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.

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.

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.

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.

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.

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.

TSH levels and TSH response to TRH as a guide to the replacement treatment of patients with thyroid carcinoma.

Forty-six patients whose thyroid glands had been ablated for thyroid carcinoma, maintained on replacement treatment with triiodothyronine (T3), have been followed by means of serial determinations of pituitary thyrotropin (TSH) levels and of the TSH response to thyrotropin-releasing hormone (TRH), in order to verify the actual dose required to suppress TSH secretion. The treatment was begun with a dose of T3 roughly related to body weight (about 1 mug/kg/day). The daily dose was sequentially adjusted according to the measured TSH levels. The dose of T3 which reduced serum TSH within the normal limits varied from 40 to 120 mug daily. More precisely, it was as low as 40-50 mug daily in five patients and as high as 90-120 mug daily in three patients. The dose, which was given according to their body weights, varied from 0.75 to 1.12 mug/kg/day. TRH administration produced a marked increase of TSH levels in those patients whose TSH was elevated, and varying responses (from no response to a response three times the normal one) in those patients whose TSH was within the normal limits. In the latter group of patients, an increase of the T3 dosage inhibited the TSH response to TRH. These data suggest that, even though 1 mug/kg/day is an adequate T3 dose for the great majority of patients, the extent of TSH suppression is not surely predictable, but must be evaluated case by case by means of serial determinations of TSH levels and of the TSH response to TRH.