METHOD FOR PREPARING BROCCOLI PROTEIN PEPTIDE, BROCCOLI PROTEIN PEPTIDE PREPARED THEREFROM AND USE THEREOF

Abstract

Provided is a method for preparing a broccoli protein peptide. The method uses a broccoli protein as the raw material, and obtains a broccoli protein peptide powder through the steps of preprocessing, enzymatic hydrolysis, terminating enzymatic hydrolysis, separation, and drying and the like. Also provided is the use of the prepared broccoli protein peptide in resisting oxidation, reducing cholesterol and lowering blood lipids.

Claims

1-12. (canceled)

13. A method for preparing a broccoli protein peptide, the method comprising: (a) adding water, anhydrous sodium sulfite, and EDTA to a raw material broccoli protein thereby making a predigestion mixture, wherein the weight of the water is 4 to 8 times that of raw material broccoli protein, the EDTA has a mass/volume ratio of 0.02-0.05 g/L, and the anhydrous sodium sulfite has a mass/volume ratio of 0.05-0.1 g/L; (b) digesting the predigestion mixture for 3-4 hours at a temperature with an enzyme selected from the group consisting of neutral protease, papain, alkaline protease, trypsin, pepsin, bromelain and compound protease thereby forming a digested mixture; (c) terminating the digestion by heating the digested mixture to from 80-90 C. for from 5-15 minutes; (d) optionally, centrifuging or filtering the mixture from (c); (e) filtering the liquid from (c) or (d) with a membrane having a pore size of 100 to 500 nm; (f) optionally, debittering the mixture from (e) by treating with active carbon or clay; and (g) concentrating and/or drying the mixture from (e) or (f), thereby obtaining the broccoli protein peptide.

14. The method of claim 13, wherein the enzyme is selected from trypsin and neutral protease, or alkaline protease and papain, or alkaline protease and neutral protease.

15. A broccoli protein peptide prepared according to the method of claim 1.

16. A method of antioxidizing, lowering cholesterol or lowering blood lipid, comprising administering the broccoli protein peptide according to claim 15.

17. A food, medicine, health care product or cosmetic that has antioxidant effects, cholesterol lowering effects or blood lipid lowering effects, comprising the broccoli protein peptide according to claim 15.

18. The method of claim 13, wherein the enzyme is neutral protease, 200-600 units of enzyme are added per gram of raw material broccoli protein, the temperature is 501 C., and the pH of the digested mixture is adjusted to from 6.0-7.0 with NaOH before terminating.

19. The method of claim 13, wherein the enzyme is papain, 1000-3000 units of enzyme are added per gram of raw material broccoli protein, the temperature is 501 C., and the pH of the digested mixture is adjusted to from 6.5-7.0 with NaOH before terminating.

20. The method of claim 13, wherein the enzyme is alkaline protease, 200-600 units of enzyme are added per gram of raw material broccoli protein, the temperature is 501 C., and the pH of the digested mixture is adjusted to from 9.0-9.5 with NaOH before terminating.

21. The method of claim 13, wherein the enzyme is trypsin, 5-15 units of enzyme are added per gram of raw material broccoli protein, the temperature is 501 C., and the pH of the digested mixture is adjusted to from 8.0-8.5 with NaOH before terminating.

22. The method of claim 13, wherein the enzyme is pepsin, 5-15 units of enzyme are added per gram of raw material broccoli protein, the temperature is 36-38 C., and the pH of the digested mixture is adjusted to from 1.5-2.5 with NaOH before terminating.

23. The method of claim 13, wherein the enzyme is bromelain, 1000-5000 units of enzyme are added per gram of raw material broccoli protein, the temperature is 401 C., and the pH of the digested mixture is adjusted to from 6.0-7.0 with NaOH before terminating.

24. The method of claim 13, wherein the enzyme is compound protease, 5-3600 units of enzyme are added per gram of raw material broccoli protein, the temperature is 501 C., and the pH of the digestion is controlled to 8.0-8.5 for the initial 0.5-1 hour and then not controlled for the remainder of the digestion.

25. The method of claim 24, wherein the compound protease is selected from trypsin and neutral protease, or alkaline protease and papain, or alkaline protease and neutral protease.

Description

EMBODIMENTS

[0079] In the following examples, the reagents and instruments used are those commonly used in the art, and can be purchased from a chemical or biological product/preparation company unless otherwise specified. The methods used in the following examples are conventional methods in the art. It will be apparent to those skilled in the art that the operation of these experiments and the corresponding results can be obtained without any doubt from the prior art or the operating manual provided by the manufacturer.

Example 1: Preparation of Raw Material Broccoli Protein

[0080] (1) Raw material crushing: a whole broccoli (20 kg) was washed, dried, crushed with a crusher, and then pressed and filtered with a gauze (300 mesh) to obtain pressed juice, 9.5 L.

[0081] (2) Heating: the pressed juice from the last step was heated to 70 C. in a pot, the protein flocculates and floated on top, until there's no green at the bottom.

[0082] (3) Separating: the heated layered pressed juice was filtered to obtain a filter cake (flocculated protein) and a filtrate.

[0083] (4) Spray drying: 2.8 L of purified water was added to the flocculated protein obtained by filtration, mixed and homogenized, and then 682 g of raw material broccoli protein was obtained by spray drying (BUCHI, Small Spray Dryer B-290).

Example 2

[0084] (1) Protein Preprocessing: 100 g of broccoli protein prepared in example 1 was taken, and 500 ml of purified water, 0.05 g of anhydrous sodium sulfite and 0.02 g of EDTA were added and stirred to form a broccoli protein pulp.

[0085] (2) Proteolysis: 0.3 g of trypsin (4000 U/g) and 0.3 g of neutral protease (800,000 U/g) were added to the preprocessed broccoli protein pulp, stirred at 50 C. in a water bath, and the pH of the pulp was controlled at 8.2 with NaOH solution, the enzymolysis lasted for 3.5 h in total.

[0086] (3) Terminating of the enzymolysis and separation: the protein pulp that had been subjected to enzymolysis was heated to 80 C. for 5 minutes to inactivate the enzyme. After the pulp was cooled to room temperature, it was centrifuged at 10,000 rpm for 10 minutes with a high-speed centrifuge, then the supernatant was filtered through a 200 nm membrane to obtain about 400 ml of supernatant containing broccoli protein peptide. At this point, free nitrogen was used as a regular indicator, and the content of free nitrogen in the supernatant was measured as 1400 mg/l according to GB12143.2-1989.

[0087] (4) Debitterizing: 0.5% (W/V) of activated carbon was added to the obtained 400 ml supernatant, and the activated carbon was removed by filtering 30 minutes after debitterizing.

[0088] (5) Concentrating: the protein peptide solution was concentrated with a three-effect falling film evaporator until the solid content of the solution was greater than 10%.

[0089] (6) Spray Drying: the broccoli protein peptide supernatant obtained in step (4) was directly dried by spray drying (BUCHI, Small Spray Dryer B-290) to obtain 30 g of broccoli protein peptide.

[0090] The various indexes of the test and analysis of the obtained broccoli protein peptide are as shown in table 1. The test refers to the national standard prescribed test method for soybean protein peptide.

TABLE-US-00001 TABLE 1 Test and analysis results of broccoli protein peptide samples No. Items of analysis Results of analysis 1 Free amino acid 6.39% 2 Molecular weight Larger than 10000 D 8.41% 3 distribution Smaller than or equal to 91.59% 10000 D 4 Urease Negative

Example 3

[0091] (1) Sample Preprocessing: six broccoli protein samples prepared in example 1 were weighed, 10 g each, and were numbered A, B, C, D, E, F, 50 ml of distilled water was added respectively, the mixture were well shaken, then 5 mg of solid anhydrous sodium sulfite and 2 mg of solid EDTA were added.

[0092] (2) Proteolysis: proteases were added to the preprocessed six broccoli protein pulp samples according to the content of table 2, the pH value and temperature of the six samples were controlled according to table 2,

TABLE-US-00002 Table 2 Formula for proteolysis No. A B C D E F Proteases 0.03 g of 0.03 g 0.03 g 0.03 g of 0.05 g 0.1 g of 2709 of of 1389 of bromelain alkaline trypsin papain neutral pepsin (500,000 protease (4000 (800,000 protease (3000 U/g) (200,000 U/g) U/g) (200,000 U/g) U/g) U/g) pH value 9.5 8.5 7.0 7.0 2.0 7.0 Temperature 50 C. 50 C. 50 C. 50 C. 37 C. 40 C.

[0093] The enzymolysis lasted for 3.5-4.0 h in total.

[0094] (3) Terminating of the enzymolysis and separation: the protein pulp A, D, C, D, E, F that had been subjected to enzymolysis were heated to 80 C. respectively for 5 minutes to inactivate the enzymes, after the temperature was decreased to room temperature, the pulps were filtered with filter clothes (400 mesh) and the supernatants were obtained. Then the filtered supernatants were filtered with a 200 nm membrane respectively to obtain supernatants containing broccoli protein peptide.

[0095] (4) The obtained broccoli protein peptide supernatants were lyophilized to obtain broccoli protein peptide samples digested with different enzymes.

Example 4

[0096] The antioxidant capacity was determined with the neutral protease and trypsin-digested peptide prepared in example 2, as well as the alkaline protease-digested peptide, the trypsin-digested peptide, the neutral protease-digested peptide, the papain-digested peptide and the pepsin-digested peptide prepared in example 3 used as samples, with soybean protein peptide and reduced glutathione used as controls (DPPH free radical scavenging rate method).

[0097] (1) DPPH (1,1-diphenyl-2-picrylhydrazyl) (25 mg) was weighed accurately, dissolved in absolute ethanol and the volume reached to 250 mL in a brown volumetric flask, DPPH solution with the concentration of 0.1 mg/mL was obtained and was kept in dark place to be supplied as standby.

[0098] (2) 1.0 mL sample solutions with different concentrations (100 mg/mL, 10 mg/mL, 1 mg/mL, 0.1 mg/mL) were taken, the solutions were placed in a 10 mL colorimetric tube respectively, 4.0 ml DPPH solution was added, the mixture was reacted at room temperature in a dark place for 30 minutes.

[0099] (3) The absorbance value was measured at 517 nm with the absolute ethanol as blank. The DPPH free radical scavenging rate was calculated according to the following formula.

DPPH free radical scavenging rate (%)=A.sub.0(A.sub.sA.sub.c)/A.sub.0100% [0100] In the formula. A.sub.0the absorbance value of 1.0 mL distilled water+3.0 mL solution [0101] A.sub.sthe absorbance value of 1.0 mL sample solution+3.0 mL DPPH solution [0102] A.sub.cthe absorbance value of 1.0 mL sample solution+3.0 mL absolute ethanol.

[0103] The experiment was repeated for three times, the average value of the scavenging rate was obtained (if the solution was turbid, the measurement was taken after centrifugation). The results are as shown in table 3.

TABLE-US-00003 TABLE 3 Results of the determination of the antioxidant effect of each protein peptide of broccoli Scavenging rate concentration Peptide 100 mg/ml 10 mg/ml 1 mg/ml 0.1 mg/ml Alkaline 87.7% 76.1% 8.6% 3.0% protease-digested peptide Trypsin-digested 85.9% 73.3% 10.4% 2.4% peptide Neutral 90.2% 77.9% 21.0% 14.1% protease-digested peptide Papain-digested 86.7% 72.3% 15.4% 1.1% peptide Pepsin-digested 78.3% 68.4% 13.4% 3.6% peptide Neutral protease and 91.9% 19.2% 23.3% 8.3% trypsin-digested peptide Soybean protein 75.6% 26.8% 10.2% 6.3% peptide Reduced glutathione 95.9% 90.9% 72.1% 30.8% (GSH)

[0104] It can be seen from table 3 that the DPPH free radical scavenging rates of the neutral protease-digested peptide, neutral protease and trypsin-digested peptide were significantly better than that of other single enzyme-digested peptides, and can reach 10% of that of reduced glutathione (the reducing power is about 10 mass % of GSH), the rate is significantly better than soybean protein peptide.

Example 5

[0105] Investigation of the influence of protease-digested peptides obtained in examples 2 and 3 on blood lipid levels of golden hamsters with hypercholesterolemia.

[0106] (1) Materials of the Experiment

[0107] (1.1) Animals

[0108] Golden hamster, male, 120, weight of 70-90 g, provided by Beijing Wei Tong Li Hua Experimental Animal Technology Co., Ltd., certificate number: SCXK (Beijing) 2006-0009.

[0109] Animals were fed in the animal houses of the pharmacological center of the Central Research Institute of Zhejiang Hisun Pharmaceutical Co., Ltd., room temperature: 20-28 C., humidity: 40-70%, ventilation times: 8-10 times per hour. The feed to the hamster was the standard feed (mice feed) provided by the experimental animal center in Zhejiang Province, the executive standard was GB14924-2001. The high fat diet was homemade (formula: 0.3% cholesterol, 20% palm oil, 79.7% basal feed).

[0110] (1.2) Drugs and Reagents

[0111] Drugs tested: The alkaline protease and neutral protease compound enzyme-digested peptide obtained in example 2, the neutral protease-digested peptide, the bromelain-digested peptide, the papain-digested peptide, the pepsin-digested peptide, the trypsin-digested peptides, alkaline protease-digested peptide obtained in example 3.

[0112] The above drugs were stored at 4 C. to 8 C. and formulated with 1% CMC (sodium carboxymethylcellulose CMC-Na) to the required concentration.

[0113] Reagents and Kits:

[0114] Ether: analytical grade, Hangzhou Chemical Reagent Co., Ltd., batch number: 20131112;

[0115] Total cholesterol (TC), batch number: 20140725;

[0116] Low density lipoprotein (LDL-C), provided by Shanghai Kehua. Bioengineering Co., Ltd.

[0117] (1.3) Instrument

[0118] Xi Sen Mei Kang automatic biochemical analyzer, model CHEX-180.

[0119] (2.) Experimental Methods and Results

[0120] (2.1) Method

[0121] With the reference to the regulations and literatures such as State Food and Drug Administration Order No. 28, Drug Registration Management Measures (Secretary: Shao Mingli issued, Oct. 1, 2007) and New Drugs (Western medicine) Preclinical Research Guidelines (Pharmacy Pharmacology Toxicology), the hamsters were allowed to acclimate for 7 days, they can drink and eat freely, light period of 10 h/14 h, on the 8th day, animals were fed with high fat feed and were fed for 3 weeks, then the animal were anesthetized with ether, weighed, then 0.5 mL blood were obtained from postorbital vein, 0.5% heparin was used for anticoagulation, centrifuged at 5000 rpm for 10 minutes, the plasma was absorbed. The levels of TC and LDL-C in the plasma were measured by automatic biochemical analyzer, and the animals were grouped based on their blood lipid level and weight, the grouping are as shown in the following table:

TABLE-US-00004 TABLE 4 Grouping situation. Adminis- Volume of Dose Number of tration adminis- Group (g .Math. kg.sup.1) animals route tration Model 8 ig (gavage) 10 ml/kg Neutral protease- 0.5 8 ig (gavage) 10 ml/kg digested peptide Alkaline protease and 0.5 8 ig (gavage) 10 ml/kg neutral protease compound enzyme digested peptide Bromelain-digested 0.5 8 ig (gavage) 10 ml/kg peptide Papain-digested 0.5 8 ig (gavage) 10 ml/kg peptide Pepsin-digested 0.5 8 ig (gavage) 10 ml/kg peptide Trypsin-digested 0.5 8 ig (gavage) 10 ml/kg peptide Alkaline protease- 0.5 8 ig (gavage) 10 ml/kg digested peptide

[0122] The hamsters in the model group were given equal volume of menstruum. During the administration period, the hamsters in the administration group and the model group were fed with high fat feed continuously. After 10 days of administration, the animals were anesthetized with ether, weighed, then 0.5 mL blood were obtained from postorbital vein, heparin was used for anticoagulation, centrifuged at 5000 rpm for 10 minutes, the plasma was absorbed. The levels of TC and LDL-C in the plasma were measured by automatic biochemical analyzer.

[0123] (2.2) Data Processing:

[0124] The results of all the experiments were expressed with, xs, the comparison between any two groups used t-test. P<0.05 was statistically significant.

[0125] (2.3) Results

[0126] As shown in table 5, compared with the model group, the neutral protease-digested peptide, the papain-digested peptide can both significantly reduce the TC (*P<0.05, **P<0.01); the papain-digested peptide can significantly reduce the LDL-C (*P<0.05, **P<0.01).

TABLE-US-00005 TABLE 5 Effect of each protein peptide of broccoli on lipid levels in hypercholesterolemia model golden hamsters after oral administration (x s, n = 8) Dose Group (g .Math. kg.sup.1) TC LDL-C Model group 10.54 1.21 2.93 0.75 Neutral protease- 0.5 8.79 1.46** 2.40 0.52 digested peptide group Alkaline protease and 0.5 9.60 1.18 2.33 0.33* neutral protease compound enzyme digested peptide group Bromelam-digested 0.5 10.06 1.75 3.05 0.93 peptide group Papain-digested 0.5 8.83 1.42** 2.20 0.41** peptide group Pepsin-digested 0.5 10.46 1.54 3.01 0.68 peptide group Trypsin-digested 0.5 11.97 1.81 3.78 0.70 peptide group Alkaline protease- 0.5 10.37 1.37 2.73 0.32 digested peptide group Compared with the model group, *P < 0.05, **P < 0.01.