Method for determining lactase activity in dairy product

Abstract

The invention belongs to the field of lactase activity detection, and particularly relates to a method for determining lactase activity in dairy products, which comprises the following steps: S1, mixing a to-be-detected sample with an ONPG substrate solution for reaction to obtain a sample mixed solution, mixing the sample mixed solution with a reaction stop solution, centrifuging, and filtering to obtain a sample to-be-detected solution; s2, mixing a to-be-detected sample with the reaction stop solution to obtain a blank mixed solution, mixing the blank mixed solution with the ONPG substrate solution for reaction, centrifuging, and filtering to obtain a blank to-be-detected solution; s3, preparing an o-nitrophenol standard working solution with gradient concentration, performing HPLC (High Performance Liquid Chromatography) analysis, and drawing a working curve according to an analysis result; and S4, carrying out HPLC analysis on the sample to-be-detected liquid and the blank to-be-detected liquid, and calculating the lactase activity in the sample to be detected according to an analysis result. The detection limit of the method provided by the invention can reach 6.5 * 10 <-2 > U / g, and dairy products with low lactase activity can be analyzed.

Description

Technical Field

[0001] The invention belongs to the field of lactase activity detection, and specifically relates to a method for determining lactase activity in dairy products. Background Art

[0002] Lactase (EC 3.2.1.23), also known as β-galactosidase or β-D-galactoside galatohydrolase, or ONPG for short, is a disaccharidase present on the surface of the microvilli of the small intestinal mucosa of mammals. Lactose in milk is hydrolyzed by lactase, and the product glucose is the energy source for metabolism of various parts of the human body, while galactose is a structural sugar necessary for the metabolism of the human brain and mucosal tissues, and is an essential substance for the development of the infant's brain. When lactase is deficient, lactose cannot be broken down and absorbed, which will cause lactose intolerance and lactose malabsorption, that is, lactase deficiency. Detection of lactase activity is a common method for clinical diagnosis of lactase deficiency.

[0003] Lactase is mainly used in the dairy industry. It can convert low-sweetness and low-solubility lactose into sweeter, more soluble monosaccharides. It reduces the possibility of lactose crystallization in ice cream, condensed milk, and evaporated milk, while increasing sweetness. Because lactase is of special importance and catalytic activity to organisms, it has broad application prospects.

[0004] Currently, there are mainly two methods for quantitative detection of lactase activity: small intestinal mucosal biopsy, ONPG test, stable isotope method, high performance liquid chromatography (differential method) and fecal reducing sugar determination. Among them, the small intestinal mucosal biopsy method for measuring lactase activity is mainly a commonly used method in medicine, and the small intestinal mucosal biopsy method is a invasive examination, which is difficult to operate and is affected by factors such as the site of the biopsy mucosa and the proficiency of the operator; the ONPG test method is mainly to decompose o-nitrophenol-β-D-galactoside into galactose and o-nitrophenol (ONP for short) in the presence of lactase. O-nitrophenol has specific absorption at 420nm, and the color depth is measured by a spectrophotometer. It has high requirements for sample clarity and is generally used for the determination of lactase active agents. The lactase content of such preparations is relatively high, and it is not suitable for samples with trace lactase such as dairy products; the stable isotope technology method is to measure the 13C-glucose concentration in the blood to reflect the small intestine's lactose tolerance. Digestive capacity, but the instrumentation required for this method is expensive, the analysis cost is high, and the isotope is also radioactive, so there are fewer application scenarios; high performance liquid chromatography (differential method) is mainly used to determine the content of galactose, glucose and lactose, the hydrolysis products of lactase, and mainly studies high-content samples such as fermented foods. It is also not suitable for samples with trace lactase such as dairy products; the fecal reducing sugar determination method uses fecal reducing sugar, which has high requirements for fecal sample retention, and the cost of Clinitest reagent is high; in addition, there are some patented methods that record the analysis method of lactase activity, which is mainly a rapid test kit method, similar to the ONPG test method, which is only applicable to the detection of preparations with high lactase content, but it is difficult to use this method to process low-content and more complex dairy matrices. Since dairy products themselves contain less lactase, the existing analysis methods of lactase activity are not suitable for dairy products with low lactase activity.

[0005] Based on this, there is an urgent need in the art for an analysis method for lactase activity in dairy products, which can solve the problem that the existing lactase activity analysis method is not suitable for dairy products with low lactase activity. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for determining lactase activity in dairy products, which can solve the problem that the existing lactase activity analysis method cannot analyze dairy products with low lactase activity.

[0007] To achieve the object of the invention, the present invention provides a method for determining lactase activity in dairy products, comprising the following steps:

[0008] S1. The sample to be tested is mixed with the o-nitrophenyl-β-D-galactoside substrate solution to obtain a sample mixture, the mixed sample solution is mixed with the reaction termination solution, and the mixture is centrifuged and filtered to obtain a sample solution to be tested;

[0009] S2. The sample to be tested is mixed with the reaction termination solution to obtain a blank mixed solution, the blank mixed solution is mixed with the o-nitrophenyl-β-D-galactoside substrate solution for reaction, and the blank test solution is obtained by centrifugation and filtration;

[0010] S3. Prepare a standard working solution of o-nitrophenol with a gradient concentration and perform HPLC analysis, and draw a working curve based on the analysis results;

[0011] S4. Perform HPLC analysis on the sample solution and the blank solution, and calculate the lactase activity in the sample according to the analysis results. If the concentration of o-nitrophenol in the sample exceeds the linear range of the working curve, the sample needs to be diluted with a buffer solution and then the step of S1 is repeated to obtain a new solution, and then the new solution is analyzed to calculate the lactase activity in the sample;

[0012] The calculation formula of lactase activity in the sample to be tested is as follows:

[0013]

[0014] Where:

[0015] X—enzyme activity of the sample to be tested, in U / g;

[0016] c—the concentration of o-nitrophenol in the sample solution, in milligrams per liter (mg / L);

[0017] c 0 —The concentration of o-nitrophenol in the blank test solution, in milligrams per liter (mg / L);

[0018] V—the volume of the sample to be tested, i.e., the sum of the volume of the sample to be tested, the volume of the o-nitrobenzene-β-D-galactoside substrate solution, and the volume of the reaction termination solution, in milliliters (mL), where the volume of the sample to be tested is converted according to 1g=1mL;

[0019] m—the mass of the sample represented by the sample test solution, in g;

[0020] M—molar mass of o-nitrophenol: 123.11 g / mol;

[0021] t——reaction time (min);

[0022] f——dilution factor of the sample to be tested.

[0023] Optionally, the reaction termination solution is a sodium carbonate-EDTA aqueous solution or a hydrochloric acid aqueous solution.

[0024] Optionally, the concentration of sodium carbonate in the sodium carbonate-EDTA aqueous solution is 40-60 g / L.

[0025] Optionally, the concentration of the hydrochloric acid aqueous solution is 3 to 8 mol / L.

[0026] Optionally, the concentration of β-D-galactoside in the β-D-galactoside substrate solution is 2.0 to 3.0 g / L.

[0027] Optionally, in step S1, the reaction time of the sample to be tested and the o-nitrophenyl-β-D-galactoside substrate solution is 5 to 15 minutes.

[0028] Optionally, in step S2, the reaction time of the sample to be tested and the reaction termination solution is 5 to 15 minutes.

[0029] Optionally, step S1 and step S2 are performed in an environment of 25.0-35.0°C.

[0030] Optionally, in steps S1 and S2, the mass volume ratio of the sample to be tested to the o-nitrophenyl-β-D-galactoside substrate solution to the reaction termination solution is (0.50-1.50) g: (2.50-7.50) mL: (1.00-3.00) mL.

[0031] Optionally, the sample to be tested and the o-nitrophenyl-β-D-galactoside substrate solution in steps S1 and S2 need to be equilibrated at a temperature of 25 to 35° C. for 5 to 15 minutes before use.

[0032] Optionally, the concentration of the o-nitrophenol gradient concentration standard solution in step S3 is 0 to 200 mg / L.

[0033] Optionally, the HPLC analysis parameters in step S4 include:

[0034] Chromatographic column: C18 column, column length 250 mm, inner diameter 4.6 μm, filler particle size 5 μm;

[0035] Column temperature: 35-45°C;

[0036] Injection volume: 10 μL;

[0037] Detector wavelength: 275nm;

[0038] Mobile phase: A: diammonium phosphate buffer; B: methanol;

[0039] Mobile phase flow rate: 0.5~1.5mL / min;

[0040] Liquid phase elution procedure:

[0041]

[0042]

[0043] Beneficial effects of the present invention:

[0044] (1) The present invention combines high performance liquid chromatography with the ONPG substrate method to avoid the defects of the high performance liquid chromatography alone that cannot analyze low lactase activity, and also avoids the defects of the ONPG method alone that cannot analyze low lactase activity. At the same time, it also avoids the defects of the spectrophotometric method that cannot analyze turbid dairy products. It has obvious advantages in the determination of lactase activity in the processing of dairy matrix and is more suitable for dairy product testing. Compared with the ONPG test method in the existing standard, the detection limit of the method of the present invention can reach 6.5×10 -2 U / g, which is more suitable for the detection of dairy products with low lactase content.

[0045] (2) The present invention uses a liquid chromatography column for separation, thereby avoiding the influence of insoluble impurities on the test results, and is suitable for the analysis of dairy product samples.

[0046] (3) Compared with the existing high performance liquid chromatography (differential method), the present invention can not only detect dairy products with lower content, but also is not affected by the background content of galactose and glucose, and can obtain more accurate and stable results.

[0047] (4) Compared with the existing kit method, the reagents used in the present invention are easy to obtain and simple to operate, and are more suitable for the analysis of lactase in dairy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a comparison chart of the colors of the gradient concentration o-nitrophenol working solutions prepared in Example 1 of the present invention.

[0049] Figure 2 The figure is a graph of the concentration point 5 mg / L of the o-nitrophenol gradient concentration standard solution prepared in the comparative example of the present invention.

[0050] Figure 3 It is the negative sample spectrum of the comparative example of the present invention.

[0051] Figure 4 It is the HPLC spectrum of the negative added sample of the comparative example of the present invention (the spiked concentration is 10 mg / L).

[0052] Figure 5 The linear chromatogram of the gradient concentration o-nitrophenol standard solution prepared in Example 1 of the present invention.

[0053] Figure 6 These are pictures of the blank sample and negative added sample of the comparative example of the present invention.

[0054] Figure 7 This is a linear curve (marked at 0-50 mg / L) measured by a spectrophotometer for the linear points of the comparative example of the present invention.

[0055] Figure 8 It is the spectrophotometric linearity graph of the negative added sample of the comparative example of the present invention (the spiked concentration is 10 mg / L). DETAILED DESCRIPTION

[0056] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0057] As used herein, the terms "comprises," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0058] "Optional" or "any one" means that the subsequently described matter or event can or cannot occur, and that the description includes instances where the event occurs and instances where it does not.

[0059] The indefinite articles "a" and "an" before the elements or components of the present invention have no limitation on the quantity requirements (i.e. the number of occurrences) of the elements or components. Therefore, "a" or "an" should be interpreted as including one or at least one, and the elements or components in the singular form also include the plural form, unless the quantity obviously refers to the singular form only.

[0060] The terms "one embodiment", "some embodiments", "exemplarily", "specific examples" or "some examples" described in the present invention mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this document, the schematic representation of the above terms does not necessarily refer to the same embodiment or example.

[0061] The numerical range described in the present invention not only includes the point values ​​listed in the embodiments, but also includes any point values ​​not listed between the numerical ranges described in the present invention. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0062] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the field.

[0063] The existing standard method and patented method cannot accurately analyze the lactase activity in dairy products with low lactase activity. Based on this, the present application provides a method for determining the lactase activity in dairy products, comprising the following steps:

[0064] S1. The sample to be tested is mixed with the o-nitrophenyl-β-D-galactoside substrate solution to obtain a sample mixture, the mixed sample solution is mixed with the reaction termination solution, and the mixture is centrifuged and filtered to obtain a sample solution to be tested;

[0065] S2. The sample to be tested is mixed with the reaction termination solution to obtain a blank mixed solution, the blank mixed solution is mixed with the o-nitrophenyl-β-D-galactoside substrate solution for reaction, and the blank test solution is obtained by centrifugation and filtration;

[0066] S3. Prepare a standard working solution of o-nitrophenol with a gradient concentration and perform HPLC analysis, and draw a working curve based on the analysis results;

[0067] S4. Perform HPLC analysis on the sample solution and the blank solution, and calculate the lactase activity in the sample according to the analysis results. If the concentration of o-nitrophenol in the sample exceeds the linear range of the working curve, the sample needs to be diluted with a buffer solution and then the step of S1 is repeated to obtain a new solution, and then the new solution is analyzed to calculate the lactase activity in the sample;

[0068] The calculation formula of lactase activity in the sample to be tested is as follows:

[0069]

[0070] Where:

[0071] X—enzyme activity of the sample to be tested, in U / g;

[0072] c—the concentration of o-nitrophenol in the sample solution, in milligrams per liter (mg / L);

[0073] c 0 —The concentration of o-nitrophenol in the blank test solution, in milligrams per liter (mg / L);

[0074] V—the volume of the sample to be tested, i.e., the sum of the volume of the sample to be tested, the volume of the o-nitrobenzene-β-D-galactoside substrate solution, and the volume of the reaction termination solution, in milliliters (mL), where the volume of the sample to be tested is converted according to 1g=1mL;

[0075] m—the mass of the sample represented by the sample test solution, in g;

[0076] M—molar mass of o-nitrophenol: 123.11 g / mol;

[0077] t——reaction time (min);

[0078] f——dilution factor of the sample to be tested.

[0079] In the present application, the o-nitrophenyl-β-D-galactoside substrate solution (ONPG) is a colorless transparent liquid, which is hydrolyzed by lactase to generate yellow o-nitrophenol (ONP), causing the solution to turn yellow. The higher the enzyme activity, the more complete the hydrolysis of ONPG, the more ONP produced, and the darker the color.

[0080] In particular, the present application compares the color of the standard working solution corresponding to the linear maximum concentration of the o-nitrophenol working curve with the color of the sample test solution by naked eye. If the color of the sample test solution is lighter than the color of the linear maximum concentration, it means that the o-nitrophenol in the sample test solution is within the linear range, and the results can be directly analyzed according to the standard curve. If the color of the sample test solution is close to or darker than the color of the standard working solution, it is necessary to dilute the sample test solution and analyze the diluted sample test solution again to calculate the lactase activity in the test sample.

[0081] In some specific embodiments, the sample test solution with too dark color is diluted because the lactase activity in the sample test solution with too dark color is too high. At this time, the ONPG substrate content is insufficient, resulting in incomplete reaction, and the final quantitative result is less than the actual value, which brings analysis errors and affects the quantitative results.

[0082] In some specific embodiments, the reaction termination solution is a sodium carbonate-EDTA aqueous solution or a hydrochloric acid aqueous solution. After the addition of these two reaction termination solutions, the pH of the reaction system is changed to inactivate the lactase in the sample to be tested, thereby terminating the reaction.

[0083] In some specific embodiments, the concentration of sodium carbonate in the sodium carbonate-EDTA aqueous solution is 40 to 60 g / L. As an example, the concentration of sodium carbonate in the sodium carbonate-EDTA aqueous solution can be 40 g / L, 45 g / L, 50 g / L, 55 g / L, or 60 g / L, as long as the concentration of sodium carbonate in the sodium carbonate-EDTA aqueous solution is within this range.

[0084] In some specific embodiments, the concentration of the hydrochloric acid aqueous solution is 3 to 8 mol / L. As an example, the concentration of the hydrochloric acid aqueous solution can be 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, as long as the concentration of the hydrochloric acid aqueous solution is within the range.

[0085] In some specific embodiments, the concentration of β-D-galactoside in the β-D-galactoside substrate solution is 2.0 to 3.0 g / L. As an example, the concentration of β-D-galactoside in the β-D-galactoside substrate solution can be 2.0 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L.

[0086] In some specific embodiments, the reaction time of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution in step S1 is 5 to 15 minutes. As an example, the reaction time of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution in step S1 can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, and 15 minutes, as long as the reaction time of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution in step S1 is within this range.

[0087] In some specific embodiments, the reaction time between the sample to be tested and the reaction termination solution in step S2 is 5 to 15 minutes. As an example, the reaction time between the sample to be tested and the reaction termination solution in step S2 can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, as long as the reaction time between the sample to be tested and the reaction termination solution in step S2 is within the range.

[0088] It can be found that the main difference between step S1 and step S2 is the reaction order of the sample to be tested and the reaction termination solution. In step S1, the mixed reaction of the sample to be tested and the reaction termination solution is carried out after the reaction of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution is completed, which can terminate the reaction. In step S2, the mixed reaction of the sample to be tested and the reaction termination solution is carried out before the reaction of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution. At this time, the lactase in the sample to be tested will be inactivated due to the influence of the reaction termination solution, and a blank test solution can be obtained after separation and filtration.

[0089] In some specific embodiments, step S1 and step S2 are performed under an environment of 25.0-35.0° C. As an example, the reaction environment temperature of step S1 and step S2 can be 25.0° C., 25.5° C., 26.0° C., 26.5° C., 27.0° C., 27.5° C., 28.0° C., 28.5° C., 29.0° C., 29.5° C., 30.0° C., 30.5° C., 31.0° C., 31.5° C., 32.0° C., 32.5° C., 33.0° C., 33.5° C., 34.0° C., 34.5° C., 35.0° C., as long as the reaction environment temperature of step S1 and step S2 is within the range.

[0090] In a specific embodiment, the reaction environment temperature of step S1 and step S2 is 29.9-30.1° C. Reacting in this temperature range can achieve the best hydrolysis effect of lactase.

[0091] In some specific embodiments, the mass volume ratio of the sample to be tested to the o-nitrophenyl-β-D-galactoside substrate solution to the reaction termination solution in S1 and S2 is (0.50-1.50) g: (2.50-7.50) mL: (1.00-3.00) mL. As an example, the selected value range of the mass-to-volume ratio of the sample to be tested can be 0.50 to 1.50g, including 0.50g, 0.55g, 0.60g, 0.65g, 0.70g, 0.75g, 0.80g, 0.85g, 0.90g, 0.95g, 1.00g, 1.05g, 1.10g, 1.15g, 1.20g, 1.25g, 1.30g, 1.35g, 1.40g, 1.45g, and 1.50g, as long as the selected value of the mass-to-volume ratio of the sample to be tested is within the range.

[0092] As an example, the volume ratio of the o-nitrobenzene-β-D-galactoside substrate solution is 1.00-3.00 mL. As an example, the volume ratio of the o-nitrobenzene-β-D-galactoside substrate solution can be 1.00 mL, 1.10 mL, 1.20 mL, 1.25 mL, 1.30 mL, 1.40 mL, 1.50 mL, 1.55 mL, 1.60 mL, 1.70 mL, 1.80 mL, 1.90 mL, 2.00 mL, 2.25 mL, 2.50 mL, 2.75 mL, 3.00 mL, as long as the volume ratio of the o-nitrobenzene-β-D-galactoside substrate solution is within the range.

[0093] As an example, the volume ratio of the reaction termination liquid is 1.00-3.00mL. As an example, the volume ratio of the reaction termination liquid can be 1.00mL, 1.10mL, 1.20mL, 1.25mL, 1.30mL, 1.40mL, 1.50mL, 1.55mL, 1.60mL, 1.70mL, 1.80mL, 1.90mL, 2.00mL, 2.25mL, 2.50mL, 2.75mL, 3.00mL, as long as the volume ratio of the reaction termination liquid is within the range.

[0094] In some specific embodiments, the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution in steps S1 and S2 need to be balanced before use, and the equilibrium temperature is 25-35°C. As an example, the equilibrium temperature can be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C, as long as the equilibrium temperature of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution is within this range.

[0095] In some specific embodiments, the equilibrium time of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution is 5 to 15 min. As an example, the equilibrium time of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, or 15 min, as long as the equilibrium time of the sample to be tested and the o-nitrobenzene-β-D-galactoside substrate solution is within this range.

[0096] In some specific embodiments, the concentration of the o-nitrophenol gradient concentration standard solution in step S3 is 0 to 200 mg / L. As an example, the concentration of the o-nitrophenol gradient concentration standard solution in step S3 can be 0 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, 200 mg / L. Of course, the concentration point outside this range can also be set according to the actual sample situation, as long as the test analysis requirements are met.

[0097] In some specific embodiments, the HPLC analysis parameters in step S4 include:

[0098] Chromatographic column: C18 column, column length 250 mm, inner diameter 4.6 μm, filler particle size 5 μm;

[0099] Column temperature: 35-45°C;

[0100] Injection volume: 10 μL;

[0101] Detector wavelength: 275nm;

[0102] Mobile phase: A: diammonium phosphate buffer; B: methanol;

[0103] Mobile phase flow rate: 0.5~1.5mL / min;

[0104] In some specific embodiments, the column temperature of the chromatographic column is 35-45°C. As an example, the column temperature of the chromatographic column can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, or 45°C, as long as the column temperature of the chromatographic column is within the range.

[0105] In some specific embodiments, the flow rate of the mobile phase is 0.5-1.5 mL / min. As an example, the flow rate of the mobile phase can be 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, as long as the flow rate of the mobile phase is within the range.

[0106] In some specific embodiments, the liquid phase elution procedure is as follows:

[0107]

[0108]

[0109] The present invention will be further described below by means of specific embodiments:

[0110] Reagent preparation

[0111] Reaction buffer preparation:

[0112] Weigh 8.8 g potassium dihydrogen phosphate (KH 2 PO 4 )、6.1g potassium dihydrogen phosphate (K 2 HPO 4 ), dissolve 0.12g anhydrous magnesium sulfate and 18.6mg EDTA in 900mL of water, use 1mol / L hydrochloric acid or sodium hydroxide solution to adjust the pH to 6.50±0.05, make up to 1000mL with water, and shake well.

[0113] ONPG substrate solution:

[0114] Dissolve 250.0 mg of o-nitrophenyl-β-D-galactoside (ONPG) in about 80 mL of reaction buffer, make up to 100 mL with reaction buffer, shake well, prepare within 2 hours before use, and preheat at 30°C for no more than 30 minutes.

[0115] Sodium carbonate-EDTA aqueous solution:

[0116] 50g of sodium carbonate (NaCO 3 ) and 37.2 g EDTA were dissolved in about 900 mL of water, made up to 1000 mL with water, and shaken well.

[0117] Standard preparation

[0118] o-Nitrophenol (ONP) stock solution (1000 mg / L):

[0119] Weigh 100 mg (accurate to 0.001 g) of o-nitrophenol (CAS No.: 824-39-5) standard into a 100 mL volumetric flask. Dissolve it in methanol and dilute to volume and mix well.

[0120] o-Nitrophenol (ONP) standard working solution (100 mg / L):

[0121] Accurately pipette 1 mL of o-nitrophenol (ONP) stock solution (1000 mg / L) (3.3.1), dilute it with methanol solution and make up to 10 mL, and prepare it before use.

[0122] Instruments and equipment

[0123] High performance liquid chromatograph: equipped with UV or PDA detector.

[0124] Analytical balance: sensitivity 0.01g; 0.01mg.

[0125] Vortex mixer: Oscillation speed not less than 2400 rpm.

[0126] Constant temperature water bath.

[0127] Centrifuge: Maximum speed is not less than 10000rpm.

[0128] Example 1

[0129] Add 1.00 g of the sample to be tested into a 50 mL centrifuge tube and equilibrate in a (30 ± 0.1) °C water bath for 10 min.

[0130] Quickly add 5.00 mL of preheated ONPG substrate solution, mix and put it in the water bath again. After accurately reacting for 10 minutes, quickly add 2.00 mL of EDTA aqueous solution with a sodium carbonate concentration of 50 g / L, mix and centrifuge, take the supernatant and filter it through a 0.22 μm aqueous filter membrane to obtain the sample test solution.

[0131] Add 1.00g of the sample to be tested into a 50mL centrifuge tube, balance in a (30±0.1)℃ water bath for 10min, add 2.00mL of 50g / L EDTA aqueous solution of sodium carbonate, and mix well. Then quickly add 5.00mL of preheated 2.5g / L ONPG substrate solution, mix, and put it into the water bath again, react for 10min accurately. Centrifuge, take the supernatant and filter it through a 0.22μm aqueous filter membrane to obtain a blank test solution.

[0132] Standard working solutions with o-nitrophenol concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L were prepared and analyzed by HPLC. A working curve was drawn based on the analysis results. The HPLC analysis parameters are as follows:

[0133] C18 chromatographic column (column length 250 mm, inner diameter 4.6 μm, filler particle size 5 μm);

[0134] Column temperature: 40°C;

[0135] Mobile Phase:

[0136] A: Diammonium hydrogen phosphate buffer (weigh 1.5 g of diammonium hydrogen phosphate aqueous solution, add 2 mL of phosphoric acid, and dilute to 1 L with distilled water);

[0137] B: methanol;

[0138] Mobile phase flow rate: 1.0 mL / min;

[0139] Injection volume: 10 μL;

[0140] Detector wavelength: o-nitrophenol (ONP) 275 nm.

[0141] Liquid phase elution procedure:

[0142]

[0143] The standard curve of o-nitrophenol obtained by analyzing the o-nitrophenol gradient concentration working solution is y=1.442694×10 5 x+8.400427×10 4 , Figure 1This is a picture of the o-nitrophenol standard working solution with gradient concentrations. The test tubes are numbered 1 to 9, with a total of 9 test tubes. The o-nitrophenol working solution concentrations corresponding to test tubes 1 to 9 are 0 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L, respectively. Figure 1 It can be seen that the color of the o-nitrophenol solution in the test tube changes with the increase of concentration. The o-nitrophenol concentration corresponding to test tube No. 8 is close to the maximum linear point of the working curve, while the o-nitrophenol concentration in the darker test tube No. 9 has exceeded the maximum linear point of the working curve. Therefore, it is also possible to quickly judge whether the sample to be tested needs to be diluted by comparing the color of the sample test solution with the color corresponding to the o-nitrophenol working solution concentration corresponding to the maximum linear point of the working curve. Figure 5 The chromatogram of the o-nitrophenol standard working solution with gradient concentration used in drawing the standard curve in Example 1 of the present invention is shown in FIG. Figure 5 It can be seen that the method provided by the present invention has good linearity and can accurately quantify samples of dairy matrix.

[0144] Then, the blank solution and the sample solution were analyzed using the same HPLC parameters as those used to draw the standard working curve, and the analysis results were substituted into the following formula for calculation:

[0145]

[0146] Where:

[0147] X—enzyme activity of the sample to be tested, in U / g;

[0148] c—the concentration of o-nitrophenol in the sample solution, in milligrams per liter (mg / L);

[0149] c 0 —The concentration of o-nitrophenol in the blank test solution, in milligrams per liter (mg / L);

[0150] V—the volume of the sample to be tested, i.e., the sum of the volume of the sample to be tested, the volume of the o-nitrobenzene-β-D-galactoside substrate solution, and the volume of the reaction termination solution, in milliliters (mL), where the volume of the sample to be tested is converted according to 1g=1mL;

[0151] m—the mass of the sample represented by the sample test solution, in g;

[0152] M—molar mass of o-nitrophenol: 123.11 g / mol;

[0153] t——reaction time (min);

[0154] f——dilution factor of the sample to be tested.

[0155] The lactase activity in the sample to be tested was calculated to be 0.13 U / g.

[0156] Example 2

[0157] 0.50 g of the sample to be tested was added to a 50 mL centrifuge tube, and the tube was equilibrated in a (25 ± 0.1) ° C water bath for 15 min. Then, 2.50 mL of preheated ONPG substrate solution with a concentration of 3 g / L was quickly added. After mixing, the tube was placed in a water bath again. After an accurate reaction time of 5 min, 1.00 mL of EDTA aqueous solution with a sodium carbonate concentration of 60 g / L was quickly added. After mixing, the tube was centrifuged and the supernatant was filtered through a 0.22 μm aqueous filter membrane to obtain the sample solution to be tested.

[0158] Add 0.50g of the sample to be tested into a 50mL centrifuge tube, equilibrate in a (25±0.1)℃ water bath for 15min, quickly add 1.00mL of EDTA aqueous solution with a sodium carbonate concentration of 60g / L, and mix well. Then quickly add 2.50mL of preheated ONPG substrate solution with a concentration of 3g / L, mix, and put it into the water bath again, react accurately for 15min. Centrifuge, take the supernatant and filter it through a 0.22μm aqueous filter membrane to obtain a blank test solution.

[0159] The HPLC parameters and working curve of Example 1 were used to analyze the test solution and blank solution, and the lactase activity in the test sample was calculated to be 8.1×10 -2 U / g.

[0160] Example 3

[0161] Add 1.50 g of the sample to be tested into a 50 mL centrifuge tube, equilibrate in a (35±0.1)°C water bath for 5 min, then quickly add 7.50 mL of preheated 2 g / L ONPG substrate solution, mix and put it into the water bath again, react accurately for 5 min, quickly add 3.00 mL of 5 mol / L hydrochloric acid aqueous solution, mix and centrifuge, take the supernatant and filter it through a 0.22 μm aqueous filter membrane to obtain the sample solution to be tested.

[0162] Add 1.50g of the sample to be tested into a 50mL centrifuge tube, balance in a (35±0.1)℃ water bath for 5min, add 3.00mL of 5mol / L hydrochloric acid aqueous solution, and mix well. Then quickly add 7.50mL of preheated 2g / L ONPG substrate solution, mix, and put it in a water bath again, react for 10min accurately. Centrifuge, take the supernatant and filter it through a 0.22μm aqueous filter membrane to obtain a blank test solution.

[0163] The sample test solution and the blank test solution were analyzed using the HPLC parameters and working curve of Example 1. During the calculation, it was found that the concentration of o-nitrophenol in the test sample exceeded the linear range of the working curve. Therefore, the sample was diluted with buffer (dilution factor was 1.5) to obtain a new sample test solution, and the new sample test solution was subjected to HPLC analysis. Finally, it was calculated that the lactase activity in the test sample was 2.07 U / g.

[0164] Verification Example

[0165] To further prove the accuracy and reliability of the method provided by the present invention, repeatability and precision tests are now carried out, as follows:

[0166] Three batches of different dairy product samples were tested using the method of Example 1, with enzyme activity of 6.5×10 -2 The recovery rate and repeatability of lactase were measured. The results are shown in Table 1 below.

[0167] Table-1 Recovery rate and relative standard deviation of three dairy products

[0168] Ibeli adult formula milk powder Daily Fresh Double Protein Milk Mengniu hydrolyzed milk Recovery rate (%) Recovery rate (%) Recovery rate (%) Positive Addition 1 92.8 99.0 94.2 Positive Addition 2 93.6 98.6 101.9 Positive Addition 3 96.3 99.1 92.3 Positive Addition 4 93.9 97.9 96.3 Positive Addition 5 95.7 98.3 97.6 Positive Addition 6 97.0 99.2 93.7 average value 94.9 98.7 96.0 RSD% 1.76 0.51 3.58

[0169] It can be seen from the analysis results in Table 1 that the method provided by the present invention is capable of analyzing a variety of different types of dairy products, and the recovery rate and relative standard deviation both meet the test analysis requirements.

[0170] Comparative Example 1

[0171] A blank sample (lactase-free) and a spiked negative sample with an o-nitrophenol conversion concentration of 10 mg / L are provided, which are recorded as blank sample and negative spiked sample (e.g. Figure 6 As shown in Table 3, samples 1 to 9 used in drawing the standard curve in Example 1, the blank samples, and the negative added samples were analyzed by the existing standard method (spectrophotometry) and the method of the present invention, and the results were shown in Table 3 and Table 4, respectively, as follows:

[0172] Table-2 Analysis results of existing standard methods

[0173] Sample name Absorbance (420nm) Concentration (mg / L) No. 1 0.0021 0 No. 2 0.0406 1 No.3 0.0577 2 No. 4 0.1831 5 No. 5 0.3441 10 No. 6 0.7182 20 No.7 1.9025 50 No. 8 4.7646 100 No. 9 4.3699 200 Blank Sample 3.0589 108.76 Negative spiked samples 3.5276 127.04

[0174] Table-3 Analysis results of the method of the present invention

[0175]

[0176]

[0177] As shown in Tables 2 and 3, the results obtained by analyzing the dairy matrix using the existing standard method and the blank sample both show positive results, and the results of the negative added sample are also quite different from the actual results of the sample. Obviously, the existing standard method is not suitable for the analysis of lactase activity in a dairy matrix with a complex matrix. The results obtained by the method provided by the present invention are very close to the actual addition amount and have strong applicability.

[0178] Furthermore, this comparative example also conducted other analyses on the two methods, wherein:

[0179] Figure 2 It is the chromatogram of the linear point concentration (5 mg / L) of the method of the present invention.

[0180] Figure 3 It is the chromatogram of negative sample tested by the method of the present invention.

[0181] Figure 4 It is the chromatogram of the negative added sample (spiked concentration is 10 mg / L) of the present invention.

[0182] Figure 7 This is the standard curve diagram of the existing standard method. It can be found that the linear point of 50 mg / L has exceeded the reading range of the spectrophotometer (the most accurate reading range of the UV spectrophotometer is between 0.3 and 0.7), but it can still be linear. However, the linear point exceeding 50 mg / L can no longer draw a curve.

[0183] Figure 8 This is a standard curve diagram of negative added samples analyzed by the existing standard method. Since the turbidity reading of the blank sample has exceeded the instrument range, the standard curve diagram of the negative added sample is more abnormal. Therefore, the dairy matrix is ​​not suitable for determination by the existing standard spectrophotometric method. The method of the present invention is suitable for the determination of lactase activity in dairy samples.

[0184] Comparative Example 2

[0185] After analyzing the verification example and comparative example 1, the differences between the present invention and the existing standard method and patent method are summarized, and the details are shown in Table 4.

[0186] Table-4 Differences between the present invention provider and existing standard methods and patented methods

[0187]

[0188] As shown in Table 4, the matrix of the prior art is mainly reagents, lactase preparations or raw materials with high lactase content, while the present invention is applicable to dairy products with low lactase activity and samples containing trace amounts of lactase. The detection limit of the present invention can reach 6.5×10 -2U / g, measured by high performance liquid chromatography (PDA detector), can ensure the accuracy, precision and reproducibility of the data. The present invention uses fewer types of reagents than the kit method, and the kit method has higher requirements for the stability control of the lactase rapid test card, and the operation is more difficult. By solving the above problems, the present invention can directly measure the content of lactase in dairy products, can accurately analyze the actual content of lactase in products claiming to contain lactase circulating on the market, and can provide services for enterprises, accurately control the final content of lactase from the production end, and regulate the market.

[0189] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for determining lactase activity in dairy products, characterized in that: The following steps are involved: S1. The sample to be tested is mixed with the o-nitrophenyl-β-D-galactoside substrate solution to obtain a sample mixture, the sample mixture is mixed with the reaction termination solution, and the mixture is centrifuged and filtered to obtain a sample solution to be tested; S2. The sample to be tested is mixed with the reaction termination solution to obtain a blank mixed solution, the blank mixed solution is mixed with the o-nitrophenyl-β-D-galactoside substrate solution for reaction, and the blank test solution is obtained by centrifugation and filtration; S3. Prepare a standard working solution of o-nitrophenol with a gradient concentration and perform HPLC analysis, and draw a working curve based on the analysis results; S4. Perform HPLC analysis on the sample solution and the blank solution, and calculate the lactase activity in the sample according to the analysis results. If the concentration of o-nitrophenol in the sample exceeds the linear range of the working curve, the sample needs to be diluted with a buffer solution and then the step of S1 is repeated to obtain a new solution, and then the new solution is analyzed to calculate the lactase activity in the sample; The calculation formula of lactase activity in the sample to be tested is as follows: Where: X—enzyme activity of the sample to be tested, in U / g; c—the concentration of o-nitrophenol in the sample solution, in milligrams per liter (mg / L); c0—the concentration of o-nitrophenol in the blank test solution, in milligrams per liter (mg / L); V—the volume of the sample to be tested, i.e., the sum of the volume of the sample to be tested, the volume of the o-nitrobenzene-β-D-galactoside substrate solution, and the volume of the reaction termination solution, in milliliters (mL), where the volume of the sample to be tested is converted according to 1g=1mL; m—the mass of the sample represented by the sample test solution, in grams (g); M—molar mass of o-nitrophenol: 123.11 g / mol; t——reaction time (min); f——dilution factor of the sample to be tested.

2. The method for determining lactase activity in dairy products according to claim 1, characterized in that: The reaction termination solution is a sodium carbonate-EDTA aqueous solution or a hydrochloric acid aqueous solution.

3. The method for determining lactase activity in dairy products according to claim 2, characterized in that: The concentration of sodium carbonate in the sodium carbonate-EDTA aqueous solution is 40-60 g / L; The concentration of the hydrochloric acid aqueous solution is 3-8 mol / L.

4. The method for determining lactase activity in dairy products according to claim 1, characterized in that: The concentration of β-D-galactoside in the β-D-galactoside substrate solution is 2.0-3.0 g / L.

5. The method for determining lactase activity in dairy products according to claim 1, characterized in that: The reaction time of the sample to be tested and the o-nitrophenyl-β-D-galactoside substrate solution in step S1 is 5 to 15 minutes; The reaction time of the sample to be tested and the reaction termination solution in step S2 is 5 to 15 minutes.

6. The method for determining lactase activity in dairy products according to claim 1, characterized in that: The steps S1 and S2 are performed in an environment of 25.0-35.0°C.

7. The method for determining lactase activity in dairy products according to claim 1, characterized in that: In the steps S1 and S2, the mass volume ratio of the sample to be tested, the o-nitrobenzene-β-D-galactoside substrate solution and the reaction termination solution is (0.50-1.50) g: (2.50-7.50) mL: (1.00-3.00) mL.

8. The method for determining lactase activity in dairy products according to claim 1, characterized in that: The sample to be tested and the o-nitrophenyl-β-D-galactoside substrate solution in steps S1 and S2 need to be equilibrated at a temperature of 25 to 35° C. for 5 to 15 minutes before use.

9. The method for determining lactase activity in dairy products according to claim 1, characterized in that: The concentration of the o-nitrophenol gradient concentration standard solution in step S3 is 0 to 200 mg / L.

10. The method for determining lactase activity in dairy products according to claim 1, characterized in that: The HPLC analysis parameters in step S4 include: Chromatographic column: C18 column, column length 250 mm, inner diameter 4.6 μm, filler particle size 5 μm; column temperature: 35-45 °C; Injection volume: 10 μL; Detector wavelength: 275nm; Mobile phase: A: diammonium phosphate buffer; B: methanol; Mobile phase flow rate: 0.5~1.5mL / min; Liquid phase elution procedure:

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