterium mixture containing three different strains. CONCLUSIONS: In the present study we successfully applied fluorescent labelling and fluorimetric detection to investigate the adhesive properties of some Lactobacillus and Bifidobacterium strains and a Bifidobacterium mixture to Caco-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The results proved that fluorescent labelling is suitable for adhesion studies and provides a reliable and safer alternative to radioactive labelling.

Genotypic and phenotypic diversity of Lactobacillus delbrueckii subsp. lactis strains of dairy origin.

Eighty-nine strains of Lactobacillus delbrueckii subsp. lactis isolated from Italian hard and semi-hard cheeses and artisan starter cultures were characterised by phenotypic and genotypic methods. Phenotypic diversity was evaluated by studying biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR and pulsed field gel electrophoresis (PFGE). Phenotypic characterisation indicated a wide variability of the acidifying activity within Lact. delbrueckii subsp. lactis. Although the data was variable, it allowed us to evidence groups of strains with different acidifying properties, especially in terms of acidification intensity. Concerning peptidase activity, Lact. delbrueckii subsp. lactis showed a homogeneously high x-prolil-dipeptidil-aminopeptidase activity and a considerable but more heterogeneous lysil-aminopeptidase activity. The increased resolution obtained by the use of two molecular typing techniques, i.e. RAPD-PCR and PFGE, allowed to widen the level of strain heterogeneity. Technological and ecological pressures are determinant in selecting Lact. delbrueckii subsp. lactis sub-populations which are more functional to the different cheese technologies.

Evaluation of bacterial communities belonging to natural whey starters for Grana Padano cheese by length heterogeneity-PCR.

AIMS: To detect bacteria present in controlled dairy ecosystems with defined composition by length-heterogeneity (LH)-PCR. LH-PCR allows to distinguish different organisms on the basis of natural variations in the length of 16S rRNA gene sequences. METHODS AND RESULTS: LH-PCR was applied to depict population structure of the lactic acid bacteria (LAB) species recoverable from Grana Padano cheese whey starters. Typical bacterial species present in the LAB community were evidenced and well discriminated. Small differences in species composition, e.g. the frequent finding of Streptococcus thermophilus and the constant presence of thermophilic lactobacilli (Lactobacillus helveticus, Lact. delbrueckii subsp. lactis/bulgaricus and Lact. fermentum) were reliably highlighted. Specificity of LH-PCR was confirmed by species-specific PCR from total DNA of the cultures. CONCLUSIONS: LH-PCR is a useful tool to monitor microbial composition and population dynamics in dairy starter cultures. When present, non-dominant bacterial species present in the whey starters, such as Strep. thermophilus, can easily be visualized and characterized without isolating and cultivating single strains. A similar approach can be applied to more complex dairy ecosystems such as milk or cheese curd. SIGNIFICANCE AND IMPACT OF THE STUDY: Community members and differences in population structure of controlled dairy ecosystems such as whey starters for hard cheeses can be evaluated and compared in a relative easy, fast, reliable and highly reproducible way.

Peptidase activity in various species of dairy thermophilic lactobacilli.

AIMS: The aim of the present work was to evaluate the enzymatic potential manifested by aminopeptidase activity of different thermophilic Lactobacillus biotypes and to measure the influence of cell growth phase on enzyme expression. METHODS AND RESULTS: The activities were evaluated by the hydrolysis of beta-naphthylamide substrates for both whole and mechanically disrupted cells of L. helveticus, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains, collected from both the exponential and the stationary growth phase. In general, activities were higher for cells in the exponential rather than in the stationary phase and the disrupted cells showed higher activities than the whole cells. The highest activity expressed by all strains corresponded to X-prolyl-dipeptidyl aminopeptidase while a moderate activity was observed towards Arg-betaNa, Lys-betaNa and Leu-betaNa. The lowest activity was observed for Pro-betaNa. CONCLUSIONS: It may be inferred that the cell structure and the cell physiology are crucial to define the level of efficiency of expression for aminopeptidase activity. The two species may be characterized by a different enzymatic system that hydrolyses N-terminal leucine. SIGNIFICANCE AND IMPACT OF THE STUDY: The differences of peptidase activities in L. helveticus and L. delbrueckii species acquires an importance to comprehend their role in the biochemical events occurring in cheese ripening.

Biodiversity in Lactobacillus helveticus strains present in natural whey starter used for Parmigiano Reggiano cheese.

AIMS: Lactobacillus helveticus is the dominant microflora of the natural whey starters used for Parmigiano Reggiano cheese making. The aim of this work was to study the biodiversity of different strains of Lact. helveticus present in six cultures and to compare them with strains of the same species previously isolated from natural whey cultures used for Grana Padano and Provolone cheeses. METHODS AND RESULTS: Twenty different biotypes of Lact. helveticus strains were identified combining the results deriving from SDS-PAGE of cell surface proteins and PCR fingerprinting using M13 as a primer. The biotypes were present in varying amounts in the six natural whey starters and the biodiversity was demonstrated not only within the whey cultures, but also between the whey cultures. CONCLUSIONS: Lact. helveticus strains isolated from Parmigiano Reggiano whey cultures analysed by PCR M13, SDS-PAGE and RFLP were distinguishable from Lact. helveticus strains of different dairy origin, namely Grana Padano and Provolone natural whey starters. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of different Lact. helveticus biotypes seems to be related to the specific ecosystem of cheese making and may be considered as one of the elements contributing to the typicality of Parmigiano Reggiano cheese.

Molecular typing of Lactobacillus delbrueckii of dairy origin by PCR-RFLP of protein-coding genes.

Thirty-five strains of Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus isolated from dairy products were typed by restriction fragment length polymorphism (RFLP) of protein-coding genes. The strains were analysed by RFLP of PCR amplified, infragenic fragments of the following housekeeping genes: beta-galactosidase, lactose permease, and proline dipeptidase. Sequencing of the variable regions of the 16S rDNA was then performed on a reduced number of strains. PCR-RFLP analysis evidenced wide strain heterogeneity. Strains were grouped into genotypes according to both subspecies assignment and infra-species genetic polymorphism. This polymorphism was related to the presence of microbial groups within subspecies populations. The low infra-species sequence polymorphism detected in the variable region of the 16S rRNA gene did not enable to group the strains with the same sensitivity reached by PCR-RFLP of protein-coding genes. PCR-RFLP of protein-coding genes applied to L. delbrueckii seems a promising tool to evaluate microbial diversity within bacterial subpopulations. Differences among bacterial subpopulations based upon molecular heterogeneity in protein-coding genes would enable to better understand the role of strains from different ecological niches.