CN114136960B - Method for batch measurement of rice resistant starch content - Google Patents
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Abstract
The invention discloses a method for measuring the content of resistant starch in rice in batches, which utilizes a double-enzyme hydrolysis method to measure the content of resistant starch in rice, namely, firstly, non-resistant starch is hydrolyzed into glucose by pancreatic alpha-amylase, then, the resistant starch is dissolved by steel balls and KOH, and then, the resistant starch is hydrolyzed into glucose by Amyloglucosidase (AMG) solution, and the glucose content is measured by a GOPOD method, so that the content of the resistant starch is calculated according to a standard curve. Compared with the defects of long time consumption, low efficiency, high cost and the like of the traditional AOAC method, the method for measuring the content of the resistant starch in batches has the advantages of low cost, good repeatability, high accuracy, batch operation and the like.
Description
Technical Field
The invention belongs to the technical field of analysis and detection of resistant starch content in rice, and particularly relates to a method for detecting resistant starch content in rice.
Background
Rice is an extremely important grain crop, more than 60% of people in China take rice as main food, more than 1/3 of people in the world take rice as main food, the GI value of the inter-rice species Glycemic index is changed to 48-93, the rice and products thereof are mainly digested in the stomach and the small intestine in an amylase enzymolysis mode, and the final product is glucose, so the rice is considered as food with high Glycemic Index (GI);
resistant Starch (RS) is a generic term for starch and starch degradation products that can avoid hydrolysis of pancreatic alpha-amylase in healthy small intestine and are not digested and absorbed, designated by Englyst in 1983. RS has similar physiological functions with dietary fiber, and has effects of regulating blood sugar and insulin sensitivity of diabetic patients.
The existing methods for measuring the content of resistant starch in rice mainly comprise a Berry method, an Englyst method, a McCleary method, a Goni method and the like, and the quantitative measurement methods of domestic resistant starch are not unified standard, and are generally the Goni method and the AOAC method. Therefore, the research on the determination method of the resistant starch has a certain practical significance.
One existing detection method uses a kit (Resistant Starch Assay Kit) provided by the company Megazyme, ireland: and weighing the rice sample, pouring the rice sample into a test tube with a screw cap, and calculating the glucose content of the sample, thereby calculating the resistant starch content of the sample. In the scheme, in the process of dissolving resistant starch in KOH solution, starch emulsification can be caused by uniform mixing of a vortex machine; the RS of the rice can form insoluble starch blocks, which affects the measurement effect; the scheme has the technical defects of higher cost and incapability of measuring the RS content of the rice in a large scale.
Disclosure of Invention
The invention aims to solve the problem of providing a method for measuring the content of resistant starch in rice in batches, which can accurately measure the content of resistant starch in rice so as to better evaluate the nutritional and functional value of starch.
In order to solve the technical problems, the invention provides a method for measuring the content of resistant starch of rice in batches, which comprises the following steps of:
1) Weighing 0.0100+/-0.0001 g of rice flour serving as a sample to be detected, placing the rice flour into a container (for example, a 2mL centrifuge tube), adding 0.4mL of mixed working solution (10 mg/mL pancreatic alpha-amylase and 3U/mL AMG), uniformly mixing, and incubating for 12+/-0.5 h in a shaking table at constant temperature of 200+/-20 r/min and 37+/-0.1 ℃;
2) Adding 0.4mL of ethanol (absolute ethanol) into the incubated product obtained in the step 1), uniformly mixing at 4+/-1 ℃, centrifuging, and pouring out supernatant;
3) Adding 0.8mL of 50% (volume%) ethanol into the precipitate obtained by pouring out the supernatant, mixing uniformly at 4+ -1deg.C, centrifuging, and pouring out the supernatant again;
4) Repeating the step 3) for another time, and drying the obtained precipitate;
5) Adding a steel ball (diameter 6 mm) and 0.2mL of KOH solution with concentration of 2M into the dried precipitate, grinding for 60+ -10 s (grinding on a grinder until no obvious lump exists), immediately placing on ice (placing in an ice box), grinding for 60+ -10 s again after 10min, and placing on ice (placing in an ice box) for 10min;
6) Adding 0.8mL of sodium acetate buffer (1.2M, pH=3.8) to the product obtained in the step 5) (taken out of ice), immediately adding 0.01mL of AMG diluent 2 (1650U/mL) after mixing, placing in a water bath at 50+/-2 ℃ for incubation for 30min, and shaking uniformly every 10min;
7) Centrifuging the product obtained in the step 6) at the temperature of 4+/-1 ℃ to obtain supernatant serving as a liquid to be detected;
taking 0.03mL of a solution to be tested (supernatant), adding 0.9mLGOPOD reagent, and uniformly mixing to obtain the solution to be tested;
adding 0.9mL of GOPOD reagent into 0.03mL of sodium maleate buffer solution, and uniformly mixing to obtain a blank reagent;
incubating the reagent to be detected and the blank reagent in a constant-temperature water bath at 50+/-2 ℃ for 20min, cooling to room temperature, and measuring at 510nm by using an ultraviolet-visible spectrophotometer, so as to obtain the absorbance value of the reagent to be detected after incubation relative to the blank reagent after incubation;
description: if the content of resistant starch in the sample to be detected is more than 10%, distilled water is required to dilute the solution to be detected by a certain multiple, and the concentration is reduced to below 10%, so that the solution can be detected in the color development range of the GOPOD reagent;
8) Substituting 0.03mL of glucose standard solution for the 0.03mL of the solution to be tested, and measuring the absorbance value of the glucose standard solution after incubation relative to a blank reagent after incubation at 510nm according to the method described in the step 7), so as to obtain a standard curve;
9) Substituting the absorbance value obtained in the step 7) into the standard curve obtained in the step 8) to obtain the glucose concentration in the liquid to be detected;
and calculating to obtain the resistant starch content in the sample to be detected by using the glucose concentration in the liquid to be detected.
As an improvement of the method for measuring the content of resistant starch in rice in batches, the invention is as follows:
in the step 8): the concentration of the gradient standard glucose solution is 200 mug/mL, 400 mug/mL, 600 mug/mL, 800 mug/mL and 1000 mug/mL respectively;
the resulting standard curve is y=3019.4x+21.482;
x represents absorbance; y represents the glucose concentration.
As a further improvement of the method for measuring the content of resistant starch in rice in batches according to the invention, step 9):
wherein: glucose concentration units are μg/mL.
As a further improvement of the method for measuring the content of resistant starch in rice in batches of the invention:
mix working solution (10 mg/mL pancreatic alpha-amylase, 3U/mL amyloglucosidase): dissolving 1g of pancreatic alpha-amylase (containing 3000U/g of enzyme) in 100mL of sodium maleate buffer (0.1M, pH 6.0), stirring, adding 1mL of AMG diluent 1 (300U/mL), uniformly mixing, centrifuging for 10min (3000 r/min), and taking supernatant; for standby, the solution is required to be prepared for use at present;
AMG diluent 1 (300U/mL): taking 0.4mL of AMG solution (containing enzyme 3300U/mL) in a centrifuge tube, diluting to 4.4mL with 0.1M sodium maleate buffer solution, and storing at 4deg.C for use
AMG diluent 2 (1650U/mL): 1mL of AMG solution (supplied by Megazyme company, cat# K-RSTAR, enzyme-containing 3300U/mL) was placed in a centrifuge tube, and 1mL of 0.1M sodium maleate buffer solution was added thereto, followed by mixing, and storage at 4℃was performed.
Description: sodium maleate buffer (0.1 m, ph 6.0): 11.6g of sodium maleate (AR, 99.0% or more, HPLC, CAS NO: 110-16-7) are taken, dissolved in 800mL of distilled water, and adjusted to pH6.0 with sodium hydroxide (4M), then 0.3g of calcium chloride is added, and the mixture is stored at 4 ℃ with distilled water to a volume of 1L for later use.
As a further improvement of the method for measuring the content of resistant starch in rice in batches of the invention:
the centrifugation in the steps 2), 3) and 7) is as follows: centrifugal force of 13000r/min for 8-12 min (at 4 ℃).
As a further improvement of the method for measuring the content of resistant starch in rice in batches of the invention:
and the drying in the step 4) is carried out for 4+/-0.5 h at 50+/-5 ℃.
As a further improvement of the method for measuring the content of resistant starch in rice in batches of the invention:
the rice was ground into fine rice, and then ground into fine powder which was sieved with a 100-mesh sieve, and dried (conventional ventilation drying) to obtain rice flour as a sample. Sealing and preserving rice flour; and (5) standby application.
According to the method for measuring the content of the resistant starch in batches, a non-resistant starch part in rice flour is hydrolyzed by a conventional double-enzyme hydrolysis method, the resistant starch is fully dissolved in a KOH solution by a steel ball grinding method, the diluted AMG solution is used for hydrolyzing the resistant starch into glucose, and finally the glucose content is measured by reducing a GOPOD color development system in an equal proportion, so that the content of the resistant starch is deduced.
In the present invention:
1. the stirring of the stirrer in the traditional method is improved to fully grind with steel balls, so that the phenomenon of RS agglomeration and emulsification of rice is avoided;
2. the incubation conditions of double-enzyme hydrolysis are improved, the solution reaction system is reduced in an equal proportion, and on the basis of adopting steel ball grinding, the RS content of rice is measured in a large scale by using a trace amount of reagent, so that the detection efficiency and repeatability of resistant starch of the rice are improved, and meanwhile, the measurement cost is saved.
In summary, the invention discloses a method for measuring the content of resistant starch in rice in batches, which utilizes a double-enzyme hydrolysis method to measure the content of resistant starch in rice, namely, firstly, non-resistant starch is hydrolyzed into glucose by pancreatic alpha-amylase, then, the resistant starch is dissolved by steel balls and KOH, and then, the resistant starch is hydrolyzed into glucose by Amyloglucosidase (AMG) solution, and the content of glucose is measured by a GOPOD method, so that the content of the resistant starch is calculated according to a standard curve. Compared with the defects of long time consumption, low efficiency, high cost and the like of the traditional AOAC method, the method for measuring the content of the resistant starch in batches has the advantages of low cost, good repeatability, high accuracy, batch operation and the like. The cost of the present invention is only about 10% of that of conventional AOACs.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a glucose standard curve measured by the GOPOD method of the present invention;
FIG. 2 is a graph showing the trend of increasing the resistant starch content over time;
FIG. 3 shows the resistant starch content of Y128 rice flour samples at different dilution concentrations of AMG.
Detailed Description
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1, a method for batch measurement of rice resistant starch content:
1. materials and instruments
Rice flour of a sample to be detected; constant temperature oscillating table, desk-top high-speed refrigerated centrifuge, mini centrifuge, oven, steel ball (diameter 6 mm), full-automatic sample rapid grinding instrument, miniature vortex mixer, ultraviolet visible spectrophotometer.
2. Reagent preparation
(1) Sodium maleate buffer (0.1 m, ph 6.0): 11.6g of sodium maleate (AR, 99.0% or more, HPLC, CAS NO: 110-16-7) are taken, dissolved in 800mL of distilled water, and adjusted to pH6.0 with sodium hydroxide (4M), then 0.3g of calcium chloride is added, and the mixture is stored at 4 ℃ with distilled water to a volume of 1L for later use.
(2) AMG diluent 1 (300U/mL): 0.4mL of AMG solution (Megazyme, cat. K-RSTAR, enzyme-containing 3300U/mL) was placed in a centrifuge tube, diluted to 4.4mL with the above-mentioned sodium maleate buffer (0.1M, pH 6.0), and stored at 4℃for use.
(3) AMG diluent 2 (1650U/mL): 1mL of AMG solution (supplied by Megazyme company, cat. K-RSTAR, containing enzyme 3300U/mL) was placed in a centrifuge tube, and 1mL of sodium maleate buffer (0.1M, pH 6.0) was added thereto, followed by mixing, and storage at 4℃was performed.
(4) Mix working solution (10 mg/mL pancreatic alpha-amylase, 3U/mL amyloglucosidase): 100mL of sodium maleate buffer (0.1M, pH 6.0) was used to dissolve 1g of pancreatic alpha-amylase (supplied by Megazyme, 3000U/g containing enzyme) and stirred for 5min, then 1mL of AMG diluent 1 (300U/mL) was added, after mixing well, centrifuged for 10min (3000 r/min), and the supernatant was removed for use, and this solution was required to be ready for use.
(5) Sodium acetate buffer (1.2 m, ph 3.8): 69.6mL of sodium acetate (AR, 99.5% or more, CN No. 81601) was placed in a 1000mL volumetric flask, 800mL of distilled water was added, the pH was adjusted to 3.8 with sodium hydroxide solution (4M), and 1L of distilled water was used to determine the volume.
(6) Potassium hydroxide solution (2M): 11.22g of potassium hydroxide (AR, not less than 85.0% and CAS NO: 1310-58-3) was placed in a plastic cup containing 90mL of distilled water, stirred with a glass rod to be sufficiently dissolved, and then 100mL of distilled water was used for constant volume.
(7) Glucose standard solutions (200. Mu.g/mL, 400. Mu.g/mL, 600. Mu.g/mL, 800. Mu.g/mL, and 1000. Mu.g/mL): 1.0000g glucose was accurately weighed and placed in a 100mL beaker and dissolved in 50mL sodium maleate buffer (0.1M, pH 6.0). After sufficiently dissolving the mixture by stirring in a glass, the mixture was transferred to a 100mL volumetric flask, and the glass rod and the 100mL beaker were repeatedly rinsed with 40mL of sodium maleate buffer (0.1M, pH 6.0) and transferred to the 100mL volumetric flask. The volume is fixed by using a rubber head dropper, and the mixture is uniformly mixed to obtain 1000 mug/mL glucose standard solution. Mu.l, 200. Mu.l, 300. Mu.l and 400. Mu.l of glucose standard solution (1000. Mu.g/mL) were aspirated into 2mL centrifuge tubes, 400. Mu.l, 300. Mu.l, 200. Mu.l and 100. Mu.l of sodium maleate buffer solution (0.1M, pH 6.0) were added respectively, and the mixture was shaken well to obtain 200. Mu.g/mL, 400. Mu.g/mL, 600. Mu.g/mL and 800. Mu.g/mL of glucose standard solution.
(8) GOPOD reagent: 50ml of GOPOD Reagent Buffer (Buffer: pH 7.4, p-hydroxybenzoic acid and sodium azide:0.09% w/v, supplied by Megazyme Co., ltd.) were set to 1L with distilled water as a diluent.
20ml of the diluent is taken to dissolve 80mg of GOPOD Reagent Enzymes (Glucose oxidanse plus peroxidase and-aminoantipyrine, available from Megazyme company, product number K-RSTAR) and is fully dissolved, transferred to the rest 980ml of diluent, fully mixed and then transferred to a bottle wrapped by aluminum foil, and stored at 2-5 ℃ in a dark place.
3. The method comprises the following specific steps:
1) Taking 10-20 g of rice, grinding into fine rice by a rice polisher, grinding into fine powder by a cyclone mill, sieving by a 100-mesh sieve, and sealing and storing the rice flour after conventional drying for 24 hours (namely, conventional ventilation drying for 24 hours at room temperature);
accurately weighing 0.0100g (+ -0.0001) rice flour in a 2mL centrifuge tube, and tapping the centrifuge tube to enable a sample to fall to the bottom; adding 0.4mL of the freshly prepared mixed working solution (10 mg/mL of pancreatic alpha-amylase and 3U/mL of AMG), fully and uniformly mixing on a miniature vortex mixer, and then placing the centrifuge tube level in a constant-temperature shaking table at 37 ℃ for 12h (swing speed 200 r/min) incubation;
2) Taking out the centrifuge tube after 12h incubation, adding 0.4mL of absolute ethyl alcohol, fully and uniformly mixing on a vortex machine, and centrifuging for 10min (13000 r/min,4 ℃);
3) The supernatant was removed, 0.8mL of 50% ethanol was added, and after thoroughly mixing on a vortex machine, the mixture was centrifuged with a centrifuge for 10min (13000 r/min,4 ℃ C.);
4) Repeating the step 3) once again, pouring out the supernatant again, and putting the centrifuge tube into a 50 ℃ oven for drying and precipitating.
5) Drying the precipitate in a centrifuge tube for about 4 hours, adding a steel ball (diameter 6 mm) and 0.2mL KOH (2M), grinding for 60 seconds on a grinder (no obvious lump), immediately placing into an ice box, grinding for 60 seconds again after 10 minutes, and placing into the ice box for 10 minutes again;
6) 0.8mL of sodium acetate buffer (1.2M, pH=3.8) was added, and immediately after thorough mixing, 0.01mL of AMG diluent 2 (1650U/mL) was added, after thorough mixing, the centrifuge tube was placed in a 50℃water bath for 30min incubation, and shaking was performed once every 10min interval.
7) Taking out the centrifuge tube, wiping the outer surface of the centrifuge tube, and centrifuging for 10min (13000 r/min,4 ℃) in the centrifuge, wherein the obtained supernatant is used as the liquid to be detected (if the content of the resistant starch in the sample is more than 10%, distilled water is required to dilute the liquid to be detected by a certain multiple, and the concentration is reduced to below 10%, so that the liquid can be detected in the color development range of the GOPOD reagent);
taking 0.03mL of liquid to be tested (supernatant) in a 2mL centrifuge tube, adding 0.9mL of GOPOD reagent, and fully and uniformly mixing to obtain the reagent to be tested;
taking 0.03mL of sodium maleate buffer solution, adding 0.9mL of GOPOD reagent, and uniformly mixing to obtain a blank reagent;
incubating the reagent to be detected and the blank reagent in a constant-temperature water bath at 50+/-2 ℃ for 20min, cooling to room temperature, and measuring at 510nm by using an ultraviolet-visible spectrophotometer, so as to obtain the absorbance value of the reagent to be detected relative to the blank reagent;
that is, the absorbance value of the test reagent after incubation relative to the blank reagent after incubation was measured at 510nm with an ultraviolet-visible spectrophotometer.
8) The absorbance value of the standard glucose solution after incubation at 510nm was measured as described in step 7) with respect to the blank reagent after incubation, to give a standard curve, y=3019.4x+21.482 (equation one), using 0.03mL of standard glucose solution (concentrations of 200. Mu.g/mL, 400. Mu.g/mL, 600. Mu.g/mL, 800. Mu.g/mL, 1000. Mu.g/mL, respectively) instead of 0.03mL of the test solution.
x represents absorbance; y represents the glucose concentration.
9) Substituting the absorbance value obtained in step 7) into the above formula y=3019.4x+21.482 to obtain the glucose concentration of the supernatant. The resistant starch content of the sample was calculated using the following formula.
Wherein: glucose concentration units are μg/mL.
Experiment 1 "resistant starch standard sample (39%)", "HJX74" and "Y128" were measured according to the steps 1) to 7) of "three, specific steps as follows" in example 1, and the absorbance value obtained in step 7) was substituted into the above formula y=3019.4x+21.482 to obtain the glucose concentration of the supernatant. And calculating the content of the resistant starch in the sample by using a formula II. Each sample was set up with 3 replicates. The final results are shown in Table 1 below.
Table 1, experimental data for each sample
The above experiment is to test the obtained data by adopting the technical scheme set by the invention, wherein the average content of resistant starch of HJX74 is 0.76%, the average content of resistant starch of Y128 is 2.12%, and the average content of resistant starch of a resistant starch standard sample is 38.64%.
Comparative experiment 1
The following tests were performed on the samples described in experiment 1 using the conventional AOAC method, and the test methods can be referred to as follows:
accurately weighing 0.1000g of each of a resistant starch standard sample (the resistant starch content is 39% provided by Megazyme company), HJX74 sample rice flour and Y128 sample rice flour, and respectively performing the following steps:
pouring directly into the test tube with screw cap, and gently beating the test tube to ensure that the sample is concentrated at the bottom. Adding 4mL of mixed working solution (10 mg/mL of pancreatic alpha-amylase and 3U/mL of amyloglucosidase) into each test tube, uniformly mixing by a vortex oscillator, discharging into an oscillating water bath according to the movement direction, and incubating for 16 hours (200 r/min) at 37 ℃;
after 16h, the test tube was removed from the shaking water bath, excess water was wiped off with a paper towel, the reaction was stopped by adding 4.0mL absolute ethanol, vortexed with a vortexer and centrifuged for 10min (3000 rpm,4 ℃); centrifuging, pouring out supernatant, adding 2mL of 50% ethanol for resuspension, swirling by a swirler, adding 6mL of 50% ethanol, mixing, centrifuging for 10min (3000 rpm,4 ℃), pouring out supernatant, repeating the resuspension and centrifuging steps, pouring out supernatant again, turning over a test tube, and sucking out excessive liquid by using absorbent paper; ice-bathing test tubes, adding a magnetic stirring rod and 2mL of KOH (2M) to each test tube, stirring for 20min with a magnetic stirrer in an ice-bath state to resuspend floc and dissolve RS; then 8mL of 1.2M sodium acetate buffer (pH 3.8) is added into each test tube, the mixture is stirred by a magnetic stirrer, 0.1mL of AMG (3300U/mL) is immediately added, the mixture is uniformly mixed, and the mixture is put into a water bath at 50 ℃ for incubation for 30min, and a vortex device is used for intermittent uniform mixing during the period;
for the resistant starch standard (resistant starch content 39%) supplied by Megazyme company, the RS content was >10%, the sample in the transfer tube was transferred to a 100mL volumetric flask, and the flask was washed several times with a small amount of washing, transferred to the volumetric flask, and finally the volume was fixed to 100mL with distilled water, and mixed well. Taking 1mL of supernatant, centrifuging for 10min (3000 rpm,4 ℃), and directly centrifuging for 10min (3000 rpm,4 ℃) for HJX74 sample rice flour and Y128 rice flour (content < 10%). 0.1mL of the supernatant was aspirated separately into test tubes, 3.0mL of GOPOD reagent was added, incubated in a constant temperature water bath at 50℃for 20min, and the absorbance value of each solution at 510nm was measured relative to the blank reagent (0.1 mL of sodium acetate buffer, pH4.5, 3.0mL of GOPOD reagent).
Resistant starch standard samples (39%), HJX74, Y128 were tested as in comparative experiment 1 above, with 3 replicates per sample. The final results are shown in Table 2 below.
Table 2, experimental data for each sample
The samples were tested according to the current AOAC method, with an average content of resistant starch of HJX74 of 0.64%, a resistant starch content of Y128 of 2.22% and a resistant starch standard sample of 37.97%.
Therefore, the GOPOD method of the invention is basically consistent with the existing AOAC method, and is similar to the true value. The conventional AOAC method (stirrer method) is limited by the size of a magnetic stirrer, and one instrument can only measure more than ten samples; the improved steel ball stirring method can be used for measuring the content of resistant starch on a large scale, and one steel ball mill can be used for measuring hundreds of samples. Both methods have the advantage that they save time costs and increase efficiency in the case of accurate values being measured.
The time of the centrifuge tube in the step 1) of the third embodiment of the comparative example 1 is changed from 12 hours to 4 hours, 8 hours and 16 hours respectively; the remainder was identical to example 1. Detecting 39% of standard samples and Y128;
the results obtained were: with increasing reaction time, the resistant starch content of both the 39% standard and the Y128 sample tended to decrease significantly, with the resistant starch content of the 39% standard incubated for 12h being closest to that measured by the AOAC method incubated for 16h, 38.64% and 37.97%, respectively. Compared with the AOAC method, the method only incubates for 12 hours to achieve the same effect as the 16 hours of incubation of the AOAC method, so the method has more advantages. See in particular fig. 2.
Comparative example 2-2, in which "AMG dilution 2 (1650U/mL)" in step 5) of "three" in example 1 was changed to "AMG dilution (containing enzyme 3300U/mL)", "AMG dilution (containing enzyme 825U/mL)", and "AMG dilution (containing enzyme 412.5U/mL)", respectively, the remainder was the same as in example 1. Detection is performed for Y128;
in FIG. 3, AMG dilution is 0, corresponding to a concentration of 3300U/mL; AMG dilution is 1, and the corresponding concentration is 1650U/mL; and the rest are analogized.
The results were: the content of the resistant starch measured is substantially unchanged when the AMG solution is diluted 0 to 3 times, i.e. when the AMG concentration is diluted from 3300U/mL to 825U/mL; when diluted 3 to 31 times, i.e., when the AMG concentration is diluted from 825U/mL to 103U/mL, the AMG concentration is too low to completely hydrolyze the resistant starch to glucose, which is different from the present invention. Therefore, when the starch is diluted to more than 3 times, the content of the resistant starch is gradually reduced, the dilution is 3 times as a critical point, and in order to reduce the experiment cost as much as possible and ensure the accuracy of the experiment to the greatest extent, the selected dilution of 1 time-AMG diluent 2 (1650U/mL) is more advantageous. The specific gradient experiment is shown in figure 3.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
Claims (7)
1. The method for measuring the content of the resistant starch of the rice in batches is characterized by sequentially carrying out the following steps:
1) Weighing 0.0100+/-0.0001 g of rice flour serving as a sample to be detected in a container, adding 0.4mL of mixed working solution, 10mg/mL of pancreatic alpha-amylase and 3U/mL of AMG, uniformly mixing, and incubating for 12+/-0.5 h in a shaking table with constant temperature oscillation at 200+/-20 r/min and 37+/-0.1 ℃;
2) Adding 0.4mL of ethanol into the incubated product obtained in the step 1), uniformly mixing at 4+/-1 ℃, centrifuging, and pouring out supernatant;
3) Adding 0.8mL of 50% ethanol into the precipitate obtained by pouring out the supernatant, uniformly mixing at 4+/-1 ℃, centrifuging, and pouring out the supernatant again;
4) Repeating the step 3) for another time, and drying the obtained precipitate;
5) Adding a steel ball and 0.2mL of KOH solution with the concentration of 2M into the dried precipitate, immediately placing the precipitate on ice after grinding for 60+/-10 s, and then grinding again for 60+/-10 s after 10min, and placing the precipitate on ice for 10min;
6) Adding 0.8mL of sodium acetate buffer solution into the product obtained in the step 5), immediately adding 0.01mL of AMG diluent 2 after uniformly mixing, and placing the mixture in a water bath with the temperature of 50+/-2 ℃ for incubation for 30min, and shaking uniformly once every 10min;
AMG dilution 2: 1mL of AMG solution is taken and placed in a centrifuge tube, 1mL of 0.1M sodium maleate buffer solution is added, and the mixture is uniformly mixed;
7) Centrifuging the product obtained in the step 6) at the temperature of 4+/-1 ℃ to obtain supernatant serving as a liquid to be detected;
adding 0.9mL of GOPOD reagent into 0.03mL of the solution to be tested, and uniformly mixing to obtain the reagent to be tested;
taking 0.03mL of sodium maleate buffer solution, adding 0.9mL of GOPOD reagent, and uniformly mixing to obtain a blank reagent;
incubating the reagent to be detected and the blank reagent in a constant-temperature water bath at 50+/-2 ℃ for 20min, cooling to room temperature, and measuring at 510nm by using an ultraviolet-visible spectrophotometer, so as to obtain the absorbance value of the reagent to be detected after incubation relative to the blank reagent after incubation;
8) Substituting 0.03mL of glucose standard solution for the 0.03mL of the solution to be tested, and measuring the absorbance value of the glucose standard solution after incubation relative to a blank reagent after incubation at 510nm according to the method described in the step 7), so as to obtain a standard curve;
9) Substituting the absorbance value obtained in the step 7) into the standard curve obtained in the step 8) to obtain the glucose concentration in the liquid to be detected;
and calculating to obtain the resistant starch content in the sample to be detected by using the glucose concentration in the liquid to be detected.
2. The method for batch measurement of rice resistant starch content according to claim 1, wherein:
in the step 8): the concentration of the gradient standard glucose solution is 200 mug/mL, 400 mug/mL, 600 mug/mL, 800 mug/mL and 1000 mug/mL respectively;
the resulting standard curve is y=3019.4x+21.482;
x represents absorbance; y represents the glucose concentration.
3. The method for batch measurement of rice resistant starch content according to claim 1 or 2, wherein:
the step 9) is as follows:
resistant starch content =；
Wherein: glucose concentration units are [ mu ] g/mL.
4. A method for batch measurement of rice resistant starch content according to claim 3, wherein:
mixing working solution: dissolving 1g of pancreatic alpha-amylase in 100mL of sodium maleate buffer solution, stirring, adding 1mL of AMG diluent 1, uniformly mixing, centrifuging for 10min, and taking supernatant;
AMG dilution 1: 0.4mL of AMG solution was placed in a centrifuge tube and diluted to 4.4mL with 0.1M sodium maleate buffer.
5. The method for batch measurement of rice resistant starch content according to claim 4, wherein:
the centrifugation in the steps 2), 3) and 7) is as follows: and (3) centrifuging for 8-12 min at 13000 r/min.
6. The method for batch measurement of rice resistant starch content according to claim 5, wherein:
and the drying in the step 4) is carried out for 4+/-0.5 h at 50+/-5 ℃.
7. The method for batch measurement of rice resistant starch content according to claim 6, wherein:
grinding rice into fine rice, grinding into 100 mesh fine powder, and drying to obtain rice flour as sample.
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| CN1719221A (en) * | 2005-06-07 | 2006-01-11 | 中国科学院亚热带农业生态研究所 | A kind of method of measuring resistance starch content |
| CN102277399A (en) * | 2011-08-10 | 2011-12-14 | 王学勇 | Preparation and application of raw material of novel resistant starch |
| CN102533938A (en) * | 2010-12-16 | 2012-07-04 | 上海市农业科学院 | Resistant starch content measuring method for rice |
| CN109142281A (en) * | 2018-09-19 | 2019-01-04 | 河北大学 | The method for detecting Potato Resistant Starch content and structure based on field flow isolation technics |
| CN111220604A (en) * | 2019-12-06 | 2020-06-02 | 东北农业大学 | Method for measuring total starch content in meat product |
| AU2020101061A4 (en) * | 2020-06-19 | 2020-07-30 | Anhui Science And Technology University | A Method for Determination of Soluble Sugar Content in Stem Samples of Sweet Sorghum |
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| CN1719221A (en) * | 2005-06-07 | 2006-01-11 | 中国科学院亚热带农业生态研究所 | A kind of method of measuring resistance starch content |
| CN102533938A (en) * | 2010-12-16 | 2012-07-04 | 上海市农业科学院 | Resistant starch content measuring method for rice |
| CN102277399A (en) * | 2011-08-10 | 2011-12-14 | 王学勇 | Preparation and application of raw material of novel resistant starch |
| CN109142281A (en) * | 2018-09-19 | 2019-01-04 | 河北大学 | The method for detecting Potato Resistant Starch content and structure based on field flow isolation technics |
| CN111220604A (en) * | 2019-12-06 | 2020-06-02 | 东北农业大学 | Method for measuring total starch content in meat product |
| AU2020101061A4 (en) * | 2020-06-19 | 2020-07-30 | Anhui Science And Technology University | A Method for Determination of Soluble Sugar Content in Stem Samples of Sweet Sorghum |
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