Method for detecting content of total glucosinolate in broccoli bud seedlings

Abstract

The invention discloses a method for detecting the content of total glucosinolate in broccoli sprouts. The detection method provided by the invention comprises the following steps: crushing a bud seedling sample, adding an ethanol solution, extracting and centrifuging to obtain an extracting solution; adding a PdCl2 solution and a sodium carboxymethyl cellulose solution into the extracting solution, standing, and detecting by using a spectrophotometer; and substituting the detected data into a formula, and calculating to obtain the total glucosinolate content in the bud seedlings. According to the characteristics of broccoli bud seedlings, high-concentration ethanol is selected as an extraction reagent, a palladium chloride method is adopted for detection, the purpose of rapid and accurate detection is achieved by optimizing extraction conditions and detection conditions, and technical support is provided for producing bud seedlings with high glucosinolate content.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, specifically relating to a method for detecting the total glucosinolate content in broccoli sprouts. Background Technology

[0002] Broccoli, also known as green cauliflower, is rich in glucosinolates, with the content of glucosinolates, particularly radicchio, far exceeding that of other Brassica vegetables. The highest content is found in the seeds, florets, and seedlings. Studies have shown that glucosinolates and their degradation products in vegetables such as broccoli can enhance the body's immunity, antioxidant, anti-mutagenic, and anti-cancer abilities. Consuming cruciferous vegetables can also alleviate chronic diseases including cardiovascular disease, diabetes, and obesity.

[0003] Methods for detecting total glucosinolates in plants mainly include colorimetric methods, high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis, and nuclear magnetic resonance spectroscopy (NMR). These methods often require complex sample pretreatment steps, or lack sufficient sensitivity for detecting low concentrations of glucosinolates, or are susceptible to interference from complex sample matrices affecting accuracy, or involve expensive instruments, limiting their application in resource-constrained laboratories. For example, Velasco et al. first applied near-infrared reflectance spectroscopy (NIRS) to the analysis and identification of glucosinolates (Velasco et al., 1998). This method can accurately detect the content of glucosinolates, but it requires high-performance instruments. Li Yanli et al. combined this method with traditional chemical methods to create an NIRS detection method (Li Yanli et al., 2003), but it requires a large number of standards to establish a model, which has limitations. Svanem et al. used low-temperature organic solvents when simultaneously extracting glucosinolates and their degradation products (Agerbirk et al., 2012). In the research experiments of Mohn et al., methanol was the best solvent for the extraction of glucosinolates (Mobn et al., 2007). In solvent extraction, boiling water (Li Donghua et al., 2013) and chloroform (Bennett, 2007) can also be used as solvents for crude extraction of glucosinolates.

[0004] In recent years, most studies on glucosinolates have focused on the identification and analysis of their monomeric structures, with less research on extraction processes. Different researchers have optimized the extraction process of glucosinolates from broccoli. Li Ning (2015) obtained the optimal extraction process for glucosinolates from dried broccoli through single-factor experiments: 90% ethanol as the extractant, a material-to-liquid ratio of 1:11, an extraction time of 1 h, and repeated extraction four times, yielding glucosinolates with a purity of 15.22 mg·g⁻¹. Jiang Min et al. (2012) used methanol as a solvent to extract glucosinolates from broccoli seeds (Jiang Min et al., 2013). Jia Zhiyong et al. (2016) obtained the optimal extraction conditions for glucosinolates from broccoli stems through single-factor and orthogonal experiments: 75% ethanol concentration, a material-to-liquid ratio of 1:15, an extraction temperature of 70℃, and an extraction time of 30 min. Under these conditions, the molar concentration of glucosinolates in the extract was 601.24 μmol / g. Deng Yanmei et al. (2013) determined the optimal conditions for extracting glucosinolates from broccoli florets using single-factor and orthogonal experiments: 75% ethanol concentration, a material-to-liquid ratio of 1:7, an extraction temperature of 80℃, and an extraction time of 20 min. The optimal conditions for extracting glucosinolates from broccoli florets were 33.71 μmol / g. He Xia et al. (2021) optimized the extraction process of total glucosinolates from broccoli florets, selecting the optimal process as follows: enzyme inactivation treatment at 80℃ for 15 min after adding 100℃ distilled water, followed by drying at 70℃ with forced air, 90% ethanol concentration, a material-to-liquid ratio of 1:9, an extraction temperature of 60℃, and an extraction time of 30 min. Under these conditions, the highest total glucosinolate content in broccoli florets was measured to be 414.98 μmol / g.

[0005] The above methods were used to study the extraction and detection processes of glucosinolates from broccoli florets, dried broccoli powder, and florets. However, due to the small size and high water content of broccoli sprouts, their tissue structure differs from that of florets and stems. Broccoli sprouts are also rich in ascorbic acid, anthocyanins, soluble sugars, and soluble proteins, which affect the extraction of total glucosinolates. Furthermore, the glucosinolate content in sprouts is much higher than that in florets. The extraction methods for total glucosinolates in florets cannot extract all the glucosinolates from broccoli sprouts, making it difficult to accurately detect the total glucosinolate content in broccoli sprouts using existing extraction methods and detection conditions.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting the total glucosinolate content in broccoli buds using a combination of palladium chloride-spectrophotometry. This method can quickly and conveniently achieve accurate detection of the total glucosinolate content in a large number of bud samples.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a method for detecting the total glucosinolate content in broccoli sprouts, comprising the following steps: S1. Crush the seedling sample, add ethanol solution, extract, centrifuge, and take the supernatant to obtain the extract; S2. Add PdCl2 solution and sodium carboxymethyl cellulose solution to the extract, let stand, and detect using a spectrophotometer; S3. Substitute the detected data into the formula to calculate the total glucosinolate content in the seedlings.

[0009] This invention, taking into account the unique characteristics of broccoli sprouts, proposes a suitable detection procedure for broccoli sprouts, overcoming the aforementioned technical problems. Specifically, this invention first directly crushes the broccoli sprouts and extracts them with an ethanol solution, which fully dissolves various glucosinolate components. The supernatant obtained after centrifugation can be directly used as the test solution, simplifying the process. Then, palladium chloride and sodium carboxymethyl cellulose are added to the supernatant. PdCl2 reacts with glucosinolate molecules under acidic conditions to form a soluble glucosinolate-palladium complex, while the addition of sodium carboxymethyl cellulose acts as a stabilizer. Finally, quantitative determination is performed at a specific wavelength using a spectrophotometer, achieving a simple, rapid, and accurate detection of glucosinolates in sprouts.

[0010] In step S1, the volume fraction of the ethanol solution is 80-85%; the amount of ethanol solution used is 15-16 ml of ethanol solution per 1 g of seedling.

[0011] In step S1, the extraction conditions are: water bath at 80-85℃ for 20-25 minutes.

[0012] In step S1, the centrifugation conditions are: a rotation speed of 8000-8500 rpm and a time of 6-7 min. By optimizing the centrifugation conditions, it is possible to retain the total glucosinolates in the supernatant while reducing the presence of impurities in the supernatant.

[0013] In step S1, the seedlings are seedlings that have germinated 7-10 days after the seeds germinate.

[0014] In step S2, the concentration of the PdCl2 solution is 4-5 mM; the amount of PdCl2 solution used is 2-3 ml of PdCl2 solution for every 1 g of seedlings.

[0015] In step S2, the concentration of the sodium carboxymethyl cellulose solution is 0.01-0.015 g / ml; the amount of sodium carboxymethyl cellulose solution used is 4-5 ml of sodium carboxymethyl cellulose solution for every 1 g of seedlings.

[0016] In step S2, the conditions for settling are: room temperature, in darkness for 2-2.5 hours.

[0017] In step S2, the detection wavelength is 420 nm.

[0018] In step S3, the formula is as follows: C 样品 = (A 样品反应后 -A 样品反应前 ) ÷ (A 标准品反应后 -A 标准品反应前 )×C 标准 ÷(W÷V; In the formula: C 样品 Concentration of total glucosinolates in broccoli sprout samples, mg / ml; A 样品反应后 : Absorbance of the sample after the reaction; A 样品反应前 : Absorbance of the sample before reaction; A 标准品反应后 : Absorbance of the standard sample after reaction; A 标准品反应前 : Absorbance of the standard before reaction; C 标准 =0.25 mg / ml, which is the total glucosinolate concentration in the standard solution; the standard is potassium glucosinolate. W: Sample mass, g; V: Volume of extraction reagent, L.

[0019] Compared with the prior art, the beneficial effects achieved by the present invention are: This invention uses high-concentration ethanol as the extraction reagent and improves the extraction efficiency of total glucosinolates from broccoli sprouts by optimizing the extraction conditions. Based on this, the invention employs the palladium chloride method to detect the extract, and by optimizing the detection conditions, achieves rapid and accurate detection, providing technical support for the production of sprouts with high glucosinolate content. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0022] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.

[0023] Example This embodiment provides a method for detecting the total glucosinolate content in broccoli sprouts, including the following steps: (1) Take 1 g of fresh broccoli sprouts that have been germinated for 10 days, grind and crush them with liquid nitrogen, add 15 ml of extraction reagent 80% ethanol, bathe in water at 80℃ for 20 min, centrifuge at 8000 rpm for 6 min, and take the supernatant, which is the total glucosinolate solution. (2) Add 2 ml of 4 mM PdCl2 solution and 4 ml of 1% sodium carboxymethyl cellulose solution to 1 ml of total glucosinolate solution, mix well and let stand at room temperature and in the dark for 2 h; (3) The solution after standing was detected by a spectrophotometer at a wavelength of 420 nm. The total glucosinolate content in the seedlings was obtained by substituting the solution into the calculation formula. The calculation formula is: C 样品 = (A 样品反应后 -A 样品反应前 ) ÷ (A 标准品反应后 -A 标准品反应前 )×C 标准 ÷(W÷V; In the formula: C 样品 Concentration of total glucosinolates in broccoli sprout samples; A 样品反应后 : Absorbance of the sample after the reaction; A 样品反应前 : Absorbance of the sample before reaction; A 标准品反应后 : Absorbance of the standard sample after reaction; A 标准品反应前 : Absorbance of the standard before reaction; C 标准 =0.25 mg / ml, which is the total glucosinolate concentration in the standard solution; the standard is potassium glucosinolate. W: Sample mass, g; V: Volume of extraction reagent, L.

[0024] Test case 1. The influence of different wavelengths on detection results The extraction reagent was 80% methanol solution (a common extraction reagent in the palladium chloride method), and the wavelengths were 420 nm, 505 nm, and 545 nm, respectively. The results are shown in Table 1.

[0025] Table 1. Detection results of total glucosinolates in broccoli shoots at different wavelengths.

[0026] As shown in Table 1, the ΔOD values ​​of the standard and the test sample before and after the reaction were the largest at a wavelength of 420 nm, followed by 505 nm and 545 nm. This indicates that the reaction product of total glucosinolates in broccoli buds and the reaction solution of palladium chloride has the largest absorbance at a wavelength of 420 nm.

[0027] 2. The effect of different extraction reagents on the detection results The extraction reagents were 60% methanol solution, 80% methanol solution, 100% methanol solution, 60% ethanol solution, 80% ethanol solution, and 95% ethanol solution, respectively, with the remaining steps being the same as in the previous example. The results are shown in Table 2.

[0028] Table 2. Results of total glucosinolate detection in broccoli shoots after treatment with different extraction reagents.

[0029] Table 2 shows that when the extraction reagent volume is 7 ml, the ΔOD of the sample before and after the reaction is as follows: 420 nm The extraction reagent with the highest value is 80% ethanol, followed by 95% ethanol, 80% methanol, 100% methanol, 60% ethanol, and 60% methanol.

[0030] Based on the above formula, the total glucosinolate content in the samples obtained from 60% methanol solution, 80% methanol solution, 100% methanol solution, 60% ethanol solution, 80% ethanol solution, and 95% ethanol solution is calculated to be 639.9530939 μg / g, 1623.782926 μg / g, 1554.110755 μg / g, 1422.912466 μg / g, 2398.930313 μg / g, and 2192.728609 μg / g, respectively.

[0031] Based on the measured data and the calculated total glucosinolate content, it was found that ethanol extraction at the same concentration was more effective than methanol extraction, indicating that total glucosinolates have higher solubility in ethanol. Furthermore, as the ethanol concentration increased, the ΔOD value measured after extraction... 420 nm The value and content gradually increased, with 80% ethanol solution showing the best extraction effect, after which they began to decrease.

[0032] 3. The effect of different extraction reagent volumes on detection results The extraction reagent volumes were 5 ml, 7 ml, and 15 ml, respectively, with the rest being the same as in the previous example. The results are shown in Table 3.

[0033] Table 3. Results of total glucosinolate detection in broccoli shoots after treatment with different extraction reagent volumes

[0034] Table 3 shows that when pretreated with 80% ethanol, the ΔOD value of 1 g of fresh broccoli sprouts after extraction with 5 ml of 80% ethanol extraction reagent was measured. 420 nm The value is the ΔOD measured after extraction with 7 ml of 80% ethanol extraction reagent. 420 nm The value was 1.05 times that of the 7 ml extraction reagent, and the volume of 5 ml extraction reagent was 71.4% of the volume of 7 ml extraction reagent, indicating that the total glucosinolate content extracted was higher when 7 ml of extraction reagent was added; the ΔOD value after extraction with 15 ml of 80% ethanol reagent was higher. 420 nm The value is the ΔOD value measured after extraction with 7 ml of 95% ethanol extraction reagent. 420 nm The value is 0.60 times that of the 7 ml extraction reagent, while the volume of 15 ml extraction reagent is 2.14 times that of the 7 ml extraction reagent; the ΔOD value after extraction with 50 ml of 80% ethanol was measured. 420 nm The value is the ΔOD measured after extraction with 7 ml of 80% ethanol extraction reagent. 420 nm The value is 0.184 times that of the 50 ml extraction reagent, which is 7.14 times the volume of the 7 ml extraction reagent.

[0035] Calculations using the formula show that the total glucosinolate content extracted by adding 5 ml, 7 ml, 15 ml, and 50 ml of extraction reagent were 1843.34 μg / g, 2446.36 μg / g, 3162.20 μg / g, and 3182.57 μg / g, respectively, with total glucosinolate concentrations of 370.14 μg / g, 351.23 μg / g, 211.66 μg / g, and 63.91 μg / g, respectively. These results indicate that adding 15 ml and 50 ml of extraction reagent yields more total glucosinolates, but the total glucosinolate content is consistent with that obtained by adding 15 ml of extraction solution, indicating lower extraction efficiency. Therefore, considering both extraction efficiency and total extraction volume, when extracting total glucosinolates from broccoli sprouts, it is recommended to crush 1 g of fresh broccoli sprouts and add them to 15 ml of extraction solution for more accurate results.

[0036] 4. Detection range Take 1 ml of each of sample 1 (95% ethanol), sample 2 (62.5 μg / ml potassium glucoside), and sample 3 (1000 μg / ml potassium glucoside), and test them according to the method described in the example. The results are shown in Table 4.

[0037] Table 4. Detection results of the determination range of total glucosinolates

[0038] The total glucosinolate content was calculated using the formula, and the total glucosinolate concentration was 0 in sample 1, 49.08 μg / ml in sample 2, and 996.42 μg / ml in sample 3. This indicates that the detection method provided by this invention can extract total glucosinolates in the range of 0-1000 μg / ml.

[0039] 5. Extraction efficiency Take 1.875 mg (sample 4) and 7.5 mg (sample 5) of black mustard glucosinolate potassium salt, add 15 ml of 95% ethanol solution for extraction, then add detection solution and react at room temperature. Detect the absorption peak of the sample at 420 nm wavelength before reaction and after standing in the dark at room temperature for 2 h. The results are shown in Table 5.

[0040] Table 5. Results of extraction efficiency of total glucosinolates

[0041] Based on the measurement results, the total glucosinolate content of sample 4 was calculated to be 1.75 mg, with an extraction efficiency of 93.3%, and the total glucosinolate content of sample 5 was 7.02 mg, with an extraction efficiency of 93.6%. The average extraction efficiency was 93.5%, indicating that the method provided by the present invention has a high extraction efficiency.

[0042] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting the total glucosinolate content in broccoli sprouts, characterized in that, Includes the following steps: S1. Crush the seedling sample, add ethanol solution, extract, centrifuge to obtain the extract; S2. Add PdCl2 solution and sodium carboxymethyl cellulose solution to the extract, let stand, and detect using a spectrophotometer; S3. Substitute the detected data into the formula to calculate the total glucosinolate content in the seedlings.

2. The detection method according to claim 1, characterized in that, In step S1, the volume fraction of the ethanol solution is 80-85%; the amount of ethanol solution used is 15-16 ml of ethanol solution per 1 g of seedling.

3. The detection method according to claim 1, characterized in that, In step S1, the extraction conditions are: water bath at 80-85℃ for 20-25 minutes.

4. The detection method according to claim 1, characterized in that, In step S1, the centrifugation conditions are: a rotation speed of 8000-8500 rpm and a time of 6-7 min.

5. The detection method according to claim 1, characterized in that, In step S1, the seedlings are seedlings that have germinated 7-10 days after the seeds germinate.

6. The detection method according to claim 1, characterized in that, In step S2, the concentration of the PdCl2 solution is 4-5 mM; The amount of PdCl2 solution used is: 2-3 ml of PdCl2 solution for every 1 g of seedlings.

7. The detection method according to claim 1, characterized in that, In step S2, the concentration of the sodium carboxymethyl cellulose solution is 0.01-0.015 g / ml; The dosage of the sodium carboxymethyl cellulose solution is as follows: add 4-5 ml of sodium carboxymethyl cellulose solution for every 1 g of seedlings.

8. The detection method according to claim 1, characterized in that, In step S2, the conditions for settling are: room temperature, in darkness for 2-2.5 hours.

9. The detection method according to claim 1, characterized in that, In step S2, the detection wavelength is 420 nm.

10. The detection method according to claim 1, characterized in that, In step S3, the formula is as follows: C 样品 =(A 样品反应后 -A 样品反应前 )÷(A 标准品反应后 -A 标准品反应前 )×C 标准 ÷(W÷V); In the formula: C 样品 Concentration of total glucosinolates in broccoli sprout samples, mg / ml; A 样品反应后 : Absorbance of the sample after the reaction; A 样品反应前 : Absorbance of the sample before reaction; A 标准品反应后 : Absorbance of the standard sample after reaction; A 标准品反应前 : Absorbance of the standard before reaction; C 标准 =0.25 mg / ml, which is the total glucosinolate concentration in the standard solution; the standard is potassium glucosinolate. W: Sample mass, g; V: Volume of extraction reagent, L.

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