BACKGROUND
lactobacilli have long been of interest to the dairy and agriculture industries, in fact, they are defined as generally regarded as safe , and some have been found as ubiquitous members of the mucosae of healthy subjects  <cit> .
some studies describe the use of lactic acid bacteria  for the treatment or prevention of infections of the intestinal and genital tracts with different extents of success  <cit> .
it is quite difficult to identify which properties of lactobacilli are required to prevent and eventually treat diseases and to determine the adequate dosage, duration, and methods of delivery.
in respect to vaginal probiotics, the protective role of lactobacilli seems to be based upon two mechanisms, namely, the specific adherence to the vaginal epithelium leading to intensive colonization of this surface, and the control of the remaining vaginal microflora through antagonism against pathogens.
as a consequence, the ability of lactobacillus to adhere to epithelial cells and mucosal surfaces is a key criterion for the selection of probiotics  <cit> .
the efficacy of the available commercial products is also strictly dependent on the viability of the probiotic strains contained in the preparations, since the amount of applied microorganism could be crucial for the effectiveness of the product  <cit> , and several studies revealed that some health food products did not satisfy the claims stated on the labels therefore minimizing the expected health benefits  <cit> .
therefore the evaluation of cell viability in conditions that mimic the practical application is a key issue in the selection of probiotics.
also the development of novel fermentation strategies to increase the final biomass yield is central to bypass one of the bottlenecks encountered in the production of starters, probiotic ingredients and medical devices.
however, since their growth is inhibited by their primary metabolic product , lactobacilli are rarely cultivated at high cellular density  in batch/fed-batch fermentation processes.
vaginal probiotics are a rather new area of investigation and, therefore, not much is known about the mechanisms, the conditions or characteristics needed to assess their efficacy.
several strains appear to be effective in colonizing and then protecting the intestine and the urogenital tract  <cit> , from infections.
commercial lactobacilli-based products such as normogin® have demonstrated to be a reliable treatment for reducing the recurrence of bacterial vaginosis  <cit> .
it has been reported that infection mechanisms are mainly due to a disestablishment of the normal resident vaginal microflora, primarily a loss of h2o2-producing lactobacilli  <cit> , although some studies do not support this hypothesis  <cit> .
in vitro studies have suggested that the re-colonization of the urinary tract by certain specific strains of lactobacilli seems to be a suitable approach to prevent infections and relapses  <cit> .
recently it has also been suggested that some probiotic bacteria could be effective not only when locally delivered  but also when assumed per os <cit> , and this establishes a link between the rate of intestinal survival and vaginal colonization  <cit> .
lactobacillus crispatus can persist in the gastrointestinal tract  <cit>  and is among the most prevalent species of the lactobacillus-dominated human vaginal microbiota  <cit> , and resistance to very low ph conditions have also been described  <cit> .
a strain of l. crispatus  isolated from the vaginal flora of a healthy woman was characterized in this study.
in particular, the ability of l. crispatus l <dig> to survive to an in vitro simulated digestion was evaluated and its physiological and metabolic requirements were investigated.
optimal growth conditions were defined, in order to obtain high density cultivations needed for potential applications of this strain as probiotic supplement.
the use of an in situ product removal fermentation process allowed a 7-fold improvement of the biomass yield compared to traditional processes, accompanied by an extremely high cellular viability .
given the necessity of probiotic preparations to deliver a certain amount of viable microbial cells the effect of different protective agents on freeze-drying procedures was also investigated.
moreover, in order to investigate on the chemical nature of the agents that are at the basis of the beneficial effect of l. crispatus l <dig> we have established the primary structure of its exopolysaccharides , since previous studies  <cit>  on bacterial adhesion showed that eps might promote the adherence of bacteria to biological surfaces, thereby facilitating the colonization of various ecological niches.
intriguingly, the eps resulted to be a mannan polysaccharide possessing a structure very similar to the one produced by candida albicans <cit> .
the eps was found to be present in adherent biofilms and might be involved in initial and permanent adhesion.
moreover the eps-induced increased expression of the human defensin hbd- <dig> in vaginal cells was also verified, identifying a possible connection with c. albicans growth inhibition  <cit> .
RESULTS
strain identification and h2o <dig> production
a lactobacillus strain isolated from human vaginal secretion was allotted to crispatus subspecies by 16s ribosomal dna sequencing  <cit>  and it was named l. crispatus l <dig>  in particular, pcr products were pooled, purified and sequenced.
in addition, the ability of  <dig> lactobacillus strains to produce h2o <dig> was evaluated.
the percentage of strains classified as strong, medium, weak and negative h2o <dig> producers was  <dig>   <dig>   <dig> and 5%, respectively.
l. crispatus l <dig> was found to be the best of the isolates in the laboratory collection.
in vitro digestion
results from shake flask experiments simulating the passage through the gastrointestinal tract showed a good resistance of l. crispatus l <dig> to the in vitro digestion process.
the bacterial dose significantly influenced results, as shown in figure 1a clearly indicating that  <dig> ⋅ <dig> cells∙ml− <dig> corresponds to the minimal required initial concentration of cells necessary to survive gastric juices.
incubation in simulated pancreatic juices  with different oxgall concentrations  did not affect viability, whereas a slight increase of the cell number within 4 h was observed.
moreover, treated cells reached a final biomass yield comparable with that of the control cells .
shakeflask experiments
a semidefined medium containing soy peptone  and yeast extract  was used to investigate the amount of biomass and lactic acid produced using different carbon sources .
the final titer of biomass produced in shake flasks was very similar in all the media analysed.
the production of lactic acid was quite high ranging between  <dig>  and  <dig>  g∙l− <dig>  and resulting in relevant yp/s ranging between  <dig>  and  <dig>  g∙g− <dig>  the yp/s on dextrins could not be calculated due to the presence of high molecular weight carbohydrates  that were not degraded and metabolized as evidenced by high performance anion exchange chromatography with pulsed amperometric detection  analyses.
l. crispatus
l <dig> in shake flasks on sdm medium supplemented with different carbon sources
8h
x/s
p/s
the concentration of biomass and lactic acid were measured in the broth after 24 h of growth.
yx/s indicates g of dry biomass produced per g of substrate; yp/s indicates g of lactic acid produced per g of substrate; μ8h indicates the specific growth rate in h− <dig> calculated in the first 8 h of growth.
values are an average of  <dig> different experiments with standard deviations ≤ 5%.
batch and microfiltration fermentation processes
glucose and sucrose were selected as carbon sources for the following batch experiments.
during these experiments l. crispatus l <dig> demonstrated a similar growth rate and final concentration of cells.
the maximum titer of biomass on the two substrates was slightly different, in particular,  <dig>  ±  <dig>  gcdw∙l− <dig> were obtained on glucose and  <dig>  ±  <dig>  gcdw∙l− <dig> on sucrose .
the final amount of lactic acid was also quite similar, and it corresponded to  <dig> and 14 g∙l− <dig> on glucose and sucrose, respectively.
product  inhibition was also studied to better characterize the physiology of l. crispatus l <dig>  increasing amounts of sodium lactate added to the sdm medium at a fixed ph lowered the initial specific growth rate .
in particular, μ appeared to be reduced by half with 45 g∙l− <dig> lactate .
in order to dilute lactic acid and overcome inhibition problems, a bioreactor with microfiltration modules was used to perform in situ product removal experiments .
a maximum of  <dig>  gcdw∙l− <dig> in 45 h of growth were produced with a final concentration of 46 g∙l− <dig> of lactic acid.
as it is shown in table  <dig>  a 7-fold improvement of the final titer of biomass was achieved by microfiltration experiments compared to previous batch processes.
moreover the total amount of lactic acid produced was equal to 148 g  with a yp/s of  <dig>  g∙g− <dig> .
all results presented are average of at least  <dig> experiments.
l. crispatus
l <dig> grown on sdm supplemented with 20 g · l
− <dig>
glucose or sucrose as main carbon sources
max
the medium contained soy peptone and yeast extract as nitrogen sources.
l. crispatus
l1
x/s
p/s
yx/s indicates g of dry biomass produced per g of substrate; yp/s indicates g of lactic acid produced per g of substrate.
values are an average of  <dig> different experiments.
eps production and purification
the epss content in the fermentation broth ranged between 200–400 mg⋅l− <dig> and the initial protein titre was estimated up to  <dig> fold.
protease was used to eliminate these major contaminants of the exopolysaccarides, and the following tangential ultrafiltration / diafiltration  was performed to further purify the product and to remove salt and other smaller contaminants.
during uf the flux decreased from  <dig>  to  <dig>  l∙m−2 h− <dig> and it increased again to  <dig>  l∙m−2 h− <dig> during the df phase that lasted until reaching a conductivity of  <dig> ms/cm.
the supernatant was concentrated  <dig> fold compared to the initial volume.
the recovery yield after membrane purification was on average 85% and the purified epss solution had a protein content that was inferior to  <dig> % w/w.
structure determination of mannan polymer
compositional and methylation analyses showed the presence of different derivatives of mannose, such as terminal manp, 2-substituted manp, 3-substituted manp, 6-substituted manp and  <dig> -substituted manp.
on this ground, it could be deemed the presence of a very intricate polymer only based on a mannose monosaccharide, in which other mannose branching residues were attached to a mannan backbone.
the polysaccharide underwent nuclear magnetic resonance  analysis and even though the 1h-  and 13c-nmr spectra appeared rather complex, it was clearly related to the mannan polysaccharides already described  <cit> .
2d nmr and degradation procedures confirmed the structure, a 6-substituted mannan backbone with small branching chains  of manp residues .
inhibition of c. albicans adhesion to vk2/e6e7
c. albicans is a constituent of the vaginal microbiota and, as opportunistic pathogen, it causes genital infections in humans.
in immuno-compromised individuals, overgrowth of the fungus results in candidiasis.
c. albicans pathogenecity depends on several virulence traits that allow the fungus to invade new tissues, evade the immune system of the host, and facilitate the infection  <cit> .
to verify the antagonist effect of l. crispatus l <dig> against c. albicans, the influence of the strain on the adhesion capacity of c. albicans to immortalized human vaginal epithelial cell line was evaluated.
the results demonstrate that there is a significantly reduced adhesion of c. albicans to vk2/e6e <dig> cells by 58 ±  <dig> , 49 ±  <dig>  and 44 ±  <dig> % in the competition, exclusion and displacement assays, respectively .
moreover, confluent cell monolayers were treated with increasing concentrations of eps, isolated and purified from l. crispatus l <dig>  and successively infected with c. albicans.
the concentration required to interfere with yeast adhesiveness was equal to  <dig>  mg∙ml− <dig>
figure 5b shows the effect of eps on the adhesion of c. albicans to vaginal epithelial cells under the conditions of exclusion, competition and displacement.
the adhesion interference was of about 48% in the exclusion assay, when the monolayers were pre-treated with  <dig>  mg∙ml− <dig> eps for 18 h and before addition of the c. albicans suspension.
in the competition and displacement tests the reduction in adherence was comparable to that obtained in the control experiment.
a set of experiments was performed to determine whether hbd- <dig> was secreted by vaginal epithelial cells treated with increasing concentrations of eps.
hbd- <dig> elisas showed that the concentration of hbd- <dig> protein was significantly high in the supernatant after 18 h treatment .
interestingly, the plateau was reached at the same concentration .
discussion
lactobacilli are the prevailing bacteria of the vaginal flora of healthy individuals that regulate the equilibrium between the resident microbiota and the vaginal environment  <cit> .
cervicovaginal microbiota not dominated by lactobacilli may facilitate transmission of hiv and other sexually transmitted infections.
l. crispatus, l. jensenii, and to a lesser extent l. gasseri, are common in the vagina of healthy women, whereas the dominance of l. iners is associated with bacterial vaginosis  <cit> .
borgdorff and colleagues  <cit>  identified six microbiome clusters and concluded that l. crispatus-dominated cervicovaginal microbiota are associated with a lower prevalence of sexually transmitted infections and a lower likelihood of genital hiv- <dig> rna shedding.
recent literature describes the identification of l. crispatus as a member of the resident beneficial flora of the vaginal mucosae  <cit> .
in agreement with this finding the strain isolated in this work from vaginal fluids of a healthy woman was found to belong to this species and named l. crispatus l <dig> .
vaginal probiotics based on lactic acid bacteria have been proposed as a valid strategy against recurrent infections.
lab use several mechanisms to create an unfriendly environment for pathogens which include the production of antimicrobial substances, such as organic acids, hydrogen peroxide and bacteriocins, and the synthesis of biofilms, in order colonize the vaginal mucosa and displace the infective agents  <cit> .
in view of a potential application of l. crispatus l <dig> as vaginal probiotic, it was interesting to characterize the properties of this new isolate due to the capacity of this strain to modify the host microenvironment and therefore possibly deliver health benefits.
the production of lactic acid and hydrogen peroxide were initially investigated and l. crispatus l <dig> demonstrated the ability to produce both metabolites, and compared to other lactobacilli  <cit>  it proved a better resistance to high concentrations of lactic acid, therefore enhancing its competition capacity.
several studies assessed the effectiveness of oral administration of vaginal probiotic bacteria  <cit> .
for this reason we monitored the resistance of l. crispatus l <dig> to a simulated digestion process by incubating the bacterium in shake flasks at ph  <dig> in the presence of pepsine.
data showed that strain survival was linked to the dose of treated bacteria, and, that with a starting concentration of  <dig> ⋅ <dig> cell∙ml− <dig> cell viability was apparently not affected by small intestine juices.
in vitro assays simulating exposure to pancreatic juices were also performed showing that, unexpectedly, l. crispatus l <dig> was unaffected by the treatment.
these data demonstrate the strain’s potential to be orally delivered.
the perspective to use this strain for biotechnological purposes prompted further studies regarding the development of a medium that could enhance strain growth and be suitable for industrial applications.
experiments were initially performed in shake flasks to identify the most suitable carbon source for maximizing the yield of biomass and lactic acid, and sucrose and glucose were chosen for further small scale batch experiments.
as shown in table  <dig> the growth rate of l. crispatus l <dig> was not affected by the two different carbon sources; a slightly lower yp/s was obtained with glucose, nevertheless, the latter is often preferred for industrial processes and therefore it was selected for the following fermentation experiments.
in order to increase the production of biomass and related product a high cell density fermentation process exploiting a microfiltration strategy was developed to keep the concentration of lactic acid below the toxic threshold for l .crispatus l <dig> .
the feeding strategy avoided the waste of carbon source and determined a 7-fold and a 4-fold increase of the final titer of biomass and lactic acid, respectively, compared to previous batch experiments .
based on earlier studies on l. bulgaricus <cit>  a higher improvement of the final biomass concentration was expected.
probably the adhesion of cells to membrane capillaries lowered transmembrane fluxes thus reducing the medium exchange rate.
however, the concentration of biomass reached was very high compared to that obtained by cultivating other lactobacilli; moreover, biomass resulted extremely viable  at the end of the experiments , valuable result for the foreseen application in medical devices/ food supplements.
adhesion seems to be one of the key factors determining the colonization of the digestive ecosystem.
consistently the surface characteristics of lactobacilli are expected to contribute in several ways to their interactions with the host gastrointestinal tract and the gut microbiota, affecting their survival, adherence to the host tissue and interactions with themselves and with other bacteria.
since eps can have important influences on these processes and on the colonization of the host  <cit>  we also have investigated the chemical nature of the eps produced by l. crispatus l <dig>  this structure resulted to be a very intricate comb-like mannan polysaccharide that has been already isolated and identified as capsule/eps/protein bound-eps in a number of microorganisms, among these in the yeast c. albicans <cit> .
we therefore hypothesised that the similarity of structure between the eps of l. crispatus l <dig> and the carbohydrate part of mannoproteins and protein bound-polysaccharides excreted by c. albicans could be in part responsible for contrasting c. albicans infections.
for this reason the ability of l. crispatus l <dig> live cells or of the purified eps to hinder growth of c. albicans was analysed by performing adhesion assays with vaginal cells.
in particular three mechanisms were evaluated:  exclusion by adhered lactobacilli or eps,  competition for and  displacement of adherent yeast cells.
live l. crispatus cells demonstrated the ability to strongly reduce the adherence of invading yeast cells in all types of assays, the most powerful being the competition modality in which adherence was decreases by 58% compared to the control.
the purified eps only inhibited yeast adhesiveness if pre-incubated with vaginal cells before the addition of c. albicans, whereas it was not efficient in competing or displacing yeast cells.
as is known, human defensins, short cysteine-rich cationic proteins, are key components of the innate immune system.
the inducible human beta-defensins are antimicrobial peptides with a broad spectrum of antibacterial and antifungal activity.
human beta-defensin  <dig>  is primarily produced by epithelial cells.
the peptide is highly inducible due to various stimuli and has a broad spectrum antimicrobial activity that is cidal for candida.
it was interesting to observe that the pre-treatment of vaginal epithelial cells with eps induced a high expression of the antimicrobial peptide hbd- <dig> against c. albicans.
the up-regulation of hbd- <dig> might represent a further mechanism of host protection against candida infections.
overall these data indicate that this molecule is at least in part responsible of the impairment of c. albicans adhesion to vaginal cells, thus also demonstrating that it has a main role in the beneficial effect of l. crispatus l <dig> as a natural, probiotic, microbicide for vaginal health.
in the light of the information above it is not surprising that l. crispatus l <dig> synthesizes a mannan polysaccharide that closely resembles the carbohydrate part of mannoproteins and protein bound-polysaccharides excreted by c. albicans.
in our opinion, this is a an important step towards the comprehension of the molecular mechanisms at the basis of the probiotic effect of l. crispatus ssp.
CONCLUSIONS
the present work describes the identification of a new human isolate named l. crispatus l <dig> and its characterization in order to demonstrate that it meets some of the criteria that identify probiotic strains, such as the ability to produce high titers of lactic acid and h2o <dig>  in view of its potential application as oral vaginal probiotic, simulated digestion treatments were performed demonstrating its suitability for oral administration.
growth optimization was initially analysed in shake flasks and following microfiltration experiments allowed reaching high yields of extremely viable biomass, a key prerequisite for probiotic preparations.
the characterization of the structure of the eps produced by l. crispatus l <dig> showed a similarity with surface molecules produced by c. albicans and the inhibition of the adherence of this yeast to vaginal cells in the presence of live l. crispatus l <dig> further suggested an important role of this bacterium as a promoter of vaginal health.
these achievements underlie the potential of l. crispatus l <dig> as orally administered vaginal probiotic, and hence the strain results interesting for biotechnological applications in medical devices and oral supplements.

 lactobacilli have long been of interest to the dairy and agriculture industries, in fact, they are defined as generally regarded as safe, and some have been found as ubiquitous members of the mucosae of healthy subjects. some studies describe the use of lactic acid bacteria for the treatment or prevention of infections of the intestinal and genital tracts with different extents of success. it is quite difficult to identify which properties of lactobacilli are required to prevent and eventually treat diseases and to determine the adequate dosage, duration, and methods of delivery. in respect to vaginal probiotics, the protective role of lactobacilli seems to be based upon two mechanisms, namely, the specific adherence to the vaginal epithelium leading to intensive colonization of this surface, and the control of the remaining vaginal microflora through antagonism against pathogens. as a consequence, the ability of lactobacillus to adhere to epithelial cells and mucosal surfaces is a key criterion for the selection of probiotics. the efficacy of the available commercial products is also strictly dependent on the viability of the probiotic strains contained in the preparations, since the amount of applied microorganism could be crucial for the effectiveness of the product, and several studies revealed that some health food products did not satisfy the claims stated on the labels therefore minimizing the expected health benefits. therefore the evaluation of cell viability in conditions that mimic the practical application is a key issue in the selection of probiotics. also the development of novel fermentation strategies to increase the final biomass yield is central to bypass one of the bottlenecks encountered in the production of starters, probiotic ingredients and medical devices. however, since their growth is inhibited by their primary metabolic product, lactobacilli are rarely cultivated at high cellular density in batch/fed-batch fermentation processes. vaginal probiotics are a rather new area of investigation and, therefore, not much is known about the mechanisms, the conditions or characteristics needed to assess their efficacy. several strains appear to be effective in colonizing and then protecting the intestine and the urogenital tract, from infections. commercial lactobacilli-based products such as normogin® have demonstrated to be a reliable treatment for reducing the recurrence of bacterial vaginosis. it has been reported that infection mechanisms are mainly due to a disestablishment of the normal resident vaginal microflora, primarily a loss of h2o2-producing lactobacilli, although some studies do not support this hypothesis. in vitro studies have suggested that the re-colonization of the urinary tract by certain specific strains of lactobacilli seems to be a suitable approach to prevent infections and relapses. recently it has also been suggested that some probiotic bacteria could be effective not only when locally delivered but also when assumed per os, and this establishes a link between the rate of intestinal survival and vaginal colonization. lactobacillus crispatus can persist in the gastrointestinal tract and is among the most prevalent species of the lactobacillus-dominated human vaginal microbiota, and resistance to very low ph conditions have also been described. a strain of l. crispatus isolated from the vaginal flora of a healthy woman was characterized in this study. in particular, the ability of l. crispatus l to survive to an in vitro simulated digestion was evaluated and its physiological and metabolic requirements were investigated. optimal growth conditions were defined, in order to obtain high density cultivations needed for potential applications of this strain as probiotic supplement. the use of an in situ product removal fermentation process allowed a 7-fold improvement of the biomass yield compared to traditional processes, accompanied by an extremely high cellular viability. given the necessity of probiotic preparations to deliver a certain amount of viable microbial cells the effect of different protective agents on freeze-drying procedures was also investigated. moreover, in order to investigate on the chemical nature of the agents that are at the basis of the beneficial effect of l. crispatus l we have established the primary structure of its exopolysaccharides, since previous studies on bacterial adhesion showed that eps might promote the adherence of bacteria to biological surfaces, thereby facilitating the colonization of various ecological niches. intriguingly, the eps resulted to be a mannan polysaccharide possessing a structure very similar to the one produced by candida albicans. the eps was found to be present in adherent biofilms and might be involved in initial and permanent adhesion. moreover the eps-induced increased expression of the human defensin hbd- in vaginal cells was also verified, identifying a possible connection with c. albicans growth inhibition.  strain identification and h2o production a lactobacillus strain isolated from human vaginal secretion was allotted to crispatus subspecies by 16s ribosomal dna sequencing and it was named l. crispatus l in particular, pcr products were pooled, purified and sequenced. in addition, the ability of lactobacillus strains to produce h2o was evaluated. the percentage of strains classified as strong, medium, weak and negative h2o producers was and 5%, respectively. l. crispatus l was found to be the best of the isolates in the laboratory collection. in vitro digestion results from shake flask experiments simulating the passage through the gastrointestinal tract showed a good resistance of l. crispatus l to the in vitro digestion process. the bacterial dose significantly influenced results, as shown in figure 1a clearly indicating that ⋅ cells∙ml− corresponds to the minimal required initial concentration of cells necessary to survive gastric juices. incubation in simulated pancreatic juices with different oxgall concentrations did not affect viability, whereas a slight increase of the cell number within 4 h was observed. moreover, treated cells reached a final biomass yield comparable with that of the control cells. shakeflask experiments a semidefined medium containing soy peptone and yeast extract was used to investigate the amount of biomass and lactic acid produced using different carbon sources. the final titer of biomass produced in shake flasks was very similar in all the media analysed. the production of lactic acid was quite high ranging between and  g∙l− and resulting in relevant yp/s ranging between and  g∙g− the yp/s on dextrins could not be calculated due to the presence of high molecular weight carbohydrates that were not degraded and metabolized as evidenced by high performance anion exchange chromatography with pulsed amperometric detection analyses. l. crispatus l in shake flasks on sdm medium supplemented with different carbon sources 8h x/s p/s the concentration of biomass and lactic acid were measured in the broth after 24 h of growth. yx/s indicates g of dry biomass produced per g of substrate; yp/s indicates g of lactic acid produced per g of substrate; μ8h indicates the specific growth rate in h− calculated in the first 8 h of growth. values are an average of different experiments with standard deviations ≤ 5%. batch and microfiltration fermentation processes glucose and sucrose were selected as carbon sources for the following batch experiments. during these experiments l. crispatus l demonstrated a similar growth rate and final concentration of cells. the maximum titer of biomass on the two substrates was slightly different, in particular,  ±  gcdw∙l− were obtained on glucose and  ±  gcdw∙l− on sucrose. the final amount of lactic acid was also quite similar, and it corresponded to and 14 g∙l− on glucose and sucrose, respectively. product inhibition was also studied to better characterize the physiology of l. crispatus l increasing amounts of sodium lactate added to the sdm medium at a fixed ph lowered the initial specific growth rate. in particular, μ appeared to be reduced by half with 45 g∙l− lactate. in order to dilute lactic acid and overcome inhibition problems, a bioreactor with microfiltration modules was used to perform in situ product removal experiments. a maximum of gcdw∙l− in 45 h of growth were produced with a final concentration of 46 g∙l− of lactic acid. as it is shown in table  a 7-fold improvement of the final titer of biomass was achieved by microfiltration experiments compared to previous batch processes. moreover the total amount of lactic acid produced was equal to 148 g with a yp/s of  g∙g−. all results presented are average of at least experiments. l. crispatus l grown on sdm supplemented with 20 g · l − glucose or sucrose as main carbon sources max the medium contained soy peptone and yeast extract as nitrogen sources. l. crispatus l1 x/s p/s yx/s indicates g of dry biomass produced per g of substrate; yp/s indicates g of lactic acid produced per g of substrate. values are an average of different experiments. eps production and purification the epss content in the fermentation broth ranged between 200–400 mg⋅l− and the initial protein titre was estimated up to fold. protease was used to eliminate these major contaminants of the exopolysaccarides, and the following tangential ultrafiltration / diafiltration was performed to further purify the product and to remove salt and other smaller contaminants. during uf the flux decreased from to  l∙m−2 h− and it increased again to  l∙m−2 h− during the df phase that lasted until reaching a conductivity of ms/cm. the supernatant was concentrated fold compared to the initial volume. the recovery yield after membrane purification was on average 85% and the purified epss solution had a protein content that was inferior to % w/w. structure determination of mannan polymer compositional and methylation analyses showed the presence of different derivatives of mannose, such as terminal manp, 2-substituted manp, 3-substituted manp, 6-substituted manp and -substituted manp. on this ground, it could be deemed the presence of a very intricate polymer only based on a mannose monosaccharide, in which other mannose branching residues were attached to a mannan backbone. the polysaccharide underwent nuclear magnetic resonance analysis and even though the 1h- and 13c-nmr spectra appeared rather complex, it was clearly related to the mannan polysaccharides already described. 2d nmr and degradation procedures confirmed the structure, a 6-substituted mannan backbone with small branching chains of manp residues. inhibition of c. albicans adhesion to vk2/e6e7 c. albicans is a constituent of the vaginal microbiota and, as opportunistic pathogen, it causes genital infections in humans. in immuno-compromised individuals, overgrowth of the fungus results in candidiasis. c. albicans pathogenecity depends on several virulence traits that allow the fungus to invade new tissues, evade the immune system of the host, and facilitate the infection. to verify the antagonist effect of l. crispatus l against c. albicans, the influence of the strain on the adhesion capacity of c. albicans to immortalized human vaginal epithelial cell line was evaluated. the results demonstrate that there is a significantly reduced adhesion of c. albicans to vk2/e6e cells by 58 ± , 49 ±  and 44 ±  % in the competition, exclusion and displacement assays, respectively. moreover, confluent cell monolayers were treated with increasing concentrations of eps, isolated and purified from l. crispatus l and successively infected with c. albicans. the concentration required to interfere with yeast adhesiveness was equal to  mg∙ml− figure 5b shows the effect of eps on the adhesion of c. albicans to vaginal epithelial cells under the conditions of exclusion, competition and displacement. the adhesion interference was of about 48% in the exclusion assay, when the monolayers were pre-treated with  mg∙ml− eps for 18 h and before addition of the c. albicans suspension. in the competition and displacement tests the reduction in adherence was comparable to that obtained in the control experiment. a set of experiments was performed to determine whether hbd- was secreted by vaginal epithelial cells treated with increasing concentrations of eps. hbd- elisas showed that the concentration of hbd- protein was significantly high in the supernatant after 18 h treatment. interestingly, the plateau was reached at the same concentration. discussion lactobacilli are the prevailing bacteria of the vaginal flora of healthy individuals that regulate the equilibrium between the resident microbiota and the vaginal environment. cervicovaginal microbiota not dominated by lactobacilli may facilitate transmission of hiv and other sexually transmitted infections. l. crispatus, l. jensenii, and to a lesser extent l. gasseri, are common in the vagina of healthy women, whereas the dominance of l. iners is associated with bacterial vaginosis. borgdorff and colleagues identified six microbiome clusters and concluded that l. crispatus-dominated cervicovaginal microbiota are associated with a lower prevalence of sexually transmitted infections and a lower likelihood of genital hiv- rna shedding. recent literature describes the identification of l. crispatus as a member of the resident beneficial flora of the vaginal mucosae. in agreement with this finding the strain isolated in this work from vaginal fluids of a healthy woman was found to belong to this species and named l. crispatus l. vaginal probiotics based on lactic acid bacteria have been proposed as a valid strategy against recurrent infections. lab use several mechanisms to create an unfriendly environment for pathogens which include the production of antimicrobial substances, such as organic acids, hydrogen peroxide and bacteriocins, and the synthesis of biofilms, in order colonize the vaginal mucosa and displace the infective agents. in view of a potential application of l. crispatus l as vaginal probiotic, it was interesting to characterize the properties of this new isolate due to the capacity of this strain to modify the host microenvironment and therefore possibly deliver health benefits. the production of lactic acid and hydrogen peroxide were initially investigated and l. crispatus l demonstrated the ability to produce both metabolites, and compared to other lactobacilli it proved a better resistance to high concentrations of lactic acid, therefore enhancing its competition capacity. several studies assessed the effectiveness of oral administration of vaginal probiotic bacteria. for this reason we monitored the resistance of l. crispatus l to a simulated digestion process by incubating the bacterium in shake flasks at ph  in the presence of pepsine. data showed that strain survival was linked to the dose of treated bacteria, and, that with a starting concentration of ⋅ cell∙ml− cell viability was apparently not affected by small intestine juices. in vitro assays simulating exposure to pancreatic juices were also performed showing that, unexpectedly, l. crispatus l was unaffected by the treatment. these data demonstrate the strain’s potential to be orally delivered. the perspective to use this strain for biotechnological purposes prompted further studies regarding the development of a medium that could enhance strain growth and be suitable for industrial applications. experiments were initially performed in shake flasks to identify the most suitable carbon source for maximizing the yield of biomass and lactic acid, and sucrose and glucose were chosen for further small scale batch experiments. as shown in table  the growth rate of l. crispatus l was not affected by the two different carbon sources; a slightly lower yp/s was obtained with glucose, nevertheless, the latter is often preferred for industrial processes and therefore it was selected for the following fermentation experiments. in order to increase the production of biomass and related product a high cell density fermentation process exploiting a microfiltration strategy was developed to keep the concentration of lactic acid below the toxic threshold for l.crispatus l. the feeding strategy avoided the waste of carbon source and determined a 7-fold and a 4-fold increase of the final titer of biomass and lactic acid, respectively, compared to previous batch experiments. based on earlier studies on l. bulgaricus a higher improvement of the final biomass concentration was expected. probably the adhesion of cells to membrane capillaries lowered transmembrane fluxes thus reducing the medium exchange rate. however, the concentration of biomass reached was very high compared to that obtained by cultivating other lactobacilli; moreover, biomass resulted extremely viable at the end of the experiments, valuable result for the foreseen application in medical devices/ food supplements. adhesion seems to be one of the key factors determining the colonization of the digestive ecosystem. consistently the surface characteristics of lactobacilli are expected to contribute in several ways to their interactions with the host gastrointestinal tract and the gut microbiota, affecting their survival, adherence to the host tissue and interactions with themselves and with other bacteria. since eps can have important influences on these processes and on the colonization of the host we also have investigated the chemical nature of the eps produced by l. crispatus l this structure resulted to be a very intricate comb-like mannan polysaccharide that has been already isolated and identified as capsule/eps/protein bound-eps in a number of microorganisms, among these in the yeast c. albicans. we therefore hypothesised that the similarity of structure between the eps of l. crispatus l and the carbohydrate part of mannoproteins and protein bound-polysaccharides excreted by c. albicans could be in part responsible for contrasting c. albicans infections. for this reason the ability of l. crispatus l live cells or of the purified eps to hinder growth of c. albicans was analysed by performing adhesion assays with vaginal cells. in particular three mechanisms were evaluated: exclusion by adhered lactobacilli or eps, competition for and displacement of adherent yeast cells. live l. crispatus cells demonstrated the ability to strongly reduce the adherence of invading yeast cells in all types of assays, the most powerful being the competition modality in which adherence was decreases by 58% compared to the control. the purified eps only inhibited yeast adhesiveness if pre-incubated with vaginal cells before the addition of c. albicans, whereas it was not efficient in competing or displacing yeast cells. as is known, human defensins, short cysteine-rich cationic proteins, are key components of the innate immune system. the inducible human beta-defensins are antimicrobial peptides with a broad spectrum of antibacterial and antifungal activity. human beta-defensin is primarily produced by epithelial cells. the peptide is highly inducible due to various stimuli and has a broad spectrum antimicrobial activity that is cidal for candida. it was interesting to observe that the pre-treatment of vaginal epithelial cells with eps induced a high expression of the antimicrobial peptide hbd- against c. albicans. the up-regulation of hbd- might represent a further mechanism of host protection against candida infections. overall these data indicate that this molecule is at least in part responsible of the impairment of c. albicans adhesion to vaginal cells, thus also demonstrating that it has a main role in the beneficial effect of l. crispatus l as a natural, probiotic, microbicide for vaginal health. in the light of the information above it is not surprising that l. crispatus l synthesizes a mannan polysaccharide that closely resembles the carbohydrate part of mannoproteins and protein bound-polysaccharides excreted by c. albicans. in our opinion, this is a an important step towards the comprehension of the molecular mechanisms at the basis of the probiotic effect of l. crispatus ssp.  the present work describes the identification of a new human isolate named l. crispatus l and its characterization in order to demonstrate that it meets some of the criteria that identify probiotic strains, such as the ability to produce high titers of lactic acid and h2o in view of its potential application as oral vaginal probiotic, simulated digestion treatments were performed demonstrating its suitability for oral administration. growth optimization was initially analysed in shake flasks and following microfiltration experiments allowed reaching high yields of extremely viable biomass, a key prerequisite for probiotic preparations. the characterization of the structure of the eps produced by l. crispatus l showed a similarity with surface molecules produced by c. albicans and the inhibition of the adherence of this yeast to vaginal cells in the presence of live l. crispatus l further suggested an important role of this bacterium as a promoter of vaginal health. these achievements underlie the potential of l. crispatus l as orally administered vaginal probiotic, and hence the strain results interesting for biotechnological applications in medical devices and oral supplements. 
a microfiltration strategy was exploited to improve the cellular yield in 2 l-fermentation processes, reaching 27 g · l− of dry biomass. a new human isolate named lactobacillus crispatus l was characterized in this work, and a preliminary evaluation of its probiotic potential is described together with a process to obtain a high productivity of viable biomass. vaginal lactic acid bacteria defend the host against pathogens through a combination of competitive exclusion, competition for nutrients, production of antimicrobial substances and through the activation of the immune system. moreover, l. crispatus l demonstrated a greater stability to high concentrations of lactic acid, compared to other lactobacilli. the paper describes the evaluation of l. crispatus l as potential vaginal probiotic and the fermentation processes to obtain high concentrations of viable cells. interestingly a higher expression of the human defensin hbd- was also observed in vaginal cells treated with the purified exopolysaccharide, indicating a possible correlation with c. albicans growth inhibition. the specific l. crispatus l exopolysaccharide was purified from the fermentation broth and characterized by nmr showing structural features and similarity to exopolysaccharides produced by pathogenic strains. in a simulated digestion process ⋅ cells∙ml− survived the gastric environment with 80% viability, without being affected by small intestine juices.
 vaginal lactic acid bacteria defend the host against pathogens through a combination of competitive exclusion, competition for nutrients, production of antimicrobial substances and through the activation of the immune system. a new human isolate named lactobacillus crispatus l was characterized in this work, and a preliminary evaluation of its probiotic potential is described together with a process to obtain a high productivity of viable biomass.  in a simulated digestion process ⋅ cells∙ml− survived the gastric environment with 80% viability, without being affected by small intestine juices. experiments on six different c sources were performed to analyze growth and organic acids production and, glucose, provided the best performances. a microfiltration strategy was exploited to improve the cellular yield in 2 l-fermentation processes, reaching 27 g · l− of dry biomass. moreover, l. crispatus l demonstrated a greater stability to high concentrations of lactic acid, compared to other lactobacilli. the specific l. crispatus l exopolysaccharide was purified from the fermentation broth and characterized by nmr showing structural features and similarity to exopolysaccharides produced by pathogenic strains. live l. crispatus l cells strongly reduced adhesion of a yeast pathogenic strain, candida albicans in particular, in adherence assays. interestingly a higher expression of the human defensin hbd- was also observed in vaginal cells treated with the purified exopolysaccharide, indicating a possible correlation with c. albicans growth inhibition.  the paper describes the evaluation of l. crispatus l as potential vaginal probiotic and the fermentation processes to obtain high concentrations of viable cells. l. crispatus l1high cell densities cultivationsimulated digestion 