The extracellular proteome of Bifidobacterium animalis subsp. lactis BB-12 reveals proteins with putative roles in probiotic effects

Probiotics are live microorganisms that exert health-promoting effects on the human host, as demonstrated for numerous strains of the genus Bifidobacterium. To unravel the proteins involved in the interactions between the host and the extensively used and well-studied probiotic strain Bifidobacterium animalis subsp. lactis BB-12, proteins secreted by the bacterium, i.e. belonging to the extracellular proteome present in the culture medium, were identified by 2-DE coupled with MALDI-TOF MS. Among the 74 distinct proteins identified, 31 are predicted to carry out their physiological role either outside the cell or on its surface. These proteins include solute-binding proteins for oligosaccharides, amino acids and manganese, cell wall-metabolizing proteins, and 18 proteins that have been described to interact with human host epithelial cells or extracellular matrix proteins. The potential functions include binding of plasminogen, formation of fimbriae, adhesion to collagen, attachment to mucin and intestinal cells as well as induction of immunomodulative response. These findings suggest a role of the proteins in colonization of the gastrointestinal tract, adhesion to host tissues, or immunomodulation of the host immune system. The identification of proteins predicted to be involved in such interactions can pave the way towards well targeted studies of the protein-mediated contacts between bacteria and the host, with the goal to enhance the understanding of the mode of action of probiotic bacteria.     

A method for the identification of proteins secreted by lactic acid bacteria grown in complex media

Lactic acid bacteria (LAB) are known for their special nutritional requirements, being usually cultured in complex media to achieve optimal growth. In this paper, a protocol based on trichloroacetic acid precipitation of peptides and proteins is presented. The method has been tested on four probiotic LAB strains grown in De Man Rogosa Sharpe (MRS) broth, a complex medium that is often used for the culture of such bacteria. This protocol allowed the detection of 19 proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10 of them being successfully identified by tandem MS. Thereafter, the 10 were found to be secreted or surface associated by bioinformatic means. In conclusion, this work supplies a method for the identification of proteins secreted by LAB, allowing discrimination between the proteins present in the MRS and those produced by probiotic LAB.     

Differential protein expression in early Atlantic cod larvae (Gadus morhua) in response to treatment with probiotic bacteria

Proteome analysis was used to study the effects of probiotic bacteria treatment on the protein expression in early Atlantic cod (Gadus morhua) larvae. The main focus was on proteins connected to immune function, growth and development. The results demonstrated that none of the identified cod larvae proteins showing up-regulation after administration of probiotic bacteria are known to have a function in immunity. In contrast, several proteins exhibiting down-regulation after exposure to the probiotic bacteria may be related to stress and immune responses. This indicates that the probiotic treated larvae were experiencing a lower level of immune-stimulation than the control group. It is therefore tempting to postulate that the probiotic bacteria mixture used in the present study reduced the environmental stress by inhibiting the growth of pathogenic bacteria. Most of the identified up-regulated proteins in the probiotic group relative to the control may be linked to growth and development. The most pronounced up-regulation of proteins was found in several muscle α-actin isoforms indicating improved growth upon probiotic bacteria treatment.     

Identification method based on PCR combined with automated ribotyping for tracking probiotic Lactobacillus strains colonizing the human gut and vagina

AIMS: A molecular methodology based on PCR-associated automated ribotyping was developed to specifically detect the Lactobacillus strains of two probiotic products (an orally administered lyophilized preparation and vaginal tablets) in human faeces and vaginal swabs. METHODS AND RESULTS: The 16S-23S rDNA sequences and the ribotype profiles of the probiotic lactobacilli were characterized and new species-specific primer sets were designed. The identification of faecal and vaginal lactobacilli isolated from subjects administered with the probiotic products was performed by using PCR with species-specific primers followed by strain-specific automated ribotyping. CONCLUSIONS: The PCR-ribotyping identification allowed to study the colonization patterns of the probiotic lactobacilli in the human gut and vagina evidencing the strains with the best survival capability. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed molecular method represents a powerful tool of strain-specific identification, useful for differentiating exogenous from indigenous strains in any microbial ecosystem and for rationally choosing probiotic bacteria with the best chance of survival in the host.     

Identification and characterization of starter lactic acid bacteria and probiotics from Columbian dairy products

AIMS: Considering the significant rise in the probiotic market in Columbia, and given the lack of reports concerning the microbial population and strain performance in products from different producers, this study aims at determining the number of viable starter bacteria and probiotics in bio-yoghurts available at the Columbian market, identifying the species and analysing the performance of the isolated strains in bile acid resistance, antagonistic activity against pathogens, and adherence capacity to human intestinal epithelial cells. METHODS AND RESULTS: Seven bio-yoghurts were analysed for the bacterial species present. Species identification was carried out using 16S rRNA gene targeted PCR. The cultured bacteria were tested for bile acid resistance, adherence to a human intestinal epithelial cell line, and antagonism against the pathogen Salmonella enterica serovar Typhimurium. A total of 17 different strains were identified. Based on plate counting, all bio-yoghurts have at least total viable cells of approximately 10(7) CFU ml(-1). Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were the most frequently isolated bacteria. Viable Bifidobacterium was only recovered from one product. However, after PCR analysis, DNA of this genus was confirmed in five out of seven products. Major differences were found for S. typhimurium antagonism. The adherence capacity to Caco-2 cells was observed in 10 of the isolated strains. In general, low survival to simulated gastric juice was observed. CONCLUSIONS: Some of the isolated strains have probiotic potential, although not all of them were present in the advised amount to exert beneficial health effects. However, the full correct scientific name of the isolated bacteria and their viable counts were not included on the product label. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the identification and functionality of starter bacteria and probiotics present in dairy products on the Columbian market.     

Taxonomic and strain-specific identification of the probiotic strain Lactobacillus rhamnosus 35 within the Lactobacillus casei group

Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.     

Genetically modified probiotics in foods

Probiotics have many potential therapeutic uses, but have not been universally accepted because of a lack of understanding of their action. Lactic acid bacteria (LAB) have been modified by traditional and genetic engineering methods to produce new varieties. Modern techniques of molecular biology have facilitated the identification of probiotic LAB strains, but only a few LAB have been modified by recombinant-DNA technology because of consumer resistance to their introduction to markets, especially in Europe.     

Utilisation of enterobacterial repetitive intergenic consensus (ERIC) sequence-based PCR to fingerprint the genomes of Bifidobacterium isolates and other probiotic bacteria

Enterobacterial repetitive intergenic consensus based on PCR (ERIC-PCR) was used to generate DNA fingerprints for bifidobacteria and other probiotic bacteria. Two primers (ERIC 1R and ERIC 2) used in ERIC-PCR revealed that all of the probiotic bacteria tested possess enterobacterial repetitive intergenic consensus sequences with the PCR products ranging from 250 bp to 5000 bp. The bacterial strains can be differentiated by comparing fingerprint patterns. The dendrogram of the fingerprints revealed that most of the bifidobacterial wild type strains fell into one cluster at similarity level of approximately 79%.     

Regulation of immune response at intestinal and peripheral sites by probiotics

The gut associated lymphoid tissue (GALT) should protect intestinal mucosa against pathogens, but also avoid hypersensitivity reactions to food proteins, normal bacterial flora and other environmental macromolecules. The interaction between epithelial cells and microflora is fundamental to establish gut mucosal barrier and GALT development. The normal colonization of intestine by commensal bacteria is thus crucial for a correct development of mucosal immune system. Probiotic bacteria are normal inhabitants of microflora and may confer health benefits to the host. The modification of the intestinal microflora towards a healthier probiotics enriched microflora may generate beneficial mucosal immunomodulatory effects and may represent a new strategy to cure intestinal and allergic diseases. The health benefits may be specific for different probiotic strains. Ongoing research is providing new insights into the probiotic beneficial effects and related mechanisms. This review represents an update of immunomodulatory activity of different probiotics and of the more accredited mechanisms underlying such activities. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Probiotic bacteria as potential detoxification tools: assessing their heavy metal binding isotherms

Dietary exposure to heavy metals may have detrimental effects on human and animal health, even at low concentrations. Specific probiotic bacteria may have properties that enable them to bind toxins from food and water. We assessed the interaction of probiotic bacteria with cadmium and lead in vitro as an initial screening step to identify strains for heavy metal decontamination in food and intestinal models. Binding isotherms for cadmium and lead were characterized for Lactobacillus rhamnosus LC-705, Propionibacterium freudenreichii subsp. shermanii JS and a mix of them used by the food industry. Differences among the strains and their combinations in binding performance at a range of concentrations between 0.1 and 100 mg.L-1 were evaluated with the Langmuir model for biosorption. The effects of pH, contact time, and viability on the binding capacities were also investigated. All strains and their combinations were found to bind cadmium and lead efficiently at low concentration ranges commonly observed in foods. However, the two strains and their combinations differed significantly in their maximum binding capacities and affinities represented by the Langmuir constants Qmax and b, respectively. The binding seemed to occur instantaneously and in a pH-dependent manner, which can be perfectly described by a segmented linear-plateau model.     

Antioxidant and Antiradical Properties of Probiotic Strains Bacillus amyloliquefaciens ssp. plantarum

The aim of the present study was to investigate the in vitro antioxidant potential of the cell-free extracts (CFE) of two probiotic bacteria Bacillus amyloliquefaciens ssp. plantarum IMV B-7142 and Bacillus amyloliquefaciens ssp. plantarum IMV B-7143 and their hepatoprotective effects. These strains are the main components of the veterinary probiotic preparation endosporyn. The CFE of probiotic bacteria were able to stabilize the 2.2-diphenyl-1-picrylhydrazyl radical to its neutral form at their cultivation during 24–48 h. But this index was more pronounced for the IMV B-7142 strain and amounted to 44.4–51.2%. The hydroxyl radical scavenging activity of the CFE of probiotic bacteria increased more than 70–80% regardless of the cultivation period (24–48 h). The antioxidant potential of probiotic strains is associated with the synthesis of the multiple biologically active molecules. The phenolic and benzoic acids-antioxidants (gallic, 4-hydroxyphenylacetic, caffeic, syringic, p-coumaric, trans-ferulic, and trans-cinnamic acids) were identified among metabolites of B. amyloliquefaciens ssp. plantarum strains. The CFE of probiotic strains were able to protect of rat hepatocytes from the toxic effects of the carbon tetrachloride (CCl₄). Post-treatment of stress-induced rat hepatocytes by CFE of the IMV B-7042 was accompanied by an increase of the catalase activity of cells by 485.2 mM/min × mg of protein, compared to stress-damaged sample. In doing so, the content of the main markers of oxidative stress: lipid hydroperoxides and malondialdehyde decreased significantly. The results suggested that CFE of both probiotic strains have potent antioxidant properties and effectively protect of stress-damaged rat hepatocytes.

     

Genome comparison of Bifidobacterium longum strains NCC2705 and CRC-002 using suppression subtractive hybridization

Because probiotic effects are strain dependent, genomic explanations of these differences will contribute to understanding their mechanisms of action. The genomic sequence of the Bifidobacterium longum probiotic strain NCC2705 was determined, but little is known about the genetic diversity between strains of this species. Suppression subtractive hybridization (SSH) is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains. The purpose of this study was to identify genetic differences between genomes of B. longum strains NCC2705 and CRC-002 using PCR-based SSH. Strain CRC-002 produces exopolysaccharides whereas NCC2705 is not known for reliable exopolysaccharide production. Thirty-five and 30 different sequences were obtained from the SSH libraries of strains CRC-002 and NCC2705, respectively. Specific CRC-002 genes found were predicted to be involved in the biosynthesis of exopolysaccharides and metabolism of other carbohydrates, and these genes were not present in the genome of strain NCC2705. The identification of an endo-1,4-beta-xylanase gene in the CRC-002 SSH library is an important difference because xylanase genes have previously been proposed as a defining characteristic of the NCC2705 strain. The results demonstrate that the SSH technique was useful to highlight potential genes involved in complex sugar metabolism that differ between the two probiotic strains.     

Fluorescent reporter systems for tracking probiotic lactic acid bacteria and bifidobacteria

In the last two decades, there has been increasing evidence supporting the role of the intestinal microbiota in health and disease, as well as the use of probiotics to modulate its activity and composition. Probiotic bacteria selected for commercial use in foods, mostly lactic acid bacteria and bifidobacteria, must survive in sufficient numbers during the manufacturing process, storage, and passage through the gastro-intestinal tract. They have several modes of action and it is crucial to unravel the mechanisms underlying their postulated beneficial effects. To track their survival and persistence, and to analyse their interaction with the gastro-intestinal epithelia it is essential to discriminate probiotic strains from endogenous microbiota. Fluorescent reporter proteins are relevant tools that can be exploited as a non-invasive marker system for in vivo real-time imaging in complex ecosystems as well as in vitro fluorescence labelling. Oxygen is required for many of these reporter proteins to fluoresce, which is a major drawback in anoxic environments. However, some new fluorescent proteins are able to overcome the potential problems caused by oxygen limitations. The current available approaches and the benefits/disadvantages of using reporter vectors containing fluorescent proteins for labelling of bacterial probiotic species commonly used in food are addressed.     

Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria

Aims:  To assess the ability of five probiotic bacteria to bind aflatoxin B₁ and to determine the key role of teichoic acids in the binding mechanism.
Methods and Results:  The strains were incubated in aqueous solutions containing aflatoxin B₁ (AFB₁). The amount of free toxin was quantified by HPLC. Stability of the bacteria–aflatoxin complex was evaluated by repeated washes with buffer. In order to understand the binding process, protoplasts, spheroplasts and cell wall components of two strains were analysed to assess their capacity to bind AFB₁. Additionally, the role of teichoic acids in the AFB₁ binding process was assessed. Lactobacillus reuteri strain NRRL14171 and Lactobacillus casei strain Shirota were the most efficient strains for binding AFB₁. The stability of the AFB₁–bacteria complex appears to be related to the binding ability of a particular strain; AFB₁ binding was also pH-dependent. Our results suggest that teichoic acids could be responsible for this ability.
Conclusions:  Our results provide information concerning AFB₁ binding by previously untested strains, leading to enhanced understanding of the mechanism by which probiotic bacteria bind AFB₁.
Significance and Impact of the Study:  Our results support the suggestion that some probiotic bacteria could prevent absorption of aflatoxin from the gastrointestinal tract.     

Validation of the official control method based on Polymerase Chain Reaction (PCR) for identification of authorised probiotic yeast in animal feed

Council Directive 70/524/EEC regulates the application of probiotic (microorganisms) additives in feeding stuffs. In the present study a method for the differentiation and strain identification of authorised probiotic Saccharomyces cereviseae strains in feeding stuffs by Polymerase Chain Reaction (PCR) was validated. Four different samples of animal feeding stuffs containing yeast at levels between 10(5) to 10(7) CFU/g were examined. Samples were enumerated on chloramphenicol glucose yeast extract agar and colonies were selected from these plates for DNA extraction and subsequent analysis. The PCR method using delta sequence primers produced an 'amplified sequence polymorphism' characteristic for the test strain. Feeds supplemented with one of four probiotic yeast strains each were analysed by seven of nine invited laboratories. All laboratories returned valid results with the exception of one laboratory that had insufficiently separated bands on the gel. The method had a good reproducibility for probiotic yeast isolates from feed of all four authorised probiotic yeast strains (APYS) CBS 493.94, APYS CNCM 1-1079, APYS CNCM 1-1077, APYS NCYC SC47 and of a commercially available yeast reference strain, NCYC 81. The PCR method is to be considered by CEN and ISO as official control method for identification of authorised probiotic Saccharomyces cerevisiae strains from feeding stuffs.     

The normal faecal microflora does not affect the adhesion of probiotic bacteria in vitro

Adhesion of probiotic microorganisms to the intestinal mucosa is considered important for many of the reported health effects. The influence of the endogenous microflora on the adhesion of four probiotic lactobacilli to immobilised intestinal mucus was investigated. It was observed that pre-treatment of the immobilised mucus with faecal extract slightly increased the adhesion of Lactobacillus GG. Pre-treatment of the immobilised mucus with faecal bacteria did not affect the adhesion of the tested strains. These results suggest that the normal microflora may not greatly affect the initial adhesion of the probiotic bacteria. This validates the results of earlier reports where the influence of the normal microflora was not taken into account.     

Binding of extracellular matrix molecules by probiotic bacteria

AIMS: The aim of this study was to investigate extracellular matrix (ECM) and mucin binding of selected bacterial isolates with probiotic features in comparison with commercially used probiotic bacteria. METHODS AND RESULTS: ECM molecules were immobilized in microtitre plates (mucin and fetuin) or on the surface of latex beads. Porcine mucin was bound by all 13 probiotic strains tested with important inter-strain differences; however, fetuin binding was similar (weak) for all 14 strains tested. Strongly positive (three) binding of bovine fibrinogen was expressed by strains from fermented food (Lactobacillus rhamnosus GG, L. casei Shirota and L. johnsonii La1) as well as by L. casei L.c., Lactobacillus sp. 2I3 and by L. plantarum LP. The other strains expressed moderate (2) or weakly positive (1) binding of bovine fibrinogen. Strongly positive (3) binding of porcine fibronectin was observed only with two strains; however, all other strains also bound this molecule. Bovine lactoferrin was bound to a higher extent than transferrins. SIGNIFICANCE AND IMPACT OF THE STUDY: Some animal strains (at least L. casei L.c. and Lactobacillus sp. 2I3) are comparable with the commercially used strains with respect to their ECM binding ability. As this feature is important for probiotic bacteria to be able to colonize intestine, these strains should be considered for their wider use in fermented feed (or probiotic preparations) for animals.     

Rapid identification of Lactobacillus and Bifidobacterium in probiotic products using multiplex PCR

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.