Marie Alminger

A standardised static in vitro digestion method suitable for food-an international consensus

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

In Vitro Models for Studying Secondary Plant Metabolite Digestion and Bioaccessibility

There is an increased interest in secondary plant metabolites, such as polyphenols and carotenoids, due to their proposed health benefits. Much attention has focused on their bioavailability, a prerequisite for further physiological functions. As human studies are time consuming, costly, and restricted by ethical concerns, in vitro models for investigating the effects of digestion on these compounds have been developed and employed to predict their release from the food matrix, bioaccessibility, and assess changes in their profiles prior to absorption. Most typically, models simulate digestion in the oral cavity, the stomach, the small intestine, and, occasionally, the large intestine. A plethora of models have been reported, the choice mostly driven by the type of phytochemical studied, whether the purpose is screening or studying under close physiological conditions, and the availability of the model systems. Unfortunately, the diversity of model conditions has hampered the ability to compare results across different studies. For example, there is substantial variability in the time of digestion, concentrations of salts, enzymes, and bile acids used, pH, the inclusion of various digestion stages; and whether chosen conditions are static (with fixed concentrations of enzymes, bile salts, digesta, and so on) or dynamic (varying concentrations of these constituents). This review presents an overview of models that have been employed to study the digestion of both lipophilic and hydrophilic phytochemicals, comparing digestive conditions in vitro and in vivo and, finally, suggests a set of parameters for static models that resemble physiological conditions.

Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P(5), D/L-Ins(2,3,4,5)P(4), D/L-Ins(2,4,5)P(3) or D/L-Ins(1,2,4)P(3), D/L-Ins(1,2)P(2) or Ins(2, 5)P(2) or D/L-Ins(4,5)P(2) to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1, 2,3,5,6)P(5) isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F(1) and F(2) from wheat bran and the myo-inositol phosphates produced by fraction F(2). Biochim. Biophys. Acta 302, 326-328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P(5), D-Ins(2,3,4,5)P(4), D-Ins(2,4,5)P(3), Ins(2,5)P(2) to finally Ins(2)P (notation 6/1/3/4/5).

Mind the gap—deficits in our knowledge of aspects impacting the bioavailability of phytochemicals and their metabolites—a position paper focusing on carotenoids and polyphenols

Various secondary plant metabolites or phytochemicals, including polyphenols and carotenoids, have been associated with a variety of health benefits, such as reduced incidence of type 2 diabetes, cardiovascular diseases, and several types of cancer, most likely due to their involvement in ameliorating inflammation and oxidative stress. However, discrepancies exist between their putative effects when comparing observational and intervention studies, especially when using pure compounds. These discrepancies may in part be explained by differences in intake levels and their bioavailability. Prior to exerting their bioactivity, these compounds must be made bioavailable, and considerable differences may arise due to their matrix release, changes during digestion, uptake, metabolism, and biodistribution, even before considering dose‐ and host‐related factors. Though many insights have been gained on factors affecting secondary plant metabolite bioavailability, many gaps still exist in our knowledge. In this position paper, we highlight several major gaps in our understanding of phytochemical bioavailability, including effects of food processing, changes during digestion, involvement of cellular transporters in influx/efflux through the gastrointestinal epithelium, changes during colonic fermentation, and their phase I and phase II metabolism following absorption.

In Vitro Bioaccessibility of β-Carotene from Heat-Processed Orange-Fleshed Sweet Potato

Orange-fleshed sweet potato (OFSP) is currently promoted in parts of sub-Saharan Africa as a biofortified staple food with large potential to provide considerable amounts of provitamin A carotenoids. However, the bioaccessibility of provitamin A carotenoids from OFSP has not been widely investigated, especially not as an effect of different preparation methods. In this study, we used an in vitro digestion model to assess the bioaccessibility of beta-carotene from differently heat-processed OFSP. The fraction of carotenoids transferred from the food matrix to a micellar phase obtained after microfiltration and to a supernatant obtained after low-speed centrifugation was investigated. The percentage of accessible all-trans-beta-carotene in the micellar phase varied between 0.5 and 1.1% in the heat-processed OFSP without fat and between 11 and 22% with the addition of 2.5% (w/w) cooking oil. In comparison with the micellar phase, the percentage of accessible all-trans-beta-carotene in the supernatant phase was significantly higher (P < 0.001), between 24 and 41% without fat and between 28 and 46% with fat. These results support the importance of fat for an improved micellarization of beta-carotene. Overall, the high in vitro bioaccessibility of beta-carotene from heat-processed OFSP indicates that sweet potato might be a promising dietary approach to combat vitamin A deficiency.

Purification and characterization of a phytate‐degrading enzyme from germinated oat (Avena sativa)

A phytate-degrading enzyme (myo-inositol hexakisphosphate phosphohydrolase) has been purified about 5,400-fold from germinated oat seedlings to apparent homogeneity. The molecular mass of the native monomeric enzyme was estimated to be about 67 kDa. Optimal pH for degradation of phytate was 5.0 and the optimal temperature 38 °C. Kinetic parameters for the hydrolysis of Na-phytate are KM 30 µM and kcat 356 s−1 at 35 °C and pH 5.0. The oat phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyses phytate in a stepwise manner. The first hydrolysis product was identified as D/L-l(1,2,3,4,5) P5. © 1999 Society of Chemical Industry

Effects of Thermal Processing on the in Vitro Bioaccessibility and Microstructure of β-Carotene in Orange-Fleshed Sweet Potato

The effects of different preparation methods on the bioaccessibility of β-carotene in orange-fleshed sweet potato (OFSP), an important food crop in sub-Saharan Africa, have been evaluated using an in vitro digestion procedure. The preparation methods included, on fresh roots, boiling followed by puréeing and oil addition (BOL) and homogenization followed by boiling and oil addition (HOM); on milled flour from freeze-dried fresh roots, cooking of porridge followed by oil addition (POA) and oil addition to flour followed by cooking of porridge (POB). The retention of all-trans-β-carotene ranged from 58% (POB) to 72% (BOL). The presence of oil during heating resulted in a significantly higher formation of 13-cis-β-carotene for the POB-treated samples than for the other samples. The efficiency of micellarization of all-trans-β-carotene after in vitro digestion was 50% (HOM), 48% (POB), 31% (POA), and 16% (BOL). Brightfield microscopy of the cell structure after processing and in vitro digestion showed a high degree of cell-wall rupture for the HOM-treated samples, whereas cells appeared intact for the BOL samples. Also, coherent anti-Stokes Raman scattering (CARS) microscopy showed smaller β-carotene bodies residing in the HOM samples than in the BOL samples after digestion. These results suggest that the in vitro bioaccessibility of β-carotene in an OFSP meal can be improved by processing methods that promote cell-wall rupture.

Oxidation of Cod Liver Oil during Gastrointestinal in Vitro Digestion

Oxidation of cod liver oil rich in long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) was investigated during a gastrointestinal (GI) in vitro digestion. The digestion stimulated TBA-reactive substances (TBARS) formation in both the gastric and intestinal steps, whereas levels of lipid hydroperoxides remained nearly constant. The presence of digestive compounds was decisive for the TBARS development because TBARS did not change when the cod liver oil was subjected only to the temperature and pH gradient of the GI model. Preformed oxidation products in the cod liver oil resulted in further elevated TBARS levels during the digestion. Addition of hemoglobin (11.5 μM) to emulsified cod liver oil dramatically increased TBARS and lipid hydroperoxide levels during GI digestion, whereas 1 mg α-tocopherol/g oil did not show any protection against oxidation. Specific concern thus needs to be taken in the design of foods containing LC n-3 PUFA to preserve these lipids and avoid harmful oxidation, both before and after consumption.

Impaired uptake of beta-carotene by Caco-2 human intestinal cells in the presence of iron.

At present, there are conflicting data regarding whether or not β-carotene has a positive effect on iron absorption. This study was undertaken to evaluate possible interactions involved in the uptake of β-carotene and iron in a human intestinal cell model (Caco-2). The Caco-2 cells were incubated with test solutions containing different amounts of ferrous chloride (10–50 µM) and β-carotene (0.3–2.5 µM) incorporated in synthetic micelles. In the absence of iron, cellular accumulation of β-carotene from synthetic micelles was proportional (r2=0.97, P <0.001) to the β-carotene concentration in the test solution. However, with addition of ferrous chloride (30 µM), the β-carotene uptake was significantly reduced (P <0.05), on average by 22%. There was also an inverse relationship between the β-carotene uptake and iron concentration in the test solution (r2=0.93, P <0.05). Iron provided in physiological amounts inhibited the uptake of β-carotene in the in vitro Caco-2 cell system.

Can Coca Leaves Contribute to Improving the Nutritional Status of the Andean Population

Background Coca leaves (Erythroxylum coca) have been promoted as a food that could address the dietary deficiencies of the Andean population, but this is based on nutrient analyses of a small sample of leaves. Objective We assessed the nutritional potential of eight samples of coca leaves grown in different regions of Peru. Methods We used AOAC techniques to measure nutrients, nutrient inhibitors (phytate, polyphenols, oxalic acid, and fiber), and alkaloid concentrations, all expressed per 100 g dry weight (DW) of the ground leaves. Minerals were measured by inductively coupled plasma-mass spectrometry in two independent laboratories. Results The leaves contained protein, 20.28 g/100 gDW, with lysine as the limiting amino acid; β-carotene, 3.51 mg/100 gDW; vitamin E, 16.72 mg/100 gDW; trace amounts of vitamin D; calcium, 990.18 and 1033.17 mg/100 gDW at two different laboratories; iron, 29.16 and 29.16 mg/100 gDW; zinc, 2.71 and 2.63 mg/100 gDW; and magnesium, 225.19 and 196.69 mg/100 gDW. Cocaine was the principal alkaloid, with a concentration of 0.56 g/100 gDW; other alkaloids were also identified. The results were compared with those for other edible leaves. The nutrient contributions of coca powder (5 g) and bread made with coca were compared with those of normal portions of alternative foods. Conclusions Two spoonfuls of coca leaf flour would satisfy less than 10% of dietary intakes for schoolchildren and adults for critical commonly deficient nutrients in the diet. Coca leaves do not provide nutritional benefits when eaten in the recommended quantities, and the presence of absorbable cocaine and other alkaloids may be potentially harmful; hence coca leaves cannot be recommended as a food.