Preparation method of broccoli sulforaphane extract

Abstract

The invention belongs to the technical field of plant natural product extraction, and particularly relates to a broccoli sulforaphane extract preparation method, which comprises: drying broccoli florets, crushing to prepare broccoli florets powder, using a phosphate buffer solution as an enzymatic hydrolysate, using ethyl acetate as an extraction solution, and using ethanol as a solvent to prepare the broccoli sulforaphane extract. Carrying out endogenous enzymolysis, ultrasonic-assisted extraction and vacuum rotary evaporation to dryness to obtain broccoli sulforaphane leaching paste, and detecting the content of sulforaphane by using an ultra-high performance liquid chromatograph. The broccoli sulforaphane is extracted by the method disclosed by the invention, and the content of the obtained sulforaphane is 0.91 + / -0.01 mg / g DW. According to the method, the dried broccoli pollen is used as a raw material, the sulforaphane in the dried broccoli pollen is extracted by using ultrasonic-assisted ethyl acetate, and technological parameters are optimized through a single-factor test and a response surface test, so that the sulforaphane which is simple in process, easy to operate and capable of obtaining high-content sulforaphane without introducing exogenous enzyme is obtained. And the method is suitable for industrial implementation of the sulforaphane extraction method.

Description

Technical Field

[0001] The invention belongs to the technical field of plant natural product extraction, and particularly relates to a method for preparing a broccoli sulforaphane extract. Background Art

[0002] Sulforaphane (1-isothiocyanate-4-methylsulfonylbutane), an isothiocyanate derivative, is widely found in cruciferous vegetables such as broccoli, cauliflower and radish. Its content is related to the variety, plant part and development stage. Taking broccoli as an example, the isothiocyanate content in broccoli florets is the highest, followed by stems and leaves. Sulforaphane is not directly present in plants, but is produced by endogenous myrosinase hydrolysis of glucoraphanin (4-methylsulfinylbutenylthioglucosinolate) under the condition of plant cell rupture.

[0003] The precursor of sulforaphane, glucoraphanin, is neither toxic nor biologically active, while its hydrolysis product SFN has rich biological activity. In the past few years, researchers have conducted extensive research on SFN to gain a deeper understanding of its protective effects in various in vivo pathologies and in vitro studies in experimental models, such as its physiological activities in terms of anticancer, antibacterial, antioxidant, and autoimmune regulation. Studies have shown that sulforaphane is considered to be the most promising anticancer agent due to its powerful and broad-spectrum anticancer activity. SFN can induce the production of type II detoxification enzymes and can also inhibit lung cancer, gastric cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, skin cancer, and other cancers through anti-proliferation mechanisms such as cell cycle arrest and apoptosis. In addition, it has been shown to have anti-inflammatory, antioxidant, intestinal microbial regulation, neuroprotective and immunomodulatory effects.

[0004] At present, the preparation methods of sulforaphane mainly include chemical synthesis and enzymatic extraction. The former requires the use of a large amount of toxic organic reagents, and the reaction process is difficult to control, with a large number of by-products, and has not been widely used in the food industry. The sulforaphane extracted from natural plants by the enzymatic extraction method is safer and can better meet the market demand for the safety of functional food ingredients. In the main enzymatic hydrolysis step of enzymatic extraction of sulforaphane, the yield of sulforaphane is often closely related to the enzymatic hydrolysis conditions. In addition, the extraction method and conditions will also affect the yield of sulforaphane. With the development of emerging technologies, the extraction methods of bioactive substances are becoming more and more diversified, such as solvent extraction, supercritical fluid extraction and preparative liquid chromatography. Although these methods have higher yields, they also have disadvantages such as low economic benefits and high energy consumption, and are not suitable for factory production.

[0005] In recent years, the research on sulforaphane has mainly focused on its active functions, and there are relatively few systematic studies on its extraction. Therefore, studying the controllable factors in its extraction method can effectively increase the production of sulforaphane and provide a theoretical basis for its industrial production.

[0006] The present invention uses dried broccoli pollen as a raw material, uses ultrasound to assist ethyl acetate to extract sulforaphane therein, and optimizes process parameters through single factor experiments and response surface experiments to obtain a sulforaphane extraction method that is simple in process, easy to operate, does not require the introduction of exogenous enzymes, can obtain high-content sulforaphane, and is suitable for factory-scale implementation. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a broccoli sulforaphane extract, which has a simple extraction process, does not require any expensive instruments, does not require the introduction of exogenous enzymes, has a low production cost, and is suitable for mass production.

[0008] To achieve the above object, the technical solution adopted by the present invention is: ① Cut the fresh broccoli into florets, dry and crush them to prepare broccoli floret pollen; ② Take 1 part of broccoli floret pollen in step ①, add 0.18 parts of L(+)-ascorbic acid, and mix with 5-25 parts of phosphate buffer with a pH of 4.5-7.5, and shake at 110 rpm / min and 15-35°C for 4-6 hours to obtain broccoli sulforaphane enzymatic hydrolyzate, wherein the units of broccoli floret pollen: L(+)-ascorbic acid: phosphate buffer are g:g:mL; ③ Add 5-15 parts of ethyl acetate to the broccoli sulforaphane hydrolysate in step ②, and perform ultrasonic-assisted extraction under the conditions of ultrasonic power of 200-500 W, ultrasonic temperature of 20-60° C., and ultrasonic time of 10-50 min, wherein the unit of the number of parts of broccoli floret: ethyl acetate is g: mL; ④ The crude broccoli sulforaphane extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste; ⑤ The broccoli sulforaphane extract in step ④ was dissolved in a certain amount of acetonitrile solution, filtered through a 0.22 μm organic filter membrane into an automatic injection bottle, and the sulforaphane content was determined by ultra-high performance liquid chromatography. The analysis conditions were as follows: the chromatographic column was Shimadzu Shim-pack GIST C18 column (4.6×250 nm, 5 μm), the DAD detector detection wavelength was 190 nm, the flow rate was 1 mL / min, the column temperature was 30°C, and the injection volume was 5 μL. The mobile phase A was ultrapure water, B was acetonitrile, and the gradient elution program was 0-1min, 70% A and 30% B; 1-9 min, 70%-50% A and 30%-50% B; 9-11 min, 50%-70% A and 50%-30% B; 11-12 min, 70% A and 30% B. The results were expressed in mg / g DW on a dry weight basis.

[0009] Preliminary options: In step ②, the pH of the phosphate buffer is 5.5, the number of portions is 15, the enzymatic hydrolysis time is 5.5 h, and the enzymatic hydrolysis temperature is 35°C; In step ③, the number of ethyl acetate portions is 15, the ultrasonic power is 250 W, the ultrasonic time is 10 min, and the ultrasonic temperature is 40°C.

[0010] Preferred solution: In step ②, the pH of the phosphate buffer is 5.5, the number of portions is 15, the enzymatic hydrolysis time is 5.5 h, and the enzymatic hydrolysis temperature is 33°C; In step ③, the number of ethyl acetate portions is 16, the ultrasonic power is 250 W, the ultrasonic time is 10 min, and the ultrasonic temperature is 41°C.

[0011] The above technical solution has the following beneficial effects: The present invention uses dried broccoli pollen as a raw material and uses ultrasound-assisted ethyl acetate to extract sulforaphane therein, thereby obtaining a sulforaphane extraction method with simple process, convenient operation, and no need to add exogenous enzymes, thereby increasing the resource utilization rate of broccoli and laying a good theoretical foundation for the development of sulforaphane functional foods. The raw materials of the invention have a wide range of sources and a simple pretreatment method; ethyl acetate is used for extraction, and the ethyl acetate has low toxicity, is volatile, and is easy to be removed under reduced pressure, and does not contain other bad odors and solvent residues; the extraction process is simple, does not require any expensive instruments, does not require the introduction of exogenous enzymes, has low production costs, and is suitable for mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a schematic diagram of the extraction process of the present invention; Figure 2 is a standard curve diagram of sulforaphane in the present invention; Figure 3 is a chromatogram of the sulforaphane standard and broccoli sample in the present invention; Figure 4 This is a graph showing the effect of different enzymatic hydrolysis times on the sulforaphane extraction content in the present invention; Figure 5 This is a graph showing the effect of different enzymatic hydrolysis temperatures on the sulforaphane extraction content in the present invention; Figure 6 This is a graph showing the effect of different enzymatic hydrolysis pH on the sulforaphane extraction content in the present invention; Figure 7 This is a graph showing the effect of different enzymatic hydrolysis material-liquid ratios on the sulforaphane extraction content in the present invention; Figure 8 This is a graph showing the effect of different ultrasonic times on the sulforaphane extraction content in the present invention; Fig. 9 This is a graph showing the effect of different ultrasonic temperatures on the sulforaphane extraction content in the present invention; Fig.10 This is a graph showing the effect of different ultrasonic powers on the sulforaphane extraction content in the present invention; Fig.11 This is a graph showing the effect of different extraction material-liquid ratios on the sulforaphane extraction content in the present invention; Fig.12 3D response surface diagram and contour diagram of the effect of interaction of different factors on sulforaphane content in the response surface test of the present invention; Different letters indicate significant differences ( p <0.05).

[0013] The following is a further description with reference to specific embodiments. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be noted that the specific embodiments described here are only used to explain the present invention and are not limited to the present invention. Example 1

[0015] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 4 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0016] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0017] (3) Sulforaphane content detection: The broccoli sulforaphane extract was dissolved in a certain amount of acetonitrile solution, filtered through a 0.22 μm organic filter membrane into an automatic injection bottle, and the sulforaphane content was determined by ultra-high performance liquid chromatography. The analysis conditions were as follows: the chromatographic column was Shimadzu Shim-pack GIST C18 column (4.6×250 nm, 5 μm), the DAD detector detection wavelength was 190 nm, the flow rate was 1 mL / min, the column temperature was 30°C, and the injection volume was 5 μL. The mobile phase A was ultrapure water, and B was acetonitrile. The gradient elution program was 0-1 min, 70% A and 30% B; 1-9 min, 70%-50% A and 30%-50% B; 9-11 min, 50%-70% A and 50%-30% B; 11-12 min, 70% A and 30% B. The results were expressed in mg / g DW on a dry weight basis. Example 2

[0018] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 4.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0019] Step (2) and step (3) are the same as in Example 1. Example 3

[0020] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0021] Step (2) and step (3) are the same as in Example 1. Example 4

[0022] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0023] Step (2) and step (3) are the same as in Example 1. Example 5

[0024] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and pass through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 6 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0025] Step (2) and step (3) are the same as in Example 1. Example 6

[0026] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 15°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0027] Step (2) and step (3) are the same as in Example 1. Example 7

[0028] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 25°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0029] Step (2) and step (3) are the same as in Example 1. Example 8

[0030] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0031] Step (2) and step (3) are the same as in Example 1. Example 9

[0032] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 45°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0033] Step (2) and step (3) are the same as in Example 1. Example 10

[0034] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 55°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0035] Step (2) and step (3) are the same as in Example 1. Embodiment 11

[0036] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 4.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0037] Step (2) and step (3) are the same as in Example 1. Example 12

[0038] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0039] Step (2) and step (3) are the same as in Example 1. Embodiment 13

[0040] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0041] Step (2) and step (3) are the same as in Example 1. Embodiment 14

[0042] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add pH 6 and 7.5 mL (solid-liquid ratio 1:15 (g / mL)) phosphate buffer, mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0043] Step (2) and step (3) are the same as in Example 1. Embodiment 15

[0044] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add phosphate buffer with a pH of 6.5 and 7.5 mL (solid-liquid ratio 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0045] Step (2) and step (3) are the same as in Example 1. Example 16

[0046] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C until the moisture content is less than 7%, crush, and pass through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 2.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:5 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0047] Step (2) and step (3) are the same as in Example 1. Embodiment 17

[0048] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:10 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0049] Step (2) and step (3) are the same as in Example 1. Embodiment 18

[0050] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0051] Step (2) and step (3) are the same as in Example 1. Embodiment 19

[0052] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 10 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:20 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0053] Step (2) and step (3) are the same as in Example 1. Embodiment 20

[0054] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 12.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:25 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0055] Step (2) and step (3) are the same as in Example 1. Embodiment 21

[0056] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 5.5 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0057] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0058] Step (3) is the same as in Example 1. Embodiment 22

[0059] Step (1) is the same as in Example 21.

[0060] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0061] Step (3) is the same as in Example 1. Embodiment 23

[0062] Step (1) is the same as in Example 21.

[0063] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 30 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0064] Step (3) is the same as in Example 1. Embodiment 24

[0065] Step (1) is the same as in Example 21.

[0066] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 40 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0067] Step (3) is the same as in Example 1. Embodiment 25

[0068] Step (1) is the same as in Example 21.

[0069] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 50 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0070] Step (3) is the same as in Example 1. Embodiment 26

[0071] Step (1) is the same as in Example 21.

[0072] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 20°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0073] Step (3) is the same as in Example 1. Embodiment 27

[0074] Step (1) is the same as in Example 21.

[0075] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 30°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0076] Step (3) is the same as in Example 1. Embodiment 28

[0077] Step (1) is the same as in Example 21.

[0078] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0079] Step (3) is the same as in Example 1. Embodiment 29

[0080] Step (1) is the same as in Example 21.

[0081] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 50°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0082] Step (3) is the same as in Example 1. Embodiment 30

[0083] Step (1) is the same as in Example 21.

[0084] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 60°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0085] Step (3) is the same as in Example 1. Embodiment 31

[0086] Step (1) is the same as in Example 21.

[0087] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0088] Step (3) is the same as in Example 1. Embodiment 32

[0089] Step (1) is the same as in Example 21.

[0090] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 250 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0091] Step (3) is the same as in Example 1. Embodiment 33

[0092] Step (1) is the same as in Example 21.

[0093] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 300 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0094] Step (3) is the same as in Example 1. Embodiment 34

[0095] Step (1) is the same as in Example 21.

[0096] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 350 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0097] Step (3) is the same as in Example 1. Embodiment 35

[0098] Step (1) is the same as in Example 21.

[0099] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 400 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0100] Step (3) is the same as in Example 1. Embodiment 36

[0101] Step (1) is the same as in Example 21.

[0102] (2) Preparation of broccoli sulforaphane crude extract: Add 2.5 mL (solid-liquid ratio 1:5 (g / mL)) of ethyl acetate solution to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extract under the conditions of ultrasonic power of 250 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0103] Step (3) is the same as in Example 1. Embodiment 37

[0104] Step (1) is the same as in Example 21.

[0105] (2) Preparation of broccoli sulforaphane crude extract: 5 mL (solid-liquid ratio 1:10 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 250 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0106] Step (3) is the same as in Example 1. Embodiment 38

[0107] Step (1) is the same as in Example 21.

[0108] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 250 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0109] Step (3) is the same as in Example 1. Embodiment 39

[0110] Step (1) is the same as in Example 21.

[0111] (2) Preparation of broccoli sulforaphane crude extract: 10 mL (solid-liquid ratio 1:20 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 250 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0112] Step (3) is the same as in Example 1. Embodiment 40

[0113] Step (1) is the same as in Example 21.

[0114] (2) Preparation of broccoli sulforaphane crude extract: 12.5 mL (solid-liquid ratio 1:25 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 250 W, ultrasonic temperature of 40°C, and ultrasonic time of 10 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0115] Step (3) is the same as in Example 1. Embodiment 41

[0116] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 25°C for 4 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0117] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 30°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0118] Step (3) is the same as in Example 1. Embodiment 42

[0119] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 45°C for 4 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0120] Step (2) is the same as in Example 41.

[0121] Step (3) is the same as in Example 1. Embodiment 43

[0122] Step (1) is the same as in Example 41.

[0123] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 50°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0124] Step (3) is the same as in Example 1. Embodiment 44

[0125] Step (1) is the same as in Example 42.

[0126] Step (2) is the same as in Example 43.

[0127] Step (3) is the same as in Example 1. Embodiment 45

[0128] Step (1) is the same as in Example 41.

[0129] (2) Preparation of broccoli sulforaphane crude extract: 5 mL (solid-liquid ratio 1:10 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0130] Step (3) is the same as in Example 1. Embodiment 46

[0131] Step (1) is the same as in Example 42.

[0132] Step (2) is the same as in Example 45.

[0133] Step (3) is the same as in Example 1. Embodiment 47

[0134] Step (1) is the same as in Example 41.

[0135] (2) Preparation of broccoli sulforaphane crude extract: 10 mL (solid-liquid ratio 1:20 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0136] Step (3) is the same as in Example 1. Embodiment 48

[0137] Step (1) is the same as in Example 42.

[0138] Step (2) is the same as in Example 47.

[0139] Step (3) is the same as in Example 1. Embodiment 49

[0140] (1) Preparation of broccoli sulforaphane enzymatic hydrolysate: Wash fresh broccoli and cut into florets, dry with hot air at 40°C to a moisture content of less than 7%, crush, and sieve through a 60-mesh sieve to prepare broccoli floret pollen. Take 0.5 g of broccoli floret pollen, add 0.09 g L(+)-ascorbic acid, add 7.5 mL of phosphate buffer with a pH of 5.5 (solid-liquid ratio of 1:15 (g / mL)), mix, and shake at 110 rpm / min and 35°C for 4 h to obtain broccoli sulforaphane enzymatic hydrolysate.

[0141] (2) Preparation of broccoli sulforaphane crude extract: 5 mL (solid-liquid ratio 1:10 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 30°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0142] Step (3) is the same as in Example 1. Embodiment 50

[0143] Step (1) is the same as in Example 49.

[0144] (2) Preparation of broccoli sulforaphane crude extract: 5 mL (solid-liquid ratio 1:10 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 50°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0145] Step (3) is the same as in Example 1. Embodiment 51

[0146] Step (1) is the same as in Example 49.

[0147] (2) Preparation of broccoli sulforaphane crude extract: 10 mL (solid-liquid ratio 1:20 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 30°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0148] Step (3) is the same as in Example 1. Embodiment 52

[0149] Step (1) is the same as in Example 49.

[0150] (2) Preparation of broccoli sulforaphane crude extract: 10 mL (solid-liquid ratio 1:20 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 50°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0151] Step (3) is the same as in Example 1. Embodiment 53

[0152] Step (1) is the same as in Example 49.

[0153] (2) Preparation of broccoli sulforaphane crude extract: 7.5 mL (solid-liquid ratio 1:15 (g / mL)) of ethyl acetate solution was added to the broccoli sulforaphane enzymatic hydrolyzate obtained in step (1), and extraction was performed under the conditions of ultrasonic power of 200 W, ultrasonic temperature of 40°C, and ultrasonic time of 20 min. After extraction, the mixed solution was refrigerated centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the residue was repeatedly extracted with an equal amount of ethyl acetate for 3 times, and the filtrate was combined to obtain a broccoli sulforaphane crude extract. The broccoli sulforaphane crude extract was concentrated to dryness by rotary evaporation at 35°C to obtain a broccoli sulforaphane extract paste.

[0154] Step (3) is the same as in Example 1. Embodiment 54

[0155] Step (1) is the same as in Example 49.

[0156] Step (2) is the same as in Example 53.

[0157] Step (3) is the same as in Example 1. Embodiment 55

[0158] Step (1) is the same as in Example 49.

[0159] Step (2) is the same as in Example 53.

[0160] Step (3) is the same as in Example 1. Embodiment 56

[0161] Step (1) is the same as in Example 49.

[0162] Step (2) is the same as in Example 53.

[0163] Step (3) is the same as in Example 1. Embodiment 57

[0164] Step (1) is the same as in Example 49.

[0165] Step (2) is the same as in Example 53.

[0166] Step (3) is the same as in Example 1.

[0167] The beneficial effects of the present invention are further illustrated by test examples below. Test Example 1 Ultra-high Performance Liquid Chromatography Results of Sulforaphane

[0168] Figure 2 The standard curve of sulforaphane is shown. The standard equation of sulforaphane is y =3258847.5025 x −8690.4645, R 2=0.9999, good fit. Figure 3 The chromatograms of sulforaphane standard and broccoli sample are shown, with a peak time of 7.06 min and good peak shape. Experimental Example 2 Effect of different enzymatic hydrolysis times on the extraction content of sulforaphane

[0169] Figure 4 The effect of enzymatic hydrolysis time on the sulforaphane extraction content was shown. Figure 4 As shown in the figure, with the increase of enzymatic hydrolysis time, the sulforaphane content showed a trend of first increasing and then decreasing. When the enzymatic hydrolysis time was 5 h, 5.5 h and 6 h, there was no significant difference in the sulforaphane content ( p >0.05), but the sulforaphane content was the highest when the enzymatic hydrolysis time was 5.5 h, which was 0.70±0.01 mg / gDW. Therefore, the enzymatic hydrolysis time of 5.5 h was selected as the zero level for the subsequent extraction process optimization experiment. Experimental Example 3 Effect of different enzymatic hydrolysis temperatures on the sulforaphane extraction content

[0170] Figure 5 The effect of enzymatic hydrolysis temperature on the extractable content of sulforaphane was demonstrated. Figure 5 It can be seen that as the enzymatic hydrolysis temperature increases, the sulforaphane content first increases significantly and then decreases significantly ( p <0.05), when the enzymatic hydrolysis temperature was 35℃, the sulforaphane content was 0.76±0.02 mg / g DW, so the enzymatic hydrolysis temperature of 35℃ was selected as the zero level for the subsequent extraction process optimization experiment. Experimental Example 4 Effect of different enzymatic hydrolysis pH on the extraction content of sulforaphane

[0171] Figure 6 The effect of enzymatic hydrolysis pH on the sulforaphane extraction content was demonstrated. Figure 6 As shown in Figure 2, pH has a significant effect on the activity of endogenous myrosinase, as shown in Figure 2. Figure 5 It can be seen that there is no significant difference in the yield of sulforaphane at pH 5.5 and 6.0 ( p >0.05), were 0.69±0.02 mg / g DW and 0.68±0.01 mg / g DW, respectively. Therefore, phosphate buffer with pH 5.5 was selected as the zero level for subsequent extraction process optimization experiments. Experimental Example 5 Effect of different enzymatic hydrolysis material-liquid ratios on the sulforaphane extraction content

[0172] Figure 7 The effect of the enzymatic hydrolysis feed-liquid ratio on the sulforaphane extraction content was demonstrated. Figure 6As shown in the figure, with the increase of the enzymatic hydrolysis liquid ratio, the sulforaphane content first increased and then decreased. When the enzymatic hydrolysis liquid ratio was 1:15 (g / mL), the sulforaphane content was the highest, which was 0.75±0.01 mg / g DW. Therefore, the enzymatic hydrolysis liquid ratio of 1:15 (g / mL) was selected as the zero level for the subsequent extraction process optimization experiment. Experimental Example 6 Effect of different ultrasonic times on the content of sulforaphane extraction

[0173] Figure 8 The effect of ultrasound time on the sulforaphane extraction content was demonstrated. Figure 8 It can be seen that there is no significant difference in the effect of ultrasound time on the content of sulforaphane extraction ( p >0.05), when the ultrasonic time was 10 min, the extracted sulforaphane content was 0.89±0.02 mg / g DW. In order to save energy, the ultrasonic time of 10 min was selected as the zero level for the subsequent extraction process optimization experiment. Experimental Example 7 Effect of different ultrasonic temperatures on the content of sulforaphane extraction

[0174] Fig. 9 The effect of ultrasound temperature on the sulforaphane extraction content was demonstrated. Fig. 9 It can be seen that as the ultrasonic temperature increases, the sulforaphane content first increases significantly and then decreases significantly ( p <0.05), when the ultrasonic temperature was 40℃, the sulforaphane content was the highest, which was 0.93±0.02 mg / g DW, so the ultrasonic temperature of 40℃ was selected as the zero level for the subsequent extraction process optimization experiment. Experimental Example 8 Effect of different ultrasonic powers on the content of sulforaphane extraction

[0175] Fig.10 The effect of ultrasound power on the sulforaphane extraction content was demonstrated. Fig.10 It can be seen that when the ultrasonic power is 250W, the highest sulforaphane content is 0.88±0.03 mg / g DW. After that, there is no significant difference in sulforaphane content with the increase of ultrasonic power ( p >0.05), and in order to save energy, an ultrasonic power of 250 W was selected as the zero level for subsequent extraction process optimization experiments. Experimental Example 9 Effect of different extraction material-liquid ratios on sulforaphane extraction content

[0176] Fig.11 The effect of the ratio of solid to liquid on the content of sulforaphane extraction was demonstrated. Fig.11 It can be seen that with the increase of the extraction material-liquid ratio, the content of sulforaphane increased significantly ( p<0.05), and then stabilized. When the extraction material-liquid ratio was 1:15 (g / mL), the sulforaphane content reached the highest, which was 0.80±0.01 mg / g DW. When the material-liquid ratio increased to 1:20 (g / mL) and 1:25 (g / mL), there was no significant difference in the change of sulforaphane content ( p >0.05), indicating that ethyl acetate has completely extracted sulforaphane in the sample, so the solid-liquid ratio of 1:15 (g / mL) was selected as the zero level for subsequent extraction process optimization experiments. Experimental Example 10 Optimization of sulforaphane extraction from broccoli by response surface methodology

[0177] The variance analysis of the single factor test results was performed to determine the three factors that had the greatest impact on the sulforaphane extraction amount: enzymatic hydrolysis temperature, extraction material-liquid ratio and ultrasonic temperature. Combining the central composite experimental design principle of Box-Benhnken, DesignExpert 13.0 was used to perform response surface experimental factor and level analysis. Taking sulforaphane content (mg / g DW) as the response value, a three-factor three-level response surface experiment was conducted. Table 1 is the response surface experimental factor level table.

[0178] Table 1. Response surface experiment factor level table level A: Enzymatic hydrolysis temperature (℃) B: Extraction temperature (℃) C: Ethyl acetate volume multiple (mL / g) -1 25 30 10 0 35 40 15 1 45 50 20 The response surface experiment design and results are shown in Table 2, where the 13th to 17th experiments were the central group experiments.

[0179] Table 2 Response surface experimental design and results project A: Enzymatic hydrolysis temperature (℃) B: Extraction temperature (℃) C: Ethyl acetate volume multiple (mL / g) Sulforaphane content (mg / g DW) Embodiment 41 -1（25） -1（30） 0（1∶15） 0.86 Embodiment 42 1（45） -1 0 0.67 Embodiment 43 -1 1（50） 0 0.85 Embodiment 44 1 1 0 0.65 Embodiment 45 -1 0（45） -1（1∶10） 0.69 Embodiment 46 1 0 -1 0.55 Embodiment 47 -1 0 1（1∶20） 0.84 Embodiment 48 1 0 1 0.65 Embodiment 49 0（35） -1 -1 0.74 Embodiment 50 0 1 -1 0.69 Embodiment 51 0 -1 1 0.83 Embodiment 52 0 1 1 0.84 Embodiment 53 0 0 0 0.90 Embodiment 54 0 0 0 0.92 Embodiment 55 0 0 0 0.91 Embodiment 56 0 0 0 0.92 Embodiment 57 0 0 0 0.90 A quadratic regression model was established based on the test results. The broccoli sulforaphane extract content (Y) was used as the response value. The coded quadratic regression equation was: Y=0.9100-0.0900A-0.0087B+0.0613C-0.0025AB-0.0125AC+0.0150BC-0.1225A^2-0.0300B^2-0.1050C^2 Table 3 Analysis of variance of quadratic regression model Sources of variance sum of squares Degrees of Freedom Mean square sum F-number P-value Significance Model 0.2203 9 0.0245 195.82 < 0.0001 ** A 0.0648 1 0.0648 518.40 < 0.0001 ** B 0.0006 1 0.0006 4.90 0.0625 C 0.0300 1 0.0300 240.10 < 0.0001 ** AB 0.0000 1 0.0000 0.2000 0.6682 AC 0.0006 1 0.0006 5.00 0.0604 BC 0.0009 1 0.0009 7.20 0.0314 * A² 0.0632 1 0.0632 505.47 < 0.0001 ** B² 0.0038 1 0.0038 30.32 0.0009 ** C² 0.0464 1 0.0464 371.37 < 0.0001 ** Residual 0.0009 7 0.0001 Lack of Fit 0.0005 3 0.0002 1.58 0.3258 Not significant Pure Error 0.0004 4 0.0001 Cor Total 0.2212 16 CV % 1.42 R² 0.9960 Adjusted R² 0.9910 Predicted R² 0.9628 Note:** p <0.01;* p <0.05 The above regression models were subjected to variance analysis as shown in Table 3. The regression equation models for sulforaphane extraction rate were all extremely significant ( p <0.01, the lack-of-fit term is not significant ( p>0.05), the model correlation coefficient was 0.9960, the adjusted determination coefficient was 0.9910, the prediction model coefficient was 0.9628, and the CV of the model was 0.0142, indicating that the model had a good fit with the actual experiment and the error was small. The model was established and could be used to optimize the analysis and prediction of the broccoli sulforaphane extraction process and its content. The results of variance analysis of each item in the model showed that according to the F value, the influence of each factor on the sulforaphane extraction content was: A (enzyme hydrolysis temperature) > C (ethyl acetate volume multiple) > B (extraction temperature). Factors A, C, A², B² and C 2 The effect on the sulforaphane extraction content was extremely significant ( p <0.01), and the factor BC had a significant effect on the content of sulforaphane extract ( p <0.05), factors B, AB and AC had no significant effect on the content of sulforaphane extraction ( p >0.05).

[0180] The steeper the slope of the response surface, the more sensitive the response value is to the change of the factor level. Conversely, the less significant the contour line is, the more elliptical it is, indicating that the interaction between the two factors is stronger. Conversely, if the contour is circular, it means that the interaction between the two factors is weaker. The contour curves and response surfaces of the three factors of enzymatic hydrolysis temperature, extraction temperature, and extraction material-liquid ratio are shown in Figure 2. Fig.12 As shown, the interaction between the volume multiple of ethyl acetate and the extraction temperature is more obvious. In contrast, the interaction between the volume multiple of ethyl acetate and the enzymatic hydrolysis temperature, and between the enzymatic hydrolysis temperature and the extraction temperature is weaker.

[0181] The optimal extraction process parameters predicted by Design Expert 13.0 software were: enzymatic hydrolysis temperature 32.694°C, extraction temperature 40.967°C, ethyl acetate volume multiple 16.775:1 mL / g, and the predicted sulforaphane content was 0.93 mg / g DW.

[0182] According to the actual situation, the enzymatic hydrolysis temperature was determined to be 33℃, the extraction temperature was 41℃, and the volume multiple of ethyl acetate was 16:1 mL / g. The response surface validation test was carried out to verify that the sulforaphane content was 0.91±0.01 mg / g DW, which was 2.15% different from the predicted value, indicating that the regression model was good and the optimization results were reasonable. In addition, under these conditions, the traditional extraction was carried out to obtain a sulforaphane content of 0.80±0.02 mg / g DW, and the ultrasound-assisted extraction increased the sulforaphane content of broccoli by 13.75%.

[0183] The process of the present invention is simple, easy to operate, and high-content sulforaphane can be obtained without adding exogenous enzymes. The present invention can be used for mass production of sulforaphane in food and biological factories.

[0184] It should be noted here that the above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a broccoli sulforaphane extract, characterized in that: The following steps are involved: (1) Cutting fresh broccoli into florets, drying and crushing the florets to prepare broccoli floret pollen; (2) using phosphate buffer as an enzymatic hydrolyzer to perform endogenous enzymatic hydrolysis on broccoli florets to obtain broccoli sulforaphane enzymatic hydrolyzate; (3) using ethyl acetate as an extractant, performing ultrasound-assisted extraction of sulforaphane in the broccoli sulforaphane enzymatic hydrolysate to obtain a broccoli sulforaphane crude extract; (4) concentrating the crude broccoli sulforaphane extract to dryness by rotary evaporation at 35° C. to obtain a broccoli sulforaphane extract paste; (5) The sulforaphane content was determined using ultra-high performance liquid chromatography.

2. The method for preparing a broccoli sulforaphane extract according to claim 1, characterized in that: The broccoli floret pollen in step (1) is obtained by the following method: washing fresh broccoli and cutting into florets, drying with hot air at 40° C. until the moisture content is less than 7%, crushing, and passing through a 60-mesh sieve to prepare broccoli floret pollen.

3. The method for preparing a broccoli sulforaphane extract according to claim 1, characterized in that: The endogenous enzymatic hydrolysis process in step (2) is as follows: 0.18 parts of L(+)-ascorbic acid are added to 1 part of broccoli floret, and mixed with 5-25 parts of phosphate buffer with a pH of 4.5-7.5, and shaken at 110 rpm / min and 15-35° C. for 4-6 hours to obtain broccoli sulforaphane enzymatic hydrolyzate, wherein the unit of the ratio of broccoli floret pollen: L(+)-ascorbic acid: phosphate buffer is g:g:mL.

4. The method for preparing a broccoli sulforaphane extract according to claim 1, characterized in that: The ultrasonic-assisted extraction process in step (3) is as follows: 5-15 parts of ethyl acetate are added to the broccoli sulforaphane hydrolysate, and ultrasonic-assisted extraction is performed under the conditions of an ultrasonic power of 200-500 W, an ultrasonic temperature of 20-60° C., and an ultrasonic time of 10-50 min, wherein the unit of the ratio of broccoli floret to ethyl acetate is g:mL.

5. The method for preparing a broccoli sulforaphane extract according to claim 1, characterized in that: The conditions for determining the content of sulforaphane by ultra-high performance liquid chromatography in step (5) are as follows: dissolving the broccoli sulforaphane extract paste in a certain amount of acetonitrile solution, filtering the solution through a 0.22 μm organic filter membrane into an automatic injection bottle, and determining the content of sulforaphane by ultra-high performance liquid chromatography. The analysis conditions are as follows: the chromatographic column is Shimadzu Shim-pack GIST C18 chromatographic column (4.6×250 nm, 5 μm), the detection wavelength of the DAD detector is 190 nm, the flow rate is 1 mL / min, the column temperature is 30°C, and the injection volume is 5 μL. The mobile phase A is ultrapure water, and B is acetonitrile. The gradient elution program is 0-1 min, 70% A and 30% B; 1-9 min, 70%-50% A and 30%-50% B; 9-11 min, 50%-70% A and 50%-30% B; 11-12 min, 70% A and 30% B. The results are based on dry weight, in mg / gDW.

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