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.     

Characterization of factors affecting attachment of Bifidobacterium species to amylomaize starch granules

AIMS: Two human-derived Bifidobacterium strains, PL1 and PL2, were tested for their ability to attach to amylomaize starch granules, and factors affecting binding were assessed. METHODS AND RESULTS: Good binding to granules was observed when the strains were grown on maltose or amylomaize starch, but not on glucose. Binding activity was localized to cell wall components and was sensitive to treatment with proteolytic enzymes. Several methodologies were employed to confirm these observations, including studies using radiolabelled cells, dot blot assays and scanning electron microscopy (SEM) analysis. CONCLUSION: Results from this study indicated that binding of strains PL1 and PL2 to amylomaize starch granules was mediated by a cell wall-associated proteinaceous factor that was induced when the strains were grown on starch or a related substrate, but not glucose. SIGNIFICANCE AND IMPACT OF THE STUDY: Attachment of probiotic strains to starch or other dietary fibres is believed to offer a selective advantage in the host intestine and may even prolong viability in adverse food environments. Therefore, characterizing the mechanisms of attachment has commercial implications in the design of synbiotic products.     

Role ofBifidobacterium longum in the induction of apoptotic deletion in the human enterocyte-like Caco-2 cell line

Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced byB. longum. This has been valuedin vitro, performing the incubation of threeB. longum strains with enterocyte-like Caco-2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells withB. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherentB. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues.     

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.     

Live/dead state is not the factor influencing adhesion ability of Bifidobacterium animalis KLDS2.0603

Two essential requirements for probiotic bifidobacteria are that they be “live” and have “colonization” ability, following FAO/WHO guideline recommendations. The amount of research on the adhesion ability of bifidobacteria compares poorly with that of other probiotic bacteria, such as lactobacilli. The aim of the present study was to determine how gastrointestinal conditions affect the adhesion ability of bifidobacteria, and to investigate the relationship between the adhesion ability and the live/dead state of bifidobacteria. The adhesion ability of Bifidobacterium animalis KLDS2.0603 that had been subjected to the digestive enzymes, pepsin, trypsin, and proteinase K, was decreased significantly, but these treatments did not significantly change the strain’s survival rates, which were 98.78%, 97.60%, and 97.63% respectively. B. animalis KLDS2.0603 subjected to LiCl retained its adhesion ability but had a lower survival rate (59.28%) than the control group (P<0.01). B. animalis KLDS 2.0603 subjected to sodium metaperiodate exhibited higher adhesion ability than the control group (P<0.01), but the bacterial cells were killed totally. The results of transmission electron microscopy and laser scanning confocal microscopy showed that live/dead state of bifidobacteria was not one of the main factors that affected the adhesion ability of bifidobacteira, and that the substances affecting the adhesion ability of bifidobacteria were on the outer surface layer of the bifidobacterial cells. Our results also indicated that the substances related to the adhesion ability of bifidobacteria are proteinaceous. The above results will help us to understand the adhesion and colonization processes of bifidobacteria in the human gastrointestinal tract.     

The protective effects of high amylose maize (amylomaize) starch granules on the survival of Bifidobacterium spp. in the mouse intestinal tract

The possibility of using high amylose maize starch granules as a delivery system for probiotic bacteria has been investigated using Bifidobacterium spp. LaftiTM 8B and LaftiTM 13B which were isolated from a healthy human. The Bifidobacterium cells were able to adhere to the amylomaize starch granules and were also able to hydrolyse the starch during growth. Initially, in vitro studies were carried out by studying the survival of strains Bifidobacterium LaftiTM 8B and LaftiTM 13B when exposed to pH 2.3, 3.5 and 6.5 as well as 0.03 and 0.05% w/v bile acids. Both strains were grown either in the absence or presence of high amylose maize starch granules, then mixed with the high amylose maize starch granules and exposed to acidic buffers or bile acid solutions. It was shown that growth in and the presence of high amylose maize starch granules led to enhanced survival of strains LaftiTM 8B and LaftiTM 13B. Subsequently, survival in vivo was monitored by measuring the faecal level of Bifidobacterium LaftiTM 8B after oral administration of the strain to mice. A sixfold better recovery of strain LaftiTM 8B from mice faeces after oral dosage was noted for cells grown in amylose-containing medium compared with controls. It was concluded that high amylose maize starch granules contributed to enhanced survival of Bifidobacterium sp. LaftiTM 8B and LaftiTM 13B.     

Effect of Encapsulation on Viability of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> CFR815j and Physiochemical Properties of Ice Cream

The health beneficial attributes of bifidobacteria and its safe association with the host gut has increased its significance as a probiotic. However delivering probiotic bifidobacteria with Minimum Biological Value (MBV) through product has always been a challenge. In the present study, an attempt was made to maintain the viability of native isolate of Bifidobacterium longum CFR 815j and deliver through ice-cream. B. longum CFR815j was microencapsulated in alginate starch capsules by emulsification followed by evaluation of bead stability in simulated gastrointestinal conditions. After incorporation in ice-cream, the effect on chemical properties, sensory parameters and meltdown characteristics of the product were also evaluated. Survival studies of B. longum revealed higher counts than 10⁷ in the product which is essential for probiotic bacteria to exhibit beneficial effect. Further, all the properties of this ice-cream were comparable to the regular ice-cream. Our studies conclude that encapsulation was able to maintain the requisite MBV of bifidobacteria in ice-cream without affecting the sensory characteristics.     

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.     

Evaluation of microencapsulation of a Bifidobacterium strain with starch as an approach to prolonging viability during storage

AIMS: To optimize a spray coating process for the production of encapsulated microspheres containing viable Bifidobacterium cells and to determine whether the readily gelatinized modified starch coating used in this study improved bacterial survival in foods or under acid conditions. METHODS AND RESULTS: An air inlet temperature of 100 degrees C was demonstrated to be optimal for the spray drying process, as it afforded good drying, low outlet temperatures (45 degrees C) and resulted in less than 1 log reduction in bifidobacteria numbers during drying. Maximum recovery yields of 30% were obtained after optimizing the air aspiration conditions. The average size of the Bifidobacterium PL1-containing starch microparticles was determined by scanning electron microscopy to be of the order of 5 microm. The starch-coated cells did not display any enhanced viability compared with free PL1 cells when exposed to acid conditions for 6 h or in two dry food preparations over 20 d storage at ambient temperature (19-24 degrees C). Determination of 1491 nucleotides of the 16S rRNA gene from PL1 indicated that it shared 97% homology with a previously sequenced Bifidobacterium ruminantium strain. CONCLUSIONS: Our data demonstrated that, although spray drying is a valuable process for encapsulating bifidobacteria, further work is required to ascertain a more appropriate coating material that will protect this strain against adverse environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of small, uniformly coated microspheres containing viable bifidobacteria using an affordable and industrially convenient process, such as spray drying, has commercial implications for the production of probiotic products. Although popular for use as a coating polymer by the food industry, this study indicated that modified starches might not be suitable for use as an encapsulating material for probiotic strains.     

Carbohydrate preference, acid tolerance and bile tolerance in five strains of Bifidobacterium

AIMS: To assess the suitability of bifidobacteria for inclusion in synbiotic products on the basis of carbohydrate preference, acid and bile tolerance. METHODS AND RESULTS: Five strains of Bifidobacterium were analysed for their carbohydrate preference from 12 substrates. Maximum growth rates were used to compare substrate preferences. Galacto-oligosaccharides and isomalto-oligosaccharides were well utilized by all the test species. Most bacteria tested could also utilize at least one type of fructan molecule. To determine transit tolerance of potentially probiotic bifidobacteria, acid and bile resistance was tested. A wide range acid resistance was found. Bile tolerance also varied. CONCLUSIONS: GOS and IMO were generally well utilized by the tested species. Other substrates were used to different degrees by the different species. Most bifidobacteria are poorly resistant to strongly acidic conditions with the exception of Bifidobacterium lactis Bb12. Bile tolerances were widely variable and it was shown that caution should be exercised when using colorimetric methods to assess bile tolerance. SIGNIFICANCE AND IMPACT OF STUDY: The study allows the comparison of the properties of bifidobacteria, allowing a cost effective screen for the best species for use in synbiotic products to allow better survival and efficacy.     

In vitro utilization of amylopectin and high-amylose maize (Amylomaize) starch granules by human colonic bacteria.

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M(r)) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.     

The mucus binding of Bifidobacterium lactis Bb12 is enhanced in the presence of Lactobacillus GG and Lact. delbrueckii subsp. bulgaricus

The ability to adhere to mucosal surfaces is related to many probiotic health effects. In the presence of Lactobacillus GG or Lact. bulgaricus, the adhesion of Bifidobacterium lactis Bb12 to a mucus model was more than doubled. Other tested lactobacilli did not affect the adhesion, nor was the adhesion of the lactobacilli influenced by the bifidobacteria. Co-aggregation between Bif. lactis Bb12 and the tested lactobacilli was insignificant and does not explain the observed effect. The results suggest that combinations of probiotics strains may have synergistic adhesion effects. Such specific strain combinations should also be assessed in clinical studies.     

The role of hemagglutination and effect of exopolysaccharide production on bifidobacteria adhesion to Caco-2 cells in vitro

It is believed that an important criterion for a potential probiotic strain is that it is capable of adhering to mucosal surfaces in the human gastrointestinal tract. The purpose of this study was to investigate a possible relationship between exopolysaccharide production and adhesion to Caco-2 cells by Bifidobacterium breve A28 and Bifidobacterium bifidum A10. In a preselection process, the hemagglutination abilities of these bacteria were determined prior to undertaking adhesion studies. B. breve A28, which produces large amounts of EPS (97.00 ± 2.00 mg/l) and has good hemagglutination abilities (+3) was found to adhere strongly to Caco-2 cells. Under gastrointestinal conditions, the high EPS producing- B. breve A28 was found to have better viability and adhesion to Caco-2 cells than the low EPS producing- B. bifidum A10. Also, B. breve A28 was found to be more effective at inhibiting Escherichia coli ATCC 11229 than B. bifidum A10. This investigation showed that high EPS production and adhesion ability may be important in the selection of bifidobacteria as probiotic strains.     

Inactivation of Adhesion and Invasion of Food-Borne Listeria monocytogenes by Bacteriocin-Producing Bifidobacterium Strains of Human Origin

Three bacteriocin-producing bifidobacterial isolates from newborns were identified as Bifidobacterium thermacidophilum (two strains) and B. thermophilum (one strain). This study was undertaken to evaluate the ability of these strains to compete with food-borne Listeria monocytogenes for adhesion and invasion sites on Caco-2 and HT-29 cells. The bifidobacteria adhered at levels ranging from 4% to 10% of the CFU added, but none of the bifidobacteria were able to invade cells. The abilities of Listeria to adhere to and to invade cells varied widely depending on the strain tested. Three groups of Listeria were identified based on invasiveness: weakly invasive, moderately invasive, and highly invasive strains. One strain from each group was tested in competition with bifidobacteria. B. thermacidophilum RBL70 was the most effective in blocking invasion of Listeria, and the decreases in invasion ranged from 38% to 90%. For all three bifidobacterial strains, contact between the cell monolayer and the bifidobacteria for 1 h before exposure to Listeria increased the degree of inhibition. Finally, visualization of competition for adhesion sites on cells by fluorescent in situ hybridization suggested that the two bacteria tended to adhere in close proximity.     

Cholesterol-lowering probiotics: in vitro selection and in vivo testing of bifidobacteria

Thirty-four strains of bifidobacteria belonging to Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium pseu-docatenulatum were assayed in vitro for the ability to assimilate cholesterol and for bile salt hydrolase (BSH) against glycocholic and taurodeoxycholic acids (GCA and TDCA). Cholesterol assimilation was peculiar characteristic of two strains belonging to the species B. bifidum (B. bifidum MB 107 and B. bifidum MB 109), which removed 81 and 50 mg of cholesterol per gram of biomass, being the median of specific cholesterol absorption by bifidobacteria 19 mg/g. Significant differences in BSH activities were not established among bifidobacterial species. However, the screening resulted in the selection of promising strains able to efficiently deconjugate GCA and TDCA. No relationship was recognized between BSH phenotype and the extent of cholesterol assimilation. On the basis of cholesterol assimilation or BSH_GCA and BSH_TDCA activities, B. bifidum MB 109 (DSMZ 23731), B. breve MB 113 (DSMZ 23732), and B. animalis subsp. lactis MB 2409 (DSMZ 23733) were combined in a probiotic mixture to be fed to hypercholesterolemic rats. The administration of this probiotic formulation resulted in a significant reduction of total cholesterol and low-density cholesterol (LDL-C), whereas it did not affect high-density cholesterol (HDL-C) and HDL-C/LDL-C ratio.     

Identification and quantification of Bifidobacterium species isolated from food with genus-specific 16S rRNA-targeted probes by colony hybridization and PCR.

A Bifidobacterium genus-specific target sequence in the V9 variable region of the 16S rRNA has been elaborated and was used to develop a hybridization probe. The specificity of this probe, named lm3 (5'-CGGGTGCTI*CCCACTTTCATG-3'), was used to identify all known type strains and distinguish them from other bacteria. All of the 30 type strains of Bifidobacterium which are available at the German culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen, 6 commercially available production strains, and 34 closely related relevant strains (as negative controls) were tested. All tested bifidobacteria showed distinct positive signals by colony hybridization, whereas all negative controls showed no distinct dots except Gardnerella vaginalis DSM4944 and Propionibacterium freudenreichii subsp. shermanii DSM4902, which gave slight signals. Furthermore, we established a method for isolation and identification of bifidobacteria from food by using a PCR assay without prior isolation of DNA but breaking the cells with proteinase K. By this method, all Bifidobacterium strains lead to a DNA product of the expected size. We also established a quick assay to quantitatively measure Bifidobacterium counts in food and feces by dilution plating and colony hybridization. We were able to demonstrate that 2.1 x 10(6) to 2.3 x 10(7) colonies/g of sour milk containing bifidobacteria hybridized with the specific nucleotide probe. With these two methods, genus-specific colony hybridization and genus-specific PCR, it is now possible to readily and accurately detect any bifidobacteria in food and fecal samples and to discriminate between them and members of other genera.     

Conjugated linoleic acid biosynthesis by human-derived Bifidobacterium species

AIMS: To assess strains of Lactobacillus, Lactococcus, Pediococcus and Bifidobacterium for their ability to produce the health-promoting fatty acid conjugated linoleic acid (CLA) from free linoleic acid. METHODS AND RESULTS: In this study, strains of Lactobacillus, Lactococcus, Pediococcus and Bifidobacterium were grown in medium containing free linoleic acid. Growth of the bacteria in linoleic acid and conversion of the linoleic acid to CLA was assessed. Of the bacteria assessed, nine strains of Bifidobacterium produced the c9, t11 CLA isomer from free linoleic acid. The t9, t11 CLA isomer was also produced by some strains, but at much lower concentrations. CONCLUSIONS: The production of CLA by bifidobacteria exhibited considerable interspecies variation. Bifidobacterium breve and B. dentium were the most efficient CLA producers among the range of strains tested, with B. breve converting up to 65% linoleic acid to c9, t11 CLA when grown in 0.55 mg ml(-1) linoleic acid. Strains also varied considerably with respect to their sensitivity to linoleic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of CLA by probiotic bifidobacteria offers a possible mechanism for some health-enhancing properties of bifidobacteria and provides novel opportunities for the development of functional foods.     

Applyed aspects of molecular biology of Bifidobacteria and Lactobacillus

The data obtained in the molecular genetic studies of the representatives of the genera Bifidobacterium and Lactobacillus are given. At present the genomes of Bifidobacterium longum, Lactobacillus acidophilus and Lactobacillus plantarum sized 2,256.640, 1,993.564 and 3,308.274 base pairs respectively, were completely sequenced. The use of different methods of gene indication and gene typing essentially widened the existing notions of the species variety of these bacteria, made it possible to change the classification of known species and to find out new ones, vegetating in different biotopes of the human body. Molecular genetic approaches also ascertained the taxonomic position of production strains of lactic-acid bacteria used in food industry, helped determine the kinetics of the passage of probiotic strains of bifidobacteria and lactobacilli along the gastrointestinal tract, as well as determine the duration of their preservation in the intestine after oral administration. In addition, the data on the evaluation of the therapeutic effect of some preparations, probiotics and synbiotics, with determination of the population level of endogenic and orally introduced exogenic bifidobacteria and lactobacilli are presented.