Role of Lactobacillus rhamnosus in Parmigiano Reggiano cheese ripening: a genotypic and post-genomic study

Abstract

Lactic Acid Bacteria (LAB) constitute an heterogeneous group of bacteria that are traditionally used to produce fermented foods. They may play different roles in cheese-making where starter LAB (SLAB) participate in the fermentation process, whereas non starter LAB (NSLAB) are implicated in the maturation of cheese. Although the role of NSLAB in ripening has not yet been clarified, different authors have suggested their importance in the cheese ageing. In Parmigiano Reggiano (PR) cheese, NSLAB, autochthonic of raw milk and arising from the environment, are the protagonists of the different biochemical processes during the production and ripening stages. Recent studies in microbiological ecology of PR cheese allowed a deeper insight of the microbial composition during the manufacturing and ripening stages of the same cheese-making process. Further, the study of samples representative of the subsequent stages of the same cheese-making by culture-independent methods led to determine the microbial succession during 24 months of PR ripening. It was demonstrated that SLAB are dominant until the 2nd month of ripening. Differently, after cheese brining, the species NSLAB, especially Lb. rhamnosus, are able to grow and increase in number, while SLAB cells undergo to autolysis. Lb. rhamnosus was shown to be the dominant species present after a lack of essential nutrients, such as sugars. Therefore, this species seems to well adapt to the absence of lactose in cheese, confirming an optimal adaptability to unfavorable growth conditions. Presumably, this characteristic was due to the ability of Lb. rhamnosus to use nitrogen fraction as an alternative energy source. So far, only few studies are available about NSLAB, and in particular about Lb. rhamnosus, in PR and the exact role of these bacteria has not been investigated in greater depth. In particular, the biotypes of the dominant species during the maturation stage of cheese processing have never been studied. Indeed, it can only be hypothesized that the technological pressure determines, at different stages, the potential development of the biotype that may have a specific biochemical role leading to certain flavors and sensorial traits of the PR cheese. For this reason, Lb. rhamnosus, the dominant species among NSLAB in PR, has been chosen as subject for the present study. This Ph.D thesis aims to better understand the role played by this species in cheese-making and ripening process, and to hypothesize the strategies used to adapt its energy metabolism to the environmental conditions met in PR cheese. To reach this issue, three different steps has been pursued. In the first step, genotypic relatedness at the intraspecies level for 66 Lb. rhamnosus strains isolated during the same cycle of PR cheesemaking, was determined. Biodiversity has been estimated by means of two DNA fingerprinting techniques, i.e., randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and repetitive extragenic palindromic-polymerase chain reaction REP-PCR analysis. It was found an intraspecies heterogeneity in Lb. rhamnosus strains which is certainly correlated to their abilities to adapt to specific environmental and technological conditions. In this regard, the detection of biotypes that mark specific steps in cheese ripening, or of those that can develop differently throughout the ripening process, suggests that they may have also specific roles closely linked to their peculiar metabolical properties. In the second step of the research the metabolic data (fermentative profiles, synthesis of organic acids and FAA) and 2-DE analyses followed by the identification of a large number of proteins were combined to highlight Lb. rhamnosus physiological mechanisms of adaptation that are responsible for survival in cheese during ripening. Cultivation was carried out under cheese-like conditions (Cheese broth) and on pasteurized and micro-filtrated milk and MRS broth As shown by fermentative profiles analysis, the major part of the Lb. rhamnosus strains had the capacity to use numerous chemical compounds as energy sources. Growth and acidification differed depending on the culture media. Compared to growth on MRS or pasteurized and micro-filtrated milk, all strains cultivated in cheese-like medium (cheese broth, CB) showed a decrease of the synthesis of D,L-lactic acid and synthesized higher levels of acetic acid. Except for one strain, the others caused an increase of the concentration of free amino acids during cultivation on CB. A proteomic approach was applied to elucidate the (i) intra-specific diversity of Lb. rhamnosus and (ii) the metabolic mechanisms involved in growth in CB which mimicked the cheese during ripening. The proteomic maps of five strains showing different metabolic traits were comparatively determined after growth on MRS and CB. The result showed that synthesis of ca. 60 - 70% of the cytosolic proteins of Lb. rhamnosus strains was not affected by the culture media. The remaining part (ca. 30-40%) of the total proteome seemed to vary depending on strains and growth media. A total of 93 protein spots which showed an increased level of synthesis during growth in MRS (46 spots) or CB (47 spot) were identified by MALDI-time of flight mass spectrometry (MALDI-TOF-MS/MS) and nano-electrospray ionization-ion trap mass spectrometry (ESI-MS/MS). Compared to cells grown in MRS, Lb. rhamnosus strains cultivated under cheese-like conditions modified the synthesis of proteins related to protein biosynthesis, nucleotide and carbohydrate metabolisms, glycolysis pathway, proteolytic activity, cell wall and exopolysaccharide biosyntheses, cellular regulation, amino acid and citrate metabolisms, oxidation/reduction processes and stress response. It was confirmed that Lb. rhamnosus strains have the capacity to activate different metabolic pathways depending on the culture media. In the last step an innovative cDNA-AFLP protocol was developed to investigate the changes in gene expression profile during the growth of one Lb. rhamnosus strain isolated in 20 month ripened PR and therefore able to growth and survive in this hostile environment. Changes in gene expression was evaluated in CB versus a rich medium (MRS). This study represents one of the few concerning bacterial transcriptomic analysis through cDNA-AFLP approaches. This technique allowed to generate unique transcript tags from reverse-transcribed messenger RNA using restriction enzymes and selective PCR amplification. Results evidenced that Lb. rhamnosus is able to modify the expression of a large part of genes when cultivated in cheese-like conditions (CB) compared to growth under in vitro optimal conditions (MRS). However, these genes still have to be recognized thus it is not possible at the moment to correlate them with proteins found with the proteomic study. For this reason further analysis will have to be focused on gel extraction followed by the identification of expressed genes. This will allow to deepen the knowledge of the basal metabolism through the identification of constitutive genes, as well as to analyze the reactions of the cells to the dairy environment by the identification of differently expressed genes in the two cultured media. Moreover the expected results could lead to the discovery of Lb. rhamnosus genes putatively involved both in the physiological mechanisms of adaptation to cheese-like substrates, that are fundamental for survival during ripening, and in the definition of organoleptic characteristics of the PR cheese.