Effect ofKluyveromyces marxianus on the growth and survival of bifidobacteria in milk

Four strains of bifidobacteria (B. bifidum 93,B. infantis ATCC 17 930,B. longum ATCC 15 707, andB. longum JR) cultivated in MRS broth modified by the addition of cysteine-hydrochloride (0.05 %) were serially subcultured in unsupplemented cow milk alone or in combination withKluyveromyces marxianus var.marxianus 269 under aerobic conditions. In monoculture, bifidobacteria did not multiply after the second subculture. In contrast, in coculture with yeast bifidobacteria reached counts about 8 log CFU/g even during 15 subcultures. In addition,K. marxianus var.marxianus 269 significantly prolonged the survival of bifidobacteria in milk at 4 °C. The most susceptible strains (B. longum Jr andB. infantis ATCC 17 930) completely lost their viability within 5 and 12 d of storage, respectively, while in coculture with yeasts all bifidobacteria cultures tested survived for at least 40 d. The results could be useful in producing kefir-like fermented milks containing bifidobacteria. The study was supported by grant no. 238/10/12596/0 of theInternal Grant Agency of Czech University of Agriculture ion Prague.     

Measurement of growth of bifidobacteria on inulofructosaccharides

A simple method for continuous measurement of growth of bifidobacteria is described. An inoculated broth in a small tube was evacuated at 100mmHg through a two-way stopcock and incubated at 37°C. Growth was periodically measured by inserting the tube into a spectrophotometer. Doubling times of three bifidobacterium strains in broth containing various inulofructosaccharide preparations were compared. Inulofructosacchandes from the Jerusalem artichoke tubers supported good growth of these bacteria.     

Human milk provides peptides highly stimulating the growth of bifidobacteria.

The large intestine of breast-fed infants is colonized predominantly by bifidobacteria, which have a protective effect against acute diarrhea. In this study we report for the first time the identification of human milk peptides that selectively stimulate the growth of bifidobacteria. Several bifidogenic peptides were purified chromatographically from pepsin-treated human milk and identified as proteolytically generated fragments from the secretory component of the soluble polyimmunoglobulin receptor and lactoferrin; both of these proteins exhibit antimicrobial effects. Hydrolysis of the identified peptides with the gastrointestinal proteases pepsin, trypsin and chymotrypsin did not lead to the loss of bifidogenic activity, indicating their potential function in vivo. Sequential comparison revealed a similar structural motif within the identified peptides. A correspondingly designed small peptide (prebiotic lactoferrin-derived peptide-I, PRELP-I) was found to stimulate the growth of bifidobacteria as effectively as the native peptides. The combination of antimicrobial and bifidobacterial growth stimulatory activity in human milk proteins leads to highly specific compounds capable of regulating the microbial composition of infants' large intestine.     

Effect of bifidobacteria on nitrites and nitrosamines

The effects of six different bifidobacteria strains were studied on two procarcinogens: nitrite and nitrosamines. Growth of bifidobacteria was not affected by nitrite concentrations below 50 μmol 1^-1. At nitrite concentrations greater than 2000 μmol 1^-1, total growth inhibition was observed. Nitrite elimination by a non-enzymic mechanism was noted for six strains of bifidobacteria. Acids produced by the bacteria seem to be involved in nitrite elimination. Nitrosamines tested had no effect on growth of bifidobacteria. Only one strain ( Bifidobacterium longum BB 536) was able to metabolize nitrosamines by an intracellular mechanism.     

Regulatory effects of bifidobacteria on the growth of other colonic bacteria

In the human large intestine bifidobacteria are a numerically important group of micro-organisms which are considered to exert a range of biological activities related to host health. One aspect is the inhibitory effect of these bacteria on other species, possibly excluding long term colonization by invasive pathogens. It has been suggested that the mechanism of inhibition carried out by bifidobacteria is related to the fermentative production of acids such as acetate and lactate. Experiments reported in this paper attemptedto address this theory. Co-culture experiments whereby Bifidobacterium infantis was incubated with Escherichia coli and Clostridium perfringens, in a varietyof fermentation systems, indicated that the bifidobacterium was able to exert an inhibitory effect not necessarily related to acid production. Further studies showed that eight species of bifidobacteria could variously excrete an anti-microbial substance with a broadspectrum of activity. Species belonging to the genera Salmonella, Listeria, Campylobacter and Shigella, as well as Vibrio cholerae, were all affected. These results show that bifidobacteria are able to exert more than one mechanism of inhibition, which may be of some importance with regard to protection against gastroenteritis.     

Specific growth rate of bifidobacteria cultured on different sugars

The ability of six bifidobacterial strains (3 of human origin and 3 isolates from fermented milk products) to utilize glucose, lactose, melezitose, sucrose, raffinose, and stachyose was determined. Dairy-related bifidobacterial strains were identified asBifidobacterium animalis (2 strains) or asB. pseudolongum (1 strain). Human strains includedB. longum (2 strains) andB. breve (1 strain). All strains fermented lactose, sucrose, raffinose, and stachyose. Melezitose was utilized only byB. longum. B. pseudolongum did not ferment either glucose or melezitose. All isolates had a higher specific growth rate on raffinose and stachyose than on glucose. Dairy strains grew slowly on glucose compared to human strains.     

Inter-species differences in the growth of bifidobacteria cultured on human milk oligosaccharides

Human milk (HM) contains as the third most abundant component around 200 different structures of human milk oligosaccharides (HMOs). HMOs are the first and irreplaceable prebiotics for infants, supporting bifidobacteria as the most important bacterial group in an infant intestine. The aim of our study was to test the growth of bifidobacteria in HM and on HMOs. Bifidobacteria were isolated from two groups of infants. The first one (eight strains) were isolated from infants who had bifidobacteria in their feces but, after a short period of time (4 to 24 days), bifidobacteria were no longer detected in their feces (disappeared bifidobacteria [DB]). The second group of bifidobacteria (eight strains) originated from infants with continual presence of bifidobacteria in their feces (persistent bifidobacteria [PB]). There were significant differences (p?Bifidobacterium bifidum and B. breve species were able to utilize HMOs, while B. adolescentis and B. longum subsp. longum species did not. The ability to grow in HM and to utilize HMOs seem to be important properties of bifidobacteria which are able to colonize infant intestinal tract.     

Patterns of growth and beta-galactosidase production by bifidobacteria

We investigated the patterns of growth and beta-galactosidase formation in the strains Bifidobacterium adolescentis GO-13, MS-42, 91-BIM, and 94-BIM, and B. bifidum No. 1, LVA-3, 791 on media with various carbon sources. The synthesis of beta-galactosidase was shown to be associated with exponential growth of the cultures involved. The maximum specific rate of beta-galactosidase synthesis of 0.20 U mg(-1) h(-1) was observed in B. bifidum LVA-3 after 3-6 h of cultivation. This value for B. adolescentis 91-BIM and 94-BIM was lower and amounted to 0.03-0.08 U mg(-1) h(-1). On the medium with lactose, the highest specific growth rates for B. bifidum LVA-3 and B. bifidum No.1 were 0.38 and 0.60 h(-1), respectively, after 3-6 h of cultivation. For B. adolescentis 91-BIM and 94-BIM, this parameter peaked at 12-15 h of cultivation at 0.13 and 0.22 h(-1), respectively. The hydrolytic activity of beta-galactosidase in the growth medium decreased during the stationary growth phase of the tested cultures.     

Effect of bile on the lipid composition and surface properties of bifidobacteria

AIM: The changes produced on the bacterial surface of Bifidobacteria cells when they are grown in bile were compared with those provoked by bile added to bacteria grown in the absence of bile. METHODS AND RESULTS: The adhesive properties, the zeta potential and the lipid composition of Bifidobacterial strains, isolated from human faeces and grown in MRS medium, were determined. Bacteria grown in MRS with bile showed a loss of adherence and autoaggregation in correlation with a decrease in the surface hydrophobicity in comparison to those grown in MRS without bile, concomitant with the absence of two glycolipids, the increase of sugar content and minor changes in fatty acid composition. The surface changes caused by bile shock on bacteria grown in bile-free medium were much less pronounced and, in addition, no effect on the lipid composition was apparent. CONCLUSIONS: The comparison of the results indicates that bile action on surface properties is related to metabolic changes. SIGNIFICANCE AND IMPACT OF THE STUDY: Long-term exposure of bacteria to bile may cause metabolic changes affecting their adhesive properties irreversibly. This may be taken as a criterion to define the probiotic properties of different strains.     

Effect of skim milk-alginate beads on survival rate of bifidobacteria

In this study, an attempt was made to increase the survival rate of bifidobacteria entrapped in alginate in the gastrointestinal tract, and to investigate the potential industrial applications, for example lyophilized capsules and yogurt. First, the protective effect of various food additives on bifidobacterial survivability was determined after exposure to simulated gastric juices and bile salts. The additives used in this study were skim milk (SM), poly dextrose (PD), soy fiber (SF), yeast extract (YE), chitosan (CS), κ-carageenan (κ-C) and whey, which were added at 0.6% concentration (w/v) to 3% alginate-bifidobacterial solution. In the simulated gastric juices and bile salts, the protective effect of 0.6% skim milk-3% alginate (SM-A) beads on the survival rate of bifidobacteria proved to be higher than the other additives. Second, the hydrogen ion permeation was detected through SM-A vessel without bifidobacterial cells at different SM concentrations (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%). There were no differences in terms of the pH decrease in SM-A vessels at 0.6%, 0.8%, and 1.0% (w/v) SM concentrations. The survival rate of bifidobacteria in SM-A beads would appear to be related to the SM buffering capacity against hydrogen ions and its tendency to reduce the pore size of bead. In this experiment, the survival rate of bifidobacteria entrapped in beads containing 0.6% SM showed the highest viability after exposure to simulated gastric juices for 3 h, thereby indicating that 0.6% SM is the optimum concentration for 3% alginate bead preparation. Third, the effect of SM-A beads on the freeze-drying and yogurt storage for 10 days was investigated. SM-A beads were found to be more efficient for freeze drying and yogurt storage than untrapped cells and the alginate bead. Consequently, the survival rate of bifidobacteria entrapped in SM-A beads was increased in simulated gastric juices, bile salts and probiotic products such as lyophilized capsules and yogurt, SM-A beads can be expected to produce high value probiotic products.     

Summer Meeting 2013: growth and physiology of bifidobacteria

Bifidobacteria are a minor fraction of the human colon microbiota with interesting properties for carbohydrate degradation. Monosaccharides such as glucose and fructose are degraded through the bifid shunt, a dedicated pathway involving phosphoketolase activity. Its stoechiometry learns that three moles of acetate and two moles of lactate are produced per two moles of glucose or fructose that are degraded. However, deviations from this 3 : 2 ratio occur, depending on the rate of substrate consumption. Slower growth rates favour the production of acetate and pyruvate catabolites (such as formate) at the cost of lactate. Interestingly, bifidobacteria are capable to degrade inulin‐type fructans (ITF) (oligofructose and inulin) and arabinoxylan‐oligosaccharides (AXOS). Beta‐fructofuranosidase activity enables bifidobacteria to degrade ITF. However, this property is strain‐dependent. Some strains consume both fructose and oligofructose, with different preferences and degradation rates. Small oligosaccharides (degree of polymerization or DP of 2–7) are taken up, in a sequential order, indicating intracellular degradation and as such giving these bacteria a competitive advantage towards other inulin‐type fructan degraders such as lactobacilli, bacteroides and roseburias. Other strains consume long fractions of oligofructose and inulin. Exceptionally, oligosaccharides with a DP of up to 20 (long‐chain inulin) are consumed by specific strains. Also, the degradation of AXOS by α‐arabinofuranosidase and β‐xylosidase is strain‐dependent. Particular strains consume the arabinose substituents, whether or not together with a consumption of the xylose backbones of AXOS, either up to xylotetraose or higher and either extra‐ or intracellularly. The production of high amounts of acetate that accompanies inulin‐type fructan degradation by bifidobacteria cross‐feeds other colon bacteria involved in the production of butyrate. However, bifidobacterial strain‐dependent differences in prebiotic degradation indicate the existence of niche‐specific adaptations and hence mechanisms to avoid competition among each other and to favour coexistence with other colon bacteria.     

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.     

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.     

Mechanism of growth inhibition by free bile acids in lactobacilli and bifidobacteria

The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated DeltapH. The bile acid concentrations that dissipated DeltapH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (DeltaPsi). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated DeltapH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4x MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8 x MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the DeltapH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2x MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane DeltapH and DeltaPsi dissipation), potassium ion, and other cellular components and eventually cell death.