Digestion Model System Research Articles

Investigation of the Bioaccessibility of Functional Ice Cream with Blueberry Enriched with Whey Protein Gel

Ice cream is a complex product obtained by blowing air through special equipment and then freezing a physicochemical mixture consisting of milk, sugar, emulsifier, stabilizer, oil, color and flavoring substances. Recently, especially with the pandemic experienced all over the world, consumers have begun to turn to functional foods that have high nutritional value and are important for health. Functional foods, in addition to its nutritional effects, are defined as foods that have health protective, corrective and/or disease risk reducing effects, depending on one or more effective ingredients, and these effects are scientifically and clinically proven. In order for a product to have functional properties, it must contain bioactive ingredients, probiotic microorganisms and also have a prebiotic effect. For this reason, our study aimed to provide functional properties to ice cream with blueberries, which are rich in phenolic compounds, and to examine the phenolic substance bioaccessibility of this functional ice cream under mouth, stomach and small intestine conditions simulated with the in vitro gastrointestinal digestion model system. In this context, functional ice cream was produced by trapping the phenolic rich blueberry fruit in six different concentrations of whey protein gel, and the amount of phenolic substance and protein amount were determined after in vitro digestion. While the phenolic substance content of protein gel ice cream in the small intestine environment was between 261-485 µg/100 g and an average of 114 µg/100 g in the control sample, in the oral environment these values were determined as 85-251 µg/100 g in protein gel ice cream and 291 µg/100 g in the control sample. As a result of our study, it was determined that the amount of gallic acid phenolic substance and bioaccessibility of ice cream samples produced with protein gel increased from the oral environment to the small intestine. In the control sample (blueberry ice cream without protein gel), it was observed that the amount of phenolic substance was highest in the oral environment and decreased as it went to the small intestine environment. According to the FAO Guidelines for Use of Nutrition Claims, samples with a whey protein gel ratio of 16%, 18% and 20% can be considered as "protein sources". Thus, in this study, functionalized in terms of protein content and phenolic substance, increased bioaccessibility and high protein ice cream production was carried out.

Gut microbiota of the small intestine as an antimicrobial barrier against foodborne pathogens: Impact of diet on the survival of S. Typhimurium and L. monocytogenes during in vitro digestion

The human gastrointestinal tract employs an assortment of chemical, enzymatic and immune barriers to impede pathogen colonization. An essential component of these barriers is the gut microbiota, which infers protection against ingested pathogens through its colonization resistance mechanisms. Specifically, the gut microbiota of the distal small intestine (ileum) renders a crucial line of defense, given that this location is regarded as an important interaction site. This study aimed to evaluate the impact of the ileal microbiota on the survival of the foodborne pathogens Salmonella enterica serotype Typhimurium and Listeria monocytogenes, utilizing an in vitro digestion model system. Moreover, the effect of diet on the gut microbiota colonization resistance mechanisms was assessed, by comparing a healthy (high fiber/low sugar) and a western diet (low fiber/high sugar). For S. Typhimurium, the results revealed that the digestion of a healthy diet led to a similar inactivation compared to the western diet, with the values of total log reduction being 0.83 and 0.82 log(CFU), respectively; yet the lack of readily accessible nutrients in the healthy diet combined with the acidic shock during gastric digestion caused the induction of stress tolerance to the pathogen. This resulted in increased pathogen survival in the presence of gut microbiota, with S. Typhimurium proliferating during the ileal phase with a maximum specific growth rate of 0.16 1/h. On the contrary, for L. monocytogenes, the healthy diet was associated with a greater inactivation than the western diet (total log reduction values: 3.08 and 1.30 log(CFU), respectively), which appeared strongly influenced by the encounter of the pathogen with the gut microbiota. Regarding the latter, the species Escherichia coli and Bacteroides thetaiotaomicron appeared to be the most prevalent in most cases. Finally, it was also demonstrated that the ileal microbiota colonization resistance mechanisms largely relied on competitive responses. The obtained knowledge of this research can contribute to the development and/or complementation of defensive strategies against pathogen infection, while also underlining the value of in vitro approaches.

Lauric acid adsorbed cellulose nanocrystals reduced the in vitro gastrointestinal digestion of oil-water pickering emulsions

An in vitro gastrointestinal tract (GIT) fate of beta-carotene-loaded oil-in-water Pickering emulsions stabilized with lauric acid-adsorbed cellulose nanocrystals (CL) was investigated. Different concentrations (0.5, 1.0, 1.5 and 2.0%, w/v) of CL and pure cellulose nanocrystals (CNC) were used. During in vitro digestion, the effects of CL and CNC and their concentrations on the lipid droplet diameter (D 4,3 ), zeta potential, microscopic structure, phase stability (creaming index), degree of lipid digestion (free fatty acid release), and bioaccessibility of loaded beta-carotene were recorded. The Pickering emulsions stabilized with levels of CL above 1.0% (w/v) were stable against lipid droplet flocculation and coalescence and impeded the increase of D 4,3 (p ≤ 0.05) throughout the GIT phases. A significant increase in the D 4,3 was recorded for the emulsion stabilized with CNC due to flocculation and coalescence of lipid droplets. Instead of electrostatic repulsion stabilization, Pickering emulsions stabilized with CL displayed steric stabilization activity at the lower pH of the gastric phase, where the lowest zeta potential of less than +10 mV was found. The Pickering emulsion stabilized with CNC had significantly higher lipid digestion (∼63%) than stabilized with CL (∼52%). Average bioaccessibilities were up to 65% for CL stabilized Pickering emulsions and 70% for CNC-stabilized Pickering emulsions. CL can maintain emulsions phase stability by retaining lipid droplet size, reducing lipid digestibility, and prolonging the release of lipophilic nutrients. Other modifications of CNC may be required to release such nutrients in the small intestine, and in vivo studies are needed to verify in vitro assays. • Modifying nanocellulose with lauric acid improved emulsion stability but delayed the release of beta-carotene for absorption in an in vitro digestion model system. • Other types of chemical modification may be necessary to deliver phytonutrients for absorption in the small intestine.

Effect of gastric pH and bile acids on the survival of Listeria monocytogenes and Salmonella Typhimurium during simulated gastrointestinal digestion

The human gastrointestinal tract (GIT) harbors numerous defensive mechanisms to impede pathogen colonization, including gastric acidity and bile acids in the small intestine. This study aims to elucidate the survival of Salmonella Typhimurium and Listeria monocytogenes under different levels of gastric pH (2.0–3.5) and bile acid concentrations (2.5 mM – 10.0 mM), during an in vitro digestion process following the ingestion of a contaminated food model system. The results showed that S. Typhimurium was more susceptible to gastric acidity than L. monocytogenes, yet more bile acid tolerant. Interestingly, the bile acids bactericidal effect towards L. monocytogenes was higher if the previous exposure to the gastric acidity was harsher. Our findings suggest that, despite the different microbial behavior of the two species in the simulated GIT, the effects of gastric acidity and bile acids alone could not prevent their survival in the intestine. Industrial relevanceS. Τyphimurium and L. monocytogenes represent biological threats that are of major concern for the food industry due to their ubiquitous nature and their ability to trigger foodborne illnesses. Their ability to survive the gastrointestinal passage is crucial for their subsequent intestinal colonization that can lead to host infection. Therefore, obtaining a deeper insight into the factors affecting pathogen survival in the gut is vital for the establishment of more efficient strategies to prevent foodborne diseases. Given the limitations and ethical constraints of conducting studies like this in vivo, the applied in vitro digestion model system along with the mathematical modelling can provide a valuable alternative that can be readily utilized in the food and pharmaceutical industry as a platform for similar digestion studies.

Digestive propensity of Aflatoxin M1 (4‐Hydroxyaflatoxin B1), an indication from in vitro digestion model system

Milk quality and safety maintenance is an important issue, as many reports showed several biological and chemical contaminants in the milk. Among all contaminants, AFM1 is detected frequently in milk samples. Therefore, our main aim was to understand the digestive fate of AFM1 in an in vitro digestion (IVD) model system by using AFM1 spiked milk samples (2 μg/L). The IVD of the milk was confirmed by SDS-PAGE and AFM1 was estimated with ELISA. ELISA results confirmed that AFM1 was detected only in the spiked undigested milk samples, but not in IVD control and spiked milk samples. This observation clearly shows that AFM1 was digested during IVD. We also assessed AFM1 at different stages of digestion and found that AFM1 was digested only at the pancreatic digestion stage. In conclusion, the present study indicates that AFM1 at 2 ug/L in the milk might be completely digested. Novelty impact statement ‘Aflatoxin M1 appears to be digested by pancreatic juice, especially by the lipase’.

Profiling of In Vitro Bioaccessibility and Intestinal Uptake of Flavonoids after Consumption of Commonly Available Green Tea Types.

The aim of this study was to profile the bioaccessibility and intestinal absorption of epicatechins and flavonols in different forms of green tea and its formulation: loose leaf tea, powdered tea, 35% catechins containing GTE, and GTE formulated with green tea-derived polysaccharide and flavonols (CATEPLUS™). The bioaccessibillity and intestinal absorption of epicatechins and flavonols was investigated by using an in vitro digestion model system with Caco-2 cells. The bioaccessibility of total epicatechins in loose leaf tea, powdered tea, GTE, and CATEPLUS™ was 1.27%, 2.30%, 22.05%, and 18.72%, respectively, showing that GTE and CATEPLUS™ had significantly higher bioaccessibility than powdered tea and loose leaf tea. None of the flavonols were detected in powdered tea and loose leaf tea, but the bioaccessibility of the total flavonols in GTE and CATEPLUS™ was 85.74% and 66.98%, respectively. The highest intestinal absorption of epicatechins was found in CATEPLUS™ (171.39 ± 5.39 ng/mg protein) followed by GTE (57.38 ± 9.31), powdered tea (3.60 ± 0.67), and loose leaf tea (2.94 ± 1.03). The results from the study suggest that formulating green tea extracts rich in catechins with second components obtained from green tea processing could enhance the bioavailability of epicatechins.

Biodetoxification of fungal mycotoxins zearalenone by engineered probiotic bacterium Lactobacillus reuteri with surface-displayed lactonohydrolase.

Zearalenone (ZEN) is one of the common mycotoxins with quite high occurrence rate and is harmful to animal and human health. Lactobacillus reuteri is known as a probiotic bacterium with active immune stimulating and high inhibitory activity against pathogenic microorganisms. In this study, we expressed the lactonohydrolase from Rhinocladiella mackenziei CBS 650.93 (RmZHD) in L. reuteri via secretion and surface-display patterns, respectively. Endogenous signal peptides from L. reuteri were first screened to achieve high expression for efficient ZEN hydrolysis. For secretion expression, signal peptide from collagen-binding protein showed the best performance, while the one from fructose-2,6-bisphosphatase worked best for surface-display expression. Both of the engineered strains could completely hydrolyze 5.0 mg/L ZEN in 8 h without detrimental effects on bacterial growth. The acid and bile tolerance assay and anchoring experiment on Caco-2 cells indicated both of the abovementioned engineered strains could survive during digestion and colonize on intestinal tract, in which the surface-displayed strain had a better performance on ZEN hydrolysis. Biodetoxification of model ZEN-contaminated maize kernels showed the surface-displayed L. reuteri strain could completely hydrolyze 2.5 mg/kg ZEN within 4 h under low water condition. The strain could also efficiently detoxify natural ZEN-contaminated corn flour in the in vitro digestion model system. The colonized property, survival capacity, and the efficient hydrolysis performance as well as probiotic functionality make L. reuteri strain an ideal host for detoxifying residual ZEN in vivo, which shows a great potential for application in feed industry.

Biologically Active Digests from Pumpkin Oil Cake Protein: Effect of Cross‐linking by Transglutaminase

AbstractThe objective of this study was to show that biologically active hydrolysates can be obtained by simulated human gastrointestinal digestion (HGD) of transglutaminase cross‐linked pumpkin oil cake protein (Tg‐C) which was previously reported as a potential functional food additive. A two‐stage in vitro digestion model system (by pepsin and α chymotrypsin and trypsin, simultaneously) was used to simulate the process of HGD on native and Tg‐C major storage pumpkin oil seed/cake protein, cucurbitin (C). The biologically active potential of the digests was evaluated, measuring the angiotensin‐converting‐I enzyme (ACE) inhibitory and anti‐oxidant capacity. The ACE inhibitory activity was determined in both final digests, with IC50 = 0.30 ± 0.04 mg/ml for C and IC50 = 0.28 ± 0.01 for Tg‐C. The anti‐oxidant potency of the examined proteins was enhanced by the digestion process. The 2,2‐diphenyl‐1‐picrylhydrazyl and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) radical cation activities and reducing power testing showed that all the hydrolysates act as a radical quencher and reducing agents. Overall, the results showed that the cross‐linking by Tg did not influence the digestion process, as well as having no effect on the biological activity of the hydrolysates. These also indicate that Tg‐C, if used as functional food additive, after food consumption can be digested and become a source of peptides exerting positive effects on human health.

Houttuynia cordata Extract Improves Physical Endurance Performance by Regulating Endothelial Production of Nitric Oxide.

Vascular function is mediated by various regulatory molecules, including endothelial nitric oxide (NO), which regulates the vasodilation of smooth muscle cells. We investigated whether standardized Houttuynia cordata extract (SHCE) could improve physical endurance performance by regulating the endothelial production of NO. For the standardization of Houttuynia cordata (HC) extract, its bioactive components were identified and quantified using ultraperformance liquid chromatography-mass spectrometry. Bioaccessibility and biological activity were measured by the in vitro digestion model system and free radical scavenging capacity, respectively. The vascular function in the endothelium was assessed by the phosphorylation of endothelial nitric oxide synthase (eNOS). A preliminary clinical trial was carried out to assess the physical endurance performance. HC extract was standardized to bioactive components, including chlorogenic acid, rutin, and quercitrin, with the concentration of 5.53, 6.09, and 16.15 mg from 1 g of dry weight, respectively. Bioaccessibility was 33.17%, 31.67%, and 11.18% for chlorogenic acid, rutin, and quercitrin, respectively. Antioxidant activities of SHCE were expressed as vitamin C equivalent antioxidant capacity in 55.81 and 17.23 mg/g of HC extract using ABTS and DPPH scavenging assay, respectively. In human aortic endothelial cells, insulin-mediated phosphorylation of eNOS was increased by SHCE in the presence of palmitate. However, the expression of blood pressure-regulating genes was not altered. The level of blood lactate concentration and the heart rate of subjects who drank SHCE were lower than those of subjects who drank plain water. Oxygen uptake from subjects drinking SHCE was slightly higher than that from those who drank plain water. This study demonstrated that SHCE decreased heart rate and blood lactate, increased oxygen uptake, and improved physical performance, presumably due to the increased NO production.

Digestive recovery of sulfur‐methyl‐l‐methionine and its bioaccessibility in Kimchi cabbages using a simulated in vitro digestion model system

Sulfur-methyl-L-methionine (SMM) has been known to provide various biological functions such as radical scavenging effect, inhibition of adipocyte differentiation, and prevention of gastric mucosal damage. Kimchi cabbages are known to be a major food source providing SMM but its bioaccessibility has not been studied. The objective of current study was to determine both the digestive stability of SMM and the amount released from Kimchi cabbages under a simulated in vitro digestion model system. The in vitro digestion model system simulating a human gastrointestinal tract was carried out for measuring digestive recovery and bioaccessibility of SMM. SMM was quantified by using high-performance liquid chromatography with a fluorescence detector. Recovery of an SMM standard after digestion was 0.68 and 0.65% for fasted and fed conditions, respectively, indicating that the digestive stability of the SMM standard was not affected by dietary energy or co-ingested food matrix. The SMM standard was also significantly stable in acidic pH (P < 0.05). The bioaccessibility of SMM from Kimchi cabbages was measured under a fasted condition, resulted in 8.83, 14.71 and 10.88%, for salivary, gastric and small intestinal phases, respectively. Results from our study suggest that SMM from Kimchi cabbages, a component of food sources, is more bioavailable than SMM by itself.

CHANGES IN PROFILING OF PHENOLIC COMPOUNDS, ANTIOXIDATIVE EFFECT AND TOTAL PHENOLIC CONTENT INSMILAX CHINAUNDERIN VITROPHYSIOLOGICAL CONDITION

The three-stage of in vitro digestion model system simulating human digestive fluids was employed to determine changes in the profile of phenolic compounds, antioxidant activity and total phenolic content by digestion. The composition of total phenolic compounds in leaf, root and stem detected by gas chromatography-mass Spectroscopy method was 44.27, 19.31 and 8.59%, respectively, and 3-hydroxy-benzoic acid was found as a predominant phenolic compound identified in all parts of Smilax china. Both 2, 2-diphenylpicrylhydrazyl free radical scavenging effect (8.66 mg/mL for IC50) and total phenolic content (2,383.55 µg gallic acid equivalent/mL) using Folin–Ciocalteu method were remarkably high in leaf of Smilax china. Bioaccessible antioxidative effect and digestive stability of total phenolic content in leaf were also significantly higher than those of other parts. PRACTICAL APPLICATIONS Our study determined the relationship among phenolic compounds' composition, antioxidative effect or total phenolic content. Positive relationship was observed between antioxidative effect and total phenolic content in each part of Smilax china under in vitro physiological condition. As a result, total phenolic content determined after digestion of each part of Smilax china could be regarded as a major contributor for predicting antioxidant activity in human body. Our results provide information that Smilax china may be a useful substance for the application of natural antioxidant.

Changes in the antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulated gastrointestinal digestion

Abstract A two-stage in vitro digestion model system (a pepsin treatment for 2 h followed by a pancreatin treatment for 2 h, both at 37 °C) was used to simulate the process of human gastrointestinal (GI) digestion to determine the changes in antioxidant activities of loach peptide previously prepared by papain digestion. Results showed that the final GI digests contained 38.1% free amino acids, with short chain peptides (