Preparation method of broccoli sprout powder rich in glucoraphanin and sulforaphane

Abstract

The present invention discloses a method for preparing broccoli sprout powder rich in glucoraphanin and sulforaphane, belonging to the field of food processing. The broccoli sprout powder preparation method comprises the following steps: screening broccoli seeds, washing, disinfecting, and soaking them, then placing them on a tray lined with filter paper. At an ambient temperature of 25-27°C, the seeds are first germinated with distilled water for 1-2 days, then sprayed with a sucrose solution every 8-12 hours, and allowed to germinate under natural light. The sprouts are harvested after germination for 4-7 days. The harvested sprouts are first steamed, immediately cooled to room temperature, and then soaked in a zinc sulfate solution. After removal and grinding, the pulp is placed in 2 volumes of distilled water and hydrolyzed with endogenous enzymes at 35-40°C. The hydrolyzed broccoli sprouts are freeze-dried and pulverized to obtain the broccoli sprout powder. This broccoli sprout powder is rich in glucoraphanin and sulforaphane and has high nutritional value.

Description

Technical Field

[0001] The invention relates to a method for preparing broccoli sprout powder rich in glucoraphanin and sulforaphane, and belongs to the field of food processing. Background Art

[0002] With the improvement of people's living standards, fresh, functional, and nutritious vegetables are widely in demand. As a new form of functional food, sprouts are characterized by simple and flexible production technology and rich nutritional value. Sprouts are suitable for indoor cultivation and can be harvested after 3 to 11 days of growth. The production process avoids the use of pesticides and herbicides and heavy metal pollution. Compared with their mature plants, sprouts have a richer content of bioactive substances, including typical nitrogen-sulfur derivatives glucosinolates (GLs) and isothiocyanates, phenols, vitamins, minerals, etc.

[0003] Broccoli sprouts contain 10 to 100 times more glucoraphanin than mature broccoli. Existing research indicates that consuming broccoli sprouts has significant health benefits and reduces the risk of chronic diseases. Existing in vitro and in vivo studies have revealed the chemical composition and biological effects of broccoli sprouts, including anticancer, antioxidant, anti-inflammatory, antidiabetic, and neuroprotective activities.

[0004] Sulforaphane, produced from glucoraphanin hydrolyzed by myrosinase, has powerful health benefits, such as anti-cancer, cardiovascular disease prevention, antioxidant, anti-inflammatory, antibacterial, and immune regulation. In addition to hydrolysis by endogenous plant enzymes, sulforaphane can also be decomposed by the human body's flora. However, due to the presence of epidermal-specific sulfonprotein (ESP) in broccoli sprouts, glucoraphanin preferentially produces sulforaphane nitrile, and the conversion rate of glucoraphanin to sulforaphane is low.

[0005] Therefore, it is of research significance to increase the content of glucoraphanin and sulforaphane in broccoli sprouts, increase the conversion rate of glucoraphanin to sulforaphane, and prepare a functional vegetable product that meets the public's diversified, convenient, and nutritious needs. Summary of the Invention

[0006] In response to the defects and shortcomings of the prior art, the present invention provides a method for preparing broccoli sprout powder rich in glucoraphanin and sulforaphane. The method comprises spraying broccoli sprouts in the growth stage with an inducer to accumulate glucoraphanin in the broccoli sprouts; then combining steaming and zinc sulfate soaking with enzymatic hydrolysis to promote the formation of sulforaphane in the broccoli sprouts; and freeze-drying to maximize the preservation of the nutrient content in the sprouts, thereby providing broccoli sprout powder rich in glucoraphanin and sulforaphane.

[0007] Sucrose, as a signaling molecule, participates in plant growth and development, causing changes in the osmotic pressure of sprouts or acting as a substrate, inducing the production of active substances in the sprouts through its signaling effect, leading to a large accumulation of glucoraphanin in broccoli sprouts. Enzyme activity is affected by factors such as temperature and metal ions. By combining steaming and zinc sulfate soaking to selectively inactivate the ESP enzyme in broccoli sprouts while retaining the activity of the MYR enzyme, the broccoli sprouts are converted more glucoraphanin into sulforaphane rather than sulforaphane nitrile, thereby increasing the sulforaphane content.

[0008] The present invention aims to provide a method for preparing broccoli sprout powder rich in glucoraphanin and sulforaphane, the method comprising the following steps:

[0009] (1) Seed pretreatment: The selected broccoli seeds were washed, sterilized, and soaked, and then placed on a tray covered with filter paper. They were germinated with distilled water for 1 to 2 days at an ambient temperature of 25 to 27°C, and then sprayed with sucrose solution every 8 to 12 hours. The seeds were germinated under natural light. The sprouts were harvested after germination for 4 to 7 days.

[0010] (2) ESP enzyme inactivation treatment: the sprouts harvested in step (1) are first steamed, then immediately cooled to room temperature, and then immersed in a zinc sulfate solution;

[0011] (3) Enzymatic hydrolysis: The sprouts after soaking in step (2) were taken out, ground in 2 volumes of distilled water, and hydrolyzed with endogenous enzymes at 35-40° C.; the hydrolyzed slurry was freeze-dried;

[0012] (4) Crushing: Crushing the freeze-dried sample obtained in step (3) to obtain broccoli sprout powder.

[0013] In one embodiment, the screening in step (1) specifically involves selecting broccoli seeds with full grains, uniform size, and germination ability.

[0014] In one embodiment, the disinfection in step (1) is performed by soaking in a 0.5% NaClO solution for 10 to 15 minutes, and then rinsing with distilled water until the pH of the solution is neutral.

[0015] In one embodiment, the soaking in step (1) refers to soaking the sterilized broccoli seeds in distilled water at 25 to 30° C. for 4 to 6 hours.

[0016] In one embodiment, the mass concentration of the sucrose solution in step (1) is 1-3%, and the spraying amount is 0.02-0.04 mL / cm 2 ; The preferred mass concentration of sucrose solution is 3%.

[0017] In one embodiment, the steaming treatment time in step (2) is 0.5 to 2 minutes, preferably 1 minute.

[0018] In one embodiment, the concentration of the zinc sulfate solution in step (2) is 2 to 10 mmol / L, preferably 6 to 8 mmol / L, and the soaking time is 1 to 2 hours.

[0019] In one embodiment, the endogenous enzyme hydrolysis time in step (3) is 20 to 60 minutes, preferably 20 minutes.

[0020] In one embodiment, the freeze-drying in step (3) is specifically to place the hydrolyzed sprouts in a -30 to -40°C refrigerator for pre-freezing for 30 to 48 hours, and then freeze-drying, the freeze-drying vacuum degree is 0.095 to 0.105 mbar, the cold trap temperature is -55 to -60°C, and the time is 35 to 40 hours; preferably, the freeze-drying is specifically to place the hydrolyzed sprouts in a -30°C refrigerator for pre-freezing for 30 hours, and then freeze-drying, the freeze-drying vacuum degree is 0.095 mbar, the cold trap temperature is -55°C, and the time is 40 hours.

[0021] The second object of the present invention is to provide a broccoli sprout powder prepared by the above method.

[0022] The third object of the present invention is to provide a use of the broccoli sprout powder described above in the preparation of food, medicine and health products.

[0023] A fourth object of the present invention is to provide a method for simultaneously increasing the content of glucoraphanin and sulforaphane in broccoli sprout powder, the method comprising the following steps:

[0024] (1) Seed pretreatment: The selected broccoli seeds were washed, sterilized, and soaked, and then placed on a tray covered with filter paper. They were germinated with distilled water for 1 to 2 days at an ambient temperature of 25 to 27°C, and then sprayed with sucrose solution every 8 to 12 hours. The seeds were germinated under natural light. The sprouts were harvested after germination for 4 to 7 days.

[0025] (2) ESP enzyme inactivation treatment: the sprouts harvested in step (1) are first steamed, then immediately cooled to room temperature, and then immersed in a zinc sulfate solution;

[0026] (3) Enzymatic hydrolysis: The sprouts after soaking in step (2) are taken out, ground in 2 volumes of distilled water, and hydrolyzed with endogenous enzymes at 35-40° C.; the hydrolyzed slurry is freeze-dried to obtain freeze-dried sprouts;

[0027] (4) Crushing: Crushing the freeze-dried sample obtained in step (3) to obtain broccoli sprout powder.

[0028] Beneficial effects of the present invention:

[0029] The present invention uses an inducer to treat broccoli sprouts, which can increase the glucoraphanin content in the sprouts by up to 4.72 times. The combined steaming and zinc sulfate soaking treatment and enzymatic hydrolysis treatment of the sprouts can increase the sulforaphane content in the sprouts by up to 2.25 times. Compared with fresh sprouts, the broccoli sprout powder has a longer storage time and maintains the original color, aroma and taste of the broccoli sprouts. It is rich in bioactive substances such as glucoraphanin and sulforaphane and is rich in nutrients. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a data graph showing the effect of different concentrations of sucrose solution treatment on the glucoraphanin content in broccoli sprouts in Example 1 and Comparative Examples 4 and 8;

[0031] Figure 2 This is a data graph showing the effect of different enzymatic hydrolysis times on the sulforaphane content in broccoli sprouts in Examples 1-2 and Comparative Example 3. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below with reference to specific examples, but the embodiments of the present invention are not limited to these examples.

[0033] The present invention relates to a determination method:

[0034] 1. Determination of glucoraphanin content

[0035] Glucoraphanin was extracted from a 0.5 g sample using 70% methanol. The mixture was stirred thoroughly and incubated in a water bath (70°C, 30 min) with constant frequency oscillation. After extraction, the mixture was centrifuged at 4°C (10,000 g, 15 min), and the supernatant was collected. The extraction was repeated once, and the supernatant was dried using a rotary evaporator at 35°C. The sample was dissolved in ultrapure water, the volume was adjusted to 2 mL, and the resulting solution was passed through a 0.22 μm membrane filter for determination.

[0036] HPLC conditions were used on an LC-20AT high-performance liquid chromatograph: C18 column (150 mm × 4.6 mm, 3 μm), detection wavelength at 230 nm. Mobile phase A consisted of 5% acetonitrile, 95% water, and 0.1% formic acid. Flow rate: 0.7 mL / min, elution time: 9 min at 25°C, injection volume: 20 μL.

[0037] Preparation of glucoraphanin standard solution: Prepare 2 mg / mL glucoraphanin stock solution in ultrapure water to prepare 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL standard solutions.

[0038] 2. Determination of sulforaphane content

[0039] Mix 0.2 g of broccoli sprout powder with 0.5 mL of ultrapure water and vortex for 1 minute; add 5 mL of ethyl acetate to the slurry, sonicate for 5 minutes at 4°C, and shake for 20 minutes; then centrifuge at 15,000 g for 10 minutes, remove the supernatant, and extract the remaining material repeatedly with 5 mL of ethyl acetate; combine the two supernatants, evaporate at 35°C, redissolve in 2 mL of 30% acetonitrile, filter through a 0.22 μm membrane, and store at -20°C.

[0040] An LC-20AT high performance liquid chromatograph was used, and the HPLC conditions were as follows: chromatographic column C18 (150 mm × 4.6 mm, 3 um), mobile phase A was water, mobile phase B was acetonitrile, gradient elution: 10% to 60% to 100% (0 to 25 to 30 min) B, column temperature was 30 °C, flow rate was 0.7 mL / min, detection wavelength was 254 nm, and injection volume was 20 uL.

[0041] Preparation of sulforaphane standard solution: Prepare a 2 mg / mL stock solution by diluting it with acetonitrile to prepare standard solutions of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, and 0.4 mg / mL.

[0042] Example 1

[0043] A method for preparing broccoli sprout powder rich in glucoraphanin and sulforaphane, the method comprising the following steps:

[0044] (1) Seed removal, disinfection, and soaking: Select broccoli seeds with full, uniform size, and germination ability. After removal and screening, rinse with distilled water 1-2 times, soak in 0.5% NaClO solution for 15 min, rinse with distilled water 5 times until the pH of the solution is neutral, and soak in 30°C distilled water for 4 h; remove floating seeds and drain excess water;

[0045] (2) Seed germination: The drained seeds from step (1) were evenly spread on a tray covered with filter paper, and the seeds were planted under natural light at an ambient temperature of 27°C. After germination for 1 day, 20 mL of a 3% sucrose solution was sprayed every 12 hours. The seedlings were harvested after germination for 4 days and stored in a -20°C refrigerator for later use.

[0046] (3) Combined steaming and zinc sulfate soaking treatment: The sprouts harvested in step (2) were first steamed for 1 min, then immediately cooled to room temperature in an ice bath, and soaked in a 6 mmol / L zinc sulfate solution for 1 h;

[0047] (4) Enzymatic hydrolysis: The sprouts after soaking in step (3) were taken out, ground in 2 volumes of distilled water, and hydrolyzed at 37°C using endogenous enzymes for 20 min;

[0048] (5) Freeze drying: The sprouts hydrolyzed in step (4) were placed in a -30°C refrigerator for pre-freezing for 30 h, and then freeze-dried. The freeze drying vacuum was 0.095 mbar, the cold trap temperature was -55°C, and the time was 40 h.

[0049] (6) Crushing: The dried sprouts in step (5) are placed in a small crusher and crushed multiple times at intervals of 30 seconds to obtain broccoli sprout powder of different mesh sizes as needed.

[0050] Example 2

[0051] The only difference from Example 1 is that the enzymatic hydrolysis time in step (3) is adjusted to 40 min and 60 min, respectively. Other parameters and conditions are the same as those in Example 1.

[0052] Comparative Example 1

[0053] The only difference from Example 1 is that the sucrose solution in step (2) is replaced with distilled water and steps (3) and (4) are not performed. Other parameters and conditions are the same as those in Example 1.

[0054] Comparative Example 2

[0055] The only difference from Example 1 is that the combined treatment of steaming and zinc sulfate soaking in step (3) is omitted, and other parameters and conditions are the same as those in Example 1.

[0056] Comparative Example 3

[0057] The only difference from Example 1 is that the enzymatic hydrolysis stage in step (4) is omitted, and other parameters and conditions are the same as those in Example 1.

[0058] Comparative Example 4

[0059] The only difference from Example 1 is that the sucrose solution in step (2) is replaced by distilled water, and the other parameters and conditions are the same as those in Example 1.

[0060] Comparative Example 5

[0061] The only difference from Example 1 is that the steaming treatment in step (3) is adjusted to only be performed for 1 minute, and the other parameters and conditions are the same as those in Example 1.

[0062] Comparative Example 6

[0063] The only difference from Example 1 is that the adjustment step (3) is to perform only zinc sulfate soaking treatment, and the other parameters and conditions are the same as those in Example 1.

[0064] Comparative Example 7

[0065] The only difference from Example 1 is that the zinc sulfate solution in step (3) is replaced by ascorbic acid solution, and the other parameters and conditions are the same as those in Example 1.

[0066] Comparative Example 8

[0067] The only difference from Example 1 is that the concentration of the sucrose solution in step (2) is adjusted to 1% and 2%, and the other parameters and conditions are the same as those in Example 1.

[0068] Result determination

[0069] 1. The broccoli sprout powder prepared in Examples 1 and 2 and Comparative Examples 1 to 8 was analyzed for glucoraphanin and sulforaphane content. The results are shown in Table 1:

[0070] Table 1. Effects of different treatments on the content of glucoraphanin and sulforaphane in broccoli sprouts

[0071]

[0072] From Table 1 and Figure 1 and Figure 2 The results showed that the present invention effectively increased the content of glucoraphanin in broccoli sprouts by treating them with sucrose. Simultaneously, the combined steaming and zinc sulfate soaking treatments inactivated the ESP enzyme in the broccoli sprouts to the greatest extent, promoting the conversion of glucoraphanin to sulforaphane. This, in turn, increased the content of sulforaphane in the broccoli sprout powder. Compared with the distilled water spraying group, the glucoraphanin content increased by 4.72 times, and the sulforaphane content increased by 2.25 times, reaching 5.47 mg / g dry weight.

[0073] 2. The ESP enzyme activity was measured in the sprouts after the combined steaming and soaking treatment in Example 1, the sprouts harvested in Comparative Example 2, the sprouts after steaming in Comparative Example 5, and the sprouts after soaking in Comparative Example 6. The results of ESP enzyme activity are shown in Table 2:

[0074] Table 2 Effects of different treatments on ESP enzyme activity in broccoli sprouts

[0075]

[0076] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing broccoli sprout powder rich in glucoraphanin and sulforaphane, characterized in that: The method comprises the following steps: (1) Seed pretreatment: The selected broccoli seeds were sterilized, soaked, and then placed on a tray covered with filter paper. They were germinated with distilled water for 1 to 2 days at an ambient temperature of 25 to 27°C. Then, they were sprayed with a 3% sucrose solution every 8 to 12 hours. The seeds were germinated under natural light and the sprouts were harvested after 4 to 7 days of germination. (2) ESP inactivation treatment: The sprouts harvested in step (1) are first steamed, then immediately cooled to room temperature, and then immersed in a zinc sulfate solution; the steaming time is 0.5 to 2 minutes; the zinc sulfate solution concentration is 2 to 10 mmol / L, and the immersion time is 1 to 2 hours; (3) Enzymatic hydrolysis: The sprouts after soaking in step (2) are taken out, ground into pulp with water, and hydrolyzed with endogenous enzymes at 35-40°C; the hydrolyzed pulp is freeze-dried to obtain a freeze-dried sample; (4) Grinding: Grinding the freeze-dried sample obtained in step (3) to obtain broccoli sprout powder.

2. The method according to claim 1, characterized in that The disinfection in step (1) is specifically performed by soaking in a 0.5% NaClO solution for 10 to 15 minutes, and then rinsing with distilled water until the pH of the solution is neutral.

3. The method according to claim 1, characterized in that The spraying amount of the sucrose solution in step (1) is 0.02~0.04mL / cm 2 .

4. The method according to claim 1, wherein The endogenous enzyme hydrolysis time in step (3) is 20 to 60 minutes.

5. The method according to claim 1, characterized in that The freeze drying in step (3) is specifically to place the hydrolyzed sprouts in a -30~-40℃ refrigerator for pre-freezing for 30~48h, and then freeze drying, the freeze drying vacuum degree is 0.095~0.105mbar, the cold trap temperature is -55~-60℃, and the time is 35~40h.

6. Broccoli sprout powder prepared by the method according to any one of claims 1 to 5.

7. Use of the broccoli sprout powder according to claim 6 in the preparation of foods, medicines and health products.

8. A method for simultaneously increasing the content of glucoraphanin and sulforaphane in broccoli sprout powder, characterized in that: The method comprises the following steps: (1) Seed pretreatment: The selected broccoli seeds were sterilized, soaked, and then placed on a tray covered with filter paper. They were germinated with distilled water for 1 to 2 days at an ambient temperature of 25 to 27°C. Then, they were sprayed with a 3% sucrose solution every 8 to 12 hours and germinated under natural light. The sprouts were harvested after 4 to 7 days of germination. (2) ESP inactivation treatment: The sprouts harvested in step (1) are first steamed, then immediately cooled to room temperature, and then immersed in a zinc sulfate solution; the steaming time is 0.5 to 2 minutes; the zinc sulfate solution concentration is 2 to 10 mmol / L, and the immersion time is 1 to 2 hours; (3) Enzymatic hydrolysis: The sprouts after soaking in step (2) are taken out, ground into pulp with water, and hydrolyzed with endogenous enzymes at 35-40°C; the hydrolyzed pulp is freeze-dried to obtain a freeze-dried sample; (4) Grinding: Grinding the freeze-dried sample obtained in step (3) to obtain broccoli sprout powder.

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