Method for detecting soybean oligosaccharide content in food

Abstract

The application discloses a kind of soybean oligosaccharide content detection methods in food, the food is soy milk, the soybean oligosaccharide is stachyose, raffinose and sucrose.Method includes the following steps: (1) preparation standard stock solution and standard working solution;(2) preparation sample solution to be measured;(3) high performance liquid chromatography analysis condition;(4) establish standard working curve;(5) result analysis.The application establishes the high performance liquid chromatography determination method of low content level soybean oligosaccharide in soy milk.The application innovatively uses derivatization method to the soybean oligosaccharide (stachyose, raffinose and sucrose) and is derived, thereby greatly improves detection sensitivity, so that simple and easy to operate, without needing to carry out multiple large volume extraction solvent extraction and rotary evaporation concentration processing, and fills the blank of soybean oligosaccharide content detection field in soy milk.The method is simple in operation, high in sensitivity, good in reproducibility and stability, can satisfy the daily detection requirement of low content level soybean oligosaccharide in soy milk for inspection organization and enterprise internal laboratory.

Description

Technical Field

[0001] This invention relates to a high-performance liquid chromatography method for the detection of soybean oligosaccharides in food, especially soy milk. Background Technology

[0002] Soybean oligosaccharides are a general term for soluble sugars in soybeans, mainly composed of raffinose, stachyose, and sucrose. Foods processed from soybeans contain a certain amount of soybean oligosaccharides. The existing standard GB / T22491-2008, "Soybean Oligosaccharides," uses a differential detection method to measure soybean oligosaccharide raw materials, which is not suitable for the accurate determination of extremely low levels of soybean oligosaccharides in food, especially soy milk. Current research reports on soybean oligosaccharides in food mainly use high-performance liquid chromatography-differential refractive index detection or ion chromatography-pulse amperometric detection. No research has been reported on low levels of soybean oligosaccharides in soy milk. Ion chromatography offers high sensitivity, but ordinary laboratories, especially industrial laboratories, generally do not have ion chromatographs. Ion chromatographs typically use conductivity detectors, and using pulse amperometric detectors would significantly increase the cost of such laboratories. Furthermore, ion chromatography is relatively unstable for detecting sugars and is easily affected by complex sample matrices. Currently, high-performance liquid chromatography-differential refractive index (HPLC-DRI) is commonly used to determine soybean oligosaccharides in food. While this detector is stable, its sensitivity is relatively poor due to its general-purpose nature. To improve sensitivity, multiple large-volume extractions with 75% ethanol are often performed, followed by combined extracts and rotary evaporation to near dryness. This process is not only complex but also time-consuming, resulting in difficulty in achieving complete dryness. The resulting viscous substance leads to low recovery rates and difficulty in membrane filtration, causing significant interference during instrumentation. Therefore, it is necessary to establish a rapid, simple, highly sensitive, stable, and easily applicable method for detecting soybean oligosaccharides in soybean milk. Summary of the Invention

[0003] The purpose of this invention is to provide a high-performance liquid chromatography (HPLC) method for detecting low levels of soybean oligosaccharides in food, especially soy milk. The technical solution for achieving this invention is as follows:

[0004] The method for separating and detecting soybean oligosaccharides in food described herein comprises the following specific steps:

[0005] a. Preparation of standard stock solutions and standard working solutions, wherein the standard stock solution has a concentration of 10.0 mg / mL, and the standard working solutions are prepared by stepwise dilution of the standard stock solutions of raffinose, stachyose, and sucrose with pure water to prepare a series of mixed standard working solutions with raffinose concentrations of 0.05 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL, and 1.00 mg / mL; stachyose concentrations of 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL, and 5.00 mg / mL; and sucrose concentrations of 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL, and 5.00 mg / mL.

[0006] b. Preparation of the sample solution: Weigh 2 g (accurate to 0.01 g) of sample into a 10 mL centrifuge tube, add 5.0 mL of water, vortex to mix, and sonicate for 10 min. Add 100 μL each of zinc acetate solution and potassium ferrocyanide solution, and dilute to the mark with water. Mix well. Transfer a portion of the sample solution to a stoppered plastic centrifuge tube, centrifuge at 10,000 r / min for 5 min, and collect the supernatant. Transfer 0.50 mL of the supernatant to a 5 mL stoppered colorimetric tube, add 200 μL of ethyl 4-aminobenzoate solution and 100 μL of sodium cyanoborohydride solution, vortex to mix, and seal the tube. Heat in a 70℃ water bath for 50 min to derivatize. After the reaction solution cools to room temperature, add the sample solution to 1 mL, filter through a 0.45 μm aqueous microporous membrane, and then analyze.

[0007] c. The instrument used was a high-performance liquid chromatograph, with a photodiode array detector; the chromatographic column was C2. 18 The column was set at 35℃, with a detection wavelength of 305 nm. The mobile phase consisted of 0.05% phosphoric acid / methanol / acetonitrile = 75 / 10 / 15 (V / V / V). The flow rate was 1 mL / min, and the injection volume was 10 μL.

[0008] d. Construction of the standard curve: Take 0.50 mL of the series of mixed standard working solutions, process them in the same way as the derivatization step, and then perform high-performance liquid chromatography (HPLC) analysis. Detect under the chromatographic conditions of step c. Plot the concentrations of the raffinose and stachyose standard working solution derivatives on the x-axis and the corresponding peak areas on the y-axis, and perform linear regression to obtain the standard curve equation.

[0009] e. Results analysis: The filtrate from step b was injected into a high-performance liquid chromatograph, and gradient elution and detection were performed under the chromatographic conditions in step c. The peak area of ​​the target analyte in the filtrate was measured, and the retention time was used for qualitative analysis. The standard curve prepared in step d was used for quantitative analysis, and the contents of raffinose, stachyose and sucrose in the sample were calculated.

[0010] Innovation of this invention:

[0011] Unlike existing high-performance liquid chromatography-differential refractive index detection and / or ion chromatography-pulse amperometric detection methods for detecting soybean oligosaccharides in food, this invention innovatively introduces ethyl 4-aminobenzoate with ultraviolet and fluorescent absorbing groups as a derivatizing agent. This transforms soybean oligosaccharides (sucrose, raffinose, and stachyose) from non-luminescent targets into targets with ultraviolet and fluorescent absorbing groups, enabling soybean oligosaccharides without ultraviolet absorption or fluorescent chromophores to be detected by ultraviolet detectors or fluorescence detectors. This not only significantly improves detection sensitivity (the detection limit of the diode array detector of this invention can be as low as 0.02 g / 100g, and the detection limit of the fluorescence detector can reach 0.0004 g / 100g), enabling accurate quantitative detection of low levels of soybean oligosaccharides in soy milk, but also eliminates the need for large sample volumes, multiple large-volume extractions with solvents (such as 75% ethanol), and the need to evaporate a large amount of extraction solvent by rotary evaporation to increase the concentration factor and improve detection sensitivity. This greatly reduces solvent consumption and operation time, making the pretreatment operation simple and easy.

[0012] This invention selects 305 nm, which is within a relatively large wavelength range, as the quantitative wavelength by comparison, thereby avoiding interference from a large number of interfering substances in the natural matrix of soy milk. With 305 nm as the quantitative wavelength, the detection requirements for extremely low levels of soybean oligosaccharides in the complex matrix of soy milk can be fully met without the need for solid phase extraction.

[0013] This invention selects a suitable type C 18 Column (150 mm long C) 18 Using a column as the stationary phase, we continuously explore and optimize the composition and ratio of various mobile phases, such as adjusting the acidity of the mobile phase and different ratios of methanol, acetonitrile, and acidic mobile phases, in order to achieve the best sensitivity and separation for soybean oligosaccharides and natural impurities in soybean milk. This allows for the detection of low levels of soybean oligosaccharides in soybean milk, a natural matrix, without the need for column pretreatment, making the pretreatment operation simple, easy to implement, and easy to promote and popularize.

[0014] This invention utilizes a derivatization reaction to transform soybean oligosaccharides, a target substance without absorbing groups, into a target substance with ultraviolet (UV) or fluorescent (FL) absorbing groups, enabling the soybean oligosaccharides to be detected by UV or FLIR detectors. This is extremely important in laboratories with limited equipment. The flexibility of this method in terms of detector equipment allows testing institutions or in-house laboratories to adjust and arrange instruments according to actual needs, promptly meeting the detection requirements for soybean oligosaccharides in soy milk. This also makes this invention more readily accepted and promoted by a wider range of enterprises.

[0015] The analytical instrument used in this invention is a common high-performance liquid chromatograph equipped with a photodiode array detector. Compared with ion chromatographs, which have a narrower audience, this method is easier to popularize and promote. At the same time, it can overcome the shortcomings of the differential refractive index detector equipped with high-performance liquid chromatograph, which has poor sensitivity and cannot meet the requirements for the detection of low content soybean oligosaccharides.

[0016] This invention innovatively introduces a derivatization reaction system, using the highly stable derivatizing agent ethyl 4-aminobenzoate to derivatize and transform soybean oligosaccharides (sucrose, stachyose, raffinose) into target compounds simultaneously possessing UV-absorbing and fluorescent groups. Ordinary liquid chromatography equipment is used in conjunction with common and inexpensive C14 chromatography. 18 This chromatographic column can achieve highly sensitive, stable, and reliable detection of soybean oligosaccharides in soy milk. Due to its significantly improved sensitivity, samples only require simple protein precipitation and dilution with water before processing. Furthermore, the target analytes possess both UV and fluorescent absorbing groups, further increasing the applicability and operational flexibility of this invention. Even under conditions of limited instrumentation, it allows for flexible use of different liquid chromatography instruments equipped with diode array detectors or fluorescence detectors. This invention provides a novel technical approach for the analysis and detection of low levels of soybean oligosaccharides in soy milk or other foods by relevant testing institutions and in-house laboratories. Attached Figure Description

[0017] The attached figure shows the chromatogram of soybean oligosaccharide standards.

[0018] Figure 1 Chromatogram of soybean oligosaccharide standards (stachyose 0.5 mg / mL, raffinose 0.5 mg / mL, sucrose 1.0 mg / mL) (photodiode array detector);

[0019] Figure 2 Chromatogram of soybean oligosaccharide standards (stachyose 0.5 mg / mL, raffinose 0.5 mg / mL, sucrose 1.0 mg / mL) (fluorescence detector). Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. This embodiment is implemented based on the technology of the present invention, and detailed implementation methods and specific operating procedures are given to illustrate the inventiveness of the present invention. However, the scope of protection of the present invention is not limited to the following embodiments.

[0021] Example 1

[0022] Unless otherwise specified, all reagents are of analytical grade, and the water is Grade I water as specified in GB / T 6682.

[0023] 1.1 Acetonitrile (HPLC)

[0024] 1.2 Methanol (HPLC)

[0025] 1.3 Glacial acetic acid (AR)

[0026] 1.4 Phosphoric acid (AR)

[0027] 1,5-4-Aminobenzoate (AR);

[0028] 1.6 Cyanoboron hydrocracking (AR);

[0029] 1.7 Zinc acetate (AR);

[0030] 1.8 Potassium ferrocyanide (AR);

[0031] 1.9 Cottonseed sugar standard (purity ≥ 98%);

[0032] 1.10 Stachyose standard (purity ≥98%);

[0033] 1.11 Ethyl 4-aminobenzoate (ABEE) solution: Dissolve 5.0 g of ethyl 4-aminobenzoate (ABEE) and 5.0 g of acetic acid in water.

[0034] Dilute to 100 mL in methanol and store at 4°C for later use.

[0035] 1.12 Sodium cyanoborohydride (NaBH3CN) solution: Dissolve 0.50 g of sodium cyanoborohydride in methanol and bring the volume to 10 mL.

[0036] Store in a refrigerator at 4°C for later use.

[0037] 1.13 Zinc acetate solution: Weigh 21.9 g of zinc acetate, add 3.0 mL of glacial acetic acid, dissolve in water and bring the volume to 100 mL.

[0038] 1.14 Potassium ferrocyanide solution: Weigh 10.6 g of potassium ferrocyanide, dissolve it in water and make up to 100 mL.

[0039] 1.15 Phosphoric acid solution (0.05%): Transfer 0.50 mL of phosphoric acid to 1000 mL of water and shake well.

[0040] 1.16 Raffinose Standard Stock Solution (10.0 mg / mL): Accurately weigh 100 mg of raffinose standard (accurate to 0.1 mg).

[0041] Dissolve and dilute to the mark with water in a 10 mL volumetric flask to prepare a raffinose standard stock solution with a mass concentration of 10.0 mg / mL. Store at 4°C for use. Shelf life is 3 months.

[0042] Stachyose standard stock solution (10.0 mg / mL): Accurately weigh 100 mg of stachyose standard (accurate to 0.1 mg) into a 10 mL volumetric flask, dissolve in water and dilute to the mark to prepare a stachyose standard stock solution with a mass concentration of 10.0 mg / mL. Store at 4°C for later use. Shelf life is 3 months.

[0043] Sucrose standard stock solution (10.0 mg / mL): Accurately weigh 100 mg of sucrose standard (accurate to 0.1 mg) into a 10 mL volumetric flask, dissolve in water and dilute to the mark to prepare a sucrose standard stock solution with a mass concentration of 10.0 mg / mL. Store at 4℃ for later use. Shelf life is 3 months.

[0044] 1.17 Working series of mixed standard solutions of raffinose, stachyose, and sucrose: Transfer an appropriate volume of solution with a concentration of 10.0 mg / mL.

[0045] Raffinose, stachyose, and sucrose standard stock solutions were diluted with water to prepare a series of mixed standard working solutions with raffinose concentrations of 0.05 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL, and 1.00 mg / mL; stachyose concentrations of 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL, and 5.00 mg / mL; and sucrose concentrations of 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL, and 5.00 mg / mL.

[0046] 2.1 High-performance liquid chromatograph: equipped with a photodiode array detector or a fluorescence detector.

[0047] 2.2 Analyze the balance.

[0048] 2.3 Ultrasonic cleaning machine.

[0049] 2.4 High-speed centrifuge.

[0050] 2.5 Vortex mixer.

[0051] 2.6 Constant temperature water bath.

[0052] 3.1 High Performance Liquid Chromatography Reference Conditions

[0053] a) Chromatographic column: C 18 Columns (4.6 × 150 mm, 5.0 μm) or equivalent performance;

[0054] b) Mobile phase: 0.05% phosphoric acid / methanol / acetonitrile = 75 / 10 / 15 (V / V / V);

[0055] c) Flow rate: 1.0 mL / min;

[0056] d) Column temperature: 35 ℃;

[0057] e) Injection volume: 10 μL;

[0058] f) Photodiode array detector: wavelength 305 nm; fluorescence detector: excitation wavelength 361 nm, emission wavelength 305 nm.

[0059] 3.2 Plotting the Standard Curve

[0060] Take 0.50 mL of the series of mixed standard working solutions, process them according to the derivatization procedure, and then perform high-performance liquid chromatography (HPLC) analysis. Detect under the chromatographic conditions in 3.1. Plot the concentrations of the sucrose, raffinose, and stachyose standard working solution derivatives on the x-axis and the corresponding peak areas on the y-axis to obtain the standard curve equation through linear regression.

[0061] 4. Sample Testing Procedures

[0062] 4.1 Sample Pretreatment

[0063] 4.1.1 Extraction

[0064] Weigh 2 g (accurate to 0.01 g) of sample into a 10 mL centrifuge tube, add 5.0 mL of water, vortex to mix, and sonicate for 10 min. Add 100 μL each of zinc acetate solution and potassium ferrocyanide solution, and dilute to the mark with water. Mix well. Transfer a portion of the sample solution to a stoppered plastic centrifuge tube, centrifuge at 10,000 r / min for 5 min, and collect the supernatant for later use.

[0065] 4.1.2 Derivatives

[0066] Transfer 0.50 mL of the supernatant to a 5 mL stoppered colorimetric tube, add 200 μL of ethyl 4-aminobenzoate solution and 100 μL of sodium cyanoborohydride solution, vortex to mix, seal the tube, and heat in a 70℃ water bath for 50 min for derivatization. After the reaction solution cools to room temperature, add water to make up to 1 mL of sample solution, and filter through a 0.45 μm aqueous microporous membrane for analysis.

[0067] 4.2 Test solution determination

[0068] The sample was analyzed according to the chromatographic conditions in 3.1. Qualitative analysis was performed based on retention time or a diode array detector (DAD) spectrum, and quantification was performed using the external standard method. The contents of sucrose, raffinose, and stachyose derivatives should be within the linear range of the standard curve; if they exceed the linear range, the sample should be diluted before analysis. Chromatograms of soybean oligosaccharide standards (stachyose 0.5 mg / mL, raffinose 0.5 mg / mL, sucrose 1.0 mg / mL) (photodiode array detector, fluorescence detector) are shown in Appendix A.

[0069] 4.3 Blank Test

[0070] Except for not weighing the sample, the determination shall be carried out in parallel with the test according to steps 4.1 to 4.2.

[0071] The content of soybean oligosaccharides in the sample is calculated using the following formula:

[0072]

[0073] In the formula:

[0074] X — The content of raffinose, stachyose and sucrose in the sample, in g / 100g;

[0075] m—mass of the sample, in grams;

[0076] c i —Concentrations of raffinose, stachyose, and sucrose in the sample solution, in mg / mL;

[0077] V – Sample final volume.

[0078] The result is expressed as the arithmetic mean of two independent measurements obtained under repeatability conditions. The blanks must be removed and the result is retained to three decimal places.

[0079] 6.1 Limits of quantitation for diode array detection: Limit of quantitation for raffinose: 0.02 g / 100g; Limit of quantitation for stachyose: 0.03 g / 100g; Limit of quantitation for sucrose: 0.03 g / 100g.

[0080] Limits of quantitation for fluorescence detectors: raffinose limit of quantitation is 0.0004 g / 100g; stachyose limit of quantitation is 0.0006 g / 100g; sucrose limit of quantitation is 0.0006 g / 100g.

[0081] 6.2 Precision: The relative standard deviation of the two independent test results of the obtained sample is no greater than 10%.

[0082] Table 1. Spiked recovery rate of stachyose in soy milk

[0083]

[0084] Table 2. Spiked recovery rate of raffinose in soy milk

[0085]

[0086] Table 3. Spiked recovery rate of sucrose in soy milk

[0087]

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting the content of soybean oligosaccharides in a food, characterized by, The food is soy milk, the soybean oligosaccharide is raffinose, stachyose and sucrose; the detection method comprises the following steps: a. Preparation of standard stock solution and standard working solution, the standard stock solution is 10.0 mg / mL, and the standard working solution is prepared by using the step-by-step dilution method, wherein the standard stock solution of raffinose, stachyose and sucrose is diluted with pure water to prepare a series of mixed standard working solutions with the concentration of raffinose being 0.05 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL and 1.00 mg / mL, the concentration of stachyose being 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL and 5.00 mg / mL, and the concentration of sucrose being 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL and 5.00 mg / mL; b. Preparation of the sample solution to be detected: 5.0 mL of water is added to the sample, vortexed and uniformly mixed, ultrasonically extracted for 10 min, 100 μL of zinc acetate solution and 100 μL of potassium ferrocyanide solution are added, water is added to the scale, mixed, and part of the sample solution is taken into a plastic centrifuge tube with a plug, centrifuged for 5 min, and the supernatant is taken for standby, 0.50 mL of the supernatant is removed, 200 μL of 4-aminobenzoic acid ethyl ester solution and 100 μL of sodium cyanoborohydride solution are added, vortexed and uniformly mixed, sealed, heated in a 70℃ water bath for 50 min, and after the reaction solution is cooled to room temperature, water is added to 1 mL, and 0.45 μm water phase microporous filter membrane is used for detection; c.The instrument used is high performance liquid chromatograph, and the detector is photodiode array detector; the chromatographic column is C 18 18-A column, the column temperature is 35°C, the detection wavelength is 305 nm; the mobile phase is 0.05% phosphoric acid / methanol / acetonitrile=75 / 10 / 15 V / V / V; the flow rate is 1 mL / min; the injection volume is 10 μL; d. Preparation of the standard curve: 0.50 mL of the series of mixed standard working solutions is taken, and after being treated in the same way as the derivation step, it is determined by high performance liquid chromatography, and under the chromatographic conditions of step c, the concentration of the sucrose, raffinose and stachyose standard working solution derivative is taken as the abscissa, and the corresponding peak area is taken as the ordinate, and linear regression is performed to obtain the standard curve equation; e. Result analysis: the filtrate in step b is injected into the high performance liquid chromatograph, elution and detection are performed under the chromatographic conditions of step c, the peak area of the target object in the filtrate is measured, the retention time is used for qualitative analysis, the standard curve prepared in step d is used for quantitative analysis, and the content of sucrose, raffinose and stachyose in the sample to be detected is calculated.

2. The method according to claim 1, wherein, The preparation of the standard stock solution in step a is specifically as follows: 100 mg of raffinose standard is accurately weighed, dissolved in a 10 mL volumetric flask with water, and diluted to the calibration mark to prepare a raffinose standard stock solution with a mass concentration of 10.0 mg / mL; 100 mg of stachyose standard is accurately weighed, dissolved in a 10 mL volumetric flask with water, and diluted to the calibration mark to prepare a stachyose standard stock solution with a mass concentration of 10.0 mg / mL; 100 mg of sucrose standard is accurately weighed, dissolved in a 10 mL volumetric flask with water, and diluted to the calibration mark to prepare a sucrose standard stock solution with a mass concentration of 10.0 mg / mL; the preparation of the standard working solution in step a is specifically as follows: a certain volume of the raffinose, stachyose and sucrose standard stock solutions with a concentration of 10.0 mg / mL is diluted with water to prepare a series of mixed standard working solutions with a raffinose concentration of 0.05 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.50 mg / mL and 1.00 mg / mL; a stachyose concentration of 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL and 5.00 mg / mL; and a sucrose concentration of 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 2.00 mg / mL and 5.00 mg / mL.

3. The method for detecting soybean oligosaccharide content in food according to claim 1, characterized in that, The preparation of the sample solution to be tested in step b is specifically as follows: 2 g of the sample is weighed into a 10 mL centrifuge tube, 5.0 mL of water is added, vortexed and mixed, ultrasonically extracted for 10 min, 100 uL of zinc acetate solution and 100 uL of potassium ferrocyanide solution are added, diluted to the calibration mark with water, mixed, and a portion of the sample solution is taken into a plastic centrifuge tube with a stopper, centrifuged at 10 000 r / min for 5 min, and the supernatant is taken for standby use. 0.50 mL of the supernatant is taken into a 5 mL colorimetric tube with a stopper, 200 μL of 4-aminobenzoic acid ethyl ester solution and 100 μL of sodium cyanoborohydride solution are added, vortexed and mixed, the tube is sealed, placed in a 70℃ water bath, and heated for derivatization for 50 min. After the reaction solution is cooled to room temperature, the sample solution is supplemented with water to 1 mL, and filtered through a 0.45 μm water phase microporous filter for testing.

4. The method according to claim 1, wherein The specification of the C18 chromatographic column in step c is 4.6 mm x 50 mm in diameter, and the particle size is 5 μm.

5. The method according to claim 1, wherein the soybean oligosaccharide content in the food is detected. The coefficient of the standard curve in step d is not less than 0.999.

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