CN117665223A - Efficient in-vitro detection method for simulating intestinal glucose release - Google Patents
Efficient in-vitro detection method for simulating intestinal glucose release Download PDFInfo
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 53
- 239000008103 glucose Substances 0.000 title claims abstract description 53
- 238000001514 detection method Methods 0.000 title claims abstract description 25
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 20
- 238000000338 in vitro Methods 0.000 title claims abstract description 11
- 235000013305 food Nutrition 0.000 claims abstract description 38
- 239000012530 fluid Substances 0.000 claims abstract description 26
- 108010019160 Pancreatin Proteins 0.000 claims abstract description 22
- 229940055695 pancreatin Drugs 0.000 claims abstract description 22
- 239000004382 Amylase Substances 0.000 claims abstract description 21
- 102000013142 Amylases Human genes 0.000 claims abstract description 21
- 108010065511 Amylases Proteins 0.000 claims abstract description 21
- 235000019418 amylase Nutrition 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims abstract description 17
- 230000001079 digestive effect Effects 0.000 claims abstract description 15
- 238000002156 mixing Methods 0.000 claims abstract description 9
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- 238000003756 stirring Methods 0.000 claims abstract description 6
- 238000005070 sampling Methods 0.000 claims abstract description 5
- 210000002249 digestive system Anatomy 0.000 claims abstract description 4
- 235000013312 flour Nutrition 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 11
- 235000007164 Oryza sativa Nutrition 0.000 claims description 10
- 235000009566 rice Nutrition 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 235000012054 meals Nutrition 0.000 claims description 5
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 4
- 229910000041 hydrogen chloride Inorganic materials 0.000 claims description 4
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 claims description 4
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 210000004051 gastric juice Anatomy 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 2
- 239000001099 ammonium carbonate Substances 0.000 claims description 2
- 235000012501 ammonium carbonate Nutrition 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 230000029087 digestion Effects 0.000 abstract description 28
- 210000000214 mouth Anatomy 0.000 abstract description 8
- 102000057297 Pepsin A Human genes 0.000 abstract description 7
- 108090000284 Pepsin A Proteins 0.000 abstract description 7
- 229940111202 pepsin Drugs 0.000 abstract description 7
- 102000038379 digestive enzymes Human genes 0.000 abstract description 6
- 108091007734 digestive enzymes Proteins 0.000 abstract description 6
- 230000001580 bacterial effect Effects 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 5
- 210000000936 intestine Anatomy 0.000 abstract description 3
- 239000012898 sample dilution Substances 0.000 abstract description 2
- 239000003792 electrolyte Substances 0.000 description 17
- 239000011550 stock solution Substances 0.000 description 17
- 239000008280 blood Substances 0.000 description 10
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- 102000004190 Enzymes Human genes 0.000 description 7
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 150000001720 carbohydrates Chemical class 0.000 description 6
- 235000014633 carbohydrates Nutrition 0.000 description 6
- 230000000291 postprandial effect Effects 0.000 description 6
- 210000002784 stomach Anatomy 0.000 description 6
- 239000012895 dilution Substances 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 150000004676 glycans Chemical class 0.000 description 5
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 102000004139 alpha-Amylases Human genes 0.000 description 4
- 108090000637 alpha-Amylases Proteins 0.000 description 4
- 229940024171 alpha-amylase Drugs 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229920002472 Starch Polymers 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 230000008569 process Effects 0.000 description 3
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- 229940099352 cholate Drugs 0.000 description 2
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000002641 glycemic effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 238000009928 pasteurization Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 238000004088 simulation Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 229920002148 Gellan gum Polymers 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- XOYLJNJLGBYDTH-UHFFFAOYSA-M chlorogallium Chemical compound [Ga]Cl XOYLJNJLGBYDTH-UHFFFAOYSA-M 0.000 description 1
- 210000004913 chyme Anatomy 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000019621 digestibility Nutrition 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000000216 gellan gum Substances 0.000 description 1
- 235000010492 gellan gum Nutrition 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 235000011837 pasties Nutrition 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
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- 230000004044 response Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of food detection, and particularly relates to a high-efficiency detection method for simulating in-vitro release of glucose in intestines. The detection method comprises the following steps: adding the food fluid into the digestive juice under stirring in 45-55deg.C water bath, adding pancreatin and amylase, mixing to obtain digestive system, timing, and sampling at different time nodes to detect glucose content. According to the detection method disclosed by the invention, simulated digestion in the oral cavity, gastric phase and intestinal phase stages is combined, pepsin is not used, the dosage of pancreatin and amylase is reduced, and the temperature of simulated digestion is raised to 45-55 ℃, so that errors caused by bacterial spoilage, sugar auxiliary materials in digestive enzymes and sample dilution are prevented.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a high-efficiency detection method for simulating in-vitro release of glucose in intestines.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Carbohydrates, particularly carbohydrates (carbohydrates) of raw sugar, represented by starch, after digestion by the human digestive system, eventually enter the blood in the form of glucose, not only the most important energy source for the human body, but also the most important cause of postprandial blood glucose elevation in the human body. Over the past several decades, with increased productivity and increased average income, consumption of carbohydrates in the diet continues to increase, resulting in excessive postprandial blood glucose rise, which is prone to obesity and other conditions.
Glycemic Index (GI) is the most important index for evaluating postprandial Glycemic response of carbohydrate foods, a specific value which can be calculated by standard unified in-vivo blood glucose test, and currently, the method for testing the GI value of foods in various countries is based on the international standard organization ISO-outlet human body specification test ISO 26642:2010. However, human blood glucose testing is ethically limited and the results are susceptible to personal variability.
The food GI value and the digestibility change level of the food in the human body can be effectively and accurately predicted by using in-vitro simulated digestion, and the nutritional ingredients such as the food GI value and the like can be effectively predicted by using simulated digestive juice and digestive enzymes to simulate the digestion processes of different organs. However, for the in vitro simulated digestion test of glucose for predicting the GI value of food, the digestive enzyme used interferes with the test result due to the inclusion of carbohydrate excipients, and the simulated digestion is a complete set of digestion process of the simulated human body, which requires digestion of oral cavity, gastric phase and intestinal phase at a simulated body temperature of 37 ℃, the simulated digestion time is long, and the food is prone to bacterial spoilage during the simulated digestion process. In addition, the food needs to be mixed with different simulated digestive juice at each digestion stage, and data errors are caused after multiple dilutions.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide an efficient in-vitro detection method for simulating the release of glucose in intestines. According to the detection method disclosed by the invention, simulated digestion in the oral cavity, gastric phase and intestinal phase stages is combined, pepsin is not used, the dosage of pancreatin and amylase is reduced, and the temperature of simulated digestion is raised to 45-55 ℃, so that errors caused by bacterial spoilage, sugar auxiliary materials in digestive enzymes and sample dilution are prevented.
The food has short stay time in the oral cavity, and the enzymolysis of the salivary amylase in the oral cavity on the polysaccharide is very limited, so that the simulation experiment omits the oral phase; food stays in the stomach for about 30 minutes, is mainly acted by hydrochloric acid and pepsin in the stomach, and salivary amylase from the oral cavity has extremely low enzyme activity under the action of low pH in the stomach, so that the digestion of polysaccharide is not obvious, and the early-stage simulated gastric juice experiment also proves that the glucose release effect of the stomach in the stomach on main foods such as starch is negligible; the pH of intestinal juice in the intestinal stage is recovered to be in a near neutral environment of partial alkali, and a large amount of pancreatin can decompose polysaccharide to release glucose, and the glucose is mainly released in the intestinal stage and absorbed into blood by a human body, so that the glucose is a main link for causing postprandial blood glucose increase of the human body. The postprandial blood sugar release is delayed to reduce blood sugar fluctuation, which is beneficial to reducing the sugar load of postprandial hyperglycemia on organisms, and has a protective effect on diabetics in particular. The purpose of the scheme is to provide a high-efficiency and rapid postprandial blood glucose release evaluation method for developing functional staple food preparation with slow-release glucose. Multiple experiments prove that links in simulated oral cavity and stomach can be omitted, and simultaneously, the glucose release of simulated digestive main polysaccharide is matched with the measuring range of a biological sensor in a 1-hour monitoring time window by adjusting the pH value, the temperature, the proportion of feed liquid and the consumption of digestive enzyme of simulated intestinal fluid, so that the purposes of immediately sampling and immediately detecting, avoiding the defects of repeated dilution and increasing the workload, shortening the detection time and improving the efficiency are achieved, and the purpose of obtaining the glucose release speed and the glucose release degree by one-time experiment is realized.
In order to achieve the above object, the present invention is realized by the following technical scheme:
in a first aspect, the present invention provides a method for efficient in vitro simulation of intestinal glucose release comprising the steps of:
adding the food fluid into the digestive juice under stirring in 45-55deg.C water bath, adding pancreatin and amylase, mixing to obtain digestive system, timing, and sampling at different time nodes to detect glucose content.
The digestive enzyme contains polysaccharide auxiliary materials such as starch, and the like, and experiments are carried out according to the enzyme activity amount in the prior art, and the auxiliary materials in the enzyme preparation also release glucose, so that the experimental result is greatly interfered. A blank control experiment shows that the reagent-grade pepsin and pancreatin have large interference on the detection of the glucose content, and the amylase interference is relatively small. Considering that the experiment mainly examines the release condition of glucose, and the protein content of the examined sample is small, pepsin is removed, and the dosage of pancreatin and amylase is reduced.
Pancreatin and amylase were used in simulated intestinal fluid. The activity of amylopsin in pancreatin is inferior to that of amylase, and additional amylase is required. Pancreatin and amylase have slightly different optimum pH, with pancreatin being about pH7.5-8.5 and amylase being about pH6.0-7.5, so pH7.5 is selected to suit both enzymes.
For a near neutral simulated intestinal juice environment, food is stirred for 1-2 hours at 37 ℃ and is easy to generate bacteria breeding spoilage with different degrees, and repeatable experimental data is difficult to obtain. Since the optimum temperature of pancreatin and amylase is 50 ℃, incubation at this temperature is equivalent to pasteurization, slowing down bacterial growth.
The numerical error of the glucose content detected by the enzyme membrane of the biosensor is larger after multiple dilutions. Therefore, the substrate dosage is adjusted, so that the sampled product is directly loaded to be detected without dilution, and the detection efficiency can be improved.
Preferably, the weight proportion of the food fluid is the main meal: aqueous liquid = 1:10-18, wherein the main food powder comprises rice flour or oat flour.
Preferably, the digestive juice consists of simulated gastric juice, simulated intestinal juice and calcium chloride solution according to the volume ratio of 1:1.9-2.1:0.0045-0.0060, and the pH value is 7.5.
Preferably, the concentration of the calcium chloride solution is 0.29-0.31M.
Preferably, the volume ratio of the food fluid to the digestive juice is 0.8-1.2:2.0-2.5.
Preferably, the simulated gastric fluid has a potassium chloride concentration of 8.6-8.7mmol/L, a potassium dihydrogen phosphate concentration of 1.1-1.2 mmol/L, a sodium bicarbonate concentration of 31-32mmol/L, a sodium chloride concentration of 58-60mmol/L, a magnesium chloride hexahydrate concentration of 0.14-0.16mmol/L, an ammonium carbonate concentration of 0.6-0.7mmol/L and a hydrogen chloride concentration of 19-20mmol/L.
Preferably, the simulated intestinal juice has a potassium chloride concentration of 8.4-8.6mmol/L, a potassium dihydrogen phosphate concentration of 0.9-1.1mmol/L, a sodium bicarbonate concentration of 106-107mmol/L, a sodium chloride concentration of 47-49mmol/L, a magnesium chloride hexahydrate concentration of 0.41-0.42mmol/L and a hydrogen chloride concentration of 10-11mmol/L.
Preferably, the pancreatin is added in an amount of 45-55U per gram of main meal.
Preferably, the amylase is added in an amount of 21000-23000U per gram of main meal.
Preferably, the glucose content is detected using a biosensing analyzer.
The beneficial effects obtained by one or more of the technical schemes of the invention are as follows:
the invention combines simulated digestion of oral cavity, gastric phase and intestinal phase into one-step simulated digestion, and only uses pancreatin and amylase, thereby avoiding data errors caused by sugar auxiliary materials in enzyme preparations and accelerating detection time.
The invention increases the temperature of simulated digestion to the optimum temperature of pancreatin and amylase, and the heat preservation at the temperature is equivalent to pasteurization, thereby slowing down bacterial breeding and improving the repeatability of data.
The invention adjusts the substrate dosage, so that the sampled product is directly loaded into the sample for detection without dilution, and the detection efficiency is improved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a graph of test data for simulating digestion of glucose release at various times in accordance with example 1 of the present invention;
FIG. 2 is a graph showing the data of comparative example 1 of gastric phase digestion of glucose release at various times in accordance with the present invention;
FIG. 3 is a graph showing the measurement data of glucose release at different times of intestinal phase digestion according to comparative example 1 of the present invention.
Detailed Description
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail below with reference to specific examples and comparative examples.
The simulated digestive juice used in the examples and comparative examples of the present invention was prepared by reference to "Astandardised static in vitro digestion method suitable for Food-an international consensus" (Food function., 2014,5,1113-1124). Simulated digestive fluids were formulated, including Simulated Saliva (SSF), simulated Gastric Fluid (SGF), and Simulated Intestinal Fluid (SIF). The simulated digestion solution contains various electrolytes, enzymes and the like, and for the convenience of experiments, 1.25 times of electrolyte stock solution is prepared according to the table 1, and the electrolyte stock solution is stored at the temperature of minus 20 ℃. During the reaction, enzyme and Ca are involved 2+ The addition of the solution and water eventually became a simulated digestive fluid with an electrolyte concentration of 1 fold. Ca cannot be added into the electrolyte stock solution 2+ Solutions, which may precipitate, are added to the final mixture of simulated digestive juice and food.
TABLE 1 preparation of digestive juice electrolyte stock solution
The rice flour used in the examples and comparative examples of the present invention was purchased commercially, and the slow-release rice flour was prepared by mixing rice flour with gellan gum and then adding GaCl 2 The preparation method comprises freeze drying and pulverizing the gel.
Example 1
0.8g of rice flour or oat flour was mixed in proportion with 11.7ml of water, water was used at 85℃to make the mixture completely pasty, and used as a food fluid for detection. The digestion solution was prepared, 8.0mL of SGF electrolyte stock solution, 15.2mL of SIF electrolyte stock solution and 0.036mL of 0.29M calcium chloride solution were added, and the mixture was adjusted to pH7.5, followed by stirring and mixing. Adding food fluid into a water bath kettle with the temperature of 50 ℃ and continuously stirring by magnetic force. Finally, 36U of pancreatin (BR, 1:4000, cas:8049-47-6, microphone, lot#: C14096797) and 16800U of alpha-amylase (BR, cas:9000-90-2, microphone, lot#: C15042243, 44U/mg) were added (1% of the system), mixed well, timing was started, and 20. Mu.L of supernatant was aspirated with a microinjector at different time nodes. The glucose content was measured directly with a biosensing analyzer (SBA-40D biosensing analyzer, academy of sciences of Shandong, biolabs of Biotechnology, calibration with glucose standard solution of 100 mg/dl). The results of the detection are shown in Table 2 and FIG. 1.
Table 2 results of the glucose level test of example 1
Comparative example 1
Glucose assays were performed as described in A standardised static in vitro digestion method suitable for Food-an international consensus (Food function., 2014,5,1113-1124).
5ml of the food fluid was mixed with 3.5ml of SSF stock solution, 0.5ml of salivary amylase solution (salivary amylase was formulated with SSF stock solution, 1500U/ml) was added, followed by 25ul and 975ul of water of 0.3M calcium chloride solution, and thoroughly mixed. The pH was adjusted to 7 and the reaction was carried out at 37℃for 2min. The samples were frozen with liquid nitrogen and the glucose content was measured using a biosensing analyzer.
10ml of the saliva digested sample was mixed with 7.5ml of SGF electrolyte stock solution, 1.6ml of pepsin solution (containing 25000U/ml pepsin) was added, then 5ul and 695ul of water of 0.3M calcium chloride solution were added, 0.2ml of 1MHCl was added to adjust pH to 3.0, reaction was carried out for 2 hours at 37℃and the sample was frozen with liquid nitrogen, and glucose content was measured by a biosensing analyzer.
20mL of chyme was mixed with 11mL of SIF electrolyte stock solution, 5.0mL of trypsin solution (pancreatin was formulated with SIF electrolyte stock solution, 800U/mL), 2.5mL of cholate (160 mM cholate), then 40ul of 0.3M calcium chloride solution and 1.31mL of water were added, 0.15mL of 1M NaOH was added, pH was adjusted to 7.0, and the mixture was reacted at 37℃for 2 hours. The samples were frozen with liquid nitrogen and the glucose content was measured using a biosensing analyzer.
Sampling at each time point was performed, frozen and inactivated samples were thawed at room temperature, centrifuged at 12000rpm×5min, and the supernatant was diluted 100-fold and assayed for glucose content using a biosensing analyzer.
The results of the detection are shown in Table 3, FIG. 2 and FIG. 3.
TABLE 3 results of the glucose level test of comparative example 1
The comparison example and the comparison example 1 show that the detection method of the example 1 can rapidly and accurately detect the glucose content in the food fluid, while the glucose content of the comparison example 1 has obvious deviation and takes a long time when detecting the slow-release rice flour, which is unfavorable for the rapid detection of the glucose.
Example 2
0.8g of rice flour or oat flour was mixed in proportion with 14.4ml of water, water was used at 82℃to make the whole paste as a food fluid for detection. The digestion solution was prepared, 12.2mL of SGF electrolyte stock solution, 25.7mL of SIF electrolyte stock solution and 0.080mL of 0.31M calcium chloride solution were added, and the mixture was adjusted to pH7.5 and stirred and mixed well. The food fluid was added to the kettle and magnetically stirred continuously at 45 ℃. Finally, 44U of pancreatin (BR, 1:4000, cas:8049-47-6, microphone, lot#: C14096797) and 18400U of alpha-amylase (BR, cas:9000-90-2, microphone, lot#: C15042243, 44U/mg) were added, mixing was carried out, timing was started, and 20 mu L of clear liquid was sucked by a microinjector at different time nodes. The glucose content was measured directly with a biosensing analyzer (SBA-40D biosensing analyzer, academy of sciences of Shandong, biolabs of Biotechnology, calibration with glucose standard solution of 100 mg/dl).
Example 3
0.8g of rice flour or oat flour was mixed in proportion with 10.4ml of water, water bath at 88℃to make a paste completely, and used as a food fluid for detection. The digestion solution was prepared, 9.5mL of SGF electrolyte stock solution, 18.5mL of SIF electrolyte stock solution and 0.049mL of 0.31M calcium chloride solution were added, and the mixture was adjusted to pH7.5, followed by stirring and mixing. The food fluid was added to the kettle and magnetically stirred continuously at 55 ℃. Finally, 38U of pancreatin (BR, 1:4000, cas:8049-47-6, microphone, lot#: C14096797) and 17600U of alpha-amylase (BR, cas:9000-90-2, microphone, lot#: C15042243, 44U/mg) were added, mixing was carried out, timing was started, and 20 mu L of clear liquid was sucked by a microinjector at different time nodes. The glucose content was measured directly with a biosensing analyzer (SBA-40D biosensing analyzer, academy of sciences of Shandong, biolabs of Biotechnology, calibration with glucose standard solution of 100 mg/dl).
Example 4
0.8g of rice flour or oat flour was mixed in proportion with 12.8ml of water, water was used at 85℃in a water bath to make the whole paste as a food fluid for detection. The digestion solution was prepared, 9.7mL of SGF electrolyte stock solution, 19.9mL of SIF electrolyte stock solution and 0.051mL of 0.30M calcium chloride solution were added, and the mixture was stirred and mixed well. The food fluid was added to a 53 ℃ water bath with continuous magnetic stirring. Finally, pancreatin (BR, 1:4000, cas:8049-47-6, microphone, lot#: C14096797) 42U, alpha-amylase (BR, cas:9000-90-2, microphone, lot#: C15042243, 44U/mg) 18000U, mixing well, starting timing, and sucking 20 μl of clear liquid with a microinjector at different time nodes. The glucose content was measured directly with a biosensing analyzer (SBA-40D biosensing analyzer, academy of sciences of Shandong, biolabs of Biotechnology, calibration with glucose standard solution of 100 mg/dl).
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. An efficient in vitro simulated intestinal glucose release detection method is characterized by comprising the following steps:
adding the food fluid into the digestive juice under stirring in 45-55deg.C water bath, adding pancreatin and amylase, mixing to obtain digestive system, timing, and sampling at different time nodes to detect glucose content.
2. The method of claim 1, wherein the digestive juice comprises simulated gastric juice, simulated intestinal juice and calcium chloride solution in a volume ratio of 1:1.9-2.1:0.0045-0.0060 and a pH of 7.5.
3. The method of claim 1, wherein the food fluid is a main meal in a weight ratio of: aqueous liquid = 1:10-18, wherein the main food powder comprises rice flour or oat flour.
4. The method according to claim 2, wherein the simulated gastric fluid has a potassium chloride concentration of 8.6 to 8.7mmol/L, a potassium dihydrogen phosphate concentration of 1.1..about.1.2 mmol/L, a sodium hydrogen carbonate concentration of 31 to 32mmol/L, a sodium chloride concentration of 58 to 60mmol/L, a magnesium chloride hexahydrate concentration of 0.14 to 0.16mmol/L, an ammonium carbonate concentration of 0.6 to 0.7mmol/L, and a hydrogen chloride concentration of 19 to 20mmol/L.
5. The method according to claim 2, wherein the simulated intestinal fluid has a potassium chloride concentration of 8.4-8.6mmol/L, a potassium dihydrogen phosphate concentration of 0.9-1.1mmol/L, a sodium bicarbonate concentration of 106-107mmol/L, a sodium chloride concentration of 47-49mmol/L, a magnesium chloride hexahydrate concentration of 0.41-0.42mmol/L, and a hydrogen chloride concentration of 10-11mmol/L.
6. The method of claim 1, wherein the volume ratio of the food fluid to the digestive juice is 0.8-1.2:2.0-2.5.
7. The method of claim 1, wherein the concentration of the calcium chloride solution is 0.29-0.31M.
8. The method of claim 1, wherein the pancreatin is added in an amount of 45-55U per gram of main meal.
9. The method of claim 1, wherein the amylase is added in an amount of 21000U to 23000U per gram of staple food powder.
10. The method of claim 1, wherein the glucose content is detected using a biosensing analyzer.
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