Prebiotic effects of oligosaccharides on selected vaginal lactobacilli and pathogenic microorganisms

The purpose of this study was to select endogenous human vaginal lactobacilli strains on the basis of the main probiotic properties observed in the vaginal environment in order to use them for the evaluation of the potential prebiotic properties of oligosaccharides. From vaginal samples of 50 women with a normal flora, 17 lactobacilli strains were first isolated because of their high level of hydrogen peroxide production. Then six strains were selected mainly for their ability (i) to adhere to vaginal cells, (ii) to produce compounds in sufficient amount, such as lactic acid, having an inhibitory action on pathogens, and less importantly, (iii) to demonstrate arginine deiminase activity. These six strains were found to belong to three distinct species: Lactobacillus crispatus, L. jensenii and L. vaginalis. One strain of each species was chosen as a potential vaginal probiotic strain with regard to our criteria. These three strains were then used to evaluate the prebiotic properties of different oligosaccharide series: two fructooligosaccharide series (FOS Actilight and FOS Raftilose) and two glucooligosaccharide series varying by their osidic linkages (alpha-1,6/alpha-1,4 GOS and alpha-1,2/alpha-1,6/alpha-1,4 GOS). The test was based on the ability of the oligosaccharides to promote the growth of the three beneficial strains selected but not of pathogenic microorganisms often encountered in urogenital infections such as Candida albicans, Escherichia coli and Gardnerella vaginalis. Oligosaccharide hydrolysis was followed by HPLC analysis. This revealed that two oligosaccharide series (FOS Actilight DP3 and all alpha-1,6/alpha-1,4 GOS DP > or = 4) were used only by the lactobacilli strains, the pathogenic microorganisms being unable to metabolise them. The selected lactobacilli and oligosaccharides are good candidates for incorporation in a formula to prevent vaginal infections.     

Fermentation of Fructooligosaccharides by Lactic Acid Bacteria and Bifidobacteria

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.     

In vitro screening of probiotic lactic acid bacteria and prebiotic glucooligosaccharides to select effective synbiotics

Probiotics and prebiotics have been demonstrated to positively modulate the intestinal microflora and could promote host health. Although some studies have been performed on combinations of probiotics and prebiotics, constituting synbiotics, results on the synergistic effects tend to be discordant in the published works. The first aim of our study was to screen some lactic acid bacteria on the basis of probiotic characteristics (resistance to intestinal conditions, inhibition of pathogenic strains). Bifidobacterium was the most resistant genus whereas Lactobacillus farciminis was strongly inhibited. The inhibitory effect on pathogen growth was strain dependent but lactobacilli were the most effective, especially L. farciminis. The second aim of the work was to select glucooligosaccharides for their ability to support the growth of the probiotics tested. We demonstrated the selective fermentability of oligodextran and oligoalternan by probiotic bacteria, especially the bifidobacteria, for shorter degrees of polymerisation and absence of metabolism by pathogenic bacteria. Thus, the observed characteristics confer potential prebiotic properties on these glucooligosaccharides, to be further confirmed in vivo, and suggest some possible applications in synbiotic combinations with the selected probiotics. Furthermore, the distinctive patterns of the different genera suggest a combination of lactobacilli and bifidobacteria with complementary probiotic effects in addition to the prebiotic ones. These associations should be further evaluated for their synbiotic effects through in vitro and in vivo models.     

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.     

Biotechnological production and application of fructooligosaccharides

Currently, prebiotics are all carbohydrates of relatively short chain length. One important group is the fructooligosaccharides (FOS), a special kind of prebiotic associated to the selective stimulation of the activity of certain groups of colonic bacteria. They have a positive and beneficial effect on intestinal microbiota, reducing the incidence of gastrointestinal infections and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of FOS, such as solid-state fermentations utilizing various agro-industrial by-products. By optimizing the culture parameters, FOS yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of FOS. This article is an overview of the results considering recent studies on FOS biosynthesis, physicochemical properties, sources, biotechnological production and applications.     

Prebiotic effectiveness of inulin extracted from edible burdock

To investigate the prebiotic potential of burdock inulin (B-INU), the in vitro and in vivo effects of B-INU on bacterial growth were studied. B-INU significantly stimulated the growth of bifidobacteria in Man-Rogosa-Sharp (MRS) medium, anaerobically. Compared with chicory inulin (C-INU), long-chain inulin (L-INU) and fructooligosaccharides (FOS), 1% (w/v) B-INU promoted the specific growth rate of beneficial bacteria. The decreases of media pH with B-INU were almost the same as that with C-INU and FOS. In vivo, B-INU significantly increased the number of lactobacilli and bifidobacteria (P<0.05) in cecal content. Mice fed with B-INU, C-INU and FOS for 14 days had greater number of cecal beneficial bacteria population than those fed with L-INU for 14 days. In addition, all fructans did not cause any side effects, such as eructation and bloating. Results indicated that inulin extracted from edible burdock showed prebiotic properties that could promote health.     

Prebiotic effects of neoagaro-oligosaccharides prepared by enzymatic hydrolysis of agarose

To investigate the prebiotic properties of neoagaro-oligosaccharides (NAOS), obtained from enzymatic hydrolysis of agarose, the in vitro and in vivo effects of NAOS on bacterial growth were studied. In vitro NAOS were found to be highly resistant to enzymes of the upper gastrointestinal tract, which remained intact after 24h incubation with different amylolytic enzymes. NAOS significantly stimulated the growth of bifidobacteria and lactobacilli in Man-Rogosa-Sharp (MRS) medium, anaerobically. Compared with fructo-oligosaccharides (FOS), 1% (w/v) NAOS promoted the specific growth rate of beneficial bacteria by about 100%. The decreases of media pH with NAOS were almost the same as that with FOS. In vivo, NAOS significantly increased the numbers of lactobacilli and bifidobacteria (P<0.05) in fresh feces or cecal content while reducing putrefactive microorganisms. Mice fed with 2.5% (w/v) NAOS for 7 days had larger increases in colonic beneficial bacteria population than those fed with even 5% (w/v) FOS for 14 days. No side effects, such as eructation and bloating, were found. Interestingly, NAOS with higher degrees of polymerization (DP) showed better prebiotic activity. These results indicated that NAOS had great prebiotic effect, which could be beneficial to the host.     

Chitosan oligosaccharides, dp 2-8, have prebiotic effect on the Bifidobacterium bifidium and Lactobacillus sp

In order to investigate the prebiotic potential of chitosan oligosaccharide (COS), prepared by enzymatic hydrolysis of fully deacetylated chitosan polymer, the effect of COS on bacterial growth was studied. The degree of polymerization (dp) of COS was determined by MALDI-ToF mass spectrometry, and the COS was found to be composed of dimer (33.6%), trimer (16.9%), tetramer (15.8%), pentamer (12.4%), hexamer (8.3%), heptamer (7.1%), and octamer (5.9%). The minimum inhibitory concentrations (MIC) of chitosan polymer against lactic acid bacteria and bifidobacteria were below 0.31%. However, this only applied to two strains, the other bacteria tested grew on MRS broth containing 5% COS. The effects of COS on the growth of bifidobacteria and lactic acid bacteria were compared with those of fructo-oligosaccharide (FOS). FOS was found to have a growth stimulatory effect on only three strains: Bifidobacterium bifidium, B. infantis and Lactobacillus casei. However, COS stimulated the growth of most Lactobacillus sp. and B. bifidium KCTC 3440. The amount of the growth and the specific growth rate of B. bifidium increased with increasing COS concentration. The cultivation time required to obtain maximum growth was reduced to about 25% in MRS broth supplemented with 0.2-0.4% COS. These results demonstrate that COS has considerable bifidogenic potential. Both cell growth and specific growth rates of L. brevis in MRS broth supplemented with 0.1% COS increased by 25%. The present study shows that COS stimulates the growth of some enteric bacteria, and that COS has potential use as a prebiotic health-food.     

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.

     

Multiple Omics Uncovers Host–Gut Microbial Mutualism During Prebiotic Fructooligosaccharide Supplementation

Fructooligosaccharide (FOS), a prebiotic well known for its health-promoting properties, can improve the human gut ecosystem most likely through changes in its microbial composition. However, the detailed mechanism(s) of action of FOS in the modulation of the gut ecosystem remain(s) obscure. Traditional methods of profiling microbes and metabolites could barely show any significant features due to the existence of large interindividual differences, but our novel microbe–metabolite correlation approach, combined with faecal immunoglobulin A (IgA) measurements, has revealed that the induction of mucosal IgA by FOS supplementation correlated with the presence of specific bacteria. Furthermore, the metabolic dynamics of butyrate, l-phenylalanine, l-lysine and tyramine were positively correlated with that of these bacteria and IgA production, whereas p-cresol was negatively correlated. Taken together, our focused intraindividual analysis with omics approaches is a powerful strategy for uncovering the gut molecular network and could provide a new vista for understanding the human gut ecosystem.     

Characterization and Antioxidant Property of Probiotic and Synbiotic Yogurts

The effect of a prebiotic (fructooligosaccharides) or a synbiotic components (prebiotic and probiotic) on the viability, proteolysis and antioxidant properties of probiotic and synbiotic yogurt during 28?days of storage at 4?°C has been investigated. Yogurt starters in conjunction with either probiotic bacteria Lactobacillus plantarum CFR 2194, Lactobacillus fermentum CFR 2192 and/or fructooligosaccharides (FOS) were used for yogurt preparation. Titratable acidity and pH of all yogurt samples followed a similar pattern of increase or decrease during storage. Proteolysis in synbiotic yogurts was found to be significantly (P?<?0.05) higher in comparison with that of control. The addition of prebiotics had no effect (P?=?0.17888) on the viability of yogurt starters during cold storage. No observable changes in the viability of probiotic cultures in probiotic groups. However, supplementation of FOS affected the growth significantly (P?<?0.05) in promoting the growth of L. plantarum and L. fermentum. Antioxidant activities, the index of nutritional value of yogurt, were monitored. Results showed that the DPPH-radical-scavenging activity (85?%) in synbiotic yogurt containing L. plantarum and FOS was significantly higher (P?<?0.05) in comparison with that of control yogurt (72?%). Total phenolics and the ferric reducing power were highest in synbiotic yogurts in comparison with that of other test samples during the entire period of storage. Addition of selected probiotics with FOS thus resulted in an improved functionality of yogurt.     

Quantification of anammox populations enriched in an immobilized microbial consortium with low levels of ammonium nitrogen and at low temperature

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).     

In vitro fermentation of sugar beet arabino-oligosaccharides by fecal microbiota obtained from patients with ulcerative colitis to selectively stimulate the growth of Bifidobacterium spp. and Lactobacillus spp

The potential prebiotic properties of arabino-oligosaccharides (AOS) derived from sugar beet pulp was studied using mixed cultures of human fecal bacteria from patients with ulcerative colitis (UC), in remission or with active disease, and in healthy controls. These results were compared to those for fructo-oligosaccharides (FOS), which are known to have a prebiotic effect. Fermentation studies were carried out using a small-scale static batch system, and changes in the fecal microbial communities and metabolites were monitored after 24 h by quantitative real-time PCR and short-chain fatty acid analysis. With a few minor exceptions, AOS affected the communities similarly to what was seen for FOS. Quantitative real-time PCR revealed that Bifidobacterium spp. and Lactobacillus spp. were selectively increased after fermentation of AOS or FOS by fecal microbiota derived from UC patients. The stimulation of growth of Lactobacillus spp. and Bifidobacterium spp. was accompanied by a high production of acetate and hence a decrease of pH. The fermentation of AOS may help improve the inflammatory conditions in UC patients through stimulation of bacteria eliciting anti-inflammatory responses and through production of acetate. AOS may therefore represent a new prebiotic candidate for reduction of the risk of flare-ups in UC patients. However, human trials are needed to confirm a health-promoting effect.     

Raw potato starch and short-chain fructo-oligosaccharides affect the composition and metabolic activity of rat intestinal microbiota differently depending on the caecocolonic segment involved

AIMS: In vitro studies have suggested that fructo-oligosaccharides (FOS) and resistant starch (two fermentable non-digestible carbohydrates) display different fermentation kinetics. This study investigated whether these substrates affect the metabolic activity and bacterial composition of the intestinal microflora differently depending on the caecocolonic segment involved. METHODS AND RESULTS: Eighteen rats were fed a low-fibre diet (Basal) or the same diet containing raw potato starch (RPS) (9%) or short-chain FOS (9%) for 14 days. Changes in wet-content weights, bacterial populations and metabolites were investigated in the caecum, proximal and distal colon and faeces. Both substrates exerted a prebiotic effect compared with the Basal diet. However, FOS increased lactic acid-producing bacteria (LAPB) throughout the caecocolon and in faeces, whereas the effect of RPS was limited to the caecum and proximal colon. As compared with RPS, FOS doubled the pool of caecal fermentation products, while the situation was just the opposite distally. This difference was mainly because of the anatomical distribution of lactate, which accumulated in the caecum with FOS and in the distal colon with RPS. Faeces reflected these impacts only partly, showing the prebiotic effect of FOS and the metabolite increase induced by RPS. CONCLUSIONS: This study demonstrates that FOS and RPS exert complementary caecocolonic effects. SIGNIFICANCE AND IMPACT OF THE STUDY: The RPS and FOS combined ingestion could be beneficial by providing health-promoting effects throughout the caecocolon.     

In vitro study of Prebiotic Properties of Levan-type Exopolysaccharides from Lactobacilli and Non-digestible Carbohydrates Using Denaturing Gradient Gel Electrophoresis

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.     

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.     

Fermentation properties of gentio-oligosaccharides

AIMS: To investigate the fermentation properties of gentio-oligosaccharides (GOS), as compared to fructo-oligosaccharides (FOS) and maltodextrin in mixed faecal culture. METHODS AND RESULTS: The substrates were incubated in 24 h batch culture fermentations of human faecal bacteria. Fluorescent in situ hybridization was used to determine changes in populations of bifidobacteria, lactobacilli, clostridia, bacteroides, streptococci and Escherichia coli. Gas and short-chain fatty acid (SCFA) production was also measured. GOS gave the largest significant increases in bifidobacteria, lactobacilli and total bacterial numbers during the incubations. However, FOS appeared to be a more selective prebiotic as it did not significantly stimulate growth of bacterial groups which were not probiotic in nature. GOS and maltodextrin produced the highest levels of SCFA. Lowest gas production was seen with GOS and highest with FOS. CONCLUSIONS: GOS possessed bifidogenic activity in vitro. Although fermentation of GOS was not as selective as FOS, gas production was lower. Gas production is often seen as an undesirable side effect of prebiotic consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided the first data on fermentation of GOS in mixed faecal culture. The study has also used molecular microbiology methods (FISH) to quantify bacterial groups. The data extend our knowledge of the selectivity of fermentation of oligosaccharides by the gut microflora.     

Fermentation of fructooligosaccharides by lactic acid bacteria and bifidobacteria

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.     

In vitro study of prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis

Batch cultures inoculated with human faeces were used to study the prebiotic properties of levan-type exopolysaccharides (EPS) from Lactobacillus sanfranciscensis as well as levan, inulin, and fructooligosaccharide (FOS). Denaturing gradient gel electrophoresis of 16S rDNA fragments generated by PCR with universal primers was used to analyse the cultures. Characteristic changes were revealed in the composition of the gut bacteria during fermentation of the carbohydrates. An enrichment of Bifidobacterium spp. was found for the EPS and inulin but not for levan and FOS. The bifidogenic effect of the EPS was confirmed by culturing on selective medium. In addition, the use of EPS and FOS resulted in enhanced growth of Eubacterium biforme and Clostridium perfringens, respectively.     

Annotation and De Novo Sequence Characterization of Extracellular β-Fructofuranosidase from <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> Strain HKF42

The genome of a fungal strain Penicillium chrysogenum strain HKF42, which can grow on 20% sucrose has been annotated for 7595 protein coding sequences. On mining of CAZymes, we could annotate a β-fructofuranosidase gene responsible for fructo-oligosaccharides (FOS) synthesis which is a known prebiotic. The enzyme activity was demonstrated and validated with the generation of FOS as kestose and nystose.