Method for cultivating broccoli seeds with high glucoraphanin content

Abstract

The invention relates to a high glucoraphanin content broccoli seed cultivation method, which comprises: S1, after broccoli enters a flowering phase, spraying a culture fertilizer to broccoli leaf surfaces for the first time, then spraying once every 3-4 days, and spraying 3-4 times in total; s2, collecting and drying the broccoli seeds which are mature after the broccoli seeds are cultured for 60-65 days to obtain broccoli seeds rich in glucoraphanin; the culture fertilizer in the step S1 is prepared from a glucoraphanin synthesis accelerant with the content of 1.2 to 1.6 mg / L, an ESP inhibitor with the content of 0.8 to 1.0 mg / L and a plant growth regulator with the content of 2.1 to 2.5 mg / L, and the glucoraphanin synthesis accelerant is prepared from selenomethionine, chitosan oligosaccharide, tryptophan and a seaweed extract according to the mass ratio of (5 to 7) to (1 to 2) to (20 to 30) to (10 to 20). The application has the effect of increasing the glucoraphanin content in the broccoli seeds.

Description

Technical Field

[0001] The present application relates to the field of broccoli seed breeding technology, and in particular to a method for breeding broccoli seeds with high glucoraphanin content. Background Art

[0002] Glucoraphanin is widely found in cruciferous vegetables, such as broccoli, kale, cauliflower and Chinese cabbage, among which broccoli seeds have the highest content of glucoraphanin. Glucoraphanin can be degraded to produce sulforaphane, which is the most powerful isothiocyanate found in vegetables so far for anti-cancer activity and is currently recognized as one of the best natural products for cancer prevention and anti-cancer effects.

[0003] Methionine in broccoli seeds is decomposed to obtain glucoraphanin, which is converted into sulforaphane by the catalytic action of myrosinase. Currently, the extraction of sulforaphane from broccoli seeds has the problem of large consumption of broccoli seeds. About 0.5-1g of sulforaphane can be extracted from each kilogram of broccoli seeds, and the yield is 0.05%-0.1%. The low yield limits the application of sulforaphane. Since the content of glucoraphanin in broccoli seeds is not high, and the epidermal specific sulfonate protein (ESP) in broccoli seeds promotes the degradation of glucoraphanin into nitrile compounds, the conversion rate of glucoraphanin to sulforaphane is reduced, resulting in a low yield of sulforaphane. Existing methods for increasing the content of glucoraphanin, such as genetic improvement and optimization of planting conditions, are time-consuming and have limited improvement effects. Summary of the invention

[0004] In order to solve the above technical problems, the present application provides a method for cultivating broccoli seeds with high glucoraphanin content.

[0005] The present application provides a method for cultivating broccoli seeds with high glucoraphanin content, which adopts the following technical scheme: A method for cultivating broccoli seeds with high glucoraphanin content, comprising the following steps: S1. When broccoli enters the flowering period, spray the broccoli leaves with culture fertilizer for the first time, and then spray once every 3-4 days, for a total of 3-4 times; S2. The mature broccoli seeds after 60-65 days of cultivation are collected and dried to obtain broccoli seeds rich in glucoraphanin; The culture fertilizer in step S1 includes a glucoraphanin synthesis promoter with a content of 1.2-1.6 mg / L, an ESP inhibitor with a content of 0.8-1.0 mg / L, and a plant growth regulator with a content of 2.1-2.5 mg / L. The glucoraphanin synthesis promoter includes selenomethionine, chitosan oligosaccharide, tryptophan, and seaweed extract in a mass ratio of (5-7):(1-2):(20-30):(10-20).

[0006] By adopting the above technical scheme, the glucoraphanin content in the broccoli seeds obtained in Examples 1.4 and 1.5 of the present application was 39.9 mg / g and 39.7 mg / g, respectively, which was 9.7 mg / g and 9.9 mg / g lower than that in Example 1.1 of the present application, and 9.4 mg / g and 9.6 mg / g lower than that in Example 1.2, respectively. It may be that when the mass ratio of selenomethionine, chitosan oligosaccharide, tryptophan and seaweed extract in the glucoraphanin synthesis promoter is (5-7): (1-2): (20-30): (10-20), the synergistic effect between the glucoraphanin synthesis promoter, the ESP inhibitor and the plant growth regulator can be fully promoted, thereby facilitating the synthesis and accumulation of glucoraphanin in broccoli seeds. In addition, the present application effectively increases the glucoraphanin content in the broccoli seeds produced by the broccoli head during the breeding process through the synergistic effect between the glucoraphanin synthesis promoter, the ESP inhibitor and the plant growth regulator, thereby obtaining broccoli seeds with a high glucoraphanin content.

[0007] Preferably, the mass ratio of the selenomethionine, the chitosan oligosaccharide, the tryptophan and the seaweed extract is 6:1.5:25:15.

[0008] By adopting the above technical scheme, the content of glucoraphanin in the broccoli seeds obtained in Example 1.3 of the present application is 53.9 mg / g, which is 4.3 mg / g higher than that in Example 1.1 and 4.6 mg / g higher than that in Example 1.2. This may be because when the mass ratio of selenomethionine, chitosan oligosaccharide, tryptophan and seaweed extract is 6:1.5:25:15, the synergistic effect between the glucoraphanin synthesis promoter, the ESP inhibitor and the plant growth regulator can be more fully promoted, thereby further increasing the content of glucoraphanin in the broccoli seeds.

[0009] Preferably, the ESP inhibitor comprises N-acetylcysteine ​​and EDTA-2Na in a mass ratio of (1-3):(5-9).

[0010] By adopting the above technical solution, the present application uses N-acetylcysteine ​​and EDTA-2Na in combination to synergistically inhibit the activity of ESP. The glucoraphanin content in broccoli seeds obtained in Examples 2.3 and 2.4 of the present application is 42.5 mg / g and 42.7 mg / g, respectively, which is 7.3 mg / g and 7.1 mg / g lower than that in Example 2.1, and 7.6 mg / g and 7.4 mg / g lower than that in Example 2.2. It may be that when the mass ratio of N-acetylcysteine ​​and EDTA-2Na in the ESP inhibitor is (1-3): (5-9), the synergistic effect between the glucoraphanin synthesis promoter, the ESP inhibitor and the plant growth regulator can be promoted, thereby increasing the glucoraphanin content in the broccoli seeds.

[0011] Preferably, the ESP inhibitor comprises N-acetylcysteine ​​and EDTA-2Na in a mass ratio of 2:7.

[0012] By adopting the above technical scheme, the glucoraphanin contents in the broccoli seeds obtained in Examples 2.1 and 2.2 of the present application were 49.8 mg / g and 50.1 mg / g, respectively, which were 3.5 mg / g and 3.6 mg / g lower than those in Example 1.3, respectively. This may be because the mass ratio of selenomethionine, chitosan oligosaccharides, tryptophan and seaweed extract was 6:1.5:25:15, and the mass ratio of N-acetylcysteine ​​and EDTA-2Na in the ESP inhibitor was 2:7, which could more fully promote the synergistic effect between the glucoraphanin synthesis promoter, the ESP inhibitor and the plant growth regulator, thereby further increasing the glucoraphanin content in the broccoli seeds.

[0013] Preferably, the plant growth regulator comprises gibberellin, naphthyl acetic acid and 6-benzylaminoadenine in a mass ratio of (2-6):(6-10):(9-13).

[0014] By adopting the above technical scheme, the glucoraphanin contents in the broccoli seeds obtained in Examples 3.3 and 3.4 of the present application were 44.3 mg / g and 44.1 mg / g, respectively, which were 6.1 mg / g and 6.3 mg / g lower than those in Example 3.1, and 6 mg / g and 6.2 mg / g lower than those in Example 3.2. This may be because when the mass ratio of gibberellin, naphthylacetic acid and 6-benzylaminoadenine in the plant growth regulator is (2-6):(6-10):(9-13), the synergistic effect between the glucoraphanin synthesis promoter, the ESP inhibitor and the plant growth regulator can be promoted, thereby increasing the glucoraphanin content in the broccoli seeds.

[0015] Preferably, the plant growth regulator comprises gibberellin, naphthyl acetic acid and 6-benzylaminoadenine in a mass ratio of 4:8:11.

[0016] By adopting the above technical scheme, the glucoraphanin contents in the broccoli seeds obtained in Examples 3.1 and 3.2 of the present application were 50.4 mg / g and 50.3 mg / g, respectively, which were 3.5 mg / g and 3.6 mg / g lower than those in Example 1.3, respectively. This may be because the mass ratio of selenomethionine, chitosan oligosaccharide, tryptophan and seaweed extract was 6:1.5:25:15, the mass ratio of N-acetylcysteine ​​and EDTA-2Na in the ESP inhibitor was 2:7, and the mass ratio of gibberellin, naphthylacetic acid and 6-benzylaminoadenine in the plant growth regulator was 4:8:11, which can more fully promote the synergistic effect between the glucoraphanin synthesis promoter, the ESP inhibitor and the plant growth regulator, thereby further increasing the glucoraphanin content in the broccoli seeds.

[0017] Preferably, the spraying method in S1 is based on the broccoli leaf surface being covered with droplets.

[0018] Preferably, the concentration of the glucoraphanin synthesis promoter in step S1 is 1.4 mg / L.

[0019] In summary, this application has the following beneficial effects: The present application effectively increases the content of glucoraphanin in broccoli seeds by giving full play to the synergistic effect of a certain proportion of glucoraphanin synthesis promoter, ESP inhibitor and plant growth regulator. The glucoraphanin synthesis promoter and plant growth regulator may increase the content of glucoraphanin in broccoli seeds by promoting the glucoraphanin synthesis process in broccoli seeds, and the ESP inhibitor may promote the accumulation of glucoraphanin by inhibiting the activity of ESP enzyme, thereby increasing the overall content of glucoraphanin in the cultivated broccoli seeds. DETAILED DESCRIPTION

[0020] The specific embodiments are merely explanations of the present application and are not limitations of the present application. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, as long as they are within the scope of the claims of the present application, they are protected by the patent law.

[0021] Material Source Broccoli seeds, variety Green Gem (LBS), were purchased from Xiamen Wenxing Vegetable Seedlings Co., Ltd.; Selenomethionine, chitosan oligosaccharide, tryptophan, gibberellin, naphthylacetic acid, N-acetylcysteine ​​and 55% anhydrous ethanol were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Seaweed extract, CAS No. 223751-74-4, active ingredient content 98%, purchased from Hubei Langbowan Biopharmaceutical Co., Ltd.; 6-Benzylaminoadenine was purchased from Sigma-Aldrich (Shanghai) Biotechnology Co., Ltd.; EDTA-2Na was purchased from Jining Yuanlian Chemical Technology Co., Ltd.

[0022] The concentration ranges of the glucoraphanin synthesis promoter, ESP inhibitor and plant growth regulator in the culture fertilizer of the present application are common knowledge that can be obtained by those skilled in the art through the prior art.

[0023] Preparation Example 1.1 The preparation method of the culture fertilizer comprises the following steps: a. Weigh 0.149 mg selenomethionine, 0.06 mg chitosan oligosaccharide, 0.596 mg tryptophan, 0.596 mg seaweed extract, 0.222 mg N-acetylcysteine ​​and 0.778 mg EDTA-2Na, respectively, and dissolve them in 990 ml of water; b. Weigh 0.43 mg of gibberellin, 0.87 mg of naphthylacetic acid and 1.2 mg of 6-benzylaminoadenine respectively and dissolve them in 10 ml of 55% ethanol solution; c. Mix the solution in step a with the solution in step b, and stir evenly to obtain a culture fertilizer.

[0024] Preparation Example 1.2 The difference from Preparation Example 1.1 is that in step a, selenomethionine is 0.204 mg, chitosan oligosaccharide is 0.029 mg, tryptophan is 0.875 mg, and seaweed extract is 0.292 mg.

[0025] Preparation Example 1.3 The difference from Preparation Example 1.1 is that in step a, selenomethionine is 0.177 mg, chitosan oligosaccharide is 0.044 mg, tryptophan is 0.737 mg, and seaweed extract is 0.442 mg.

[0026] Preparation Example 1.4 The difference from Preparation Example 1.1 is that in step a, selenomethionine is 0.059 mg, chitosan oligosaccharide is 0.147 mg, tryptophan is 0.295 mg, and seaweed extract is 0.884 mg.

[0027] Preparation Example 1.5 The difference from Preparation Example 1.1 is that in step a, selenomethionine is 0.254 mg, chitosan oligosaccharide is 0.005 mg, tryptophan is 1.01 mg, and seaweed extract is 0.127 mg.

[0028] Preparation Example 2.1 The difference from Preparation Example 1.3 is that in step a, N-acetylcysteine ​​is 0.1 mg and EDTA-2Na is 0.9 mg.

[0029] Preparation Example 2.2 The difference from Preparation Example 1.3 is that in step a, N-acetylcysteine ​​is 0.375 mg and EDTA-2Na is 0.625 mg.

[0030] Preparation Example 2.3 The difference from Preparation Example 1.3 is that in step a, N-acetylcysteine ​​is 0.048 mg and EDTA-2Na is 0.952 mg.

[0031] Preparation Example 2.4 The difference from Preparation Example 1.3 is that in step a, N-acetylcysteine ​​is 0.625 mg and EDTA-2Na is 0.375 mg.

[0032] Preparation Example 3.1 The difference from Preparation Example 1.3 is that in step b, the amount of gibberellin is 0.238 mg, the amount of naphthylacetic acid is 1.19 mg, and the amount of 6-benzylaminoadenine is 1.07 mg.

[0033] Preparation Example 3.2 The difference from Preparation Example 1.3 is that in step b, the amount of gibberellin, the amount of naphthylacetic acid, and the amount of 6-benzylaminoadenine are 0.6 mg, 0.6 mg, and 1.3 mg.

[0034] Preparation Example 3.3 The difference from Preparation Example 1.3 is that in step b, the amount of gibberellin is 0.139 mg, the amount of naphthylacetic acid is 0.278 mg, and the amount of 6-benzylaminoadenine is 2.08 mg.

[0035] Preparation Example 3.4 The difference from Preparation Example 1.3 is that in step b, the amount of gibberellin is 0.8 mg, the amount of naphthylacetic acid is 0.2 mg, and the amount of 6-benzylaminoadenine is 1.5 mg.

[0036] Example 1.1 The method for cultivating broccoli seeds with high glucoraphanin content comprises the following steps: S1. When broccoli enters the flowering period, spray the broccoli leaves with fertilizer for the first time, until the broccoli leaves are covered with droplets, and then spray once every 3 days, for a total of 4 times; S2. The mature broccoli seeds after 60 days of cultivation are collected and dried to obtain broccoli seeds rich in glucoraphanin; The culture fertilizer in step S1 is the culture fertilizer in Preparation Example 1.1.

[0037] Examples 1.2-1.5 The culture fertilizers used in Examples 1.2-1.5 are the culture fertilizers in Preparation Examples 1.2-1.5, respectively.

[0038] Examples 2.1-2.4 The culture fertilizers used in Examples 2.1-2.4 are the culture fertilizers in Preparation Examples 2.1-2.4, respectively.

[0039] Examples 3.1-3.4 The culture fertilizers used in Examples 3.1-3.4 are the culture fertilizers in Preparation Examples 3.1-3.4, respectively.

[0040] Performance Testing Referring to patent CN105566409B, a method for extracting and isolating glucoraphanin from broccoli seeds includes the following steps: (1) Take 200 g of dried broccoli seeds and place them in an oven at 105°C for 60 min. Then grind them and add 2000 mL of ethanol solution. Heat to 70°C and extract for 30 min. Filter the filtrate to obtain the glucoraphanin solution. The obtained glucoraphanin solution is concentrated on a rotary evaporator to a volume of 1 / 10 of the original glucoraphanin solution to obtain a glucoraphanin concentrate. (2) Fill the pretreated ADS-17 macroporous adsorption resin column, pass the glucoraphanin concentrate obtained in step (1) through the resin column at a rate of 5 BV / h, and adsorb it with the macroporous adsorption resin, wherein the volume of the glucoraphanin concentrate passing through the resin column is 20 BV; (3) The macroporous adsorption resin adsorbing glucoraphanin was washed with deionized water and then eluted with a 20 BV volume, 95% molar concentration ethanol solution, wherein the elution rate was 5 BV / h through the resin column to obtain a resin eluate; (4) The resin eluate obtained in step (3) was concentrated to a volume of 1 / 30 of the original volume on a rotary evaporator, and silica gel having the same weight as the volume of the concentrate was added and mixed, and the ethanol therein was evaporated to obtain silica gel adsorbing glucoraphanin; (5) Wet column packing was performed by wetting 200-800 mesh silica gel with petroleum ether and loading it into a glass column. The silica gel was allowed to settle naturally, and then nitrogen was passed through to compact the silica gel. The petroleum ether was released from the bottom of the glass column, and then the silica gel adsorbed with glucoraphanin was filled into the top of the silica gel column. The volume ratio of petroleum ether to silica gel and silica gel adsorbed with glucoraphanin was 30:10:1. (6). First, 10 column volumes of petroleum ether were used for elution at a rate of 6 BV / h, then 10 column volumes of a mixed solvent of ethyl acetate and petroleum ether in a volume ratio of 1:1 were used for elution at a rate of 6 BV / h, and finally 10 column volumes of a mixed solvent of ethyl acetate and ethanol in a volume ratio of 9:1 were used for elution at a rate of 6 BV / h. Different eluents were collected respectively, and the eluents were concentrated and freeze-dried at a freeze-drying temperature of -50°C to obtain glucoraphanin product.

[0041] The glucoraphanin content of the mature broccoli seeds obtained in Examples 1.1-1.5, Examples 2.1-2.4 and Examples 3.1-3.4 of the present application was measured.

[0042]

[0043] As can be seen from Table 1, the glucoraphanin content of Example 1.3 of the present application is 53.9 mg / g, indicating that the method for cultivating broccoli seeds with high glucoraphanin content provided by the present application has a good effect on increasing the glucoraphanin content in broccoli seeds.

[0044] Compared with the content of glucoraphanin in Example 1.1, the content of glucoraphanin in Examples 1.4 and 1.5 of the present application was reduced by 9.7 mg / g and 9.9 mg / g, respectively. Compared with the content of glucoraphanin in Example 1.2, the content of glucoraphanin in Examples 1.4 and 1.5 of the present application was reduced by 9.4 mg / g and 9.6 mg / g, respectively. This indicates that when the mass ratio of selenomethionine, chitosan oligosaccharide, tryptophan and seaweed extract in the glucoraphanin synthesis promoter is (5-7):(1-2):(20-30):(10-20), it has a good effect on increasing the content of glucoraphanin in broccoli seeds.

[0045] The glucoraphanin content in Example 1.3 of the present application is increased by 4.3 mg / g and 4.6 mg / g respectively compared with that in Example 1.1 and Example 1.2, indicating that a mass ratio of selenomethionine, chitosan oligosaccharides, tryptophan and seaweed extract of 6:1.5:25:15 has a better effect on increasing the glucoraphanin content in broccoli seeds.

[0046] Compared with Example 2.1, the glucoraphanin content in Examples 2.3 and 2.4 of the present application was reduced by 7.3 mg / g and 7.1 mg / g, respectively. Compared with Example 2.2, the glucoraphanin content in Examples 2.3 and 2.4 of the present application was reduced by 7.6 mg / g and 7.4 mg / g, respectively. This indicates that when the mass ratio of N-acetylcysteine ​​to EDTA-2Na in the ESP inhibitor is (1-3):(5-9), it has a good effect on increasing the glucoraphanin content in broccoli seeds.

[0047] Compared with Examples 2.1 and 2.2, the glucoraphanin content in Example 1.3 of the present application is increased by 3.5 mg / g and 3.6 mg / g, respectively, indicating that when the mass ratio of selenomethionine, chitosan oligosaccharides, tryptophan and seaweed extract is 6:1.5:25:15, and the mass ratio of N-acetylcysteine ​​and EDTA-2Na is 2:7, it has a better effect on increasing the glucoraphanin content in broccoli seeds.

[0048] Compared with Example 3.1, the glucoraphanin content in Examples 3.3 and 3.4 of the present application was reduced by 6.1 mg / g and 6.3 mg / g, respectively. Compared with Example 3.2, the glucoraphanin content in Examples 3.3 and 3.4 of the present application was reduced by 6 mg / g and 6.2 mg / g, respectively. This indicates that when the mass ratio of gibberellin, naphthylacetic acid and 6-benzylaminoadenine in the plant growth regulator is (2-6):(6-10):(9-13), it has a good effect on increasing the glucoraphanin content in broccoli seeds.

[0049] Compared with Examples 3.1 and 3.2, the glucoraphanin content in Example 1.3 of the present application is increased by 3.5 mg / g and 3.6 mg / g, respectively, indicating that when the mass ratio of selenomethionine, chitosan oligosaccharides, tryptophan and seaweed extract is 6:1.5:25:15, the mass ratio of N-acetylcysteine ​​and EDTA-2Na is 2:7, and the mass ratio of gibberellin, naphthylacetic acid and 6-benzylaminoadenine in the plant growth regulator is 4:8:11, it has a better effect on increasing the glucoraphanin content in broccoli seeds.

Claims

1. A method for cultivating broccoli seeds with high glucoraphanin content, characterized in that: The following steps are involved: S1. When broccoli enters the flowering period, spray the broccoli leaves with culture fertilizer for the first time, and then spray once every 3-4 days, for a total of 3-4 times; S2. The mature broccoli seeds after 60-65 days of cultivation are collected and dried to obtain broccoli seeds rich in glucoraphanin; The culture fertilizer in step S1 includes a glucoraphanin synthesis promoter with a content of 1.2-1.6 mg / L, an ESP inhibitor with a content of 0.8-1.0 mg / L, and a plant growth regulator with a content of 2.1-2.5 mg / L. The glucoraphanin synthesis promoter includes selenomethionine, chitosan oligosaccharide, tryptophan, and seaweed extract in a mass ratio of (5-7):(1-2):(20-30):(10-20).

2. The method for cultivating broccoli seeds with high glucoraphanin content according to claim 1, characterized in that: The mass ratio of the selenomethionine, the chitosan oligosaccharide, the tryptophan and the seaweed extract is 6:1.5:25:

15.

3. The method for cultivating broccoli seeds with high glucoraphanin content according to claim 1, characterized in that: The ESP inhibitor comprises N-acetylcysteine ​​and EDTA-2Na in a mass ratio of (1-3):(5-9).

4. The method for cultivating broccoli seeds with high glucoraphanin content according to claim 3, characterized in that: The ESP inhibitor comprises N-acetylcysteine ​​and EDTA-2Na in a mass ratio of 2:

7.

5. The method for cultivating broccoli seeds with high glucoraphanin content according to claim 1, characterized in that: The plant growth regulator comprises gibberellin, naphthylacetic acid and 6-benzylaminoadenine in a mass ratio of (2-6):(6-10):(9-13).

6. The method for cultivating broccoli seeds with high glucoraphanin content according to claim 5, characterized in that: The plant growth regulator comprises gibberellin, naphthylacetic acid and 6-benzylaminoadenine in a mass ratio of 4:8:

11.

7. The method for cultivating broccoli seeds with high glucoraphanin content according to claim 1, characterized in that: The spraying method in S1 is based on the broccoli leaf surface being covered with droplets.

8. The method for cultivating broccoli seeds with high glucoraphanin content according to claim 1, characterized in that: The concentration of the glucoraphanin synthesis promoter in step S1 is 1.4 mg / L.

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