BACKGROUND
the contamination of raw milk cheese production chains, using bifidobacteria as fecal indicators, was previously studied in two different cheese processes in france  <cit> .
it has been demonstrated in that study that the species bifidobacterium pseudolongum was an efficient indicator of fecal contamination from animal origin, since this species is predominant in cow feces and has never been isolated in human feces.
in addition, as an indicator, b. pseudolongum was more sensitive than e. coli along the two different cheeses processes.
on the other side, bifidobacteria are also bacteria that are often desired in food products for their technological properties as thickening and stabilizing agents or for their health promoting effects.
for these reasons, bifidobacteria and lactic acid bacteria are commonly added to fermented food such as fermented milk or yoghurt but also in food supplements.
the positive effects encountered with bifidobacteria can be mitigated by the difficulty of some strains or species to resist or adapt to an industrial process.
they prefer to grow in the absence of oxygen, which can be a challenge in industry.
in addition, some strains are not able to survive the adverse conditions found in the stomach and duodenum making it difficult to exert any positive effect on the host.
the previous study of a production chain of l’etoile du vercors  showed an increase of total bifidobacteria along the process and led to the isolation of two other bifidobacterium populations, b. crudilactis and b. mongoliense <cit> .
these populations were able to grow along the cheese production chain.
the description of the species b. crudilactis was based on one of those populations  <cit> .
the other population could be phylogenetically identified as b. mongoliense <cit>  that was recently described based on two isolates obtained from a traditional fermented mare’s milk in mongolia.
the aims of the study were  to further detect the two bifidobacteria populations along the l’etoile du vercors cheese production chain using 16s rdna-restriction fragment length polymorphism,  to evaluate the potential of b. mongoliense as a new food supplement or as a potential new probiotic by the study of several phenotypic characteristics such as sugar fermentation profile, growth temperature profile and aptitude to survive in adverse condition such as acidic environment or environment rich in bile salts and  to perform a genotypic classification using dna-dna hybridization and mlsa  to identify if those new populations belong to specific clusters.
RESULTS
validation of the pcr-rflp method on pure isolates of bifidobacteria
sixty-four strains belonging to  <dig> different species were already tested in a previous study using pcr-rflp  <cit> .
eight additional strains of bifidobacterium crudilactis and four french isolates identified as b. mongoliense were analyzed in this part of the work .
the rflp patterns observed  with alui was named v ,  with taqi were called ix  or x .
the patterns combination v-x was attributed to b. crudilactis while the v-ix combination was observed for b. mongoliense.
in vitro
detection of bifidobacterium crudilactis and b. mongoliense in the l’etoile du vercors cheese process and in commercialized cheeses
- l’etoile du vercors cheese process .
production steps: a, raw milk; b, after addition of rennet; c, after removal from the mold; d, ripening ; n = number of cheese sample.
out of the  <dig> samples from the l’etoile du vercors cheese process  analyzed by pcr-rflp,  <dig>  were v-x type positive  and  <dig>  were v-ix type  positive.
their average counts were respectively  <dig>  ±  <dig>  and  <dig>  ±  <dig>  log cfu ml- <dig>
bifidobacterium crudilactis was detected in 82%  and 77%  of the samples, but in only 9% of the samples at step c. the species b. mongoliense was detected in only 18% of the total samples .
as observed for b. crudilactis, a very low percentage of samples were positive at step c , while the highest percentage was found at stage d .
the mean counts of b. crudilactis and of b. mongoliense were studied at each step of the production chain .
the b. crudilactis species presented highly significant variations .
a marked decrease at step c  was followed by a high increase at step d .
the mean counts of b. mongoliense were low  at the different steps of the production chain .
- commercialized raw milk cheeses .
bifidobacterium crudilactis
and
b. mongoliense
in  <dig> trade st marcellin cheeses and of  <dig> other trade raw milk cheeses
log cfu ml
- <dig>
or g
-1
- <dig>
or g
-1
*, other retail cheese with a manufacturing process similar to the one in vercors .
thirteen commercialized raw milk cheeses were analyzed: five trade cheeses from the plant in vercors and eight other trade raw milk cheeses with a manufacturing process similar to the one in vercors .
bifidobacterium crudilactis was detected in four st marcellin cheeses from the vercors’ plant , with counts equal to  <dig> log cfu g- <dig> in two samples and  <dig> log cfu g- <dig> in the two others, but not in the picodon cheese from this plant.
b. mongoliense was detected in four cheeses out of five from this plant.
this species was found in the picodon cheese  and in three st marcellin samples .
in the eight other commercialized cheeses manufactured with a similar process to the one in vercors, b. crudilactis was isolated from only three cheeses  while b. mongoliense was isolated from five cheeses , including one cheese made with pasteurized milk .
phenotypic and genotypic characterization of bifidobacterium crudilactis and b. mongoliense strains isolated from the cheeses
the species bifidobacterium crudilactis was described by  <cit>  on the basis of  <dig> strains out of  <dig> isolates from raw milk and two raw milk cheese production chains.
of the  <dig> isolates, one hundred and thirty-seven were obtained from  <dig> raw milk cheeses produced in the same french raw milk cheese factory in the vercors area, from april  <dig> to february  <dig>  <cit> .
another isolate was taken from a raw milk sample in another french raw milk cheese factory in the courtenay area.
the three remaining isolates came from three commercialized french raw milk cheeses .
the description of bifidobacterium mongoliense <cit>  was based on the study of two strains isolated from a traditional mare’s milk product from mongolia.
all strains analyzed in this study are presented in table  <dig>  using thirteen b. mongoliense strains isolated from french raw milk cheeses a more complete description of the species was made.
six of these strains were isolated from six raw milk cheeses at different steps of vercors’ plant .
three strains  were obtained from three commercialized cheeses from the vercors’ factory .
one strain  came from a raw milk cheese sample during ripening at day  <dig> in another french raw milk cheese factory in the courtenay area, and three others  from three commercialized french raw milk cheeses .
all cheeses containing b. crudilactis and b. mongoliense strains were made from raw cow milk, except for the two picodon cheeses, which were made from raw goat milk.
the strain fr41/ <dig> was retained as representative of the b. mongoliense french isolates since it was phenotypically the closest to all other french isolates.
phenotypic characterization
the major differential characteristics between the species bifidobacterium crudilactis, b. mongoliense, and the type strain of b. psychraerophilum are presented in table  <dig>
bifidobacterium mongoliense
,
b. crudilactis
and
b. psychraerophilum
lmg 21775
t
t
legend of table 4: bm: b. mongoliense, bc: b. crudilactis, bp: b. psychraerophilum; a, growth within 14 days; b, within 8 days; c, within 48 h; d, mean of  <dig> experiments performed in the laboratory; nt, not tested.
other phenotypic characteristics of the french b. mongoliense strains: acidification by ≥ 90% of the strains of ribose, galactose, glucose, esculin, cellobiose, maltose, melibiose and raffinose and were positive for α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, tyrosine arylamidase and histidine arylamidase.
none  ferment glycerol, erythritol, d-arabinose, l-xylose, adonitol, β-methyl-xyloside, l-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, inulin, xylitol, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, gluconate, 2-keto-gluconate, 5-keto-gluconate.
all strains  were negative for urease, indole production, nitrate reduction, arginine dihydrolase, β-galactosidase-6-phosphate, β-glucuronidase, β-n-acetylglucosaminidase, acide glutamique decarboxylase, α-fucosidase, acide pyroglutamique arylamidase, glutamyl arylamidase.
bifidobacterium psychraerophilum is one of the genetically closest species also able to multiply at low temperatures.
the  <dig> french strains identified as b. mongoliense were able to multiply from 10°c within 14 days to 41°c within 8 days.
the fr41/ <dig> strain, representative of the  <dig> strains, is also able to form colonies on tpy agar under aerobic conditions at 37°c within 3 days.
the colonies reach a reduced diameter under aerobic conditions compared to the diameter, up to 1 mm, observed after 3 days under anaerobic conditions.
in tpy, the minimum initial ph for growth of fr41/ <dig> was  <dig>  within 48 h .
similar growth characteristics were observed by  <cit>  except that no growth of b. mongoliense yit 10443t occurred at 10°c, nor at 40°c.
resistance to gastric, pancreatic juices and bile salts of bifidobacterium mongoliense strains fr 49/f/ <dig> and fr41/2
gastric juice
at ph  <dig>  a decrease of at least  <dig> log was observed for both studied strains after 1 h of incubation.
at ph  <dig>  a good survival of both strains was observed, even after 5 h of incubation, with no significant decrease in bifidobacteria counts .
bifidobacterium mongoliense
strains fr 49/f/ <dig> and fr 41/2
a
b
c
decrease in viable cell counts  after exposure: ato low ph  solutions during 1-5 h at 37°c, bto pancreatic juice solution during 1-5 h at 37°c, cto bile salts  during 48 h at 37°c.
na: not applicable.
nd: not determined.
pancreatic juice
both strains had a good survival in the presence of pancreatic juice .
a slight decrease was observed for both of them.
the strain fr49/f/ <dig> presented a decrease of respectively  <dig> ,  <dig> ,  <dig>  and  <dig>  log after 2 h, 3 h, 4 h and 5 h. the strain fr41/ <dig> presented a decrease of  <dig>  log after 5 h.
bile salts
in presence of a concentration of 1% bile salts, a decrease of  <dig>  and  <dig>  log was respectively observed for the strains fr49/f/ <dig> and fr41/ <dig> after 48 h. the strain fr49/f/ <dig> was less sensitive to lower bile salts concentration with a decrease of  <dig>  log and  <dig>  log in presence of bile salts at a concentration of respectively  <dig> % and  <dig> %.
the sensitivity of the strain fr41/ <dig> to lower bile salts concentration was the same than in presence of 1% with a decrease of  <dig>  log and  <dig>  log with a concentration of bile salts of respectively  <dig> % and  <dig> % .
genotypic characteristics
all data concerning bifidobacterium crudilactis  were given by  <cit> .
the 16s rdna sequences of b. mongoliense strains fr41/2t, fr49/f/ <dig>  and fr101/h/ <dig> were continuous stretches of 1452 bp respectively .
there was 100% identity between these sequences after alignment with clustalx  <cit> , and a high phylogenetic relationship  between the 16s rrna gene sequence of fr41/ <dig> and that of b. mongoliense yit 10443t.
the dna g + c content of the representative strain fr41/ <dig> was  <dig> % .
the average dna g + c content of six strains in the group was  <dig> % , which is also the g + c content of the b. mongoliense type strain .
those values were higher than the g + c contents of b. psychraerophilum lmg 21775t  and b. crudilactis lmg 23609t , using the tm method.
however, similar dna g + c contents are found with the same method in the genus bifidobacterium, for the species b. cuniculi, b. gallinarum, b. pseudolongum subsp.
pseudolongum, b. pseudolongum subsp.
globosum and b. scardovii <cit> .
dna-dna hybridizations were performed with dnas of fr41/ <dig>  other french b. mongoliense isolates and the type strains of b. mongoliense, b. minimum and b. subtile, the closest phylogenetically related species .
the dna-dna relatedness  between b. mongoliense dsm 21395t and fr41/ <dig> and between fr41/ <dig> and the other french isolates  confirmed identification of these isolates as b. mongoliense.
strain fr41/ <dig> showed low dna-dna similarity to the type strains of b. minimum and b. subtile .
bifidobacterium mongoliense
strains, and between fr41/ <dig> and the type strains of
b. mongoliense, b. minimum
and
b subtile
legend of table 6: dsmz, deutsche sammlung von mikroorganismen und zellkulturen gmbh, göttingen, germany.
the strain bifidobacterium mongoliense  was deposited in the dsmz by dr. koichi watanabe, yakult central institute for microbial research,  <dig>  yaho, kunitachi, tokyo 186– <dig>  japan.
strains clustering using a multilocus approach
analysis of mlsa  results of b. mongoliense fr41/ <dig> and fr49/f/ <dig> and b. crudilactis fr62/b/3t, based on concatenated sequences of the housekeeping genes clpc, fusa, gyrb, iles, purf, rplb and rpob, revealed a cluster for these strains, that was separated from other bifidobacterium strains from the mlst and ncbi database .
an allelic profile was determined for the  <dig> strains , according to the method of  <cit>  and was compared to the allelic profile of previously studied strains  <cit> .
the same sequence type was observed for the strains fr41/ <dig> and fr49/f/ <dig>  there were different by only one nucleic acid for the allele purf and rplb.
bifidobacterium crudilactis
and
b. mongoliense
discussion
it appears from our results that the species bifidobacterium crudilactis and b. mongoliense are commonly present in st-marcellin and other cheeses having a similar manufacturing process such as picodon, camembert, brie and reblochon.
b. crudilactis and b. mongoliense were detected in respectively 77% and 30% of cheeses from l’etoile du vercors plant after 21 days of ripening.
in addition, they were present in more than half of analyzed retail cheeses.
their presence and their source are not explained yet but they probably contribute to organoleptic and technologic properties of those cheeses.
during the l’etoile du vercors cheese process, a very low percentage of samples were positive at step c  with respectively 9% and 2% of positive samples for bifidobacterium crudilactis and b. mongoliense.
an explanation for the low percentage could be the presence of competitive flora at this stage associated with a low ph .
however, during ripening , the conditions seemed more favorable for those species with respectively 77% and 30% of positive samples for b. crudilactis and b. mongoliense.
the ripening temperature, 12°c, used in the vercors factory enables these species to grow along the production chain.
b. crudilactis was isolated at higher levels  than b. mongoliense in the vercors’ factory.
these levels were still higher in the four commercialized st marcellin cheeses .
bifidobacterium mongoliense was detected in only 30% of the samples during ripening of the vercors’ production chain  but was present at much higher levels in three of the commercialized st marcellin cheeses  and in the picodon cheese made with goat raw milk  from the vercors area.
these results suggest that both species, when present along the chain, continue to multiply in the cheeses after production, during storage.
b. mongoliense was detected in one cheese made from pasteurized milk.
the most probable explanation is a cross contamination along the process or commercialization leading to the presence of this bacterial species.
interestingly, the other species, b. crudilactis was detected in thermized milk in our study  and in thermized milk from canada  <cit> .
this suggests that b. crudilactis has the potential to resist to some heat treatment.
those properties should be better investigated, as they are important when these bacteria are incorporated in food products where heating steps are present.
the resistance to gastric and pancreatic juices, and bile salts of these bifidobacteria encourages further study for their potential to survive the transit through the human intestinal tract.
the stomach normally produces around  <dig>  l of hcl per day at a ph of  <dig>  and a stomach ph can drop as low as  <dig>   <cit> .
it is therefore important, when selecting probiotic strains candidates, to choose strains able to survive this barrier.
usually, acid resistance of probiotic strains is estimated for different ph values ranging from  <dig>  to  <dig> .
incubation time can vary from  <dig> to 360 minutes at 37°c, according to the fact that food usually remains in the stomach for about 90 minutes.
in our study, a poor survival of strains fr49/f/ <dig> and fr41/ <dig> was observed in gastric juice at ph =  <dig>  after 1 h of incubation.
however, the survival of the strains was excellent at ph  <dig>  those results are quite encouraging knowing the limitation of the used method, namely hcl acidified media do not take into account the influence of diet or non-acidic gastric constituents on the microorganisms during their gastric transit making that test usually too severe.
for example, the strain lactobacillus gg cannot survive in static models, but is able to reach colon in high number in vivo <cit> .
the strains assessed in our study should be further tested in a dynamic in vitro model such as the shime or m-shime system  <cit>  for confirmation of their survival through the transit.
pancreatic secretions are amongst the most aggressive fluids in the human body.
in our case, using a commercial pancreatic solution at ph  <dig>  a good resistance of both strains was observed even after 5 h incubation.
physiological concentrations of bile salts are between  <dig>   <cit>  and  <dig> %  <cit> .
this is why bile solutions containing bile salts at concentrations of  <dig> ,  <dig>  and 1% were tested in our study.
usually, bacteria are exposed to bile salts from  <dig> to 96 h. bile salts secretions in vivo are highly variable and difficult to predict.
in our study, the strain fr 49/f/ <dig> showed a better resistance than the fr 41/ <dig> with  <dig> % bile salts.
in the other conditions, a decrease of approximately  <dig> logarithms was observed.
it is important to note that bile salts solutions and animal bile extracts are more aggressive for bacteria than human bile  <cit> .
according to the fact that human bile is not easily available, testing of bile salts is a good compromise even if the conditions tested are probably more aggressive than in reality.
the best compromise would have been to use bovine bile extract, which is less toxic than bile salts, but still more toxic than human bile.
the results obtained by mlsa are in accordance with the other genomic data presented in this study and allow a temporarily classification of b. crudilactis and b. mongoliense in two different clusters, well separated from the other bifidobacteria clusters.
if, in the future, additional genome sequences are available on public database such as ncbi or on bifidobacterium mlst  database , more closely related bifidobacterium species such as b. bohemicum, b. minimum, and b. psychraerophilum could be included in this analysis as well.
this method was already successfully used for species identification in several genera such as vibrio or bifidobacterium <cit> .
however, one of the next steps will be to continue the genetic analysis of the full genome sequences to better understand the evolution of those strains and be able to explain their specific phylogenetic characteristics.
origin of the species b. crudilactis and b. mongoliense is not known.
in a previous paper  <cit> , b. pseudolongum was chosen as an efficient fecal indicator since its mean counts remained stable along both studied processes.
in addition, b. pseudolongum defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces.
in the case of b. crudilactis and b. mongoliense, the fecal origin is not demonstrated.
CONCLUSIONS
the species b. crudilactis and b. mongoliense were detected in raw milk cheeses from the vercors’ area.
the origin of these bacteria is not known yet, but their presence in the cheeses probably contributes to specific organoleptic and technologic characteristics of those cheeses.
this study allowed to report that these bacteria are able to grow under 12°c during the manufacturing process of the cheeses, which has never been described for bifidobacteria, and which is a very interesting technological asset.
in addition, the bacteria were able to resist to low ph and aggressive fluids such as pancreatic juice and biliar salts.
because of the special characteristics of these bifidobacteria, further investigations should be performed to better characterize their technologic potential or their beneficial effect as possible new probiotics.
the genotypic analyses using total dna-dna hybridization, gc% content, 16s rrna gene sequencing and mlsa further confirmed the classification of b. crudilactis and b. mongoliense into two different clusters, well separated from other bifidobacteria clusters.

 the contamination of raw milk cheese production chains, using bifidobacteria as fecal indicators, was previously studied in two different cheese processes in france. it has been demonstrated in that study that the species bifidobacterium pseudolongum was an efficient indicator of fecal contamination from animal origin, since this species is predominant in cow feces and has never been isolated in human feces. in addition, as an indicator, b. pseudolongum was more sensitive than e. coli along the two different cheeses processes. on the other side, bifidobacteria are also bacteria that are often desired in food products for their technological properties as thickening and stabilizing agents or for their health promoting effects. for these reasons, bifidobacteria and lactic acid bacteria are commonly added to fermented food such as fermented milk or yoghurt but also in food supplements. the positive effects encountered with bifidobacteria can be mitigated by the difficulty of some strains or species to resist or adapt to an industrial process. they prefer to grow in the absence of oxygen, which can be a challenge in industry. in addition, some strains are not able to survive the adverse conditions found in the stomach and duodenum making it difficult to exert any positive effect on the host. the previous study of a production chain of l’etoile du vercors showed an increase of total bifidobacteria along the process and led to the isolation of two other bifidobacterium populations, b. crudilactis and b. mongoliense. these populations were able to grow along the cheese production chain. the description of the species b. crudilactis was based on one of those populations. the other population could be phylogenetically identified as b. mongoliense that was recently described based on two isolates obtained from a traditional fermented mare’s milk in mongolia. the aims of the study were to further detect the two bifidobacteria populations along the l’etoile du vercors cheese production chain using 16s rdna-restriction fragment length polymorphism, to evaluate the potential of b. mongoliense as a new food supplement or as a potential new probiotic by the study of several phenotypic characteristics such as sugar fermentation profile, growth temperature profile and aptitude to survive in adverse condition such as acidic environment or environment rich in bile salts and to perform a genotypic classification using dna-dna hybridization and mlsa to identify if those new populations belong to specific clusters.  validation of the pcr-rflp method on pure isolates of bifidobacteria sixty-four strains belonging to different species were already tested in a previous study using pcr-rflp. eight additional strains of bifidobacterium crudilactis and four french isolates identified as b. mongoliense were analyzed in this part of the work. the rflp patterns observed with alui was named v, with taqi were called ix or x. the patterns combination v-x was attributed to b. crudilactis while the v-ix combination was observed for b. mongoliense. in vitro detection of bifidobacterium crudilactis and b. mongoliense in the l’etoile du vercors cheese process and in commercialized cheeses - l’etoile du vercors cheese process. production steps: a, raw milk; b, after addition of rennet; c, after removal from the mold; d, ripening; n = number of cheese sample. out of the samples from the l’etoile du vercors cheese process analyzed by pcr-rflp, were v-x type positive and were v-ix type positive. their average counts were respectively  ±  and  ±  log cfu ml- bifidobacterium crudilactis was detected in 82% and 77% of the samples, but in only 9% of the samples at step c. the species b. mongoliense was detected in only 18% of the total samples. as observed for b. crudilactis, a very low percentage of samples were positive at step c, while the highest percentage was found at stage d. the mean counts of b. crudilactis and of b. mongoliense were studied at each step of the production chain. the b. crudilactis species presented highly significant variations. a marked decrease at step c was followed by a high increase at step d. the mean counts of b. mongoliense were low at the different steps of the production chain. - commercialized raw milk cheeses. bifidobacterium crudilactis and b. mongoliense in trade st marcellin cheeses and of other trade raw milk cheeses log cfu ml - or g -1 - or g -1 *, other retail cheese with a manufacturing process similar to the one in vercors. thirteen commercialized raw milk cheeses were analyzed: five trade cheeses from the plant in vercors and eight other trade raw milk cheeses with a manufacturing process similar to the one in vercors. bifidobacterium crudilactis was detected in four st marcellin cheeses from the vercors’ plant, with counts equal to log cfu g- in two samples and log cfu g- in the two others, but not in the picodon cheese from this plant. b. mongoliense was detected in four cheeses out of five from this plant. this species was found in the picodon cheese and in three st marcellin samples. in the eight other commercialized cheeses manufactured with a similar process to the one in vercors, b. crudilactis was isolated from only three cheeses while b. mongoliense was isolated from five cheeses, including one cheese made with pasteurized milk. phenotypic and genotypic characterization of bifidobacterium crudilactis and b. mongoliense strains isolated from the cheeses the species bifidobacterium crudilactis was described by on the basis of strains out of isolates from raw milk and two raw milk cheese production chains. of the isolates, one hundred and thirty-seven were obtained from raw milk cheeses produced in the same french raw milk cheese factory in the vercors area, from april to february. another isolate was taken from a raw milk sample in another french raw milk cheese factory in the courtenay area. the three remaining isolates came from three commercialized french raw milk cheeses. the description of bifidobacterium mongoliense was based on the study of two strains isolated from a traditional mare’s milk product from mongolia. all strains analyzed in this study are presented in table  using thirteen b. mongoliense strains isolated from french raw milk cheeses a more complete description of the species was made. six of these strains were isolated from six raw milk cheeses at different steps of vercors’ plant. three strains were obtained from three commercialized cheeses from the vercors’ factory. one strain came from a raw milk cheese sample during ripening at day in another french raw milk cheese factory in the courtenay area, and three others from three commercialized french raw milk cheeses. all cheeses containing b. crudilactis and b. mongoliense strains were made from raw cow milk, except for the two picodon cheeses, which were made from raw goat milk. the strain fr41/ was retained as representative of the b. mongoliense french isolates since it was phenotypically the closest to all other french isolates. phenotypic characterization the major differential characteristics between the species bifidobacterium crudilactis, b. mongoliense, and the type strain of b. psychraerophilum are presented in table  bifidobacterium mongoliense, b. crudilactis and b. psychraerophilum lmg 21775 t t legend of table 4: bm: b. mongoliense, bc: b. crudilactis, bp: b. psychraerophilum; a, growth within 14 days; b, within 8 days; c, within 48 h; d, mean of experiments performed in the laboratory; nt, not tested. other phenotypic characteristics of the french b. mongoliense strains: acidification by ≥ 90% of the strains of ribose, galactose, glucose, esculin, cellobiose, maltose, melibiose and raffinose and were positive for α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, tyrosine arylamidase and histidine arylamidase. none ferment glycerol, erythritol, d-arabinose, l-xylose, adonitol, β-methyl-xyloside, l-sorbose, rhamnose, dulcitol, inositol, mannitol, sorbitol, inulin, xylitol, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, gluconate, 2-keto-gluconate, 5-keto-gluconate. all strains were negative for urease, indole production, nitrate reduction, arginine dihydrolase, β-galactosidase-6-phosphate, β-glucuronidase, β-n-acetylglucosaminidase, acide glutamique decarboxylase, α-fucosidase, acide pyroglutamique arylamidase, glutamyl arylamidase. bifidobacterium psychraerophilum is one of the genetically closest species also able to multiply at low temperatures. the french strains identified as b. mongoliense were able to multiply from 10°c within 14 days to 41°c within 8 days. the fr41/ strain, representative of the strains, is also able to form colonies on tpy agar under aerobic conditions at 37°c within 3 days. the colonies reach a reduced diameter under aerobic conditions compared to the diameter, up to 1 mm, observed after 3 days under anaerobic conditions. in tpy, the minimum initial ph for growth of fr41/ was within 48 h. similar growth characteristics were observed by except that no growth of b. mongoliense yit 10443t occurred at 10°c, nor at 40°c. resistance to gastric, pancreatic juices and bile salts of bifidobacterium mongoliense strains fr 49/f/ and fr41/2 gastric juice at ph  a decrease of at least log was observed for both studied strains after 1 h of incubation. at ph  a good survival of both strains was observed, even after 5 h of incubation, with no significant decrease in bifidobacteria counts. bifidobacterium mongoliense strains fr 49/f/ and fr 41/2 a b c decrease in viable cell counts after exposure: ato low ph solutions during 1-5 h at 37°c, bto pancreatic juice solution during 1-5 h at 37°c, cto bile salts during 48 h at 37°c. na: not applicable. nd: not determined. pancreatic juice both strains had a good survival in the presence of pancreatic juice. a slight decrease was observed for both of them. the strain fr49/f/ presented a decrease of respectively,, and log after 2 h, 3 h, 4 h and 5 h. the strain fr41/ presented a decrease of log after 5 h. bile salts in presence of a concentration of 1% bile salts, a decrease of and log was respectively observed for the strains fr49/f/ and fr41/ after 48 h. the strain fr49/f/ was less sensitive to lower bile salts concentration with a decrease of log and log in presence of bile salts at a concentration of respectively % and %. the sensitivity of the strain fr41/ to lower bile salts concentration was the same than in presence of 1% with a decrease of log and log with a concentration of bile salts of respectively % and %. genotypic characteristics all data concerning bifidobacterium crudilactis were given by. the 16s rdna sequences of b. mongoliense strains fr41/2t, fr49/f/ and fr101/h/ were continuous stretches of 1452 bp respectively. there was 100% identity between these sequences after alignment with clustalx, and a high phylogenetic relationship between the 16s rrna gene sequence of fr41/ and that of b. mongoliense yit 10443t. the dna g + c content of the representative strain fr41/ was %. the average dna g + c content of six strains in the group was %, which is also the g + c content of the b. mongoliense type strain. those values were higher than the g + c contents of b. psychraerophilum lmg 21775t and b. crudilactis lmg 23609t, using the tm method. however, similar dna g + c contents are found with the same method in the genus bifidobacterium, for the species b. cuniculi, b. gallinarum, b. pseudolongum subsp. pseudolongum, b. pseudolongum subsp. globosum and b. scardovii. dna-dna hybridizations were performed with dnas of fr41/ other french b. mongoliense isolates and the type strains of b. mongoliense, b. minimum and b. subtile, the closest phylogenetically related species. the dna-dna relatedness between b. mongoliense dsm 21395t and fr41/ and between fr41/ and the other french isolates confirmed identification of these isolates as b. mongoliense. strain fr41/ showed low dna-dna similarity to the type strains of b. minimum and b. subtile. bifidobacterium mongoliense strains, and between fr41/ and the type strains of b. mongoliense, b. minimum and b subtile legend of table 6: dsmz, deutsche sammlung von mikroorganismen und zellkulturen gmbh, göttingen, germany. the strain bifidobacterium mongoliense was deposited in the dsmz by dr. koichi watanabe, yakult central institute for microbial research, yaho, kunitachi, tokyo 186– japan. strains clustering using a multilocus approach analysis of mlsa results of b. mongoliense fr41/ and fr49/f/ and b. crudilactis fr62/b/3t, based on concatenated sequences of the housekeeping genes clpc, fusa, gyrb, iles, purf, rplb and rpob, revealed a cluster for these strains, that was separated from other bifidobacterium strains from the mlst and ncbi database. an allelic profile was determined for the strains, according to the method of and was compared to the allelic profile of previously studied strains. the same sequence type was observed for the strains fr41/ and fr49/f/ there were different by only one nucleic acid for the allele purf and rplb. bifidobacterium crudilactis and b. mongoliense discussion it appears from our results that the species bifidobacterium crudilactis and b. mongoliense are commonly present in st-marcellin and other cheeses having a similar manufacturing process such as picodon, camembert, brie and reblochon. b. crudilactis and b. mongoliense were detected in respectively 77% and 30% of cheeses from l’etoile du vercors plant after 21 days of ripening. in addition, they were present in more than half of analyzed retail cheeses. their presence and their source are not explained yet but they probably contribute to organoleptic and technologic properties of those cheeses. during the l’etoile du vercors cheese process, a very low percentage of samples were positive at step c with respectively 9% and 2% of positive samples for bifidobacterium crudilactis and b. mongoliense. an explanation for the low percentage could be the presence of competitive flora at this stage associated with a low ph. however, during ripening, the conditions seemed more favorable for those species with respectively 77% and 30% of positive samples for b. crudilactis and b. mongoliense. the ripening temperature, 12°c, used in the vercors factory enables these species to grow along the production chain. b. crudilactis was isolated at higher levels than b. mongoliense in the vercors’ factory. these levels were still higher in the four commercialized st marcellin cheeses. bifidobacterium mongoliense was detected in only 30% of the samples during ripening of the vercors’ production chain but was present at much higher levels in three of the commercialized st marcellin cheeses and in the picodon cheese made with goat raw milk from the vercors area. these results suggest that both species, when present along the chain, continue to multiply in the cheeses after production, during storage. b. mongoliense was detected in one cheese made from pasteurized milk. the most probable explanation is a cross contamination along the process or commercialization leading to the presence of this bacterial species. interestingly, the other species, b. crudilactis was detected in thermized milk in our study and in thermized milk from canada. this suggests that b. crudilactis has the potential to resist to some heat treatment. those properties should be better investigated, as they are important when these bacteria are incorporated in food products where heating steps are present. the resistance to gastric and pancreatic juices, and bile salts of these bifidobacteria encourages further study for their potential to survive the transit through the human intestinal tract. the stomach normally produces around  l of hcl per day at a ph of and a stomach ph can drop as low as. it is therefore important, when selecting probiotic strains candidates, to choose strains able to survive this barrier. usually, acid resistance of probiotic strains is estimated for different ph values ranging from to. incubation time can vary from to 360 minutes at 37°c, according to the fact that food usually remains in the stomach for about 90 minutes. in our study, a poor survival of strains fr49/f/ and fr41/ was observed in gastric juice at ph =  after 1 h of incubation. however, the survival of the strains was excellent at ph  those results are quite encouraging knowing the limitation of the used method, namely hcl acidified media do not take into account the influence of diet or non-acidic gastric constituents on the microorganisms during their gastric transit making that test usually too severe. for example, the strain lactobacillus gg cannot survive in static models, but is able to reach colon in high number in vivo. the strains assessed in our study should be further tested in a dynamic in vitro model such as the shime or m-shime system for confirmation of their survival through the transit. pancreatic secretions are amongst the most aggressive fluids in the human body. in our case, using a commercial pancreatic solution at ph  a good resistance of both strains was observed even after 5 h incubation. physiological concentrations of bile salts are between and %. this is why bile solutions containing bile salts at concentrations of, and 1% were tested in our study. usually, bacteria are exposed to bile salts from to 96 h. bile salts secretions in vivo are highly variable and difficult to predict. in our study, the strain fr 49/f/ showed a better resistance than the fr 41/ with % bile salts. in the other conditions, a decrease of approximately logarithms was observed. it is important to note that bile salts solutions and animal bile extracts are more aggressive for bacteria than human bile. according to the fact that human bile is not easily available, testing of bile salts is a good compromise even if the conditions tested are probably more aggressive than in reality. the best compromise would have been to use bovine bile extract, which is less toxic than bile salts, but still more toxic than human bile. the results obtained by mlsa are in accordance with the other genomic data presented in this study and allow a temporarily classification of b. crudilactis and b. mongoliense in two different clusters, well separated from the other bifidobacteria clusters. if, in the future, additional genome sequences are available on public database such as ncbi or on bifidobacterium mlst database, more closely related bifidobacterium species such as b. bohemicum, b. minimum, and b. psychraerophilum could be included in this analysis as well. this method was already successfully used for species identification in several genera such as vibrio or bifidobacterium. however, one of the next steps will be to continue the genetic analysis of the full genome sequences to better understand the evolution of those strains and be able to explain their specific phylogenetic characteristics. origin of the species b. crudilactis and b. mongoliense is not known. in a previous paper, b. pseudolongum was chosen as an efficient fecal indicator since its mean counts remained stable along both studied processes. in addition, b. pseudolongum defined a contamination from animal origin since this species is predominant in cow dung and has never been isolated in human feces. in the case of b. crudilactis and b. mongoliense, the fecal origin is not demonstrated.  the species b. crudilactis and b. mongoliense were detected in raw milk cheeses from the vercors’ area. the origin of these bacteria is not known yet, but their presence in the cheeses probably contributes to specific organoleptic and technologic characteristics of those cheeses. this study allowed to report that these bacteria are able to grow under 12°c during the manufacturing process of the cheeses, which has never been described for bifidobacteria, and which is a very interesting technological asset. in addition, the bacteria were able to resist to low ph and aggressive fluids such as pancreatic juice and biliar salts. because of the special characteristics of these bifidobacteria, further investigations should be performed to better characterize their technologic potential or their beneficial effect as possible new probiotics. the genotypic analyses using total dna-dna hybridization, gc% content, 16s rrna gene sequencing and mlsa further confirmed the classification of b. crudilactis and b. mongoliense into two different clusters, well separated from other bifidobacteria clusters. 
crudilactis and b. mongoliense into two different clusters well separated from other bifidobacteria clusters. the study of a production chain of raw milk cheeses led to the isolation of two bifidobacterium populations: b. crudilactis and b. mongoliense, that were able to grow along the production chain. the aims of this study were to further detect and characterize these bacteria along the process and evaluate the ability of some strains to survive or grow in adverse conditions. according to the observed characteristics such as survival in adverse conditions and their ability to grow under 12°c during the manufacturing process of the cheeses, which has never been described for bifidobacteria and which is a very interesting technological asset, these b. crudilactis and b. mongoliense strains should be further investigated for a potential use in new food or in food supplements. using pcr coupled with restriction fragment length polymorphism, b. crudilactis and b. mongoliense were detected in respectively 77% and 30% of st marcellin cheeses from production chain after 21 days of ripening. they were present in more than half of all analyzed retail cheeses with counts going from to log cfu g- for b. crudilactis and to log cfu g- for b. mongoliense. the genotypic analyses using total dna-dna hybridization, gc% content, 16s rrna gene sequencing and multilocus sequencing analysis confirmed the classification of bifidobacterium. bifidobacterium mongoliense was sensitive to ph  with an observed decrease of at least log for both studied strains after 1 h incubation. good survival was observed for the same strains in presence of pancreatic juice with a decrease of less than one log.
 the study of a production chain of raw milk cheeses led to the isolation of two bifidobacterium populations: b. crudilactis and b. mongoliense, that were able to grow along the production chain. the aims of this study were to further detect and characterize these bacteria along the process and evaluate the ability of some strains to survive or grow in adverse conditions.  using pcr coupled with restriction fragment length polymorphism, b. crudilactis and b. mongoliense were detected in respectively 77% and 30% of st marcellin cheeses from production chain after 21 days of ripening. they were present in more than half of all analyzed retail cheeses with counts going from to log cfu g- for b. crudilactis and to log cfu g- for b. mongoliense. bifidobacterium mongoliense was sensitive to ph  with an observed decrease of at least log for both studied strains after 1 h incubation. at ph  no significant decrease was observed. good survival was observed for the same strains in presence of pancreatic juice with a decrease of less than one log. survival of strain fr49/f/ was better than fr41/ with a decrease of logarithms and almost logarithms. the genotypic analyses using total dna-dna hybridization, gc% content, 16s rrna gene sequencing and multilocus sequencing analysis confirmed the classification of bifidobacterium. crudilactis and b. mongoliense into two different clusters well separated from other bifidobacteria clusters.  according to the observed characteristics such as survival in adverse conditions and their ability to grow under 12°c during the manufacturing process of the cheeses, which has never been described for bifidobacteria and which is a very interesting technological asset, these b. crudilactis and b. mongoliense strains should be further investigated for a potential use in new food or in food supplements. bifidobacteriacrudilactismongolienseraw milk cheesessequencingpcr-rflpdna-dna hybridizationmlsa 