Method for determining total amount of soybean isoflavones in food

Abstract

The application discloses a method for determining total soybean isoflavone in food, which realizes quantitative conversion of various soybean isoflavone glycosides in the sample into corresponding aglycones by extraction under alkaline conditions and hydrolysis of glucosidase; the method realizes determination of low-concentration soybean isoflavone in the sample and effectively shortens the detection period by applying ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry; and methodological research shows that the method has a lower detection limit and higher accuracy. Since the application realizes determination of total quantity of 12 kinds of soybean isoflavones in the sample by only 3 kinds of soybean isoflavone aglycones, the use cost of standard products is reduced to only 1 / 3 of the original on the basis of originally needing to use at least 6 kinds of isoflavone standard products. In addition, total soybean isoflavone is calculated in terms of aglycone, which can more accurately reflect the actual biological activity level on the one hand, and can also avoid the inconsistency of the total isoflavone result caused by the non-uniform use of glycosides or aglycones on the other hand.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical detection, and particularly relates to determination of total soy isoflavone content in soy protein-containing food. BACKGROUND

[0002] Soy isoflavones are mainly derived from legume beans of leguminous plants, and have a structure similar to that of estrogen, so they can bind to estrogen receptors and produce estrogen-like activity after being absorbed in the body. Due to the estrogen-like effect, soy isoflavones are also known as phytoestrogens and are used to improve menopausal syndrome. In addition, studies have shown that soy isoflavones have antioxidant, obesity prevention, diabetes prevention, cancer risk reduction, blood sugar reduction, and osteoporosis prevention effects, and are therefore widely promoted in the health product industry. However, excessive intake of soy isoflavones can also have negative effects, especially for infants using soy-based formula.

[0003] As the only and primary protein source allowed in GB 10765-2021 (National Food Safety Standard Infant Formula) soy-based infant formula, soy protein isolate of soybean origin often contains a certain amount of soy isoflavones. Due to the lack of relevant data, it was previously believed that the use of soy-based infant formula was safe, however, recent studies have shown that when infants are exposed to a certain concentration of soy isoflavones during the sensitive window period of development, the estrogen-like effect produced will have an adverse effect on reproductive system development and have the risk of causing related diseases in adulthood. Considering the widespread eating habits of soybeans and their products in China and other Asian countries, and due to the fact that soy protein isolate is a rare plant protein that can replace animal protein and is widely used in the food industry and dietary supplements, the risk of excessive intake of phytoestrogens is increasing, and accurate and rapid determination of the content of soy isoflavones in food not only provides reasonable dietary guidance for consumers, but also promotes the development of the soy deep processing industry chain. Soy protein isolate with low isoflavone residues will further ensure the safety of soy-based infant formula.

[0004] Soy isoflavones can be divided into two categories: one is free aglycone, including daidzein, glycitein, and genistein; the other is conjugated, with aglycone combined with glucose, glucose acetyl, and glucose malonyl, respectively. The above free and conjugated types total 12, of which the conjugated type accounts for about 97%-98% of the total isoflavones in soybeans. Due to the poor stability of acetyl and malonyl isoflavone glycosides, it is difficult to obtain the corresponding standard, which in turn causes difficulties in the quantification of this type of isoflavones. Therefore, most methods only detect 3 aglycones (daidzein, glycitein, and genistein) and 3 glycosides (daidzin, glycitin, and genistin), which cannot truly reflect the actual content level of soy isoflavones in the sample.

[0005] The determination methods of soybean isoflavones recorded by the American Association of Analytical Chemists (AOAC) and the United States Pharmacopoeia (USP) all adopt the standard atlas comparison method, and through the introduction of the "conversion factor", the acetyl and malonyl soybean isoflavone glycosides are quantitatively detected by taking daidzin, glycitein and genistin as standard products. However, the above method needs to ensure the full consistency of the chromatographic conditions, including the selection of the specifications and brands of the chromatographic column, so as to ensure the relatively consistent retention time, and when the sample matrix is complex, a larger result deviation is often caused. Other methods such as AOAC 2001.10 hydrolyze the acetyl and malonyl soybean isoflavone glycosides by saponification, so as to obtain the corresponding daidzin, glycitein and genistin, and six standard products are used for quantitative determination. The method can more accurately determine the total amount of isoflavones, but the determination deviation of the sample with a lower content is larger.

[0006] At present, the domestic standard methods include GB / T 23788-2009 "Determination of soybean isoflavones in health food - high performance liquid chromatography method" and QB / T 5397-2019 "Determination of isoflavone content in soybean food", both of which determine three aglycones (daidzein, glycitein and genistein) and three glycosides (daidzin, glycitein and genistin) in the sample by high performance liquid chromatography combined with ultraviolet detection. Since the polar mixed solvents such as methanol and water are generally used for extraction by oscillation or ultrasonic method, and also due to the lack of acetyl and malonyl soybean isoflavone glycoside standard products, it is impossible to effectively determine the other six main isoflavones, so that the determination result is lower than the true result.

[0007] In view of the limitations and inaccuracy of the above methods, it is necessary to develop and establish a determination method which can truly reflect the content level of soybean isoflavones in the sample and has the characteristics of accuracy, simplicity and rapidness.

[0008] Based on the above research, the present application aims to realize the accurate determination of total isoflavones in soybean related food and provide strong technical support for food development and food safety supervision. SUMMARY

[0009] The present application provides a method for determining the total amount of soybean isoflavones in food, which can realize the quantitative conversion of various isoflavone glycosides in the sample into corresponding aglycones by extraction under alkaline conditions combined with the hydrolysis of glucosidase. The use of ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry can realize the determination of low-concentration soybean isoflavones in the sample and effectively shorten the detection period.

[0010] Technical scheme: In order to achieve the above purpose, the technical scheme adopted by the present application is:

[0011] A method for determining the total amount of soybean isoflavones in food, comprising the following steps:

[0012] S1, the sample to be measured is extracted and treated using an alkaline methanol solution to obtain an extract;

[0013] S2, β-glucosidase is added to the extract for enzymatic hydrolysis, and after conversion, pure methanol is added for dissolution to obtain a test solution;

[0014] S3, the test solution is detected by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry to obtain the content of isoflavone compounds in the sample to be measured, including daidzein, glycitein and genistein.

[0015] The principle of the determination method is that the sample to be measured (also referred to as a test sample in the present application) is extracted by an alkaline methanol solution. During the extraction process, the acyl groups on the malonyl and acetyl soybean isoflavone glycosides in the sample are hydrolyzed and converted into corresponding isoflavone glycosides (daidzin, glycitin and genistin). The extract is enzymatically hydrolyzed by β-glucosidase in an acidic buffer solution, and the glucose groups on the isoflavone glycoside molecules are hydrolyzed and removed. All isoflavone glycosides in the solution are converted into corresponding aglycones (daidzein, glycitein and genistein). The enzymatic hydrolysate is mixed and diluted with pure methanol, separated by a reversed-phase chromatographic column, detected by high-performance liquid chromatography-tandem mass spectrometry, and quantified by an external standard method.

[0016] Optionally, in an embodiment, in step S1, the alkaline methanol solution is an 80% methanol aqueous solution containing 3 g / L sodium hydroxide.

[0017] Optionally, in an embodiment, the extraction process includes adding 80 mL of the alkaline methanol solution to the sample to be measured containing 50-500 ug of total soybean isoflavones, ultrasonically extracting for 20 min, neutralizing with 4 mL of glacial acetic acid, and then diluting to 100 mL with 80% methanol solution.

[0018] Optionally, in an embodiment, in step S1, the total amount of isoflavones in the sample to be measured ranges from 50 ug to 500 ug in terms of aglycone.

[0019] Optionally, in an embodiment, step S2 specifically includes mixing 1 volume equivalent of the extract with 4 volume equivalents of acetic acid buffer containing 5 active units of glucosidase, shaking for enzymatic hydrolysis at 50°C for 120 min, cooling, and diluting to 10 volume equivalents with methanol.

[0020] Further optionally, in an embodiment, in step S2, the concentration of the acetic acid buffer is 0.1 mol / L, and pH is 5.0.

[0021] Optionally, in an embodiment, the enzymatic sample solution is filtered with a 0.2 μιη polypropylene (PP) filter head before use in step S2.

[0022] Optionally, in an embodiment, the detection condition of the liquid chromatography in the ultra-performance liquid chromatography-tandem mass spectrometry in step S3 comprises:

[0023] Chromatographic column: C18 column, 50 mm x 2.1 mm, 1.8 μιη, or equivalent sub-2 μιη ultra-performance chromatographic column.

[0024] Optionally, in an embodiment, the other detection condition of the liquid chromatography further comprises:

[0025] Mobile phase: A phase, 0.1% (v / v) formic acid solution; B phase, acetonitrile solution containing 0.1% (v / v) formic acid;

[0026] Gradient elution: 0 min-12.0 min, 92%-75% A; 12.0 min-13.0 min, 75% A; 13.0 min-13.1 min, 75%-92% A; 13.1 min-15.0 min, 92% A;

[0027] Flow rate: 0.4 mL / min;

[0028] Column temperature: 40°C;

[0029] Injection volume: 1 μΐ.

[0030] Optionally, in an embodiment, the mass spectrometry condition in the high-performance liquid chromatography-tandem mass spectrometry in step S3 comprises: electrospray ion source, ESI + Positive ion.

[0031] Optionally, in an embodiment, the mass spectrometry condition further comprises:

[0032] Dry gas temperature: 300°C;

[0033] Dry gas flow rate: 10 L / min;

[0034] Atomizer pressure: 20 psi;

[0035] Syringe gas temperature: 250°C;

[0036] Syringe gas flow rate: 11 L / min;

[0037] Capillary voltage: 2000 V.

[0038] Optionally, in an embodiment, in step S3, the high performance liquid chromatography-tandem triple quadrupole mass spectrometry adopts a multiple reaction monitoring (MRM) mode, and the selection ion parameter conditions of the isoflavone compounds include:

[0039] Daidzein, retention time 9±0.5 min, parent ion 255.1 m / z, daughter ion 199.1 m / z, capillary acceleration voltage 4 V, collision energy 26 V;

[0040] Glycitein, retention time 10±0.5 min, parent ion 285.1 m / z, daughter ion 270.1 m / z, capillary acceleration voltage 4 V, collision energy 30 V;

[0041] Genistein, retention time 12±0.5 min, parent ion 271.1 m / z, daughter ion 215.1 m / z, capillary acceleration voltage 4 V, collision energy 30 V.

[0042] Optionally, in an embodiment, the detection processing in step S3 specifically includes the following steps:

[0043] S31, making a standard curve

[0044] The standard samples of the isoflavone components daidzein, glycitein, and genistein are respectively dissolved in pure methanol as standard solutions, and different gradient concentrations are prepared by using 80% methanol solution. The standard series solutions are sequentially injected into the liquid chromatography-tandem mass spectrometer, and the corresponding peak areas are measured. The concentration of each isoflavone in the standard working solution is taken as the abscissa, and the peak area is taken as the ordinate, and a standard curve is drawn.

[0045] S32, isoflavone component determination

[0046] The test solution obtained in step S2 is injected into the ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer for detection, and the corresponding peak area is obtained. According to the standard curve, the concentration of each isoflavone in the test solution is obtained.

[0047] Optionally, in an embodiment, in step S31, the preparation method of the standard solution includes:

[0048] S311, the soy isoflavone standard is dissolved in pure methanol to obtain a 100 μg / mL soy isoflavone standard stock solution;

[0049] S312, the soy isoflavone standard stock solution is dissolved in 80% methanol solution to obtain a 5-10 μg / mL soy isoflavone standard intermediate solution;

[0050] S313, the soy isoflavone standard intermediate solution is dissolved in 80% methanol solution to obtain a 0.05-0.5 μg / mL soy isoflavone mixed standard solution.

[0051] Optionally, in an embodiment, in step S312, the daidzein standard intermediate solution is 5 μg / mL, the glycitein standard intermediate solution is 5 μg / mL, and the genistein standard intermediate solution is 10 μg / mL.

[0052] Optionally, in an embodiment, in step S313, the daidzein concentration in the soy isoflavone mixed standard solution is 0.2 μg / mL, the glycitein concentration is 0.05 μg / mL, and the genistein concentration is 0.5 μg / mL.

[0053] Optionally, in an embodiment, the content of each component of soy isoflavones (daidzein (X1), glycitein (X2), genistein (X3)) in the sample is calculated according to formula (1):

[0054]

[0055] In the formula:

[0056] X i X - the content of a single component of soy isoflavones in the sample, in units of milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0057] C i C - the concentration of each isoflavone obtained according to the standard working curve, in units of micrograms per milliliter (μg / mL);

[0058] V - the total volume of sample dilution, in units of milliliters (mL);

[0059] m - the mass of the sample, in units of grams (g).

[0060] Optionally, in an embodiment, the total content of soy isoflavones in the sample is calculated according to formula (2):

[0061] X = X1+ X2+ X3 (2)

[0062] In the formula:

[0063] X - the total content of soy isoflavones in the sample, in units of milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0064] X1 - the content of daidzein in the sample, in units of milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0065] X2 - the content of glycitein in the sample, in units of milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0066] X3 - content of the test genistein, in milligrams per gram (mg / g) or milligrams per milliliter (mg / mL).

[0067] The application provides a method for determining the total amount of soybean isoflavones in food, which is suitable for food containing soybean isoflavone components, including the determination of soybean isoflavones in health food with soybean isoflavones as main functional components or with soybean protein isolate added, and soy-based infant formula food.

[0068] Beneficial effects: the method for determining the total amount of soybean isoflavones in food by using ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry has the following advantages:

[0069] (1) Since the glycoside type in isoflavones has hydrophilicity and relatively large molecular weight, it is difficult to be directly absorbed after entering the human body, and when soybean isoflavone glycosides enter the human body, they are hydrolyzed into aglycone under the action of intestinal glucosidase and rapidly absorbed in the small intestine to exhibit biological activity, so it is more scientific to detect the total amount of aglycone in the sample.

[0070] (2) The reduction of the detection target will help to shorten the instrument running time and reduce the detection cost. By combining the hydrolysis of glucosidase under alkaline conditions, the quantitative conversion of various isoflavone glycosides in the sample into corresponding aglycone can be realized. The use of ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry can realize the determination of low concentration soybean isoflavones in the sample and effectively eliminate the interference of sample matrix.

[0071] (3) Since only 3 types of soybean isoflavone aglycone are used to realize the determination of the total amount of 12 soybean isoflavones, the use cost of standard products is reduced to only 1 / 3 of the original 6 isoflavone standard products.

[0072] (4) The total soybean isoflavone is calculated as aglycone, which can more accurately reflect the actual biological activity level, and can also avoid the difference in result expression caused by the use of glycoside or aglycone.

[0073] (5) The methodological study shows that the method has a low detection limit and high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 The structural formula of various soybean isoflavone aglycone and glycoside.

[0075] Figure 2 The total ion flow chromatogram of soybean isoflavone (soybean isoflavone, daidzein, genistein).

[0076] Figure 3 The standard curve of soybean isoflavone.

[0077] Figure 4 Standard curve of daidzein.

[0078] Figure 5 Standard curve of genistein.

[0079] Figure 6 Reaction schematic diagram of isoflavone glycosides hydrolyzed into isoflavone aglycone and glucose under the action of β-glucosidase.

[0080] Figure 7 Response value comparison of acetyl and malonyl soybean isoflavone glycosides in the sample to extraction with different concentrations of sodium hydroxide in 80% methanol solution: (A) acetyl soybean isoflavone glycosides, (B) malonyl soybean isoflavone glycosides.

[0081] Figure 8 Response value comparison of soybean isoflavone glycosides and corresponding aglycone in the sample to extraction with different concentrations of sodium hydroxide in 80% methanol solution: (A) soybean isoflavone glycosides, (B) soybean isoflavone aglycone. DETAILED DESCRIPTION

[0082] Soybean Isoflavones are mainly derived from legume beans of leguminous plants, and are a class of active substances synthesized by secondary metabolism after leguminous plants grow to a certain stage. The structural feature of 3-benzopyranone is the mother nucleus of a class of flavonoids, which can be divided into two types according to the type: one type is aglycone, including soybean aglycone, daidzein, and genistein; the other type is glycoside combined with glucose, including soybean glycoside, daidzein glycoside, genistein glycoside, and corresponding acetyl and malonyl soybean isoflavone glycosides. Thus, the currently known soybean isoflavones include 12 different substances, as shown in the table. Figure 1 When each type of soybean isoflavone glycoside enters the human body, it is hydrolyzed into the corresponding aglycone under the action of intestinal glucosidase and is rapidly absorbed in the small intestine to exhibit its biological activity. Therefore, detecting the total amount of aglycone in the sample is more scientific. At the same time, the reduction of the target object will help to shorten the instrument running time, eliminate interference, and reduce the detection cost.

[0083] Currently, AOAC 2008.03 and USP use high performance liquid chromatography to analyze soybean products and isoflavone dietary supplements. The acetyl and malonyl isoflavone glycosides are qualitatively determined by using standard chromatograms and the relative retention times of three types of glycosides and aglycone standards, and are quantitatively determined by the relationship of molecular weight conversion. The above method has the following problems: (1) the total amount is calculated by a “conversion factor”, which is not the true determination result, and cannot be accurately determined when the matrix is complex or the chromatographic retention is offset, resulting in deviation; (2) the operation condition is harsh, and the chromatographic condition needs to be fully consistent, which requires a relatively high requirement for the operator and is not conducive to the promotion of the method; (3) the single needle chromatography running time of AOAC 2008.03 is 60 min, and the gradient elution time of USP is 74 min. AOAC 2001.10 is a high performance liquid chromatography method, which converts malonyl and acetyl isoflavone glycosides into isoflavone glycosides by saponification, and can achieve accurate quantification by using 6 standard products. However, when the isoflavone content in the sample is low, the deviation is large, and the chromatographic elution time is also long, which is 44.5 min.

[0084] The domestic method GB / T 23788-2009 and QB / T5397-2019 quantitatively determine 6 isoflavones in the sample by using 3 glycosides and 3 aglycone standards, and define the total amount of the 6 substances as the total isoflavone, which does not reflect the amount of malonyl and acetyl isoflavones, resulting in a low determination result. At the same time, there are reports on the determination of soybean isoflavones by liquid chromatography-mass spectrometry, but they are generally limited to the detection of 6 substances, i.e. soybean aglycone, daidzein, genistein, soybean glycoside, daidzein glycoside, and genistin, and do not systematically detect the total amount of actual isoflavones.

[0085] The application provides a method for determining the total amount of soybean isoflavones in food. Through extraction under alkaline conditions and hydrolysis by glucosidase, quantitative conversion of various soybean isoflavone glycosides in the sample into corresponding aglycones is realized. Through application of ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry, determination of low-concentration soybean isoflavones in the sample is realized, and the detection period is effectively shortened. The methodological study shows that the method has a low detection limit and high accuracy. Since the application realizes determination of the total amount of 12 kinds of soybean isoflavones in the sample through only 3 kinds of soybean isoflavone aglycones, the use cost of standard products is reduced to only 1 / 3 of the original based on the original need of using at least 6 kinds of isoflavone standard products. In addition, the total soybean isoflavones are calculated based on the aglycone, which can more accurately reflect the actual biological activity level, and can also avoid the inconsistency of the total isoflavone results caused by the use of glycosides or aglycones. Soybean isoflavones are used as phytoestrogens to improve menopausal syndrome, but excessive soybean isoflavones can also have negative effects. Considering that soybeans and their products have a universal eating habit in China and other Asian countries, and soybean protein isolate is widely used in the food industry and dietary supplements as a few plant proteins that can replace animal proteins, the risk of excessive intake of phytoestrogens is increasing. The application aims to realize accurate determination of the total isoflavones in soy-based formula food, provide strong technical support for food development and food safety supervision, provide reasonable dietary guidance for consumers, and promote the development of the soybean deep processing industry chain, such as the production of soybean protein isolate with low isoflavone residue.

[0086] The application will be further described below in combination with the drawings and examples. According to the following examples, the application can be better understood. However, it is easy for those skilled in the art to understand that the specific material ratio, process conditions and results described in the examples are only used to illustrate the application, and should not and will not limit the application described in detail in the claims.

[0087] Examples

[0088] 1. Reagents and materials

[0089] Note: Unless otherwise specified, the reagents used in the method of this example are analytical pure, and the water is first-grade water specified in GB / T 6682.

[0090] 1.1 Reagents

[0091] Acetonitrile (CH3CN): chromatographically pure.

[0092] Methanol (CH3OH): chromatographically pure.

[0093] Formic acid (HCOOH): chromatographically pure.

[0094] Ice acetic acid (CH3COOH).

[0095] Sodium hydroxide (NaOH).

[0096] Anhydrous sodium acetate (CH3COONa).

[0097] β-glucosidase: derived from almonds, activity units >4U / mg.

[0098] 1.2 Reagent Preparation

[0099] Alkaline methanol solution: Weigh 3g of sodium hydroxide, add 200mL of water to dissolve it, and dilute to 1000mL with methanol and mix well.

[0100] Methanol solution (80%, volume ratio): Measure 800 mL of methanol, add 200 mL of water, and mix well.

[0101] Sodium acetate buffer solution (0.1 mol / L, pH 5.0): Weigh 8.20 g of anhydrous sodium acetate, dissolve it in 900 mL of water, adjust the pH to 5.0 ± 0.1 with glacial acetic acid, and dilute with water to 1000 mL.

[0102] Acetonitrile solution: Measure 1 mL of formic acid, dilute to 1000 mL with acetonitrile, mix well, and degas by sonication.

[0103] Formic acid solution (0.1%, volume ratio): Measure 1 mL of formic acid, dilute to 1000 mL with water, mix well, and degas by sonication.

[0104] β-glucosidase solution: Weigh an appropriate amount of β-glucosidase and dissolve it in sodium acetate buffer to make the enzyme concentration 1.25 U / mL. Prepare immediately before use.

[0105] 1.3 Standard Products

[0106] Daidaidzein Standard (C 15 H 10 O4 (CAS: 486-66-8) Standard: Purity ≥ 98%, or standard that has been certified by the state and granted a standard substance certificate.

[0107] Soy flavonoid standard (C 16 H 12 O5, CAS: 40957-83-3) Standard: Purity ≥ 98%, or standard that has been certified by the state and granted a standard substance certificate.

[0108] Dysgenin Standard (C 15 H 10 O5 (CAS:446-72-0) Standard: Purity ≥98%, or standard that has been certified by the state and granted a standard substance certificate.

[0109] 1.4 Preparation of Standard Solutions

[0110] (1) Soy isoflavone standard stock solution (100 μg / mL): 20 mg of each of soy isoflavone standard (daidzein, glycitein, genistein) was precisely weighed into a 200 mL brown volumetric flask, and an appropriate amount of methanol was added, and ultrasonically dissolved, and allowed to cool, and then diluted to the mark with methanol, and mixed, to give a concentration of 100 μg / mL, and stored in a refrigerator at 4°C, and the storage period was 6 months.

[0111] (2) Soy isoflavone standard intermediate solution

[0112] Daidzein standard intermediate solution (5 μg / mL): 5 mL of daidzein standard stock solution was precisely transferred, and diluted with 80% methanol solution and diluted to 100 mL. Stored in a refrigerator at 4°C in the dark, and the storage period was one week.

[0113] Glycitein standard intermediate solution (5 μg / mL): 5 mL of glycitein standard stock solution was precisely transferred, and diluted with 80% methanol solution and diluted to 100 mL. Stored in a refrigerator at 4°C in the dark, and the storage period was one week.

[0114] Genistein standard intermediate solution (10 μg / mL): 5 mL of genistein standard stock solution was precisely transferred, and diluted with 80% methanol solution and diluted to 50 mL. Stored in a refrigerator at 4°C in the dark, and the storage period was one week.

[0115] (3) Soy isoflavone mixed standard solution: 4 mL, 1 mL, and 5 mL of daidzein, glycitein, and genistein standard intermediate solutions, respectively, were precisely transferred into a 100 mL brown volumetric flask, and diluted with 80% methanol solution and diluted to the mark. The concentration of daidzein in the soy isoflavone mixed standard solution was 0.2 μg / mL, the concentration of glycitein was 0.05 μg / mL, and the concentration of genistein was 0.5 μg / mL. Prepared immediately before use.

[0116] (4) Soy isoflavone standard series solution: 0.5 mL, 1 mL, 2 mL, 5 mL, and 10 mL of soy isoflavone standard solution were precisely transferred, and diluted with 80% methanol solution to 10 mL. At the standard series concentration, the concentration of daidzein was 0.010 μg / mL, 0.020 μg / mL, 0.040 μg / mL, 0.10 μg / mL, and 0.20 μg / mL, respectively; the concentration of glycitein was 0.0025 μg / mL, 0.0050 μg / mL, 0.010 μg / mL, 0.025 μg / mL, and 0.050 μg / mL, respectively; and the concentration of genistein was 0.025 μg / mL, 0.05 μg / mL, 0.10 μg / mL, 0.25 μg / mL, and 0.50 μg / mL, respectively. (*The soy isoflavone mixed standard solution was used, and no dilution was performed).

[0117] 2. Instruments and equipment

[0118] Ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometer: equipped with an electrospray ionization source (ESI).

[0119] Analytical balance: sensitivity of 0.01 g, 0.001 g, 0.00001 g.

[0120] Ultrasonic cleaner.

[0121] pH meter: accuracy of 0.01.

[0122] Centrifuge.

[0123] Tissue homogenizer.

[0124] Constant temperature shaking water bath.

[0125] 3. Analysis steps

[0126] 3.1 Preparation of test sample

[0127] Solid samples need to be crushed, ground, and uniformly mixed. Liquid samples are shaken and mixed before determination.

[0128] Accurately weigh an appropriate amount of the mixed sample (the total amount of isoflavones in the test sample is 50-500 μg in terms of aglycone) in a 100 mL brown volumetric flask, add 80 mL of alkaline methanol solution, ultrasonic extract for 20 min, add 4 mL of glacial acetic acid, mix well, and dilute to the mark with 80% methanol solution, and mix well.

[0129] Take an appropriate amount of the extract, centrifuge to make it clear, and accurately transfer 1 mL of the supernatant into a 15 mL centrifuge tube, add 4 mL of glucosidase solution (containing 5 U of enzyme), mix well. Shake the enzyme solution at 50°C in a water bath for 120 min, take it out and cool to room temperature, add 5 mL of pure methanol, mix well and ultrasonic for 5 min. Take an appropriate amount, filter with a 0.2 μm polypropylene (PP) filter head and reserve for use.

[0130] Note: Avoid strong light during operation.

[0131] 3.2 Instrument reference conditions

[0132] 3.2.1 Liquid chromatography reference conditions

[0133] Chromatographic column: C 18 18 column, 50 mm x 2.1 mm, 1.8 μm, or equivalent chromatographic column.

[0134] Mobile phase: A phase, formic acid solution (0.1%); B phase, acetonitrile solution containing 0.1% formic acid.

[0135] Gradient elution: 0 min~12.0 min, 92%~75% A; 12.0 min~13.0 min, 75% A; 13.0 min~13.1 min, 75%~92% A; 13.1 min~15.0 min, 92% A.

[0136] Flow rate: 0.4 mL / min.

[0137] Column temperature: 40℃.

[0138] Injection volume: 1 μL.

[0139] 3.2.2 Mass spectrometry reference conditions

[0140] Ionization mode: ESI + .

[0141] Dry gas temperature: 300℃

[0142] Dry gas flow rate: 10 L / min

[0143] Atomizer pressure: 20 psi

[0144] Sheath gas temperature: 250℃

[0145] Sheath gas flow rate: 11 L / min

[0146] Capillary voltage: 2000 V.

[0147] Multiple reaction monitoring (MRM) mode, acceleration voltage and collision energy are shown in Table 1.

[0148] Table 1 Ion pair parameter conditions

[0149]

[0150] In some examples, the retention time of daidzein is 8.89 min, the retention time of biochanin A is 9.81 min, and the retention time of genistein is 11.97 min.

[0151] 3.3 Preparation of standard curve

[0152] The standard series solution was injected into the liquid chromatography-tandem mass spectrometer in turn from low concentration to high concentration, and the corresponding peak area was determined. The concentration of each isoflavone in the standard working solution was taken as the abscissa, and the peak area was taken as the ordinate to draw the standard curve, as shown in Figures 2-4 Table 2 shows the statistics of each standard curve.

[0153] Table 2 Standard curve verification

[0154]

[0155]

[0156] 3.4 Determination of sample solution

[0157] The sample solution was injected into the liquid chromatography-tandem mass spectrometer to obtain the corresponding peak area, and the concentration of each isoflavone in the sample solution was obtained according to the standard curve.

[0158] The test results are shown in Table 1. Figure 5 As shown in Table 1, the peak areas of daidzein, glycitein and genistein were 109919, 40885 and 31693 respectively. The concentrations of daidzein, glycitein and genistein were C1=0.1372 μg / mL, C2=0.02095 μg / mL and C2=0.2696 μg / mL respectively by substituting the peak area values into the respective standard curves.

[0159] 4 Expression of analysis results

[0160] 4.1 The content of each component of soy isoflavones (daidzein (X1), glycitein (X2), genistein (X3)) in the sample was calculated according to formula (1):

[0161]

[0162] In the formula:

[0163] X i The content of a single component of soy isoflavones in the sample, in units of milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0164] C i The concentration of each isoflavone obtained according to the standard working curve, in units of micrograms per milliliter (μg / mL);

[0165] V - the total volume of sample dilution, in units of milliliters (mL);

[0166] m - the mass of the sample, in units of grams (g).

[0167] According to the test results of 3.4, the content of a single component of soy isoflavones in the sample was as follows: daidzein 0.1212 mg / g, glycitein 0.01851 mg / g, and genistein 0.2382 mg / g.

[0168] 4.2 The total content of soy isoflavones in the sample was calculated according to formula (2):

[0169] X = X1 + X2 + X3 (2)

[0170] In the formula:

[0171] X — Total content of soybean isoflavones in the sample, in milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0172] Xi — Content of daidzein in the sample, in milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0173] X2— Content of glycitein in the sample, in milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0174] X3— Content of genistein in the sample, in milligrams per gram (mg / g) or milligrams per milliliter (mg / mL);

[0175] The calculation result is expressed as the arithmetic mean of two independent measurement results obtained under repeatability conditions, and the result is rounded to three significant digits (or two digits after the decimal point).

[0176] Substitute the result in Step 4.1 into Formula (2) to obtain the total content of soybean isoflavones in the sample as 0.3779 mg / g.

[0177] 5 Precision

[0178] The absolute difference between the two independent measurement results obtained under repeatability conditions shall not exceed 15% of the arithmetic mean.

[0179] Take the determination of three samples as an example:

[0180] Table 3 Determination results of soybean isoflavones in samples

[0181]

[0182] a Based on the relative standard deviation of six samples determined in parallel

[0183] Example 2 Selection of hydrolysis conditions

[0184] Hydrolysis effect of glucosidase on soybean isoflavones: β-glucosidase (EC 3.2.1.21), also known as glucose hydrolase, can efficiently hydrolyze the glucose group at the non-reducing end of glycoside molecules through a double substitution mechanism of glycosylation and deglycosylation, converting glycosides into aglycones, as shown in the following reaction formula: Figure 6 Therefore, β-glucosidase is widely used in hydrolysis processes in the food industry.

[0185] The method of hydrolysis experiment is as follows: through experimental condition optimization, it is confirmed that the enzyme activity of almond-derived glucosidase is optimal under the condition of pH=5 and temperature of 50°C. Under this condition, 1 mL of sample solution extracted by 80% methanol is mixed with 4 mL of acetate buffer containing 5 active units of β-glucosidase, and then hydrolysis is carried out after dilution with methanol. Mass spectrometry is used for detection, and the hydrolysis rate is calculated by comparing the mass spectrometry response value changes of various soybean isoflavone glycosides at different hydrolysis times. Hydrolysis rate (%) = 100% - (mass spectrometry absolute response value of each type of isoflavone glycoside after enzymolysis / mass spectrometry absolute response value of each type of isoflavone glycoside in the sample without enzymolysis treatment).

[0186] As shown in Table 4, through comparison of hydrolysis experiments, the hydrolysis activity of almond-derived glucosidase on malonyl and acetyl soybean isoflavone glycosides is relatively weak. The reason may be that the acetyl and malonyl side chains on the sugar group of this type of isoflavone glycoside form a steric hindrance effect, which hinders the formation of a sugar group-enzyme covalent intermediate and thus cannot rapidly release the aglycone. If the acetyl and malonyl groups on the glucosyl group are first removed by chemical hydrolysis reaction, then ordinary soybean isoflavone glycosides can be hydrolyzed into the corresponding aglycone by using less enzyme amount and in a relatively short time, thereby realizing the detection of total isoflavones in the sample.

[0187] Table 4 Hydrolysis rate (%) of almond-derived glucosidase on 12 types of soybean isoflavones

[0188]

[0189] Since the ester bond in the molecule of malonyl and acetyl soybean isoflavone glycoside is easily hydrolyzed under alkaline conditions, when 0.3% sodium hydroxide is contained in the 80% methanol solution, the acetyl group on the acetyl soybean isoflavone glycoside molecule is basically completely hydrolyzed after ultrasonic extraction for 20 min, reaching more than 99.8% ( Figure 7 -A); at the same time, under this condition, the malonyl group of malonyl soybean isoflavone glycoside is also hydrolyzed, reaching more than 99.6% ( Figure 7 -B). At the same time, when malonyl and acetyl soybean isoflavone glycosides are hydrolyzed, the response value of isoflavone glycosides in the sample is significantly higher than that of the sample directly extracted by 80% ( Figure 8 -A), indicating that malonyl and acetyl soybean isoflavone glycosides are all hydrolyzed and converted into the corresponding isoflavone glycosides under alkaline conditions; at the same time, the response values of various soybean isoflavone aglycones originally present in the sample under different alkaline concentration conditions are basically the same as the response values obtained under the 80% methanol extraction condition ( Figure 8 -B), and the RSD of the response values obtained under the 6 different extraction solvents is less than 2%, indicating that the soybean isoflavone aglycones in the sample do not degrade and remain stable under the extraction conditions of different concentrations of alkali.

[0190] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for measuring the total amount of soy isoflavones in a food product, characterized by, The method comprises the following steps: S1, adding 80 mL of an alkaline methanol solution to a sample to be tested, ultrasonic extraction for 20 minutes, then adding 4 mL of glacial acetic acid and diluting to 100 mL with 80% methanol solution to obtain an extraction solution, wherein the alkaline methanol solution is an 80% methanol aqueous solution containing 3 g / L sodium hydroxide; S2, adding β-glucosidase to the extraction solution for enzymolysis, then adding pure methanol for mixing and dissolving to obtain a test solution, wherein the isoflavone compounds in the test solution include daidzein, glycitein and genistein; specifically, 1 volume equivalent of the extraction solution is mixed with 4 volume equivalents of an acetic acid buffer containing 5 active units of glucosidase, and the mixture is shaken for enzymolysis at 50°C for 120 min, then cooled and diluted to 10 volume equivalents with pure methanol; S3, detecting the test solution by using ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry to obtain the content of isoflavone compounds in the sample to be tested.

2. The method of claim 1, wherein, In step S1, the total amount of isoflavones in the sample to be tested is 50-500 μg in terms of aglycone.

3. The method of claim 1, wherein, In step S2, the concentration of the acetic acid buffer is 0.1 mol / L, and the pH is 5.

0.

4. The method of claim 1, wherein, In step S2, the sample solution after the enzymatic digestion is filtered with a 0.2 µ polypropylene filter head for storage.

5. The method of claim 1, wherein, In step S3, the detection conditions of the liquid chromatography in the ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry include: Column: C18 column, 50 mm x 2.1 mm, 1.8 μ m.

6. The method of claim 1, wherein, In step S3, the mass spectrometry conditions in the high performance liquid chromatography-tandem triple quadrupole mass spectrometry include: electrospray ion source, ESI + Positive ion.

7. The method of claim 1, wherein, In step S3, the ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry adopts a multiple reaction monitoring mode, and the selected ion parameter conditions of the isoflavone compounds include: Daidzein, retention time 9 ± 0.5 min, parent ion 255.1 m / z , daughter ion 199.1 m / z , capillary acceleration voltage 4 V, collision energy 26 V; Daidzein, retention time 10 ± 0.5 min, parent ion 285.1 m / z , daughter ion 270.1 m / z , capillary acceleration voltage 4 V, collision energy 30 V; Genistein, retention time 12 ± 0.5 min, parent ion 271.1 m / z , daughter ion 215.1 m / z , capillary acceleration voltage 4 V, collision energy 30 V.

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