Method for detecting glycosylglycerol in dairy product

Abstract

The invention belongs to the field of analysis and detection, and particularly relates to a method for detecting glycosylglycerol in dairy products. The method is realized by the following steps: firstly, pre-treating a sample; preparing a standard stock solution; and an ion chromatography method is adopted for detection. According to the detection method provided by the invention, the glycosylglycerol in the dairy product can be effectively detected, the matrix effect is greatly reduced, a complicated pretreatment process is not needed, the specificity is high, the detection time is greatly reduced, the repeatability is good, and a technical support and a quality control guarantee are provided for the application of monitoring the glycosylglycerol in the dairy product; according to the detection method provided by the invention, the adsorption of the target component on the ion exchange resin can be reduced, the retention time is shortened, the peak trailing is reduced, and the detection sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the field of analytical detection, and particularly relates to a method for detecting glucosylglycerol in dairy products. Background Art

[0002] Glucosylglycerol, with the molecular formula C9H18O8, is a class of naturally occurring small active isoglycoside molecules in nature. It can be synthesized by a variety of green plants (such as lilies, resurrection plants), algae (such as various cyanobacteria), and some heterotrophic organisms, and plays an important role in the resistance and adaptation of living organisms to adverse environmental conditions. As a class of glycoside compounds formed by the connection of glycerol molecules and glucose molecules through glycosidic bonds, glucosylglycerol has functions such as skin moisturization, promoting the synthesis of collagen in dermal cells, and increasing the metabolic rate of aging cells. In addition, it has good biocompatibility, and has stability to biological macromolecules, high moisture retention, non-cariogenicity, and potential health care and therapeutic effects, and is widely used in industries such as cosmetics, food industry, and pharmaceutical field.

[0003] In the food industry, glucosylglycerol is often used as an emulsifier, stabilizer, humectant, etc., which can improve the quality and taste and extend the shelf life of food. Chinese Patent Application CN118759092A discloses a method for detecting glucosylglycerol in cosmetics, which simplifies the pretreatment steps and shortens the detection time. However, in the prior art, there is no relevant record of detecting glucosylglycerol in dairy products, and due to the completely different matrices of dairy products and cosmetics, the applicability between methods is poor and detection cannot be carried out. Summary of the Invention

[0004] Aiming at the problems existing in the prior art, the present invention provides a method for detecting glucosylglycerol in dairy products.

[0005] The technical solution adopted by the present invention to achieve the above purpose is as follows: The present invention provides a method for detecting glucosylglycerol in dairy products, comprising the following steps: (1) Sample pretreatment: Weigh a liquid dairy product, first add chloroform, vortex and shake, and then remove the chloroform layer; then add a methanol aqueous solution, vortex and shake to disperse the sample evenly, and perform ultrasonic extraction; after the ultrasonic extraction is completed, centrifuge, take the supernatant, add pure water, and filter through a filter membrane to obtain a sample to be detected; (2) Standard stock solution: Accurately weigh glucosylglycerol and dissolve it with a methanol aqueous solution; Standard working solution: Accurately pipette an appropriate amount of the standard stock solution and dilute it with water to form a series of working solutions with different concentrations; (3) Use ion chromatography for detection.

[0006] Preferably, in step (1), the ratio of the dairy product to chloroform is 1 g:10; the ratio of the dairy product to the aqueous methanol solution is 1 g:10 mL; the volume concentration of the aqueous methanol solution is 10%; the volume ratio of the supernatant to pure water is 1:1.

[0007] Preferably, in step (1), the ultrasonic extraction is carried out at a temperature of 40 °C for 60 min; the centrifugation is carried out at 5000 r / min for 10 min.

[0008] Preferably, in step (2), the volume concentration of the aqueous methanol solution is 10%; the concentration of the standard stock solution is 1000 mg / L; the concentrations of the standard working solutions are 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mg / L.

[0009] Preferably, in step (3), the ion chromatography conditions are as follows: chromatographic column: Hamilton RCX-30 anion exchange chromatographic column, 4.6 × 250 mm, particle size 7 μm; eluent A is 50 mmol / L sodium hydroxide + 500 mmol methanol, eluent B is 50 mmol / L sodium hydroxide + 500 mmol / L sodium acetate; flow rate: 1 mL / min; column temperature: 30 °C; injection volume: 20 μL; gradient elution; detector: integrated pulsed amperometric detector; gold working electrode.

[0010] Preferably, during the detection process, the detection potential waveform is: 。

[0011] Preferably, the specific program of the gradient elution is: 。

[0012] The beneficial effects of the present invention are as follows: (1) The detection method provided by the present invention can effectively detect glucosylglycerol in dairy products, greatly reduce the matrix effect, and does not require a complex pretreatment process, has strong specificity, greatly reduces the detection time, and has good repeatability, providing technical support and quality control guarantee for the monitoring of the application of glucosylglycerol in dairy products; (2) The detection method provided by the present invention can reduce the adsorption of the target component on the ion exchange resin, shorten the retention time, reduce peak tailing, and improve the detection sensitivity. Description of the Drawings

[0013] Figure 1 is the standard curve; Figure 2 is the ion chromatogram of glucosylglycerol; Figure 3Ion chromatogram of glucosylglycerol for Comparative Example 1. Detailed implementation manners

[0014] The technical solutions of the present invention will be further explained and illustrated below through specific examples.

[0015] Example 1 1 Materials and methods 1.1 Main reagents and instruments Glucosylglycerol (CAS 22160-26-5, w≥99%), Qingdao Zhongke Lanzhi Biotechnology Development Co., Ltd.; methanol (chromatographic grade), Fisher Company, USA; experimental water, ultrapure water, filtered through a 0.22 μm aqueous filter membrane before injection.

[0016] Ion chromatograph, Metrohm Company, Switzerland; numerically controlled ultrasonic cleaner: KQ-500DB, Kunshan Ultrasonic Instruments Co., Ltd.; organic needle filter: nylon 66 (0.22μm), Shanghai Anpel Laboratory Technologies Inc.

[0017] 1.2 Preparation of standard solutions Standard stock solution (1000 mg / L): Accurately weigh 10 mg of glucosylglycerol, dissolve it with 10% methanol water (V:V = 1:9), transfer it to a 10 mL volumetric flask, and make up to the mark. Store it in the dark in a refrigerator at 4 °C.

[0018] Standard working solutions: Accurately pipette an appropriate amount of the standard stock solution and dilute it with water to prepare working solutions with concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mg / L.

[0019] 1.3 Experimental methods Ion chromatography conditions: Chromatographic column: Hamilton RCX-30 anion exchange chromatographic column (4.6 × 250 mm, particle size 7 μm); eluent: A (50 mmol / L sodium hydroxide), B (50 mmol / L sodium hydroxide + 500 mmol / L sodium acetate); flow rate: 1 mL / min; column temperature: 30 °C; injection volume: 20 μL; detector: integrated pulsed amperometric detector; gold working electrode. The detection potential waveform is shown in Table 1, and the gradient elution settings are shown in Table 2.

[0020] Table 1 Detection potential waveform Table 2 Eluent gradient settings 1.4 Sample preparation and sample pretreatment Weigh 1.0 g (accurate to 0.1 mg) of liquid dairy products and place them in a 50 mL centrifuge tube. First, add 10 mL of chloroform, vortex and shake, then remove the chloroform layer. Add 10 mL of 10% methanol-water (V:V = 1:9) solution, vortex and shake to disperse the sample evenly, and ultrasonically extract for 60 min at a water temperature of 40 °C. After the ultrasonic extraction is completed, place it in a centrifuge and centrifuge at 5000 r / min for 10 min. Take 1 mL of the supernatant, add 1 mL of pure water, and filter through a 0.22 μm organic filter membrane for testing.

[0021] 1.5 Linear equations, ranges, and detection limits Prepare standard solutions with mass concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mg / L. Using the peak area of the target as the ordinate and the mass concentration as the abscissa, establish a linear equation. The linear equations, detection limits, and correlation coefficients of the target are shown in Table 3. The correlation coefficient is 0.9997, and the linear relationship is good within the prepared mass concentration range, meeting the quantitative requirements of the target. The standard curve is as Figure 1 shown. The detection spectrum is as Figure 2 shown.

[0022] Table 3 Linear ranges and linear equations of glucosylglycerol During the detection process, the specific retention time, etc. are shown in Table 4.

[0023] Table 4 1.6 Recovery rates and precisions Prepare blank samples according to 1.5, conduct a standard addition recovery experiment at three spiking levels, perform GC-MS analysis on the instrument according to the experimental conditions, parallelly determine each concentration level 6 times, and calculate the recovery rates and precisions, as shown in Table 5. The results show that the standard addition recovery rates of glucosylglycerol are 95.9% - 108.3%, and the relative standard deviations are 2.5% - 5.0%, meeting the detection requirements.

[0024] Table 5 Method precisions and standard addition recovery rates Comparative Example 1 The other experimental procedures are the same as those in Example 1.

[0025] Sample pretreatment: Weigh 1.0 g (accurate to 0.1 mg) of liquid dairy products, place them in a 50 mL centrifuge tube, add 10 mL of 10% methanol-water (V:V = 1:9) solution, vortex to disperse the sample evenly, and ultrasonically extract for 60 min at a water temperature of 40 °C. After the ultrasonic extraction is completed, place it in a centrifuge and centrifuge at 5000 r / min for 10 min. Take 1 mL of the supernatant, add 1 mL of pure water, and filter through a 0.22 μm organic filter membrane for testing.

[0026] After adopting this pretreatment process, after detection, in addition to the peaks of the target substances, there are impurity peaks in the chromatogram, and the matrix effect is still about 80%; as Figure 3 shown.

[0027] Comparative Example 2 Other experimental procedures are the same as those in Example 1.

[0028] Ion chromatography conditions: Chromatographic column: Hamilton RCX-30 anion exchange chromatographic column (4.6 × 250 mm, particle size 7 μm); Eluent: A (50 mmol / L sodium hydroxide), B (50 mmol / L sodium hydroxide + 500 mmol / L sodium acetate); Flow rate: 1 mL / min; Column temperature: 30 °C; Injection volume: 20 μL; Detector: Integrating pulsed amperometric detector; Gold working electrode.

[0029] After changing the eluent, the retention time becomes about 2 - 3 min longer, and peak tailing appears, affecting the detection sensitivity.

[0030] Effect Example For 10 batches of liquid dairy product samples, the test solutions were prepared by the method provided in Example 1, and injected and determined according to the analysis conditions provided in Example 1. The target compounds were not detected in the samples to be tested. Through the spiking experiment for detection, under the same added amount, the method provided in Example 1 could detect them all; 8 batches were detected in Comparative Example 1, and 8 batches were detected in Comparative Example 2.

Claims

1. A method for detecting glucosylglycerol in dairy products, characterized in that, It includes the following steps: (1) Sample pretreatment: Weigh a liquid dairy product. First, add chloroform, vortex and shake, and then remove the chloroform layer. Then add an aqueous methanol solution, vortex and shake to disperse the sample evenly, and perform ultrasonic extraction. After the ultrasonic extraction is completed, centrifuge, take the supernatant, add pure water, and filter through a filter membrane to obtain the sample to be tested; (2) Standard stock solution: Accurately weigh glucosylglycerol and dissolve it in an aqueous methanol solution; Standard working solution: Accurately pipette an appropriate amount of the standard stock solution and dilute it with water to form a series of working solutions with different concentrations; (3) Use ion chromatography for detection.

2. The detection method according to claim 1, wherein In step (1), the ratio of the dairy product to chloroform is 1 g:10; the ratio of the dairy product to the aqueous methanol solution is 1 g:10 mL; the volume concentration of the aqueous methanol solution is 10%; the volume ratio of the supernatant to pure water is 1:

1.

3. The detection method according to claim 2, characterized in that In step (1), the ultrasonic extraction is carried out at a temperature of 40 °C for 60 min; the centrifugation is carried out at 5000 r / min for 10 min.

4. The detection method according to any one of claims 1 to 3, characterized in that, In step (2), the volume concentration of the aqueous methanol solution is 10%; the concentration of the standard stock solution is 1000 mg / L; the concentrations of the standard working solutions are 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0 mg / L.

5. The detection method according to claim 1 or 4, characterized in that In step (3), the ion chromatography conditions are as follows: Chromatographic column: Hamilton RCX-30 anion exchange chromatographic column, 4.6 × 250 mm, particle size 7 μm; Eluent A is 50 mmol / L sodium hydroxide + 500 mmol / L methanol, and eluent B is 50 mmol / L sodium hydroxide + 500 mmol / L sodium acetate; Flow rate: 1 mL / min; Column temperature: 30 °C; Injection volume: 20 μL; Gradient elution; Detector: Integrating pulsed amperometric detector; Gold working electrode.

6. The detection method according to claim 5, wherein During the detection process, the detection potential waveform is: 。 7. The detection method according to claim 5 or 6, characterized in that The specific program of the gradient elution is: 。

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