Fatty acid bioaccessibility and structural breakdown from in vitro digestion of almond particles.

Previous studies have shown that the size of almond particles influences lipid bioaccessibility during digestion. However, the extent of structural breakdown of almond particles during gastric digestion and its impact on lipid bioaccessibility is unclear. In this study, in vitro digestion of almond particles was conducted using a dynamic model (Human Gastric Simulator) and a static model (shaking water bath). Structural breakdown of particles during the gastric phase occurred only in the Human Gastric Simulator, as evidenced by a reduction in particle size (15.89 ± 0.68 mm2 to 12.19 ± 1.29 mm2, p < 0.05). Fatty acid bioaccessibility at the end of the gastric phase was greater in the Human Gastric Simulator than in the shaking water bath (6.55 ± 0.85% vs. 4.54 ± 0.36%, p < 0.01). Results showed that the in vitro model of digestion which included peristaltic contractions (Human Gastric Simulator) led to breakdown of almond particles during gastric digestion which increased fatty acid bioaccessibility

Brush border digestion : development of a physiologically relevant in vitro model : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physiology at Massey University, Manawatū, New Zealand

Chapter Two has, in part, been published in the Springer publication, Reviews of Physiology, Biochemistry and Pharmacology, but the Author retains the right to publish the material in any collection consisting of the Author's own works: Hooton, D., Lentle, R., Monro, J., Wickham, M., & Simpson, R. (2015). The secretion and action of brush border enzymes in the mammalian small intestine. In: B. Nilius, T. Gudermann, R. Jahn, R. Lill, O. Petersen, & P. de Tombe (Eds.), Reviews of Physiology, Biochemistry and Pharmacology (vol 168, pp. 59-118). Cham: Springer. doi: 10.1007/112_2015_24The majority of current in vitro digestion methods either exclude the small intestinal brush border (BB) phase of digestion or do not incorporate the entire array of BB enzymes that are required to achieve terminal endogenous digestion in vivo. Accordingly, the digestate, and its derivitives, may not be representative of the digestive process in vivo. In order to improve the fidelity of the in vitro digestion process this thesis developed a physiologically relevant small intestinal BB phase using enzymes isolated from rat small intestinal mucosal tissue. The activities of BB enzymes were assessed and compared with known values, and under conditions physiologically representative of the small intestine. Although there were significant differences in BB enzyme activities depending on pH, enzyme solubilisation, and upon prolonged exposure to biliopancreatic secretions the BB preparation was deemed suitable for use in an in vitro digestion method. A rationale for the composition of the BB digestive phase was developed based on published physiological data, and was validated using glycosylated polyphenolic compounds as substrates. Liquid chromatography mass spectrometry (LC-MS) was used to assess the derivatisation products of BB digestion. In the absence of biliopancreatic secretions the onion flesh polyphenolic compounds quercetin-4ʹ-glucoside and isorhamnetin-4ʹ-glucoside, but not quercetin-3-glucoside or quercetin-34ʹ-diglucoside were hydrolysed. The positive control quercetin-3-glucoside was hydrolysed, and the negative control quercetin-3-rutinoside was not hydrolysed. The deglycosylation of quercetin-3-glucoside was monitored under conditions representative of the small intestine, i.e. incorporating bile and pancreatin, while at the appropriate pH. Quercetin-3-glucoside was significantly deglycosylated in BB treatments (no treatment or pancreatin alone) compared to BB treatments with bile (bile alone or pancreatin and bile). The mammalian digestive system is equipped to hydrolyse macronutrients from their polymeric form through to monomers and oligomers suitable for absorption across the epithelial layer. As such the inactivation or degradation of some BB enzymes during the BB digestive phase by bile or pancreatin was not unexpected, and does not preclude its use as an in vitro tool in the future

The in vitro assessment of the bioavailability of iron in New Zealand beef : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Physiology at Massey University, Palmerston North, New Zealand /

The bioavailability of iron in New Zealand beef either alone or as part of a 'typical' New Zealand meal was investigated. The solubility of iron and its in vitro absorption by mouse intestinal tissue were used to evaluate iron bioavailability. The solubility of haem and/or non-haem iron in meat (beef longissimus muscle), vegetables and meat-plus-vegetables was investigated. Samples were cooked and then subjected to in vitro gastrointestinal digestion with pepsin followed by a combination of pancreatic enzymes and bile. Cooking at 65°C for 90 minutes reduced the soluble iron concentration in meat by 81% and reduced the haem iron concentration by 27%, which coincided with a 175% increase in non-haem iron concentrations. However, gastrointestinal digestion increased the solubility of iron in cooked meat (333%), vegetables (367%) and meat-plus-vegetables (167%). A proportion (35%) of the haem iron in the meat was broken down by the action of pancreatic enzymes leading to a 46% increase in non-haem iron concentrations, although this was not the case for the meat-plus-vegetables. Validation studies showed that mouse intestinal segments mounted in Ussing chambers maintained integrity and viability, and were responsive to glucose, theophylline and carbachol. Intestinal tissue from iron deficient mice was then used in the Ussing chambers to investigate the absorption of iron from ferrous gluconate and the soluble fractions of meat, vegetables and meat-plus-vegetables after gastrointestinal digestion. Results indicated a trend towards a higher absorption of iron from meat and ferrous gluconate, compared to vegetables and meat-plus-vegetables. However, iron absorption results were difficult to interpret due to the wide variation in the data. This variation was possibly due to errors associated with the sample processing and the analysis of iron, which was by inductively coupled-mass spectroscopy. Overall, the present study showed that before estimations can be made on the bioavailability of food iron, the effects of the cooking and gastrointestinal digestion processes must be considered. Further, the use of in vitro gastrointestinal digestion followed by the use of Ussing chambers to assess intestinal absorption is a potentially valuable system for assessing mineral bioavailability

Gastrointestinal endogenous proteins as a source of bioactive peptides : Doctor of Philosophy in Nutritional Sciences at Riddet Institute, Massey University, Palmerston North, New Zealand

Gastrointestinal endogenous proteins (GEP) were investigated as a source of bioactive peptides. In silico and in vitro methods were used singly or in combination to study GEP-derived peptides after simulated digestion. The presence of bioactive peptides after in vivo digestion was determined using a porcine model. Bioactivity of the peptides was assessed using selected in vitro bioactivity assays, and peptides were characterised using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. In the in silico study, twenty six different GEP and seven dietary proteins were subjected to simulated in silico gastrointestinal (SIGIT) digestion. The predicted resultant peptides possessing amino acid sequences identical to those of known bioactive peptides were identified by screening them against an online database of bioactive peptides (BIOPEP). The predicted number of bioactive peptides released after the SIGIT digestion of GEP ranged from 1 (secretin) to 39 (mucin-5AC), while those for dietary proteins ranged from 1 (gliadin) to 55 (myosin). Angiotensin-I-converting enzyme (ACE-I) inhibitory peptide sequences were found in abundance in both GEP and dietary proteins. The GEP mucin-5AC and the dietary protein myosin were predicted to release the highest number of ACE-I inhibitory peptides (38 and 49 peptides respectively), and were found to be comparable in their potential to release ACE-I inhibitory peptides. Following SIGIT digestion of eleven representative GEP, nineteen novel GEP-derived peptide sequences were selected by applying quantitative structure-activity relationship rules, and were chemically synthesised. Two novel peptides with the amino acid sequences RPCF and MIM, showing dipeptidyl peptidase IV (DPP-IV) inhibitory activity and five novel antioxidant (2,2-diphenyl-1-picrylhydrazyl (DPPH)- inhibitory and, or ferric reducing antioxidant power (FRAP) activity) peptides with amino acid sequences CCK, RPCF, CRPK, QQCP and DCR were identified. These results indicate that GEP may contain novel bioactive peptide sequences. The potential release of bioactive peptides, from four GEP (trypsin, lysozyme, mucin, and serum albumin) and a dietary protein (chicken albumin), in the gastrointestinal tract (GIT) was investigated using an in vitro digestion model. The in vitro digests were screened for ACE-I-, renin-, platelet-activating factor acetylhydrolase (PAF-AH)-, and DPP-IV-inhibition, and antioxidant activity. All four in vitro GEP digests showed ACE-I inhibition comparable to that of the positive control captopril. In comparison to the unfractionated digests, the enriched fractions (<3 and <10 kDa) of lysozyme and serum albumin showed greater renin-, PAF-AH-, and DPP-IV-inhibition, and antioxidant potential. Over 190 peptide sequences were identified from these fractions using mass spectrometry. Stomach chyme (SC) and jejunal digesta (JD) were collected from growing pigs that were fed a protein-free diet for a period of 3 days. The peptides extracted from SC and JD samples were characterized by SDS-PAGE, and their ACE-I-, DPPH-, and microsomal lipid peroxidation (MLP)- inhibition, FRAP activity determined. Potential bioactive peptides responsible for bioactivity were identified using mass spectrometry. SDS-PAGE analysis showed that all of the samples contained a heterogeneous mixture of peptides. Porcine JD samples inhibited ACE-I and DPPH, while SC samples inhibited MLP. Characterization studies identified over 180 peptide sequences from the enriched fractions of SC and JD samples that showed the highest activity. Further, a porcine serum albumin peptide sequence (FAKTCVADESAENCDKS) was found to be a sub-sequence of a larger sequence identified in the in vitro digest of human serum albumin. There was considerable inter-animal variation for the bioactivities. This may be attributed to sampling effects and, or natural variations in the gut contents, thus underlining the complexity involved in in vivo release of bioactive peptides. Together, the results indicate: 1) GEP contain abundant encrypted bioactive peptide sequences; 2) GEP-derived bioactive peptides display a range of bioactivities; 3) GEP-derived bioactive peptides are released during gastrointestinal digestion in pigs; 4) GEP may contain numerous novel bioactive peptide sequences encoded within their primary sequence. In conclusion, the evidence reported here suggests that, like the dietary proteins, GEP are also a potentially rich source of exogenously-derived bioactive peptides in the gastrointestinal tract. Beyond their primary functions, GEP may act as an important cryptomic source of bioactive peptides, given that the amount of GEP secreted into the gut is equal to or greater than the dietary protein ingested per day, and that up to 80% of GEP are known to be digested

Aryl hydrocarbon receptor activation during in vitro and in vivo digestion of raw and cooked broccoli (brassica oleracea var. Italica)

Broccoli is rich in glucosinolates, which can be converted upon chewing and processing into Aryl hydrocarbon Receptor (AhR) ligands. Activation of AhR plays an important role in overall gut homeostasis but the role of broccoli processing on the generation of AhR ligands is still largely unknown. In this study, the effects of temperature, cooking method (steaming versus boiling), gastric pH and further digestion of broccoli on AhR activation were investigated in vitro and in ileostomy subjects. For the in vitro study, raw, steamed (t = 3 min and t = 6 min) and boiled (t = 3 min and t = 6 min) broccoli were digested in vitro with different gastric pH. In the in vivo ileostomy study, 8 subjects received a broccoli soup or a broccoli soup plus an exogenous myrosinase source. AhR activation was measured in both in vitro and in vivo samples by using HepG2-Lucia™ AhR reporter cells. Cooking broccoli reduced the AhR activation measured after gastric digestion in vitro, but no effect of gastric pH was found. Indole AhR ligands were not detected or detected at very low levels both after intestinal in vitro digestion and in the ileostomy patient samples, which resulted in no AhR activation. This suggests that the evaluation of the relevance of glucosinolates for AhR modulation in the gut cannot prescind from the way broccoli is processed, and that broccoli consumption does not necessarily produce substantial amounts of AhR ligands in the large intestine

Digestibility of resistant starch containing preparations using two in vitro models

Background: Resistant starch (RS) is known for potential health benefits in the human colon. To investigate these positive effects it is important to be able to predict the amount, and the structure of starch reaching the large intestine. Aim of the study: The aim of this study was to compare two different in vitro models simulating the digestibility of two RS containing preparations. Methods: The substrates, high amylose maize (HAM) containing RS type 2, and retrograded long chain tapioca maltodextrins (RTmd) containing RS type 3 were in vitro digested using a batch and a dynamic model, respectively. Both preparations were characterized before and after digestion by using X-Ray and DSC, and by measuring their total starch, RS and protein contents. Results: Using both digestion models, 60-61g/100g of RTmd turned out to be indigestible, which is very well in accordance with 59g/100g found in vivo after feeding RTmd to ileostomy patients. In contrast, dynamic and batch in vitro digestion experiments using HAM as a substrate led to 58g/100g and 66g/100g RS recovery. The degradability of HAM is more affected by differences in experimental parameters compared to RTmd. The main variations between the two in vitro digestion methods are the enzyme preparations used, incubation times and mechanical stress exerted on the substrate. However, for both preparations dynamically digested fractions led to lower amounts of analytically RS and a lower crystallinity. Conclusions: The two in vitro digestion methods used attacked the starch molecules differently, which influenced starch digestibility of HAM but not of RTm

Behaviour of milk protein ingredients and emulsions stabilised by milk protein ingredients in the simulated gastrointestinal tract : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology, Massey University, Manawatu, New Zealand

Milk clotting behaviours in the stomach impact the digestion rates of protein and fat. A variety of milk protein products are applied as functional ingredients in many foods. This research was conducted to investigate the digestion behaviours of various commercial dairy ingredients and lipids in emulsions stabilised by these ingredients using a dynamic in vitro digestion model, i.e., a human gastric simulator (HGS), with a focus on the effect of different structures of clots formed in dairy ingredients during gastric digestion on hydrolysis of proteins and/or lipids. Skim milk powder (SMP), milk protein concentrate (MPC) 4851, MPC 4861, sodium caseinate, whey protein isolate (WPI) and heated (90°C, 20 min) WPI were used in the present study. Results showed that SMP and MPC 4851, which contained casein micelles, formed ball-like clots with a relatively dense network after 10 min of gastric digestion. These clots did not disintegrate after 220 min of digestion. MPC 4861 and sodium caseinate generated clots at around 40 min, and a loose, fragmented structure was observed at the end of the gastric digestion due to a lacking micellar structure of caseins. No clot was observed in WPI or heated WPI after 220 min gastric digestion, although aggregation occurred at around 40 min in heated WPI. These differences in coagulation behaviours apparently affected the rate of gastric emptying and protein hydrolysis by pepsin in the gastric system. In SMP and MPC 4851, the gastric emptying and hydrolysis of caseins was much slower than that observed in MPC 4861 and sodium caseinate. The most rapid gastric emptying of proteins was observed in the WPI samples both with and without heating. This is attributed to the formation of varied structured clots at different times under the gastric conditions. The effect of protein concentration on the gastric behaviour of these dairy ingredients in solution was then examined, with a particular emphasis on the structure of clots. SMP and MPC 4851 have been selected as model protein ingredients. Their gastric behaviours were investigated over a protein concentration range of 0.5-5.0% (w/w). The results showed that the digestion behaviour of SMP and MPC 4851 followed a similar pattern. The rate of pH changes in the emptied digesta during digestion was protein concentration dependent. With an increase in protein concentration, the decrease in pH slowed. The protein concentration had no apparent impact on the casein clotting time. Clots were formed in the first 10 min of digestion in all samples. However, in both SMP and MPC 4851, when protein concentration was lower than 2.0% (w/w) the clots consisted of small protein pieces with a loose, porous and open structure after a 220 min digestion. Whereas a cheese ball-like clot with a denser network was observed at the end of gastric digestion when the protein concentration varied from 2.0% to 5.0% (w/w). Such a difference in the structure apparently affected the rate of protein hydrolysis. A more rapid hydrolysis (P < 0.05) of the clotted protein was observed when protein concentration was lower than 2.0% (w/w) compared to the samples containing a higher proportion of protein (2.0%-5.0%, w/w). To study the effect of different coagulation behaviours on the digestion of oil droplets in oil-in-water emulsions, these dairy ingredients (with the exception of SMP) were used to prepare an oil-in-water emulsion (20.0% soy oil and 4.0% protein, w/w). They were digested under the dynamic gastric conditions using the HGS. The gastric digesta was emptied at 20 min intervals. Then all digesta were mixed to investigate the lipid digestion under the small intestinal conditions. Changes in physicochemical properties of emulsions, involving the particle size, the microstructure, the oil content of the emptied gastric digesta and the amount of free fatty acids (FFAs) released during the small intestine stage, were determined using an in vitro small intestinal digestion model. Aggregation of MPC 4851-stabilised emulsion took place after 5 min of digestion in the HGS with the largest size. The aggregates remained in the stomach and did not disappear during the whole gastric digestion. The hydrolysis of the aggregated network by pepsin was largely slowed by the reduced ability of the simulated gastric fluid (SGF, containing pepsin) to diffuse into the larger sized aggregates. MPC 4851-stabilised emulsion thus resulted in the slowest release of oil droplets into the small intestine. In comparison, MPC 4861 and sodium caseinate-stabilised emulsions aggregated in the stomach at approximately 40 min, forming smaller sized aggregates. These aggregates disintegrated at the mid and late-stages of digestion in these two emulsions. Therefore, MPC 4861 and sodium caseinate-stabilised emulsions had a more rapid delivery of oil droplets into the small intestine. In relation to the WPI-stabilised emulsions both with and without heating, the aggregations formed at a similar time to that which was observed in MPC 4861 and sodium caseinate-stabilised-emulsions; i.e., at approximately 40 min. However, they had the smallest sized aggregates amongst all samples and they disintegrated quickly with further digestion. WPI-stabilised emulsions both with and without heating had the fastest gastric emptying and hydrolysis by pepsin in the early and mid-stages of the gastric digestion process. Thus, the highest level of oil content contained in the emptied gastric digesta was produced from both WPI-stabilised emulsions. In the mixed gastric digesta, which were subjected to the small intestinal digestion, the oil contents contained in the different emulsion samples varied. This difference impacted the extent of lipid digestion by pancreatic lipase. The sample with a higher oil content released a greater amount of FFAs compared to the sample with a lower oil content. The extent of lipid digestion of different emulsion samples adhered to the following pattern: MPC 4851-stabilised emulsion < MPC 4861-stabilised emulsion < sodium caseinate-stabilised emulsion, WPI-stabilised emulsions both with and without heating. Overall, the gastric behaviours of dairy ingredients either in solutions or emulsions were affected by the formation of structured clots/aggregates. The differences in clotting/aggregation times and their structures were greatly dependent on the component and structure of protein, the processing prior to digestion and the susceptibility to proteases. These differences in protein coagulation/aggregation behaviour impacted the rates of protein hydrolysis and gastric emptying. The oil content and protein composition of the gastric digesta transferred into small intestine and the extent of lipid digestion in small intestine were also affected. These results are important in an application perspective. They provide useful information for the design and development of healthier food products by allowing greater control over the manipulation of protein bioavailability, which subsequently provides greater control over lipid metabolism

In Vitro Digestion Rate and Estimated Glycemic Index of Oat Flours from Typical and High β-Glucan Oat Lines

The in vitro starch digestion rate and estimated glycemic index (GI) of oat flours and oat starches from typical and high β-glucan oat lines were evaluated along with the impact of heating on starch digestion. Flour from oat lines (\u27Jim\u27, \u27Paul\u27, IA95, and N979 containing 4.0, 5.3, 7.4, and 7.7% β-glucan, respectively) was digested by pepsin and porcine pancreatin. To determine the impact of heating on starch digestion, oat slurries were prepared by mixing oat flour and water (1:8 ratio) and heating for 10 min prior to digestion. Viscosity, as measured on a Rapid Visco Analyzer, increased with increases in concentration and molecular weight of β-glucan. The in vitro starch digestion of oat flours and a control, white bread made from wheat flour, increased as the digestion time increased. Starch digestion of oat flour was slower than that of the control (p \u3c 0.05). Heat treatment of oat-flour slurries increased the starch digestion from a range of 31–39% to a range of 52–64% measured after 180 min of in vitro digestion. There were no differences in starch digestibility among oat starches extracted from the different oat lines. The GI, estimated by starch hydrolysis of oat flours, ranged from 61 to 67, which increased to a range of 77–86 after heating. Oat-flour slurries prepared from IA95 and N979 lines with high β-glucan concentrations had lower GI values than did slurries made from Jim and Paul lines. Starch digestion was negatively correlated with β-glucan concentrations in heated oat-flour slurries (R2 = 0.92). These results illustrate that the oat soluble fiber, β-glucan, slowed the rate of starch digestion. This finding will help to develop new food products with low GI by using oat β-glucan

Fruit carotenoids affect the bioaccessibility and cellular uptake of ß-carotene from orange fleshed sweet potato

Context and motivation of the work: To fight against vitamin A deficiency in developing countries, consumption of orange fleshed sweet potato (OFSP) which contains high concentrations of beta-carotene (provitamin A) is currently promoted. Indeed, clinical studies show that the vitamin A status was increased in children fed with OFSP (1). However, information on how well provitamin A was absorbed by intestinal cells is unclear and depends of bioaccessibility. Bioaccessibility of carotenoids depends on various factors included food matrix, food preparation method, presence of fiber and dietary fat or other microconstituants in the food (2).This last point and especially the interaction between carotenoids could be well documented. Moreover, few studies reports informations about the interaction through food matrix. The objective of this study is to assess the effect of carotenoids from fruits juices on the bioaccessibility and cellular uptake of BC from OFSP by using the in vitro digestion coupled with Caco-2 cell culture model. Materiel and methods: To simulate gastric and small intestinal phases of digestion, boiled OFSP (5g) mixed with fruit juice samples (30 ml) in saline solution were incubated at 37 °C (pH 4, pepsin, 30 minutes).Then, the pH was adjusted 6 and bile extract and pancreatin were added for 30 minutes. Micelles were collected in aqueous fraction after centrifugation and filtration (0.22?m). A micellarisation test without food matrix was also investigated (equimolar quantity of carotenoids, 5 ml of 100 mg/ml bile, 17h, 37 °C). Digestat from in vitro digestion were incubated (1h30) on Caco-2 TC7cells cultivated 21 days on wells in order to study the intestinal absorption. Extraction and HPLC analysis of carotenoids from micelles and from OFSP food products were carried out according previous study (3). Results: Effect of fruit juice on the micellarization of beta-carotene from OFSP showed that addition of Pink grapefruit reduces BC micellarization whereas kiwi juice and Clementine increased all-trans BC, but decreased 13-cis BC. Lycopene, beta-cryptoxanthine and lutein carotenoids as standard carotenoids were added to OFSP instead of fruit juices during in vitro digestion and confirmed some carotenoid interactions particularly for lycopene. Micellarization test showed that lycopene decreased and BCX increased. Cellular uptake of all- trans BC from digested OFSP (36%) and 13-cis BC (13 %) significantly decreased in presence of fruit juices. Conclusion: This study shows that carotenoids from fruit juice modify the bioaccessibility and cellular uptake of BC from OFSP. However, further investigations are needed to identify other components responsible of interactions in order to show efficacy of OFSP for maintaining adequate vitamin A status in a meal. (Texte intégral

In vitro versus in situ evaluation of the effect of phytase supplementation on calcium and phosphorus solubility in soya bean and rapeseed meal broiler diets

1. In vitro assays provide a sensitive and economic tool to evaluate dietary effects, but have limitations. In this study, the effect of phytase supplementation on solubility, and presumed availability, of calcium (Ca) and phosphorus (P) in soybean meal (SBM) and rapeseed meal (RSM) based diets was evaluated both in situ and by a 2-step in vitro digestion assay that simulated the gastric and small intestine (SI) phases of digestion