Anion-π interactions in active centers of superoxide dismutases.

We investigated 1060 possible anion-π interactions in a data set of 41 superoxide dismutase active centers. Our observations indicate that majority of the aromatic residues are capable to form anion-π interactions, mainly by long-range contacts, and that there is preference of Trp over other aromatic residues in these interactions. Furthermore, 68% of total predicted interactions in the dataset are multiple anion-π interactions. Anion-π interactions are distance and orientation dependent. We analyzed the energy contribution resulting from anion-π interactions using ab initio calculations. The results showed that, while most of their interaction energies lay in the range from -0 to -4kcalmol-1, those energies can be up to -9kcalmol-1 and about 34% of interactions were found to be repulsive. Majority of the suggested anion-π interacting residues in ternary complexes are metal-assisted. Stabilization centers for these proteins showed that all the six residues found in predicted anion-π interactions are important in locating one or more of such centers. The anion-π interacting residues in these proteins were found to be highly conserved. We hope that these studies might contribute useful information regarding structural stability and its interaction in future designs of novel metalloproteins.

[Production of Antimicrobial Polypeptides by Propionibacterium freudenreichii RVS-4-irf].

Propionibacterium freudenreichii RVS-4-irf is a probiotic bacterium producing antimicrobial exometabolites applicable for foodstuff protection. Production of antimicrobial factors other than lowmolecular propionates was observed in media with and without trypton. A method was developed for production of the fraction of high-molecular mass exopolymers from the culture liquid. Their polypeptide nature was confirmed using proteinase K. The preparation of extracellular polypeptides from P. freudenreichii RVS-4-irf exhibited species-specific activity in suppression of fungal and bacterial growth.

Metabolic engineering of acid resistance elements to improve acid resistance and propionic acid production of Propionibacterium jensenii.

Propionic acid (PA) and its salts are widely used in the food, pharmaceutical, and chemical industries. Microbial production of PA by propionibacteria is a typical product-inhibited process, and acid resistance is crucial in the improvement of PA titers and productivity. We previously identified two key acid resistance elements-the arginine deaminase and glutamate decarboxylase systems-that protect propionibacteria against PA stress by maintaining intracellular pH homeostasis. In this study, we attempted to improve the acid resistance and PA production of Propionibacterium jensenii ATCC 4868 by engineering these elements. Specifically, five genes (arcA, arcC, gadB, gdh, and ybaS) encoding components of the arginine deaminase and glutamate decarboxylase systems were overexpressed in P. jensenii. The activities of the five enzymes in the engineered strains were 26.7-489.0% higher than those in wild-type P. jensenii. The growth rates of the engineered strains decreased, whereas specific PA production increased significantly compared with those of the wild-type strain. Among the overexpressed genes, gadB (encoding glutamate decarboxylase) increased PA resistance and yield most effectively; the PA resistance of P. jensenii-gadB was more than 10-fold higher than that of the wild-type strain, and the production titer, yield, and conversion ratio of PA reached 10.81 g/L, 5.92 g/g cells, and 0.56 g/g glycerol, representing increases of 22.0%, 23.8%, and 21.7%, respectively. We also investigated the effects of introducing these acid resistance elements on the transcript levels of related enzymes. The results showed that the expression of genes in the engineered pathways affected the expression of the other genes. Additionally, the intracellular pools of amino acids were altered as different genes were overexpressed, which may further contribute to the enhanced PA production. This study provides an effective strategy for improving PA production in propionibacteria; this strategy may be useful for the production of other organic acids. Biotechnol. Bioeng. 2016;113: 1294-1304. © 2015 Wiley Periodicals, Inc.

Influence of ensiling, exogenous protease addition, and bacterial inoculation on fermentation profile, nitrogen fractions, and ruminal in vitro starch digestibility in rehydrated and high-moisture corn.

Exogenous protease addition may be an option to increase proteolysis of zein proteins and thus starch digestibility in rehydrated and high-moisture corn (HMC) ensiled for short periods. In addition, microbial inoculation may accelerate fermentation and increase acid production and thus increase solubilization of zein proteins. Four experiments were performed to evaluate the effect on fermentation profile, N fractions, and ruminal in vitro starch digestibility (ivSD) of the following: (1) rehydration and ensiling of dry ground corn; (2) exogenous protease addition to rehydrated un-ensiled and ensiled corn; (3) exogenous protease addition or inoculation in rehydrated ensiled corn; and (4) exogenous protease addition or inoculation in HMC. Experiments 1, 2, and 3 were performed with 7 treatments: dry ground corn (DGC); DGC rehydrated to a targeted dry matter content of 70% (REH); REH treated with exogenous protease (REH+); REH ensiled for 30 d (ENS); ENS treated with exogenous protease (ENS+); ENS treated with a microbial inoculant containing Lactobacillus plantarum, Lactobacillus casei, Enterococcus faecium, and Pediococcus sp. (ENSI); and ENS treated with exogenous protease and microbial inoculant (ENSI+). Experiment 1 compared DGC, REH, and ENS with ivSD being greater for ENS (64.9%) than DGC and REH (51.7% on average). Experiment 2 compared REH and ENS without or with exogenous protease addition (REH+ and ENS+, respectively). Ensiling and exogenous protease addition increased ivSD, but exogenous protease addition was more effective in ENS than REH (6.4 vs. 2.6 percentage unit increase). Experiment 3 compared the effects of exogenous protease addition and inoculation in ENS corn (ENS, ENS+, ENSI, and ENSI+). The addition of protease, but not inoculant, increased ivSD. Inoculation reduced pH and acetate, propionate, and ethanol concentrations, and increased lactate and total acid concentrations. In experiment 4, 8 treatments were a combination of HMC noninoculated or inoculated with 1 of 3 microbial inoculants and with or without exogenous protease addition. The inoculant treatments contained (1) Lactobacillus buchneri 40788 and Pediococcus pentosaceus, (2) L. buchneri 40788, and (3) a mixture of P. pentosaceus and Propionibacterium freudenreichii. Protease, but not inoculation, increased ivSD by 7.5 percentage units (44.4 vs. 51.9%). Protease addition increased ivSD in rehydrated corn and HMC. Microbial inoculation improved fermentation profiles but did not affect ivSD.

Recrystallized S-layer protein of a probiotic Propionibacterium: structural and nanomechanical changes upon temperature or pH shifts probed by solid-state NMR and AFM.

Surface protein layers (S layers) are common constituents of the bacterial cell wall and originate from the assembly of strain-dependent surface layer proteins (Slps). These proteins are thought to play important roles in the bacteria's biology and to have very promising technological applications as biomaterials or as part of cell-host cross-talk in probiotic mechanism. The SlpA from Propionibacterium freudenreichii PFCIRM 118 strain was isolated and recrystallized to investigate organization and assembly of the protein using atomic force microscopy and solid-state (1)H and (13)C-nuclear magnetic resonance. SlpA was found to form hexagonal p1 monolayer lattices where the protein exhibited high proportions of disordered regions and of bound water. The lattice structure was maintained, but softened, upon mild heating or acidification, probably in relation with the increasing mobilities of the disordered protein regions. These results gave structural insights on the mobile protein regions exposed by S layer films, upon physiologically relevant changes of their environmental conditions.

Effects of Propionibacterium strains on ruminal fermentation, nutrient digestibility and methane emissions in beef cattle fed a corn grain finishing diet.

Twenty ruminally cannulated beef heifers were fed a high corn grain diet in a randomized block design to determine the effect of three direct fed microbial (DFM) strains of Propionibacterium on ruminal fermentation, nutrient digestibility and methane (CH4) emissions. The heifers were blocked in five groups on the basis of BW and used in five 28-day periods. Dietary treatments included (1) Control and three strains of Propionibacterium (2) P169, (3) P5, and (4) P54. Strains were administered directly into the rumen at 5×109 CFU with 10 g of a maltodextrin carrier in a gel capsule; Control heifers received carrier only. All heifers were fed the basal diet (10 : 90 forage to concentrate, dry matter basis). Rumen contents were collected on days 15 and 18, ruminal pH was measured continuously between days 15 and 22, enteric CH4 emissions were measured between days 19 and 22 and diet digestibility was measured from days 25 to 28. Mean ruminal pH was 5.91 and was not affected by treatments. Similarly, duration of time that pH<5.8 and 5.6 was not affected by treatment. Likewise, total and major volatile fatty acid profiles were similar among all treatments. No effects were observed on dry matter intake and total tract digestibility of nutrients. Total enteric CH4 production (g/day) was not affected by Propionibacterium strains and averaged 139 g/day. Similarly, mean CH4 yield (g CH4/kg of dry matter intake) was similar for all the treatments. The relative abundance of total Propionibacteria in the rumen increased with administration of DFM and were greater 3 h post-dosing relative to Control, but returned to baseline levels before feeding. Populations of Propionibacterium P169 were higher at 3 and 9 h as compared with the levels at 0 h. In conclusion, moderate persistency of the inoculated strains within the ruminal microbiome and pre-existing high propionate production due to elevated levels of starch fermentation might have reduced the efficacy of Propionibacterium strains to increase molar proportion of propionate and subsequently reduce CH4 emissions.

Great interspecies and intraspecies diversity of dairy propionibacteria in the production of cheese aroma compounds.

Flavor is an important sensory property of fermented food products, including cheese, and largely results from the production of aroma compounds by microorganisms. Propionibacterium freudenreichii is the most widely used species of dairy propionibacteria; it has been implicated in the production of a wide variety of aroma compounds through multiple metabolic pathways and is associated with the flavor of Swiss cheese. However, the ability of other dairy propionibacteria to produce aroma compounds has not been characterized. This study sought to elucidate the effect of interspecies and intraspecies diversity of dairy propionibacteria on the production of aroma compounds in a cheese context. A total of 76 strains of Propionibacterium freudenreichii, Propionibacterium jensenii, Propionibacterium thoenii, and Propionibacterium acidipropionici were grown for 15 days in pure culture in a rich medium derived from cheese curd. In addition, one strain each of two phylogenetically related non-dairy propionibacteria, Propionibacterium cyclohexanicum and Propionibacterium microaerophilum were included. Aroma compounds were analyzed using headspace trap-gas chromatography-mass spectrometry (GC-MS). An analysis of variance performed on GC-MS data showed that the abundance of 36 out of the 45 aroma compounds detected showed significant differences between the cultures. A principal component analysis (PCA) was performed for these 36 compounds. The first two axes of the PCA, accounting for 60% of the variability between cultures, separated P. freudenreichii strains from P. acidipropionici strains and also differentiated P. freudenreichii strains from each other. P. freudenreichii strains were associated with greater concentrations of a variety of compounds, including free fatty acids from lipolysis, ethyl esters derived from these acids, and branched-chain acids and alcohols from amino acid catabolism. P. acidipropionici strains produced less of these compounds but more sulfur-containing compounds from methionine catabolism. Meanwhile, branched-chain aldehydes and benzaldehyde were positively associated with certain strains of P. jensenii and P. thoenii. Moreover, the production of compounds with a common origin was correlated. Compound abundance varied significantly by strain, with fold changes between strains of the same species as high as in the order of 500 for a single compound. This suggests that the diversity of dairy propionibacteria can be exploited to modulate the flavor of mild cheeses.

Effect of supplementing direct-fed microbials on broiler performance, nutrient digestibilities, and immune responses.

Direct-fed microbials (DFM) are used to improve livestock health and performance. The effects of 2 DFM products, a blend of 3 Bacillus strains (DFMB) and a Propionibacteriumspp. (DFMP), on broiler performance, nutrient utilization, and immune responses were investigated. Day-old (n = 120) male broilers were divided into 24 groups of 5 birds and fed 3 wheat-based diets in mash form (8 groups per diet) from d 1 to 22. The control diet was fed without or with 7.5 × 10(4) cfu/g of either DFMB or DFMP. From d 19 to 21 fecal samples were collected for determination of total tract apparent retention (TTAR) of nutrients and AMEn. On d 21, feed intake and BW were determined. On d 22, 5 birds per treatment were killed by cervical dislocation to collect jejunal and ileal contents for determination of digesta viscosity and apparent ileal digestibility (AID) of nutrients, respectively, and ileum, cecal tonsil, and spleen tissues for Toll-like receptors (TLR) and cytokine expressions. Compared with the control, DFM did not affect BW gain and feed intake but DFMP reduced G:F (P < 0.01). Compared with the control (2,875 kcal/kg), birds fed on DFMB and DFMP had higher AMEn (2,979 and 2,916 kcal/kg, respectively; P < 0.05), whereas both DFM reduced the AID of DM (P < 0.001) and CP (P < 0.01). Furthermore, DFMP reduced TTAR of NDF (29.0 vs. 18.4%; P < 0.001), whereas both DFM increased TTAR of DM and fat (P < 0.001). Supplementing DFMP downregulated ileal expression of TLR-2b, IL-2, IL-4, IL-6, IL-10, and IL-13, whereas DFMB downregulated TLR-2b, IL-2, IL-4, and IL-6 in all 3 tissues, IL-10 in the spleen, and upregulated IL-13 in the spleen. In conclusion, the DFM did not improve performance but increased the AMEn of diet by possibly increasing DM and fat retention. Overall, both DFM showed an antiinflammatory effect in the ileum, but DFMB had more effects on local and systemic immunity than DFMP.

A highly stable protein chimera built from fragments of different folds.

Proteins increased in complexity during the course of evolution. Domains as well as subdomain-sized fragments were recruited and adapted to form new proteins and novel folds. This concept can be used in engineering to construct new proteins. We previously reported the combination of fragments from two ancient protein folds, a flavodoxin-like and a (ßα)8-barrel protein. Here we report two further attempts at engineering a chimeric protein from fragments of these folds. While one of the constructs showed a high tendency to aggregate, the other turned out to be a highly stable, well-structured protein. In terms of stability against heat and chemical denaturation this chimera, named NarLHisF, is superior to the earlier presented CheYHisF. This is the second instance of a chimera build from two different protein folds, which demonstrates how easily recombination can lead to the development and diversification of new proteins--a mechanism that most likely occurred frequently in the course of evolution. Based on the results of the failed and the successful chimera, we discuss important considerations for a general design strategy for fold chimeras.

The elusive 5'-deoxyadenosyl radical in coenzyme-B12-mediated reactions.

Vitamin B(12) and its biologically active counterparts possess the only examples of carbon-cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B(12) (dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car-Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5'-deoxyadenosyl radical (dAdo(•)) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories that help understand how B(12) enzymes catalyze coenzyme activation and control highly reactive radical intermediates.

The production of conjugated α-linolenic, γ-linolenic and stearidonic acids by strains of bifidobacteria and propionibacteria.

Conjugated fatty acids are regularly found in nature and have a history of biogenic activity in animals and humans. A number of these conjugated fatty acids are microbially produced and have been associated with potent anti-carcinogenic, anti-adipogenic, anti-atherosclerotic and anti-diabetogenic activities. Therefore, the identification of novel conjugated fatty acids is highly desirable. In this study, strains of bifidobacteria and propionibacteria previously shown by us and others to display linoleic acid isomerase activity were assessed for their ability to conjugate a range of other unsaturated fatty acids during fermentation. Only four, linoleic, α-linolenic, γ-linolenic and stearidonic acids, were converted to their respective conjugated isomers, conjugated linoleic acid (CLA), conjugated α-linolenic acid (CLNA), conjugated γ-linolenic acid (CGLA) and conjugated stearidonic acid (CSA), each of which contained a conjugated double bond at the 9,11 position. Of the strains assayed, Bifidobacterium breve DPC6330 proved the most effective conjugated fatty acid producer, bio-converting 70% of the linoleic acid to CLA, 90% of the α-linolenic acid to CLNA, 17% of the γ-linolenic acid to CGLA, and 28% of the stearidonic acid to CSA at a substrate concentration of 0.3 mg mL⁻¹. In conclusion, strains of bifidobacteria and propionibacteria can bio-convert linoleic, α-linolenic, γ-linolenic and stearidonic acids to their conjugated isomers via the activity of the enzyme linoleic acid isomerase. These conjugated fatty acids may offer the combined health promoting properties of conjugated fatty acids such as CLA and CLNA, along with those of the unsaturated fatty acids from which they are formed.

Identification, typing and characterisation of Propionibacterium strains from healthy mucosa of the human stomach.

Forty two Propionibacterium isolates were recovered from biopsy samples of the gastric mucosa of eight out of 12 healthy people. Of these, 41 were identified as belonging to Propionibacterium acnes; the remaining isolate was identified as belonging to Propionibacterium granulosum. Repetitive extragenic palindromic (REP)-PCR typing suggested that up to four strains might be present in the mucosa of the same individual. Sequence analysis of either recA, tly or camp5 genes of P. acnes isolates revealed two distinct phylogenetic lineages. As per the recA, most isolates belonged to type I, while the remainder of the isolates belonged to type II. Phenotypic analyses of representative isolates showed the different strains to have diverse biochemical properties. For example, large differences were seen in carbohydrate fermentation patterns, the results of qualitative and quantitative enzymatic profiling, and survival at acidic pH. In contrast, the patterns of resistance/susceptibility to a series of 16 antibiotics were rather similar, with no atypical resistances observed. The examined strains showed limited-if any-enzymatic activities that could be ultimately related to pathogenicity (lipolytic, proteolytic or haemolytic activity). This suggests that, in the gastric ecosystem, some Propionibacterium spp. genotypes and/or phenotypes can be considered true commensals.

Kinetic analysis and pH-shift control strategy for propionic acid production with Propionibacterium Freudenreichii CCTCC M207015.

The production of propionic acid by Propionibacterium freudenreichii CCTCC M207015 was investigated in a 7.5-l stirred-tank fermentor. Batch fermentations by P. freudenreichii CCTCC M207015 at various pH values ranging from 5.5 to 7.0 were studied. Based on the analysis of the time course of specific cell growth rate (mu (x)) and specific propionic acid formation rate (mu (p)), a two-stage pH-shift control strategy was proposed. At first 48 h, pH was controlled at 6.5 to obtain the maximal mu (x), subsequently pH 6.0 was used to maintain high mu (p) to enhance the production of propionic acid. By applying this pH-shift control strategy in propionic acid fermentation, the maximal propionic acid and glucose conversion efficiency had a significant improvement and reached 19.21 g/l and 48.03%, respectively, compared with those of constant pH operation (14.58 g/l and 36.45%). Fed-batch fermentation with pH-shift control strategy was also applied to produce propionic acid; the maximal propionic acid yield and glucose conversion efficiency reached 25.23 g/l and 47.76%, respectively.

Electrochemical regulation of the end-product profile in Propionibacterium freudenreichii ET-3 with an endogenous mediator.

The end-product profile of the glucose fermentation by Propionibacterium freudenreichii ET-3 changed on an electrochemical treatment, in which the culture vessel was filled with a carbon felt anode. Acetate and propionate were produced as final end products in a molar ratio of 2:3 without any electrochemical treatments at the point of the consumption of lactate as an intermediate of the glucose fermentation. The ratio was changed to 1:1 at the point of the lactate consumption by the electrochemical incubation at an electrode potential of 0.4 V versus Ag|AgCl for 100 h. During further electrochemical incubation, propionate was oxidized to acetate as a final end-product in the microbe-containing anode chamber. 1,4-Dihydroxy-2-naphthoic acid produced by P. freudenreichii ET-3 itself would receive electrons from the metabolic pathway and serve as an electron transfer mediator from the microbial cells to the electrode.

Structural studies on exopolysaccharides produced by three different propionibacteria strains.

The exopolysaccharides produced by three propionibacteria strains, Propionibacterium freudenreichii 109, Propionibacterium freudenreichii 111, and Propionibacterium thoenii 126, grown on whey-based media, were found to be charged heteropolymers, composed of D-glucose, D-mannose, and D-glucuronic acid in molar ratios of 2:2:1. By means of methylation analysis, mass spectrometry, partial acid hydrolysis, and 1D/2D NMR (1H and 13C) studies, it was determined that all three exopolysaccharides contain the same branched, pentasaccharide repeating unit: [Formula: see text].

Comparison of amplified ribosomal DNA restriction analysis, peptidoglycan hydrolase and biochemical profiles for rapid dairy propionibacteria species identification.

Species of dairy propionibacteria are used as cheese-ripening cultures as well as probiotics. However, no rapid identification methods are currently available. With this in mind, the present study compared three methods, (i) carbohydrate fermentation, (ii) ARDRA (amplified ribosomal DNA restriction analysis) and (iii) peptidoglycan hydrolase (PGH) activity profiles to improve the identification of Propionibacterium thoenii, Propionibacterium jensenii, Propionibacterium acidipropionici and Propionibacterium microaerophilum. The species Propionibacterium freudenreichii and Propionibacterium cyclohexanicum have previously been shown to be easily distinguishable from the other species. Principal component analysis of the carbohydrate fermentation profiles of 113 P. thoenii, P. jensenii, P. acidipropionici and P. microaerophilum strains correctly classified 85% of the strains based on the fermentation of seven carbohydrates. Regarding PGH profiles, optimized conditions of PGH-renaturing SDS-PAGE were applied to 34 of the strains. The PGH profiles of P. acidipropionici and P. microaerophilum were indistinguishable from one another, but were easily distinguished from P. jensenii and P. thoenii. However, four strains exhibited atypical profiles. Hence, in general, the PGH profiles were shown to be conserved within a species, with some exceptions. Four endonucleases were tested for ARDRA and the four species differentiated by combining the profiles obtained with MspI and HaeIII. P. freudenreichii and P. cyclohexanicum profiles were also performed but showed wide differences. Consequently, ARDRA was shown to be the most appropriate method for rapidly distinguishing strains of propionibacteria. Carbohydrate fermentation and peptidoglycan hydrolase activity profiles are useful as complementary identification tools, since about 15% of the 34 strains tested showed atypical profiles.

Towards a model to explain the intragenic complementation in the heteromultimeric protein propionyl-CoA carboxylase.

Mutations in the PCCA or PCCB genes coding for alpha and beta subunits of propionyl CoA carboxylase can cause propionic acidemia. To understand the molecular basis of the intragenic complementation previously reported at the PCCB locus, we now examine the complementation behaviour of four carboxy-terminal and 11 amino-terminal naturally occurring mutant alleles both using cell fusion and reconstructing the complementation event by transfecting the mutant cDNAs to generate multimeric hybrid proteins. Alleles carrying mutations p.R410W and p.W531X are able to complement with 10 out of 11 amino-terminal mutations assayed. Only the unstable p.R512C, p.L519P and p.G112D mutants fail to complement. The results analyzed in the framework of the crystal structure of the homologous 12S transcarboxylase from Propionibacterium shermanii show that all mutant alleles studied are located at beta subunits interfaces, complementing alleles at the inter-trimer interface, where the catalysis probably happens, and non-complementing alleles at the intra-trimer interface, probably disrupting the trimer formation. Our results also show a remarkable stabilization effect when p.R410W is cotransfected with p.G246V. We propose a model for intragenic complementation requiring the production of two different beta subunits carrying carboxy and amino-terminal mutations that allow regenerating functional active sites and in which a stabilization effect between subunits could be relevant to ameliorate the biochemical phenotype of each mutation separately.

Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in Propionibacterium freudenreichii.

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.