Validation of a dual in vivo-in vitro assay for predicting the digestibility of nutrients in humans.

BACKGROUND: The validation of a dual in vivo-in vitro digestibility assay ('dual digestibility assay') for separately predicting the upper-tract, hindgut and total tract digestibility of nutrients in humans, as estimated using organic matter digestibility (OMD), is described. Human upper-tract OMD was predicted using an animal (rat) model with digesta from the terminal ileum collected from rats fed one of four complete human diets (wheat bran diet, pectin diet, mixed low-fibre diet, mixed high-fibre diet). Large intestinal OMD was predicted using an in vitro hindgut fermentation assay employing a human faecal inoculum and with the rat ileal digesta as the substrate. RESULTS: A comparison of total tract OMD of the four diets from a human balance study (OMDhuman ) with that predicted using the dual digestibility assay (OMDdual ) showed no significant differences (P > 0.05). OMDhuman and OMDdual were highly correlated (r = 0.953, P = 0.047). CONCLUSION: The dual digestibility assay accurately predicts the uptake of dietary nutrients (as grams of organic matter) in humans over the total tract. The assay is able to separately quantify the digestibility of nutrients in the upper and lower digestive tracts. The validation of the dual digestibility assay needs to be extended to a wider range of human diets.

Optimisation of inoculum concentration and incubation duration for an in vitro hindgut dry matter digestibility assay.

The aim was to optimise inoculum concentration and incubation duration for a published in vitro hindgut digestibility assay using ileal digesta (sampled from the chicken or rat) pertaining to a mixed human diet as the substrate. The study also sought to investigate the digestibility of the inoculum itself and the importance of correcting for this in the in vitro hindgut digestion assay. For two assays, hindgut dry matter digestibility (DMD) generally increased with inoculum concentration. A sharp increase in DMD observed at high inoculum concentrations may have been related to problems with filtering the inoculum. An inoculum concentration of 160 g/L was considered optimal based on close agreement of observed values with previously published in vivo hindgut dry matter digestibility for similar diets. One of the methods was chosen for optimisation of the duration of incubation. Ileal substrate organic matter digestibility (OMD) increased with increasing time of incubation for all diets. An incubation duration of 18 h using a mean inoculum digestibility value for calculation purposes was considered optimal based on observed in vivo hindgut DMD values in humans, but there was little difference in estimated in vitro hindgut DMD between 18 and 24h incubation durations. Although considerably lower than the OM digestibility of the substrate (no less than 51% after 48 h), the OM digestibility of the inoculum (13% after 48 h) itself was of significance in calculating estimated digestibility. The optimised assay gave realistic hindgut OMD values ranging from 55% to 79% (Wheat Bran Diet and Pectin Diet, respectively) using an 18-h incubation duration.

Low-dose whey protein-enriched water beverages alter satiety in a study of overweight women.

AIM: To determine the effect of low-dose whey protein-enriched water beverages on postprandial satiety and energy intake (EI). METHODS: Fifty overweight and mildly obese women were given 500 mL water-based beverages on 4 different occasions in a double blind, cross-over study. The beverages were reasonably matched for colour, flavour, sweetness and contained 0% (water control, 0 g, 8 kJ), 1% (5 g, 93 kJ), 2% (10 g, 178 kJ) and 4% (20 g, 348 kJ) whey protein by weight (ClearProtein8855™). Following a standard evening meal and breakfast, beverages were consumed 120 min before an ad libitum lunch at which EI was measured. Feelings associated with hunger and fullness were also measured using visual analogue scales (VAS). RESULTS: 46 participants completed all 4 beverage conditions. There was a significant effect of beverage preload on hunger (beverage×time; P=0.0074), where each of the 1%, 2% and 4% w/w protein beverages decreased hunger compared to the water control (P<0.05). Suppression of hunger was also maintained for longer following the protein beverages (Friedman test, P=0.013). Fullness (beverage×time; P=0.0020) and satisfaction (beverage×time; P=0.0356) were both increased by the 1% and 4% protein beverages (P<0.05). EI at lunch decreased by up to 8 percent (control vs 4% protein, delta=-247 kJ, Tukey's post hoc, P>0.05) when escalating protein doses were added to the water preload (water control, 3028 kJ; 1%, 3080 kJ; 2%, 2924 kJ; 4%, 2781 kJ), only partial compensation for the added energy. CONCLUSIONS: These low-dose, whey protein-enriched water beverages significantly altered short term postprandial satiety, however the effect was not sufficient to impact on food intake when assessed 2 h after consumption.

Predicted apparent digestion of energy-yielding nutrients differs between the upper and lower digestive tracts in rats and humans.

The apparent digestibility of energy-yielding nutrients (carbohydrate, protein, and fat) was predicted in the human upper digestive tract and large bowel separately for 4 diverse diets containing either a single dietary fiber source [wheat bran and pectin (PE) diets] or mixed fiber sources [low-fiber (LF) and high-fiber (HF) diets). A human balance study was undertaken to determine fecal energy and nutrient excretion and a rat model was used to predict human ileal energy and nutrient excretion. Total tract energy digestibility ranged from 92 (HF diet) to 96% (PE diet and LF diet), while at the ileal level it ranged from 79 to 86% for the HF diet to the LF diet. The predicted upper-tract digestion of starch, sugars, and fat was high, with ileal digestibilities exceeding 90% for all diets. Nonstarch polysaccharides were poorly digested in the upper tract for all diets except in the PE diet. The daily quantity of protein excreted at the ileal level was between 2 (HF diet) and 5 (PE diet) times higher than that at the fecal level. The large differences between fecal and ileal nutrient loss highlight that fecal digestibility data alone provide incomplete information on nutrient loss. There is a need to be able to routinely determine the uptake of energy in the upper and lower digestive tracts separately.

The effect of hydrolysis time on amino acid analysis.

Determining the amino acid content of a protein involves the hydrolysis of that protein, usually in acid, until the protein-bound amino acids are released and made available for detection. Both the variability in the ease of peptide bond cleavage and differences in the acid stability of certain amino acids can significantly affect determination of a protein's amino acid content. By using multiple hydrolysis intervals, a greater degree of accuracy can be obtained in amino acid analysis. Correction factors derived by linear extrapolation of serial hydrolysis data are currently used. Compartmental modeling of the simultaneous hydrolysis (yield) and degradation (decay) of amino acids by nonlinear multiple regression of serial hydrolysis data has also been validated and applied to determine the amino acid composition of various biological samples, including egg-white lysozyme, human milk protein, and hair. Implicit in the routine application of serial hydrolysis in amino acid analysis, however, is an understanding that correction factors, derived either linearly or through the more accurate nonlinear multiple regression approach, need to be determined for individual proteins rather than be applied uniformly across all protein types.