Determination method of starch dissolution rate

Abstract

The invention discloses a starch dissolution rate determination method, which comprises: S1, adding a Termamyl SC 4X enzyme preparation to a gelatinized starch sample, and carrying out a hydrolysis reaction; s2, centrifuging and filtering the hydrolysate obtained in the step S1 to obtain a test sample; s3, performing high performance liquid detection on the test sample to respectively obtain the contents of glucose, maltose, maltotriose and glucan; and S4, calculating the starch dissolution rate according to the contents of glucose, maltose, maltotriose and glucan. According to the determination method of the starch dissolution rate, a gelatinized starch sample and a Termamyl SC 4X enzyme preparation are subjected to a hydrolysis reaction, and the starch dissolution rate is calculated by determining the contents of glucose, maltose, maltotriose and glucan. The method overcomes the problems of batch difference of a multi-component enzyme system carried by malt and uncertainty of a malt enzyme system on hydrolysis of aged starch in a traditional determination method, and realizes the effect of predicting the starch leaching rate in production through a starch leaching rate determination result in a laboratory.

Description

Technical Field

[0001] The invention relates to the technical field of beer brewing, and in particular to a method for determining starch dissolution rate. Background Art

[0002] Rice, corn and barley are the main auxiliary materials for beer production. The starch dissolution rate (extraction rate) is one of the important indicators for testing the quality of auxiliary material batches, which directly reflects the starch utilization rate.

[0003] Application No. 201510430635.x discloses a method for detecting rice leaching rate, comprising the following steps: A. rice gelatinization, weighing a rice sample and gelatinizing the starch in the rice; B. saccharification, weighing malt and adding the rice sample treated in step A to form a mixture; C. determination of the leaching rate of the mixture, using the agreed saccharification method and the density bottle method to measure the leaching rate of the mixture; D. calculation.

[0004] The above rice extraction rate detection method is to perform saccharification and enzymatic hydrolysis after mixing with malt, and determine the starch extraction rate by the agreement saccharification method and the density bottle method. The agreement saccharification method and the density bottle method are conventional methods for determining the starch extraction rate or dissolution rate. However, in actual production, it is found that for raw materials with the same starch dissolution rate measured by the agreement saccharification method, when the production conditions are not adjusted, the starch dissolution results are far apart, which affects the normal production of beer. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the present invention provides a method for determining the starch dissolution rate, wherein a hydrolyzate is prepared by adding a fixed enzyme preparation, and the contents of glucose, maltose, maltotriose and glucan are determined by high performance liquid chromatography, and the starch extraction rate is calculated by reverse calculation through the total sugar content of the degradable part. The determination method is simple and the result is stable, and the starch extraction rate in actual production can be predicted through the laboratory determination results.

[0006] In order to solve the above technical problems, the present invention provides a method for determining starch dissolution rate, comprising the following steps:

[0007] S1. Add Termamyl SC 4X enzyme preparation to the gelatinized starch sample to perform hydrolysis reaction;

[0008] S2, centrifuging and filtering the hydrolyzate obtained in step S1 to obtain a test sample;

[0009] S3, performing high performance liquid chromatography on the test samples to obtain the contents of glucose, maltose, maltotriose, and dextran respectively;

[0010] S4. Calculate the starch dissolution rate based on the content of glucose, maltose, maltotriose and dextran.

[0011] As a preferred embodiment, the usage ratio of the starch sample to the Termamyl SC 4X enzyme preparation in S1 is (0.9-1.1):(1.8-2.2).

[0012] As a preferred embodiment, the concentration of the Termamyl SC 4X enzyme preparation in the hydrolysis reaction system is 450-500 ppm.

[0013] As a preferred embodiment, the starch sample in S1 is a starch sample obtained by gelatinization of rice, corn or barley.

[0014] As a preferred embodiment, the temperature of the hydrolysis reaction in S1 is 88° C.-92° C., and the time is 2-2.5 h.

[0015] As a preferred embodiment, the step S3 further comprises the step of adding a mobile phase to the test sample.

[0016] As a more preferred embodiment, the pH value of the system after the test sample and the mobile phase are mixed is 4.0.

[0017] The pH was maintained at 4.0 to ensure that any residual enzyme activity in the test samples would not be further hydrolyzed.

[0018] As a preferred embodiment, the mobile phase is a 4.8-5.2 mM sulfuric acid solution.

[0019] As a preferred embodiment, the conditions for the HPLC detection in S3 are a flow rate of 0.6 mL / min, a temperature of 65° C., an injection volume of 10 μL, and a running time of 30 min.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The starch dissolution rate determination method of the present invention overcomes the batch difference problem of the multi-component enzyme system carried by malt itself by subjecting the gelatinized starch sample to a hydrolysis reaction with the Termamyl SC 4X enzyme preparation, thereby achieving the purpose of guiding actual production through laboratory determination results.

[0022] (2) The starch dissolution rate determination method of the present invention detects the contents of glucose, maltose, maltotriose and dextran by HPLC, and then reversely calculates the starch dissolution rate, thereby overcoming the disadvantage that in the traditional determination method, there is uncertainty as to whether aged starch is involved in the calculation, which affects the accuracy of the determination result.

[0023] (3) The starch dissolution rate determination method of the present invention is to obtain the polysaccharide content by HPLC detection of the hydrolyzate, and calculate the starch extraction rate by the polysaccharide content. The determination method is simple to operate and is convenient for timely detection of the starch dissolution rate; at the same time, the determination result is reliable and suitable for industrial promotion.

[0024] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the results of the determination of starch dissolution rate when different fixed enzyme preparations are used for enzymatic hydrolysis;

[0026] Figure 2 It is the result of starch dissolution rate determination of different batches of raw materials;

[0027] Figure 3 This is a graph showing the results of starch dissolution rate determination using the method of Example 1 of the present application and the agreed saccharification method for different batches of raw materials;

[0028] Figure 4 This is a comparison chart of the starch dissolution rate measurement results of the same batch of corn starch using the method of Example 1 of the present application and the agreed saccharification method. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the invention is further described below with reference to specific illustrations. However, the invention is not limited to the following implementation cases.

[0030] It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with this technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantive technical significance. Any structural modification, change in proportion or adjustment of size, without affecting the effects and purposes that can be achieved by the present invention, should still fall within the scope of the technical contents disclosed by the present invention.

[0031] Rice, corn and barley are the main auxiliary materials for beer production. The starch dissolution rate of rice, corn and barley is one of the important indicators for testing the quality of auxiliary material batches, which directly reflects the starch utilization rate.

[0032] In the prior art, malt is added to the gelatinized starch sample, the starch is saccharified by the multi-component enzyme system carried by the malt itself, and the starch extraction rate is measured by the agreement saccharification method and the density bottle method. However, in actual production, it is found that for raw materials with similar starch dissolution rates, the actual dissolution results are far apart, making the laboratory test results and the actual production situation very different.

[0033] Through long-term research, it was found that in actual production, during the cooling process of gelatinized starch, the starch molecules will rearrange and form insoluble precipitates through hydrogen bonds. This process is the reverse process of gelatinization. In essence, the starch molecules dissolve and swell during the gelatinization process, and then rearrange and combine to form a substance similar to the structure of natural starch, which is called aged starch. The malt enzyme system used in production is basically unable to hydrolyze aged starch, so the laboratory test results are far from the actual production dissolution results.

[0034] In order to solve the impact of the presence of aged starch on actual production, we considered replacing the enzymatic hydrolysis of malt in production with enzymatic hydrolysis of fixed enzyme preparations, but there is still a difference between the laboratory test results and the actual production. The reason is that aged starch not only comes from the conversion in production, but also partly from natural formation. However, due to the uncertainty of whether the malt enzyme system can hydrolyze aged starch, the agreed saccharification method cannot effectively evaluate the contribution of the presence of natural aged starch to the starch dissolution rate. Even if malt is replaced with fixed enzymes in production, the laboratory test results still cannot guide actual production.

[0035] In order to solve the above technical problems, the inventors of the present application changed their thinking and no longer used the traditional agreed saccharification method and density bottle method to measure the starch dissolution rate. Instead, they added fixed enzyme preparations to the gelatinized starch. First, they overcame the batch differences in the enzyme systems carried by different batches of malt, which affected the measurement results. Second, the presence of fixed enzyme preparations can hydrolyze part of the aged starch and hydrolyze the gelatinized starch as much as possible, ensuring the consistency between the laboratory measurement results and the actual production.

[0036] In actual production, not all fixed enzyme preparations can be used in this application. This application uses α-thermoresistant amylase-Novozymes (SC type), β-glucanase-Novozymes, neutral protease-Novozymes (product name: Neutrase0.8L BrewQ Novozymes protease (neutral 0.8LBrewQ type)), xylanase-Novozymes, glucoamylase-Novozymes and saccharifying enzyme-Danisco (purchased from Genenco (China) Bioengineering Co., Ltd., product model DIAZYME CN4) to participate in the hydrolysis reaction. The results are as follows: Figure 1As shown, among the obtained results, only the α-thermostable amylase Novozymes (Termamyl SC 4X enzyme preparation) was equivalent to the conventional test results, so the present application selected Termamyl SC 4X enzyme preparation as the enzyme preparation for subsequent experiments.

[0037] Example 1

[0038] A method for determining starch dissolution rate comprises the following steps:

[0039] S1. Add Termamyl SC 4X enzyme preparation to the gelatinized starch sample to carry out hydrolysis reaction, wherein the starch sample is a gelatinized starch sample of rice, corn or barley, the dosage ratio of the starch sample to the Termamyl SC 4X enzyme preparation is 1:2, and the concentration of the Termamyl SC 4X enzyme preparation in the hydrolysis reaction system is 500ppm; the temperature of the hydrolysis reaction is 90°C and the time is 2h.

[0040] Specifically:

[0041] 1) Take 25g of gelatinized starch sample, add tap water to 100g and record the actual weight. The sample weight deviation is ±0.10g;

[0042] 2) Add Termamyl SC 4X enzyme preparation to the sample. The concentration of Termamyl SC 4X enzyme preparation in the system is 500 ppm (1 g Termamyl SC 4X is dissolved in 50 ml, and 625 ul is added to the sample). Keep at 90° C. for 2 h.

[0043] S2. Centrifuge and filter the hydrolyzate obtained in step S1 to obtain a test sample.

[0044] Specifically: the hydrolyzate was centrifuged and passed through a 0.45 μm filter to obtain a test sample.

[0045] S3. Add mobile phase to the test sample, the mobile phase is 5mM sulfuric acid solution, the pH value of the system after the test sample and the mobile phase are mixed is 4.0, and the pH value is maintained at 4.0 to ensure that the residual enzyme activity in the test sample will not be further hydrolyzed. Perform high performance liquid chromatography on the test sample to obtain the contents of glucose, maltose, maltotriose, and dextran.

[0046] Specifically: add HPLC mobile phase (or 5 mm H2SO4 solution) to the sample until the fill line, cover the flask with paraffin film, shake vigorously to mix the sample evenly, and filter an aliquot of the hydrolyzate into an HPLC bottle for HPLC detection.

[0047] Liquid phase conditions:

[0048] Column: HPX-87H column and protective column

[0049] Flow rate: 0.60 ml / min

[0050] Mobile phase: 0.005M H2SO4

[0051] Temperature: 65°C (column temperature)

[0052] Injection volume: 10.0ul

[0053] Run time: 30 minutes

[0054] Standard sample preparation:

[0055] Dextran (Dp4+) 3.25 g 3.25% (w / v)

[0056] Maltotriose (Dp3) 0.85 g 0.85% (w / v)

[0057] Maltose (Dp2) 2.0 g 2.0% (w / v)

[0058] Glucose (Dp1) 2.0 g 2.0% (w / v)

[0059] Fructose 0.5g 0.5%(w / v)

[0060] Glycerol 1.0g 1.0%(w / v)

[0061] Lactic acid (85%) 1.0ml 0.85% (v / v)

[0062] Acetic acid (glacial acetic acid) 0.50ml 0.50%(v / v)

[0063] Ethanol (200 proof alcohol concentration) 16.0 ml 16.0% (v / v)

[0064] S4. Calculate the starch dissolution rate based on the content of glucose, maltose, maltotriose and dextran.

[0065] Example 2

[0066] The difference from Example 1 is that Termamyl SC 4X enzyme preparation is added to the gelatinized starch sample, the concentration of Termamyl SC 4X enzyme preparation in the hydrolysis reaction system is 450 ppm (1 g Termamyl SC 4X is dissolved in 45 ml, and 625 ul is added to the sample), and the temperature is maintained at 90° C. for 2 h. The remaining steps are the same as in Example 1.

[0067] In this example, the immobilized enzyme preparation is at the lowest concentration. Below this concentration, hydrolysis cannot be completed, resulting in different results.

[0068] Example 3

[0069] The difference from Example 1 is that Termamyl SC 4X enzyme preparation is added to the gelatinized starch sample, the concentration of Termamyl SC 4X enzyme preparation in the hydrolysis reaction system is 450 ppm (1 g Termamyl SC 4X is dissolved in 45 ml, and 625 ul is added to the sample), and the temperature is maintained at 88° C. for 2.5 h. The remaining steps are the same as in Example 1.

[0070] In this embodiment, the immobilized enzyme preparation is at the lowest concentration. Due to the structural stability of aged starch, this temperature (88°C) is the lowest limit. Below this temperature, the optimal effect of the immobilized enzyme preparation cannot be achieved, and at this temperature, the hydrolysis time needs to be extended to 2.5h to achieve the optimal hydrolysis effect.

[0071] Example 4

[0072] The difference from Example 1 is that a mobile phase is added to the test sample, the mobile phase is a 4.5 mM sulfuric acid solution, and the pH value of the system after the test sample and the mobile phase are mixed is 4.0. The pH value is maintained at 4.0 to ensure that the residual enzyme activity in the test sample will not be further hydrolyzed. The remaining steps are the same as in Example 1.

[0073] In this embodiment, the mobile phase is at the lowest concentration, which is the lower limit of the mobile phase concentration. Below this concentration, the optimal carrying effect cannot be achieved, the components are not fully separated, and the target polysaccharide amount cannot be accurately obtained, resulting in deviations in the calculation results.

[0074] Example 5

[0075] Taking Example 1 as an example, the dissolution rates of different batches of raw starch were measured, and the raw materials involved are shown in Table 1.

[0076] Table 1

[0077]

[0078]

[0079] The starch dissolution rate was measured using the method in Example 1. The results are shown in Table 2.

[0080] Table 2

[0081]

[0082]

[0083] in,

[0084] %Sol or %SI = sum([DP1], [DP2], [DP3], [DP4+]) x (flask volume / 100) / (sample weight x %DS)

[0085] %Sac or %GI = [DP1] / sum([DP1], [DP2], [DP3], [DP4+]

[0086] DS is the moisture content of the raw material (provided by the supplier when purchasing the raw material);

[0087] SI is starch dissolution rate;

[0088] GI is the proportion of monosaccharides after enzymatic hydrolysis of starch raw materials;

[0089] weight is the sample weight;

[0090] Volumn is the volume of the flask.

[0091] The corresponding relationship between starch dissolution rate and raw materials was measured, such as Figure 2 As shown. Among them, Budweiser rice Jilin Haicheng 20240116 and Budweiser rice Jilin Haicheng 20231202 are Budweiser rice produced by Jilin Haicheng on different dates; cornstarch COFCO Yushu 2021010732, corn starch COFCO Yushu 2024010631, corn starch COFCO Yushu 2024011032, corn starch COFCO Yushu 2024010633, corn starch COFCO Yushu 2024010634, and cornstarch COFCO Yushu 2024011232 are corn starch produced by COFCO Yushu on different dates. Figure 2It can be seen that when the raw materials are Budweiser rice Jilin Haicheng 20240116 and Budweiser rice Jilin Haicheng 20231202, the corresponding starch dissolution rates are 104.04 and 103.31 respectively; when the raw materials are corn starch COFCO Yushu 2021010732, corn starch COFCO Yushu 2024010631, corn starch COFCO Yushu 2024011032, corn starch COFCO Yushu 2024010633, corn starch COFCO Yushu 2024010634, and corn starch COFCO Yushu 2024011232, the corresponding starch dissolution rates are 109.55, 107.85, 107.16, 114.39, 113.45, and 110.30 respectively. It can be seen that the method of the present application can determine the dissolution rate of raw starch from different batches, can significantly analyze the differences in raw materials between different batches from the same manufacturer, and abandons the negative impact of the complex and unstable malt enzyme system in the malt enzymatic hydrolysis process, and the measurement results are more reliable.

[0092] The method of the present application uses high performance liquid chromatography to directly determine the sugar components in the raw materials and calculate the final starch dissolution rate. The method is easy to operate and has high accuracy. In addition, the method uses a single fixed enzyme preparation, which can avoid the differences between malt batches and achieve the effect of guiding industrial production with laboratory results.

[0093] Different batches of raw materials were respectively subjected to the starch dissolution rate determination using the method of Example 1 of the present application and the agreed saccharification method. The determination results are as follows: Figure 3 As shown, the red bar graph is the leaching rate measured by the present invention, and the blue dot graph is the leaching rate measured by the conventional saccharification method. Figure 3 It can be seen that when the raw materials are Canadian wheat, A wheat, French wheat and corn granules, the starch extraction rate measured by the agreed saccharification method is greater than that measured by the method of the present invention, indicating that the method of the present invention is not suitable for the determination of the starch dissolution rate of malt and corn granules. For different batches of corn produced by COFCO Yushu, the values ​​measured by the agreed saccharification method are basically the same, while the differences between different batches can be seen by the method of the present invention. In addition, for some batches, the starch extraction rate measured by the method of the present invention is greater than the value measured by the agreed saccharification method. The reason is that the present invention uses a fixed enzyme preparation for enzymatic hydrolysis, which can hydrolyze part of the aged starch, while the malt in the agreed saccharification method cannot hydrolyze the aged starch or the hydrolysis effect is very poor, so that the measured starch extraction rate is relatively small.

[0094] It can be seen that the starch dissolution rate is measured by the method of the present application, and the measurement results are relatively stable and reliable.

[0095] The same batch of corn starch Feitian (purchased from Henan Feitian Biotechnology Co., Ltd., batch chn-2023-00210) and corn starch COFCO (purchased from COFCO Biotechnology Co., Ltd., batch chn-2023-00211) were measured using the method in Example 1 of the present application and the traditional agreed saccharification method, respectively. The measurement results are as follows Figure 4 As shown, the blue bar graph is the starch dissolution rate data measured by the conventional saccharification method, and the red bar graph is the data measured by the method of this application. Figure 4 It can be seen that the use of the agreed saccharification method cannot clearly determine the impact of aged starch, resulting in unclear differences in the results; while the method of the present application can hydrolyze part of the aged starch, and the result obtained is the actual content of the extract that can be hydrolyzed into reducing sugars, and the measurement result is more reliable.

[0096] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for determining starch dissolution rate, characterized in that: The steps include: S1. Add Termamyl SC 4X enzyme preparation to the gelatinized starch sample to perform hydrolysis reaction; S2, centrifuging and filtering the hydrolyzate obtained in step S1 to obtain a test sample; S3, performing high performance liquid chromatography on the test samples to obtain the contents of glucose, maltose, maltotriose, and dextran respectively; S4. Calculate the starch dissolution rate based on the content of glucose, maltose, maltotriose and dextran.

2. The method for determining starch dissolution rate according to claim 1, wherein The dosage ratio of the starch sample to the Termamyl SC 4X enzyme preparation in the S1 is (0.9-1.1):(1.8-2.2).

3. The method for determining starch dissolution rate according to claim 1, characterized in that: The concentration of the Termamyl SC 4X enzyme preparation in the hydrolysis reaction system is 450ppm-550ppm.

4. The method for determining starch dissolution rate according to claim 1, wherein The starch sample in S1 is a starch sample obtained by gelatinization of rice, corn or barley.

5. The method for determining starch dissolution rate according to claim 1, characterized in that: The temperature of the hydrolysis reaction in S1 is 88°C-92°C, and the time is 2h-2.5h.

6. The method for determining starch dissolution rate according to claim 1, characterized in that: The S3 comprises the step of adding a mobile phase to the test sample.

7. The method for determining starch dissolution rate according to claim 6, characterized in that: The pH value of the system after the test sample and the mobile phase were mixed was 4.

0.

8. The method for determining starch dissolution rate according to claim 7, characterized in that: The mobile phase is a 4.8mM-5.2mM sulfuric acid solution.

9. The method for determining starch dissolution rate according to claim 1, characterized in that: The conditions for the HPLC detection in S3 are as follows: flow rate of 0.6 mL / min, temperature of 65° C., injection volume of 10 μL, and running time of 30 min.

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