Method for detecting chymosin activity based on HPLC (High Performance Liquid Chromatography) by taking polypeptide as substrate and application

Abstract

The invention provides an HPLC (High Performance Liquid Chromatography)-based chymosin activity detection method taking polypeptide as a substrate and application, and relates to the technical field of enzyme activity detection. The method comprises the following steps: mixing 1 mg / mL-2. 5 mg / mL of a substrate polypeptide solution and 0.002 U / mL-0. 02 U / mL of a test solution for enzymolysis, centrifuging to take a supernatant, carrying out HPLC (High Performance Liquid Chromatography) quantitative analysis, and calculating the chymosin activity according to the peak area of the product polypeptide, the sequence of the substrate polypeptide is as shown in SEQ ID No. 1. According to the method for determining the activity of the chymosin based on the HPLC and taking the polypeptide as the substrate, the activity of the chymosin in a chymosin-containing product can be quantitatively detected through the specific 8-peptide substrate of the chymosin, and the method which is easy and convenient to obtain raw materials, high in operability, high in accuracy and good in repeatability is developed for quality judgment of a product taking the chymosin as a main component.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme activity detection, and in particular relates to a chymosin activity detection method based on HPLC with a polypeptide as a substrate and an application thereof. Background Art

[0002] Chymosin exists in the stomachs of ruminant mammals as a precursor to prochymosin. In recent years, the sources of chymosin have become increasingly diverse, primarily categorized into three types: animal-derived chymosin, extracted from the stomachs of cattle, pigs, and sheep; plant-derived chymosin, derived from fig leaves and pineapple fruit; and microbial-derived chymosin, produced by molds and yeasts. In dairy processing, chymosin is widely used in cheese and yogurt production and is an essential ingredient in cheesemaking. In medicine, chymosin can be used to treat gastrointestinal diseases, improve gastric mucosal lesions, and regulate gastric acid concentration. Furthermore, chymosin plays an important role in the production of imitation cheeses, the extraction of whey peptides, and the manufacture of cheese-containing beverages. Therefore, measuring chymosin activity is a crucial indicator for evaluating the quality of food and pharmaceutical products.

[0003] In food science, rennet activity is measured according to official methods specified by the International Organization for Standardization (ISO 23058) and the International Dairy Federation (IDF 199:2006). This method assesses rennet activity by measuring the time required for visible flocculation of a standard milk matrix prepared with a calcium chloride solution (0.5 g / L, pH approximately 6.5). The clotting time of the rennet sample is then compared with a bovine rennet reference standard with a defined enzyme composition and known clotting activity. However, this method relies on visual determination of the clotting endpoint, which is highly subjective and ambiguous, resulting in limited accuracy. Furthermore, the standard milk matrix exhibits significant natural variability in composition, making it difficult to obtain consistent, uniform batches. The chymosin potency assay method in the national standards for commercially available chymosin-containing drugs approved by the State Food and Drug Administration is essentially the same: precisely measure 10 mL of substrate solution into a test tube, incubate in a constant-temperature water bath at 30°C ± 0.5°C for 10 minutes, accurately add 1 mL of standard solution, shake well, and immediately start the timer. Use a glass rod to continuously draw the solution down the tube wall to form a uniform thin layer. When visible particles appear in the thin layer, coagulation is confirmed. The time from the addition of the standard solution to the onset of coagulation is accurately recorded. The assay is repeated three times, with the relative standard deviation of the three measurements not exceeding 5%. The average value is taken as the coagulation time of the standard solution. However, the current method for determining chymosin activity is affected by multiple factors, such as substrate source, temperature, pH, metal ions, and endpoint determination, resulting in significant measurement errors and poor reliability of the test results. Summary of the Invention

[0004] To address the above-mentioned technical issues, the first objective of the present invention is to provide an HPLC-based method for determining chymosin activity using a peptide as a substrate. This method, based on the amino acid sequence of κ-casein (κ-CN), designs an octapeptide substrate specific for chymosin. This method, characterized by simple raw material acquisition, robust operability, high accuracy, and excellent reproducibility, has been established.

[0005] The second object of the present invention is to provide an application of the chymosin activity determination method in evaluating products with chymosin as the main component.

[0006] The technical solutions of the present invention are as follows: The present invention provides a chymosin activity assay method based on HPLC using a polypeptide as a substrate. A 1 mg / mL to 2.5 mg / mL substrate polypeptide solution and a 0.002 U / mL to 0.02 U / mL test sample solution are mixed and hydrolyzed, and the supernatant is collected by centrifugation and quantitatively analyzed by HPLC. The chymosin activity is calculated based on the peak area of ​​the product polypeptide. The sequence of the substrate polypeptide is shown in SEQ ID No. 1.

[0007] As an embodiment, the sequence of the product polypeptide is shown as SEQ ID No.2.

[0008] As an embodiment, the solvent of the substrate polypeptide solution is phosphate buffer, the solvent of the test solution is phosphate buffer, and the pH value of the phosphate buffer is 6.0-7.0.

[0009] As an embodiment, the volume ratio of the substrate polypeptide solution to the test solution is 20:1~30:1.

[0010] As an embodiment, the temperature of the enzymatic hydrolysis is 30-40° C., and the time of the enzymatic hydrolysis is 20 min-60 min.

[0011] As an embodiment, the HPLC chromatographic conditions are as follows: 20 mM phosphoric acid solution as phase A, acetonitrile as phase B, and gradient elution with 17% to 60% phase B.

[0012] As an embodiment, the method for calculating chymosin activity includes a relative method or a direct method.

[0013] As an embodiment, the relative method includes: establishing a standard curve of the enzyme activity of a chymosin standard and the peak area of ​​the product polypeptide, and calculating the chymosin activity of the test sample based on the peak area of ​​the test sample product polypeptide.

[0014] As an embodiment, the absolute method includes: Step 1: Determine the linear relationship between the peak area and mass concentration of the 50 μg / ml product peptide; Step 2: Using 0.02 U / mL chymosin standard solution as the mother solution, perform gradient dilution, mix with 2.5 mg / mL of the substrate polypeptide solution according to the volume ratio, perform enzymatic hydrolysis in a water bath, centrifuge and collect the supernatant, and measure the peak area of ​​each standard product polypeptide by HPLC; Step 3, converting the peak area of ​​each standard product polypeptide measured in step 2 into mass concentration based on the linear relationship obtained in step 1, and calculating the amount of product generated per minute during enzymatic hydrolysis to obtain the product generation rate; Step 4, plotting a curve with the product formation rate as the ordinate and the activity of the chymosin standard solution as the abscissa to obtain a linear relationship between the product formation rate and the activity of the chymosin standard solution; Step 5, mixing 1-2.5 mg / ml of the substrate polypeptide solution as shown in SEQ ID No. 1 and 0.002 U / mL-0.02 U / mL of the test solution in the volume ratio and performing enzymatic hydrolysis in a water bath. After boiling in a water bath, centrifugation is performed to obtain the supernatant, and the peak area of ​​the test product polypeptide is determined by HPLC. The peak area of ​​the test product polypeptide is converted to mass concentration according to the linear relationship obtained in step 1, and the amount of product generated per minute during enzymatic hydrolysis is calculated to obtain the test product generation rate; Step 6: Based on the linear relationship obtained in step 4, the test sample product generation rate obtained in step 5 is converted into the test sample chymosin activity.

[0015] The present invention also provides an application of the chymosin activity determination method in evaluating products with chymosin as a main component.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes HPLC technology to quantitatively detect chymosin activity in products containing chymosin using a chymosin-specific octapeptide substrate. Furthermore, the method utilizes readily available raw materials, is highly operable, and offers high accuracy and reproducibility. This invention develops a new, highly reliable and accurate method for evaluating products containing chymosin as a primary ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a comparison chart of the results before and after hydrolysis of the substrate polypeptide; Figure 2 HPLC detection diagram of different types of product peptides; Figure 3 HPLC detection diagram of gradient elution with different proportions of phase B in the mobile phase; Figure 4 HPLC detection diagram of different column temperatures; Figure 5 This is the HPLC detection diagram of different test sample components without interference with the determination; Figure 6This is a linear relationship diagram between different enzymatic hydrolysis times and products; Figure 7 is the relationship between different substrate peptide concentrations and product formation rates; Figure 8 is the standard curve of chymosin standard concentration and product polypeptide peak area; Figure 9 This is the HPLC test chart of the chymosin activity assay of the test sample; Figure 10 This is the standard curve of the activity of the chymosin standard and the rate of product polypeptide formation. DETAILED DESCRIPTION

[0018] The present invention provides an HPLC-based method for determining chymosin activity using a polypeptide as a substrate. A 1 mg / mL to 2.5 mg / mL substrate polypeptide solution is mixed with a 0.002 U / mL to 0.02 U / mL test sample solution and enzymatically hydrolyzed. The supernatant is then centrifuged and quantitatively analyzed by HPLC. The chymosin activity is calculated based on the peak area of ​​the product polypeptide. The sequence of the substrate polypeptide is shown in SEQ ID No. 1: HLSFMAIP. Using the octapeptide HLSFMAIP as a substrate, the present invention establishes a method for detecting chymosin activity with high operability, accuracy, and reproducibility. As an optional embodiment, the substrate polypeptide is artificially synthesized.

[0019] In the present invention, chymosin specifically cleaves the peptide bond between phenylalanine (Phe)₁₀ and methionine (Met)₁₀. Therefore, it is speculated that enzymatic hydrolysis of the substrate octapeptide will yield two tetrapeptide product peaks, namely product polypeptide 1 (HLSF) as shown in SEQ ID No. 2 and product polypeptide 2 (MAIP) as shown in SEQ ID No. 3. In the present invention, based on the peak patterns of the two product polypeptide peaks after enzymatic hydrolysis, the sequence of the product polypeptide is preferably product polypeptide 1 (HLSF). As an optional embodiment, the product polypeptide is artificially synthesized.

[0020] In the present invention, the solvent of the substrate polypeptide solution, the test solution or the product polypeptide solution is a phosphate buffer, and the pH value of the phosphate buffer is 6.0-7.0, preferably 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. The phosphate buffer is composed of sodium dihydrogen phosphate and sodium hydrogen phosphate in a mass ratio of 1 to 9:1, and the mass ratio is preferably 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1 or 9:1. In the phosphate buffer of the present invention, deformation, aggregation or degradation of polypeptide or chymosin can be avoided, and the components in the buffer do not affect the detection of the content of the enzymatic hydrolysis product, and the specificity is good. In the present invention, the volume ratio of the substrate polypeptide solution to the test solution is 20:1 to 30:1, preferably 20:1, 22:1, 24:1, 26:1, 28:1 or 30:1. The concentration of the substrate polypeptide solution is 1 mg / mL to 2.5 mg / mL, preferably 1 mg / mL, 1.5 mg / mL, 2 mg / mL or 2.5 mg / mL. The concentration of the test solution is 0.002 U / mL to 0.02 U / mL, preferably 0.002 U / mL, 0.004 U / mL, 0.006 U / mL, 0.008 U / mL, 0.01 U / mL, 0.012 U / mL, 0.014 U / mL, 0.016 U / mL, 0.018 U / mL or 0.02 U / mL. In the present invention, the concentration of the product polypeptide solution is 0.03 mg / mL to 0.2 mg / mL, preferably 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.10 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL or 0.2 mg / mL.

[0021] In the present invention, the enzymatic hydrolysis temperature is 30-40°C, preferably 30°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C; the enzymatic hydrolysis time is 20 min-60 min, preferably 20 min, 30 min, 40 min, 50 min or 60 min. As an optional embodiment, the enzymatic hydrolysis is completed in a water bath. In the present invention, the substrate polypeptide solution is preheated at the enzymatic hydrolysis temperature for 5-20 min, and then mixed with the test sample solution in the above-mentioned volume ratio for enzymatic hydrolysis. In the present invention, the enzymatic hydrolysis product is centrifuged to obtain the supernatant, the centrifugal speed is 8000 rpm-15000 rpm, and the centrifugal time is 3-10 min.

[0022] In the present invention, the HPLC chromatographic conditions are as follows: 20 mM phosphoric acid solution as phase A, acetonitrile as phase B, and gradient elution of 17% to 60% phase B. The gradient elution program is preferably 0 to 2 min 17% B, 2 to 10 min 17% B to 60% B, 10 to 10.1 min 60% B to 17% B, and 10.1 to 15 min 17% B. The chromatographic column is a C18 chromatographic column with a specification of 4.6 mm × 150 mm, 3 μm.

[0023] In the present invention, the method for calculating chymosin activity includes relative and absolute methods. The relative method comprises adding phosphate buffer to a chymosin standard (chymosin reference) to prepare chymosin standard solutions of varying concentrations: 0.0020 U / mL, 0.004 U / mL, 0.0061 U / mL, 0.0088 U / mL, 0.0101 U / mL, 0.0122 U / mL, and 0.0162 U / mL. The chymosin standard solutions of varying concentrations are then mixed with 1-2.5 mg / mL of the substrate polypeptide represented by SEQ ID No. 1 at the specified volume ratios and hydrolyzed in a water bath. After boiling in a water bath, the supernatant is centrifuged and the peak area of ​​the product polypeptide is measured by HPLC. A standard curve is plotted with the concentration of the chymosin standard solution as the horizontal axis and the peak area of ​​the product polypeptide represented by SEQ ID No. 2 as the vertical axis. Then, 1-2.5 mg / mL of the substrate polypeptide as shown in SEQ ID No. 1 and 0.002 U / mL-0.02 U / mL of the test solution were mixed in the volume ratio and hydrolyzed in a water bath. After boiling in a water bath, the supernatant was collected by centrifugation and the peak area of ​​the product polypeptide was determined by HPLC. The chymosin activity of the test sample was calculated according to the standard curve method.

[0024] In the present invention, the absolute method includes: Step 1: Determine the linear relationship between the peak area and mass concentration of the 50 μg / ml product peptide (HLSF).

[0025] Step 2: Add phosphate buffer to the chymosin standard to prepare 0.0020 U / mL, 0.004 U / mL, 0.0061 U / mL, 0.0088 U / mL, 0.0101 U / mL, 0.0122 U / mL, and 0.0162 U / mL chymosin standard solutions of different activities. Take the above-mentioned chymosin standards of different concentrations and 2.5 mg / mL of the substrate polypeptide solution shown in SEQ ID No. 1 according to the volume ratio and mix them in a water bath for 30 minutes. After boiling in a water bath, centrifuge and take the supernatant. HPLC determines the peak area of ​​each standard product polypeptide.

[0026] Step 3: Convert the peak area of ​​each standard product polypeptide measured in step 2 into mass concentration according to the linear relationship obtained in step 1, and calculate the amount of product generated per minute during 30 minutes of enzymatic hydrolysis to obtain the product generation rate.

[0027] Step 4: draw a curve with the product generation rate as the ordinate and the activity of the chymosin standard solution as the abscissa to obtain a linear relationship between the product generation rate and the activity of the chymosin standard solution.

[0028] Step 5: A 1-2.5 mg / ml solution of the substrate polypeptide as shown in SEQ ID No. 1 and a 0.002 U / mL-0.02 U / mL test solution were mixed in the volume ratio and enzymatically hydrolyzed in a water bath. After boiling in a water bath, the supernatant was centrifuged and the peak area of ​​the product polypeptide was measured by HPLC. The peak area of ​​the product polypeptide was converted to mass concentration according to the linear relationship obtained in step 1, and the amount of product generated per minute during 30 minutes of enzymatic hydrolysis was calculated to obtain the product generation rate.

[0029] Step 6: Based on the linear relationship obtained in step 4, the product formation rate obtained in step 5 is converted into the chymosin activity of the test sample.

[0030] Since the chymosin activity detection method of the present invention can accurately detect the chymosin activity in medicines and chymosin food additives, the present invention also provides a chymosin activity determination method for evaluating products with chymosin as the main component.

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is 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.

[0032] Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available. If specific conditions of use are not specified, they are usually carried out under conventional conditions or under conditions recommended by the company.

[0033] 1. Test materials Ultrapure water (Feld (Beijing) Scientific Instrument Co., Ltd.); acetonitrile (Beijing J&K Technology Co., Ltd.); phosphoric acid (Beijing J&K Technology Co., Ltd.); sodium dihydrogen phosphate (Beijing Tongguang Fine Chemical Co., Ltd.); disodium hydrogen phosphate (Beijing Tongguang Fine Chemical Co., Ltd.); rennet standard (China Food and Drug Inspection Institute); substrate peptide (HLSFMAIP, GenScript Biotech Co., Ltd.), product peptide 1 (HLSF, GenScript Biotech Co., Ltd.); product peptide 2 (MAIP, GenScript Biotech Co., Ltd.); commercially available drug 1, commercially available drug 2, commercially available drug 3; commercially available rennet food additive.

[0034] 2. Main reagents and instruments High-performance liquid chromatography (HPLC) with a C18 column (4.6 mm × 150 mm, 3 μm); a water bath; a centrifuge; a microbalance; a microbalance; a magnetic stirrer; and a vortexer.

[0035] Example 1 Accurately weigh 10.14 g of sodium dihydrogen phosphate and 6.8 g of sodium hydrogen phosphate, add 900 ml of water, and sonicate to dissolve. Adjust the pH to 6.3 ± 0.05. Place in a 1000 ml volumetric flask, add water to the mark, and shake to obtain phosphate buffer. Accurately weigh 25 mg of the substrate peptide with the sequence HLSFMAIP and dilute to a concentration of 2.5 mg / mL with an appropriate amount of phosphate buffer. Use a pipette to transfer 480 μL of substrate peptide solution into a 1.5 mL centrifuge tube. After preheating in a 30°C water bath for 10 min, add 20 μL of 0.2 mg / mL test solution, vortex and mix quickly, and centrifuge. Incubate the tube in a 30°C water bath for 30 min to inactivate the enzyme. After the reaction, boil the tube in boiling water for 10 min to terminate the enzymatic hydrolysis reaction. Cool to room temperature and centrifuge at 12,000 rpm for 5 min. Take the supernatant and perform gradient elution at a column temperature of 40°C with 20 mM phosphoric acid water as phase A and acetonitrile as phase B under the conditions of 17% B from 0 to 2 min, 17% B to 60% B from 2 to 10 min, 60% B to 17% B from 10 to 10.1 min, and 17% B from 10.1 to 15 min. The peak area of ​​the product peptide was determined by HPLC.

[0036] Example 2 Accurately weigh 51.67 g of sodium dihydrogen phosphate and 5.6 g of sodium hydrogen phosphate, add 900 ml of water, and sonicate to dissolve. Adjust the pH to 6.1 ± 0.05. Place the solution in a 1000 ml volumetric flask, add water to the mark, and shake to obtain phosphate buffer. Accurately weigh 25 mg of the substrate peptide with the sequence HLSFMAIP and dilute it to a concentration of 1 mg / mL with an appropriate amount of phosphate buffer. Pipette 480 μL of the substrate peptide solution into a 1.5 mL centrifuge tube. Preheat the solution in a 35°C waterbath for 10 min. Add 20 μL of the 0.04 mg / mL test sample solution, vortex briefly, and centrifuge. Incubate the tube in a 35°C waterbath for 40 min. Afterward, inactivate the enzyme by boiling the tube in water for 10 min. Cool the tube to room temperature, centrifuge at 10,000 rpm for 7 min, and collect the supernatant. Determine the peak area of ​​the product peptide by HPLC under the chromatographic conditions described in Example 1.

[0037] Example 3 Accurately weigh 15.19 g of sodium dihydrogen phosphate and 6.4 g of sodium hydrogen phosphate, add 900 ml of water, and sonicate to dissolve. Adjust the pH to 6.5 ± 0.05. Place the solution in a 1000 ml volumetric flask, add water to the mark, and shake the phosphate buffer. Accurately weigh 25 mg of the substrate peptide with the sequence HLSFMAIP and dilute it to a 5 mg / mL concentration with an appropriate amount of phosphate buffer. Pipette 480 μL of the substrate peptide solution into a 1.5 mL centrifuge tube. Preheat the solution in a 38°C waterbath for 10 min. Add 20 μL of the 3 mg / mL test sample solution, vortex thoroughly, and centrifuge. Incubate the tube in a 38°C waterbath for 55 min. Afterward, inactivate the enzyme by boiling the tube in water for 10 min. Cool the tube to room temperature, centrifuge at 15,000 rpm for 5 min, and collect the supernatant. Determine the peak area of ​​the product peptide by HPLC at a column temperature of 40°C using the chromatographic conditions described in Example 1.

[0038] Test Example 1 1. Substrate Selection Based on the amino acid sequence of κ-casein (κ-CN), the following chymosin peptide substrates were designed (see Table 1). Based on the chymosin activity assay method of Example 1, 1 mg each of the 2-, 4-, 6-, 8-, 10-, 12-, 14-, and 16-peptides were weighed and added to pH 6.3 phosphate buffer to prepare a 1 mg / ml substrate solution. 90 μL of each of these peptide substrate solutions was incubated in a 30°C water bath for 10 min. 10 μL of a freshly prepared 0.02 mg / ml chymosin standard solution was then added to the above peptide substrate solutions. After enzymatic hydrolysis in a 30°C water bath for 15 min, the solution was then boiled in water for 5 min, and the supernatant was centrifuged and analyzed by HPLC under the chromatographic conditions of Example 1. The results showed that the hydrolysis effect of substrates 2-peptide, 4-peptide, 6-peptide, 10-peptide, 12-peptide, 14-peptide and 16-peptide was not as obvious as that of 8-peptide under the condition of 0.02 mg / ml chymosin. If the peptide chain was too long or too short, it would be difficult to accurately measure the chymosin activity. Therefore, 8-peptide was used as the substrate. Among them, the control results before and after hydrolysis of 2-peptide, 4-peptide, 6-peptide and 8-peptide are shown in Figure 2. Figure 1 shown.

[0039] Table 1 Chymosin polypeptide substrates

[0040] 2. Substrate Selection Since chymosin can specifically cleave the peptide bond between phenylalanine (Phe) 105 and methionine (Met) 106, it is speculated that two 4-peptide product peaks will be obtained after enzymatic hydrolysis of the substrate 8-peptide, namely product polypeptide 1 (HLSF) and product polypeptide 2 (MAIP). Based on the chymosin activity detection method in Example 1, the supernatant of product polypeptide 1 (HLSF) and product polypeptide 2 (MAIP) was centrifuged at 1 mg / ml and the supernatant was obtained. This conclusion was confirmed after HPLC analysis under the chromatographic conditions of Example 1. According to the peak shape of the two product polypeptide peaks after enzymatic hydrolysis (such as Figure 2 As described above), it was finally decided to use the product peptide 1 (HLSF) for quantification.

[0041] 3. Optimization of Chromatographic Conditions Based on the chymosin activity detection method of Example 1, 20 mM phosphoric acid water was used as phase A, acetonitrile was used as phase B, and a 10%-90% gradient elution of phase B was used. However, a good separation effect was not obtained for the substrate 8 peptides and the product polypeptide 1. Therefore, the mobile phase ratio was further adjusted, and a 17%-60% gradient elution of phase B was used (see Table 2), which achieved the best separation between the substrate and the product (e.g., Figure 3 Then, based on the chymosin activity detection method in Example 1, the effects of column temperatures of 20°C, 30°C, and 40°C on peak shape were investigated. The results showed that the effect was best when the column temperature was 40°C (as shown in FIG. Figure 4 shown).

[0042] Table 2 17%-60% phase B gradient elution

[0043] 4. Test sample interference determination Take commercially available drug 1, commercially available drug 2, commercially available drug 3 and food additive rennet as test samples, weigh 10 mg of commercially available drug 1 (capacity: 198 mg / pill, specification: greater than 40 U / pill), 10 mg of commercially available drug 2 (capacity: 164 mg / pill, specification: greater than 40 U / pill), 50 mg of commercially available drug 3 (greater than 40 U / g), and 1 mg of food additive rennet (12,000 / g), and add 100 ml of pH 6.3 phosphate buffer to fully dissolve them based on the rennet activity detection method in Example 1. Take 450 μL of pH 6.3 phosphate buffer and place it in a centrifuge tube, add 50 μL of the above test sample solution to each tube, and mix them evenly. After 30°C water bath for 30 min, boil in water bath for 10 min, centrifuge to obtain the supernatant, and perform HPLC determination under the chromatographic conditions of Example 1 (as shown in Figure 2). Figure 5 The results showed that the blank solution did not interfere with the detection of the product, and the preparation itself and other components in the buffer did not affect the detection of the enzymatic hydrolysis product content, indicating that the method had good specificity.

[0044] 5. Optimization of enzymatic hydrolysis time Based on the chymosin activity detection method of Example 1, 360 μL of 2.5 mg / ml substrate was accurately pipetted, 540 μL of pH 6.3 phosphate buffer was added, and 7 parallel portions were prepared. The mixture was vortexed and mixed, and the mixture was bathed in a 30°C water bath for 10 min. 100 μL of approximately 0.02 mg / ml chymosin standard solution was added to each portion. The mixture was enzymatically digested for 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively. The mixture was then bathed in boiling water for 10 min, centrifuged, and the supernatant was collected. HPLC analysis was performed under the chromatographic conditions of Example 1 (e.g., Figure 6 The results showed that a standard curve was drawn with time as the horizontal axis and the product peptide peak area as the vertical axis. The curve equation was a first-order linear equation, that is, a zero-order reaction. The product formation rate remained basically constant (slope) and was not affected by time or substrate concentration within 60 min.

[0045] 6. Optimization of substrate concentration Based on the chymosin activity detection method of Example 1, 180, 240, 360, 450, and 480 μl of 2.5 mg / ml substrate were accurately pipetted, and 300, 240, 120, 30, and 0 μl of pH 6.3 phosphate buffer were added, respectively. The mixture was vortexed and mixed, and the mixture was incubated in a 30°C water bath for 10 min. 20 μl of 0.01 mg / ml chymosin standard (881 U / g) solution was added to each sample. The mixture was enzymatically hydrolyzed for 30 min, and then incubated in a boiling water bath for 10 min. The supernatant was centrifuged and the supernatant was determined by HPLC under the chromatographic conditions of Example 1 (e.g., Figure 7 The results show that, since this method aims to measure chymosin activity, the substrate concentration should be as high as possible to maintain a relatively stable product formation rate. A curve is plotted with substrate concentration as the abscissa and product formation rate as the ordinate. When the substrate concentration is between 2.25 and 2.5 mg / ml, the product formation rate remains relatively stable.

[0046] 7. Determination of enzyme activity of test sample (relative method) Based on the chymosin activity assay method of Example 1, 9.19 mg of chymosin standard (enzyme activity 881 U / g) was accurately weighed and diluted to a 500 mL volumetric flask to obtain a chymosin standard solution. 250, 500, 750, 1000, 1250, 1500, and 2000 μL of the chymosin standard solution were pipetted, and 1750, 1500, 1250, 1000, 750, 500, and 0 μL of pH 6.3 phosphate buffer were added, respectively, and vortexed to mix. The chymosin activity gradient is shown in Table 3. 960 μL of 2.5 mg / mL substrate polypeptide was accurately pipetted, incubated at 30°C for 10 min, and then 40 μL of the chymosin standard solution of different concentrations was added. After enzymatic digestion for 30 min, the mixture was boiled in boiling water for 10 min, and the supernatant was centrifuged and analyzed by HPLC under the chromatographic conditions of Example 1. The standard curve was drawn with the concentration of chymosin standard as the horizontal axis and the peak area of ​​the product 4 peptides as shown in the sequence SEQ ID NO.2 as the vertical axis (as shown in FIG. Figure 8 shown).

[0047] Table 3 Chymosin activity gradient of chymosin standard solution

[0048] Based on the chymosin activity detection method of Example 1, 10 mg each of commercial drug 1 (batch 1, batch 2, batch 3), commercial drug 2 (batch 1, batch 2, batch 3), 50 mg of commercial drug 3, and 1000 mg of commercial chymosin food additive were accurately weighed and placed in a 250 ml volumetric flask. They were dissolved and diluted to the mark with pH 6.3 phosphate buffer. Accurately pipette 960 μL of 2.5 mg / ml substrate solution, and after 10 min in a 30°C water bath, accurately pipette 40 μL of each of the above test sample solutions, enzymatically hydrolyze for 30 min, and then boil in a boiling water bath for 10 min. The supernatant was centrifuged and assayed by HPLC. The chymosin activity of each test sample was determined according to the standard curve method (as shown in Table 4, Figure 9 shown).

[0049] Table 4 Determination results of chymosin activity of test samples

[0050] 8. Determination of enzyme activity of test sample (absolute method) 8.1 Definition of chymosin activity units (determination of the conversion coefficient between the activity of chymosin reference material and the detection method of the present invention) Accurately weigh 10 mg of the product peptide 1 (HLSF) into a 10 ml volumetric flask, dissolve it to the mark with pH 6.3 phosphate buffer, and dilute it to 50 μg / ml as a reference solution. HPLC analysis of the peak area of ​​the product peptide at this concentration using a freshly prepared 50 μg / ml solution of the product peptide 1 (HLSF) was performed under the chromatographic conditions described in Example 1. A linear relationship between the peak area and mass concentration of the product peptide at 50 μg / ml was obtained.

[0051] Based on the chymosin activity detection method in Example 1, 960 μL of 2.5 mg / ml substrate polypeptide was accurately pipetted and 35, 30, 25, 20, 15, 10, and 0 μL of pH 6.3 phosphate buffer were added, respectively. The mixture was vortexed and mixed, and the mixture was incubated in a 30°C water bath for 10 min. 5, 10, 15, 20, 25, 30, and 40 μL of 0.018 mg / ml (0.0162 U / mL) chymosin standard solution were added, respectively. After enzymatic hydrolysis for 30 min, the mixture was boiled in water bath for 10 min, and the supernatant was centrifuged. The peak area of ​​each standard product polypeptide was determined by HPLC under the chromatographic conditions described in Example 1.

[0052] Based on the linear relationship between the peak area and mass concentration of the 50 μg / ml product polypeptide, the peak area of ​​each standard product polypeptide obtained after the reaction of the chymosin standard and the substrate polypeptide was converted into mass concentration, and the product generation rate of 30 minutes of enzymatic hydrolysis was calculated. The activity (U) of the chymosin standard was used as the horizontal axis and the product generation rate (μmol / min) was used as the vertical axis. The curve results are shown in the figure below. Figure 10 As shown, R 2 The value is greater than 0.99, and the slope is 5.0078. This indicates that under the given conditions of the detection method of the present invention, the enzyme activity conversion coefficient is 1 chymosin activity unit per minute of enzymatic hydrolysis producing 5 μmol of product polypeptide, and the unit chymosin activity conversion coefficient is obtained.

[0053] 8.2 Determination of enzyme activity The enzymatic hydrolysis reaction and HPLC determination were carried out according to the test sample preparation and enzymatic hydrolysis conditions of Example 1. Based on the linear relationship between the peak area and mass concentration of the 50 μg / ml product polypeptide, the peak area of ​​the test sample product polypeptide was converted into mass concentration, and the amount of product generated per minute during enzymatic hydrolysis was calculated to obtain the test sample product generation rate. The test sample chymosin activity was then converted based on the unit enzyme activity conversion coefficient (see Table 4).

[0054] Test Example 2: Verification of the accuracy of the detection method 1. Accurately weigh approximately 10 mg (881 U / g) of chymosin standard into a mortar. Add a small amount of pH 6.3 phosphate buffer solution and grind until evenly dispersed. Transfer the solution to a 500 mL volumetric flask in small amounts with pH 6.3 phosphate buffer solution and dilute to the mark. Prepare S1-S7 according to Table 3.

[0055] 2. Accurately weigh approximately 5 mg of the contents of the commercially available drug 2 capsule into a 250 ml volumetric flask. Dissolve and dilute to the mark with enzyme reaction buffer (pH 6.3 phosphate buffer). Mix the commercially available drug 2 solution with the S3-S5 chymosin standard solutions of varying concentrations in a 1:1 ratio to prepare the commercially available drug 2 spiked solutions. The chymosin standard solutions are categorized as high, medium, and low based on their concentrations. The commercially available drug 2 spiked solutions are prepared according to Table 5.

[0056] Table 5 Preparation of spiked solution for commercially available drug 2 capsules

[0057] 3. Determination of Recovery Rate Peptide control: Accurately weigh 10 mg of the product peptide 1 (HLSF) into a 10 ml volumetric flask, dissolve it in pH 6.3 phosphate buffer, and dilute to the mark. Dilute to 50 μg / ml for use as a control.

[0058] Based on the chymosin activity detection method of Example 1, the samples were mixed with 20 μL of 0.02 mg / mL concentration of commercial drug 2 capsule solution or commercial drug 2 capsule spiked solution, respectively, and enzymatically hydrolyzed for 30 min, boiled in a boiling water bath for 10 min, centrifuged, and the supernatant was determined by HPLC. The chymosin activity of commercial drug 2 capsules is shown in Table 6.

[0059] Table 6 Rennet activity of test capsules

[0060] The recovery rate calculation formula is: The above results show that at the three levels of high, medium and low, the spiked recoveries of the nine spiked solutions were all between 80% and 120%, indicating that the detection method of the present invention has good accuracy.

[0061] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for determining chymosin activity based on HPLC using a polypeptide as a substrate, characterized in that: 1 mg / mL~2.5 mg / mL substrate peptide solution and 0.002 U / mL~0.02 U / mL test sample solution were mixed and hydrolyzed, and the supernatant was collected by centrifugation and quantitatively analyzed by HPLC. The chymosin activity was calculated based on the peak area of ​​the product peptide. The sequence of the substrate polypeptide is shown as SEQ ID No.

1.

2. The method for detecting chymosin activity according to claim 1, wherein The sequence of the product polypeptide is shown in SEQ ID No.

2.

3. The method for detecting chymosin activity according to claim 1, wherein The solvent of the substrate polypeptide solution is phosphate buffer, and the solvent of the test solution is phosphate buffer; the pH value of the phosphate buffer is 6.0-7.

0.

4. The method for detecting chymosin activity according to claim 1, wherein The volume ratio of the substrate polypeptide solution to the test solution is 20:1~30:

1.

5. The method for detecting chymosin activity according to claim 1, wherein The enzymatic hydrolysis temperature is 30-40° C., and the enzymatic hydrolysis time is 20 min-60 min.

6. The method for detecting chymosin activity according to claim 1, wherein The HPLC chromatographic conditions are as follows: 20 mM phosphoric acid solution as phase A, acetonitrile as phase B, and gradient elution of phase B from 17% to 60%, wherein the gradient elution is 17% B from 0 to 2 min, 17% B to 60% B from 2 to 10 min, 60% B to 17% B from 10 to 10.1 min, and 17% B from 10.1 to 15 min.

7. The method for detecting chymosin activity according to claim 1, wherein The method for calculating chymosin activity includes a relative method or an absolute method.

8. The method for detecting chymosin activity according to claim 7, wherein: The relative method includes: establishing a standard curve of the activity concentration of the chymosin standard solution and the peak area of ​​the product polypeptide, and calculating the chymosin activity of the test sample according to the peak area of ​​the test sample product polypeptide.

9. The method for detecting chymosin activity according to claim 7, wherein: The jus cogens mentioned include: Step 1: Determine the linear relationship between the peak area and mass concentration of the 50 μg / ml product peptide; Step 2: Using 0.02 U / mL chymosin standard solution as the mother solution, perform gradient dilution, mix with 2.5 mg / mL of the substrate polypeptide solution according to the volume ratio, perform enzymatic hydrolysis in a water bath, centrifuge and collect the supernatant, and measure the peak area of ​​each standard product polypeptide by HPLC; Step 3, converting the peak area of ​​each standard product polypeptide measured in step 2 into mass concentration based on the linear relationship obtained in step 1, and calculating the amount of product generated per minute during enzymatic hydrolysis to obtain the product generation rate; Step 4, plotting a curve with the product formation rate as the ordinate and the activity of the chymosin standard solution as the abscissa to obtain a linear relationship between the product formation rate and the activity of the chymosin standard solution; Step 5, mixing 1-2.5 mg / ml of the substrate polypeptide solution as shown in SEQ ID No. 1 and 0.002 U / mL-0.02 U / mL of the test solution in the volume ratio and performing enzymatic hydrolysis in a water bath. After boiling in a water bath, centrifugation is performed to obtain the supernatant, and the peak area of ​​the test product polypeptide is determined by HPLC. The peak area of ​​the test product polypeptide is converted to mass concentration based on the linear relationship obtained in step 1, and the amount of product generated per minute during enzymatic hydrolysis is calculated to obtain the product generation rate; Step 6: Based on the linear relationship obtained in step 4, the test sample product generation rate obtained in step 5 is converted into the test sample chymosin activity.

10. Use of the method for determining chymosin activity according to any one of claims 1 to 9 in evaluating products with chymosin as the main ingredient.

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