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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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rod Herman and John Zhang reports a relationship with Corteva Agriscience LLC that includes: employment. The Authors are employed by Corteva Agriscience which develops and markets transgenic seed., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2020 Society of Chemical Industry.)

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 13 C-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 13 C-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 13 C-delta over baseline values could be used as indicators of a higher risk for gastric disease., (Copyright © 2020 Asociación Mexicana de Gastroenterología. Publicado por Masson Doyma México S.A. All rights reserved.)

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 β-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.

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 HA 1 and HA 2 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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.

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 Mg 2+ , Co 2+ , Cu 2+ and Zn 2+ 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

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.

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).

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.

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., (© 2016 S. Karger AG, Basel.)

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.

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.

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.

Foods of plant origin contain flavonoids. In the adzuki bean, (+)-catechin, quercetin 3-O-rutinoside (rutin), and quercetin 7-O-β-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.

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.

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.

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.

In the present study, structural changes in the milk protein α-lactalbumin (α-LA) and its proteolysis were investigated for the potential formation of protein-fatty acid complexes during in vivo gastric digestion. Capsule endoscopy allowed visualisation of the digestion of the test drinks, with nasogastric tubes allowing sampling of the gastric contents. A total of ten healthy volunteers had nasogastric tubes inserted into the stomach and ingested test drinks containing 50 g/l of sucrose and 25 g/l of α-LA with and without 4 g/l of oleic acid (OA). The samples of gastric contents were collected for analysis at 3 min intervals. The results revealed a rapid decrease in the pH of the stomach of the subjects. The fasting pH of 2·31 (SD 1·19) increased to a pH maxima of pH 6·54 (SD 0·29) after ingestion, with a subsequent decrease to pH 2·22 (SD 1·91) after 21 min (n 8). Fluorescence spectroscopy and Fourier transform IR spectroscopy revealed partial protein unfolding, coinciding with the decrease in pH below the isoelectric point of α-LA. The activity of pepsin in the fasting state was found to be 39 (SD 12) units/ml of gastric juice. Rapid digestion of the protein occurred: after 15 min, no native protein was detected using SDS-PAGE; HPLC revealed the presence of small amounts of native protein after 24 min of gastric digestion. Mirocam® capsule endoscopy imaging and video clips (see the online supplementary material) revealed that gastric peristalsis resulted in a heterogeneous mixture during gastric digestion. Unfolding of α-LA was observed during gastric transit; however, there was no evidence of a cytotoxic complex being formed between α-LA and OA.

Background: Endotracheal intubation increases the risk for microaspiration of secretions around the tube cuff. Pepsin has been used as a biomarker for gastric aspiration. Amylase is a newer proposed biomarker for aspiration of oral contents., Objective: To assess the presence of pepsin and amylase in paired oral-tracheal secretions of adult patients treated with mechanical ventilation., Methods: In this descriptive study, paired samples of oral and tracheal secretions were obtained from adult patients at baseline and again within 4 hours when a need for endotracheal suctioning was assessed. Assays of pepsin and amylase were processed in a specialty diagnostic laboratory., Results: The sample consisted of 10 men and 3 women with a median age of 56 years. The majority were intubated with a subglottic suction endotracheal tube (9 patients, 69%), receiving synchronized intermittent mandatory ventilation (10 patients; 77%), and receiving enteral feedings (11 patients; 85%) through a tube distally placed in the stomach (8 patients; 67%). Pepsin was present in oral secretions of 9 patients (69%), and in tracheal specimens of 7 patients (54%) at one or both sampling times. Amylase was detected in all patients' oral secretions and in tracheal secretions of 5 patients (38%) at one or both sampling times., Conclusions: Many patients had pepsin, amylase, or both in tracheal aspirates. Pepsin was more commonly detected than was amylase. Although the relationship of this finding to long-term outcomes was not assessed, findings indicate that microaspiration of oral and gastric secretions occurs frequently., (©2014 American Association of Critical-Care Nurses.)

To study the potential probiotic characteristics such as decrease of pH, microbial viability, and tolerance to simulated digestive steps of fermented soy beverage ("soy yogurt") produced with lactobacilli isolated from cocoa fermentation (Lactobacillus fermentum TcUESC01 and Lactobacillus plantarum TcUESC02) during fermentation and refrigerated storage. The sensory acceptance of the yogurts was also tested. Samples of soy yogurt produced with L. fermentum TcUESC01 or L. plantarum TcUESC02 were collected during fermentation (0, 4, 8, and 12 h) and refrigerated storage (1, 9, 18, and 27 d), and submitted to pH and bacterial viability determinations. Tolerance to simulated digestion steps was done with refrigerated storage samples at 9 °C. Simulated digestion was performed in 3 successive steps: exposure to pepsin-HCl solution, bile shock, and simulated small intestinal juice. During storage, a decrease in pH and lactobacillus viability was observed. L. fermentum TcUESC01 showed to be more resistant than L. plantarum TcUESC02 to simulated gastrointestinal digestion. All soy yogurts showed acceptable hedonic scores (greater than 5 in a 9-point hedonic scale ranging from "like extremely" to "dislike extremely") in sensory evaluation for flavor, aroma, color, consistency, and overall impression. L. plantarum TcUESC02 and, especially, L. fermentum TcUESC01 showed potential probiotic characteristics when considering pH, cell viability, and tolerance to simulated digestive steps and did not affect the sensory characteristics when supplemented to soy yogurt during storage., (© 2014 Institute of Food Technologists®)

Digestion of nutrients is an essential function of the newborn infant gut to allow growth and development and understanding infant digestive function is essential to optimize nutrition and oral drug delivery. Ethical considerations prohibit invasive in vivo trials and as a consequence in vitro assays are often conducted. However, the choice of in vitro model parameters are not supported by an exhaustive analysis of the literature and do not mimic precisely the digestive conditions of the infant. This review contains a compilation of the studies which characterized the gastroduodenal conditions in full-term or preterm infants of variable postnatal age from birth up to six months. Important data about healthy full-term infants are reported. The enzymatic (type of enzymes and level of activity) and nonenzymatic (milk-based diet, frequency of feeding, bile salt concentrations) conditions of digestion in infants are shown to differ significantly from those in adults. In addition, the interindividual and developmental variability of the digestive conditions in infants is also highlighted.

Background & Aims: Non-coagulation of protein from enteral nutrition (EN) in the stomach is considered to improve gastric emptying and may result in reduced upper gastrointestinal complications such as reflux and aspiration pneumonia. For the development of a new EN protein mixture with reduced gastric coagulation, the coagulating properties of individual proteins, a novel blend of four proteins (P4 protein blend) and commercial EN products were investigated., Methods: A semi-dynamic, computer controlled setup was developed to mimic gastric digestion. The coagulation behaviour of 150 ml protein solutions and EN products was investigated. These were heat-treated calcium caseinate, sodium caseinate, whey, soy and pea protein, and the P4 protein blend comprising of the latter four (all solutions 6% w/v protein), four new enteral nutrition product varieties (New Nutrison® .0 or 1.5 kcal/ml, with and without MultiFibre MF6™) based on the P4 protein blend and two other commercially available casein dominant EN products (T1 and T2)., Results: Calcium caseinate and sodium caseinate yielded a total wet coagulate of 43.5 ± 0.7 g and 52.7 ± 6.2 g, respectively. Whey, soy, pea and the P4 protein blend did not produce any measurable coagulate. T1 and T2 resulted in a total wet coagulate of 37.5 ± 0.8 g and 57.3 ± 0.8 g, respectively, while all new EN product varieties based on the P4 protein blend did not produce any measurable coagulate., Conclusions: The P4 protein blend renders EN product varieties non-coagulating after in vitro gastric digestion., (Copyright © 2012 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background: Because reflux of gastric juice into the oropharynx must precede its aspiration into the lungs, it is reasonable to hypothesize that the detection of pepsin (the major gastric enzyme in gastric juice) in oral secretions may provide a relatively noninvasive method of predicting risk for aspiration., Objective: To describe the incidence of pepsin in oral and tracheal secretions collected concurrently from a sample of 50 gastric-fed patients undergoing mechanical ventilation., Methods: An exploratory descriptive design with a convenience sample from 4 medical and surgical intensive care units. An oral secretion and a tracheal secretion were collected concurrently from each patient (yielding a sample of 50 oral and 50 tracheal secretions). The tracheal secretions were obtained via the inline suction system with an attached sputum trap; oral secretions were obtained via a Yankauer suction tip with an attached sputum trap. All specimens were assayed for pepsin by the Western blot method., Results: Oral secretions from 10 patients (20%) and tracheal secretions from 2 patients (4%) were pepsin-positive. Both patients with pepsin-positive tracheal secretions also had pepsin-positive oral secretions. Pepsin was not found in the tracheal secretions from the remaining 8 patients with pepsin-positive oral secretions., Conclusions: Although reflux of gastric juice into the oropharynx must precede its aspiration into the lungs, individual reflux events do not necessarily lead to aspiration. Thus, it is reasonable that we found pepsin 5 times more often in oral secretions than in tracheal secretions.

Background: The effect of a water-saving irrigation regime on yield, chemical composition, rumen in situ dry matter disappearance (DMD) and neutral detergent fiber disappearance (NDFD), along with 7 h in vitro starch degradability (7 h IVSD), in maize hybrids selected for whole-plant silage making was investigated. A plot experiment was conducted in a continental climate location and four commercial maize hybrids (FAO class 700) were used in a completely randomized design with a factorial arrangement of irrigation treatments (fully irrigated (FI) and water-saving regime (WS)) and four replicates/treatment. The total amount of irrigation water was 494 mm in FI plots and 367 mm in WS plots, the latter achieved by skipping irrigations at vegetative growth stage, silking and blistering., Results: Whole-plant yield, chemical composition, DMD, NDFD and 7 h IVSD slightly differed among hybrids and were not influenced by irrigation treatments. Plant dry matter content was lower in FI than WS plots (320 vs. 341 g kg⁻¹) , respectively; P < 0.05). Differences among hybrids were recorded for starch and acid detergent fiber contents (P < 0.05)., Conclusions: The lack of differences on yield and nutritive value in tested maize hybrids grown under different water supply suggests the water-saving regime could be suitable for an optimal use of available water in maize management., (© 2013 Society of Chemical Industry.)

An oil body dispersion (11.3% fat) was prepared by wet disintegration of walnuts and was then subjected to a two-step model of in vitro digestion. In a gastric environment, proteolysis by pepsin led to the destabilization and coalescence of the oil bodies. Aggregation of the coalesced oil bodies was apparent under a confocal microscope, with aggregates up to 275 μm in size. Pepsin-resistant peptides and proteins remained at the surface of the oil bodies, and some were further resistant to intestinal proteases. Under intestinal conditions, the hydrolysis of walnut triglycerides led to the spontaneous formation of a new type of multiple emulsions, ranging from 2 to 45 μm in size and with protein material inside the inner water droplets. Transmission electron microscopy revealed the presence of a liquid-crystalline phase of bile salts and lipolytic products at the surface of the oil droplets and some bile salt crystals at the surface of the inner water droplets.

Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17-41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is digested by nonhuman enzymes. The degradation of LF in the GI tract should be taken into consideration when health effects are proposed, because LF has now been approved by the European Food Safety Authority as a dietary supplement in food products., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Arginine kinase (AK) is expressed in a wide variety of species, including human food sources (seafood) and pests (cockroaches and moths), and has been reported as a novel allergen. However, there has been little research on the allergenicity of AK in crustaceans. In this study the physicochemical properties of AK from mud crab (Scylla paramamosain) were investigated., Results: Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and inhibition enzyme-linked immunosorbent assay revealed that purified AK was unstable in thermal processing and in acid buffer. Under simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions, purified AK was much more readily degraded by pepsin than by trypsin or chymotrypsin. The unpurified AK in crab myogen degraded more markedly than purified AK. In addition, in two-phase gastrointestinal digestion, AK was rapidly degraded by pepsin but resistant to trypsin and chymotrypsin digestion, while tropomyosin derived from mud crab was resistant to pepsin digestion but digested readily by trypsin or chymotrypsin. Further study of serum samples obtained from crab-allergic human patients indicated that the allergenicity of AK was markedly reduced by digestion with SGF but not SIF., Conclusion: AK is an important food allergen despite its unstable physicochemical properties of digestibility., (Copyright © 2012 Society of Chemical Industry.)

Magnetic bead cellulose was prepared by a suspension method from the mixture of viscose and magnetite using thermal sol-gel transition and regeneration of cellulose. The prepared magnetic particles after their activation with divinyl sulfone were shown to be suitable magnetic carrier for immobilization of α-chymotrypsin and for its application in proteomic studies. The specific activity of the immobilized proteinase was high; its activity did not change in the course of storage. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH and temperature dependence of the activity, self-cleavage activity, and possibility of repeated use. α-Chymotrypsin immobilized to magnetic bead cellulose was used for the proteolytic digestion of porcine pepsin A and human gastric juice and a possibility of direct use of enzyme reaction products for matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis was shown.

The hydrolysis of PEtOx is studied to evaluate the potential toxicity of partially hydrolyzed polymers that might interfere with its increasing popularity for biomedical applications. The hydrolysis of PEtOx is studied in the presence of digestive enzymes (gastric and intestinal) and at 5.8 M hydrochloric acid as a function of temperature (57, 73, 90, and 100 °C). It is found that PEtOx undergoes negligible hydrolysis at 37 °C and that thermal and solution properties are not altered when up to 10% of the polymer backbone is hydrolyzed. Mucosal irritation and cytotoxicity is also absent up to 10% hydrolysis levels. In conclusion, PEtOx will not decompose at physiological conditions, and partial hydrolysis will not limit its biomedical applications., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many infant formulas are enriched with lactoferrin (Lf) because of its claimed beneficial effects on health. Native bovine Lf (bLf) is known to inhibit in vitro replication of human enteroviruses, a group of pathogenic viruses that replicate in the gut as their primary infection site. On the basis of a model digestion and human gastrointestinal enzymes, we hypothesized that bLf could retain its antiviral properties against enterovirus in the gastrointestinal tract, either as an intact protein or through bioactive peptide fragments released by digestive enzymes. To test our hypothesis, bLf was digested with human gastric juice and duodenal juice in a 2-step in vitro digestion model. Two gastric pH levels and reduction conditions were used to simulate physiological conditions in adults and infants. The antiviral activity of native bLf and of the digested fractions was studied on echovirus 5 in vitro, using various assay conditions, addressing several mechanisms for replication inhibition. Both native and digested bLf fractions revealed a significant inhibitory effect, when added before or simultaneously with the virus onto the cells. Furthermore, a significant stronger sustained antiviral effect was observed when bLf was fully digested in the gastric phase with fast pH reduction to 2.5, compared with native bLf, suggesting the release of antiviral peptides from bLf during the human digestion process. In conclusion, this study demonstrates that bLf may have a role in the prevention of human gastrointestinal virus infection under physiological conditions and that food containing bLf may protect against infection in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

An aqueous suspension of almond oil bodies (about 10% lipids) was prepared and subjected to in vitro gastric (with pepsin) and intestinal (with bile salts and pancreatin) digestion, simulating fasting conditions. The physicochemical and structural changes of the almond oil body emulsion were examined. The almond oil body emulsion behaved similarly to a protein-stabilized emulsion, with flocculation of the oil bodies occurring under gastric conditions. Proteins, peptides, and phospholipids covered the surface of the oil bodies throughout gastric digestion. Under intestinal conditions, bile salts displaced the interfacial peptides and phospholipids, and disrupted the flocs. Gastric pepsinolysis of almond proteins was a prerequisite for their digestion in the duodenum. The oil body membrane had a negative impact on the efficiency of gastric digestion, and long chain fatty acids, the main lipolytic products, accumulated at the surface of the oil bodies and therefore limited the activity of pancreatic lipase.

There is a growing interest in the optimization of dietary emulsions for monitoring postprandial lipid metabolism in the frame of preventing metabolic diseases. Using various emulsions, we investigated in a systematic scheme the combination of (i) in vitro gastrointestinal lipolysis and (ii) absorption and metabolism of lipolysis media in Caco-2 cells. Four emulsions based on either milk fat olein (OL) or rapeseed oil (RA) as the dispersed phase and either lecithin (LE) or sodium caseinate (CA) as the emulsifier were tested. After a sequential incubation of these emulsions with gastric and pancreatic enzymes, lipolysis media were incubated with Caco-2 cells, after dilution (1 : 20) to maintain the barrier integrity. Both gastric and duodenal lipolysis levels were similar to values reported in vivo and the rates of lipolysis were higher with LE-stabilized emulsions than with CA-stabilized emulsions (P < 0.05). TAG secretion by Caco-2 cells was found to be higher using (i) duodenal vs. gastric media (P < 0.001) and (ii) emulsions stabilized with CA vs. LE (P < 0.01). Consistently, gene expression of both FABP2 and FATP4 induced by the duodenal media was (i) higher than that with gastric media (P < 0.001) and (ii) faster than that with model mixed micelles. Using gastric media, TAG secretion of Caco-2 cells after 12 h was higher with RA than with OL (P < 0.001). Moreover, the RA-CA emulsion increased the size of secreted lipoprotein particles (514 nm vs. 61 to 130 nm; P < 0.01). In conclusion, it was possible to observe distinct responses in the lipid metabolism of Caco-2 cells incubated with lipolysis media obtained from different dietary emulsions digested by gastrointestinal lipases in vitro.

Reflux of gastroduodenal contents and delayed gastric emptying are the most common and serious problems after esophagectomy with gastric reconstruction. However, attempts to reduce the above symptoms, surgically as well as non-surgically, had no or limited effect. To address this issue, we performed retrosternal gastric reconstruction with duodenal diversion plus Roux-en-Y anastomosis (RY) in eight patients with thoracic esophageal cancer and compared the outcomes with control patients who underwent standard reconstruction. The procedure is simple, safe, and not associated with any postoperative complications. The pancreatic amylase concentrations in the gastric juice samples on postoperative day 2 were slightly lower in the non-RY group than in the RY group (1884 ± 2152 vs. 25,790 ± 23,542IU/mL, respectively, P= 0.07). Postoperative endoscopic examination showed neither reflux esophagitis nor residual gastric content in the RY group. Quality of life assessed by the Dysfunction After Upper Gastrointestinal Surgery-32 questionnaire postoperatively was significantly better in the RY group than in the non-RY group for 'decreased physical activity,''symptoms of reflux,''nausea and vomiting,' and 'pain.' The results of this pilot study suggest that gastric reconstruction with duodenal diversion plus RY seems effective in improving both the reflux and delayed gastric emptying. The benefits of this procedure need to be further assessed in a large-scale, randomized controlled trial., (© 2011 Copyright the Authors. Journal compilation © 2011, Wiley Periodicals, Inc. and the International Society for Diseases of the Esophagus.)

Scope: Four Bet v 1 homologous food allergens from celeriac (rApi g 1), apple (rMal d 1), peach (rPru p 1) and hazelnut (rCor a 1), were used to probe the structural responsiveness of the Bet v 1 scaffold to gastric digestion conditions and its impact on allergenicity., Methods and Results: Low pH induced conformational changes of all homologues, which was reduced at physiological ionic strength for all except rPru p 1 as observed by circular dichroism (CD)-spectroscopy. The homologues were rapidly digested by pepsin, losing their IgE binding activity, although the kinetics and patterns of digestion varied subtly between homologues, rApi g 1 being the most stable. We have demonstrated for the first time that gastric phosphatidyl-choline (PC) induced conformational changes in all homologues but only rMal d 1 penetrated the PC vesicles as detected by fluorescence polarization, slowing its digestion and retaining more of its allergenic activity. PC enhanced basophil activation of all digested allergens except rApi g 1., Conclusion: The Bet v 1 scaffold is generally susceptible to low pH and pepsinolysis and interacts with PC vesicles, properties which can explain effects of the gastric environment on their allergenicity. These data show the importance of including surfactants in model digestion systems., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An in vitro digestion model mimicking the gastric phase of the human gastrointestinal tract coupled with SDS-PAGE and MALDI-TOF mass spectroscopy was employed to study the hydrolysis profiles of whey proteins in solution and adsorbed at the oil-water interface. The objective of this work was to understand the differences in hydrolysis behaviour of whey protein isolates once adsorbed at the interface, and comparisons were carried out with pure beta-lactoglobulin and alpha-lactalbumin fractions. In solution, while beta-lactoglobulin appeared to be resistant to enzymatic treatment, alpha-lactalbumin was fully degraded. Adsorption of both proteins at the oil-water interface affected their conformational structure and susceptibility to peptic hydrolysis. Adsorbed beta-lactoglobulin was hydrolyzed into small polypeptides and in contrast, the resistance of alpha-lactalbumin to pepsin increased upon adsorption at the interface. In addition, changes in the particle size distribution of the droplets during pepsin hydrolysis mainly depended on the original protein concentration. The results suggested that exchanges occur at the interface between adsorbed and non-adsorbed protein, that is to say that either some protein desorb from the interface and does not fully recover its structure in solution, or that hydrolysis of the protein at the interface induces further adsorption and hydrolysis of the protein in solution. These mechanisms have important implications in the digestibility of the proteins., (2010 Elsevier Inc. All rights reserved.)

The tolerance of lactic acid bacteria (LAB) from kefir grains to gastrointestinal tract conditions was evaluated in vitro. The effects of pH values and bile salts on the viability of LAB were investigated. The results demonstrated that pH value showed a significant effect on the viability. The viable counts exhibited a reduction of 1.5 to 2 log cycles in 0.3% to 0.5% bile salts after 4 h. The viability of LAB exposure to sequential simulated gastric and intestinal juices was assessed by response surface model (RSM). RSM indicated that the gastric pH and gastric contact time significantly affected the viability (P < 0.05), while the effect of intestinal contact time was not significant. Moreover, RSM revealed the interactions of pH and gastric contact time, and of pH and intestinal contact time. The LAB cells, temporarily damaged by the low pH of gastric juice (pH < 2), could recover in the intestinal juice; and the longer the intestinal contact time, the higher the viability of LAB. RSM proved to be a useful and accurate method to predict the viability of LAB under certain laboratory conditions by the model validation. This study indicated that LAB from kefir grains exhibited excellent tolerance to sequential simulated gastrointestinal tract conditions, and that kefiran possessed a significant protective effect on LAB in hostile environments.

A novel endo-beta-1,4-glucanase, AC-EG65, with a molecular mass of 65 kDa, was purified from the gastric juice of the mollusc, Ampullaria crossean, by ammonium sulfate fractionation, anion exchange, gel filtration, hydrophobic interaction and a second round of anion exchange chromatography. AC-EG65 showed specific carboxymethyl cellulose hydrolytic activity of 13.3 U/mg protein and the optimal pH and temperature of the activity were pH 5.5-6.5 and 50-55 degrees C, respectively. From the cDNA library of A. crossean stomach tissue, eight endo-beta-1,4-glucanase genes with high similarity were successfully cloned based on the partial amino acid sequences of AC-EG65 and were classified into 3 groups: eg65-a, eg65-b, and eg65-c. The open reading frames of the groups eg65-a, eg65-b, and eg65-c were 2142 bp, 2171 bp, and 2169 bp in length, encoding 713, 723 and 722 amino acids, respectively. The eight deduced proteins consisted of a family II carbohydrate-binding module (CBM2) and a glycosyl hydrolase family 9 (GHF9) catalytic domain. More than 98% amino acid identities were shared within the same group and more than 87% sequence identities among the groups. The endogenous origins of these EGase genes were supported by PCR amplification using ovary genomic DNA as template.

Aims: To isolate acid- and bile-resistant Saccharomyces cerevisiae strains directly from food samples and to preliminarily select them on the basis of fundamental probiotic properties., Methods and Results: A rapid screening method allowed the isolation and selection of 20 acid- and bile-resistant yeasts from foods, avoiding time-consuming isolation steps. The strains were characterized for their specific survival in simulated gastric juice and in intestinal fluid after pre-exposure at low pH. Ten isolates demonstrated a satisfactory survival percentage in intestinal fluid after pre-exposure to gastric juice and appreciable lipolytic and proteolytic properties, as demonstrated by the API-ZYM test. By using molecular methods five strains were identified as Saccharomyces cerevisiae, three as Candida spp., one as Candida pararugosa and one as Pichia spp. The Saccharomyces cerevisiae strains showed considerable probiotic properties, achieving a 80< % <90 survival through the simulated gastrointestinal tract, as well as interesting glucosidase activities., Conclusions: The research represents an efficient strategy to select and identify Saccharomyces cerevisiae strains with desirable acid and bile resistances., Significance and Impact of the Study: This paper reports the direct selection of potentially probiotic yeasts from foods and provides indications about the ability of Saccharomyces cerevisiae strains to survive conditions simulating the human gastrointestinal tract.

Gastroesophageal reflux and extraesophageal reflux have been postulated to be involved in the pathogenesis of otitis media. This is supported by recent studies revealing the presence of gastric pepsin in the middle ear space of children with otitis media but not in control patients without otitis media. Reflux's role in otitis media appears to be most pronounced in younger children and those with purulent effusions.

The lipase inhibitory activity of ethanol extracts obtained from some marine algae collected on the Tunisian coast was evaluated. Caulerpa prolifera extract markedly reduced both dog gastric (DGL) and human pancreatic lipase (HPL) activities. Generally, the inhibition reached 100% after 40 to 60 min of incubation depending on lipase types and on substrates used. Moreover, the inhibitory effect of C. prolifera extract on lipases appeared to be accelerated by adding bile salts, which likely modified the interface and allowed the inhibitory compound to inactivate the lipase. The separation of C. prolifera extract by thin-layer chromatography (TLC) resulted in eight fractions showing efficient inhibition rate against DGL, compared to the crude extract. In the case of HPL, TLC fractionation reduced the inhibitory rates, suggesting that the effect of algal extract on lipases may be caused by a synergetic action of several compounds within the extract. High-performance liquid chromatograph separation resulted in isolation of a major compound displaying high inhibition capacity of HPL activity. Caulerpa prolifera extract may therefore be useful in developing antiobesity drugs.

We characterized major digestive enzymes in Panulirus argus using a combination of biochemical assays and substrate-(SDS or native)-PAGE. Protease and amylase activities were found in the gastric juice while esterase and lipase activities were higher in the digestive gland. Trypsin-like activity was higher than chymotrypsin-like activity in the gastric juice and digestive gland. Stability and optimal conditions for digestive enzyme activities were examined under different pHs, temperature and ionic strength. The use of protease inhibitors showed the prevalence of serine proteases and metalloproteases. Results for serine proteases were corroborated by zymograms where several isotrypsins-like (17-21 kDa) and isochymotrypsin-like enzymes (23-38 kDa) were identified. Amylases (38-47 kDa) were detected in zymograms and a complex array of non-specific esterases isoenzymes was found in the digestive gland. Isoenzyme polymorphism was found for trypsin, amylase, and esterase. This study is the first to evidence the biochemical bases of the plasticity in feeding habits of P. argus. Distribution and properties of enzymes provided some indication on how the digestion takes place and constitute baseline data for further studies on the digestion physiology of spiny lobsters.

Consumption of coffee beverages has been reported to cause gastric irritation in some consumers as a result of increased gastric acid secretion. In the complex mechanisms of gastric acid secretion, the activity and expression of the H+,K+-ATPase is regulated by transmitters, such as histamine, acetylcholine, gastrin, somatostatin, and their corresponding receptors. Here, we report the effect of three coffee constituents, chlorogenic acid, caffeine, and N-methyl pyridinium ions, on the expression of the histamine receptor H2, the acetylcholine receptor M3, the gastrin receptor, the somatostatin receptor, and the H+,K+-ATPase. Human gastric cancer cells were exposed to chlorogenic acid, caffeine, or N-methyl pyridinium in their coffee brew-representative concentrations as well as to physiological stimulators of gastric acid secretion. Gene expression levels of receptor proteins and those of the H+,K+-ATPase were measured at different time points by real-time PCR. Expression of prosecretory receptors significantly increased between one and one-half to twofold after treatment with chlorogenic acid or caffeine compared to control cells at the same time point. Chlorogenic acid and caffeine also increased the H+,K+-ATPase gene expression twofold higher compared to control cells. In contrast, N-methyl pyridinium downregulated the expression of the prosecretory gastrin receptor significantly, by -27%. In conclusion, chlorogenic acid, caffeine, and N-methyl pyridinium impair the expression of gastric acid secretion-related proteins in a time-dependent manner. Future work will be aimed at the elucidation of the cooperative interplay of individual components using recombinates of single coffee constituents.

Aim: To determine the efficacy of gastric juice polymerase chain reaction (PCR) for the detection of H pylori infection in comparison with histology and gastric antral biopsy PCR in patients on a proton pump inhibitor (PPI)., Methods: Eighty-five consecutive patients with dyspeptic symptoms were enrolled. Gastric biopsies for histology, PCR and gastric juice were collected at endoscopy for PCR of the H pylori urease C gene (ure C). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, positive and negative likelihood ratio for PCR of gastric juice for the H pylori ure C gene was compared to histology and gastric antral biopsy H pylori ure C PCR in patients with and without PPI., Results: Gastric juice PCR was positive in 66 (78%) patients. Histology showed H pylori associated gastritis in 57 (67%). Gastric biopsy PCR was positive in 72 (85%). In patients not taking PPI, the sensitivity, specificity, PPV, NPV, accuracy and positive and negative likelihood ratio for gastric juice PCR were 89%, 72%, 91%, 67%, 90%, 85%, 3.1 and 0.1 respectively. In patients on PPI these values were 86%, 100%%, 100%, 29%, 86%, 9.5 and 1.4, respectively., Conclusion: Gastric juice PCR for the diagnosis of H pylori infection has increased sensitivity compared to histology with PPI. The use of gastric juice PCR is recommended to confirm H pylori status in patients taking PPIs.

A microelectrochemical pH-stat with an automatic feedback function was fabricated. The operation of the device is based on the nonstandard use of an electrochemical three-electrode system with a pH-sensitive reference electrode, a Ag/AgCl working electrode, and an iridium auxiliary electrode that functions as an actuator to adjust the solution pH. The combination of the electrodes caused a negative feedback in response to a pH change. The shift of the potential of the pH-sensitive reference electrode caused an overpotential on the Ag/AgCl working electrode, which then caused a significant current increase. This led to the electrolysis of water on the auxiliary electrode and the rapid recovery of the pH. The negative feedback function to stabilize the initial state could be confirmed for changes to both the acidic and basic directions. The performance of the pH-stat was characterized in the titration of acetic acid or ammonia. The charge generated in the feedback process changed linearly with respect to the concentration. The pH-stat was also used in the determination of urea by urease and that of the activities of trypsin and pepsin while maintaining the optimum pH for the enzymes. The pH to be fixed could be changed by changing the working electrode potential. Moreover, the two pH-stats could be used to form a pH gradient in a microflow channel by fixing the pH values at two positions.

Objective: The objective of this study was to study the association between pepsin in tracheal aspirate samples and the development of bronchopulmonary dysplasia in preterm infants., Methods: Serial tracheal aspirate samples were collected during the first 28 days from mechanically ventilated preterm neonates. Bronchopulmonary dysplasia was defined as the need for supplemental oxygen at 36 weeks' postmenstrual age. An enzymatic assay with a fluorescent substrate was used to detect pepsin. Total protein was measured by the Bradford assay to correct for the dilution during lavage. Immunohistochemistry using antibody against human pepsinogen was performed in 10 lung tissue samples from preterm infants., Results: A total of 256 tracheal aspirate samples were collected from 59 preterm neonates. Pepsin was detected in 234 (91.4%) of 256 of the tracheal aspirate samples. Twelve infants had no bronchopulmonary dysplasia, 31 infants developed bronchopulmonary dysplasia, and 16 infants died before 36 weeks' postmenstrual age. The mean pepsin concentration was significantly lower in infants with no bronchopulmonary dysplasia compared with those who developed bronchopulmonary dysplasia or developed bronchopulmonary dysplasia/died before 36 weeks' postmenstrual age. Moreover, the mean pepsin level was significantly higher in infants with severe bronchopulmonary dysplasia compared with moderate bronchopulmonary dysplasia. The mean pepsin level in tracheal aspirate samples from the first 7 days was also lower in infants with no bronchopulmonary dysplasia compared with those who developed bronchopulmonary dysplasia or developed bronchopulmonary dysplasia/died before 36 weeks' postmenstrual age. Pepsinogen was not localized in the lung tissues by immunohistochemistry., Conclusion: The concentration of pepsin was increased in the tracheal aspirate of preterm infants who developed bronchopulmonary dysplasia or died before 36 weeks' postmenstrual age. Recovery of pepsin in tracheal aspirate samples is secondary to gastric aspiration, not by hematogenous spread or local synthesis in the lungs. Chronic aspiration of gastric contents may contribute in the pathogenesis of bronchopulmonary dysplasia.

Next to health promoting effects, the functional aspect of probiotic strains also involves their capacity to reach the colon as viable metabolically active cells. The present study aimed to assess the potential of 24 probiotic product isolates and 42 human reference strains of Bifidobacterium to survive gastrointestinal transit under in vitro conditions. The survival capacity of exponential and stationary phase cultures upon exposure to gastric and small intestinal juices was determined using a recently developed microplate-based assay in combination with the LIVE/DEAD BacLight Bacterial Viability kit. All 66 strains tested displayed a considerable loss in viability during exposure to an acidic pepsin containing solution (pH 2.0). Among the 10 taxa tested, cultures of B. animalis ssp. lactis appeared to be most capable to survive gastric transit. Although to a lesser extent, the presence of bile salts also affected the viability of most of the strains tested. Except for 3 strains, all 66 strains showed bile salt hydrolase activity using an agar-based assay. In contrast, the bifidobacterial strains used in this study appeared to possess a natural ability to survive the presence of pancreatin (pH 8.0). Although the effect was not significant, a slightly enhanced tolerance to gastrointestinal transit was observed when cells were in the stationary phase, especially when exposed to acid, compared with cells being in the exponential phase. Survival in the gastrointestinal tract appeared to be largely strain-dependent and hence implies that different strains will likely display a different behavior in functionality. The assay used in this study allows an initial assessment of strains for use as probiotic cultures prior to selecting potential candidate strains for further investigation in vivo.

Objective: We sought to confirm the finding of pepsin/pepsinogen in the middle ear fluid of children with otitis media in a larger sample size using a sensitive and specific pepsin assay., Study Design and Setting: We evaluated 152 children (225 ear samples) in a prospective study at a tertiary care children's hospital. The presence of pepsin in middle ear aspirates was determined using enzymatic assay., Results: Of the patients, 14.4 percent (22 of 152) had detectable pepsin activity in one or both of the ear samples with no pepsin activity detected in control serum. Average pepsin concentration in the samples was 96.6 +/- 170.8 ng/ml, ranging from 13 to 687 ng/ml. Pepsin concentration in the middle ear of children younger than 1.0 year was significantly higher than in older age groups., Conclusion and Significance: Results indicate that pepsin/pepsinogen is present in the middle ears of children with otitis media, although not at the high rate previously reported. Gastric reflux may be one causative factor in the pathogenesis of otitis media.