Method for detecting content of 5-hydroxymethylfurfural in lactobacillus fermented cow milk product

Abstract

The invention discloses a method for detecting the content of 5-hydroxymethylfurfural in a lactobacillus fermented cow milk product, and relates to the technical field of food detection. The invention provides a method for detecting the content of 5-hydroxymethylfurfural in a lactobacillus fermented cow milk product, which comprises the following steps: detecting the chromaticity DE value of the lactobacillus fermented cow milk product by using a colorimeter, and substituting the chromaticity DE value into an equation to obtain the content of 5-hydroxymethylfurfural, the equation is as follows: Y is equal to 0.059 X < 2 >-1.2971 X + 5.5086; in the formula, X is a chroma DE value, and Y is the content of 5-hydroxymethylfurfural. The content of the 5-hydroxymethylfurfural is rapidly detected through the colorimeter, and the detection method is rapid, simple, convenient and effective.

Description

Technical Field

[0001] The invention relates to the technical field of food detection, and in particular to a method for detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product. Background Art

[0002] Lactic acid bacteria fermented milk is one of the most widely used products among lactic acid bacteria fermented foods. After lactic acid bacteria fermentation, milk produces a large amount of lactic acid bacteria metabolites such as extracellular polysaccharides, active peptides, and lactic acid, which can play a wide range of metabolic and regulatory roles. It has physiological functions such as regulating intestinal flora, enhancing the body's immunity, alleviating non-alcoholic fatty liver, anti-oxidation, and lowering blood lipids and cholesterol. During the lactic acid bacteria fermentation and the molding of finished products, especially when treated at high temperatures, milk is very likely to undergo the Maillard reaction, generating a series of aromatic and color substances, of which 5-hydroxymethylfurfural is one of the representative products. Relevant studies have shown that as one of the intermediate products of the Maillard reaction, the content of 5-hydroxymethylfurfural has a certain correlation with its color change. Therefore, 5-hydroxymethylfurfural can be used as one of the important indicators to measure the sensory quality of lactic acid bacteria fermented milk products.

[0003] The prior art discloses a method for detecting 5-hydroxymethylfurfural (Shiqiao, Luo Yifan, Xu Qiankun, et al. Rapid detection of chestnut shell-based 5-hydroxymethylfurfural and process parameter optimization [J]. Guangdong Chemical Industry, 2024, 51 (24): 32-35). The method uses ultraviolet three-wavelength spectrophotometry to detect the content of 5-hydroxymethylfurfural in chestnut shell extract. In this method, water is used to replace glacial acetic acid as the sample dilution solvent, which greatly reduces the detection cost. However, it is necessary to determine the detection wavelength based on the substances contained in the sample. The detection wavelengths of different samples are different, and the detection wavelength of lactic acid bacteria fermented milk cannot be determined.

[0004] Chinese patent CN104931594A discloses a method for detecting the content of 5-hydroxymethylfurfural in Schisandra chinensis, which uses high performance liquid chromatography to determine 5-hydroxymethylfurfural in Schisandra chinensis. The detection method comprises the following steps: (A) preparation of a test solution; (B) preparation of a reference solution; (C) determination by high performance liquid chromatography, respectively accurately aspirating the reference solution and the test solution, injecting them into a liquid chromatograph, determining them according to certain chromatographic conditions, quantifying them by external standard method, and calculating them. The method for detecting 5-hydroxymethylfurfural by high performance liquid chromatography in the invention has high sensitivity and good reproducibility.

[0005] However, the current determination of 5-hydroxymethylfurfural content mostly uses high performance liquid chromatography and UV-visible spectrophotometry. These determination methods require the purchase of corresponding standards and special reagents, and have problems such as cumbersome operation and high testing costs.

[0006] Currently, there is no method for detecting the content of 5-hydroxymethylfurfural in lactic acid bacteria fermented milk in the prior art. Therefore, there is an urgent need to provide a method for detecting the content of 5-hydroxymethylfurfural in lactic acid bacteria fermented milk that is simple to operate and has high accuracy. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: On the one hand, the present invention provides a method for detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product, which comprises using a colorimeter to detect the chromaticity DE value of the lactic acid bacteria fermented milk product, substituting the chromaticity DE value into an equation to obtain the content of 5-hydroxymethylfurfural; The equation is: Y=0.059X 2 -1.2971X+5.5086; Wherein, X is the chromaticity DE value, and Y is the 5-hydroxymethylfurfural content.

[0009] Specifically, the test mode of the colorimeter is the reflection mode, and the detection parameters are: the aperture plate specification is 6-25 mm, and the number of captures is 1-5 times.

[0010] According to some embodiments of the present invention, the specifications of the aperture plate are 6 mm, 10 mm, and 25 mm.

[0011] Furthermore, the aperture plate has a specification of 6 mm and the number of capture times is 3 times.

[0012] In another aspect, the present invention provides a method for evaluating the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product, the evaluation method comprising the following steps: (1) Use a colorimeter to detect the color DE value of lactic acid bacteria fermented milk products; (2) Detection of 5-hydroxymethylfurfural in lactic acid bacteria fermented milk products by high performance liquid chromatography; (3) The correlation between the 5-hydroxymethylfurfural content detected by HPLC and the colorimetric DE value detected by colorimeter was analyzed, and an evaluation equation was constructed; (4) The colorimetric DE value of the lactic acid bacteria fermented milk product was measured by a colorimeter and substituted into the evaluation equation to obtain the content of 5-hydroxymethylfurfural.

[0013] Specifically, the test mode of the colorimeter is the reflection mode, and the detection parameters are that the aperture plate specification is 6-25 mm, and the number of acquisitions is 1-5 times. Preferably, the aperture plate specification is 6 mm, 10 mm, 25 mm, and the number of acquisitions is 3 times.

[0014] Specifically, the high performance liquid chromatography column is an octadecylsilane bonded silica gel chromatography column; Furthermore, the high performance liquid chromatography column is an Agilent Zorbax SB-Aq chromatography column.

[0015] Furthermore, the specifications of the HPLC column are 250 mm×4.6 mm, 5 μm.

[0016] Specifically, the mobile phase for the high performance liquid chromatography detection is formic acid solution-acetonitrile.

[0017] Furthermore, the volume ratio of the formic acid solution to acetonitrile is 98:2.

[0018] Furthermore, the mobile phase for the high performance liquid chromatography detection is 0.1% formic acid solution-acetonitrile.

[0019] Furthermore, the detection conditions of the high performance liquid chromatography are: Flow rate 0.9-1.2 mL / min; Injection volume 10-15 μL; Column temperature 25-35°C; The detection wavelength is 284 nm.

[0020] Furthermore, the detection conditions of the high performance liquid chromatography are: Flow rate: 1.0 mL / min; Injection volume: 10 μL; Column temperature: 30°C; The detection wavelength is 284 nm.

[0021] Furthermore, the correlation analysis method in step (3) is selected from any one of Pearson correlation coefficient, Spearman rank correlation coefficient, Kendall correlation coefficient, and partial correlation analysis; Furthermore, the method of correlation analysis in step (3) is Spearman rank correlation coefficient.

[0022] According to some embodiments of the present invention, the evaluation equation constructed in step (3) is Y=0.059X 2 -1.2971X+5.5086.

[0023] In yet another aspect, the present invention provides application of the above detection method in quality detection of lactic acid bacteria fermented milk.

[0024] Specifically, the application is to evaluate whether the lactic acid bacteria fermented milk produces the Maillard reaction by detecting the content of 5-hydroxymethylfurfural in the lactic acid bacteria fermented milk.

[0025] The beneficial effects of the present invention are: The method for quickly detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product based on the DE value of a colorimeter described in the present invention can quickly detect the content of 5-hydroxymethylfurfural through the DE value. The detection method is fast, simple and effective, and avoids the use of complex, cumbersome and costly detection methods such as high performance liquid chromatography and spectrophotometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are the results of DE value determination in 18 different batches of lactic acid bacteria fermented milk products.

[0027] Figure 2 These are the results of determination of 5-hydroxymethylfurfural content in 18 different batches of lactic acid bacteria fermented milk products.

[0028] Figure 3 is the regression equation of DE value and 5-hydroxymethylfurfural content of lactic acid bacteria fermented milk products. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0030] Experimental instruments: The colorimeter was purchased from X-Rite, USA, model number is Ci7600.

[0031] The manufacturer of experimental materials: The lactic acid bacteria fermented milk is produced by Jiangzhong Pharmaceutical Co., Ltd.

[0032] Example 1 Step 1: Use a colorimeter to detect the chromaticity DE value 18 batches of lactic acid bacteria fermented milk products of different batches were collected, 50g of each batch was taken and placed in the colorimetric dish of the powder test sample of the colorimeter, the measurement mode was selected as the reflection mode, the detection parameters were: the aperture plate was 6mm, the number of extractions was 3 times, and the standard white plate was used as the reference standard to perform colorimetric value detection and calculate the DE value of lactic acid bacteria fermented milk products. The results are shown in Table 1 and Figure 1 As shown: Table 1

[0033] Step 2: Determination of 5-hydroxymethylfurfural content by high performance liquid chromatography 1) Preparation of reference solution: Accurately weigh about 0.6048 mg of 5-hydroxymethylfurfural reference substance, place it in a 100 mL volumetric flask, add methanol, sonicate to dissolve it and make up to volume to obtain a 5-hydroxymethylfurfural reference substance solution with a concentration of about 6.048 μg / mL.

[0034] 2) Preparation of test solution: Accurately weigh 5 g of lactic acid bacteria fermented milk spray powder, place it in a 50 mL volumetric flask, add about 40 mL of purified water, sonicate for 10 min to dissolve it, cool, dilute to the scale with purified water, shake well, filter through a 0.45 μm microporous filter membrane to obtain the test solution of lactic acid bacteria fermented milk product.

[0035] 3) Liquid chromatography conditions: An Agilent Zorbax SB-Aq column (250 mm × 4.6 mm, 5 μm) was used for chromatographic separation, with 0.1% formic acid solution-acetonitrile (98:2) as the mobile phase for elution, a flow rate of 1.0 mL / min, an injection volume of 10 μL, a column temperature of 30 °C, and a detection wavelength of 284 nm.

[0036] 4) Sampling analysis: Using the above-mentioned liquid chromatography conditions, the 5-hydroxymethylfurfural reference solution and the 18 batches of lactic acid bacteria fermented milk product test solution were subjected to liquid chromatography detection, and the external standard method was used for quantitative calculation of the 5-hydroxymethylfurfural content in the lactic acid bacteria fermented milk product. The results are shown in Tables 2 and Figure 2 As shown: Table 2

[0037] Step 3 Correlation analysis between 5-hydroxymethylfurfural content and DE value SPSS software was used to perform Spearman correlation analysis on the content of 5-hydroxymethylfurfural in lactic acid bacteria fermented milk products and the colorimetric DE value detected by the colorimeter, and a regression equation was constructed; Spearman correlation analysis showed that there was a significant correlation between the 5-hydroxymethylfurfural content and the DE value (P < 0.05), and the regression equation was Y = 0.059X 2 -1.2971X+5.5086, R 2 =0.9114( Figure 3 ).

[0038] Example 2 Step 1: Use a colorimeter to detect the chromaticity DE value Take 3 batches of lactic acid bacteria fermented milk products of different batches, take 50g of each batch, put them into the colorimetric dish of the powder test sample of the colorimeter, select the reflection mode for the measurement mode, the detection parameters are the aperture plate specification of 6mm, and the number of captures is 3 times. Use the standard white plate as the reference standard, perform the colorimetric value detection, and calculate the DE value of the lactic acid bacteria fermented milk product.

[0039] Step 2: Determination of 5-hydroxymethylfurfural content by high performance liquid chromatography 1) Preparation of reference solution: Accurately weigh about 0.6048 mg of 5-hydroxymethylfurfural reference substance, place it in a 100 mL volumetric flask, add methanol, sonicate to dissolve it and make up to volume to obtain a 5-hydroxymethylfurfural reference substance solution with a concentration of about 6.048 μg / mL.

[0040] 2) Preparation of test solution: Accurately weigh 5 g of lactic acid bacteria fermented milk spray powder, place it in a 50 mL volumetric flask, add about 40 mL of purified water, sonicate for 10 min to dissolve it, cool, dilute to the scale with purified water, shake well, filter through a 0.45 μm microporous filter membrane to obtain the test solution of lactic acid bacteria fermented milk product.

[0041] 3) Liquid chromatography conditions: An Agilent Zorbax SB-Aq column (250 mm × 4.6 mm, 5 μm) was used for chromatographic separation, with 0.1% formic acid solution-acetonitrile (98:2) as the mobile phase for elution, a flow rate of 1.2 mL / min, an injection volume of 10 μL, a column temperature of 35 °C, and a detection wavelength of 284 nm.

[0042] 4) Sampling analysis: Using the above-mentioned liquid chromatography conditions, liquid chromatography was performed on the 5-hydroxymethylfurfural reference solution and the three batches of lactic acid bacteria fermented milk product test solutions, and the external standard method was used for quantification to calculate the content of 5-hydroxymethylfurfural in the lactic acid bacteria fermented milk product.

[0043] Step 3: Calculate the 5-hydroxymethylfurfural content According to the above regression equation (Y=0.059X 2 -1.2971X+5.5086), the DE values ​​of three batches of lactic acid bacteria fermented milk products were entered, and the 5-hydroxymethylfurfural content was calculated as the predicted value. The 5-hydroxymethylfurfural content of three batches of lactic acid bacteria fermented milk products measured by high performance liquid chromatography was compared, and the results are shown in Table 3: Table 3

[0044] The results showed that the predicted values ​​of 5-hydroxymethylfurfural content in three batches of lactic acid bacteria fermented milk products were 1.9435, 2.1267, and 2.0697 μg / g, respectively, and the measured values ​​were 1.9427, 2.1256, and 2.0693 μg / g, respectively, with error values ​​of 0.0008, 0.0011, and 0.0004 μg / g, respectively, indicating that the method for detecting 5-HMF of the present invention has high accuracy.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product, characterized in that: In order to use a colorimeter to detect the chromaticity DE value of lactic acid bacteria fermented milk products, the chromaticity DE value was substituted into the equation to obtain the content of 5-hydroxymethylfurfural; The equation is: Y=0.059X 2 -1.2971X+5.5086; Wherein, X is the chromaticity DE value, and Y is the 5-hydroxymethylfurfural content.

2. The detection method according to claim 1, characterized in that: The test mode of the colorimeter is the reflection mode, and the detection parameters are: the aperture plate specification is 6-25 mm, and the number of captures is 1-5 times.

3. A method for evaluating the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product, characterized in that: The evaluation method comprises the following steps: (1) Use a colorimeter to detect the color DE value of lactic acid bacteria fermented milk products; (2) Detection of 5-hydroxymethylfurfural in lactic acid bacteria fermented milk products by high performance liquid chromatography; (3) The correlation between the 5-hydroxymethylfurfural content detected by HPLC and the colorimetric DE value detected by colorimeter was analyzed, and an evaluation equation was constructed; (4) The colorimetric DE value of the lactic acid bacteria fermented milk product was measured by a colorimeter and substituted into the evaluation equation to obtain the content of 5-hydroxymethylfurfural.

4. The evaluation method according to claim 3, characterized in that: The high performance liquid chromatography column is an octadecylsilane bonded silica gel column; the mobile phase is formic acid solution-acetonitrile.

5. The evaluation method according to claim 4, characterized in that: The HPLC column was an Agilent ZorbaxSB-Aq column; the mobile phase was 0.1% formic acid solution-acetonitrile; the volume ratio of formic acid solution to acetonitrile was 98:

2.

6. The evaluation method according to claim 3, characterized in that: The detection conditions of the high performance liquid chromatography are: Flow rate 0.9-1.2 mL / min; Injection volume 10-15 μL; Column temperature 25-35°C; The detection wavelength is 284 nm.

7. The evaluation method according to claim 3, characterized in that: The detection conditions of the high performance liquid chromatography are: The flow rate was 1.0 mL / min; Injection volume: 10 μL; Column temperature: 30°C; The detection wavelength is 284 nm.

8. The evaluation method according to claim 3, characterized in that: The correlation analysis method in step (3) is selected from any one of Pearson correlation coefficient, Spearman rank correlation coefficient, Kendall correlation coefficient, and partial correlation analysis.

9. The evaluation method according to claim 8, characterized in that: The method of correlation analysis in step (3) is Spearman rank correlation coefficient.

10. Application of the detection method according to any one of claims 1 to 2 in the quality detection of lactic acid bacteria fermented milk.

Images