Starch content rapid detection kit and application thereof

Abstract

The invention provides a rapid starch content detection kit and application thereof, and belongs to the technical field of food detection.The kit comprises a reaction microwell plate with glucose oxidase and horse radish peroxidase fixed in reaction microwells, amylase working fluid containing alpha-amylase, BSA and amylase diluent, and a reaction microwell plate with glucose oxidase and horse radish peroxidase fixed in reaction microwells, the saccharifying enzyme working solution consists of saccharifying enzyme, BSA (Bovine Serum Albumin) and saccharifying enzyme diluent; and the substrate solution contains tetramethyl benzidine. The starch content detection scheme provided by the invention has the characteristics of simple treatment steps, short time consumption, high specificity, high sensitivity, high accuracy and the like.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a rapid starch content detection kit and its application. Background Technology

[0002] Starch is a polysaccharide formed by the polymerization of glucose monomers and is one of the most important energy storage substances in plant tissues. It is widely found in the roots, stems, leaves, and seeds of plants, and is an important component of human food and a major source of energy for the human body.

[0003] Starch is widely used in fields such as biology, food science, medicine, and industrial production as a stabilizer, thickener, colloid forming agent, humectant, or emulsifier. Therefore, determining the starch content in a sample is of great significance.

[0004] Currently, there are many methods for determining starch content, mainly including the iodine-alkali method, gravimetric method, optical rotation method, and enzyme-colorimetric method. While the iodine-alkali method has high sensitivity, the detection error is relatively large, making it more suitable for samples with high starch content. The gravimetric method is cumbersome and time-consuming. The optical rotation method is affected by the properties and state of the sample, leading to significant differences in results. The enzymatic hydrolysis method has specificity and selectivity, providing accurate, reliable, and reproducible results, but it is also cumbersome and time-consuming. The enzyme-colorimetric method combines enzymatic hydrolysis with colorimetry, offering a simple and rapid operation, but requires a large variety of expensive reagents that are difficult to store, thus limiting its application. Therefore, a new method is needed to achieve a simple, rapid, and accurate detection of starch content in samples. Summary of the Invention

[0005] In view of this, the present invention aims to provide a rapid method and reagent kit for detecting starch content, so as to achieve rapid, low-cost and accurate and reliable detection of starch content.

[0006] In a first aspect, the present invention provides a rapid starch content detection kit, which includes at least the following: The reaction microplate has glucose oxidase and horseradish peroxidase immobilized in its reaction microwells, and the mass ratio of the two is (25-100):(2-20). A working solution of amylase containing α-amylase, BSA and amylase diluent, wherein the amylase diluent is a 0.01 mol / L disodium hydrogen phosphate-citrate buffer at pH 5.5; A working solution for saccharifying enzyme, consisting of saccharifying enzyme, BSA, and saccharifying enzyme diluent, wherein the saccharifying enzyme diluent is a 0.05 mol / L disodium hydrogen phosphate-citrate buffer at pH 4.0; Substrate solution containing tetramethylbenzidine (TMB).

[0007] Preferably, the mass ratio of glucose oxidase to horseradish peroxidase immobilized in the reaction wells is 12.5:1. In some embodiments of the present invention, the reaction wells are prepared by mixing the enzyme diluent, glucose oxidase, and horseradish peroxidase to prepare an enzyme mixture reagent, adding the enzyme mixture reagent to each reaction well, and freeze-drying. The enzyme diluent is a pH 5.0 disodium hydrogen phosphate-citrate buffer with added 0.5% BSA, 2.5% mannitol, 2.5% sorbitol, 0.1% glycine, and 0.1% preservative Proclin 950. In the enzyme mixture reagent, the final concentration of glucose oxidase is 0.025 mg / mL, and the final concentration of horseradish peroxidase is 0.002 mg / mL. This kit of the present invention immobilizes glucose oxidase and horseradish peroxidase in the wells at the most suitable amount using industrial methods, eliminating the need for sample addition steps during user operation, reducing operational errors, and better ensuring the stability of the detection results.

[0008] Preferably, the amylase working solution consists of α-amylase, BSA, preservative proclin 950, and amylase dilution, wherein the concentration of BSA is 1.5%, the concentration of preservative proclin 950 is 0.1%, and the concentration of α-amylase is 40 U / mL.

[0009] Preferably, the saccharifying enzyme working solution consists of saccharifying enzyme, BSA, preservative proclin 950, and saccharifying enzyme dilution solution, wherein the concentration of BSA is 1.5%, the concentration of preservative proclin 950 is 0.1%, and the concentration of saccharifying enzyme is 1.25 KU / mL.

[0010] Preferably, the substrate solution consists of disodium ethylenediaminetetraacetate, TMB, and 0.01M disodium hydrogen phosphate-citric acid buffer at pH 4.0, with the concentration of disodium ethylenediaminetetraacetate being 0.01% and the concentration of TMB being 0.05%.

[0011] The above kit may also include standards, stop solutions and sample diluents. Preferably, the standards are prepared from D-glucose and standard diluents, the stop solution is a 1 mol / L hydrochloric acid solution, and both the standard diluent and the sample diluent are 0.01 M disodium hydrogen phosphate-citrate buffer at pH 5.0.

[0012] Secondly, the present invention provides the application of the above-mentioned rapid starch content detection kit in detecting the starch content of samples, wherein the samples include, but are not limited to, food, grains, feed, etc.

[0013] Thirdly, the present invention provides a method for rapid detection of starch content, which utilizes the rapid starch content detection kit provided by the present invention, and specifically includes the following steps: S1. Add amylase working solution to the pretreated sample to be tested and react, then add saccharifying enzyme working solution and react, centrifuge and take the supernatant, and dilute it to obtain the sample test solution. S2. Add substrate solution and sample solution or standard to the reaction microwell, incubate in the dark, and after terminating the reaction, measure the absorbance value at wavelengths of 450nm / 630nm. S3. Establish a standard curve, and calculate the starch content based on the absorbance value of the sample solution and the standard curve. The calculation formula is as follows: ; Where a is the intercept in the standard curve equation, b is the slope in the standard curve equation, Y is the absorbance value of the sample solution, C is the sample dilution factor, 0.9 is the conversion factor, and X is the starch content in the sample in g / 100g.

[0014] In step S1 above, the pretreatment includes the following operations: after grinding and sieving the sample, it is treated with hexane and ethanol solutions in sequence, centrifuged and the precipitate is retained.

[0015] Preferably, the ratio of amylase working solution to saccharifying enzyme working solution is 1:1 (v / v).

[0016] The enzyme mixture pre-fixed in the microwells catalyzes the color development. After the reaction is terminated, the absorbance value is measured at 450 nm. A standard curve is established based on the concentration and absorbance value of the standard. The glucose content in the sample is then calculated using the absorbance value of the sample, and finally converted into starch content.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: The kit of this invention uses an enzyme catalysis method. When detecting samples, it is only necessary to add the standard or the prepared diluted sample and substrate solution to the reaction microwell. After the colorimetric reaction is terminated, the absorbance value is measured. The sample processing steps are simple and time-saving, and it has the characteristics of high specificity, high sensitivity and high accuracy.

[0018] This invention pre-fixes some reagents in the reaction microwells, ensuring reagent stability while simplifying the sample addition process. The small amount of reaction system saves raw materials, and detection can be performed directly after the reaction is complete, avoiding waste. The kit uses TMB as the chromogenic substrate, which is stable and non-toxic.

[0019] In the kit of this invention, all reagents are prepared using disodium hydrogen phosphate-citric acid buffer. By simply adjusting the ratio, buffers of various concentrations and pH values ​​can be prepared to meet the needs of the entire experiment. The preparation is simpler and the cost is relatively low. Attached Figure Description

[0020] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0021] Figure 1 This is a schematic diagram illustrating the composition of the rapid starch content detection kit provided in an embodiment of the present invention; Figure 2 The left image shows the phenomenon of starch hydrolysis detection in an embodiment of the present invention, and the right image shows the phenomenon after the reaction is terminated. Figure 3 This is a partial diagram showing the phenomena observed in the detection of starch hydrolysis using iodine reagent in Example 3 of the present invention; Figure 4 This is the standard curve graph established in Embodiment 6 of the present invention. Detailed Implementation

[0022] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, 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 pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0024] To address the technical problems of cumbersome operation, numerous and expensive reagents in existing enzymatic methods for starch content determination, this invention provides a rapid starch content detection kit and detection method. Through improvements to the reaction microplate and reagents, the detection kit not only has high sensitivity and simple composition, but also has short sample processing time, convenient operation, good stability, and lower detection cost.

[0025] like Figure 1 As shown, this embodiment of the invention provides a rapid starch content detection kit, which includes the following: Box 1, and reaction microplate 3 and reagents fixed inside the box by base 2; The reaction microplate consists of a plastic support and separate reaction microwell strips. Each reaction microwell contains an enzyme mixture that can catalyze the glucose reaction and ultimately make TMB appear colored. The mixture includes glucose oxidase and horseradish peroxidase in a mass ratio of (25-100):(2-20). The reagents include standard 4, 10× sample diluent 5, amylase working solution 6, saccharifying enzyme working solution 7, tetramethylbenzidine working solution (i.e., substrate solution) 8, and stop solution 9; among which, the standard is D-glucose solution, the stop solution is 1 mol / L hydrochloric acid solution, and the 10× sample diluent is a 10-fold concentrated solution of the sample diluent.

[0026] Furthermore, in some embodiments of the present invention, the mass ratio of glucose oxidase to horseradish peroxidase is 12.5:1, and the preparation method of the reaction microwells is as follows: the mixed enzyme diluent, glucose oxidase and horseradish peroxidase are mixed to prepare an enzyme mixture reagent, wherein the mixed enzyme diluent is a pH 5.0 disodium hydrogen phosphate-citrate buffer with 0.5% BSA, 2.5% mannitol, 2.5% sorbitol, 0.1% glycine and 0.1% preservative proclin 950 added, the concentration of glucose oxidase is 0.025 mg / mL and the concentration of horseradish peroxidase is 0.002 mg / mL; then 50 μL of enzyme mixture reagent is added to each reaction microwell, and the mixture is freeze-dried to obtain the final product.

[0027] Furthermore, in some embodiments of the present invention, the amylase working solution is composed of α-amylase, BSA, preservative and amylase diluent, wherein the amylase diluent is a 0.01 mol / L disodium hydrogen phosphate-citric acid buffer at pH 5.5, the BSA concentration is 1.5%, the concentration of the preservative proclin 950 is 0.1%, and the concentration of α-amylase is 40 U / mL.

[0028] Furthermore, in some embodiments of the present invention, the saccharifying enzyme working solution is composed of saccharifying enzyme, BSA, preservative and saccharifying enzyme diluent, wherein the saccharifying enzyme diluent is a 0.05 mol / L disodium hydrogen phosphate-citrate buffer at pH 4.0, the BSA concentration is 1.5%, the preservative proclin 950 concentration is 0.1%, and the saccharifying enzyme concentration is 1.25 KU / m.

[0029] Furthermore, in some embodiments of the present invention, the sample diluent is a 0.01M disodium hydrogen phosphate-citrate buffer with a pH of 5.0, and is supplemented with proclin 950 at a final concentration of 0.1% (v / v).

[0030] This invention also provides a detection method based on the above-mentioned rapid starch content detection kit, specifically: (1) Sample pretreatment: Mix about 0.1g of sample with n-hexane, shake and centrifuge and retain the precipitate; add ethanol solution to the precipitate, shake and centrifuge and retain the precipitate; add 0.5mL of amylase working solution, mix well and boil in water for 5min, then add 0.5mL of saccharifying enzyme working solution and boil in water at 60℃ for 20min; centrifuge and take the supernatant, dilute with sample diluent to obtain the sample solution to be tested; (2) Add the standard or the sample solution to the reaction microwell, then add the substrate solution and mix well. React in the dark. After the reaction is terminated, measure the absorbance value at 450 nm. Establish a standard curve based on the concentration and absorbance value of the standard. Then use the absorbance value of the sample to calculate the glucose content in the sample and finally convert it into starch content.

[0031] Furthermore, in some embodiments of the present invention, the reaction conditions for step (2) are: reaction at room temperature (25°C) for 15 minutes or more, or reaction at 40°C for 5 minutes or more.

[0032] With the combined use of the amylase working solution and the saccharifying enzyme working solution of this invention, sample processing can be completed in a short time (25 min). The reasons include: amylase hydrolysis is more effective under slightly acidic conditions; the concentration used is the optimal concentration after comparative adjustment; the micro-reaction system used, with an added volume of only 0.5 mL of amylase solution, allows for faster temperature rise in a water bath, and the operation of adding and mixing followed immediately by boiling in a water bath ensures that the sample reacts in a relatively uniform system with the largest contact area, preventing sample precipitation and accumulation that would slow down the reaction rate; the addition of a saccharifying enzyme dilution with a pH of 4 to the amylase working solution with a pH of 5.5 creates a mixed reaction system with a pH that provides the optimal reaction environment for the saccharifying enzyme.

[0033] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0034] Example 1 This example provides a reaction microplate and demonstrates the effect of changes in the amounts of glucose oxidase and horseradish peroxidase in the reaction microplates on the detection. The specific experiment is as follows: (1) Preparation of reagents.

[0035] Since glucose oxidase has a pH range of 3.5–6.5, with an optimal pH of around 5.0, and its pH range of 4.5–7.0 is basically the same; the optimal pH of horseradish peroxidase varies slightly depending on the hydrogen donor, but is mostly around 5; in addition, TMB can maintain high sensitivity at a pH of around 4.0; taking all factors into consideration, the pH of the standard diluent or sample diluent can be set to 5.0, the pH of the glucose oxidase and horseradish peroxidase diluents can be set to 5.0, and the pH of the TMB diluent can be set to 4.0.

[0036] Standard dilution: Prepare a 0.01M disodium hydrogen phosphate-citrate buffer solution with a pH of 5.0, and add proclin950 to make the final volume fraction (v / v) 0.1%.

[0037] Standard: Weigh 1.0000g of D-glucose, add 10mL of standard diluent, and dissolve to a concentration of 100mg / mL. Continue to dilute with standard diluent to 0.1mg / mL and 0.0125mg / mL as positive controls (P), and the standard diluent of 0mg / mL as negative control (N).

[0038] Substrate solution: Prepare a 0.01M disodium hydrogen phosphate-citrate buffer solution with pH 4.0, add disodium ethylenediaminetetraacetate to make the final mass concentration (m / v) 0.01%, and add TMB hydrochloride to make the final mass concentration (m / v) 0.05%.

[0039] Mixed enzyme dilution: Prepare a pH 5.0 disodium hydrogen phosphate-citrate buffer, add BSA to achieve a final mass concentration (m / v) of 0.5%, add mannitol to achieve a final mass concentration (m / v) of 2.5%, add sorbitol to achieve a final mass concentration (m / v) of 2.5%, add glycine to achieve a final mass concentration (m / v) of 0.1%, and add preservative proclin 950 to achieve a final volume fraction (v / v) of 0.1%.

[0040] Glucose oxidase solution: Weigh 10 mg of glucose oxidase powder (250 U / mg), add 2 mL of mixed enzyme diluent to dissolve it, and the concentration is 5 mg / mL. Then dilute it with mixed enzyme diluent to 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL and 0.0125 mg / mL for use.

[0041] Horseradish peroxidase solution: Weigh 20 mg of horseradish peroxidase powder (250 U / mg), add 2 mL of mixed enzyme diluent to dissolve it, and the concentration is 10 mg / mL. Then dilute it with mixed enzyme diluent to 0.5 mg / mL, 0.1 mg / mL, 0.02 mg / mL and 0.004 mg / mL for use.

[0042] (2) Detection.

[0043] Take an appropriate amount of clean microwells and determine the absorbance using a matrix titration experiment. The absorbance value of the blank control, close to 0, is used as the reference standard. The higher the P / N value, the more sensitive the reagent and the better the effect. Add 25 μL of the diluted horseradish peroxidase to each well, then 25 μL of the diluted glucose oxidase to each well, followed by 50 μL of substrate solution to each well, and finally 50 μL of the standard solution at the above concentration to each well. After mixing, cover with a light-proof plate and incubate at room temperature (25℃) for 30 minutes. Add 50 μL of stop solution (1 mol / L hydrochloric acid solution), and measure the absorbance at 450 nm / 630 nm wavelengths using an automated microplate reader. The results are shown in Table 1.

[0044] Table 1. Detection results at different enzyme concentrations

[0045] The data in Table 1 show that when the glucose oxidase concentration was 0.05 mg / mL and the horseradish peroxidase concentration was 0.004 mg / mL, the blank control was 0.0052, which was very close to 0, and the P / N value was also the highest.

[0046] (3) Preparation of reaction micropores.

[0047] The reaction micropores are prepared according to the optimal concentration ratio obtained in step (2), and the preparation method is as follows: Take an appropriate amount of the mixed enzyme dilution solution, add glucose oxidase to make a final concentration of 0.025 mg / mL, add horseradish peroxidase to make a final concentration of 0.002 mg / mL, mix well and prepare an enzyme mixture reagent; then take an appropriate amount of clean microwells, add the prepared enzyme mixture reagent into each well (50 μL), freeze dry it using freeze-drying technology and store it for later use.

[0048] Example 2 Based on the rapid starch content detection kit provided by this invention, this example verifies the temperature and time conditions required for its detection. The specific experimental procedure is as follows: (1) Preparation of required reagents.

[0049] Substrate solution: Prepare a 0.01M disodium hydrogen phosphate-citrate buffer solution with pH 4.0, add disodium ethylenediaminetetraacetate to bring the final mass concentration (m / v) to 0.01%, and add TMB hydrochloride to bring the final mass concentration (m / v) to 0.05%.

[0050] Standard: Weigh 1.0000g of D-glucose and add 10mL of standard diluent (same as in Example 1). After dissolution, the concentration is 100mg / mL. Continue to dilute with standard diluent to 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, and 2μg / mL to prepare standard solutions. The standard diluent of 0mg / mL is used as a blank.

[0051] (2) Detection under different reaction conditions.

[0052] Take an appropriate amount of reaction microwells (preparation conditions as in Example 1), add 50 μL of substrate solution to each well, then add 100 μL of the above standard or blank control to each well, mix well, and cover with a light-proof plate. Incubate at room temperature (25℃) for 5, 10, 15, 20, 25, and 30 minutes. Add 50 μL of stop solution (1 mol / L hydrochloric acid solution), and measure the absorbance at 450 nm / 630 nm wavelengths using an automated microplate reader. The results are shown in Table 2. The reaction phenomena are as follows. Figure 2 As shown.

[0053] Table 2 Optimization of reaction time at room temperature (25℃)

[0054] Take an appropriate amount of reaction microwells (preparation conditions are the same as in Example 1), add 50 μL of substrate solution to each well, then add 100 μL of the above standard or blank control to each well, mix well, and then cover with a light-proof plate. Incubate at 40°C for 5, 10, and 15 minutes, add 50 μL of stop solution, and measure the absorbance value at 450 nm / 630 nm wavelength using an automated microplate reader. The results are shown in Table 3.

[0055] Table 3 Optimization of reaction time at 40℃

[0056] The data in Tables 2 and 3 show that the reaction tends to stabilize after 15 minutes at room temperature (25℃), and when the temperature is increased to 40℃, the reaction can be completed in just 5 minutes.

[0057] Example 3 This example provides an amylase working solution whose pH can accommodate the working conditions of both amylase and saccharifying enzyme.

[0058] (1) Determination of amylase dilution solution.

[0059] Prepare the following three amylase dilution solutions: Amylase dilution 1: 0.01 mol / L disodium hydrogen phosphate-citric acid buffer, pH 5.5; Amylase dilution 2: 0.01 mol / L disodium hydrogen phosphate-citric acid buffer, pH 6.0; Amylase dilution 3: 0.01 mol / L disodium hydrogen phosphate-citric acid buffer, pH 6.5.

[0060] Amylase solutions: Weigh out 1.000g of α-amylase powder (40KU / g) in 3 portions, add 10mL of the above 3 diluents to each portion, vortex at 2500 rpm for 10 minutes, centrifuge at 4000 rpm for 10 minutes, take the supernatant and dilute with the corresponding diluents to obtain 5 amylase solutions of different concentrations.

[0061] Three groups of 0.1000g soluble starch samples were weighed, corresponding to three dilution solutions, with 15 tubes in each group. Five different concentrations of amylase solution were prepared for each dilution solution, with three tubes for each concentration, for comparing the hydrolysis time of different enzymes. 0.45mL of the dilution solution was transferred to the corresponding sample tube, followed by 0.05mL of the corresponding concentration of amylase solution. After vortexing and mixing, the tubes were immediately placed in a boiling water bath for 5min, 10min, and 15min, respectively. Iodine reagent was used to determine whether the starch was completely hydrolyzed. The results are shown in Table 4, and some phenomena are observed. Figure 3 Tube 1 shows the result after diluting amylase solution 1 80 times and incubating in water for 15 minutes; tube 2 shows the result after diluting amylase solution 1 40 times and incubating in water for 5 minutes; tube 3 shows the result after diluting amylase solution 1 40 times and incubating in water for 10 minutes; tube 4 shows the result after diluting amylase solution 1 40 times and incubating in water for 15 minutes; tube 5 shows the result after diluting amylase solution 1 20 times and incubating in water for 5 minutes; tube 2 shows the result after diluting amylase solution 1 20 times and incubating in water for 10 minutes; tube 7 shows the result after diluting amylase solution 1 20 times and incubating in water for 15 minutes; tube 8 shows the result after diluting amylase solution 1 10 times and incubating in water for 5 minutes; tube 9 shows the result after diluting amylase solution 1 10 times and incubating in water for 10 minutes; and tube 10 shows the result after diluting amylase solution 1 10 times and incubating in water for 15 minutes.

[0062] Table 4 Comparison of amylase dilution, concentration, and reaction time

[0063] Note: √ indicates complete hydrolysis, × indicates incomplete hydrolysis.

[0064] The above results indicate that 0.01 mol / L disodium hydrogen phosphate-citric acid buffer solutions with a pH of 5.5-6.5 can all serve as reaction buffer systems for amylase, showing little difference in enzymatic hydrolysis efficiency and meeting experimental requirements. Given that the optimal pH for saccharifying enzymes is around 4.0-4.5, this example uses a 0.01 mol / L disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.5 as the amylase dilution solution. Furthermore, Table 4 shows that when α-amylase powder is treated and used as described in this example, depending on its concentration, the sample can be completely hydrolyzed within 5-15 minutes; moreover, using a 10-fold diluted amylase solution, a boiling water bath for 5 minutes completely hydrolyzes 0.1000 g of soluble starch.

[0065] (2) Preparation of amylase working solution.

[0066] Weigh 1.000 g (40 KU / g) of α-amylase powder into a 15 mL centrifuge tube, add 10 mL of 0.01 mol / L disodium hydrogen phosphate-citric acid buffer (pH 5.5), vortex at 2500 rpm for 10 minutes, centrifuge at 4000 rpm for 10 minutes, and collect 9 mL of the supernatant. Add this supernatant to 891 mL of 0.01 mol / L disodium hydrogen phosphate-citric acid buffer (pH 5.5), add BSA to achieve a final mass concentration (m / v) of 1.5%, and add proclin 950 preservative to achieve a final volume fraction (v / v) of 0.1%. The resulting amylase working solution (40 U / mL) can be stored at 4 °C.

[0067] (3) Stability test of amylase working solution.

[0068] The prepared amylase working solution (40 U / mL) was divided into two portions and stored separately at 4℃ and 37℃, respectively, to detect the difference in activity. Specifically, two tubes containing 0.1000 g of soluble starch sample were weighed, and 0.5 mL of the amylase working solution (40 U / mL) stored at 4℃ and 37℃, respectively, were added. After vortexing and mixing, the tubes were immediately placed in a boiling water bath for 5 min. Iodine reagent was used to test whether the starch was completely hydrolyzed. The results are shown in Table 5. The results indicate that this amylase working solution can be stably stored for a certain period of time.

[0069] Table 5. Comparison of amylase working solution activity at 4℃ and 37℃.

[0070] Note: √ indicates complete hydrolysis, × indicates incomplete hydrolysis.

[0071] Example 4 Based on the amylase working solution in Example 3, this example further provides a compatible saccharifying enzyme working solution, and the specific experimental procedure is as follows: (1) Determination of the dilution solution for saccharifying enzyme.

[0072] Prepare the following dilution of saccharifying enzyme: The saccharifying enzyme dilution solution was 1:0.01 mol / L disodium hydrogen phosphate-citric acid buffer, pH 4.0. Saccharifying enzyme dilution 2: 0.05 mol / L disodium hydrogen phosphate-citrate buffer, pH 4.0; Saccharifying enzyme dilution 3: 0.1 mol / L disodium hydrogen phosphate-citric acid buffer, pH 4.0.

[0073] Standard: Weigh 1.0000g of D-glucose and add 10mL of standard diluent (same as in Example 1). After dissolving, the concentration is 100mg / mL. Continue to dilute with standard diluent to 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, and 2μg / mL to prepare standard solutions. The standard diluent of 0mg / mL is used as a blank control. Amylase working solution: Weigh 1.000 g of α-amylase powder into a 15 mL centrifuge tube, add 10 mL of 0.01 mol / L disodium hydrogen phosphate-citric acid buffer with pH 5.5, vortex at 2500 rpm for 10 minutes, centrifuge at 4000 rpm for 10 minutes, take 9 mL of the supernatant, add it to 891 mL of 0.01 mol / L disodium hydrogen phosphate-citric acid buffer with pH 5.5, add BSA to make the final mass concentration (m / v) reach 1.5%, add preservative proclin 950 to make the final volume fraction (v / v) 0.2%, and finally obtain amylase working solution (40 U / mL).

[0074] Saccharifying enzyme solution: Weigh 3 portions of saccharifying enzyme powder (100KU / g), each 1.000g, add 10mL of the above 3 saccharifying enzyme diluents, vortex at 2500 rpm for 10 minutes, centrifuge at 4000 rpm for 10 minutes, take the supernatant and dilute with the corresponding diluents to obtain 4 saccharifying enzyme solutions of different concentrations.

[0075] Weigh 0.1000g of soluble starch sample into 3 groups, corresponding to 3 dilution solutions, 12 tubes per group. For each dilution solution, prepare 4 different concentrations of amylase solution, 3 tubes per concentration, for comparison of different saccharification times. Add 0.5mL of amylase working solution (40U / mL) to the corresponding sample tubes, vortex to mix, and immediately place in a boiling water bath for 5 minutes. Remove and slightly cool with cool water, then add 0.5mL of different concentrations of amylase solution, mix well, and incubate at 60℃ for 10 minutes, 20 minutes, and 40 minutes (mixing appropriately during incubation). After incubation, centrifuge at 4000 rpm for 1 minute, collect the supernatant, and dilute with sample dilution solution (same as standard dilution solution) at a ratio of 1:10000 before testing.

[0076] Take an appropriate amount of reaction microwells (preparation conditions are the same as in Example 1), add 50 μL of substrate solution to each well, then add 100 μL of the above-mentioned test samples to each well, mix well, attach a light-proof plate, incubate at 40°C for 5 minutes, then add 50 μL of stop solution (1 mol / L hydrochloric acid solution), and measure the absorbance values ​​at 450 nm / 630 nm wavelengths using an automatic microplate reader. The results are shown in Table 6.

[0077] Table 6 Comparison of saccharifying enzyme dilution, concentration, and reaction time

[0078] As can be seen from the data in Table 6, when the saccharifying enzyme powder is processed and used in the manner described in this example, and 0.01 mol / L disodium hydrogen phosphate-citrate buffer at pH 4.0 is used as the saccharifying enzyme diluent, a higher concentration of saccharifying enzyme and a longer working time are required to completely saccharify the sample. When 0.05 mol / L disodium hydrogen phosphate-citrate buffer at pH 4.0 and 0.1 mol / L disodium hydrogen phosphate-citrate buffer at pH 4.0 are used as the saccharifying enzyme diluents, the 8-fold diluted saccharifying enzyme working solution can completely saccharify the sample in 20 minutes.

[0079] (2) Preparation of saccharifying enzyme working solution.

[0080] Weigh 1.000 g (100 KU / g) of glucoamylase powder into a 15 mL centrifuge tube, add 10 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer (pH 4.0), vortex at 2500 rpm for 10 minutes, centrifuge at 4000 rpm for 10 minutes, and collect 9 mL of the supernatant. Add this supernatant to 63 mL of 0.05 mol / L disodium hydrogen phosphate-citric acid buffer (pH 4.0), add BSA to achieve a final mass concentration (m / v) of 1.5%, and add proclin 950 preservative to achieve a final volume fraction (v / v) of 0.1%. The resulting glucoamylase working solution (1.25 KU / mL) can be stored at 4 °C.

[0081] (3) Stability test of the saccharifying enzyme working solution.

[0082] The prepared amylase working solution (1.25 KU / mL) was divided into two portions and stored separately at 4℃ and 37℃, respectively, to detect the difference in activity. The specific procedure was as follows: Weigh 0.1000 g of soluble starch sample into two tubes, add the amylase working solution (40 U / mL) stored at 4℃, vortex to mix, and immediately place in a boiling water bath for 5 min. After slightly cooling with cool water, add 0.5 mL of the amylase working solution (1.25 KU / mL) stored at 4℃ or 37℃, mix well, and incubate in a 60℃ water bath for 20 min (mixing occasionally). After incubation, centrifuge at 4000 rpm for 1 minute, collect the supernatant, dilute with sample diluent at a ratio of 1:10000, and then analyze. The activity was evaluated by sample recovery rate, and the results are shown in Table 7. The results indicate that this amylase working solution can be stably stored for a certain period of time.

[0083] Table 7 Comparison of the activity of glucoamylase working solution at 4℃ and 37℃

[0084] Example 5 This example provides a rapid starch content detection kit, which includes at least the following: Reaction microplate: The reaction microplate consists of a plastic support and separate reaction microwell strips, and each reaction microwell is prepared according to the method in Example 1.

[0085] Standards: A series of D-glucose solutions of different concentrations prepared from standard dilution (same as in Example 1) and D-glucose.

[0086] 10× Sample Diluent: A 10-fold concentrate of the sample diluent, wherein the sample diluent is the same as the standard diluent in Example 1.

[0087] Amylase working solution: The preparation method is the same as in Example 3.

[0088] Saccharifying enzyme working solution: The preparation method is the same as in Example 4.

[0089] Substrate liquid: The preparation method is the same as in Example 1.

[0090] Termination solution: 1 mol / L hydrochloric acid solution.

[0091] Example 6 The rapid starch content detection kit described in Example 5 was used to test various real samples, and the detection results were compared with the starch content results obtained from testing these real samples according to the GB022939-2023 standard to evaluate the reliability of the kit and method of the present invention.

[0092] (1) Sample preparation.

[0093] Take samples of different types of food, grains, feed, etc., grind them, and pass them through a 40-mesh sieve for later use.

[0094] (2) Sample processing.

[0095] Weigh 0.1g of the sample with known starch content; add 1mL of n-hexane, vortex and centrifuge, discard the supernatant and retain the precipitate; add 1mL of 85% ethanol solution, vortex and centrifuge, discard the supernatant and retain the precipitate; add 0.5mL of amylase working solution, vortex and mix well, then immediately place in a boiling water bath for 5 minutes; remove and cool slightly in cold water, then add 0.5mL of saccharifying enzyme working solution, vortex and mix well, then place in a water bath at 60℃ for 20 minutes (it can be removed and vortexed twice during the process), then centrifuge at 4000r / min for 1min, take the supernatant and dilute it 500-10000 times with the sample diluent before testing, this factor is the sample dilution factor.

[0096] (3) Detection.

[0097] Bring all reagents in the kit, except for the amylase and glucoamylase working solutions, to room temperature, with the reaction wells warmed as needed. Place the reaction wells on the microplate holder and add 50 μL of substrate solution to all wells. Add 100 μL of standard solution or sample solution to the corresponding reaction wells and tap the edge of the holder to mix. Cover with a light-proof plate and incubate at 40°C for 5 minutes. Add 50 μL of stop solution to each well and measure the absorbance at 450 nm / 630 nm using an automated microplate reader.

[0098] (4) Establishment of standard curve.

[0099] A standard curve was constructed with the concentration of the standard sample as the x-axis and the corresponding absorbance value as the y-axis, yielding a linear equation (see ①). The linear graph is shown below. Figure 4 .

[0100] ①; Where a is the intercept, b is the slope, x is the concentration of the standard / sample test solution in μg / mL, and y is the absorbance value of the standard / sample test solution.

[0101] (5) Calculation of results.

[0102] Based on the absorbance value of the sample solution and linear equation ①, the starch content in the sample solution can be calculated in g / 100g. The calculation formula is shown in ②. The calculation results are shown in Table 8.

[0103] ②; Where a is the intercept in Equation ①, b is the slope in Equation ①, Y is the absorbance value of the sample solution to be tested, C is the sample dilution factor, 0.9 is the conversion factor, and X is the starch content in the sample to be tested, in g / 100g.

[0104] Table 8 Detection results of actual samples

[0105] The above results indicate that the starch content of the samples detected using the kit of this invention is very close to the content detected by the national standard GB5009.9-2023.

[0106] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A starch content rapid detection kit, characterized in that, At least comprising the following: a reaction microplate, glucose oxidase and horseradish peroxidase are fixed in the reaction microcavity of the reaction microplate, and the mass ratio of the two is (25-100):(2-20); amylase working solution containing α-amylase, BSA and amylase diluent, wherein the amylase diluent is 0.01 mol / L disodium hydrogen phosphate-citric acid buffer solution with pH 5.5; glucoamylase working solution containing glucoamylase, BSA and glucoamylase diluent, wherein the glucoamylase diluent is 0.05 mol / L disodium hydrogen phosphate-citric acid buffer solution with pH 4.0; substrate solution containing tetramethyl benzidine.

2. The starch content rapid detection kit according to claim 1, characterized in that, The preparation method of the reaction microcavity is as follows: mixing enzyme diluent, glucose oxidase and horseradish peroxidase to prepare enzyme mixed reagent, adding the enzyme mixed reagent into each reaction microcavity, and freeze-drying to obtain the reaction microcavity; wherein the enzyme diluent is disodium hydrogen phosphate-citric acid buffer solution with pH 5.0 and added with 0.5% BSA, 2.5% mannitol, 2.5% sorbitol, 0.1% glycine and 0.1% preservative proclin 950, the concentration of the glucose oxidase is 0.025 mg / mL, and the concentration of the horseradish peroxidase is 0.002 mg / mL.

3. The starch content rapid detection kit according to claim 1, characterized in that, The amylase working solution is composed of α-amylase, BSA, preservative proclin 950 and amylase diluent, the concentration of the BSA is 1.5%, the concentration of the preservative proclin 950 is 0.1%, and the concentration of the α-amylase is 40 U / mL.

4. The starch content rapid detection kit according to claim 1, characterized in that, The glucoamylase working solution is composed of glucoamylase, BSA, preservative proclin 950 and glucoamylase diluent, the concentration of the BSA is 1.5%, the concentration of the preservative proclin 950 is 0.1%, and the concentration of the glucoamylase is 1.25 KU / mL.

5. The starch content rapid detection kit according to claim 1, characterized in that, The substrate solution is disodium hydrogen phosphate-citric acid buffer solution with pH 4.0 and 0.01 M, added with 0.01% disodium ethylenediaminetetraacetate and 0.05% tetramethyl benzidine.

6. The starch content rapid detection kit according to claim 1, characterized in that, The kit further comprises at least one of a standard, a termination solution and a sample diluent, the standard is prepared from D-glucose and standard diluent, the termination solution is 1 mol / L hydrochloric acid solution, and the standard diluent and the sample diluent are both 0.01 M disodium hydrogen phosphate-citric acid buffer solution with pH 5.

0.

7. The starch content rapid detection kit according to any one of claims 1-6 is applied to detecting the starch content of a sample.

8. Use according to claim 6, characterized in that, The sample includes food, grain and feed.

9. A method for rapid detection of starch content, characterized by, The starch content rapid detection kit according to claim 1 is used for detection, and the detection includes the following steps: S1, adding amylase working solution into the pretreated sample to be detected for reaction, then adding glucoamylase working solution for reaction, centrifuging to obtain upper clear liquid, and diluting to obtain sample to be tested liquid; S2, adding substrate solution and sample to be tested liquid or standard into the reaction microcavity, incubating in dark, measuring absorbance value at 450 nm / 630 nm wavelength after termination of reaction. S3, a standard curve is established, and the starch content is calculated according to the absorbance value of the sample to be tested and the standard curve, and the calculation formula is: ； Wherein, a is the intercept in the standard curve equation, b is the slope in the standard curve equation, Y is the absorbance value of the sample to be tested, C is the sample dilution multiple, 0.9 is the conversion coefficient, and X is the starch content in the sample to be tested and the unit is g / 100g.

10. The method for rapid detection of starch content according to claim 9, characterized in that, The pretreatment in step S1 includes: after the sample is ground and sieved, the sample is treated with n-hexane and ethanol solution in sequence, centrifuged and the precipitate is reserved.

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