Torsten Bohn

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.

Total phenolics, flavonoids, anthocyanins and antioxidant activity following simulated gastro-intestinal digestion and dialysis of apple varieties: Bioaccessibility and potential uptake

In the present study, an in vitro model simulating gastrointestinal (GI) digestion, including dialysability, was adapted to assess free soluble polyphenols from apples (four varieties). Results indicated that polyphenol release was mainly achieved during the gastric phase (ca. 65% of phenolics and flavonoids), with a slight further release (<10%) during intestinal digestion. Anthocyanins present after the gastric phase (1.04-1.14mg/100g) were not detectable following intestinal digestion. Dialysis experiments employing a semipermeable cellulose membrane, presenting a simplified model of the epithelial barrier, showed that free soluble dialysable polyphenols and flavonoids were 55% and 44% of native concentrations, respectively, being approximately 20% and 30% lower than that of the GI digesta. Similar results were found for the antioxidant capacity of dialysable antioxidants, being 57% and 46% lower compared to total antioxidants in fresh apples (FRAP and ABTS test, respectively). It is suggested that some polyphenols are bound to macromolecular compounds that are non-dialysable, that the presented method allowed the study of free soluble polyphenols available for further uptake, and that both chemical extraction and concentrations in final digesta would overestimate polyphenol availability.

Carotenoids, inflammation, and oxidative stress—implications of cellular signaling pathways and relation to chronic disease prevention

Several epidemiologic studies have shown that diets rich in fruits and vegetables reduce the risk of developing several chronic diseases, such as type 2 diabetes, atherosclerosis, and cancer. These diseases are linked with systemic, low-grade chronic inflammation. Although controversy persists on the bioactive ingredients, several secondary plant metabolites have been associated with these beneficial health effects. Carotenoids represent the most abundant lipid-soluble phytochemicals, and in vitro and in vivo studies have suggested that they have antioxidant, antiapoptotic, and anti-inflammatory properties. Recently, many of these properties have been linked to the effect of carotenoids on intracellular signaling cascades, thereby influencing gene expression and protein translation. By blocking the translocation of nuclear factor κB to the nucleus, carotenoids are able to interact with the nuclear factor κB pathway and thus inhibit the downstream production of inflammatory cytokines, such as interleukin-8 or prostaglandin E2. Carotenoids can also block oxidative stress by interacting with the nuclear factor erythroid 2-related factor 2 pathway, enhancing its translocation into the nucleus, and activating phase II enzymes and antioxidants, such as glutathione-S-transferases. In this review, which is organized into in vitro, animal, and human investigations, we summarized current knowledge on carotenoids and metabolites with respect to their ability to modulate inflammatory and oxidative stress pathways and discuss potential dose-health relations. Although many pathways involved in the bioactivity of carotenoids have been revealed, future research should be directed toward dose-response relations of carotenoids, their metabolites, and their effect on transcription factors and metabolism.

Lycopene from heat-induced cis -isomer-rich tomato sauce is more bioavailable than from all- trans -rich tomato sauce in human subjects

Lycopene is present mainly as cis-isomers in human serum and tissues whereas all-trans-lycopene predominates in tomato products, suggesting that all-trans-lycopene is isomerised in the body or is less bioavailable. The objectives of the present study were to develop processing conditions for tomatoes to obtain products with different cis-trans-lycopene isomer distribution and to assess their bioavailability. Healthy adult subjects (n 12) were recruited for this randomised cross-over trial. Each intervention was preceded by a 2-week washout period. Two tomato sauces, one rich in all-trans-lycopene (32.5 mg total lycopene/100 g sauce; 5 % cis-isomers), the other high in cis-lycopene (26.4 mg total lycopene/100 g sauce; 45 % cis-isomers), were produced by different heat-processing techniques. Each sauce (150 g) was served in a standardised meal at 08.00 hours after overnight fasting. Plasma TAG-rich lipoprotein fractions over 9.5 h following test-meal consumption as a measure of lycopene absorption were obtained and expressed as baseline-corrected area under the concentration v. time curves (AUC), using HPLC-electrochemical detection. AUC values adjusted for the amount lycopene consumed showed that total, total cis-, and all-trans-lycopene responses were significantly higher from the cis-isomer-rich sauce, compared with the all-trans-rich sauce, being 7.30 (sem 1.45) v. 4.74 (sem 1.08) nmol x h/l (P = 0.002), 3.80 (sem 0.76) v. 1.98 (sem 0.37) nmol x h/l (P = 0.0005) and 3.50 (sem 0.76) v. 2.76 (sem 0.76) nmol x h/l (P = 0.01), respectively. The present study demonstrates significant lycopene bioavailability from cis-lycopene-rich tomato sauce and highlights the importance of considering isomer-distribution for lycopene bioavailability. Furthermore, processing parameters can be controlled to alter isomer patterns of tomato products and influence lycopene bioavailability.

Development of a multi-class method for the quantification of veterinary drug residues in feedingstuffs by liquid chromatography-tandem mass spectrometry

A simple multi-residue method was developed for detecting and quantifying 33 analytes from 13 classes of antibiotics (tetracyclines (3), quinolones (7), penicillins (3), ionophore coccidiostats (7), macrolides (3), sulfonamides (1), quinoxalines (2), phenicols (2), lincosamides (1), diaminopyrimidines (1), polypeptides (1), streptogramins (1) and pleuromutilins (1)) in animal feeds. Extraction and clean-up procedures were optimized with spiked piglet feed. Samples were extracted by ultrasonic-assisted extraction with a mixture of methanol/acetonitrile/McIlvaine buffer at pH 4.6 (37.5/37.5/25, v/v/v) containing 0.3% of EDTA-Na(2), followed by a clean up using a dispersive solid-phase extraction (d-SPE) with PSA (primary secondary amine). Detection of antibiotics was achieved by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) within 28 min using both positive and negative ESI mode. Average recoveries ranged from 51% (oxytetracycline) to 116% (tilmicosin) with associated relative standard deviations of 7.3% and 9.0% and an overall mean of 87%. Limits of quantification ranged from 3.8 ngg(-1) (lincomycin) to 65.0 ngg(-1) (bacitracin). Following optimization, the method was further verified for bovine and lamb feeding stuffs; negative matrix effects were evaluated and overcome by a standard addition method.

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.

Comparison of 3 spectrophotometric methods for carotenoid determination in frequently consumed fruits and vegetables.

UNLABELLED Carotenoids are C-40 tetraterpenoid compounds with potential health beneficial effects. Major dietary sources include a variety of fruits and vegetables. Rapid screening methods are therefore desired, but their accuracy varies depending on the carotenoid profile and the matrix of the plant food. In the present study, 3 different methods were compared, all based on a rapid extraction protocol and spectrophotometric measurements to determine the total amount carotenoids present in fruits and vegetables (n = 28), either with or without chlorophyll. Published methods (a) Lichtenthaler and (b) Hornero-Méndez and Mínguez-Mosquera were compared with a newly developed method (method c) based on the average molar absorption coefficient (135310 Lcm(-1)mol(-1)) and wavelength (450 nm in acetone), for the 5 predominant carotenoid species (beta-carotene, zeaxanthin, lycopene, lutein, beta-cryptoxanthin) in the investigated foods. All results were compared to HPLC (method d). To avoid overestimating carotenoid concentrations due to chlorophyll A and B presence, the effect of saponification was studied for all methods. Overall, saponification led to significant carotenoid losses (12.6 +/- 0.9%). Methods a, b, c, and d yielded 5.1 +/- 0.4 mg/100 g, 4.6 +/- 0.5 mg/100 g, 4.3 +/- 0.5 mg/100 g, and 4.2 +/- 0.5 mg/100 g total carotenoids, respectively, with method a leading to significant higher mean concentrations compared to all other methods (P < 0.001, Bonferroni) with methods b and c being not significantly different and highly correlated compared to HPLC (> r = 0.95). Similar results were found when stratifying for chlorophyll content and fruits compared with vegetables, however, accuracy varied for individual fruits, highlighting the limitation to use the same method for all plant foods. PRACTICAL APPLICATION This study presents a comparison of various rapid spectrophotometric measurements to determine total carotenoid content in various fruits and vegetables and could aid in the selection of the appropriate method for individual plant foods with different carotenoid profile and matrices.

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.

Effects of the Endocrine Disruptors Atrazine and PCB 153 on the Protein Expression of MCF-7 Human Cells

Polychlorinated biphenyls (PCBs) and a number of pesticides can act as endocrine disrupting compounds (EDCs). These molecules exhibit hormonal activity in vivo, and can therefore interact and perturb normal physiological functions. Many of these compounds are persistent in the environment, and their bioaccumulation may constitute a significant threat for human health. Physiological abnormalities following exposure to these xenobiotic compounds go along with alterations at the protein level of individual cells. In this study, MCF-7 cells were exposed to environmentally relevant concentrations of atrazine, PCB153 (100 ppb, respectively), 17-beta estradiol (positive control, 10 nM) and a negative control (solvent) for t = 24 h (n = 3 replicates/exposure group). After trizol extraction and protein solubilization, protein expression levels were studied by 2D-DIGE. Proteins differentially expressed were excised, trypsin-digested, and identified by MALDI-ToF-ToF, followed by NCBInr database search. 2D-DIGE experiments demonstrated that 49 spots corresponding to 29 proteins were significantly differentially expressed in MCF-7 cells (>1.5-fold, P < 0.05, Students paired t test). These proteins belonged to various cellular compartments (nucleus, cytosol, membrane), and varied in function; 88% of proteins were down-regulated during atrazine exposure, whereas 75% of proteins were up-regulated by PCB153. Affected proteins included those regulating oxidative stress such as superoxide dismutase and structural proteins such as actin or tropomyosin, which may explain morphological changes of cells already observed under the microscope. This study highlights the susceptibility of human cells to compounds with endocrine disrupting properties.

Carotenoids, polyphenols and micronutrient profiles of Brassica oleraceae and plum varieties and their contribution to measures of total antioxidant capacity.

The consumption of phytochemicals such as carotenoids and polyphenols within whole fruits and vegetables has been associated with decreased incidence of various inflammation and oxidative stress related chronic diseases, which may be due to direct antioxidant effects, or indirect mechanisms such as affecting signal transduction/gene expression. Within the present study, we investigated the antioxidant composition of two major groups of vegetables and fruits, Brassica oleraceae and prunus spp., and estimated their contribution to antioxidant capacity. For this purpose, 17 plum and 27 Brassica varieties were collected in Luxembourg, and analysed for their individual polyphenol and carotenoid profile, vitamin C, dietary fibre, and minerals/trace elements, and their correlation with markers of antioxidant capacity (FRAP, ABTS, Folin-Ciocalteu). Total carotenoid and polyphenol content varied considerably between the different Brassica and plum varieties, with highest concentrations in the variety Kale (13.3 ± 0.58 mg/100g wet weight) and Cherry plum (1.96 ± 0.28 mg/100g) for carotenoids; and Kale (27.0 ± 0.91 mg/100g) and Kirks plum (185 ± 14 mg/100g) for polyphenols. In developed multiple linear-regression-models for Brassica, flavonoids, anthocyanins, lutein and vitamin C were found to be the best predictors of antioxidant capacity as assessed by FRAP (R(2)=0.832) and flavonoids, neochlorogenic acid and vitamin C as assessed by ABTS (R(2)=0.831); while for plums these were selenium, total sugars, chlorogenic acid and vitamin C (R(2)=0.853), and selenium, chlorogenic acid and flavonoids for FRAP (R(2)=0.711). When considering Brassica and plum consumption in Luxembourg, it is estimated that both contribute to an antioxidant intake equivalent to 26 and 6 mg per day of ascorbic acid equivalents, respectively.