CN115201430A - Method for measuring glycemic index of rice in vitro simulation manner - Google Patents
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Abstract
The invention discloses a method for in-vitro simulation determination of a rice glycemic index, and belongs to the technical field of food measurement. The in-vitro simulation method comprises the steps of simulating oral chewing and gastrointestinal digestion, and constructing a detection model, so that the real glycemic index GI of the rice can be more accurately predicted. The method solves the problems that the in vivo test determination method needs a man-made test object, has high technical difficulty, high cost and long time, and has poor popularization and operability; the defects that the Goni in-vitro simulation determination method neglects the influence of the oral chewing process on rice digestion, cannot accurately reflect the real digestion condition of the rice and has poor discrimination on different types of rice are overcome; the physical property analyzer based multiple extrusion device simulates the real chewing process of human beings, fully considers the influence of the chewing process on the rice glycemic index, further determines the digestion rate of the rice in the simulated gastrointestinal digestion environment, calculates the rice glycemic index, has more real, objective and accurate results, and has high discrimination on different types of rice.
Description
Technical Field
The invention belongs to the technical field of food measurement, and particularly relates to a method for in-vitro simulated determination of a rice glycemic index.
Background
Rice is one of the important staple food grains. In recent years, the incidence of blood sugar-related metabolic diseases such as diabetes and obesity has been rapidly increasing with the improvement in the living standard of residents and the change in dietary structure. The main component of rice is starch, the digestion and absorption of the starch are closely related to the metabolism of blood sugar, and the incidence of diseases such as diabetes, obesity and the like is directly influenced. Therefore, much attention has been paid to the influence of rice on blood glucose metabolism.
The Glycemic Index (GI) is totally called as a blood glucose generation index, can reflect the speed and the capability of increasing blood glucose of food compared with glucose, and can help special people to select proper food and reduce the incidence rate of diseases such as diabetes, adiposis and the like. According to the rising speed of blood sugar, the food can be divided into high-sugar-rising food (GI > 70), medium-sugar-rising food (55 ≦ GI ≦ 70), and low-sugar-rising food (GI < 55). During the digestion process of the rice, the starch can be hydrolyzed into glucose, and the blood sugar concentration is increased. Therefore, the accurate evaluation of the glycemic index of the rice is of great significance for guiding consumers to formulate reasonable rice eating modes according to specific health management requirements.
At present, the glycemic index of a surrogate food is usually determined by taking a healthy adult as a subject, fasting for 12 hours, then taking 50 grams of the food to be measured, taking blood to measure the blood glucose concentration at regular time, calculating the ratio of the area under the blood glucose line of the surrogate food to the area under the blood glucose line of a standard food (white bread) according to a blood glucose-time curve, and multiplying the ratio by 100. The method has high requirements on the testee, is expensive, time-consuming and labor-consuming, and is not favorable for the rapid detection of the glycemic index of food. Therefore, there is a need to develop rapid and accurate in vitro testing methods.
Currently, goni's method is mostly used in an in vitro simulation method for determining the glycemic index of food. Weighing 50mg of sample, firstly reacting in simulated gastric fluid for 1 hour, then adjusting the pH value to 6.9, adding amylase, simulating small intestine digestion for 3 hours, and sampling every 30 minutes to determine the hydrolysis rate of the sample. The sample Hydrolysis Index (HI) is calculated from the sample hydrolysis rate curve and the sample glycemic index is calculated from the relationship between the sample hydrolysis index and the sample glycemic index. The method can be used for measuring the glycemic index of food under in vitro simulation condition. Compared with in vivo tests, the simulation method has strong operability and low cost.
The glycemic index of edible rice is closely related to the chewing degree of the oral cavity of a user, namely the digestion of the rice in the gastrointestinal tract can be obviously influenced by the granular state and the rheological property of the rice after the rice is chewed by the oral cavity. However, the existing Goni method for determining the glycemic index of rice through in-vitro simulation has the defects that the sample pretreatment mode is simple, the difference with the actual rice ingestion digestion process is large, and the influence of the oral chewing process on the rice digestion is ignored. In addition, the Goni method has a small amount of test samples and poor representativeness. Therefore, there is a limit to the Goni method for determining the glycemic index of rice by in vitro simulation.
Disclosure of Invention
In view of the defects of the existing rice glycemic index determination technology, the invention focuses on an innovative method, establishes a determination method which can fully reflect the influence of the oral chewing process on rice digestion and can more accurately predict the true glycemic index of rice under the in-vitro simulation condition, and achieves the aim by simulating oral chewing, gastrointestinal digestion, constructing a mathematical model and the like.
Aiming at the problems that the conventional rice glycemic index determination in vivo test determination method needs a human test object, has high technical difficulty, high cost, long time and poor popularization and operability; the Goni in-vitro simulation determination method has the advantages that the amount of the determined samples is too small, the pretreatment of the samples is simple, the influence of the oral chewing process on the digestion of the rice is ignored, the real digestion condition of the rice cannot be accurately reflected, the distinguishing degree of the glycemic indexes of different types of rice is poor, and the method is closer to the real ingestion process of the rice and can more accurately determine the glycemic indexes of the rice.
The method is based on a multiple extrusion device of a physical property analyzer, simulates the real chewing process of human beings, fully considers the influence of the chewing process on the rice glycemic index, further determines the digestion rate of the rice in the simulated gastrointestinal digestion environment on the basis, and calculates the rice glycemic index, so that the result is closer to the real condition, is more objective and accurate, and has high distinguishing degree of different types of rice.
The invention provides a method for in vitro simulation determination of a rice glycemic index, which comprises the following steps:
(1) Mixing the cooked rice with low-temperature amylase and buffer solution, and then placing the mixture in an extrusion device for circular reciprocating extrusion;
(2) Transferring the extruded rice into simulated gastric juice to simulate gastric digestion;
(3) After simulated digestion of the stomach is finished, adjusting the pH, adding pancreatin, amyloglucosidase and transferase, uniformly mixing, and simulating digestion of the small intestine; sampling at different time, centrifuging and collecting supernatant; measuring the glucose content in the supernatant by using a Glucose (GOPOD) detection kit, calculating the starch hydrolysis rate in the rice according to the starch hydrolysis rate = (the generation amount of glucose is multiplied by 0.9/the total amount of starch) multiplied by 100%, and obtaining a starch hydrolysis rate-time curve; obtaining the area under the line AUC from the starch hydrolysis rate-time curve Rice-in vitro ；
(4) Taking white bread with the same quality as rice as a reference, measuring the starch hydrolysis rate of the white bread according to the processes of the steps (1) to (3), and obtaining a starch hydrolysis rate-time curve; obtaining the area under the line AUC from the starch hydrolysis rate-time curve White bread-in vitro (ii) a Using AUC Rice-in vitro 、AUC White bread-in vitro Calculating the starch hydrolysis index HI, HI = (AUC) in cooked rice Rice-in vitro /AUC White bread-in vitro )×100％；
(5) Calculating the rice glycemic index according to a starch hydrolysis index-glycemic index HI-GI detection model;
the construction process of the HI-GI detection model is as follows:
selecting healthAn adult subject is fasted for a period of time, ingests the same amount of different types of cooked rice, takes blood once every 20-40min, and measures the blood sugar concentration; comparing with white bread of cooked rice, and obtaining the area AUC under blood glucose concentration-time curve within 0-120min after cooked rice and white bread are eaten Rice-body 、AUC White bread-in-vivo Calculating the rice glycemic index (GI = AUC) under the in vivo test conditions Rice-body /AUC White bread-in-vivo ) (ii) a Meanwhile, starch Hydrolysis Indexes (HI) of different types of rice are measured through the steps (1) to (4), and the HI is linearly related to a glycemic index GI under in-vivo test conditions to obtain an HI-GI detection model.
In one embodiment of the present invention, in the step (1), the mass ratio of the cooked rice to the low-temperature amylase is (25-35): (0.005-0.02). Particularly preferably (25-35): 0.01.
in one embodiment of the present invention, in step (1), the enzymatic activity of the low temperature amylase is 2000U/g.
In one embodiment of the present invention, in the step (1), the cooked rice and the buffer solution are used in an amount of (25-35) g: (3-5) mL.
In one embodiment of the present invention, in step (1), the buffer solution may specifically be a phosphate buffer solution with pH = 6.8.
In one embodiment of the present invention, in the step (1), the number of times of the cyclic reciprocal pressing is 20 to 30 times. Preferably 25-30 times.
In one embodiment of the present invention, in the step (1), the temperature environment for extrusion is a constant temperature of 36 ℃.
In one embodiment of the present invention, in the step (1), the extrusion may be performed in a multi-extrusion apparatus of a physical property analyzer.
In one embodiment of the present invention, in the step (1), the extrusion is performed at a speed of 5 to 10mm/s and an extrusion distance of 85 to 95mm.
In one embodiment of the present invention, in step (2), the simulated gastric fluid comprises sodium chloride, pepsin, hydrochloric acid with pH = 1.2; the dosage conditions are 2-3g of sodium chloride: 1-2g pepsin: 900mL of hydrochloric acid having pH = 1.2.
In one embodiment of the present invention, in step (2), the apparatus for simulating gastrointestinal digestion comprises a 2L reaction vessel, 15cm in diameter, equipped with a 5cm diameter paddle, the paddle being 3cm from the bottom and the paddle rotating at 50rpm.
In one embodiment of the present invention, in step (2), the simulated gastric digestion is performed for 2-3 hours.
In one embodiment of the present invention, in the step (3), the mass ratio of pancreatin to cooked rice is (8-12): (25-35). Particularly preferably (9-12): (25-35); further preferably (9-10): (25-35).
In one embodiment of the present invention, in step (3), the enzymatic activity of pancreatin is 8 × USP.
In one embodiment of the present invention, in step (3), the amyloglucosidase is used in an amount of (1.5-2.5) mL/(25-35) g relative to cooked rice.
In one embodiment of the present invention, in the step (3), the enzyme activity of amyloglucosidase is 260U/mL.
In one embodiment of the present invention, in the step (3), the mass ratio of the transferase to the cooked rice is (0.02 to 0.03): (25-35).
In one embodiment of the present invention, in step (3), the enzyme activity of transferase is 300U/mg.
In one embodiment of the present invention, in step (3), the pH is adjusted to 6.0 to 7.0.
In one embodiment of the invention, the different types of rice include different rice materials and rice cooked under cooking conditions.
Compared with the existing evaluation method, the method provided by the invention has the following obvious advantages and progresses:
(1) The method for determining the glycemic index of the rice through in-vitro simulation provided by the invention can fully reflect the influence of oral processing on rice digestion and blood sugar change in the rice ingestion process by simulating oral chewing in the early stage.
(2) Compared with the existing in vitro simulation method, the method for determining the rice glycemic index through in vitro simulation provided by the invention has the advantages of large test sample amount, strong sample representativeness, high obtained result discrimination, high accuracy and good stability.
Detailed Description
The cooked rice according to the present invention is: weighing rice sample, washing for 3 times, adding water (rice water ratio) at a certain mass ratio, and steaming for 30min. Slightly stirring the rice with a spoon, and stewing for 10min.
Example 1
Step 1: weighing 30g of rice, placing the rice in a multiple extrusion device of a physical property analyzer, keeping the temperature of the device constant at 37 ℃, and adding 3.6mL of phosphate buffer solution with the pH value of 6.8 and containing 0.01g of low-temperature amylase (the enzyme activity is 2000U/g);
step 2: operating a multi-extrusion device to perform reciprocating extrusion for 25 cycles at the operating speed of 5mm/s and the extrusion distance of 93mm, and simulating the oral chewing digestion of rice;
and step 3: then transferring the extruded rice into simulated gastric juice to simulate stomach digestion for 2 hours; the simulated gastric fluid contains 2g of sodium chloride, 2g of pepsin (the enzyme activity is 200U/mg) and 900mL of hydrochloric acid with the pH value of 1.2; the simulated gastrointestinal digestion device comprises a 2L reaction vessel with a diameter of 15cm, a stirring paddle with a diameter of 5cm, a distance of 3cm from the bottom of the stirring paddle, and a rotation speed of 50rpm of the stirring paddle.
And 4, step 4: adding sodium phosphate to adjust the pH of the system to 6.8, adding 9g of pancreatin (the enzyme activity is 8 multiplied by USP), 2mL of amyloglucosidase (the enzyme activity is 260U/mL) and 25mg of transferase (the enzyme activity is 300U/mg), quickly and uniformly dispersing, simulating small intestine digestion, sampling at 0min,30min,60min,90min,120min,150min,180min for 2mL, centrifuging at 10000rpm, taking supernate, measuring the glucose content in the supernate by using a Glucose (GOPOD) detection kit, and calculating the starch hydrolysis rate (S% = (the glucose generation amount is multiplied by 0.9/the total amount of starch) multiplied by 100%) in the rice; the hydrolysis rate of white bread during simulated gastrointestinal digestion was determined using 30g of white bread as a control.
And 5: according to the starch hydrolysis rate-time in riceArea under the curve (AUC) Rice-in vitro ) Area under the line of the hydrolysis ratio-time curve (AUC) of white bread versus control White bread-in vitro ) Ratio, calculate starch hydrolysis index in rice (HI = (AUC) Rice-in vitro /AUC White bread-in vitro )×100％)；
Step 6: the rice glycemic index was calculated according to the starch Hydrolysis Index (HI) -Glycemic Index (GI) mathematical model (GI =37.17+ 0.575hi).
The establishment process of the HI-GI mathematical model is as follows:
selecting 10-20 healthy adult subjects, fasting for 12 hr, taking 50g of different types of cooked rice, collecting blood every 30min, measuring blood glucose concentration, comparing 50g of white bread, and analyzing AUC (blood glucose concentration-time curve) under blood glucose concentration-time curve within 0-120min after cooked rice and white bread are eaten Rice-body 、AUC White bread-in-vivo The rice glycemic index (GI = AUC) under the in vivo test conditions was calculated Rice-in-body rice /AUC White bread-in-vivo ). Then, by analyzing the correlation of the starch Hydrolysis Index (HI) determined by steps 1-5 for different types of rice with the Glycemic Index (GI) under in vivo test conditions, a fitting equation (GI =37.17+ 0.575HI) was obtained.
Specifically, a HI-GI mathematical model is constructed by selecting rice starch Hydrolysis Index (HI) and in-vivo test Glycemic Index (GI) under the condition of 50g of rice water with different ratios, and corresponding HI and GI results are shown in Table 1.
TABLE 1 Rice starch Hydrolysis Index (HI) and in vivo Glycemic Index (GI) for different Rice Water ratios
| Ratio of rice to water (mass ratio) | Index of starch hydrolysis in Rice (HI) | In vivo test 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 |
Obtaining a HI-GI mathematical model through fitting: GI =37.17+0.575hi (r = 0.917).
Example 2 in vitro simulation of the glycemic index of rice
Other procedures referring to example 1, the conditions of the amounts of the corresponding low temperature amylase and pancreatin and the number of cycles of the reciprocal extrusion are shown in Table 2.
Step 1: weighing 30g of rice, placing the rice in a multiple extrusion device of a physical property analyzer, controlling the device to keep the constant temperature of 37 ℃, and adding 3.6mL of phosphate buffer solution with a certain amount of low-temperature amylase and pH = 6.8;
step 2: running a multiple extrusion device to perform reciprocating extrusion for a certain number of times of circulation, wherein the running speed is 5mm/s, the extrusion distance is 93mm, and the oral chewing digestion of rice is simulated;
and step 3: transferring the extruded rice into simulated gastric juice to simulate gastric digestion for 2 hours; the simulated gastric fluid contains 2g of sodium chloride, 2g of pepsin and 900mL of hydrochloric acid with pH value of 1.2; the simulated gastrointestinal digestion device comprises a 2L reaction vessel with a diameter of 15cm, a stirring paddle with a diameter of 5cm, a distance of 3cm from the bottom of the stirring paddle, and a rotating speed of 50rpm of the stirring paddle.
And 4, step 4: adding sodium phosphate to adjust the pH of the system to 6.8, adding a certain amount of pancreatin, 2mL of amyloglucosidase and 25mg of transferase, quickly and uniformly dispersing, simulating digestion of small intestine, sampling at 0min,30min,60min,90min,120min,150min,180min for 2mL and 10000rpm, centrifuging, taking supernate, measuring the glucose content in the supernate by using a Glucose (GOPOD) detection kit, and calculating the starch hydrolysis rate (S% = (glucose production amount multiplied by 0.9/total amount of starch) multiplied by 100%) in the rice; the hydrolysis rate of white bread during simulated gastrointestinal digestion was determined using 30g of white bread as a control.
And 5: according to the area under the starch hydrolysis rate-time curve (AUC) in rice Cooked rice ) Area under the line of the hydrolysis ratio-time curve (AUC) of white bread versus control White bread ) Ratio, calculate starch hydrolysis index (HI = (AUC) in rice Cooked rice /AUC White bread )×100％)；
Step 6: the rice glycemic index was calculated according to the starch Hydrolysis Index (HI) -Glycemic Index (GI) mathematical model (GI =37.17+ 0.575HI).
TABLE 2 evaluation of the accuracy of different in vitro simulation conditions
Example 3 validation experiment
The glycemic index GI of the hot and cold cooked rice was determined by the method corresponding to condition 2 in example 2 In vitro 1 Glycemic index, GI, by comparison with in vivo assay data In vivo And glycemic index GI measured by Goni method In vitro 2 And comparing to verify the accuracy and discrimination of the method.
In vivo test data GI In vivo Obtaining: selecting 20 healthy adult subjects, fasting for 12 hr, taking 50g of different types of cooked rice, collecting blood every 30min, measuring blood glucose concentration, comparing 50g of white bread, and analyzing AUC (blood glucose concentration-time curve) under blood glucose concentration-time curve within 0-120min after cooked rice and white bread are eaten Cooked rice 、AUC White bread Calculating the Glycemic Index (GI) of the cooked rice under the in vivo test conditions In vivo ＝AUC Cooked rice /AUC White bread )。
GI measured by Goni method In vitro 2 Obtaining: 50mg of rice was weighed, 5mL of water was added, and homogenization was performed for 1min, then 10mL of HCl-KCl buffer solution with pH of 1.5 was added, and mixing was performed uniformly. 0.2mL of pepsin solution (1 g of pepsin with 200U/mg enzyme activity dissolved in 10mL of HCl-KCl buffer solution) was added, and after 1 hour of reaction in a 40 ℃ water bath shaker, the solution was adjusted to 25mL with pH6.5 of a maleate triester buffer solution. Then, after 5mL of an α -amylase solution (enzyme activity 2.6UI, maleate triester buffer solution) was added, 1mL was sampled every 30min until 3 hours. The sample is taken out and put into boiling water to be heated for 5min for enzyme deactivation. Then, adding 3mL of 0.4M acetate buffer solution with pH4.75, adding 60 μ L of amyloglucosidase (the enzyme activity is 260U/mg), reacting at 60 ℃ for 45min, degrading the hydrolyzed starch into glucose, measuring the glucose content by using a glucose (GOPOD method) detection kit, and calculating the starch hydrolysis rate (S% = (glucose production amount x 0.9/total starch amount) × 100%) in the rice; the hydrolysis rate of white bread during simulated gastrointestinal digestion was determined according to the above method using 50mg white bread as a control. According to the area under the starch hydrolysis rate-time curve (AUC) in rice Cooked rice ) Area under the line of the hydrolysis ratio-time curve (AUC) of white bread versus control White bread ) Ratio, calculating starch Hydrolysis Index (HI) in rice In vitro 2 ＝(AUC Cooked rice /AUC White bread ) X 100%); according to a starch Hydrolysis Index (HI) -Glycemic Index (GI) mathematical model (GI) In vitro 2 ＝39.71+0.549HI In vitro 2 ) And calculating the glycemic index of the rice.
TABLE 3 evaluation of the accuracy of the method for determining the glycemic index of different cooked rice
| Sample (I) | GI In vitro 1 | GI In vitro 2 | GI In vivo |
| Hot rice | 79.82 | 84.62 | 81.03 |
| Cold rice | 71.25 | 80.34 | 73.86 |
Note: the hot rice refers to rice which is not cooled and regenerated; the cold rice is rice cooled to room temperature and retrogradation occurs.
Claims (10)
1. The method for measuring the glycemic index of rice in vitro is characterized by comprising the following steps:
(1) Mixing the cooked rice with low-temperature amylase and buffer solution, and then placing the mixture in an extrusion device for circular reciprocating extrusion;
(2) Transferring the extruded rice into simulated gastric juice to simulate gastric digestion;
(3) After simulated digestion of the stomach, adjusting the pH, adding pancreatin, amyloglucosidase and transferase, mixing uniformly, and simulating digestion of the small intestine;sampling at different time, centrifuging and collecting supernatant; determining the glucose content in the supernatant by using a glucose detection kit, calculating the starch hydrolysis rate in the rice according to the starch hydrolysis rate = (the glucose production amount is multiplied by 0.9/the total amount of starch) multiplied by 100%, and obtaining a starch hydrolysis rate-time curve; obtaining the area under the line AUC from the starch hydrolysis rate-time curve Rice-in vitro ；
(4) Taking white bread with the same quality as rice as a reference, measuring the starch hydrolysis rate of the white bread according to the processes of the steps (1) to (3), and obtaining a starch hydrolysis rate-time curve; obtaining the area under the line AUC from the starch hydrolysis rate-time curve White bread-in vitro (ii) a Using AUC Rice-in vitro 、AUC White bread-in vitro Calculating the starch hydrolysis index HI in the rice;
(5) And calculating the glycemic index of the rice according to a HI-GI (starch hydrolysis index-glycemic index) detection model.
2. The method of claim 1, wherein the HI-GI detection model is constructed as follows:
selecting healthy adult subjects, fasting for a period of time, taking the same amount of different types of cooked rice, taking blood once every 20-40min, and determining blood glucose concentration; comparing with white bread of cooked rice, and obtaining the area AUC under blood glucose concentration-time curve within 0-120min after cooked rice and white bread are eaten Rice-body 、AUC White bread-in-vivo Calculating the rice glycemic index GI = AUC under the in vivo test condition Rice-body /AUC White bread-in-vivo (ii) a Meanwhile, measuring starch hydrolysis indexes HI of different types of rice through the steps (1) to (4), and performing linear correlation on the HI and the glycemic index GI under in-vivo test conditions to obtain an HI-GI detection model.
3. The method according to claim 1, wherein in the step (1), the mass ratio of the cooked rice to the low-temperature amylase is (25-35): (0.005-0.02); the dosage conditions of the cooked rice and the buffer solution are (25-35) g: (3-5) mL.
4. The method according to claim 1, wherein the enzyme activity of the low temperature amylase in step (1) is 2000U/g.
5. The method according to claim 1, wherein the number of the cyclic reciprocal compressions in the step (1) is 20 to 30.
6. The method according to claim 1, wherein in the step (1), the extrusion is performed at a speed of 5 to 10mm/s and an extrusion distance of 85 to 95mm.
7. The method according to claim 1, wherein in step (2), the simulated gastric fluid comprises sodium chloride, pepsin, hydrochloric acid with pH = 1.2; the dosage conditions are 2-3g of sodium chloride: 1-2g pepsin: 900mL of hydrochloric acid having pH = 1.2.
8. The method according to claim 1, wherein in the step (3), the mass ratio of the pancreatin to the rice is (8-12): (25-35); the dosage condition of the amyloglucosidase relative to the rice is (1.5-2.5) mL/(25-35) g; the mass ratio of the transferase to the cooked rice is (0.02-0.03): (25-35).
9. The method of claim 1, wherein in step (3), the enzymatic activity of pancreatin is 8 × USP; the enzyme activity of the amyloglucosidase is 260U/mL; the enzyme activity of the transferase is 300U/mg.
10. The method of any one of claims 1-9, wherein the different types of rice include different rice materials or rice cooked under different cooking conditions.
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| CN116287335B (en) * | 2023-02-21 | 2024-01-30 | 浙江大学 | Method for evaluating intestinal microecological regulation effect of arabinoxylans and application thereof |
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