Membrane concentration technology-based amylase activity detection method and application thereof

Abstract

The invention discloses an amylase activity detection method based on secondary membrane concentration, and belongs to the field of biotechnology detection. The method comprises the following steps: carrying out first membrane concentration through tangential flow filtration, primarily enriching amylase and replacing a buffer solution; performing secondary membrane concentration through centrifugal ultrafiltration to efficiently prepare a high-concentration enzyme solution; and finally, carrying out activity detection on the concentrated solution. According to the method disclosed by the invention, two membrane concentration steps are synergistically combined and are preferably carried out in the calcium-containing buffer solution, so that the technical problems of large activity loss, low recovery rate and serious interference when trace amylase is concentrated from complex sugar-containing matrixes such as cane sugar and agar are effectively solved. The method has the advantages of simple operation, high sensitivity and good reproducibility, and is suitable for raw material quality control, production process monitoring and product stability evaluation in the fields of food industry and enzyme preparations.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a method for detecting amylase activity based on membrane concentration technology and its application. Background Technology

[0002] In this study, we focus on the application of a secondary membrane concentration-based method for detecting amylase activity in dairy systems. Amylase activity in dairy products such as yogurt significantly impacts product texture and stability, especially when room temperature storage or the use of modified starches (such as HPDSP). Even extremely low concentrations of α-amylase can cause significant viscosity changes and texture deterioration during storage. Therefore, establishing a method to effectively extract and concentrate trace amounts of amylase from samples is crucial for accurately assessing its activity and controlling product quality.

[0003] Current conventional enzyme activity detection methods are often limited by detection sensitivity and matrix interference when dealing with complex matrices (such as yogurt), making accurate quantification difficult. Secondary membrane concentration technology, by combining ultrafiltration membranes with different molecular weight cutoffs, can achieve efficient enrichment and purification of amylase in samples, and is particularly suitable for the detection of trace enzyme activity in dairy products. This method not only helps to eliminate interference from sugars, proteins, and other components, but also effectively maintains enzyme activity stability, providing a reliable sample basis for subsequent activity assays.

[0004] Although membrane concentration technology has been applied in biomolecular separation, its applicability and stability in acidic, protein-rich, and high-viscosity systems (such as yogurt) still require systematic evaluation. Furthermore, the effects of enzyme source characteristics (such as the differences in acid resistance and thermal stability between α-amylases derived from Aspergillus oryzae and Bacillus licheniformis) and process conditions (including concentration pressure, membrane material, and pretreatment methods) on recovery rates and enzyme activity retention still need further optimization and validation. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides a method for concentrating amylase from a sugar-containing matrix, comprising the following steps:

[0006] 1) Dissolve the sugar matrix containing amylase in a buffer solution to obtain the first solution;

[0007] 2) Filter and concentrate the first solution to obtain the second solution;

[0008] 3) Filter and concentrate the second solution to obtain a concentrated amylase solution.

[0009] This application also provides a method for detecting amylase activity, using the Megazyme kit to detect the activity of the concentrated amylase solution obtained in the above method.

[0010] This application also provides the application of the above methods or the above detection methods in the quality control of raw materials in the food industry, the monitoring of enzyme preparation production processes, or the evaluation of product storage stability.

[0011] Therefore, this study aims to establish a method for detecting amylase activity based on secondary membrane concentration, so as to improve the sensitivity and accuracy of amylase detection in complex dairy product systems such as yogurt, and provide technical support for raw material quality control, production process monitoring and product storage stability research.

[0012] The beneficial effects of this application include, but are not limited to: (1) High activity recovery rate: The two-step membrane concentration process is mild, and the enzyme activity loss can be controlled within 20%, which is significantly better than the precipitation method. (2) High sensitivity and anti-interference ability: It effectively separates amylase from interfering components such as sugar and protein in the matrix, and is especially suitable for detecting trace amylase activity as low as 1 mU / mL. (3) Simple and universal operation: No complex large-scale equipment is required. It can be carried out in a routine laboratory and can be widely used in different matrices such as sucrose, agar, and dairy products. (4) Good reproducibility: The method has a high degree of standardization, is less affected by subjective operation factors, and the detection results are reliable. Attached Figure Description

[0013] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0014] Figure 1 This is a schematic diagram of the amylase concentration process. Detailed Implementation

[0015] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0016] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0017] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0018] This application provides a method for concentrating amylase from a sugar-containing matrix, comprising the following steps:

[0019] 1) Dissolve the sugar matrix containing amylase in a buffer solution to obtain the first solution;

[0020] 2) Filter and concentrate the first solution to obtain the second solution;

[0021] 3) Filter and concentrate the second solution to obtain a concentrated amylase solution.

[0022] In some embodiments, the sugar matrix may be selected from sucrose or agar.

[0023] In some embodiments, the amylase may be α-amylase. In some embodiments, preferably, the amylase may be derived from Aspergillus oryzae.

[0024] In some embodiments, the buffer solution in step 1) may be a PBS buffer. Preferably, in some embodiments, the buffer solution may be a 20 mM PBS buffer. More preferably, in some embodiments, the buffer solution may contain 0.5–5 mM calcium chloride. For example, the buffer solution may contain 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5 mM calcium chloride. Any range characterized by combinations of the above-mentioned values ​​is also included, which will not be elaborated here. In some embodiments, more preferably, the buffer solution may contain 1 mM calcium chloride.

[0025] In some embodiments, the pH of the buffer solution in step 1) can be 6.0 to 7.2. For example, in step 1), the pH of the buffer solution can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, or 7.2. Any range characterized by combinations of the above-mentioned values ​​is also included, which will not be elaborated here.

[0026] In some embodiments, the amylase content in the first solution is 0.5–3 mU / mL. For example, the amylase content in the first solution is 0.5, 0.75, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3 mU / mL. Any range characterized by combinations of the above values ​​is also included, which will not be elaborated here. In some embodiments, preferably, the amylase content in the first solution is 1 mU / mL.

[0027] In some embodiments, the sugar matrix content in the first solution can be 0.01–0.1 g / mL. For example, the sugar matrix content in the first solution can be 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1 g / mL. Any range characterized by combinations of the above values ​​is also included, which will not be elaborated here. In some embodiments, preferably, the sugar matrix content in the first solution can be 0.05 g / mL.

[0028] In some embodiments, in step 2), the first solution may be concentrated by filtration using a tangential flow filtration device and an ultrafiltration membrane. Preferably, in some embodiments, the ultrafiltration membrane may be a polyethersulfone membrane with a molecular weight cutoff of 10 kDa.

[0029] In some embodiments, in step 2), the volume of the second solution can be 5% to 20% of the volume of the first solution. For example, in step 2), the volume of the second solution can be 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, or 20% of the volume of the first solution. Any range characterized by combinations of the above values ​​is also included, which will not be elaborated here. In some embodiments, preferably, in step 2), the volume of the second solution can be 10% of the volume of the first solution.

[0030] In some embodiments, in step 3), a 5μm filter membrane can be used for filtration.

[0031] In some embodiments, in step 3), a centrifugal ultrafiltration device can be used for concentration. In some embodiments, preferably, the ultrafiltration membrane of the centrifugal ultrafiltration device can be an ultrafiltration membrane with a molecular weight cutoff of 10 kDa.

[0032] In some embodiments, in step 3), the volume of the concentrated amylase solution is 5% to 20% of the volume of the second solution. For example, in step 3), the volume of the second solution can be 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, or 20% of the volume of the first solution. Any range characterized by combinations of the above values ​​is also included, which will not be elaborated here. In some embodiments, preferably, in step 3), the volume of the concentrated amylase solution is 10% of the volume of the second solution.

[0033] In some embodiments, the first solution may be pre-filtered using a pre-filter membrane before step 2).

[0034] In some embodiments, before step 2), the first solution needs to be left to stand at 4°C for 4 hours, and then centrifuged at 4°C, 8000 rpm for 20 minutes.

[0035] In some embodiments, the method excludes the use of organic solvent precipitation for concentration.

[0036] This application also provides a method for detecting amylase activity, using the Megazyme kit to detect the activity of the concentrated amylase solution obtained in the above method.

[0037] This application also provides the application of the above methods or the above detection methods in the quality control of raw materials in the food industry, the monitoring of enzyme preparation production processes, or the evaluation of product storage stability.

[0038] The present invention aims to overcome the shortcomings of existing technologies in efficiently concentrating trace amounts of amylase from complex sugar matrices while maintaining a high activity recovery rate, and provides a simple and highly sensitive method for detecting amylase activity based on secondary membrane concentration.

[0039] To achieve the above objectives, the present invention adopts the following technical solution:

[0040] A method for detecting amylase activity based on secondary membrane concentration is proposed, the core of which lies in a two-step purification and enrichment strategy involving primary and secondary membrane concentration of the sample. The primary membrane concentration preferably employs tangential flow filtration (TFF) using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa (such as a polyethersulfone (PES) membrane). Under mild pressure (e.g., ≤2 bar), a large volume of sample is initially concentrated and buffer is replaced, effectively removing small-molecule sugar impurities. The secondary membrane concentration preferably employs centrifugal ultrafiltration using an ultrafiltration membrane with a matching molecular weight cutoff (such as a 10 kDa ultrafiltration tube). This provides a final, highly efficient concentration of the primary concentrate, ultimately yielding a sample suitable for high-sensitivity activity detection.

[0041] Furthermore, to maintain the stability of amylase throughout the concentration process, calcium ions (e.g., 1 mM CaCl2) should be added to the buffer solution as an enzyme stabilizer, and organic solvent precipitation methods (e.g., OrgoSol) that can lead to irreversible loss of enzyme activity should be avoided. TM Buffer).

[0042] This invention provides a method for efficiently concentrating amylase from sucrose or agar while maintaining its activity, comprising the following steps:

[0043] 1. Sample pretreatment: Dissolve the sucrose or agar sample containing amylase in phosphate buffer (PBS, pH 6.0–7.2), and add calcium ions (Ca). 2+ To stabilize the enzyme structure;

[0044] 2. Membrane filtration concentration: Tangential flow filtration is performed using a polyethersulfone (PES) membrane with a molecular weight cutoff of 10 kDa to gradually concentrate the product to the target volume;

[0045] 3. Secondary concentration: The enzyme concentration is further increased by using a 10kDa centrifugal ultrafiltration tube;

[0046] 4. Enzyme activity assay: The enzyme activity was calculated by measuring the A400 absorbance using the Megazyme kit at 40°C.

[0047] 5. Avoidance of precipitation method: This invention explicitly excludes the use of organic solvent precipitation methods (such as OrgoSol). TM Buffer), because it causes a significant decrease in enzyme activity.

[0048] Preferred solution:

[0049] • Use PBS buffer containing 1 mM CaCl2;

[0050] • During the concentration process, the pressure should be controlled within 2 Pa (efficiency is highest at 1.5 Pa, and 2 Pa is the condition to ensure normal operation of membrane filtration);

[0051] • Large particulate impurities are removed using a 5μm pre-filtration membrane (Aladdin, aqueous phase) before concentration;

[0052] • Avoid using CaCl2 for amylase concentration in agar, as it easily forms gels and affects the concentration effect.

[0053] Beneficial effects

[0054] 1. High enzyme activity retention rate, with less than 20% enzyme activity loss during concentration;

[0055] 2. Simple to operate, requiring no complex equipment, suitable for conventional laboratories;

[0056] 3. It can effectively separate amylase from sugar background, avoiding false negative results;

[0057] 4. Suitable for the detection and concentration of low concentrations of amylase (e.g., 1 mU / mL).

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0059] Example

[0060] Example 1: Concentration and detection of amylase in sucrose;

[0061] [Experimental Objective]

[0062] Concentration of amylase in sucrose

[0063]

Experimental Methods

[0064] 20mM PBS buffer preparation:

[0065] Prepare initial solution a: 2.4 g NaH2PO4, 1 L water

[0066] Preparation of initial solution b: 284 mg Na₂HPO₄, 100 mL water

[0067] Prepare final solution c: Take 941 mL of solution a, add approximately 59 mL of solution b to adjust the pH to 6.0, and add water to bring the volume to 1 L. Filter using a 0.45 μm filter membrane.

[0068] 100mM calcium chloride is prepared by using 1.11g of water per 100mL.

[0069] Preparation of 1mM calcium chloride: 10mL of 100mM calcium chloride, 1L of solution c.

[0070] Preparation of sucrose sample concentrate a0:

[0071] ASa: α-Amylase is derived from Aspergillus oryzae powder, ~30 U / mg. Microbial amylases are extracellular enzymes that can be used in various industrial applications, such as bread and malt syrup production, soy sauce and soybean paste fermentation, etc. The molecular weight of α-amylase isolated from Aspergillus oryzae is 51 kDa (sedimentation and diffusion) and 49 kDa (gel filtration).

[0072] Dissolve 10g of sucrose in 200mL of phosphate buffer (calcium chloride) (with 1mU / mL ASa added). The resulting solution is the original positive sample solution. Concentrate the solution using a PES-10kDa membrane (prepare 800mL of PBS to replace the sucrose). Concentrate the solution to approximately 20mL.

[0073] Dissolve 10g of sucrose in 200mL of phosphate buffer (calcium chloride) to obtain the original negative sample solution. Concentrate the solution using a PES-10kDa membrane (prepare 800mL of PBS to replace the sucrose). Concentrate the solution to approximately 20mL.

[0074] Preparation of secondary concentrate b0:

[0075] 20 mL of concentrate a0 was filtered through a 5 μL filter membrane and then concentrated to 2 mL using a 10 kDa centrifuge filter tube.

[0076] Test the amylase activity.

[0077] Protein precipitation C0 (Activity-preserving protein precipitation and concentration kit):

[0078] Protein precipitation was performed on the secondary concentrate b0 using the following method to obtain protein precipitate c0.

[0079] 1. OrgoSol TM The buffer was stored at -20°C for 1 hour.

[0080] 2. Take 10 mL of OrgoSol TM The buffer was stored at -20℃ for 20 minutes.

[0081] 3. Add 1 mL of protein solution and mix well.

[0082] 4. Add 5 μl of SEED and mix well.

[0083] 5. Store immediately at -20℃ for 3 hours.

[0084] Centrifuge at 15,000g for 15 minutes at 1-4℃.

[0085] 7. After centrifugation, immediately remove the test tube from the centrifuge and pour the supernatant into a beaker. Be careful not to spill any white particles.

[0086] 8. Turn the test tube over on a clean paper towel for 5 seconds, then transfer the test tube to an ice bucket.

[0087] Amylase activity assay:

[0088] 1. Dissolve the protein precipitate c0 in 200 μL of PBS buffer to obtain the final solution d0, which is used for enzyme activity detection.

[0089] 2. Amylase activity assay: Using the Megazyme kit, take 200 μL of HR substrate and 200 μL of protein extraction solution, react at 40℃ for 20 min-48 h, and measure A using a microplate reader. 400 Absorbance value.

[0090] 3. Calculation of amylase activity: Units (CU) / gΔA 400 ×0.313×Dilution factor

[0091] Sample Description

[0092] a1: The original solution of the positive sample is used to prepare concentrated solution a0, secondary concentrated solution b0 is prepared, protein precipitate c0 is prepared, and the final solution d0 is obtained.

[0093] a2: Take 200 μL of amylase solution (ASa 1 mU / mL buffer) to prepare protein precipitate c0, obtaining the final solution d0. a3: 200 μL of amylase solution (ASa 2 mU / mL buffer).

[0094] a4: The original solution of the positive sample is used to prepare concentrated solution a0, and then a secondary concentrated solution b0 is prepared to obtain the final solution d0.

[0095] a5: Positive sample original solution, prepare concentrated solution a0, prepare secondary concentrated solution b0, to obtain final solution d0, and store for 2 weeks. a6: Negative sample original solution, prepare concentrated solution a0, prepare secondary concentrated solution b0, to obtain final solution d0.

[0096] Control b: Control, ASa 1mU / mL.

[0097] Table 1: Comparison of Sucrose Concentration Results

[0098] serial number sample absorbance amylase activity Concentration ratio a / b 1 Positive sucrose concentration-precipitation-reconstituted solution a1 1.830 0.57 112％ 2 Amylase-precipitate-reconstituted solution a2 1.601 0.50 98％ 3 amylase solution a3 2.287 0.72 141％ 4 Positive sucrose concentrate a4 2.458 0.77 151％ 5 Positive sucrose concentrate can be stored for 2 weeks. 1.718 0.54 106％ 6 Negative sucrose concentrate a6 0.126 0.04 = None 8％ 4 Compare with b 1.625 0.51 100％

[0099] Example 2: Concentration and detection of amylase in agar;

[0100] [Experimental Objective]

[0101] Agar Concentrate

[0102]

Experimental Methods

[0103] Preparation of agar sample concentrate a0:

[0104] ASa: α-Amylase is derived from Aspergillus oryzae powder, ~30 U / mg. Microbial amylases are extracellular enzymes that can be used in various industrial applications, such as bread and malt syrup production, soy sauce and soybean paste fermentation, etc. The molecular weight of α-amylase isolated from Aspergillus oryzae is 51 kDa (sedimentation and diffusion) and 49 kDa (gel filtration).

[0105] Dissolve 10g of agar in 200mL of phosphate buffer (with 1mU / mL ASa added). After dissolution, incubate at 4℃ for 4 hours, then centrifuge at 8000rpm for 20 minutes at 4℃ to obtain the original positive sample solution. Concentrate the solution using a PES-10kDa membrane (prepare 800mL of PBS according to the specified ratio to replace the sucrose). Concentrate the solution to approximately 20mL.

[0106] Dissolve 10g of agar in 200mL of phosphate buffer (calcium chloride). After dissolution, incubate at 4℃ for 4 hours, then centrifuge at 8000rpm for 20 minutes at 4℃ to obtain the original negative sample solution. Filter and concentrate the solution using a PES-10kDa membrane (prepare 800mL of PBS according to the specified ratio to replace the sucrose). Concentrate the solution to approximately 20mL.

[0107] Preparation of secondary concentrate b0:

[0108] 20 mL of concentrate a0 was filtered through a 5 μL filter membrane and then concentrated to 2 mL using a 100 kDa centrifuge filter tube to obtain sample a2. The concentrate was then concentrated to 2 mL using a 10 kDa centrifuge filter tube to obtain sample a1.

[0109] Amylase activity assay:

[0110] 4. Take 200 μL of the sample solution prepared above for enzyme activity detection.

[0111] 5. Amylase activity assay: Using the Megazyme kit, take 200 μL of HR substrate and 200 μL of protein extraction solution, react at 40℃ for 20 min-48 h, and measure A using a microplate reader. 400 Absorbance value.

[0112] 6. Calculation of amylase activity: Units (CU) / gΔA 400 ×0.313×Dilution factor

[0113] Table 2: Comparison of Agar Concentration Results

[0114] Sample Description

[0115] a1: The original solution of the positive sample was used to prepare concentrated solution a0. A 100kDa centrifuge filter tube was used to prepare secondary concentrated solution b0, and then a 10kDa centrifuge filter tube was used to prepare secondary concentrated solution b0.

[0116] a2: The original solution of the positive sample was used to prepare concentrated solution a0, and then a secondary concentrated solution b0 was prepared using a 100kDa centrifuge filter tube. a3: The original solution of the positive sample was used to prepare concentrated solution a0, and then a secondary concentrated solution b0 was prepared using a 100kDa centrifuge filter tube and a 10kDa centrifuge filter tube. The filtrate was collected.

[0117] Control b: Control, ASa 1mU / mL.

[0118] a4: Original solution of negative sample, preparation of concentrated solution a0, preparation of secondary concentrated solution b0.

[0119] Control b: Control

[0120] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0121] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0122] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0123] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and are considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A method for concentrating amylase from a sugar-containing matrix, characterized in that, Includes the following steps: 1) Dissolve the sugar matrix containing amylase in a buffer solution to obtain the first solution; 2) Filter and concentrate the first solution to obtain the second solution; 3) Filter and concentrate the second solution to obtain a concentrated amylase solution.

2. The method as described in claim 1, characterized in that, The sugar-containing matrix is ​​selected from sucrose or agar; And / or, the amylase is α-amylase, preferably, the amylase is derived from Aspergillus oryzae.

3. The method as described in claim 1, characterized in that, In step 1), the buffer solution is PBS buffer, preferably 20 mM PBS buffer, and more preferably, the buffer solution contains 0.5–5 mM calcium chloride. More preferably, the buffer solution contains 1 mM calcium chloride; And / or, in step 1), the pH of the buffer solution is 6.0 to 7.

2.

4. The method as described in claim 1, characterized in that, The amylase content in the first solution is 0.5–3 mU / mL. Preferably, the amylase content in the first solution is 1 mU / mL; And / or, the sugar matrix content in the first solution is 0.01 to 0.1 g / mL, preferably, the sugar matrix content in the first solution is 0.05 g / mL.

5. The method as described in claim 1, characterized in that, In step 2), the first solution is filtered and concentrated using a tangential flow filtration device and an ultrafiltration membrane. Preferably, the ultrafiltration membrane is a polyethersulfone membrane with a molecular weight cutoff of 10 kDa. And / or, in step 2), the volume of the second solution is 5% to 20% of the volume of the first solution, preferably, in step 2), the volume of the second solution is 10% of the volume of the first solution.

6. The method as described in claim 1, characterized in that, In step 3), a 5μm filter membrane is used for filtration; And / or, in step 3), a centrifugal ultrafiltration device is used for concentration, preferably, the ultrafiltration membrane of the centrifugal ultrafiltration device is an ultrafiltration membrane with a molecular weight cutoff of 10 kDa; And / or, in step 3), the volume of the concentrated amylase solution is 5% to 20% of the volume of the second solution, preferably, in step 3), the volume of the concentrated amylase solution is 10% of the volume of the second solution.

7. The method as described in claim 1, characterized in that, Before proceeding to step 2), the first solution is pre-filtered using a pre-filter membrane; And / or, before proceeding to step 2), the first solution needs to be allowed to stand at 4°C for 4 hours, and then centrifuged at 4°C, 8000 rpm for 20 minutes.

8. The method as described in claim 1, characterized in that, The method excludes the use of organic solvent precipitation for concentration.

9. A method for detecting amylase activity, characterized in that, The activity of the concentrated amylase solution in the methods described in claims 1 to 8 was detected using the Megazyme kit.

10. The application of the method described in claims 1 to 8 or the detection method described in claim 9 in the quality control of raw materials in the food industry, the monitoring of enzyme preparation production process, or the evaluation of product storage stability.

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