体外消化实验.doc

体外模拟消化（口腔、胃、小肠）：1、 参考Gawlik-Dziki等（2009）方法（The effect of simulated digestion in vitro on bioactivity of wheat bread with Tartary buckwheat flavones addition）2.4.1. Preparation of simulated saliva, gastric and intestinal fluids Simulated saliva solution was prepared by dissolve of 2.38 gNa2HPO4, 0.19 g KH2PO4 and 8 g NaCl in 1 l of distilled water. The solution was adjusted to pH 6.75 and a-amylase (EC 3.2.1.1.) was added to obtain 200 U of enzyme activity. For the gastric digestion, 0.32% pepsin (from porcine stomach mucosa, pepsin A, EC 3.4.23.1) dilution in 0.03 M NaCl, pH 1.2 was prepared. Simulated intestinal. juice was prepared by dilution of 0.05 g of pancreatin and 0.3 g of bile extract in 35 ml 0.1 M NaHCO3.2.4.1. Preparation of simulated saliva, gastric and intestinal fluidsSimulated saliva solution was prepared by dissolve of 2.38 g Na2HPO4, 0.19 g KH2PO4 and 8 g NaCl in 1 l of distilled water. The solution was adjusted to pH 6.75 and -amylase (EC 3.2.1.1.) was added to obtain 200 U of enzyme activity. For the gastric digestion, 0.32% pepsin (from porcine stomach mucosa, pepsin A, EC 3.4.23.1) dilution in 0.03 M NaCl, pH 1.2 was prepared. Simulated intestinal juice was prepared by dilution of 0.05 g of pancreatin and 0.3 g of bile extract in 35 ml 0.1 M NaHCO3.2、 参考Versantvoort等（2005）（Applicability of an in vitro digestion model in assessing the bioaccessibility of mycotoxins from food）和Hur等（2009）的方法（Influence of initial emulsifier type on microstructural changes occurring in emulsified lipids during in vitro digestion）Table 1Constituents and concentrations of the various synthetic juices of the in vitro digestion model representing fed conditionsSaliva 唾液Gastric juice 胃液Duodenal juice 十二指肠液Bile juice 胆汁Inorganic solution10 ml KCl 89.6 g/l 15.7 ml NaCl 175.3g/l40 ml NaCl 175.3g/l30 ml NaCl 175.3g/l10 ml KSCN 20g/l 3.0 ml NaH2PO4 88.8 g/l40 ml NaHCO3 84.7 g/l68.3 ml NaHCO3 84.7 g/l10 ml NaH2PO4 88.8 g/l 9.2 ml KCl 89.6 g/l10 ml KH2PO4 8g/l4.2 ml KCl 89.6 g/l10 ml NaSO4 57g/l 18 ml CaCl2 2H2O 22.2 g/l6.3 ml KCl 89.6 g/l150 l HCl 37% g/g1.7 ml NaCl 175.3 g/l 10 ml NH4Cl 30.6 g/l10 ml MgCl2 5 g/l20 ml NaHCO3 84.7 g/l 6.5 ml HCl 37%g/g180 l HCl 37%g/gOrganic solution 8 ml urea 25 g/l10 ml glucose 65 g/l4 ml urea 25 g/l10 ml urea 25 g/l10 ml glucuronic acid (葡萄糖醛酸) 2g/l3.4 ml urea 25g/l10 ml glucosamine hydrochloride (盐酸氨基葡萄糖) 33g/lAdd to mixture organic+ inorganic solution290 mg -amylase 1g BSA9ml CaCl2 2H2O 22.2 g/l10ml CaCl2 2H2O 22.2 g/l15 mg uric acid 2.5 g pepsin1g BSA1.8 g BSA25 mg mucin3 g mucin9 g pancreatin30 g bile1.5 g lipasepH 6.8 0.21.30 0.028.1 0.28.2 0.2The inorganic and organic solutions are augmented to 500ml with distilled water. After mixing of the inorganic and organic solutions, some further constituents are added and dissolved. If necessary, the pH of the juices is adjusted to the appropriate interval.另外，抗性淀粉含量测定：1、 参考Li等（2008）方法（Characterization of maize amylose-extender (ae) mutant starches. Part I: Relationship between resistant starch contents and molecular structures）2.3. Resistant starch (RS) contentRS contents of the ae-mutant starch samples were determined using the AOAC Method 991.43 for total dietary fiber (AOAC, 2003) and Englysts method (1992) for comparison. For the AOAC 991.43 method, starch (1.0 g, dry-starch basis, dsb) was suspended in a Mes-tris buffer solution (0.05 M, 40 ml) and hydrolyzed with 500 u of a-amylase from Bacillus licheniformis (Sigma Chemical, Cat. No. A3403) in a boiling water-bath for 30 min with stir. The sample was then digested with protease from Bacillus licheniformis (5 mg, Sigma Chemical, Cat. No. P3910) at 60 C in a shaker waterbath for 30 min. The sample dispersion was adjusted to pH 4.44.6 by adding HCl and then hydrolyzed with amyloglucosidase (300 U, Sigma chemical, Cat. No. A9913) at 60 C in a shaker water-bath for 30 min. The digested sample was filtered through a celite layer in a crucible and washed twice with 15 ml of 78% ethanol, twice with 15 ml of 100% ethanol and rinsed with 15 ml of acetone. The remaining sample was dried in an oven at 100 C overnight. The resistant starch content was calculated using the equation: （%）RS content = Remaining sample weight (g, dsb)/initial sample weight (g, dsb) 100%For RS determined using Englysts method (1992), Starch (1.000 g, db) in 20 mL of sodium acetate buffer (0.1 M, pH 5.2) was cooked in a boiling water-bath for 30 min. The starch dispersion was cooled down to 37 C, mixed with an enzyme solution (5 mL) consisting of pancreatin extract and amyloglucosidase, and incubated in a water-bath at 37 C. The pancreatin extract was prepared as follows; 3.0 g of pancreatin (Sigma, Cat. No. P7545) was suspended in 20 mL deionized water, stirred for 10 min at room temperature, and centrifuged at 1500g for 10 min. The enzyme solution was prepared by mixing 13.5 ml supernatant of pancreatin extract, 210 U amyloglucosidase (Sigma, Cat. No. A7095), and 1.0 mL deionized water. The rapid digestible starch (RDS) was defined as the total starch digested