The In Vitro Digestive Process:

Design in a Single Batch

The in vitro simulator of the digestive process in a single batch was designed to evaluate the bioaccessibility of flaxseed lignans. The upper gastrointestinal tract was simulated according to the methodol­

ogy reported by Sumeri et al. (2008) with modifications; these changes altered the time of passage of the bolus through the stomach and through the small intestine.

Moreover, instead of directly adding the sample, a corresponding bolus of food and saliva mixture was drawn. For this, artifi­

cial saliva was prepared according to Arvisenet et al. (2008). In this in vitro sim­

ulation conditions were recreated that occur during fermentation in the colon, keeping the whole process in the same bio­

reactor. The large intestine stage was per­

formed according to methodology reported by Possemiers (2004) and De Boever

(2000), with the difference that all steps occurring in the colon (ascending, trans­

verse and descending colon), were per­

formed in a single reactor. Other in vitro colon simulators have long fermentation times, of between 1 and 14 days (Yoo and Chen, 2006), which is not suitable to esti­

mate the bioavailability, but this involves shorter fermentation times in the colon.

Figure 4.1 shows a diagram of the in vitro simulation conditions of the entire diges­

tive process.

An example of the use of in vitro diges­

tion process in a single batch was the com­

plete digestion process of Gouda cheese, which contained 15 g linseed meal per kg of cheese, resulting in the detection of several lignans (Fig. 4.2). During in vitro digestion of the small intestine secoisolariciresinol diglucoside (SDG) was identified; this plant lignan was probably released by pancreatic enzymes in the intestine. Eeckhaut (2008)

Fig. 4.1. A scheme of the digestive process in vitro in a single batch.

Bioaccessibility and Bioavailability of Bioactive Compounds 53

detected no lignan in the small intestine, indicating that lactic acid bacteria present in cheese promoted its release. By the action of intestinal bacteria, SDG is metabolized to enterodiol (ED) and enterolactone (EL); how­

ever aglycone secoisolariciresinol (SECO) was not detected, probably because it is fast

metabolized into ED. In Fig. 4.2, it can be observed that the SDG and ED content decreases along the colon in vitro digestive process and, at the same time, the content of EL is increased. The bioavailability calcu­

lated for lignans SDG, ED and EL was 1.59%, 0.99% and 2.42%, respectively.

140

Cheese (µg/g)

120 100 80 60 40 20 0

SDGSECO EDEL

MasticationStomach

Small intestine 2

Small intestine 1

Small intestine 3

Small intestine 4

Large intestine 1

Large intestine 2

Large intestine 3

Large intestine 4

Fig. 4.2. The profile of lignans secoisolariciresinol diglucoside (SDG), aglycone secoisolariciresinol (SECO), enterodiol (ED) and enterolactone (EL) during in vitro digestion of cheese fortified with flaxseed.

References

Aherne, S.A., Jiwan, M.A., Daly, T. & O’Brien, N.M., 2009. Geographical location has greater impact on carotenoid content and bioaccessibility from tomatoes than variety. Plant Foods for Human Nutrition 64, 250–256.

Aherne, S.A., Daly, T., Jiwan, M.A., O’Sullivan, L. & O’Brien, N.M., 2010. Bioavailability of beta­carotene isomers from raw and cooked carrots using an in vitro digestion model coupled with a human intestinal Caco­2 cell model. Food Research International 43, 1449–1454.

Argyri, K., Komaitis, M. & Kapsokefalou, M., 2006. Iron decreases the antioxidant capacity of red wine under conditions of in vitro digestion. Food Chemistry 96, 281–289.

Argyri, K., Birba, A., Miller, D.D., Komaitis, M. & Kapsokefalou, M. 2009. Predicting relative concen­

trations of bioavailable iron in foods using in vitro digestion: new developments. Food Chemistry 113, 602–607.

Argyri, K., Theophanidi, E., Kapna, A., Staikidou, C., Pounis, G., Komaitis, M., Georgiou, C. &

Kapsokefalou, M., 2011. Iron or zinc dialyzability obtained from a modified in vitro digestion procedure compare well with iron or zinc absorption from meals. Food Chemistry 127, 716–721.

Arvisenet, G., Billy, L., Poinot, P., Vigneau, E., Bertrand, D. & Prost, C., 2008. Effect of apple particle state on the release of volatile compounds in a new artificial mouth device. Journal of Agricultural and Food Chemistry 56, 3245–3253.

Bermudez­Soto, M.J., Tomas­Barberan, F.A. & Garcia­Conesa, M.T., 2007. Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to in vitro gastric and pancreatic digestion. Food Chemistry 102, 865–874.

Bhagavan, H.N. & Chopra, R.K., 2007. Plasma coenzyme Q10 response to oral ingestion of coenzyme Q10 formulations. Mitochondrion 7, S78–S88.

Bouayed, J., Hoffmann, L. & Bohn, T., 2011. Total phenolics, flavonoids, anthocyanins and antioxidant activity following simulated gastro­intestinal digestion and dialysis of apple varieties:

Bioaccessibility and potential uptake. Food Chemistry 128, 14–21.

Bouayed, J., Deusser, H., Hoffmann, L. & Bohn, T., 2012. Bioaccessible and dialysable polyphenols in selected apple varieties following in vitro digestion vs. their native patterns. Food Chemistry 131, 1466–1472.

Brandon, E.F.A., Oomen, A.G., Rompelberg, C.J.M., Versantvoort, C.H.M., van Engelen, J.G.M. & Sips, A., 2006. Consumer product in vitro digestion model: Bio accessibility of contaminants and its application in risk assessment. Regulatory Toxicology and Pharmacology 44, 161–171.

Carnachan, S.M., Bootten, T.J., Mishra, S., Monro, J. & Sims, I.M., 2012. Effects of simulated digestion in vitro on cell wall polysaccharides from kiwifruit (Actinidia spp.). Food Chemistry 133, 132–139.

Chiu, Y.­T. & Stewart, M., 2012. Comparison of konjac glucomannan digestibility and fermentability with other dietary fibers in vitro. Journal of Medicinal Food 15, 120–125.

Choi, M.H., Kim, G.H., Park, K.H., Kim, Y.S. & Shim, S.M., 2011. Bioaccessibility of total sugars in carbonated beverages and fermented milks. Journal of the Korean Society for Applied Biological Chemistry 54, 778–782.

Cilla, A., Laparra, J.M., Alegria, A., Barbera, R. & Farre, R., 2008. Antioxidant effect derived from bioac­

cessible fractions of fruit beverages against H2O2­induced oxidative stress in Caco­2 cells. Food Chemistry 106, 1180–1187.

Cilla, A., Perales, S., Lagarda, M.J., Barberá, R., Clemente, G. & Farré, R., 2011. Influence of storage and in vitro gastrointestinal digestion on total antioxidant capacity of fruit beverages. Journal of Food Composition and Analysis 24, 87–94.

Connolly, M.L., Lovegrove, J.A. & Tuohy, K.M., 2012. In vitro fermentation characteristics of whole grain wheat flakes and the effect of toasting on prebiotic potential. Journal of Medicinal Food 15, 33–43.

De Boever, P., Deplancke, B. & Verstraete, W., 2000. Fermentation by gut microbiota cultured in a simu­

lator of the human intestinal microbial ecosystem is improved by supplementing a soygerm pow­

der. Journal of Nutrition 130, 2599–2606.

De Noni, I. & Cattaneo, S., 2010. Occurrence of b­casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro­intestinal digestion. Food Chemistry 119, 560–566.

Dona, A.C., Pages, G., Gilbert, R.G. & Kuchel, P.W., 2010. Digestion of starch: In vivo and in vitro kinetic models used to characterise oligosaccharide or glucose release. Carbohydrate Polymers 80, 599–617.

Eeckhaut, E., Struijs, K., Possemiers, S., Vincken, J.P., De Keukeleire, D. & Verstraete, W., 2008.

Metabolism of the lignan macromolecule into enterolignans in the gastrointestinal lumen as determined in the simulator of the human intestinal microbial ecosystem. Journal of Agricultural and Food Chemistry 56, 4806–4812.

Ercan, P. & El, S.N., 2011. Changes in content of coenzyme Q10 in beef muscle, beef liver and beef heart with cooking and in vitro digestion. Journal of Food Composition and Analysis 24, 1136–1140.

García­Sartal, C., Romarís­Hortas, V., Barciela­Alonso, M.d.C., Moreda­Piñeiro, A., Dominguez­

Gonzalez, R. & Bermejo­Barrera, P., 2011. Use of an in vitro digestion method to evaluate the bio­

accessibility of arsenic in edible seaweed by inductively coupled plasma­mass spectrometry.

Microchemical Journal 98, 91–96.

Garrett, D.A., Failla, M.L. & Sarama, R.J., 1999. Development of an in vitro digestion method to assess carotenoid bioavailability from meals. Journal of Agricultural and Food Chemistry 47, 4301–4309.

Gawlik­Dziki, U., Dziki, D., Baraniak, B. & Lin, R., 2009. The effect of simulated digestion in vitro on bioactivity of wheat bread with Tartary buckwheat flavones addition. LWT ­ Food Science and Technology 42, 137–143.

Bioaccessibility and Bioavailability of Bioactive Compounds 55

Gil­Izquierdo, A., Gil, M.I., Ferreres, F. & Tomas­Barberan, F.A., 2001. In vitro availability of flavonoids and other phenolics in orange juice. Journal of Agricultural and Food Chemistry 49, 1035–1041.

Glahn, R.P., Lai, C. & VanCampen, D.R., 1997. Application of an in vitro digestion/Caco­2 cell model to improve iron availability of infant formula. Faseb Journal 11, 3503.

Goicoechea, E., Brandon, E.F.A., Blokland, M.H. & Guillén, M.D., 2011. Fate in digestion in vitro of several food components, including some toxic compounds coming from omega­3 and omega­6 lipids. Food and Chemical Toxicology 49, 115–124.

Hur, S.J., Park, S.J. & Jeong, C.H., 2011. Effect of buckwheat extract on the antioxidant activity of lipid in mouse brain and its structural change during in vitro human digestion. Journal of Agricultural and Food Chemistry 59, 10699–10704.

Kapsokefalou, M. & Miller, D.D., 1991. Effects of meat and selected food components on the valence of nonheme iron during in vitro digestion. Journal of Food Science 56, 352.

Kulkarni, S.D., Acharya, R., Rajurkar, N.S. & Reddy, A.V.R., 2007. Evaluation of bioaccessibility of some essential elements from wheatgrass (Triticum aestivum L.) by in vitro digestion method.

Food Chemistry 103, 681–688.

Kwasniak, J., Falkowska, L. & Kwasniak, M., 2012. The assessment of organic mercury in Baltic fish by use of an in vitro digestion model. Food Chemistry 132, 752–758.

Laurent, C., Besancon, P. & Caporiccio, B., 2007. Flavonoids from a grape seed extract interact with digestive secretions and intestinal cells as assessed in an in vitro digestion/Caco­2 cell culture model. Food Chemistry 100, 1704–1712.

Liang, L., Wu, X., Zhao, T., Zhao, J., Li, F., Zou, Y., Mao, G. & Yang, L., 2012. In vitro bioaccessibility and antioxidant activity of anthocyanins from mulberry (Morus atropurpurea Roxb.) following simulated gastro­intestinal digestion. Food Research International 46, 76–82.

Mahler, G.J., Shuler, M.L. & Glahn, R.P., 2009. Characterization of Caco­2 and HT29­MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability. Journal of Nutritional Biochemistry 20, 494–502.

Makelainen, H.S., Makivuokko, H.A., Salminen, S.J., Rautonen, N.E. & Ouwehand, A.C., 2007. The effects of polydextrose and xylitol on microbial community and activity in a 4­stage colon simula­

tor. Journal of Food Science 72, M153–M159.

Makivuokko, H.A., Saarinen, M.T., Ouwehand, A.C. & Rautonen, N.E., 2006. Effects of lactose on colon microbial community structure and function in a four­stage semi­continuous culture system.

Bioscience Biotechnology and Biochemistry 70, 2056–2063.

Minekus, M., Marteau, P., Havenaar, R. & Huisintveld, J.H.J., 1995. A multicompartmental dynamic computer­controlled model simulating the stomach and small­intestine. Alternatives to Laboratory Animals 23, 197–209.

Molly, K., Woestyne, M.V. & Verstraete, W., 1993. Development of a 5­step multichamber reactor as a simulation of the human intestinal microbial ecosystem. Applied Microbiology and Biotechnology 39, 254–258.

Moreda­Pineiro, J., Alonso­Rodriguez, E., Romaris­Hortas, V., Moreda­Pineiro, A., Lopez­Mahia, P., Muniategui­Lorenzo, S., Prada­Rodriguez, D. & Bermejo­Barrera, P., 2012. Assessment of the bio­

availability of toxic and non­toxic arsenic species in seafood samples. Food Chemistry 130, 552–560.

Parada, J. & Aguilera, J.M., 2007. Food microstructure affects the bioavailability of several nutrients.

Journal of Food Science, 72, R21–R32.

Possemiers, S., Verthe, K., Uyttendaele, S. & Verstraete, W., 2004. PCR­DGGE­based quantification of stability of the microbial community in a simulator of the human intestinal microbial ecosystem.

Fems Microbiology Ecology 49, 495–507.

Reyes, L.H., Encinar, J.R., Marchante­Gayón, J.M., Alonso, J.I.G. & Sanz­Medel, A., 2006. Selenium bioaccessibility assessment in selenized yeast after “in vitro” gastrointestinal digestion using two­

dimensional chromatography and mass spectrometry. Journal of Chromatography A 1110, 108–116.

Sumeri, I., Arike, L., Adamberg, K. & Paalme, T., 2008. Single bioreactor gastrointestinal tract simula­

tor for study of survival of probiotic bacteria. Applied Microbiology and Biotechnology 80, 317–324.

Yoo, J.Y. & Chen, X.D., 2006. GIT physicochemical modeling ­ A critical review. International Journal of Food Engineering 2, 12.

© CAB International 2013. Natural Antioxidants and Biocides from

56 Wild Medicinal Plants (eds C.L. Céspedes et al.)