The yacon (Smallanthus sonchifolius) tuber was examined with regard to
its prebiotic effects compared with commercialized fructo-oligosaccharides (FOS). A feed
containing 10% yacon tuber, which is equivalent to 5% commercialized FOS in terms of the
amount of fructo-oligosaccharides (GF2, GF3 and GF4), was administrated to rats for 28
days. The yacon diet changed the intestinal microbial communities beginning in the first
week, resulting in a twofold greater concentration of cecal short-chain fatty acids
(SCFAs). The SCFA composition differed, but the cecal pH in rats fed yacon tuber was equal
to that in rats fed FOS. Serum triglycerides were lower in rats fed yacon compared with
rats fed FOS and the control diet. Cecal size was greater with the yacon tuber diet
compared with the control diet. The abundant fermentation in the intestines created a
selective environment for the intestinal microbiota, which included Lactobacillus
acidophilus, Bifidobacterium pseudolongum, Bifidobacterium animalis and
Barnesiella spp. according to identification with culture-independent
analysis, 16S rRNA gene PCR-DGGE combined with cloning and sequencing.
Barnesiella spp. and B. pseudolongum were only found
in the rats fed the yacon diet, while L. acidophilus and B.
animalis were found in abundance in rats fed both the yacon and FOS diets. The
genus Barnesiella has not previously been reported to be associated with
yacon or FOS fermentation. We concluded that the physiological and microbiological effects
of the yacon tuber were different from those of FOS. Differences in cecal size, blood
triglycerides and microbial community profiles including their metabolites (SCFAs) between
the yacon tuber and FOS were shown to be more greatly affected by the yacon tuber rather
intestinal microbes; Smallanthus sonchifolius; yacon; DGGE; fructo-oligosaccharides
The developing intestinal microbiota of breast-fed infants is considered to play an important role in the priming of the infants' mucosal and systemic immunity. Generally, Bifidobacterium and Lactobacillus predominate the microbiota of breast-fed infants. In intervention trials it has been shown that lactobacilli can exert beneficial effects on, for example, diarrhea and atopy. However, the Lactobacillus species distribution in breast-fed or formula-fed infants has not yet been determined in great detail. For accurate enumeration of different lactobacilli, duplex 5′ nuclease assays, targeted on rRNA intergenic spacer regions, were developed for Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus. The designed and validated assays were used to determine the amounts of different Lactobacillus species in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructo-oligosaccharides in a 9:1 ratio (OSF). A breast-fed group (BF) was studied in parallel as a reference. During the 6-week intervention period a significant increase was shown in total percentage of fecal lactobacilli in the BF group (0.8% ± 0.3% versus 4.1% ± 1.5%) and the OSF group (0.8% ± 0.3% versus 4.4% ± 1.4%). The Lactobacillus species distribution in the OSF group was comparable to breast-fed infants, with relatively high levels of L. acidophilus, L. paracasei, and L. casei. The SF-fed infants, on the other hand, contained more L. delbrueckii and less L. paracasei compared to breast-fed infants and OSF-fed infants. An infant milk formula containing a specific mixture of prebiotics is able to induce a microbiota that closely resembles the microbiota of BF infants.
Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.
Our study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal microbial community in rats, as well as on a number of cecal parameters, which may be influenced by a changed microbiota.
Principal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks), while no effects of apple juice, purée or pomace on microbial composition in cecum were observed. Administration of either 0.33 or 3.3% apple pectin in the diet resulted in considerable changes in the DGGE profiles.
A 2-fold increase in the activity of beta-glucuronidase was observed in animals fed with pectin (7% in the diet) for four weeks, as compared to control animals (P < 0.01). Additionally, the level of butyrate measured in these pectin-fed animal was more than double of the corresponding level in control animals (P < 0.01). Sequencing revealed that DGGE bands, which were suppressed in pectin-fed rats, represented Gram-negative anaerobic rods belonging to the phylum Bacteroidetes, whereas bands that became more prominent represented mainly Gram-positive anaerobic rods belonging to the phylum Firmicutes, and specific species belonging to the Clostridium Cluster XIVa.
Quantitative real-time PCR confirmed a lower amount of given Bacteroidetes species in the pectin-fed rats as well as in the apple-fed rats in the four-week study (P < 0.05). Additionally, a more than four-fold increase in the amount of Clostridium coccoides (belonging to Cluster XIVa), as well as of genes encoding butyryl-coenzyme A CoA transferase, which is involved in butyrate production, was detected by quantitative PCR in fecal samples from the pectin-fed animals.
Our findings show that consumption of apple pectin (7% in the diet) increases the population of butyrate- and β-glucuronidase producing Clostridiales, and decreases the population of specific species within the Bacteroidetes group in the rat gut. Similar changes were not caused by consumption of whole apples, apple juice, purée or pomace.
Edible brown algae are used as major food material in Far East Asian countries, particularly in South Korea and Japan. They contain fermentable dietary fibers, alginic acid (uronic acid polymer) and laminaran (β-1,3-glucan), that are fermented into organic acids by intestinal bacteria. To clarify the effect of edible algae on the intestinal environment, the cecal microbiotas of rats fed diets containing no dietary fiber (control) or 2% (wt/wt) sodium alginate or laminaran for 2 weeks were analyzed using FLX amplicon pyrosequencing with bar-coded primers targeting the bacterial 16S rRNA gene. The most abundant phylum in all groups was Firmicutes. Specifically, Allobaculum was dominant in all diet groups. In addition, Bacteroides capillosus (37.1%) was abundant in the alginate group, while Clostridium ramosum (3.14%) and Parabacteroides distasonis (1.36%) were only detected in the laminaran group. Furthermore, rats fed alginate showed simplified microbiota phylotypes compared with others. With respect to cecal chemical compounds, laminaran increased cecal organic acid levels, particularly propionic acid. Alginate increased total cecal organic acids. Cecal putrefactive compounds, such as indole, H2S, and phenol, were decreased by both alginate and laminaran. These results indicate that edible brown algae can alter the intestinal environment, with fermentation by intestinal microbiota.
Butyric acid, one of the key products formed when β-glucans are degraded by the microbiota in the colon, has been proposed to be important for colonic health. Glutamine bound to the fibre may have similar effects once it has been liberated from the fibre in the colon. Both β-glucans and glutamine are found in high amounts in malted barley. Lactobacillus rhamnosus together with malt has been shown to increase the formation of butyric acid further in rats.
To investigate whether Lactobacillus rhamnosus 271, Lactobacillus paracasei 87002, Lactobacillus plantarum HEAL 9 and 19, and Bifidobacterium infantis CURE 21 affect the levels of short-chain fatty acids and glutamine in caecum and portal blood of rats fed barley malt.
The experimental diets were fed for 12 days. The daily dose of the probiotic strain was 1×109 colony forming units and the intake of fibre 0.82 g/day.
The malt mostly contained insoluble fibre polymers (93%), consisting of glucose and xylose (38–41 g/kg) and some arabinose (21 g/kg). The fibre polysaccharides were quite resistant to fermentation in the rats, regardless of whether or not probiotics were added (25–30% were fermented). Caecal and portal levels of acetic acid decreased in the rats after the addition of L. plantarum HEAL 9 and L. rhamnosus 271, and also the levels of butyric acid. Viable counts of Lactobacillus, Bifidobacterium and Enterobacteriaceae were unaffected, while the caecal composition of Lactobacilli was influenced by the type of strain administrated. Portal levels of glutamine were unchanged, but glycine levels increased with L. plantarum HEAL 9 and 19 and phenylalanine with L. rhamnosus 271.
Although the probiotic strains survived and reached the caecum, except B. infantis CURE 21, there were no effects on viable counts or in the fermentation of different fibre components, but the formation of some bacterial metabolites decreased. This may be due to the high proportion of insoluble fibres in the malt.
dietary fibre; probiotics; short-chain fatty acids; amino acids; microbiota
This study aimed at investigating the fecal microbiotas of children with celiac disease (CD) before (U-CD) and after (T-CD) they were fed a gluten-free diet and of healthy children (HC). Brothers or sisters of T-CD were enrolled as HC. Each group consisted of seven children. PCR-denaturing gradient gel electrophoresis (DGGE) analysis with V3 universal primers revealed a unique profile for each fecal sample. PCR-DGGE analysis with group- or genus-specific 16S rRNA gene primers showed that the Lactobacillus community of U-CD changed significantly, while the diversity of the Lactobacillus community of T-CD was quite comparable to that of HC. Compared to HC, the ratio of cultivable lactic acid bacteria and Bifidobacterium to Bacteroides and enterobacteria was lower in T-CD and even lower in U-CD. The percentages of strains identified as lactobacilli differed as follows: HC (ca. 38%) > T-CD (ca. 17%) > U-CD (ca. 10%). Lactobacillus brevis, Lactobacillus rossiae, and Lactobacillus pentosus were identified only in fecal samples from T-CD and HC. Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus gasseri were identified only in several fecal samples from HC. Compared to HC, the composition of Bifidobacterium species of T-CD varied, and it varied even more for U-CD. Forty-seven volatile organic compounds (VOCs) belonging to different chemical classes were identified using gas-chromatography mass spectrometry-solid-phase microextraction analysis. The median concentrations varied markedly for HC, T-CD, and U-CD. Overall, the r2 values for VOC data for brothers and sisters were equal to or lower than those for unrelated HC and T-CD. This study shows the effect of CD pathology on the fecal microbiotas of children.
Ageing changes gut microbiota composition and alters immune system function. Probiotics, prebiotics and synbiotics may improve the health status of elderly individuals by modifying the intestinal environment and the microbiota composition, and by stimulating the immune system. In this work, we studied the effects of synbiotic supplementation on the gut microbiota of healthy elderly volunteers. Fifty-one elders were randomly assigned to consume either a synbiotic dietary supplement or a placebo in addition to their usual diet for a 2-week period. The synbiotic product consisted of the probiotic Lactobacillus acidophilus NCFM and the prebiotic lactitol and was ingested twice a day, with a total daily dose of 10 g lactitol and 2 × 1010 cells of probiotic bacteria. Before, during and after the intervention period fecal quantities of six phylogenetic bacterial groups were determined using quantitative PCR, and relative changes in total microbiota composition were assessed by percent guanine-plus-cytosine profiling. The microbiota profiles showed certain relative changes within the microbial community, and indicated an increase of bifidobacteria levels during synbiotic supplementation. Quantification by PCR confirmed the in changes in the microbiota composition; for example increases in total levels of endogenous bifidobacteria and lactobacilli were recorded. Throughout the 6-week study period there was a decrease unrelated to intervention in the Blautia coccoides–Eubacterium rectale bacterial group levels and Clostridium cluster XIVab levels, but this decrease appeared to be halted during the synbiotic intervention. In conclusion, putatively beneficial changes in microbiota were observed in the elderly subjects supplemented with the synbiotic product.
Lactobacillus acidophilus; NCFM; Lactitol; Synbiotic; Elderly; Microbiota
Obesity leads to changes in the gut microbial community which contribute to the metabolic dysregulation in obesity. Dietary fat and fiber affect the caloric density of foods. The impact of dietary fat content and fiber type on the microbial community in the hind gut is unknown. Effect of dietary fat level and fiber type on hindgut microbiota and volatile fatty acid (VFA) profiles was investigated. Expression of metabolic marker genes in the gut, adipose tissue and liver was determined. A 2×2 experiment was conducted in pigs fed at two dietary fat levels (5% or 17.5% swine grease) and two fiber types (4% inulin, fermentable fructo-oligosaccharide or 4% solka floc, non-fermentable cellulose). High fat diets (HFD) resulted in a higher (P<0.05) total body weight gain, feed efficiency and back fat accumulation than the low fat diet. Feeding of inulin, but not solka floc, attenuated (P<0.05) the HFD-induced higher body weight gain and fat mass accumulation. Inulin feeding tended to lead to higher total VFA production in the cecum and resulted in a higher (P<0.05) expression of acyl coA oxidase (ACO), a marker of peroxisomal β-oxidation. Inulin feeding also resulted in lower expression of sterol regulatory element binding protein 1c (SREBP-1c), a marker of lipid anabolism. Bacteria community structure characterized by DGGE analysis of PCR amplified 16S rRNA gene fragments showed that inulin feeding resulted in greater bacterial population richness than solka floc feeding. Cluster analysis of pairwise Dice similarity comparisons of the DGGE profiles showed grouping by fiber type but not the level of dietary fat. Canonical correspondence analysis (CCA) of PCR- DGGE profiles showed that inulin feeding negatively correlated with back fat thickness. This study suggests a strong interplay between dietary fat level and fiber type in determining susceptibility to obesity.
The emergence and spread of antibiotic resistance in pathogens have led to a restriction on the use of antibiotic growth promoters (AGPs) in animal feed in some countries. The potential negative after-effects of a ban on AGPs could be mitigated by improving animal intestinal health with prebiotic dietary fibers such as xylo-oligosaccharides (XOS). However, the mechanism(s) by which an antibiotic or prebiotic contributes to the health and growth of animals are not well understood. Here, we evaluated XOS and virginiamycin (VIRG)-mediated changes in gut microbiota of broiler chickens using pyrosequencing of the 16S rRNA gene.
There was a significant change in the relative abundance of certain bacteria, but the overall microbial diversity was not affected by treatment with either XOS or VIRG. Supplementation of HXOS (2 g XOS/kg diet) increased the proportion of Lactobacillus genus in the cecum, whereas Propionibacterium and Corynebacterium genera were enriched in the ileum of VIRG (16 mg/kg) treated birds. Furthermore, an increase in the cecal concentrations of acetate and propionate was observed in HXOS- and VIRG-fed chickens, respectively. These two groups of birds had better feed conversion efficiencies in comparison with the control group from day 7 to 21. In addition, temporal variations in the gut microbiota were evident in the chickens of different ages.
Treatments with XOS or VIRG modified the relative abundance but not the presence or absence of specific microbial genus. The increase in both Lactobacillus spp. and acetate production in the cecum of HXOS-treated chickens may promote intestinal health.
Electronic supplementary material
The online version of this article (doi:10.1186/s40168-015-0079-4) contains supplementary material, which is available to authorized users.
Chicken; Gut microbiome; Xylo-oligosaccharides; Virginiamycin; Pyrosequencing
Human subjects consumed biscuits containing either galacto-oligosaccharides or fructo-oligosaccharides in a double-blinded, crossover study. The impact of supplementing the diet with three biscuits per day on the fecal microbiota was evaluated by selective culture of particular bacterial groups, measurement of β-galactosidase activity, and nucleic acid-based analytical methods (PCR-denaturing gradient gel electrophoresis [PCR-DGGE] and fluorescent in situ hybridization). The composition of the bifidobacterial populations was monitored at the level of species (PCR-DGGE) and strains (pulsed-field gel electrophoresis of DNA digests), and representative cultures were tested quantitatively for their ability to use galacto-oligosaccharides. Technical improvements to DGGE analysis of the microbiota were made by the use of an internal standard that allowed valid comparisons of fragment staining intensities to be made between profiles, the use of S1 nuclease digestion to remove single-stranded DNA to facilitate cloning of DNA sequences cut from gels, and the extraction of RNA to be used as the template in reverse transcription-PCR-DGGE. RNA-DGGE profiles were markedly different (Dice's similarity coefficient, 58.5%) from those generated by DNA-DGGE. Neither the sizes of the bacterial populations nor the DNA-DGGE profiles of the microbiota were altered by the consumption of the biscuits, but the RNA-DGGE profiles were altered by the detection or increased staining intensity of 16S rRNA gene sequences originating from Bifidobacterium adolescentis and/or Colinsella aerofaciens in the feces of 11 of 15 subjects. β-Galactosidase activity was elevated in the feces of some subjects as a result of biscuit consumption. Subjects differed in the ability of the bifidobacterial strains harbored in their feces to use galacto-oligosaccharides. Our observations suggest that a phylogenetic approach to analysis of the gut ecosystem may not always be optimal and that a more physiological (biochemical) method might be more informative.
Obesity is rising at an alarming rate globally. Different fermentable carbohydrates have been shown to reduce obesity. The aim of the present study was to investigate if two different fermentable carbohydrates (inulin and β-glucan) exert similar effects on body composition and central appetite regulation in high fat fed mice.
Thirty six C57BL/6 male mice were randomized and maintained for 8 weeks on a high fat diet containing 0% (w/w) fermentable carbohydrate, 10% (w/w) inulin or 10% (w/w) β-glucan individually. Fecal and cecal microbial changes were measured using fluorescent in situ hybridization, fecal metabolic profiling was obtained by proton nuclear magnetic resonance (1H NMR), colonic short chain fatty acids were measured by gas chromatography, body composition and hypothalamic neuronal activation were measured using magnetic resonance imaging (MRI) and manganese enhanced MRI (MEMRI), respectively, PYY (peptide YY) concentration was determined by radioimmunoassay, adipocyte cell size and number were also measured. Both inulin and β-glucan fed groups revealed significantly lower cumulative body weight gain compared with high fat controls. Energy intake was significantly lower in β-glucan than inulin fed mice, with the latter having the greatest effect on total adipose tissue content. Both groups also showed an increase in the numbers of Bifidobacterium and Lactobacillus-Enterococcus in cecal contents as well as feces. β- glucan appeared to have marked effects on suppressing MEMRI associated neuronal signals in the arcuate nucleus, ventromedial hypothalamus, paraventricular nucleus, periventricular nucleus and the nucleus of the tractus solitarius, suggesting a satiated state.
Although both fermentable carbohydrates are protective against increased body weight gain, the lower body fat content induced by inulin may be metabolically advantageous. β-glucan appears to suppress neuronal activity in the hypothalamic appetite centers. Differential effects of fermentable carbohydrates open new possibilities for nutritionally targeting appetite regulation and body composition.
The impact of nonstarch polysaccharides (NSP) differing in their functional properties on intestinal bacterial community composition, prevalence of butyrate production pathway genes, and occurrence of Escherichia coli virulence factors was studied for eight ileum-cannulated growing pigs by use of terminal restriction fragment length polymorphism (TRFLP) and quantitative PCR. A cornstarch- and casein-based diet was supplemented with low-viscosity, low-fermentability cellulose (CEL), with high-viscosity, low-fermentability carboxymethylcellulose (CMC), with low-viscosity, high-fermentability oat β-glucan (LG), and with high-viscosity, high-fermentability oat β-glucan (HG). Only minor effects of NSP fractions on the ileal bacterial community were observed, but NSP clearly changed the digestion in the small intestine. Compared to what was observed for CMC, more fermentable substrate was transferred into the large intestine with CEL, LG, and HG, resulting in higher levels of postileal dry-matter disappearance. Linear discriminant analysis of NSP and TRFLP profiles and 16S rRNA gene copy numbers for major bacterial groups revealed that CMC resulted in a distinctive bacterial community in comparison to the other NSP, which was characterized by higher gene copy numbers for total bacteria, Bacteroides-Prevotella-Porphyromonas, Clostridium cluster XIVa, and Enterobacteriaceae and increased prevalences of E. coli virulence factors in feces. The numbers of butyryl-coenzyme A (CoA) CoA transferase gene copies were higher than those of butyrate kinase gene copies in feces, and these quantities were affected by NSP. The present results suggest that the NSP fractions clearly and distinctly affected the taxonomic composition and metabolic features of the fecal microbiota. However, the effects were more linked to the individual NSP and to their effect on nutrient flow into the large intestine than to their shared functional properties.
A study was designed to elucidate effects of selected carbohydrates on composition and activity of the intestinal microbiota. Five groups of eight rats were fed a western type diet containing cornstarch (reference group), sucrose, potato starch, inulin (a long- chained fructan) or oligofructose (a short-chained fructan). Fructans are, opposite sucrose and starches, not digestible by mammalian gut enzymes, but are known to be fermentable by specific bacteria in the large intestine.
Animals fed with diets containing potato starch, or either of the fructans had a significantly (p < 0.05) higher caecal weight and lower caecal pH when compared to the reference group, indicating increased fermentation. Selective cultivation from faeces revealed a higher amount of lactic acid bacteria cultivable on Rogosa agar in these animals. Additionally, the fructan groups had a lower amount of coliform bacteria in faeces. In the inulin and oligofructose groups, higher levels of butyrate and propionate, respectively, were measured.
Principal Component Analysis of profiles of the faecal microbiota obtained by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR amplified bacterial 16S rRNA genes as well as of Reverse Transcriptase-PCR amplified bacterial 16S rRNA resulted in different phylogenetic profiles for each of the five animal groups as revealed by Principal Component Analysis (PCA) of band patterns.
Even though sucrose and cornstarch are both easily digestible and are not expected to reach the large intestine, the DGGE band patterns obtained indicated that these carbohydrates indeed affected the composition of bacteria in the large gut. Also the two fructans resulted in completely different molecular fingerprints of the faecal microbiota, indicating that even though they are chemically similar, different intestinal bacteria ferment them. Comparison of DNA-based and RNA-based profiles suggested that two species within the phylum Bacteroidetes were not abundant in numbers but had a particularly high ribosome content in the animals fed with inulin.
Thylakoid membranes derived from green leaf chloroplasts affect appetite-regulating
hormones, suppress food intake, reduce blood lipids and lead to a decreased body weight in
animals and human subjects. Thylakoids also decrease the intestinal in
vitro uptake of methyl-glucose in the rat. The aim of this study was to
investigate the effect of dietary thylakoids on the gut microbiota composition, mainly the
taxa of lactobacilli and bifidobacteria, in rats fed either a thylakoid-enriched diet or a
control diet for 10 d. At the same time, a glucose-tolerance test in the same rats was
also performed. Food intake was significantly decreased in the thylakoid-fed rats compared
with the control-fed rats over the 10-d study. An oral glucose tolerance test after 10 d
of thylakoid- or control-food intake resulted in significantly reduced plasma insulin
levels in the thylakoid-fed rats compared with the control-fed rats, while no difference
was observed for blood glucose levels. Analysis of gut bacteria showed a significant
increase of lactobacilli on the ileal mucosa, specifically Lactobacillus
reuteri, in the rats fed the thylakoid diet compared with rats fed the control
diet, while faecal lactobacilli decreased. No difference in bifidobacteria between the
thylakoid and control groups was found. Analyses with terminal restriction fragment length
polymorphism and principal component analysis of faeces demonstrated different microbial
populations in the thylakoid- and control-fed animals. These findings indicate that
thylakoids modulate the gut microbial composition, which might be important for the
regulation of body weight and energy metabolism.
Colon; Small intestine; Obesity; Lactobacilli; Quantitative PCR; MW, modified Wilkins–Chalgren; OGTT, oral glucose tolerance test; PCA, principal component analysis; qPCR, quantitative PCR; T-RFLP, terminal restriction fragment length polymorphism
Several substances, including glutamine and propionic acid but in particular butyric acid, have been proposed to be important for colonic health. β-Glucans lead to the formation of comparatively high amounts of butyric acid, and germinated barley foodstuff obtained from brewer’s spent grain (BSG), containing high amounts of β-glucans and glutamine, has been reported to reduce the inflammatory response in the colon of patients with ulcerative colitis. The present study examines how 3 barley products, whole grain barley, malt, and BSG, affect SCFA in the hindgut and serum of rats and whether the addition of Lactobacillus rhamnosus 271 to each of these diets would have further effects. Amino acids in plasma and the cecal composition of the microbiota were also analyzed. The butyric acid concentration in the distal colon and serum was higher in the malt groups than in the other groups as was the serum concentration of propionic acid. The concentrations of propionic and butyric acids were higher in the cecum and serum of rats given L. rhamnosus than in those not given this strain. The proportion of plasma glutamine and the cecal number of bifidobacteria were lower in the malt groups than in the other groups. L. rhamnosus decreased the number of cecal bifidobacteria, whereas plasma glutamine was unaffected. We conclude that malt together with L. rhamnosus 271 had greater effects on propionic and butyric acid concentrations in rats than the other barley products. This is interesting when developing food with effects on colonic health.
The ability to predictably engineer the composition of bowel microbial communities (microbiota) using dietary components is important because of the reported associations of altered microbiota composition with medical conditions. In a synecological study, weanling conventional Sprague-Dawley rats (21 days old) were fed a basal diet (BD) or a diet supplemented with resistant starch (RS) at 5%, 2.5%, or 1.25% for 28 days. Pyrosequencing of 16S rRNA genes and temporal temperature gradient electrophoresis (TTGE) profiles in the colonic digesta showed that rats fed RS had altered microbiota compositions due to blooms of Bacteroidetes and Actinobacteria. The altered microbiota was associated with changes in colonic short-chain fatty acid (SCFA) concentrations, colonic-tissue gene expression (Gsta2 and Ela1), and host physiology (serum metabolite profiles and colonic goblet cell numbers). Comparisons between germ-free and conventional rats showed that transcriptional and serum metabolite differences were mediated by the microbiota and were not the direct result of diet composition. Altered transcriptomic and physiological responses may reflect the young host's attempts to maintain homeostasis as a consequence of exposure to a new collection of bacteria and their associated biochemistry.
Knowledge of the trophisms that underpin bowel microbiota composition is required in order to understand its complex phylogeny and function. Stable-isotope (13C)-labeled inulin was added to the diet of rats on a single occasion in order to detect utilization of inulin-derived substrates by particular members of the cecal microbiota. Cecal digesta from Fibruline-inulin-fed rats was collected prior to (0 h) and at 6, 12, 18 and 24 h following provision of the [13C]inulin diet. RNA was extracted from these cecal specimens and fractionated in isopycnic buoyant density gradients in order to detect 13C-labeled nucleic acid originating in bacterial cells that had metabolized the labeled dietary constituent. RNA extracted from specimens collected after provision of the labeled diet was more dense than 0-h RNA. Sequencing of 16S rRNA genes amplified from cDNA obtained from these fractions showed that Bacteroides uniformis, Blautia glucerasea, Clostridium indolis, and Bifidobacterium animalis were the main users of the 13C-labeled substrate. Culture-based studies of strains of these bacterial species enabled trophisms associated with inulin and its hydrolysis products to be identified.
B. uniformis utilized Fibruline-inulin for growth, whereas the other species used fructo-oligosaccharide and monosaccharides. Thus, RNA–stable-isotope probing (RNA-SIP) provided new information about the use of carbon from inulin in microbiota metabolism.
Lactobacillus jensenii, L. iners, L. crispatus and L. gasseri are the most frequently occurring lactobacilli in the vagina. However, the native species vary widely according to the studied population. The present study was performed to genetically determine the identity of Lactobacillus strains present in the vaginal discharge of healthy and bacterial vaginosis (BV) intermediate Mexican women.
In a prospective study, 31 strains preliminarily identified as Lactobacillus species were isolated from 21 samples collected from 105 non-pregnant Mexican women. The samples were classified into groups according to the Nugent score criteria proposed for detection of BV: normal (N), intermediate (I) and bacterial vaginosis (BV). We examined the isolates using culture-based methods as well as molecular analysis of the V1–V3 regions of the 16S rRNA gene. Enterobacterial repetitive intergenic consensus (ERIC) sequence analysis was performed to reject clones.
Clinical isolates (25/31) were classified into four groups based on sequencing and analysis of the 16S rRNA gene: L. acidophilus (14/25), L. reuteri (6/25), L. casei (4/25) and L. buchneri (1/25). The remaining six isolates were presumptively identified as Enterococcus species. Within the L. acidophilus group, L. gasseri was the most frequently isolated species, followed by L. jensenii and L. crispatus. L. fermentum, L. rhamnosus and L. brevis were also isolated, and were placed in the L. reuteri, L. casei and L. buchneri groups, respectively. ERIC profile analysis showed intraspecific variability amongst the L. gasseri and L. fermentum species.
These findings agree with previous studies showing that L. crispatus, L. gasseri and L. jensenii are consistently present in the healthy vaginal ecosystem. Additional species or phylotypes were detected in the vaginal microbiota of the non-pregnant Mexican (Hispanic-mestizo) population, and thus, these results further our understanding of vaginal lactobacilli colonisation and richness in this particular population.
Lactobacilli; Mexican population; 16S rRNA; Species identification; Vaginal microbiota; Bacterial vaginosis
The intestinal microbiota of broiler chickens and the microbiota in the litter have been well studied, but the interactions between these two microbiotas remain to be determined. Therefore, we examined their reciprocal effects by analyzing the intestinal microbiotas of broilers reared on fresh pine shavings versus reused litter, as well as the litter microbiota over a 6-week cycle. Composite ileal mucosal and cecal luminal samples from birds (n = 10) reared with both litter conditions (fresh versus reused) were collected at 7, 14, 21, and 42 days of age. Litter samples were also collected at days 7, 14, 21, and 42. The microbiotas were profiled and compared within sample types based on litter condition using PCR and denaturing gradient gel electrophoresis (PCR-DGGE). The microbiotas were further analyzed using 16S rRNA gene clone libraries constructed from microbiota DNA extracted from both chick intestinal and litter samples collected at day 7. Results showed significant reciprocal effects between the microbiotas present in the litter and those in the intestines of broilers. Fresh litter had more environmental bacteria, while reused litter contained more bacteria of intestinal origin. Lactobacillus spp. dominated the ileal mucosal microbiota of fresh-litter chicks, while a group of bacteria yet to be classified within Clostridiales dominated in the ileal mucosal microbiota in the reused-litter chicks. The Litter condition (fresh versus reused) seemed to have a more profound impact on the ileal microbiota than on the cecal microbiota. The data suggest that the influence of fresh litter on ileal microbiota decreased as broilers grew, compared with temporal changes observed under reused-litter rearing conditions.
We are exposed to millions of microbial and dietary antigens via the gastrointestinal tract, which likely play a key role in type 1 diabetes (T1D). We differentiated the effects of these two major environmental factors on gut immunity and T1D. Diabetes-prone BioBreeding (BBdp) rats were housed in specific pathogen-free (SPF) or germ-free (GF) conditions and weaned onto diabetes-promoting cereal diets or a protective low-antigen hydrolyzed casein (HC) diet, and T1D incidence was monitored. Fecal microbiota 16S rRNA genes, immune cell distribution, and gene expression in the jejunum were analyzed. T1D was highest in cereal-SPF (65%) and cereal-GF rats (53%) but inhibited and delayed in HC-fed counterparts. Nearly all HC-GF rats remained diabetes-free, whereas HC-fed SPF rats were less protected (7 vs. 29%). Bacterial communities differed in SPF rats fed cereal compared with HC. Cereal-SPF rats displayed increased gut CD3+ and CD8α+ lymphocytes, ratio of Ifng to Il4 mRNA, and Lck expression, indicating T-cell activation. The ratio of CD3+ T cells expressing the Treg marker Foxp3+ was highest in HC-GF and lowest in cereal-SPF rats. Resident CD163+ M2 macrophages were increased in HC-protected rats. The cathelicidin antimicrobial peptide (Camp) gene was upregulated in the jejunum of HC diet–protected rats, and CAMP+ cells colocalized with CD163. A cereal diet was a stronger promoter of T1D than gut microbes in association with impaired gut immune homeostasis.
CPT-11 is a drug used as chemotherapy for colorectal cancer. CPT-11 causes toxic side-effects in patients. CPT-11 toxicity has been attributed to the activity of intestinal microbiota, however, intestinal microbiota may also have protective effects in CP!-11 chemotherapy. This study aimed to elucidate mechanisms through which microbiota and dietary fibres could modify host health. Rats bearing a Ward colon carcinoma were treated with a two-cycle CPT-11/5-fluorouracil therapy recapitulating clinical therapy of colorectal cancer. Animals were fed with a semi-purified diet or a semi-purified diet was supplemented with non-digestible carbohydrates (isomalto-oligosaccharides, resistant starch, fructo-oligosaccharides, or inulin) in 3 independent experiments. Changes in intestinal microbiota, bacteria translocating to mesenteric lymphnodes, cecal GUD activity, and cecal SCFA production, and the intestinal concentration of CPT-11 and its metabolites were analysed. Non-digestible carbohydrates significantly influenced feed intake, body weight and other indicators of animal health. The identification of translocating bacteria and their quantification in cecal microbiota indicated that overgrowth of the intestine by opportunistic pathogens was not a major contributor to CPT-11 toxicity. Remarkably, fecal GUD activity positively correlated to body weight and feed intake but negatively correlated to cecal SN-38 concentrations and IL1-β. The reduction in CPT-11 toxicity by non-digestible carbohydrates did not correlate to stimulation of specific bacterial taxa. However, cecal butyrate concentrations and feed intake were highly correlated. The protective role of intestinal butyrate production was substantiated by a positive correlation of the host expression of MCT1 (monocarboxylate transporter 1) with body weight as well as a positive correlation of the abundance of bacterial butyryl-CoA gene with cecal butyrate concentrations. These correlations support the interpretation that the influence of dietary fibre on CPT-11 toxicity is partially mediated by an increased cecal production of butyrate.
Prebiotics are non-digestible food ingredients believed to beneficially affect host health by selectively stimulating the growth of the beneficial bacteria residing in the gut. Such beneficial bacteria have been reported to protect against pathogenic infections. However, contradicting results on prevention of Salmonella infections with prebiotics have been published. The aim of the present study was to examine whether S. Typhimurium SL1344 infection in mice could be prevented by administration of dietary carbohydrates with different structures and digestibility profiles. BALB/c mice were fed a diet containing 10% of either of the following carbohydrates: inulin, fructo-oligosaccharide, xylo-oligosaccharide, galacto-oligosaccharide, apple pectin, polydextrose or beta-glucan for three weeks prior to oral Salmonella challenge (107 CFU) and compared to mice fed a cornstarch-based control diet.
The mice fed with diets containing fructo-oligosaccharide (FOS) or xylo-oligosaccharide (XOS) had significantly higher (P < 0.01 and P < 0.05) numbers of S. Typhimurium SL1344 in liver, spleen and mesenteric lymph nodes when compared to the mice fed with the cornstarch-based control diet. Significantly increased amounts (P < 0.01) of Salmonella were detected in ileal and fecal contents of mice fed with diets supplemented with apple pectin, however these mice did not show significantly higher numbers of S. Typhimyrium in liver, spleen and lymph nodes than animals from the control group (P < 0.20).
The acute-phase protein haptoglobin was a good marker for translocation of S. Typhimurium in mice. In accordance with the increased counts of Salmonella in the organs, serum concentrations of haptoglobin were significantly increased in the mice fed with FOS or XOS (P < 0.001). Caecum weight was increased in the mice fed with FOS (P < 0.01), XOS (P < 0.01), or polydextrose (P < 0.001), and caecal pH was reduced in the mice fed with polydextrose (P < 0.001). In vitro fermentation in monocultures revealed that S. Typhimurium SL1344 is capable of fermenting FOS, beta-glucan and GOS with a corresponding decline in pH.
Supplementing a cornstarch-based rodent diet with 10% FOS or XOS was found to increase the translocation of S. Typhimurium SL1344 to internal organs in mice, while 10% apple pectin was found to increase the numbers of S. Typhimurium in intestinal content and feces.
Dietary fiber reduces the intestinal absorption of nutrients and the blood concentrations of cholesterol and triglycerides.
We wished to test the hypothesis that high-viscosity (HV) and low-viscosity preparations of barley and oat β-glucan modify the expression of selected genes of lipid-binding proteins in the intestinal mucosa and reduce the intestinal in vitro uptake of lipids.
Five different β-glucan extracts were separately added to test solutions at concentrations of 0.1–0.5% (wt/wt), and the in vitro intestinal uptake of lipids into the intestine of rats was assessed. An intestinal cell line was used to determine the effect of β-glucan extracts on the expression of intestinal genes involved in lipid metabolism and fatty acid transport.
All extracts reduced the uptake of 18:2 when the effective resistance of the unstirred water layer was high. When the unstirred layer resistance was low, the HV oat β-glucan extract reduced jejunal 18:2 uptake, while most extracts reduced ileal 18:2 uptake. Ileal 18:0 uptake was reduced by the HV barley extract, while both jejunal and ileal cholesterol uptakes were reduced by the medium-purity HV barley extract. The inhibitory effect of HV barley β-glucan on 18:0 and 18:2 uptake was more pronounced at higher fatty acid concentrations. The expression of genes involved in fatty acid synthesis and cholesterol metabolism was down-regulated with the HV β-glucan extracts. β-Glucan extracts also reduced intestinal fatty-acid-binding protein and fatty acid transport protein 4 mRNA.
The reduced intestinal fatty acid uptake observed with β-glucan is associated with inhibition of genes regulating intestinal uptake and synthesis of lipids. The inhibitory effect of β-glucan on intestinal lipid uptake raises the possibility of their selective use to reduce their intestinal absorption.
PMID: 19716281 CAMSID: cams3668
β-Glucan; Cholesterol; Fatty acids; Intestinal lipid uptake; Sterol regulatory element-binding protein; Fatty acid synthesis
Consumption of prebiotics may modulate gut microbiota, subsequently affecting the bacterial composition, metabolite profile, and human health. Previous studies indicate that also changes in intestinal integrity may occur. In order to explore this further we have investigated the effect of the putative prebiotic xylo-oligosaccharides (XOS) on the gut microbiota and intestinal integrity in male Wistar rats. As changes in intestinal integrity may be related to the expected bifidogenic effect of XOS, we additionally addressed effects of supplementation with a commensal Bifidobacterium pseudolongum (BIF) isolated from the same breed of laboratory rats.
Changes in faecal and caecal bacterial composition determined by 16S rRNA gene sequencing and quantitative PCR for selected bacterial groups revealed that the overall bacterial composition did not differ markedly between the control (CON), XOS, and BIF groups, when correcting for multiple comparisons. However as hypothesised, the relative abundance of Bifidobacterium spp. was increased in XOS-fed rats as compared to CON in faecal samples after the intervention. Also Lactobacillus spp. was increased in both the XOS and BIF groups in caecum content compared to CON. Intestinal permeability determined in vivo by FITC-dextran permeability and in vitro using extracted caecum water in trans-epithelial resistance (TER) assay showed no effect on intestinal integrity in either the XOS or the BIF groups. However, the expression of occludin, which is part of the tight junction complex, was increased in the XOS group compared to the CON group.
Supplementation with XOS or a commensal Bifidobacterium pseudolongum had very limited effects on intestinal integrity in rats as only significant change in expression of a single tight junction protein gene was found for the XOS group.
Xylooligosaccharides; Bifidobacterium; Gut microbiota; Intestinal integrity