Candidate Probiotic Strains Research Articles

Isolation and characterization of probiotic strains for improving oral health

Probiotics have been proven effective for preventing caries. In contrast, the effect of probiotics on improving oral diseases such as gingivitis, periodontitis and halitosis has been less explored. ObjectiveTo perform a screening of lactic acid bacteria, according to international guidelines for the evaluation of probiotics, in order to select candidate probiotic strains for preventing oral disorders. Study designThe strains were isolated from healthy children and were subjected to a variety of in vitro tests in order to show their functionality. The safety of the strains was assessed by determining antibiotic susceptibility and production of lactic acid. ResultsForty-six of the 100 new isolates were assigned to lactic acid bacteria genera after a biochemical characterization. Most of the new isolated strains seem to be resistant to oral conditions, have great ability to form aggregates and have high antagonistic activity against oral pathogens. None of the strains produced unpleasant volatile compounds. The strains showed high ability to adhere to oral tissues and they do not present any antibiotic resistance. Moreover, an increased risk of developing caries due to their ability to produce lactic acid was discarded in seven pre-selected probiotic candidates. ConclusionsThese lactic acid bacteria show promising properties to be used as potential probiotics for improving oral health.

Lactococcus lactisNCC 2287 Alleviates Food Allergic Manifestations in Sensitized Mice by Reducing IL-13 Expression Specifically in the Ileum

Objective. Utilizing a food allergy murine model, we have investigated the intrinsic antiallergic potential of the Lactococcus lactis NCC 2287 strain. Methods. BALB/c mice were sensitized at weekly intervals with ovalbumin (OVA) plus cholera toxin (CT) by the oral route for 7 weeks. In this model, an oral challenge with a high dose of OVA at the end of the sensitization period leads to clinical symptoms. Lactococcus lactis NCC 2287 was given to mice via the drinking water during sensitization (prevention phase) or after sensitization (management phase). Results. Lactococcus lactis NCC 2287 administration to sensitized mice strikingly reduced allergic manifestations in the management phase upon challenge, when compared to control mice. No preventive effect was observed with the strain. Lactococcus lactis NCC 2287 significantly decreased relative expression levels of the Th-2 cytokine, IL-13, and associated chemokines CCL11 (eotaxin-1) and CCL17 (TARC) in the ileum. No effect was observed in the jejunum. Conclusion/Significance. These results taken together designate Lactococcus lactis NCC 2287 as a candidate probiotic strain appropriate in the management of allergic symptoms.

Substantiating efficacy of probiotics

Even if over 600 probiotic human intervention trials have been performed, not all health promoting effects attributed to probiotics have been substantiated or supported by well conducted clinical trials as yet. However, in evaluating the success met in the area or the validity of the results reached so far, several points should be kept in mind. The targeted health benefits are multiple and diverse, and they deal with populations as different as healthy or at risk infants, adults or elderly. Quite often, the studied disease corresponds to a syndrome rather than a single/simple manifestation from which the underlying causes or drivers are not fully understood. As a consequence, recognized disease risk factors and validated biomarkers are lacking and this hampers fulfilling the present requirements of regulatory authorities. In addition, key questions such as determining the right window of intervention have not always been addressed. While this might not be too difficult to assess when aiming at reducing the risk of diarrhea f.ex. it is quite challenging in the case of allergic diseases: when to optimally intervene in life? When to intervene in the disease course? An additional level of complexity comes from the huge diversity of probiotic candidate strains while it is altogether proven that purported health benefits are strain specific. This renders selection of the best probiotic candidate rather difficult especially since the mechanisms of action of probiotic strains in the highly complex ecosystem(s) they act on has only started to be unraveled. Therefore, dedicated screening strategies are requested. The steps and gaps that need to be filled in order to better substantiate probiotic effects and try to meet the challenge of conciliating science/research and regulation are being evaluated in different working groups such as the ILSI Probiotic Task Force.

Physiology and genetics of candidate probiotic strains relevant to animal health and production

Physiology and genetics of candidate probiotic strains relevant to animal health and production

Isolation and identification of a bacterium from marine shrimp digestive tract: A new degrader of starch and protein

It is a practical approach to select candidate probiotic bacterial stains on the basis of their special traits. Production of digestive enzyme was used as a trait to select a candidate probiotic bacterial strain in this study. In order to select a bacterium with the ability to degrade both starch and protein, an ideal bacterial strain STE was isolated from marine shrimp (Litopenaeus vannamei) intestines by using multiple selective media. The selected isolate STE was identified on the basis of its morphological, physiological, and biochemical characteristics as well as molecular analyses. Results of degradation experiments confirmed the ability of the selected isolate to degrade both starch and casein. The isolate STE was aerobic, Gram-negative, rod-shaped, motile and non-spore-forming, and had catalase and oxidase activities but no glucose fermentation activity. Among the tested carbon/nitrogen sources, only Tween40, alanyl-glycine, aspartyl-glycine, and glycyl-l-glutamic acid were utilized by the isolate STE. Results of homology comparison analyses of the 16S rDNA sequences showed that the isolate STE had a high similarity to several Pseudoalteromonas species and, in the phylogenetic tree, grouped with P. ruthenica with maximum bootstrap support (100%). In conclusion, the isolate STE was characterized as a novel strain belonging to the genus Pseudoalteromonas. This study provides a further example of a probiotic bacterial strain with specific characteristics isolated from the host gastrointestinal tract.

Technological properties of candidate probiotic Lactobacillus plantarum strains

The study of new probiotic strains for their technological relevance and use in dairy products is important for trade and industry. Eight Lactobacillus plantarum strains isolated from Bulgarian cheeses and selected for their potential probiotic properties were characterized. In vitro tests with the API ZYM system revealed high aminopeptidase and phosphatase activity, and weak lipolytic activity. The L. plantarum strains showed also a weak proteolytic activity and were characterized as slow variants on the base of their coagulation ability. They maintained high viability in fermented milk over extended shelf-times at refrigerated temperature and demonstrated a good adaptation to 6% NaCl. Among the preservatives, only calcium propionate did not affect the growth of L. plantarum. The highest concentrations used of potassium sorbate (0.5 and 1%) and nisaplin (0.02%) decreased the bacterial growth. One L. plantarum strain was tested as an adjunct to commercially available formula for cream cheese. This candidate probiotic culture withstood the technological processing and retained high number of 10 7 cfu g −1 at the end of the 3 months storage period at 4 °C.

Lactic acid bacteria as novel adjuvant systems for prevention and treatment of atopic diseases

Allergic diseases have become the new epidemic in westernized countries. Several factors including reduction of microbial contact early in life may have contributed to this dramatic development. As conventional immunotherapy has obvious limitations, there is a need for the development of new prophylactic and therapeutic treatment approaches. In this respect, lactic acid bacteria have received increasing attention as potential treatment tools against allergy. In this review, different treatment strategies with probiotics will be discussed in light of the recent literature. Supplementation trials with probiotics have shown to reduce allergic manifestations in children. New treatment approaches use selected probiotic strains as mucosal antigen delivery vehicles for recombinant allergens. The most recent studies focus on the optimal intervention time to enhance the treatment efficacy with probiotics for primary disease prevention. Probiotics may serve as attractive adjuvant systems for improved allergy treatment. Recent work supports the value of well conducted clinical and experimental studies that compare different treatment regimes and identify the best candidate probiotic strains for therapeutic immunomodulation. Such data, as well as understanding the mechanisms of immune modulation in pathogenic and therapeutic settings, are fundamental to be able to recommend probiotic use in allergy treatment.

Usefulness of a set of simple in vitro tests for the screening and identification of probiotic candidate strains for dairy use

A set of simple in vitro tests (identification by species-specific PCR, genetic diversity, phage sensitivity, growth and viability in milk, resistance to salts and flavor compounds, bacterial interactions, tolerance to simulated gastric juice and bile, bile salts deconjugation, hydrophobicity and β-galactosidase and antibacterial activities), that can be carried out in almost every laboratory of microbiology, mainly in developing countries where there is often limited access to sophisticated techniques, allowed us to identify, among 19 intestinal human isolates, a potential candidate for new probiotic dairy foods for the local market. Lactobacillus gasseri LgF 37/1 performed well in the culture media used for the enumeration of probiotic bacteria in argentinian dairy products. This strain showed also high tolerance to the technological challenges assessed, bile salts resistance, the capacity to produce bacteriocin-like metabolites, to inhibit pathogenic bacteria, to deconjugate bile salts and high hydrophobicity. Further in vivo research should be carried out with this strain before claiming probiotic properties for it. However, the use of a set of simple in vitro techniques proved to be important to determine which strains should undergo future and more complex studies.

Lactic acid bacteria isolated from canine faeces

Lactic acid bacteria (LAB) were isolated and sequenced from the faeces of healthy dogs. Five of these strains were selected and further characterized to clarify the potential of these strains as probiotics for canine. LAB were found in 67% (14/21) of the canine faeces samples when plated on Lactobacilli Selective Media without acetic acid. Out of 13 species identified with partial 16S rRNA gene sequencing, Lactobacillus fermentum LAB8, L. mucosae LAB12, L. rhamnosus LAB11, L. salivarius LAB9 and Weissella confusa LAB10 were selected as candidate probiotic strains based on their frequency, quantity in faeces, growth density, acid tolerance and antimicrobial activity. The minimal inhibitory concentration values of these isolates were determined for 14 antibiotics. L. salivarius LAB9, W. confusa LAB10 and L. mucosae LAB12 were viable in pH 2 for 4 h (mLBS), indicating tolerance to acidity and thus the potential to survive in gastrointestinal tract of the canine. The LAB8-LAB12 strains showed antimicrobial activity against Micrococcus luteus A1 NCIMB86166. Thirteen different LAB species were found from the faecal microbiota of the healthy canines. Five acid tolerant and antimicrobially active LAB strains with the capacity to grow to high densities both aerobically and anaerobically were chosen to serve as candidate probiotics. The selected LAB strains are among the first host-specific LAB with antimicrobial activity isolated from canines that could serve as potential probiotics for canine use.

Recommendations for Improved Use of the Murine TNBS-Induced Colitis Model in Evaluating Anti-inflammatory Properties of Lactic Acid Bacteria: Technical and Microbiological Aspects

Probiotic bacteria have been shown to exert promising beneficial effects in different types of intestinal disorders, including chronic inflammation. In this context, animal models of inflammatory bowel disease are useful in studying the possible prophylactic role of candidate probiotic strains. This study aimed at evaluating the critical technological and microbiological parameters as well as the robustness of the murine trinitrobenzene sulfonic acid (TNBS)-induced model of colitis, after intragastric administration of lactic acid bacteria (LAB) preparations. A standardized methodology was applied to assess the protective effect achieved by various bacterial concentrations and culture conditions of the reference strain Lactobacillus plantarum NCIMB 8826. Not only was protection found to vary in function in different levels of colitis, but also repeated experiments showed a clear bacterial dose-dependent attenuation of colitis. The physiological stage of bacteria was shown to impact as well, with substantial, mild, or reduced improvement of inflammatory scores for exponentially growing, stationary-phase, or killed bacteria, respectively. A recombinant strain, secreting murine interleukin-10 (IL-10) and previously reported to successfully treat colitis in two different models of murine colitis (dextran sulfate sodium [DSS] and IL-10-deficient mice), was used to validate the final experimental conditions. In conclusion, we identified and optimized some of the key parameters that need to be controlled in order to ensure reliable comparison of results generated over a long period of time or independent experiments. The recommendations for an improved model presented here will prove to be helpful for reproducible, independent comparison of the anti-inflammatory potential of wild-type or recombinant candidate probiotic strains, whether administered as pure cultures or as blends.

Evaluation of candidate probiotic strains for gilthead sea bream larvae (Sparus aurata) using an in vivo approach

The aim of this study was to evaluate the effect of six bacterial strains on gilthead sea bream larvae (Sparus aurata). Six bacterial strains isolated from well-performing live food cultures were identified by sequencing fragments of their 16s rDNA genome to the genus level as Cytophaga sp., Roseobacter sp., Ruergeria sp., Paracoccus sp., Aeromonas sp. and Shewanella sp. Survival rates of gilthead sea bream larvae transferred to seawater added these bacterial strains at concentrations of 6 +/- 0.3 x 10(5) bacteria ml(-1) were similar to those of larvae transferred to sterilized seawater and showed an average of 86% at 9 days after hatching, whereas, survival rates of larvae transferred to filtered seawater were lower (P < 0.05), and showed an average of 39%, 9 days after hatching. Several bacterial strains isolated from well-performing live food cultures showed a positive effect for sea bream larvae when compared with filtered seawater. The approach used in this study could be applied as an in vivo evaluation method of candidate probiotic strains used in the rearing of marine fish larvae.

Selection of enterococci for potential canine probiotic additives

Enterococci are important inhabitants of animal intestine and are widely used in probiotic products. A potentially successful probiotic strain is expected to have several desirable properties in order to be able to exert its beneficial effects. Forty enterococcal isolates from dog faeces were tested for characters believed to be important for probiotic strains; bacteriocin production, resistance or tolerance to antibiotics, low pH, bile tolerance and adhesive activity. The total count of enterococci was found to be 3.3–7.3 log 10 CFU/g of faeces. Most identified strains were Enterococcus faecium. All strains were sensitive to vancomycin, ampicillin, penicillin and chloramphenicol. Thirty-three percentage of strains were resistant to erythromycin and 28% to tetracycline. Among 40 isolates, 75% showed a broad inhibitory spectrum only against Gram-positive indicator bacteria. Seven strains with broad bacteriocin activity were selected for further assays. In the presence of 1% bile, the survival rate of selected strains ranged between 72 and 98%. Survival of strains at pH 3.0 was found in the range between 76 and 87% after 3 h. The adhesion of the tested strains to intestinal mucus ranged from 4 to 11% for canine mucus and from 5 to 8% for human mucus. E. faecalis EE4 and E. faecium EF01 showed the best probiotic properties. It indicates that they could be used as new candidate probiotic strains after in vivo testing.

Use of Mouse Models To Evaluate the Persistence, Safety, and Immune Modulation Capacities of Lactic Acid Bacteria

Recent clinical and experimental observations showed that specific probiotic microorganisms may provide therapeutic benefits in inflammatory bowel disease. However, a rigorous screening for new candidate probiotic strains with optimized therapeutic properties necessitates also determining possible adverse interactions with the host, particularly in individuals who are not healthy. We have evaluated the persistence of strains of lactic acid bacteria (LAB) in the digestive tracts of mice, their immunomodulation capacity, and their safety in healthy animals and in a colitis model. Following daily administration of 10(9) CFU of viable LAB orally, intragastrically, or intrarectally, the animals' feces were examined for bacterial excretion and cytokines were quantified in intestinal samples by quantitative reverse transcription-PCR. The level of bacterial translocation was assessed in healthy mice and in mice suffering from colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Irrespective of the route of administration, the potential probiotic strain Lactobacillus plantarum NCIMB8826 was found to persist for up to 10 days in the digestive tracts of mice. This strain did not induce detrimental effects in healthy or in TNBS-treated animals, as was reflected by the absence of weight loss, intestinal inflammation, modification of cytokine levels in the ileum and colon (healthy mice), and bacterial dissemination (healthy and colitic animals). Moreover, the translocation of endogenous microflora to the mesenteric lymph nodes and spleen was greatly reduced in the TNBS-treated mice after administration of LAB. This property, together with the strain's persistence capacity and innocuousness renders L. plantarum NCIMB8826 an attractive candidate as a probiotic to be used in the prevention or treatment of chronic inflammation.

Gastrointestinal microbial community shifts observed following oral administration of a Lactobacillus fermentum strain to mice

The indigenous gastrointestinal microbiota acts as an integral defense against the colonisation of orally introduced microbes. Whilst this can be important in host protection, some introduced species, including lactobacilli, can have a positive impact on existing microbial communities. The interaction of a candidate probiotic strain of Lactobacillus fermentum within the gastrointestinal tract was monitored in a mouse model and its effect on the indigenous microbiota observed. L. fermentum KLD was administered via oro-gastric doses to mice with both a specific pathogen-free (SPF) and an ampicillin-depleted gut microbiota, containing no detectable lactobacilli. Its persistence was monitored by detection in faecal homogenates using culturing methods and polymerase chain reaction with L. fermentum specific primers. Microbial population shifts were observed using denaturing gradient gel electrophoresis (DGGE). L. fermentum KLD was detected within the gastrointestinal tract of SPF mice for up to 36 h, and for greater than 11 days in the ampicillin-treated mice. The administration resulted in substantial changes within the host Lactobacillus levels, determined by DGGE of 16S rDNA from faecal samples. Denaturing gradient profiles, from faecal samples collected at a range of pre- and post-dose intervals of groups of 10 SPF mice, indicated that several other constituents of the gastrointestinal community also fluctuated following dosing. These included Bifidobacterium and Eubacterium, which increased following KLD administration. The indigenous microbiota affected the persistence of L. fermentum KLD and in SPF mice the administration of this strain induced significant shifts in the indigenous microbial community.

Gastrointestinal microbial community shifts observed following oral administration of a Lactobacillus fermentum strain to mice

The indigenous gastrointestinal microbiota acts as an integral defense against the colonisation of orally introduced microbes. Whilst this can be important in host protection, some introduced species, including lactobacilli, can have a positive impact on existing microbial communities. The interaction of a candidate probiotic strain of Lactobacillus fermentum within the gastrointestinal tract was monitored in a mouse model and its effect on the indigenous microbiota observed. L. fermentum KLD was administered via oro-gastric doses to mice with both a specific pathogen-free (SPF) and an ampicillin-depleted gut microbiota, containing no detectable lactobacilli. Its persistence was monitored by detection in faecal homogenates using culturing methods and polymerase chain reaction with L. fermentum specific primers. Microbial population shifts were observed using denaturing gradient gel electrophoresis (DGGE). L. fermentum KLD was detected within the gastrointestinal tract of SPF mice for up to 36 h, and for greater than 11 days in the ampicillin-treated mice. The administration resulted in substantial changes within the host Lactobacillus levels, determined by DGGE of 16S rDNA from faecal samples. Denaturing gradient profiles, from faecal samples collected at a range of pre- and post-dose intervals of groups of 10 SPF mice, indicated that several other constituents of the gastrointestinal community also fluctuated following dosing. These included Bifidobacterium and Eubacterium, which increased following KLD administration. The indigenous microbiota affected the persistence of L. fermentum KLD and in SPF mice the administration of this strain induced significant shifts in the indigenous microbial community.

Probiotics prevent zonulin-mediated intestinal barrier dysfunction secondary to bacterial colonization

Background: In the mammalian host, the intestinal epithelium along with indigenous microflora serves as a powerful protective barrier. During pathologic conditions, this ecosystem gets disrupted leading to adverse consequences. We have previously shown that Gram (-) normal gut flora can adhere, invade, and trigger necrotizing enterocolitis-like changes in weanling rabbits. Recently, we described a novel eukaryotic protein, zonulin, and its regulatory role in gut permeability via tight junction disassembly. In this study, we evaluated the effect of a candidate probiotic strain, Lactobacillus plantarum strain PP-217 (Lp) on zonulin-mediated changes in the gut mucosa. Methods: Explant cultures from rabbit intestine were used to investigate the ability of a normal flora E. coli to induce zonulin release. Polarity of zonulin release, changes in transepithelial electrical resistance (TEER), transport of inulin, and transcytosis of E. coli were studied in the snapwell system using rabbit and rhesus monkey gut. Zonulin concentration was measured by sandwich ELISA. Results: E. coli induced significant release of zonulin from rabbit organ cultures (1.68 2.2 ng/mg protein.mrrr') compared to uninfected controls. Co-infection with Lp, resulted in a 3-8-fold reduction in zonulin release. Cultures of E. coli alone did not produce zonulin. In the snapwell systems, addition of 10 E. colilml to the apical side of the intestinal segments resulted in zonulin release from the luminal side, but not from the basolateral (BL) side, a drop in TEER (ll 20-57 n crrr'), an increased passage of inulin (11-34 ~mol.h/ crrr'), and transcytosis of E. coli to the BL side (3.3 XlO 1.8XlO in 1-6 hr). Lp reduced the release of zonulin (10-100 fold), prevented the drop in TEER (>50%), reduced inulin transcytosis (>75%), and caused a 4-100fold blockage of E. coli transcytosis. Addition of FIIO, a synthetic octapeptide analogue of zonulin provided similar protection in gut permeability. Conclusions: These results demonstrate that normal flora bacteria can induce zonulin release by the small intestine resulting in a reduction in barrier function and increased bacterial transcytosis. Candidate probiotic strains have the ability to prevent such changes. While the mechanism of action of probiotics in this system remains unidentified, the implications and its potential use can range from enteric bacterial infections to a myriad of conditions involving reduced intestinal permeability. Supported by NIH grants HD 29735 & 34974.