Potential probiotic properties of phytase-producing Lactobacillus salivarius FC113

Probiotics have known efficacy as dietary supplements. Here, Lactobacillus strain F113 was characterized for probiotic use. Strain FC113 was selected as having the highest phytase activity (403.6 U) among tested strains showing acid tolerance and nitrite production. FC113 was tentatively identified as Lactobacillus salivarius based on an API 50 CHL assay and 16S rRNA gene analysis. The production of interleukin (IL)-1α and tumor necrosis factor (TNF)-α was measured in in vitro culture experiments. Cytokine production by L. salivarius FC113 at 1?×?10⁷ CFU/ml was approximately 175.5?±?36.40 pg/mL IL-1α and 353.5?±?61.79 pg/mL TNF-α. L. salivarius FC113 was profoundly resistant to artificial gastric juice (pH 2.5, 1 % pepsin), and persisted for 24 h in artificial bile acid. According to results obtained with an API ZYM kit, L. salivarius FC113 did not generate carcinogenic enzymes. L. salivarius F113 had an inhibitory effect on food-borne pathogens, and adhered strongly to HT-29 human intestinal epithelial cell lines. These results show that L. salivarius FC113 has probiotic characteristics, and exhibits high feed bioavailability in the host animal, in addition to an immune-stimulating effect.     

Intestinal and immunological effects of daily oral administration of Lactobacillus salivarius CECT5713 to healthy adults

There is an increasing interest in the intestinal and immunological effects of probiotics. The aim of the present study is to evaluate the tolerance and beneficial effects in healthy adults of the strain, Lactobacillus salivarius CECT5713 isolated from breast milk. A phase II, randomized, double-blinded, placebo-controlled human clinical trial was carried out in 40 healthy adults. The Probiotic group received a daily dose of 2 × 10⁸ CFU of L. salivarius CECT5713 in capsules during 4 weeks while volunteers of the control received only a placebo. Gastrointestinal and immunological parameters were analyzed. Results showed that L. salivarius CECT5713 was well tolerated and no adverse effects were detected. Consumption of the probiotic strain increased fecal lactobacilli counts (7.9 ± 0.1 vs. 7.05 ± 0.2 CFU/g feces, P = 0.001). Also, an improvement in the frequency of defecation (P = 0.04) was observed. Probiotic treatment induced significantly the percentage of NK cells and monocytes, as well as the plasmatic levels of immunoglobulins M, A and G, and the regulatory cytokine IL-10 (72.3 ± 11.7 in probiotic group vs. 27.3 ± 6.4 pg/mL in control group, P < 0.01). Thus, it can be concluded that daily administration of L. salivarius CECT5713 to healthy adults is safe and improve gut microbiota and different parameters related to immune response.     

Salivaricin P, One of a Family of Two-Component Antilisterial Bacteriocins Produced by Intestinal Isolates of Lactobacillus salivarius

Lactobacillus salivarius DPC6005, a porcine intestinal isolate, produces a two-component bacteriocin, salivaricin P, with homology to ABP-118 produced by a human probiotic L. salivarius strain. Indeed, molecular characterization revealed that while the peptides Sln1 and ABP-118α are identical, their companion peptides (Sln2 and ABP-118β, respectively) differ by two amino acids. This observation suggests that two-component bacteriocins may be a common feature of intestinal L. salivarius strains.     

Evaluation of Caco-2 and human intestinal epithelial cells as in vitro models of colonic and small intestinal integrity

Although the colonic cell line Caco-2 is widely used as a model of the small intestinal barrier function, it has limitations such as overestimated transepithelial electrical resistance (TEER) compared to in vivo conditions. Therefore, we investigated Human Intestinal Epithelial Cells (HIECs) as an alternative in vitro model.We explored whether cell seeding number of HIEC-6, and the number of incubation days for HIEC and Caco-2 cells had an impact on TEER, and tight junction expression was examined for both cell lines via immunofluorescence in the presence and absence of probiotic bacteria.We observed no significant difference in TEER readings for either cell lines when cultured for different days. Further, the HIEC TEER readings did not change with increased seeding number and were not significantly different from a control with no cells. HIECs expressed Claudin-1 and Zonula Occludens-1 but not Occludin. Caco-2 co-culture with probiotic bacteria demonstrated a significant increase in TEER, particularly for the lactobacillus strains, whereas HIEC TEER did not respond to bacterial co-incubation.Our study shows that although HIECs express certain TJ proteins, a significant TEER was not observed, likely due to the embryonic origin of the cells, which limits the application of this cell line as a suitable model for small intestinal barrier function.     

Probiotics Can Generate FoxP3 T-Cell Responses in the Small Intestine and Simultaneously Inducing CD4 and CD8 T Cell Activation in the Large Intestine

Most studies on probiotics aim to restore intestinal homeostasis to reduce immune-pathology in disease. Of equal importance are studies on how probiotics might prevent or delay disease in healthy individuals. However, knowledge on mechanisms of probiotic actions in healthy individuals is scarce. To gain more insight in how different bacterial strains may modulate the healthy intestinal immune system, we investigated the effect of the food derived bacterial strains L. plantarum WCFS1, L. salivarius UCC118, and L. lactis MG1363, on the intestinal regulatory immune phenotype in healthy mice. All three bacterial strains induced an upregulation of activity and numbers of CD11c⁺ MHCII⁺ DCs in the immune-sampling Peyer’s Patches. Only L. salivarius UCC118 skewed towards an immune regulatory phenotype in the small intestinal lamina propria (SILP). The effects were different in the large intestine lamina propria. L. salivarius UCC118 induced activation in both CD4 and CD8 positive T-cells while L. plantarum WCFS1 induced a more regulatory phenotype. Moreover, L. plantarum WCFS1 decreased the Th1/Th2 ratio in the SILP. Also L. lactis MG1363 had immunomodulatory effects. L. lactis MG1363 decreased the expression of the GATA-3 and T-bet in the SILP. As our data show that contradictory effects may occur in different parts of the gut, it is recommended to study effects of probiotic in different sites in the intestine. Our strain-specific results suggest that unspecified application of probiotics may not be very effective. Our data also indicate that selection of specific probiotic strain activities on the basis of responses in healthy mice may be a promising strategy to specifically stimulate or suppress immunity in specific parts of the intestine.     

Functional role of <Emphasis Type="Italic">oppA</Emphasis> encoding an oligopeptide-binding protein from <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> Ren in bile tolerance

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and some L. salivarius strains are considered as probiotics. Bile tolerance is a crucial property for probiotic bacteria to survive the transit through the GIT and exert their beneficial effects. In this work, the functional role of oppA encoding an oligopeptide transporter substrate-binding protein from L. salivarius Ren in bile salt tolerance was investigated. In silico analysis revealed that the oppA gene encodes a 61.7-kDa cell surface-anchored hydrophilic protein with a canonical lipoprotein signal peptide. Homologous overexpression of OppA was shown to confer 20-fold higher tolerance to 0.5 % oxgall in L. salivarius Ren. Furthermore, the recombinant strain exhibited 1.8-fold and 3.6-fold higher survival when exposed to the sublethal concentration of sodium taurocholate and sodium taurodeoxycholate, respectively, while no significant change was observed when exposed to sodium glycocholate and sodium glycodeoxycholate (GDCA). Our results indicate that OppA confers specific resistance to taurine-conjugated bile salts in L. salivarius Ren. In addition, the OppA overexpression strain also showed significant increased resistance to heat and salt stresses, suggesting the protective role of OppA against multiple stresses in L. salivarius Ren.     

Modulatory Effects of <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> on Intestinal Mucosal Immunity of Piglets

Recent studies have demonstrated that lactobacilli or their cell components can improve certain immune function in animals. The aim of this study is to investigate the effects of porcine lactobacilli on the intestinal mucosal immunity of piglets. Neonatal piglets were used as a model and were orally administrated with Lactobacillus salivarius B1 isolated from the duodenal mucosa of a healthy piglet. The feces of the piglets were collected on days 7, 14, and 21 for intestinal microflora analysis. On day 28, the piglets were sacrificed, and their intestinal mucosa samples were immediately collected to investigate the changes in intestinal morphological and immunocompetent cells. Finally, the expression of cytokines and TLRs was detected in the different intestinal segments. The results indicate that L. salivarius B1 can partially ameliorate the microflora of the feces and increase the number of intestinal immunocompetent cells, as the intraepithelial lymphocyte (P < 0.05), and the IgA-producing cells (P < 0.01) in the lactobacilli-treated group were all increased compared with those in the control group. Enhanced expression of the cytokine IL-6 gene was also observed in the ileum (P < 0.05). Moreover, L. salivarius B1 can also upregulate the expression of TLR2 in the intestinal tract at the gene and protein levels (P < 0.05). The results demonstrate that L. salivarius B1 is beneficial for the maturation of the intestinal mucosal immune system and elicited local immunomodulatory activities. In addition, the modulatory effects of L. salivarius B1 on mucosal immunity mainly depend on its extracellular components.     

Unusual genome complexity in Lactobacillus salivarius JCM1046

Background

Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046.

Results

JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM.

Conclusion

Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-771) contains supplementary material, which is available to authorized users.     

Antagonistic activity of Lactobacillus acidophilus RY2 isolated from healthy infancy feces on the growth and adhesion characteristics of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAggEC) infection is an important cause of acute diarrhea, affecting children in developing countries and travelers visiting tropical or subtropical areas. Three probiotics can exert bacteriostatic or bactericidal effects on human and animal intestinal pathogens, the efficiency of probiotics on EAggEC infection remains unclear. In this study, the antagonistic activity of probiotic bacteria isolated from infant faeces was examined against several EAggEC stains. While three isolates, Lactobacillus acidophilus RY2, Lactobacillus salivarius MM1 and Lactobacillus paracasei En4 were shown to significantly inhibit the growth of EAggEC. In addition, the antagonistic activity of the Lactobacillus species was maintained despite heating (100 °C, 15 min) of cell free culture supernatant (CFCS). The antagonistic activity of the CFCS however, could be reduced following lactate dehydrogenase treatment and at pH 7.2. Furthermore, in an adhesion–inhibition assay, L. acidophilus RY2 was shown to be more effective than L. salivarius MM1 and L. paracasei EN4. This study suggests that L. acidophilus RY2 could be used as a probiotic organism against EAggEC.     

Screening for lactobacilli with probiotic properties in the infant gut microbiota

Lactobacilli are believed to be beneficial for the human hosts and are currently being evaluated as potentially probiotic bacteria. In this study, Lactobacillus strains were isolated from infant faeces and were examined in vitro for potential probiotic properties. Faecal specimens from 63 healthy, full-term infants were collected at 4, 30 and 90 days after delivery. Seventy-four Lactobacillus strains were isolated and one or more different phenotypes from each infant (n = 44) were selected for further testing. The bacterial isolates were identified mainly as L. gasseri, L. crispatus, Lactobacillus paracasei, L. salivarius, L. fermentum after amplification and sequencing of 16s rRNA gene. The strains were examined for acid and bile tolerance, adhesion to Caco-2 cells, antibiotic susceptibility and antimicrobial activity against selected enteric pathogens. The great majority of the isolated lactobacilli were susceptible to ampicillin, amoxicillin/clavulanic acid, tetracycline, erythromycin, cephalothin, chloramphenicol and rifampicin. Resistance to vancomycin or bacitracin was detected to 34% of the strains. Twenty strains out of forty-four exhibited significant tolerance to bile salts. Those strains were subsequently tested for resistance to low pH conditions (pH 2 and 3). Interestingly, 85% (17 strains) of the tested lactobacilli remained unaffected at pH 3 after 3 h of incubation, 6 strains were found resistant at pH 2 after 1.5 h and only 2 strains found resistant after 3 h of incubation. Two of the strains were able to adhere to Caco-2 cells. In conclusion, two isolates fulfilled the in vitro probiotic criteria and are good candidates for further in vivo evaluation.     

Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10⁷ CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.     

Functional and Probiotic Attributes of an Indigenous Isolate of Lactobacillus plantarum

Background

Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.

Methods and Findings

An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.

Conclusion

The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.     

Complete Genome Sequence of Lactobacillus salivarius CECT 5713, a Probiotic Strain Isolated from Human Milk and Infant Feces

Lactobacillus salivarius is a homofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. L. salivarius CECT 5713, a strain isolated simultaneously from breast milk and infant feces of a healthy mother-infant pair, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays. Here, we report its complete and annotated genome sequence.In the last years, culture-dependent and -independent analyses of the bacterial diversity of human milk and colostrum have revealed that these biological fluids are a source of live staphylococci, streptococci, lactic acid bacteria, and bifidobacteria in the infant gut (5, 6, 8, 9, 11, 13), where they play a key role in the initiation and development of the gut microbiota (12). In a previous study, we isolated L. salivarius CECT 5713 from human milk and infant feces of a mother-child pair (10). Subsequent studies revealed that this strain was a good probiotic candidate since it achieved high survival rates when exposed to the gastrointestinal tract conditions, showed a strong adherence to intestinal cells, stimulated the expression of mucin-encoding genes, produced antimicrobial compounds (lactate, acetate, and hydrogen peroxide), and displayed in vivo and in vitro immunomodulatory, anti-inflammatory, and antibacterial properties against pathogenic bacteria (2, 10, 15). Moreover, oral administration of L. salivarius CECT 5713 appears to be an efficient alternative for the treatment of infectious mastitis in lactating women (7). Similarly, studies with other L. salivarius strains in animal models and clinical trials have demonstrated their probiotic function and, particularly, their anti-inflammatory effects (3, 14, 16).In order to interrogate the genome sequence of L. salivarius CECT 5713 with regard to its probiotic properties, the complete genome sequence was determined by a whole-genome shotgun strategy using pyrosequencing technology (454 Life Sciences, Banford, CT). The initial draft assembly provided by 454 Life Sciences was based on 444,604 high-quality pyrosequencing reads, which assembled into 59 contigs. The genome sequence of L. salivarius UCC118 (1), a well-characterized probiotic strain, was used to order these contigs into large scaffolds.The genome of L. salivarius CECT 5713 consists of a circular chromosome of 1,828,169 bp, two plasmids (pHN1, 44,581 bp; pHN2, 20,426 bp), and a megaplasmid (pHN3, 242,962 bp). The overall GC content of the chromosome is 32.93%, similar to that of the megaplasmid but lower than those of the plasmids (>38%). The entire genome of CECT 5713 contains 1,558 protein-, 87 tRNA-, and 51 rRNA-encoding genes. A comparison between the genomes of L. salivarius CECT 5713 and UCC118 revealed the presence of 52 protein-encoding genes that are exclusive for CECT 5713, including genes encoding a 6-phospho-β-glucosidase and three collagen-binding proteins, which may explain the high potential for competitive exclusion of pathogens displayed by this strain. The genes responsible for the bacteriocin activity of L. salivarius CECT 5713 are located in pHN3. This megaplasmid contains six open reading frames (ORFs) closely related, but not identical, to the genes responsible for the biosynthesis of salivaricin ABP-118, a two-component class II bacteriocin (4), in L. salivarius UCC118. Globally, several features of the L. salivarius CECT 5713 genome suggest a strong probiotic potential in humans.     

Characterization of a Bacteriocin-Like Substance Produced by a Vaginal Lactobacillus salivarius Strain

A novel bacteriocin-like substance produced by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328 with activity against Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used for the design of a probiotic to prevent urogenital infections.     

Persistence of the Oral Probiotic Streptococcus salivarius M18 Is Dose Dependent and Megaplasmid Transfer Can Augment Their Bacteriocin Production and Adhesion Characteristics

Bacteriocin-producing probiotic Streptococcus salivarius M18 offers beneficial modulatory capabilities within the oral microbiome, apparently through potent inhibitory activity against potentially deleterious bacteria, such as Streptococcus pyogenes. The oral cavity persistence of S. salivarius M18 was investigated in 75 subjects receiving four different doses for 28 days. Sixty per cent of the subjects already had some inhibitor-producing S. salivarius in their saliva prior to probiotic intervention. Strain M18’s persistence was dependent upon the dose, but not the period of administration. Culture analysis indicated that in some individuals the introduced strain had almost entirely replaced the indigenous S. salivarius, though the total numbers of the species did not increase. Selected subjects showing either high or low probiotic persistence had their salivary populations profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Analysis indicated that while certain bacterial phenotypes were markedly modulated, the overall composition of the oral microbiome was not modified by the probiotic treatment. Megaplasmids encoding bacteriocins and adhesion factors were transferred in vitro to generate a transconjugant S. salivarius exhibiting enhanced antimicrobial production and binding capabilities to HEp-2 cells. Since no widespread perturbation of the existing indigenous microbiota was associated with oral instillation and given its antimicrobial activity against potentially pathogenic streptococci, it appears that application of probiotic strain M18 offers potential low impact alternative to classical antibiotic prophylaxis. For candidate probiotic strains having relatively poor antimicrobial or adhesive properties, unique derivatives displaying improved probiotic performance may be engineered in vitro by megaplasmid transfer.     

Using In Vitro Immunomodulatory Properties of Lactic Acid Bacteria for Selection of Probiotics against Salmonella Infection in Broiler Chicks

Poultry is known to be a major reservoir of Salmonella. The use of lactic acid bacteria has become one of successful strategies to control Salmonella in poultry. The purpose of this study was to select lactic acid bacteria strains by their in vitro immunomodulatory properties for potential use as probiotics against Salmonella infection in broiler chicks. Among 101 isolated lactic acid bacteria strains, 13 strains effectively survived under acidic (pH 2.5) and bile salt (ranging from 0.1% to 1.0%) conditions, effectively inhibited growth of 6 pathogens, and adhered to Caco-2 cells. However, their in vitro immunomodulatory activities differed significantly. Finally, three strains with higher in vitro immunomodulatory properties (Lactobacillus plantarum PZ01, Lactobacillus salivarius JM32 and Pediococcus acidilactici JH231) and three strains with lower in vitro immunomodulatory activities (Enterococcus faecium JS11, Lactobacillus salivarius JK22 and Lactobacillus salivarius JM2A1) were compared for their inhibitory effects on Salmonella adhesion and invasion to Caco-2 cells in vitro and their antimicrobial effects in vivo. The former three strains inhibited Salmonella adhesion and invasion to Caco-2 cells in vitro, reduced the number of Salmonella in intestinal content, spleen and liver, reduced the levels of lipopolysaccharide-induced TNF-α factor (LITAF), IL-1β, IL-6 and IL-12 in serum and increased the level of IL-10 in serum during a challenge study in vivo more efficiently than the latter three strains. These results suggest that in vitro immunomodulatory activities could be used as additional parameters to select more effective probiotics as feed supplements for poultry.     

Probiotic properties of Lactobacillus isolates originating from porcine intestine and feces

In this study, a total of 94 lactic acid bacterial (LAB) isolates of porcine small intestinal and fecal origin were screened for their probiotic properties. The aim was to evaluate whether their isolation site and putative species identity play a role in these characteristics and whether either of these can be used as a predictive factor for the probiotic potential of bacterial isolates. The isolates were preliminarily identified by partial 16S rRNA gene sequencing and characterized in vitro for their pH and bile tolerance, adhesion capacity towards porcine enterocytes isolated from five intestinal sites and for antimicrobial activity towards five indicator pathogens. The interdependence of these characteristics was statistically evaluated. The isolates tolerated low pH and bile well. Adherence to the enterocytes of different origins did not correlate with the strain isolation site. In general, higher adherence was observed to colon cells in comparison to the small intestinal enterocytes. Culture filtrates of the isolates caused a decrease of up to three orders of magnitude in the intestinal pathogen cell numbers. The inhibition was mostly due to lactic and other organic acids. The predominating phylotypes identified were Lactobacillus reuteri and Lactobacillus salivarius, of which the former generally had the best adhesion capacity, whereas the latter appeared to be the best inhibitor. Based on the results, several strains of the pig Lactobacillus isolates tested may function as promising candidates for use in probiotic products. However, it was not possible to use the isolation site or the species identity of the isolates as reliable preliminary screening factors.     

Detection and Specific Enumeration of Multi-Strain Probiotics in the Lumen Contents and Mucus Layers of the Rat Intestine After Oral Administration

Although the detection of viable probiotic bacteria following their ingestion and passage through the gastrointestinal tract (GIT) has been well documented, their mucosal attachment in vivo is more difficult to assess. In this study, we investigated the survival and mucosal attachment of multi-strain probiotics transiting the rat GIT. Rats were administered a commercial mixture of the intestinal probiotics Lactobacillus acidophilus LA742, Lactobacillus rhamnosus L2H and Bifidobacterium lactis HN019 and the oral probiotic Streptococcus salivarius K12 every 12 h for 3 days. Intestinal contents, mucus and faeces were tested 6 h, 3 days and 7 days after the last dose by strain-specific enumeration on selective media and by denaturing gradient gel electrophoresis. At 6 h, viable cells and DNA corresponding to all four probiotics were detected in the faeces and in both the lumen contents and mucus layers of the ileum and colon. Viable probiotic cells of B. lactis and L. rhamnosus were detected for 7 days and L. acidophilus for 3 days after the last dose. B. lactis and L. rhamnosus persisted in the ileal mucus and colon contents, whereas the retention of L. acidophilus appeared to be relatively higher in colonic mucus. No viable cells of S. salivarius K12 were detected in any of the samples at either day 3 or 7. The study demonstrates that probiotic strains of intestinal origin but not of oral origin exhibit temporary colonisation of the rat GIT and that these strains may have differing relative affinities for colonic and ileal mucosa.     

Lead biosorption efficiency of Levilactobacillus brevis MZ384011 and Levilactobacillus brevis MW362779: A response surface based approach

Lead (Pb) is a substantial contaminant in the environment and a potent toxin for living organisms. Current study describes probiotic characteristics of Pb-biosorbing lactic acid bacteria (LAB), and response surface methodology (RSM) based optimization of physical conditions for maximum Pb biosorption. A total of 18 LAB, isolated from carnivore feces (n = 8) and human breast milk (n = 9), along with one reference strain Lactobacillus acidophilus ATCC4356 were included in the study. Pb biosorption was strain specific. Eight strains, demonstrating ≥ 70 % lead biosorption, were selected for further testing. The lactobacillus-Pb complex was found to be stable and strains had a negative surface charge. The strains displayed good probiotic properties with the survival rate of 71–90 % in simulated gastric environment, 36–69 % in intestinal condition (1.8 % bile salts) and 55–72 % hydrophobicity. On the basis of excellent probiotic ability, Levilactobacillus brevis MZ384011 and Levilactobacillus brevis MW362779 were selected for optimization of physical conditions of Pb biosorption through RSM. Maximum biosorption was observed at pH 6 in 60 min at a cell density of 1 g/L. L. brevis MZ384011 and L. brevis MW362779 are recommended for experimentation on Pb toxicity amelioration and safety evaluation in in-vivo setting.     

Inhibition of Staphylococcus aureus adherence to Caco-2 cells by lactobacilli and cell surface properties that influence attachment

Staphylococcus aureus is an opportunistic pathogen that can colonize human and animal intestinal tracts, causing certain gastrointestinal diseases. The adherence of enteric pathogens to host intestinal epithelial cells is important for their pathogenesis. In the present study, Lactobacillus salivarius and Lactobacillus plantarum were investigated in vitro to examine their ability to competitively exclude S. aureus. Various factors involved in attachment, including bacterial status and cell concentration, growth phase, competition patterns, and surface-layer protein extracts, were also investigated. Live lactobacilli in the mid-log growth phase exhibited maximum inhibitory activity when lactobacilli were pre- or co-incubated with S. aureus. However, the inhibitory activity was significantly reduced when the lactobacilli were inactivated by heating or treated with LiCl. Furthermore, both lactobacilli possessed certain cell surface properties, such as hydrophobicity, autoaggregation, and coaggregation ability. L. salivarius and L. plantarum strongly inhibited S. aureus adherence to Caco-2 cells and their inhibition activity was significantly influenced by several factors that affect adhesion inhibition.