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.

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.

Relationship between growth behaviour, micro and macroscopic morphologies and freezing sensitivity of the ripening starter Geotrichum candidum is strain specific and mostly related to the morphotypes: the arthrospores/hyphae parameter.

Microscopic conformation, growth behaviour and freezing sensitivity of seven strains of Geotrichum candidum, a ripening starter, were studied and compared according to their macroscopic morphotypes. It has been shown that the thallus forming units (TFU)×ml-1/OD600nm ratio as a function of time is an interesting parameter to follow G. candidum sporulation through the growth behaviour. Microscopic conformation, growth behaviour and freezing sensitivity are clearly strain specific and mostly related to their corresponding morphotypes "yeast", "mould" or "intermediate". The two "mould" strains that sporulate weakly (UCMA103, UCMA499) showed a low survival rate to freezing stress whereas the "yeast" strains expressed a significant resistance owing to the arthrospore abundance. Interestingly, one strain (UCMA96) which appeared on solid medium in accord with the "mould" morphotype respond similarly to freezing stress.

In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli.

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.

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.

Response to environmental stress as a global phenomenon in biology: the example of microorganisms.

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.

Safety assessment of dairy microorganisms: Geotrichum candidum.

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.

Safety assessment of dairy microorganisms: the Lactobacillus genus.

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.

Cryotolerance of Lactobacillus delbrueckii subsp. bulgaricus CFL1 is modified by acquisition of antibiotic resistance.

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.

Development of primers for detecting dominant yeasts in smear-ripened cheeses.

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.

Levels of cystathionine gamma lyase production by Geotrichum candidum in synthetic media and correlation with the presence of sulphur flavours in cheese.

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).

Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese.

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.

In vitro screening of potential probiotic activities of selected lactobacilli isolated from unpasteurized milk products for incorporation into soft cheese.

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 degrees 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 10(7) 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.

Numbers and strains of lactobacilli in some probiotic products.

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.

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.

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.

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.

Nystatin and osmotica as chemical enhancers of the phenotypic adaptation to freeze-thaw stress in Geotrichum candidum ATCC 204307.

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.

Safety and efficacy of probiotic lactobacilli in promoting growth in post-weaning Swiss mice.

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.