Gastric Protein Digestion Research Articles

Impact of microstructure of whey protein gels on in vitro gastric protein digestion is sustained after oral structural breakdown by mastication

Gastric protein digestion can be influenced by food micro- and macrostructure. Understanding the interplay between microstructure and macrostructural breakdown during oral processing is crucial to enhance the nutritional value of protein-rich foods. This study compared the in vitro gastric protein digestion of whey protein gels differing in microstructure before and after mastication. Whey protein isolate was mixed with κ-carrageenan to obtain heat-induced gels differing in microstructure (homogeneous, coarse stranded, protein continuous, bi-continuous). Gel boli were collected from 14 participants. The number, size and total surface area of bolus fragments were determined. In vitro gastric protein digestion was quantified following the INFOGEST 2.0 protocol with minor modifications. Before mastication, coarse stranded gels showed the highest digestion rate (1.07 mmol L−1∙g dry matter−1/h), while homogeneous, protein continuous and bi-continuous gels showed lower and similar digestion rates (0.82–0.87 mmol L−1∙g dry matter−1/h). After mastication, the total surface area of coarse stranded gels increased 7.9-fold leading to a 1.9-fold increase in digestion rate. In contrast, a 3.4-fold increase in total surface area of bi-continuous gels caused a 2.8-fold increase in digestion rate. The total surface area of homogeneous and protein continuous gels increased 3.1- to 3.6-fold upon mastication resulting in a 1.7- to 1.9-fold increase in digestion rate. The increase in protein hydrolysis did not correlate with the degree of structural breakdown after mastication across gels differing in microstructure. We conclude that the impact of microstructure of whey protein gels on in vitro gastric protein digestion is sustained after oral structural breakdown by mastication.

Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model

BackgroundBovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine. ObjectivesThis study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk. MethodsNine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultra-high-temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined. ResultsDuring the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than in pigs fed raw and PNH milk. For example, the gastric release of total essential AA was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that in pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk). ConclusionsHeat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AA entering the small intestine was mainly increased by homogenization.

The concept of enzymatic disintegration of nutrients in the upper gastrointestinal tract

The aim of the study was to study the effect of salivary amylase on improving gastric protein digestion, as well as the effect of gastric protein hydrolysis on improving fat digestion. The work consisted of two parts, in the first part, in two series, the hydrolysis of proteins under the influence of gastric juice in the presence of starch and the effect of saliva amylase on the change in the total proteolytic activity of gastric juice when using substrates of a mixture of starch and proteins were investigated. In the second part of the work, the lipolytic activity of pancreatic juice with the studied proteins was studied in the presence and absence of bile. From the data obtained, it was found that the use of a mixture of starch with proteins helps to reduce the hydrolysis of proteins by gastric juice. The use of salivary amylase promotes an increase in protein hydrolysis by reducing the formation of starch-protein complexes that prevent the digestion of proteins by gastric juice. All the studied proteins, except gelatin, have an inhibitory effect on lipase in the pancreatic juice; the degree of inhibitory effect of each protein is expressed differently. It depends on their degree of digestion in gastric juice. Thus, the preliminary hydrolysis of proteins by pepsins in the stomach contributes not only to the further improvement of their hydrolysis under the influence of proteolytic enzymes of pancreatic juice, but also to the hydrolysis of fats under the influence of pancreatic lipase. It is concluded that the sequence of nutrient breakdown by enzymes of saliva, stomach and pancreas is primarily aimed at enzymatic disintegration between polysaccharides and proteins, as well as proteins and fats, in order to reduce their interaction and the formation of physical complexes. Thus, secondarily, improve the digestibility of nutrients in the upper gastrointestinal tract. The conceptual assumption of enzymatic disintegration of nutrients in the upper gastrointestinal tract allows us to substantiate the existing sequence of initial digestion of polysaccharides by saliva, as well as the initial digestion of proteins by gastric juice.

Aggregation structure induced by heat treatments mediated the gastric digestion behavior of soybean protein.

The common belief that heat treatment enhances the gastric digestion of proteins is largely based on findings from animal proteins and may not apply to all proteins, particularly plant proteins. Here, we compared the digestion characteristics of soybean protein isolates (SPI) in an in vitro semi-dynamic digestion model and found distinct effects of heat treatment on the digestion properties of plant proteins. The results revealed that heat-treated SPIs formed clots during the early stages of digestion, although the clots gradually became smaller and looser as digestion progressed, the systems remained turbid at the end of gastric digestion, indicating the lag in their emptying. Furthermore, heat treatment altered the rheological properties of SPI, resulting in increased viscosity and slower gastric emptying. These effects became more pronounced with increasing heat treatment temperatures. The fluorescence spectrum analysis indicated that heat treatment altered its conformation. This led to protein unfolding and exposure of hydrophobic groups, facilitating the formation of larger aggregates during digestion. Additionally, heat treatment exposed more cleavage sites for gastric proteases, increasing the extent of hydrolysis. Elevated levels of free amino acids and a smaller molecular weight distribution further corroborated these findings. These findings contribute to a deeper understanding of the gastric digestion characteristics of plant proteins and the relationship between protein aggregation structure and the digestion process.

Interplay between microstructure, mechanical properties, macrostructure breakdown and in vitro gastric digestion of whey protein gels

Gastric digestion of proteins is influenced by multiple factors including microstructure, mechanical properties and structure breakdown during mastication. The interplay between these factors affects protein digestion but is underexplored. This study aimed to investigate the contribution of microstructure, mechanical properties and macrostructure breakdown on in vitro whey protein gastric digestion. Whey protein isolate (WPI) was mixed with different types of polysaccharides (κ-carrageenan, ι-carrageenan, pectin) at various concentrations to obtain heat- or acid-induced gels with distinct microstructures (homogeneous, coarse stranded, protein continuous and bi-continuous) and Young's moduli (E, 19–165 kPa). Structural breakdown during mastication was mimicked crudely by cutting single gel cylinders into several smaller cubes to increase the total surface area by a factor of 2.65. In vitro gastric digestion was measured using the INFOGEST 2.0 protocol with minor modifications. Homogeneous heat-induced WPI/κ-carrageenan gels showed the highest digestion rate followed by protein continuous, coarse stranded and bi-continuous heat-induced WPI/κ-carrageenan gels with similar E. A 1.47-fold increase in E decreased the digestion rate of acid-induced homogeneous WPI/ι-carrageenan gels by a factor of 0.22. In contrast, a 1.13–1.83-fold increase in E barely changed the digestion rate of acid-induced protein continuous WPI/pectin gels. A 2.65-fold increase in total surface area increased the digestion rate of all gels by a factor of 1.35–2.54 depending on microstructure and mechanical properties. We conclude that the microstructure of protein gels affects in vitro protein gastric digestion and the impact of Young's modulus on in vitro protein gastric digestion depends strongly on the microstructure of protein gels.

Bitter Peptides YFYPEL, VAPFPEVF, and YQEPVLGPVRGPFPIIV, Released during Gastric Digestion of Casein, Stimulate Mechanisms of Gastric Acid Secretion via Bitter Taste Receptors TAS2R16 and TAS2R38.

Eating satiating, protein-rich foods is one of the key aspects of modern diet, although a bitter off-taste often limits the application of some proteins and protein hydrolysates, especially in processed foods. Previous studies of our group demonstrated that bitter-tasting food constituents, such as caffeine, stimulate mechanisms of gastric acid secretion as a signal of gastric satiation and a key process of gastric protein digestion via activation of bitter taste receptors (TAS2Rs). Here, we tried to elucidate whether dietary non-bitter-tasting casein is intra-gastrically degraded into bitter peptides that stimulate mechanisms of gastric acid secretion in physiologically achievable concentrations. An in vitro model of gastric digestion was verified by casein-fed pigs, and the peptides resulting from gastric digestion were identified by liquid chromatography–time-of-flight-mass spectrometry. The bitterness of five selected casein-derived peptides was validated by sensory analyses and by an in vitro screening approach based on human gastric parietal cells (HGT-1). For three of these peptides (YFYPEL, VAPFPEVF, and YQEPVLGPVRGPFPIIV), an upregulation of gene expression of TAS2R16 and TAS2R38 was observed. The functional involvement of these TAS2Rs was verified by siRNA knock-down (kd) experiments in HGT-1 cells. This resulted in a reduction of the mean proton secretion promoted by the peptides by up to 86.3 ± 9.9% for TAS2R16kd (p < 0.0001) cells and by up to 62.8 ± 7.0% for TAS2R38kd (p < 0.0001) cells compared with mock-transfected cells.

Gastric protein digestion of goat and cow milk infant formula and human milk under simulated infant conditions

The protein digestion kinetics of goat milk infant formula (GMF) is previously shown to be more comparable to that of human milk (HM) than cow milk infant formula (CMF). To evaluate whether gastric behaviour contributes to differences in protein digestion kinetics, fresh HM, a GMF and a CMF were subjected to in vitro gastric digestion simulating infant conditions. Coagulation behaviour, particle size distribution and viscosity of the digesta were evaluated. After centrifugation of the digesta, total solids and protein distribution, and protein hydrolysis in the cream, serum and pellet fraction were investigated. The GMF and CMF were in general similar with respect to physicochemical and protein breakdown properties. However, a number of notable differences in physicochemical behaviour were observed, which may contribute to faster initial protein digestion of GMF. HM behaved differently from both formulas. These differences provide new insights into the possibilities for improvement of infant formulas.

Ontogeny of digestive tract of Stellate sturgeon (Acipenser stellatus) from hatching to juvenile stage: Digestive enzymes activity, stomach and proximal intestine

This study was an attempt to evaluate ontogenic development of digestive enzymes activity, histomorphological changes of stomach and proximal intestine of Stellate sturgeon (Acipenser stellatus) from hatching up to 50 days post hatching (dph). The digestive enzymes activity of pepsin, trypsin, chymotrypsin, alkaline phosphatase, amylase and lipase was considered. Pepsin activity was detected in one-day-old prelarvae followed by a sharp increase at the beginning of larval stage (i.e., 8 dph) onward. However, few fluctuations coincided with changes of food types consumed by the fish. The highest chymotrypsin activity was observed at 1 dph. During larvae period, the enzyme activity showed two surges that occurred at 15 and 31 dph. Although descending trends for trypsin (with the highest activity at 8 dph) and amylase (with the highest activity at 24 dph) were observed, lipase and alkaline phosphatase revealed an incremental trend during early growth stages. At 10 dph, functional stomach was observed with epithelia containing some crypts. Intestinal morphometric indices such as intestinal folds were measurable at 4 dph; epithelium, however, appeared immediately after hatching. Pearson's correlation coefficient revealed positively related activities of trypsin, chymotrypsin and amylase. They all reflected negative relationships with pepsin, alkaline phosphatase and lipase. The findings suggest that gastric digestion of proteins become dominant during the ontogenetic development of Stellate digestive tract. Over this period, the fish relied on energy dense lipid molecules to meet their metabolic demands while they spare protein for further tissue synthesis/maintenance. Moreover, pepsin seems to be more responsive to the type of food consumed by the Stellate and might be a useful indicative of the nutritional status of the fish. The concluding remarks are discussed in light of the significance of various histological changes and their interrelations with ontogeny of digestive enzymes activity.

'The Rate at Which Digested Protein Enters the Small Intestine Modulates the Rate of Amino Acid Digestibility throughout the Small Intestine of Growing Pigs

BackgroundActinidin, a cysteine protease in kiwifruit (KF), increases both the gastric digestion and gastric-emptying rate of beef muscle protein. ObjectiveThis study aimed to determine the relation between the rate of digested nitrogen entering the small intestine (SI; a function of the extent of gastric digestion and gastric-emptying rate) and the disappearance of amino acids (AAs) in different parts of the SI at set times postfeeding. MethodsMale 9-wk-old pigs (n = 90; mean ± SD body weight: 28 ± 2.9 kg) were fed a diet containing 14% beef for 3 d. The beef-based diet was supplemented with green KF pulp (containing actinidin), gold KF pulp supplemented with actinidin, or gold KF pulp alone (no actinidin). The KF or actinidin amounts corresponded to the intake of 2 KFs/human meal. On day 3, pigs were killed at 0.5, 1, 3, 5, and 7 h postprandially. Stomach chyme was analyzed to determine the rate of digested nitrogen entering the SI. Apparent AA digestibility at set times was determined in the proximal, medial, and distal SI. Polynomial and correlation analyses were conducted. ResultsThe rate of digested nitrogen entering the SI was higher (P < 0.001) with actinidin (e.g., >44% at 5 h postprandially). Actinidin also increased the apparent AA digestibility at the proximal and medial SI (P ≤ 0.05) at set times (e.g., 42% and 15% greater for arginine, respectively), but not in general for the distal SI (P > 0.05). At the proximal SI, apparent AA digestibility was correlated more strongly with the digested nitrogen entering the SI (r = 0.73, P < 0.001; n = 57) than with gastric emptying (r = 0.64, P < 0.001) or gastric protein digestion (r = 0.57, P < 0.001). Similar trends were observed for the medial SI. ConclusionThe rate of digested nitrogen entering the SI is an accurate predictor of the rate of AA digestibility and the location of AA absorption in the pig SI.

Premature Infants have Lower Gastric Digestion Capacity for Human Milk Proteins than Term Infants.

Whether premature infants have lower gastric protein digestive capacity than term infants and the extent to which human milk proteases contribute to overall gastric digestion are unknown and were investigated in this study. Human milk and infant gastric samples were collected from 16 preterm (24-32 wk gestational age) and 6 term (38-40 wk gestational age) mother-infant pairs within a range of 5 to 42 days postnatal age. For each pair, an aliquot of human milk was adjusted to pH 4.5 and incubated for 2 hours at 37 °C to simulate the gastric conditions without pepsin (milkinc). Their gastric protein digestion capacity was measured as proteolysis (free N-terminals) and protease activities. Two-way analysis of variance followed by Tukey post hoc test was applied to compare measurements between preterm and term infants as well as among human milk, milkinc, and gastric samples. Measurements of gastric protein digestion were significantly lower in preterm infants than term infants. Overall milk protease activity did not differ between human milk samples from term- and preterm-delivering mothers. As protease activity did not increase with simulated gastric incubation, milk proteases likely contributed minimally to gastric digestion. Preterm infants have lower gastric protein digestion capacity than term infants, which could impair nutrient acquisition. Human milk proteases contribute minimally to overall gastric digestion. The limited activity of milk proteases suggests that these enzymes cannot compensate for the premature infant's overall lower gastric protein digestion.

Simulation of the gastric digestion of proteins of meat bolus using a reaction-diffusion model.

A reaction-diffusion mathematical model has been developed to predict the gastric digestion of meat proteins. The model takes into account pepsin diffusion and proton diffusion in bolus particles and the pH buffering capacity of meat. The computations show that the size of bolus particles and the change in gastric pH have a substantial effect on the percentage of protein digested in the stomach and that the pH buffering capacity of meat has to be accounted for to properly calculate the gastric digestibility of meat. The intensity of surface transfers between stomach fluid and bolus particles has a significant impact on protein digestibility, whereas the variation in pepsin content in the stomach between individuals appears to have little effect on protein digestibility. From a nutritional standpoint, the simulations show that meat protein digestibility is high under normal physiological stomach conditions. However, in a situation where masticatory capacity, hydrochloric acid secretion and gastric motor function performances are reduced, such as with advancing age, protein digestibility rapidly decreases, ultimately leading to near-zero digestibility value in the stomach in extreme cases.

Interactions between acid and proteins under in vitro gastric condition - a theoretical and experimental quantification.

The gastric digestion of proteins is influenced by the pH and the gastric pH fluctuates after food consumption. However, the dynamics of gastric pH still need to be quantitatively understood. Proteins in food strongly influences the gastric pH. Therefore, we studied the interaction between acid and proteins, including the buffer reaction and the acid diffusion in protein gels. The buffer capacity of proteins stems from its content of ionizable amino acid side groups. Based on this, we set up a model and method to parameterize the buffer capacity of proteins. Moreover, the liberated carboxyl and amino groups during enzymatic hydrolysis of protein can also contribute to the buffer capacity. While we expected protons to diffuse faster than pepsin, we found that the penetration distance of acid is comparable to that of pepsin. The buffer reaction caused the acid to concentrate tenfold in the gel compared to the bulk acid concentration. Therefore, we postulated that the buffer reaction reduces acid diffusivity in gels.

Rapid simulated gastric fluid digestion of in-seed/grain proteins expressed in genetically engineered crops

The speed of simulated gastric digestion of proteins expressed in genetically engineered (GE) crops is commonly used to inform the allergenicity risk assessment. However, persistence of purified proteins in simulated gastric fluid (SGF) is poorly correlated with the allergenic status of proteins. It has been proposed that the plant or food matrix may affect the digestion of proteins and should be considered in interpreting digestion results. Here the SGF digestion of several GE proteins both as purified preparations and in soybean, corn, and cotton seed/grain extracts (in-matrix) are compared. Cry1F, Cry1Ac, phosphinothricin acetyltransferase (PAT), aryloxyalkanoate dioxygenase-1 (AAD-1), aryloxyalkanoate dioxygenase-12 (AAD-12), and double mutant 5-enol pyruvylshikimate-3-phosphate synthase (2mEPSPS) were all found to rapidly digest both as purified protein preparations and in seed/grain extracts from GE crops expressing these proteins. Based on these results, purified protein from microbial sources is a suitable surrogate for proteins in-matrix when conducting SGF digestion studies.

Gastric digestion of protein through pancreozyme action optimizes intestinal forms for absorption, mucin formation and villus integrity

Recovery of dietary protein proceeds through two phases of digestion before suitable forms exist for absorption. The first phase engages the gastric system where low pH weakens overall structures allowing pepsin to disrupt hydrophobic bonding and enhance aqueous compatibility. Secondly, trypsin, chymotrypsin and elastase proteolysis in conjunction with carboxypeptidases A and B cooperate to form a mixture of free amino acids and peptides that progressively arise in the small intestinal lumen. Free amino acids are dominated by the aromatic, aliphatic and basic ones while resulting peptides largely involve the nonessentials.Motility convectively transfers digestion products to the unstirred water layer of the upper villus where filtration limits entry to low molecular weight solutes that can be further digested by underlying enzymes to optimize membrane absorption. Mucin oligosaccharides are credited for creating a microenvironment having reduced pH’s ∼5.5–6.0 that favors enzymes finalizing digestion as well as absorption of two type peptides. This pH is speculated to optimize peptide forms having either a zwitterion-like charge for proton gradient transfer or be non-dissociated and passively diffused. A high frequency of peptides having either glycine or proline can be rationalized as providing particularly favorable electronic terms for peptide absorption by either approach while being non-competitive.The upper villus has first access to absorbed products where goblet cells assure continuance of mucin and continuity of the unstirred water layer. Glutamine is their dominant nutrient for synthesis of mucin oligosaccharides while also providing glutamic acid for either formation of its associated protein or is consumed for energy. Dietary cystine and threonine are frequently limiting, and their assurance is necessary for the mucin core while ready availability of glycine and/or serine together with proline also foster mucin formation. Unused absorbed products descend the villus in venules adjacent to the surface and provide nutrition as well as information about the lumen for adaptation of replacement cells. Sustaining the villus with absorbed nutrients takes priority for continuation of operational efficiency before their entry into the portal system and subsequent body use.

Dietary Actinidin from Kiwifruit (Actinidia deliciosa cv. Hayward) Increases Gastric Digestion and the Gastric Emptying Rate of Several Dietary Proteins in Growing Rats1,2

Dietary actinidin influences the extent to which some dietary proteins are digested in the stomach, and it is hypothesized that the latter modulation will in turn affect their gastric emptying rate (GE). In this study, the effect of dietary actinidin on GE and gastric digestion of 6 dietary protein sources was determined in growing rats. Each dietary protein source [beef muscle, gelatin, gluten, soy protein isolate (SPI), whey protein isolate, and zein] was included in 2 semisynthetic diets as the sole nitrogen source. For each protein source, 1 of the 2 diets contained actinidin [76.5 U/g dry matter (DM)] in the form of ground freeze-dried green kiwifruit (Actinidia deliciosa cv. Hayward), whereas the other diet contained freeze-dried gold kiwifruit (Actinidia chinensis cv. Hort16A), which is devoid of actinidin (3.4 U/g DM). For both diets, dietary kiwifruit represented 20% of the diet on a DM basis. The real-time GE was determined in rats gavaged with a single dose of the diets using magnetic resonance spectroscopy over 150 min (n = 8 per diet). Gastric protein digestion was determined based on the free amino groups in the stomach chyme collected from rats fed the diets (n = 8 per diet) that were later killed. GE differed across the protein sources [e.g., the half gastric emptying time (T½) ranged from 157 min for gluten to 266 min for zein] (P < 0.05). Dietary actinidin increased the gastric digestion of beef muscle (0.6-fold), gluten (3.2-fold), and SPI (0.6-fold) and increased the GE of the diets containing beef muscle (43%T½) and zein (23%T½;P < 0.05). There was an inverse correlation between gastric protein digestion and DM retained in the stomach (r = −0.67;P < 0.05). In conclusion, dietary actinidin increased gastric protein digestion and accelerated the GE for several dietary protein sources. GE may be influenced by gastric protein digestion, and dietary actinidin can be used to modulate GE and protein digestion in the stomach of some dietary protein sources but not others.

Actinidin Enhances Gastric Protein Digestion As Assessed Using an in Vitro Gastric Digestion Model

Consumption of kiwifruit has long been claimed anecdotally to assist in gastric digestion. This has generally been assumed to be due to the presence of the proteolytic enzyme actinidin; however, there is little published evidence supporting this assumption. This paper reports the findings of an in vitro study that examined the effect of kiwifruit proteases (actinidin) on the digestion of a range of common food proteins under simulated gastric conditions. An extract from green kiwifruit containing actinidin was prepared. Several protein sources derived from soy, meat, milk, and cereals were incubated in the presence or absence of the kiwifruit extract using an in vitro digestion system consisting of incubation with pepsin at pH 1.9, simulating gastric digestion in humans. The digests were subjected to gel electrophoresis (SDS-PAGE). For some protein sources, simulated digestion in the presence of kiwifruit extract resulted in a substantially greater loss of intact protein and different peptide patterns from those seen after digestion with pepsin alone. As an example, the addition of actinidin extract enhanced the digestion of alpha-, beta-, and kappa-caseins in sodium caseinate by 37, 33, and 48%, respectively. Under simulated gastric conditions, kiwifruit extract containing actinidin enhanced the digestion of some, but not all, food proteins over and above that found with pepsin alone.

A Possible Application of Monosodium Glutamate to Nutritional Care for Elderly People

Recently, it has been clarified that glutamate (Glu) can stimulate the umami taste as well as the visceral sensation to help the gastric protein digestion. Our survey suggests the possibility that the amount of free Glu in hospital foods is lower than that in ordinary foods. In the present study, monosodium glutamate (MSG) was supplemented to meals for 11 elderly inpatients during 2 months, and the fortification effects on their nutritional status, general condition, and quality of life (QOL) were investigated. The degree of recognition was improved, and peripheral lymphocytes were increased, even when there was no change in nutritional intake or protein nutritional status. Based on these results, we concluded that appropriate utilization of Glu for nutritional care of the elderly people would be useful for improving QOL.

Acid-induced unfolding kinetics in simulated gastric digestion of proteins

Stability in simulated gastric fluid (SGF) is used as one element in a weight-of-evidence evaluation aimed at determining if a novel protein present in food represents an allergenic risk. The SGF assay is designed to measure the susceptibility of a protein to the enzyme pepsin under acidic conditions, and results are often considered to be primarily a reflection of the vulnerability of a protein to pepsin hydrolysis. However, proteins may not be susceptible to protease cleavage in their native folded conformation, and several studies confirm that protein unfolding is often a prerequisite for pepsinolysis. Here, we explore protein unfolding kinetics as a limiting factor in the digestion of proteins in SGF. Theoretical digestion patterns generated using consecutive and simultaneous exponential models, and the fit of a two stage consecutive exponential model (unfolding followed by pepsinolysis) to laboratory data, support unfolding kinetics as playing a critical role in determining the speed at which many proteins digest in SGF. Results also support modeling of the terminal phase of digestion with a single exponential decline as being a good stability estimate for the most recalcitrant protein form.

Stimulatory effect of ingested protein and/or free amino acids on the secretion of the gastro-endocrine hormone cholecystokinin and on tryptic activity, in early-feeding herring larvae, Clupea harengus

In the larvae of many marine teleosts, the stomach is absent until they approach or attain meta- morphosis. Consequently, the formation of chyme con- taining specific free amino acids from the gastric digestion of protein, which are believed to be signals initiating the release of the digestive hormone cholecystokinin (CCK), is lacking. CCK, when secreted into the blood circulation from specialized intestinal cells, stimulates gallbladder motility and is a key factor causing the release of pan- creatic digestive enzymes into the gut lumen. Using first- feeding Atlantic herring larvae (Clupea harengus )a s a model, the aim of the present study was to determine if a CCK response together with tryptic activity could be elicited in larvae ingesting dietary protein and/or FAA. Larvae were tube fed single lamellar liposome vesicles (SLV) containing: (1) physiological saline (PS), (2) bovine serum albumin (BSA), (3) specific free amino acids (FAA), or (4) a ratio (1:1) of BSA and FAA. The CCK and trypsin levels were then assayed (radio-immunoas- say) at 0, 15, 60 and 120 min after tube feeding. A marked CCK response was elicited in all treatments compared to the PS control at 15 and 30 min and was significant (p<0.05) at 120 min after tube feeding. Larvae tube fed the FAA treatment exhibited CCK levels that increased linearly from 1.6 to 5.6 fmol mg -1 dry weight (DW) after 2 h of digestion, although this response was below the BSA and BSA:FAA treatments. The BSA and BSA:FAA treatments, after 15 min of digestion, showed a rapid CCK increase, over the PS and the FAA liposome treat- ments, to 8.1 and 5.4 fmol mg -1 DW, respectively. At the end of the assay, BSA and BSA:FAA demonstrated similar levels (10.2 and 9.2 fmol mg -1 DW, respectively). Larvae tube fed the PS control or the FAA liposome treatment did not demonstrate any appreciable increase in tryptic activity during the 2 h digestion period (0.03-0.071 and 0.03-0.048 mU mg -1 DW, respectively). In contrast, the BSA:FAA treatment increased from 0.03-0.148 mU mg -1 DW 1 h after feeding, which was significantly (p<0.05) higher than the PS and FAA liposomes, and then decreased markedly (0.085 mU mg -1 DW) after 2 h of digestion. The larvae tube fed BSA liposomes, however, demonstrated steadily increasing tryptic activity throughout the sampling period, attaining 0.255 mU mg -1 DW after 2 h, which was significantly (p<0.05) more than all the other treatments. The results showed that ingested liposomes containing FAA or the protein BSA or a combination of these two nutrients ef- fectively stimulated CCK production in first-feeding herring larvae. In contrast, liposomes containing only physiological saline did not elicit a CCK response. In addition, liposomes containing BSA stimulated tryptic activity in herring larvae, which was not observed in fish fed liposomes that included only FAA or PS. This suggests that a suitable protein substrate is required to regulate protein digestion.

Studies on gastric digestion of protein and carbohydrate, gastric secretion and exocrine pancreatic secretion in the growing pig.

Six pigs, initially of 35 kg mean live weight, were each fitted with a re-entrant cannula. This was formed on either side of a short pouch of duodenum into which the pancreatic duct opened and which contained a simple cannula linked to the centre of the re-entrant cannula. Each pig received two diets: diet A was based on wheat starch, sucrose and casein, while diet B was based on barley and soya-bean meal. The diets were given in equal amounts at 12 h intervals. Digesta and pancreatic juice were collected continuously during three 12 h periods for each pig on each diet. Mean duodenal output: dietary intake values for diets A and B respectively were: digesta 1.80, 2.86; dry matter 1.05, 1.03; nitrogen 1.05, 1.06; trichloroacetic acid (TCA)-soluble N 7.69, 9.10; glucose 0.97, 0.89. For diet A the proportion of TCA-soluble N in total N rose from 13 to 50% during 12 h, while it was approximately 50% throughout 12 h for diet B. Mean total pepsin (EC 3.4.23.1) activities (units/24 h) were 760449 (diet A) and 1 466 571 (diet B). Salivary and gastric secretions were calculated to be approximately 4 and 8 kg/24 h for diets A and B respectively. Mean flows in pancreatic juice (g/24 h) for diets A and B respectively were: juice 1204, 2182; protein 10.94, 12.10; N 1.98, 2.14; ash 9.46, 17.31; sodium 3.88, 6.91; potassium 0.23, 0.54; calcium 0.031, 0.046; phosphorus 0.024, 0.026. Mean total enzyme activities (units x 10(-3)/24 h) for diets A and B respectively were: trypsin (EC 3.4.21.4) 138, 114; chymotrypsin (EC 3.4.21.1) 84, 84; carboxypeptidase A (EC 3.4.2.1) 5, 4; carboxypeptidase B (EC 3.4.2.2) 15, 17; amylase (EC 3.2.1.1) 1061, 981. It was calculated that the minimum amount of endogenous N from saliva and gastric secretion was 0.3-0.6 g in 24 h. This assumes no absorption of N occurred anterior to the duodenal cannula.