Method for predicting GI value of starch-based food

Abstract

The invention discloses a method for predicting the GI value of starch-based food, and belongs to the technical field of food digestion detection. According to the method, the dialysis bag is added into the in-vitro small intestine digestion simulation system, and the glucose in the digestion simulation process is transferred out, so that the influence of the glucose generated in the digestion simulation 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 method can be applied to the field of functional low-GI food development, and scientific guidance is provided for functional food and food accessory diet development.

Description

Technical Field

[0001] The invention relates to a method for predicting the GI value of starch-based food, and belongs to the technical field of food digestion detection. Background Art

[0002] With economic development and improved living standards, public health awareness has significantly increased, and the demand for dietary nutrition quality has continued to rise. The big health industry has gradually become one of the national strategic industries. However, problems such as obesity, diabetes and sub-health are becoming increasingly prominent, which is closely related to the digestion process of daily diet in the gastrointestinal tract. The gastrointestinal tract is a complex digestive system, including multiple chambers such as the mouth, stomach, small intestine, and large intestine, which are responsible for breaking down food, absorbing nutrients, and excreting waste. In particular, carbohydrates are broken down into glucose in the human digestive system to provide energy for various metabolic activities. In order to explore the effects of different foods on blood sugar, Jenkins et al. proposed the concept of glycemic index (GI). GI value has become an important indicator for distinguishing the functionality of carbohydrates. Although human blood sugar testing can truly reflect the GI value of food, due to its high cost, complex operation, ethical issues and individual differences, it is difficult to meet the high-throughput GI screening needs of the food industry, which limits the research and development and promotion of low-GI foods.

[0003] Based on this, researchers have developed a GI value prediction method based on an in vitro digestive system. Static in vitro digestion tests (such as the Englyst method) have gradually become the mainstream of application due to their simple operation, low cost and good repeatability. The Englyst method simulates gastrointestinal digestion in vitro and measures the glucose concentration of digestion products in combination with colorimetry or liquid chromatography to calculate the GI value. However, the Englyst method and other similar methods have operational problems, such as cumbersome operation, time-consuming measurement process, data dependence on statistical software, and the inability to dynamically reflect the real process of "digestion and absorption" in the human body, and the prediction results are not accurate enough. In addition, some patents have disclosed different methods for predicting in vitro GI values, such as calculating GI values ​​through transglycosylation or enzyme digestion models, but these methods generally involve complicated mathematical calculations and reference sample comparisons, and there are unreasonable steps, which limit the actual application effect.

[0004] In summary, the products of the enzymatic hydrolysis of food in the above in vitro method (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 in 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.

[0005] Therefore, there is an urgent need for an efficient, simple and accurate method to determine the hydrolysis rate of starch during in vitro digestion simulation, so as to evaluate the digestion patterns of starch-based food samples. Summary of the invention

[0006] To solve the above problems, the present invention adds a dialysis bag to the in vitro simulated small intestine digestion system to transport out the glucose in the simulated digestion process, thereby reducing the impact of glucose generated during the process on the digestive system and more realistically restoring the digestive effect of starch-based foods in the small intestine.

[0007] A first object of the present invention is to provide a method for predicting the GI value of starch-based foods, comprising the steps of:

[0008] (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;

[0009] (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;

[0010] (3) Simulating small intestinal digestion: mixing a gastric digestion simulator, intestinal electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, sodium hydroxide solution and water, adjusting the pH to 6.5-7.5, to obtain a mixture 3; adding the mixture 3 to a dialysis bag with a cutoff of 20-40 kDa, placing it in a dialysis solution for dialysis, and obtaining a small intestinal digestion simulator in the dialysis bag;

[0011] (4) detecting the starch hydrolysis curve of the small intestinal digestion simulation substance, calculating the eGI value of the in vitro simulated digestion experiment of the sample to be tested; substituting the eGI value into the standard curve to obtain the GI value of the sample to be tested;

[0012] The standard curve is: y=1.116x-11.985, y represents the actual GI value of the sample to be tested, and x represents the eGI value of the in vitro simulated digestion experiment of the sample to be tested.

[0013] In one embodiment, the starch-based food comprises different kinds of starch; the different kinds of starch include tapioca starch, corn starch, sweet potato starch, potato starch, slowly digestible starch, resistant starch, and the like.

[0014] In one embodiment, the composition of salivary electrolyte stock solution (SSF) is: KCI, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, (NH4)2CO3, pH 6.6-7.1.

[0015] In one embodiment, the composition of the gastric electrolyte stock solution (SGF) is: KCI, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, (NH4)2CO3, pH 2.5-3.5.

[0016] In one embodiment, the composition of the intestinal electrolyte stock solution (SIF) is: KCI, KH2PO4, NaHCO3, NaCl, MgCl2(H2O)6, (NH4)2CO3, pH 6.5-7.5.

[0017] In one embodiment, the sample to be tested includes starch-based food; the starch-based food includes but is not limited to rice, noodles, biscuits, bread, and may also be different types of starch.

[0018] In one embodiment, in step (1), the sample to be tested is prepared as a suspension with a concentration of 5% to 15% g / mL; the amount of α-amylase added 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; and the homogenization treatment is performed at 36.5 to 37.5°C and 153 to 255N for 5 to 20s.

[0019] 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; in step (3), the amount of pancreatic enzyme added is 3500-4500U / g starch-based food; the amount of saccharifying enzyme added is 1000-1500U / g starch-based food; in step (3), the volume ratio of the gastric digestion simulation material, the intestinal fluid electrolyte stock solution and the mixture 3 is 15-20:8-12:35-40; the stirring reaction in step (2) and step (3) is carried out at 36.5-37.5°C and 50-150r / min for 1.5-2.5h.

[0020] In one embodiment, the calculation method of the eGI value of the in vitro simulated digestion experiment in step (4) is:

[0021] According to the starch hydrolysis curve of the small intestinal digestion simulation, the hydrolysis index HI was calculated by definite integral from 0 to 180 minutes, and the hydrolysis index was substituted into the eGI-HI formula to calculate the eGI value;

[0022] The eGI-HI formula is eGI=39.71+0.549HI.

[0023] In one embodiment, the steps of simulating oral digestion are:

[0024] Prepare 3-7 mL of the sample to be tested (10%, g / 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.

[0025] In one embodiment, the steps of simulating gastric digestion are:

[0026] 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 / L CaCl2, 670-720 μL of ultrapure water and 0.1-0.3 mL of 0.5-1.5 mol / L hydrochloric acid solution, mix well and adjust the solution pH to 3.0, react at 36.5-37.5°C and 50-150 r / min for 1.5-2.5 h to obtain a gastric digestion simulation.

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

[0028] Add 10-12 mL SIF electrolyte stock solution, 4.0-6.0 mL pancreatic enzyme (4000 U), 0.05-0.15 mL saccharifying enzyme (0.2 mL saccharifying enzyme per gram of dry starch), 35-45 μL 0.1-0.5 mol / L CaCl2, 0.10-0.20 mL 0.5-1.5 mol / L sodium hydroxide solution and 1.11-1.51 mL ultrapure water to 15-25 mL gastric digestion simulation, mix well, adjust the solution pH to 7.0, and react at 36.5-37.5°C and 50-150 r / min for 1.5-2.5 h;

[0029] The simulated small intestinal digestion reaction was carried out in a dialysis bag; the dialysis bag was placed in 50-70 times the dialysate.

[0030] A second object of the present invention is to provide a method for simulating small intestinal digestion and absorption of starch-based food, comprising the steps of:

[0031] (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;

[0032] (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;

[0033] (3) Simulating small intestinal digestion: mixing a gastric digestion simulator, intestinal electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, sodium hydroxide solution and water, adjusting the pH to 6.5-7.5, to obtain a mixture 3; adding the mixture 3 to a dialysis bag with a cutoff of 20-40 kDa, placing it in a dialysis solution for dialysis, and obtaining a small intestinal digestion simulator in the dialysis bag;

[0034] When simulating small intestinal digestion, the dialysis bag is placed in the dialysate, and the volume ratio of the mixture 3 to the dialysate is 1:50-80; wherein the dialysate is an intestinal fluid electrolyte stock solution.

[0035] In one embodiment, 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; and the homogenization treatment is performed at 36.5 to 37.5°C and 153 to 255N for 5 to 20s.

[0036] In one embodiment, the volume ratio of the oral simulated chewing material, the gastric fluid electrolyte stock solution and the mixture 2 in step (2) is 8-10:5-7.5:15-20; the amount of pancreatic enzyme added in step (3) is 3500-4500 U / g starch-based food; the amount of saccharifying enzyme added is 1000-1500 U / g starch-based food; the volume ratio of the gastric digestion simulation material, the intestinal fluid electrolyte stock solution and the mixture 3 in step (3) is 15-20:8-12:35-40.

[0037] 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.

[0038] The third object of the present invention is to provide an application of any of the above methods in the field of food, medicine or feed, including: detecting the digestion and absorption effects of food, medicine or feed.

[0039] The fourth 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 a dialysis bag is added to the simulated digestion system, comprising the steps of:

[0040] (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;

[0041] (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;

[0042] (3) Simulating small intestinal digestion: mixing a gastric digestion simulator, intestinal electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, sodium hydroxide solution and water, adjusting the pH to 6.5-7.5, to obtain a mixture 3; adding the mixture 3 to a dialysis bag with a cutoff of 20-40 kDa, placing it in a dialysis solution for dialysis, and obtaining a small intestinal digestion simulator in the dialysis bag;

[0043] When simulating small intestinal digestion, the dialysis bag is placed in the dialysis solution, and the volume ratio of the mixture 3 to the dialysis solution is 1:5-80; wherein the dialysis solution is an intestinal fluid electrolyte stock solution.

[0044] Beneficial Effects of the Invention

[0045] The present invention adds a dialysis bag to the in vitro simulated small intestine digestion system to transport out the glucose in the simulated digestion process, thereby reducing the influence of the glucose generated during the process on the digestive system and more realistically restoring the digestive effect of starch-based foods in the small intestine.

[0046] Specifically:

[0047] (1) The present invention eliminates the influence of glucose generation on the process of enzyme-catalyzed reaction, takes into account the absorption of glucose, truly reflects the process of human body's digestion and absorption, embodies the real enzymatic reaction and digestion and absorption in the human body, and ensures real-time and accurate quantitative measurement.

[0048] (2) The present invention simplifies the traditional in vitro method for determining the glycemic index of starch-based foods, removes unreasonable or unnecessary steps, and is easier to operate. At the same time, it overcomes the defect of the traditional technology that the isolated point test cannot reflect the overall digestion situation.

[0049] (3) The data analysis of the method for determining the glycemic index of starch-based foods in vitro does not rely on professional statistical software, which significantly reduces the data analysis time and ensures the accuracy of the data.

[0050] (4) The linear correlation between the in vitro digestion parameters obtained in the present invention and the GI value is above 0.98, and the prediction accuracy is higher.

[0051] In summary, the present invention discloses a simple and efficient in vitro test detection technology, which can accurately predict the glycemic index (GI) of starch-based foods. This method uses starch-based foods with known GI as standard references to establish a mathematical function relationship between the in vivo GI index and the parameters of the in vitro simulated gastrointestinal digestion test (Simulated In Vitro Gastrointestinal Digestion Assay) to achieve accurate prediction of the GI index of the starch-based food to be tested. Based on the physicochemical properties of food, the present invention combines knowledge from multiple fields such as enzymology, biochemistry, food chemistry and digestion reaction kinetics simulation to construct a new generation of simulated dynamic small intestine simulated digestion and absorption and real-time monitoring system. The system can be applied to the development of functional low-GI foods, explore the analysis method of in vitro simulated starch staple food digestion samples that are closer to the digestion, transport and absorption of the human small intestine, and provide scientific guidance for the development of functional foods and food supplements, and is used to quickly detect and screen food samples with low glycemic index (Low GI). BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The digestion and hydrolysis curves of starch samples with different added amounts of glucose in Example 1;

[0053] Figure 2 : is the digestion and hydrolysis curve of starch samples under different dialysate volumes in Example 2;

[0054] Figure 3 The hydrolysis curves of the starch samples with and without the dialysis bag in Example 3;

[0055] Figure 4 are the hydrolysis curves of different starch-based foods in Example 4;

[0056] Figure 5 There is a linear relationship between the actual GI value of the sample and the predicted eGI value. DETAILED DESCRIPTION

[0057] 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.

[0058] raw material:

[0059] 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.

[0060] Table 1 Preparation of electrolyte stock solution for simulated digestion fluid

[0061]

[0062]

[0063] Note: “ / ” means not to add.

[0064] Dialysis bags SP131345 cellulose dialysis bag (20000) 20 kDa, SP131381 cellulose dialysis bag (50000) 50 kDa Dalton, and SP131417 cellulose dialysis bag (100000) 100 kDa Dalton were purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0065] Pancreatin was purchased from Sigma-Aldrich (8 U / mg, P7545, EC 232-468-9);

[0066] Glucoamylase was purchased from Sigma-Aldrich (260 U / mL, A7095, EC 3.2.1.3);

[0067] α-Amylase was purchased from Sigma-Aldrich (16 U / mg, A3176, EC 647-015-00-4);

[0068] Barley, corn, rice, rice starch, pea starch, high amylose corn starch, regular corn starch, and potato starch are all commercially available.

[0069] Noodles, white steamed buns, white bread, and glutinous rice were all commercially available; Shutang Rice No. 2 was purchased from the Labor Union Committee of Chongqing Academy of Agricultural Sciences; Indica rice was purchased from Jinjian Rice Industry; and Thai glutinous rice was purchased from Thailand Zhengming Co., Ltd.

[0070] The intelligent mechanized beating homogenizing device was purchased from Ningbo Licheng Instrument Co., Ltd., and the product model is LC-11L.

[0071] 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.

[0072] Detection Methods

[0073] 1. Calculation method of starch hydrolysis rate in in vitro simulation:

[0074] 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.

[0075] 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.

[0076] 2. Calculation method of digestibility in mice:

[0077] 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;

[0078] Starch hydrolysis rate (%) = 1-unhydrolyzed starch mass / intragastric starch mass × 100%.

[0079] Example 1: Detecting the Effect of Glucose on Digestive Enzymes during Digestion

[0080] Corn starch (referred to as starch in the following embodiments) was used as a raw material, and glucose was added to obtain 5 test samples (the starch content in the 5 groups of samples was consistent), which were as follows:

[0081] Control: starch sample;

[0082] Sample 1: starch + 10% w / w glucose;

[0083] Sample 2: starch + 20% w / w glucose;

[0084] Sample 3: starch + 40% w / w glucose;

[0085] Sample 4: starch + 50% w / w glucose.

[0086] 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:

[0087] (1) Prepare 0.5 mol / L sodium acetate buffer (pH 5.2) and 90% (v / v) ethanol aqueous solution;

[0088] 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;

[0089] (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;

[0090] (3) The glucose oxidase-peroxidase (GOPOD) method was used to determine the glucose content released (i.e., in the supernatant) at each time point.

[0091] 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.

[0092] 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.

[0093] Example 2: Screening of dialysis conditions

[0094] Glucose in the system can be removed by dialysis, so appropriate dialysis conditions were screened based on a 20 kDa dialysis bag.

[0095] The amount of starch used in different samples was the same. Glucose was used as the detection object. Appropriate dialysis conditions were screened and different volumes of dialysis fluid (in this embodiment, the dialysis fluid was sodium acetate buffer solution) were set according to the volume of the simulated digestion system, respectively as follows:

[0096] Control: starch;

[0097] Sample 1: starch + 5 times the volume of dialysate;

[0098] Sample 2: starch + 10 times the volume of dialysate;

[0099] Sample 3: starch + 20 times the volume of dialysate;

[0100] Sample 4: starch + 50 times the volume of dialysate;

[0101] Sample 5: starch + 75 times the volume of dialysate;

[0102] By simulating the enzymatic digestion reaction activity of the small intestine in vitro, the rate and digestibility of reducing sugar released from the starch to be tested under different volumes of dialysate are measured. The steps are as follows:

[0103] (1) Prepare 0.5 mol / L sodium acetate buffer (pH 5.2) and 90% (v / v) ethanol aqueous solution;

[0104] 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;

[0105] (2) Weigh 0.20 g of sample powder 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 mixture. Place it in a 37°C water bath for 30 min. Add 2 mL of mixed enzyme and mix well. Transfer the mixture in the centrifuge tube to a dialysis bag (20 kDa). Then place it in different volumes of dialysis solution and start the reaction in a 37°C shaking water bath to generate an enzymatic solution. When the reaction reaches 0, 20, 30, 60, 90, 120, 160, 180, 240, 300 and 420 min, use a near-infrared spectrometer (ReactRaman) to analyze the glucose dialyzed. TM The glucose concentration in the dialysate was measured by an in-situ Raman spectrometer (802), and the glucose content released by the starch enzymatic reaction system at each time point was obtained.

[0106] The results are as follows Figure 2 As shown, the results show that when the dialysate volume is small, the transport rate of glucose is relatively slow, and the final transport amount is not much different from that of native starch, but the early transport amount is slightly increased; with the increase in the volume of dialysate, the dialysis transport rate of glucose gradually accelerates, and the glucose content in the system gradually decreases.

[0107] When the dialysate volume increased to 50 times, the dialysis transport rate tended to balance, so that the glucose generated in each stage could be quickly transported to the external dialysate. The dialysis transport rate of 75 times the volume was the same as that of 50 times, indicating that the dialysis effect was best under the condition of 50 times the volume. Therefore, it was decided to use 50 times the volume of dialysate in the subsequent simulated small intestinal digestion experiment.

[0108] Example 3: A method for simulating small intestinal digestion of starch-based food

[0109] 1. A method for simulating the digestion of starch-based food in the small intestine, simulating the enzymatic digestion reaction activities in the human mouth, stomach and small intestine through in vitro tests, and measuring the rate of reducing sugar release from the starch to be tested and the digestibility of the food:

[0110] (1) Simulating oral digestion: prepare 5 mL of the sample to be tested (corn starch, 10%, g / mL), 3.5 mL of SSF electrolyte stock solution with a pH of 7.0, vortex and oscillate to mix, add 0.5 mL of SSF electrolyte stock solution containing 1500 U / mL α-amylase, 25 μL of 0.3 mol / L CaCl2 solution and 975 μL of ultrapure water, and mix well; transfer to an intelligent mechanized beating homogenizer, apply a force of 204 N for 10 s to obtain an oral simulated chewing material;

[0111] (2) Simulated gastric digestion: Take 10 mL of simulated oral 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 solution, 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;

[0112] (3) Simulated small intestinal digestion: Take 20 mL of gastric digestion simulant, 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 solution, 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 small intestinal digestion products;

[0113] The simulated small intestinal digestion was reacted in a dialysis bag. At the beginning of the reaction, the dialysis bag was placed in a 50-fold volume of dialysis solution (the dialysis solution in this embodiment was an intestinal electrolyte stock solution) and dialyzed for 280 minutes.

[0114] 2. Characterization of in vivo and in vitro simulated digestion products

[0115] (1) In vitro starch digestion curve

[0116] During the dialysis process, the glucose concentration in the dialysate was detected by near-infrared spectrometer at different time points, and the in vitro starch digestibility was calculated.

[0117] In vitro starch digestion curve results Figure 3As shown, the results show that the use of dialysis bags can speed up the hydrolysis process of the entire starch. Compared with not using dialysis bags, 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 95.3% after adding adsorbents. This shows that the use of dialysis bags in the digestion simulation process 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.

[0118] (2) Starch digestion results in vivo

[0119] 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.

[0120] 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 corn starch digestibility (95.3%) with the in vivo corn starch digestibility of mice, it can be seen that after using the dialysis bag, the starch digestibility is higher than that without the dialysis bag, which is closer to the actual digestion in vivo.

[0121] Table 2 Starch digestion results in vivo

[0122]

[0123] The above results show that the dialysis bag 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.

[0124] Comparative Example 1: Using Dialysis Bags with Different Molecular Weights

[0125] On the basis of Example 2, the size of the dialysis bag was changed to 10, 50, and 100 kDa, and the dialysis effect of glucose was tested.

[0126] The results showed that when using 10, 50, and 100 kDa dialysis bags, the starch hydrolysis rate in the dialysis bags was greatly reduced; the dialysis rate of 10 kDa for glucose was slower, while the dialysis bags of 50 and 100 kDa would dialyze out both pancreatic amylase and amyloglucosidase, resulting in a decrease in the hydrolysis rate of glucose.

[0127] Example 4: A method for predicting the GI values ​​of different starch-based foods in vitro

[0128] 1. Constructing the GI value prediction equation

[0129] White bread, white steamed bread, noodles, Shutangmi No. 2, indica rice and glutinous rice were cooked, and white bread was used as a control. The starch hydrolysis rate curve was obtained according to the method of Example 3.

[0130] The starch hydrolysis rate curve is as follows Figure 4 As shown, the hydrolysis index (HI) was calculated by definite integral calculation of the hydrolysis rate curve of different samples (Shutangmi No. 2, noodles, white steamed bread, glutinous rice, and indica rice) from 0 to 180 minutes, and the eGI value of the in vitro simulated digestion experiment of each starch-based food was calculated by the formula (eGI=39.71+0.549HI).

[0131] The eGI values ​​and true GI values ​​of the in vitro simulated digestion experiments of various starch-based foods were fitted (as shown in Table 3), and the functional relationship between the glycemic index GI value of the starch-based food samples and the eGI value of the in vitro digestion experiment was established. The results are as follows: Figure 5 The results showed that for the five starch-based food standards, the eGI values ​​of the in vitro digestion experiment showed a linear relationship with the true GI values ​​(y = 1.116x-11.985, R 2 =0.9858). It can be seen that the linear correlation between the test parameters of the in vitro simulated digestion experiment obtained by the method of Example 3 and the predicted GI value is above 0.98, and the prediction accuracy is very high.

[0132] Table 3 eGI and actual GI values ​​of different samples

[0133] Predicting eGI value Actual GI value Shutangmi 68.4 65 Noodle 85.9 82 Steamed bun 89.7 89 Sticky rice 96.3 94 Indica Rice 98.7 100

[0134] 2. Verification of GI value prediction equation

[0135] Take Thai glutinous rice, cook it, obtain its hydrolysis rate curve according to the above method, and calculate the eGI value (96.3). Substitute the eGI value into the equation y=1.116x-11.985 to obtain its true GI value of 95.5, which is within 2% of the Thai glutinous rice GI value of 94 provided by the Chinese Institute of Food Fermentation Industries (for details, see the official website of the Chinese Institute of Food Fermentation Industries). It can be seen that the GI value predicted by the method of the present invention is accurate and reliable.

[0136] 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 predicting the GI value of starch-based foods, 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: Mix a gastric digestion simulant, intestinal electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, sodium hydroxide solution and water, and adjust the pH to 6.5-7.5 to obtain a mixture 3; add the mixture 3 to a dialysis bag with a cutoff of 20-40 kDa, place it in a dialysis solution for dialysis, and obtain a small intestinal digestion simulant in the dialysis bag; the volume ratio of the mixture 3 to the dialysis solution is 1:50-80; (4) detecting the starch hydrolysis curve of the small intestinal digestion simulation substance, calculating the eGI value of the in vitro simulated digestion experiment of the sample to be tested; substituting the eGI value into the standard curve to obtain the GI value of the sample to be tested; The standard curve is: y=1.116x-11.985, y represents the actual GI value of the sample to be tested, and x represents the eGI value of the in vitro simulated digestion experiment of the sample to be tested.

2. The method according to claim 1, characterized in that In step (1), the sample to be tested is prepared as a suspension with a concentration of 5% to 15% g / mL; the amount of α-amylase added is 1000 to 2000 U / g starch-based food; the volume ratio of the sample suspension to be tested, the salivary electrolyte stock solution and the mixture 1 is 1 to 5:1 to 3.5:8 to 10; and the homogenization treatment is performed at 36.5 to 37.5° C. and 153 to 255 N for 5 to 20 seconds.

3. 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; in step (3), the amount of pancreatic enzyme added is 3500-4500U / g starch-based food; the amount of saccharifying enzyme added is 1000-1500U / g starch-based food; in step (3), the volume ratio of the gastric digestion simulation material, the intestinal fluid electrolyte stock solution and the mixture 3 is 15-20:8-12:35-40; in step (2) and step (3), the stirring reaction is carried out at 36.5-37.5°C and 50-150r / min for 1.5-2.5h.

4. The method according to claim 1, characterized in that: The calculation method of the eGI value of the in vitro simulated digestion experiment in step (4) is: According to the starch hydrolysis curve of the small intestinal digestion simulation, the hydrolysis index HI was calculated by definite integral from 0 to 180 minutes, and the hydrolysis index was substituted into the eGI-HI formula to calculate the eGI value; The eGI-HI formula is eGI=39.71+0.549HI.

5. 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, intestinal electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, sodium hydroxide solution and water, adjusting the pH to 6.5-7.5, to obtain a mixture 3; adding the mixture 3 to a dialysis bag with a cutoff of 20-40 kDa, placing it in a dialysis solution for dialysis, and obtaining a small intestinal digestion simulator in the dialysis bag; When simulating small intestinal digestion, the dialysis bag is placed in the dialysate, and the volume ratio of the mixture 3 to the dialysate is 1:50-80; wherein the dialysate is an intestinal fluid electrolyte stock solution.

6. The method according to claim 5, 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 sample suspension to be tested, the salivary electrolyte stock solution and the mixture 1 is 1 to 5:1 to 3.5:8 to 10; and the homogenization treatment is performed at 36.5 to 37.5° C. and 153 to 255 N for 5 to 20 seconds.

7. The method according to claim 5, 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; in step (3), the amount of pancreatic enzyme added is 3500-4500U / g starch-based food; the amount of saccharifying enzyme added is 1000-1500U / g starch-based food; in step (3), the volume ratio of the gastric digestion simulation material, the intestinal fluid electrolyte stock solution and the mixture 3 is 15-20:8-12:35-40.

8. The method according to claim 5, 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 the dialysis bag to the 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, intestinal electrolyte stock solution, pancreatic enzyme, saccharifying enzyme, CaCl2, sodium hydroxide solution and water, adjusting the pH to 6.5-7.5, to obtain a mixture 3; adding the mixture 3 to a dialysis bag with a cutoff of 20-40 kDa, placing it in a dialysis solution for dialysis, and obtaining a small intestinal digestion simulator in the dialysis bag; When simulating small intestinal digestion, the dialysis bag is placed in the dialysis solution, and the volume ratio of the mixture 3 to the dialysis solution is 1:5-80; wherein the dialysis solution is an intestinal fluid electrolyte stock solution.

Images