Bioaccessibility and metabolism of flaxseed lignans evaluated in a single batch simulator of digestive process

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1 Bioaccessibility and metabolism of flaxseed lignans evaluated in a single batch simulator of digestive process Claudia Fuentealba, Ociel Muñoz Universidad Austral de Chile, Instituto de ciencia y tecnología de los alimentos, Valdivia, Chile (claudia.fuentealba.c@gmail.com) ABSTRACT Secoisolariciresinol diglucoside (SDG) is the most abundant lignan in flaxseed, which can be metabolized to the mammalian lignans, enterodiol (ED) and enterolactona (EL) by human intestinal microflora. The flaxseed lignan and its mammalian metabolites are known to have a number of potential health benefits. The aim of this study was evaluate the metabolic changes of SDG from whole flaxseed during the digestive process by an in vitro model in a single batch. The digestive simulator includes chewing, stomach, small and large intestine. Flaxseed was mixed with artificial saliva for 20 s, this bolus is carried out in a bioreactor with sterile water. For stomach simulation, ph was adjusted to 2.0, and a stomach solution containing pepsin was added. The content of the bioreactor was then neutralized to ph 6.0 to simulate the small intestine, bile salt and pancreatin solutions were added. Finally, concentrated brain heart infusion broth was added to the bioreactor with the intestinal bacteria from fecal matter of 20 healthy donors. Lignans were not detected during mastication; SDG is released in the stomach which corresponds to 0.12% of total SDG in flaxseed. In the small intestine SECO was found, the low content of SDG released in the stomach is rapidly converted to SECO. The bioaccesibility of SECO was 7.6%. In the ascending colon, ED and EL was found which remains constant for 36 h. Therefore, intestinal bacteria are able to metabolize SECO during colon stages. The fermentative capacity was measured to verify the metabolic activity of bacteria. After 24 h of incubation, the concentration of SCFA increased particularly acetic, isovaleric and butiric acid. These results indicate that carbohydrate fermentation occurs, and probably the bacteria are consuming the soluble fiber in flaxseed and do not have enough time to metabolize more lignans. Keywords: Bioaccesibility; flaxseed; lignans; digestive simulation INTRODUCTION Lignans are phenolic compounds present in edible plants. Secoisolariciresinol (SECO) is present in flaxseed as a glycoside. Secoisolariciresinol diglucoside (SDG) is the most abundant lignan in flaxseed, which can be metabolized to the mammalian lignans, enterodiol and enterolactona by human intestinal microflora [1, 2]. The flaxseed lignan and its mammalian metabolites are known to have a number of potential health benefits, including decreased formation of breast, prostate and colon cancers attributed to (anti)-estrogenic and antioxidant properties [2, 3]. Several in vitro gastrointestinal models have been designed. The simulator of the human intestinal microbial ecosystem (SHIME) involves five or six bioreactors with controlled conditions of ph simulating the stomach, small intestine, ascending, transverse and descending colon [4, 5]. The TNO gastrointestinal model (TIM) is four computer-controlled chambers simulating the conditions of stomach, duodenum, jejunum and ileum. TIM includes simulation of peristaltic movements, controlled squeezing, absorption of nutrients and water in the small intestine compartments and simulation of gastric emptying rates and intestinal transit times [6, 7]. Both simulators show faults in the absorption of metabolites and fluids, and colonization of microorganism in the colon, among others [7]. In addition, both models don t incorporate into the simulation the mastication to obtain a digestive complete process. Recently, a new system using a single bioreactor to study the passage in stomach and small intestine was designed. This system simulates the upper gastrointestinal tract, which can be used to determine the survival of probiotics from different food matrix. The simulation of the stomach and small intestine is carried out in a flask with stirring, combining acid and gastric solutions, specifically pepsin to simulate stomach and pancreatin and bile salts in neutral ph for the small intestine. This model is a more realistic replication of the conditions of the upper gastrointestinal tract [8, 9]. To simulate the digestive process in the colon, EnteroMix simulator consists of four reactors that recreate the conditions of the

2 ascending colon, transverse, descending and sigmoid using the same fecal inoculum obtained from one or more donors, which was developed for the study the effects of fermentation of carbohydrates in the microbial composition of the colon [10, 11]. With regard to the metabolism of flax lignans, Eeckhaut et al. (2008) [12] estimated the bioavailability of SDG in the upper gastrointestinal tract through an artificial stomach and intestinal digestion, also investigated the microbial fermentation in the colon to determine the metabolism of SDG in the large intestine, using SHIME. The SDG was released from the SDG-oligomer in the large intestine and SECO was released by microbial action in the ascending colon, which then becomes to ED and EL in the transverse colon. However, this study was conducted with a flax lignan concentrate (40%) as food matrix in gastric stimulation, a situation that is far from the actual conditions, omitting the effect of other nutritional components of flaxseed. The aim of this study was evaluated the metabolic changes of SDG from whole flaxseed during the digestive process by an in vitro model in a single batch. The digestive simulator includes chewing, stomach, small and large intestine. MATERIALS & METHODS The artificial saliva was prepared by dissolving 5.21 g L -1 NaHCO 3, 0.88 g L -1 NaCl, 0.48 g L -1 KCl, 0.44 g L -1 CaCl 2 2H 2 O, 1.04 g L -1 K 2 HPO4, 2.16 g L -1 mucin and 13 g L -1 of units of porcine α-amylase in deionized water, the ph was adjusted to % NaN 3 was added as a preservative. 50 g of flaxseed was mixed with 50 ml of artificial saliva and stirred gently for 20 s. 5 ml of sample for subsequent HPLC analysis of lignans was taken. 700 ml of distilled water in a bioreactor was sterilized (121 º C, 15 min), ph was adjusted to 2.0 at 37 C. 20 ml of the solution to the stomach (50 mg porcine gastric mucosa pepsin in 20 ml of 0.1 M HCl) was added. ph was adjusted again to 2.0 and inoculated with 200 ml of sample, corresponding to the saliva and flaxseed mixture from the previous stage. After 1 h the ph was changed to 6.0, 34 ml of bile salts (7.5 g of bovine bile in 50 ml of distilled water) and 50 ml of pancreatic juice (2 g pancreatic pancreatin pig dissolved in 50 ml of 0.02 M phosphate buffer at ph 7.5) was added. For 4 h the ph was gradually changed from 6.0 to 7.5 and nitrogen flow was injected to create anaerobic conditions. The bioreactor was maintained at 37 º C, with constant stirring at 100 rpm, with sporadic N 2 flow. The ph was adjusted with 2 M HCl and 1 M NaOH. 5 samples were taken every 1 h. Approximately 1 g of human fecal sample was dissolved in 10 ml of sterile phosphate buffer (0.1 mol L -1, ph 7.0) containing 10 mg of sodium thioglycolate as reducing agent. The samples were centrifuged (5000 rpm for 5 min) to remove solid material and the supernatant was recovered. This procedure was repeated with 20 donors (informed consent) to form a mixture of all samples, which were stored at 4 C. 50 ml of the solution of intestinal bacteria were incubated with 150 ml of brain heart broth under anaerobic conditions for 24 h at 36 C to increase the number of colonies. 150 ml of brain heart broth concentrate to 1 L, previously sterilized (121 º C, 15 min), was added to the bioreactor, then the bioreactor was inoculated with 50 ml of intestinal bacteria previously enriched. The ph was maintained at 5.5, 6.0, 6.5 and 7.0 per 12 h for each ph value.4 samples were taken every 12 h. A constametric model 3200 LDC analytical pump and a UV-Visible analytical detector model SM4000 LDC were used. Separation was achieved on a Luna C8 column (250 x 4.6 mm id, 5 µm particle size; Phenomenex Inc.) operated at 20 C. The detection was made at a wavelength of 280 nm. The chromatograms were analyzed using Clarity software, and the injection volume was 20 µl. The mobile phase was fase A: 1% aqueous acetic acid/acetonitrile (85:15 v/v); and fase B: acetonitrile. The gradient was: 100% A 0 min, 76% A 11 min, 60% A min, y 100% A min. The flow rate was 1 ml min -1. Then, the bioaccesibility was calculated as (free SDG) / (total SDG). Fermentative capacity was evaluated by short-chain fatty acid (SCFA). 900 μl of batch culture fermentation sample with 100 μl of formic acid was centrifuged at rpm for 5 min. Supernatant was filtered through 0.45 μm filter into new 1.5 ml eppendorf tube. 500 μl of filtered supernatant was added to a GC vial. 1 μl of sample was injected into GC (GC-2010 Shimadzu) equipped with a flame ionization detector and a capillary column (30 m x 0.32 mm) The carrier gas was He at a column flow rate of 2,4 ml min -1 with a split ratio of 1:100. The column temperature was 105 ºC. RESULTS & DISCUSSION The single batch simulator of digestive process was designed for evaluate flaxseed lignan bioaccesibility. The upper gastrointestinal tract was executed according to Sumeri el al (2008) [8] methodology with modifications. The mastication process was added to the simulation, so the bolus was a mixture of the whole flaxseed and saliva. Artificial saliva was prepared according to Arvisenet (2008) [13]. No crush was

3 simulated because usually flaxseed is swallowed intact. The stages of colon were performed according to previous reports [4, 5] with modifications. The main change was that we kept the same bioreactor which avoids using another vessel. Other digestive simulators have running times from one to fourteen days per cycle [7], which is not suitable for normal digestive system. Although digestion times are relative to each person, so in this study we chose to work with the lowest levels, thereby simulating the digestive process of a normal person with a faster metabolism. Lignans were not detected during mastication, therefore α-amylase had no effect on the lignan release or the chewing time was not enough. SDG is released in the stomach which corresponds to 0.12% of bioaccesibility. However, this lignan is not detected during the other stages of digestive simulation. The acid conditions of the stomach are not sufficient to release a large amount of SDG. Nevertheless, SDG was not released because the lignan is an esterified ester polymer that is composed of glutaric acid 3-hydroxy-3- methyl (HMGA) and other phenolic compounds such as glycosides of p-coumaric acid and feluric. Also, SDG is complexed with insoluble fiber, gums, polysaccharides and mucilage associated with the shell [14-16]. Similar results were obtained from other authors, whom claimed that the role of the lignan macromolecule should be seen as a delivery system in the large intestine [12]. In the small intestine was found increasing SECO (Figure 1). Probably, the low content of SDG released in the stomach is rapidly converted to SECO by enzymes present in the small intestine or SECO is released directly from flaxseed. These results differ from those obtained by Eeckhaut et al (2008) [12], which detected no SECO in small intestine, SECO was only detected in ascending colon by the action of intestinal bacteria. The bioaccesibility of SECO was 7.6%. SECO could be absorbed in the small intestine, and then only a fraction could reach the large intestine. SECO was detected in human urine indicating that this lignan is absorbed by epithelium gut [17]. In the ascending colon, ED and EL was detected, which remains constant for 36 hours, therefore intestinal bacteria are able to metabolize SECO in the ascending, transverse and descending colon with a constant rate. Finally, in the last 12 h of fermentation increases the content of enterolignans, which corresponds to 53 mg g -1 flaxseed. The bioavailability of ED and EL was 1.6% for both lignans. This last increment in enterolignans content may be due to SECO has begun to accumulate in the reactor and much of the fiber in flaxseed has been consumed by bacteria, thus the microflora are more available to the metabolism of lignans. Figure 1. Simulation of the digestive process for whole flaxseed. The amount of enterolignans is relatively low compared to the total that could have been released. It is for this reason that the fermentative capacity of the bacteria from fecal samples in the early stages of the large intestine was evaluated. After 24 h of incubation (Figure 2), the concentration of SCFA increased particularly acetic, isovaleric, propionic and isobutyric acid. The acetic acid corresponds to 85% of total SCFA. The isovaleric, propionic and isobutyric acid correspond to 6.7, 4.0 and 2.3%, respectively These results indicate that carbohydrate fermentation occurs, and probably the bacteria are consuming the soluble fiber in flaxseed and do not have enough time to metabolize more SECO. (Anti)-estrogenic activity is associated with mammalians lignans and antioxidant activity is associated with plant lignans, so if the metabolism in the colon does not occur in a complete degree the potential health benefits may correspond mainly for its

4 antioxidant activities. Further experiments are needed, including a study with several probiotic bacteria which may metabolize flax lignans, Figure 2. SCFA content after colon fermentation for whole flaxseed. CONCLUSION Consuming whole flaxseed confer nutritional benefits due lignans SDG and SECO are released, so they can be absorbed by the human gut. Furthermore, intestinal bacteria are able to metabolize these plants lignans to enterolignans. Even if the bioaccesibility was not as high as expected these results are satisfactory. Further experiments are needed, including a study with several probiotic bacteria which may metabolize flax lignans, increasing the bioaccesibility for SDG in whole flaxseed. ACKNOWLEDGMENTS This research was supported by the project FONDECYT , for which the authors are deeply indebted. C. Fuentealba obtained the CONICYT doctoral fellowship in REFERENCES [1] Charlet, S. Bensaddek,L. Raynaud, S. Gillet, F. Mesnard, F. & Fliniaux, M.A An HPLC procedure for the quantification of anhydrosecoisolariciresinol. Application to the evaluation of flax lignan content. Plant Physiology and Biochemistry, 40, [2] Hu, C. Yuan, Y.V. & Kitts, D.D Antioxidant activities of the flaxseed lignan secoisolariciresinol diglucoside, its aglycone secoisolariciresinol and the mammalian lignans enterodiol and enterolactone in vitro. Food Chemical Toxicology, 45, [3] Kitts, D.D. Yuan, Y.V. Wijewickreme, A.N. & Thompson, L.U Antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian lignan metabolites enterodiol and enterolactone. Molecular and Cellular Biochemistry, 202, [4] Possemiers, S. Verthé, K. Uyttendaele, S. & Verstraete, W PCR-DGGE-based quantification of stability of the microbial community in a simulator of the human intestinal microbial ecosystem. FEMS Microbiology Ecology 49: [5] De Boever, P. Deplancke, B. & Verstraete, W Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder. Journal of Nutrition 130, [6] Minekus, M. Marteau, P. Havennar, R. & Huis, H A multicompartmental dynamic computer-controlled model simulating the stomach and small-intestine. Alternatives to Laboratory Animals 23 (2), [7] Yoo, M. & Chen, X GIT physicochemical modeling A critical review. International Journal of Food Engineering 2 (4), Art. 4 [8] Sumeri, I. Arike, L. Adamberg, K. & Paalme, T Single bioreactor gastrointestinal tract simulator for study of survival of probiotic bacteria. Appl. Microbiol. Biotechnol. 80,

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