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.

Meat and fish serve as important protein sources in the companion animal diet; however, limited protein digestibility data are available for assessing protein digestibility differences among good-quality protein sources. Beef loin, pork loin, chicken breast, pollock fillet, and salmon fillet were evaluated for composition, protein digestibility, and AA bioavailability using the immobilized digestive enzyme assay, cecectomized rooster assay, and ileally cannulated dog assay. Pollock contained the greatest amount of CP, total essential AA (TEAA), and total nonessential AA (TNEAA; DM basis; 96.9, 38.6, and 50.3%, respectively). Salmon contained the next greatest amounts (92.8, 36.4, and 44.6%), followed by chicken (90.3, 36.1, 43.2%). Beef had the least CP content (82.7%), but had slightly greater TEAA and TNEAA concentrations (33.9, 42.0%) compared with pork (86.2, 33.6, 41.3%). Immobilized digestive enzyme assay values were greatest for pollock fillet (0.71) and least for chicken breast (0.52). Beef loin, pork loin, and salmon fillet were similar (0.63, 0.62, and 0.64, respectively). Standardized TEAA and TNEAA digestibility coefficients, evaluated using the cecectomized rooster assay, were greatest (P < 0.05) for pollock fillet (90.4 and 89.8%, respectively) and least (P < 0.05) for chicken breast (86.6 and 85.9%, respectively) and salmon fillet (87.8 and 86.4%, respectively). Dogs assigned to a 5 x 5 Latin square design were fed 5 diets, with each test substrate as the major protein source. No significant differences (P > 0.05) were found in ileal digestibility of protein. Values ranged from 88.9% for chicken to 90.5% for pork loin and pollock fillet. Ileal TEAA and TNEAA coefficients were not different among test substrates, with values between 91.7 and 92.7%, and 88.8 and 90.4%, respectively. Total tract CP apparent digestibility values ranged from 94.4 to 94.8%, with no differences noted among treatments. Despite marked differences in composition and predicted and standardized digestibility values, when the protein sources were added to diets at a concentration of approximately 30% (25% of total energy intake), no differences in test protein substrates were noted in either ileal or total tract nutrient digestibility.

A galactoglucomannan oligosaccharide (GGMO) obtained from fiberboard production was evaluated as a dietary supplement for dogs. The GGMO substrate contained increased concentrations of oligosaccharides containing mannose, xylose, and glucose, with the mannose component accounting for 35% of DM. Adult dogs assigned to a 6 × 6 Latin square design were fed 6 diets, each containing a different concentration of supplemental GGMO (0, 0.5, 1, 2, 4, and 8%) that replaced dietary cellulose. Total tract DM and OM apparent digestibilities increased (P < 0.001) linearly, whereas total tract CP apparent digestibility decreased (P < 0.001) linearly as dietary GGMO substrate concentration increased. Fecal concentrations of acetate, propionate, and total short-chain fatty acids increased (P ≤ 0.001) linearly, whereas butyrate concentration decreased (P ≤ 0.001) linearly with increasing dietary concentrations of GGMO. Fecal pH decreased (P ≤ 0.001) linearly as dietary GGMO substrate concentration increased, whereas fecal score increased quadratically (P ≤ 0.001). Fecal phenol (P ≤ 0.05) and indole (P ≤ 0.01) concentrations decreased linearly with GGMO supplementation. Fecal biogenic amine concentrations were not different among treatments except for phenylethylamine, which decreased (P < 0.001) linearly as dietary GGMO substrate concentration increased. Fecal microbial concentrations of Escherichia coli, Lactobacillus spp., and Clostridium perfringens were not different among treatments. A quadratic increase (P ≤ 0.01) was noted for Bifidobacterium spp. as dietary GGMO substrate concentration increased. The data suggest positive nutritional properties of supplemental GGMO when incorporated in a good-quality dog food.

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.

Fermentable carbohydrates may enhance the ability of the gastrointestinal tract to defend against pathogenic infection. We hypothesized that a mannose-rich, galactoglucomannan oligosaccharide-arabinoxylan (GGMO-AX) complex would positively impact immune status and prevent weight loss resulting from acute coccidiosis (Eimeria acervulina) infection of chicks. Using a completely randomized design, 1-d-old commercial broiler chicks (n = 160; 4 replications/treatment; 5 chicks/replication) were assigned to one of 4 corn-soybean meal-based diets containing supplemental GGMO-AX (0, 1, 2, or 4%) that replaced dietary cellulose. On d 9 posthatch, an equal number of chicks on each diet were inoculated with either distilled water (sham control) or E. acervulina (1 × 10(6) oocysts). All birds were euthanized on d 7 postinoculation (PI) for collection of cecal contents and duodenal tissue. Overall, BW gain of chicks was not affected by diet PI, whereas infection decreased (P < 0.01) weight gain on d 0 to 7 PI. Feed intake was not affected by dietary treatment, but infection decreased (P < 0.01) feed intake on d 0 to 7 PI. Overall, infection, but not diet, decreased (P < 0.01) G:F on d 0 to 7 PI. Cecal propionate concentrations were independently affected by infection and diet, while butyrate concentrations were affected only by infection (P = 0.02). Cecal Bifidobacterium spp. populations were affected (P < 0.01) by diet, with the 2% GGMO-AX resulting in the highest cfu/g of cecal contents (on a DM basis). Messenger RNA expression of all duodenal cytokines evaluated was affected by infection status (P ≤ 0.02) but not by dietary treatment alone. Supplementing 4% GGMO-AX consistently resulted in the greatest fold change in proinflammatory cytokine expression, while inhibiting antiinflammatory cytokine expression, which indicates a more robust innate immune response. Despite decreasing performance, 4% dietary GGMO-AX improved select fermentation indices and the innate intestinal immune response to an acute E. acervulina infection.

Fermentable carbohydrates may enhance the ability of the gastrointestinal tract to defend against a pathogenic infection. We hypothesized that a galactoglucomannan oligosaccharide-arabinoxylan (GGMO-AX) complex would positively affect immune status and prevent colonization and shedding in Salmonella typhimurium-infected chicks. Using a completely randomized design, 1-d-old commercial broiler chicks (n = 240 chicks; 4 replications/treatment; 5 chicks/replication) were assigned to 1 of 6 dietary treatments differing in concentration of GGMO-AX (0, 1, 2, or 4%) or containing 2% Safmannan or 2% short-chain fructooligosaccharides. Cellulose was used to make diets iso-total dietary fiber. On d 10 posthatch, an equal number of chicks on each diet were inoculated with either phosphate-buffered saline (sham control) or Salmonella typhimurium (1 × 10(8) cfu). All birds were euthanized on d 10 postinoculation (PI) for collection of intestinal contents and select tissues. Body weight gain and feed intake of chicks were greater (P < 0.05) in infected chicks PI for all time periods, except for weight gain on d 0 to 3 PI. Gain:feed was affected (P < 0.05) by diet, with Safmannan-fed chicks having the highest G:F and 1% GGMO-AX-fed chicks having the lowest. The GGMO-AX substrate demonstrated effects similar to a prebiotic substrate as indicated by increased cecal short-chain fatty acid concentrations, decreased cecal pH, and increased populations of Lactobacillus spp. and Bifidobacteria spp. as dietary GGMO-AX concentration increased. Excreta Salmonella typhimurium populations on d 5 and 10 PI, and ileal and cecal Salmonella typhimurium populations, tended to be affected (P < 0.10) by the main effect of diet. Messenger RNA expression of IFN-γ in the cecal tonsils was the only cytokine independently affected by infection and diet (P < 0.01). Chicks fed 2 and 4% GGMO-AX had similar expressions of IFN-γ and IL-1β, regardless of infection, suggesting that Salmonella typhimurium virulence was suppressed. Dietary supplementation with GGMO-AX resulted in prebiotic-like effects but did not limit Salmonella typhimurium intestinal colonization or shedding, but possibly decreased the virulence of Salmonella typhimurium within the digestive tract.

It is of interest to discover new fermentable carbohydrate sources that function as prebiotics. This study evaluated the hydrolytic digestibility, fermentative capacity, and microbiota modulating properties of Temulose molasses, four hydrolyzed fractions of Temulose molasses, short-chain fructooligosaccharides (scFOS), and a yeast cell wall preparation (Safmannan). These substrates resisted in vitro hydrolytic digestion. Each substrate was fermented in vitro using dog fecal inoculum, and fermentation characteristics were quantified at 0 and 12 h. All Temulose molasses substrates decreased pH by at least 0.64 unit and resulted in greater (P < 0.05) butyrate and total short-chain fatty acid (SCFA) production compared to scFOS and Safmannan. Temulose molasses substrates resulted in higher (P < 0.01) or equal Bifidobacterium spp. concentrations compared to scFOS. Temulose molasses substrate and its fractions demonstrated prebiotic characteristics as indicated by low hydrolytic digestibility, high fermentability, and enhanced growth of microbiota considered to be beneficial to health.

"Temulose" is the trade name for a water-soluble molasses produced on a large scale (300-400 tonnes per year) as a byproduct of the fiberboard industry. The feedstock for Temulose is predominantly a single species of pine ( Pinus taeda ) grown and harvested in stands in southeastern Texas. Because of the method of production, the molasses was predicted to consist of water-soluble hemicelluloses, mainly arabinoxylan-type and galactoglucomannan-type oligosaccharides, plus minor components of lignin, but no detailed structural study had been reported. The structure and composition of the molasses has now been deduced by a combination of MALDI-TOF mass spectrometry, size exclusion chromatography, proton and (13)C NMR techniques, and classic carbohydrate analysis. Limited acid hydrolysis released a series of galactoglucomannan oligosaccharides (GGMO) that were selectively recovered from the acid-labile arabinogalactan by precipitation with ethanol. The precipitate was named "Temulose brown sugar" because of its appearance, and is shown to consist of GGMO with a degree of polymerization (DP) from 4 to 13, with the major component being DP 5-8. The structure of these oligosaccharides is a β-1,4-linked backbone of Man and Glc residues, with occasional α-1,6 branching by single galactosyl units.