Probiotic characteristics and in vitro compatibility of a combination of Bifidobacterium breve M-16 V,Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536

The consumption of probiotic-based products has risen greatly in recent decades. Due to their probiotic characteristics, microorganisms such as lactobacilli and bifidobacteria are in daily use in the production of food supplements. In the present study, three bifidobacterial strains (Bifidobacterium breve M-16 V, Bifidobacterium longum subsp. infantis M-63 and Bifidobacterium longum subsp. longum BB536) were tested for growth compatibility, resistance to antimicrobial agents, antibacterial activity against pathogens, resistance to gastric acidity, bile salt hydrolysis and adhesion to the human intestinal epithelial cell line HT29. All of these strains were resistant to gentamycin, but none showed in vitro growth incompatibility or the presence of known resistance determinants. B. breve M-16 V had the best probiotic characteristics and, indeed, was the only strain possessing antibacterial activity against Escherichia coli and Klebsiella pneumoniae. All strains were resistant to simulated gastric juice, while only B. longum subsp. longum BB536 and B. breve M-16 V showed a bile salt hydrolytic activity. Interestingly, a strong adhesion to HT29 cells was observed in all Bifidobacterium strains. In conclusion, B. breve M-16 V, B. longum subsp. longum BB536 and B. longum subsp. infantis M-63 showed several promising characteristics as probiotic strains.     

婴幼儿粪便中双歧杆菌的分离及其菌株特性研究

为筛选具有潜在益生功能的双歧杆菌,本研究采用改良MRS培养基,从婴幼儿粪便中分离到5株菌株:AR499,AR667,AR668,AR669和AR610。通过16S r DNA测序分别鉴定为两歧双歧杆菌,短双歧杆菌,动物双歧杆菌,长双歧杆菌和假小链双歧杆菌。对其耐酸耐胆盐能力和黏附性能进行测试,结果表明,菌株AR499耐酸性较好,菌株AR610有较强的耐胆盐能力,菌株AR499的自动聚集能力和细胞表面疏水性都较高。实验表明,菌株AR499可作为一株耐受性和黏附性能较好的益生菌进一步深入研究,以期应用于优良双歧杆菌产品的开发。     

Growth-promoting effects of lactoferrin on L. acidophilus and Bifidobacterium spp.

We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.     

Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA

This study aimed at developing a novel multiplex polymerase chain reaction (PCR) primer set for identification of the potentially probiotic Bifidobacterium species B. adolescentis, B. animalis subsp. animalis (B. animalis), B. bifidum, B. breve, B. longum biovar infantis (B. infantis), B. animalis subsp. lactis B. lactis, B. longum biovar longum (B. longum) and B. pseudolongum. The primer set comprised specific and conserved primers and was derived from the integrated sequences of 16S and 23S rRNA genes and the rRNA intergenic spacer region (ISR) of each species. It could detect and identify type strains and isolates from pharmaceuticals or dairy products corresponding to the eight Bifidobacterium species with high specificity. It was also useful for screening of the related strains from natural sources such as the gastro-intestinal tract and feces. We suggest that the assay system from this study is an efficient tool for simple, rapid and reliable identification of Bifidobacterium species for which probiotic strains are known.     

Bifidobacterium microbiota and parameters of immune function in elderly subjects

Faecal and serum samples were collected over a period of 6 months from 55 institutionalized elderly subjects, who were enrolled in a double-blind placebo-controlled study. Participants were randomized in one of the three treatment groups: intervention (two probiotic Bifidobacterium longum strains: 2C and 46), placebo and commercial control (Bifidobacterium lactis Bb-12). The faecal Bifidobacterium microbiota was characterized by genus and species-specific PCR. Serum levels of the cytokines IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 were determined by enzyme-linked immunosorbent assay. Each participant harboured on average approximately three different bifidobacterial species. The most frequently detected species were B. longum, Bifidobacterium adolescentis and Bifidobacterium bifidum. Depending on the treatment, the intervention resulted in specific changes in the levels of certain Bifidobacterium species, and positive correlations were found between the different species. Negative correlations were observed between the levels of Bifidobacterium species and the pro-inflammatory cytokine TNF-alpha and the regulatory cytokine IL-10. The presence of faecal B. longum and Bifidobacterium animalis correlated with reduced serum IL-10. The anti-inflammatory TGF-beta1 levels were increased over time in all three groups, and the presence of Bifidobacterium breve correlated with higher serum TGF-beta1 levels. This indicates that modulation of the faecal Bifidobacterium microbiota may provide a means of influencing inflammatory responses.     

Bile salt toxicity to some bifidobacteria strains: role of conjugated bile salt hydrolase and pH

The purpose of this work was to study some aspects of bile salt toxicity towards bifidobacteria. A strain (Bifidobacterium coryneforme ATCC 25911) was selected for its lack of conjugated bile salt hydrolase activity (CBSH-), and was used with three deconjugating strains (CBSH+), for study of their growth and viability in the presence of two dihydroxylated conjugated bile salts (tauro- and glyco-deoxycholic acids). The presence of the glycoconjugate induced a more significant growth inhibition for the four strains than the tauroconjugate. The viability of the strains was measured at several pH levels. Glycodeoxycholic acid, but not taurodeoxycholic acid, exerted a lethal effect, which increased at low pH. This phenomenon was more pronounced for the CBSH- strain. We explain some of these results using an hypothesis based on the consequence of dissociation of conjugated and deconjugated bile salts, and the value of their pKa.     

Manufacturing of fermented goat milk with a mixed starter culture of Bifidobacterium animalis and Lactobacillus acidophilus in a controlled bioreactor

AIMS: This work was undertaken to study the feasibility and the characteristics of a fermented product made of goat milk, using a mixed starter culture of Bifidobacterium animalis and Lactobacillus acidophilus under controlled conditions, and to determine their survival in the fermented milk during refrigerated storage. METHODS AND RESULTS: Goat milk was inoculated with Lact. acidophilus and Bif. animalis mixed starter, fermented in a glass bioreactor with controlled temperature (37 degrees C) and anaerobiosis, and monitored for growth and acidification. The fermented milk was then stored for 10 days under refrigeration, and monitored daily for starter microflora survival and pH changes. Lact. acidophilus viable counts reached a maximum of 7.1 x 10(8) colony-forming units (CFU) ml(-1), and Bif. animalis a maximum of 6.3 x 10(7) CFU ml(-1) by 20 h of fermentation. During refrigerated storage, both strains exhibited a good survival, with viable numbers remaining essentially constant throughout the experiment, whereas the pH of the fermented milk dropped slightly. CONCLUSIONS: Mixed cultures of Bif. animalis and Lact. acidophilus may be used to produce fermented goat milk with high counts of both probiotic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Goat milk fermented with Bif. animalis and Lact. acidophilus can be manufactured as an alternative probiotic dairy product.     

Specific detection of bifidobacterium strains in a pharmaceutical probiotic product and in human feces by polymerase chain reaction

For PCR specific detection of the strains Bifidobacterium longum Y 10, B. infantis Y 1 and B. breve Y 8 used in a new probiotic product (VSL-3), strains-specific rDNA primers have been developed. Spacer regions between the 16S and 23S rRNA genes (ITS) of the three strains were amplified by PCR with conserved primers and the nucleotide sequence of these ITSs were determined. On the basis of their comparison with the rDNA sequences retrieved from GenBank, we designed new primers which specifically recognize the species B. breve and the two strains B. infantis Y 1 and B. breve Y 8. Specificity of these primers was confirmed through the analysis of 60 bifidobacteria strains belonging to the more representative human species. The feasibility of this PCR method was investigated in commercial VSL-3 product and fecal samples collected from 4 patients affected by inflammatory bowel deseases and two healthy subjects before and after the VSL-3 administration. By PCR analysis of different VSL-3 commercial batches we were successful in differentiating and quantifying the strains B. longum Y 10, B. infantis Y 1 and B. breve Y 8. B. infantis Y 1 and B. breve Y 8 could be detected at high concentration in fecal specimens of both patients and subjects treated with the probiotic preparation, showing a different colonization behaviour. Seven days after the VSL-3 treatment suspension, no patients and subjects harbored B. infantis Y 1 and B. breve Y 8, indicating a transient presence of these exogenous strains.     

A colony immunoblotting method for quantitative detection of a Bifidobacterium animalis probiotic strain in human faeces

A colony immunoblotting method has been developed to allow detection of the probiotic Bifidobacterium animalis strain DN-173 010 in human faecal samples. Rabbits were immunized with heat-killed DN-173 010 bacteria resulting in the production of an antiserum highly specific for bacteria belonging to Bif. animalis species. Of the 89 strains representative of 29 different bifidobacterial species tested, only the 15 strains of the Bif. animalis species could be detected with the antiserum. In Western immunoblotting the serum reacts with a protein of 45-kDa apparent molecular weight. None of the bacteria classically encountered in human faecal samples and able to grow on non-selective Columbia blood agar (enterobacteria, Bacteroides or Lactobacillus for instance) reacted with the antiserum. Taking advantage of the high specificity of the antiserum and of the absence of Bif. animalis bacteria in faeces samples of five human volunteers, we demonstrated that strain DN-173 010 survives the intestinal transit. Being based on a combination of semiselective cultivation and colony immunoblotting techniques, the method allowed detection of the Bif. animalis strain even when it represented only one thousandth of the total bifidobacterial population.     

Adaptation and response of Bifidobacterium animalis subsp. lactis to bile: a proteomic and physiological approach

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

Screening of species-specific lactic acid bacteria for veal calves multi-strain probiotic adjuncts

The selection of promising specific species of lactic acid bacteria with potential probiotic characteristics is of particular interest in producing multi species-specific probiotic adjuncts in veal calves rearing. The aim of the present work was to select and evaluate in?vitro the functional activity of lactic acid bacteria, Bifidobacterium longum and Bacillus coagulans strains isolated from veal calves in order to assess their potential use as multi species-specific probiotics for veal calves. For this purpose, bacterial strains isolated from faeces collected from 40 healthy 50-day-calves, were identified by RiboPrinter and 16s rRNA gene sequence. The most frequent strains belonged to the species B. longum, Streptococcus bovis, Lactobacillus animalis and Streptococcus macedonicus. Among these, 7 strains were chosen for testing their probiotic characteristics in?vitro. Three strains, namely L. animalis SB310, Lactobacillus paracasei subsp. paracasei SB137 and B. coagulans SB117 showed varying individual but promising capabilities to survive in the gastrointestinal tract, to adhere, to produce antimicrobial compounds. These three selected species-specific bacteria demonstrated in?vitro, both singularly and mixed, the functional properties needed for their use as potential probiotics in veal calves.     

蒙古族儿童源益生特性双歧杆菌的筛选及鉴定

【目的】双歧杆菌在人和动物胃肠道中发挥着重要的生理作用,然而双歧杆菌能否耐受胃酸、肠液及胆汁酸是影响活菌制剂益生效果的关键因素,本研究旨在从蒙古族儿童粪便中分离筛选出具有良好益生特性的双岐杆菌。【方法】本文采用双岐杆菌选择性培养基对样品进行分离纯化,并对菌株进行生理生化鉴定,以耐受人工胃肠液、耐受牛胆盐为手段对各菌株的益生特性进行评价,并且对B. animalisV9进行了16S rDNA分子生物学鉴定。【结果】本文从12位健康蒙古族儿童粪便中分离得到11株双歧杆菌,经传统生理生化试验鉴定为5株B. adolescentis：A1、H3、G4、A8、V10;3株B. longum：C6、C7、D11;1株B. pseudocatenlatum：B2;1株B. bifidum：G5;1株B. animalis：V9。B. animalis V9具有较强的耐酸性,在pH2.0的人工胃液中厌氧培养3h后存活率为92.4%,而其它10株双歧杆菌在此条件下的存活率均小于31.25%;B. animalis V9在pH2.0的人工胃液中厌氧培养3h后接入pH8.0的人工肠液中消化8h,存活率为99.7%,并且可以耐受0.3%的牛胆盐。进一步对V9菌株进行16S rDNA分子生物学鉴定,发现与Bifidobacterium animalis subsp. lactic BB12的同源性为99%。【结论】本研究结果显示B. animalis V9来源安全,并且具有良好耐酸、耐胆盐益生特性,有望在乳制品及保健类产品中得到广泛的应用。     

The viability of bifidobacteria introduced into kimchi

The viability of bifidobacteria in mul-kimchi, a type of kimchi with added water, was investigated under various conditions. When a mul-kimchi preparation was inoculated with five strains of Bifidobacterium at a concentration of 10(7) cfu ml-1, Bif. longum JK-2 showed the highest viability, maintaining a population of 10(6) cfu ml-1 after 1 week at 4 degrees C. The influence of NaCl concentration and initial pH on viability was further investigated in mul-kimchi inoculated with Bif. longum JK-2; NaCl concentrations greater than 3% (w/w) reduced viability considerably. In kimchi started with an initial pH of 6.5, the cells showed the highest survival. When mul-kimchi containing 2% NaCl (w/w) was inoculated with 10(8) cfu ml-1 Bif. longum JK-2, there was a 10-fold reduction in viability during 10 d of incubation at 4 degrees C. These results demonstrate acceptable levels of the organism in the product, suggesting the possible use of selected strains of bifidobacteria in commercial kimchi production.     

Acid-bile, antibiotic resistance and inhibitory properties of propionibacteria isolated from Turkish traditional home-made cheeses

In this study, a total of 29 Propionibacterium spp. were isolated from traditional home-made Turkish cheese samples. As a result of the identification, isolates were identified as Propionibacterium freudenreichii subsp. freudenreichii (15 strains), Propionibacterium jensenii (12), and Propionibacterium thoenii (2). All isolates and 5 reference strains were examined for their abilities to survive at pH 2.0, 3.0, 4.0, 5.0 and in the presence of 0.06, 0.15 and 0.30% bile salts, their influence on the growth of food-borne and spoilage bacteria, as well as their sensitivity against 11 selected antibiotics. Only seven propionibacteria strains survived in both the acidic and bile salt environments. Propionibacterium spp. strains strongly inhibited growth of the Escherichia coli ATCC 11229 and Shigella sonnei Mu:57 strains (91%). All propionibacteria strains were sensitive to a majority of the antibiotics used in the investigations. Overall, dairy propionibacteria showed high antibacterial activity, resistance to pH 4.0, 5.0, high resistance to bile salts and will provide an alternative source to Lactobacillus and Bifidobacterium as probiotic culture.     

Evaluation of the PCR method for identification of Bifidobacterium species

AIMS: Bifidobacterium species are known for their beneficial effects on health and their wide use as probiotics. Although various polymerase chain reaction (PCR) methods for the identification of Bifidobacterium species have been published, the reliability of these methods remains open to question. METHODS AND RESULTS: In this study, we evaluated 37 previously reported PCR primer sets designed to amplify 16S rDNA, 23S rDNA, intergenic spacer regions, or repetitive DNA sequences of various Bifidobacterium species. CONCLUSIONS: Ten of 37 experimental primer sets showed specificity for B. adolescentis, B. angulatum, B. pseudocatenulatum, B. breve, B. bifidum, B. longum, B. longum biovar infantis and B. dentium. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that published Bifidobacterium primer sets should be re-evaluated for both reproducibility and specificity for the identification of Bifidobacterium species using PCR. Improvement of existing PCR methods will be needed to facilitate identification of other Bifidobacterium strains, such as B. animalis, B. catenulatum, B. thermophilum and B. subtile.     

The F1F0-ATPase of Bifidobacterium animalis is involved in bile tolerance

Adaptation and tolerance to bile stress are important factors for the survival of bifidobacteria in the intestinal tract. Bifidobacterium animalis is a probiotic microorganism which has been largely applied in fermented dairy foods due to its technological properties and its health-promoting effects for humans. The effect of the presence of bile on the activity and expression of F₁F₀-ATPase, the pool of ATP and the intracellular pH of B. animalis IPLA 4549 and its mutant with acquired resistance to bile B. animalis 4549dOx was determined. The bile-resistant mutant tolerated the acid pH better than its parent strain. Bile induced the expression of the F₁F₀-ATPase and increased the membrane-bound H⁺-ATPase activity, in both parent and mutant strains. In acidic conditions (pH 5.0), the expression and the activity of this enzyme were higher in the mutant than in the parent strain when cells were grown in the absence of bile. Total ATP content was higher for the mutant in the absence of bile, whereas the presence of bile induced a decrease of intracellular ATP levels, which was much more pronounced for the parent strain. At pH 4.0, and independently on the presence or absence of bile, the mutant showed a higher intracellular pH than its parent strain. These findings suggest that the bile-adapted B. animalis strain is able to tolerate bile by increasing the intracellular ATP reserve, and by inducing proton pumping by the F₁F₀-ATPase, therefore tightly regulating the internal pH, and provide a link between the physiological state of the cell and the response to bile.     

Cell envelope changes in Bifidobacterium animalis ssp. lactis as a response to bile

Bifidobacterium animalis ssp. lactis is a probiotic frequently used as adjunct culture in fermented dairy products. In order to ensure its proper function at the intestinal level, this bacterium has to be tolerant to physiological concentrations of bile. This study examined the influence of bile on the fatty acid composition and the membrane characteristics of B. animalis IPLA 4549 and its mutant with acquired resistance to bile, B. animalis 4549dOx. Bile adaptation triggers in B. animalis 4549dOx a decrease in membrane fluidity and in the protein : phospholipid ratio, as well as a shift in the fatty acid composition of the cell. Remarkably, the presence of bile in the growth medium induced similar changes in both B. animalis cells. Furthermore, transmission electron microscopy analysis showed that bile promotes a severe distortion of the cell surface. This study provides new insights of the action of bile on the cell envelope of bifidobacteria.     

Adhesion of Bifidobacterium spp. to human intestinal mucus

Twenty-four Bifidobacterium strains were examined for their ability to bind to immobilized human and bovine intestinal mucus glycoproteins. Each of the tested bacteria exhibited its characteristic adhesion to human and bovine fecal mucus. No significant differences were found among the taxonomic species. Among the tested bacteria, B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. infantis, B. longum and B. pseudocatenulatum adhered to human fecal mucus better than bovine fecal mucus, while the binding of B. animalis and B. lactis was not preferential. These results suggest that the mucosal adhesive properties of bifidobacteria may be a strain dependent feature, and the mucosal binding of the human bifidobacteria may be more host specific.