体外消化实验

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 bioactivityof 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 thegastric 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 α-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 thebioaccessibility 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 solution 10 ml KCl 89.6g/l15.7 ml NaCl 175.3g/l40 ml NaCl175.3g/l30 ml NaCl175.3g/l10 ml KSCN20g/l3.0 ml NaH2PO4 88.8g/l40 ml NaHCO384.7 g/l68.3 ml NaHCO384.7 g/l10 ml NaH2PO488.8 g/l9.2 ml KCl 89.6 g/l10 ml KH2PO48g/l4.2 ml KCl 89.6 g/l 10 ml NaSO457g/l18 ml CaCl2· 2H2O22.2 g/l6.3 ml KCl89.6 g/l150 μl HCl 37%g/g1.7 ml NaCl175.3 g/l10 ml NH4Cl 30.6 g/l10 ml MgCl2 5g/l20 ml NaHCO384.7 g/l6.5 ml HCl 37%g/g180 μl HCl37%g/gOrganic solution 8 ml urea 25 g/l 10 ml glucose 65 g/l 4 ml urea 25 g/l 10 ml urea 25 g/l10 ml glucuronic acid(葡萄糖醛酸) 2g/l3.4 ml urea 25g/l10 ml glucosaminehydrochloride (盐酸氨基葡萄糖) 33g/lAdd to mixtureorganic+ inorganic solution290 mgα-amylase1g BSA9mlCaCl2· 2H2O22.2 g/l10ml CaCl2· 2H2O22.2 g/l15 mg uric acid 2.5 g pepsin 1g BSA 1.8 g BSA25 mg mucin 3 g mucin 9 g pancreatin 30 g bile1.5 g lipasepH 6.8 ± 0.2 1.30 ± 0.02 8.1 ± 0.2 8.2 ± 0.2 The 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. PartI: 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 Englyst’s 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 Bacilluslicheniformis (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 ºC in a shaker waterbath for 30 min. The sample dispersion was adjusted to pH 4.4–4.6 by adding HCl and then hydrolyzed with amyloglucosidase (300 U, Sigma chemical, Cat. No. A9913) at 60 ºC 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 ºC 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 Englyst’s 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 ºC, mixed with an enzyme solution (5 mL) consisting of pancreatin extract and amyloglucosidase, and incubated in a water-bath at 37 ºC. 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 within the first 20 min, and the slowly digestible starch (SDS) was the starch digested between 20 and 120 min (Englyst et al., 1992). The resistant starch content was calculated as follows:（%）RS content = 100%×(total starch – RDS - SDS) (g, dsb)/total starch (g, dsb)澳大利亚NI 消化系统图片GI20。