Method for testing inhibition rate of oil-soluble lipid digestion inhibitor and application

Abstract

The invention provides a method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor and application, the method comprises the steps of solution preparation, enzyme addition incubation, cooling extraction and centrifugal determination, and belongs to the technical field of food detection. According to the method, the oil-PNPB-inhibitor and the enzyme are used as raw materials, the catalytic process on an oil-water interface is simulated, the effect of inhibiting lipid digestion is exerted, the inhibition rate of the oil-soluble lipid digestion inhibitor can be accurately determined, and the method has the characteristics of high sensitivity, small error, rapid determination, small solvent consumption, small interference of external environmental factors and the like, and is suitable for industrial production. Theoretical reference is provided for research of a determination method of the lipid digestion inhibitor, efficient determination of the inhibition effect of the oil-soluble lipid digestion inhibitor is facilitated, and a foundation can be laid for improvement of a traditional determination method.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and in particular relates to a method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor and an application thereof. Background Art

[0002] Obesity is a chronic metabolic disease caused by excessive calorie intake and fat metabolism disorders. Due to the modern lifestyle and the increase in the consumption of foods rich in fat and carbohydrates, the prevalence of diabetes and obesity has risen sharply, and up to 90% of type 2 diabetes patients in the world are overweight or obese. At present, obesity has replaced malnutrition and become the most serious threat to public health and survival in the 21st century. Reducing obesity is a way to slow down the incidence of type 2 diabetes. Therefore, it can inhibit the catalytic decomposition of triglycerides by lipase, regulate the production of triglycerides, and prevent the absorption of fat to achieve the effect of lowering lipids. Based on the above principle, artificial enzyme inhibitors such as orlistat were developed to achieve the purpose of lipid-lowering. However, artificial enzyme inhibitors such as orlistat have many side effects, such as allergies, abdominal pain, indigestion, vomiting, etc. Compared with artificial synthetic drugs, natural enzyme inhibitors have less adverse effects on the body. The effective ingredients of flavor molecules are widely available, safe, low-toxic, and environmentally friendly. At the same time, studies have reported that flavor molecules have lipid-lowering effects, but oil-soluble compounds such as flavor molecules are difficult to dissolve in water, so the determination of their inhibition rate has limitations. At present, the commonly used test methods for determining the inhibition rate of lipid digestion inhibitors are the copper soap method, automatic titration method and PNPB method. However, the automatic titration method has problems such as large solvent consumption and long measurement time; the copper soap method is greatly affected by external environmental factors. When the indoor temperature is different, after the addition of the ketone reagent, the reaction rate of copper ions and free fatty acids in the organic layer will change with the change of temperature, resulting in different amounts of products generated within the same measurement time, and ultimately leading to different measurement results under the same conditions. In addition, pyridine reagent is required for the preparation of ketone reagents. Pyridine is a low-toxic substance and should be avoided as much as possible; the PNPB method is only suitable for water-soluble solvents, the oil phase does not participate in the reaction and has a narrow range of application. Therefore, it is urgent to develop a stable method to evaluate the inhibitory effect of inhibitors. Summary of the invention

[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method and application for testing the inhibition rate of an oil-soluble lipid digestion inhibitor, the steps of which include solution preparation, enzyme incubation, cooling extraction, and centrifugal determination, and belong to the field of food detection technology. The present invention uses oil-PNPB-inhibitors and enzymes as raw materials, simulates the catalytic process at the oil-water interface, exerts the effect of inhibiting lipid digestion, and can accurately determine the inhibition rate of oil-soluble lipid digestion inhibitors. It has the characteristics of high sensitivity, small error, rapid determination, small solvent consumption, and little interference from external environmental factors. It not only provides a theoretical reference for the study of the determination method of lipid digestion inhibitors, but also helps to efficiently determine the inhibitory effect of oil-soluble lipid digestion inhibitors, and can also lay the foundation for the improvement of traditional determination methods.

[0004] Technical solution: A method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor, comprising the following steps: S1. The emulsion, bile salt and oil-PNPB-inhibitor solution were mixed uniformly and homogenized for 3-10 min to obtain a mixed solution; S2. Add Tris-HCl buffer and enzyme solution to the mixed solution, incubate at 37°C and 200 rpm for 35 min to prepare a test solution; S3. The test solution was heated at a constant temperature of 70-80°C for 15-20 min, and then placed in an ice bath; S4. Add the extractant and vortex for 2-10 min, then sonicate for 2-10 min, centrifuge at 8000-8500 rpm for 3-10 min, and obtain the supernatant; S5. The ultraviolet absorbance of the supernatant was measured at 307 nm; The oil-PNPB-inhibitor solution is a mixed solution of inhibitor, p-nitrophenylbutyrate and oil. Furthermore, the inhibitor is a small molecule compound that is poorly soluble in water. Furthermore, the poorly water-soluble small molecule compounds include but are not limited to fragrance molecules. Furthermore, the fragrance molecules include but are not limited to eugenol, carvone, and caryophyllene. Furthermore, the oil includes soybean oil, peanut oil, corn oil and rapeseed oil. Furthermore, the concentration of the inhibitor in the mixture of p-nitrophenylbutyrate and oil is 2-4 mg / mL, and the concentration of p-nitrophenylbutyrate in the oil is 5-20 mM. Furthermore, the ice bath time in step S3 is 10-20 min. Furthermore, the extractant in step S4 includes but is not limited to dichloromethane, ethyl acetate, toluene, and n-octanol. Application of any of the above test methods in determining the inhibition rate of oil-soluble lipid digestion inhibitors. Beneficial effects: 1. The test method provided by the present invention combines and improves the copper soap method and the PNPB method. The ice bath treatment significantly improves the defect that the extractant is easily volatilized by heat and causes errors in the copper soap method, and eliminates the influence of the external temperature on the measurement process, thereby reducing errors and making the experimental results more accurate. In addition, the step of reacting with copper ions is omitted in the present invention, thereby reducing the errors caused by this step. In addition, pyridine reagent is required when preparing ketone reagent. Pyridine is a low-toxic substance. The present invention omits the step of preparing ketone reagent, which saves time and cost while being relatively safe and environmentally friendly. 2. The test method provided by the present invention greatly reduces the overall solvent consumption of the automatic titration method, shortens the reaction time, and can carry out multiple groups of experiments at the same time, so that the determination efficiency is significantly improved; in addition, the application scope of the PNPB method is expanded, and the limitation of being only used for water-soluble substances is solved. The addition of oil can better simulate the in vitro digestion process of lipids, which is convenient for the inhibition rate test of oil-soluble lipid digestion inhibitors, and has stronger practicality and a wider range of applications. 3. The method provided by the present invention utilizes PNPB to be hydrolyzed by lipase to generate a color-developing substance PNP, and measures the absorbance at the optimal wavelength of 307nm to reflect the inhibitory effect of oil-soluble lipid digestion inhibitors. It has the characteristics of high sensitivity, high accuracy, high precision and rapid measurement, which helps to lay a good data foundation for the detection of lipid digestion inhibitors and promote the further development of research on lipid digestion inhibitor detection methods. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the standard curve of PNP concentration and absorbance at 307 nm in Example 1; Figure 2 The absorbance results of PNPB-soybean oil solutions of different concentrations in Example 1; Figure 3 is the PNP release amount of PNPB-soybean oil solution of different concentrations in Example 1; Figure 4 The stability of the PNPB enzymatic hydrolysis product PNP in Example 3; Figure 5 The PNP and FFA release amounts of the 10 mM PNPB-soybean oil solution in Example 4 and Example 5; Figure 6 It is the inhibitor inhibition rate of Example 6, Example 7 and Example 8. Figure 7 This is the inhibition rate of the inhibitor in different oils of Example 7. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Example 1 Concentration optimization test of PNPB-oil phase solution in emulsion system S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of oil phase solution: Weigh 0.22 mL of p-nitrophenyl butyrate (PNPB) and dissolve it in 50 mL of soybean oil. Prepare 5, 10, 15, 20, and 25 mM PNPB-soybean oil solutions by proportional dilution method and set aside; S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.1 mL of the oil phase solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Add 0.5 mL of Tris-HCl buffer and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of ethyl acetate and vortex for 3 min, then sonicate in a cell crusher for 2 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. The test process of Example 1 was repeated 3 times. Figure 1 and Figure 2 It can be seen that the concentration of PNP-ethyl acetate solution is 0.1-0.5mM as the horizontal axis and the average value of absorbance is the vertical axis, and the standard curve y=1.8406x+0.046(R 2=0.9913), the absorbance values ​​of 5mM PNPB-soybean oil solution in Example 1 were 0.319, 0.327, and 0.332, respectively; the absorbance values ​​of 10mM PNPB-soybean oil solution were 0.540, 0.547, and 0.551, respectively; the absorbance values ​​of 15mM PNPB-soybean oil solution were 0.655, 0.666, and 0.672, respectively; the absorbance values ​​of 20mM PNPB-soybean oil solution were 0.877, 0.895, and 0.921, respectively; and the absorbance values ​​of 25mM PNPB-soybean oil solution were 1.122, 1.125, and 1.141, respectively. Depend on Figure 1 and Figure 3 It can be seen that according to the standard equation, the amount of PNP released from 5mM PNPB-soybean oil solution is 0.148mM, 0.153mM, and 0.155mM, respectively; the amount of PNP released from 10mM PNPB-soybean oil solution is 0.268mM, 0.272mM, and 0.274mM, respectively; the amount of PNP released from 15mM PNPB-soybean oil solution is 0.331mM, 0.337mM, and 0.340mM, respectively; the amount of PNP released from 20mM PNPB-soybean oil solution is 0.451mM, 0.461mM, and 0.475mM, respectively; the amount of PNP released from 25mM PNPB-soybean oil solution is 0.585mM, 0.586mM, and 0.595mM, respectively. The results showed that the consumption of PNPB in the 10mM PNPB-soybean oil solution was small and the absorbance values ​​of the blank group and the inhibitor group were within the absorbance range determined by the standard curve. Therefore, the 10mM PNPB-soybean oil solution was selected for application. Example 2 Stability test of product PNP S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of oil phase solution: 0.088 mL of p-nitrophenyl butyrate (PNPB) was dissolved in 50 mL of soybean oil; S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.1 mL of the oil phase solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Add 0.5 mL of Tris-HCl buffer and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of ethyl acetate and vortex for 3 min, then sonicate in a cell crusher for 2 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. The test process of Example 2 was repeated for 5 days, 3 times per day. Figure 4 It can be seen that the release amount of PNP measured in Example 2 does not change significantly with the extension of the measurement time and tends to be stable. The release amounts on the first day are: 0.268mM, 0.283mM, 0.288mM, the release amounts on the second day are: 0.268mM, 0.275mM, 0.278mM, the release amounts on the third day are: 0.274mM, 0.277mM, 0.277mM, the release amounts on the fourth day are: 0.268mM, 0.272mM, 0.274mM, the release amounts on the fifth day are: 0.278mM, 0.284mM, 0.285mM, the release amounts for 5 days tend to be stable, and the standard deviation between groups is within ±5%, indicating that the structure of the PNPB enzymatic hydrolysis product PNP is stable and not easily affected by the external environment, and can be used as a colorimetric substance for determining the release amount of PNP. Example 3 Product PNP sample recovery experiment S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of oil phase solution: 0.088 mL of p-nitrophenyl butyrate (PNPB) was dissolved in 50 mL of soybean oil; (6) Preparation of PNP-Tris-HCl buffer: weigh 0.0168 g PNP and dissolve it in 40 mL Tris-HCl buffer (base). (7) Preparation of 0.224 mM PNP-Tris-HCl buffer: weigh 2.24 mL base, add 7.76 mL Tris-HCl buffer and dissolve evenly. (8) Preparation of 0.280 mM PNP-Tris-HCl buffer: weigh 2.80 mL of the standard, add 7.20 mL of Tris-HCl buffer, and dissolve evenly; (9) Preparation of 0.336 mM PNP-Tris-HCl buffer: weigh 3.36 mL of the standard, add 6.64 mL of Tris-HCl buffer, and dissolve evenly; S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.1 mL of the oil phase solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Add 0.5 mL of PNP-Tris-HCl buffer of different concentrations and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of ethyl acetate and vortex for 3 min, then sonicate in a cell crusher for 2 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. Table 1 Results of the sample recovery test of PNP in Example 2 As can be seen from Table 1, the average recovery rate of PNP in Example 2 is 109.939%, and the recovery rates of the PNP-Tris-HCl buffer group with a concentration of 0.224mM are 110.714%, 111.111%, and 114.286%, the recovery rates of the PNP-Tris-HCl buffer group with a concentration of 0.280mM are 110.714%, 105.000%, and 110.357%, and the recovery rates of the PNP-Tris-HCl buffer group with a concentration of 0.336mM are 111.688%, 106.169%, and 109.416%. The standard deviation of the recovery rates of the three groups is 2.819%, which is within ±5%, indicating that the present invention uses PNP as a color developing substance measured by an ultraviolet spectrophotometer to reflect the inhibitory effect of the lipid digestion inhibitor, which is practical and reliable. Example 4 Comparative experiment of product PNP and product FFA (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of oil phase solution: p-nitrophenyl butyrate (PNPB) was dissolved in soybean oil to prepare a 10 mM oil phase solution; S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.05, 0.10, 0.15, 0.20, and 0.25 mL of the oil phase solution were added respectively and homogenized at 10,000 rpm for 3 min to prepare an emulsion; S3. Add 0.5 mL of Tris-HCl buffer and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of ethyl acetate and vortex for 3 min, then sonicate in a cell crusher for 2 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. Depend on Figure 5 It can be seen that according to the standard equation, in the PNPB-soybean oil mixed solution, the amount of PNP released by 0.2% PNPB in the system is 0.0790mmol, 0.0865mmol, and 0.0870mmol, respectively, the amount of PNP released by 0.4% PNPB is 0.1340mmol, 0.1360mmol, and 0.1370mmol, respectively, the amount of PNP released by 0.6% PNPB is 0.1710mmol, 0.1715mmol, and 0.1625mmol, respectively, the amount of PNP released by 0.8% PNPB is 0.2240mmol, 0.2230mmol, and 0.2245mmol, respectively, and the amount of PNP released by 1.0% PNPB is 0.2745mmol, 0.2780mmol, and 0.2780mmol, respectively. The results show that the consumption of PNPB in the PNPB-soybean oil mixed solution is proportional to the amount of PNP generated. In addition, proportionally increasing the amount of PNPB-oil system can provide more substrates to keep the reaction going. And at a soybean oil-PNPB concentration of 10 mM, the amount of bile salt and emulsifier is sufficient to maintain the emulsification ability, form a stable emulsion interface, and ensure the continuous action of lipase. Example 5 Comparative experiment of product PNP and product FFA S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of 0.1 M NaOH solution: weigh 1 g of NaOH and dissolve it in deionized water to make up to 250 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, and then centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of oil phase solution: Pipette 0.088 mL of p-nitrophenyl butyrate (PNPB) and dissolve it in 50 mL of soybean oil for later use; S2. 0.3 g of sodium caseinate and 8.5 mL of Tris-HCl (pH = 7.00) buffer were mixed and then 0.5, 1.0, 1.5, 2.0, and 2.5 mL of the oil phase solution were added, respectively, and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Take 41mL of buffer solution into a beaker, add 2mL of emulsion and 2mL of bile salt, adjust the pH to 7.00 with NaOH solution, heat in a water bath until the temperature rises to 37℃, add 1mL of enzyme solution, start titration, and record the results after 35 minutes of reaction. Table 2 FFA and PNP release in Example 4 and Example 5 From Table 2, Figure 5 It can be seen that according to the NaOH consumption, in the PNPB-soybean oil mixed solution, the amount of FFA released by 0.2% soybean oil in the system is 0.049mmol, 0.081mmol, and 0.057mmol, respectively, the amount of FFA released by 0.4% soybean oil is 0.104mmol, 0.105mmol, and 0.117mmol, respectively, the amount of FFA released by 0.6% soybean oil is 0.151mmol, 0.140mmol, and 0.150mmol, respectively, the amount of FFA released by 0.8% soybean oil is 0.205mmol, 0.200mmol, and 0.203mmol, respectively, and the amount of FFA released by 1.0% soybean oil is 0.243mmol, 0.237mmol, and 0.226mmol, respectively. The results show that in this system, the amount of both the product PNP and the product FFA continues to increase, and the substrate soybean oil and PNPB will not have competitive inhibition, resulting in the amount of one of the substrates decreasing as the system expands. In addition, the fitting curve results show that the soybean oil consumption in the PNPB-soybean oil mixed solution is proportional to the FFA production, which is similar to the slope of the PNPB consumption and PNP production in the PNPB-soybean oil mixed solution, indicating that the two reactants PNPB and soybean oil react with lipase simultaneously and generate similar amounts of PNP and FFA respectively. The amount of FFA produced can be replaced by the amount of PNP produced, and the method is authentic and reliable. Example 6 A method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor comprises the following steps: S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of blank oil phase solution: 0.088 mL of p-nitrophenyl butyrate (PNPB) was dissolved in 50 mL of soybean oil and set aside; (6) Preparation of oil-PNPB-inhibitor solution: Weigh 0.016 g of eugenol and dissolve it in 4 mL of the blank oil phase solution to prepare a 4 mg / mL oil-PNPB-inhibitor solution for later use; S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.1 mL of the oil-PNPB-inhibitor solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Add 0.5 mL of Tris-HCl buffer and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of ethyl acetate and vortex for 3 min, then sonicate in a cell crusher for 2 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. Example 7 A method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor comprises the following steps: S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of blank oil phase solution: 0.088 mL of p-nitrophenyl butyrate (PNPB) was dissolved in 50 mL of soybean oil and set aside; (6) Preparation of oil-PNPB-inhibitor solution: Weigh 0.016 g of carvone and dissolve it in 4 mL of blank oil phase solution to prepare a 4 mg / mL oil-PNPB-inhibitor solution for later use; S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.1 mL of the oil-PNPB-inhibitor solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Add 0.5 mL of Tris-HCl buffer and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of ethyl acetate and vortex for 3 min, then sonicate in a cell crusher for 2 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. Example 8 A method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor comprises the following steps: S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of blank oil phase solution: 0.088 mL of p-nitrophenyl butyrate (PNPB) was dissolved in 50 mL of soybean oil and set aside; (6) Preparation of oil-PNPB-inhibitor solution: Weigh 0.016 g of caryophyllene and dissolve it in 4 mL of the blank oil phase solution to prepare a 4 mg / mL oil-PNPB-inhibitor solution for later use; S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.1 mL of the oil-PNPB-inhibitor solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Add 0.5 mL of Tris-HCl buffer and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of ethyl acetate and vortex for 3 min, then sonicate in a cell crusher for 2 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. Performance Testing (1) Inhibitor inhibition rate The inhibitor inhibition rates of Examples 6, 7 and 8 were calculated based on the measurement results of the blank oil phase group. The preparation processes of Examples 6, 7 and 8 were repeated 3 times. Depend on Figure 6 It can be seen that Example 6 is an inhibitor eugenol group, and its inhibition rates are 18.76%, 17.17%, and 16.99% respectively; Example 7 is an inhibitor carvone group, and its inhibition rates are 10.97%, 9.73%, and 8.85% respectively; Example 8 is an inhibitor caryophyllene group, and its inhibition rates are 23.89%, 22.12%, and 21.95%. The inhibition rates of different inhibitors are significantly different, and the method is universal. Example 9 The inhibition rate test method of different oil-soluble lipid digestion inhibitors comprises the following steps: S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of blank group oil phase solution: 0.088 mL of p-nitrophenyl butyrate (PNPB) was taken and dissolved in 50 mL of corn oil, peanut oil, linseed oil, sunflower oil, rapeseed oil, rice oil, and soybean oil respectively for later use; (6) Preparation of oil-PNPB-inhibitor solution: Weigh 0.016 g of eugenol and dissolve it in 4 mL of the blank oil phase solution to prepare a 4 mg / mL oil-PNPB-inhibitor solution for later use; S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.1 mL of the oil-PNPB-inhibitor solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Add 0.5 mL of Tris-HCl buffer and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of ethyl acetate and vortex for 3 min, then sonicate in a cell crusher for 2 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. Performance Testing (1) Inhibitor inhibition rate in different oils The inhibitor inhibition rates of different oils in Example 9 were calculated based on the measurement results of the blank oil phase group, and the preparation process of Example 9 was repeated 3 times. Depend on Figure 7It can be seen that Example 9 is a corn oil group, and its inhibition rates are 13.00%, 15.93%, and 16.32% respectively; Example 9 is a peanut oil group, and its inhibition rates are 21.81%, 15.99%, and 20.43% respectively; Example 9 is a linseed oil group, and its inhibition rates are 15.74%, 16.59%, and 16.93% respectively; Example 9 is a sunflower oil group, and its inhibition rates are 13.44%, 17.04%, and 16.01% respectively; Example 9 is a rapeseed oil group, and its inhibition rates are 16.37%, 18.08%, and 16.80% respectively; Example 9 is a rice oil group, and its inhibition rates are 16.41%, 16.52%, and 15.54% respectively; Example 9 is a soybean oil group, and its inhibition rates are 18.76%, 17.17%, and 16.99% respectively. The inhibition rates of different oil inhibitors were significantly different, and the method was universal. Comparative Example 1 The difference between this comparative example and Example 6 is that the copper soap method is used for determination. A method for determining the inhibition rate of an oil-soluble lipid digestion inhibitor using a copper soap method comprises the following steps: S1. Solution preparation (1) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (2) Preparation of emulsifier solution: Weigh 1.2 g sodium caseinate and dissolve it in 50 mL Tris-HCl (pH = 7.00) buffer, and dilute to 100 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of blank oil phase solution: 0.088 mL of p-nitrophenyl butyrate (PNPB) was dissolved in 50 mL of soybean oil and set aside; (6) Preparation of oil-PNPB-inhibitor solution: Weigh 0.016 g of eugenol and dissolve it in 4 mL of the blank oil phase solution to prepare a 4 mg / mL oil-PNPB-inhibitor solution for later use; (7) Preparation of ketone reagent solution: Weigh 2.5 g of copper acetate and dissolve it in 50 mL of deionized water. Stir and dissolve at 60°C. Adjust the pH to 6.10 with pyridine. S2. 10 mL of the emulsifier solution and 0.125 mL of the bile salt solution were mixed and then 0.1 mL of the oil-PNPB-inhibitor solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Add 0.5 mL of Tris-HCl buffer and 1 mL of enzyme solution to 2 mL of the emulsion, and incubate at 37°C and 200 rpm in a shaker for 35 min to prepare a test solution; S4. The test solution was heated at 80°C for 15 min and then cooled in an ice bath for 10 min. S5. Add 1.5 mL of isooctane and vortex for 3 min, take 1 mL of the supernatant and add 1 mL of ketone reagent solution and vortex for 10 min, centrifuge at 8500 rpm for 3 min, and obtain the supernatant; S6. The ultraviolet absorbance of the supernatant was measured at 307 nm. Comparative Example 2 The difference between this comparative example and Example 6 is that the automatic titration method is used for determination. A method for determining the inhibition rate of an oil-soluble lipid digestion inhibitor using an automatic titration method comprises the following steps: S1. Solution preparation (3) Preparation of Tris-HCl buffer: weigh 6 g Tris, 1.46 g sodium chloride, and 1.108 g calcium chloride, dissolve in deionized water, make up to 1000 mL, and adjust the pH to 7.00 with hydrochloric acid; (4) Preparation of 0.1 M NaOH solution: weigh 1 g of NaOH and dissolve it in deionized water to make up to 250 mL for later use; (3) Preparation of enzyme solution: Weigh 0.4 g of porcine pancreatic lipase, dissolve in 40 mL of Tris-HCl (pH = 7.00) buffer, homogenize at 10000 rpm for 3 min, and then centrifuge at 7000 rpm for 10 min, and take the supernatant for later use; (4) Preparation of bile salt solution: Weigh 0.34 g of porcine bile salt and dissolve it in 10 mL of Tris-HCl (pH = 7.00) buffer for later use; (5) Preparation of blank oil phase solution: 0.088 mL of p-nitrophenyl butyrate (PNPB) was dissolved in 50 mL of soybean oil and set aside; (6) Preparation of oil-PNPB-inhibitor solution: Weigh 0.016 g of eugenol and dissolve it in 4 mL of the blank oil phase solution to prepare a 4 mg / mL oil-PNPB-inhibitor solution for later use; S2. 0.5 g sodium caseinate and 8.5 mL Tris-HCl (pH = 7.00) buffer were mixed and then 1 mL of the oil phase solution was added and homogenized at 10000 rpm for 3 min to obtain an emulsion; S3. Take 36mL of buffer solution into a beaker, add 2mL of emulsion and 2mL of bile salt, adjust the pH to 7.00 with NaOH solution, heat in a water bath until the temperature rises to 37℃, add 1mL of enzyme solution, start titration, and record the results after 35 minutes of reaction. Performance Testing (1) Inhibitor inhibition rate The inhibitor inhibition rates of Example 6, Comparative Example 1 and Comparative Example 2 were calculated based on the measurement results of the blank oil phase group. The preparation processes of Example 6, Comparative Example 1 and Comparative Example 2 were repeated 3 times. Table 3 Inhibitor inhibition rate of Example 6, Comparative Example 1 and Comparative Example 2 As shown in Table 2, Comparative Example 1 uses the copper soap method to determine the lipid inhibition effect of eugenol. The inhibition rates of eugenol are 16.47%, 13.79%, and 17.01%, respectively. The average inhibition rate is 15.76%, and the standard deviation is 1.72%, indicating that the determination results of Comparative Example 1 deviate from the actual values, and the copper soap method has a greater impact on the experimental results under a high room temperature environment. The determination method provided by the present invention further improves the technical solution, eliminates the influence of the external environment on the experiment, and omits the steps related to the error, reduces the error, and the result is more accurate and the error is smaller. In addition, the present invention avoids the use of toxic reagent pyridine, which is green and environmentally friendly. In Comparative Example 2, the lipid inhibition effect of eugenol was determined by automatic titration. The inhibition rates of eugenol were 23.95%, 14.92%, and 16.78%, respectively. The average inhibition rate was 18.55%, and the standard deviation was 4.77%, which was significantly higher than that of Example 6 and Comparative Example 1, indicating that the experimental results determined by the automatic titration method had a large error. The automatic titration method of Comparative Example 2 consumed a large amount of solvent during the determination process and the experimental time was long. The determination method provided by the present invention further improved the technical solution, greatly reduced the solvent consumption, and the results were more accurate and the error was smaller. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor, characterized in that: The following steps are involved: S1. The emulsion, bile salt and oil-PNPB-inhibitor solution were mixed uniformly and homogenized for 3-10 min to obtain a mixed solution; S2. Add Tris-HCl buffer and enzyme solution to the mixed solution, incubate at 37°C and 200 rpm for 35 min to prepare a test solution; S3. The test solution was heated at a constant temperature of 70-80°C for 15-20 min, and then placed in an ice bath; S4. Add the extractant and vortex for 2-10 min, then sonicate for 2-10 min, centrifuge at 8000-8500 rpm for 3-10 min, and obtain the supernatant; S5. The ultraviolet absorbance of the supernatant was measured at 307 nm; The oil-PNPB-inhibitor solution is a mixed solution of inhibitor, p-nitrophenylbutyrate and oil.

2. The method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor according to claim 1, characterized in that: The inhibitor is a small molecule compound that is poorly soluble in water.

3. The method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor according to claim 2, characterized in that: The poorly water-soluble small molecule compounds include but are not limited to fragrance molecules.

4. The method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor according to claim 3, characterized in that: The fragrance molecules include but are not limited to eugenol, carvone, and caryophyllene.

5. The method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor according to claim 1, characterized in that: The oils include soybean oil, peanut oil, corn oil, and rapeseed oil.

6. The method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor according to claim 1, characterized in that: The concentration of the inhibitor in the mixture of p-nitrophenylbutyrate and oil is 2-4 mg / mL, and the concentration of p-nitrophenylbutyrate in the oil is 5-20 mM.

7. The method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor according to claim 1, characterized in that: The ice bath time in step S3 is 10-20 min.

8. The method for testing the inhibition rate of an oil-soluble lipid digestion inhibitor according to claim 1, characterized in that: The extractant in step S4 includes but is not limited to dichloromethane, ethyl acetate, toluene, and n-octanol.

9. Use of the test method according to any one of claims 1 to 8 in determining the inhibition rate of an oil-soluble lipid digestion inhibitor.

Images