Simulated Gastric Fluid Digestion Research Articles

Evaluation of protein‐phytochemical interactions in a hypoallergenic edible peanut matrix (813.10)

Allergenic peanut proteins (PP) are stable to digestion, remain immunoreactive and trigger IgE‐mediated allergic reactions in PP sensitive individuals. In this study, a hypoallergenic peanut protein‐phytochemical matrix (PPPM), produced by complexing cranberry juice (CB) with light roast peanut flour, was evaluated to determine the nature of protein‐phytochemical interactions and the stability of the binding post‐ingestion. PPPM or unmodified peanut flour were subjected to a simulated gastric fluid digestion. Aliquots of the enzymatic mixture were taken at different time points and inhibited with either sodium bicarbonate (pH ~7‐8) or heat (pH ~2) to stop the digestive process. Digested PP fragments and undigested PP were examined on SDS‐PAGE and screened for their peanut‐specific IgE binding capacity using Western blotting, respectively. SDS‐PAGE revealed that PP were more soluble at a low pH than at a basic pH. PP treated with CB had reduced solubility at both acidic and basic pH, compared to untreated PP. However digestion of both PPPM and unmodified peanut flour resulted in formation of highly soluble small molecular weight PP fragments at either pH. Examination of the Western blots revealed that digested PP fragments or undigested PP from PPPM exhibited altered IgE binding, compared to proteins from unmodified peanut flour. Modification of PP with CB phytochemicals led to the formation of insoluble complexes with altered IgE binding to the protein epitopes, suggesting altered PP digestion and IgE binding capacity.Grant Funding Source: Supported by Everett W. Byrd Endowment and NC State appropriations for NCSU at Kannapolis

Physicochemical behaviour of WPI-stabilized emulsions in in vitro gastric and intestinal conditions

Most studies on the in vitro lipid digestion of protein-stabilized emulsions have been carried out under simulated gastric and intestinal conditions. In this study, the digestion behaviour of whey protein isolate (WPI)-stabilized emulsions was examined under simulated intestinal fluid (SIF) conditions (pH 7.5, 2.5mg bile salts/mL and 0.8mg pancreatin/mL) after the emulsions had been digested in a model simulated gastric fluid (SGF) containing pepsin (pH 1.6 and 3.2mg pepsin/mL) for different times. The droplet size, ζ-potential, microstructure, surface protein and amount of free fatty acids released were examined. The results indicated that WPI emulsions did not undergo pronounced changes in droplet size and microstructure during SGF digestion followed by coalescence during the subsequent SIF digestion. When WPI emulsions were treated with SGF, α-lactalbumin and a portion of β-lactoglobulin proteins adsorbed at the interface were hydrolysed by pepsin, resulting in small peptides being produced as characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis. In general, digestion in SGF containing pepsin accelerated coalescence of the emulsion droplets during subsequent digestion in SIF containing pancreatic lipase. However, the changes in the size, the microstructure and the proteolysis of the interfacial proteins of the emulsions under gastric conditions did not influence the rate and the extent of lipid digestion in the subsequent intestinal environment.

Influence of gastric digestive reaction on subsequent in vitro intestinal digestion of sodium caseinate-stabilized emulsions

In this study, in vitro intestinal lipid digestion and the physicochemical and microstructural changes of sodium caseinate-stabilized emulsions were examined after the emulsions had been digested in a model simulated gastric fluid containing pepsin for different times. The average size, size distribution, microstructure, proteolysis of interfacial proteins and lipolysis of the emulsion droplets were monitored as a function of digestion time. The emulsion droplets underwent extensive droplet flocculation, with some coalescence together with proteolysis of interfacial proteins, in simulated gastric fluid, resulting in changes in the droplet size and the microstructure of the emulsions. In general, digestion in simulated gastric fluid containing pepsin accelerated coalescence of the emulsion droplets during subsequent digestion in simulated intestinal fluid containing pancreatic lipase. However, the changes in the size, the microstructure and the proteolysis of the interfacial proteins of the emulsions under gastric conditions did not influence the rate and the extent of lipid digestion in the subsequent intestinal environment.

Synthesis of a New [6]-Gingerol Analogue and Its Protective Effect with Respect to the Development of Metabolic Syndrome in Mice Fed a High-Fat Diet

To determine the effects of a [6]-gingerol analogue (6G), a major chemical component of the ginger rhizome, and its stable analogue after digestion in simulated gastric fluid, aza-[6]-gingerol (A6G), on diet-induced body fat accumulation, we synthesized 6G and A6G. Mice were fed either a control regular rodent chow, a high-fat diet (HFD), or a HFD supplemented with 6G and A6G. Magnetic resonance imaging adiposity parameters of the 6G- and A6G-treated mice were compared with those of control mice. Supplementation with 6G and A6G significantly reduced body weight gain, fat accumulation, and circulating levels of insulin and leptin. The mRNA levels of sterol regulatory element-binding protein 1c (SREBP-1c) and acetyl-CoA carboxylase 1 in the liver were significantly lower in mice fed A6G than in HFD control mice. Our findings indicate that A6G, rather than 6G, enhances energy metabolism and reduces the extent of lipogenesis by downregulating SREBP-1c and its related molecules, which leads to the suppression of body fat accumulation.

Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides

Mucosal delivery of peptide/protein therapeutics via the oral route is a desirable strategy in human immunotherapy. A key step for enhancing the bioavailability of orally administered therapeutics is to protect them from enzymatic digestion in the gastrointestinal tract. Here, we generated transgenic rice seeds accumulating allergen-derived T cell epitopes, a model tolerogen for the control of pollen allergy, in either ER-derived protein body-I (PB-I) or protein storage vacuole protein body-II (PB-II). Compared with PB-II-localized or chemically synthesized forms, PB-I-localized T cell epitopes showed higher resistance to enzymatic digestion in simulated gastric fluid. Moreover, the dose of T cell epitope required for suppression of allergen-specific IgE in mice was about 20-fold lower when fed in PB-I localized form than when unprotected. These findings demonstrate the potential of bioencapsulation in PB-I for broad applications as a viable strategy to achieve efficient mucosal delivery of oral peptide/protein therapeutics.

The effect of simulated gastrointestinal digestion on shrimp Penaeus vannamei allergenicity

Pen a 1 is a major shrimp allergen that induces an allergy. The aim of this study is to assess the effect of simulated gastrointestinal fluids on the allergenicity of Pen a 1. Purified Pen a 1 from shrimp (Penaeus vannamei) was subjected to digestion in SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) for a set time. The allergenicity of digestive Pen a 1 was analyzed by immunoblotting and Ci-ELISA, using pool sera from patients with shrimp specific IgE. The results showed that Pen a 1 exhibited a decrease in allergenicity with increasing digestion time in the SGF and SIF. However, Pen a l exhibited strong resistance to digestive fluids, and all yielded fragments (33 kD, 23 kD, and 14 kD) showed allergic activity. Therefore, anti-digestion may be an important factor for Pen a 1 to induce an allergy.

Probing novel allergenic proteins of commonly consumed legumes

Leguminous crops are the main source of protein in Asian subcontinent including India and their proteins may induce allergic reactions in sensitized individuals. Pepsin resistance of proteins is a characteristic feature of most of the allergens. Simulated gastric fluid (SGF) assay as validated by digestion of purified known allergenic and non-allergenic proteins was the basis of this study. Purified allergenic proteins were stable to SGF digestion contrary to rapidly digested non-allergenic proteins. Crude proteins extracts (CPE) of soybean, peanut, chickpea, black gram, kidney bean and Bengal gram were digested in vitro to detect their non-digestible proteins. Six proteins from soybean and seven from peanut remained undigested after SGF digestion. Likewise, seven proteins from chickpea (70, 64, 55, 45, 35, 20 and 18 kDa), ten from black gram (47, 30, 29, 28, 26, 24, 22, 16, 14 and 12 kDa), five from kidney bean (45, 29, 24, 20 and 6.5 kDa) and one from Bengal gram (20 kDa) remained undigested in SGF. Most of the proteins stable in SGF for more than 2 min showed similarity with characterized allergens on the basis of their molecular weights as in case of soybean, peanut, chickpea and black gram. Also, soybean and chickpea stable proteins showed IgE binding property with respective allergic patient’s sera. The non-digestible proteins from the chickpea, black gram, kidney bean and Bengal gram are being reported for the first time by our group. IgE binding of SGF resistant soybean and chickpea proteins is being reported first time as well.

Salt-Soluble Proteins from Wheat-Derived Foodstuffs Show Lower Allergenic Potency than Those from Raw Flour

Salt-soluble proteins from wheat flour have been described as main allergens associated with both baker's asthma and food allergy. However, most studies have used raw flour as starting material, thus not considering potential changes in allergenic properties induced by the heat treatment and other industrial processing to produce wheat-derived foodstuffs. Salt extracts from different commercial wheat-derived products were obtained and their allergenic properties investigated by IgE-immunodetection, ELISA assays, and skin prick test. The IgE-binding capacity of salt-soluble proteins from commercial breads and cooked pastas was reduced around 50% compared with that of raw flour, the reduction being less dramatic in noncooked pastas and biscuits. Several wheat-derived foodstuffs showed major IgE-binding components of 20 and 35 kDa, identified as avenin-like and globulin proteins, respectively. These proteins, as well as most flour and bread salt-soluble proteins, were hydrolyzed when subjected to simulated gastrointestinal digestion. However, the digested products still exhibited a residual IgE-binding capacity. Therefore, processing of wheat flour to obtain derived foodstuffs decreases the IgE binding-capacity of the major salt-soluble wheat proteins. Moreover, simulated gastric fluid digestion further inactivates some heat-resistant IgE-binding proteins.

Differential influence of the degree of processing on immunogenicity following proteolysis of casein and β-lactoglobulin

Simulated gastric fluid digestion was used to investigate how heat processing and fat content influence effects of proteolytic digestion on milk protein immunogenicity in milk with varying fat content and degree of processing. Changes in antigenicity and allergenicity of casein and β-lactoglobulin (β-LG) were demonstrated, using polyclonal antibodies and milk-allergic patient sera in immunoblot and enzyme-linked immunosorbent assay (ELISA) inhibition. Heat-induced changes in ultra-high temperature processed (UHT) milk increased stability of IgE and IgG casein epitopes to digestive proteolysis, but had little effect on specific IgE or IgG binding. Native κ-casein may be more resistant to digestion, but this was abrogated by heat treatment. Although β-LG's digestive stability decreased in UHT milk, immunogenicity was retained after hydrolysis in both processed (UHT) and non-processed (semi-skimmed) milks. Differences in fat levels in three milk matrices which had been processed similarly did not appear to influence digestibility. The possible clinical significance of these findings remains is discussed.

Site Specific Delivery of Microencapsulated Fish Oil to the Gastrointestinal Tract of the Rat

The aim of this study was to design food grade matrices to deliver microencapsulated fish oil to the large bowel of the rat where the potential exists to retard inflammation and cancer development. Digestion in simulated gastric fluid and intestinal fluid demonstrated that only 4-6% of oil was released from the following dried emulsion formulations: 50% fish oil encapsulated in heated casein-glucose-dried glucose syrup (1:1:1) (Cas-Glu-DGS-50); 25% fish oil in casein-modified resistant starch (Hylon VII) (1:1) (Cas-Hylon-25); or 25% fish oil in Cas-Glu-Hylon (1:1:1) (Cas-Glu-Hylon-25). A short-term gavage study (0-12 h) with fish oil and Cas-Glu-DGS-50 demonstrated the appearance of fish oil long chain (LC) n-3 polyunsaturated fatty acids (PUFA) into the plasma indicating specific small intestinal absorption with little LC n-3 PUFA reaching the large bowel. In a 2-week-long term, daily gavage study, the bioavailability of fish oil and fish oil in Cas-Glu-DGS-50 or Cas-Hylon-25 demonstrated that fish oil and Cas-Glu-DGS-50 LC n-3 PUFA were incorporated into the tissue of the small intestine and colon, whereas Cas-Hylon-25 was resistant to degradation in the small intestine. The use of modified Hylon VII for targeted colonic delivery was confirmed in the final short-term gavage study (0-14 h) using Cas-Glu-Hylon-25 with [(14)C]-trilinolenin as a marker incorporated into the microcapsules, where up to 60% of the labeled oil reached the large bowel. Depending on the microencapsulating matrix employed, fish oil can be delivered selectively to the small intestine or to a high degree to the large bowel.

Effect of Polyglycerol Esters of Fatty Acids on the Physicochemical Properties and Stability of β-Carotene Emulsions during Digestion in Simulated Gastric Fluid

The effects of six different polyglycerol esters of fatty acids (PGEs) and two different particle sizes produced using various processing parameters on the physicochemical properties and stability of the β-carotene emulsions during digestion in simulated gastric fluid (SGF) were investigated. β-Carotene emulsions were prepared by high-pressure homogenization using β-carotene (0.1% w/w) in soybean oil as the oil phase and 1% (w/w) PGE in Milli-Q water as the water phase. The particle size of β-carotene emulsions was measured by a laser diffraction technique, and the stability of emulsions was interpreted in terms of the increase in particle size and span value of emulsion droplets and the retention of β-carotene during digestion in SGF. The average particle size ranges of emulsions were 0.17 to 0.27 μm for fine emulsions and 1.16 to 1.59 μm for coarse emulsions. In the prepared β-carotene emulsions, the particle size decreased with increasing polymerization of the glycerol in PGEs, and the higher polymerization of the glycerol also increased the stability of emulsions during digestion in SGF. Although the β-carotene content in the emulsions significantly decreased with increasing digestion period, loss of β-carotene was more severe in unstable emulsions than in stable emulsions, suggesting that the particles incorporated into droplets could provide some protective barrier for decreasing the β-carotene degradation. Therefore, β-carotene emulsions stabilized by PGEs with high polymerization of the glycerol may be useful for further applications in food and drug formulations. Decaglycerol monooleate (MO750) was demonstrated to be the most effective emulsifier in stabilizing β-carotene emulsions in this study.

Computational analysis of the relationship between allergenicity and digestibility of allergenic proteins in simulated gastric fluid

BackgroundSafety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types.ResultsThe model presented in this paper is as effective as SGF digestion experiments, but more stable and reproducible. On the basis of this model, food allergens are satisfactorily classified as ACS and NACS types by their pathways for sensitization; the former are relatively resistant to gastric fluid digestion while the later are relatively labile.ConclusionThe results suggest that it is better to classify allergens into ACS and NACS types when understanding the relationship between their digestibility and allergenicity and the digestibility of a target foreign protein is a parameter for evaluating its allergenicity during safety assessments of GM food.

Quantitative measurement of protein digestion in simulated gastric fluid

The digestibility of novel proteins in simulated gastric fluid is considered to be an indicator of reduced risk of allergenic potential in food, and estimates of digestibility for transgenic proteins expressed in crops are required for making a human-health risk assessment by regulatory authorities. The estimation of first-order rate constants for digestion under conditions of low substrate concentration was explored for two protein substrates (azocoll and DQ-ovalbumin). Data conformed to first-order kinetics, and half-lives were relatively insensitive to significant variations in both substrate and pepsin concentration when high purity pepsin preparations were used. Estimation of digestion efficiency using densitometric measurements of relative protein concentration based on SDS–PAGE corroborated digestion estimates based on measurements of dye or fluorescence release from the labeled substrates. The suitability of first-order rate constants for estimating the efficiency of the pepsin digestion of novel proteins is discussed. Results further support a kinetic approach as appropriate for comparing the digestibility of proteins in simulated gastric fluid.

Peanut protein allergens: Gastric digestion is carried out exclusively by pepsin

Background A major characteristic of many food allergens, including Ara h 1, a major peanut allergen, is their resistance to gastric digestion. One estimate of the allergenic potential of a possible protein allergen is its stability under simulated gastric conditions. Objective Because the rate and extent of digestion of allergenic proteins will affect the severity of any subsequent allergic response, it is important to correlate protein allergen digestion in simulated gastric fluid with that in actual gastric fluid. Methods A major peanut allergen, Ara h 1, was digested in vitro by using both pepsin and porcine gastric fluid. Several comparisons between the 2 sets of proteolytic conditions were assessed including pH optima and the effect of temperature, denaturants, and specific enzyme inhibitors. Results In vitro digestion of Ara h 1 with pepsin and porcine gastric fluid resulted in virtually identical hydrolysis patterns as observed on SDS-PAGE. The protease activity of both pepsin and gastric fluid were inhibited at high pH and in the presence of pepstatin. However, both remained active in 4 mol/L urea and at 60°C. Conclusions Protein digestion in the porcine stomach is carried out by pepsin. In vivo gastric digestion is modeled accurately by peptic hydrolysis. Digestion conditions in vivo are comparable to experimental conditions in vitro provided that the acidic nature of the stomach contents is optimal for characterization of the allergen under standard pepsin digestion conditions. Additional experimentation using crude food extracts, both in the presence and absence of a complete meal, is needed to elucidate the complete physiologic nature of food allergen digestion.

Estabilidad de alimento sólido marcado con diferentes radiofármacos para vaciamiento gástrico

Sulfur colloid 99mTc-SC, the radiopharmaceutical of choice for solid gastric emptying studies, is not available in our country. It has led us to assess the solid binding stability of seven alternative radiopharmaceuticals that could present adequate fixation to it a priori. The stability of labelled solid food with seven colloidal 99mTc-radiopharmaceuticals of different sizes and nature (MAA, tin colloid, rhenium sulphide macrocolloid, albumin microcolloid, sulfur nanocolloid, albumin nanocolloid and rhenium sulfur nanocolloid) has been studied by measuring their dissociated activity after two hours digestion in simulated gastric fluid (kept 120' in agitation, in HCl 0.1 M at 37). The survey also assesses radiopharmaceutical labelling stability after two hours digestion in identical conditions by measuring their radiochemical purity in ITLC. In these conditions, MAA, rhenium sulphide macrocolloid, albumin microcolloid and albumin microcolloid present the best behaviour, with an activity linked to food over 90 % of the previously fixed activity. According to the results, there is no relationship between the radiopharmaceutical size and nature and the stability of its binding to the solid food. Because rhenium sulphide macrocolloid is no longer manufactured and the other three radiopharmaceuticals which have a binding stability to the solid food over 90 % do not include digestive explorations amongst their indications, nowadays, there is a serious legal limitation to carry out this type of studies in our country.

Analysis of avocado allergen (Prs a 1) IgE-binding peptides generated by simulated gastric fluid digestion

Background Resistance to pepsin digestion has been claimed to be a characteristic of food allergens that can induce severe adverse reactions. Moreover, pepsin treatment is included in protocols to evaluate the potential allergenicity of transgenic foods. Allergenic plant class I chitinases, such as avocado Prs a 1, are the panallergens involved in the latex-fruit syndrome. Previous reports indicated their susceptibility to simulated gastric fluid (SGF) digestion. Objective We sought to evaluate the IgE-binding capacity and the in vivo reactivity of the SGF products of the avocado allergen Prs a 1. Methods Patients with a clinical history of latex-fruit allergy syndrome, a positive skin prick test (SPT) response to Prs a 1, and specific IgE to avocado were selected. Untreated and SGF-digested Prs a 1 samples were analyzed by means of IgE and IgG immunoblotting, IgE immunoblotting and ELISA-inhibition assays, and SPTs. Peptides from SGF-digested samples were fractionated by means of HPLC, characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization analysis, and tested for in vivo reactivity with SPTs. Results Neither protein staining nor IgE immunoblotting with a pool of sera from allergic patients resulted in the detection of any band after SDS-PAGE separation of an SGF-digested sample of Prs a 1. However, this sample showed a similar inhibitory potency to that of untreated Prs a 1 in both immunoblot- and ELISA-inhibition assays (up to 70% inhibition of the IgE binding to crude avocado extract) and induced positive SPT responses in 5 of 8 allergic patients. Peptides from SGF-digested Prs a 1 were separated by means of HPLC, and 4 of them reached more than 50% inhibition values when using avocado extract as the solid phase in ELISA-inhibition assays. Reactive peptides were located both in the N-terminal hevein-like domain and in the catalytic domain of Prs a 1. Those corresponding to the hevein-like domain (approximately 5100 d) produced positive SPT responses in 5 of 8 allergic patients, whereas 2 peptides located in the catalytic domain (approximately 1400 and 2500 d) were reactive in 2 or 3 of the 8 patients. Conclusion Prs a 1 was extensively degradated when subjected to SGF digestion. However, the resulting peptides, particularly those corresponding to the hevein-like domain, were clearly reactive both in vitro and in vivo.

Immunogenic properties of rapidly digested food proteins following gavage exposure of mice: a comparison of ovalbumin with a potato acid phosphatase preparation

The ability of food proteins to resist digestion in simulated gastric fluid (SGF) correlates with allergenic potential. The purpose of the current investigations was to determine whether this association is due solely to the failure of unstable proteins to elicit an immune response when administered orally. We have examined immune responses induced in BALB/c mice by gavage administration of ovalbumin (OVA) and a crude potato protein extract (PPE) containing acid phosphatase activity. The stability of OVA and PPE in SGF was measured using sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The ability of these proteins to stimulate specific IgG and IgE antibody production in mice following parenteral (intraperitoneal; ip) or oral (gavage) exposure was compared using enzyme-linked immunosorbent and homologous passive cutaneous anaphylaxis assays, respectively. Both OVA and PPE induced specific IgG antibody responses when administered either by gavage or by ip injection. Parenteral, but not gavage, exposure to OVA was associated with robust IgE antibody responses. Administration of PPE failed to stimulate strong IgE production via either route of exposure. Differential stability in SGF was observed, with PPE being digested extremely rapidly (within 1 min), whereas OVA was more resistant. The strong association reported by others between stability in SGF and allergenic potential is unlikely to be solely due to orally-ingested labile proteins failing to provoke immune responses due to degradation in the stomach.

Plants as a natural source of concentrated mineral nutritional supplements

Edible plants enhanced with minerals were tested to determine whether these plants could be used as a new source of mineral dietary supplements that provide essential minerals in a more available form than current, inorganically based mineral supplements. A select cultivar of Brassica juncea was identified that can be cultivated under hydroponic conditions to contain high levels of nutritionally important minerals such as Cr, Fe, Mn, Se, and Zn. Sequential extraction, simulated gastric fluid digestion, and simulated intestinal fluid digestion were used to assess the degree of solubility and potential availability of each metal examined. Results from these solubility experiments indicate that the accumulated trace elements achieve greater soluble concentrations than those provided in popular mineral supplements. The consistent high concentration of minerals in the edible plant tissue allows processing small quantities of these enriched plants into capsules or tablets that supply 100% of the recommended daily intake of these elements in soluble form from a natural, vegetative source.