Method for detecting content of starch in fermented grains by double-enzyme method
Through the dual-enzyme detection method, amylase and saccharase catalyzed reactions, the problem of insufficient sensitivity and safety of starch detection in the prior art was solved, and high sensitivity, accuracy and safe starch content analysis was achieved.
Patent Information
- Application Number
- CN202411071740.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-06
- Publication Date
- 2024-12-06
AI Technical Summary
The existing methods of starch detection of wine mash use chemical methods, which are insufficient in sensitivity and accuracy, and the strong acids and alkalis used pose a potential threat to the environment and operators' health.
The two-enzyme detection method was used to measure the changes of the product and quantitatively analyze the starch content by measuring the changes of the product, including sample pretreatment, enzymatic decomposition and chromogenic reaction steps, and DNS color developer and UV spectrophotometer Or a microplate reader for measurement.
It improves the sensitivity and accuracy of detection, simplifies operation, reduces the use of chemical reagents, improves safety and detection reliability, and is suitable for the analysis of complex samples.
Smart Images
Figure CN119082260A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of fermented grains analysis and detection, in particular to a method for detecting starch content in fermented grains by using a double enzyme method. Background Art
[0002] Fermented grains are a key intermediate product in the winemaking process. They refer to a mixture of yeast, sugars and other compounds formed during the fermentation stage. They play an important role in wine production and affect the flavor, aroma and quality of the final product. Fermented grain starch testing is one of the important indicators for evaluating the wine yield during the production process.
[0003] The current detection of starch in mash is carried out using chemical methods. The sensitivity and accuracy of chemical methods for detecting starch may be limited, and the test results may not be reliable enough and the accuracy is not high enough. In addition, some reagents used in chemical methods (such as acidic and alkaline reagents) may pose potential risks to the environment and the health of operators. For example, the chemical method commonly used at present requires a large amount of strong acid and alkali. Acid needs to be added and heated in an electric furnace for reflux for 30 minutes, and titration is performed on the electric furnace. On the one hand, the large-scale use of strong acids and alkalis is not good for the environment, and workers who inhale too much strong acids and alkalis will endanger their health. On the other hand, workers are prone to explosion during the reflux and titration process, which endangers the physical and mental safety of workers.
[0004] Therefore, it is urgently necessary to develop a method with higher sensitivity, more accurate and reliable detection results, less use of chemical reagents and better safety. Summary of the invention
[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a method for detecting starch content in fermented grains by a double enzyme method, which is as follows:
[0006] A method for detecting starch content in fermented grains by double enzyme method comprises the following steps:
[0007] (1) Sample pretreatment: The fermented grains sample is dried to constant weight and ground into powder;
[0008] (2) Sampling: Take a sample, add purified water to a boiling water bath, and cool to room temperature;
[0009] (3) Prepare reagents: 1% amylase solution, 0.25% saccharifying enzyme solution, and 1 mg / mL glucose standard solution;
[0010] (4) pH adjustment: adjust the pH of the sample solution to 4.5-5;
[0011] (5) Amylase hydrolysis: add 1% amylase solution, bathe in 60°C water for 1 h, filter, and adjust to volume;
[0012] (6) Saccharifying enzyme hydrolysis: Take the above amylase hydrolyzate, add 0.25% saccharifying enzyme solution, incubate in a 60°C water bath for 0.5 h, and adjust to volume.
[0013] (7) Use DNS colorimetric reagent for color development and measure the absorbance at 540 nm using a UV spectrophotometer or microplate reader.
[0014] Furthermore, the drying is performed at 40-65°C.
[0015] Furthermore, the grinding into powder is powder that passes through a 40-mesh sieve.
[0016] Furthermore, the step (2) is to take a sample, add 30-50 times the amount of purified water and boil it in a boiling water bath for 15-20 minutes.
[0017] Furthermore, the 1% amylase solution in step (3) is prepared by the following steps: 1 g of amylase is dissolved in purified water and the volume is adjusted to 100 mL in a volumetric flask, centrifuged, and the supernatant is collected.
[0018] Furthermore, the 0.25% saccharifying enzyme solution in step (3) is prepared by the following steps: 0.25 g of saccharifying enzyme is dissolved in purified water and the volume is adjusted to 100 mL in a volumetric flask, centrifuged, and the supernatant is collected.
[0019] Furthermore, the 1 mg / mL glucose standard solution in step (3) is prepared by the following steps: weigh 0.1 g of anhydrous glucose (dried at 105° C. for more than 2 h) and put it into a conical flask or beaker, add purified water to dissolve and dilute to a 100 mL volumetric flask.
[0020] Furthermore, the step (5) specifically includes adding 5-10 mL of 1% amylase solution, bathing at 60° C. for 50 min-60 min, filtering, and diluting to 50 mL with purified water.
[0021] Furthermore, the step (6) is to add 1 mL of the above amylase hydrolyzate to 1 mL of 0.25% saccharifying enzyme solution, place in a 60° C. water bath for 30 min-120 min, and then dilute to 10 mL with purified water.
[0022] In addition, in order to determine the optimal parameters and step effects, the applicant has also conducted sufficient exploratory experiments, and now discloses some of the experimental contents, as follows:
[0023] 1. Effect of pH value of amylase:
[0024] In this experiment, 0.1g of fermented grains sample was weighed, 3mL of water was added and boiled in a water bath for 20min, and cooled to room temperature to explore the effect of different pH on the activity of amylase. The pH was set from 4, 4.5, 5, 5.5, 6, 6.5, and 7 to determine the optimal pH of amylase; 10mL of 1% amylase was added and placed in a 60℃ water bath for 1h. After the hydrolysis was completed, the volume was adjusted to 50mL, 1mL of enzymatic hydrolyzate was added with 2mL DNS and boiled in a water bath for 5min, 9mL of water was added, the OD value was recorded, and the optimal pH of amylase was analyzed. The results are as follows Figure 1 shown.
[0025] Depend on Figure 1 It can be seen that as the pH value increases, the OD value rises and then decreases, and the activity of amylase first increases and then gradually decreases. When the pH is 4.5, the activity of amylase reaches the maximum. Therefore, the optimal pH of amylase is 4.5.
[0026] 2. Effect of amylase treatment time
[0027] In this experiment, 0.1g of fermented grains sample was weighed, 3mL of water was added and boiled in a water bath for 20min, the pH value of the sample solution was adjusted to 4.5-4.6, 10mL of 1% amylase was added, and it was placed in a 60℃ water bath for different time periods to explore the optimal hydrolysis time of amylase. The hydrolysis time was set from 30min, 40min, 50min, 60min, and 70min to analyze the optimal time for amylase hydrolysis. After the hydrolysis was completed, the volume was fixed to 50mL, 1mL of enzymatic hydrolyzate was added with 2mL DNS and boiled in a water bath for 5min, 9mL of water was added, the OD value was recorded, and the optimal time for amylase hydrolysis was determined. The results are as follows Figure 2 shown.
[0028] Depend on Figure 2 It can be seen that the OD value continues to rise when the hydrolysis time of amylase is from 30min to 50min. Therefore, it can be concluded that as the hydrolysis time increases, the degree of hydrolysis gradually increases; when the hydrolysis time is 50min, it reaches the maximum value, and the OD value remains constant when the hydrolysis time is further extended. Therefore, it can be concluded that the hydrolysis time of amylase is 50min.
[0029] 3. Effect of pH value on saccharifying enzyme
[0030] In this test, 0.1 g of fermented grains sample was weighed, 3 mL of water was added and boiled in a water bath for 20 min, the pH value of the sample solution was adjusted to 4.5-4.6, 10 mL of 1% amylase was added, and the solution was placed in a 60°C water bath for 50 min and the volume was fixed to 50 mL.
[0031] To investigate the effect of different pH on the activity of saccharifying enzyme, the pH values were set at 4, 4.2, 4.4, 4.6, 4.8, and 5. The amylase hydrolysate was adjusted to the corresponding pH. 1 mL of amylase hydrolysate was taken and 1 mL of 0.25% saccharifying enzyme was added respectively. The solution was placed in a 60°C water bath for 30 min. After the hydrolysis was completed, the volume was fixed to 10 mL. 1 mL of hydrolysate was added with 2 mL of DNS in a boiling water bath for 5 min. 9 mL of water was added and the OD value was recorded. The glucose concentration was read from the standard curve and the starch content was calculated to determine the optimal pH of the saccharifying enzyme. The calculation formula was as follows: starch content (%) = glucose concentration * dilution factor (mL) * 0.9 / weighed sample weight (mg); Figure 3 shown.
[0032] Depend on Figure 3 It can be seen that the pH between 4-5 has no significant effect on the content of starch hydrolyzed by saccharifying enzyme. Therefore, when using saccharifying enzyme to hydrolyze starch, it is sufficient to keep the pH between 4-5; since the optimal pH of amylase is 4.5-4.6 and the optimal pH of saccharifying enzyme is 4-5, there is no need to adjust the pH during saccharifying enzyme hydrolysis.
[0033] 4. Effect of saccharifying enzyme concentration and starch content
[0034] In this test, 0.1 g of fermented grains sample was weighed, 3 mL of water was added and boiled in a water bath for 20 min, the pH value of the sample solution was adjusted to 4.5-4.6, and the sample solution was placed in a 60°C water bath for 50 min. After the hydrolysis was completed, the volume was fixed to 50 mL.
[0035] To explore the effect of different concentrations of saccharifying enzyme on the content of hydrolyzed starch, 1 mL of amylase hydrolyzate was added with 1 mL of saccharifying enzyme of different concentrations, and placed in a 60°C water bath for 30 min. The degree of starch hydrolysis by saccharifying enzyme of different concentrations was analyzed. The concentration of saccharifying enzyme was set to 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, and 0.3%. After the hydrolysis was completed, the volume was fixed to 10 mL. 1 mL of enzymatic hydrolyzate was added with 2 mL DNS in a boiling water bath for 5 min, 9 mL of water was added, the OD value was recorded, the glucose concentration was read from the standard curve, the starch content was calculated, and the optimal concentration of saccharifying enzyme was determined. The calculation formula was: starch content (%) = glucose concentration * dilution factor (mL) * 0.9 / weighed sample weight (mg); the results are as follows Figure 4 shown.
[0036] Depend on Figure 4 It can be seen that with the increase of saccharifying enzyme concentration, the starch content gradually increases. When the saccharifying enzyme concentration reaches 0.25%, the starch content no longer increases. Therefore, the optimal concentration of saccharifying enzyme is 0.25%.
[0037] 5. Effect of saccharifying enzyme addition amount and starch content
[0038] In this test, 0.1 g of fermented grains sample was weighed, 3 mL of water was added and boiled in a water bath for 20 min, the pH value of the sample solution was adjusted to 4.5-4.6, and the sample solution was placed in a 60°C water bath for 50 min. After the hydrolysis was completed, the volume was fixed to 50 mL.
[0039] To explore the effect of different amounts of saccharifying enzyme added on the content of hydrolyzed starch, 1 mL of amylase hydrolysate was added with different amounts of 2.5% saccharifying enzyme, and placed in a 60°C water bath for 30 min. The addition amounts were set to 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.9 mL, and 1 mL. After the hydrolysis was completed, the volume was fixed to 10 mL, 1 mL of hydrolysate was added with 2 mL DNS in a boiling water bath for 5 min, 9 mL of water was added, the OD value was recorded, the glucose concentration was read from the standard curve, the starch content was calculated, and the optimal amount of saccharifying enzyme added was determined. The calculation formula was: starch content (%) = glucose concentration * dilution factor (mL) * 0.9 / weighed sample weight (mg); the results are as follows Figure 5 shown.
[0040] Based on the hydrolysis of 30min, different gradient addition amounts were set. Figure 5 It can be seen that as the amount of saccharifying enzyme added gradually increases, the starch content also gradually increases; when the amount of saccharifying enzyme added is 0.9 mL, the starch content reaches the maximum value, and the starch content no longer increases with the continued addition of saccharifying enzyme, but remains constant. It can be concluded that the amount of amylase added is 0.9 mL. In the actual hydrolysis process, the amount of saccharifying enzyme added is 1 mL, so as to facilitate the subsequent constant volume, etc.
[0041] 6. Effect of hydrolysis time
[0042] In this test, 0.1 g of fermented grains sample was weighed, 3 mL of water was added and boiled in a water bath for 20 min, the pH value of the sample solution was adjusted to 4.5-4.6, and the sample solution was placed in a 60°C water bath for 50 min. After the hydrolysis was completed, the volume was fixed to 50 mL.
[0043] To explore the optimal time for saccharifying enzyme hydrolysis, 1 mL of amylase hydrolysate was added with 1 mL of 0.25% saccharifying enzyme, and placed in a 60°C water bath for different times. The hydrolysis time was set to 30 min, 60 min, 90 min, and 120 min. After the hydrolysis was completed, the volume was adjusted to 10 mL. 1 mL of hydrolysate was added with 2 mL of DNS in a boiling water bath for 5 min, and 9 mL of water was added. The OD value was recorded, the glucose concentration was read from the standard curve, the starch content was calculated, and the optimal hydrolysis time was determined. The calculation formula was: starch content (%) = glucose concentration * dilution factor (mL) * 0.9 / weighed sample weight (mg); the results are as follows Figure 6 shown.
[0044] Depend on Figure 6 It can be seen that as the saccharifying enzyme hydrolysis time gradually increases, the starch content remains almost unchanged; therefore, the saccharifying enzyme hydrolysis time is 30 minutes.
[0045] 7. Determine the optimal sample weight
[0046] This experiment explored the effect of double enzyme hydrolysis on different weights of fermented grains samples. Weigh different weights of fermented grains samples, add 3mL of water and boil in a water bath for 20min, adjust the pH value of the sample solution to 4.5-4.6, add 10mL of 1% amylase, and place in a 60℃ water bath for 50min. After the hydrolysis is completed, the volume is adjusted to 50mL, 1mL of amylase hydrolysate is added with 1mL of 0.25% saccharifying enzyme, and placed in a 60℃ water bath for 30min. After the hydrolysis is completed, the volume is adjusted to 10mL, 1mL of enzymatic hydrolysate is added with 2mL DNS and boiled in a water bath for 5min, 9mL of water is added, the OD value is recorded, the glucose concentration is read from the standard curve, the starch content is calculated, and the enzymatic hydrolysis effect of samples of different weights is analyzed. The calculation formula is: starch content (%) = glucose concentration * dilution factor (mL) * 0.9 / weighed sample weight (mg). The results are as follows Figure 7 shown.
[0047] Depend on Figure 7 It can be seen that the starch content remains almost unchanged when 0.1g-0.5g of the sample is weighed. Therefore, in this experiment, 0.1g of the mash sample is weighed.
[0048] Compared with the prior art, the technical effects created by the present invention are embodied in:
[0049] The dual enzyme method for hydrolyzing starch from fermented grains adopted in the present invention mainly has the following functions: the method utilizes amylase and saccharifying enzyme to catalyze the reaction and quantitatively analyzes the starch content in the sample by measuring the change of the product. Compared with the traditional method, the present invention has the advantages of simple operation, high sensitivity, wide application range, high safety factor, etc.
[0050] The dual enzyme method for hydrolyzing fermented grains starch of the present invention has the following beneficial effects:
[0051] 1. High selectivity and specificity: The enzymatic method uses specific enzymes (such as amylase) to decompose starch. These enzymes have high selectivity and specificity for the target substance. Unlike the chemical method, the enzymatic method can accurately react to starch, reducing the interference of other substances, thereby improving the accuracy of detection.
[0052] 2. High sensitivity: Enzymatic methods can detect very low concentrations of starch. Enzymatic reactions usually have high sensitivity and can detect trace amounts of starch, making them suitable for the analysis of low-concentration samples.
[0053] 3. Accurate quantification: The enzymatic method can quantify the starch content by measuring the amount of the enzyme reaction product. The enzyme-catalyzed reaction is usually proportional to the concentration of starch, so the starch content can be accurately quantified by measuring the amount of the reaction product (such as glucose).
[0054] 4. Easy operation: The enzymatic method is relatively easy to operate, usually including steps such as mixing the enzyme with the sample, incubating the reaction and measuring the results. Compared with the chemical method, the enzymatic method does not require complicated reagent preparation or cumbersome operation steps, and the results are often read automatically by the instrument, saving operation time and labor intensity.
[0055] 5. Environmentally friendly: Enzymatic methods usually do not involve toxic or hazardous chemicals, and the enzymes used are mostly of biological origin, so they are environmentally friendly. The handling and disposal process is relatively safe, with less health risks to operators.
[0056] 6. High reproducibility: Due to the specificity and stability of the enzyme reaction, enzymatic detection usually has high reproducibility. Multiple determinations under the same conditions can obtain consistent results, ensuring the reliability of the experimental data.
[0057] 7. Applicable to complex samples: Enzymatic methods can effectively detect starch in complex sample matrices, such as fermented grains, despite the presence of a variety of other components and by-products. The specificity of the enzyme can reduce the interference of other substances in the sample, thereby obtaining accurate starch content data.
[0058] 8. Efficiency and automation: It can process multiple samples at the same time and reduce human errors through standardized processes, which is suitable for large-scale sample analysis.
[0059] 9. Strong operability: The enzyme method can be carried out under conventional laboratory conditions without the need for special experimental conditions or equipment. It is also easy to combine with other analytical techniques (such as photometer, electrochemical analysis, etc.), further enhancing the functionality and flexibility of the detection.
[0060] 10. Clear data interpretation: The amount of product (such as glucose) generated by the enzyme reaction usually has a clear relationship with the concentration of starch, and the data interpretation is intuitive and easy to understand. This makes the analysis and reporting of results easier and more intuitive. BRIEF DESCRIPTION OF THE DRAWINGS
[0061] Figure 1 This is a graph showing the effect of pH on amylase.
[0062] Figure 2 This is a graph showing the results of amylase treatment time.
[0063] Figure 3 This is a graph showing the effect of pH on saccharifying enzyme.
[0064] Figure 4 This is a graph showing the relationship between saccharifying enzyme concentration and starch content.
[0065] Figure 5 This is a graph showing the relationship between the amount of saccharifying enzyme added and the starch content.
[0066] Figure 6 This is a graph showing the relationship between hydrolysis time and starch content.
[0067] Figure 7 This is a graph showing the relationship between sample weight and starch content.
[0068] Figure 8 This is the flow chart of the double enzyme method for hydrolyzing fermented grains starch in this application. DETAILED DESCRIPTION
[0069] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.
[0070] Example:
[0071] Take 12 different fermentation batches of in-tank / out-tank fermented grains samples and divide them into 12 groups. Examples 1-12 are each measured for 12 different fermented grains samples, and then compared with the results obtained by using the local standard DB34 / T2264-2014. The specific results are as follows:
[0072] Table 1 Comparison of starch content extracted from local standards and enzymatic fermented grains
[0073] Example DB34 / T2264-2014 Enzymatic method Example 1 68.61％ 69.56％ Example 2 51.11％ 52.76％ Example 3 59.44％ 59.92％ Example 4 62.37％ 62.77％ Example 5 58.59％ 57.80％ Example 6 49.17％ 49.57％ Example 7 61.49％ 62.55％ Example 8 51.94％ 51.28％ Example 9 51.77％ 50.72％ Example 10 57.02％ 57.82％ Embodiment 11 51.53％ 50.72％ Example 12 51.77％ 51.46％
[0074] Finally, it should be pointed out that the above embodiments are only representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.
Claims
1. A method for detecting starch content in fermented grains by double enzyme method, characterized in that: The steps include: (1) Sample pretreatment: The fermented grains sample is dried to constant weight and ground into powder; (2) Sampling: Take a sample, add purified water to a boiling water bath, and cool to room temperature; (3) Prepare reagents: 1% amylase solution, 0.25% saccharifying enzyme solution, and 1 mg / mL glucose standard solution; (4) pH adjustment: adjust the pH of the sample solution to 4.5-5; (5) Amylase hydrolysis: add 1% amylase solution, bathe in 60°C water for 1 h, filter, and adjust to volume; (6) Saccharifying enzyme hydrolysis: Take the above amylase hydrolyzate, add 0.25% saccharifying enzyme solution, incubate in a 60°C water bath for 0.5 h, and adjust to volume. (7) Use DNS colorimetric reagent for color development and measure the absorbance at 540 nm using a UV spectrophotometer or microplate reader.
2. The method for detecting starch content in fermented grains by double enzyme method according to claim 1, characterized in that: The drying is performed at 40-65°C.
3. The method for detecting starch content in fermented grains by double enzyme method according to claim 1, characterized in that: The powder is ground into powder that passes through a 40-mesh sieve.
4. The method for detecting starch content in fermented grains by double enzyme method according to claim 1, characterized in that: The step (2) specifically comprises taking a sample, adding 30-50 times the amount of purified water and boiling it in a water bath for 15-20 minutes.
5. The method for detecting starch content in fermented grains by double enzyme method according to claim 1, characterized in that: The 1% amylase solution in step (3) is prepared by the following steps: 1 g of amylase is dissolved in purified water and the volume is fixed to a 100 mL volumetric flask, centrifuged, and the supernatant is collected.
6. The method for detecting starch content in fermented grains by double enzyme method according to claim 1, characterized in that: The 0.25% saccharifying enzyme solution in step (3) is prepared by the following steps: 0.25 g of saccharifying enzyme is dissolved in purified water and the volume is fixed to a 100 mL volumetric flask, centrifuged, and the supernatant is collected.
7. The method for detecting starch content in fermented grains by double enzyme method according to claim 1, characterized in that: The 1 mg / mL glucose standard solution in step (3) is prepared by the following steps: weigh 0.1 g of anhydrous glucose and put it into a conical flask or beaker, add purified water to dissolve it and make up to 100 mL of the volumetric flask.
8. The method for detecting starch content in fermented grains by double enzyme method according to claim 1, characterized in that: The step (5) specifically includes adding 5-10 mL of 1% amylase solution, bathing in a 60° C. water bath for 50-60 min, filtering, and diluting to 50 mL with purified water.
9. The method for detecting starch content in fermented grains by double enzyme method according to claim 1, characterized in that: The step (6) is specifically to add 1 mL of the above amylase hydrolyzate to 1 mL of 0.25% saccharifying enzyme solution, place in a 60° C. water bath for 30-120 min, and then dilute to 10 mL with purified water.