Valentina Bernini

Dynamics of whole and lysed bacterial cells during Parmigiano-Reggiano cheese production and ripening.

ABSTRACT Microbial succession during Parmigiano-Reggiano cheesemaking was monitored by length heterogeneity PCR (LH-PCR), considering the intact and lysed cells at different stages of cheese production and ripening. When starter species underwent autolysis, species coming from milk were able to grow. For the first time, the LH-PCR technique was applied to study a fermented food.

Comparative genomics and transcriptional analysis of prophages identified in the genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei.

ABSTRACT Lactobacillus gasseri ATCC 33323, Lactobacillus salivarius subsp. salivarius UCC 118, and Lactobacillus casei ATCC 334 contain one (LgaI), four (Sal1, Sal2, Sal3, Sal4), and one (Lca1) distinguishable prophage sequences, respectively. Sequence analysis revealed that LgaI, Lca1, Sal1, and Sal2 prophages belong to the group of Sfi11-like pac site and cos site Siphoviridae, respectively. Phylogenetic investigation of these newly described prophage sequences revealed that they have not followed an evolutionary development similar to that of their bacterial hosts and that they show a high degree of diversity, even within a species. The attachment sites were determined for all these prophage elements; LgaI as well as Sal1 integrates in tRNA genes, while prophage Sal2 integrates in a predicted arginino-succinate lyase-encoding gene. In contrast, Lca1 and the Sal3 and Sal4 prophage remnants are integrated in noncoding regions in the L. casei ATCC 334 and L. salivarius UCC 118 genomes. Northern analysis showed that large parts of the prophage genomes are transcriptionally silent and that transcription is limited to genome segments located near the attachment site. Finally, pulsed-field gel electrophoresis followed by Southern blot hybridization with specific prophage probes indicates that these prophage sequences are narrowly distributed within lactobacilli.

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).

Recovery and differentiation of long ripened cheese microflora through a new cheese-based cultural medium

A partial picture of the typical microflora of PDO Parmigiano Reggiano cheese was achieved by studying the cultivability of lactic acid bacteria associated with its manufacturing and ripening. A comprehensive sampling design allowed for the analysis of the cheese microflora during its production over 20 months of ripening. An innovative cheese agar medium (CAM) was prepared after testing 18 formulations all based on grated Parmigiano Reggiano ripened cheese. During cheese manufacturing and ripening, different samples were sampled and their microflora was recovered using CAM in comparison with other traditional media. Colonies which formed units from the different agar media tested were picked and isolated; the phylogenetic positions of 154 isolated strains were studied at level of species by 16S-rRNA gene sequencing. CAM seems to be able to recover the minority population coming from milk and whey starter, hardly estimable, during the first hours of production, on traditional media.

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.

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 Simpsons 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.

Genetic Characterization of the Bifidobacterium breve UCC 2003 hrcA Locus

ABSTRACT The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes and transcriptional regulators, including the DnaJ and the HrcA proteins. Genome analysis of Bifidobacterium breve UCC 2003 revealed a second copy of a dnaJ gene, named dnaJ2, which is flanked by the hrcA gene in a genetic constellation that appears to be unique to the actinobacteria. Phylogenetic analysis using 53 bacterial dnaJ sequences, including both dnaJ1 and dnaJ2 sequences, suggests that these genes have followed a different evolutionary development. Furthermore, the B. breve UCC 2003 dnaJ2 gene seems to be regulated in a manner that is different from that of the previously characterized dnaJ1 gene. The dnaJ2 gene, which was shown to be part of a 2.3-kb bicistronic operon with hrcA, was induced by osmotic shock but not significantly by heat stress. This induction pattern is unlike those of other characterized dnaJ genes and may be indicative of a unique stress adaptation strategy by this commensal microorganism.

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug-1. Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products.

Can the development and autolysis of lactic acid bacteria influence the cheese volatile fraction? The case of Grana Padano

In this study, the relationship between the dynamics of the growth and lysis of lactic acid bacteria in Grana Padano cheese and the formation of the volatile flavor compounds during cheese ripening was investigated. The microbial dynamics of Grana Padano cheeses that were produced in two different dairies were followed during ripening. The total and cultivable lactic microflora, community composition as determined by length heterogeneity-PCR (LH-PCR), and extent of bacterial lysis using an intracellular enzymatic activity assay were compared among cheeses after 2, 6 and 13months of ripening in two dairies. The evolution of whole and lysed microbiota was different between the two dairies. In dairy 2, the number of total cells was higher than that in dairy 1 in all samples, and the number of cells that lysed during ripening was lower. In addition, at the beginning of ripening (2months), the community structure of the cheese from dairy 2 was more complex and was composed of starter lactic acid bacteria (Lactobacillus helveticus and Lactobacillus delbrueckii) and NSLAB, possibly arising from raw milk, including Lactobacillus rhamnosus/Lactobacillus casei and Pediococcus acidilactici. On the other hand, the cheese from dairy 1 that ripened for 2months was mainly composed of the SLAB L. helveticus and L. delbrueckii. An evaluation of the free-DNA fraction through LH-PCR identified those species that had a high degree of lysis. Data on the dynamics of bacterial growth and lysis were evaluated with respect to the volatile profile and the organic acid content of the two cheeses after 13months of ripening, producing very different results. Cheese from dairy 1 showed a higher content of free fatty acids, particularly those deriving from milk fat lipolysis, benzaldehyde and organic acids, such as pGlu and citric. In contrast, cheese from dairy 2 had a greater amount of ketones, alcohols, hydrocarbons, acetic acid and propionic acid. Based on these results, we can conclude that in the first cheese, the intracellular enzymes that were released from lysis were mainly involved in aroma formation, whereas in the second cheese, the greater complexity of volatile compounds may be associated with its more complex microbial composition caused from SLAB lysis and NSLAB (mainly L. rhamnosus/L. casei) growth during ripening.

Identification of dairy lactic acid bacteria by tRNAAla–23S rDNA-RFLP

The aim of this study was to evaluate the potential of target tRNA(Ala)-23S ribosomal DNA for identification of lactic acid bacteria strains associated with dairy ecosystem. For this purpose tRNA(Ala)-23S ribosomal DNA Restriction Fragment Length Polymorphism (tRNA(Ala)-23S rDNA-RFLP) was compared with two widely used DNA fingerprinting methods - P1 Random Amplified Polymorphic DNA (RAPD), (GTG)5 repetitive extragenic palindromic PCR (rep-PCR) - for their ability to identify different species on a set of 10 type and 34 reference strains. Moreover, 75 unknown isolates collected during different stages of Grana Padano cheese production and ripening were identified using tRNA(Ala)-23S rDNA-RFLP and compared to the RFLP profiles of the strains in the reference database. This study demonstrated that the target tRNA(Ala)-23S rDNA has high potential in bacterial identification and tRNA(Ala)-23S rDNA-RFLP is a promising method for reliable species-level identification of lactic acid bacteria (LAB) in dairy products.