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It is hypothesized that the digestible indispensable amino acid score (DIAAS) can be determined based on dynamic in vitro gastrointestinal digestion experiments as replacement for invasive animal studies. We determined the in vitro DIAAS for immature herring eggs (roe) proteins in comparison with reference proteins. The true ileal digestibility of protein and indispensable amino acids (IAA) was measured under human conditions simulated in a gastrointestinal model (tiny-TIM). The in vitro true ileal digestibility of ovalbumin, cooked and raw chicken egg white, and casein was similar to that found in humans ( r 2 = 0.96), providing a casual observation to support the validity of tiny-TIM. The digestibility of the immature herring egg proteins was 71% to 92%. The highest IAA digestibility was found for immature whole herring egg protein (55%-80%) in comparison to immature herring egg membrane and immature de-membraned herring protein (50%-70%). The DIAAS as recommended by FAO for children and adults, but measured in vitro, were 91% for immature whole herring egg protein (lysine first limiting), 71% for immature herring egg membrane protein (histidine first limiting), and 88% for immature herring egg de-membraned protein (sulfur AA first limiting). True ileal protein and amino acid digestibility can be determined in a dynamic gastrointestinal model, such as tiny-TIM, which can be used for estimating the DIAAS. Immature herring egg proteins, a previously underutilized resource, were determined to be an important and valuable source of IAA for human consumption. [ABSTRACT FROM AUTHOR]

Galacto-oligosaccharides (GOS) are considered to be prebiotic, altliough the contribution of specific members of the microbiota to GOS fermentation and the exact microbial metabolites that are produced upon GOS fermentation are largely unknown. We aimed to determine this using uniformly 13C-labeled GOS. The normal (control) medium and unlabeled or 13C-labeled GOS was added to a dynamic, validated, in vitro model of the large-intestine containing an aduit-type microbiota. Liquid-chromatography MS was used to measure the incorporation of C label into metabolites. 16S-rRNA stable isotope probing coupled to a phylogenetic micro-array was used to determine label incorporation in microbial biomass. The priman/ members within the complex microbiota that were directly involved in GOS fermentation were shown to be Bifidobacterium longum, B. bifidum, B. catenulatum, Lactobacillus gasseh, and L. salivarius, in line with the prebiotic effect of GOS, although some other species incorporated label also. GOS fermentation led to an increase in acetate (+49%) and lactate (+23%) compared with the control. Total organic acid production was 8.50 and 7.52 mmol/g of carbohydrate fed for the GOS and control experiments, respectively. At the same time, the cumulative production of putrefactive metabolites (branched-chain fatty acids and ammonia) was reduced by 55%. Cross-feeding of metabolites from primary GOS fermenters to other members of the microbiota was observed. Our findings support a prebiotic role for GOS and its potential to act as a synbiotic in combination with certain probiotic strains. [ABSTRACT FROM AUTHOR]

A polymerase chain reaction (PCR)-based method was developed for the identification of isolates of Bifidobacterium at the species level. Using two Bifidobacterium-specific primers directed against the 16S ribosomal gene (Bif164 and Bif662), a PCR product was obtained from the type strains of 12 different bifidobacterial species that have been found in the human alimentary tract. After purification of the PCR products, the DNA was restricted with five different restriction enzymes. The size of the different restriction fragments was used as a fingerprint for the identification of individual bifidobacterial species. The amplified ribosomal DNA restriction analysis method was subsequently used to speciate bifidobacterial isolates from child’s feces and from an in vitro model of the large intestine. [Copyright &y& Elsevier]

Protein hydrolysates are an important part of the human diet. Often, they are prepared from milk, soy, or collagen. In the present study, four different collagen hydrolysates were tested, varying in the average molecular weight and the animal source. Three types of samples, the dissolved start products, in vitro generated dialysates (containing the digested components that are potentially available for small intestinal absorption), and human serum collected after product ingestion, were analyzed using LC-MS to compare the state of the hydrolysates before and after absorption, i.e., uptake into the blood. It was found that the composition of the collagen hydrolysates prior to and after ingestion was highly complex and dynamic, which made it challenging to predefine a strategy for a targeted analysis. Therefore, we implemented a new analytical approach to first map hydrolysate data sets by performing non-targeted LC-MS analysis followed by non-targeted and targeted data analysis. It was shown that the insight gained by following such a top down (data) analytical workflow could be crucial for defining a suitable targeted setup and considering data trends beyond the defined targets. After having defined and performed a limited targeted analysis, it was found that, in our experimental setup, Hyp-Gly and especially Pro-Hyp contributed significantly as carrier to the total Hyp increase in blood after ingestion of collagen hydrolysate. [ABSTRACT FROM AUTHOR]

Carbohydrates, including starches, are an important energy source for humans, and are known for their interactions with the microbiota in the digestive tract. Largely, those interactions are thought to promote human health. Using 16S ribosomal RNA (rRNA)-based stable isotope probing (SIP), we identified starch-fermenting bacteria under human colon-like conditions. To the microbiota of the TIM-2 in vitro model of the human colon 7.4 g l−1 of [U-13C]-starch was added. RNA extracted from lumen samples after 0 (control), 2, 4 and 8 h was subjected to density-gradient ultracentrifugation. Terminal-restriction fragment length polymorphism (T-RFLP) fingerprinting and phylogenetic analyses of the labelled and unlabelled 16S rRNA suggested populations related to Ruminococcus bromii, Prevotella spp. and Eubacterium rectale to be involved in starch metabolism. Additionally, 16S rRNA related to that of Bifidobacterium adolescentis was abundant in all analysed fractions. While this might be due to the enrichment of high-GC RNA in high-density fractions, it could also indicate an active role in starch fermentation. Comparison of the T-RFLP fingerprints of experiments performed with labelled and unlabelled starch revealed Ruminococcus bromii as the primary degrader in starch fermentation in the studied model, as it was found to solely predominate in the labelled fractions. LC-MS analyses of the lumen and dialysate samples showed that, for both experiments, starch fermentation primarily yielded acetate, butyrate and propionate. Integration of molecular and metabolite data suggests metabolic cross-feeding in the system, where populations related to Ruminococcus bromii are the primary starch degrader, while those related to Prevotella spp., Bifidobacterium adolescentis and Eubacterium rectale might be further involved in the trophic chain. [ABSTRACT FROM AUTHOR]

16S rRNA-based stable isotope probing (SIP) and nuclear magnetic resonance (NMR) spectroscopy-based metabolic profiling were used to identify bacteria fermenting glucose under conditions simulating the human intestine. The TIM-2 in vitro model of the human intestine was inoculated with a GI tract microbiota resembling that of the small intestine, to which subsequently 4, 20 or 40 mM of [U-13C]-glucose were added. RNA was extracted from lumen samples after 0 (control), 1, 2 and 4 h and subjected to density-gradient ultracentrifugation. Phylogenetic analysis of unlabeled 16S rRNA revealed a microbial community dominated by lactic acid bacteria and Clostridium perfringens. Distinct 13C-incorporation into bacterial RNA was only observed for the 40-mM addition. 16S rRNA fingerprinting showed an activity drop of Lactobacillus fermentum after glucose addition, while Streptococcus bovis and C. perfringens were identified as the most active glucose-fermenters. Accordingly, NMR analysis identified lactate, acetate, butyrate and formate as the principal fermentation products, constituting up to 91% of the 13C-carbon balance. RNA-SIP combined with metabolic profiling allowed us to detect differential utilization of a general model carbohydrate, indicating that this approach holds great potential to identify bacteria involved in the fermentation of dietary relevant oligo- and polymeric carbohydrates in the human intestine. [ABSTRACT FROM AUTHOR]

The release characteristics of a unique blend of carotenoid beadlets designed to increase bioavailability were tested using the dynamic gastrointestinal model TIM‐1. Individual carotenoid bioaccessibility peaks were observed over approximately 3–4 hr in the order of lutein and zeaxanthin first, followed by lycopene, and then finally α‐ and β‐carotene; when tested as a beadlet blend or when the beadlets were compressed into tablets. Bioaccessibility measurements of 7%–20% were similar to those previously reported in literature and comparable between the two formulations, beadlet blend and tablet formulations. Total recovery of carotenoids from all compartments ranged from 70% to 90% for all carotenoids, except lycopene where almost 50% was unrecoverable after digestion in the TIM system. [ABSTRACT FROM AUTHOR]

A validated in vitro model of the large intestine ( TIM-2), set up with human or pig faeces, was used to evaluate the impact of potentially probiotic Lactobacillus amylovorus DSM 16698, administered alone (i), in the presence of prebiotic galactooligosaccharides ( GOS) (ii), and co-administered with probiotic Bifidobacterium animalis ssp. lactis Bb-12 ( Bb-12) (iii) on GOS degradation, microbial growth ( L. amylovorus, lactobacilli, bifidobacteria and total bacteria) and metabolite production. High performance anion exchange chromatography revealed that GOS degradation was more pronounced in TIM-2 inoculated with pig faeces than with human faeces. Denaturing gradient gel electrophoresis profiling of PCR-amplified 16S r RNA genes detected a more complex Lactobacillus spp. community in pig faecal material than in human faecal inoculum. According to 16S r RNA gene-targeted q PCR, GOS stimulated the growth of lactobacilli and bifidobacteria in faecal material from both materials. The cumulative production of short chain fatty acids and ammonia was higher ( P < 0.05) for pig than for human faeces. However, lactate accumulation was higher ( P < 0.05) in the human model and increased after co-administration with GOS and Bb-12. This study reinforced the notion that differences in microbiota composition between target host organisms need to be considered when animal data are extrapolated to human, as is often done with pre- and probiotic intervention studies. [ABSTRACT FROM AUTHOR]

Abstract: A high-performance liquid chromatography–high resolution Fourier transform ion cyclotron resonance mass spectrometry (HPLC–FTICR-MS) method was developed to investigate the metabolism of ginsenosides in in vitro models of the gastro-intestinal tract. The metabolites were identified by high-resolution tandem mass spectrometry. Degradation and bioconversion routes of the different ginsenosides at acidic (gastric) conditions and in the presence of intestinal microbiota were elaborated. Besides hydrolysis (deglycosylation) also hydration reactions occurred at acidic conditions. The results illustrate the value of metabolite profiling by HPLC–FTICR-MS for understanding of the mechanisms in bioavailability of herbal drugs and their metabolites. [Copyright &y& Elsevier]