Detection method of 5-hydroxymethylfurfural in lactic acid bacteria fermented milk products

Abstract

The present invention discloses a method for detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product, and relates to the technical field of food detection. The present invention provides a method for detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product, comprising using a colorimeter to detect the colorimetric DE value of the lactic acid bacteria fermented milk product, and substituting the colorimetric DE value into an equation to obtain the content of 5-hydroxymethylfurfural; the equation is: Y = 0.059X 2 ‑1.2971X+5.5086; where X is the colorimetric DE value and Y is the 5-hydroxymethylfurfural content. The present invention uses a colorimeter to quickly detect the 5-hydroxymethylfurfural content. This detection method is fast, simple, and effective.

Description

Technical Field

[0001] The present 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 lactic acid bacteria-fermented foods. Fermentation by lactic acid bacteria produces a large number of lactic acid bacteria metabolites, including exopolysaccharides, bioactive peptides, and lactic acid. These products exert a wide range of metabolic and regulatory effects, including modulating intestinal flora, enhancing immunity, alleviating non-alcoholic fatty liver disease, providing antioxidant benefits, and lowering blood lipids and cholesterol. During the lactic acid bacteria fermentation and product forming process, particularly during high-temperature processing, milk is susceptible to the Maillard reaction, generating a series of aromatic and color compounds. 5-Hydroxymethylfurfural is a representative example. Studies have shown that the content of 5-Hydroxymethylfurfural, an intermediate product of the Maillard reaction, correlates with color changes. Therefore, 5-Hydroxymethylfurfural can be used as an important indicator of the sensory quality of lactic acid bacteria-fermented milk products.

[0003] A method for detecting 5-hydroxymethylfurfural is disclosed in the prior art (Shi Qiao, 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 instead of glacial acetic acid as the sample dilution solvent, which greatly reduces the detection cost. However, the detection wavelength needs to be determined according to the substances contained in the sample. The detection wavelength of different samples is 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. The method employs high-performance liquid chromatography (HPLC) to determine the content of 5-hydroxymethylfurfural in Schisandra chinensis. The method comprises the following steps: (A) preparing a test solution; (B) preparing a reference solution; and (C) performing HPLC determination. The reference solution and the test solution are each precisely aspirated and injected into a liquid chromatograph. The determination is performed under specific chromatographic conditions, and the quantification is performed using an external standard method. The method employing HPLC for the detection of 5-hydroxymethylfurfural in this invention exhibits 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] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides a method for detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product, comprising using a colorimeter to detect the color DE value of the lactic acid bacteria fermented milk product, and substituting the color DE value into an equation to obtain the content of 5-hydroxymethylfurfural;

[0010] The equation is: Y=0.059X 2 -1.2971X+5.5086;

[0011] Wherein, X is the color DE value, and Y is the 5-hydroxymethylfurfural content.

[0012] 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.

[0013] According to some embodiments of the present invention, the specifications of the aperture plate are 6 mm, 10 mm, and 25 mm.

[0014] Furthermore, the aperture plate specification is 6 mm, and the number of acquisitions is 3 times.

[0015] 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:

[0016] (1) Use a colorimeter to detect the color DE value of lactic acid bacteria fermented milk products;

[0017] (2) Detection of 5-HMF in lactic acid bacteria fermented milk by high performance liquid chromatography;

[0018] (3) Correlation analysis was performed between the 5-hydroxymethylfurfural content detected by HPLC and the colorimetric DE value detected by colorimeter, and an evaluation equation was constructed;

[0019] (4) The colorimeter was used to detect the color DE value of the lactic acid bacteria fermented milk product, and the DE value was substituted into the evaluation equation to obtain the content of 5-hydroxymethylfurfural.

[0020] Specifically, the test mode of the colorimeter is the reflection mode, the detection parameters are the aperture plate specification of 6-25mm, and the number of acquisitions is 1-5. Preferably, the aperture plate specifications are 6mm, 10mm, and 25mm, and the number of acquisitions is 3.

[0021] Specifically, the high performance liquid chromatography column is an octadecylsilane bonded silica gel chromatography column;

[0022] Furthermore, the high performance liquid chromatography column is an Agilent Zorbax SB-Aq chromatography column.

[0023] Furthermore, the specifications of the HPLC column are 250 mm×4.6 mm, 5 μm.

[0024] Specifically, the mobile phase for the high performance liquid chromatography detection is formic acid solution-acetonitrile.

[0025] Furthermore, the volume ratio of the formic acid solution to acetonitrile is 98:2.

[0026] Furthermore, the mobile phase for the high performance liquid chromatography detection is 0.1% formic acid solution-acetonitrile.

[0027] Furthermore, the detection conditions of the high performance liquid chromatography are:

[0028] Flow rate 0.9-1.2 mL / min;

[0029] Injection volume 10-15 μL;

[0030] Column temperature 25-35°C;

[0031] The detection wavelength is 284 nm.

[0032] Furthermore, the detection conditions of the high performance liquid chromatography are:

[0033] The flow rate was 1.0 mL / min;

[0034] Injection volume: 10 μL;

[0035] Column temperature 30°C;

[0036] The detection wavelength is 284 nm.

[0037] 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;

[0038] Furthermore, the method of correlation analysis in step (3) is Spearman rank correlation coefficient.

[0039] According to some embodiments of the present invention, the evaluation equation constructed in step (3) is Y=0.059X 2 -1.2971X+5.5086.

[0040] In another aspect, the present invention provides application of the above detection method in quality detection of lactic acid bacteria fermented milk.

[0041] 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.

[0042] The beneficial effects of the present invention are:

[0043] The method for rapidly detecting the content of 5-hydroxymethylfurfural in a lactic acid bacteria fermented milk product based on the DE value of a colorimeter disclosed herein can rapidly detect the content of 5-hydroxymethylfurfural by the DE value. This method is rapid, 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

[0044] Figure 1 The results show the DE value determination results of 18 different batches of lactic acid bacteria fermented milk products.

[0045] Figure 2 The results are as follows: 5-HMF content determination results in 18 different batches of lactic acid bacteria fermented milk products.

[0046] Figure 3 is the regression equation of DE value and 5-hydroxymethylfurfural content of lactic acid bacteria fermented milk products. DETAILED DESCRIPTION

[0047] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0048] Experimental instruments:

[0049] The colorimeter was purchased from X-Rite, USA, model Ci7600.

[0050] Purchaser of experimental materials:

[0051] Lactic acid bacteria fermented milk is produced by Jiangzhong Pharmaceutical Co., Ltd.

[0052] Example 1

[0053] Step 1: Use a colorimeter to detect the color DE value

[0054] Eighteen batches of lactic acid bacteria fermented milk products were collected, and 50g of each batch was placed in the colorimeter powder test sample cuvette. 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. The color value was tested and the DE value of the lactic acid bacteria fermented milk products was calculated. The results are shown in Table 1 and Figure 1 As shown:

[0055] Table 1

[0056]

[0057] Step 2: Determination of 5-HMF content by HPLC

[0058] 1) Preparation of Reference Solution: Accurately weigh approximately 0.6048 mg of 5-HMF reference standard into a 100 mL volumetric flask. Add methanol, sonicate to dissolve, and dilute to volume to obtain a 5-HMF reference solution with a concentration of approximately 6.048 μg / mL.

[0059] 2) Preparation of test solution: Accurately weigh 5 g of lactic acid bacteria fermented milk spray powder and place it in a 50 mL volumetric flask. Add approximately 40 mL of purified water and sonicate for 10 min to dissolve it. Allow to cool, then dilute to the mark with purified water. Shake well and filter through a 0.45 μm microporous membrane to obtain the lactic acid bacteria fermented milk product test solution.

[0060] 3) Liquid chromatography conditions: Chromatographic separation was performed using an Agilent Zorbax SB-Aq column (250 mm × 4.6 mm, 5 μm). The mobile phase was 0.1% formic acid solution-acetonitrile (98:2). The flow rate was 1.0 mL / min, the injection volume was 10 μL, the column temperature was 30°C, and the detection wavelength was 284 nm.

[0061] 4) Sampling analysis: Using the above-mentioned liquid chromatography conditions, liquid chromatography was performed on the 5-hydroxymethylfurfural reference solution and the 18 batches of lactic acid bacteria fermented milk product test solution. The external standard method was used for quantitative determination and the content of 5-hydroxymethylfurfural in the lactic acid bacteria fermented milk product was calculated. The results are shown in Tables 2 and 3. Figure 2 As shown:

[0062] Table 2

[0063]

[0064] Step 3 Correlation analysis between 5-hydroxymethylfurfural content and DE value

[0065] 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 obtained as Y = 0.059X 2 -1.2971X+5.5086, R 2 =0.9114( Figure 3 ).

[0066] Example 2

[0067] Step 1: Use a colorimeter to detect the color DE value

[0068] Three different batches of lactic acid bacteria-fermented milk products were sampled, 50g each, and placed in a colorimeter powder test sample cuvette. The measurement mode was selected as reflectance, with a 6mm aperture plate and three acquisitions. Colorimetric values ​​were measured using a standard white plate as a reference, and the DE value of the lactic acid bacteria-fermented milk products was calculated.

[0069] Step 2: Determination of 5-HMF content by HPLC

[0070] 1) Preparation of Reference Solution: Accurately weigh approximately 0.6048 mg of 5-HMF reference standard into a 100 mL volumetric flask. Add methanol, sonicate to dissolve, and dilute to volume to obtain a 5-HMF reference solution with a concentration of approximately 6.048 μg / mL.

[0071] 2) Preparation of test solution: Accurately weigh 5 g of lactic acid bacteria fermented milk spray powder and place it in a 50 mL volumetric flask. Add approximately 40 mL of purified water and sonicate for 10 min to dissolve it. Allow to cool, then dilute to the mark with purified water. Shake well and filter through a 0.45 μm microporous membrane to obtain the lactic acid bacteria fermented milk product test solution.

[0072] 3) Liquid chromatography conditions: Chromatographic separation was performed using an Agilent Zorbax SB-Aq column (250 mm × 4.6 mm, 5 μm). The mobile phase was 0.1% formic acid solution-acetonitrile (98:2). The flow rate was 1.2 mL / min, the injection volume was 10 μL, the column temperature was 35°C, and the detection wavelength was 284 nm.

[0073] 4) Sampling Analysis: Using the above-mentioned liquid chromatography conditions, liquid chromatography was performed on the 5-hydroxymethylfurfural reference solution and three batches of lactic acid bacteria fermented milk product test solutions. The external standard method was used for quantification and calculation of the 5-hydroxymethylfurfural content in the lactic acid bacteria fermented milk product.

[0074] Step 3 Calculate the 5-hydroxymethylfurfural content

[0075] According to the above regression equation (Y=0.059X 2 The DE values ​​of three batches of lactic acid bacteria fermented milk products were used to calculate the 5-HMF content, which was the predicted value. This was compared with the actual 5-HMF content of the three batches of lactic acid bacteria fermented milk products measured by HPLC. The results are shown in Table 3:

[0076] Table 3

[0077]

[0078] 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, while 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.

[0079] 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 principles of the present invention should be included in the scope of protection 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 color DE value of lactic acid bacteria fermented milk products, the color 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 color 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-HMF in lactic acid bacteria fermented milk by high performance liquid chromatography; (3) Correlation analysis was performed between the 5-hydroxymethylfurfural content detected by HPLC and the colorimetric DE value detected by colorimeter, and an evaluation equation was constructed; (4) The colorimeter was used to detect the color DE value of the lactic acid bacteria fermented milk product, and the DE value was substituted into the evaluation equation to obtain the content of 5-hydroxymethylfurfural.

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

5. The evaluation method according to claim 4, wherein: 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, wherein: The detection conditions of the described 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, wherein: The detection conditions of the described 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, wherein: 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. Use of the detection method according to any one of claims 1 to 2 in the quality detection of lactic acid bacteria fermented milk.

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