Survival of bifidobacteria administered to calves

Twenty-five bifidobacteria were isolated from feces of calves. Isolates were identified, and their functional properties and antimicrobial activity were determined. From 10 strains with suitable properties rifampicin-resistant mutants (RRBs) were prepared and mixture of RRBs was administered to 2-d-old calves. These strains were identified by sequencing as Bifidobacterium animalis ssp. animalis (6 strains), B. thermophilum (2 strains), B. choerinum (1 strain) and B. longum ssp. suis (1 strain). The control group was without probiotic treatment. Survival ability of administered bifidobacteria was monitored in fecal samples by cultivation on modified TPY agar supplemented with mupirocin, acetic acid, and rifampicin. Administered bifidobacteria survived in gastrointestinal tract of calves for at least 60 d. Other bacteria were also determined after cultivation using fluorescence in situ hybridization (FISH). Bifidobacteria and lactobacilli dominated in fecal microflora. Significantly lower amounts of E. coli and higher amounts of bifidobacteria and total anaerobes were found in the treated group relative to the control group.     

Distribution of bifidobacteria in the gastrointestinal tract of calves

Development of gastrointestinal microflora of calves with special reference to bifidobacteria was investigated; fecal bacteria were enumerated in calves aged 3 days to 7 weeks. Bacteria were detected by using selective media, bifidobacteria using modified TPY agar with an addition of mupirocin and acetic acid and by fluorescence in situ hybridization (FISH). Bifidobacteria were dominant group of fecal flora of calves after 7 d of life, constituting 10 % of total bacterial counts. The highest bacterial concentrations were observed in rumen, cecum, and colon, the lowest in abomasum and duodenum. Bifidobacteria and lactobacilli exhibited the highest survival ability during stomach passage and dominated in all parts of the digestive tract. Bifidobacteria counts determined by FISH were significantly higher than those provided by cultivation. Modified TPY agar was highly selective and suitable for bifidobacteria isolation but FISH was shown to be a more precise method for their enumeration. Our results show that gastrointestinal microflora of calves in the milk-feeding period is similar to breast-fed infants with respect to the occurrence of bifidobacteria as a dominant bacterial group. The use of Bifidobacterium strains offers a promising way for providing beneficial effectors for calves in the milk-feeding period.     

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.     

Optimization of culture conditions of probiotic bifidobacteria for maximal adhesion to hexadecane

Culture growth conditions were optimized for adhesion to hexadecane of the probiotic Bifidobacterium bifidum HI 39 and HI 48. Among three growth media used, MILS lactose broth was the best medium to obtain maximum cell adhesion, followed by MRS and TPY lactose broth for B. bifidum HI 39 and HI 48. Increasing the incubation time from 6 to 18 h resulted in a gradual increase in percentage adhesion at 37 °C of both organisms in MILS, MRS and TPY media. Thereafter, incubation up to 48 h showed a marked reduction in adhesion of B. bifidum HI 39 and B. bifidum HI 48. When the test cultures were grown at pH values from 5.0 to 8.0 in MILS lactose broth at 37 °C for 18 h, there was a gradual enhancement in cell adhesion up to pH 7.0; but higher pH values retarded the bacterial adhesion. The study showed that the optimum conditions for adhesion to hexadecane of the selected bifidobacterial strains were pH 7.0 and incubation at 37 °C for 18 h in MILS broth.     

Folate production by bifidobacteria as a potential probiotic property

The ability of 76 Bifidobacterium strains to produce folate was investigated. In order to evaluate folic acid productivity, bifidobacteria were cultivated in the folate-free semisynthetic medium SM7. Most of the tested strains needed folate for growth. The production and the extent of vitamin accumulation were not a function of species but were distinctive features of individual strains. Six strains among the 17 that grew without folate produced significantly higher concentrations of vitamin (between 41 and 82 ng ml(-1)). The effects of exogenous folate and p-aminobenzoic acid (PABA) concentrations on folate production were evaluated. In contrast to most of the other strains, the folate yield of B. adolescentis MB 239 was not negatively affected by either PABA or exogenous folic acid. Folate production by B. adolescentis MB 239 was studied in the pH range of the colonic environment, and a comparison of folate production on raffinose, lactose, and fructo-oligosaccharides, which belong to three important groups of fermentable intestinal carbon sources, was established. Differences in folate biosynthesis by B. adolescentis MB 239 were not observed as a function either of the pH or of the carbon source. Fecal culture experiments demonstrated that the addition of B. adolescentis MB 239 may increase the folate concentration in the colonic environment.     

Characterization of lactobacilli towards their use as probiotic adjuncts in poultry

AIMS: A total of 112 strains of lactic acid bacteria of duck origin were studied for their use as a probiotic feed supplement. METHODS AND RESULTS: In vitro studies included aggregation, co-aggregation, cell surface hydrophobicity and adhesion activities on poultry crop cells and human Hep2-cells. Additionally, growth with bile acids (chicken bile, ox gall and taurocholic acid) and tolerance to acidic pH were tested. Among all the isolates, two strains (Lactobacillus animalis TMW 1.972 and Lactobacillus salivarius TMW 1.992) were selected for a survival test in poultry. Monitoring and differentiation of these strains was achieved by selective detection as rifampicin and erythromycin double-resistant mutants. After a single feed administration, both micro-organisms were shown to persist in the crop and caecum of ducks for a period of 18 and 22 days, respectively. For identification of Lact. animalis and Lact. salivarius, two specific PCRs targeted against 16S rDNA were developed. CONCLUSIONS: Within the autochtoneous microflora of ducks, two strains of lactobacilli exhibited strong potential as probiotic adjuncts. The results indicate that the natural gut microflora of poultry serves as an excellent source for optimal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: A general strategy for the selection of probiotic strains is presented. The suggested sequence of tests allows identification of the most promising candidates within complex ecosystems or large strain collections with minimal expenditure.     

Adhesion of bifidobacteria to granular starch and its implications in probiotic technologies

Adhesion of 19 Bifidobacterium strains to native maize, potato, oat, and barley starch granules was examined to investigate links between adhesion and substrate utilization and to determine if adhesion to starch could be exploited in probiotic food technologies. Starch adhesion was not characteristic of all the bifidobacteria tested. Adherent bacteria bound similarly to the different types of starch, and the binding capacity of the starch (number of bacteria per gram) correlated to the surface area of the granules. Highly adherent strains were able to hydrolyze the granular starches, but not all amylolytic strains were adherent, indicating that starch adhesion is not a prerequisite for efficient substrate utilization for all bifidobacteria. Adhesion was mediated by a cell surface protein(s). For the model organisms tested (Bifidobacterium adolescentis VTT E-001561 and Bifidobacterium pseudolongum ATCC 25526), adhesion appeared to be specific for alpha-1,4-linked glucose sugars, since adhesion was inhibited by maltose, maltodextrin, amylose, and soluble starch but not by trehalose, cellobiose, or lactose. In an in vitro gastric model, adhesion was inhibited both by the action of protease and at pH values of < or =3. Adhesion was not affected by bile, but the binding capacity of the starch was reduced by exposure to pancreatin. It may be possible to exploit adhesion of probiotic bifidobacteria to starch granules in microencapsulation technology and for synbiotic food applications.     

Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens

AIM: To develop in vitro assays for comparing the antagonistic properties and anti-oxidative activity of probiotic Lactobacillus and Bifidobacterium strains against various entero- and urinary pathogens. METHODS AND RESULTS: The antagonistic activity of five probiotic lactobacilli (Lactobacillus rhamnosus GG, Lactobacillus fermentum ME-3, Lactobacillus acidophilus La5, Lactobacillus plantarum 299v and Lactobacillus paracasei 8700:2) and two bifidobacteria (Bifidobacterium lactis Bb12, Bifidobacterium longum 46) against six target pathogens was estimated using different assays (solid and liquid media, anaerobic and microaerobic cultivation) and ranked (low, intermediate and high). Bacterial fermentation products were determined by gas chromatography, and the total anti-oxidative activity of probiotics was measured using linolenic acid test. Pyelonephritic Escherichia coli was highly suppressed by GG and both bifidobacteria strains. Lactobacilli strains 8700:2, 299v and ME-3 were the most effective against Salmonella enterica ssp. enterica in microaerobic while ME-3 and both bifidobacteria expressed high activity against Shigella sonnei in anaerobic milieu. Lact. paracasei, Lact. rhamnosus and Lact. plantarum strains showed intermediate antagonistic activity against Helicobacter pylori under microaerobic conditions on solid media. The highest anti-oxidative activity was characteristic for Lact. fermentum ME-3 (P < 0.05). No efficient antagonist against Clostridium difficile was found. The positive correlations between the pH, lactic acid production and anti-microbial activity for all tested probiotics were assessed. CONCLUSIONS: Developed experimental assays enable to compare the anti-microbial and -oxidative activity of Lactobacillus and/or Bifidobacterium probiotics, which have been claimed to possess the ability of suppressing the growth of various enteric and urinary pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening Lactobacillus and Bifidobacterium sp. strains according to their activity in various environmental conditions could precede the clinical efficacy studies for adjunct treatment with probiotics in cure of different gastrointestinal and urinary tract infections.     

Susceptibility of bifidobacteria to lysozyme as a possible selection criterion for probiotic bifidobacterial strains

Resistance or susceptibility of bifidobacteria to lysozyme and growth of bifidobacteria in human milk were tested. Susceptible bifidobacterial strains stopped their growth almost immediately after the addition of lysozyme (400 μg/ml), moderately susceptible strains exhibited reduced growth rate, and growth curves of resistant strains were not affected. Strains of human origin were more resistant to lysozyme than animal strains. While strains of B. bifidum grew well in human milk samples, the growth B. animalis strains was inhibited after inoculation to human milk. The resistance to lysozyme seems to be a promising criterion for the selection of new probiotic bifidobacterial strains.     

Presence of bifidobacteria in the rumen of calves fed different rations.

A study was made on the numbers and species of bifidobacteria present in the rumen of calves fed high-roughage and high-concentrate diets. With the roughage ration the bifidobacteria were not detectable in a 10(-3) dilution, whereas with the concentrate ration their number was high, usually in the order of 10(8) to 10(9)/ml of rumen fluid. The species most represented, identified by means of deoxyribonucleic acid-deoxyribonucleic acid hybridization tests, included Bifidobacterium ruminale, Bifidobacterium globosum, and an apparently new species.     

Stationary-phase acid and heat treatments for improvement of the viability of probiotic lactobacilli and bifidobacteria

AIMS: To investigate whether sublethal treatments of stationary-phase probiotic cultures enhance their survival during lethal treatments and to adapt these treatments to the fermenter-scale production of probiotic cultures. METHODS AND RESULTS: Conditions for acid and heat pretreatments were screened for three Lactobacillus and two Bifidobacterium strains. Strains were sublethally treated both at laboratory scale and at fermenter scale in a strain-specific manner and exposed to a subsequent lethal treatment. At laboratory scale viability improvement was detected in each strain. However, improvement was more pronounced in the Lactobacillus than in the Bifidobacterium strains. At fermenter scale three strains were tested: for the two Lactobacillus strains a marked improvement in viability was obtained whereas for the Bifidobacterium strain the improvement was either minor or not detected. CONCLUSIONS: Development of treatments for viability enhancement of probiotic strains is feasible, but strain-specific optimization is necessary to obtain notable improvements. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain-specific treatments were developed for the viability enhancement of stationary-phase probiotic cells both at laboratory and fermenter scale. These results can be utilised in the production of probiotic cultures with improved viability.     

Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile

Bifidobacterium breve 46, Bifidobacterium lactis 8:8 and Bifidobacterium longum 6:18 and three reference strains B. breve CCUG 24611, B. lactis JCM 10602, and Bifidobacterium pseudocatenulatum JCM 1200 were examined for acid and bile tolerance, prebiotic utilization and antimicrobial activity against four Clostridium difficile (CD) strains including the hypervirulent strain, PCR ribotype NAP1/027. B. lactis 8:8 and B. lactis JCM 10602 exhibited a high tolerance in MRSC broth with pH 2.5 for 30 min. B. breve 46 and B. lactis 8:8 remained 100% viable in MRSC broth with 5% porcine bile after 4 h. All six strains showed a high prebiotic degrading ability (prebiotic score) with galactooligosaccharides (GOS), isomaltooligosaccharides (IMOS) and lactulose as carbon sources and moderate degradation of fructooligosaccharides (FOS). Xylooligosaccharides (XOS) was metabolized to a greater extent by B. lactis 8:8, B. lactis JCM 10602, B. pseudocatenulatum JCM 1200 and B. longum 6:18 (prebiotic score >50%). All strains exhibited extracellular antimicrobial activity (AMA) against four CD strains including the CD NAP1/027. AMA of B. breve 46, B. lactis 8:8 and B. lactis JCM 10602 strains was mainly ascribed to a combined action of organic acids and heat stable, protease sensitive antimicrobial peptides when cells were grown in MRSC broth with glucose and by acids when grown with five different prebiotic-non-digestible oligosaccharides (NDOs). None of C. difficile strains degraded five prebiotic-NDOs. Whole cells of B. breve 46 and B. lactis 8:8 and their supernatants inhibited the growth and toxin production of the CD NAP1/027 strain.     

Supercritical CO2 interpolymer complex encapsulation improves heat stability of probiotic bifidobacteria

The probiotic industry faces the challenge of retention of probiotic culture viability as numbers of these cells within their products inevitably decrease over time. In order to retain probiotic viability levels above the therapeutic minimum over the duration of the product’s shelf life, various methods have been employed, among which encapsulation has received much interest. In line with exploitation of encapsulation for protection of probiotics against adverse conditions, we have previously encapsulated bifidobacteria in poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex microparticles under supercritical conditions. The microparticles produced had suitable characteristics for food applications and also protected the bacteria in simulated gastrointestinal fluids. The current study reports on accelerated shelf life studies of PVP:PVAc-CA encapsulated Bifidobacterium lactis Bb12 and Bifidobacterium longum Bb46. Samples were stored as free powders in glass vials at 30 °C for 12 weeks and then analysed for viable counts and water activity levels weekly or fortnightly. Water activities of the samples were within the range of 0.25–0.43, with an average a _w  = 0.34, throughout the storage period. PVP:PVAc-CA interpolymer complex encapsulation retained viable levels above the recommended minimum for 10 and 12 weeks, for B. longum Bb46 and B. lactis Bb12, respectively, thereby extending their shelf lives under high storage temperature by between 4 and 7 weeks. These results reveal the possibility for manufacture of encapsulated probiotic powders with increased stability at ambient temperatures. This would potentially allow the supply of a stable probiotic formulation to impoverished communities without proper storage facilities recommended for most of the currently available commercial probiotic products.     

The evaluation of a mupirocin-based selective medium for the enumeration of bifidobacteria from probiotic animal feed

In this study, MRS medium supplemented with cysteine hydrochloride and mupirocin, termed Bifidobacterium selective medium (BSM) was found to be elective for bifidobacteria but inhibitory to a wide range of non-bifidobacteria strains commonly included in probiotic animal feed. Bacilli, lactobacilli, lactococci and streptococci failed to form colonies on BSM and enterococci, pediococci and propionibacteria formed colonies <0.5 mm in diameter. Bifidobacteria formed colonies >1 mm in size and could be readily distinguished. The addition of nystatin to BSM further inhibited Saccharomyces cerevisiae. BSM was successfully used to enumerate the bifidobacteria components, confirmed through fructose-6-phophate-phosphoketolase detection, present in two commercial probiotic feeds. The medium is recommended for the enumeration of bifidobacteria from animal feeds especially when not a numerically dominant component.     

Occurrence of bifidobacteria in faeces of calves fed milk or a combined diet

The development of faecal bacteria composition in calves fed milk or a combined diet was investigated from 4 to 21 days of age. On day 7, bifidobacteria in faeces of milk-fed calves already increased from about 7.6 to 9.2 log CFU/g and did not change until the end of the study, whereas in calves fed the combined diet bifidobacteria only moderately increased to 7.9 log CFU/g and decreased slowly until day 21. The counts of bifidobacteria in calves on a combined diet were significantly (p < 0.01) lower compared to those in milk-fed calves. Bifidobacterial counts determined by cultivation or by fluorescence in situ hybridisation (FISH) did not differ significantly. Our results showed that the occurrence of bifidobacteria in calf faeces is highly dependent on the diet composition. Faecal bacteria flora of calves fed exclusively by milk is rich in bifidobacteria, but in calves on a combined diet coliforms dominated.     

Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing

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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.     

Isolation, growth on prebiotics and probiotic potential of novel bifidobacteria from pigs

Bifidobacteria were isolated from the faeces of pigs of various ages and examined for their potential use as probiotics in combination with di- and oligosaccharides. Ninty-six per cent of the isolates were found to have characteristics in common with Bifidobacterium boum, B. thermophilum and B. choerinum. B. thermophilum was most commonly isolated from sows, whereas most of the other strains were isolated from piglets. A few strains of each species were able to grow in the presence of air. A microplate assay was modified to allow comparison of growth on different substrates. Di- and oligosaccharides considered to promote bifidobacteria were screened for their ability to support growth of selected isolates in vitro. Growth on these substrates varied within and between species. Of the fructose oligosaccharides tested, Actilight P supported the best growth of the widest range of strains. The strains which grew best on the disaccharide lactulose were related to B. choerinum and some of these strains grew on xylo-oligosaccharides. It seems that prebiotic di- and oligosaccharides may have both a species and intra-species/strain selective effect. B. choerinum appeared to be well adapted to the gut of pre-weaned piglets.     

Safety evaluation of probiotic bifidobacteria by analysis of mucin degradation activity and translocation ability

Although probiotic-containing nutrient formulas for infants and toddlers have become very popular, some adverse effects related to translocation of probiotic strains have been reported. We assessed the safety of probiotic bifidobacteria that have been used in clinical investigations and proven to have beneficial effects, by analyzing mucin degradation activity and translocation ability. Mucin degradation activities of three probiotic bifidobacteria strains; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63, were evaluated by three in vitro tests comprising growth in liquid medium, SDS-PAGE analysis of degraded mucin residues, and degradation assay in Petri dish. All test strains and control type strains failed to grow in the liquid medium containing mucin as the only carbon source, although good growth was obtained from fecal sample. In the SDS-PAGE analyses of mucin residues and observation of mucinolytic zone in agar plate, the three test strains also showed no mucin degradation activity as the type strains, although fecal sample yielded positive results. In another study, a high dose of B. longum BB536 was administered orally to conventional mice to examine the translocation ability. No translocation into blood, liver, spleen, kidney and mesenteric lymph nodes was observed and no disturbance of epithelial cells and mucosal layer in the ileum, cecum and colon was detected, indicating that the test strain had no translocation ability and induced no damage to intestinal surface. These results resolve the concern about bacterial translocation when using bifidobacteria strains as probiotics, which have been tested in various clinical trials, supporting the continuous use of these probiotic strains without anxiety.