Kit for detecting starch content and detection method

Abstract

The invention relates to the technical field of food analysis and detection, in particular to a kit for detecting starch content and a detection method. The kit comprises 4000 to 7000 U / mL of heat-resistant alpha-amylase, 3000 to 6000 U / mL of amyloglucosidase, a GOPOD color developing reagent, a GOPOD color developing reagent buffer solution, a 1.0 mg / mL D-glucose standard solution and a standardized starch sample, and all the reagents are subpackaged in sealed bottles. The kit for detecting the starch content and the detection method have the advantages of simplicity and convenience in operation, rapidness in detection, accurate result, good stability and the like; the application range is wide, and the total starch content in various food, feed, plants and cereal products can be detected.

Description

Technical Field

[0001] This invention relates to the field of food analysis and testing technology, and more specifically, to a reagent kit and method for detecting starch content. Background Technology

[0002] Starch is a major source of energy for humans, constituting a significant component of grains, tubers, and various processed foods. Its content is a crucial indicator for food quality control, nutritional evaluation, and product development. Common methods for starch detection include acid hydrolysis, enzymatic hydrolysis, and polarimetry. Currently, the most accurate and specific starch determination methods are largely based on enzymatic hydrolysis combined with colorimetric analysis. This involves converting starch into D-glucose using amylase and amyloglucosidase. The resulting D-glucose is then oxidized by glucose oxidase (GOD) to produce D-gluconic acid and hydrogen peroxide. The latter, in the presence of peroxidase (POD), reacts with the chromogenic substrate phenol to form a red quinone imine compound. The starch content is calculated by measuring the absorbance of this colored compound. Despite the high accuracy and specificity of enzymatic hydrolysis, existing detection methods still have the following shortcomings in practical applications:

[0003] (1) Poor reagent stability: especially enzyme reagents, which usually have a shelf life of only about one month at 4°C, making it difficult to meet the actual needs of use;

[0004] (2) Cumbersome operation: The types of reagents are scattered, there are many operation steps, and the dependence on the experimental personnel is high;

[0005] (3) In complex food matrices, the detection results are easily affected by fluctuations in reaction conditions and background interference, resulting in insufficient repeatability and stability.

[0006] Therefore, it is necessary to provide a starch content determination kit and its detection method that has a complete reaction system, is easy to operate, and is suitable for batch detection, so as to improve detection efficiency and data reliability. Summary of the Invention

[0007] The purpose of this invention is to provide an integrated, highly stable starch content detection kit and detection method to solve the problems of poor reagent stability, cumbersome operation, difficulty in detecting resistant starch, and poor repeatability of detection results in the prior art.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A kit for detecting starch content includes 4000-7000 U / mL thermostable α-amylase, 3000-6000 U / mL amylase, GOPOD chromogenic reagent, GOPOD chromogenic reagent buffer, 1.0 mg / mL D-glucose standard solution, and standardized starch sample, with each reagent aliquoted into sealed bottles.

[0010] The GOPOD colorimetric reagent is a mixture of 3000-7000 U / mL glucose oxidase, 2000-6000 U / mL horseradish peroxidase, and 0.4-0.6 mmol / L 4-aminoantipyrine.

[0011] The GOPOD colorimetric reagent buffer solution is a 40-60 mmol / L phosphate buffer solution with a pH of 7.4.

[0012] According to a preferred embodiment of the present invention, 0.02% by mass of Proclin 300 is added to the heat-resistant α-amylase, amyloglucosidase, GOPOD colorimetric reagent, and GOPOD colorimetric reagent buffer.

[0013] According to a preferred embodiment of the present invention, 0.05-0.12% by mass of 4-hydroxybenzoic acid is added to the heat-resistant α-amylase, amyloglucosidase, and GOPOD colorimetric reagent.

[0014] According to a preferred embodiment of the present invention, the GOPOD colorimetric reagent buffer is a 50 mmol / L phosphate buffer solution with pH 7.4.

[0015] This invention also provides a method for detecting starch content, comprising the following steps:

[0016] (1) Mix the food sample to be tested with ethanol solution to fully disperse the starch in the sample, and add heat-resistant α-amylase diluted 30 times with HEPES buffer for preliminary hydrolysis;

[0017] (2) After initial hydrolysis, add acetic acid-sodium acetate buffer solution to adjust the pH of the solution to acidic, add amyloglucosidase solution, and perform secondary hydrolysis in a water bath at 50-60℃;

[0018] (3) Dissolve the GOPOD colorimetric reagent in the GOPOD colorimetric reagent buffer to obtain the GOPOD working solution, and mix it with the reaction solution obtained in step (2); at the same time, prepare a reagent blank and a mixture of 0.1 mL glucose standard solution and GOPOD working solution;

[0019] (4) Use a spectrophotometer at a wavelength of 510 nm, zero the sample with a reagent blank, and measure the absorbance value; and calculate the starch content in the sample based on the molecular weight conversion relationship.

[0020] According to a preferred embodiment of the present invention, the preliminary hydrolysis in step (1) is carried out at 75-85°C.

[0021] According to a preferred embodiment of the present invention, the ethanol solution in step (1) is an aqueous ethanol solution with a volume concentration of 80%.

[0022] According to a preferred embodiment of the present invention, the mixture obtained in step (3) is incubated in a water bath at 50-55°C for 20 minutes before testing.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The reagent kit for detecting starch content of the present invention, by adding enzyme stabilizers and preservatives, extends the shelf life of the reagent from one month in traditional methods to more than one year, greatly reducing reagent waste and the hassle of frequent preparation. It replaces the toxic and harmful additives in traditional reagent kits, reducing the opportunity for laboratory personnel to directly contact harmful chemicals and improving experimental safety. The reagents are pre-packaged, simplifying the operation steps, reducing the labor intensity of testing personnel, and improving work efficiency. It provides standardized quality control materials and standard curve materials, reducing operational differences between laboratories and between laboratory personnel, and improving the repeatability and accuracy of test results.

[0025] 2. The method for detecting starch content of the present invention can complete the analysis of a single sample within 70 minutes and 20 samples within 2 hours, which greatly improves the detection efficiency; it has the advantages of simple operation, rapid detection, accurate results, and good stability; it has a wide range of applications and can detect the total starch content in various foods, feeds, plant and grain products (natural or processed). Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The images show the results of the detection method of the present invention after detecting wheat flour, corn starch, and potato starch.

[0028] Figure 2 A comparison of the detection results of wheat flour, corn starch, and potato starch using the detection method of this invention and the official AOAC method;

[0029] Figure 3Line graph showing repeated determinations of a 71.9% starch matrix sample;

[0030] Figure 4 This describes the stability changes of the reagent kit of the present invention over extended storage time;

[0031] Figure 5 The standard curve for the detection of glucose using GOPOD colorimetric reagent. Detailed Implementation

[0032] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used that do not specify the manufacturer are all commercially available conventional products.

[0033] Example 1

[0034] The kit for detecting starch content is available in various specifications, including 10, 20, 50, or 100 tests for total starch content. Taking the 50-test kit as an example, the kit includes the following reagent vials:

[0035] Reagent bottle 1: Heat-resistant α-amylase (5 mL) with an enzyme activity of 4000-7000 U / mL, containing 0.05% 4-hydroxybenzoic acid and 0.02% Proclin 300 by mass. It can be stably stored at 4℃ for more than 1 year.

[0036] Reagent bottle 2: Amylase with an enzyme activity of 3000-6000 U / mL (5mL), containing 0.05% 4-hydroxybenzoic acid and 0.02% Proclin 300 by mass. It can be stored stably at 4℃ for more than 1 year.

[0037] Reagent bottle 3: GOPOD colorimetric reagent buffer - 50 mmol / L phosphate buffer solution (10×, 25 mL, pH 7.4), with 0.02% Proclin 300 added, can be stably stored at 4℃ for more than 1 year;

[0038] Reagent Bottle 4: GOPOD colorimetric reagent (5 vials, 10 kits / vial, store at 4℃ / -20℃): a mixture of 3000-7000 U / mL glucose oxidase, 2000-6000 U / mL horseradish peroxidase and 0.4-0.6 mmol / L 4-aminoantipyrine, with the addition of 0.02% Proclin 300 and 0.05% 4-hydroxybenzoic acid. It can be stably stored at 4℃ for more than 1 year.

[0039] Reagent bottle 5: D-glucose standard solution (5 mL, 1.0 mg / mL), can be stably stored for more than 2 years at room temperature;

[0040] Reagent bottle 6: Standardized corn starch quality control sample, can be stably stored for more than 2 years at room temperature.

[0041] The HEPES buffer, acetate-sodium acetate buffer, and ethanol solution used in the test are not included in the kit and should be prepared fresh for each use.

[0042] Example 2: Method for detecting starch content

[0043] (1) Preparation of reagents

[0044] 80% ethanol solution (by volume): Add 20 mL of deionized water to 80 mL of anhydrous ethanol, shake well, and store at room temperature.

[0045] HEPES buffer (50 mmol / L, pH 7.0): Weigh 1.19 g HEPES, add 80 mL of deionized water, adjust the pH to 7.0 with NaOH solution, bring the volume to 100 mL, add 0.074 g anhydrous calcium chloride, stir to dissolve, and store at 4°C. The calcium ions in the buffer are activators of heat-stable α-amylase.

[0046] Preparation of acetic acid-sodium acetate buffer (200 mmol / L, pH 4.5): Measure 11.6 mL of glacial acetic acid, add 800 mL of deionized water, adjust the pH to 4.5 with 1 mol / L NaOH solution, bring the volume to 1000 mL, add 0.56 g of anhydrous calcium chloride, stir to dissolve, and store at 4℃.

[0047] GOPOD working solution: Take one vial of GOPOD colorimetric reagent, dissolve it in 50 mL of phosphate buffer solution, shake well, and store at 4℃ (use within 24 h).

[0048] (2) Weigh 100 mg of the sample to be tested into the bottom of the test tube, add 0.2 mL of 80% ethanol aqueous solution, shake well, add 0.1 mL of reagent bottle 1 (thermostable α-amylase) and 2.9 mL of HEPES buffer, shake well, react in an 85℃ water bath for 10 min, shake the test tube vigorously every 2~3 min to ensure the solution is uniform, so that the α-amylase hydrolyzes the starch into maltose.

[0049] (3) Add 4.6 mL of acetate-sodium acetate buffer to the above test tube, add 0.2 mL of reagent bottle 2 (amylase), shake well, and react at 50℃ for 30 min to hydrolyze maltose into glucose.

[0050] (4) Transfer all the liquid in the above test tube to a 100 mL volumetric flask, and make up to 100 mL with acetic acid-sodium acetate buffer. Take 2 mL of liquid from the volumetric flask, centrifuge at 10000×g for 3 min at room temperature, and record it as sample A. Take another test tube A, add 0.1 mL of sample A and 3 mL of GOPOD working solution, and react at 50℃ for 20 min.

[0051] Simultaneously prepare D-glucose standard control and blank control. D-glucose standard control: Take another test tube B, add 0.1 mL of D-glucose standard solution and 3 mL of reagent GOPOD working solution, and react at 50℃ for 20 min. Blank control: Take another test tube C, add 0.1 mL of deionized water and 3 mL of GOPOD working solution, and react at 50℃ for 20 min.

[0052] (5) After the reaction is completed, cool to room temperature and then perform absorbance detection (complete within 1 hour). In a 96-well plate, take 0.2 mL of the reaction solution from tubes A, B, and C respectively, place them in an ELISA reader, and measure the absorbance at 510 nm. The difference between the absorbance value of the test sample A and the absorbance value of the blank control is recorded as dA.

[0053] The determination of the absorbance value of the D-glucose standard control can be used to convert the absorbance value of the test sample into the amount of glucose generated in the entire reaction. That is, in the above reaction 3, the mass of D-glucose contained in 0.1 mL of 1.0 mg / mL D-glucose standard solution is 100 μg. This indicates that the amount of glucose generated in the test sample in the reaction (μg) = (100 μg (D-glucose)) / absorbance value of glucose standard control × dA. The ratio of the mass of D-glucose (100 μg) to its absorbance is denoted as F.

[0054] The normal range for absorbance measurement using an ELISA reader is between 0 and 2.5. If the absorbance exceeds this range, the reaction solution after reaction 3 can be diluted with deionized water by a factor of D.

[0055] (6) Calculation of results:

[0056]

[0057]

[0058] In the formula:

[0059] dA: The difference between the absorbance value of the sample to be tested and the absorbance value of the blank control;

[0060] F: A conversion factor that converts the absorbance value of the sample to D-glucose content, representing how many μg of D-glucose corresponds to each unit of absorbance;

[0061] 100 / 0.1: Volume correction factor. At the start of reaction 3, 0.1 mL of the test sample is taken from 100 mL of total solution for the reaction.

[0062] D: The dilution factor of the reaction solution after reaction 3 is completed;

[0063] W: Weight of the sample to be tested (100 mg);

[0064] 162 / 180: Molecular weight conversion factor. Starch is 162 g / mol and glucose is 180 g / mol. The measured amount of glucose is converted into the amount of starch in the sample according to the ratio of relative molecular mass.

[0065] 1 / 1000: Converts μg to mg.

[0066] To verify the detection performance of this kit, the starch content of various samples was determined and compared using the detection method of this invention and conventional methods.

[0067] Example 3 Accuracy Experiment

[0068] Common samples such as wheat flour, corn starch, and potato starch were selected, and their starch content was determined using the kits and detection methods of Example 1 and Example 2 of this invention, as well as the AOAC official method (996.11). The results of the kit's detection of wheat flour, corn starch, and potato starch are shown in the figures below. Figure 1 As shown, A is the reaction solution after the hydrolysis of maltose to glucose is complete; B is the color of the reaction solution after the colorimetric reaction is complete. The comparison results of the measured starch content are as follows: Figure 2 As shown in the figure. The results showed that there was no significant difference in the determination results between the two methods (P>0.05), and the correlation coefficient reached 0.998, indicating that the kit of the present invention has high accuracy.

[0069] In addition, compared with traditional methods, the detection time of this kit is shortened from more than 16 hours to less than 70 minutes, and the efficiency is improved by more than 90%.

[0070] Example 4: Precision Experiment of the Method of the Invention

[0071] The same corn starch matrix sample (containing 71.9% starch) was tested six times using the detection method described in Example 2. The results are shown in Table 1. Figure 3 As shown in the figure. Based on the results, the relative standard deviation (RSD) of the starch content was ≤2%, indicating that this kit has good repeatability and precision.

[0072] Table 1. Repeated determination values ​​of corn starch matrix samples Sequence 1 2 3 4 5 6 Starch content (%) 70.012 70.77857 69.23571 69.42857 71.62714 70.04

[0073] Example 5 Stability Experiment

[0074] The kit was stored at 4°C, and performance was tested using a 71.9% starch matrix sample as the substrate after 0, 1, 3, 6, 9, and 12 months of storage. Results (Table 2 and...) Figure 4 The results showed that the kit could be stored at 4°C for more than one year without significant changes in any performance indicators, indicating that the kit has good stability.

[0075] Table 2. Changes in the stability of the test kit with increasing storage time Months of storage 0 1 3 6 9 12 Starch content (%) 71.7672 71.7464 71.5224 70.4208 70.6248 71.8896

[0076] Example 6: Detection Limit and Linear Range

[0077] Accurately weigh 12 mg of D-glucose and dissolve it in 10 mL of pure water to prepare a 1.2 mg / mL glucose standard stock solution. Serially dilute the stock solution with pure water to concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. Take seven test tubes and add 100 μL of glucose standard solution of different concentrations to each tube. Add 3.0 mL of GOPOD colorimetric reagent to each tube, mix thoroughly, and incubate at 50 ℃ for 20 min. After the reaction, rapidly cool to room temperature. Use a 0 mg / mL glucose solution as a blank control and measure the absorbance (OD) at a wavelength of 510 nm. 510 The results are shown in Table 3.

[0078] Table 3. Absorbance values ​​corresponding to different D-glucose concentrations. Glucose concentration (mg / mL) 0 0.2 0.4 0.6 0.8 1.0 1.2 OD 510 ]] 0.036 0.184667 0.329333 0.467667 0.588 0.713333 0.857

[0079] The limit of detection for this kit is 0.09 μg / mL (reagent), equivalent to 0.09 g / 100 g total starch content (sample weight 100 mg, extraction volume 10.2 mL). Under the action of glucose oxidase and horseradish peroxidase, glucose is oxidized to hydrogen peroxide, which reacts with the chromogenic agent to form a red product. The absorbance at 510 nm effectively reflects the glucose content. This method exhibits good linearity in the detection range of 0-120 μg D-glucose per assay. Figure 5 ).

[0080] The above description is only a partial embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A reagent kit for detecting starch content, characterized in that, The reagents include 4000-7000 U / mL heat-resistant α-amylase, 3000-6000 U / mL amylase, GOPOD chromogenic reagent, GOPOD chromogenic reagent buffer, 1.0 mg / mL D-glucose standard solution, and standardized starch sample. Each reagent is aliquoted into a sealed bottle. The GOPOD colorimetric reagent is a mixture of 3000-7000 U / mL glucose oxidase, 2000-6000 U / mL horseradish peroxidase, and 0.4-0.6 mmol / L 4-aminoantipyrine. The GOPOD colorimetric reagent buffer solution is a 40-60 mmol / L phosphate buffer solution with a pH of 7.

4.

2. The reagent kit for detecting starch content according to claim 1, characterized in that, The thermostable α-amylase, amyloglucosidase, GOPOD chromogenic reagent, and GOPOD chromogenic reagent buffer all contain 0.02% Proclin 300 by mass.

3. The reagent kit for detecting starch content according to claim 1, characterized in that, The heat-resistant α-amylase, amyloglucosidase, and GOPOD colorimetric reagent contain 0.05-0.12% by mass of 4-hydroxybenzoic acid.

4. The reagent kit for detecting starch content according to claim 1, characterized in that, The GOPOD colorimetric reagent buffer solution is a 50 mmol / L phosphate buffer solution with a pH of 7.

4.

5. The method for detecting starch content according to claim 1, characterized in that, Includes the following steps: (1) Mix the food sample to be tested with an ethanol solution to fully disperse the starch in the sample, and add heat-resistant α-amylase diluted with HEPES buffer for preliminary hydrolysis; (2) After initial hydrolysis, add acetic acid-sodium acetate buffer solution to adjust the pH of the solution to acidic, add amyloglucosidase solution, and perform secondary hydrolysis in a water bath at 50-60℃; (3) Dissolve the GOPOD colorimetric reagent in the GOPOD colorimetric reagent buffer to obtain the GOPOD working solution, and mix it with the reaction solution obtained in step (2); at the same time, prepare a reagent blank and a mixture of 0.1 mL glucose standard solution and GOPOD working solution; (4) Use a spectrophotometer at a wavelength of 510 nm, zero the sample with a reagent blank, and measure the absorbance value; and calculate the starch content in the sample based on the molecular weight conversion relationship.

6. The method for detecting starch content according to claim 5, characterized in that, The preliminary hydrolysis in step (1) is carried out at 75-85℃.

7. The method for detecting starch content according to claim 5, characterized in that, In step (1), the ethanol solution is an ethanol aqueous solution with a volume concentration of 80%.

8. The method for detecting starch content according to claim 5, characterized in that, The mixture obtained in step (3) is incubated in a water bath at 50-55℃ for 20 minutes before testing.

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