Quantitative detection method of betacyanin

Abstract

The invention relates to a quantitative detection method of betacyanin. The invention provides a betacyanin quantitative detection method which comprises the following steps: (1) establishing a standard curve: preparing a gradient standard solution from a betacyanin standard sample, and adding the gradient standard solution on an elisa plate to measure a light absorption value; obtaining a standard curve according to the absorbance, and obtaining a linear regression equation according to the standard curve and the Lambert-Beer law; (2) quantitative detection: pre-washing a to-be-detected sample to obtain an extracting solution, removing impurities of the extracting solution, diluting to obtain a loading solution, adding the loading solution to the ELISA plate, detecting to obtain a light absorption value of the to-be-detected sample, and substituting the light absorption value of the to-be-detected sample into the linear regression equation for calculation to obtain the concentration of the betacyanin in the to-be-detected sample. Finally, the content of betacyanin in the sample is obtained through calculation according to the formula # imgabs0 #, the quantitative detection method is faster, simpler, more convenient and more accurate, the method can be applied to the samples containing grease, and batch determination of the samples is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of quantitative detection of betacyanin, and particularly relates to a method for quantitatively detecting betacyanin. Background Art

[0002] Betacyanin is a natural water-soluble plant pigment, mainly existing in red beets of Chenopodiaceae, bougainvillea of Amaranthaceae, petals of Portulacaceae, cactus fruits of Cactaceae, pericarp and pulp of pitaya, pokeberry berries of Phytolaccaceae, and cockscomb flowers. Betacyanin has antibacterial, anti-inflammatory, anti-cancer, and antioxidant biological activities, and can not only be widely applied in the fields of food and cosmetics, but also play an important role in the treatment of diseases related to oxidative stress, inflammation, and dyslipidemia.

[0003] There are few reports on the content determination method of betacyanin. Currently, first, the detection method of betacyanin mainly measures the absorbance value and then directly calculates the absolute content of betacyanin according to the Lambert-Beer law and the molar extinction coefficient (betacyanin ε = 56600 - 60000 L·mol -1 ·cm -1 ,) and molar mass. However, the physical meaning of the molar absorptivity (ε) is: when the concentration of the absorbing substance is 1 mol / L, the thickness of the absorption cell is 1 cm, and light of a certain wavelength passes through, the absorbance value A caused. The value of ε depends on the wavelength of the incident light and the light absorption characteristics of the absorbing substance, and is affected by the solvent and temperature. Therefore, the method of calculating the content of betacyanin using the molar extinction coefficient in the literature cannot accurately quantify; second, in samples with more oil, it is often impossible to directly extract betacyanin with an aqueous solution. Currently, there is no optimized pretreatment technical step, and it is difficult to accurately quantify the content of betacyanin. Third, the existing methods can measure 1 - 6 samples at a time and cannot achieve batch detection. Therefore, the detection method of betacyanin content is a problem that must be solved in the development and application of betacyanin. Summary of the Invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for quantitatively detecting betacyanin, and the method for quantitatively detecting betacyanin provided by the present invention is faster, simpler, more accurate, and can achieve batch determination of the betacyanin content in beets and oil-containing samples.

[0005] The technical solution for the present invention to solve the above technical problems is as follows:

[0006] The purpose of the present invention is to provide a method for quantitatively detecting betacyanin, comprising the following steps:

[0007] (1) Standard curve establishment: Take standard samples of betacyanin to prepare gradient standard solutions, add the gradient standard solutions to an ELISA plate to measure absorbance values; draw a standard curve with a slope of a and an intercept of b based on the absorbance, and obtain a linear regression equation according to the standard curve and the Lambert-Beer law;

[0008] (2) Quantitative detection: Take a sample to be tested, perform pre-washing and extraction to obtain an extract with a volume of V, then successively remove impurities and dilute the extract to obtain a sample loading solution. Add the sample loading solution to an ELISA plate to detect the absorbance value A of the sample to be tested. Substitute the absorbance value of the sample to be tested into the linear regression equation for calculation, and the concentration of betacyanin in the sample to be tested can be obtained. Finally, according to the mass m of the sample to be tested, the volume V of the extract, and the dilution factor D of the sample loading solution, use the formula to calculate the content ω of betacyanin in the sample, that is, complete the quantitative detection of betacyanin.

[0009] Preferably, when diluting the extract, if the concentration of the extract is greater than 30 mg / L, it needs to be diluted; if it is less than 30 mg / L, it can be directly measured without dilution, and at this time D = 1.

[0010] The beneficial effects of the present invention are as follows: The present invention calculates the concentration value of the sample to be tested by establishing a linear equation based on the Lambert-Beer law (A = Kbc). The method of the present invention is faster, simpler, more accurate, and can realize batch determination of samples.

[0011] Based on the above technical solutions, the present invention can be further improved as follows.

[0012] Further, the sample to be tested includes at least one of beet and oil mixture.

[0013] Further, the oil mixture includes at least one of ice cream, cream, and ice cream bar.

[0014] Further, when the sample to be tested is beet, the pre-washing and extraction steps of the beet are specifically as follows:

[0015] Weigh and crush the beet and mix it evenly. Add pre-cooled ethanol to the beet to submerge the sample, mix it evenly, centrifuge it, and discard the supernatant to obtain a precipitate; add pre-cooled pure water to the beet precipitate, ultrasonicate it, and then centrifuge it to retain the supernatant, which is the sample loading solution.

[0016] Further, when the sample to be tested is an oil mixture, the pre-washing and extraction steps of the oil mixture are specifically as follows:

[0017] After adding pre-cooled ethanol to the oil mixture and submerging it, mix well, centrifuge, and discard the supernatant to obtain a precipitate; add pre-cooled pure water and ethyl acetate to the precipitate of the oil mixture, submerge, ultrasonicate, and then centrifuge to obtain the intermediate layer supernatant; take the intermediate layer supernatant, add dichloromethane, mix well, and centrifuge to obtain the pigment supernatant, which is the sample loading solution.

[0018] The further beneficial effects of the above are as follows: Betacyanin is a water-soluble red natural pigment. In the extraction process of the present invention, ethyl acetate is used to achieve the effect of extracting and separating oils, and chloroform is used to further degrease and demulsify.

[0019] Further, in step (1), the concentration of the standard solution is 0 - 30 mg / L.

[0020] Preferably, when the sample to be tested is oil, the concentration of the standard solution is 0 - 15 mg / L.

[0021] The further beneficial effects of the above are as follows: Generally, when the sample exceeds 30 mg / L, it is outside the linear range.

[0022] Further, in step (1), the absorbance value is 535 nm.

[0023] Further, the dosage ratio of the beet to the pre-cooled ethanol is 1:10 - 20; the dosage ratio of the beet precipitate to the pre-cooled pure water is 1:10 - 40.

[0024] Further, the dosage ratio of the oil mixture to the pre-cooled ethanol is 1:10 - 20.

[0025] Further, the dosage ratio of the precipitate of the oil mixture, the pre-cooled pure water, and the ethyl acetate is 1:5 - 12:5 - 12; the dosage ratio of the intermediate layer supernatant to the dichloromethane is 1:0.5 - 1. Description of the Drawings

[0026] Figure 1 It is the standard curve graph drawn based on the absorbance of the betanin standard product at 535 nm in Example 1 of the present invention;

[0027] Figure 2 It is the standard curve graph drawn based on the absorbance of the betanin standard product at 535 nm in Example 2 of the present invention. Detailed Embodiments

[0028] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be purchased through regular channels.

[0029] Example 1: Quantitative Detection of Betalain in Beet

[0030] (1) Take the standard sample of betalain to prepare 8 standard solutions, the concentrations of which are 0 mg / L, 0.4 mg / L, 0.8 mg / L, 1.6 mg / L, 2.0 mg / L, 4.0 mg / L, 7.5 mg / L, 15 mg / L, and 30 mg / L. Add the above 8 standard solutions to the ELISA plate, 200 ul / well, and measure the absorbance at 535 nm; draw a standard curve based on the absorbance at 535 nm, and according to the standard curve and the Lambert-Beer law, the linear regression equation is y=0.0604x+0.0045(R 2 =0.9996), where y is the absorbance value, x is the concentration of the liquid to be tested, in mg / L. Figure 1 As shown;

[0031] (2) Take 0.25g of the thin slices of the sample of Subet beet and crush and mix them evenly; add 10ml of precooled 2-4°C ethanol to the sample of Subet beet, immerse the sample, and ultrasonicate at 400kHZ and 400w for 10min. After ultrasonication, centrifuge for the first time and discard the supernatant to obtain the beet precipitate; add 10ml of precooled 2-4°C pure water, immerse the sample for 10min, and centrifuge for the second time (4-6°C, 8000rpm, 10min) after ultrasonication to obtain the supernatant of the sample; repeat the above steps until it is colorless, combine the supernatants, and prepare 20ml of extract;

[0032] (3) Take 200ul of the extract as the loading solution and add it to the ELISA plate. Measure the absorbance at 535nm. Substitute the absorbance value of the sample to be tested into the linear regression equation y=0.0604x+0.0045(R 2 =0.9996), the calculation shows that the betalain content of the sample was 0.6947mg / g, 0.6835mg / g, 0.6807mg / g, 0.6797mg / g, 0.6896mg / g, 0.6895mg / g, 0.6865mg / g, with an average content of 0.6863mg / g and an RSD of 0.7832% after 7 repeated measurements under reproducible conditions; the betalain content was 0.6134mg / g, 0.6760mg / g, 0.6863mg / g, 0.6790mg / g after 4 repeated measurements under reproducible conditions, with an average content of 0.6637mg / g and an RSD of 5.092%. The quantitative detection of betalain is completed.

[0033] (4) Spiked recovery rate: Take a sample of Sobet beet, weigh 25.4498g, add 13.911mg betalain, mix well, and spike the theoretical value of 0.5466mg / g as the spiked recovery sample to be tested. Weigh 0.2545g of the sample to be tested, prepare the sample according to 2.1, dilute 2 times, prepare the sample solution, measure at 535nm, and the betalain content of the sample is 1.2245mg / g. The spiked recovery rate is 102.6%, which is within the range of 80-110%, which meets the requirements of the "Guidelines for Confirmation and Validation of Chemical Analysis Methods for Conformity Assessment" (GB 27417-2017) and the "Laboratory Quality Control Specification for Physical and Chemical Testing of Foods" GBT27404-2008.

[0034] Example 2: Quantitative detection of betalain in ice cream

[0035] (1) Establishment of standard curve: Prepare 7 standard solutions with concentrations of 0 mg / L, 0.75 mg / L, 1.5 mg / L, 3.0 mg / L, 4.0 mg / L, 7.5 mg / L and 15 mg / L, respectively. Take 10 ml of each standard solution, add 10 ml of ethyl acetate and 4 ml of dichloromethane, mix well and centrifuge, take 5 ml of the pigment layer to prepare a gradient standard solution; add the 7 standard solutions to the ELISA plate, 200 ul / well, and measure the absorbance at 535 nm; draw a standard curve based on the absorbance at 535 nm, and derive the linear regression equation y=0.0589x-0.0005(R) according to the standard curve and Lambert-Beer law. 2 =1.000), where y is the absorbance value, x is the concentration of the liquid to be tested, in mg / L. Figure 2 As shown;

[0036] (2) Sample preparation: Weigh 200g of ice cream purchased from Mixue Ice City, add 0.0057g of betalain standard, mix well, and use it as the spike recovery sample. The spike theoretical value is 0.0285mg / g; the ice cream sample without betalain is used as the blank sample. Weigh about 1g of the blank sample and the spike recovery sample to be tested, and repeat 3 times, respectively: 1.0073g, 1.0100g, 1.0072g, 1.0254g, 1.0015g, 1.0081g;

[0037] (3) Add 10 ml of pre-cooled ethanol at 2 - 4°C to the ice cream sample to be tested to submerge the sample. For the first centrifugation (at 4 - 6°C, 8000 rpm, 10 min), discard the supernatant to obtain the precipitate. Add 10 ml of pre-cooled pure water at 2 - 4°C and 10 ml of ethyl acetate to the precipitate, submerge and ultrasonicate for 10 min. After ultrasonication, perform the second centrifugation (at 4 - 6°C, 8000 rpm, 10 min), and take 8 ml of the clear liquid in the middle pigment layer as the extract. Add 4 ml of dichloromethane to the 8 ml of pigment clear liquid, shake well, perform the third centrifugation (at 4 - 6°C, 8000 rpm, 10 min), and take 5 ml of the clear liquid in the pigment layer to obtain the sample loading solution. Add 200 μl of the supernatant of the sample to be tested to the microplate, and measure the absorbance at 535 nm. The average value of the absorbance of the sample blank is measured as 0.1014, the absorbance of the spiked recovery sample is measured as 0.2482, and the average value of the corrected absorbance after deducting the blank is 0.1468;

[0038] (4) Add the supernatant of the sample to be tested to the microplate to detect the absorbance of the sample to be tested at 535 nm, which is 0.2482. The absorbance of the sample blank is 0.1015. Therefore, the absorbance of the sample after deducting the blank is 0.1467. Substitute the absorbance of the sample to be tested into the linear regression equation y = 0.0589x - 0.0005 (R 2 = 1.000) for calculation, and the concentration of the sample solution to be tested can be obtained as 2.5002 mg / L. The mass of the sample is 1.0116 g. Further, the content of betacyanin in the sample to be tested can be obtained as 0.02471 mg / g, that is, the quantitative detection of betacyanin in ice cream is completed. The theoretical value of betacyanin in the spiked recovery sample is 0.0285 mg / g, and the spiked recovery rate is 86.70%.

[0039] In summary, the correlation coefficient of the linear equation of this method is greater than 0.99; the within-laboratory coefficient of variation is 5.092%, which is within the range of 3.8 - 5.3%. The spiked recovery rates are 102.6% and 86.70%, which are within the range of 80 - 110%. The linear equation, detection limit, quantification limit, precision, and spiked recovery rate all meet the requirements of "General Requirements for the Validation and Verification of Chemical Analysis Methods in Conformity Assessment" (GB 27417 - 2017) and "Quality Control Codes for Laboratories - Physical and Chemical Tests of Foods" (GBT 27404 - 2008). It can simultaneously measure 1 - 90 samples at a time, and can achieve the batch determination of the content of betacyanin in beets and oil-containing samples, indicating that the provided quantitative detection method for betacyanin is faster, simpler, and more accurate.

[0040] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A quantitative detection method for betacyanin, characterized in that, The method includes the following steps: (1) Establishing a standard curve: Taking a standard sample of betacyanin to prepare a gradient standard solution, adding the gradient standard solution to an enzyme-linked immunosorbent assay (ELISA) plate to measure the absorbance value; Drawing a standard curve with a slope of a and an intercept of b based on the absorbance, and obtaining a linear regression equation according to the standard curve and the Lambert-Beer law; (2) Quantitative detection: Take the sample to be tested, pre-wash and extract it to obtain an extract with a volume of V. Then, successively remove impurities and dilute the extract to obtain a sample loading solution. Add the sample loading solution to an enzyme-linked immunosorbent assay (ELISA) plate to detect the absorbance value A of the sample to be tested. Substitute the absorbance value of the sample to be tested into the linear regression equation for calculation, and the concentration of betacyanin in the sample to be tested can be obtained. Finally, according to the mass m of the sample to be tested, the volume V of the extract, and the dilution factor D of the sample loading solution, calculate the content ω of betacyanin in the sample through the formula to complete the quantitative detection of betacyanin.

2. The quantitative detection method of betacyanin according to claim 1, wherein The sample to be tested includes at least one of beet and oil mixture.

3. The quantitative detection method of betacyanin according to claim 2, wherein The oil mixture includes at least one of ice cream, cream, and ice lolly.

4. The quantitative detection method of betacyanin according to claim 2, characterized in that When the sample to be tested is beet, the pre-washing and extraction steps of the beet are as follows: Weigh and crush the beet and mix it evenly. Add pre-cooled ethanol to the beet to submerge the sample, mix it evenly, centrifuge, and discard the supernatant to obtain a precipitate. Add pre-cooled pure water to the beet precipitate, ultrasonicate, and then centrifuge. Retain the supernatant, which is the sample loading solution.

5. The quantitative detection method of betacyanin according to claim 3, characterized in that, When the sample to be tested is an oil mixture, the pre-washing and extraction steps of the oil mixture are as follows: Add pre-cooled ethanol to the oil mixture to submerge it, mix it evenly, centrifuge, and discard the supernatant to obtain a precipitate. Add pre-cooled pure water and ethyl acetate to the precipitate of the oil mixture to submerge it, ultrasonicate, and then centrifuge to obtain a middle-layer clear liquid. Take the middle-layer clear liquid, add dichloromethane, mix it evenly, and then centrifuge to obtain a pigment clear liquid, which is the sample loading solution.

6. The quantitative detection method of betacyanin according to claim 1, wherein In step (1), the concentration of the standard solution is 0 - 30 mg / L.

7. A quantitative detection method for betacyanin according to claim 1, characterized in that, In step (1), the absorbance value is 535 nm.

8. A quantitative detection method for betacyanin according to claim 4, characterized in that, The dosage ratio of the beet to the pre-cooled ethanol is 1:10 - 20; the dosage ratio of the beet precipitate to the pre-cooled pure water is 1:10 - 40.

9. The quantitative detection method of betacyanin according to claim 5, characterized in that, The dosage ratio of the oil mixture to the pre-cooled ethanol is 1:10 - 20.

10. A method for quantitatively detecting betacyanin according to claim 5, characterized in that, The dosage ratio of the precipitate of the oil mixture, the pre-cooled pure water, and the ethyl acetate is 1:5 - 12:5 - 12; the dosage ratio of the middle-layer clear liquid to the dichloromethane is 1:0.5 - 1.

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