Susceptibility and adaptive response to bile salts in Propionibacterium freudenreichii: physiological and proteomic analysis

Tolerance to digestive stresses is one of the main factors limiting the use of microorganisms as live probiotic agents. Susceptibility to bile salts and tolerance acquisition in the probiotic strain Propionibacterium freudenreichii SI41 were characterized. We showed that pretreatment with a moderate concentration of bile salts (0.2 g/liter) greatly increased its survival during a subsequent lethal challenge (1.0 g/liter, 60 s). Bile salts challenge led to drastic morphological changes, consistent with intracellular material leakage, for nonadapted cells but not for preexposed ones. Moreover, the physiological state of the cells during lethal treatment played an important role in the response to bile salts, as stationary-phase bacteria appeared much less sensitive than exponentially growing cells. Either thermal or detergent pretreatment conferred significantly increased protection toward bile salts challenge. In contrast, some other heterologous pretreatments (hypothermic and hyperosmotic) had no effect on tolerance to bile salts, while acid pretreatment even might have sensitized the cells. Two-dimensional electrophoresis experiments revealed that at least 24 proteins were induced during bile salts adaptation. Identification of these polypeptides suggested that the bile salts stress response involves signal sensing and transduction, a general stress response (also triggered by thermal denaturation, oxidative toxicity, and DNA damage), and an alternative sigma factor. Taken together, our results provide new insights into the tolerance of P. freudenreichii to bile salts, which must be taken into consideration for the use of probiotic strains and the improvement of technological processes.     

Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in<Emphasis Type="Italic"> Propionibacterium freudenreichii</Emphasis>

Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.     

Changes in Protein Synthesis and Morphology during Acid Adaptation of Propionibacterium freudenreichii

Survival of bacteria in changing environments depends on their ability to adapt to abiotic stresses. Microorganisms used in food technology face acid stress during fermentation processes. Similarly, probiotic bacteria have to survive acid stress imposed within the stomach in order to reach the intestine and play a beneficial role. Propionibacteria are used both as cheese starters and as probiotics in human alimentation. Adaptation to low pH thus constitutes a limit to their efficacy. Acid stress adaptation in the probiotic SI41 strain of Propionibacterium freudenreichii was therefore investigated. The acid tolerance response (ATR) was evidenced in a chemically defined medium. Transient exposure to pH 5 afforded protection toward acid challenge at pH 2. Protein neosynthesis was shown to be required for optimal ATR, since chloramphenicol reduced the acquired acid tolerance. Important changes in genetic expression were observed with two-dimensional electrophoresis during adaptation. Among the up-regulated polypeptides, a biotin carboxyl carrier protein and enzymes involved in DNA synthesis and repair were identified during the early stress response, while the universal chaperonins GroEL and GroES corresponded to a later response. The beneficial effect of ATR was evident at both the physiological and morphological levels. This study constitutes a first step toward understanding the very efficient ATR described in P. freudenreichii.     

Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis.

The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria.     

Pyrophosphate is a source of phosphoryl groups for Escherichia coli protein phosphorylation

Abstract Stress tolerance and cross-protection in Enterococcus faecalis ATCC19433 were examined after exposure to bile salts, acid or heat shock. Bile salts and heat adapted cells demonstrated induced homologous tolerance and cross-resistance. No cross-protection of heat adapted cells against acid stress is observed and pretreatment with bile salts even sensitized the cells to this challenge. Whole-cell protein extract analysis revealed that each treatment induced a battery of stress proteins. Some of these polypeptides are induced by more than one treatment. The greatest overlap is observed between bile salts and heat treatments. Eighteen stress proteins, including DnaK and GroEL, are common between these stresses.     

Bile salts and cholesterol induce changes in the lipid cell membrane of Lactobacillus reuteri

AIMS: The objective of this study was to evaluate the effect of bile salts and cholesterol in the lipid profile of Lactobacillus reuteri CRL 1098 and to determine the relationship existing between these changes: the in vitro removal of cholesterol and the tolerance of the cells to acid and cold stress. METHODS AND RESULTS: Lactobacillus reuteri CRL 1098 was grown in the following media: MRS (deMan Rogosa Sharpe; MC, control medium), MB (MC with bile salts), MCH (MC with sterile cholesterol) and MBCH (MC with bile salts and cholesterol). Fatty acids were determined by analytical gas-liquid chromatography, and phospholipids and glycolipids by colorimetric techniques. The cells from different culture media were subjected to cold and acid stress. The MB cultures displayed a decrease in phospholipids and a low ratio of saturated : unsaturated fatty acids. The presence of the unusual C18 : 0,10-OH and C18 : 0,10-oxo fatty acids was the prominent characteristic of the bile salts growing cells. The relative increase in glycolipids and the changes in the fatty acids profiles of the MB cells would be responsible for the cholesterol remotion. The changes induced by bile salts in the lipid profile did not improve the tolerance of L. reuteri CRL 1098 to freezing and acid stress. CONCLUSIONS: The changes in lipid profiles reported in this study would play a key role in the response of Lactobacilli to environmental stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides useful information about the effect of bile salts on the cell membrane of L. reuteri, a probiotic enterolactobacillus. The steady-state response of the cells subjected to bile stress seems to be the appropriate model for evaluating the bacterial behaviour in detergent-containing gastrointestinal tracts, where the bile salts stress would presumably be continuous.     

Emmental Cheese Environment Enhances Propionibacterium freudenreichii Stress Tolerance

Dairy propionibacteria are actinomycetales found in various fermented food products. The main species, Propionibacterium freudenreichii, is generally recognized as safe and used both as probiotic and as cheese starter. Its probiotic efficacy tightly depends on its tolerance towards digestive stresses, which can be largely modulated by the ingested delivery vehicle. Indeed, tolerance of this bacterium is enhanced when it is consumed within a fermented dairy product, compared to a dried probiotic preparation. We investigated both stress tolerance and protein neosynthesis upon growth in i) chemically defined or ii) aqueous phase of Emmental cheeses. Although the same final population level was reached in both media, a slower growth and an enhanced survival of CIRM BIA 1 strain of P. freudenreichii subsp. shermanii was observed in Emmental juice, compared to chemically defined medium. This was accompanied by differences in substrates used and products released as well as overexpression of various early stress adaptation proteins in Emmental juice, compared to chemically defined medium, implied in protein folding, in aspartate catabolism, in biosynthesis of valine, leucine and isoleucine, in pyruvate metabolism in citrate cycle, in the propionate metabolism, as well as in oxidoreductases. All these changes led to a higher digestive stress tolerance after growth in Emmental juice. Mechanisms of stress adaptation were induced in this environment, in accordance with enhanced survival. This opens perspectives for the use of hard and semi-hard cheeses as delivery vehicle for probiotics with enhanced efficacy.     

Adaptation and Response of Bifidobacterium animalis subsp. lactis to Bile: a Proteomic and Physiological Approach

Bile salts are natural detergents that facilitate the digestion and absorption of the hydrophobic components of the diet. However, their amphiphilic nature makes them very inhibitory for bacteria and strongly influences bacterial survival in the gastrointestinal tract. Adaptation to and tolerance of bile stress is therefore crucial for the persistence of bacteria in the human colonic niche. Bifidobacterium animalis subsp. lactis, a probiotic bacterium with documented health benefits, is applied largely in fermented dairy products. In this study, the effect of bile salts on proteomes of B. animalis subsp. lactis IPLA 4549 and its bile-resistant derivative B. animalis subsp. lactis 4549dOx was analyzed, leading to the identification of proteins which may represent the targets of bile salt response and adaptation in B. animalis subsp. lactis. The comparison of the wild-type and the bile-resistant strain responses allowed us to hypothesize about the resistance mechanisms acquired by the derivative resistant strain and about the bile salt response in B. animalis subsp. lactis. In addition, significant differences in the levels of metabolic end products of the bifid shunt and in the redox status of the cells were also detected, which correlate with some differences observed between the proteomes. These results indicate that adaptation and response to bile in B. animalis subsp. lactis involve several physiological mechanisms that are jointly dedicated to reduce the deleterious impact of bile on the cell's physiology.     

The effect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92

Bile tolerance is an important criterion in the selection of microbial strains for probiotic use. The survival and morphological changes of a potential probiotic strain, Lactobacillus acidophilus M92, in the presence of bile salts were examined. Lactobacillus acidophilus M92 has shown a satisfactory degree of tolerance against oxgall and individual bile salts tested, especially to taurocholate. The higher resistance of L. acidophilus M92 against taurine-conjugated bile salts relative to deconjugated and glycine-conjugated bile salts was attributed to its reaction to the stronger acidity of the former. Furthermore, bile salt hydrolase (BSH) was active when L. acidophilus M92 was grown in the presence of sodium taurocholate. The rate of BSH activity was highest at the exponential growth phase. It was hypothesised that BSH activity may be important for the bile salt resistance of this strain. The colonial and cellular morphology may also be a valuable parameter in the selection of bile salt-resistant Lactobacillus strains for probiotic use. Smooth (S) and rough (R) colonies, appeared in the original L. acidophilus M92 bacterial culture and demonstrated a different degree of bile tolerance. Rough colonies were more sensitive to bile salts than smooth ones. The R colony cells assumed a round form, probably induced by gaps in the cell wall caused by the cytotoxicity of glycodeoxycholate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biological effect of extracellular peptide factor from Luteococcus japonicus subsp. casei on probiotic bacteria

The biological effect of the extracellular peptide reactivating factor (RF) from Luteococcus casei on cells of probiotic cultures was studied. The RF showed the protective and reactivating effects on the Bifidobacterium bifidum cells under the action of bile salts and an acidic stress. Also, it acted as a cryoprotector during lyophilisation and long-term culture storage. The RF and the L. casei culture liquid (CL) were shown to have bifidogenic properties. The degree of protection and reactivation of lactic-acid bacteria under the action of bile salts depended on the particular strain properties. The maximum degree of protection (more than thirteen-fold) and reactivation (close to three-fold) was found in Lactobacillus casei, while the minimum values were characteristic of Lactobacillus reuterii. The resistance of lactobacilli to bile was increased in the row of L. acidophilus, L. casei, L. plantarum, L. rhamnosus, and L. reuterii correlating with the RF protection degree.     

Screening for potential probiotic from spontaneously fermented non-dairy foods based on in vitro probiotic and safety properties

The aim of this study was to screen potential probiotic lactic acid bacteria from Chinese spontaneously fermented non-dairy foods by evaluating their probiotic and safety properties. All lactic acid bacteria (LAB) strains were identified by 16S rRNA gene sequencing. The in vitro probiotic tests included survival under low pH and bile salts, cell surface hydrophobicity, auto-aggregation, co-aggregation, antibacterial activity, and adherence ability to cells. The safety properties were evaluated based on hemolytic activity and antibiotic resistance profile. The salt tolerance, growth in litmus milk, and acidification ability were examined on selected potential probiotic LAB strains to investigate their potential use in food fermentation. A total of 122 strains were isolated and identified at the species level by 16S rRNA gene sequencing and included 62 Lactobacillus plantarum, 40 Weissella cibaria, 12 Lactobacillus brevis, 6 Weissella confusa, and 2 Lactobacillus sakei strains. One W. cibaria and nine L. plantarum isolates were selected based on their tolerance to low pH and bile salts. The hydrophobicity, auto-aggregation, co-aggregation, and antagonistic activities of these isolates varied greatly. All of the 10 selected strains showed multiple antibiotic resistance phenotypes and no hemolytic activity. The highest adhesion capacity to SW480 cells was observed with L. plantarum SK1. The isolates L. plantarum SK1, CB9, and CB10 were the most similar strains to Lactobacillus rhamnosus GG and selected for their high salt tolerance and acidifying activity. The results revealed strain-specific probiotic properties were and potential probiotics that can be used in the food industry.     

Importance of microbial defence systems to bile salts and mechanisms of serum cholesterol reduction

An important feature of the intestinal microbiota, particularly in the case of administered probiotic microorganisms, is their resistance to conditions in the gastrointestinal tract, particularly tolerance to and growth in the presence of bile salts. Bacteria can use several defence mechanisms against bile, including special transport mechanisms, the synthesis of various types of surface proteins and fatty acids or the production of exopolysaccharides. The ability to enzymatically hydrolyse bile salts occurs in a variety of bacteria. Choloylglycine hydrolase (EC 3.5.1.24), a bile salt hydrolase, is a constitutive intracellular enzyme responsible for the hydrolysis of an amide bond between glycine or taurine and the steroid nucleus of bile acids. Its presence was demonstrated in specific microorganisms from several bacterial genera (Lactobacillus spp., Bifidobacterium spp., Clostridium spp., Bacteroides spp.). Occurrence and gene arrangement encoding this enzyme are highly variable in probiotic microorganisms. Bile salt hydrolase activity may provide the possibility to use the released amino acids by bacteria as sources of carbon and nitrogen, to facilitate detoxification of bile or to support the incorporation of cholesterol into the cell wall. Deconjugation of bile salts may be directly related to a lowering of serum cholesterol levels, from which conjugated bile salts are synthesized de novo. Furthermore, the ability of microorganisms to assimilate or to bind ingested cholesterol to the cell wall or to eliminate it by co-precipitation with released cholic acid was also documented. Some intestinal microflora produce cholesterol reductase that catalyses the conversion of cholesterol to insoluble coprostanol, which is subsequently excreted in faeces, thereby also reducing the amount of exogenous cholesterol.     

Cholesterol Assimilation by Lactic Acid Bacteria and Bifidobacteria Isolated from the Human Gut

The objective of this study was to evaluate the effect of human gut-derived lactic acid bacteria and bifidobacteria on cholesterol levels in vitro. Continuous cultures inoculated with fecal material from healthy human volunteers with media supplemented with cholesterol and bile acids were used to enrich for potential cholesterol assimilators among the indigenous bacterial populations. Seven potential probiotics were found: Lactobacillus fermentum strains F53 and KC5b, Bifidobacterium infantis ATCC 15697, Streptococcus bovis ATCC 43143, Enterococcus durans DSM 20633, Enterococcus gallinarum, and Enterococcus faecalis. A comparative evaluation regarding the in vitro cholesterol reduction abilities of these strains along with commercial probiotics was undertaken. The degree of acid and bile tolerance of strains was also evaluated. The human isolate L. fermentum KC5b was able to maintain viability for 2 h at pH 2 and to grow in a medium with 4,000 mg of bile acids per liter. This strain was also able to remove a maximum of 14.8 mg of cholesterol per g (dry weight) of cells from the culture medium and therefore was regarded as a candidate probiotic.     

Stress proteins and cross-protection by heat shock and salt stress in Bacillus subtilis.

Bacillus subtilis induced a set of general stress proteins in response to a salt or heat stress. Cells subjected to a mild heat stress showed a protective response which enabled them to survive otherwise lethal temperatures (e.g. 52 degrees C). In a similar way bacteria were enabled to survive toxic concentrations of NaCl by pretreatment with lower salt concentrations. A mild heat shock induced a cross-protection against lethal salt stress. The pretreatment of cells with low salt, however, was less effective in the induction of thermotolerance than a preceding mild heat stress. Three stress proteins were identified on the basis of their N-terminal amino acid sequences as homologues of GroEL, DnaK and ClpP of Escherichia coli. The role of general and specific stress proteins in the induction of thermotolerance/salt tolerance and cross-protection is discussed.     

Method for direct selection of potentially probiotic Bifidobacterium strains from human feces based on their acid-adaptation ability

A method for direct selection of acid-resistant Bifidobacterium strains was developed by prolonged exposure of human feces to homologous lethal stress conditions. The recovered strains were intrinsically resistant to acidic gastric conditions (pH 2.0) and also showed good tolerance to high concentrations of bile salts and NaCl. Bifidobacterium breve and Bifidobacterium adolescentis were, respectively, the infant- and adult-type bifidobacterial species showing the highest ability to develop an acid-tolerant phenotype. Therefore, this procedure could be applicable to the direct selection of Bifidobacterium strains with improved stability in adverse environments and, probably, contribute to expand the spectra of probiotic species of human origin currently marketed.     

Probiotic characteristics of Lactobacillus strains isolated from cheese and their antibacterial properties against gastrointestinal tract pathogens

In the present study, four Lactobacillus strains from the cheese were analyzed for its probiotic potential against enteropathogenic bacteria. The probiotic properties of the selected strains were also analyzed and the selected bacterial strains showed high tolerance in bile salts and organic acid. The strain L. plantarum LP049 showed maximum survival rate (92 ± 4.2% and 93.3 ± 2%) after 3 h of treatment at 0.25% (w/v) bile salts and 0.25% (w/v) organic acid concentrations. The ability of the Lactobacillus strains to adhere to human epithelial cells (HT-29 cell lines) was evaluated and L. plantarum LP049 showed maximum adhesion property (19.2 ± 1.1%) than other tested strains. The Lactobacillus strains produced lactic acid at various concentrations. Compared with other strains, maximum level of lactic acid (3.1 g/L), hydrogen peroxide (4.31 mM) and bacteriocin (31 AU/mg) was detected in LB049. The inhibitory activity of culture supernatant against various bacterial pathogens was observed. The zone of inhibition ranged between 6 ± 2 mm and 23 ± 2 mm. The cell free extract showed activity against, Escherichia coli (ATCC 10536), Salmonella enteritidis (ATCC 13076), Shigella flexneri (ATCC 29903), and Enterococcus faecium (ATCC 8459). Consequently, L. plantarum LP049 may be considered as a potential candidate for the production of novel bioactive metabolites for therapeutic and bio-protective applications.     

Investigation of biomarkers of bile tolerance in Lactobacillus casei using comparative proteomics

The identification of cell determinants involved in probiotic features is a challenge in current probiotic research. In this work, markers of bile tolerance in Lactobacillus casei were investigated using comparative proteomics. Six L. casei strains were classified on the basis of their ability to grow in the presence of bile salts in vitro. Constitutive differences between whole cell proteomes of the most tolerant strain (L. casei Rosell-215), the most sensitive one (L. casei ATCC 334), and a moderately tolerant strain (L. casei DN-114 001) were investigated. The ascertained subproteome was further studied for the six strains in both standard and bile stressing conditions. Focus was on proteins whose expression levels were correlated with observed levels of bile tolerance in vitro, particularly those previously reported to be involved in the bile tolerance process of lactobacilli. Analysis revealed that 12 proteins involved in membrane modification (NagA, NagB, and RmlC), cell protection and detoxification (ClpL and OpuA), as well as central metabolism (Eno, GndA, Pgm, Pta, Pyk, Rp1l, and ThRS) were likely to be key determinants of bile tolerance in L. casei and may serve as potential biomarkers for phenotyping or screening purposes. The approach used enabled the correlation of expression levels of particular proteins with a specific probiotic trait.     

Genomic and physiological analyses of an indigenous strain, <Emphasis Type="Italic">Enterococcus faecium</Emphasis> 17OM39

The human gut microbiome plays a crucial role in human health and efforts need to be done for cultivation and characterisation of bacteria with potential health benefits. Here, we isolated a bacterium from a healthy Indian adult faeces and investigated its potential as probiotic. The cultured bacterial strain 17OM39 was identified as Enterococcus faecium by 16S rRNA gene sequencing. The strain 17OM39 exhibited tolerance to acidic pH, showed antimicrobial activity and displayed strong cell surface traits such as hydrophobicity and autoaggregation capacity. The strain was able to tolerate bile salts and showed bile salt hydrolytic (BSH) activity, exopolysaccharide production and adherence to human HT-29 cell line. Importantly, partial haemolytic activity was detected and the strain was susceptible to the human serum. Genomics investigation of strain 17OM39 revealed the presence of diverse genes encoding for proteolytic enzymes, stress response systems and the ability to produce essential amino acids, vitamins and antimicrobial compound Bacteriocin-A. No virulence factors and plasmids were found in this genome of the strain 17OM39. Collectively, these physiological and genomic features of 17OM39 confirm the potential of this strain as a candidate probiotic.