Method for simulating small intestine to digest and absorb starch-based food

Abstract

The invention discloses a method for simulating small intestines to digest and absorb starch-based food, and belongs to the technical field of food digestion detection. According to the method, the glucose adsorbent is added into the in-vitro small intestine digestion simulation system, so that the influence of glucose generated in the digestion process on the digestion system is reduced, and the digestion effect of the starch-based food in the small intestine is more truly reduced; the pitch-based spherical activated carbon is added in the simulated digestion process, so that glucose can be adsorbed, the enzyme reaction process is effectively improved, the hydrolysis rate of starch is increased, and the starch is more in line with the digestion and absorption mechanism of a human body on the starch.

Description

Technical Field

[0001] The invention relates to a method for simulating small intestine digestion and absorption of starch-based food, belonging to the technical field of food digestion detection. Background Art

[0002] With the development of social economy and the improvement of residents' living standards, the public's health awareness continues to increase, consumers' demand for dietary nutritional quality is gradually increasing, and the potential market for healthy food continues to expand. Carbohydrates, as one of the most important nutrients in food, are metabolized and absorbed by the human digestive system as glucose molecules, providing the main energy source for various metabolic activities. In order to explore the changes in blood sugar in the body, Jenkins et al. from the University of Toronto proposed the concept of glycemic index (GI). The glycemic index is an important indicator for distinguishing functional carbohydrate foods. The method of determining the glycemic index in vivo usually involves human subjects, which is characterized by high cost, long procedures, low efficiency, and inability to conduct large-scale carbohydrate food GI value testing. These defects limit its application in modern breeding or food industry. For example, in vivo testing cannot meet the extensive testing needs of these industries.

[0003] To solve this problem, researchers have developed a series of in vitro simulation test methods to achieve rapid, efficient and accurate estimation of the GI value of food. Among them, the most representative in vitro simulated digestion gastrointestinal detection method was published in the literature "Measurement of rapidly available glucose (RAG) in plant foods: a potential invitro predictor of the glycaemic response" and was established by KN Englyst and his colleagues. The Englyst method involves mixing the test food with digestive enzymes at 37°C and measuring the resulting glucose level by colorimetry. Subsequently, the in vitro method was improved using advanced instruments (including high performance liquid chromatography (HPLC)) to accurately measure the glucose released by enzyme-catalyzed hydrolysis of starch. With the help of relevant mathematical models, the GI value of the test carbohydrate food can be predicted based on the amount of glucose released within 20 minutes. However, the in vitro Englyst method still faces many problems and doubts.

[0004] For example, Garsetti et al. used the Englyst in vitro digestion method (GI ~ 35-60) to evaluate biscuit products, and then compared it with the in vivo determination, and found that the ex vivo in vitro digestion method had almost no predictive value. Similarly, Brand-Miller et al. used the ex vivo Englyst in vitro digestion method to test three medium glycemic index food samples. The results showed that the GI values ​​of these three foods were all <55, and they were classified as low glycemic index foods. This shows that there are still major problems and difficulties in the rationalization of the Englyst in vitro digestion method.

[0005] Following the Englyst method, researchers continued to explore in vitro simulated digestion prediction technology for gastrointestinal testing. In patent CN101784898B, researchers improved the Englyst method by using low-temperature homogenization of food and solidified or semi-solidified food samples to ensure the integrity of food molecules. At the same time, simulated enzyme digestion of food produces various products including protein, fat and sugar alcohol. After HPLC measurement of sample sugar, a computer data model is used to simulate the GI index to improve prediction accuracy.

[0006] In patent number CN106979997B, researchers disclosed a transglycosylation enzyme reagent, which is mixed with the food to be tested for a sugar-producing reaction, and the food is broken up with the help of ultrasonic vibrations. After a simulated digestion reaction, the starch, native sugars and dietary fiber in the food obtain a chyme-like transglycosylation product. By time-series determination of the glucose concentration in this product, a sugar-producing reaction curve can be drawn. By using numerical methods and the conversion calculation of the glycemic index corresponding to the transglycosylation function, the GI value of the tested sample can be quickly evaluated. Although this method greatly shortens the in vitro detection time and simplifies the required advanced scientific instruments and manpower, its subsequent data processing involves a large amount of mathematical integration and computer software program applications, which are complicated to operate and difficult to master.

[0007] In patent number CN202110458696.2, researchers published a method for in vitro determination of the glycemic index of carbohydrate foods. This method simulates the enzymatic digestion reaction activities of the human mouth, stomach and small intestine through in vitro experiments, measures the rate of reducing sugar release and digestibility of the carbohydrate food to be tested, and uses the in vitro digestion reaction kinetic equation and data fitting model to obtain relevant in vitro digestion test parameters. In vitro digestion experiments are carried out on standard carbohydrate food standards with known GI values ​​to clarify and construct the functional relationship between the glycemic index GI of the standard sample and the in vitro digestion test parameters. According to the in vitro digestion parameters of the carbohydrate food to be tested, the glycemic index of the sample to be tested, i.e., the GI value, is predicted by comparing the functional relationship between the GI value of the standard sample and its in vitro digestion test parameters. Although this method greatly shortens the detection time of the sample to be tested and simplifies the required advanced scientific instruments and resource investment, its data processing involves the testing and data processing of a large number of reference samples, and the operation is cumbersome and lacks universality. More importantly, this method does not take into account the digestion and absorption of glucose generated during the digestion test, which has a certain impact on the enzyme reaction process.

[0008] In summary, the products of the enzymatic hydrolysis of food in the above in vitro methods (mainly glucose) do not take into account their absorption. Glucose is in an accumulation state during the in vitro simulated digestion and hydrolysis process, which is not the case of digestion and absorption by the human body. It cannot reflect the real enzymatic reaction and digestion and absorption in the human body, so it is impossible to obtain real-time and accurate quantitative measurement. There are many defects in in vitro tests for the blood glucose production of carbohydrate foods. Therefore, there is an urgent need for an efficient, simple and accurate method to determine the glucose production of starch-based foods, so as to quickly evaluate the digestion rules of starch-based food samples. Summary of the invention

[0009] To solve the above problems, the present invention reduces the influence of glucose produced during enzymatic hydrolysis on the digestive system by adding a glucose adsorbent into an in vitro simulated small intestine digestive system, and more realistically restores the digestion and absorption effect of starch-based foods in the small intestine.

[0010] The present invention provides a method for simulating small intestine digestion and absorption of starch-based food, comprising the steps of:

[0011] (1) Simulating oral digestion: mixing the sample to be tested, salivary electrolyte stock solution, α-amylase, CaCl2 and water to obtain a mixture 1, and homogenizing to obtain an oral simulated chewing material;

[0012] (2) Simulating gastric digestion: mixing oral simulated chewing material, gastric electrolyte stock solution, CaCl2, hydrochloric acid and water to adjust the pH to 2.5-3.5 to obtain mixture 2, stirring and reacting to obtain a gastric digestion simulator;

[0013] (3) Simulating small intestinal digestion: mixing a gastric digestion simulator, an intestinal fluid electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, a sodium hydroxide solution and water, adjusting the pH to 6.5 to 7.5 to obtain a mixture 3, stirring the mixture for reaction, and obtaining a small intestinal digestion simulator;

[0014] The sample to be tested is obtained by mixing starch-based food and asphalt-based spherical activated carbon.

[0015] In one embodiment, starch-based food samples include, but are not limited to, rice, noodles, biscuits, and bread.

[0016] In one embodiment, the composition of the salivary electrolyte stock solution is: KCI, KH2PO4, NaHCO3, NaCI, MgCl2(H2O)6, (NH4)2CO3, pH 6.6-7.1.

[0017] In one embodiment, the composition of the gastric juice electrolyte stock solution is: KCI, KH2PO4, NaHCO3, NaCI, MgCl2(H2O)6, (NH4)2CO3, pH 2.5-3.5.

[0018] In one embodiment, the composition of the intestinal electrolyte stock solution is: KCI, KH2PO4, NaHCO3, NaCI, MgCl2(H2O)6, (NH4)2CO3, pH 6.5-7.5.

[0019] In one embodiment, the mass ratio of starch-based food to asphalt-based spherical activated carbon in the sample to be tested is 1:1.25-2.

[0020] In one embodiment, the sample to be tested in step (1) is prepared as a suspension with a concentration of 5% to 15% g / 100 mL; the amount of α-amylase added in step (1) is 1000 to 2000 U / g starch-based food; and the volume ratio of the suspension of the sample to be tested, the salivary electrolyte stock solution and the mixture 1 is 1 to 5:1 to 3.5:8 to 10.

[0021] In one embodiment, the homogenization treatment in step (1) is performed at 36.5-37.5° C. and 153-255 N beating for 5-20 seconds.

[0022] Preferably, the homogenization process is 204N beating for 10s.

[0023] In one embodiment, in step (2), the volume ratio of the oral simulated chewing material, the gastric fluid electrolyte stock solution and the mixture 2 is 8-10:5-7.5:15-20.

[0024] In one embodiment, in step (3), the amount of pancreatic enzyme added is 3500-45000 U / g starch-based food; the amount of saccharifying enzyme added is 1000-1500 U / g starch-based food.

[0025] In one embodiment, in step (3), the volume ratio of the gastric digestion simulant, the intestinal fluid electrolyte stock solution and the mixture 3 is 15-20:8-12:35-40.

[0026] In one embodiment, the stirring reaction in step (2) and step (3) is carried out at 36.5-37.5°C and 50-150 r / min for 1.5-2.5 hours.

[0027] In one embodiment, the steps of simulating oral digestion are:

[0028] Prepare 3-7 mL of the sample suspension (10%, g / 100 mL), add 3-4 mL of SSF electrolyte stock solution with a pH of 7.0, vortex to mix, add 0.2-0.7 mL of SSF electrolyte stock solution containing 1000-2000 U / mL α-amylase, 20-30 μL of 0.1-0.5 mol / L CaCl2 and 950-1000 μL of ultrapure water, and mix; transfer to an intelligent mechanized beating homogenizer, apply a force of 153-255 N for 5-15 s to obtain an oral simulated chewing material.

[0029] In one embodiment, the step of simulating gastric digestion is: take 5-15 mL of oral simulated chewing material, add 5-10 mL of SGF electrolyte stock solution, 1.2-2.0 mL of SGF electrolyte stock solution without added porcine pepsin, 3-8 μL of 0.1-0.5 mol / LCaCl2, 670-720 μL of ultrapure water and 0.1-0.3 mL of 0.5-1.5 mol / L hydrochloric acid solution, adjust the solution pH to 3.0 after mixing, and react at 36.5-37.5°C and 50-150 r / min for 2 hours to obtain a gastric digestion simulator.

[0030] In one embodiment, the step of simulating small intestinal digestion is as follows: 15-25 mL of gastric digestion simulant is taken, 10-12 mL of SIF electrolyte stock solution, 4-6 mL of pancreatic enzyme (4000 U), 0.5-1.5 mL of saccharifying enzyme (0.2 mL of saccharifying enzyme per gram of dry starch), 35-45 μL of 0.1-0.5 mol / L CaCl2, 0.10-0.20 mL of 0.5-1.5 mol / L sodium hydroxide solution and 1.11-1.51 mL of ultrapure water are added, the pH of the solution is adjusted to 7.0 after mixing, and the reaction is carried out at 36.5-37.5° C. and 50-150 r / min for 1.5-2.5 hours to obtain a small intestinal digestion simulant.

[0031] The second object of the present invention is to provide an application of any of the methods described in the field of food, medicine or feed, wherein the application includes: detecting the digestion and absorption effects of food, medicine or feed.

[0032] The third object of the present invention is to provide a method for improving the accuracy of in vitro simulated small intestinal digestion and absorption of starch-based foods, wherein asphalt-based spherical activated carbon is added to the simulated digestion system, comprising the steps of:

[0033] (1) Simulating oral digestion: mixing the sample to be tested, salivary electrolyte stock solution, α-amylase, CaCl2 and water to obtain a mixture 1, and homogenizing to obtain an oral simulated chewing material;

[0034] (2) Simulating gastric digestion: mixing oral simulated chewing material, gastric electrolyte stock solution, CaCl2, hydrochloric acid and water to adjust the pH to 2.5-3.5 to obtain mixture 2, stirring and reacting to obtain a gastric digestion simulator;

[0035] (3) Simulating small intestinal digestion: mixing a gastric digestion simulator, an intestinal fluid electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, a sodium hydroxide solution and water, adjusting the pH to 6.5 to 7.5 to obtain a mixture 3, stirring the mixture for reaction, and obtaining a small intestinal digestion simulator;

[0036] The sample to be tested is obtained by mixing starch-based food and asphalt-based spherical activated carbon.

[0037] In one embodiment, the mass ratio of starch-based food to asphalt-based spherical activated carbon in the sample to be tested is 1:1.25-2.

[0038] In one embodiment, the sample to be tested in step (1) is prepared as a suspension with a concentration of 5% to 15% g / 100 mL; the amount of α-amylase added in step (1) is 1000 to 2000 U / g starch-based food; and the volume ratio of the suspension of the sample to be tested, the salivary electrolyte stock solution and the mixture 1 is 1 to 5:1 to 3.5:8 to 10.

[0039] In one embodiment, the homogenization treatment in step (1) is 153-255N for 5-20s.

[0040] In one embodiment, in step (2), the volume ratio of the oral simulated chewing material, the gastric fluid electrolyte stock solution and the mixture 2 is 8-10:5-7.5:15-20.

[0041] In one embodiment, in step (3), the amount of pancreatic enzyme added is 3500-4500 U / g starch-based food; the amount of saccharifying enzyme added is 1000-1500 U / g starch-based food.

[0042] In one embodiment, in step (3), the volume ratio of the gastric digestion simulant, the intestinal fluid electrolyte stock solution and the mixture 3 is 15-20:8-12:35-40.

[0043] In one embodiment, the stirring reaction in step (2) and step (3) is carried out at 36.5-37.5°C and 50-150 r / min for 1.5-2.5 hours.

[0044] Beneficial effects of the present invention:

[0045] The present invention reduces the influence of glucose generated during digestion on the digestive system by adding a glucose adsorbent to the in vitro simulated small intestine digestion system, and more realistically restores the digestion effect of starch-based foods in the small intestine. The present invention adds asphalt-based spherical activated carbon during the simulated digestion process, which can absorb glucose, effectively improve the enzyme reaction process, and increase the hydrolysis rate of starch, making it more in line with the digestion and absorption mechanism of starch by the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The effect of different glucose contents on starch hydrolysis rate;

[0047] Figure 2 The effect of different adsorbents on the adsorption of glucose;

[0048] Figure 3 The effect of different adsorbent contents on glucose production;

[0049] Figure 4 is the hydrolysis rate of starch after adding adsorbent. DETAILED DESCRIPTION

[0050] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0051] raw material:

[0052] Preparation of electrolyte stock solutions: The compositions of salivary electrolyte stock solutions (SSF), gastric electrolyte stock solutions (SGF) and intestinal electrolyte stock solutions (SIF) are shown in Table 1.

[0053] Table 1 Preparation of electrolyte stock solution for simulated digestion fluid

[0054]

[0055]

[0056] Note: “ / ” means not to add.

[0057] Asphalt-based spherical activated carbon was purchased from Shenzhen Global Greenland New Materials Co., Ltd.

[0058] Bentonite was purchased from Shandong Huawei Bentonite Co., Ltd.

[0059] Concanavalin was purchased from Shanghai Yili Biotechnology Co., Ltd.

[0060] Activated carbon powder was purchased from Shenzhen Global Greenland New Materials Co., Ltd.

[0061] Pancreatin was purchased from Sigma-Aldrich (8 U / mg, P7545, EC 232-468-9);

[0062] Glucoamylase was purchased from Sigma-Aldrich (260 U / mL, A7095, EC 3.2.1.3);

[0063] α-Amylase was purchased from Sigma-Aldrich (16 U / mg, A3176, EC 647-015-00-4);

[0064] Barley, corn, rice, rice starch, pea starch, high amylose corn starch, regular corn starch, and potato starch are all commercially available.

[0065] The intelligent mechanized beating homogenizing device was purchased from Ningbo Licheng Instrument Co., Ltd., and the product model is LC-11L.

[0066] The starch used in the embodiments and comparative examples is corn starch, and other starches may also be used. The type of starch does not affect the effect of simulated digestion.

[0067] Detection Methods

[0068] 1. Calculation method of starch hydrolysis rate in in vitro simulation:

[0069] Starch hydrolysis rate (%) = glucose content × 0.9 × 100 / S; where S is the total mass of starch, and 0.9 is the stoichiometric constant of starch extracted from the glucose content.

[0070] Glucose content detection method: Take 3 portions of supernatant (6 μL) from each centrifuge tube of the sample to be tested and place them in an ELISA plate, then add 200 μL of glucose determination reagent, place the ELISA plate in an ELISA reader, measure the absorbance of the supernatant at 520 nm, and calculate the glucose content based on the glucose standard curve; ELISA test conditions: oscillate for 10 seconds, keep constant temperature at 37°C, and keep warm for 10 minutes.

[0071] 2. Calculation method of digestibility in mice:

[0072] The contents of the mouse ileum were taken, washed with water, and dried to obtain unhydrolyzed starch. The in vivo starch hydrolysis rate was calculated by comparing the mass of unhydrolyzed starch with that of starch administered by oral administration;

[0073] Starch hydrolysis rate (%) = 1-unhydrolyzed starch mass / intragastric starch mass × 100%.

[0074] Example 1: Detecting the Effect of Glucose on Digestive Enzymes during Digestion

[0075] Corn starch (hereinafter referred to as starch) was used as the raw material, and glucose was added to obtain 5 test samples (the starch content in the 5 groups of samples was consistent), which are as follows:

[0076] Control: starch sample;

[0077] Sample 1: starch + 10% w / w glucose;

[0078] Sample 2: starch + 20% w / w glucose;

[0079] Sample 3: starch + 40% w / w glucose;

[0080] Sample 4: starch + 50% w / w glucose.

[0081] To test the effect of glucose on digestive enzymes during digestion, an in vitro test was performed to simulate the enzymatic digestion reaction activity of the small intestine and measure the rate of reducing sugar release and digestibility of the starch to be tested. The steps are as follows:

[0082] (1) Prepare 0.5 mol / L sodium acetate buffer (pH 5.2) and 90% (v / v) ethanol aqueous solution;

[0083] Preparation of mixed enzyme: Disperse 4.5 g pancreatin in 40 mL deionized water, shake in a water bath at 37°C for 10 min, and centrifuge (speed: 5000 × g, time: 10 min) to obtain pancreatin supernatant; take 27 mL pancreatin supernatant into a centrifuge tube, add 3.2 mL saccharifying enzyme to obtain mixed enzyme;

[0084] (2) Weigh the sample (ensuring that the starch content in the sample is 0.20 g) and add it to a centrifuge tube containing 4 mL of deionized water, mix well, add 4 mL of sodium acetate buffer solution and 10 glass beads to the above mixture, place it in a 37°C water bath for insulation for 30 min, then add 2 mL of mixed enzyme, mix well, place it in a 37°C shaking water bath to continue the reaction, and generate an enzymatic solution; when the reaction reaches 0, 5, 10, 15, 20, 30, 60, 90, and 120 min, measure 0.1 mL of the enzymatic solution and place it in a centrifuge tube containing 0.9 mL of 90% ethanol, and centrifuge (speed 10000g, time 5 min) to obtain a supernatant;

[0085] (3) The glucose oxidase-peroxidase (GOPOD) method was used to determine the glucose content released (i.e., in the supernatant) at each time point.

[0086] The digestion curves of samples with different glucose contents are shown in Figure 2. Figure 1 As shown, within the first 5 minutes, the digestion rate of the control group (containing only starch) was the fastest; as the amount of glucose added increased, the digestion rate of starch slowed down, and as the hydrolysis time increased, the overall digestibility of starch also decreased.

[0087] The results showed that the presence of glucose does affect the enzymatic digestion and hydrolysis process of starch, thereby affecting the efficiency and progress of the enzyme-catalyzed hydrolysis reaction, and ultimately affecting the overall digestion results of starch.

[0088] Example 2: Screening of adsorbents

[0089] Taking glucose as the detection object, the glucose adsorption performance of different adsorbents: asphalt-based spherical activated carbon, bentonite, and concanavalin was detected. The steps are as follows: a phosphate buffer solution was used to simulate the conditions of the human intestine. A 3.0 g / L glucose solution was prepared with phosphate buffer (pH = 7.4), and 50 mL was transferred to a 100 mL conical flask, and bentonite, concanavalin, and asphalt-based spherical activated carbon were added (1:1 by mass ratio with starch), respectively. The flasks were placed in a constant temperature water bath at 37°C and oscillated at a speed of 150 r / min for 12 h. The supernatant was taken (centrifuged or filtered), and the content was determined by the glucose oxidase method, a glucose oxidase kit, and an automatic biochemical analyzer. The adsorption rate and adsorption amount of bentonite, concanavalin, and asphalt-based spherical activated carbon for glucose were calculated.

[0090] The results are as follows Figure 2 As shown, the results show that the adsorption amount increases with the extension of adsorption time. Among them, asphalt-based spherical activated carbon and bentonite reached adsorption equilibrium after 5 hours, and the equilibrium adsorption amounts were 71 mg / g and 41 mg / g, respectively; concanavalin reached adsorption equilibrium after 8.3 hours, and the equilibrium adsorption amount was 55 mg / g.

[0091] Among them, in the initial stage of adsorption, the asphalt-based spherical activated carbon has more surface adsorption sites, stronger adsorption force and faster adsorption rate. After comprehensive consideration, asphalt-based spherical activated carbon was selected as the adsorbent for glucose for subsequent research.

[0092] Example 3: Adsorbent dosage

[0093] On the basis of Example 1, the sample to be tested is changed to:

[0094] Sample 1: starch + asphalt-based spherical activated carbon (mass ratio 1:0.5);

[0095] Sample 2: starch + asphalt-based spherical activated carbon (mass ratio 1:0.75);

[0096] Sample 3: starch + asphalt-based spherical activated carbon (mass ratio 1:1.25);

[0097] Sample 4: starch + asphalt-based spherical activated carbon (mass ratio 1:1.5);

[0098] Sample 5: starch + asphalt-based spherical activated carbon (mass ratio 1:2).

[0099] Sample 6: starch + asphalt-based spherical activated carbon (mass ratio 1:3).

[0100] The release rate and digestibility of reducing sugars in the samples were tested as follows:

[0101] (1) Prepare 0.5 mol / L sodium acetate buffer (pH 5.2) and 90% (v / v) ethanol aqueous solution;

[0102] Preparation of mixed enzyme: Disperse 4.5 g pancreatin in 40 mL deionized water, shake in a water bath at 37°C for 10 min, and centrifuge (speed: 5000 × g, time: 10 min) to obtain pancreatin supernatant; take 27 mL pancreatin supernatant into a centrifuge tube, add 3.2 mL saccharifying enzyme to obtain mixed enzyme;

[0103] (2) Weigh the sample (ensuring that the starch content in the sample is 0.20 g) and add it to a centrifuge tube containing 4 mL of deionized water, mix well, add 4 mL of sodium acetate buffer solution and 10 glass beads to the above mixture, place it in a 37°C water bath for insulation for 30 min, then add 2 mL of mixed enzyme, mix well, place it in a 37°C shaking water bath to continue the reaction, and generate an enzymatic solution; when the reaction reaches 0, 20, 30, 60, 90, 120, 160, 180, 240, 300, 360 and 420 min, measure 0.1 mL of the enzymatic solution and place it in a centrifuge tube containing 0.9 mL of 90% ethanol, and centrifuge (speed 10000g, time 5 min) to obtain a supernatant;

[0104] (3) The glucose oxidase-peroxidase (GOPOD) method was used to determine the glucose content in the supernatant at each time point.

[0105] The results of glucose content test are as follows Figure 3 As shown in the figure, the results show that when the addition amount of asphalt-based spherical activated carbon is low (0.5-1.25 times), the adsorption amount of glucose is low; as the addition amount of asphalt-based spherical activated carbon increases, the glucose content in the system further decreases. When the addition amount of asphalt-based spherical activated carbon reaches 2 times, the adsorption effect begins to tend to equilibrium, making the glucose content in the system almost undetectable (less than 3%), and the glucose adsorption effect is the best, which is better than the addition amount of 1.5 times and 3 times.

[0106] Asphalt-based spherical activated carbon has high adsorption selectivity, and its characteristics determine that it will not adsorb starch and will not lead to low digestibility. Therefore, the optimal addition amount of asphalt-based spherical activated carbon is twice the mass of starch, which can be effectively used for rapid and efficient adsorption of glucose in enzyme-catalyzed hydrolysis experiments.

[0107] Example 4: A method for simulating small intestinal digestion and absorption of starch-based foods

[0108] A method for simulating small intestinal digestion and absorption of starch-based food, comprising:

[0109] 1. Prepare samples:

[0110] Starch and asphalt-based spherical activated carbon were mixed in a mass ratio of 1:2 to obtain a sample to be tested.

[0111] 2. Through in vitro tests, the enzymatic digestion reaction activities in the human mouth, stomach and small intestine are simulated to measure the rate at which the tested starch releases reducing sugars;

[0112] (1) Simulating oral digestion: prepare 5 mL of the sample suspension (10%, g / mL; i.e., 1 g of the sample is dispersed in 10 mL of water), add 3.5 mL of SSF electrolyte stock solution with a pH of 7.0, vortex and mix, add 0.5 mL of SSF electrolyte stock solution containing 1500 U / mL α-amylase, 25 μL of 0.3 mol / L CaCl2 and 975 μL of ultrapure water, mix; transfer to an intelligent mechanized beating homogenizer, apply a force of 204 N for 10 s, and obtain an oral simulated chew;

[0113] (2) Simulated gastric digestion: Take 10 mL of oral simulated chewing material, add 7.5 mL of SGF electrolyte stock solution, 1.6 mL of SGF electrolyte stock solution without porcine pepsin, 5 μL of 0.3 mol / L CaCl2, 695 μL of ultrapure water and 0.2 mL of 1.0 mol / L hydrochloric acid solution, mix well and adjust the solution pH to 3.0, react at 37 °C and 100 r / min for 2 h to obtain a gastric digestion simulator;

[0114] (3) Simulated small intestinal digestion: Take 20 mL of gastric digestion simulator, add 11 mL of SIF electrolyte stock solution, 5.0 mL of pancreatic enzyme (4000 U), 0.1 mL of saccharifying enzyme (0.2 mL of saccharifying enzyme per gram of dry starch), 40 μL of 0.3 mol / L CaCl2, 0.15 mL of 1.0 mol / L sodium hydroxide solution and 1.31 mL of ultrapure water, mix well and adjust the solution pH to 7.0, react at 37°C and 100 r / min for 2 h to obtain a small intestinal digestion simulator;

[0115] After the start of the simulated small intestinal digestion reaction, the adsorbent was taken out at 0, 15, 30, 45, 60, 90, 120 and 150 min, respectively, and placed in a pure water system (40 mL) to heat and release glucose to obtain a dilution solution; 0.1 mL of the dilution solution was measured and placed in a centrifuge tube containing 0.9 mL of 90% ethanol, and centrifuged (speed 10000×g, time 5 min) to obtain a supernatant as the sample solution to be tested;

[0116] (5) Detection of digestion products: The glucose oxidase-peroxidase (GOPOD) method was used to determine the glucose content in the dilution at each time point. The operating conditions were as follows: 3 portions (6 μL) of the supernatant were taken from each centrifuge tube and placed on an ELISA plate. 200 μL of glucose assay reagent was then added. The ELISA plate was placed in an ELISA reader and the absorbance of the supernatant at 520 nm was determined. The test conditions of the ELISA reader were as follows: oscillation for 10 s, constant temperature at 37°C, and insulation for 10 min.

[0117] 3. Characterization of simulated digestive products in vivo and in vitro

[0118] (1) In vitro starch digestion curve

[0119] The starch digestion curve results are as follows Figure 4 As shown, the results show that the addition of asphalt-based spherical activated carbon accelerates the hydrolysis process of the entire starch. Compared with the case without adding adsorbent, the hydrolysis rate and hydrolysis rate in each time period are higher. The 3h starch hydrolysis rate is 78.3%, and the hydrolysis rate is 91.8% after adding adsorbent, indicating that the addition of adsorbent can effectively improve the enzyme reaction process and increase the hydrolysis rate of starch; in addition, the enzymatic reaction system after removing glucose is more in line with the human body's digestion and absorption mechanism of glucose.

[0120] (2) Starch digestion results in vivo

[0121] C57BL / 6 male mice aged 6 to 8 weeks were selected as experimental subjects and divided into 8 groups. The mice were fasted for 12 hours before the experiment and fed with the test solution (barley, corn, rice, rice starch, pea starch, high-amylose corn starch, ordinary corn starch and potato starch) at a dose of 300 mg / kg body weight by oral gavage according to the grouping. The starch digestion in the small intestine of the mice was detected after 3 hours.

[0122] The results are shown in Table 2. The results show that the digestibility of cereals reached 97.2% to 98.3%, the digestibility of common starch reached more than 87.8%, and the digestibility of common corn starch was 96.8%. Comparing the in vitro digestibility of corn starch (91.8%) with the digestibility of corn starch in mice, it can be seen that after adding the adsorbent, the starch digestibility is higher than that without adding the adsorbent, which is closer to the actual digestion in vivo.

[0123] Table 2 Starch digestion results in vivo

[0124]

[0125] The above results show that adsorbents can not only effectively accelerate the hydrolysis process of starch in vitro, but also simulate the starch digestion and metabolism process in the small intestine in vivo more realistically; by introducing adsorbents into the in vitro simulated digestive system, a small intestinal digestion model that is closer to the real physiological conditions can be constructed, which has important application potential, provides a theoretical basis for the development of more accurate digestion and absorption models in the future, and helps to deeply understand the influence mechanism of starch-based foods on digestion and absorption and blood sugar levels.

[0126] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for simulating small intestinal digestion and absorption of starch-based food, characterized in that: Includes steps: (1) Simulating oral digestion: mixing the sample to be tested, salivary electrolyte stock solution, α-amylase, CaCl2 and water to obtain a mixture 1, and homogenizing to obtain an oral simulated chewing material; (2) Simulating gastric digestion: mixing oral simulated chewing material, gastric electrolyte stock solution, CaCl2, hydrochloric acid and water to adjust the pH to 2.5-3.5 to obtain mixture 2, stirring and reacting to obtain a gastric digestion simulator; (3) Simulating small intestinal digestion: mixing a gastric digestion simulator, an intestinal fluid electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, a sodium hydroxide solution and water, adjusting the pH to 6.5 to 7.5 to obtain a mixture 3, stirring the mixture for reaction, and obtaining a small intestinal digestion simulator; The sample to be tested is obtained by mixing starch-based food and asphalt-based spherical activated carbon.

2. The method according to claim 1, characterized in that The mass ratio of starch-based food to asphalt-based spherical activated carbon in the test sample is 1:1.25~2.

3. The method according to claim 1, characterized in that The sample to be tested in step (1) is prepared as a suspension with a concentration of 5% to 15% (g / mL); the amount of α-amylase added in step (1) is 1000 to 2000 U / g starch-based food; the volume ratio of the suspension of the sample to be tested, the salivary electrolyte stock solution and the mixture 1 is 1 to 5:1 to 3.5:8 to 10.

4. The method according to claim 1, characterized in that: In step (1), the homogenization treatment is carried out at 36.5-37.5°C and 153-255N for 5-20s.

5. The method according to claim 1, characterized in that In step (2), the volume ratio of the oral simulated chewing material, the gastric fluid electrolyte stock solution and the mixture 2 is 8-10:5-7.5:15-20.

6. The method according to claim 1, characterized in that In step (3), the amount of pancreatic enzyme added is 3500-45000 U / g starch-based food; the amount of saccharifying enzyme added is 1000-1500 U / g starch-based food.

7. The method according to claim 1, characterized in that In step (3), the volume ratio of the gastric digestion simulant, the intestinal fluid electrolyte stock solution and the mixture 3 is 15-20:8-12:35-40.

8. The method according to claim 1, characterized in that The stirring reaction in step (2) and step (3) is carried out at 36.5-37.5°C and 50-150 r / min for 1.5-2.5 hours.

9. Use of the method according to any one of claims 1 to 8 in the field of food, medicine or feed, characterized in that: include: Test the digestion and absorption effects of food, medicine or feed.

10. A method for improving the accuracy of in vitro simulated small intestinal digestion and absorption of starch-based foods, characterized in that: Adding asphalt-based spherical activated carbon into a simulated digestion system includes the following steps: (1) Simulating oral digestion: mixing the sample to be tested, salivary electrolyte stock solution, α-amylase, CaCl2 and water to obtain a mixture 1, and homogenizing to obtain an oral simulated chewing material; (2) Simulating gastric digestion: mixing oral simulated chewing material, gastric electrolyte stock solution, CaCl2, hydrochloric acid and water to adjust the pH to 2.5-3.5 to obtain mixture 2, stirring and reacting to obtain a gastric digestion simulator; (3) Simulating small intestinal digestion: mixing a gastric digestion simulator, an intestinal fluid electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, a sodium hydroxide solution and water, adjusting the pH to 6.5 to 7.5 to obtain a mixture 3, stirring the mixture for reaction, and obtaining a small intestinal digestion simulator; The sample to be tested is obtained by mixing starch-based food and asphalt-based spherical activated carbon in a mass ratio of 1:1.25-2.

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