U.S. patent application number 15/571625 was filed with the patent office on 2019-02-07 for method for preparing broccoli protein peptide, broccoli protein peptide prepared therefrom and use thereof.
The applicant listed for this patent is ZHEJIANG HISUN PHARMACEUTICAL CO., LTD., ZHEJIANG TELEY BIOTECHNOLOGY CO., LTD.. Invention is credited to Haiming LIN, Huan QI, Hailing TANG, Jidong WANG, Yongdong WANG, Hui ZHANG, Xiaohe ZHENG.
Application Number | 20190038694 15/571625 |
Document ID | / |
Family ID | 57218456 |
Filed Date | 2019-02-07 |
United States Patent
Application |
20190038694 |
Kind Code |
A2 |
WANG; Jidong ; et
al. |
February 7, 2019 |
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.
Inventors: |
WANG; Jidong; (Zhejiang,
CN) ; QI; Huan; (Zhejiang, CN) ; ZHENG;
Xiaohe; (Zhejiang, CN) ; ZHANG; Hui;
(Zhejiang, CN) ; WANG; Yongdong; (Zhejiang,
CN) ; TANG; Hailing; (Zhejiang, CN) ; LIN;
Haiming; (Zhejiang, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZHEJIANG HISUN PHARMACEUTICAL CO., LTD.
ZHEJIANG TELEY BIOTECHNOLOGY CO., LTD. |
Taizhou City, Zhejiang
Linhai City, Zhejiang |
|
CN
CN |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180147248 A1 |
May 31, 2018 |
|
|
Family ID: |
57218456 |
Appl. No.: |
15/571625 |
Filed: |
May 3, 2016 |
PCT Filed: |
May 3, 2016 |
PCT NO: |
PCT/CN2016/080880 PCKC 00 |
371 Date: |
November 3, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 33/185 20160801;
A23L 33/18 20160801; A23V 2002/00 20130101; A61P 3/06 20180101;
C12Y 304/22032 20130101; A61K 38/011 20130101; A61K 36/31 20130101;
C12Y 304/00 20130101; C12Y 304/22002 20130101; C12Y 304/21004
20130101; C12Y 304/25001 20130101; A61P 39/06 20180101; C12P 21/06
20130101; C12Y 304/23 20130101 |
International
Class: |
A61K 36/31 20060101
A61K036/31; C12P 21/06 20060101 C12P021/06; A61K 38/01 20060101
A61K038/01; A61P 3/06 20060101 A61P003/06; A61P 39/06 20060101
A61P039/06; A23L 33/185 20060101 A23L033/185 |
Foreign Application Data
Date |
Code |
Application Number |
May 4, 2015 |
CN |
201510216844.4 |
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.degree. 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 50.+-.1.degree. 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 50.+-.1.degree. 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 50.+-.1.degree. 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 50.+-.1.degree. 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.degree. 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 40.+-.1.degree. 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 50.+-.1.degree. 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
TECHNICAL FIELD
[0001] The present invention relates to a broccoli protein peptide,
specifically, it relates to the preparation method for broccoli
protein peptide and use of the obtained broccoli protein
peptide.
TECHNICAL BACKGROUND
[0002] Broccoli (Brassica oleracea var. italia) is a variant of
cruciferous Brassica cabbage, which is originally from Italy.
Broccoli is rich in bioactive substances and nutrients, which is
known as the "vegetable crown". The nutritional ingredient of
broccoli is not only high in content, but also very comprehensive,
which mainly includes proteins, carbohydrates, fats, minerals,
vitamin C and carotene and so on. According to the analysis, every
100 g fresh broccoli bulb, contains 3.5 g-4.5 g protein, which is 3
times of that in cauliflower, 4 times of that in tomato. Patent
ZL200910091131.4 discloses a broccoli leaf protein and its
preparation method, wherein, a flocculated protein is obtained by
flocculating broccoli juice by adding acid with heating or adding
acid and flocculant, then broccoli leaf protein is obtained by
drying the flocculated protein.
[0003] With the deepening of the research on nutrition and so on,
the biological function of protein peptide has been paid more and
more attention. Protein peptide is a kind of compound whose
molecular structure is between amino acid and protein. It is the
structure and function fragment of protein, and has dual function
of regulating the physiological function of the body and providing
nutrition for the body. Protein peptide has a variety of biological
activities, such as: immune regulation, anti-thrombosis,
antihypertensive, cholesterol-lowering, inhibition of bacteria and
viruses, anti-cancer effect, anti-oxidation and scavenging free
radicals, improving element absorption and mineral transportation,
promoting growth and so on. Modern nutrition research found that
the protein absorbed by human body are mainly digested and absorbed
in the form of oligopeptide after the proteins have been hydrolyzed
by the enzyme in the digestive tract, the proportion of the
proteins absorbed in the form of free amino acid is very small (Li
Jianrong, Feng Ping. Progress in Study of Functional Polypeptides.
Food Science, 2004, 25 (11): 415-419), so the polypeptide can be
absorbed and used by the body more easily and faster than the
protein and free amino acids, and the protein is absorbed in the
form of a polypeptide, and it is also beneficial to maximize the
use of its biological active function. Mature protein peptide
products in foreign markets are corn protein peptide breakfast
drinks from Japan, milk protein peptides anti-alcohol drinks from
South Korea. In the market of China, the products are soybean
protein peptide immunoregulation health care products, milk protein
peptide nutritional supplements and so on.
[0004] In view of the high protein content of broccoli, the
production of broccoli protein peptide using broccoli as a raw
material can provide a new way for the high value-added utilization
of broccoli. The invention selects different enzymes to hydrolyze
broccoli protein, and screens out the broccoli protein peptide
powder with high specificity, the powder has anti-oxidation and
cholesterol-lowering and lipid-lowering activities, and can be used
to develop health food with related functions. In addition, the
broccoli protein peptide that we developed can also be developed as
protein supplements or can also be used as raw materials for the
production of cosmetics, food additives, beverages and so on.
SUMMARY OF THE INVENTION
[0005] One object of the present invention is to provide a method
for preparing a broccoli protein peptide and use thereof.
[0006] In one aspect, the present invention provides a method for
preparing a broccoli protein peptide, comprising:
[0007] (a) protein preprocessing: water that is 4-8 times of weight
of broccoli protein is added to the raw material broccoli protein
to form a protein pulp, and anhydrous sodium sulfite with the
mass-volume ratio of 0.05-0.1 g/l and EDTA with the mass-volume
ratio of 0.02-0.05 g/l are further added to the protein pulp;
[0008] (b) proteolysis: neutral protease (200-600 units per gram of
raw material) is added to the pulp obtained in step (a), the
temperature of the solution is controlled at 50.+-.1.degree. C.,
then the pH of the resulting solution is adjusted to 6.0-7.0 with
NaOH, the time of enzymolysis is 3-4 hours;
[0009] (c) terminating the enzymolysis: the enzymolysis solution
obtained in step (b) is heated to 80-90.degree. C. for 5-15 minutes
to inactivate the enzyme, the solution is cooled to room
temperature;
[0010] (d) optionally, the liquid obtained in step (c) is
centrifuged or filtered;
[0011] (e) the liquid obtained in step (c) or (d) is filtered with
a membrane having a pore size of 100 to 500 nm;
[0012] (f) optionally, the filtrate obtained in step (e) is
subjected to a debittering treatment with activated carbon or
clay;
[0013] (g) the liquid obtained in step (e) or (f) is concentrated
and/or dried to obtain a broccoli protein peptide.
[0014] Wherein, anhydrous sodium sulfate is added in the step (a)
in order to break the disulfide bond in the protein, metal ions can
be well complexed by adding EDTA, thereby reducing the impact of
the metal ions on the protease activity in the subsequent
steps.
[0015] The broccoli protein peptide prepared by this method has a
good antioxidant effect, and its DPPH free radical scavenging rate
can reach 10% of that of the reduced glutathione (about the
reducing power of 10 mass % of GSH). In addition, the obtained
broccoli protein peptide prepared according to the method also
significantly reduces total cholesterol in a living organism and
therefore can be used in the fields of food, medicine, health care
products, or cosmetics and so on.
[0016] In another aspect, the present invention also provides
another method for preparing a broccoli protein peptide,
comprising:
[0017] (a) protein preprocessing: water that is 4-8 times of weight
of broccoli protein is added to the raw material broccoli protein
to form a protein pulp, and anhydrous sodium sulfite with the
mass-volume ratio of 0.05-0.1 g/l and EDTA with the mass-volume
ratio of 0.02-0.05 g/l are further added to the protein pulp;
[0018] (b) proteolysis: papain (1000-3000 units per gram of raw
material) is added to the pulp obtained in step (a), the
temperature of the solution is controlled at 50.+-.1.degree. C.,
then the pH of the resulting solution is adjusted to 6.5-7.0 with
NaOH, the time of enzymolysis is 3-4 hours;
[0019] (c) terminating the enzymolysis: the enzymolysis solution
obtained in step (b) is heated to 80-90.degree. C. for 5-15 minutes
to inactivate the enzyme, the solution is cooled to room
temperature;
[0020] (d) optionally, the liquid obtained in step (c) is
centrifuged or filtered;
[0021] (e) the liquid obtained in step (c) or (d) is filtered with
a membrane having a pore size of 100 to 500 nm;
[0022] (f) optionally, the filtrate obtained in step (e) is
subjected to a debittering treatment with activated carbon or
clay;
[0023] (g) the liquid obtained in step (e) or (f) is concentrated
and/or dried to obtain a broccoli protein peptide.
[0024] Wherein, anhydrous sodium sulfate is added in the step (a)
in order to break the disulfide bond in the protein, metal ions can
be well complexed by adding EDTA, thereby reducing the impact of
the metal ions on the protease activity in the subsequent
steps.
[0025] The broccoli protein peptide prepared by this method has a
good cholesterol lowering effect and a good blood lipid lowering
effect, therefore can be used in the fields of food, medicine,
health care products and so on.
[0026] In a third aspect, the present invention further provides a
method for the preparation of a broccoli protein peptide,
comprising:
[0027] (a) protein preprocessing: water that is 4-8 times of weight
of broccoli protein is added to the raw material broccoli protein
to form a protein pulp, and anhydrous sodium sulfite with the
mass-volume ratio of 0.05-0.1 g/l and EDTA with the mass-volume
ratio of 0.02-0.05 g/l are further added to the protein pulp;
[0028] (b) proteolysis: alkaline protease (200-600 units per gram
of raw material) is added to the pulp obtained in step (a), the
temperature of the solution is controlled at 50.+-.1.degree. C.,
then the pH of the resulting solution is adjusted to 9.0-9.5 with
NaOH, the time of enzymolysis is 3-4 hours;
[0029] (c) terminating the enzymolysis: the enzymolysis solution
obtained in step (b) is heated to 80-90.degree. C. for 5-15 minutes
to inactivate the enzyme, the solution is cooled to room
temperature;
[0030] (d) optionally, the liquid obtained in step (c) is
centrifuged or filtered;
[0031] (e) the liquid obtained in step (c) or (d) is filtered with
a membrane having a pore size of 100 to 500 nm;
[0032] (f) optionally, the filtrate obtained in step (e) is
subjected to a debittering treatment with activated carbon or
clay;
[0033] (g) the liquid obtained in step (e) or (f) is concentrated
and/or dried to obtain a broccoli protein peptide.
[0034] Wherein, anhydrous sodium sulfate is added in the step (a)
in order to break the disulfide bond in the protein, metal ions can
be well complexed by adding EDTA, thereby reducing the impact of
the metal ions on the protease activity in the subsequent
steps.
[0035] In a fourth aspect, the present invention further provides a
method for preparing a broccoli protein peptide, comprising:
[0036] (a) protein preprocessing: water that is 4-8 times of weight
of broccoli protein is added to the raw material broccoli protein
to form a protein pulp, and anhydrous sodium sulfite with the
mass-volume ratio of 0.05-0.1 g/l and EDTA with the mass-volume
ratio of 0.02-0.05 g/l are further added to the protein pulp;
[0037] (b) proteolysis: trypsin (5-15 units per gram of raw
material) is added to the pulp obtained in step (a), the
temperature of the solution is controlled at 50.+-.1.degree. C.,
then the pH of the resulting solution is adjusted to 8.0-8.5 with
NaOH, the time of enzymolysis is 3-4 hours;
[0038] (c) terminating the enzymolysis: the enzymolysis solution
obtained in step (b) is heated to 80-90.degree. C. for 5-15 minutes
to inactivate the enzyme, the solution is cooled to room
temperature;
[0039] (d) optionally, the liquid obtained in step (c) is
centrifuged or filtered;
[0040] (e) the liquid obtained in step (c) or (d) is filtered with
a membrane having a pore size of 100 to 500 nm;
[0041] (f) optionally, the filtrate obtained in step (e) is
subjected to a debittering treatment with activated carbon or
clay;
[0042] (g) the liquid obtained in step (e) or (f) is concentrated
and/or dried to obtain a broccoli protein peptide.
[0043] Wherein, anhydrous sodium sulfate is added in the step (a)
in order to break the disulfide bond in the protein, metal ions can
be well complexed by adding EDTA, thereby reducing the impact of
the metal ions on the protease activity in the subsequent
steps.
[0044] In a fifth aspect, the present invention further provides a
method for preparing a broccoli protein peptide, comprising:
[0045] (a) protein preprocessing: water that is 4-8 times of weight
of broccoli protein is added to the raw material broccoli protein
to form a protein pulp, and anhydrous sodium sulfite with the
mass-volume ratio of 0.05-0.1 g/l and EDTA with the mass-volume
ratio of 0.02-0.05 g/l are further added to the protein pulp;
[0046] (b) proteolysis: pepsin (5-15 units per gram of raw
material) is added to the pulp obtained in step (a), the
temperature of the solution is controlled at 36-38.degree. C., then
the pH of the resulting solution is adjusted to 1.5-2.5 with NaOH,
the time of enzymolysis is 3-4 hours;
[0047] (c) terminating the enzymolysis: the enzymolysis solution
obtained in step (b) is heated to 80-90.degree. C. for 5-15 minutes
to inactivate the enzyme, the solution is cooled to room
temperature;
[0048] (d) optionally, the liquid obtained in step (c) is
centrifuged or filtered;
[0049] (e) the liquid obtained in step (c) or (d) is filtered with
a membrane having a pore size of 100 to 500 nm;
[0050] (f) optionally, the filtrate obtained in step (e) is
subjected to a debittering treatment with activated carbon or
clay;
[0051] (g) the liquid obtained in step (e) or (f) is concentrated
and/or dried to obtain a broccoli protein peptide.
[0052] Wherein, anhydrous sodium sulfate is added in the step (a)
in order to break the disulfide bond in the protein, complex metal
ions can be well complexed by adding EDTA, thereby reducing the
effect of the metal ions on the protease activity in the subsequent
steps.
[0053] In a sixth aspect, the present invention further provides a
method for preparing a broccoli protein peptide, comprising:
[0054] (a) protein preprocessing: water that is 4-8 times of weight
of broccoli protein is added to the raw material broccoli protein
to form a protein pulp, and anhydrous sodium sulfite with the
mass-volume ratio of 0.05-0.1 g/l and EDTA with the mass-volume
ratio of 0.02-0.05 g/l are further added to the protein pulp;
[0055] (b) proteolysis: bromelain (1000-5000 units per gram of raw
material) is added to the pulp obtained in step (a), the
temperature of the solution is controlled at 40.+-.1.degree. C.,
then the pH of the resulting solution is adjusted to 6.0-7.0 with
NaOH, the time of enzymolysis is 3-4 hours;
[0056] (c) terminating the enzymolysis: the enzymolysis solution
obtained in step (b) is heated to 80-90.degree. C. for 5-15 minutes
to inactivate the enzyme, the solution is cooled to room
temperature;
[0057] (d) optionally, the liquid obtained in step (c) is
centrifuged or filtered;
[0058] (e) the liquid obtained in step (c) or (d) is filtered with
a membrane having a pore size of 100 to 500 nm;
[0059] (f) optionally, the filtrate obtained in step (e) is
subjected to a debittering treatment with activated carbon or
clay;
[0060] (g) the liquid obtained in step (e) or (f) is concentrated
and/or dried to obtain a broccoli protein peptide.
[0061] Wherein, anhydrous sodium sulfate is added in the step (a)
in order to break the disulfide bond in the protein, complex metal
ions can be well complexed by adding EDTA, thereby reducing the
impact of the metal ions on the protease activity in the subsequent
steps.
[0062] In a seventh aspect, the present invention further provides
a method for preparing a broccoli protein peptide, comprising:
[0063] (a) protein preprocessing: water that is 4-8 times of weight
of broccoli protein is added to the raw material broccoli protein
to form a protein pulp, and anhydrous sodium sulfite with the
mass-volume ratio of 0.05-0.1 g/l and EDTA with the mass-volume
ratio of 0.02-0.05 g/l are further added to the protein pulp;
[0064] (b) proteolysis: compound protease (5-3600 units per gram of
raw material) is added to the pulp obtained in step (a), the
temperature of the solution is controlled at 50.+-.1.degree. C.,
then the pH of the solution is controlled at 8.0-8.5 within 0.5-1
hour, then the pH is no longer controlled, the time of enzymolysis
is 3-4 hours;
[0065] (c) terminating the enzymolysis: the enzymolysis solution
obtained in step (b) is heated to 80-90.degree. C. for 5-15 minutes
to inactivate the enzyme, the solution is cooled to room
temperature;
[0066] (d) optionally, the liquid obtained in step (c) is
centrifuged or filtered;
[0067] (e) the liquid obtained in step (c) or (d) is filtered with
a membrane having a pore size of 100 to 500 nm;
[0068] (f) optionally, the filtrate obtained in step (e) is
subjected to a debittering treatment with activated carbon or
clay;
[0069] (g) the liquid obtained in step (e) or (f) is concentrated
and/or dried to obtain a broccoli protein peptide.
[0070] Wherein, anhydrous sodium sulfate is added in the step (a)
in order to break the disulfide bond in the protein, metal ions can
be well complexed by adding EDTA, thereby reducing the effect of
the metal ions on the protease activity in the subsequent
steps.
[0071] Wherein the compound protease is selected from trypsin and
neutral protease, or alkaline protease and papain, or alkaline
protease and neutral protease.
[0072] The broccoli protein peptide prepared by this method (for
example, using a compound protease of trypsin and neutral protease)
has a good antioxidant effect, and its DPPH free radical scavenging
rate can reach 10% of reduced glutathione (about the reducing power
of 10 mass % of the GSH), therefore it can be used in the fields of
food, medicine, health care products or cosmetics and so on.
[0073] In the method of the present invention, the "raw material",
"broccoli protein" and "raw material broccoli protein" have the
same meaning.
[0074] In an eighth aspect, the present invention provides a
broccoli protein peptide prepared by the methods for preparing a
broccoli protein peptide according to the present invention.
[0075] The broccoli protein peptide prepared by the method
described in the present invention has the following
advantages:
[0076] (1) The broccoli protein peptide prepared in the present
invention has high purity, and the content of TCA (trichloroacetic
acid) acid soluble protein of the broccoli protein peptide is more
than 90%, the molecular weight distribution less than 10000 is
above 90%, and the free amino acid represents 5-8%.
[0077] (2) The technical solution of the present invention is
stable, the mass parameters such as the content of acid soluble
protein, free amino acid and molecular weight distribution of the
prepared broccoli protein peptide do not change significantly, thus
suitable for industrial production.
[0078] The present invention will be further described by the
following examples. It should be noted that these examples are
provided for illustrating the present invention but not
limiting.
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.degree. 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.degree. 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.degree. 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 2709 0.03 g of 0.03 g of 0.03 g of 1389 0.05 g
of 0.1 g of alkaline trypsin papain neutral pepsin bromelain
protease (4000 U/g) (800,000 U/g) protease (200,000 U/g) (3000 U/g)
(500,000 U/g) (200,000 U/g) pH value 9.5 8.5 7.0 7.0 2.0 7.0
Temperature 50.degree. C. 50.degree. C. 50.degree. C. 50.degree. C.
37.degree. C. 40.degree. 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, B, C, D, E, F that had been subjected to
enzymolysis were heated to 80.degree. 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.s-A.sub.c)/A.sub.0.times.100%
[0100] In the formula, A.sub.0--the absorbance value of 1.0 mL
distilled water+3.0 mL DPPH solution [0101] A.sub.s--the absorbance
value of 1.0 mL sample solution+3.0 mL DPPH solution [0102]
A.sub.c--the 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
concentration Scavenging rate 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 peptide 86.7%
72.3% 15.4% 1.1% Pepsin-digested peptide 78.3% 68.4% 13.4% 3.6%
Neutral protease and 91.9% 79.2% 23.3% 8.3% trypsin-digested
peptide Soybean protein peptide 75.6% 26.8% 10.2% 6.3% 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.degree. 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.degree. C. to 8.degree. 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; Total
cholesterol (TC), batch number: 20140725; [0115] Low density
lipoprotein (LDL-C), provided by Shanghai Kehua Bioengineering Co.,
Ltd.
[0116] (1.3) Instrument
[0117] Xi Sen Mei Kang automatic biochemical analyzer, model
CHEX-180.
[0118] (2.) Experimental Methods and Results
[0119] (2.1) Method
[0120] 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. Number Admin- Volume of
Dose of istration admin- Group (g kg.sup.-1) animals route
istration Model -- 8 ig (gavage) 10 ml/kg Neutral protease-digested
0.5 8 ig (gavage) 10 ml/kg 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 peptide 0.5 8 ig (gavage) 10 ml/kg Pepsin-digested
peptide 0.5 8 ig (gavage) 10 ml/kg Trypsin-digested peptide 0.5 8
ig (gavage) 10 ml/kg Alkaline protease- 0.5 8 ig (gavage) 10 ml/kg
digested peptide
[0121] 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.
[0122] (2.2) Data Processing:
[0123] The results of all the experiments were expressed with,
x.+-.s, the comparison between any two groups used t-test.
P<0.05 was statistically significant.
[0124] (2.3) Results
[0125] 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 kg.sup.-1) TC
LDL-C Model group -- 10.54 .+-. 1.21 2.93 .+-. 0.75 Neutral
protease-digested 0.5 8.79 .+-. 1.46** 2.40 .+-. 0.52 peptide group
Alkaline protease and 0.5 9.60 .+-. 1.18 2.33 .+-. 0.33* neutral
protease compound enzyme digested peptide group Bromelain-digested
0.5 10.06 .+-. 1.75 3.05 .+-. 0.93 peptide group Papain-digested
peptide 0.5 8.83 .+-. 1.42** 2.20 .+-. 0.41** group Pepsin-digested
peptide 0.5 10.46 .+-. 1.54 3.01 .+-. 0.68 group Trypsin-digested
peptide 0.5 11.97 .+-. 1.81 3.78 .+-. 0.70 group Alkaline
protease-digested 0.5 10.37 .+-. 1.37 2.73 .+-. 0.32 peptide group
Compared with the model group, *P < 0.05, **P < 0.01.
* * * * *