A. Delacroix-Buchet

Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel Electrophoresis

ABSTRACT Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and Staphylococcus. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.

Molecular Fingerprinting of Dairy Microbial Ecosystems by Use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis

ABSTRACT Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of ∼150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.

Biodiversity of Bacterial Ecosystems in Traditional Egyptian Domiati Cheese

ABSTRACT Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type.

Tool for quantification of staphylococcal enterotoxin gene expression in cheese.

ABSTRACT Cheese is a complex and dynamic microbial ecosystem characterized by the presence of a large variety of bacteria, yeasts, and molds. Some microorganisms, including species of lactobacilli or lactococci, are known to contribute to the organoleptic quality of cheeses, whereas the presence of other microorganisms may lead to spoilage or constitute a health risk. Staphylococcus aureus is recognized worldwide as an important food-borne pathogen, owing to the production of enterotoxins in food matrices. In order to study enterotoxin gene expression during cheese manufacture, we developed an efficient procedure to recover total RNA from cheese and applied a robust strategy to study gene expression by reverse transcription-quantitative PCR (RT-qPCR). This method yielded pure preparations of undegraded RNA suitable for RT-qPCR. To normalize RT-qPCR data, expression of 10 potential reference genes was investigated during S. aureus growth in milk and in cheese. The three most stably expressed reference genes during cheese manufacture were ftsZ, pta, and gyrB, and these were used as internal controls for RT-qPCR of the genes sea and sed, encoding staphylococcal enterotoxins A and D, respectively. Expression of these staphylococcal enterotoxin genes was monitored during the first 72 h of the cheese-making process, and mRNA data were correlated with enterotoxin production.

Involvement of a Prophage in the Lysis of Lactococcus lactis subsp. cremoris AM2 during Cheese Ripening

Lactococcus lactis subsp. cremoris AM2 strain was previously shown to lyse early and extensively during cheese ripening. This strain is lysogenic and contains a prophage named ΦAM2. Lysis of strain AM2 and its prophage-cured derivative AM2-C in Saint-Paulin pressed-type cheese was monitored by the following parameters: cell viability, morphological changes of bacteria observed by electron microscopy and release of intracytoplasmic peptidases. Proteolysis was quantified by measuring soluble nitrogen (SN), phosphotungstic acid soluble nitrogen (PTA-N) and free amino acids. By contrast to the wild type strain AM2 which lyses early and extensively, its prophage-cured derivative AM2-C lyses only slowly and to a limited extent in cheese. These results indicate that the prophage ΦAM2 is involved in the lytic behaviour of L. lactis AM2 during cheese ripening. In addition, the comparison of two isogenic strains with similar enzymatic potential but different ability to lyse demonstrates that starter strain lysis results in a higher free amino acids production rate and a decrease of bitter taste.

Inactivation of lactococcal aromatic aminotransferase prevents the formation of floral aroma compounds from aromatic amino acids in semi-hard cheese

The enzymatic conversion of aromatic amino acids to aroma compounds plays a role in the formation of an undesirable floral aroma in Cheddar-like cheeses. In lactococci, the first step of aromatic amino acid degradation is a transamination, catalysed by an aromatic aminotransferase (AraT). We observed previously that in vitro, araT inactivation prevented degradation of aromatic amino acids and decreased degradation of Met and Leu. In this study we evaluated the effect of araT inactivation in Lactococcus lactis on flavour development in St. Paulin-type cheese. The degradation of amino acids was monitored by using radiolabelled amino acids and the volatile compounds formed were analysed by GC-MS. The development of cheese odour was also evaluated by sniffing. We confirmed that the availability of an α-ketoacid acceptor for transamination is the first limiting factor for amino acid conversion to aroma compounds in cheese. In the presence of α-ketoglutarate, araT inactivation greatly prevented formation of floral aroma compounds from aromatic amino acids while it did not affect the formation of volatile aroma compounds from branched-chain amino acids and methionine. However, the sensory analysis by sniffing did not reveal any significant effect of the gene inactivation although the odour of cheese made with the mutant tended to be less floral than that of cheese made with the wild type strain.

Assessment of the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11 during growth by flow cytometry

Aims:  The aim of this study was to improve knowledge about the dynamics of the physiological states of Lactococcus lactis ssp. cremoris SK11, a chain‐forming bacterium, during growth, and to evaluate whether flow cytometry (FCM) combined with fluorescent probes can assess these different physiological states.

Cell growth and resistance of Lactococcus lactis subsp. lactis TOMSC161 following freezing, drying and freeze-dried storage are differentially affected by fermentation conditions

To investigate the effects of fermentation parameters on the cell growth and on the resistance to each step of the freeze‐drying process of Lactococcus lactis subsp. lactis TOMSC161, a natural cheese isolate, using a response surface methodology.

The influence of starter and adjunct lactobacilli culture on the ripening of washed curd cheeses

Ten strains of lactobacillus from the CNRZ collection were tested as adjunct culture in miniature washed curd cheeses manufactured under controlled bacteriological conditions with two different starters, Lactococcus lactis subsp. lactis IL 416 and Lactococcus lactis subsp. cremoris AM2. Lactobacilli growth seemed to be dependent on the Lactobacillus strain but was not influenced by the starter strain or counts. Lactococci counts were higher in the miniature cheeses with AM2 starter and added lactobacilli than in the control cheeses without lactobacilli. Gross composition and hydrolysis of s1 casein were similar for miniature cheeses with and without lactobacilli. In the miniature cheeses manufactured with IL416 starter, the lactobacilli adjunct slightly increased the soluble nitrogen content, but that was not verified in the AM2 miniature cheeses. Phosphotungstic acid nitrogen content increased in miniature cheeses manufactured with IL416 when Lactobacillus plantarum 1572 and 1310 adjunct cultures were added. That was also verified for several Lactobacillus strains, specially Lactobacillus casei 1227, for miniature cheeses manufactured with AM2 starter. Free fatty acid content increased in miniature cheeses made with lactobacilli adjuncts 1310, 1308 and 1219 with IL416 starter, and with strains 1218, 1244 and 1308 for miniature cheeses with AM2 starter. These results indicate that production of soluble nitrogen compounds as well as free fatty acid content could be influenced by the lactobacilli adjunct, depending on the starter strain.

Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis subsp. lactis TOMSC161, Isolated from a Nonscalded Curd Pressed Cheese

ABSTRACT Lactococcus lactis is a lactic acid bacterium used in the production of many fermented foods, such as dairy products. Here, we report the genome sequence of L. lactis subsp. lactis TOMSC161, isolated from nonscalded curd pressed cheese. This genome sequence provides information in relation to dairy environment adaptation.