Molecular approaches to uncover phage-lactic acid bacteria interactions in a model community simulating fermented beverages.

Food microbial diversity and fluxes during the fermentation processes are well studied whereas phages-bacteria interactions are still poorly described in the literature. This is especially true in fermented beverages, and especially in cider, which is an alcoholic fermented apple beverage. The transcriptomic and proteomic responses of the lactic acid bacterium (LAB) Liquorilactobacillus mali UCMA 16447 to a lytic infection by phage UCMA 21115, both isolated from cider, were investigated, in order to get a better understanding of phages-bacteria interactions in such fermented beverage. During phage infection, 122 and 215 genes were differentially expressed in L. mali UCMA 16447 strain at T15 and T60 respectively, when compared to the uninfected condition. The same trends were confirmed by the proteomic study, with a total of 28 differentially expressed proteins found at T60. Overall, genes encoding cellular functions, such as carbohydrate metabolism, translation, and signal transduction, were downregulated, while genes involved in nucleotide metabolism and in the control of DNA integrity were upregulated in response to phage infection. This work also highlighted that phage infection repressed many genes involved in bacterial cell motility, and affected glycolysis.

Phage community involvement in fermented beverages: an open door to technological advances?

Bacteriophages (phages) are considered the most abundant biological entities on Earth. An increasing interest in understanding phage communities, also called viromes or phageomes, has arisen over the past decade especially thanks to the development and the accessibility of Next Generation Sequencing techniques. Despite the increasing amount of available metagenomic data on microbial communities in various habitats, viromes remain poorly described in the scientific literature particularly when it comes to fermented food and beverages such as wine and cider. In this review, a particular attention is paid to the current knowledge on phage communities, with a special focus on fermented food viromes and the methodological tools available to undertake their study. There is a striking lack of available data on the fermented foods and beverages viromes. As far as we know, and although a number of phages have been isolated from wine, no general study has to date been carried out to assess the diversity of viromes in fermented beverages and their possible interactions with microbiota throughout the fermentation process. With the aim of establishing connections between the currently used technologies to carry out the analysis of viromes, possible applications of current knowledge to fermented beverages are examined.

Pseudomonas crudilactis sp. nov., isolated from raw milk in France.

Strains belonging to the Pseudomonas genus have been isolated worldwide from various biotic (humans, animals and plant tissues) and abiotic (food, soil, water and air) environments. Raw milk provides a favorable environment for the growth of a broad spectrum of microorganisms, including Pseudomonas. Here we present the description of Pseudomonas sp. UCMA 17988 isolated from raw milk, which was previously reported to produce new antimicrobial lipopeptides. MultiLocus Sequence Analysis of four housekeeping genes (16S rRNA, gyrB, rpoD and rpoB), whole genome sequence comparison (orthoANI value, original ANI value and dDDH value), microscopy, FAME analysis, and biochemical tests were performed. Digital DNA-DNA hybridization and average nucleotide identity values between strain UCMA 17988 and its closest relatives, P. helmanticensis CECT 8548T (46.9%, 92.07%) and P. baetica CECT 7720T (26.8%, 88.50%), rate well below the designed threshold for assigning prokaryotic strains to the same species. In conclusion, strain UCMA 17988 belongs to a novel species, for which the name Pseudomonas crudilactis sp. nov (type strain UCMA 17988T = DSM 109949T = LMG 31804T) is proposed.

Phages for biocontrol in foods: What opportunities for Salmonella sp. control along the dairy food chain?

Controlling the presence of pathogenic bacteria, such as Salmonella sp., in dairy products production is a burning issue since contamination with Salmonella can occur at any stage of the production chain. The use of Salmonella-phages applied as control agents has gained considerable interest. Nonetheless, Salmonella-phage applications specifically intended for ensuring the safety of dairy products are scarce. This review identifies recent advances in the use of Salmonella-phages that are or could be applied along the dairy food chain, in a farm-to-fork approach. Salmonella-phages can be promising tools to reduce the shedding of Salmonella in cattle, and to reduce and control Salmonella occurrence in postharvest food (such as food additives), and in food processing facilities (such as biosanitizing agents). These control measures, combined with existing methods and other biocontrol agents, constitute new opportunities to reduce Salmonella occurrence along the dairy food production, and consequently to alleviate the risk of Salmonella contamination in dairy products.

Reduced growth of Listeria monocytogenes in two model cheese microcosms is not associated with individual microbial strains.

Two model antilisterial microbial communities consisting of two yeasts, two Gram positive and two Gram negative bacteria, and originating from Livarot cheese smear were previously designed. They were used in the present study to analyse the impact of microbial population dynamics on growth of Listeria monocytogenes in cheese microcosm. Specific culture media and PCR primers were developed for simultaneous culture-dependent and real-time PCR quantification of strains belonging to Marinomonas sp., Paenibacillus sp., Staphylococcus equorum, Arthrobacter arilaitensis, Pseudomonas putida, Serratia liquefaciens, Candida natalensis, and Geotrichum candidum, in cheese microcosms. All strains were enumerated after 3, 5, 8 and 14 days at 15 °C. They established well at high counts in all cheese microcosms. Growth dynamics for all strains in presence of L. monocytogenes WSLC 1685 were compared to those of microbial communities obtained by omitting in turn one of the six members of the initial community. The growth of the microbial strains was neither markedly disturbed by Listeria presence nor by the removal of each strain in turn. Furthermore, these communities had a significant reducing effect on growth of L. monocytogenes independently of pH, as confirmed by mathematical modelling. A barrier effect was observed, that could be explained by specific competition for nutrients.

Diversity and assessment of potential risk factors of Gram-negative isolates associated with French cheeses.

The goal of this study was to identify at the species level a large collection of Gram-negative dairy bacteria isolated from milks or semi-hard and soft, smear-ripened cheeses (cheese core or surface samples) from different regions of France. The isolates were then assessed for two risk factors, antibiotic resistance and volatile and non-volatile biogenic amine production in vitro. In total, 173 Gram-negative isolates were identified by rrs and/or rpoB gene sequencing. A large biodiversity was observed with nearly half of all Gram-negative isolates belonging to the Enterobacteriaceae family. Overall, 26 different genera represented by 68 species including potential new species were identified among the studied Gram-negative isolates for both surface and milk or cheese core samples. The most frequently isolated genera corresponded to Pseudomonas, Proteus, Psychrobacter, Halomonas and Serratia and represented almost 54% of the dairy collection. After Pseudomonas, Chryseobacterium, Enterobacter and Stenotrophomonas were the most frequently isolated genera found in cheese core and milk samples while Proteus, Psychrobacter, Halomonas and Serratia were the most frequently isolated genera among surface samples. Antibiotic resistance profiles indicated that resistances to the aminosid, imipemen and quinolon were relatively low while more than half of all tested isolates were resistant to antibiotics belonging to the monobactam, cephem, fosfomycin, colistin, phenicol, sulfamid and some from the penam families. Thirty-six% of isolates were negative for in vitro biogenic amine production. Among biogenic amine-producers, cadaverine was the most frequently produced followed by isoamylamine, histamine and putrescine. Only low levels (<75 mg/l) of tyramine were detected in vitro.

Impact of Gram-negative bacteria in interaction with a complex microbial consortium on biogenic amine content and sensory characteristics of an uncooked pressed cheese.

The impact of Gram-negative bacteria on sensory characteristics and production of volatile compounds as well as biogenic amines (BA) in the core of an uncooked pressed type model cheese was investigated in the presence of a defined complex microbial consortium. Eleven strains of Gram-negative bacteria, selected on the basis of their biodiversity and in vitro BA-production ability, were individually tested in a model cheese. Four out of 6 strains of Enterobacteriaceae (Citrobacter freundii UCMA 4217, Klebsiella oxytoca 927, Hafnia alvei B16 and Proteus vulgaris UCMA 3780) reached counts close to 6 log CFU g⁻¹ in the model cheese. In core of cheeses inoculated with Gram-negative bacteria, only slight differences were observed for microbial counts (Enterococcus faecalis or Lactobacillus plantarum count differences below 1 log CFU g⁻¹), acetate concentration (differences below 200 mg kg⁻¹) and texture (greater firmness) in comparison to control cheeses. Cheese core colour, odour and volatile compound composition were not modified. Although ornithine, the precursor of putrescine, was present in all cheeses, putrescine was only detected in cheeses inoculated with H. alvei B16 and never exceeded 2.18 mmol kg⁻¹ cheese dry matter. Cadaverine was only detected in cheeses inoculated with H. alvei B16, K. oxytoca 927, Halomonas venusta 4C1A or Morganella morganii 3A2A but at lower concentrations (<1.05 mmol kg⁻¹ cheese dry matter), although lysine was available. Only insignificant amounts of the detrimental BA histamine and tyramine, as well as isopentylamine, tryptamine or phenylethylamine, were produced in the cheese model by any of the Gram-negative strains, including those which produced these BA at high levels in vitro.

Investigation of the Phageome and Prophages in French Cider, a Fermented Beverage.

Phageomes are known to play a key role in the functioning of their associated microbial communities. The phageomes of fermented foods have not been studied thoroughly in fermented foods yet, and even less in fermented beverages. Two approaches were employed to investigate the presence of phages in cider, a fermented beverage made from apple, during a fermentation process of two cider tanks, one from an industrial producer and one from a hand-crafted producer. The phageome (free lytic phages) was explored in cider samples with several methodological developments for total phage DNA extraction, along with single phage isolation. Concentration methods, such as tangential flow filtration, flocculation and classical phage concentration methods, were employed and tested to extract free phage particles from cider. This part of the work revealed a very low occurrence of free lytic phage particles in cider. In parallel, a prophage investigation during the fermentation process was also performed using a metagenomic approach on the total bacterial genomic DNA. Prophages in bacterial metagenomes in the two cider tanks seemed also to occur in low abundance, as a total of 1174 putative prophages were identified in the two tanks overtime, and only two complete prophages were revealed. Prophage occurrence was greater at the industrial producer than at the hand-crafted producer, and different dynamics of prophage trends were also observed during fermentation. This is the first report dealing with the investigation of the phageome and of prophages throughout a fermentation process of a fermented beverage.

Phages Shape Microbial Dynamics and Metabolism of a Model Community Mimicking Cider, a Fermented Beverage.

Model microbial communities are often studied to better understand interactions and fluxes during fermentation processes. However, models that take into account the potential impact of bacteriophages (phages), which are recognized as drivers of microbial communities, are scarce, especially in fermented foods. This study aimed at investigating the behavior of a cider model microbial community, which was subjected to disturbance in the presence or absence of phages and at two different temperatures (25 °C and 15 °C). The model microbial community was composed of three lactic acid bacteria (LAB) strains belonging to the species Liquorilactobacillus mali, Leuconostoc mesenteroides and Oenococcus oeni, and of a Saccharomyces uvarum yeast strain. Two phages were selected, targeting L. mali and Ln. mesenteroides strains. In order to follow the behavior of the microbial community model, the phages and microbial strains were enumerated at several time points, and the metabolic signatures (sugar consumption, production of organic acids and volatile organic compounds) of the model microbial community were monitored. At 25 °C, the community with phages (P) was significantly closer to the control condition (C) than to the condition without phages (D). Microbial levels were similar between conditions C and P, which were characterized by high concentrations of compounds such as 2-phenylethanol, ethyl octanoate and isoamyl alcohol, and more globally by a more complex metabolic signature than that of condition D. In condition D, L. mali and Ln. mesenteroides were dominant while S. uvarum and O. oeni were less present, and this condition was characterized by a high concentration of ethyl lactate. At 15 °C, condition P differed from conditions C and D, as Ln. mesenteroides was not detected while the other strains all reached approximately the same levels. The metabolic range of condition P was less important than for conditions C and D. The current study showed that the influence of phages on the model microbial community dynamics and metabolisms after a disturbance phenomenon was temperature-dependent.

Contribution of omics to biopreservation: Toward food microbiome engineering.

Biopreservation is a sustainable approach to improve food safety and maintain or extend food shelf life by using beneficial microorganisms or their metabolites. Over the past 20 years, omics techniques have revolutionised food microbiology including biopreservation. A range of methods including genomics, transcriptomics, proteomics, metabolomics and meta-omics derivatives have highlighted the potential of biopreservation to improve the microbial safety of various foods. This review shows how these approaches have contributed to the selection of biopreservation agents, to a better understanding of the mechanisms of action and of their efficiency and impact within the food ecosystem. It also presents the potential of combining omics with complementary approaches to take into account better the complexity of food microbiomes at multiple scales, from the cell to the community levels, and their spatial, physicochemical and microbiological heterogeneity. The latest advances in biopreservation through omics have emphasised the importance of considering food as a complex and dynamic microbiome that requires integrated engineering strategies to increase the rate of innovation production in order to meet the safety, environmental and economic challenges of the agri-food sector.

Surface microbial consortia from Livarot, a French smear-ripened cheese.

The surface microflora (902 isolates) of Livarot cheeses from three dairies was investigated during ripening. Yeasts were mainly identified by Fourier transform infrared spectroscopy. Geotrichum candidum was the dominating yeast among 10 species. Bacteria were identified using Biotype 100 strips, dereplicated by repetitive extragenic palindromic PCR (rep-PCR); 156 representative strains were identified by either BOX-PCR or (GTG)(5)-PCR, and when appropriate by 16S rDNA sequencing and SDS-PAGE analysis. Gram-positive bacteria accounted for 65% of the isolates and were mainly assigned to the genera Arthrobacter , Brevibacterium , Corynebacterium , and Staphylococcus . New taxa related to the genera Agrococcus and Leucobacter were found. Yeast and Gram-positive bacteria strains deliberately added as smearing agents were sometimes undetected during ripening. Thirty-two percent of the isolates were Gram-negative bacteria, which showed a high level of diversity and mainly included members of the genera Alcaligenes , Hafnia , Proteus , Pseudomonas , and Psychrobacter . Whatever the milk used (pasteurized or unpasteurized), similar levels of biodiversity were observed in the three dairies, all of which had efficient cleaning procedures and good manufacturing practices. It appears that some of the Gram-negative bacteria identified should now be regarded as potentially useful in some cheese technologies. The assessment of their positive versus negative role should be objectively examined.

Corrigendum: Characterization of Milkisin, a Novel Lipopeptide With Antimicrobial Properties Produced By Pseudomonas sp. UCMA 17988 Isolated From Bovine Raw Milk.

[This corrects the article DOI: 10.3389/fmicb.2018.01030.].

Purification, Characterization and Thermodynamic Assessment of an Alkaline Protease by Geotrichum Candidum of Dairy Origin.

BACKGROUND: Alkaline proteases is the important group of enzymes having numerous industrial applications including dairy food formulations. OBJECTIVES: The current study deals with the purification and characterization of an alkaline serine protease produced by Geotrichum candidum QAUGC01, isolated from indigenous fermented milk product, Dahi. MATERIAL AND METHODS: In total twelve G. candidum strains were screened for their proteolytic activity by using standard protease assay. The protease production from G. candidum QAUGC01 was optimized by varying physio-chemical conditions. The protease was purified by using two-step method: ammonium sulfate precipitation and gel filtration chromatography. Protease was further characterized by studying various parameter like temperature, pH, modulators, metal ions and organic solvent. A thermodynamic study was also carried out to explore the half-life of protease. RESULTS: The G. candidum grew profusely at 25 °C and at an initial pH of 4.0 for 72 h of incubation producing 26.21 U/ml maximum extracellular protease. Protease revealed that Vmax and Km was 26.25 U.ml-1.min-1 and 0.05 mg.mL-1, respectively using casein as substrate. The enzyme was stable at a temperature range (25-45 °C) and pH (8-9). Residual enzyme activity was strongly inhibited in the presence of PMSF (7.5%). The protease could hydrolyze proteinaceous substrates, casein (98%) and BSA (95%). The thermodynamic studies explored that the half-life of the enzyme that was 106.62 min, 38.72 min and 15.71 min at 50, 60 and 70 °C, respectively. CONCLUSIONS: Purified protease from G. candidum GCQAU01 is an ideal candidate for industrial application.

Characterization of Milkisin, a Novel Lipopeptide With Antimicrobial Properties Produced By Pseudomonas sp. UCMA 17988 Isolated From Bovine Raw Milk.

Biosurfactants such as lipopeptides are amphiphilic compounds produced by microorganisms such as bacteria of the genera of Pseudomonas and Bacillus. Some of these molecules proved to have interesting antimicrobial, antiviral, insecticide, and/or tensioactive properties that are potentially useful for the agricultural, chemical, food, and pharmaceutical industries. Raw milk provides a physicochemical environment that is favorable to the multiplication of a broad spectrum of microorganisms. Among them, psychrotrophic bacterial species, especially members of the genus Pseudomonas, are predominant and colonize milk during cold storage and/or processing. We isolated the strain Pseudomonas sp. UCMA 17988 from raw cow milk, with antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica Newport. Antimicrobial molecules involved in the antagonistic activity of this strain were characterized. A mass spectrometry analysis highlighted the presence of four lipopeptides isoforms. The major isoform (1409 m/z), composed of 10 carbons in the lipidic chain, was named milkisin C. The three other isoforms detected at 1381, 1395, and 1423 m/z, that are concomitantly produced, were named milkisin A, B, and D, respectively. The structure of milkisin, as confirmed by nuclear magnetic resonance analyses, is closely related to amphisin family. Indeed, the peptidic chain was composed of 11 amino acids, 6 of which are conserved among the family. In conclusion, Pseudomonas sp. UCMA 17988 produces new members of the amphisin family which are responsible for the antagonistic activity of this strain.

A PCR-based method for identification of lactobacilli at the genus level.

We developed a polymerase chain reaction (PCR)-based method for the identification of lactobacilli at the genus level. One specific primer, LbLMA1-rev, was designed by analysing similarities between the nucleotide sequence of the spacer between the 16S and 23S rRNA genes in a number of Lactobacillus strains. Amplification with LbLMA1-rev and R16-1, a universal primer, generated a PCR product for 23 Lactobacillus species. Electrophoresis did not reveal any discrete bands when Escherichia coli, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Carnobacterium pissicola, Pediococcus pentosaceus, Bifidobacterium bifidum, Weissella confusa, Enterococcus hirae, Staphylococcus aureus or Listeria monocytogenes DNA were used as template.

Resin straw as an alternative system to securely store frozen microorganisms.

Freezing of prokaryotic and eukaryotic microorganisms is the main interest in the study of cold stress responses of living organisms. In parallel, applications which arise from this approach are of two types: (i) optimization of the frozen starters used in food processing; and (ii) improvement of the ex situ preservation of microorganisms in collections. Currently, cryopreservation of microorganisms in collections is carried out in cryotubes, and bibliographical references related to freezing microorganisms packaged in straws are scarce. In this context, a preliminary study was completed to evaluate the technological potential of ionomeric resin straws compared to polycarbonate cryo-tubes. Survival under freezing stress was tested on three microorganisms selected for their biotechnological interest: two lactic acid bacteria, Lactococcus lactis subsp. cremoris and Lactobacillus delbrueckii subsp. bulgaricus and a deuteromycete fungus, Geotrichum candidum. The stress was carried out by repeated freezing-thawing cycles to artificially accelerate the lethal effect of freezing on the microorganisms. Two main results were obtained: (i) the survival rate values (per freezing-thawing cycle) seems to depend on the thermal type of the studied microorganism, and (ii) there was no, under our experimental conditions, significant difference between straws and tubes. However, conservation in the resin straws lead to a slight increase in the survival of L. cremoris and G. candidum compared to microtubes. In those conditions, straws seems an alternative system to securely store frozen microorganisms with three main characteristics: (i) a high resistance to thermal stress, (ii) a safe closing by hermetic weld, and (iii) a system for inviolable identification.

Intra-species chromosome-length polymorphism in Geotrichum candidum revealed by pulsed field gel electrophoresis.

Geotrichum candidum is an ascomycetous anamorph yeast-like fungus found in various habitats. It is a component of the natural flora of milk and is used as a maturing agent for both soft and hard cheeses. This microorganism displays phenotypic variability and may act as an opportunist pathogen, causing geotrichosis. Cytological analysis of G. candidum strain ATCC 204307 showed this strain to have eight chromosomes. We prepared chromosomal DNA from 13 strains of G. candidum differing in habitat and morphotype. We used pulsed field gel electrophoresis (PFGE) in two sets of conditions to determine the size of the chromosomal DNA molecules. The strains investigated had five to eight chromosomes, 0.6 to 4.5 Mb in size. We estimated genome size in these 13 strains to be between 11 and 19 Mb. Pulsed-field gel electrophoresis profiles showed a high degree of polymorphism, indicating considerable variability between strains. Genome size and the presence of large chromosomes appeared to be correlated with morphotype. Strains with a mold-like or intermediate morphotype tended to have larger genomes than strains with a yeast-like morphotype did.

Traditional cheeses: rich and diverse microbiota with associated benefits.

The risks and benefits of traditional cheeses, mainly raw milk cheeses, are rarely set out objectively, whence the recurrent confused debate over their pros and cons. This review starts by emphasizing the particularities of the microbiota in traditional cheeses. It then describes the sensory, hygiene, and possible health benefits associated with traditional cheeses. The microbial diversity underlying the benefits of raw milk cheese depends on both the milk microbiota and on traditional practices, including inoculation practices. Traditional know-how from farming to cheese processing helps to maintain both the richness of the microbiota in individual cheeses and the diversity between cheeses throughout processing. All in all more than 400 species of lactic acid bacteria, Gram and catalase-positive bacteria, Gram-negative bacteria, yeasts and moulds have been detected in raw milk. This biodiversity decreases in cheese cores, where a small number of lactic acid bacteria species are numerically dominant, but persists on the cheese surfaces, which harbour numerous species of bacteria, yeasts and moulds. Diversity between cheeses is due particularly to wide variations in the dynamics of the same species in different cheeses. Flavour is more intense and rich in raw milk cheeses than in processed ones. This is mainly because an abundant native microbiota can express in raw milk cheeses, which is not the case in cheeses made from pasteurized or microfiltered milk. Compared to commercial strains, indigenous lactic acid bacteria isolated from milk/cheese, and surface bacteria and yeasts isolated from traditional brines, were associated with more complex volatile profiles and higher scores for some sensorial attributes. The ability of traditional cheeses to combat pathogens is related more to native antipathogenic strains or microbial consortia than to natural non-microbial inhibitor(s) from milk. Quite different native microbiota can protect against Listeria monocytogenes in cheeses (in both core and surface) and on the wooden surfaces of traditional equipment. The inhibition seems to be associated with their qualitative and quantitative composition rather than with their degree of diversity. The inhibitory mechanisms are not well elucidated. Both cross-sectional and cohort studies have evidenced a strong association of raw-milk consumption with protection against allergic/atopic diseases; further studies are needed to determine whether such association extends to traditional raw-milk cheese consumption. In the future, the use of meta-omics methods should help to decipher how traditional cheese ecosystems form and function, opening the way to new methods of risk-benefit management from farm to ripened cheese.

Biodiversity of the Surface Microbial Consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen Cheeses.

Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious "house" flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.

Diversity and dynamics of lactobacilli populations during ripening of RDO Camembert cheese.

The diversity and dynamics of Lactobacillus populations in traditional raw milk Camembert cheese were monitored throughout the manufacturing process in 3 dairies. Culture-dependent analysis was carried out on isolates grown on acidified de Man - Rogosa - Sharpe agar and Lactobacillus anaerobic de Man Rogosa Sharpe agar supplemented with vancomycin and bromocresol green media. The isolates were identified by polymerase chain reaction - temperature gradient gel electrophoresis (PCR-TGGE) and (or) species-specific PCR and (or) sequencing, and Lactobacillus paracasei and Lactobacillus plantarum isolates were characterized by pulsed field gel electrophoresis (PFGE). Milk and cheese were subjected to culture-independent analysis by PCR-TGGE. Presumed lactobacilli were detected by plate counts throughout the ripening process. However, molecular analysis of total DNA and DNA of isolates failed to detect Lactobacillus spp. in certain cases. The dominant species in the 3 dairies was L. paracasei. PFGE analysis revealed 21 different profiles among 39 L. paracasei isolates. Lactobacillus plantarum was the second most isolated species, but it occurred nearly exclusively in one dairy. The other species isolated were Lactobacillus parabuchneri, Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus helveticus, a Lactobacillus psittaci/delbrueckii subsp. bulgaricus/gallinarum/crispatus group, Lactobacillus rhamnosus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, Lactobacillus brevis, Lactobacillus kefiri, and Lactobacillus perolens. Lactobacilli diversity at the strain level was high. Dynamics varied among dairies, and each cheese exhibited a specific picture of species and strains.