Validation of a Combined In Vivo/In Vitro Ileal Fermentation Assay in the Growing Pig to be Used as a Model for Adult Humans.

BACKGROUND: An in vivo/in vitro ileal fermentation assay using growing pigs has been developed but not yet formally validated. OBJECTIVES: This study aimed to validate the in vivo/in vitro ileal fermentation assay by comparing in vitro fermentation values with those obtained in vivo in growing pigs. The effect of raising pigs under different environmental conditions was also investigated. METHODS: Thirty piglets (1.59 ± 0.31 kg body weight, mean ± standard deviation) were subjected to 1 of 3 treatments: artificially reared (AR) (nonfarm, laboratory housing conditions) from postnatal day (PND) 7 (AR group), inoculated orally with human infant fecal extracts from birth until PND 8 and AR (AR+ group), or conventionally reared on a farm (control group). Starting at PND 7, the AR and AR+ pigs received human infant formula for 3 wk, followed by a human-type diet for 5 wk. Control pigs were weaned on the farm and, on PND 63, relocated to the laboratory animal facility. From PND 63, all pigs received a human-type diet. On PND 78, pigs were killed, after which ileal digesta were collected to perform an in vitro ileal fermentation (in vitro organic matter [OM] fermentability and organic acid production) and to determine digesta microbial composition and dietary OM fermentability in vivo. RESULTS: The rearing regimen resulted in only a few differences in ileal microbial taxonomic composition. The rearing regimen generally did not affect the in vitro production of individual organic acids. The in vivo and in vitro OM fermentability of proximal ileal digesta (19.7 ± 2.04%; mean ± SEM) was similar (P > 0.05) for the AR and control pigs but not for the AR+ pigs. CONCLUSIONS: The control-rearing regimen was preferred over AR or AR+ because of ease of implementation. The in vitro ileal fermentation assay accurately predicted the in vivo OM fermentability.

The kinetics of amino acid disappearance in the small intestine is related to the extent of amino acids absorbed in growing pigs.

This study evaluated the importance of a correction for amino acids (AA) released into the hindgut on a measure of AA absorption kinetics and tested whether AA absorption kinetics are related to the extent of AA absorption using the growing pig as a model for humans. Thirty-six nine-week-old pigs (22·3 kg) received a diet containing whey protein as the sole protein source for 8 d. Pigs received their last meal containing the indigestible marker titanium dioxide before being euthanised at 1, 2, 3, 4, 6 and 12 h post-feeding. The entire content of each gastrointestinal tract (GIT) region was collected to determine AA released into the hindgut, and the kinetics and extent of AA absorption (uncorrected and corrected for AA entering the hindgut). Amounts of AA released into the hindgut increased over time (e.g. 33 and 180 mg of Glu for 4 and 6 h post-feeding). The corrected apparent amount of each AA absorbed from the GIT lumen after 4 h post-feeding was generally lower (P ≤ 0·05) than the uncorrected counterpart. Differences in both the kinetics and extent of AA absorption were observed across AA. For example, the time to reach half of the apparent AA absorption (T50) was 1·5 and 3·4 h for Met and Arg, respectively, whereas their extent of apparent absorption was 93 and 73 %. Negative correlations between parameters related to kinetics and the extent of apparent absorption were observed (e.g. for T50 r = -0·81; P < 0·001). The kinetics of AA absorption is related to the extent of AA absorption.

Differences in small intestinal apparent amino acid digestibility of raw bovine, caprine, and ovine milk are explained by gastric amino acid retention in piglets as an infant model.

Background: The rate of stomach emptying of milk from different ruminant species differs, suggesting that the small intestinal digestibility of nutrients could also differ across these milk types. Objective: To determine the small intestinal amino acid (AA) digestibility of raw bovine, caprine, and ovine milk in the piglet as an animal model for the infant. Methods: Seven-day-old piglets (n = 12) consumed either bovine, caprine, or ovine milk diets for 15 days (n = 4 piglets/milk). On day 15, fasted piglets received a single meal of fresh raw milk normalized for protein content and containing the indigestible marker titanium dioxide. Entire gastrointestinal tract contents were collected at 210 min postprandially. Apparent AA digestibility (disappearance) in different regions of the small intestine was determined. Results: On average, 35% of the dietary AAs were apparently taken up in the small intestine during the first 210 min post-feeding, with 67% of the AA digestibility occurring in the first quarter (p ≤ 0.05) and 33% in the subsequent two quarters. Overall, except for isoleucine, valine, phenylalanine, and tyrosine, the small intestinal apparent digestibility of all AAs at 210 min postprandially in piglets fed ovine milk was, on average, 29% higher (p ≤ 0.05) than for those fed bovine milk. Except for lysine, there was no difference in the apparent digestibility (p > 0.05) of any AAs between piglets fed caprine milk or ovine milk. The apparent digestibility of alanine was higher (p ≤ 0.05) in piglets fed caprine milk than those fed bovine milk. When apparent digestibility was corrected for gastric AA retention, only small differences in the small intestinal apparent digestibility of AAs were observed across milk types. Conclusion: Bovine, caprine and ovine milk had different apparent small intestinal AA digestibility at 210 min postprandially. When corrected for gastric AA retention, the differences in apparent digestibility across species largely disappeared. The apparent AA digestibility differed across small intestinal locations.

In Vitro Ileal Fermentation is Affected More by the Fiber Source Fermented than the Ileal Microbial Composition in Growing Pigs.

Background: The fermentation of undigested material in the ileum is quantitatively important. However, the respective contributions of the microbial composition and the substrate to ileal fermentation are unclear. Objective: This aim was to investigate the contribution of microbial composition and fiber source to in vitro ileal fermentation outcomes. Methods: Thirteen ileal cannulated female pigs (Landrace/Large White; 9-wk-old; 30.5 kg body weight) were given diets containing black beans, wheat bread, chickpeas, peanuts, pigeon peas, sorghum, or wheat bran as the sole protein source for 7 d (100 g protein/kg dry matter diet). On day 7, ileal digesta were collected and stored at -80°C for microbial analysis and in vitro fermentation. For each diet, a pooled ileal inoculum was prepared to ferment different fiber sources (cellulose, pectin, arabinogalactan, inulin, fructooligosaccharides, and resistant starch) for 2 h at 37°C. Organic matter fermentability and organic acid production were determined following in vitro fermentation. Data were analyzed using a 2-way ANOVA (inoculum × fiber). Results: Forty-five percent of the identified genera in the digesta differed across diets. For instance, the number of Lactococcus was 115-fold greater (P ≤ 0.05) in the digesta of pigs fed the pigeon pea diet than for pigs fed the wheat bran diet. For both in vitro organic matter fermentability and organic acid production, there were significant (P ≤ 0.05) interactions between the inoculum and the fiber source. For instance, pectin and resistant starch resulted in 1.6- to 31-fold more (P ≤ 0.05) lactic acid production when fermented by the pigeon pea inoculum than other inocula. For specific fiber sources, statistically significant correlations were found between the number of bacteria from certain members of the ileal microbial community and fermentation outcomes. Conclusions: Both the fiber source fermented and the ileal microbial composition of the growing pig affected in vitro fermentation; however, the effect of the fiber source was predominant.Curr Dev Nutr 2023;x:xx.

Available Lysine in Foods as Determined in Adult Ileostomates.

BACKGROUND: When foods are subjected to heat and pressure, a proportion of lysine is structurally altered and some will revert to lysine because of acid hydrolysis during amino acid analysis. Altered lysine molecules may be partly absorbed but are not utilized post-absorption. OBJECTIVES: A guanidination-based bioassay has been developed to determine true ileal digestible reactive lysine but it was only used in animal models (pig and rat). The objective of this study was to apply the assay and determine whether there is a difference between true ileal digestible total lysine and true ileal digestible reactive lysine in adult human ileostomates. METHODS: Six cooked or processed foods were analyzed for total lysine and reactive lysine. Six adults with a fully functioning ileostomy (4 women and 2 men; age range: 41-70 y; BMI: 20.8-28.1) participated. Foods for which total lysine > reactive lysine (cooked black beans, toasted wheat bread, and processed wheat bran), as well as a protein-free diet, were consumed by the ileostomates (n = 5 to 8; test food meals contained 25 g protein) and ileal digesta was collected. Each food was ingested twice by each participant, and the digesta was pooled. The order of foods for each participant was determined according to a Youden square. True ileal digestible total lysine and true ileal digestible reactive lysine were determined and a 2-way ANOVA model was used to analyze data. RESULTS: True ileal digestible reactive lysine was significantly lower than true ileal digestible total lysine for cooked black beans, toasted wheat bread, and processed wheat bran by 89%, 55%, and 85%, respectively (P< 0.05). CONCLUSIONS: True ileal digestible reactive lysine was lower than true ileal digestible total lysine, similar to that previously reported in pigs and rats, demonstrating the importance of determining the true ileal digestible reactive lysine contents of processed foods.

Cooked Rice-Based and Wheat-Based Food Structure Influenced Digestion Kinetics and Glycemic Response in Growing Pigs.

BACKGROUND: How starch-based food structure can affect the rate and extent of digestion in the small intestine and resulting glycemic response is not properly understood. One possible explanation is that food structure influences gastric digestion, which subsequently determines digestion kinetics in the small intestine and glucose absorption. However, this possibility has not been investigated in detail. OBJECTIVES: Using growing pigs as a digestion model for adult humans, this study aimed to investigate how physical structure of starch-rich foods affects small intestinal digestion and glycemic response. METHODS: Male growing pigs (21.7 ± 1.8 kg, Large White × Landrace) were fed one of the 6 cooked diets (250-g starch equivalent) with varying initial structures (rice grain, semolina porridge, wheat or rice couscous, or wheat or rice noodle). The glycemic response, small intestinal content particle size and hydrolyzed starch content, ileal starch digestibility, and portal vein plasma glucose were measured. Glycemic response was measured as plasma glucose concentration collected from an in-dwelling jugular vein catheter for up to 390 min postprandial. Portal vein blood samples and small intestinal content were measured after sedation and euthanasia of the pigs at 30, 60, 120, or 240 min postprandial. Data were analyzed with a mixed-model ANOVA. RESULTS: The plasma glucose Δmaxoverall and iAUCoverall for couscous and porridge diets (smaller-sized diets) were higher than that of intact grain and noodle diets (larger-sized diets): 29.0 ± 3.2 compared with 21.7 ± 2.6 mg/dL and 5659 ± 727 compared with 2704 ± 521 mg/dL⋅min, for the smaller-sized and larger-sized diets, respectively (P < 0.05). Ileal starch digestibility was not significantly different between the diets (P ≥ 0.05). The iAUCoverall was inversely related to the starch gastric emptying half-time of the diets (r = -0.90, P = 0.015). CONCLUSIONS: Starch-based food structure affected the glycemic response and starch digestion kinetics in the small intestine of growing pigs.

Effects of the maize-derived protein zein, and the milk proteins casein, whey, and α-lactalbumin, on subjective measures of satiety and food intake in normal-weight young men.

Protein is considered to be the most satiating food macronutrient and the satiating effect may be dependent on the source of the protein. The maize-derived protein zein and milk protein casein have been shown previously to lower stomach emptying rate more than dairy whey protein, but the effect of zein on satiety has not been evaluated. The objective was to compare the satiating effects of zein and casein, with whey protein and its protein component α-lactalbumin. The study was a randomised crossover design with thirteen normal-weight men (mean age 27.8 years and mean BMI 24.4 kg/m2) consuming isoenergetic (∼4000 kJ, ∼990 kcal) preload mixed meals enriched with Zein, Casein, whey protein isolate (Whey), α-lactalbumin (ALac), or maltodextrin carbohydrate (Carb). Consumption of an ad libitum standardised test meal of chicken fried rice and water provided 360 min following ingestion of the preload meal was measured, and subjective feelings of appetite (hunger, fullness, desire to eat, and prospective food consumption) were assessed using 100-mm visual analogue scales (VAS). There were no differences among the five preload mixed meals in the amount of chicken fried rice consumed at the ad libitum test meal (mean ± sem: 531.6 ± 35.0 g, p = 0.47) or total (preload + test meal) energy intakes (mean ± sem: 5780.5 ± 146.0 kJ, p = 0.29). The subjective VAS appetite ratings and total area under the curve responses for hunger, fullness, desire to eat, and prospective food consumption, were not different following consumption of all five preload mixed meals (p > 0.05). The findings indicate that the effects of zein and casein on satiety were not different from the satiating effects of whey protein and α-lactalbumin.

Gastric digestion of cow milk, almond milk and oat milk in rats.

In this study, gastric digestion of isocaloric and iso-macronutrient cow milk, almond milk and oat milk were compared in rats euthanized at different post-feeding times. The cow milk separated into a curd phase and a liquid phase in the rat stomach. This coagulation of the cow milk led to higher (P < 0.05) protein and lipid retention in the stomach compared with almond milk and oat milk. Almond milk oil bodies aggregated, creamed and rapidly layered in the stomach. This induced a faster (P < 0.05) gastric emptying of proteins (T1/2 = 36 min) compared with cow milk (T1/2 = 89 min) and oat milk (T1/2 = 55 min), and a slower gastric emptying of almond lipids than of almond proteins. In contrast, no significant physical change during the digestion of oat milk was found, with both the proteins and the lipids being steadily emptied from the stomach. This in vivo study provides information on the gastric digestion and emptying (and thereby nutritional characteristics) of plant-based milks compared with animal-based milks, that will be useful for the design of novel plant-based drinks.

Influence of food macrostructure on the kinetics of acidification in the pig stomach after the consumption of rice- and wheat-based foods: Implications for starch hydrolysis and starch emptying rate.

How the stomach can serve as a biochemical environment for starch digestion and the implications on starch emptying are not well-understood. Biochemical changes during gastric digestion of cooked wheat- and rice-based diets of varying particle size and microstructure were investigated using a growing pig model. In larger-particle size diets (rice grain, rice noodle, pasta), pH >3 was maintained in the proximal stomach digesta even until 240 min digestion, resulting in extended remaining amylase activity and accumulation of maltose from starch hydrolysis in the stomach. In smaller-particle size diets (couscous, rice couscous, semolina porridge), gastric acidification occurred faster to produce homogeneous intragastric pH and deactivated amylase. The hypothesis of the study was that food macrostructure would impact gastric acidification kinetics, and the resulting biochemical environment for starch hydrolysis in the stomach may further affect the mechanisms of food breakdown in the stomach and gastric emptying of starch.

Comparison of True Ileal Amino Acid Digestibility between Adult Humans and Growing Pigs.

BACKGROUND: It is not feasible to determine the true ileal amino acid (AA) digestibility of protein sources in humans on a routine basis, and the growing pig has been recommended as an animal model for this purpose but requires further validation. OBJECTIVES: To determine and compare true ileal AA digestibility between adult human ileostomates and growing cannulated pigs for a range of food proteins. METHODS: Seven protein sources (black beans, bread, collagen, pigeon peas, wheat bran, whey protein isolate, and zein) that spanned the range of digestibilities typically seen in foods were evaluated. Six female growing pigs received each of the protein sources, as well as a protein-free diet, and digesta were collected via ileal T-cannula. Adult human ileostomates consumed the same protein sources (5-8 ileostomates, depending on the protein source), as well as a protein-free diet, and digesta were collected. Titanium dioxide and celite were included in the diets as indigestible markers. True ileal AA digestibility coefficients were determined. RESULTS: There was a significant effect of protein source (P ≤ 0.001) for all AAs. The effect of species was not significant (P > 0.05) except for total lysine (but not for available lysine). When analyzed within diets, the statistically significant species effect for true lysine digestibility was found for black beans only. Pig and human digestibility values were generally highly and significantly (P ≤ 0.05) correlated. A linear regression equation derived for true ileal AA digestibility (given as coefficients) determined in the human and pig for the overall mean of all AAs was (y = human, x = pig) y = 1.00x - 0.010, with the slope not statistically significant (P > 0.05) from unity and the intercept not different (P > 0.05) from zero. CONCLUSIONS: True ileal AA digestibility values determined in the growing pig can be directly used for predicting digestibility in adult humans.

Structural changes in milk from different species during gastric digestion in piglets.

This study investigated the structural and physicochemical changes that occur in milk, a naturally designed complex structured emulsion, during gastric digestion using the bottle-fed piglet as an animal model. The gastric digestions of cow, goat, and sheep milk were compared in male piglets euthanized at different postfeeding times to collect the stomach chyme. The cow and noncow milks separated into curd (aggregated caseins) and liquid (mostly soluble whey) phases in the piglet's stomach. For milk from all the species, the curd remained longer in the stomach because of its slow disintegration, whereas the liquid phase emptied readily. The majority of the fat globules were found to be entrapped within the protein network of the curd. The rate of release of fat globules was strongly dependent on the breakdown of the surrounding protein network of the curd. The consistency of the gastric curds changed as digestion progressed, with goat and sheep milk curds having relatively softer curd consistency and less fused protein networks, especially toward the end of digestion. This might have led to the lower protein and fat retention in the goat and sheep milk curds and relatively faster gastric emptying of these nutrients from goat and sheep milk in comparison to cow milk. This in vivo study provided new and enhanced understanding of the mechanisms of the gastric digestion of milk from different species. It may have implications for developing bioinspired structures for the controlled digestion and delivery of nutrients.

The Complementarity of Amino Acids in Cooked Pulse/Cereal Blends and Effects on DIAAS.

The aim was to study the complementary effect between cereals and pulses on protein quality. The values for the digestible indispensable amino acid score (DIAAS) in cooked cereals and pulses, given alone, and blends of cooked cereals and pulses, were determined. True ileal digestibility (TID) values of amino acids for adult humans were obtained. It is difficult to determine ileal amino acid digestibility in humans directly, and for this reason, the growing pig is often used to obtain such values, as a preferred animal model. Seven growing pigs fitted with a T-cannula at the terminal ileum were allotted to a 7 × 6 incomplete Latin square with seven semi-synthetic diets (cooked mung bean, adzuki bean, millet, adlay, mung bean + millet, adzuki bean + adlay, and an N-free diet) and six 7-day periods. The mean TID values for crude protein differed significantly (p < 0.05), with millet having the highest digestibility (89.4%) and the adzuki bean/adlay mixture having the lowest (79.5%). For lysine, adzuki bean had the highest TID (90%) and millet had the lowest (70%). For the mean of all the amino acids, there was a significant (p < 0.05) effect of diet, with the TID ranging from 72.4% for the adzuki bean/adlay mixture to 89.9% for the adzuki beans. For the older child, adolescent, and adult, the DIAAS (%) was 93 for mung beans, 78 for adzuki beans, 22 for millet, 16 for adlay, and 66 for mung beans + millet, and 51 for adzuki beans + adlay. For mung beans, valine was first-limiting, and the SAA for adzuki beans, while lysine was first-limiting for the other foods. Chinese traditional diets, containing both cereals and pulses, are complementary for most, but not all of the indispensable amino acids.

Correction: Tracking physical breakdown of rice- and wheat-based foods with varying structures during gastric digestion and its influence on gastric emptying in a growing pig model.

Correction for 'Tracking physical breakdown of rice- and wheat-based foods with varying structures during gastric digestion and its influence on gastric emptying in a growing pig model' by Joanna Nadia et al., Food Funct., 2021, DOI: 10.1039/D0FO02917C.

Fatty Acids from Different Fat Sources and Dietary Calcium Concentration Differentially Affect Fecal Soap Formation in Growing Pigs.

BACKGROUND: Calcium (Ca) can complex with free fatty acids in the gastrointestinal tract (GIT), leading to the formation of insoluble unabsorbable Ca-fatty acid soaps, contributing to the proposed effect of Ca on weight loss in humans. OBJECTIVES: We determined the effect of dietary Ca concentration and the individual long-chain fatty acids on Ca-fatty acid soap formation and fatty acid digestibility. METHODS: Nine-week-old crossbreed male pigs (n = 144; mean ± SD body weight: 21.7 ± 0.15 kg) were used as an animal model for digestion in the adult human. The animals received purified diets containing 4 Ca concentrations (0, 2, 4, and 6 g/kg diet) and 4 fat sources (tallow, palmolein oil, soybean oil, and olive oil) in a completely randomized design. Fatty acids, Ca, and Ca-fatty acid soaps were determined in feces (n = 9 per diet). RESULTS: Increasing dietary Ca led to a 4-fold increase (P ≤ 0.05) in excreted palmitic and stearic acid when diets contained tallow or palmolein oil as the major fat source. More than 80% of these excreted fatty acids were present as soaps. For the tallow-based diets, increasing dietary Ca led to a decrease in stearic acid digestibility from 91% to 66% (P ≤ 0.01) and in palmitic acid digestibility from 96% to 83% (P ≤ 0.01). For the olive oil- and soybean oil-based diets dietary Ca did not (P > 0.05) influence fatty acid excretion. CONCLUSIONS: Ca-fatty acid soap formation led to decreased fat absorption in the GIT of growing pigs, which supports the hypothesis that higher dietary Ca concentrations reduce fat absorption.

Tracking physical breakdown of rice- and wheat-based foods with varying structures during gastric digestion and its influence on gastric emptying in a growing pig model.

There is currently a limited understanding of the effect of food structure on physical breakdown and gastric emptying of solid starch-based foods during gastric digestion. Moisture uptake, pH, particle size, rheological, and textural properties of six solid starch-based diets from different sources (Durum wheat and high amylose white rice) and of different macrostructures (porridge, native grain, agglomerate/couscous, and noodle) were monitored during 240 min of gastric digestion in a growing pig model. Changes in the physical properties of the gastric digesta were attributed to the influence of gastric secretions and gastric emptying, which were both dependent on the buffering capacity and initial macrostructure of the diets. Differences between the proximal and distal stomach regions were found in the intragastric pH and texture of the gastric digesta. For example, rice couscous, which had the smallest particle size and highest buffering capacity among the rice-based diets, had the shortest gastric emptying half-time and no significant differences between proximal and distal stomach digesta physical properties. Additionally, a relationship between gastric breakdown rate, expressed as gastric softening half-time from texture analysis, and gastric emptying half-time of dry matter was also observed. These findings provide new insights into the breakdown processes of starch-based solid foods in the stomach, which can be beneficial for the development of food structures with controlled rates of breakdown and gastric emptying during digestion.

Development of an Assay to Determine the Amount of Ca-Fatty Acid Soaps in Feces.

BACKGROUND: The quantification of fecal Ca-fatty acid soaps is important to understand how fatty acids behave in the gastrointestinal tract. OBJECTIVE: As current methods to extract Ca-fatty acid soaps from feces give low recoveries an accurate assay to determine the amount of fatty acid soaps in feces was developed. METHOD: Ca-fatty acid soaps are determined indirectly after non-soap fatty acid compounds have been extracted from the feces. Synthetic Ca-fatty acid soaps of different chain lengths (C12-C18) and degree of saturation (C18:0-C18:2) were incubated with several solvents to find the solvents that least-solubilize the Ca-fatty acid soaps. A three-step extraction was devised using extractions with hexane, hexane-isopropanol and water either at room temperature or at 60°C, 37°C, or 80°C, respectively. Feces were spiked with free fatty acids, Ca-fatty acid soaps, Na-fatty acid salts, and phospholipids. RESULTS: All of the free fatty acids and phospholipids and almost all of the Na-fatty acid salts were removed and 98% of Ca-lauric acid soap, 99% of Ca-stearic acid soap, and 93% of oleic acid soap were recovered. CONCLUSIONS: The method is suitable for determining fatty acids in the form of Ca-fatty acid soaps in feces. HIGHLIGHTS: New method to determine fecal Ca-fatty acid soaps. Consistent and high recovery of fatty acid-soaps.

Spatial-temporal changes in pH, structure and rheology of the gastric chyme in pigs as influenced by egg white gel properties.

The hypothesis is that the characteristics of ingested protein gels influences the subsequent in vivo gastric digestion process. Three egg white gels (EWGs) of identical composition but differing in structure and texture were prepared and fed to pigs. Sampling throughout a 6 h postprandial period, and at different locations in the stomach of the pigs, enabled a detailed spatial-temporal mapping of the pH, dry matter content, particle size and rheological properties. The results showed different gastric acidification kinetics implying an effect of the gel structure and/or texture. The most elastic and cohesive gel resulted in the highest median particle size and the most viscoelastic chyme. Distal and proximal regions of the stomach did not differ in terms of dry matter content, particle size distribution or rheological properties. These results demonstrate the consequences of protein food structure on gastric chyme properties, and thus suggest an effect on the digestion process.