Simulated gastric fluid assay for estimating the digestibility of newly expressed proteins in GE crops: Missteps in development and interpretation.

Digestive stability of a food protein in simulated gastric fluid (SGF) continues to be considered a risk factor for allergy, even though the current science does not support this belief. Methodological shortcomings of the adaption of the SGF assay for use with purified proteins has been cited as a reason to discount results that do not conform to this belief. Missteps in conducting and interpreting the results of SGF assays are reviewed here. However, these methodological shortcomings do not invalidate the conclusion that allergenic proteins are not systematically more stable to digestion than non-allergens. The growing evidence for the dual allergen exposure hypothesis, whereby sensitization to food allergens is primarily caused by dermal and inhalation exposure to food dust, and tolerization against food allergy is primarily induced by gut exposure in food, likely explains why the digestive stability of a protein is not a risk factor for allergenicity.

Anisakis simplex (s.l.) resistance to the action of gastric enzymes depends upon previous treatments applied to infected fish mince and affects antigen release.

BACKGROUND: Freezing is considered the most suitable technological treatment to avoid Anisakis infection from eating raw or undercooked fish but modifications of their cuticles upon freezing may reduce their resistance to gastric fluids, provoking a greater release of allergens. This work aimed to study the relationship between freezing-induced modifications of Anisakis simplex s.l., antigen recognition, and resistance to oral and gastric digestion in spiked fish mince. RESULTS: (i) Differences between non-treated larvae and larvae that survived freezing / thawing were studied in terms of respiratory capacity, survival in simulated gastric fluid (SGF), recognition of antigens and allergens. (ii) Untreated (i.e. chilled) mince containing live larvae, mince frozen at two freezing rates, with a negative (uninfected) mince and a positive mince (infected with broken larvae) as controls, were subjected to the oral and gastric phases of a simulated digestion process. Anisakis able to survive freezing showed lower resistance to gastric fluid (i.e. faster mortality as compared to controls). Untreated larvae released significantly more antigens than freeze-surviving larvae but only after 96 h in SGF. In treatments rendering complete larvae mortality, the highest loss of larvae integrity was found upon fast freezing. There was a positive correlation between antigen release and the number of ruptures of larvae after the oral digestion phase, whereas a more complex trend was observed after oral plus gastric digestion phases. CONCLUSION: These results suggest a new factor to consider for sensitized patients and suggest that the numbers of L3 should be reduced before industrial freezing to minimize risk. © 2020 Society of Chemical Industry.

Association of cagA+ Helicobacter pylori strains with high urease activity and dyspepsia in Mexican adults. / Asociación de cepas de Helicobater pylori cagA+ con alta actividad de ureasa y dispepsia en adultos mexicanos.

INTRODUCTION AND AIMS: Helicobacter pylori (H. pylori) is associated with a higher risk of peptic ulcer and gastric cancer. The sole presence of the bacterium is not a determinant of clinical outcome, but rather the interaction of strain type and host factors determines the risk of disease. Our aim was to study the association between bacterial load, strain type, and gastric symptoms in H. pylori-positive subjects. MATERIALS AND METHODS: In a community survey, a diagnostic 13C-urea breath test for H. pylori was performed on 302 volunteers that were not taking antibiotics, antacids, or proton pump inhibitors one month prior to the test. The breath test produced 25 H. pylori-positive subjects, between 25-74 years of age, who then took a gastric symptoms survey and were tested for the presence of the cagA genotype in gastric juice, using the Entero-test®. Bacterial load was determined as a measure of urease activity, utilizing the delta over baseline value, obtained in the 13C-urea breath test. RESULTS: A total of 48% of the H. pylori-positive subjects were cagA+. A positive association was found between cagA status and high gastric urease activity (P<.0001) and the latter was significantly associated with the presence of symptoms (P<.0001). CONCLUSION: Gastric urease activity was strongly associated with dyspeptic symptoms and cagA+ H. pylori. Elevated 13C-delta over baseline values could be used as indicators of a higher risk for gastric disease.

Mimicking the passage of avian influenza viruses through the gastrointestinal tract of chickens.

In contrast to human influenza viruses that replicate in the respiratory tract and are airborne transmitted, avian viruses also replicate in gut epithelial cells and are transmitted via the fecal-oral route. On this route, the virus is exposed to destructive fluids of the digestive tract, which are acidic and contain the proteases pepsin (gizzard) or chymotrypsin and trypsin (intestine). Only the latter enzyme activates virus by cleaving hemagglutinin (HA) into HA1 and HA2 subunits. We mimicked the passage of viruses through the gastrointestinal tract by treating them with digestive fluids from chicken and determined titers and integrity of HA by western-blot. Gizzard fluid completely inactivated virions and degrades HA even at a high dilution, but only if the pH was kept acidic. If the fluid is diluted with neutral buffer (mimicking virus uptake with seawater) particles were more resistant. Virions containing an uncleaved HA were even activated suggesting that gastric juice contains a trypsin-like protease. Undiluted intestinal fluid inactivated particles and destroyed HA, but diluted fluid activated virions. A virus isolated from the duck´s intestine is more tolerant against intestinal fluid compared to fowl plague virus suggesting that the former is better adapted to grow in the intestine. We also demonstrate that influenza viruses replicate to high titers in a novel chicken epithelial gut cell line. While viruses with a monobasic HA cleavage site require addition of trypsin, these cells effectively process HA with a polybasic cleavage site, which could be blocked with an inhibitor of the cellular furin protease.

INFOGEST static in vitro simulation of gastrointestinal food digestion.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.

Sea Buckthorn Oil as a Valuable Source of Bioaccessible Xanthophylls.

Sea buckthorn oil, derived from the fruits of the shrub, also termed seaberry or sandthorn, is without doubt a strikingly rich source of carotenoids, in particular zeaxanthin and ß-carotene. In the present study, sea buckthorn oil and an oil-in-water emulsion were subjected to a simulated gastro-intestinal in vitro digestion, with the main focus on xanthophyll bioaccessibility. Zeaxanthin mono- and di-esters were the predominant carotenoids in sea buckthorn oil, with zeaxanthin dipalmitate as the major compound (38.0%). A typical fatty acid profile was found, with palmitic (49.4%), palmitoleic (28.0%), and oleic (11.7%) acids as the dominant fatty acids. Taking into account the high amount of carotenoid esters present in sea buckthorn oil, the use of cholesterol esterase was included in the in vitro digestion protocol. Total carotenoid bioaccessibility was higher for the oil-in-water emulsion (22.5%) compared to sea buckthorn oil (18.0%) and even higher upon the addition of cholesterol esterase (28.0% and 21.2%, respectively). In the case of sea buckthorn oil, of all the free carotenoids, zeaxanthin had the highest bioaccessibility (61.5%), followed by lutein (48.9%), making sea buckthorn oil a potential attractive source of bioaccessible xanthophylls.

Comparative study of the bioaccessibility of the colorless carotenoids phytoene and phytofluene in powders and pulps of tomato: microstructural analysis and effect of addition of sunflower oil.

The objective was to assess the potential bioavailability of phytoene (PT) and phytofluene (PTF) from tomato powders used as raw materials for supplements as compared to the pulp of a common tomato and a cherry tomato. PT and PTF are attracting much interest nowadays as they can provide health and cosmetic benefits. PT and PTF levels in the more concentrated powder were up to 1000 times higher than in the tomatoes. The bioaccessibility from the powders was lower as compared to the tomato fruits and increased markedly when sunflower oil was added. However, the best source of potentially absorbable PT and PTF (0.5 and 2 mg g-1 respectively) was by far the powder with higher levels of them. This result could be due to the higher carotenoid concentration in the powder, the reduction of the particle sizes, and the rupture of cell structures compared to the pulps.

Digestive enzymes of the Californian two-spot octopus, Octopus bimaculoides (Pickford and McConnaughey, 1949).

Octopus bimaculoides is an important commercially fished species in the California Peninsula with aquaculture potential; however, to date limited information is available regarding its digestive physiology. The objective of this study was focused on biochemically characterizing the main enzymes involved in the digestion of O. bimaculoides. Optimum pH, temperature and thermostability were determined for amylases, lipases, trypsin and chymotrypsin; optimum pH and protease inhibitor effect were assessed for acidic and alkaline proteases, and the effect of divalent ions on trypsin and chymotrypsin activity was evaluated in enzymatic extracts from the digestive (DG) and salivary glands (SG) and crop gastric juices (GJ). High amylase activity was detected in GD and GJ whereas this activity is very low in other cephalopods. Salivary glands had the greatest activity in most of the enzyme groups, showing the importance of this organ in digestion. Optimum pH was different depending on the organ and enzyme analyzed. The optimum pH in DG was 3 showing the predominance of acidic proteases in the digestion process. All enzymes were resistant and stable at high temperatures in contrast with other marine species. Trypsin and chymotrypsin activity were highly incremented with the presence of Mg2+, Co2+, Cu2+ and Zn2+ in some tissues. The inhibitor assay showed the importance of serine proteases, metalloproteases and aspartic proteases in the digestive process of this species. This study is the first in assessing the main digestive enzymes of O. bimaculoides and in remarking the importance of other digestive enzyme groups besides proteases in octopuses.

Comparison of lipases for in vitro models of gastric digestion: lipolysis using two infant formulas as model substrates.

The aim of this study was to find a lipase suitable as a surrogate for Human Gastric Lipase (HGL), since the development of predictive gastrointestinal lipolysis models are hampered by the lack of a lipase with similar digestive properties as HGL. Three potential surrogates for HGL; Rhizopus Oryzae Lipase (ROL), Rabbit Gastric Lipase (RGL) and recombinant HGL (rHGL), were used to catalyze the in vitro digestion of two infant formulas (a medium-chain triacylglyceride enriched formula (MC-IF) and a predominantly long-chain triacylglyceride formula (LC-IF)). Digesta were withdrawn after 0, 5, 15, 30, 60 min of gastric digestion and after 90 or 180 min of intestinal digestion with or without the presence of pancreatic enzymes, respectively. The digesta were analyzed by scanning electron microscopy and gas chromatography to quantify the release of fatty acids (FAs). Digestions of both formulas, catalyzed by ROL, showed that the extent of gastric digestion was higher than expected from previously published in vivo data. ROL was furthermore insensitive to FA chain length and all FAs were released at the same pace. RGL and rHGL favoured the release of MC-FAs in both formulas, but rHGL did also release some LC-FAs during digestion of MC-IF, whereas RGL only released MC-FAs. Digestion of a MC-IF by HGL in vivo showed that MC-FAs are preferentially released, but some LC-FAs are also released. Thus of the tested lipase rHGL replicated the digestive properties of HGL the best and is a suitable surrogate for HGL for use in in vitro gastrointestinal lipolysis models.

Novel ciliate lipases for enzyme replacement during exocrine pancreatic insufficiency.

AIM AND OBJECTIVES: Exocrine pancreatic insufficiency caused by inflammation or pancreatic tumors results in nutrient malfunction by a lack of digestive enzymes and neutralization compounds. Despite satisfactory clinical results with current enzyme therapies, a normalization of fat absorption in patients is rare. An individualized therapy is required that includes high dosage of enzymatic units, usage of enteric coating, and addition of gastric proton pump inhibitors. The key goal to improve this therapy is to identify digestive enzymes with high activity and stability in the gastrointestinal tract. METHODS: We cloned and analyzed three novel ciliate lipases derived from Tetrahymena thermophila. Using highly precise pH-STAT-titration and colorimetric methods, we determined stability and lipolytic activity under physiological conditions in comparison with commercially available porcine and fungal digestive enzyme preparations. We measured from pH 2.0 to 9.0, with different bile salts concentrations, and substrates such as olive oil and fat derived from pig diet. RESULTS: Ciliate lipases CL-120, CL-130, and CL-230 showed activities up to 220-fold higher than Creon, pancreatin standard, and rizolipase Nortase within a pH range from pH 2.0 to 9.0. They are highly active in the presence of bile salts and complex pig diet substrate, and more stable after incubation in human gastric juice compared with porcine pancreatic lipase and rizolipase. CONCLUSIONS: The newly cloned and characterized lipases fulfilled all requirements for high activity under physiological conditions. These novel enzymes are therefore promising candidates for an improved enzyme replacement therapy for exocrine pancreatic insufficiency.

A Novel Phytase Derived from an Acidic Peat-Soil Microbiome Showing High Stability under Acidic Plus Pepsin Conditions.

Four novel phytases of the histidine acid phosphatase family were identified in two publicly available metagenomic datasets of an acidic peat-soil microbiome in northeastern Bavaria, Germany. These enzymes have low similarity to all the reported phytases. They were overexpressed in Escherichia coli and purified. Catalytic efficacy in simulated gastric fluid was measured and compared among the four candidates. The phytase named rPhyPt4 was selected for its high activity. It is the first phytase identified from unculturable Acidobacteria. The phytase showed a longer half-life than all the gastric-stable phytases that have been reported to date, suggesting a strong resistance to low pH and pepsin. A wide pH profile was observed between pH 1.5 and 5.0. At the optimum pH (2.5) the activity was 2,790 µmol/min/mg at the physiological temperature of 37°C and 3,989 µmol/min/mg at the optimum temperature of 60°C. Due to the competent activity level as well as the high gastric stability, the phytase could be a potential candidate for practical use in livestock and poultry feeding.

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion.

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.

Phytase production by solid-state fermentation of groundnut oil cake by Aspergillus niger: A bioprocess optimization study for animal feedstock applications.

This investigation deals with the use of agro-industrial waste, namely groundnut oil cake (GOC), for phytase production by the fungi Aspergillus niger NCIM 563. Plackett-Burman design (PBD) was used to evaluate the effect of 11 process variables and studies here showed that phytase production was significantly influenced by glucose, dextrin, distilled water, and MgSO4 · 7H2O. The use of response surface methodology (RSM) by Box-Behnken design (BBD) of experiments further enhanced the production by a remarkable 36.67-fold from the original finding of 15 IU/gds (grams of dry substrate) to 550 IU/gds. This is the highest solid-state fermentation (SSF) phytase production reported when compared to other microorganisms and in fact betters the best known by a factor of 2. Experiments carried out using dried fermented koji for phosphorus and mineral release and also thermal stability have shown the phytase to be as efficient as the liquid enzyme extract. Also, the enzyme, while exhibiting optimal activity under acidic conditions, was found to have significant activity in a broad range of pH values (1.5-6.5). The studies suggest the suitability of the koji supplemented with phytase produced in an SSF process by the "generally regarded as safe" (GRAS) microorganism A. niger as a cost-effective value-added livestock feed when compared to that obtained by submerged fermentation (SmF).

Disintegration kinetics of food gels during gastric digestion and its role on gastric emptying: an in vitro analysis.

The understanding of the disintegration and gastric emptying of foods in the stomach is important for designing functional foods. In this study, a dynamic stomach model (human gastric simulator, HGS) was employed to investigate the disintegration and subsequent emptying of two differently structured whey protein emulsion gels (soft and hard gels).The gels were mechanically ground into fragments to reproduce the particle size distribution of an in vivo gel bolus. The simulated gel bolus was prepared by mixing gel fragments and artificial saliva, and exposed to 5 hours of simulated gastric digestion in the presence and absence of pepsin. Results showed that regardless of pepsin, the soft gel always disintegrated faster than the hard gel. The presence of pepsin significantly accelerated the disintegration of both gels. In particular, it enhanced abrasion of the soft gel into fine particles (<0.425 mm) after 180 min of processing. The emptying of the gels was influenced by the combined effects of the original particle size of the gel boluses and their disintegration kinetics in the HGS. In the presence or absence of pepsin, the larger particles of the soft gel emptied slower than the hard one during the first 120 min of process. However, in the presence of pepsin, the soft gel emptied faster than the hard one after 120 min because of a higher level of disintegration. These findings highlight the role of food structure, bolus properties and biochemical effects on the disintegration and gastric emptying patterns of gels during gastric digestion.

Impact of extraneous proteins on the gastrointestinal fate of sunflower seed (Helianthus annuus) oil bodies: a simulated gastrointestinal tract study.

In this study, we examined the physicochemical nature of sunflower seed oil bodies (in the absence and presence of added protein) exposed to gastrointestinal conditions in vitro: crude oil bodies (COB); washed oil bodies (WOB); whey protein isolate-enriched oil bodies (WOB-WPI); and, sodium caseinate enriched-oil bodies (WOB-SC). All oil body emulsions were passed through an in vitro digestion model that mimicked the stomach and duodenal environments, and their physicochemical properties were measured before, during, and after digestion. Oil bodies had a positive charge under gastric conditions because the pH was below the isoelectric point of the adsorbed protein layer, but they had a negative charge under duodenal conditions which was attributed to changes in interfacial composition resulting from adsorption of bile salts. Oil bodies were highly susceptible to flocculation and coalescence in both gastric and duodenal conditions. SDS-PAGE analysis indicated degradation of oleosin proteins (ca. 18-21 kDa) to a greater or lesser extent (dependent on the emulsion) during the gastric phase in all emulsions tested; there is evidence that some oleosin remained intact in the crude oil body preparation during this phase of the digestion process. Measurements of protein displacement from the surface of COBs during direct exposure to bile salts, without inclusion of a gastric phase, indicated the removal of intact oleosin from native oil bodies.

The Effects of Broccoli Sprout Extract Containing Sulforaphane on Lipid Peroxidation and Helicobacter pylori Infection in the Gastric Mucosa.

BACKGROUND/AIMS: The aims of this study were to investigate whether a broccoli sprout extract containing sulforaphane (BSES) inhibited the Helicobacter pylori infection density and exerted an antioxidative effect on gastric mucosal damage. METHODS: The enrolled subjects were randomized in a double-blinded manner into three groups. Finally, 33 H. pylori (+) BSES treatment subjects (group A), 28 H. pylori (+) placebo subjects (group B), and 28 H. pylori (-) BSES treatment subjects (group C) were studied. H. pylori infection density was indirectly quantified by a (13)C-urea breath test (UBT), and the ammonia concentration in gastric juice aspirates was measured through gastroscopic examination. Malondialdehyde (MDA), an oxidative damage biomarker, and reduced glutathione (GSH), an antioxidant biomarker, were measured in the gastric mucosa by an enzyme-linked immunosorbent assay. RESULTS: BSES treatment did not significantly affect the UBT values or ammonia concentration in group A (p=0.634 and p=0.505, respectively). BSES treatment did significantly reduce mucosal MDA concentrations in group A (p<0.05) and group C (p<0.001), whereas the gastric mucosal GSH concentrations did not differ before and after treatment in any of the groups. CONCLUSIONS: BSES did not inhibit the H. pylori infection density. However, BSES prevented lipid peroxidation in the gastric mucosa and may play a cytoprotective role in H. pylori-induced gastritis.

Refining in silico simulation to study digestion parameters affecting the bioaccessibility of lipophilic nutrients and micronutrients.

Despite the considerable number of in vivo and in vitro studies on the digestive fate of lipophilic nutrients, micronutrients, and bioactives, the effects of the structure and composition of foods on the physicochemical mechanisms of luminal digestion are still poorly understood. Studying them is indeed complex because the number of parameters is high and many of them are interdependent. To solve this problem, an in silico simulation based on a multi-agent system was recently proposed to study the intestinal bioaccessibility of lipophilic nutrients and micronutrients from a single oil droplet. The roles of lipolysis and solubilization in bile salt were included. The effects of several food and digestion parameters were in line with those reported in the experimental literature. The goal of the research reported in this new article was to include more digestion parameters in the simulation in order to make it more realistic against complex cases. This was done in one specific digestion condition reflecting in vitro experiments, using droplets of tricaprylin or triolein containing vitamin A. The structure and principles of the original model were kept, with independent local modifications in order to study each factor separately. First, a gastric step was added where lipolysis took place, and only a marginal effect on the following intestinal step was found. Then, the chemical form of vitamin A, either non-hydrolyzed retinyl ester or retinyl ester instantly hydrolyzed into retinol, was investigated by considering different localizations in the droplet, resulting in a higher bioaccessibility for the retinol. The case of a mixture of tricaprylin and triolein indicated an influence of the oil phase viscosity. The consideration of mixed micelles compared to simple bile salt micelles was also investigated, and resulted in a higher vitamin A bioaccessibility, especially with triolein. Finally, a full model including the most influential parameters was tested to simulate the digestion of triglyceride-limonene mixtures, giving bioaccessibility trends in very good agreement with the literature.

Ex vivo digestion of carp muscle tissue--ACE inhibitory and antioxidant activities of the obtained hydrolysates.

In the digestive tract of humans, bioactive peptides, i.e. protein fragments impacting the physiological activity of the body, may be released during the digestion of food proteins, including those of fish. The aim of the study was to establish the method of human ex vivo digestion of carp muscle tissue and evaluate the angiotensin I-converting enzyme inhibitory and antioxidant activities of hydrolysates obtained after digestion. It was found that the hydrolysates of carp muscle tissue obtained with the three-stage method of simulated ex vivo digestion showed ACE inhibitory as well as antioxidative activities. It was demonstrated that the degree of hydrolysis depended on the duration of individual stages and the degree of comminution of the examined material. Although the applied gastric juices initiated the process of hydrolysis of carp muscle tissue, the duodenal juices caused a rapid increase in the amount of hydrolysed polypeptide bonds. The antihypertensive and antioxidative activities of the hydrolysates of carp muscle tissue increased together with progressive protein degradation. However, the high degree of protein hydrolysis does not favour an increase in the activity of free radical scavenging. The presented results are an example of the first preliminary screening of the potential health-promoting biological activity of carp muscle tissue in an ex vivo study.

Quercetin 7-O-glucoside suppresses nitrite-induced formation of dinitrosocatechins and their quinones in catechin/nitrite systems under stomach simulating conditions.

Foods of plant origin contain flavonoids. In the adzuki bean, (+)-catechin, quercetin 3-O-rutinoside (rutin), and quercetin 7-O-ß-D-glucopyranoside (Q7G) are the major flavonoids. During mastication of foods prepared from the adzuki bean, the flavonoids are mixed with saliva and swallowed into the stomach. Here we investigated the interactions between Q7G and (+)-catechin at pH 2, which may proceed in the stomach after the ingestion of foods prepared from the adzuki bean. Q7G reacted with nitrous acid producing nitric oxide (˙NO) and a glucoside of 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone. (+)-Catechin reacted with nitrous acid producing ˙NO and 6,8-dinitrosocatechin. The production of the dinitrosocatechin was partly suppressed by Q7G, and the suppression resulted in the enhancement of Q7G oxidation. 6,8-Dinitrosocatechin reacted further with nitrous acid generating the o-quinone, and the quinone formation was effectively suppressed by Q7G. In the flavonoids investigated, the suppressive effect decreased in the order Q7G≈quercetin>kaempferol>quercetin 4'-O-glucoside>rutin. Essentially the same results were obtained when (-)-epicatechin was used instead of (+)-catechin. The results indicate that nitrous acid-induced formation of 6,8-dinitrosocatechins and the o-quinones can be suppressed by flavonols in the stomach, and that both a hydroxyl group at C3 and ortho-hydroxyl groups in the B-ring are required for efficient suppression.

Investigation of the reactions of acrylamide during in vitro multistep enzymatic digestion of thermally processed foods.

This study investigated the fate of acrylamide in thermally processed foods after ingestion. An in vitro multistep enzymatic digestion system simulating gastric, duodenal and colon phases was used to understand the fate of acrylamide in bakery and fried potato products. Acrylamide levels gradually decreased through gastric, duodenal and colon phases during in vitro digestion of biscuits. At the end of digestion, acrylamide reduction was between 49.2% and 73.4% in biscuits. Binary model systems composed of acrylamide and amino acids were used to understand the mechanism of acrylamide reduction. High-resolution mass spectrometry analyses confirmed Michael addition of amino acids to acrylamide during digestion. In contrast to bakery products, acrylamide levels increased significantly during gastric digestion of fried potatoes. The Schiff base formed between reducing sugars and asparagine disappeared rapidly, whereas the acrylamide level increased during the gastric phase. This suggests that intermediates like the Schiff base that accumulate in potatoes during frying are potential precursors of acrylamide under gastric conditions.