Method for detecting anti-aging new food raw materials in food

Abstract

The invention discloses a method for detecting anti-aging new food raw materials in foods, the foods comprise milk and dairy products, beverages, wines, cocoa products, chocolates and chocolate products (including cocoa butter substitute chocolates and chocolate products), candies, frozen drinks and the like, and the anti-aging new food raw materials are sodium hyaluronate. The method comprises the following steps: (1) preparing a standard stock solution and a standard working solution; (2) preparing a to-be-detected sample solution; (3) setting high performance liquid chromatography analysis conditions; (4) establishing a standard working curve; and (5) analyzing a result. The invention establishes a high performance liquid chromatography determination method for anti-aging new food raw materials in food. According to the method, the anti-aging new food raw material sodium hyaluronate in the food is determined by innovatively adopting a high-pressure acid hydrolysis-precolumn derivatization method, so that the detection sensitivity is improved, the detection cost is reduced, the operation is simple and easy to implement, and a brand new technical thought is provided for analysis and detection of sodium hyaluronate in the food. The method is simple to operate, high in sensitivity, good in reproducibility and stability, low in cost and beneficial to batch detection of related products.

Description

Technical Field

[0001] The invention relates to a high-pressure acid hydrolysis-pre-column derivatization-high performance liquid chromatography detection method of sodium hyaluronate, a new anti-aging food raw material in food. Background Art

[0002] Hyaluronic acid (HA), also known as hyaluronic acid, is a high molecular weight straight-chain viscous polysaccharide produced by polymerization reaction of N-acetylglucosamine and glucuronic acid through β-1,3 glycosidic bonds. At present, industrially produced hyaluronic acid often exists in the form of its sodium salt, namely sodium hyaluronate. In China, the public's awareness of sodium hyaluronate mainly stays in medical beauty and cosmetics, but sodium hyaluronate foods are common abroad. At present, there are more than 2,000 foods containing sodium hyaluronate on sale worldwide, and dietary supplements containing sodium hyaluronate have long been widely popular in the international market. Oral sodium hyaluronate can beautify the skin, promote wound healing and repair, anti-oxidation, relieve osteoarthritis, etc.

[0003] In 2008, my country approved sodium hyaluronate as a new resource food. As a raw material for health food, it proved the safety of edible sodium hyaluronate. In January 2021, the National Health Commission approved sodium hyaluronate as a new food raw material for use in ordinary foods, and stipulated its scope of use: milk and dairy products, beverages, alcohol, cocoa products, etc., and stipulated the recommended maximum usage. With the expansion of the scope of application of sodium hyaluronate, sodium hyaluronate has appeared more and more in the food field, and many brands have regarded it as one of the important "selling points" of their products. In the promotion of sodium hyaluronate foods, "Q elasticity", "water light" and "hydration" often appear, mainly to hydrate the skin, but the actual content of sodium hyaluronate in these oral liquids, sparkling water and other foods that claim to add sodium hyaluronate is completely unknown. There are too many conceptual propaganda on the market now, and oral sodium hyaluronate is still a new thing. The rapid expansion of the market may lead to phenomena such as "OEM" and "OEM", and even inaccurate labeling and other violations.

[0004] At present, the classic method for detecting the content of sodium hyaluronate is the sulfuric acid-carbazole method, which uses strong acid sulfuric acid to degrade sodium hyaluronate into glucuronic acid, which then reacts with carbazole to form a colored complex, which is then determined under ultraviolet spectrophotometry. 2 SO 4It is a strong acid, and the degradation process has not only acid effect but also thermal effect. Too high concentration can easily cause polysaccharide carbonization, and too low concentration can easily lead to incomplete hydrolysis. Many ingredients in food, such as preservatives, emulsifiers, stabilizers, etc., may also react with carbazole sulfate reagent to develop color at 530 nm, resulting in poor method specificity and accuracy. CN113287708A discloses a method for detecting the content of sodium hyaluronate in solid beverages, using organic reagents to flocculate protein, preliminarily separate the protein in the solid beverage, and then use liquid phase method to test sodium hyaluronate. This technology is only applicable to solid beverages with simpler matrix food matrix, the pretreatment is too simple, and the detection wavelength of 201nm, an ultra-low band, cannot effectively resist interference, so it cannot meet the detection requirements of complex matrices of multiple types of food in the announcement. CN114646708A relates to a method for determining the content of sodium hyaluronate, using 12 mol / mL hydrochloric acid, that is, pure concentrated hydrochloric acid to degrade sodium hyaluronate in solid beverages, and programming the temperature for at least 13h under normal pressure, and using high performance liquid chromatography-evaporative light scattering to detect sodium hyaluronate. This technology is only applicable to raw material samples, and pure concentrated hydrochloric acid is required for hydrolysis under normal pressure. At the same time, the hydrolysis time is too long, requiring 13 hours, which is not conducive to the determination of large quantities of actual samples. The use of an evaporative light scattering detector, a universal detector, is not only susceptible to contamination by complex food matrices, resulting in poor stability, but also susceptible to interference from complex food matrices, affecting the accuracy of the results. CN 116026952 A relates to a method for detecting sodium hyaluronate, which uses hyaluronidase to enzymolyze soft candy samples, separates them with a cation exchange chromatography column, and detects them at a low wavelength of 232 nm. This technology is only applicable to a single food matrix of soft candy. The hyaluronidase used is currently produced by only one enterprise, and its price is very expensive. For batch product testing, the cost is very high. The cost of hyaluronidase used in a single sample is as high as 500 yuan or more. At the same time, it must be estimated according to the content of sodium hyaluronate in the sample. Insufficient enzyme dosage can easily lead to inaccurate content determination. At the same time, the storage of the enzyme is relatively harsh. It must be used up within a short time after opening to avoid enzyme failure. The use of expensive cation exchange chromatography columns has increased the cost of the method. In addition, detection at a low wavelength of 232 nm cannot effectively eliminate the interference of other impurities in complex food matrices. CN 113917011 B discloses a method for detecting the content of sodium hyaluronate and its use. After the sample to be tested is treated with a borax sulfuric acid solution, it is separated by ion chromatography and detected by a pulsed amperometric detector.This technology uses borax sulfuric acid solution to degrade sodium hyaluronate in food, which not only has acid effect but also thermal effect, and too high concentration easily causes carbonization of food matrix, which makes its release incomplete, and too low concentration easily leads to incomplete hydrolysis, and acid hydrolysis under normal pressure often cannot effectively degrade sodium hyaluronate wrapped in complex food, and also cannot effectively guarantee the accuracy and stability of the result, and in addition, this technology uses ion chromatograph that is not commonly used in enterprise laboratory, and needs to be equipped with special pulse amperometric detector for detection, and needs to use expensive ion exchange chromatographic column, which undoubtedly greatly increases the cost of this technology, and is not conducive to the application and popularization of this invention. Therefore, it is necessary to establish a kind of low cost, simple operation, high sensitivity, high stability, easy to popularize and apply sodium hyaluronate detection method in food. The present invention innovatively adopts high pressure sterilization pot process in combination with acid hydrolysis to first convert sodium hyaluronate into small molecule substances, and then introduces a derivatization reaction system, and adopts a derivatization agent 4-aminobenzoic acid ethyl ester with good stability to derive, and sodium hyaluronate is converted into a target with ultraviolet absorption group, and common liquid chromatography instrument is used to cooperate with common cheap C. 18 The chromatographic column can complete high-sensitivity, high-stability and high-reliability detection in food. The present invention provides a new technical idea for the analysis and detection of sodium hyaluronate, a new anti-aging food raw material, in food by relevant inspection agencies and internal laboratories of enterprises. Summary of the invention

[0005] The object of the present invention is to provide a high performance liquid chromatography detection method for sodium hyaluronate, a new anti-aging food raw material in food. The technical scheme for implementing the present invention is as follows: The method for separating and detecting sodium hyaluronate, a new anti-aging food raw material in food, comprises the following specific steps: a. Prepare standard stock solution and standard working solution. The standard stock solution is 10.0 mg / mL. Pipette appropriate amount of standard substance stock solution with a concentration of 1000 μg / mL to prepare standard series solutions with concentrations of 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1 000 μg / mL. Take 2.0 mL of each of the above sample solutions in a 10 mL stoppered colorimetric tube, add 2 mL of 4.0 mol / L hydrochloric acid solution, vortex to mix, seal, and place in a 121 ℃ high pressure sterilizer for hydrolysis for 60 min. After cooling to room temperature, adjust the acidity to neutral with 40% sodium hydroxide solution, transfer the solution to a 10 mL volumetric flask, dilute to the mark with water, shake well, and prepare a series of standard working solutions of sodium hyaluronate synchronous hydrolysis with concentrations of 2.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL, and 100.0 μg / mL, respectively. Transfer 0.50 mL of the above standard working solution to a 5 mL stoppered colorimetric tube, add 200 μL of ethyl 4-aminobenzoate solution and 50 μL of sodium cyanoborohydride solution, vortex mix, and seal the tube. Heat in a 70°C water bath for derivatization for 50 min. After derivatization, cool to room temperature, add water to 2.00 mL, and pass through a 0.45 μm aqueous microporous filter for testing.

[0006] b. Prepare the sample solution to be tested: weigh 1.0 g of the sample (accurate to 0.001 g) and add 2 mL of water to a 10 mL stoppered colorimetric tube to fully dissolve and mix (the alcohol sample needs to be heated to evaporate the ethanol first), add 2 mL of 4.0 mol / L hydrochloric acid solution, vortex to mix, seal, and place in a 121 ℃ high pressure sterilizer for hydrolysis for 60 min. After cooling to room temperature, adjust the acidity to neutral with 40% sodium hydroxide solution, transfer the solution to a 10 mL volumetric flask, dilute to the mark with water, and shake well. Transfer the sample solution to a stoppered plastic centrifuge tube, centrifuge at 10,000 r / min for 5 min, and take the supernatant for use. Transfer 0.50 mL of the supernatant to a 5 mL stoppered colorimetric tube, add 200 μL of ethyl 4-aminobenzoate solution and 50 μL of sodium cyanoborohydride solution, vortex to mix, and seal the tube. Heat in a 70℃ water bath for derivatization for 50 min. After derivatization, cool to room temperature, add water to 2.00 mL, and filter through a 0.45 μm aqueous microporous filter for testing.

[0007] c. The instrument used is a high performance liquid chromatograph, the detector is a photodiode array detector; the chromatographic column is C 18The column temperature was 35 °C and the detection wavelength was 307 nm. The mobile phase was 0.05% phosphoric acid / methanol / acetonitrile = 75 / 15 / 10 (V / V / V). The flow rate was 1 mL / min and the injection volume was 10 μL.

[0008] d. Drawing of standard curve: The sodium hyaluronate standard curve is subjected to simultaneous hydrolysis and derivatization for high performance liquid chromatography determination. According to the chromatographic conditions of step c, the concentration of the sodium hyaluronate hydrolysis derivative is used as the abscissa and the corresponding peak area is used as the ordinate, and a linear regression is performed to obtain the standard curve equation.

[0009] e. Result analysis: Take the filtrate in step b and inject it into a high performance liquid chromatograph, perform gradient elution and detection under the chromatographic conditions of step c, measure the peak area of ​​the target substance in the filtrate, qualitatively determine the retention time, and quantitatively determine the content of sodium hyaluronate in the sample to be tested based on the standard curve prepared in step d.

[0010] Compared with the prior art, the present invention has the following innovative features: Sodium hyaluronate has strong polarity and weak ultraviolet absorption. The present invention innovatively uses 4-aminobenzoic acid ethyl ester with ultraviolet absorption groups as a derivatizing agent, so that the target sodium hyaluronate is transformed from a target without a luminescent group to a target with an ultraviolet absorption group, which can be detected by a commonly used ultraviolet detector. This not only greatly improves the detection sensitivity, but also the detection limit can reach 3.3 mg / kg, which can fully meet the detection requirements of sodium hyaluronate with low or higher added content in food.

[0011] As a high molecular weight polysaccharide, the degradation of sodium hyaluronate is currently mainly concentrated in enzymatic hydrolysis or acid hydrolysis. The reaction conditions of hyaluronidase are mild, and it can specifically and selectively cut β-1,4-glycosidic bonds. However, only bacterial hyaluronidase (represented by streptococcus hyaluronidase) has the best enzymatic hydrolysis. Currently, only one company can produce it. However, bacterial hyaluronidase is very expensive, and the cost is high for batch product testing. At present, the cost of hyaluronidase used in a single sample is as high as 500 yuan or more. At the same time, it must be estimated based on the content of sodium hyaluronate in the sample. Insufficient enzyme dosage can easily lead to inaccurate content determination. At the same time, the storage of enzymes is relatively harsh. After opening, it must be used up within a short time to avoid enzyme failure, which invisibly increases the cost of this method. The acid hydrolysis method currently used has been shown in experiments that it is easy to be incompletely hydrolyzed under normal pressure, or it is easy to cause carbonization of complex food matrices, which leads to the inability to release sodium hyaluronate in the sample, and even the phenomenon of the target sodium hyaluronate cannot be detected. In view of the deficiencies in existing research, the present invention innovatively adopts a high-pressure sterilization pot device, a high-temperature and high-pressure process combined with an acid hydrolysis method, which effectively ensures the complete release and hydrolysis of the target substance sodium hyaluronate in a complex food matrix, thereby maximizing the accuracy and stability of the measurement results.

[0012] The present invention compares the effects of two derivatizing agents, phenylmethylpyrazolone (PMP) and ethyl para-aminobenzoate (ABEE), and innovatively uses ethyl para-aminobenzoate as a derivatizing agent for the first time in the detection method of sodium hyaluronate in complex food matrices. The quantitative wavelength is 307 nm, which effectively avoids the interference of other substances in the complex matrix of food, thereby ensuring the accuracy of the measurement results. The test results show that the products of the PMP derivatization method and the ABEE derivatization method have good ultraviolet absorption characteristics (the PMP derivatization product has a strong absorption peak at 245nm, and the ABEE derivatization product has a strong absorption peak at 307nm), but the sensitivity of the ABEE derivatization product is higher than that of the PMP derivatization product. In addition, the PMP derivatization method is to dissolve the sugar substance in a sodium hydroxide solution, and the acid needs to be neutralized after the reaction is completed, and the excess derivatization agent must be removed by multiple extractions with chloroform, otherwise the peak broadening of the excess derivatization agent will affect the quantification of the target. In addition to ultraviolet absorption characteristics, ABEE derivatives also have high fluorescence intensity. When the instrument is tight, fluorescence detectors can be used for measurement. At the same time, the peak of excess derivatization reagents is the last to appear, which will not interfere with the separation and quantification of the target compound sodium hyaluronate. In addition, the PMP derivatization reaction time (100 min) is 1 times longer than the ABEE derivatization time (50 min), which is not conducive to the actual operation of large batches of samples. In principle, the present invention innovatively selects ABEE derivatives as the derivatization agent for sodium hyaluronate hydrolyzate, taking into account simple and fast operation, high sensitivity, and strong anti-interference.

[0013] The present invention adopts the acid degradation method, which uses the hydrogen ions in the solution to protonate the oxygen atoms on the glycosidic bond, thereby causing the glycosidic bond to break. The degradation rate is fast and the molecular weight of the final product is small. At the same time, the acid degradation method has low cost and is conducive to batch related product testing. Inorganic strong acids such as hydrochloric acid and sulfuric acid can cause sodium hyaluronate to produce a strong degradation reaction. The present invention investigates hydrochloric acid (HCl) and sulfuric acid (H 2 SO 4 ), trifluoroacetic acid (CF 3 The results showed that among the three inorganic acids, hydrochloric acid had the best effect in hydrolyzing sodium hyaluronate. 2 SO 4 It is a strong acid. The degradation process has not only acid effect but also thermal effect. Too high concentration can easily cause carbonization of polysaccharides, while too low concentration can easily lead to incomplete hydrolysis. Trifluoroacetic acid has weak oxidizing property and mainly reacts with -CF 3The strong electronegativity of the group attracts hydrogen ions, thereby catalyzing the hydrolysis of polysaccharides. It is volatile, and a hydrolysis tube or headspace bottle must be used to completely seal the hydrolysis process during hydrolysis, otherwise it will affect the hydrolysis effect and the reproducibility of the test. Hydrochloric acid has oxidizing and strong acidity, which can promote the degradation of sodium hyaluronate into small molecules. As a hydrolysis reagent, hydrochloric acid can meet the requirements of a general stoppered tube, and the results are reproducible. This study selected hydrochloric acid for hydrolysis. The present invention explores and optimizes different acidic reagents, acid concentrations, and acid hydrolysis times to achieve the best hydrolysis effect for sodium hyaluronate in food. In addition, during the experiment, the sodium hyaluronate standard solution was hydrolyzed synchronously to correct the loss during the hydrolysis process.

[0014] The food matrix is ​​complex. The present invention selects the appropriate type C by comparing four different brands of chromatographic columns. 18 Column (250 mm long C 18 The invention relates to a chromatographic column, wherein the composition and proportion of various mobile phases are continuously explored and optimized, such as the adjustment of the acidity of the mobile phase and the different proportions of the three mobile phases, namely, methanol, acetonitrile and acid mobile phase. Among the four mobile phase systems, namely, methanol-phosphoric acid (0.05%), acetonitrile-phosphoric acid (0.05%), methanol / acetonitrile-phosphoric acid (0.05%) and methanol / acetonitrile-ammonium acetate (50 mmol / L, pH 4.0), the 0.05% phosphoric acid solution-methanol / acetonitrile system is finally selected so that the sodium hyaluronate in the food can achieve the best separation from the impurity interference peaks in the sample, and can also be fully separated from sugar substances such as glucose, sucrose, fructose, lactose, maltose and the like that may exist in the sample at the same time, indicating that the present invention has good specificity.

[0015] The present invention also innovatively studies the peak area of ​​sodium hyaluronate hydrolyzed derivatives with molecular weights ranging from thousands to millions of Daltons added to food as functional factors. The results show that the peak areas of sodium hyaluronate hydrolyzed derivatives with different molecular weights are consistent at the same concentration level, and the addition of sodium hyaluronate with different molecular weights in actual samples will not affect the accuracy of its content determination results.

[0016] The sodium hyaluronate derivatives produced by the derivatization method of the present invention can maintain stability for a long time at room temperature. The peak areas of sodium hyaluronate derivatives of different concentrations such as 5.0 μg / mL, 20.0 μg / mL, and 50.0 μg / mL remain basically unchanged within 72 hours at room temperature, and the relative standard deviations are 0.6%, 0.2%, and 0.7%, respectively. It can be seen that the sodium hyaluronate hydrolysis derivatives of the method of the present invention have good stability, which is particularly important for testing large quantities of actual food samples, and effectively ensures the accuracy and stability of the sodium hyaluronate determination results in large quantities of samples under long-term operation of the instrument.

[0017] The analytical instrument used in the present invention is a common high performance liquid chromatograph equipped with a UV detector, rather than expensive instruments such as liquid chromatography-mass spectrometry, which makes it easier to popularize and promote the application of the method. At the same time, the chromatographic column used is a common and inexpensive C 18 Chromatographic columns, rather than expensive cation exchange columns, greatly reduce the cost of detection. BRIEF DESCRIPTION OF THE DRAWINGS Chromatogram of sodium hyaluronate standard.

[0019] Figure 1 Chromatogram of sodium hyaluronate standard (50 mg / L); Figure 2 Chromatogram of actual sample of sodium hyaluronate; Figure 3 Chromatograms of hydrolyzed derivatives of sodium hyaluronate with different molecular weights; Figure 4 The chromatograms showing the effects of different sugars on the determination of hydrolyzed derivatives of sodium hyaluronate; Figure 5 Stability of hydrolyzed derivatives of sodium hyaluronate. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with specific embodiments and drawings of the specification. This implementation case is implemented based on the technology of the present invention, and a detailed implementation method and specific operation process are now given to illustrate that the present invention is creative, but the protection scope of the present invention is not limited to the following embodiments. Example

[0021] Unless otherwise specified, all reagents were of analytical grade and water was grade 1 water as specified in GB / T 6682.

[0022] 1.1 Acetonitrile (HPLC); 1.2 Methanol (HPLC); 1.3 Glacial acetic acid (AR); 1.4 Phosphate (AR); 1.5 ethyl 4-aminobenzoate (AR); 1.6 Cyanohydroboration (AR); 1.7 hydrochloric acid (AR); 1.8 sodium hydroxide (AR); 1.9 Sodium hyaluronate (CAS: 9067-32-17, molecular weight 1500kDa, purity 95.34%); 1.10 4-aminobenzoic acid ethyl ester (ABEE) solution: Dissolve 5.0 g of 4-aminobenzoic acid ethyl ester (ABEE) and 5.0 g of acetic acid. Add methanol to a volume of 100 mL and store in a 4°C refrigerator for later use; 1.11 Cyanoborohydride (NaBH3CN) solution: 0.5 g NaBH 3 CN was dissolved in methanol to a volume of 10 mL. Store in a 4°C refrigerator until use.

[0023] 1.12 Sodium hydroxide solution (mass fraction 40%): weigh 40 g of sodium hydroxide into a 100 mL volumetric flask and add water to Fill to the mark and shake well.

[0024] 1.13 Hydrochloric acid solution (4.0 mol / L): Pipette 16.6 mL of hydrochloric acid into a 50 mL volumetric flask and dilute to the mark with water. Shake well.

[0025] 1.14 Phosphoric acid solution (0.05%): Pipette 0.50 mL of phosphoric acid into 1000 mL of water and shake well.

[0026] 1.15 Sodium hyaluronate standard stock solution: Accurately weigh 10 mg of sodium hyaluronate standard (accurate to 0.1 mg) and place Place in a 10 mL volumetric flask, heat with water to dissolve and dilute to the mark to prepare a standard stock solution with a mass concentration of 1000 μg / mL. Store in a 4°C refrigerator for future use. The validity period is 6 months.

[0027] 1.16 Synchronous hydrolysis of sodium hyaluronate standard solution. Pipette appropriate amount of 1000 μg / mL standard substance stock solution to prepare standard series solutions with concentrations of 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1 000 μg / mL, take 2.0 mL of each sample solution into a 10 mL stoppered colorimetric tube, add 2 mL of 4.0 mol / L hydrochloric acid solution, vortex to mix, seal, and place in a 121℃ autoclave for hydrolysis for 60 min. After cooling to room temperature, adjust the acidity to neutral with 40% sodium hydroxide solution, transfer the solution to a 10 mL volumetric flask, dilute to the mark with water, and shake well. Pipette 0.50 mL of the supernatant into a 5 mL stoppered colorimetric tube, add 200 μL of ethyl 4-aminobenzoate solution and 50 μL of sodium cyanoborohydride solution, vortex to mix, and seal the tube. Heat in a 70°C water bath for 50 min. After derivatization, cool to room temperature, add water to 2.00 mL, and filter through a 0.45 μm aqueous microporous filter for testing.

[0028] 2.1 High performance liquid chromatograph: equipped with UV detector.

[0029] 2.2 Analytical balance: with a sensitivity of 0.01 g and 0.01 mg.

[0030] 2.3 Ultrasonic cleaner.

[0031] 2.4 High-speed centrifuge: speed not less than 10,000 r / min.

[0032] 2.5 Vortex mixer.

[0033] 2.6 Constant temperature water bath.

[0034] 2.7 High temperature and high pressure sterilizer.

[0035] 3.1 HPLC reference chromatographic conditions a) Chromatographic column: C 18 Column (4.6 × 250 mm, 5.0 μm) or equivalent.

[0036] b) Mobile phase: 0.05% phosphoric acid / methanol / acetonitrile = 75 / 15 / 10 (V / V / V).

[0037] c) Flow rate: 1.0 mL / min.

[0038] d) Column temperature: 35 °C.

[0039] e) Injection volume: 10 μL.

[0040] f) Photodiode array detector: wavelength 307 nm.

[0041] 3.2 Standard curve drawing The sodium hyaluronate standard curve is subjected to simultaneous hydrolysis and derivatization for high performance liquid chromatography determination. According to the chromatographic conditions of step c, the concentration of the sodium hyaluronate hydrolysis derivative is used as the abscissa and the corresponding peak area is used as the ordinate, and a linear regression is performed to obtain the standard curve equation.

[0042] 4.1 Sample pretreatment 4.1.1 Extraction Weigh 2 g (accurate to 0.01 g) of sample into a 10 mL centrifuge tube, add 5.0 mL of water, vortex mix, ultrasonically extract for 10 min, add 100 uL of zinc acetate solution and potassium ferrocyanide solution, dilute to scale with water, and mix. Take part of the sample solution into a plastic centrifuge tube with a stopper, centrifuge at 10,000 r / min for 5 min, and take the supernatant for use. Weigh 1.0 g of sample (accurate to 0.001 g) into a 10 mL stoppered colorimetric tube, add 2 mL of water to fully dissolve and mix (wine samples must be heated to evaporate ethanol first), add 2 mL of 4.0 mol / L hydrochloric acid solution, vortex mix, seal, and place in a 121 ℃ autoclave for hydrolysis for 60 min. After cooling to room temperature, adjust the acidity to neutral with 40% sodium hydroxide solution, transfer the solution to a 10 mL volumetric flask, dilute to scale with water, and shake well. Transfer the sample to a plastic centrifuge tube with a stopper, centrifuge at 10,000 r / min for 5 min, and take the supernatant for later use.

[0043] 4.1.2 Derivatives Pipette 0.50 mL of the supernatant into a 5 mL stoppered colorimetric tube, add 200 μL of ethyl 4-aminobenzoate solution and 50 μL of sodium cyanoborohydride solution, vortex to mix, and seal the tube. Heat in a 70°C water bath for 50 min. After derivatization, cool to room temperature, add water to 2.00 mL, and filter through a 0.45 μm aqueous microporous filter for testing.

[0044] 4.2 Sample measurement The samples to be tested shall be determined according to the chromatographic conditions in 3.1, and the qualitative analysis shall be based on the retention time or the diode array detector (DAD) spectrum, and the quantitative analysis shall be based on the external standard method. The content of sodium hyaluronate derivatives in the sample shall be within the linear range of the standard curve. If it exceeds the linear range, it shall be diluted before analysis. The sodium hyaluronate standard (50 mg / L) and the sodium hyaluronate chromatogram in the actual sample, the specific experimental chromatogram, and the stability are shown in Appendix A.

[0045] 4.3 Blank test Carry out the measurement in parallel with the test according to steps 4.1 to 4.2, except that the sample is not weighed.

[0046] The content of sodium hyaluronate in the sample is calculated according to the following formula:

[0047] Where: X——the content of sodium hyaluronate in the sample, mg / kg; m——the weight of the sample, g; c i ——Concentration of sodium hyaluronate in the sample solution, μg / mL; C0 ——Concentration of sodium hyaluronate in blank test solution, μg / mL; V——sample fixed volume, mL.

[0048] It is expressed as the arithmetic mean of two independent measurement results obtained under repeatability conditions. The result must deduct the blank and be retained to three decimal places. 6.1 Detection limit and quantification limit: The detection limit of sodium hyaluronate is 1.0 mg / kg, and the quantification limit is 3.3 mg / kg.

[0049] 6.2 Precision: The relative standard deviation of two independent test results of the sample obtained is not greater than 10%.

[0050] Table 1 Spiked recoveries and relative standard deviations of sodium hyaluronate in different samples

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for detecting new anti-aging food raw materials in food, characterized in that: The food is milk and dairy products, beverages, alcohol, cocoa products, chocolate and chocolate products (including cocoa butter substitute chocolate and products), candy, frozen drinks, etc. The raw material of the new anti-aging food is sodium hyaluronate. The detection method includes: a. Prepare standard stock solution and standard working solution. The standard stock solution is 1000 μg / mL. Pipette appropriate amount of standard substance stock solution with a concentration of 1000 μg / mL to prepare a standard series of solutions with concentrations of 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1 000 μg / mL. Take 2.0 mL of each of the above sample solutions in a 10 mL stoppered colorimetric tube, add 4.0 mol / L hydrochloric acid solution, vortex mix, seal, place in a 121 ℃ autoclave for hydrolysis for 60 min, adjust the acidity to neutral with sodium hydroxide solution, and then dilute to 10 mL with water to prepare a series of standard working solutions of sodium hyaluronate synchronous hydrolysis with concentrations of 2.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL, and 100.0 μg / mL. Take 0.50 mL of the above standard working solution, add 200 μL of ethyl 4-aminobenzoate solution and 50 μL of sodium cyanoborohydride solution, and place in a 70 ℃ water bath for heating and derivatization for 50 min. After derivatization, cool to room temperature, add water to 2.00 mL, and pass through a 0.45 μm aqueous microporous filter for testing; b. Prepare the sample solution to be tested: add 2 mL of water to the sample and fully dissolve and mix (the alcohol sample needs to be heated to evaporate the ethanol first), add 4.0 mol / L hydrochloric acid solution and vortex to mix, seal, place in a 121 ℃ high pressure sterilizer for hydrolysis for 60 min, adjust the acidity to neutral with sodium hydroxide solution, and then dilute to 10 mL with water. Centrifuge the sample solution for 5 min, remove 0.50 mL of the supernatant, add 200 μL of 4-aminobenzoic acid ethyl ester solution and 50 μL of sodium cyanoborohydride solution, place in a 70 ℃ water bath and heat for derivatization for 50 min, cool to room temperature after derivatization, add water to 2.00 mL, and pass through a 0.45 μm aqueous microporous filter for testing; c. The instrument used is a high performance liquid chromatograph, the detector is a photodiode array detector; the chromatographic column is C 18 Column, column temperature was 35 °C, detection wavelength was 307 nm; mobile phase was 0.05% phosphoric acid / methanol / acetonitrile = 75 / 15 / 10 (V / V / V); flow rate: 1 mL / min; injection volume: 10 μL; d. Drawing of standard curve: The sodium hyaluronate standard curve is subjected to simultaneous hydrolysis and derivatization for high performance liquid chromatography determination. According to the chromatographic conditions of step c, the concentration of the hydrolysis derivative of the sodium hyaluronate standard working solution is used as the horizontal coordinate, and the corresponding peak area is used as the vertical coordinate. Linear regression is performed to obtain the standard curve equation; e. Result analysis: Take the filtrate in step b and inject it into a high performance liquid chromatograph, perform gradient elution and detection under the chromatographic conditions of step c, measure the peak area of ​​the target substance in the filtrate, qualitatively determine the retention time, and quantitatively determine the content of sodium hyaluronate in the sample to be tested based on the standard curve prepared in step d.

2. The method for detecting new anti-aging food raw materials in food according to claim 1, characterized in that: The specific steps for preparing the standard stock solution described in a are as follows: accurately weigh 10 mg (accurate to 0.1 mg) of sodium hyaluronate standard, place it in a 10 mL volumetric flask, heat it with water to dissolve and dilute to the scale line to prepare a standard stock solution with a mass concentration of 1000 μg / mL; the specific steps for preparing the standard working solution described in a are as follows: respectively pipette an appropriate amount of the standard substance stock solution with a concentration of 1000 μg / mL to prepare a standard series of solutions with concentrations of 10 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 250 μg / mL, 500 μg / mL, and 1 000 μg / mL, take 2.0 mL of each of the above sample solutions in a 10 mL stoppered colorimetric tube, add 2 mL of 4.0 mol / L hydrochloric acid solution, vortex mix, seal, and place in a 121°C high pressure sterilizer for hydrolysis for 60 min. After cooling to room temperature, adjust the acidity to neutral with 40% sodium hydroxide solution, transfer the solution to a 10 mL volumetric flask, dilute to the mark with water, shake well, and prepare a series of standard working solutions of sodium hyaluronate synchronous hydrolysis with concentrations of 2.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL, 50.0 μg / mL, and 100.0 μg / mL, respectively. Pipette 0.50 mL of the above standard working solution into a 5 mL stoppered colorimetric tube, add 200 μL of ethyl 4-aminobenzoate solution and 50 μL of sodium cyanoborohydride solution, vortex mix, seal the tube, and heat in a 70°C water bath for derivatization for 50 min. After derivatization, cool to room temperature, add water to 2.00 mL, and pass through a 0.45 μm aqueous microporous filter for testing.

3. The new anti-aging food raw material in food according to claim 1 of The detection method is characterized in that The specific steps for preparing the sample solution described in b are as follows: weigh 1.0 g of the sample (accurate to 0.001 g) and add 2 mL of water to a 10 mL stoppered colorimetric tube to fully dissolve and mix (the alcohol sample needs to be heated to evaporate the ethanol first), add 2 mL of 4.0 mol / L hydrochloric acid solution, vortex to mix, seal, and place in a 121 ℃ high pressure sterilizer for hydrolysis for 60 min; take out and cool to room temperature, adjust the acidity to neutral with 40% sodium hydroxide solution, transfer the solution to a 10 mL volumetric flask, dilute to the mark with water, and shake well; transfer the sample solution to a stoppered plastic centrifuge tube, centrifuge at 10,000 r / min for 5 min, and take the supernatant for use. Transfer 0.50 mL of the supernatant to a 5 mL stoppered colorimetric tube, add 200 μL of ethyl 4-aminobenzoate solution and 50 μL of sodium cyanoborohydride solution, vortex to mix, and seal the tube. Place in a 70℃ water bath for heating and derivatization for 50 min. After derivatization, cool to room temperature, add water to 2.00 mL, and filter through a 0.45 μm aqueous microporous filter for testing.

4. The method for detecting new anti-aging food raw materials in food according to claim 1, characterized in that: c 18 The column specifications are 4.6 mm × 250 mm inner diameter, and the particle size is 5 μm.

5. The method for detecting new anti-aging food raw materials in food according to claim 1, characterized in that: The standard curve coefficient described in d is not less than 0.

999.

6. The method for detecting new anti-aging food raw materials in food according to claim 1, characterized in that: The food is milk and dairy products, beverages, alcohol, cocoa products, chocolate and chocolate products (including cocoa butter substitute chocolate and products) as well as candy and frozen drinks. The raw material of the new anti-aging food is sodium hyaluronate.

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