1. A method for detecting capsaicin content by using botrytis cinerea, which is characterized by comprising the following steps: inoculating botrytis cinerea into V8 culture medium containing capsaicin with different concentrations, and establishing a linear standard curve of capsaicin concentration and diameter of botrytis cinerea plaque; extracting capsaicin in a sample to be detected, and preparing a liquid to be detected; inoculating the botrytis cinerea into a V8 culture medium containing the solution to be detected for culture, and calculating the content of capsaicin in the sample to be detected according to the diameter of the botrytis cinerea bacterial plaque to be detected in the culture time and a linear standard curve.

2. The method according to claim 1, wherein the cultivation time is 3 to 4 days.

3. The method according to claim 1, wherein the linear standard curve of capsaicin concentration and gray mold plaque diameter at 4d culture is:

y-1.699 x +49.44 equation (1);

y is capsaicin concentration, and x is gray mold plaque diameter.

4. The method according to claim 1, wherein the Botrytis cinerea is a Botrytis cinerea suspension, and the inoculation concentration of the Botrytis cinerea suspension is 0.5-1.5 x 10⁵one/mL.

5. The method as claimed in claim 4, wherein the Botrytis cinerea suspension is obtained by shaking cultured Botrytis cinerea in a staining solution, the staining solution comprises maltose and peptone, and the mass ratio of the maltose to the peptone is (5-10): (1-3).

6. The method of claim 5, wherein the culturing of the botrytis cinerea comprises: the botrytis cinerea is inoculated in a V8 culture medium and cultured for 8-13 days at the temperature of 23-27 ℃.

7. The method as claimed in claim 1, wherein the method for extracting capsaicin from the sample to be tested comprises the following steps: and ultrasonically extracting a sample to be detected by using methanol, filtering, concentrating, and performing constant volume by using the methanol to obtain the liquid to be detected.

8. The method according to claim 7, wherein the temperature of the ultrasonic extraction is 45-55 ℃ and the time is 40-50 min.

9. The method of claim 7, wherein the mass-to-volume ratio of the sample to be tested to the methanol ultrasound is (8-13): 12-17) g/mL.

10. The method according to claim 7, wherein the temperature of the concentration is 66-73 ℃.

Background

Capsaicin (Capsaicin) is an alkaloid specific to fruits of plants belonging to the genus Capsicum of the family Solanaceae and capable of causing burning pain in mammals consuming products related to capsicum. Generally, capsaicin accounts for more than 80% of the capsaicinoid content of the pepper fruits and is the main determinant substance for the formation of the peppery taste of the peppers. The capsaicin has the functions of diminishing inflammation, resisting oxidation, resisting tumors, reducing fat and the like, and has wide application value in the fields of medicine, feed addition and the like.

At present, the extraction method of capsaicin comprises a supercritical extraction method, a solvent extraction method, an enzyme dissolution method, an ultrasonic-assisted extraction method, a microwave-assisted method and the like, wherein the ultrasonic-assisted extraction method has high efficiency and low equipment cost, and is widely applied at present. The capsaicin detection method mainly comprises artificial taste (Schwarval index quantification), spectrophotometry, High Performance Liquid Chromatography (HPLC) and the like. However, High Performance Liquid Chromatography (HPLC) is used more frequently for detecting the content of capsaicin due to its higher accuracy. However, the HPLC detection is limited by the cost of the detection instrument and the complexity of the detection process, and thus the application of the HPLC detection in capsaicin detection is limited. Therefore, it is desirable to provide a cost-effective and simple-procedure method for measuring capsaicin content.

Disclosure of Invention

In view of this, the present invention aims to provide a method for detecting capsaicin content by using botrytis cinerea, which has the characteristics of cost saving, simple process and high accuracy.

In order to achieve the above purpose, the invention provides the following technical scheme:

a method for detecting capsaicin content by using botrytis cinerea comprises the following steps: inoculating botrytis cinerea into V8 culture medium containing capsaicin with different concentrations, and establishing a linear standard curve of capsaicin concentration and diameter of botrytis cinerea plaque; extracting capsaicin in a sample to be detected, and preparing a liquid to be detected; inoculating the botrytis cinerea into a V8 culture medium containing the solution to be detected for culture, and calculating the content of capsaicin in the sample to be detected according to the diameter of the botrytis cinerea bacterial plaque to be detected in the culture time and a linear standard curve.

Preferably, the culture time is 3-4 d.

Preferably, the linear standard curve of capsaicin concentration and gray mold plaque diameter when cultured for 4d is:

y-1.699 x +49.44 equation (1);

y is capsaicin concentration, and x is gray mold plaque diameter.

Preferably, the botrytis cinerea is a botrytis cinerea suspension, and the inoculation concentration of the botrytis cinerea suspension is 0.5-1.5 multiplied by 10⁵one/mL.

Preferably, the botrytis cinerea suspension is obtained by shaking cultured botrytis cinerea in a staining solution, wherein the staining solution comprises maltose and peptone, and the mass ratio of the maltose to the peptone is (5-10) to (1-3).

Preferably, the method for culturing the botrytis cinerea comprises: the botrytis cinerea is inoculated in a V8 culture medium and cultured for 8-13 days at the temperature of 23-27 ℃.

Preferably, the method for extracting capsaicin from the sample to be detected comprises the following steps: and ultrasonically extracting a sample to be detected by using methanol, filtering, concentrating, and performing constant volume by using the methanol to obtain the liquid to be detected.

Preferably, the temperature of ultrasonic extraction is 45-55 ℃, and the time is 40-50 min.

Preferably, the mass-volume ratio of the sample to be detected to the methanol ultrasonic wave is (8-13): 12-17) g/mL.

Preferably, the concentration temperature is 66-73 ℃.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for detecting capsaicin content by using botrytis cinerea, which comprises the following steps: inoculating botrytis cinerea into V8 culture medium containing capsaicin with different concentrations, and establishing a linear standard curve y of the concentration of the capsaicin and the diameter of the botrytis cinerea plaque, wherein y is the concentration of the capsaicin and x is the diameter of the botrytis cinerea plaque, and the linear standard curve y is-1.699 x + 49.44; extracting capsaicin in a sample to be detected, and preparing a liquid to be detected; inoculating the botrytis cinerea into a V8 culture medium containing the solution to be detected for culture, and calculating the content of capsaicin in the sample to be detected according to the diameter of the botrytis cinerea bacterial plaque to be detected in the culture time and a linear standard curve. The method for measuring the capsaicin content has the characteristics of low cost, simple and convenient operation, economy, practicability and high accuracy.

Drawings

FIG. 1 is a graph showing the difference in growth of Botrytis cinerea on V8 medium containing capsaicin at different concentrations in example 1;

FIG. 2 is the colony diameters of Botrytis cinerea on the medium of different capsaicin concentrations V8 in example 1;

FIG. 3 is a linear relationship between capsaicin concentration and Botrytis cinerea colony diameter.

Detailed Description

The invention provides a method for detecting capsaicin content by using botrytis cinerea, which comprises the following steps: inoculating botrytis cinerea into V8 culture medium containing capsaicin with different concentrations, and establishing a linear standard curve of capsaicin concentration and diameter of botrytis cinerea plaque; extracting capsaicin in a sample to be detected, and preparing a liquid to be detected; inoculating the botrytis cinerea into a V8 culture medium containing the solution to be detected for culture, and calculating the content of capsaicin in the sample to be detected according to the diameter of the botrytis cinerea bacterial plaque to be detected in the culture time and a linear standard curve.

In the invention, the Botrytis cinerea is obtained by separating and purifying field pathogenic plants, and is proved to be Botrytis cinerea through sequencing.

In the present invention, the kind of the sample to be measured is not particularly limited. In an embodiment of the present invention, the types of the samples to be tested include pepper fruits, further specifically, hangzhou pepper No. 12, spicy No. 2 and P1622.

In the present invention, the culture time is preferably 3 to 4 days, and more preferably 4 days. In the method, if the culture time of the botrytis cinerea exceeds 4 days, the size of the botrytis cinerea plaque on a 0ug/ml culture medium exceeds a culture dish, and the effective diameter cannot be measured, so that the culture time of the botrytis cinerea is 3-4 days.

In the present invention, the linear standard curve of capsaicin concentration and gray mold plaque diameter at 4d culture is: y-1.699 x +49.44 equation (1); y is capsaicin concentration, and x is gray mold plaque diameter.

In the invention, the botrytis cinerea is a botrytis cinerea suspension, and the inoculation concentration of the botrytis cinerea suspension is preferably 0.5-1.5 multiplied by 10⁵one/mL, more preferably 1X 10⁵one/mL.

In the invention, the botrytis cinerea suspension is obtained by shaking cultured botrytis cinerea in a staining solution, the staining solution preferably comprises maltose and peptone, and the mass ratio of the maltose to the peptone is preferably (5-10): 1-3), and more preferably 8: 2.

In the present invention, the method for culturing the botrytis cinerea preferably comprises: the botrytis cinerea is inoculated in a V8 culture medium, and is preferably cultured at 23-27 ℃ for 8-13 days, and more preferably at 25 ℃ for 10 days.

In the present invention, the method for extracting capsaicin from the sample to be tested preferably comprises: and ultrasonically extracting a sample to be detected by using methanol, filtering, concentrating, and performing constant volume by using the methanol to obtain the liquid to be detected.

In the invention, the temperature of ultrasonic extraction is preferably 45-55 ℃, more preferably 50 ℃, and the time is preferably 40-50 min, more preferably 45 min. In the invention, the long ultrasonic time is beneficial to the extraction of capsaicin, but the extraction time is consumed if the ultrasonic time is too long; the extraction temperature is low, the extraction of capsaicin is insufficient, and the capsaicin is easy to degrade when the extraction temperature is high.

In the invention, the mass-to-volume ratio of the sample to be detected to the methanol ultrasonic wave is preferably (8-13): 12-17) g/mL, and more preferably 10:15 g/mL. In the present invention, capsaicin extracted by methanol has higher extraction efficiency than other organic reagents.

In the invention, the concentration temperature is preferably 66-73 ℃, and more preferably 70 ℃.

In the invention, the method also preferably comprises freeze drying and crushing the sample to be detected before the methanol ultrasonic extraction.

In the present invention, filtration is also preferably included after the concentration, and as an embodiment, filtration with a 0.45 μm filter is used.

The raw material sources not mentioned in the present invention are not particularly limited, and conventional commercial products in the art may be used.

The invention is not limited to the mechanical equipment which is not mentioned, and the conventional mechanical equipment in the field can be adopted.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation of Linear Standard Curve for capsaicin concentration and Gray mold plaque diameter

1. Preparing capsaicin:

preparing a capsaicin mother solution: capsaicin (60.0mg) and methanol (1mL) were mixed to form a capsaicin stock solution preparation, which was stored at a low temperature of 4 ℃.

2. Highly pathogenic gray mold species (Botrytis cinerea, isolated and purified from field-diseased plants) were selected for the experiments, and the propagation conditions of the species were as follows:

1) preparation of V8 medium: v8(360mL), calcium carbonate (2.0g), agar (20.0 g). Mixing the components, adding distilled water to form 1000mL of solution preparation, and placing the solution preparation at 4 ℃ for later use after high temperature and high pressure (121 ℃, 15 minutes);

2) preparation of V8 dip dyeing solution: maltose (8.0g), peptone (2.0 g). Mixing the components, adding distilled water to form 200mL of solution preparation, and placing the solution preparation at 4 ℃ for later use after high temperature and high pressure (121 ℃, 15 minutes);

3) propagation culture conditions: melting the solid culture medium at high temperature, pouring 10mL of the solid culture medium into a 90mm disposable sterile culture dish, and inoculating a loop after solidification with the botrytis cinerea. Culturing at 25 deg.C for 10 days.

3. The identification method for inhibiting the growth of botrytis cinerea by capsaicin comprises the following steps:

1) scraping propagating Botrytis cinerea from V8 culture medium, adding dip dyeing solution, shaking to release spore, filtering with two layers of gauze, adding dip dyeing solution, diluting to spore concentration of 1 × 10⁵Per mL;

2) preparing V8 solid culture medium (10 mL/culture dish) with capsaicin concentrations of 0. mu.g/mL, 15. mu.g/mL, 30. mu.g/mL and 45. mu.g/mL respectively;

3) the 4 media containing capsaicin at different concentrations in step 2 were inoculated with spores at a concentration of 1X 10⁵1 mu L of botrytis cinerea liquid per mL;

4) after culturing for 4 days, measuring the diameters of the gray mold bacterial plaques on the culture medium by using a ruler;

5) as can be seen from FIGS. 1-3, different concentrations of capsaicin inhibited Botrytis cinerea, the higher the concentration of capsaicin, the more pronounced the inhibition, and the concentration of capsaicin was linearly related to the diameter of Botrytis cinerea. Performing linear trend analysis on the diameters of the gray mold plaque and the concentration of the capsaicin by using Excel 2007 to obtain a conversion formula y of-1.699 x +49.44 and a correlation coefficient R²＝0.995。

Example 2

Extraction of Hangzhou pepper No. 12 capsaicin

1) Picking fresh No. 12 capsicum fruits of Hangzhou peppers with the green maturity of 10g, putting the fruits into a freeze dryer for freeze drying, grinding the fruits until the crushed fruits are put into a 50mL centrifuge tube, and preserving the crushed samples at 4 ℃ for later use;

2) adding 15mL of methanol into a 50mL tube filled with a sample, sealing the tube by using a preservative film, and then performing ultrasonic oscillation extraction for 45min under the condition of water bath at 50 ℃;

3) filtering the extractive solution with filter paper, concentrating at 70 deg.C to below 1mL with rotary evaporator, and diluting to 1mL with methanol;

4) filtering the extractive solution with 0.45 μm filter membrane;

5) preparing 10 mu L of extracting solution into a 10mLV8 culture medium, inoculating botrytis cinerea, culturing for 4 days, measuring diameters of botrytis cinerea plaques on the culture medium by using a ruler, wherein the diameters of the botrytis cinerea plaques are 27.75mm, and calculating the concentration of capsaicin in pepper fruits according to a formula y of-1.699 x +49.44 to be 2.29 mu g/mL, the content of capsaicin in a V8 culture medium to be 22.93 mu g, the concentration of the capsaicin in the extracting solution to be 2.29mg/mL, and the content of the capsaicin in Hangzhou pepper No. 12 fruits to be 2.29mg/10 g.

Example 3

The procedure was the same as in example 1, except that the pepper fruit was spicy No. 2. The measured diameter of the gray mold plaque is 6.25mm, the concentration of capsaicin in the pepper fruits is 38.82 mu g/mL according to the formula y being-1.699 x +49.44, and the capsaicin content in the pepper spicy No. 2 fruits is converted into 38.82mg/10 g.

Example 4

The procedure was as in example 1, except that the pepper fruit was P1622. The measured diameter of the gray mold plaque was 14.25mm, and the concentration of capsaicin in the pepper fruits was 25.23 μ g/mL calculated according to the formula y-1.699 x +49.44, which was converted to 25.23mg/10g of capsaicin in the pepper P1622 fruits.

Comparative example 1

After capsaicin was extracted from 10g of Hangzhou pepper 12 fruits (method see example 2), the capsaicin content was measured using a high performance liquid chromatography HPLC (Waters Corp., Milford, MA, USA) system: the mobile phase is methanol + water (65+35, volume ratio), the type of a chromatographic column is 4.6mm multiplied by 250mm, the sample amount of each sample is 10 mu L, the flow rate is 1mL/min, the ultraviolet detection wavelength is 280nm, the capsaicin peak time is about 15min, and the capsaicin concentration in Hangzhou pepper 12 pepper fruits is 2.07mg/10g through peak area conversion.

Comparative example 2

After capsaicin was extracted from 10g of spicy No. 2 fruits (method reference example 2), the capsaicin content was measured using a high performance liquid chromatography HPLC (Waters corp., Milford, MA, USA) system: the mobile phase is methanol + water (65+35, volume ratio), the model of the chromatographic column is 4.6mm multiplied by 250mm, the sample amount of each sample is 10 mu L, the flow rate is 1mL/min, the ultraviolet detection wavelength is 280nm, the capsaicin peak-off time is about 15min, and the capsaicin concentration in spicy No. 2 pepper fruits is 34.77mg/10g through peak area conversion.

Comparative example 3

After extraction of capsaicin from 10g P1622 fruits (method reference example 2), the capsaicin content was measured using a high performance liquid chromatography HPLC (Waters corp., Milford, MA, USA) system: the mobile phase is methanol + water (65+35, volume ratio), the model of the chromatographic column is 4.6mm multiplied by 250mm, the sample amount of each sample is 10 mu L, the flow rate is 1mL/min, the ultraviolet detection wavelength is 280nm, the capsaicin peak time is about 15min, and the capsaicin concentration in the P1622 pepper fruit is 28.39mg/10g through peak area conversion.

Example 5

The results of the capsaicin contents in the pepper fruits of examples 2 to 4 and comparative examples 1 to 3 are shown in Table 1.

TABLE 1 capsaicin contents in pepper fruits of examples 2-4 and comparative examples 1-3

As can be seen from Table 1, the capsaicin values measured by the method of the present invention are relatively close to those measured by High Performance Liquid Chromatography (HPLC). Therefore, the method for detecting capsaicin has high accuracy, and has the advantages of low cost and simple and convenient operation compared with a High Performance Liquid Chromatography (HPLC) method.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.