Pretreatment diluent and detection method for detecting oil-based matrix products

Abstract

The present invention relates to a pretreatment diluent for detecting oil-based matrix products. The diluent comprises water, Tween, and a calcium salt, wherein the mass ratio of the calcium salt to water is 1-30:99-70, and the mass ratio of the Tween to the calcium salt is 1-30:99-70. The present invention also relates to a pretreatment method for detecting oil-based matrix products, a method for detecting the oil content of oil-based matrix products, and a method for performing sterility testing on oil-based matrix products. Compared with existing methods, the method of the present invention is simple to operate and takes less time, without loss or loss of raw materials, thereby improving the accuracy of the experiment and reducing the operation time.

Description

Technical Field

[0001] The present invention belongs to the field of oil-based matrix product detection, and more particularly relates to a pre-treatment diluent and a detection method for oil-based matrix products. Background Art

[0002] For products with oil-based matrices, such as creams, two key tests are oil content and sterility. Crude fat content is a key quality indicator in the standards for grains, oilseeds, and feed products, serving as a crucial basis for evaluating product quality and organizing production. There are over ten methods for determining crude fat content both domestically and internationally. GB5009.6-2016, "National Food Safety Standard - Determination of Fat in Foods," specifies three methods for determining oil content in foods: Soxhlet extraction, acid hydrolysis, and alkaline hydrolysis. Soxhlet extraction is currently the most widely used method and is recognized as a classic method for determining fat content, primarily used for fat determination in foods. The Soxhlet extraction method, as specified in GB5009.6-2016, involves weighing the sample, placing it in an evaporating dish filled with quartz sand, and evaporating it to dryness in a boiling water bath. After evaporation, the sample is dried in an electric forced-air drying oven at 100°C ± 5°C for 30 minutes. The sample is then removed, ground into a fine powder, and transferred to a filter paper cartridge. The Soxhlet extraction method, as specified in GB5009.6-2016, "National Food Safety Standard - Determination of Fat in Foods," requires evaporating the sample to dryness in a boiling water bath, followed by drying and grinding. This process requires ensuring that the sample is evaporated to dryness in a boiling water bath, which can be time-consuming. Determining the evaporation time requires experience, leading to poor evaporation results and prolonged extraction times.

[0003] Isopropyl myristate is often used to dissolve water-insoluble drugs or their components in sterility testing. For example, in the article "Study on Sterility Testing Methods for Several Antibiotic Eye Ointments," the ointment base is difficult to dissolve, making direct sterility testing impossible. The article describes the pretreatment method as follows: Add 100 mL of isopropyl myristate to 2 g of eye ointment, shake to dissolve the sample, and heat in a 45°C water bath to complete dissolution. However, other articles report that isopropyl myristate cannot completely dissolve creams. In the article "Establishment of a Sterility Testing Method for Minocycline Hydrochloride Ointment," isopropyl myristate, 10% Tween-80 peptone solution, and 20% ethanol solution were used as solvents, but none of them were able to disperse and dissolve minocycline hydrochloride ointment. Sterility testing requires passing the sample through a filter. Currently, there is no effective solution for testing highly viscous samples like creams that are difficult to filter through.

[0004] Some literature reports that water-insoluble creams can be emulsified and separated using a surfactant such as Tween to separate the sample. For example, in the document "Study on Sterility Test Method for Compound Sulfadiazine Zinc Cream," the sample pretreatment method is to add Tween-80 sodium chloride-peptone buffer to the sample to emulsify it, centrifuge it, and then take the precipitate for inspection. However, for creams with more complex formulas, especially those containing thickeners, the effect of using Tween alone for emulsification and separation is not obvious, resulting in deviations in the test results. Therefore, it is necessary to develop a method that can more accurately detect oil-based products with high viscosity and difficulty in separation. Summary of the Invention

[0005] In response to the above-mentioned defects and problems of the prior art, the present invention has developed a pretreatment diluent for detecting oil-based matrix products, as well as a corresponding pretreatment method for detecting oil-based matrix products, a method for detecting the oil content of oil-based matrix products, and a method for performing sterility testing on oil-based matrix products.

[0006] A first aspect of the present invention provides a pretreatment diluent for detecting oil-based matrix products, characterized in that the diluent comprises a calcium salt aqueous solution and Tween, wherein the mass ratio of calcium salt to water in the calcium salt aqueous solution is in the range of 1-30:99-70, and the volume ratio of Tween to the calcium salt aqueous solution in the diluent is in the range of 1-30:99-70.

[0007] Optionally, the mass ratio of calcium salt to water in the calcium salt aqueous solution is in the range of 9-20:91-80, and the volume ratio of Tween to the calcium salt aqueous solution in the diluent is in the range of 4-15:96-85.

[0008] Optionally, the Tween is selected from one or more of Tween-80, Tween-60, Tween-40, and Tween-20.

[0009] Optionally, the calcium salt is selected from one or more of calcium chloride, calcium bromide, calcium iodide, calcium nitrate, and calcium sulfate.

[0010] Optionally, the oil-based matrix product is a cream.

[0011] The inventors of the present invention have found through a large number of experiments that when Tween alone is added to separate the oil phase and the water phase, the boundary between the oil phase and the water phase is not obvious after low-speed centrifugation; by further adding a calcium salt, such as calcium chloride, a complete oil layer can be obtained, which can be further processed.

[0012] Another aspect of the present invention provides a pre-treatment method for detecting oil-based matrix products, characterized in that the oil-based matrix product is treated with the above-mentioned diluent.

[0013] Another aspect of the present invention provides a method for detecting the fat content of a fat-based product, characterized in that the fat-based product is treated with the above-mentioned diluent before detection.

[0014] Optionally, the detection method comprises the following steps:

[0015] (1) Sample processing: add the sample to be tested into the above diluent, mix well and centrifuge, and remove the oil layer in the centrifuge tube;

[0016] (2) Determination of oil content: Place the oil layer into the extraction cylinder of a Soxhlet extractor, connect it to a receiving bottle, and extract; evaporate the extract to dryness and weigh it.

[0017] Preferably, in the method for detecting the oil content of an oil-based matrix product, the mass ratio of calcium salt to water in the calcium salt aqueous solution is in the range of 9-20:91-80, and the volume ratio of Tween to the calcium salt aqueous solution in the diluent is in the range of 4-15:96-85.

[0018] Another aspect of the present invention provides a method for sterility testing of oil-based matrix products, characterized in that the above-mentioned diluent is used for treatment before testing.

[0019] Optionally, the detection method comprises the following steps:

[0020] (1) Add the sample to be tested to the above diluent, mix well and centrifuge, remove the oil layer in the centrifuge tube, and smear it on the culture medium;

[0021] (2) filtering the solution in the middle layer of the centrifuge tube, and rinsing each filter membrane with a buffer solution after filtration, wherein the filter membrane is inoculated in the culture medium;

[0022] (3) Use buffer to dissolve the precipitate at the bottom of the centrifuge tube, inoculate it into the culture medium after dissolution, and then test it for sterility.

[0023] Preferably, in the method for performing sterility testing on an oil-based matrix product, the mass ratio of calcium salt to water in the calcium salt aqueous solution is in the range of 9-20:91-80, and the volume ratio of Tween to the calcium salt aqueous solution in the diluent is in the range of 4-15:96-85; more preferably, the volume ratio of Tween to the calcium salt aqueous solution in the diluent is in the range of 4-6:96-94.

[0024] For the sterility test of grease-based matrix products, water is taken after low-speed centrifugation and checked by membrane filtration. The inventors found that it was impossible to pass through the filter membrane. Therefore, an attempt was made to dilute the sample with isopropyl myristate, and water is taken after low-speed centrifugation and checked by membrane filtration. The filtration rate was too slow, and the method was still undesirable. The diluent was changed to Tween-80, and the same operating process could not meet the demand for filtration. The inventors, through a large number of experiments, found that the reasonable proportion of Tween and calcium salts could make the sample dissolution of the viscosity of the emulsifiable paste high and not penetrating the filter, thereby maintaining the safety of the raw material and improving the speed of filtration.

[0025] The detection object applicable to the detection method and pre-treatment diluent of the present invention is a grease-based matrix product. Preferably, the detection object is a cream that is not easily soluble in water. Preferably, the detection object is a cream containing a thickener. More preferably, the detection object is a product containing grease, an emulsifier, a thickener, and a moisturizer. In an embodiment of the present invention, the detection object is a sterile cream described in patent application 202211104373.4, which is incorporated herein by reference in its entirety. Those skilled in the art can arbitrarily select the detection object within the scope of the detection object to which the present invention is applicable, and are not bound by the specific embodiments of the present invention.

[0026] Compared with the prior art, the present invention provides a kind of oil-based matrix product, such as pre-treatment diluent and pre-treatment and detection method of cream product. The inventor prepares a diluent by rationally matching Tween and calcium salt, which is dissolved and used immediately, so that the oil and water of samples with high viscosity and difficult separation such as cream are separated, which not only maintains the integrity of the oil raw material, but also improves the speed of sample processing and the speed of filtration. The detection method of the present invention utilizes this diluent to collect the oil components in the sample more quickly, avoids the omission of the oil components and shortens the experimental time, and solves the problem of difficult dissolution of samples in traditional samples during sterility testing, and culture and inspection. After verification by a systematic method, the microorganisms in the cream can be collected more completely, which can avoid missed detection and improve the accuracy of the test results. Compared with the prior art method, the method of the present invention is simple to operate and takes less time, without loss and loss of raw materials, which not only improves the accuracy of the experiment, but also reduces the operation time and avoids the occurrence of false negative results. DETAILED DESCRIPTION

[0027] The present invention is further described below with reference to the examples. It should be understood that the examples are only used to further illustrate and explain the present invention and are not intended to limit the present invention.

[0028] Unless otherwise defined, technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in experiments or applications, the materials and methods are described herein below. In the event of conflict, the present specification, including definitions, will control. The materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting.

[0029] The test methods, detection methods, and conventional experimental reagent preparation methods used in the examples of the present invention are all in accordance with conventional operations in the art unless otherwise specified. The sample tested in the examples of the present invention is the cream described in patent application 202211104373.4.

[0030] Example 1 Determination of oil content

[0031] 1. Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 5:95.

[0032] 2. Preparation of Tween-Calcium Chloride Solution: Take Tween and calcium chloride solutions, and prepare Tween-Calcium Chloride solution in a volume ratio of 1:99, and mix well.

[0033] 3. Sample treatment: Weigh 5 g of sample into a 50 mL centrifuge tube, accurate to 0.001 g, add 45 mL of Tween-calcium chloride solution, mix well and centrifuge (4000 r / min, 20 min), and remove the upper oil layer in the centrifuge tube.

[0034] 4. Determination of Oil Content: Place the oil layer on the sample in an evaporating dish, add approximately 20g of quartz sand, and evaporate to dryness in a boiling water bath. Wipe the evaporating dish and the glass rod with the sample with ether-soaked cotton wool. Place the cotton wool in a filter paper tube. Dry in an electric forced-air oven at 100°C ± 5°C for 30 minutes. Remove, grind, and transfer the entire sample to a filter paper tube. Place the filter paper tube in the extraction tube of a Soxhlet extractor. Connect the tube to a receiving flask that has been dried to a constant weight. Add anhydrous ether to the top of the extractor condenser in a hot water bath to continue extraction. At the end of the extraction, collect one drop of the extract with a frosted glass rod. The absence of oil stains on the frosted glass rod indicates that the extraction is complete. Remove the receiving flask and recover the anhydrous ether or petroleum ether. When 1-2mL of solvent remains in the receiving flask, evaporate it to dryness in a water bath. Dry it again at 100°C ± 5°C for 1 hour. Cool it in a desiccator for 0.5 hour, and weigh it. Repeat this process until a constant weight is reached. The fat content of the sample was calculated using the relevant formula in GB5009.6-2016 “National Food Safety Standard - Determination of Fat in Foods”.

[0035] Example 2 Determination of oil content

[0036] 1. Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 8:92.

[0037] 2. Preparation of Tween-Calcium Chloride Solution: Take Tween and calcium chloride solutions, and prepare Tween-Calcium Chloride solution in a volume ratio of 3:97, and mix well.

[0038] 3. Sample treatment: Weigh 5 g of sample into a 50 mL centrifuge tube, accurate to 0.001 g, add 45 mL of Tween-calcium chloride solution, mix well and centrifuge (4000 r / min, 20 min), and remove the upper oil layer in the centrifuge tube.

[0039] 4. Determination of Oil Content: Place the oil layer on the sample in an evaporating dish, add approximately 20g of quartz sand, and evaporate to dryness in a boiling water bath. Wipe the evaporating dish and the glass rod with the sample with ether-soaked cotton wool. Place the cotton wool in a filter paper tube. Dry in an electric forced-air oven at 100°C ± 5°C for 30 minutes. Remove, grind, and transfer the entire sample to a filter paper tube. Place the filter paper tube in the extraction tube of a Soxhlet extractor. Connect the tube to a receiving flask that has been dried to a constant weight. Add anhydrous ether to the top of the extractor condenser in a hot water bath to continue extraction. At the end of the extraction, collect one drop of the extract with a frosted glass rod. The absence of oil stains on the frosted glass rod indicates that the extraction is complete. Remove the receiving flask and recover the anhydrous ether or petroleum ether. When 1-2mL of solvent remains in the receiving flask, evaporate it to dryness in a water bath. Dry it again at 100°C ± 5°C for 1 hour. Cool it in a desiccator for 0.5 hour, and weigh it. Repeat this process until a constant weight is reached. The fat content of the sample was calculated using the relevant formula in GB5009.6-2016 “National Food Safety Standard - Determination of Fat in Foods”.

[0040] Example 3 Determination of oil content

[0041] 1. Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 10:90;

[0042] 2. Preparation of Tween-Calcium Chloride Solution: Take Tween and calcium chloride solutions, and prepare Tween-Calcium Chloride solution in a volume ratio of 5:95, and mix well.

[0043] 3. Sample treatment: Weigh 5 g of sample into a 50 mL centrifuge tube, accurate to 0.001 g, add 45 mL of Tween-calcium chloride solution, mix well and centrifuge (4000 r / min, 20 min), and remove the upper oil layer in the centrifuge tube.

[0044] 4. Determination of Oil Content: Place the oil layer on the sample in an evaporating dish, add approximately 20g of quartz sand, and evaporate to dryness in a boiling water bath. Wipe the evaporating dish and the glass rod with the sample with ether-soaked cotton wool. Place the cotton wool in a filter paper tube. Dry in an electric forced-air oven at 100°C ± 5°C for 30 minutes. Remove, grind, and transfer the entire sample to a filter paper tube. Place the filter paper tube in the extraction tube of a Soxhlet extractor. Connect the tube to a receiving flask that has been dried to a constant weight. Add anhydrous ether to the top of the extractor condenser in a hot water bath to continue extraction. At the end of the extraction, collect one drop of the extract with a frosted glass rod. The absence of oil stains on the frosted glass rod indicates that the extraction is complete. Remove the receiving flask and recover the anhydrous ether or petroleum ether. When 1-2mL of solvent remains in the receiving flask, evaporate it to dryness in a water bath. Dry it again at 100°C ± 5°C for 1 hour. Cool it in a desiccator for 0.5 hour, and weigh it. Repeat this process until a constant weight is reached. The fat content of the sample was calculated using the relevant formula in GB5009.6-2016 “National Food Safety Standard - Determination of Fat in Foods”.

[0045] Example 4 Determination of oil content

[0046] 1. Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 12:88;

[0047] 2. Preparation of Tween-Calcium Chloride Solution: Take Tween and calcium chloride solutions, and prepare Tween-Calcium Chloride solution in a volume ratio of 7:93, and mix well.

[0048] 3. Sample treatment: Weigh 5 g of sample into a 50 mL centrifuge tube, accurate to 0.001 g, add 45 mL of Tween-calcium chloride solution, mix well and centrifuge (4000 r / min, 20 min), and remove the upper oil layer in the centrifuge tube.

[0049] 4. Determination of Oil Content: Place the oil layer on the sample in an evaporating dish, add approximately 20g of quartz sand, and evaporate to dryness in a boiling water bath. Wipe the evaporating dish and the glass rod with the sample with ether-soaked cotton wool. Place the cotton wool in a filter paper tube. Dry in an electric forced-air oven at 100°C ± 5°C for 30 minutes. Remove, grind, and transfer the entire sample to a filter paper tube. Place the filter paper tube in the extraction tube of a Soxhlet extractor. Connect the tube to a receiving flask that has been dried to a constant weight. Add anhydrous ether to the top of the extractor condenser in a hot water bath to continue extraction. At the end of the extraction, collect one drop of the extract with a frosted glass rod. The absence of oil stains on the frosted glass rod indicates that the extraction is complete. Remove the receiving flask and recover the anhydrous ether or petroleum ether. When 1-2mL of solvent remains in the receiving flask, evaporate it to dryness in a water bath. Dry it again at 100°C ± 5°C for 1 hour. Cool it in a desiccator for 0.5 hour, and weigh it. Repeat this process until a constant weight is reached. The fat content of the sample was calculated using the relevant formula in GB5009.6-2016 “National Food Safety Standard - Determination of Fat in Foods”.

[0050] Example 5 Determination of oil content

[0051] 1. Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 15:85.

[0052] 2. Preparation of Tween-Calcium Chloride Solution: Take Tween and calcium chloride solutions, and prepare Tween-Calcium Chloride solution in a volume ratio of 10:90, and mix well.

[0053] 3. Sample treatment: Weigh 5 g of sample into a 50 mL centrifuge tube, accurate to 0.001 g, add 45 mL of Tween-calcium chloride solution, mix well and centrifuge (4000 r / min, 20 min), and remove the upper oil layer in the centrifuge tube.

[0054] 4. Determination of Oil Content: Place the oil layer on the sample in an evaporating dish, add approximately 20g of quartz sand, and evaporate to dryness in a boiling water bath. Wipe the evaporating dish and the glass rod with the sample with ether-soaked cotton wool. Place the cotton wool in a filter paper tube. Dry in an electric forced-air oven at 100°C ± 5°C for 30 minutes. Remove, grind, and transfer the entire sample to a filter paper tube. Place the filter paper tube in the extraction tube of a Soxhlet extractor. Connect the tube to a receiving flask that has been dried to a constant weight. Add anhydrous ether to the top of the extractor condenser in a hot water bath to continue extraction. At the end of the extraction, collect one drop of the extract with a frosted glass rod. The absence of oil stains on the frosted glass rod indicates that the extraction is complete. Remove the receiving flask and recover the anhydrous ether or petroleum ether. When 1-2mL of solvent remains in the receiving flask, evaporate it to dryness in a water bath. Dry it again at 100°C ± 5°C for 1 hour. Cool it in a desiccator for 0.5 hour, and weigh it. Repeat this process until a constant weight is reached. The fat content of the sample was calculated using the relevant formula in GB5009.6-2016 “National Food Safety Standard - Determination of Fat in Foods”.

[0055] Example 6 Determination of oil content

[0056] Weigh 5g of sample to the nearest 0.001g and place it in an evaporating dish. Add approximately 20g of quartz sand and evaporate to dryness in a boiling water bath. Dry in an electric forced-air drying oven at 100°C ± 5°C for 30 minutes. Remove, grind, and transfer the entire sample to a filter paper tube. Wipe the evaporating dish and the glass rod with sample using absorbent cotton soaked in ether. Place the cotton in the filter paper tube. Place the filter paper tube in the extraction tube of a Soxhlet extractor and connect it to a receiving flask that has been dried to a constant weight. Add anhydrous ether to the top of the extractor condenser in a hot water bath to extract. At the end of the extraction, collect one drop of extract with a frosted glass rod. The absence of oily spots on the frosted glass rod indicates that the extraction is complete. Remove the receiving flask and recover the anhydrous ether or petroleum ether. When 1-2mL of solvent remains in the receiving flask, evaporate it to dryness in a water bath. Dry it again at 100°C ± 5°C for 1 hour. Cool it in a desiccator for 0.5 hours, and weigh it. Repeat this process until a constant weight is reached. The fat content of the sample was calculated using the relevant formula in GB5009.6-2016 “National Food Safety Standard - Determination of Fat in Foods”.

[0057] Example 7 Sample sterility test pretreatment

[0058] 1) Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 9:91, sterilize.

[0059] 2) Preparation of Tween-Calcium Chloride Solution: Prepare a Tween-Calcium Chloride solution by mixing sterilized Tween and the sterilized calcium chloride solution described above in a volume ratio of 1:99. Mix well and set aside.

[0060] Test sample control group

[0061] Take 20 unsterilized samples and perform the same procedure on each tube as follows:

[0062] Weigh 20 g of each sample into a sterilized 250 mL blue-cap bottle, add 180 mL of Tween-calcium chloride solution, mix well, and divide into four 50 mL sterile centrifuge tubes. Place in a 37°C water bath for 30 min, then centrifuge (4000 rpm / min, 20 min). Take out the centrifuge tubes and sterilize the surface. Take two centrifuge tubes of oil layers and spread them on tryptic soy peptone agar medium and Sabouraud glucose agar medium respectively; filter the middle layer solutions of the two centrifuge tubes respectively, and after filtration, rinse each filter membrane with 100 mL of pH 7.0 sterile sodium chloride peptone buffer solution for a total of three rinses, inoculate one filter membrane with thioglycollate fluid culture medium, and the other filter membrane with tryptic soy peptone liquid culture medium; use 4 mL of pH 7.0 sterile sodium chloride peptone buffer-peptone buffer to dissolve the bottom precipitate in each centrifuge tube. After dissolution, inoculate 4 mL of the solution in one of the centrifuge tubes into a test tube containing 50 mL of thioglycollate fluid culture medium, and inoculate 4 mL of the solution in the other centrifuge tube into a test tube containing 50 mL of tryptic soy peptone liquid culture medium.

[0063] Example 8 Sample sterility test pretreatment

[0064] 1) Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 9:91, sterilize.

[0065] 2) Preparation of Tween-Calcium Chloride Solution: Prepare a Tween-Calcium Chloride solution by mixing sterilized Tween and the sterilized calcium chloride solution described above in a volume ratio of 3:97. Mix well and set aside.

[0066] Test sample control group

[0067] Take 20 unsterilized samples and perform the same procedure on each tube as follows:

[0068] Weigh 20 g of each sample into a sterilized 250 mL blue-cap bottle, add 180 mL of Tween-calcium chloride solution, mix well, and divide into four 50 mL sterile centrifuge tubes. Place in a 37°C water bath for 30 min, then centrifuge (4000 rpm / min, 20 min). Take out the centrifuge tubes and sterilize the surface. Take two centrifuge tubes of oil layers and spread them on tryptic soy peptone agar medium and Sabouraud glucose agar medium respectively; filter the middle layer solutions of the two centrifuge tubes respectively, and after filtration, rinse each filter membrane with 100 mL of pH 7.0 sterile sodium chloride peptone buffer solution for a total of three rinses, inoculate one filter membrane with thioglycollate fluid culture medium, and the other filter membrane with tryptic soy peptone liquid culture medium; use 4 mL of pH 7.0 sterile sodium chloride peptone buffer-peptone buffer to dissolve the bottom precipitate in each centrifuge tube. After dissolution, inoculate 4 mL of the solution in one of the centrifuge tubes into a test tube containing 50 mL of thioglycollate fluid culture medium, and inoculate 4 mL of the solution in the other centrifuge tube into a test tube containing 50 mL of tryptic soy peptone liquid culture medium.

[0069] Example 9 Sample sterility test pretreatment

[0070] 1) Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 9:91, sterilize.

[0071] 2) Preparation of Tween-Calcium Chloride Solution: Prepare a Tween-Calcium Chloride solution by mixing sterilized Tween and the sterilized calcium chloride solution described above in a volume ratio of 5:95. Mix well and set aside.

[0072] Test sample control group

[0073] Take 20 unsterilized samples and perform the same procedure on each tube as follows:

[0074] Weigh 20 g of each sample into a sterilized 250 mL blue-cap bottle, add 180 mL of Tween-calcium chloride solution, mix well, and divide into four 50 mL sterile centrifuge tubes. Place in a 37°C water bath for 30 min, then centrifuge (4000 rpm / min, 20 min). Take out the centrifuge tubes and sterilize the surface. Take two centrifuge tubes of oil layers and spread them on tryptic soy peptone agar medium and Sabouraud glucose agar medium respectively; filter the middle layer solutions of the two centrifuge tubes respectively, and after filtration, rinse each filter membrane with 100 mL of pH 7.0 sterile sodium chloride peptone buffer solution for a total of three rinses, inoculate one filter membrane with thioglycollate fluid culture medium, and the other filter membrane with tryptic soy peptone liquid culture medium; use 4 mL of pH 7.0 sterile sodium chloride peptone buffer-peptone buffer to dissolve the bottom precipitate in each centrifuge tube. After dissolution, inoculate 4 mL of the solution in one of the centrifuge tubes into a test tube containing 50 mL of thioglycollate fluid culture medium, and inoculate 4 mL of the solution in the other centrifuge tube into a test tube containing 50 mL of tryptic soy peptone liquid culture medium.

[0075] Example 10 Sample sterility test pretreatment

[0076] 1) Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 9:91, sterilize.

[0077] 2) Preparation of Tween-Calcium Chloride Solution: Prepare a Tween-Calcium Chloride solution by mixing sterilized Tween and the sterilized calcium chloride solution described above in a volume ratio of 7:93. Mix well and set aside.

[0078] Test sample control group

[0079] Take 20 unsterilized samples and perform the same procedure on each tube as follows:

[0080] Weigh 20 g of each sample into a sterilized 250 mL blue-cap bottle, add 180 mL of Tween-calcium chloride solution, mix well, and divide into four 50 mL sterile centrifuge tubes. Place in a 37°C water bath for 30 min, then centrifuge (4000 rpm / min, 20 min). Take out the centrifuge tubes and sterilize the surface. Take two centrifuge tubes of oil layers and spread them on tryptic soy peptone agar medium and Sabouraud glucose agar medium respectively; filter the middle layer solutions of the two centrifuge tubes respectively, and after filtration, rinse each filter membrane with 100 mL of pH 7.0 sterile sodium chloride peptone buffer solution for a total of three rinses, inoculate one filter membrane with thioglycollate fluid culture medium, and the other filter membrane with tryptic soy peptone liquid culture medium; use 4 mL of pH 7.0 sterile sodium chloride peptone buffer-peptone buffer to dissolve the bottom precipitate in each centrifuge tube. After dissolution, inoculate 4 mL of the solution in one of the centrifuge tubes into a test tube containing 50 mL of thioglycollate fluid culture medium, and inoculate 4 mL of the solution in the other centrifuge tube into a test tube containing 50 mL of tryptic soy peptone liquid culture medium.

[0081] Example 11 Sample sterility test pretreatment

[0082] 1) Preparation of calcium chloride solution: Dissolve calcium chloride and purified water in a mass ratio of 9:91, sterilize.

[0083] 2) Preparation of Tween-Calcium Chloride Solution: Prepare a Tween-Calcium Chloride solution by mixing sterilized Tween and the sterilized calcium chloride solution described above in a volume ratio of 1:9. Mix well and set aside.

[0084] Test sample control group

[0085] Take 20 unsterilized samples and perform the same procedure on each tube as follows:

[0086] Weigh 20 g of each sample into a sterilized 250 mL blue-cap bottle, add 180 mL of Tween-calcium chloride solution, mix well, and divide into four 50 mL sterile centrifuge tubes. Place in a 37°C water bath for 30 min, then centrifuge (4000 rpm / min, 20 min). Take out the centrifuge tubes and sterilize the surface. Take two centrifuge tubes of oil layers and spread them on tryptic soy peptone agar medium and Sabouraud glucose agar medium respectively; filter the middle layer solutions of the two centrifuge tubes respectively, and after filtration, rinse each filter membrane with 100 mL of pH 7.0 sterile sodium chloride peptone buffer solution for a total of three rinses, inoculate one filter membrane with thioglycollate fluid culture medium, and the other filter membrane with tryptic soy peptone liquid culture medium; use 4 mL of pH 7.0 sterile sodium chloride peptone buffer-peptone buffer to dissolve the bottom precipitate in each centrifuge tube. After dissolution, inoculate 4 mL of the solution in one of the centrifuge tubes into a test tube containing 50 mL of thioglycollate fluid culture medium, and inoculate 4 mL of the solution in the other centrifuge tube into a test tube containing 50 mL of tryptic soy peptone liquid culture medium.

[0087] Experimental Example 1 Oil content determination results

[0088] The sample processing time, extraction completion time, and measured oil content of Examples 1-6 were statistically analyzed. The results are shown in Table 1 below.

[0089] Table 1

[0090]

[0091] As can be seen from Table 1, the sample processing time and extraction completion time for Examples 1-5 were significantly reduced compared to Example 6, which used the national standard Soxhlet extraction method. Furthermore, the oil content detection results were not significantly different from those of the national standard Soxhlet extraction method. The inventors unexpectedly discovered that the sample processing time and extraction completion time for Examples 3-5 were further reduced, and the oil content detection results were closer to those of the national standard method. Therefore, Examples 3-5 are considered preferred embodiments of the present invention.

[0092] Experimental Example 2 Sample Sterility Test Pretreatment Experiment Results

[0093] The filtration speed and time in Examples 7-11 were statistically analyzed and shown in Table 2 below.

[0094] Table 2

[0095]

[0096] In this experiment, the products of Examples 7 and 8 could not emulsify and stratify the samples well and could not be filtered; the product of Example 9 had the best separation effect and a faster filtration speed; the products of Examples 10 and 11 could also pass through the filter, but the filtration speed was slower than that of Example 9.

[0097] It should be understood that the present invention disclosed is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the term used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0098] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A method for detecting the oil content of an oil-based matrix product, characterized in that: Before testing, the oil-based matrix product is treated with a pre-treatment diluent for testing oil-based matrix products; The pretreatment diluent for detecting oil-based matrix products comprises a calcium salt aqueous solution and Tween, wherein the mass ratio of calcium salt to water in the calcium salt aqueous solution is in the range of 1-30:99-70, and the volume ratio of Tween to the calcium salt aqueous solution in the diluent is in the range of 1-30:99-70; The detection method comprises the following steps: (1) Sample processing: add the sample to be tested into the pre-treatment diluent for detecting oil-based matrix products, mix well and centrifuge, and remove the oil layer in the centrifuge tube; (2) Determination of oil content: Place the oil layer into the extraction cylinder of the Soxhlet extractor, connect it to the receiving bottle, and extract; evaporate the extract to dryness and weigh it; The oil-based matrix product is an ointment.

2. The method for detecting the grease content of a grease-based matrix product according to claim 1, wherein: The mass ratio of calcium salt to water in the calcium salt aqueous solution is in the range of 9-20:91-80, and the volume ratio of Tween to the calcium salt aqueous solution in the diluent is in the range of 4-15:96-85.

3. The method for detecting the grease content of a grease-based matrix product according to claim 1 or 2, wherein: The Tween is selected from one or more of Tween-80, Tween-60, Tween-40 and Tween-20.

4. The method for detecting the grease content of a grease-based matrix product according to claim 1 or 2, wherein: The calcium salt is selected from one or more of calcium chloride, calcium bromide, calcium iodide, calcium nitrate and calcium sulfate.

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