Comparison of Fructooligosaccharide Utilization by Lactobacillus and Bacteroides Species

The utilization of 1-kestose (GF₂) and nystose (GF₃), the main components of fructooligosaccharides (FOS), by Lactobacillus and Bacteroides species was examined. Of seven Lactobacillus and five Bacteroides strains that utilized FOS, L. salivarius, L. rhamnosus, L. casei, and L. gasseri utilized only GF₂, whereas L. acidophilus and all the Bacteroides strains utilized both GF₂ and GF₃. Only the strains able to utilize both GF₂ and GF₃ had β-fructosidase activity in the culture supernatants. The culture supernatants of the Lactobacillus strains had higher β-fructosidase activity for GF₂ than for GF₃, whereas those of the Bacteroides strains had higher activity for GF₃ than for GF₂. Furthermore, β-fructosidase activity of the culture supernatants of the Lactobacillus cells grown in the GF₃ medium was much higher than that of the cells grown in the GF₂ medium, whereas the activity of the culture supernatants of the Bacteroides cells grown in the GF₃ medium was almost the same as that of the cells grown in the GF₂ medium. These results indicate that Lactobacillus species metabolize FOS in a different way from that of Bacteroides species.     

Metabolism of fructooligosaccharides by Lactobacillus paracasei 1195

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [(3)H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) alpha-1,2 linked to two, three, or four fructose (F) units (GF(2), GF(3), and GF(4), respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF(2) and GF(3) was rapid, whereas little GF(4) uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.     

Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight inulin and inulin oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with beta-(2-->1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10(7)) with beta-(2-->1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Deconjugation of bile salts by Bacteroids and Clostridium

Deconjugation of bile salts by four strains of Bacteroides and four strains of Clostridium was studied by use of resting cells and cell-free culture supernatants. Bacteroids strains yielded active cells but showed relatively low bile salt hydrolase (BSH) activity in the culture supernatants while the reverse was the case for the spore-forming clostridial strains. BSH was formed constitutively and was oxygen insensitive. The optimum pH was between 4.5 and 5.0. Marked substrate specificity was found in two strains, one Clostridium and one Bacteroides, which showed restricted activity against taurine conjugates. Bacteroides in general attacked the taurine conjugates of dihydroxy bile acids more readily than the trihydroxy taurine conjugates. Deconjugated bile acid moieties were further modified by some resting cells, depending on the bacterial strain while no enzymatic activity other than that of BSH was found in the culture supernatants. Cells of B. fragilis 2536 performed 7 alpha-dehydrogenation when the pH of the medium allowed the reaction, and this oxidative process was markedly enhanced in the presence of an abundant supply of oxygen as a terminal electron acceptor. C. perfringens PB 6K produced the 3- keto product in addition to the 3 beta-hydroxy derivative of the liberated bile acids and the formation of the latter derivative seemed to take place without preliminary deconjugation.     

Characterization of a Novel Fructosyltransferase from Lactobacillus reuteri That Synthesizes High-Molecular-Weight Inulin and Inulin Oligosaccharides

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with β-(2→1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10⁷) with β-(2→1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.     

Fructooligosaccharides metabolism and effect on bacteriocin production in Lactobacillus strains isolated from ensiled corn and molasses

Fructo- (FOS) and galacto-oligosaccharides have been used to promote the growth of probiotics, mainly those from Lactobacillus genus. However, only few reports have evaluated the effect of prebiotics on bacteriocins activity and production. In this work, we characterized the effect of FOS supplementation on the growth, lactic and acetic acids production, and antimicrobial activity of crude extracts obtained from Lactobacillus strains isolated from ensiled corn and molasses. Seven out of 28 isolated Lactobacillus, belonging to Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus brevis, showed antimicrobial activity against Listeria innocua. Among them, the strain L. plantarum LE5 showed antimicrobial activity against Listeria monocytogenes and Enteroccocus faecalis; while the L. plantarum LE27 strain showed antimicrobial effect against L. monocytogenes, E. faecalis, Escherichia coli and Salmonella enteritidis. This antimicrobial activity in most of the cases was obtained only after FOS supplementation. In summary, these results show the feasibility to increase the antimicrobial activity of Lactobacillus bacteriocins by supplementing the growth medium with FOS.     

Lactic acid bacteria fermentation of human milk oligosaccharide components, human milk oligosaccharides and galactooligosaccharides

Human milk contains about 7% lactose and 1% human milk oligosaccharides (HMOs) consisting of lactose with linked fucose, N-acetylglucosamine and sialic acid. In infant formula, galactooligosaccharides (GOSs) are added to replace HMOs. This study investigated the ability of six strains of lactic acid bacteria (LAB), Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Streptococcus thermophilus and Leuconostoc mesenteroides subsp. cremoris, to digest HMO components, defined HMOs, and GOSs. All strains grew on lactose and glucose. N-acetylglucosamine utilization varied between strains and was maximal in L. plantarum; fucose utilization was low or absent in all strains. Both hetero- and homofermentative LAB utilized N-acetylglucosamine via the Embden-Meyerhof pathway. Lactobacillus acidophilus and L. plantarum were the most versatile in hydrolysing pNP analogues and the only strains releasing mono- and disaccharides from defined HMOs. Whole cells of all six LAB hydrolysed oNP-galactoside and pNP-galactoside indicating β-galactosidase activity. High β-galactosidase activity of L. reuteri, L. fermentum, S. thermophilus and L. mesenteroides subsp. cremoris whole cells correlated to lactose and GOS hydrolysis. Hydrolysis of lactose and GOSs by heterologously expressed β-galactosidases confirmed that LAB β-galactosidases are involved in GOS digestion. In summary, the strains of LAB used were not capable of utilizing complex HMOs but metabolized HMO components and GOSs.     

Metabolism of Fructooligosaccharides by Lactobacillus paracasei 1195

Fermentation of fructooligosaccharides (FOS) and other oligosaccharides has been suggested to be an important property for the selection of bacterial strains used as probiotics. However, little information is available on FOS transport and metabolism by lactic acid bacteria and other probiotic bacteria. The objectives of this research were to identify and characterize the FOS transport system of Lactobacillus paracasei 1195. Radiolabeled FOS was synthesized enzymatically from [³H]sucrose and purified by column and thin-layer chromatography, yielding three main products: glucose (G) α-1,2 linked to two, three, or four fructose (F) units (GF₂, GF₃, and GF₄, respectively). FOS hydrolysis activity was detected only in cell extracts prepared from FOS- or sucrose-grown cells and was absent in cell supernatants, indicating that transport must precede hydrolysis. FOS transport assays revealed that the uptake of GF₂ and GF₃ was rapid, whereas little GF₄ uptake occurred. Competition experiments showed that glucose, fructose, and sucrose reduced FOS uptake but that other mono-, di-, and trisaccharides were less inhibitory. When cells were treated with sodium fluoride, iodoacetic acid, or other metabolic inhibitors, FOS transport rates were reduced by up to 60%; however, ionophores that abolished the proton motive force only slightly decreased FOS transport. In contrast, uptake was inhibited by ortho-vanadate, an inhibitor of ATP-binding cassette transport systems. De-energized cells had low intracellular ATP concentrations and had a reduced capacity to accumulate FOS. These results suggest that FOS transport in L. paracasei 1195 is mediated by an ATP-dependent transport system having specificity for a narrow range of substrates.     

Prebiotics enhance survival and prolong the retention period of specific probiotic inocula in an in vivo murine model

AIM: To identify novel prebiotics that could be used to maintain persistence of three representative probiotic strains in vivo. METHODS AND RESULTS: Test mice were treated with prebiotics soybean oligosaccharide (SOS), fructooligosaccharide (FOS) or inulin, followed by probiotics Lactobacillus acidophilus LAFTI L10 (L10), Bifidobacterium lactis LAFTI B94 (B94) or Lactobacillus casei L26 LAFTI (L26). Faecal samples were then collected and analysed using selective medium and PCR analysis to determine the presence of the probiotic strains. In contrast to the control groups, in mice fed prebiotics, the survival and retention time of the test probiotics was increased extensively. SOS and FOS prolonged the retention period of L10 from 24 to 30 h. Of the three prebiotics, FOS gave the best result with B94, prolonging the retention period from 3 to > or =10 days. Of the three prebiotics, inulin gave the best result for L26, prolonging the retention period from 2 to > or =6 days. CONCLUSIONS: The prebiotics SOS, FOS and inulin significantly enhance survival and prolong the retention period of L10, B94 and L26 in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results demonstrate the potential use of FOS, inulin and SOS as prebiotics in conjunction with the probiotic strains L10, B94 and L26 for new synbiotic products.     

Inhibitory activity of vaginal Lactobacillus bacteria on yeasts causing vulvovaginal candidiasis

Growing frequency of therapeutical failures of vulvovaginal candidiasis, resulting from resistance of certain species of Candida to imidazole agents, raises interest in the use of probiotics from Lactobacillus genera as prophylaxis. Unfortunately, little is known about inhibitory mechanisms of Lactobacillus on Candida. The aim of this study was to compare the activity of selected Lactobacillus species, representing the physiological vaginal flora, against Candida as well as investigation whether their inhibitory activity against Candida is related strictly to hydrogen peroxide and lactic acid production. 125 strains from vaginal smears of healthy women were classified by making use of phenotypic and genotypic methods. The majority of strains belonged to L. acidophilus: L. acidophilus sensu stricto, L. crispatus, L. gasseri and L. johnsonii as well as L. fermentum and L. plantarum species. Culture supernatants of selected 25 strains representing the isolated species were examined for their inhibitory activity against the growth of Candida albicans and C. glabrata. The results showed that the strongest and the fastest activity against C. albicans was demonstrated by L. delbrueckii strains, producing the largest quantities of hydrogen peroxide. On the other hand, extended activity, demonstrable after 24 hours, was shown by non-H2O2 producing L. plantarum supernatants. Growth of C. glabrata was not inhibited by any of the examined strains of Lactobacillus. Comparison of activity of live active cultures of Lactobacillus strains and their mixtures with this of pure H2O2 and lactic acid has shown that pure chemical compounds were less active than the cultures. This suggests that mixtures of Lactobacillus strains are in cooperation with each other using many different metabolites.     

Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins

Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and β-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly β-casein, while degradation of α_s-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.     

Hydrogen peroxide produced by Lactobacillus species as a regulatory molecule for vaginal microflora

A total of 33 strains of Lactobacillus belonging to 9 species, isolated from vagina, were tested for production of hydrogen peroxide. We observed that the following species: L. delbrueckii, L. acidophilus, L. crispatus, L. johnsonii and L. gasseri dominated over other species in secretion of hydrogen peroxide to the growth medium. Concentration of this substance amounted from 0.05 to 1.06 mM (in case of strong aeration the concentration increased up to 1.8 mM). Moreover, killing properties of the pure hydrogen peroxide exerted toward Escherichia coli and Candida albicans were less prominent than these of the supernatants of cultures of Lactobacillus strains producing H2O2.     

Protoheme,a dispensable growth factor forBacteroides fragilis grown by batch and continuous culture in a basal medium

The growth yields of 10 strains ofBacteroides fragilis isolated from a variety of clinical sites were determined in (a) basal medium, (b) basal medium plus heme, and (c) basal medium plus heme and menadione. The molar growth yield values, expressed as a function of glucose (Y_G) and ATP produced (Y_ATP) for 24 h and 48 h were used for a comparison of different strains. Considerable variation occurred among strains, but in general only the results from 24-h grown cells were reproducible. After this period, the microscopic appearance of cells changed dramatically from well-formed, intact cells to large collections of extracellular vesicles and lysed cells. All strains were stimulated by heme, but marked differences occurred among strains. The addition of heme and menadione to the basal medium increased the Y_G values of some strains, whereas others were unaffected. Heme-cultured cells produced acetate, propionate, and succinate as major metabolic end products and possessed cytochrome b, menaquinone-10, and fumarate reductase activity. Strain NCTC 9343 grown without added heme by continuous culture or batch culture produced cells that were morphologically and biochemically similar. Under both conditions these cells lacked cytochromes, menaquinones, and fumarate reductase activity, but produced high levels of lactate and fumarate together with lower levels of acetate, propionate, and succinate.     

Selected prebiotics support the growth of probiotic mono-cultures in vitro

The aim of the present study was to identify suitable prebiotics for synergistic combinations with representative probiotics from the lactobacilli and bifidobacterium genera. Eleven different types of commercially available carbohydrates and compounds were screened in basal media for their effects on the growth of the probiotic cultures Lactobacillus acidophilus LAFTI L10 (L10), Bifidobacterium animalis lactis LAFTI B94 (B94) and Lactobacillus casei LAFTI L26 (L26). Growth of these probiotics was investigated by measuring optical density (OD) levels over a period of 48 h. The results of this study showed that all three probiotic strains were able to utilize a range of carbohydrates. Maximal growth of L10 was observed in basal medium supplemented with soybean oligosaccharide (SOS), followed by fructooligosaccharide (FOS) and inulin, while maximal growth of B94 was observed in basal medium supplemented with SOS, followed by raffinose, FOS, beta-glucan hydrolysate, inulin and Fibregum TAN. L26 was shown to exhibit maximal growth in basal medium supplemented with FOS followed by inulin, SOS, beta-glucan hydrolysate and beta-glucan concentrate. The results of this study indicate that several carbohydrates/compounds can enhance the growth of these representative probiotic strains in vitro.     

Carbohydrate catabolic flexibility in the mammalian intestinal commensal <Emphasis Type="Italic">Lactobacillus ruminis</Emphasis> revealed by fermentation studies aligned to genome annotations

Background

Lactobacillus ruminis is a poorly characterized member of the Lactobacillus salivarius clade that is part of the intestinal microbiota of pigs, humans and other mammals. Its variable abundance in human and animals may be linked to historical changes over time and geographical differences in dietary intake of complex carbohydrates.

Results

In this study, we investigated the ability of nine L. ruminis strains of human and bovine origin to utilize fifty carbohydrates including simple sugars, oligosaccharides, and prebiotic polysaccharides. The growth patterns were compared with metabolic pathways predicted by annotation of a high quality draft genome sequence of ATCC 25644 (human isolate) and the complete genome of ATCC 27782 (bovine isolate). All of the strains tested utilized prebiotics including fructooligosaccharides (FOS), soybean-oligosaccharides (SOS) and 1,3:1,4-β-D-gluco-oligosaccharides to varying degrees. Six strains isolated from humans utilized FOS-enriched inulin, as well as FOS. In contrast, three strains isolated from cows grew poorly in FOS-supplemented medium. In general, carbohydrate utilisation patterns were strain-dependent and also varied depending on the degree of polymerisation or complexity of structure. Six putative operons were identified in the genome of the human isolate ATCC 25644 for the transport and utilisation of the prebiotics FOS, galacto-oligosaccharides (GOS), SOS, and 1,3:1,4-β-D-Gluco-oligosaccharides. One of these comprised a novel FOS utilisation operon with predicted capacity to degrade chicory-derived FOS. However, only three of these operons were identified in the ATCC 27782 genome that might account for the utilisation of only SOS and 1,3:1,4-β-D-Gluco-oligosaccharides.

Conclusions

This study has provided definitive genome-based evidence to support the fermentation patterns of nine strains of Lactobacillus ruminis, and has linked it to gene distribution patterns in strains from different sources. Furthermore, the study has identified prebiotic carbohydrates with the potential to promote L. ruminis growth in vivo.

     

Cloning,expression and functional validation of a β-fructofuranosidase from Lactobacillus plantarum

Fructooligosaccharides (FOS) are prebiotics that selectively stimulate the growth and activity of lactobacilli and bifidobacteria. These strains metabolize FOS with endogenous β-fructofuranosidase. In this study, a β-fructofuranosidase gene from Lactobacillus plantarum ST-III designated sacA was cloned into Escherichia coli, and the properties of the recombinant protein (SacA) were examined. The sacA gene encodes a peptide of 501 amino acids with a predicted molecular weight of 56.7 kDa. Sequence alignment revealed the presence of three highly conserved motifs, NDPNG, RDP and EC, indicating that the enzyme belongs to glycoside hydrolase family 32. The predicted three-dimensional structure of the SacA enzyme was similar to β-fructofuranosidases of bifidobacteria, such that it contained a five-blade β-propeller module and a β-sandwich domain with one additional N-terminal α-helix. The optimal reaction temperature and pH of the enzyme were 37 °C and 6.0, respectively. Substrate hydrolysis and kinetic parameters demonstrated that β-fructofuranosidase from L. plantarum ST-III liberated fructosyl residues from the non-reducing terminus of fructans, such as sucrose, FOS, levan or inulin, and FOS was the preferred substrate. The expression of the sacA gene in a non-FOS-fermenting strain, Lactobacillus rhamnosus GG, enabled the recombinant strain to metabolize FOS and sucrose.     

Apparent Inhibition of beta-Fructosidase Secretion by Tunicamycin May Be Explained by Breakdown of the Unglycosylated Protein during Secretion

Suspension-cultured carrot (Daucus carota) cells synthesize and secrete β-fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of β-fructosidase as measured by the accumulation of the radioactive protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated β-fructosidase is synthesized and is associated with an endoplasmic-reticulum-rich microsomal fraction. Pulse-chase experiments show that the unglycosylated β-fructosidase does not remain in the cells and appears to be secreted in the same way as glycosylated β-fructosidase; however, no radioactive, unglycosylated β-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete β-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated β-fructosidase. In the presence of tunicamycin, there is no accumulation of β-fructosidase activity or unglycosylated β-fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of β-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall.     

Chemical and physical differentiation of superoxide dismutases in anaerobes.

Superoxide dismutase activity in crude or partially purified cell extracts from several species and strains of obligate anaerobe Bacteroides was inhibited instantaneously by NaN3 and was inactivated rapidly upon incubation with H2O2. The extent of NaN3 inhibition varied from 41 to 93%, and the half-life of the enzymatic activity in 5 mM H2O2 ranged from 1.2 to 6.1 min, depending upon the organism tests. When grown in a defined medium containing 59Fe, Bacteroides fragilis (VPI 2393) incorporated radiolabel into a 40,000-molecular-weight NaN3- and H2O2-sensitive superoxide dismutase but did not incorporate 54Mn into that protein under similar growth conditions. The anaerobe Actinomyces naeslundii (VPI 9985) incorporated 54Mn but not 59Fe into a NaN3-insensitive and H2O2-resistant superoxide dismutase. The apparent molecular weight of the superoxide dismutase from this and several other Actinomyces spp. was estimated to be 110,000 to 140,000. Comparison of these data with studies of homogeneous metallosuperoxide dismutases suggests that the Bacteroides spp. studied contain a ferrisuperoxide dismutase, whereas Actinomyces spp. contain a managanisuperoxide dismutase.