Human milk oligosaccharide consumption by probiotic and human-associated bifidobacteria and lactobacilli.

Human milk contains high concentrations of nondigestible complex oligosaccharides (human milk oligosaccharides; HMO) that reach the colon and are subsequently fermented by the infant gut microbiota. Using a high-throughput, low-volume growth determination, we evaluated the ability of 12 lactobacilli and 12 bifidobacteria strains, including several commercial probiotics, to ferment HMO and their constituent monomers. Of the 24 strains tested, only Bifidobacterium longum ssp. infantis ATCC 15697 and Bifidobacterium infantis M-63 were able to ferment 3'-sialyllactose, 6'-sialyllactose, 2'-fucosyllactose, and 3'-fucosyllactose. Bifidobacterium infantis M-63 degraded almost 90% of the 2'-fucosyllactose but left most of the fucose in the supernatant, as detected by HPLC. Among bifidobacteria, only the B. infantis strains and Bifidobacterium breve ATCC 15700 were able to ferment lacto-N-neotetraose (LNnT). Among lactobacilli, Lactobacillus acidophilus NCFM was found to be the most efficient at utilizing LNnT. The extracellular ß-galactosidase (lacL, LBA1467) of L. acidophilus NCFM cleaves the terminal galactose of LNnT for growth, leaving lacto-N-triose II in the media as detected by HPLC. Inactivation of lacL abolishes growth of L. acidophilus NCFM on LNnT. These results contribute to our knowledge of HMO-microbe interactions and demonstrate the potential for synbiotic combinations of pre- and probiotics.

Prebiotic Galactooligosaccharide Metabolism by Probiotic Lactobacilli and Bifidobacteria.

Galactooligosaccharides (GOS) are bifidogenic and lactogenic prebiotics; however, GOS utilization is strain-dependent. In this study, commercially available bifidobacteria and lactobacilli probiotic strains were evaluated for growth in the presence of GOS. Several bifidobacteria and lactobacilli grew on GOS; however, the specific GOS oligomers utilized for growth differed. A subset of probiotic bifidobacteria and lactobacilli revealed three different GOS utilization profiles delineated by the degrees of polymerization (DP) of GOS: (1) utilization of 2 DP GOS, (2) utilization of ≤3 DP GOS, and (3) utilization of all DP GOS. Specifically, Lactobacillus acidophilus NCFM (LA_NCFM) was found to efficiently consume all GOS oligomers. Extracellular ß-galactosidase activity in the cell-free supernatant of LA_NCFM correlated with accumulation of galactose. In a LacL-deficient LA_NCFM strain, GOS utilization was abolished. This is the first report of LacL's role in GOS metabolism in LA_NCFM. In vitro GOS utilization should be considered when GOS are delivered with probiotic bifidobacteria and lactobacilli.

In vitro impact of human milk oligosaccharides on Enterobacteriaceae growth.

Human milk oligosaccharides (HMOs) function as prebiotics in the infant gut by selecting for specific species of bifidobacteria and bacteroides, but little is known about their potential utilization by Enterobacteriaceae, the relative numbers of which have been linked to the onset of necrotizing enterocolitis in preterm infants. In this study, the in vitro growth of purified HMOs and other related carbohydrates was evaluated using individual strains of Enterobacteriaceae and an Enterobacteriaceae consortia enriched from piglet feces. None of the Enterobacteriaceae strains grew on 2'-fucosyllactose, 6'-sialyllactose, or lacto-N-neotetraose (LNnT); however, several strains were capable of utilizing galactooligosaccharides, maltodextrin, and the mono- and disaccharide components of HMOs for growth. The enriched fecal consortia also did not grow on 2'-fucosyllactose or 6'-sialyllactose, but there was limited growth on LNnT. It was concluded that 2'-fucosyllactose and 6'-sialyllactose supplementation of preterm infant formula should not lead to an increase in Enterobacteriaceae; however, supplementation with LNnT may require further study.

Galacto-oligosaccharides may directly enhance intestinal barrier function through the modulation of goblet cells.

SCOPE: Here we have tested the hypothesis that prebiotic galacto-oligosaccharides (GOS) may enhance mucosal barrier function through direct modulation of goblet cell function. METHODS AND RESULTS: Human adenocarcinoma-derived LS174T cells, which exhibit an intestinal goblet cell-like phenotype, were used to examine the non-prebiotic effects of GOS on goblet cell functions. LS174T cells were treated with GOS, and the expression of goblet cell secretory product genes mucin 2 (MUC2), trefoil factor 3 (TFF3), resistin-like molecule beta (RETNLB) and the Golgi-sulfotransferase genes, carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), was determined by real-time quantitative RT-PCR. In addition, the abundance of CHST5, TFF3 and RETNLB was confirmed by Western blot analysis. Following treatment with GOS for 72 h, the expression of MUC2 was significantly upregulated 2-4-fold, CHST5 and RETNLB, 5-7-fold, and TFF3 2-4-fold. Western blot analysis demonstrated increased abundance of RETNLB, TFF3 and CHST5. Addition of the Th2 cytokine IL-13 along with GOS resulted in synergistic induction of RETNLB and CHST5. IL-8 secretion was not affected by GOS treatment, suggesting that the effects of GOS are not mediated through an inflammatory pathway. CONCLUSION: Collectively, the data indicate that GOS may enhance mucosal barrier function through direct stimulation of intestinal goblet cells.

Fecal metabolomics of healthy breast-fed versus formula-fed infants before and during in vitro batch culture fermentation.

Nontargeted metabolomics analyses were used (1) to compare fecal metabolite profiles of healthy breast-fed (BF) and formula-fed (FF) infants before and during in vitro fermentation in batch culture and (2) to evaluate fecal metabolomics in assessing infant diet. Samples from healthy BF (n = 4) or FF (n = 4) infants were individually incubated at 37( °)C in anaerobic media containing 1% (wt/vol) galactooligosaccharides, 6'-sialyllactose, 2'-fucosyllactose, lacto-N-neotetraose, inulin, and gum arabic for up to 6 h, and supernatants were analyzed using GC/MS and LC/MS/MS to assess changes in various compounds. Comparison of over 250 metabolites prior to incubation showed that BF samples contained higher relative concentrations (P ≤ 0.05) of 14 compounds including human milk oligosaccharides and other metabolites presumably transferred through breast feeding (linoelaidate, myo-inositol) (P ≤ 0.05). Conversely, feces from FF infants contained 41 identified metabolites at higher levels (P ≤ 0.05) with many indicative of carbon limitation and protein fermentation. Our data are consistent with the notion that carbon-limited cultures catabolize protein and amino acids to obtain energy, whereas the provision of fermentable carbohydrate creates anabolic conditions relying on amino acids for bacterial growth. Results also suggest that fecal metabolomics can be a useful tool for studying interactions among diet, microbes, and host.

In vitro fermentation characteristics of select nondigestible oligosaccharides by infant fecal inocula.

This study sought to determine the fermentation potential of human milk oligosaccharides by mixed cultures of fecal microbiota from breast-fed (BF; n = 4) and formula-fed (FF; n = 4) infants. Infant fecal inocula were incubated with galactooligosaccharide (GOS), gum arabic (GA), HP inulin (HP), 2'-fucosyllactose (2'FL), 6'-sialyllactose (6'SL), and lacto-N-neotetraose (LNnt). GOS, 2'FL, and LNnT had a lower pH than other substrates after 3 h (P < 0.05). Total short chain fatty acids were greater in FF compared to BF infants at 6 h (P = 0.03) and 12 h (P = 0.01). GOS, 2'FL, and LNnT led to more lactate than 6'SL, HP, and GA (P < 0.05). Bifidobacteria populations were greater (P = 0.02) in FF at 6 and 12 h. Overall, GOS, 2'FL, and LNnT were rapidly fermented by infant fecal inocula, 6'SL and HP had intermediate fermentability, while GA had little fermentation. Inocula from FF infants fermented substrates more rapidly than inocula from BF infants, which should be accounted for when evaluating substrate fermentability. These data will aid in future infant formulas to promote optimal gut health in FF infants.

Effect of a viscous fiber-containing nutrition bar on satiety of patients with type 2 diabetes.

To assess the satiety-promoting effect of a novel viscous fiber-containing nutrition bar, overweight and obese adult subjects with type 2 diabetes (n=99) were randomized into a double blind, crossover study. They were fed a 300kcal lunch consisting of viscous fiber-containing nutrition bars (VF) or commercial nutrition control bars designed for people with diabetes (CH). VF resulted in a 27.1% increase in fullness (p<0.05), a 15.8% decrease in prospective consumption (p<0.001), and a 14.2% decrease in hunger (p<0.001) in the 120-240min post-lunch areas under the curve (AUC) compared to CH, but no differences were observed for nausea or thirst (p>0.05). Similar results were noted for 0-300min AUC values. VF were associated with greater frequencies and intensities of abdominal distention (p<0.001) and flatulence (p<0.001), and greater frequency of stools (p<0.001) compared to CH, but there were no differences in mean or maximum (loosest) stool consistency (p>0.05). Overall, these results suggest that VF could be a useful tool in weight management of type 2 diabetes.

Gamma-cyclodextrin lowers postprandial glycemia and insulinemia without carbohydrate malabsorption in healthy adults.

OBJECTIVE: Preliminary in vitro and animal studies have shown that gamma-cyclodextrin (GCD) is a slowly and completely digestible carbohydrate. The objective of this study was to determine the glycemic and insulinemic responses to GCD in humans. Breath hydrogen excretion was measured simultaneously to evaluate carbohydrate malabsorption. METHODS: Healthy adult subjects (N = 32) received 50 g of carbohydrate from GCD or a rapidly digested maltodextrin (MD) in a double-masked, randomized, crossover design. Plasma glucose (fingerstick) and serum insulin (venous) concentrations were measured at baseline and at 15, 30, 45, 60, 90, 120, 150, and 180 min postprandially. Breath hydrogen excretion was monitored hourly for 8 h postprandially. The severity of gastrointestinal symptoms (nausea, cramping, distension, flatulence) was rated by the subjects on a ranked scale for two 24-h periods postprandially. RESULTS: The mean baseline-adjusted peak plasma glucose concentration was 47% lower (P < 0.001), and the mean baseline-adjusted peak serum insulin concentration was decreased by 45% (P < 0.001) after subjects consumed GCD compared with MD. Positive incremental area under the curve (0-120 min) was reduced 45% for plasma glucose and 49% for serum insulin by GCD compared with MD (P < 0.001 in each case). There were no differences between GCD and MD in the proportion of positive breath hydrogen tests and both carbohydrates were equally well tolerated. CONCLUSIONS: GCD effectively lowers postprandial glycemia and insulinemia compared with MD, without resulting in appreciable carbohydrate malabsorption or gastrointestinal intolerance.

Fructooligosaccharides and Lactobacillus acidophilus modify gut microbial populations, total tract nutrient digestibilities and fecal protein catabolite concentrations in healthy adult dogs.

The objective of this research was to determine whether fructooligosaccharides (FOS) and (or) Lactobacillus acidophilus (LAC) affected concentrations of gut microbial populations, fermentative end products and nutrient digestibilities in healthy adult dogs. Two experiments were performed using 40 adult dogs (20 dogs/experiment). Dogs in each experiment were randomly assigned to one of 4 treatments. Twice daily, treatments were given orally via gelatin capsules: 1) 2 g sucrose + 80 mg cellulose; 2) 2 g FOS + 80 mg cellulose; 3) 2 g sucrose + 1 x 10(9) colony forming units (cfu) LAC; or 4) 2 g FOS + 1 x 10(9) cfu LAC. Data were analyzed by the General Linear Models procedure of SAS. In Experiment 1, FOS resulted in lower (P = 0.08) Clostridium perfringens and greater fecal butyrate (P = 0.06) and lactate (P < 0.05) concentrations. In Experiment 2, FOS supplementation increased (P < 0.05) bifidobacteria, increased lactobacilli (P = 0.08), increased fecal lactate (P = 0.06) and butyrate (P < 0.05), and decreased (P < 0.05) fecal ammonia, isobutyrate, isovalerate and total branched-chain fatty acid concentrations. Dogs fed LAC had the highest fecal concentrations of hydrogen sulfide and methanethiol in Experiment 1 and dimethyl sulfide in Experiment 2, whereas dogs fed FOS had the lowest concentrations of these compounds. Overall, FOS appeared to enhance indices of gut health by positively altering gut microbial ecology and fecal protein catabolites, whereas LAC was more effective when fed in combination with FOS rather than fed alone.

Fructooligosaccharides and Lactobacillus acidophilus modify bowel function and protein catabolites excreted by healthy humans.

The objective of this experiment was to determine whether supplementation with fructooligosaccharides (FOS) and (or) Lactobacillus acidophilus (LAC) affected bowel function and fermentative end-product concentrations in feces of healthy humans. Subjects (n = 68) were enrolled in a randomized, double-blind, placebo-controlled, parallel study design. After a 4-wk baseline period, subjects consumed one of the following treatments twice daily for 4 wk: 1) 3 g sucrose + 80 mg cornstarch; 2) 3 g FOS + 80 mg cornstarch; 3) 3 g sucrose + 1 x 10(9) colony-forming units (cfu) LAC; or 4) 3 g FOS + 1 x 10(9) cfu LAC. Subjects completed 7-d bowel function forms and 3-d dietary records before collection of fresh stool samples at wk 4, 6 and 8. Statistical analyses were performed on differences from baseline using the General Linear Models procedure of SAS. Fructooligosaccharides decreased fecal ammonia (P = 0.07) and isovalerate (P = 0.12) concentrations at wk 6. At wk 8, FOS tended (P = 0.11) to increase fecal putrescine concentrations. Lactobacillus decreased fecal organic matter percentage at wk 6 (P < 0.05) and 8 (P = 0.07). At wk 6 and 8, LAC increased (P < 0.05) fecal 2-methylindole, total indole, and total indole and phenol concentrations. At wk 8, LAC decreased fecal agmatine (P = 0.08) and phenylethylamine (P < 0.05) concentrations. In conclusion, FOS and LAC modified several metabolites associated with gut health, with FOS tending to be beneficial (decreased fecal protein catabolites) and LAC being negative (increased fecal protein catabolites).

Probiotics and prebiotics: A brief overview.

Probiotics and prebiotics are 2 food ingredients that confer physiologic effects through the gastrointestinal tract. Probiotics have been defined as viable microorganisms that (when ingested) have a beneficial effect in the prevention and treatment of specific pathologic conditions. These microorganisms are believed to exert biological effects through a phenomenon known as colonization resistance, whereby the indigenous anaerobic flora limits the concentration of potentially pathogenic (mostly aerobic) flora in the digestive tract. Other modes of action, such as supplying enzymes or influencing enzyme activity in the gastrointestinal tract, may also account for some of the other physiologic effects that have been attributed to probiotics. Conversely, prebiotics are nondigestible food ingredients that beneficially affect host health by selectively stimulating the growth and/or activity of 1 or a limited number of bacteria in the colon. The prebiotic, fructooligosaccharide (FOS), is found naturally in many foods, such as wheat, onions, bananas, honey, garlic, or leeks. They can also be isolated from chicory root or synthesized enzymatically from sucrose. Fermentation of FOS in the colon results in a large number of physiologic effects including increasing the numbers of bifidobacteria in the colon, increasing calcium absorption, increasing fecal weight, shortening of gastrointestinal transit time, and possibly lowering blood lipid levels. Other effects that have been observed in animal models include an increase in cecal weight and an increase in fecal nitrogen excretion. The increase in bifidobacteria has been assumed to benefit human health by producing compounds to inhibit potential pathogens, by reducing blood ammonia levels, and by producing vitamins and digestive enzymes.