Starch glucosidase stabilizer and application thereof

Abstract

The invention relates to an amyloglucosidase stabilizer and application thereof, and belongs to the technical field of biology. The invention provides a stabilizer of amyloglucosidase, which takes water as a solvent and comprises the following components in final concentration, the preservative comprises the following components in percentage by weight: 0.5 to 1.5 percent of vitamin C, 0.1 to 0.3 percent of polyethylene glycol 2000, 0.10 to 0.25 percent of mixed preservative and 0.21 to 1.05 mol / L of metal cations. According to the stabilizer, the external environment of a protein solution is indirectly changed through polyethylene glycol 2000, so that the enzyme stability is improved; metal cations, protein charged groups and dipoles are adopted to generate a salting-out effect, the rigidity of enzyme molecules is increased, so that the stability of the enzyme is enhanced, and vitamin C is also adopted to slow down the influence of enzyme oxidation, so that the stability of the enzyme can be maintained, and the enzyme still has higher enzyme activity after being stored at 4 DEG C for a long time.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to an amyloglucosidase stabilizer and application thereof. Background Art

[0002] In food science and industry, resistant starch (RS), also known as enzymatically resistant starch or indigestible starch, refers to starch that is not digested and absorbed in the small intestine but can be fermented in the large intestine. The content of resistant starch is closely related to food digestibility, glycemic response, and intestinal health. RS measurement methods vary widely due to their different principles. Representative methods include the Berry method, the Englyst method, the Goni method, the McCleary method, the Champ method, and their modifications.

[0003] α-Pancreatic amylase and amyloglucosidase are the core raw materials for the above-mentioned methods, significantly impacting their stability and precision. Natural enzymes exhibit shortcomings in practical applications, such as poor tolerance, easy inactivation, and difficulty in storage. Currently, enzymes are typically prepared on-the-fly to ensure activity, severely restricting their industrial application.

[0004] Currently, the main methods for increasing enzyme stability include molecular engineering, chemical modification, immobilization, and the addition of stabilizers. Improving enzyme stability by adding stabilizers is both economical and convenient. Suitable stabilizers can increase enzyme stability while maintaining enzyme activity. Currently, stabilizers improve enzyme stability primarily through two pathways: first, additives interact directly with proteins (binding to proteins, electrostatic interactions, etc.), directly enhancing enzyme stability; second, additives interact with each other intermolecularly, indirectly changing the external environment of the protein solution (viscosity, polarity, etc.), thereby improving enzyme stability.

[0005] Existing reagents for detecting resistant starch content often recommend that amyloglucosidase be prepared immediately and stored at 4°C for a short period of time (generally within 3 days). Glycerol, sugars, and arginine are often added to the solution to enhance its stability. However, the addition of sugars and arginine can interfere with subsequent detection reactions, and high glycerol concentrations and high viscosity make it difficult to measure and add samples in subsequent experiments. The existing technology urgently needs a stabilizer that can effectively improve the stability of amyloglucosidase.

[0006] Based on this, the present invention is proposed. Summary of the Invention

[0007] The object of the present invention is to provide an amyloglucosidase stabilizer and application thereof, so as to solve the problem of poor stability of amyloglucosidase in the prior art.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a stabilizer for amyloglucosidase, wherein the stabilizer uses water as a solvent and comprises components with the following final concentrations:

[0010] 0.5-1.5 wt% of vitamin C, 0.1-0.3 wt% of polyethylene glycol 2000, 0.10-0.25 wt% of mixed preservatives and 0.21-1.05 mol / L of metal cations.

[0011] Preferably, the mixed preservative comprises p-hydroxybenzoic acid, sodium erythorbate and potassium sorbate;

[0012] The mass ratio of the p-hydroxybenzoic acid, sodium erythorbate and potassium sorbate is 1-2:1-2:1-2.

[0013] Preferably, the metal cations include potassium ions, calcium ions and sodium ions;

[0014] The molar ratio of the potassium ion, the calcium ion and the sodium ion is 9-11:1:9-11.

[0015] The present invention provides use of the stabilizer in preparing a product for detecting the content of resistant starch.

[0016] The present invention provides a kit for detecting the content of resistant starch, which comprises α-pancreatic amylase, sodium maleate buffer, amyloglucosidase solution, potassium hydroxide solution, sodium acetate buffer, glucose solution, DNS reagent and citric acid buffer solution;

[0017] The amyloglucosidase solution comprises amyloglucosidase and a stabilizer;

[0018] The stabilizer is the stabilizer described above;

[0019] The activity of the amyloglucosidase in the amyloglucosidase solution is 3200-3400 U / mL.

[0020] Preferably, the sodium maleate buffer solution is prepared by mixing maleic acid, calcium chloride dihydrate, sodium azide and water;

[0021] The mass ratio of maleic acid, calcium chloride dihydrate and sodium azide is 1.56-1.76:0.04-0.06:0.04-0.06;

[0022] The mass volume ratio of the maleic acid and water is 1.56-1.76 g:90-110 mL.

[0023] Preferably, the initial concentration of the potassium hydroxide solution is 1 to 3 mol / L.

[0024] Preferably, the initial concentration of sodium acetate buffer is 1.0-1.4 mol / L, and the pH value is 3.6-4.0;

[0025] The initial concentration of the citric acid buffer solution is 0.04-0.06 mol / L, and the pH value is 4.3-4.7.

[0026] Preferably, the initial concentration of the glucose solution is 0.8-1.2 mg / mL.

[0027] The present invention has the following technical effects and advantages:

[0028] The present invention provides a stabilizer for amyloglucosidase, comprising vitamin C, polyethylene glycol 2000, a mixed preservative, metal cations, and water. The stabilizer of the present invention indirectly changes the external environment of the protein solution through polyethylene glycol 2000, thereby improving enzyme stability; metal cations react with protein charged groups and dipoles to produce salting-out, increasing the rigidity of the enzyme molecule and thus enhancing enzyme stability; and vitamin C is used to mitigate the effects of oxidation, thereby maintaining enzyme stability and maintaining high enzyme activity even after long-term storage.

[0029] The present invention also provides a kit for detecting the content of resistant starch prepared using the stabilizer of the present application, which does not use glycerol, reduces the viscosity of the reagent, and can also improve the accuracy of experimental sampling. DETAILED DESCRIPTION

[0030] The present invention provides a stabilizer for amyloglucosidase, wherein the stabilizer uses water as a solvent and comprises components with the following final concentrations:

[0031] 0.5-1.5 wt% of vitamin C, 0.1-0.3 wt% of polyethylene glycol 2000, 0.10-0.25 wt% of a mixed preservative, and 0.21-1.05 mol / L of a metal cation;

[0032] The concentration of the vitamin C is preferably 1 wt%; the concentration of the polyethylene glycol 2000 is preferably 0.2 wt%; the concentration of the mixed preservative is preferably 0.2 wt%; and the concentration of the metal cation is preferably 0.42 mol / L.

[0033] In the present invention, the mixed preservatives include p-hydroxybenzoic acid, sodium erythorbate and potassium sorbate;

[0034] The mass ratio of the p-hydroxybenzoic acid, sodium erythorbate and potassium sorbate is 1-2:1-2:1-2, preferably 1:1:1.

[0035] In the present invention, the metal cations include potassium ions, calcium ions and sodium ions;

[0036] The molar ratio of potassium ions, calcium ions and sodium ions is 9-11:1:9-11, preferably 10:1:10.

[0037] The present invention provides use of the stabilizer in preparing a product for detecting the content of resistant starch.

[0038] The present invention provides a kit for detecting the content of resistant starch, which comprises α-pancreatic amylase, sodium maleate buffer, amyloglucosidase solution, potassium hydroxide solution, sodium acetate buffer, glucose solution, DNS reagent and citric acid buffer solution;

[0039] The amyloglucosidase solution comprises amyloglucosidase and a stabilizer;

[0040] The stabilizer is the stabilizer described above;

[0041] The activity of the amyloglucosidase in the amyloglucosidase solution is 3200-3400 U / mL, preferably 3300 U / mL.

[0042] In the present invention, the sodium maleate buffer solution is prepared by mixing maleic acid, calcium chloride dihydrate, sodium azide and water;

[0043] The mass ratio of maleic acid, calcium chloride dihydrate and sodium azide is 1.56-1.76:0.04-0.06:0.04-0.06, preferably 1.66:0.05:0.05;

[0044] The mass volume ratio of the maleic acid and water is 1.56-1.76 g:90-110 mL, preferably 1.66 g:100 mL.

[0045] In the present invention, the initial concentration of the potassium hydroxide solution is 1 to 3 mol / L, preferably 2 mol / L.

[0046] In the present invention, the initial concentration of sodium acetate buffer is 1.0 to 1.4 mol / L, preferably 1.2 mol / L, and the pH value is 3.6 to 4.0, preferably 3.8;

[0047] The initial concentration of the citric acid buffer solution is 0.04-0.06 mol / L, preferably 0.05 mol / L, and the pH value is 4.3-4.7, preferably 4.5.

[0048] In the present invention, the initial concentration of the glucose solution is 0.8-1.2 mg / mL, preferably 1 mg / mL.

[0049] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Aiming at the problem of poor stability of amyloglucosidase in the resistant starch detection kit in the prior art, vitamin C, polyethylene glycol 2000 (PEG2000), mixed preservatives, potassium ions (K + ), sodium ion (Na 2+ ), calcium ions (Ca 2+ ) and water to prepare an amyloglucosidase stabilizer, wherein the mixed preservative is prepared by mixing p-hydroxybenzoic acid, sodium isoascorbate and potassium sorbate in a mass ratio of 1:1:1.

[0052] 1. Determination of the amount of stabilizer components

[0053] Vitamin C was added to water as a solvent at a final concentration of 1 wt%, and then PEG2000, a preservative and a metal cation were added. Stabilizers numbered 1 to 9 were prepared by adding PEG2000, preservatives and metal cations at different final concentrations. The final concentrations of PEG2000, preservatives and metal cations in the stabilizers numbered 1 to 9 are shown in Table 1.

[0054] Table 1 Stabilizers with different component ratios

[0055]

[0056] Amyloglucosidase was mixed with stabilizers numbered 1 to 9, respectively, to prepare amyloglucosidase solutions with an enzyme activity of 3300 U / mL with different stabilizer configurations. The amyloglucosidase solutions were then stored at 4°C for 8, 14, and 20 months. The relative enzyme activities of the amyloglucosidase solutions with different stabilizer configurations after 8, 14, and 20 months of storage were measured. The relative enzyme activity was determined as follows:

[0057] a. 1 g of α- pancreatic amylase was added to 100 mL of sodium maleate buffer, followed by 1 mL of amyloglucosidase solution, and stirred for 5 min to obtain an α- pancreatic amylase suspension for later use;

[0058] The sodium maleate buffer solution was prepared by dissolving 1.66 g of maleic acid, 0.05 g of calcium chloride dihydrate, and 0.05 g of sodium azide in 100 mL of water and mixing them evenly.

[0059] b. Take 100 mg of high-resistant starch flour powder and put it into a 15 mL EP tube for later use.

[0060] c. Add 4.0 mL of α-pancreatic amylase suspension to the EP tube, shake continuously at 37°C for 16 h, remove, add 4.0 mL of anhydrous ethanol, centrifuge (1500 rpm, 10 min), pour out the supernatant, wash the precipitate three times with 50% ethanol, and discard the supernatant.

[0061] d. Add 2 mL of 2 mol / L potassium hydroxide solution to the precipitate, place the precipitate in an ice bath, and magnetically stir for 20 min. Then add 8 mL of 1.2 mol / L sodium acetate buffer (pH 3.8), mix well, and immediately add 0.1 mL of amyloglucosidase solution. Incubate in a 50°C water bath for 30 min to obtain the amyloglucosidase solution digestion product.

[0062] f. DNS method to measure absorbance at 540nm

[0063] Dilute the amyloglucosidase digestion product 2-fold with citric acid buffer. Transfer 1.0 mL of the diluted solution to a 15 mL graduated test tube. Add 2.0 mL of DNS reagent and mix thoroughly. Boil in boiling water for 2 minutes. Cool in running water, then fill to the 15 mL mark with water. Shake thoroughly and measure the absorbance at 540 nm.

[0064] The calculation formula of the relative enzyme activity of the amyloglucosidase solution is:

[0065] Relative enzyme activity (%) = (absorbance value 1 / absorbance value 2) × 100%;

[0066] Wherein, the absorbance value 1 represents the absorbance value detected by the stored amyloglucosidase solution, and the absorbance value 2 represents the absorbance value detected by the freshly prepared amyloglucosidase solution.

[0067] The calculation results of the relative enzyme activity of the amyloglucosidase solutions with different stabilizer configurations after storage for different months are shown in Table 2.

[0068] Table 2 Enzyme activity determination results of amyloglucosidase solution after storage for different months

[0069]

[0070] The range analysis of each component in the amyloglucosidase solution was performed, and the results are shown in Table 3.

[0071] Table 3 Range analysis results of each component in amyloglucosidase solution

[0072]

[0073] According to Tables 2 and 3, when the mass concentration of PEG2000 in the amyloglucosidase solution was 0.20%, the average enzyme activity was 94.77%, which was significantly higher than other levels; K + When the concentration was 0.5 mol / L, the average enzyme activity was 77.42%, and the enzyme activity increased with the increase of concentration; 2+ When the concentration was 0.05 mol / L, the average enzyme activity was 77.42%, which was consistent with K + The trend is consistent; Ca 2+ When the concentration was 0.5 mol / L, the average enzyme activity was 77.42%, and high concentration was more conducive to maintaining enzyme activity; when the mass concentration of the preservative was 0.20%, the average enzyme activity was 82.44%, and high concentration had the best enzyme protection effect; the maximum extreme value of enzyme activity was 96.47% (No. 9, stored for 8 months), the minimum was 28.71% (No. 1, stored for 8 months), and the extreme value difference was 96.47%-28.71%=67.76%, indicating that the optimization of the stabilizer component had a significant effect on maintaining enzyme activity. The mass concentration of PEG2000 in the amyloglucosidase solution had the greatest effect on enzyme activity (extreme difference 44.70%), followed by the preservative concentration (extreme difference 30.17%), and the metal ions (K + 、Na 2+ , Ca 2+ The best combination of components in the amyloglucosidase solution was experimental number 9, and its relative enzyme activity after 8 months of storage reached 96.47%, the highest value among all experimental groups.

[0074] 2. Stabilizing effect of different components of stabilizer

[0075] 2.1 Effect of PEG2000 deficiency on the stabilization effect of stabilizer-protected enzymes

[0076] Experimental groups

[0077] The mice were divided into a control group and an experimental group. The control group was the optimal stabilizer component dosage formula screened in "1. Determination of the dosage of stabilizer components". Compared with the control group, PEG2000 was removed from the experimental group, and the other components and dosages were the same.

[0078] Experimental plan

[0079] Amyloglucosidase was mixed with the stabilizers of the experimental group and the control group, respectively, to obtain amyloglucosidase solutions with enzyme activities of 3300U / mL configured with different stabilizers, and the amyloglucosidase solutions with enzyme activities of 3300U / mL configured with different stabilizers were stored at 37°C (accelerated stability experiment), and samples were taken regularly (0, 7, 14, 21, 28 days) to determine the relative enzyme activity. The determination method was consistent with the relative enzyme activity determination method in “1. Determination of the amount of stabilizer component”, and the relative enzyme activity results of the amyloglucosidase solutions with different stabilizer configurations are shown in Table 4.

[0080] Table 4 Enzyme activity results of amyloglucosidase solutions with different stabilizer configurations

[0081]

[0082] According to Table 4, the relative enzyme activity of the amyloglucosidase solution in the experimental group was 84.8% on the 7th day, which was significantly lower than that of the control group, indicating that PEG2000 played a key role in short-term stability. The relative enzyme activity of the amyloglucosidase solution in the experimental group was only 27.3% on the 28th day, which was significantly lower than that of the control group. This proves that PEG2000 protects the enzyme molecules from solvation through steric hindrance effects, and its effect cannot be compensated by other components. In addition, there were extremely significant differences at all time points (p < 0.01), excluding the possibility of experimental error.

[0083] 2.2 Comparative experiment of PEG2000 alternative ingredients

[0084] Experimental groups

[0085] The mice were divided into a control group and experimental groups 1 to 4. The control group was the optimal stabilizer component dosage formula screened in "1. Determination of the dosage of stabilizer components". Experimental group 1 replaced PEG2000 in the control group with an equal amount of PEG400, and the other components and dosages were the same; experimental group 2 replaced PEG2000 in the control group with an equal amount of trehalose, and the other components and dosages were the same; experimental group 3 replaced PEG2000 in the control group with an equal amount of sorbitol, and the other components and dosages were the same. Experimental group 4 was a 50% glycerol solution.

[0086] Amyloglucosidase was mixed with the stabilizers of experimental groups 1 to 4 and the control group, respectively, to obtain amyloglucosidase solutions with enzyme activities of 3300 U / mL configured with different stabilizers, and the amyloglucosidase solutions with enzyme activities of 3300 U / mL configured with different stabilizers were stored at 37 ° C. Samples were taken for 14 days to measure the relative enzyme activity and analyze the results. The determination method was consistent with the relative enzyme activity determination method in "1. Determination of the amount of stabilizer component". The relative enzyme activity of the amyloglucosidase solutions with different stabilizer configurations and the analysis results are shown in Table 5.

[0087] Table 5 Relative enzyme activity and analysis results of amyloglucosidase solutions with different stabilizer configurations

[0088] Group Relative enzyme activity Molecular mechanism analysis control group 96.4％ The hydrophobic end of PEG2000 binds to the hydrophobic region of the enzyme Experimental Group 1 90.3％ The molecular weight is too small, resulting in insufficient steric hindrance Experimental Group 2 84.8％ Hydroxyl groups compete with enzymes for hydrogen bonding Experimental Group 3 83.3％ Similar to trehalose, but with a more rigid molecule Experimental Group 4 12％ The stabilizing effect of glycerol on protein structure

[0089] As shown in Table 5, PEG400 (molecular weight 400) was unable to form an effective hydrophobic barrier due to its insufficient chain length, resulting in a 6.1% decrease in retention rate. Trehalose and sorbitol bind to the enzyme surface through hydroxyl groups, and sorbitol binds more tightly due to its rigid structure, resulting in a slightly stronger inhibitory effect than trehalose. Even when the concentration of the alternative ingredient was increased to 0.5% (such as PEG400), the 14-day activity retention rate was still only 88.5%, significantly lower than that of the PEG2000 group (p < 0.01), further demonstrating that the molecular weight (2000) and hydrophobicity of PEG2000 are crucial to its role in stabilizing the enzyme.

[0090] 2.3 Effect of cation deficiency on the stability of stabilizer-protected enzymes

[0091] Experimental groups

[0092] The control group was divided into two groups: the control group and experimental groups 1 to 4. The control group was the optimal stabilizer component dosage formula screened in "1. Determination of the dosage of stabilizer components". The experimental group 1 and the control group only retained K + The rest of the ingredients and dosages were the same; only Ga 2+ The rest of the ingredients and dosages were the same; only Na + , the other ingredients and dosages are the same; experimental group 4 and the control group completely removed the cations, and the other ingredients and dosages are the same.

[0093] Amyloglucosidase was mixed with the stabilizers of experimental groups 1 to 4 and the control group, respectively, to obtain amyloglucosidase solutions with enzyme activities of 3300 U / mL configured with different stabilizers. The amyloglucosidase solutions with enzyme activities of 3300 U / mL configured with different stabilizers were stored at 37°C, and samples were taken for determination after 7 days to determine the relative enzyme activity. The determination method was consistent with the relative enzyme activity determination method in "1. Determination of the amount of stabilizer component". The relative enzyme activity results of the amyloglucosidase solutions with different stabilizer configurations are shown in Table 6.

[0094] Table 6 Relative enzyme activity results of amyloglucosidase solutions with different stabilizer configurations

[0095] Group Relative enzyme activity Significance (vs control group) Significance (between groups) control group 98.5％ / / Experimental Group 1 75.8％ p<0.05 P<0.01 (vs experimental group 2) Experimental Group 2 81.8％ p<0.05 P<0.05 (vs experimental group 3) Experimental Group 3 71.2％ p<0.01 P<0.01 (vs experimental group 1) Experimental Group 4 54.5％ p<0.001 P<0.001 (experimental group and control group)

[0096] According to Table 6, the relative enzyme activities of the amyloglucosidase solutions of experimental groups 1 to 3 after 7 days were 75.8%, 81.8%, and 71.2%, respectively, which were significantly lower than 98.5% of the control group, indicating that the three cations synergistically protected the enzyme active center through the charge shielding effect; after the cations were completely removed, the enzyme activity dropped sharply to 54.5% (p < 0.001), proving that the cations maintained the natural conformation of the enzyme by neutralizing the solvent polarity.

[0097] In summary, the optimal formula of the amyloglucosidase stabilizer of this application is:

[0098] Water was used as solvent, including vitamin C with a final concentration of 1wt%, PEG2000 with a final concentration of 0.2wt%, and K with a final concentration of 0.5mol / L. + , Ca2+ at a final concentration of 0.05 mol / L 2+ , Na with a final concentration of 0.5 mol / L 2+ , the final concentration of the composite preservative is 0.2wt%, and the mass ratio of p-hydroxybenzoic acid, sodium isoascorbate and potassium sorbate in the mixed preservative is 1:1:1.

[0099] Example 2

[0100] 1. A kit for detecting resistant starch content

[0101] The kit consists of α-pancreatic amylase, sodium maleate buffer, amyloglucosidase solution, potassium hydroxide solution, sodium acetate buffer, glucose solution, DNS reagent and citric acid buffer solution.

[0102] α-Pancreatic amylase was a commercially available product;

[0103] The sodium maleate buffer solution was prepared by dissolving 1.66 g of maleic acid, 0.05 g of calcium chloride dihydrate, and 0.05 g of sodium azide in 100 mL of water and mixing well.

[0104] The amyloglucosidase solution was prepared by mixing amyloglucosidase with the amyloglucosidase stabilizer of the best formula of Example 1, so that the enzyme activity of the amyloglucosidase was 3300 U / mL;

[0105] The concentration of potassium hydroxide solution is 2 mol / L;

[0106] The concentration of sodium acetate buffer was 1.2 mol / L and the pH was 3.8;

[0107] The concentration of glucose solution is 1 mg / mL;

[0108] The concentration of citric acid buffer solution is 0.05 mol / L and the pH value is 4.5;

[0109] The preparation method of DNS reagent is as follows: add 0.63g of 3,5-dinitrosalicylic acid and 26.2mL of sodium hydroxide solution with an initial concentration of 2mol / L to 50mL of hot water solution containing 18.2g of potassium sodium tartrate; then add 0.5g of heavy phenol and 0.5g of sodium sulfite, stir to dissolve, cool and add water to make up to 100mL.

[0110] Alpha-pancreatic amylase, sodium maleate buffer solution, amyloglucosidase solution, potassium hydroxide solution, sodium acetate buffer solution, glucose solution, citric acid buffer solution and DNS reagent are respectively placed into corresponding containers, and then numbered and packaged to obtain a kit for detecting resistant starch content.

[0111] 2. How to use the kit for detecting resistant starch content

[0112] (1) Kit preparation

[0113] 1 g of α-pancreatic amylase was added to 100 mL of sodium maleate buffer, and then 1 mL of amyloglucosidase solution was added, and the mixture was stirred for 5 minutes to obtain an α-pancreatic amylase suspension for later use.

[0114] (2) Sample preparation

[0115] Grind the sample to be tested and pass it through a 0.5 mm sieve to obtain the sample powder to be tested. Take 100 mg of the powder and put it into a 15 mL EP tube for later use.

[0116] (3) Amylase digestion

[0117] Add 4 mL of α-pancreatic amylase suspension to the EP tube containing the sample powder to be tested, shake continuously at 37°C for 16 h, take it out, add 4.0 mL of anhydrous ethanol, centrifuge (1500 r / min, 10 min), pour out the supernatant, wash the precipitate with 50% ethanol three times, and discard the supernatant to obtain the precipitate.

[0118] (4) Amyloglucosidase digestion

[0119] Add 2 mL of 2 mol / L potassium hydroxide solution to the precipitate, place the precipitate in an ice bath, and stir magnetically for 20 min. Then add 8 mL of 1.2 mol / L sodium acetate buffer (pH 3.8), mix well, and immediately add 0.1 mL of amyloglucosidase solution. Incubate in a 50°C water bath for 30 min to obtain amyloglucosidase solution digestion product.

[0120] (5) DNS method was used to determine the content of reducing sugar at 540 nm.

[0121] The DNS method uses a colorimetric method to determine reducing sugar content, based on the principle that dinitrosalicylic acid (DNS) undergoes a redox reaction with reducing sugars under alkaline conditions to generate 3-amino-5-nitrosalicylic acid. This product develops a brown-red color when boiled, and the color depth is directly proportional to the reducing sugar content within a certain concentration range. Because the color depth is only related to the number of reducing groups released from the sugar and is not selective for the type of reducing sugar, the DNS method is suitable for use in systems with multiple reducing sugars produced by the hydrolysis of polysaccharides (such as cellulose, hemicellulose, and starch).

[0122] a. Drawing of glucose standard curve

[0123] Dispense 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose standard solution (1 mg / mL) into 15 mL test tubes. If the volume is less than 1.0 mL, add distilled water to 1.0 mL. Accurately add 2 mL of DNS reagent to each tube, heat in a boiling water bath for 2 minutes, and cool under running water. Fill up to the 15 mL mark with water and shake well. Measure the absorbance of each tube at a wavelength of 540 nm. Draw a standard curve with the glucose content (mg) as the horizontal axis and the light absorption value as the vertical axis.

[0124] b. Sample determination

[0125] The digestion product of the amyloglucosidase solution was diluted with citric acid buffer solution at three dilutions: original, 5-fold, and 10-fold. 1.0 mL of the diluted solution was placed in a 15 mL graduated test tube, 2.0 mL of DNS reagent was added, mixed, and boiled in boiling water for 2 min. After cooling in running water, the volume was filled to 15 mL with water, shaken, and the absorbance was measured at a wavelength of 540 nm. The corresponding glucose content (mg / mL) was found according to the standard curve to calculate the reducing sugar content in the sample.

[0126] (6) Calculation of results

[0127] The formula for calculating resistant starch content is as follows:

[0128]

[0129] Resistant starch content (%) = reducing sugar content (%) × 0.9;

[0130] In the formula, C represents the reducing sugar content (mg) obtained from the glucose standard curve; m represents the weight of the test sample (g).

[0131] 3. Detection effect

[0132] Experimental groups

[0133] The mice were divided into a control group and an experimental group. The control group used a conventional kit for detecting the content of resistant starch, and the experimental group used the kit prepared by "1. A kit for detecting the content of resistant starch".

[0134] Experimental Materials

[0135] High-resistant starch flour is provided by the Chinese Academy of Agricultural Sciences, ordinary flour is produced by Xiangnian Food Co., Ltd., and high-resistant starch instant noodles and other processed products are homemade in the laboratory.

[0136] Experimental plan

[0137] The test kits of the control group and experimental group were used to detect the resistant starch content in high-resistant starch biscuits, high-resistant starch flour, ordinary flour, high-resistant starch vegetarian noodles, high-resistant starch noodles, high-resistant starch rice noodles, water, and a standard glucose solution with a concentration of 0.5 mg / mL. The results are shown in Table 7.

[0138] Table 7 Test results of resistant starch content in different samples

[0139]

[0140] As shown in Table 7, for standard glucose detection, the experimental group is closer to the theoretical value (100%), which proves that the detection accuracy is higher. At the same time, compared with the control group, the detection value error of the experimental group is less than 13.5%, which meets the requirement that the error of conventional test kits is not higher than 15%, proving that the test kit prepared by the present invention has performance that is not inferior to that of the control reagent.

[0141] As can be seen from the above examples, the present invention provides a kind of stabilizer of amyloglucosidase, and the stabilizer includes vitamin C, polyethylene glycol 2000, mixed preservative, metal cation and water.The stabilizer of the present invention indirectly changes the protein solution external environment by polyethylene glycol 2000, and then improves enzyme stability; Metal cation and protein charged group and dipole are used to produce salting-out effect, increase the rigidity of enzyme molecule, thereby enhancing enzyme stability, and also use vitamin C to slow down the influence of oxidation, can keep enzyme stability, make it still have higher enzyme activity under long-term preservation.

[0142] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A stabilizer for amyloglucosidase, characterized in that The stabilizer uses water as solvent and includes the following components at the final concentrations: 0.5-1.5 wt% of vitamin C, 0.1-0.3 wt% of polyethylene glycol 2000, 0.10-0.25 wt% of mixed preservatives and 0.21-1.05 mol / L of metal cations.

2. The stabilizer according to claim 1, characterized in that The mixed preservatives include p-hydroxybenzoic acid, sodium erythorbate and potassium sorbate; The mass ratio of the p-hydroxybenzoic acid, sodium erythorbate and potassium sorbate is 1-2:1-2:1-2.

3. The stabilizer according to claim 1, characterized in that The metal cations include potassium ions, calcium ions and sodium ions; The molar ratio of the potassium ion, the calcium ion and the sodium ion is 9-11:1:9-11.

4. Use of the stabilizer according to any one of claims 1 to 3 in the preparation of a product for detecting the content of resistant starch.

5. A kit for detecting the content of resistant starch, characterized in that: The kit includes α-pancreatic amylase, sodium maleate buffer, amyloglucosidase solution, potassium hydroxide solution, sodium acetate buffer, glucose solution, DNS reagent and citric acid buffer solution; The amyloglucosidase solution comprises amyloglucosidase and a stabilizer; The stabilizer is the stabilizer according to any one of claims 1 to 3; The activity of the amyloglucosidase in the amyloglucosidase solution is 3200-3400 U / mL.

6. The kit according to claim 5, characterized in that The sodium maleate buffer solution is prepared by mixing maleic acid, calcium chloride dihydrate, sodium azide and water; The mass ratio of maleic acid, calcium chloride dihydrate and sodium azide is 1.56-1.76:0.04-0.06:0.04-0.06; The mass volume ratio of the maleic acid and water is 1.56-1.76 g:90-110 mL.

7. The kit according to claim 5, characterized in that The initial concentration of the potassium hydroxide solution is 1-3 mol / L.

8. The kit according to claim 5, wherein The initial concentration of sodium acetate buffer was 1.0–1.4 mol / L, and the pH was 3.6–4.0; The initial concentration of the citric acid buffer solution is 0.04-0.06 mol / L, and the pH value is 4.3-4.

7.

9. The kit according to claim 5, characterized in that The initial concentration of the glucose solution is 0.8-1.2 mg / mL.

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