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The quantitative (n = 260) and qualitative (n = 24) microbial compositions of raw milk samples were assessed using culture analyses and a molecular inventory based on the rDNA sequences of 1697 isolates. A statistical analysis was also performed to correlate farm practices with the microbial populations present in the milk samples. Ripening bacteria and Pseudomonas were the dominant groups. Milk samples were distinguishable by the different proportions of their microbial groups, which varied among producers. Individual practices influenced the levels of technologically important microorganisms, for example, pre-dipping of teats reduced levels of lactococci. Standard plate counts correlated with the levels of Gram-negative bacteria, Pseudomonas, lactococci and ripening bacteria, depending on the milking season. Lactic acid bacteria and yeasts, which were poorly represented, nevertheless seemed to constitute a resilient basal flora. Qualitative species diversity was considerable, with more than 100 bacterial species identified, dominated by Gram-positive bacteria, notably staphylococci and Corynebacterium.

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.

This study aimed to relate the acquisition of different antibiotic resistances and the corresponding physiological responses to cold stress of Lactobacillus delbrueckii subsp. bulgaricus strain CFL1. Six resistant mutants were spontaneously obtained and studied depending on the target of the antibiotic: (i) bacitracin and vancomycin (Bac(R), Van(R), wall synthesis), (ii) novobiocin (Nov(R), DNA replication), and (iii) kanamycin, spiramycin, streptomycin (Kan(R), Spi(R), Str(R), RNA translation). The mutations modified the growth and the cold stress response at three different physiological levels: (i) Van(R) and Spi(R) mutants showed significant lower growth rates compared to the wild type strain. (ii) Van(R) and Bac(R) mutants displayed a slightly higher resistance to a freezing-thawing challenge whereas Str(R) and Spi(R) mutants were more sensitive compared to the wild type. (iii) The recovery of acidification activity after freezing and during frozen storage was improved by considering the Nov(R) strain, but not with the Van(R) and Spi(R) mutants. Thus, acquisition of some antibiotic resistance by spontaneous mutation led to modification of the cold stress response. The hypothesis of a unique cellular thermostat is discussed regarding the diversity of the tested antibiotics.

Food industry tends towards the use of controlled microorganisms in order to improve its technologies including frozen starter production. The fungus Geotrichum candidum, which is currently found in various environments, is widely used as ripening agent in some specific cheese making process. In order to optimize the cryopreservation of this microorganism, freezing experiments were carried out using a Peltier cooler-heater incubator, which permits to control the temperature downshift from +20 to -10 degrees C in time period ranges from 20 to 40min depending on the experiments. Concomitantly, study of the effect of an industrial ice nucleator protein derived from Pseudomonas syringae (SNOMAX) on the dynamic of freezing of G. candidum was carried out. Our results showed that the addition of this protein in the microbiological suspension has different complementary effects: (i) the synchronization of the different samples nucleation, leading to an homogeneous and earlier freezing, (ii) the increase of the freezing point temperature from -8.6 to -2.6 degrees C, (iii) a significant decrease of the lethality of G. candidum cells subjected to a freezing-thawing cycles challenge.

This study was completed on an artificial microbial community (consortium) including three microorganisms of dairy interest, two mesophilic (Lactococcus lactis subsp. cremoris and Geotrichum candidum) and one thermophilic (Lactobacillus delbrueckii subsp. bulgaricus). The aim of this work was to study the stress responses of the food microbial consortium subjected to a freeze–thaw challenge (–20°C, 10 min / +25°C, 3 min) combined with physiological cryoadaptation (cryotolerance). Our results show that induction of cryotolerance by near-freezing temperature pretreatment of G. candidum and L. cremoris within the microbial community is similar to the one obtained with pure cultures. However, cryotolerance of L. bulgaricus was only induced within the consortium in milk. An interspecies cryoprotection was thus identified. This acquisition is correlated with the cell envelope extracts of G. candidum and/or the extracellular fractions of the two adapted mesophilic microorganisms. Therefore, in our experimental c...

The numbers and types of Lactobacillus strains in probiotic feed or food products for sale in Europe were compared with the information stated on the product labels. The labels of all 10 products gave information on the genus and species of Lactobacillus, and seven gave information on the numbers of a specified strain(s). Lactobacilli were recovered using a recently validated method, developed as part of the European Community Project SMT4 CT98-2235 for the official control of probiotic microorganisms used as feed additives. Polymerase chain reaction (PCR)-based methods were used to assess the accuracy of labelling with regard to genus and species, and pulsed-field gel electrophoresis (PFGE) was used to identify strains. Five products were mislabelled with respect to the numbers and three with respect to species of lactobacilli. In four cases, the specified strains were not detected. Four fermented milks sold under three trademarks contained the same strain, which was named differently on each label. As safety and functionality of probiotics are strain dependent, these results demonstrated the need to control lactobacilli present in commercially probiotic human food products, not only at the species but also at the strain level, to ensure their quality and protect the consumer. The usefulness of the official methods developed for animal feed additives to identify lactobacilli in probiotic foods for humans was demonstrated.

The aim was to select potentially probiotic lactobacilli from 88 strains isolated from unpasteurized milk and cheese products, and to incorporate these bacteria in a viable state into a soft cheese, without changing its quality. The survival of these bacteria was assessed in acidic and bile conditions, after freezing at −80 °C. Four strains from unpasteurized Camembert – two Lactobacillus plantarum strains and two Lb. paracasei/casei strains – were identified and typed by PCR and PFGE and were found to display potentially probiotic characteristics in addition to resistance to low pH and bile. These characteristics were resistance to lysozyme, adhesion to CACO-2 cells, antimicrobial effects against common foodborne pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli), innocuity following the ingestion of high doses by mice and appropriate antibiotic susceptibility profiles. The potential of Lb. plantarum strain UCMA 3037 for incorporation into a soft cheese (Pont-l'Eveque registered designation of origin (RDO)) was investigated. This strain grew well and survived in sufficient numbers (more than 107 cfu/g throughout the shelf-life of the product) in the cheese. This strain did not change the quality score of the product until the best before date (75 days after manufacture). Thus, unpasteurized Camembert is a natural source of potentially probiotic lactobacilli, which could be used as an additive in the development of potentially probiotic soft cheeses. Further work is required to demonstrate the persistence and efficacy of these strains in the human host upon ingestion.

Comparaison de methodes culture-dependante et culture-independante pour l'analyse moleculaire de la diversite des lactobacilles dans le Camembert de Normandie. La contribution de deux approches culture-dependante et culture-independante a ete etudiee dans le but d'apprecier la biodiversite et l'evolution des populations de lactobacilles au cours de la transformation de lait cru en camembert AOC dans trois fromageries. Une analyse par electrophorese en gradient de temperature (TGGE) a ete effectuee comparativement sur l'ADN microbien total et sur l'ADN extrait a partir d'isolats. Les profils TGGE obtenus a partir de l'ADN total de laits et de fromages ont montre que Lactobacillus paracasei subsp. paracasei etait une espece dominante au sein des trois fromageries et que Lb. plantarum co-dominait dans l'un des trois ateliers. Apres etude des profils TGGE des isolats, ces deux especes demeurent majoritaires mais Lactobacillus delbrueckii susbp. lactis, Lb. acidophilus, Lb. delbrueckii susbp. bulgaricus et Lb. casei subsp. casei sont egalement mis en evidence. Les deux approches ont conduit a l'obtention d'informations complementaires.

During the last fifteen years, the Lactobacillus genus has evolved and contains to date more than 80 species. They are present in raw milk and dairy products such as cheeses, yoghurts and fermented milks. Quality assurance programmes associated with research, development, production and validation of the health or technological benefits of these bacteria require their relevant isolation, counting and identification. This review presents the different selective media to isolate lactobacilli, and the numerous different available tools to characterise lactobacilli at genus, species or strain level using either culture-dependent methods: phenotypical, molecular or global methods, or using new culture-independent advanced molecular methods. Enzymes used for PFGE, hybridisation probes and PCR-based method primers are listed in seven tables. In conclusion, the main advantages and disadvantages associated with these techniques are presented.

A collaborative study comprising 20 laboratories in 12 European Countries to validate an enumeration method to be used as an official control method in the framework of Council Directive 70/524/EEC for probiotic lactobacilli used as feed additives was carried out. A spread plate method following BS ISO 15214:1998 using four different agars, MRS, acidified MRS, MRS with triphenyl tetrazolium chloride (TTC) and LAMVAB (a selective medium) was validated. Precision data in terms of repeatability ( r ) and reproducibility ( R ) of the method for each medium using different feeding stuffs with a high and a low inoculation level were determined. Lactobacilli were present in the samples in mixtures with other probiotics. The enumeration of lactobacilli on all agars showed a relative standard deviation (RSD r ) between 1.2% and 3.4% and a RSD R between 2.2% and 5.2%. MRS agar was preferred, followed by acidified MRS and MRS+TTC agar. LAMVAB gave about 0.5 log 10 cfu g −1 lower counts than the MRS media. For routine analysis the use of MRS agar is recommended depending on the presence and concentration of other probiotics such as enterococci, pediococci and yeast, acidified MRS or MRS+TTC agar are recommended. Pediococci and yeast were able to grow on LAMVAB. The methodology is not applicable to mineral feed. The results are intended for consideration for adaptation as CEN and ISO standards.

International audience; 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.

The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening. Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St. Nectaire and Reblochon. Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese. We assessed the genetic diversity of G. candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance. Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France. Sixty-four isolates were characterized. We found high genetic diversity of G. candidum even within the same cheesemaking regions. Strains did not group according to region. All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne. Strains varied in d -mannitol assimilation, and none used citrate as the sole source of carbon. Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St. Nectaire were filamentous. The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St. Nectaire cheesemaking facility. This study reveals an enormous diversity of G. candidum that has been empirically selected through the centuries by the cheesemakers of France.

Le recensement des especes naturellement presentes dans les fromages au lait cru et/ou l'etude de l'equilibre des flores au cours de l'affinage necessitent de disposer de milieux fiables. Or, il existe relativement peu de milieux de denombrement specifiques pour ces micro-organismes dans le domaine laitier. Trois milieux ont ete developpes. La performance de ces milieux, en terme de selectivite et d'electivite, a ete evaluee pour 25 echantillons de fromages (Camembert, Pont l'Eveque, Livarot, Comte, Saint-Nectaire, Salers) et pour 4 echantillons de lait cru, en comparaison avec les milieux Chapman et TSA/NaCl. Le milieu CRBM permettait la recuperation des bacteries d'affinage dans leur ensemble; supplemente en bacitracine, il selectionnait les staphylocoques tandis que la furazolidone permettait de denombrer les bacteries coryneformes, y compris les Micrococcaceae. Le milieu CRBM et le milieu CRBM modifie par addition d'antibiotiques etaient plus selectifs vis-a-vis de flores indesirables (enterobacteries, Bacillus, streptocoques, levures-moisissures). Ils presentaient par ailleurs une meilleure electivite vis-a-vis des bacteries d' affinage, la diversite des especes recensees etant plus importante. Pour tous les fromages etudies, la specificite des milieux etait satisfaisante. L'utilisation des milieux CRBM apparait interessante pour la caracterisation microbiologique des laits ou des fromages au lait cru.

This study was conducted to investigate the ability of cryoprotective chemicals to induce phenotypic cryoadaptation in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T. Tolerance to negative temperature stress (freezing at -20 degrees C and thawing at 37 degrees C) was induced by pretreatment with Me(2)SO, glycerol, lactose, sucrose, and trehalose. Interestingly, Me(2)SO has a significantly greater cryoprotective effect than glycerol. Furthermore, lactose, sucrose, and trehalose, often referred to as osmotica, were shown to have greater cryoadaptive than cryoprotective properties. These results suggest that bacteria such as L. delbrueckii ssp. bulgaricus could be phenotypically adapted to freezing and thawing by an osmotic stress applied prior to freeze-thaw stress.

La maitrise des bacteries lactiques (LAB) impliquees dans le domaine agroalimentaire necessite une meilleure connaissance de leur comportement au froid (congelation des ferments, affinage a basse temperature positive, conservation des produits finis). L'etude des reponses au stress hypothermique chez les bacteries lactiques implique de devoir prendre en consideration : (i) le niveau d'abaissement de la temperature (positive ou negative), (ii) la duree de l'epreuve a basse temperature qui permet de distinguer un « choc d'une « acclimatation », (iii) les differents types thermiques. De plus, l'analyse de la connection entre le stress genere par le transfert des bacteries lactiques dans un environnement a basse temperature et la reponse qui lui est associee, engendre trois domaines d'investigation: (i) la reponse de la bacterie au plan physiologique (croissance et cryotolerance), (ii) les modifications biochimiques engendrees (degre de desaturation des acides gras et profil proteique), (iii) la mise en place des systemes de controle de la reponse. Les informations recentes en genetique moleculaire concernant les reponses au stress hypothermique des bacteries lactiques semblent insuffisamment correlees a une meilleure comprehension des phenomenes biochimiques ou physiologiques tels que la cryotolerance.

Geotrichum candidum excretes D-3-phenyllactic acid, which inhibits the growth of Listeria monocytogenes. It was found to inhibit a range of gram-positive bacteria found in humans and foodstuffs, such as Staphylococcus aureus and Enterococcus faecalis, and gram-negative bacteria from humans, such as Providencia stuartii and Klebsiella oxytoca. Scanning electron microscope studies on the effect of D-3-phenyllactic acid on L. monocytogenes showed that it caused changes in bacterial behavior and structure. The bacteria formed aggregates and secreted polysaccharides; their cell walls lost their rigidity, causing the cells to swell. Finally the bacteria broke down completely and the cells disintegrate.

Geotrichum candidum can produce and excrete compounds that inhibit Listeria monocytogenes . These were purified by ultrafiltration, centrifugal partition chromatography, thin-layer chromatography, gel filtration, and high-pressure liquid chromatography, and analyzed by liquid chromatography-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation. Two inhibitors were identified: d -3-phenyllactic acid and d -3-indollactic acid.

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.

Thirty-nine samples of refrigerated raw milk were collected on site from 20 dairy farms. Thirty per cent of teats and udders were cotton swabbed in each herd. The presence of Lactococcus spp., yeasts and Pseudomonas spp. was evaluated. The level of contamination of samples varied greatly from one farm to another. Average Lactococcus spp. counts were 3.4 × 10 2 cfu mL −1 in milk, 2.6 × 10 3 cfu per total area on teats and 7.7 × 10 2 per 25cm 2 on udders. Average yeasts counts were of 6.9 × 10, 4.8 × 10 2 and 3.0 × 10 2 for milk, teats and udders, respectively, while average Pseudomonas spp. counts were of 5.8 × 10 2 , 2.1 × 10 4 and 6.2 × 10 3 . Variations in the level of Lactococcus spp. and yeasts in milk were not linked with their levels on teats and udders.

The concentrations of seven types of microorganism in the milk produced by farms using high quality milking procedures were monitored monthly. The most commonly encountered bacterium was Pseudomonas, whose concentration varied greatly between samples, but lactococci, lactobacilli and yeasts were also present at more stable concentrations. Staphylococcus aureus and β-glucuronidase-positive Escherichia coli were occasionally detected. Listeria monocytogenes was found in the samples from only one farm, while Yersinia enterocolitica was never detected. There were seasonal variations in the concentrations of lactobacilli and yeasts over the 2 years monitored. The changes in certain bacteria (Lactococcus, Lactobacillus) on any given farm were very similar from one year to the next. The microbiological characteristics of the milk from the farms examined seemed to be fairly constant. The profiles of some bacteria used for cheesemaking were relatively stable over time. It thus seems that the milk provided by different producers could be typed.

The interactions between 14 isolates of Geotrichum candidum and 14 isolates of Brevibacterium linens were analyzed to understand their behavior in cheese making. Mixed cultures were grown in a peptone broth containing various NaCl concentrations and various pH. The pH and the concentration of NaCl affected the interactions. According to the pH, in many strain pairs, B. linens strongly stimulated the growth of G. candidum , and this interaction varied depending on the pH. Inhibition occurred only at pH 5.0. In fewer pairs, G. candidum moderately stimulated the growth of B. linens , and the optimal pH for this stimulation and the inhibition were 7.0 and 5.0, respectively. The number of pairs in which the growth of G. candidum was increased by the presence of B. linens was highest with 1.5% added NaCl. With increasing NaCl concentration, stimulation of the growth of B. linens by G. candidum decreased because of the sensitivity of G . candidum and resistance of B. linens to NaCl. Brevibacterium linens isolates were similarly grown in the presence of filtrates of stationary phase cultures of G. candidum . The filtrates exhibited effects analogous to, but larger than, those of the strains from which the filtrates were prepared.

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.

Any modification of the environment that leads to a physiological, genetic, or epigenetic adaptive response in microorganisms may be considered as a stress. Historically, forms of stresses affecting biological structures were classified either as non-thermal, such as osmotic, oxidative, or acid stress or as thermal stress, hot or cold. Currently, the classification in biology is as abiotic, including physical and chemical stress, or biotic. The aim of this mini-review is to show, through the example of microorganisms, that the response to stress can be considered, in biology, as a global phenomenon, which can be extended to anthropogenic pressure.

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.

Lactobacilli are Gram positive rods belonging to the Lactic Acid Bacteria (LAB) group. Their phenotypic traits, such as each species' obligate/facultative, homo/heterofermentation abilities play a crucial role in souring raw milk and in the production of fermented dairy products such as cheese, yoghurt and fermented milk (including probiotics). An up to date safety analysis of these lactobacilli is needed to ensure consumer safety. Lactobacillus genus is a heterogeneous microbial group containing some 135 species and 27 subspecies, whose classification is constantly being reshuffled. With the recent use of advanced molecular methods it has been suggested that the extreme diversity of the Lactobacillus genomes would justify recognition of new subgeneric divisions. A combination of genotypic and phenotypic tests, for example DNA-based techniques and conventional carbohydrate tests, is required to determine species. Pulsed-Field gel Electrophoresis (PFGE) has been successfully applied to strains of dairy origin and is the most discriminatory and reproducible method for differentiating Lactobacillus strains. The bibliographical data support the hypothesis that the ingestion of Lactobacillus is not at all hazardous since lactobacillemia induced by food, particularly fermented dairy products, is extremely rare and only occurs in predisposed patients. Some metabolic features such as the possible production of biogenic amines in fermented products could generate undesirable adverse effects. A minority of starter and adjunct cultures and probiotic Lactobacillus strains may exceptionally show transferable antibiotic resistance. However, this may be underestimated as transferability studies are not systematic. We consider that transferable antibiotic resistance is the only relevant cause for caution and justifies performing antibiotic-susceptibility assays as these strains have the potential to serve as hosts of antibiotic-resistance genes, with the risk of transferring these genes to other bacteria. However, as a general rule, lactobacilli have a high natural resistance to many antibiotics, especially vancomycin, that is not transferable. Safety assessment requirements for Lactobacillus strains of technological interest should be limited to an antibiotic profile and a study to determine whether any antibiotic resistance(s) of medical interest detected is (or are) transferable. This agrees with the recent EFSA proposal suggesting attribution of a QPS status for 32 selected species of lactobacilli.

Geotrichum candidum is a ubiquitous filamentous yeast-like fungus commonly isolated from soil, air, water, milk, silage, plant tissues, digestive tract in humans and other mammals. This species is widely used as adjunct culture in the maturation of cheese. The genus Geotrichum is composed of 18 species. A recent taxonomic revision concluded that the old Galactomyces geotrichum/G. candidum complex contained four separate species of which Galactomyces candidus sp. nov./G. candidum. M13 primer can be used for identifying species of the Geotrichum genus. Used in combination, RAPD-PCR and RAM-PCR permit strains to be differentiated. The species can be unambiguous differentiated from the two species most frequently described in human pathology: Geotrichum clavatum (reclassified Saprochaete clavata) and Geotrichum capitatum (reclassified Magnusiomyces capitatus/Saprochaete capitata). Sources of exposure are food ingestion – cheese consumption playing a major role – inhalation and contact. A bibliographic survey was conducted to assess corresponding hazards and risks. G. candidum infections (mainly pulmonary or bronchopulmonary, but also cutaneous, oral, disseminates) are very rare: fewer than 100 cases reported between 1842 and 2006. Moreover, cases were not all confirmed by repeated isolations and demonstration of the fungus' presence in tissues, a prerequisite to establish a true diagnosis of geotrichosis. Immunocompromised population was recently shown as a target for opportunistic infection. The most effective treatments include either azole drogs as ketonazole, iconazole and clotrimazole, or polyene antibiotics as amphotericin B, nystatin and pimaricin, or voriconazole–amphotericin B association. Less than 1 case/year of disease was possibly caused by G. candidum and it never included dairy products or foodborne infection. The risk of developing an infection due to G. candidum in connection with its technological use and consumption of dairy products is virtually nil. For these reasons, G. candidum should be proposed for QPS status.

PCR primers were developed for the specific detection of Clavispora lusitaniae, Debaryomyces hansenii var hansenii, Geotrichum candidum, Kluyveromyces lactis and K. marxianus and Yarrowia lipolytica, yeast species commonly found on the surface of smear cheese. Forty eight representative strains frequently found in smear cheeses or taxonomically related to the target yeasts were used as templates, to validate the designed primers. The specific and selective detection of these yeasts was effective in situ, in Livarot smear, without yeast isolation and culture and was comparable with data obtained with a conventional method. The primers described here have thus potential for PCR studies applied to cheese. It should also be possible to use some of these primers with other substrates.

International audience; The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.

Geotrichum candidum is a cheese-ripening agent with the potential to produce sulphur flavour compounds in soft cheeses. We aimed to develop an alternative test for predicting the aromatic (sulphur flavours) potential of G. candidum strains in soft cheese. Twelve strains of G. candidum with different levels of demethiolase activity (determined by a chemical method) in YEL-met (yeast extract, lactate methionine) medium were studied. We investigated cgl (cystathionine gamma lyase) gene expression after culture in three media - YEL-met, casamino acid and curd media - and then carried out sensory analysis on a Camembert cheese matrix. We found no correlation between demethiolase activity in vitro and cgl gene expression. Sensory analysis (detection of sulphur flavours) identified different aromatic profiles linked to cgl expression, but not to demethiolase activity. The RT-PCR technique described here is potentially useful for predicting the tendency of a given strain of G. candidum to develop sulphur flavours in cheese matrix. This is the first demonstration that an in vitro molecular approach could be used as a predictive test for evaluating the potential of G. candidum strains to generate sulphur compounds in situ (Camembert cheese matrix).

International audience; 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.

The wide genotypic and phenotypic diversity of Geotrichum candidum strains does not facilitate its classification as yeast or a yeast-like fungus that is still a matter of debate. Whatever its classification, G. candidum possesses many different metabolic pathways that are of particular interest to the dairy industry. G. candidum is of importance in the maturation of cheese, and much is known about its direct contribution to cheese ripening and flavour formation. Its diverse metabolic potential means that G. candidum can play an important role in the ripening of many soft and semi-hard cheeses and make a positive contribution to the development of taste and aroma. It may also influence the growth of other microorganisms, both valuable and detrimental. The significance of the presence of G. candidum in cheese depends on the particular type of production and on the presence of biotypes featuring specific types of metabolism. However, in situ metabolic pathways involved in cheese ripening and their regulations are mainly unknown. The information available provides a good understanding of the potential of G. candidum strains that are used in cheese manufacture, and permits a better choice of strain depending on the characteristics required. The biochemical activities of G. candidum and its application in the dairy industry are presented in this review.

Lactobacillus rhamnosus MA27/6B and L. acidophilus MA27/6R are strains used in feed as probiotics. Their safety profiles and growth-stimulating properties were investigated via in vivo studies on young Swiss mice. After repeated administrations of different probiotic preparations in drinking water, safety parameters determined from liver, spleen and total weight remained unchanged. The growth-stimulating properties of viable or dead lactobacilli were studied after supplementation in drinking water. The feed intake (FI), water intake and body weight gain (WG) of the animals were compared to those of control mice. The lactobacilli supplementation of a sub-optimal diet made of barley allow recording of measurable growth performance of mice. It significantly increased WG compared to control groups (P < 0.01), by +28.9% and +31.7% for L. rhamnosus MA27/6B and L. acidophilus MA27/6R, respectively. This WG was correlated with a decrease in the consumption index. The effect of the dose ingested was also investigated: 10(8) lactobacilli CFU/mouse/day produced greater WG than 10(2), 10(4) or 10(6) CFU/mouse/day. No significant differences in growth performance parameters were observed between mice fed with 10(8) cells of viable or nonviable preparations. The mouse assay described could be used as a preliminary criterion when screening candidate probiotics for growth performance properties.

Geotrichum candidum is a yeast-like fungus used as ripening starter in cheese making. The present study focused on chemical stress pretreatments affecting survival of G. candidum ATCC 204307 to freeze-thaw stress. Cryotolerance of G. candidum cells was induced by pretreatment with NaCl, CaCl2, or MgCl2, indicating heterologous phenotypic adaptation to freeze-thaw stress (- 20 to 25 degrees C) by osmotic stress. Furthermore, the nystatin, an antifungal compound, was shown to be a cryotolerance inducer.

International audience; 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.

Aims: Assessment of genetic diversity within the species Geotrichum candidum and development of tools to trace the strains that play an important role in the agro food industry. Methods and Results: RAM-PCR and RAPD-PCR techniques were assessed for their ability to discriminate 57 strains of various morphotypes, substrates and geographical origin. The techniques were complementary and, when combined, allowed us to discriminate isolates. Moreover, we established a link between a taxon and its occupation of an ecological niche, which should not be confused with the substrate of isolation. Conclusions: We observed a high degree of diversity, which could be linked to the variety of the ecological niches chosen and to the high degree of morphological polymorphism encountered within the species. Significance and Impact of the Study: Used in combination, RAM-PCR and RAPD-PCR permit traceability and monitoring systems for G. candidum strains during food processing.

International audience

Aims: Enumeration of total, active or viable probiotic micro-organisms from liquid or solid commercial feedstuffs was studied during processing and storage. Methods and Results: After sample preparation, an epifluorescence microscopy technique and a plating method were investigated comparatively. It was shown that (i) on the day of manufacture, active or viable bacteria were in equivalent amounts and that viable numbers then decreased, depending on the different processing and storage factors enhancing ABNC production, (ii) the amount of total and active lactobacilli remained close and quite stable for months at a high level (>108 active fluorescent units). Conclusions: Processing and storage promoted ABNC cells in the products tested. Consequently, both techniques should be used to evaluate the viable-dead-active status of bacteria for which functional properties are claimed. Significance and Impact of the Study: Enumeration of the whole probiotic bacterial population should be take into account for guidelines and labelling since non-viable bacteria could have a probiotic effect.

The effect of cold stress on Geotrichum candidum was investigated at chill and freezing temperatures. Specific growth rates were determined at various temperatures and plotted according to the Ratkowsky and Arrhenius equations. The obtained profiles led to the determination of characteristics including the activation energy and notional minimum temperatures. At temperatures below the optimum single linear slopes were observed. At freezing temperatures, the loss of viability of cell populations was proportional to the number of freezing-thawing cycles. Nevertheless, the ability of G. candidum to survive this challenge depended on the physiological conditions prior to the freezing stress. The loss of viability was growth phase specific. Cells harvested in stationary phase showed a higher resistance compared to those obtained with cells in exponential phase. Furthermore, the cells of G. candidum could be adapted to the freeze-thaw challenge by pre-treatment at chill temperatures. This phenomenon known as cryotolerance was a function of the duration of the preincubation exposure.

We have investigated the capacities of Geotrichum candidum strains to produce sulphides from methionine. This attribute is very important in cheese technology because of the flavouring potential of sulphur compounds. A spectrophotometric procedure using 5,5′-dithiobis(2-nitrobenzoic acid) to determine sulphides was tested on a collection of G. candidum strains, and confirmed by gas chromatography–mass spectrometry. The strains were distinguished on the basis of their ability to produce methanethiol. Gas chromatography–mass spectrometry also made it possible to identify other sulphides, such as dimethyl disulphide, dimethyl trisulphide and dimethyl sulphide. Using sonicated cells, the specific production of these four sulphides was studied in presence of L-[S-methyl-2H]methionine. Both methanethiol and dimethyl sulphide were produced from methionine, but two different pathways were used by G. candidum.

International audience; Randomly amplified polymorphic DNA (RAPD) has been used for the rapid typing of Lactococcus lactis strains isolated from raw milk from the Camembert region of Normandy. It is thought that the diversity and perhaps the area strain specificity due to climatic and geographical factors of such wild-type lactococcal strains could contribute to the flavour differences and specific features detected for the same product in different areas. The patterns from 58 isolates were analysed by UPGMA dendrograms. At a similarity level of 50%, four RAPD clusters were distinguished. Clusters 1 and 2 contained strains of subspecies lactis and cluster 3 contained strains related to the C2 strain which is genetically cremoris but phenotypically lactis. The type strain of cremoris subspecies was significantly differentiated from these strains with primers P2 and P3. Thus, there was a real genetic diversity in pattern, making it possible to detect potential typical RAPD fragments.

International audience; A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L. lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency.

International audience; Thirty-eight Lactococcus strains, isolated from raw milk produced in two dairy areas in Normandy, were identified at the phenotypic level. Only Lactococcus lactis strains with the lactis phenotype were found in the milk samples. Most strains fermented lactose (97%) and showed proteinase activity (76%). Isolates were characterized by RAPD technique and rRNA gene restriction analysis. More L. lactis strains with the lactis genotype were found in the first area, while L. lactis strains with the cremoris genotype predominated in the second area. RAPD was more efficient than rRNA gene restriction analysis in differentiating between strains with the subsp. lactis genotype. For L. lactis with the subsp. cremoris genotype, the second method gave a better result but there was poor discrimination between strains. Plasmid profiles were determined. Patterns ranged in size from 1.3 to 16.5 kbp, and 29 different profiles were found. Six groups of strains were determined, five of which were specific for the area of origin. It is suggested that the region of manufacture could influence organoleptic properties of cheeses because of different Lactococcus strains in the raw milk used for cheese making.

D-3-Phenyllactic acid is a compound with anti-Listeria activity which is produced and secreted by the yeastlike fungus, Geotrichum candidum. This compound has a bactericidal effect independent of the physiological state of Listeria monocytogenes when added at a concentration of 7 mg/ml to tryptic soy broth supplemented with yeast extract (TSB-YE). An initial L. monocytogenes population of 10(5) CFU/ml was reduced 100-fold (2 log) after 4 days of culture at 25 degrees C in TSB-YE containing D-3-phenyllactic acid. The Listeria population was reduced 1,000-fold (3 log) when the compound was added during the exponential growth phase, and was reduced to less than 10 CFU/ml when it was added during the stationary phase. D-3-Phenyllactic acid had a bacteriostatic effect in UHT whole milk, reducing the population by 4.5 log, to give fewer cells than in the control after 5 days of culture. The results obtained with L. monocytogenes at concentrations of 10(5) and 10(3) CFU/ml in cheese curds were less conclusive. D-3-Phenyllactic acid was 10 times less active than nisin in our experimental conditions (TSB-YE at 25 degrees C).

The diversity of the prokaryotes that have been studied, combined with the many different effects of low temperature, has led to an extensive literature concerning cold stress responses in mesophilic bacteria. The aim of this review is to discuss the effects of cold on the behavior of bacteria. The following three responses will be described: (i) biochemical modifications consisting first of membrane fatty acid desaturation and second of the synthesis of cold stress proteins, (ii) physiological responses of the cells to permit growth at low temperatures above 0 degrees C and cryotolerance at lower temperatures, and (iii) control of the cold shock response at a transcriptional and/or translational level. This paper reviews knowledge, most of which has been acquired in the last 10 years, in the field of cold stress responses. It is hoped that these data will help to focus attention on the metabolic responses associated with environmental disturbance.

International audience; Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens, yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas, coliforms, Escherichia coli, enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml-1), lactococci (760 cfu ml-1) and Micrococcaceae (720 cfu ml-1) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts < 100 cfu ml-1. Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml-1. About 80% of supplies had < or = 10 E. coli cfu ml-1 and all samples had < or = 1 Cl. perfringens cfu ml-1. Two of the tested milks were positive for salmonellas (2.9%), four were positive for Listeria monocytogenes (5.8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1.4%).