A method for in vitro simulation of measuring the glycemic index of rice

Abstract

The invention discloses a method for simulating and determining the glycemic index of rice in vitro, and belongs to the technical field of food measurement. The in vitro simulation method of the invention comprises simulating oral chewing, gastrointestinal digestion, and constructing a detection model, which can more accurately predict the real glycemic index GI of rice. The method of the invention solves the problem that the in vivo test determination method needs to use human as the test subject, has high technical difficulty, high cost, long time, poor scalability and operability; it also makes up for the defects of the Goni in vitro simulation determination method that ignores the influence of the oral chewing process on the digestion of rice, cannot accurately reflect the real digestion of rice, and has poor discrimination of different types of rice; based on the multiple extrusion device of the physical property analyzer to simulate the real chewing process of humans, the influence of the chewing process on the glycemic index of rice is fully considered, and the digestion rate of rice in the simulated gastrointestinal digestion environment is further determined, and the glycemic index of rice is calculated, and the result is more real, objective, and accurate, and the discrimination of different types of rice is high.

Description

Technical Field

[0001] The invention belongs to the technical field of food measurement, and in particular relates to a method for simulating in vitro determination of the glycemic index of rice. Background Art

[0002] Rice is one of the important staple foods. In recent years, with the improvement of residents' living standards and changes in dietary structure, the incidence of blood sugar-related metabolic diseases such as diabetes and obesity has risen sharply. The main component of rice is starch. The digestion and absorption of starch is closely related to blood sugar metabolism and directly affects the incidence of diseases such as diabetes and obesity. Therefore, the impact of rice on blood sugar metabolism has received widespread attention.

[0003] The full name of the glycemic index (GI) is the blood sugar production index, which reflects the speed and ability of food to increase blood sugar compared with glucose. This indicator can help special groups choose appropriate foods and reduce the incidence of diseases such as diabetes and obesity. According to the speed of blood sugar increase, foods can be divided into high-glycemic foods (GI>70), medium-glycemic foods (55≤GI≤70), and low-glycemic foods (GI<55). During the digestion of rice, starch can be hydrolyzed into glucose, which increases blood sugar concentration. Therefore, accurately evaluating the glycemic index of rice is of great significance for guiding consumers to formulate reasonable ways of eating rice according to specific health management requirements.

[0004] At present, the determination of the glycemic index usually requires healthy adults as test subjects, who first fast for 12 hours, then take in 50 grams of the food to be tested, take blood samples regularly to measure blood glucose concentration, and calculate the ratio of the area below the blood glucose line of the substitute food to the area below the blood glucose line of the standard food (white bread) based on the blood glucose-time curve, and then multiply it by 100 to get the glycemic index of the substitute food. This method has high requirements on the subjects, is expensive, time-consuming and labor-intensive, and is not conducive to the rapid detection of the glycemic index of food. Therefore, it is necessary to develop a fast and accurate in vitro test method.

[0005] At present, the method for in vitro simulation of food glycemic index mostly adopts Goni's method. This method weighs 50 mg of sample, first reacts in simulated gastric juice for 1 hour, then adjusts the pH to 6.9, adds amylase, simulates small intestinal digestion for 3 hours, and samples every 30 minutes to determine the sample hydrolysis rate. The sample hydrolysis index (HI) is calculated from the sample hydrolysis rate curve, and the sample glycemic index is calculated based on the relationship between the sample hydrolysis index and the sample glycemic index. This method can measure the glycemic index of food under in vitro simulation conditions. Compared with in vivo tests, this simulation method is highly operational and low cost.

[0006] The glycemic index of edible rice is closely related to the degree of oral chewing of the eater, that is, the particle state and rheological properties of rice after oral chewing can have a significant impact on its digestion in the gastrointestinal tract. However, the current Goni method for in vitro simulation of the glycemic index of rice has defects. The sample pretreatment method of this method is simple, which is quite different from the actual rice intake and digestion process, and ignores the effect of the oral chewing process on rice digestion. In addition, the Goni method tests a small number of samples and has poor representativeness. Therefore, the Goni method for in vitro simulation of the glycemic index of rice has certain limitations. Summary of the invention

[0007] In view of the defects of the current rice glycemic index determination technology, the present invention focuses on innovative methods to establish a determination method that can fully reflect the impact of the oral chewing process on rice digestion under in vitro simulation conditions and can more accurately predict the true glycemic index of rice, including simulating oral chewing, gastrointestinal digestion, and constructing a mathematical model to achieve this goal.

[0008] The present invention aims to solve the problems that the current in vivo test method for measuring the glycemic index of rice needs to use humans as test subjects, which is technically difficult, costly, time-consuming, and has poor scalability and operability; and the Goni in vitro simulation test method has too small a test sample amount and simple sample pretreatment, and ignores the influence of the oral chewing process on rice digestion, and cannot accurately reflect the actual digestion of rice, and has poor discrimination of the glycemic index of different types of rice. The present invention provides a method that is closer to the actual rice intake process and can more accurately measure the glycemic index of rice.

[0009] This method is based on the multiple extrusion device of the physical property analyzer, simulating the real chewing process of humans and fully considering the impact of the chewing process on the glycemic index of rice. On this basis, it further determines the digestion rate of rice in a simulated gastrointestinal digestive environment and calculates the glycemic index of rice. Therefore, the result is closer to the real situation, more objective and accurate, and has a high degree of distinction between different types of rice.

[0010] The present invention provides a method for simulating in vitro determination of the glycemic index of rice, comprising the following steps:

[0011] (1) mixing rice with low-temperature amylase and a buffer solution, and then placing the mixture in an extrusion device for cyclic reciprocating extrusion;

[0012] (2) transferring the extruded rice into simulated gastric fluid to simulate gastric digestion;

[0013] (3) After the simulated gastric digestion is completed, the pH is adjusted, pancreatic enzyme, amyloglucosidase and transferase are added, and after mixing, small intestinal digestion is simulated; sampling is performed at different times, centrifuged, and the supernatant is collected; and the glucose content in the supernatant is determined using a glucose (GOPOD method) detection kit, and the starch hydrolysis rate in the rice is calculated according to the starch hydrolysis rate = (glucose production × 0.9 / total starch amount) × 100%, and the starch hydrolysis rate-time curve is obtained; the area under the line AUC is obtained according to the starch hydrolysis rate-time curve 米饭-体外 ;

[0014] (4) Using white bread of the same mass as rice as a control, the starch hydrolysis rate in the white bread is determined according to the process of steps (1) to (3), and a starch hydrolysis rate-time curve is obtained; and the area under the line AUC is obtained according to the starch hydrolysis rate-time curve. 白面包-体外 ; Using AUC 米饭-体外 , AUC 白面包-体外 The ratio of AUC to calculate the starch hydrolysis index HI in rice, HI = (AUC 米饭-体外 / AUC 白面包-体外 )×100%;

[0015] (5) Calculate the glycemic index of rice according to the starch hydrolysis index-glycemic index HI-GI detection model;

[0016] Among them, the construction process of the HI-GI detection model is as follows:

[0017] Healthy adult subjects were selected to fast for a period of time and consume the same amount of different types of rice. Blood was collected every 20-40 minutes to measure blood glucose concentration. White bread of the same mass as rice was used as a control to obtain the area under the blood glucose concentration-time curve (AUC) within 0-120 minutes after rice and white bread were consumed. 米饭-体内 , AUC 白面包-体内 , calculate the glycemic index of rice under in vivo test conditions (GI = AUC 米饭-体内 / AUC 白面包-体内 ); at the same time, the starch hydrolysis index (HI) of different types of rice is measured through the above steps (1)-(4), and the HI is linearly correlated with the glycemic index GI under in vivo test conditions to obtain a HI-GI detection model.

[0018] In one embodiment of the present invention, in step (1), the mass ratio of rice to low-temperature amylase is (25-35): (0.005-0.02), and preferably (25-35): 0.01.

[0019] In one embodiment of the present invention, in step (1), the enzyme activity of the low-temperature amylase is 2000 U / g.

[0020] In one embodiment of the present invention, in step (1), the amount of rice and buffer solution is (25-35) g: (3-5) mL.

[0021] In one embodiment of the present invention, in step (1), the buffer solution may specifically be a phosphate buffer solution with a pH of 6.8.

[0022] In one embodiment of the present invention, in step (1), the number of cyclic reciprocating extrusion is 20-30 times, preferably 25-30 times.

[0023] In one embodiment of the present invention, in step (1), the temperature environment of extrusion is a constant temperature of 36°C.

[0024] In one embodiment of the present invention, in step (1), extrusion can be performed in a multiple extrusion device of a physical property analyzer.

[0025] In one embodiment of the present invention, in step (1), the extrusion operation speed is 5-10 mm / s, and the extrusion distance is 85-95 mm.

[0026] In one embodiment of the present invention, in step (2), the simulated gastric fluid includes sodium chloride, pepsin, and hydrochloric acid with a pH value of 1.2; the dosage conditions are 2-3 g sodium chloride: 1-2 g pepsin: 900 mL hydrochloric acid with a pH value of 1.2.

[0027] In one embodiment of the present invention, in step (2), the device for simulating gastrointestinal digestion comprises a 2L reaction vessel with a diameter of 15 cm, equipped with a stirring paddle with a diameter of 5 cm, the distance between the stirring paddle and the bottom is 3 cm, and the stirring paddle rotates at 50 rpm.

[0028] In one embodiment of the present invention, in step (2), the time for simulating gastric digestion is 2-3 hours.

[0029] In one embodiment of the present invention, in step (3), the mass ratio of pancreatic enzyme to rice is (8-12): (25-35). Specifically, it is preferably (9-12): (25-35); and more preferably (9-10): (25-35).

[0030] In one embodiment of the present invention, in step (3), the activity of pancreatic enzyme is 8×USP.

[0031] In one embodiment of the present invention, in step (3), the amount of amyloglucosidase used relative to rice is (1.5-2.5) mL / (25-35) g.

[0032] In one embodiment of the present invention, in step (3), the enzyme activity of amyloglucosidase is 260 U / mL.

[0033] In one embodiment of the present invention, in step (3), the mass ratio of transferase to rice is (0.02-0.03):(25-35).

[0034] In one embodiment of the present invention, in step (3), the enzyme activity of the transferase is 300 U / mg.

[0035] In one embodiment of the present invention, in step (3), the pH is adjusted to 6.0-7.0.

[0036] In one embodiment of the present invention, the different types of rice include rice made from different rice materials and cooked under different conditions.

[0037] Compared with the existing evaluation methods, the method provided by the present invention has the following obvious advantages and progress:

[0038] (1) The in vitro simulated glycemic index determination method of rice provided by the present invention can fully reflect the effect of oral processing on rice digestion and blood sugar changes during rice intake by simulating oral chewing in the early stage. Compared with the existing in vitro simulation method, it solves the problem of simple pre-treatment and failure to fully consider the effect of oral chewing. It can more truly reflect the process of human body intake and digestion and absorption of rice, and the obtained results are more accurate and have strong reference value.

[0039] (2) Compared with the existing in vitro simulation methods, the in vitro simulation method for determining the glycemic index of rice provided by the present invention has a large test sample volume, strong sample representativeness, high discrimination and accuracy of the obtained results, and good stability. DETAILED DESCRIPTION

[0040] The rice involved in the present invention is: weigh the rice sample, wash it three times, add water according to a certain mass ratio (rice-water ratio), steam and cook for 30 minutes, stir the rice lightly with a rice spoon, and then simmer for 10 minutes.

[0041] Example 1

[0042] Step 1: Weigh 30 g of cooked rice and place it in the multi-extrusion device of the physical property analyzer. The control device maintains a constant temperature of 37° C., and add 3.6 mL of pH 6.8 phosphate buffer solution containing 0.01 g of low-temperature amylase (enzyme activity of 2000 U / g);

[0043] Step 2: Run the multi-extrusion device for 25 reciprocating cycles at a speed of 5 mm / s and an extrusion distance of 93 mm to simulate oral chewing and digestion of rice;

[0044] Step 3: The extruded rice was then transferred to simulated gastric juice to simulate gastric digestion for 2 hours; the simulated gastric juice contained 2 g of sodium chloride, 2 g of pepsin (enzyme activity of 200 U / mg), and 900 mL of hydrochloric acid with a pH of 1.2; the simulated gastrointestinal digestion device included a 2 L reaction vessel with a diameter of 15 cm, equipped with a 5 cm diameter stirring paddle, the stirring paddle was 3 cm away from the bottom, and the stirring paddle speed was 50 rpm.

[0045] Step 4: sodium phosphate was added to adjust the pH value of the system to 6.8, 9 g pancreatic enzyme (enzyme activity was 8×USP), 2 mL amyloglucosidase (enzyme activity was 260 U / mL), and 25 mg transferase (enzyme activity was 300 U / mg) were added, and after rapid and uniform dispersion, small intestinal digestion was simulated, and 2 mL of samples were taken at 0 min, 30 min, 60 min, 90 min, 120 min, 150 min, and 180 min, respectively. The mixture was centrifuged at 10,000 rpm, and the supernatant was taken. The glucose content in the supernatant was determined by a glucose (GOPOD method) detection kit, and the starch hydrolysis rate in the rice was calculated (S%=(glucose production×0.9 / total starch amount)×100%); 30 g white bread was used as a control to determine the hydrolysis rate of white bread during simulated gastrointestinal digestion.

[0046] Step 5: According to the area under the starch hydrolysis rate-time curve (AUC 米饭-体外 ) compared with the area under the hydrolysis rate-time curve (AUC 白面包-体外 ) ratio, calculate the starch hydrolysis index (HI) in rice 米饭-体外 / AUC 白面包-体外 )×100%);

[0047] Step 6: Calculate the glycemic index of rice according to the starch hydrolysis index (HI)-glycemic index (GI) mathematical model (GI=37.17+0.575HI).

[0048] Among them, the establishment process of the HI-GI mathematical model is as follows:

[0049] First, 10-20 healthy adult subjects were selected, fasted for 12 hours, and consumed 50g of different types of rice. Blood was drawn every 30 minutes to measure blood glucose concentration, and 50g of white bread was used as a control. The area under the blood glucose concentration-time curve (AUC) within 0-120 minutes after rice and white bread were analyzed. 米饭-体内 , AUC 白面包-体内 , calculate the glycemic index of rice under in vivo test conditions (GI = AUC 米-体内饭 / AUC 白面包-体内Then, by analyzing the correlation between the starch hydrolysis index (HI) of different types of rice measured by steps 1-5 and the glycemic index (GI) under in vivo test conditions, a fitting equation (GI=37.17+0.575HI) was obtained.

[0050] Specifically, the hydrolysis index (HI) of rice starch under different rice-water ratios of 50 g and the glycemic index (GI) of the in vivo test were used to construct a HI-GI mathematical model. The corresponding HI and GI results are shown in Table 1.

[0051] Table 1 Rice starch hydrolysis index (HI) and in vivo glycemic index (GI) under different rice-water ratios

[0052] Rice-water ratio (mass ratio) Starch hydrolysis index (HI) in rice In vivo test of rice glycemic index (GI) 1：1 73.14 78.32 1：1.2 74.34 80.82 1：1.5 75.54 81.12 1：2 79.56 81.98 1：3 81.32 83.28 1：5 81.65 85.16

[0053] Through fitting, the HI-GI mathematical model was obtained: GI = 37.17 + 0.575HI (r = 0.917).

[0054] Example 2 In vitro simulation of glycemic index of rice

[0055] The other processes refer to Example 1, and the corresponding dosage conditions of low-temperature amylase and pancreatin, as well as the number of reciprocating extrusion cycles are shown in Table 2.

[0056] Step 1: Weigh 30 g of rice and place it in the multi-extrusion device of the physical property analyzer. The control device maintains a constant temperature of 37° C., and add 3.6 mL of pH=6.8 phosphate buffer solution containing a certain amount of low-temperature amylase;

[0057] Step 2: Run the multi-extrusion device to reciprocate and extrude for a certain number of cycles, with an operating speed of 5 mm / s and an extrusion distance of 93 mm to simulate the oral chewing and digestion of rice;

[0058] Step 3: The extruded rice was then transferred to simulated gastric juice to simulate gastric digestion for 2 hours; the simulated gastric juice contained 2 g of sodium chloride, 2 g of pepsin, and 900 mL of hydrochloric acid with a pH of 1.2; the simulated gastrointestinal digestion device included a 2 L reaction vessel with a diameter of 15 cm and a 5 cm diameter stirring paddle. The stirring paddle was 3 cm away from the bottom and the stirring paddle speed was 50 rpm.

[0059] Step 4: Add sodium phosphate to adjust the pH of the system to 6.8, add a certain amount of pancreatic enzyme, 2mL amyloglucosidase, and 25mg transferase, quickly disperse them evenly, simulate small intestinal digestion, take 2mL of samples at 0min, 30min, 60min, 90min, 120min, 150min, and 180min, centrifuge at 10000rpm, take the supernatant, and use a glucose (GOPOD method) detection kit to determine the glucose content in the supernatant, and calculate the starch hydrolysis rate in the rice (S% = (glucose production × 0.9 / total starch) × 100%); use 30g white bread as a control to determine the hydrolysis rate of white bread in the simulated gastrointestinal digestion process.

[0060] Step 5: According to the area under the starch hydrolysis rate-time curve (AUC 米饭 ) compared with the area under the hydrolysis rate-time curve (AUC 白面包 ) ratio, calculate the starch hydrolysis index (HI) in rice 米饭 / AUC 白面包 )×100%);

[0061] Step 6: Calculate the glycemic index of rice according to the starch hydrolysis index (HI)-glycemic index (GI) mathematical model (GI=37.17+0.575HI).

[0062] Table 2 Accuracy evaluation of different in vitro simulation conditions

[0063]

[0064] Example 3 Verification Experiment

[0065] The glycemic index GI of hot rice and cold rice was determined by the method corresponding to condition 2 in Example 2. 体外1 , through the in vivo test data glycemic index GI 体内 And the glycemic index GI measured by the Goni method 体外2 A comparison was made to verify the accuracy and discrimination of the method of the present invention.

[0066] In vivo test data GI 体内 Acquisition: 20 healthy adult subjects were selected, fasted for 12 hours, and consumed 50g of different types of rice. Blood was collected every 30 minutes to measure blood glucose concentration. 50g of white bread was used as a control. The area under the blood glucose concentration-time curve (AUC) within 0-120 minutes after rice and white bread were analyzed. 米饭 , AUC 白面包 , calculate the glycemic index (GI) of rice under in vivo test conditions 体内 =AUC 米饭 / AUC 白面包 ).

[0067] GI measured by Goni method 体外2 Obtaining: Weigh 50 mg of rice, add 5 mL of water, homogenize for 1 min, then add 10 mL of pH 1.5 HCl-KCl buffer solution and mix well. Add 0.2 mL of pepsin solution (1 g of pepsin with an enzyme activity of 200 U / mg is dissolved in 10 mL of HCl-KCl buffer solution), react in a 40°C water bath shaker for 1 h, and then add pH 6.5 maleic acid triester buffer solution to 25 mL. Next, add 5 mL of α-amylase solution (enzyme activity of 2.6 UI, maleic acid triester buffer solution), and sample 1 mL every 30 min until 3 h. The sample was placed in boiling water and heated for 5 min to inactivate the enzyme. Then, 3 mL of 0.4 M pH 4.75 acetate buffer solution was added, and 60 μL of amyloglucosidase (enzyme activity of 260 U / mg) was added to react at 60°C for 45 min to degrade the hydrolyzed starch into glucose. The glucose content was determined using a glucose (GOPOD method) detection kit, and the starch hydrolysis rate in the rice was calculated (S% = (glucose production × 0.9 / total starch amount) × 100%). According to the above method, 50 mg of white bread was used as a control to determine the hydrolysis rate of white bread in the simulated gastrointestinal digestion process. According to the area under the starch hydrolysis rate-time curve in rice (AUC 米饭 ) compared with the area under the hydrolysis rate-time curve (AUC 白面包 ) ratio, calculate the starch hydrolysis index (HI) in rice 体外2 =(AUC 米饭 / AUC 白面包 )×100%); According to the starch hydrolysis index (HI)-glycemic index (GI) mathematical model (GI 体外2 =39.71+0.549HI 体外2 ) to calculate the glycemic index of rice.

[0068] Table 3 Evaluation of the accuracy of different rice glycemic index determination methods

[0069] sample <![CDATA[GI 体外1 ]]> <![CDATA[GI 体外2 ]]> <![CDATA[GI 体内 ]]> Hot rice 79.82 84.62 81.03 Cold rice 71.25 80.34 73.86

[0070] Note: Hot rice refers to rice that has not been cooled and regenerated; cold rice refers to rice that has been cooled to room temperature and regenerated.

Claims

1. A method for in vitro simulation of measuring the glycemic index of rice, characterized in that: The process includes the following: (1) mixing rice with low-temperature amylase and a buffer solution, and then placing the mixture in an extrusion device for cyclic reciprocating extrusion; (2) Transferring the extruded rice into simulated gastric fluid to simulate gastric digestion; (3) After the simulated gastric digestion is completed, the pH is adjusted, pancreatic enzyme, amyloglucosidase and transferase are added, and after mixing, the small intestinal digestion is simulated; sampling is performed at different times, centrifuged, and the supernatant is collected; the glucose content in the supernatant is determined using a glucose detection kit, and the starch hydrolysis rate in the rice is calculated according to the starch hydrolysis rate = (glucose production × 0.9 / total starch amount) × 100%, and the starch hydrolysis rate-time curve is obtained; the area under the line AUC is obtained based on the starch hydrolysis rate-time curve 米饭-体外 ; (4) Using white bread of the same mass as rice as a control, the starch hydrolysis rate in the white bread was determined according to the process of steps (1) to (3), and a starch hydrolysis rate-time curve was obtained; the area under the line AUC was obtained based on the starch hydrolysis rate-time curve. 白面包-体外 ; Using AUC 米饭-体外 , AUC 白面包-体外 The starch hydrolysis index HI in rice is calculated by the ratio of (5) Calculate the glycemic index of rice based on the starch hydrolysis index-glycemic index HI-GI detection model; The construction process of the HI-GI detection model is as follows: Healthy adult subjects were selected to fast for a period of time and consume the same amount of different types of rice. Blood was collected every 20-40 minutes to measure blood glucose concentration. White bread of the same mass as rice was used as a control to obtain the area under the blood glucose concentration-time curve (AUC) within 0-120 minutes after rice and white bread were consumed. 米饭-体内 , AUC 白面包-体内 , calculate the glycemic index of rice under in vivo test conditions GI = AUC 米饭-体内 / AUC 白面包-体内 ; At the same time, the starch hydrolysis index HI of different types of rice is measured through the above steps (1)-(4), and the HI is linearly correlated with the glycemic index GI under in vivo test conditions to obtain a HI-GI detection model; In step (1), the mass ratio of rice to low-temperature amylase is (25-35): (0.005-0.02); every 25-35 g of rice is mixed with 3-5 mL of buffer solution; In step (2), the simulated gastric fluid includes sodium chloride, pepsin, and hydrochloric acid with a pH of 1.2; The dosage conditions are 2-3g sodium chloride: 1-2g pepsin: 900mL hydrochloric acid with pH=1.2; In step (3), the mass ratio of pancreatic enzyme to rice is (8-12): (25-35); every 1.5-2.5 mL of amyloglucosidase is mixed with 25-35 g of rice; and the mass ratio of transferase to rice is (0.02-0.03): (25-35).

2. The method according to claim 1, characterized in that In step (1), the enzyme activity of the low-temperature amylase is 2000 U / g.

3. The method according to claim 1, characterized in that In step (1), the number of reciprocating extrusion cycles is 20-30 times.

4. The method according to claim 1, characterized in that In step (1), the extrusion operation speed is 5-10 mm / s and the extrusion distance is 85-95 mm.

5. The method according to claim 1, characterized in that In step (3), the enzymatic activity of pancreatic enzyme is 8×USP; the enzymatic activity of amyloglucosidase is 260 U / mL; and the enzymatic activity of transferase is 300 U / mg.

6. The method according to any one of claims 1 to 5, characterized in that: The different types of rice include rice made from different rice materials or rice cooked under different conditions.

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