Kit for detecting milk alkaline phosphatase activity or adulteration with raw milk and use thereof

Abstract

The application discloses a kit for detecting milk alkaline phosphatase activity or adulteration of raw milk and application thereof. The kit comprises a chromogenic reagent and a standard color card for indicating alkaline phosphatase activity concentration or raw milk proportion, wherein the standard color card comprises at least two color blocks with different color depths, and each color block corresponds to a corresponding alkaline phosphatase activity concentration or raw milk proportion. The application also discloses application of the kit in detection of milk alkaline phosphatase activity or adulteration of raw milk, which comprises the following steps: (1) contacting the chromogenic reagent with a milk sample to be detected to perform a chromogenic reaction; and (2) comparing the chromogenic result with the color blocks of the standard color card to determine the alkaline phosphatase activity concentration or the range of raw milk proportion contained in the milk sample. The application is suitable for closedness inspection of a milk production line, quality sampling inspection and rapid screening of product milk, and has application prospects in the inspection of the milk production line and the quality sampling inspection and rapid screening of the product milk.

Description

Technical Field

[0001] This invention relates to a kit for detecting alkaline phosphatase activity or the proportion of raw milk, and more particularly to a kit for detecting alkaline phosphatase activity in dairy products or the proportion of raw milk mixed in, and a method for rapidly and semi-quantitatively detecting alkaline phosphatase activity or the proportion of raw milk using the kit, belonging to the field of alkaline phosphatase detection in dairy products. Background Technology

[0002] Microorganisms and pathogens in raw milk are important indicators for evaluating milk quality and safety, and their content directly affects the quality and safety of dairy products. Higher microbial content makes milk more prone to spoilage and may even lead to pasteurization failure, resulting in excessive bacteria levels in the finished milk.

[0003] Alkaline phosphatase (ALP, EC 3.1.3.1) is a naturally occurring enzyme that primarily associates with the milk fat globule membrane (MFGM) in cow's milk. ALP is a thermostable enzyme, slightly more so than other pathogenic microorganisms in raw milk. Therefore, a significant decrease in ALP activity after heat treatment of cow's milk indicates the inactivation of other pathogenic microorganisms. This characteristic allows ALP to serve as a marker of the effectiveness of heat treatment in pasteurized milk; a positive ALP test result suggests incomplete pasteurization or contamination from raw milk. Currently, commonly used ALP detection methods include colorimetric, fluorescence, and luminescence methods. While these methods offer high sensitivity and precision, they require specialized equipment, relatively complex procedures, and lengthy testing times, making them unsuitable for the rapid and convenient needs of dairy production.

[0004] Alkaline phosphatase is only one indicator of dairy product quality; other indicators, such as total bacterial count, are also needed to determine if sterilization is up to standard. However, for rapid screening, alkaline phosphatase can be used alone for quick assessment. In this case, qualitative accuracy of the alkaline phosphatase test is required. Therefore, a simple and effective rapid detection technology is needed to determine the sterilization level and contamination from raw milk leakage in dairy products, thereby improving the efficiency of simultaneous testing of large batches of samples and reducing testing costs. Summary of the Invention

[0005] One objective of this invention is to provide a kit for detecting alkaline phosphatase activity in dairy products or the proportion of raw milk added.

[0006] The second objective of this invention is to apply the kit for detecting alkaline phosphatase activity in dairy products or the proportion of raw milk contained therein to the detection of alkaline phosphatase activity in dairy products or the proportion of raw milk contained therein.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention includes:

[0008] One aspect of the present invention provides a kit for detecting alkaline phosphatase activity in dairy products or the proportion of raw milk, comprising: a colorimetric reagent and a standard colorimetric card indicating the concentration of alkaline phosphatase activity or the proportion of raw milk.

[0009] In a preferred embodiment of the present invention, the colorimetric reagent is a reagent that makes alkaline phosphatase colorimetric.

[0010] In a preferred embodiment of the present invention, the standard colorimetric card indicating alkaline phosphatase activity concentration or raw milk ratio contains at least two color blocks of different shades, each color block corresponding to a corresponding alkaline phosphatase activity concentration or raw milk ratio.

[0011] In a preferred embodiment of the present invention, the standard colorimetric card indicating alkaline phosphatase activity concentration or raw milk ratio comprises 5-10 color blocks of different shades; preferably, the standard colorimetric card indicating alkaline phosphatase activity concentration or raw milk ratio comprises 7, 8 or 9 color blocks of different shades.

[0012] For reference, the present invention provides a method for preparing a standard colorimetric card indicating alkaline phosphatase activity concentration or raw milk ratio, comprising: adding alkaline phosphatase standard at different activity concentrations or raw milk at different ratios to liquid milk containing no alkaline phosphatase or with alkaline phosphatase completely inactivated to obtain a series of standard milk samples containing alkaline phosphatase at different activity concentrations or raw milk at different ratios; adding colorimetric reagents to the series of standard milk samples to perform corresponding colorimetric reactions to obtain the color blocks displayed for each; designating the displayed color blocks as the standard colors of the colorimetric card, and labeling the alkaline phosphatase activity concentration or the proportion of raw milk added under the corresponding color blocks to obtain the standard colorimetric card.

[0013] In a preferred embodiment of the present invention, alkaline phosphatase standard at different activity concentrations or different proportions of raw milk are added to liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase in a gradient-increasing manner to obtain a series of standard milk samples containing alkaline phosphatase at different activity concentrations or different proportions of raw milk; preferably, alkaline phosphatase standard at different activity concentrations or different proportions of raw milk are added to liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase in a multiplicative-increasing manner to obtain a series of standard milk samples containing alkaline phosphatase at different activity concentrations or different proportions of raw milk; more preferably, alkaline phosphatase standard at different activity concentrations or different proportions of raw milk are added to liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase in a manner that results in a series of standard milk samples containing alkaline phosphatase at different activity concentrations or different proportions of raw milk; The concentration of ALP standard at 350 mU / L serves as the starting point for the concentration gradient. Alkaline phosphatase standards with different activity concentrations are added in increments of 350, 1050, 3500, 10500, 35000, 105000, and 350000 mU / L to obtain a series of standard milk samples containing different activity concentrations of alkaline phosphatase. Alternatively, liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase is added with a concentration gradient starting at 0% raw milk content. Different proportions of raw milk are added in increments of 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, and 100% to obtain a series of standard milk samples containing different proportions of raw milk.

[0014] In a preferred embodiment of the present invention, the reagent kit includes at least one brown reagent bottle.

[0015] In a preferred embodiment of the present invention, the colorimetric reagent is selected from either phenolphthalein sodium phosphate solution or disodium phenyl phosphate solution.

[0016] In a preferred embodiment of the present invention, the phenolphthalein sodium phosphate solution comprises ammonia, ammonium chloride, tetrasodium phenolphthalein diphosphate, and water; wherein, the preparation method of the phenolphthalein sodium phosphate solution comprises dissolving tetrasodium phenolphthalein diphosphate in an NH3-NH4Cl buffer solution to obtain the phenolphthalein sodium phosphate solution, wherein the NH3-NH4Cl buffer solution is prepared from ammonia, ammonium chloride, and water; preferably, the concentration of the phenolphthalein sodium phosphate solution is 0.5-1.5 g / L; more preferably, the concentration of the phenolphthalein sodium phosphate solution is 1 g / L; preferably, the pH of the NH3-NH4Cl buffer solution is 9.0-10.0; more preferably, the pH of the NH3-NH4Cl buffer solution is 9.8.

[0017] In a preferred embodiment of the present invention, the disodium phenyl phosphate solution comprises 2,6-dibromobenzoquinone chloroimide, Na₂CO₃, disodium phenyl phosphate, ethanol, borate buffer, and water; wherein the preparation method of the disodium phenyl phosphate solution comprises: preparing a disodium phenyl phosphate-borate buffer matrix by disodium phenyl phosphate and borate buffer; preparing a Gibb phenol reagent by disodium 2,6-dibromobenzoquinone chloroimide and ethanol; preparing a Na₂CO₃ solution by disodium Na₂CO₃ and water; a certain amount of n-butanol is added during use; preferably, the concentration of the disodium phenyl phosphate-borate buffer matrix is ​​0.4-1.2 g / L; more preferably, the concentration of the disodium phenyl phosphate-borate buffer matrix is ​​0.8 g / L; preferably, the pH of the borate buffer matrix is ​​9.0-11.0; more preferably, the pH of the borate buffer matrix is ​​9.8; preferably, the volume fraction of the Na₂CO₃ solution is 14%.

[0018] Another aspect of the present invention is to apply the kit for detecting alkaline phosphatase activity or raw milk ratio to the detection of alkaline phosphatase activity or raw milk ratio in dairy products.

[0019] In a preferred embodiment of the present invention, the specific steps of using the kit for detecting alkaline phosphatase activity or the proportion of raw milk in dairy products include: (1) contacting the colorimetric reagent with the dairy product sample to be tested to perform a colorimetric reaction; (2) comparing the colorimetric result with the color blocks of the standard colorimetric card indicating the concentration of alkaline phosphatase activity or the proportion of raw milk to determine the range of alkaline phosphatase activity concentration or the proportion of raw milk in the dairy product sample.

[0020] In a preferred embodiment of the present invention, in step (1), the volume ratio of phenolphthalein sodium phosphate solution to the sample to be tested is 0.5-4:1; preferably, the volume ratio of phenolphthalein sodium phosphate solution to the sample to be tested is 1:1; the volume ratio of disodium phenyl phosphate-borate buffer matrix to the sample to be tested is 3-12:1; preferably, the volume ratio of disodium phenyl phosphate-borate buffer matrix to the sample to be tested is 5:1; the volume ratio of Na2CO3 solution to the sample to be tested is 0.5-5:1; preferably, the volume ratio of Na2CO3 solution to the sample to be tested is 1:1; the volume ratio of Gibb phenol reagent to the sample to be tested is 0.1-0.5:1; preferably, the volume ratio of Gibb phenol reagent to the sample to be tested is 0.3:1; the volume of n-butanol added is 1-5 mL; preferably, the volume of n-butanol is 2 mL.

[0021] In a preferred embodiment of the present invention, the colorimetric reaction in step (1) is carried out in a water bath; preferably, the water bath temperature is 36-44°C; more preferably, the water bath temperature is 40°C.

[0022] This invention provides a kit for detecting alkaline phosphatase activity or the proportion of raw milk in dairy products and its applications. Compared with other detection methods, the detection does not require complicated pretreatment steps, making it simple, fast, and efficient; the detection does not require large instruments and equipment, and the results are simple and intuitive; the testing personnel do not need specialized training, resulting in low equipment and labor costs; moreover, the kit is portable, easy to operate, and applicable to the airtightness inspection of dairy production lines, quality sampling and rapid screening of dairy products, providing a favorable approach for dairy product quality control. Attached Figure Description

[0023] Figure 1 Results of the phenolphthalein sodium phosphate reagent colorimetric reaction of UHT milk samples with different concentrations of ALP standard added;

[0024] Figure 2 Results of the colorimetric reaction of disodium phthalate reagent on UHT milk samples with different concentrations of ALP standard added;

[0025] Figure 3 A standard colorimetric card for the colorimetric reaction of phenolphthalein sodium phosphate reagent based on ALP standards;

[0026] Figure 4 A standard colorimetric card for the colorimetric reaction of disodium phthalate reagent based on ALP standards;

[0027] Figure 5 Results of the colorimetric reaction of phenolphthalein sodium phosphate reagent on UHT milk samples with different proportions of raw milk added;

[0028] Figure 6 Results of the colorimetric reaction of disodium phthalate reagent with UHT milk samples containing different proportions of raw milk;

[0029] Figure 7 A standard colorimetric card for the colorimetric reaction of phenolphthalein sodium phosphate reagent based on the amount of raw milk added;

[0030] Figure 8 A standard colorimetric card for the colorimetric reaction of disodium phthalate reagent based on the amount of raw milk added;

[0031] Figure 9 The results show the expiration date determination of the sodium phenolphthalein phosphate colorimetric reagent.

[0032] Figure 10 The results show the expiration date of the sodium phthalate colorimetric reagent.

[0033] Figure 11 The results of the colorimetric reactions of milk samples under different heat treatment conditions;

[0034] Figure 12 The results are for commercially available samples 1-9; among them, Figure 12 -A represents the colorimetric result of phenolphthalein sodium phosphate reagent; Figure 12 -B represents the colorimetric result of sodium phthalate disodium phosphate colorimetric reagent;

[0035] Figure 13 The test results are for commercially available samples 10-17; among them, Figure 13 -A represents the colorimetric result of phenolphthalein sodium phosphate reagent; Figure 13 -B represents the colorimetric result of sodium phenyl phosphate colorimetric reagent. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, it should be understood that the embodiments described are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but such modifications or substitutions all fall within the protection scope of the present invention.

[0037] Example 1: Preparation of a kit for detecting alkaline phosphatase activity

[0038] 1. Preparation of colorimetric reagents

[0039] Preparation of phenolphthalein sodium phosphate colorimetric reagent: Prepare an NH3-NH4Cl buffer solution by mixing an appropriate amount of ammonia water and ammonium chloride solution. Mix 80 mL of 1 mol / L ammonia water and 20 mL of 1 mol / L ammonium chloride solution to prepare an NH3-NH4Cl buffer solution with a pH of 9.8. Weigh out tetrasodium phenolphthalein diphosphate and dissolve it in the NH3-NH4Cl buffer solution to obtain a 1 g / L phenolphthalein sodium phosphate solution. Store the prepared reagent in a brown bottle protected from light at 2-6°C.

[0040] Preparation of disodium phenyl phosphate colorimetric reagent: Weigh disodium phenyl phosphate and dissolve it in borate solution to prepare a borate-disodium phenyl phosphate-borate solution with a concentration of 8 g / L and a borate buffer pH of 9.8. Then dilute 10 times with water to prepare a buffer matrix, preparing fresh each time. Store the prepared reagent in a brown bottle protected from light at 2-6°C and use within 7 days. Weigh 2,6-dibromobenzoquinone chloroimine and dissolve it in 95% ethanol, storing in a brown bottle in a refrigerator and using within 7 days. The concentration of the Gibb phenol reagent is 10 g / L. Weigh sodium carbonate and dissolve it in water, preparing fresh each time, with a Na₂CO₃ solution volume fraction of 14%.

[0041] 2. Establishment of Standard Colorimetric Cards

[0042] (1) Standard colorimetric card for detecting ALP concentration and activity

[0043] Liquid milk containing no ALP or with completely inactivated ALP, such as ultra-high temperature (UHT) milk, is used to form milk samples containing ALP standards of different concentrations of activity by adding ALP standards of different concentrations of activity.

[0044] Add sodium phenolphthalein phosphate solution to the milk sample at a ratio of 1:1, heat in a 40°C water bath for 10 minutes, remove and let stand at room temperature for 1 hour to obtain the colors displayed by different concentrations of ALP. Define the displayed colors as the standard colors of the colorimetric card, and label the ALP concentration activity at the corresponding colors to obtain the sodium phenolphthalein phosphate colorimetric standard colorimetric card.

[0045] Add disodium phenyl phosphate-borate buffer matrix to the milk sample at a ratio of 1:5, heat in a 40°C water bath for 10 minutes, remove and add Na2CO3 solution at a ratio of 1:1 to the sample and Gibb phenol colorimetric reagent at a ratio of 0.3:1, shake well, add 2 mL of n-butanol and let stand for 2 minutes. The color displayed on the n-butanol layer is determined as the standard color of the colorimetric card. Mark the ALP concentration and activity at the corresponding color to obtain the disodium phenyl phosphate colorimetric standard colorimetric card.

[0046] The concentration gradient started at 350 mU / L of ALP standard activity in the milk sample, and increased incrementally, for example, forming a concentration gradient of 350, 1050, 3500, 10500, 35000, 105000, and 350000 mU / L. The standard colorimetric card consisted of seven color blocks indicating the milk sample. Based on the CSB05-1426-2001 paint film color standard sample card, the colorimetric results are shown in Table 1 and... Figure 1 , Figure 2 As shown, a standard colorimetric card is established as follows: Figure 3 , Figure 4 As shown.

[0047] Table 1. Colorimetric results of ALP activity at different concentrations

[0048]

[0049] (2) Standard colorimetric card for detecting raw milk content

[0050] Liquid milk containing no ALP or with completely deactivated ALP (such as UHT milk) is mixed with different proportions of raw milk to form milk samples containing different proportions of raw milk.

[0051] Add sodium phenolphthalein phosphate solution to the milk sample at a ratio of 1:1, heat in a 40°C water bath for 10 minutes, remove and let stand at room temperature for 1 hour to obtain the color displayed by different proportions of raw milk. Define the color displayed by the color as the color of the color card, and mark the proportion of raw milk under the corresponding color to obtain the sodium phenolphthalein phosphate color standard color card.

[0052] Add disodium phenyl phosphate-borate buffer matrix to the milk sample at a ratio of 1:5, heat in a 40°C water bath for 10 minutes, remove and add Na2CO3 solution at a ratio of 1:1 to the sample and Gibb phenol colorimetric reagent at a ratio of 0.3:1, shake well, add 2 ml of n-butanol and let stand for 2 minutes. The color displayed on the n-butanol layer is determined as the standard color of the colorimetric card. Mark the proportion of raw milk under the corresponding color to obtain the disodium phenyl phosphate colorimetric standard colorimetric card.

[0053] Starting with 0% raw milk, the concentration gradient was established at 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, and 100%. The standard colorimetric card consisted of 99 color blocks indicating the presence of different colors in the dairy product. Based on the CSB05-1426-2001 standard color chart for lacquer films, the color development results are shown in Table 2. Figure 5 , Figure 6 As shown, a standard colorimetric card is established as follows: Figure 7 , Figure 8 As shown.

[0054] Table 2. Colorimetric results for different raw milk ratios

[0055]

[0056] 3. Determination of the shelf life of colorimetric reagents

[0057] NH3-NH4Cl buffer solution and sodium phenolphthalein phosphate solution were prepared at different times and stored in brown bottles at 0-6℃. ALP standards (activity 35 U / mgP, 8.5 mgP / mL) were measured and placed in 15 mL centrifuge tubes. UHT milk with 0 ALP activity was added to prepare a standard milk sample with an ALP activity of 350,000 mU / mL. This was serially diluted to prepare a series of standard milk samples with ALP activities of 350,000, 105,000, 35,000, 10,500, 3,500, 1050, and 350 mU / mL. Sodium phenolphthalein phosphate colorimetric solution was placed in a centrifuge tube, and the sample to be tested was added at a ratio of 1:1. The mixture was shaken well and heated in a 40℃ water bath for 10 minutes. After removal, it was left at room temperature for 1 hour, and the color change was observed.

[0058] Sodium phenyl phosphate-borate buffer and Gibb's phenol reagent were prepared at different times and stored in brown bottles at 0-6℃. Different volumes of raw milk were measured and placed in 15mL centrifuge tubes. UHT milk with ALP activity of 0% was added to prepare a series of milk samples with raw milk ratios of 0%, 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, and 100%. The sodium phenyl phosphate-borate buffer was placed in another centrifuge tube, and the sample to be tested was added at a ratio of 5:1. After shaking, the mixture was heated in a 40℃ water bath for 10 minutes. After removal, Na₂CO₃ solution (1:1 ratio with the sample), Gibb's phenol reagent (0.3:1 ratio), and 2mL of n-butanol were added. The mixture was shaken and allowed to stand for 2 minutes. The color change of the n-butanol layer was then observed.

[0059] The measurement results are as follows Figure 9 , Figure 10 As shown, in the phenolphthalein sodium phosphate colorimetric system, the color development of the standard at different times was completely consistent; in the disodium phenyl phosphate colorimetric system, the color development changed after 7 days, but a gradient change still existed, and the color development showed no difference after 30 days. Therefore, the shelf life of the phenolphthalein sodium phosphate colorimetric reagent is at least 180 days; the shelf life of the disodium phenyl phosphate-borate buffer matrix and Gibb phenol reagent should not exceed 7 days, and it is best to prepare them immediately before use.

[0060] Experimental Example 2: Detection Test of Milk Samples Under Different Heat Treatments

[0061] Prepare pasteurized milk (provided by Modern Farming) that has undergone different heat treatments, with sterilization temperatures of 72℃, 75℃, 80℃, 85℃, 90℃, 100℃, 105℃, 110℃ and 120℃, and sterilization time of 15s for each. Also prepare one portion each of raw milk before pasteurization and UHT milk sterilization.

[0062] Take the sodium phenolphthalein phosphate colorimetric solution into a centrifuge tube, add the sample to be tested at a ratio of 1:1, shake well, and heat in a 40℃ water bath for 10 minutes. After removing from the heat, let it stand at room temperature for 1 hour, observe the color change, and compare it with the standard colorimetric card.

[0063] Transfer the sodium phenyl phosphate-borate buffer matrix to another centrifuge tube, add the sample to be tested at a ratio of 5:1, shake well, and heat in a 40℃ water bath for 10 minutes. After removing from the heat, add Na₂CO₃ solution at a ratio of 1:1 to the sample and Gibb phenol reagent at a ratio of 0.3:1, shake well, add 2 ml of n-butanol, let stand for 2 minutes, observe the color of the n-butanol layer, and compare it with the standard colorimetric card.

[0064] The test results are as follows Figure 11As shown, in the sodium phenolphthalein phosphate colorimetric system, the quality control samples of raw milk appeared pale rose, while the milk samples from 72℃ to 120℃ all appeared white (without color change). In the sodium phenyl phosphate colorimetric system, the quality control samples of raw milk appeared deep sea blue, while the milk samples from 72℃ to 120℃ all appeared ochre yellow. Therefore, it can be determined that the ALP activity of the milk samples from 72℃ to 120℃ was below 10500 mU / L, and the raw milk penetration was less than 1%, which can be basically judged as sufficient sterilization, and there was no raw milk leakage contamination.

[0065] Test Example 3: Detection Test of Commercially Available Liquid Milk Samples

[0066] Nine samples of commercially available liquid milk (randomly selected from the sample library of the dairy innovation team) were purchased and constituted the first group, with sample numbers 1-9; eight samples of liquid milk from a dairy processing company (Shanghai) were taken and constituted the second group, with sample numbers 10-17; and one sample of raw milk was prepared as a positive quality control.

[0067] Take the sodium phenolphthalein phosphate colorimetric solution into a centrifuge tube, add the sample to be tested at a ratio of 1:1, shake well, and heat in a 40℃ water bath for 10 minutes. After removing from the heat, let it stand at room temperature for 1 hour, observe the color change, and compare it with the standard colorimetric card.

[0068] Transfer the sodium phenyl phosphate-borate buffer matrix to another centrifuge tube, add the sample to be tested at a ratio of 5:1, shake well, and heat in a 40℃ water bath for 10 minutes. After removing from the heat, add Na₂CO₃ solution at a ratio of 1:1 to the sample and Gibb phenol reagent at a ratio of 0.3:1, shake well, add 2 ml of n-butanol, let stand for 2 minutes, observe the color of the n-butanol layer, and compare it with the standard colorimetric card.

[0069] The test results of samples 1-9 are as follows Figure 12 As shown, in the phenolphthalein sodium phosphate colorimetric system, the positive control sample appeared pale rose, while samples 1-9 were all white (no color change); in the disodium phenyl phosphate colorimetric system, the positive control sample appeared deep sea blue, while samples 1-9 were all ochre yellow. Therefore, it can be determined that the ALP activity of the samples was all below 10500 mU / L, and the raw milk penetration was less than 1%, which can be basically judged as sufficient sterilization and no raw milk leakage contamination.

[0070] The test results for samples 10-17 are as follows: Figure 13As shown, in the sodium phenolphthalein phosphate colorimetric system, samples 10 and 14 were light pink, while samples 11, 12, 13, 15, 16, and 17 were all white (no color change). In the sodium phenyl phosphate colorimetric system, the raw milk quality control samples appeared deep sea blue, samples 10 and 14 were military vehicle green, and samples 11, 12, 13, 15, 16, and 17 were ochre yellow. Therefore, it can be determined that the ALP activity of samples 11, 12, 13, 15, 16, and 17 was all below 10500 mU / L, and the raw milk penetration was less than 1%, which can be basically judged as sufficient sterilization and no raw milk leakage contamination. The ALP activity of samples 10 and 14 was about 10500 mU / L, and the raw milk penetration was about 2% to 3%, which can be basically judged as insufficient sterilization or the presence of a small amount of raw milk leakage contamination.

Claims

1. A kit for detecting alkaline phosphatase activity in dairy products or the proportion of raw milk added, characterized in that, include: Colorimetric reagents and standard colorimetric cards indicating alkaline phosphatase activity concentration or raw milk ratio; The colorimetric reagent is a reagent that makes alkaline phosphatase develop color; the standard colorimetric card indicating the alkaline phosphatase activity concentration or the proportion of raw milk contains at least two color patches of different shades, each color patch corresponding to the corresponding alkaline phosphatase activity concentration or the proportion of raw milk.

2. The reagent kit according to claim 1, characterized in that, The standard colorimetric card indicating alkaline phosphatase activity concentration or raw milk ratio contains 5-10 color blocks of different shades; preferably, the standard colorimetric card indicating alkaline phosphatase activity concentration or raw milk ratio contains 7, 8 or 9 color blocks of different shades.

3. The reagent kit according to claim 1, characterized in that, The method for preparing the standard colorimetric card indicating alkaline phosphatase activity concentration or raw milk ratio includes: adding alkaline phosphatase standard at different activity concentrations or raw milk at different ratios to liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase to obtain a series of standard milk samples containing alkaline phosphatase at different activity concentrations or raw milk ratios; adding colorimetric reagents to the series of standard milk samples to carry out corresponding colorimetric reactions to obtain the color blocks displayed; designating the displayed color blocks as the standard colors of the colorimetric card, and labeling the alkaline phosphatase activity concentration or raw milk ratio under the corresponding color blocks to obtain the standard colorimetric card.

4. The reagent kit according to claim 3, characterized in that, Different concentrations of alkaline phosphatase standard or different proportions of raw milk are added to liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase in a gradient-increasing manner to obtain a series of standard milk samples containing different concentrations of alkaline phosphatase or different proportions of raw milk; preferably, different concentrations of alkaline phosphatase standard or different proportions of raw milk are added to liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase in a multiple-increasing manner to obtain a series of standard milk samples containing different concentrations of alkaline phosphatase or different proportions of raw milk; more preferably, the liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase is added to the milk sample containing 350 mU / L... The concentration of ALP standard is used as the starting point for the concentration gradient. Different concentrations of alkaline phosphatase standard are added in multiples of 350, 1050, 3500, 10500, 35000, 105000, and 350000 mU / L to obtain a series of standard milk samples containing different concentrations of alkaline phosphatase. Alternatively, liquid milk containing no alkaline phosphatase or with completely inactivated alkaline phosphatase is added with the concentration gradient starting at 0% raw milk content, in multiples of 0.25%, 0.5%, 1%, 2%, 3%, 4%, 5%, and 100% to obtain a series of standard milk samples containing different proportions of raw milk.

5. The kit according to any one of claims 1-4, characterized in that, The kit includes at least one brown reagent bottle.

6. The reagent kit according to claim 1, characterized in that, The colorimetric reagent is selected from either phenolphthalein sodium phosphate solution or disodium phenyl phosphate solution; The phenolphthalein sodium phosphate solution comprises ammonia, ammonium chloride, tetrasodium phenolphthalein diphosphate, and water; Preferably, the method for preparing the sodium phenolphthalein phosphate solution includes dissolving tetrasodium phenolphthalein diphosphate in an NH3-NH4Cl buffer solution to obtain the sodium phenolphthalein phosphate solution, wherein the NH3-NH4Cl buffer solution is prepared from ammonia, ammonium chloride, and water; more preferably, the concentration of the sodium phenolphthalein phosphate solution is 0.5-1.5 g / L; most preferably, the concentration of the sodium phenolphthalein phosphate solution is 1 g / L; the pH of the NH3-NH4Cl buffer solution is preferably 9.0-10.0; more preferably, the pH of the NH3-NH4Cl buffer solution is 9.8; The sodium phenyl phosphate solution comprises 2,6-dibromobenzoquinone chloroimine, Na2CO3, sodium phenyl phosphate, ethanol, borate buffer, and water; Preferably, the method for preparing the disodium phenyl phosphate solution includes: preparing a disodium phenyl phosphate-borate buffer matrix by disodium phenyl phosphate and borate buffer; preparing a Gibb phenol reagent by disodium phenyl phosphate chloroimine and ethanol; preparing a Na2CO3 solution by disodium phenyl phosphate and water; and adding n-butanol during use. More preferably, the concentration of the disodium phenyl phosphate-borate buffer matrix is ​​0.4-1.2 g / L; more preferably, the concentration of the disodium phenyl phosphate-borate buffer matrix is ​​0.8 g / L; more preferably, the pH of the borate buffer matrix is ​​9.0-11.0; more preferably, the pH of the borate buffer matrix is ​​9.8; and the volume fraction of the Na2CO3 solution is preferably 14%.

7. The application of the kit according to claim 1 in detecting alkaline phosphatase activity or the proportion of raw milk in dairy products.

8. The application according to claim 7, characterized in that, include: (1) The colorimetric reagent is brought into contact with the dairy sample to be tested to carry out a colorimetric reaction; (2) Compare the color development results with the color blocks of the standard colorimetric card indicating the concentration of alkaline phosphatase activity or the proportion of raw milk to determine the range of alkaline phosphatase activity concentration or the proportion of raw milk in the dairy sample.

9. The application according to claim 8, characterized in that, In step (1), the volume ratio of phenolphthalein sodium phosphate solution to the sample to be tested is 0.5-4:1; preferably, the volume ratio of phenolphthalein sodium phosphate solution to the sample to be tested is 1:1; the volume ratio of disodium phenyl phosphate-borate buffer matrix to the sample to be tested is 3-12:1; preferably, the volume ratio of disodium phenyl phosphate-borate buffer matrix to the sample to be tested is 5:1; the volume ratio of Na2CO3 solution to the sample to be tested is 0.5-5:1; preferably, the volume ratio of Na2CO3 solution to the sample to be tested is 1:1; the volume ratio of Gibb phenol reagent to the sample to be tested is 0.1-0.5:1; preferably, the volume ratio of Gibb phenol reagent to the sample to be tested is 0.3:1; the volume of n-butanol added is 1-5 mL; preferably, the volume of n-butanol is 2 mL.

10. The application according to claim 8, characterized in that, The colorimetric reaction in step (1) is carried out in a water bath; preferably, the water bath temperature is 36-44°C; more preferably, the water bath temperature is 40°C.

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