A novel bio-enzyme detection method for dihydromyricetin

Abstract

The application discloses a novel dihydromyricetin biological enzyme detection method, relates to the technical field of sample tea quantitative analysis and detection, and has the technical solution as follows: S1, a gradient concentration dihydromyricetin solution is prepared; S2, a purine nucleoside phosphorylase reaction solution is obtained; S3, the absorbance value of a specified wavelength is measured by ultraviolet spectrometry, a standard working curve is drawn, and a curve equation is obtained; S4, a sample tea with a specified mass is weighed and placed in pure water, a to-be-measured solution is obtained through water bath boiling, the to-be-measured solution is measured and diluted to a certain concentration, and a diluted to-be-measured sample solution is obtained; S5, the absorbance value of a specified wavelength is measured by ultraviolet spectrometry, data are recorded, the working curve equation is substituted, and dihydromyricetin content information in the sample tea is obtained. The method has the characteristics of high sensitivity, high precision, short detection time and simple operation, and has the effect of accurate quantitative analysis without separation of target components in a complex environment.

Description

Technical Field

[0001] This invention relates to the field of quantitative analysis and detection technology of dihydromyricetin in compound tea or vine tea containing dihydromyricetin from plant sample sources, and more specifically, it relates to a novel bioenzyme detection method for dihydromyricetin. Background Technology

[0002] Analytical techniques have long been considered indispensable in the development of natural sciences, and the analysis of different samples or components relies on the improvement of analytical equipment or methods. Due to the complexity and low concentration of chemical components in plant-derived samples, the quantitative analysis of target components in these samples has been a focus of attention in fields such as chemistry and traditional Chinese medicine. Currently, chromatographic analysis techniques have developed rapidly in many fields, solving the problem of component analysis for most complex samples. However, due to the high cost of equipment and consumables, complex pretreatment procedures, and relatively long single-sample detection times, most small and medium-sized enterprises cannot afford them. High-performance liquid chromatography (HPLC), as an important component of chromatographic analysis, is an effective method for the quantitative analysis of target components in the field of heat-sensitive organic matter analysis. This method mainly utilizes the adsorption-desorption process between different packed columns and eluents to achieve the separation and quantitative analysis of target components.

[0003] Currently, the general method for analyzing dihydromyricetin, a natural chemical component from plant sources, using high-performance liquid chromatography (HPLC) is as follows: First, a suitable chromatographic column, such as a C18 column, is selected; then, a suitable eluent ratio is selected; finally, the quantitative analysis of the target chromatographic component—dihydromyricetin—is completed. While this method can effectively quantify the content of natural dihydromyricetin in plant sources, it is time-consuming (e.g., baseline flushing, selection of eluent types and ratios), expensive (all eluents are chromatographically pure), and expensive equipment (imported / domestic HPLC instruments cost approximately 400,000 / 100,000 RMB), which is detrimental to the development of small and medium-sized enterprises.

[0004] To address this, we have developed a novel bio-enzyme detection method for dihydromyricetin. This method eliminates the need for separation of the target component—natural dihydromyricetin—and allows for direct quantitative analysis of dihydromyricetin in plant-derived samples. It boasts advantages such as low cost, short detection time, simple operation, high sensitivity and accuracy, no sample pretreatment required for complex samples, and portable equipment (UV spectrophotometry). This bio-enzyme detection method enables small and medium-sized enterprises (SMEs) to promptly differentiate crude product specifications during procurement, reducing potential market risks. It also provides an effective basis for price differentiation. Furthermore, in cases where consumers have doubts about purchased goods, on-site testing can be conducted without the need for professional testing institutions. Summary of the Invention

[0005] The purpose of this invention is to provide a novel bioenzyme detection method for dihydromyricetin, which is efficient, simple and convenient. This method has the advantages of low detection limit, high sensitivity and precision, short detection time and low cost.

[0006] The above-mentioned technical objective of this invention is achieved through the following technical solution: a novel bioenzyme detection method for dihydromyricetin, specifically comprising the following steps:

[0007] S1: Weigh a certain amount of dihydromyricetin standard, dissolve it, and add purified water to make up to volume to prepare dihydromyricetin solutions with gradient concentrations.

[0008] S2: Weigh a quantitative amount of purine nucleoside phosphorylase, dissolve it in a quantitative amount of phosphate buffer (pH≈7.5) to obtain a purine nucleoside phosphorylase stock solution, and dilute it to a quantitative concentration to obtain a purine nucleoside phosphorylase reaction solution.

[0009] S3: Take quantitative amounts of dihydromyricetin solutions of varying concentrations, add quantitative amounts of purine nucleoside phosphorylase reaction solution, mix thoroughly to form a biological enzyme reaction solution, and react for 3-5 minutes. Measure the absorbance at a specified wavelength using ultraviolet light, plot a standard working curve, and obtain the curve equation.

[0010] S4: Weigh the specified mass of sample tea and put it into pure water. Use a water bath to boil the sample tea to obtain the test solution. Measure the test solution and dilute it to a certain concentration to obtain the diluted test sample solution.

[0011] S5: Quantitatively pipette the diluted sample solution to be tested and mix it with a quantitative amount of purine nucleoside phosphorylase reaction solution, and react for 3-5 minutes. Measure the absorbance value at a specified wavelength using ultraviolet spectrophotometry, record the data, and substitute it into the working curve equation to obtain the dihydromyricetin content information in the tea sample to be tested.

[0012] The method is characterized by the following: in S1, the concentration gradient of dihydromyricetin solution ranges from 1.0 μmol / L to 1000 μmol / L; in S2, the concentration of purine nucleoside phosphorylase reaction solution ranges from 10 mU / mL to 50 mU / mL; and in S3, the standard working curve R... 2 >0.98, with a specified UV wavelength of 326±2nm; the detection limit for dihydromyricetin in S4 is ≥5.0μmol / L; the recovery rate in S5 is ≥100.0% with a specified UV wavelength of 326±2nm; the relative standard deviation is <3.0%.

[0013] In summary, the present invention has the following beneficial effects:

[0014] 1. This method can detect, reduce detection costs and detection time in complex environments, while retaining the advantages of sensitivity and accuracy, easy-to-use equipment, and simple operation.

[0015] 2. This method eliminates the need for SMEs to send samples to testing institutions for analysis, providing a reliable basis for their quality assurance. In competition with similar products, this testing method offers advantages such as lower equipment purchase costs, lower equipment maintenance costs, and relatively lower requirements for operator expertise compared to liquid chromatography column methods, giving it significant potential in the market.

[0016] 3. This method applies biological enzymes to the food industry, providing a new path for the advancement of food testing technology and promoting the country's monitoring of the food market. At the same time, the use of this method can accelerate product upgrading and innovation. Attached Figure Description

[0017] Figure 1 This is a graph showing the original experimental data of adding xanthine in the embodiments of the present invention;

[0018] Figure 2 This is a graph of the original experimental data without the addition of xanthine in the embodiments of the present invention;

[0019] Figure 3 This is a data processing diagram corresponding to the addition of xanthine in an embodiment of the present invention;

[0020] Figure 4 This is the data processing diagram corresponding to the absence of xanthine in the embodiments of the present invention;

[0021] Figure 5 This is the data processing diagram corresponding to Table 3 in the embodiments of the present invention;

[0022] Figure 6 This is the data processing diagram corresponding to Table 4 in the embodiments of the present invention;

[0023] Figure 7 This is the data processing diagram corresponding to Table 5 in the embodiments of the present invention;

[0024] Figure 8 This is the data processing diagram corresponding to Table 6 in the embodiments of the present invention;

[0025] Figure 9 This is a data processing diagram of the standard error curve in an embodiment of the present invention;

[0026] Figure 10 This is the curve graph corresponding to Table 19 in the embodiments of the present invention. Detailed Implementation

[0027] The following is in conjunction with the appendix Figure 1-10 The present invention will be described in further detail below.

[0028] Example: A novel bioenzyme detection method for dihydromyricetin.

[0029] Exploration and changes in methodological model factors (XAN, purine nucleoside phosphorylase, buffer, oxygen, time):

[0030] 1. Effect of xanthine (XAN) on the absorption peak of the model dihydromyricetin (DMY) + purine nucleoside phosphorylase + xanthine (XAN) at 326±2nm.

[0031] Variable XAN:

[0032] (1) 0.1 mol / L sodium pyrophosphate buffer: Weigh 1.3295 g of sodium pyrophosphate and 0.0087 g of ethylenediaminetetraacetic acid, dissolve them in purified water, and make up to 50 mL. Adjust the pH to ≈ 7.5 with phosphoric acid.

[0033] (2) 8.8 U / mL purine nucleoside phosphorylase stock solution: Accurately weigh 0.0010 g of purine nucleoside phosphorylase with a specification of 8.8 U / mg and dissolve it in 1 mL of 0.1 mol / L sodium pyrophosphate buffer.

[0034] (3) 50 mU / mL purine nucleoside phosphorylase: Measure 0.113 mL of 8.8 U / mL purine nucleoside phosphorylase and bring the volume to 20 mL.

[0035] (4) 3mmol / L Xan: Accurately weigh 0.0045g of xanthine solid and dissolve it in 20mL of purified water by sonication. (Store protected from light)

[0036] (5) 1000 μmol / L: Weigh 0.0320 g DMY into a beaker, add an appropriate amount of purified water, then transfer it to a 100 mL volumetric flask and dilute to volume. Then dilute with purified water to prepare DMY test sample solutions of (900 μmol / L, 800 μmol / L, 700 μmol / L, 600 μmol / L, 500 μmol / L, 400 μmol / L, 300 μmol / L, 200 μmol / L, 100 μmol / L).

[0037] Model 1:

[0038] Reference solution: 0.4 mL purified water + 1.6 mL 50 mmol / mL purine nucleoside phosphorylase + 1 mL 1 mmol / L xan;

[0039] Reaction solution: 0.4 mL DMY of various concentrations + 1.6 mL 50 mmol / mL purine nucleoside phosphorylase + 1 mL 1 mmol / L Xan;

[0040] Table 1: Effect of adding XAN on the working curve

[0041]

[0042]

[0043] Note: 50 mU / mL purine nucleoside phosphorylase

[0044] Modeling 2:

[0045] Reference solution: 1.4 mL purified water + 1.6 mL 50 mU / mL purine nucleoside phosphorylase;

[0046] Reaction solution: 0.4 mL DMY solution of various concentrations + 1.6 mL 50 mU / mL purine nucleoside phosphorylase + 1 mL purified water;

[0047] Table 2: Effect of not including XAN on the working curve

[0048]

[0049] Note: 50 mU / mL purine nucleoside phosphorylase

[0050] Conclusion: The experimental results show that both models can obtain a special peak at 326±2nm. Compared with Model 1 (with XAN), Model 2 (without XAN) has a better linear fit and higher stability and accuracy. Therefore, Model 2 (without XAN) is the best choice for the experiment.

[0051] 2. Effect of purine nucleoside phosphorylase on the absorption peak of model DMY+ purine nucleoside phosphorylase at 326±2nm.

[0052] Variable purine nucleoside phosphorylase:

[0053] Standard curves for high, medium, and low enzyme concentrations:

[0054] Table 3: Purine nucleoside phosphorylase = 50 mU / mL

[0055]

[0056]

[0057] Table 4: Purine nucleoside phosphorylase = 30 mU / mL

[0058]

[0059] Table 5: Purine nucleoside phosphorylase = 10 mU / mL

[0060]

[0061]

[0062] Conclusion: The experimental results of different concentrations of purine nucleoside phosphorylase show that changes in enzyme concentration affect the standard curve R.2 The value of the enzyme has little impact, but the detection limit of the standard curve gradually increases with increasing enzyme concentration. Therefore, it provides us with more options when detecting extremely low concentrations of dihydromyricetin (DMY).

[0063] 3. Effect of buffer solution on the absorption peak of DMY+ purine nucleoside phosphorylase at 326±2nm.

[0064] Variable buffer solution:

[0065] Table 6: Effect of variable buffer solution on the uptake peak of model DMY+ purine nucleoside phosphorylase

[0066]

[0067] Conclusion: The data show that the buffer solution has a significant impact on the enzyme activity of the model DMY+ enzyme, therefore the buffer solution must be used for enzyme preparation.

[0068] 4. Effect of dissolved oxygen on the absorption peak of model DMY+purine nucleoside phosphorylase at 326±2nm.

[0069] Variable oxygen content:

[0070] Modeling:

[0071] Reference solution: 1.4 mL purified water + 1.6 mL purine nucleoside phosphorylase (50 mU / mL);

[0072] Reaction solution: 0.4 mL DMY solution of various concentrations + 1.6 mL purine nucleoside phosphorylase + 1 mL purified water.

[0073] Table 7: Normal purified water (without oxygenation treatment)

[0074]

[0075] Table 8: Purified water with oxygen added

[0076]

[0077]

[0078] Conclusion: The absorbance value under oxygenated conditions showed a decreasing trend. Therefore, high oxygen content in water is not conducive to the establishment of the DMY+purine nucleoside phosphorylase model.

[0079] 5. Effect of reaction time on the absorption peak of model DMY+ purine nucleoside phosphorylase at 326±2 nm

[0080] Variable reaction time:

[0081] Table 9: Effect of reaction time

[0082]

[0083]

[0084] Note: DMY: 1000 μmol / L purine nucleoside phosphorylase: 30 mU / mL

[0085] Table 10: Effect of reaction time (parallel experiments)

[0086]

[0087] Conclusion: The data shows that the absorbance value decreases continuously with the extension of time, indicating that extending the reaction time is not conducive to the establishment of the DMY+purine nucleoside phosphorylase model.

[0088] 6. Relative standard deviation and recovery rate of the DMY+purine nucleoside phosphorylase model method:

[0089] (1) Relative standard deviation

[0090] Weigh approximately 1g of green tea, soy sauce tea, and honeysuckle into 500mL round-bottom flasks, add about 200-300mL of purified water, and incubate in a constant temperature water bath at 90℃ for 20 minutes.

[0091] The green tea and soy sauce tea solutions were mixed with 800 μmol / L DMY standard sample solution at a ratio of 1:1. The absorbance was measured three times at 326±2 nm. The actual concentration was calculated by substituting the results into the standard curve.

[0092] Mix green tea, soy sauce tea, and honeysuckle solution in a ratio of 1:1:1, then mix with an 800 μmol / L DMY standard sample solution (the absorbance of the 800 μmol / L DMY standard sample solution has been measured) in a ratio of 1:9. Measure the absorbance three times, substitute it into the standard curve, and calculate the actual concentration.

[0093] The average value, individual absolute deviations, and relative standard deviations of the solutions were calculated based on their actual concentrations.

[0094] Table 16: Relative Standard Deviation of DMY in Green Tea (%)

[0095]

[0096] Table 17: Relative Standard Deviation of DMY in Maotai Tea (%)

[0097]

[0098]

[0099] Table 18: Relative Standard Deviation (%) of DMY in Green Tea + Soy Sauce Tea + Honeysuckle

[0100]

[0101] (2) Recovery rate

[0102] Table 19: Standard Curve

[0103] DMY concentration (μmol / L) Absorbance (au) 1000 2.154 800 1.836 600 1.44 400 1.027 200 0.546 100 0.279 80 0.169 60 0.157 40 0.099

[0104] (1) Weigh about 1g of vine tea and transfer it to a 1000mL round-bottom flask. Add 500mL of purified water and place in a constant temperature water bath at 90℃ for 20min.

[0105] (2) The tea solution was diluted 40 times to obtain a low-concentration sample. Its absorbance was measured to be 0.151 nm. Substituting this into the standard curve, the actual concentration was found to be 50.45454545 μmol / L.

[0106] (3) Prepare DMY standard sample solutions of 800 μmol / L, 600 μmol / L, 400 μmol / L, 200 μmol / L and 100 μmol / L respectively.

[0107] Table 20: Absorbance corresponding to different DMY standard sample solution concentrations

[0108]

[0109] The tea sample was mixed with the DMY standard sample solution at a ratio of (1:1).

[0110] Table 21: Absorbance of tea sample mixed with DMY standard sample solution

[0111]

[0112] Application Cases and Analysis:

[0113] Laboratory testing of vine tea products:

[0114] Weigh 1g of vine tea into 625mL of water, boil in a water bath at 90℃ for 20min to obtain a solution with a theoretically estimated DMY concentration of 1000μmol / L. Then, take the solution with a theoretically estimated DMY concentration of 1000μmol / L and dilute it to 600μmol / L, 500μmol / L, 400μmol / L, 300μmol / L, and 200μmol / L, respectively. Repeat the above operation for a second boiling.

[0115] Modeling:

[0116] Reference solution: 1.4 mL purified water + 1.6 mL 30 mU / mL purine nucleoside phosphorylase;

[0117] Reaction solution: 0.4 mL DMY solution + 1.6 mL 30 mU / mL purine nucleoside phosphorylase + 1 mL purified water.

[0118] The actual concentration within the measurement range is calculated using a high-concentration standard curve.

[0119] That is, y = 0.0023x + 0.1813

[0120] Actual concentration X = (y - 0.1813) / 0.0023

[0121] Assuming the extraction rate of vine tea is 20%, the volume used is V = [(m / M) / c] * 109 mL, and the mass of dihydromyricetin in the vine tea is m = cVM.

[0122] Table 22: First water extraction of vine tea

[0123]

[0124] Add 625 mL of water to the tea leaves from the first brew and continue boiling in a 90°C water bath for 20 minutes. Then dilute the tea leaves sequentially and measure the absorbance.

[0125] The actual concentration within the measurement range is calculated using a low-concentration standard curve.

[0126] That is, y = 0.00219x - 0.00248.

[0127] Table 23: Second water extraction of vine tea

[0128]

[0129] Table 24: Testing Results of Products Submitted by Enterprises

[0130]

[0131] Table 25: Testing Results of Products Submitted by Enterprises (Part 2)

[0132]

[0133]

[0134] Table 26: Three Test Items for Products Submitted by Enterprises

[0135]

[0136]

[0137] Table 27: Testing of Products Submitted by Enterprises (Part 4)

[0138]

[0139] Table 28: Five Test Items for Products Submitted by Enterprises

[0140]

[0141]

[0142] Table 29: Six Test Results for Products Submitted by Enterprises

[0143]

[0144]

[0145] Table 30: Seven Test Items for Products Submitted by Enterprises

[0146]

[0147] Table 31: Eight Test Items for Products Submitted by Enterprises

[0148]

[0149]

[0150] Table 32: Nine Test Items for Products Submitted by Enterprises

[0151]

[0152]

[0153] Table 33: Detection of Complex Samples

[0154]

[0155] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A novel bioenzyme detection method for dihydromyricetin, characterized by: Specifically, the following steps are included: S1: Weigh a certain amount of dihydromyricetin standard, dissolve it, and add purified water to make up to volume to prepare dihydromyricetin solutions with gradient concentrations. S2: Weigh a quantitative amount of purine nucleoside phosphorylase, dissolve it in a quantitative amount of phosphate buffer to obtain a purine nucleoside phosphorylase stock solution, and dilute it to a quantitative concentration to obtain a purine nucleoside phosphorylase reaction solution. S3: Take quantitative amounts of dihydromyricetin solutions of varying concentrations, add quantitative amounts of purine nucleoside phosphorylase reaction solution, mix thoroughly to form a biological enzyme reaction solution, and react for 3-5 minutes. Measure the absorbance at a specified wavelength using ultraviolet light, plot a standard working curve, and obtain the curve equation. S4: Weigh the specified mass of sample tea and put it into pure water. Use a water bath to boil the sample tea to obtain the test solution. Measure the test solution and dilute it to a certain concentration to obtain the diluted test sample solution. S5: Quantitatively pipette the diluted sample solution to be tested and mix it with a quantitative amount of purine nucleoside phosphorylase reaction solution, and react for 3-5 minutes. Measure the absorbance value at a specified wavelength using ultraviolet spectrophotometry, record the data, and substitute it into the working curve equation to obtain the dihydromyricetin content information in the tea sample to be tested.

2. The novel bioenzyme detection method for dihydromyricetin according to claim 1, characterized in that: The concentration gradients of dihydromyricetin solution in S1 ranged from 1.0 μmol / L to 1000 μmol / L; the concentration of purine nucleoside phosphorylase reaction solution in S2 ranged from 10 mU / mL to 50 mU / mL; and the standard working curve R in S3... 2 >0.98, with a specified ultraviolet wavelength of 326±2nm; The detection limit for dihydromyricetin in S4 was ≥5.0 μmol / L; the recovery rate in S5 was ≥100.0% at a specified ultraviolet wavelength of 326±2 nm, with a relative standard deviation of <3.0%.

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