CN111733210A - Method for rapidly determining rice enzymolysis quantity in high flux manner - Google Patents
Method for rapidly determining rice enzymolysis quantity in high flux manner Download PDFInfo
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Abstract
The invention provides a method for quickly determining the enzymolysis quantity of rice with high flux, which at least comprises the following steps: (1) pretreatment: placing the rice flour and the sodium acetate buffer solution in a 96-hole deep-hole plate, preheating and centrifuging; the rice flour refers to powdery rice; (2) in-vitro digestion reaction: adding a digestion buffer solution into the 96-hole deep-hole plate obtained in the step (1) by using a multi-channel pipette, timing and stirring, sampling at regular intervals, placing a sample into a 96-hole PCR plate, adding alcohol into the 96-hole PCR plate to finish reaction, and obtaining reaction samples at each time point; (3) and (4) measuring the enzymolysis quantity. The invention has the advantages of simple operation and low cost, and can quickly and efficiently simulate the digestion condition in the rice body so as to obtain the digestion characteristic in the rice body.
Description
Technical Field
The invention belongs to the field of medical treatment and health and food health, and relates to a method for rapidly determining the enzymolysis quantity of rice with high flux.
Background
Rice (Oryza sativa L.) is one of the most important food crops in the world and provides a large amount of energy for human activities, but excessive intake of starch by diabetics may cause life risks, and in addition, excessive intake of starch by ordinary people may cause obesity and other complications. The risk of excessive consumption of diabetes patients can be effectively reduced by increasing the content of resistant starch in rice, and meanwhile, the resistant starch serving as dietary fiber can improve the health-care effect of probiotics in intestinal tracts. Resistant Starch (Rs) refers to Starch and Starch degradation products that are not absorbed in the small intestine of healthy individuals.
Increasing the content of slowly digestible starch and resistant starch in rice is important for improving the nutritional quality of rice so as to meet the current demands of people. Breeders have been working on breeding rice varieties with high resistant starch content. Although some progress has been made, there has been no breakthrough progress, one of the reasons for which is phenotypic analysis. The method for measuring the resistant starch has the disadvantages of complicated process, low efficiency, poor controllability and high cost. This has always restricted the development of new varieties of rice with high resistant starch content.
The existing methods for measuring digestion mainly comprise an in-vivo measurement method and an in-vitro measurement method, wherein a human body is used as a test object, the cost is high, certain influence is also exerted on the human body, and the method is inconvenient and unrealistic as a conventional measurement method. At present, researchers at home and abroad mainly pay attention to the research of an in-vitro determination method, and the basic principle of the method is that most of researchers at home and abroad use enzymes to simulate in-vivo environment to hydrolyze starch and determine the enzymolysis quantity of rice. The literature reports mainly include: the Englyst method, the Guraya method and modifications thereof. The former people have made great progress on the rice digestion method, but have some defects, are difficult to realize large-scale dynamic measurement, and have high cost, complicated experiments and the like.
Disclosure of Invention
In order to overcome the defects in the rice digestion characteristic analysis aspect at present, the invention provides a method for quickly determining the rice enzymolysis with high flux, which has the advantages of simple operation, less time consumption, high flux, low cost and the like.
In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:
a method for rapidly determining the enzymolysis quantity of rice in high flux at least comprises the following steps:
(1) pretreatment: placing the rice flour and the sodium acetate buffer solution in a 96-hole deep-hole plate, preheating and centrifuging; the rice flour refers to powdery rice;
(2) in-vitro digestion reaction: adding a digestion buffer solution into the 96-hole deep-hole plate obtained in the step (1) by using a multi-channel pipette, timing and stirring, sampling at regular intervals, placing a sample into a 96-hole PCR plate, adding alcohol into the 96-hole PCR plate to finish reaction, and obtaining reaction samples at each time point;
(3) and (3) measuring the enzymolysis amount of the rice flour: transferring the reaction sample at each time point to a 96-hole enzyme label plate by using a multi-channel pipettor, measuring the glucose concentration of the reaction sample at each time point, and obtaining the rice enzymolysis amount in each sampling according to the formula (I);
the enzymatic yield is 0.9/sample mass 100% (I).
The invention also provides application of the method for rapidly determining the enzymolysis quantity of the rice at high flux in simulating the digestion condition in the rice body.
Compared with the prior art, the invention has the following beneficial effects:
the invention has the advantages of simple operation and low cost, and can quickly and efficiently simulate the digestion condition in the rice body so as to obtain the digestion characteristic in the rice body. The digestion method in the prior art is operated according to a kit method of megazyme company, and the method of the invention is different from the prior art in digestion modes, reaction systems, reaction containers, reaction terminating methods and the like. The invention provides effective technical support for rapidly analyzing the rice digestion characteristics, and can obviously improve the efficiency of screening the rice beneficial to human health.
Drawings
FIG. 1 is a graph showing the analysis of the digestion characteristics of 12 rice varieties (A is raw rice flour, B is gelatinized rice flour, and C is retrogradation rice flour).
FIG. 2 shows the physicochemical properties of rice (a is the apparent amylose content, b is the water content, c is the protein content, d is the gel consistency) for 12 rice varieties.
FIG. 3 is an RVA profile of rice flour of 12 rice varieties.
FIG. 4 is DSC spectra of rice flour of 12 rice varieties.
Detailed Description
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ conventional techniques in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, gene mutation techniques, and related fields.
The method for rapidly determining the enzymolysis quantity of the rice with high flux in one embodiment of the invention at least comprises the following steps:
(1) pretreatment: placing the rice flour and the sodium acetate buffer solution in a 96-hole deep-hole plate, preheating and centrifuging; the rice flour refers to powdery rice;
(2) in-vitro digestion reaction: adding a digestion buffer solution into the 96-hole deep-hole plate obtained in the step (1) by using a multi-channel pipette, timing and stirring, sampling at regular intervals, placing a sample into a 96-hole PCR plate, adding alcohol into the 96-hole PCR plate to finish reaction, and obtaining reaction samples at each time point;
(3) and (3) measuring the enzymolysis amount of the rice flour: transferring the reaction sample at each time point to a 96-hole enzyme label plate by using a multi-channel pipettor, measuring the glucose concentration of the reaction sample at each time point, and obtaining the rice enzymolysis amount in each sampling according to the formula (I);
the enzymatic yield is 0.9/sample mass 100% (I).
The 96 deep-hole plate is matched with the multi-channel pipettor, so that the sampling and the measurement can be conveniently and quickly carried out, and the purpose of high flux is achieved.
In one embodiment, a deep well plate with a format of 2.2ml by 96 wells may be used, for example, 12.5CM long by 8.5CM wide by 4.3CM high.
Optionally, the multichannel pipettor is a 12-channel pipettor.
In the step (1), during preheating, a magnetic stirrer is used for stirring the rice flour and the sodium acetate buffer solution. The current universal simulation external digestion uses the horizontal shaking table of controlling the temperature to realize, and the use of magnetic stirrers cooperation deep hole board can improve experimental efficiency greatly.
Optionally, in the step (1), the rice flour is selected from raw rice flour, gelatinized rice flour or retrograded rice flour.
In step (1), it is necessary to ensure that the solid particles are completely centrifuged and precipitated after centrifugation to ensure that the reaction starts when the digestion buffer is added, and the solid particles are slowly discharged when the buffer is added to prevent the bottom precipitate from being blown up.
The preparation method of the rice flour under different states comprises the following steps:
the preparation method of the raw rice flour comprises the following steps: grinding the polished rice into powder, sieving with a sieve with aperture of 0.15mm (100 meshes), and oven drying for balancing.
The preparation method of the gelatinized rice flour comprises the following steps: the raw rice flour is placed in a 96-hole deep-hole plate, then is placed in a water bath kettle with a magnetic stirring function, and is sealed by using a latex sealing cover of the 96-hole deep-hole plate. Heating and cooking while continuously stirring. After completion, the 96-well deep-well plate was taken out and cooled to 37 ℃.
The preparation method of the retrogradation rice flour comprises the following steps: putting the gelatinized rice flour into a refrigerator at 4 ℃ for 7 days.
In one embodiment, in step (2), the stirring manner is magnetic stirring.
In the step (1) or the step (2), the rotating speed of the magnetic stirrer is 600 rpm. The stirring effect is ensured.
The magnetic rotor of the magnetic stirrer used in the invention needs to be kept rotating all the time during the digestion process to ensure the consistency of the digestion process and the repeatability of the measurement result.
In the step (2), the in vitro digestion reaction is carried out in a water bath environment at 37 ℃. Simulating the in vivo environment.
Preferably, in the step (2), during sampling, the 96-well deep-well plate is taken out from the water bath environment, centrifuged, and after a quantitative sample is sucked, an equal amount of sodium acetate buffer is added into the 96-well deep-well plate, and the 96-well deep-well plate is placed into the water bath environment at 37 ℃ to restart timing.
Further, in the step (2), the reaction should be terminated by rapid centrifugation during centrifugation. The speed of the rapid centrifugation was 2000 rpm.
In the in vitro digestion experiment of rice flour (or starch), two types are mainly included, one type is that only two time points are respectively made: 20 minutes and 120 minutes; the other is to make multiple time points and finally fit the data of each time point into a digestion curve. However, in any type, there are multiple time points measured by the same sample, and since the method for stopping the reaction is to inactivate the enzyme in the reaction system, the data of the next time point cannot be measured by the reaction system after the time point is measured, and only one sample needs to be weighed at each time point, which greatly increases the workload of previous weighing, wastes the sample, chemical reagent and digestive enzyme, and increases the experiment cost. If the experiment is carried out at multiple time points and multiple times of the experiment are needed at each time point, the workload and the cost are greatly increased. The operation method of the invention can greatly reduce the workload.
In the step (2), the alcohol is 50% alcohol.
And blowing and beating the mixture evenly by using a multichannel pipettor when adding the alcohol. After sampling, the sample is mixed with 50% alcohol thoroughly, which leads to poor repeatability. The blowing and the sucking are required to be repeatedly carried out for more than 5 times.
Further, in the step (3), according to the absorbance of the reaction sample and the formula (II), determining the glucose concentration of the reaction sample;
glucose concentration [ (sample absorbance-blank absorbance)/(standard absorbance-blank absorbance) ]. + 17+ n 0.02(II)
n is the concentration of the last measurement
The last measurement refers to the last measurement of the same set of experiments. For example, the concentrations of three points of 10min, 20min and 30min are respectively C1, C2 and C3, and n is C1 in C2. Then n is 0 when testing for C1.
In one embodiment, the sodium acetate buffer comprises the following components in the following content ratio based on the total amount of the sodium acetate buffer:
CaCl23-5mmol/L
MgCl20.3-1mmol/L
NaN3100-500mg/L
the solvent is water.
The sodium acetate buffer can be prepared by the following method: add 11.8mL of glacial acetic acid to 850mL of DI water and adjust the pH to 6.0. 4mL of 1M CaC was addedl2Solution (dissolved in 1.109g CaCl2 to 10mL deionized water) while adding 1mL 0.49M MgCl2Solution (995.79 mg MgCl added)2·6H2O to 10mL of deionized water), 200mg of NaN was added3. The volume is up to 1000 mL.
In one embodiment, the digestion buffer comprises the following components in parts by weight based on the total amount of the digestion buffer:
pancreatin 10-15U/ml
Amyloglucosidase 200-500U/ml
The solvent was the sodium acetate buffer described above.
The digestion buffer must be ready for use.
The method for rapidly determining the enzymolysis quantity of the rice through high flux can be used for simulating the digestion condition in the rice body.
Selecting 12 rice varieties with different amylose contents, including high amylose content varieties Tianfeng B, Teqing, SIR3611 and Gui Dynasty No. 2, low amylose content varieties Nipponbao, Wuyun Ning No. 7, Minghui 63 and 9311, and glutinous rice varieties Gui Dynasty glutinous, Suyunuo, Yan spongnuo No. 4 and Yan nong glutinous No. 2-32. The examples which follow refer to rice material from this material. 20ml of digestion buffer was used for 100 samples. The enzymes used include pancreatin, which is available from Sigma, and transamyloglucosidase, which is available from megazyme.
EXAMPLE 1 raw Rice flour digestion
(1) Sample preparation: grinding the polished rice into powder, sieving with a sieve with aperture of 0.15mm (100 meshes), and oven drying for balancing. 20mg of rice flour was accurately weighed in a 96-well deep-well plate, and 300ul of distilled water was added.
(2) Sodium acetate buffer preparation: add 11.8mL of glacial acetic acid to 850mL of DI water and adjust the pH to 6.0. 4mL of 1M CaCl was added2Solution (dissolving 1.109g CaCl)2To 10mL of deionized water) while adding 1mL of 0.49MMgCl2Solution (995.79 mg MgCl added)2·6H2O to 10mL of deionized water), 200mg of NaN was added3. The volume is up to 1000 mL. Storing in a refrigerator at 4 deg.C.
(3) Digestion buffer: 20ml of sodium acetate buffer was weighed out, 68mg of pancreatin (4U/mg) and 1.33ml of amylotransglucosidase (0.3U/ul) were added, and shaking was carried out at 37 ℃ and 200rpm for 20 minutes. Centrifuge at 3000rpm for 5 minutes. The supernatant was transferred to a new vessel.
(4) Sample pretreatment: putting the raw rice flour into a 96-hole deep-hole plate, then putting the raw rice flour into a 37 ℃ water bath kettle, magnetically stirring at 600rpm, adding 1.2ml of sodium acetate buffer solution, preheating for 5 minutes, centrifuging at 2000rpm for 2 minutes, separating solid particles from the bottom, transferring the solid particles into the water bath kettle, and keeping the temperature at 37 ℃ without magnetic stirring.
(5) In-vitro digestion reaction: after the step (4) is completed, a 12-channel pipette is used to suck 200ul of the digestion buffer solution in the step (3) and rapidly add the digestion buffer solution into the 96-hole deep-hole plate, and stirring is started at 600rpm after the addition, and then timing is started.
(6) Sampling and detecting the concentration of glucose: the raw rice flour is sampled at 20, 40, 60, 90, 120, 180 and 240 minutes after the digestion reaction is started, the deep-well plate is taken out when the time point is reached, the raw rice flour is centrifuged at 2000rpm for 2 minutes, 20ul of digestion reaction liquid is sucked each time and transferred to a 96-well PCR plate, 20ul of sodium acetate buffer solution is added into the 96-well plate, and the raw rice flour is placed into a water bath kettle to restart timing. The reaction was terminated by adding 180ul of 50% ethanol to the PCR plate (repeatedly pipetting 5 times or more using a 12-channel pipette). And storing in a refrigerator at the temperature of-20 ℃, transferring the sample to a 96-hole enzyme label plate by using a 12-channel pipette after each time point is finished, and adding a standard sample with the glucose concentration of 1mg/ml into each enzyme label plate. Adding 200ul GOPOD, mixing (adding GOPOD, repeatedly sucking with 12-channel pipette for more than 5 times), and reacting at 50 deg.C for 20 min. The absorbance was measured using a microplate reader at a wavelength of 510 nm. And calculating the glucose concentration of the sample according to the light absorption value of the standard sample.
Example 2 analysis of digestion characteristics of gelatinized Rice flour
(1) Preparing gelatinized rice flour: the raw rice flour is placed in a 96-hole deep-hole plate, then is placed in a water bath kettle with a magnetic stirring function, and is sealed by using a latex sealing cover of the 96-hole deep-hole plate. Heating and cooking for 30 minutes while continuously stirring. After completion, the 96-well deep-well plate was taken out and cooled to 37 ℃.
(2) Sodium acetate buffer preparation: add 11.8mL of glacial acetic acid to 850mL of DI water and adjust the pH to 6.0. 4mL of 1M CaCl was added2Solution (dissolving 1.109g CaCl)2To 10mL of deionized water) while adding 1mL of 0.49MMgCl2Solution (995.79 mg MgCl added)2·6H2O to 10mL of deionized water),adding 200mg NaN3. The volume is up to 1000 mL. Storing in a refrigerator at 4 deg.C.
(3) Digestion buffer: 20ml of sodium acetate buffer was weighed out, 68mg of pancreatin (4U/mg) and 1.33ml of amylotransglucosidase (0.3U/ul) were added, and shaking was carried out at 37 ℃ and 200rpm for 20 minutes. Centrifuge at 3000rpm for 5 minutes. The supernatant was transferred to a new vessel.
(4) Sample pretreatment: placing gelatinized rice flour and sodium acetate buffer solution into a 96-hole deep-hole plate, placing the gelatinized rice flour and the sodium acetate buffer solution into a 37-DEG C water bath kettle, magnetically stirring at 600rpm, adding 1.2ml of the sodium acetate buffer solution, preheating for 5 minutes, centrifuging at 2000rpm for 2 minutes, separating solid particles from the bottom, transferring the solid particles into the water bath kettle, keeping the temperature at 37 ℃ without magnetic stirring.
(5) In-vitro digestion reaction: after the step (4) is completed, a 12-channel pipette is used to suck 200ul of the digestion buffer solution in the step (3) and rapidly add the digestion buffer solution into the 96-hole deep-hole plate, and stirring is started at 150rpm after the addition, and then timing is started.
(6) Sampling and detecting the concentration of glucose: sampling gelatinized rice flour 10, 20, 40, 60, 90, 120 and 180 minutes after the digestion reaction is started, taking out a deep-well plate when the time point is reached, centrifuging at 2000rpm for 2 minutes, sucking 20ul of digestion reaction liquid each time, transferring the digestion reaction liquid to a 96-well PCR plate, adding 20ul of sodium acetate buffer solution into the 96-well deep-well plate, and putting the 96-well plate into a water bath kettle to restart timing. The reaction was stopped by adding 180ul of 50% ethanol to the PCR plate. And storing in a refrigerator at the temperature of-20 ℃, transferring the sample to a 96-hole enzyme label plate by using a 12-channel pipette after each time point is finished, and adding a standard sample with the glucose concentration of 1mg/ml into each enzyme label plate. Adding 200ul GOPOD, mixing (adding GOPOD, repeatedly sucking with 12-channel pipette for more than 5 times), and reacting at 50 deg.C for 20 min. The absorbance was measured using a microplate reader at a wavelength of 510 nm. And calculating the glucose concentration of the sample according to the light absorption value of the standard sample.
Example 3 retrogradation rice flour digestion characterisation analysis
(1) Preparing the retrogradation rice flour: putting the gelatinized rice flour into a refrigerator at 4 ℃ for 7 days to prepare the retrogradation rice flour. (2) Sodium acetate buffer preparation: add 11.8mL of glacial acetic acid to 850mL of DI water and adjust the pH to 6.0. 4mL of 1M CaCl was added2Solution (dissolving 1.109g CaCl)2To 10mL of deionized water) while adding 1mL of 0.49M MgCl2Solution (995.79 mgMgCl added)2·6H2O to 10mL of deionized water), 200mg of NaN was added3. The volume is up to 1000 mL. Storing in a refrigerator at 4 deg.C.
(3) Digestion buffer: 20ml of sodium acetate buffer was weighed out, 68mg of pancreatin (4U/mg) and 1.33ml of amylotransglucosidase (0.3U/ul) were added, and shaking was carried out at 37 ℃ and 200rpm for 20 minutes. Centrifuge at 3000rpm for 5 minutes. The supernatant was transferred to a new vessel.
(4) Sample pretreatment: putting the retrogradation rice flour and the sodium acetate buffer solution into a 96-hole deep-hole plate, putting the plate into a 37 ℃ water bath kettle, magnetically stirring at 600rpm, adding 1.2ml of the sodium acetate buffer solution, preheating for 5 minutes, centrifuging at 2000rpm for 2 minutes, separating solid particles from the bottom, transferring the solid particles into the water bath kettle, and keeping the temperature at 37 ℃ without magnetic stirring.
(5) In-vitro digestion reaction: after the step (4) is completed, a 12-channel pipette is used to suck 200ul of the digestion buffer solution in the step (3) and rapidly add the digestion buffer solution into the 96-hole deep-hole plate, and stirring is started at 150rpm after the addition, and then timing is started.
(6) Sampling and detecting the concentration of glucose: sampling gelatinized rice flour 10, 20, 40, 60, 90, 120 and 180 minutes after the digestion reaction is started, taking out a deep-well plate when the time point is reached, centrifuging at 2000rpm for 2 minutes, sucking 20ul of digestion reaction liquid each time, transferring the digestion reaction liquid to a 96-well PCR plate, adding 20ul of sodium acetate buffer solution into the 96-well deep-well plate, and putting the 96-well plate into a water bath kettle to restart timing. The reaction was stopped by adding 180ul of 50% ethanol to the PCR plate. And storing in a refrigerator at the temperature of-20 ℃, transferring the sample to a 96-hole enzyme label plate by using a 12-channel pipette after each time point is finished, and adding a standard sample with the glucose concentration of 1mg/ml into each enzyme label plate. Adding 200ul GOPOD, mixing (adding GOPOD, repeatedly sucking with 12-channel pipette for more than 5 times), and reacting at 50 deg.C for 20 min. The absorbance was measured using a microplate reader at a wavelength of 510 nm. And calculating the glucose concentration of the sample according to the light absorption value of the standard sample.
As shown in FIG. 1, the rice flour of the material with high amylose content has a relatively slow hydrolysis rate, which is consistent with the existing reports, and the rice flour after gelatinization has a faster hydrolysis rate compared with the original rice flour, which is consistent with the previous research reports. The method is proved to be reliable and effective.
As shown in fig. 2, 3 and 4, different rice types including indica and japonica, waxy and non-waxy were selected for this experiment. The physical and chemical indexes of each material and the variety of cooked material are measured at the same time, including apparent amylose content [1], water content [2], protein content [3], gum consistency [1], RVA [4], DSC [5 ]. The results show that the selected materials have large difference of physical and chemical quality, and the method can be proved to be suitable for different rice materials.
The references used for the assay are as follows:
1.Tan,Y.F.；Li,J.X.；Yu,S.B.；Xing,Y.Z.；Xu,C.G.；Zhang,Q.F.The threeimportant traits for cooking and eating quality of rice grains are controlledby a single locus in an elite rice hybrid,Shanyou 63.Theor.Appl.Genet.1999,99,642-648.
2.Zhang C,Chen S,Ren X,et al.Molecular Structure and PhysicochemicalProperties of Starches from Rice with Different Amylose Contents Resultingfrom Modification of OsGBSSI Activity.J Agric Food Chem.2017；65(10):2222-2232.
3.Association of Official Analytical Chemists(AOAC).Protein(crude)inanimal feed.Combustion method(990.03).Standard methods of the AOAC,15th ed.；Association of Official Analytical Chemists:Arlington,VA,USA,1995.
4.Zhu,L.J.；Liu,Q.Q.；Sang,Y.J.；Gu,M.H.；Shi,Y.C.Underlying reasons forwaxy rice flours having different pasting properties.Food Chem.2010,120,94-100.
5.Zhang,C.Q.；Zhu,L.J.；Shao,K.；Gu,M.H.；Liu,Q.Q.Toward underlyingreasons for rice starches having low viscosity and high amylose:physiochemical and structural characteristics.J.Sci.Food Agric.2013,93,1543-1551.
the above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (10)
1. A method for rapidly determining the enzymolysis quantity of rice in high flux is characterized by at least comprising the following steps:
(1) pretreatment: placing the rice flour and the sodium acetate buffer solution in a 96-hole deep-hole plate, preheating and centrifuging; the rice flour refers to powdery rice;
(2) in-vitro digestion reaction: adding a digestion buffer solution into the 96-hole deep-hole plate obtained in the step (1) by using a multi-channel pipette, timing and stirring, sampling at regular intervals, placing a sample into a 96-hole PCR plate, adding alcohol into the 96-hole PCR plate to finish reaction, and obtaining reaction samples at each time point;
(3) and (3) measuring the enzymolysis amount: transferring the reaction sample at each time point to a 96-hole enzyme label plate by using a multi-channel pipettor, measuring the glucose concentration of the reaction sample at each time point, and obtaining the enzymolysis quantity of the rice at each sampling time according to the formula (I);
the enzymatic yield is 0.9/sample mass 100% (I).
2. The method for rapidly determining the enzymatic hydrolysis content of rice in high throughput according to claim 1, wherein in the step (1), the rice flour and the sodium acetate buffer solution are stirred by using a magnetic stirrer during preheating.
3. The method for rapidly determining the enzymatic hydrolysis content of rice in high throughput according to claim 1, wherein in the step (1), the rice flour is selected from raw rice flour, gelatinized rice flour or retrogradation rice flour.
4. The method for high-throughput rapid determination of the enzymatic hydrolysis amount of rice according to claim 1, wherein in the step (2), the stirring manner is magnetic stirring.
5. The method for rapid high-throughput determination of the enzymatic hydrolysis amount of rice according to claim 2 or 4, wherein the rotation speed of magnetic stirring is 600 rpm.
6. The method for high-throughput rapid determination of the enzymatic hydrolysis amount of rice according to claim 1, wherein in the step (2), the in vitro digestion reaction is carried out in a water bath environment at 37 ℃.
7. The method for high-throughput rapid determination of the enzymatic hydrolysis amount of rice according to claim 6, wherein in the step (2), when sampling, the 96-well deep-well plate is taken out from the water bath environment, centrifuged, and after a quantitative sample is sucked, an equal amount of sodium acetate buffer solution is added into the 96-well deep-well plate, and the 96-well deep-well plate is placed into the water bath environment at 37 ℃ to restart timing.
8. The method for high-throughput rapid determination of the enzymatic hydrolysis amount of rice according to claim 1, wherein in the step (2), the alcohol is 50% alcohol.
9. The method for high-throughput rapid determination of the enzymatic hydrolysis amount of rice according to claim 1, wherein in the step (3), the glucose concentration of the reaction sample is determined according to the formula (II) based on the absorbance of the reaction sample;
glucose concentration [ (sample absorbance-blank absorbance)/(standard absorbance-blank absorbance) ]. + 17+ n 0.02(II)
n is the concentration measured last time.
10. Use of the method for rapid high-throughput determination of enzymatic activity of rice according to any one of claims 1 to 9 for simulating digestion in rice.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181028A (en) * | 2007-12-03 | 2008-05-21 | 浙江大学 | Rice for diabetic patients and cultivation method thereof |
| CN108226407A (en) * | 2018-01-02 | 2018-06-29 | 扬州大学 | A kind of method of quick detection rice digestion characteristics |
| CN108519377A (en) * | 2018-04-13 | 2018-09-11 | 上海市农业科学院 | A method of screening has excellent flavour feature rice plant |
| CN109061162A (en) * | 2018-09-17 | 2018-12-21 | 东北农业大学 | A kind of method of high throughput assay Starch Hydrolysis enzyme inhibition activity |
| CN109251959A (en) * | 2018-09-17 | 2019-01-22 | 东北农业大学 | A kind of method of in-vitro simulated stomach and intestine starch digestion |
| CN110261332A (en) * | 2019-05-30 | 2019-09-20 | 扬州大学 | A kind of method of simple grain rice measurement content of amylose in rice |
-
2020
- 2020-06-15 CN CN202010543579.1A patent/CN111733210A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181028A (en) * | 2007-12-03 | 2008-05-21 | 浙江大学 | Rice for diabetic patients and cultivation method thereof |
| CN108226407A (en) * | 2018-01-02 | 2018-06-29 | 扬州大学 | A kind of method of quick detection rice digestion characteristics |
| CN108519377A (en) * | 2018-04-13 | 2018-09-11 | 上海市农业科学院 | A method of screening has excellent flavour feature rice plant |
| CN109061162A (en) * | 2018-09-17 | 2018-12-21 | 东北农业大学 | A kind of method of high throughput assay Starch Hydrolysis enzyme inhibition activity |
| CN109251959A (en) * | 2018-09-17 | 2019-01-22 | 东北农业大学 | A kind of method of in-vitro simulated stomach and intestine starch digestion |
| CN110261332A (en) * | 2019-05-30 | 2019-09-20 | 扬州大学 | A kind of method of simple grain rice measurement content of amylose in rice |
Non-Patent Citations (6)
| Title |
|---|
| LIN L.等: "Relationships between amylopectin molecular structures and functional properties of different-sized fractions of normal and high-amylose maize starches", 《FOOD HYDROCOLLOIDS》 * |
| ZHU L.等: "Digestibility and physicochemical properties of rice (Oryza sativa L.) flours and starches differing in amylose content", 《CARBOHYDRATE POLYMERS》 * |
| 杨黛 等: "抗性淀粉检测方法的研究进展", 《科技信息》 * |
| 满建民 等: "体内外消化高直链抗性水稻淀粉的波谱特性", 《食品科学》 * |
| 赵凯 等: "缓慢消化淀粉测定过程中的影响因素分析", 《食品科学》 * |
| 黄继红: "《抗性淀粉生产技术及其应用》", 31 January 2017 * |
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