NL2032572A - Method for determining capsaicin content by botrytis cinerea - Google Patents
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Abstract
The present disclosure provides a method for determining a capsaicin content by Botrytis cinerea, and relates to the technical field of food detection. The method for determining a capsaicin content by B. cinerea provided by the present disclosure includes the steps of: inoculating the B. cinerea on V8 Juice Agars supplemented with different concentrations of capsaicin to establish a linear standard curve of a capsaicin concentration versus a B. cinerea plaque diameter, y = —l.699x + 49.44, where the y is the capsaicin concentration, and the X is the B. cinerea plaque diameter; extracting the capsaicin from an unknown sample, and preparing a test solution; inoculating the B. cinerea on a V8 Juice Agar supplemented with the test solution for culture, and calculating the capsaicin content in the unknown sample according to a B. cinerea plaque diameter in a culture period and the linear standard curve.
Description
METHOD FOR DETERMINING CAPSAICIN CONTENT BY BOTRYTIS CINEREA
The present disclosure relates to the technical field of food detection, in particular to a method for determining a capsaicin content by Botrytis cinerea.
Capsaicin is an alkaloid that is unique to the fruits of Cap- sicum plants in Solanaceae and can cause a burning sensation when mammals eat pepper-related products. Generally speaking, capsaicin occupies more than 80% of the capsaicinoids in pepper fruit, and it is the main determinant of the formation of peppery taste in a pepper. Capsaicin has anti-inflammatory, anti-oxidant, anti-tumor and fat-reducing functions, and has a wide range of applications in medicine, feed additives and other fields.
At present, extraction methods of capsaicin include super- critical fluid extraction, solvent extraction, enzymatic lysis, ultrasonic-assisted extraction, and microwave-assisted extraction.
Among them, ultrasonic-assisted extraction has high efficiency and low equipment cost, and is widely used at present. Capsaicin de- tection methods mainly include human tasting (Scoville scale quan- tification), spectrophotometry, high performance liquid chromatog- raphy (HPLC), and the like. However, HPLC is widely used in the determination of capsaicin content due to higher accuracy thereof.
However, use of HPLC in capsaicin detection is limited due to the limitation of the cost of detection equipment and the cumbersome detection process. Therefore, it is necessary to provide a cost- saving and simple method for determining a capsaicin content.
In view of this, an objective of the present disclosure is to provide a method for determining a capsaicin content by B. ciner- ea, which is characterized by cost saving, simple process and high accuracy.
To achieve the above objective, the present disclosure pro- vides the following technical solution:
A method for determining a capsaicin content by B. cinerea is provided, including the steps of: inoculating the B. cinerea on V8
Juice Agars supplemented with different concentrations of capsai- cin to establish a linear standard curve of a capsaicin concentra- tion versus a B. cinerea plaque diameter; extracting the capsaicin from an unknown sample, and preparing a test solution; inoculating the B. cinerea on a V8 Juice Agar supplemented with the test solu- tion for culture, and calculating the capsaicin content in the un- known sample according to a B. cinerea plaque diameter in a cul- ture period and the linear standard curve.
Preferably, the culture may last for 3-4 days.
Preferably, on Day 4 of the culture, the linear standard curve of the capsaicin concentration versus the B. cinerea plaque diameter is: y = -1.699x + 49.44 Equation (1); where the y is the capsaicin concentration, and the x is the
B. cinerea plaque diameter.
Preferably, the B. cinerea may be a B. cinerea suspension, and an inoculation concentration of the B. cinerea suspension may be 0.5-1.5 x 10° cells/mL.
Preferably, the B. cinerea suspension may be obtained by shaking cultured B. cinerea cells in an impregnating solution, the suspension may include maltose and peptone, the maltose and the peptone may have a mass ratio of (5-10):(1-3).
Preferably, a culture method of the B. cinerea cells may in- clude the step of: inoculating the B. cinerea on the V8 Juice Agar to culture at 23-27°C for 8-13 days.
Preferably, a method for extracting the capsaicin from the unknown sample may include steps of: conducting ultrasonic extrac- tion on the unknown sample with methanol, filtering and concen- trating, and diluting a resulting sample solution with the metha- nol to obtain the test solution.
Preferably, the ultrasonic extraction may be conducted at 45- 55°C for 40-50 min.
Preferably, the unknown sample and the methanol may have a mass-volume ratio of (8-13):(12-17) g/mL during the ultrasonic ex- traction.
Preferably, the concentration may be conducted at 66-73°C.
Compared with the prior art, the present disclosure has the following beneficial effects:
The present disclosure provides a method for determining a capsaicin content by B. cinerea, including the steps of: inoculat- ing the B. cinerea on V8 Juice Agars supplemented with different concentrations of capsaicin to establish a linear standard curve of a capsaicin concentration versus a B. cinerea plaque diameter, y = -1.699x + 49.44, where the y is the capsaicin concentration, and the x is the B. cinerea plaque diameter; extracting the capsa- icin from an unknown sample, and preparing a test solution; inocu- lating the B. cinerea on a V8 Juice Agar supplemented with the test solution for culture, and calculating the capsaicin content in the unknown sample according to a B. cinerea plaque diameter in a culture period and the linear standard curve. The method provid- ed by the present disclosure is characterized by low cost, simple and convenient operation, economy and practicability, and high ac- curacy.
FIG. 1 illustrates a growth difference of B. cinerea on the
V8 Juice Agars supplemented with different concentrations of cap- saicin in Example 1;
FIG. 2 illustrates the colony diameter of B. cinerea on V8
Juice Agars at different capsaicin concentrations in Example 1;
FIG. 3 illustrates a linear relationship between capsaicin concentration and colony diameter of B. cinerea.
The present disclosure provides a method for determining a capsaicin content by B. cinerea, including the steps of: inoculat- ing the B. cinerea on V8 Juice Agars supplemented with different concentrations of capsaicin to establish a linear standard curve of a capsaicin concentration versus a B. cinerea plaque diameter; extracting the capsaicin from an unknown sample, and preparing a test solution; inoculating the B. cinerea on a V8 Juice Agar sup- plemented with the test solution for culture, and calculating the capsaicin content in the unknown sample according to a B. cinerea plaque diameter in a culture period and the linear standard curve.
In the present disclosure, a gray mold pathogen is obtained by isolation and purification from diseased plants in the field and is confirmed to be B. cinerea after sequencing.
In the present disclosure, there is no particular limitation on types of the unknown sample. In a specific example of the pre- sent disclosure, the types of the unknown sample may include pep- per fruits, and may further be Capsicum annuum cv. Hangjiao No. 12, C. annuum cv. Xinxiang No. 2, and C. annuum cv. P1622.
In the present disclosure, the culture may last preferably for 3-4 days, and more preferably 4 days. In the present disclo- sure, on conditions that the culture period of the B. cinerea ex- ceeds 4 days, a size of a B. cinerea plague on 0 ug/mL medium may exceed that of a Petri dish, and an effective diameter cannot be measured. Therefore, the culture period of the B. cinerea selected in the present disclosure is 3-4 days.
In the present disclosure, on Day 4 of the culture, a linear standard curve of capsaicin concentration versus B. cinerea plaque diameter is: y = -1.699x + 49.44 Equation (1); the y is the capsa- icin concentration, and the x is the B. cinerea plaque diameter.
In the present disclosure, the B. cinerea may preferably be a
B. cinerea suspension, and an inoculation concentration of the B. cinerea suspension may preferably be 0.5-1.5 x 10° cells/mL, and more preferably 1x10° cells/mL.
In the present disclosure, the B. cinerea suspension may be obtained by shaking cultured B. cinerea cells in an impregnating solution, the suspension may preferably include maltose and pep- tone, the maltose and the peptone may preferably have a mass ratio of (5-10):(1-3), and more preferably 8:2.
In the present disclosure, a culture method of the B. cinerea cells may preferably include the step of: inoculating the B. ci- nerea on the V8 Juice Agar to preferably culture at 23-27°C for 8- 13 days and more preferably at 25°C for 10 days.
In the present disclosure, a method for extracting the capsa-
icin from the unknown sample may preferably include steps of: con- ducting ultrasonic extraction on the unknown sample with methanol, filtering and concentrating, and diluting a resulting sample solu- tion with the methanol to obtain the test solution. 5 In the present disclosure, the ultrasonic extraction may preferably be conducted at 45-55°C for 40-50 min and more prefera- bly at 50°C for 45 min. In the present disclosure, long ultrasonic time is beneficial to capsaicin extraction, but if the ultrasonic time is too long, extraction time will be consumed; a low extrac- tion temperature results in insufficient capsaicin extraction, and a high extraction temperature results in easy degradation of the capsaicin.
In the present disclosure, the unknown sample and the metha- nol may preferably have a mass-volume ratio of (8-13):(12-17) g/mL and more preferably 10:15 g/mL during the ultrasonic extraction.
In the present disclosure, extraction of the capsaicin with the methanol has higher extraction efficiency than that with other or- ganic reagents.
In the present disclosure, the concentration may preferably be conducted at €6-73°C, and more preferably 70°C.
In the present disclosure, before the ultrasonic extraction with methanol, it may be preferable to include freeze-drying and pulverizing the unknown sample.
In the present disclosure, the concentration may preferably be followed by filtration. As a possible implementation, the fil- tration may be performed with a 0.45 um filter membrane.
The present disclosure does not specifically limit sources of unmentioned raw materials, as long as commercially available prod- ucts conventional in the art may be used.
The present disclosure does not specifically limit sources of unmentioned mechanical equipment, as long as mechanical equipment conventional in the art may be used.
The technical solution provided by the present disclosure will be described in detail below with reference to examples, but they should not be construed as limiting the protection scope of the present disclosure.
Example 1
Plotting of a linear standard curve of capsaicin concentra- tion versus B. cinerea plaque diameter 1. Preparation of capsaicin:
Preparation of a capsaicin stock solution: Capsaicin (60.0 mg) and methanol (1 mL) were mixed to form a capsaicin stock solu- tion, which was cryopreserved at 4°C. 2. A highly pathogenic Botrytis strain (B. cinerea, isolated and purified from diseased plants in the field) was selected for the experiment. The multiplication conditions of the strain were as follows: 1) V8 Juice Agar preparation: V8 (360 mL), calcium carbonate (2.0 g), and agar (20.0 g). The above components were mixed and distilled water was added to form 1,000 mL of solution formula- tion; the solution formulation was autoclaved (at 121°C for 15 min), and then placed at 4°C for later use. 2) Preparation of a V8 impregnating solution: maltose (8.0 g) and peptone (2.0 g). The above components were mixed and distilled water was added to form 200 mL of solution formulation; the solu- tion formulation was autoclaved (at 121°C for 15 min), and then placed at 4°C for later use. 3) Multiplication culture conditions: The solid medium was molten at a high temperature, 10 mL of the molten solid medium was poured into a 90 mm disposable sterilized Petri dish, and B. ci- nerea was inoculated with an inoculation loop after solidifica- tion. The B. cinerea was cultured at 25°C for 10 days for later use. 3. Identification method of capsaicin inhibiting the growth of B. cinerea: 1) The multiplied B. cinerea cells were scraped from the V8
Juice Agar, and the impregnating solution was added to fully shake to release the spores; after filtering through two layers of gauze, the impregnating solution was added to dilute to a spore concentration of 1 x 10° cells/mL; 2) V8 Juice Agars (10 mL/dish) with capsaicin concentrations of 0 pg/mL, 15 pg/mL, 30 pg/mL and 45 pg/mL were prepared, respec- tively;
3) 1 HL each of B. cinerea suspension with a spore concentra- tion of 1 x 10° cells/mL was inoculated in four culture media sup- plemented with different concentrations of capsaicin in step 2; 4) separately, a scale was used to measure the B. cinerea plaque diameter on the culture medium 4 days after culture; and 5) from FIGS. 1 to 3, different concentrations of capsaicin had an inhibitory effect on B. cinerea; higher capsaicin concen- tration showed more obvious inhibitory effect, and the capsaicin concentration was linearly related to the B. cinerea plaque diame- ter; a linear trend analysis of the B. cinerea plaque diameter versus the capsaicin concentration was conducted by using Excel 2007, and a conversion equation was obtained: y = -1.699x + 49.44,
R“ = 0.995.
Example 2
Extraction of capsaicin from C. annuum cv. Hangjiao No. 12 1) 10 g of fresh fruits of C. annuum cv. Hangjiao No. 12 were picked at the green ripe stage, freeze-dried in a freeze dryer, ground and pulverized to obtain a sample; the sample was placed in a 50 mL centrifuge tube and stored at 4°C for later use; 2) 15 mL of methanol was added to the 50 mL tube with the sample, sealed with a cling film, and extracted in a 50°C water bath by ultrasonic vibration for 45 min; 3) the extract was filtered through filter paper and concen- trated in a rotary evaporator to less than 1 mL at 70°C, and the concentrate was diluted to 1 mL with methanol; 4) the extract was filtered through a 0.45 pm filter mem- brane; and 5) 10 pL of the extract was pipetted to prepare 10 mL of V8
Juice Agar, and B. cinerea was inoculated; after culturing for 4 days, a scale was used to measure the B. cinerea plague diameter on the culture medium, and the B. cinerea plaque diameter was 27.75 mm; according to the equation y = -1.699x + 49.44, the cap- saicin concentration in the fruit was calculated to be 2.29 pg/mL, the capsaicin content in the V8 Juice Agar was 22.93 ug, the cap- saicin concentration in the extract was 2.29 mg/mL, and the capsa- icin content in the fruit of C. annuum cv. Hangjiao No. 12 was 2.29 mg/10 g .
Example 3
The operation steps were the same as those in Example 1, ex- cept that the pepper fruit was C. annuum cv. Xinxiang No. 2. The measured B. cinerea plaque diameter was 6.25 mm; according to the equation y = -1.699x + 49.44, the capsaicin concentration in the pepper fruit was calculated to be 38.82 pg/mL; after conversion, the capsaicin content in the fruit of C. annuum cv. Xinxiang No. 2 was 38.82 mg/10 g.
Example 4
The operation steps were the same as those in Example 1, ex- cept that the pepper fruit was C. annuum cv. P1622. The measured
B. cinerea plaque diameter was 14.25 mm; according to the equation y = -1.699x + 49.44, the capsaicin concentration in the pepper fruit was calculated to be 25.23 ug/mL; after conversion, the cap- saicin content in the fruit of C. annuum cv. P1622 was 25.23 mg/10 dg.
Comparative Example 1
After extracting capsaicin from 10 g of the fruit of C. annu- um cv. Hangjiao No. 12 (refer to Example 2 for the method), the capsaicin content was determined by using an HPLC system (Waters
Corp., Milford, MA, USA): the mobile phase was methanol + water (65 + 35, volume ratio), the chromatographic column model was 4.6 x 250 mm, the sample size was 10 pL for each sample, the flow rate was 1 mL/min, the UV detection wavelength was 280 nm, and the peak time of capsaicin was around 15 min; after peak area conversion, the capsaicin concentration in the fruit of C. annuum cv. Hangjiao
No. 12 was 2.07 mg/10 g.
Comparative Example 2
After extracting capsaicin from 10 g of the fruit of C. annu- um cv. Xinxiang No. 2 (refer to Example 2 for the method), the capsaicin content was determined by using an HPLC system (Waters
Corp., Milford, MA, USA): the mobile phase was methanol + water (65 + 35, volume ratio), the chromatographic column model was 4.6 x 250 mm, the sample size was 10 pL for each sample, the flow rate was 1 mL/min, the UV detection wavelength was 280 nm, and the peak time of capsaicin was around 15 min; after peak area conversion, the capsaicin concentration in the fruit of C. annuum cv. Xinxiang
No. 2 was 34.77 mg/10 g.
Comparative Example 3
After extracting capsaicin from 10 g of the fruit of C. annu- um cv. P1622 (refer to Example 2 for the method), the capsaicin content was determined by using an HPLC system (Waters Corp., Mil- ford, MA, USA): the mobile phase was methanol + water (65 + 35, volume ratio), the chromatographic column model was 4.6 x 250 mm, the sample size was 10 uL for each sample, the flow rate was 1 mL/min, the UV detection wavelength was 280 nm, and the peak time of capsaicin was around 15 min; after peak area conversion, the capsaicin concentration in the fruit of C. annuum cv. P1622 was 28.39 mg/10 g.
Example 5
The results of the capsaicin content in pepper fruits in Ex- amples 2 to 4 and Comparative Examples 1 to 3 are shown in Table 1.
Table 1 The capsaicin content in pepper fruits in Examples 2 to 4 and Comparative Examples 1 to 3 rep ee ee
Example 2 | Example 3 Example 4
Example 1 Example 2 Example 3
Capsaicin content 2.29 38.82 25.23 2.07 34.77 26.39 (mg/10 8}
It can be seen from Table 1 that the capsaicin values deter- mined by the method provided by the present disclosure are close to those determined by HPLC. Visibly, the method for determining a capsaicin content provided by the present disclosure has high ac- curacy and has the advantages of low cost and simple operation compared with HPLC.
The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of or- dinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present dis- closure.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103018242A (en) * | 2013-01-05 | 2013-04-03 | 西南大学 | Rapid colorimetric determination method for capsaicin content of capsicum products |
| CN105548553A (en) * | 2016-02-04 | 2016-05-04 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatography test strip for rapidly detecting capsaicinoids as well as preparation method and application thereof |
| AU2021101167A4 (en) * | 2020-03-12 | 2021-05-06 | Zhejiang Academy Of Agricultural Sciences | Botrytis cinerea inhibitor and use thereof |
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| CN110150330A (en) * | 2017-12-29 | 2019-08-23 | 云南宏绿辣素有限公司 | A kind of compound botanical source bactericide and preparation method thereof |
| CN108181397A (en) * | 2017-12-29 | 2018-06-19 | 浙江农林大学 | Hangzhou chili capsaicine concentration extraction measuring method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103018242A (en) * | 2013-01-05 | 2013-04-03 | 西南大学 | Rapid colorimetric determination method for capsaicin content of capsicum products |
| CN105548553A (en) * | 2016-02-04 | 2016-05-04 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatography test strip for rapidly detecting capsaicinoids as well as preparation method and application thereof |
| AU2021101167A4 (en) * | 2020-03-12 | 2021-05-06 | Zhejiang Academy Of Agricultural Sciences | Botrytis cinerea inhibitor and use thereof |
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