CN108541804A - A method of preparing goldbeater's skin nutrition peptide microballoon using hydrolyzed wheat protein - Google Patents
A method of preparing goldbeater's skin nutrition peptide microballoon using hydrolyzed wheat protein Download PDFInfo
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- CN108541804A CN108541804A CN201810227811.3A CN201810227811A CN108541804A CN 108541804 A CN108541804 A CN 108541804A CN 201810227811 A CN201810227811 A CN 201810227811A CN 108541804 A CN108541804 A CN 108541804A
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- peptide
- goldbeater
- microballoon
- glutamine
- hydrolyzed wheat
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
Abstract
The present invention provides a kind of methods preparing goldbeater's skin nutrition peptide microballoon using hydrolyzed wheat protein, belong to hydrolyzed wheat protein deep process technology field.This method includes:(1) configuration hydrolyzed wheat protein feed liquid (2) selects Protamex compound proteases, Alcalase alkali protease stepwise discretizations, centrifugal ultrafiltration to obtain small molecule target peptide;(3) small molecule target peptide is dried to obtain wheat glutamine peptide;(4) goldbeater's skin nutrition peptide microballoon is made by sodium alginate Chitosan-coated in wheat glutamine peptide.The goldbeater's skin nutrition peptide microballoon of the present invention reduces the absorption rate that glutamine enters body, to play more preferably enteral nutrition effect by controlling glutamine from the rate of release in the microballoon.The goldbeater's skin nutrition peptide microballoon of the present invention can improve animal mucosal structure, improve digestive organs function, increase the intestinal villus length of ileum, duodenum etc., improve intestinal environment, promote the growth and development of intestinal villus, increase absorption of the enteron aisle to nutriment.
Description
Technical field
The invention belongs to hydrolyzed wheat protein deep process technology field, it is related to a kind of preparing goldbeater's skin using hydrolyzed wheat protein
The method of nutrition peptide microballoon.
Background technology
Glutamine is one kind " conditionally essential amino acid " under human body stress situation, is that intestinal mucosa cell metabolism is required
Nutriment, for maintain intestinal mucosal epithelial structural intergrity it is particularly significant.It is especially tight in fatigue, infection, wound etc.
Under weight stress situation, the intracellular glutamine of intestinal mucosal epithelial is exhausted quickly.When enteron aisle lacks glutamine or food, digestion
When the stimulations such as liquid, atrophy can occur for intestinal mucosa, and villus is thinning, shortens or even falls off, intestinal immunity is badly damaged.It is clinical
It was verified that parenteral offer glutamine can effectively prevent intestinal mucosa atrophy, enhancing intestinal cell activity to improve intestine immunity
Function maintains normal bowel mucous membrane weight, structure and protein content, reduces enteric bacteria and endotoxin translocation.
China is Wheat Production and consumption big country.Currently, wheat processing by-product-hydrolyzed wheat protein (being commonly called as Gluten)
Yield at 150,000 tons or so, account for 30% of world wide production or more.As a kind of unique albumen, hydrolyzed wheat protein is tight
Close tridimensional network and amino acid combination causes its dissolubility extremely low, limits the performance of functional characteristic.It is worth note
Meaning, hydrolyzed wheat protein amino acid composition Glutamic Acid content are up to 35% or more, and most of in the form of glutamine
In the presence of so that glutamine peptide is prepared by biological enzymolysis technology as raw material using hydrolyzed wheat protein and is possibly realized, is also wheat
Protein hydrolysate higher value application provides new thinking.
Modern times digestion theory thinks that oligopeptide of the molecular weight less than 1000Da is easy to directly be digested and assimilated by body, has and turns
Running speed degree is fast, and consume energy low feature, can especially eliminate the absorption competition with free amino acid, greatly improve the suction of protein
Receive utilization rate.But when there are the glutamine peptide of high concentration, some glutamine peptide is unable to fully utilize, the present invention
Using hydrolyzed wheat protein as raw material, organically combined with microsphere preparation technology using hydrolyzed wheat protein enzymolysis, to realize height
The oligomeric wheat peptide of glutamine and goldbeater's skin nutrition peptide microballoon are effectively prepared, and are confirmed through animal experiment, compared to high glutamine
Oligomeric wheat peptide, the goldbeater's skin nutrition peptide microballoon have enteral nutrition effect outstanding.The present invention is for widening hydrolyzed wheat protein
Application range improves hydrolyzed wheat protein added value and has important practical significance.
Invention content
The present invention provides a kind of preparation methods of the wheat glutamine peptide microballoon with enteral nutrition.
The present invention is realized by following technical proposal:
A method of goldbeater's skin nutrition peptide microballoon being prepared using hydrolyzed wheat protein, is included the following steps:
(1) hydrolyzed wheat protein feed liquid is configured
Hydrolyzed wheat protein raw material is added water into a concentration of 5%-10% of albumen quality, adjustment suspension temperature is 60 DEG C,
And using the method for magnetic agitation, 30-45min is persistently stirred, and unit for uniform suspension is formed, it is spare;
(2) stepwise discretization
The pH value of suspension is 7.0 in regulating step (1), maintains temperature 50 C, addition suspension quality 1.5-2.5%'s
After Protamex compound proteases carry out enzymolysis 1h, by reaction solution, enzyme deactivation work 15min, adjusting pH value are under 95 DEG C of water bath conditions
8.5, the Alcalase alkali proteases of temperature 60 C, addition reaction solution quality 1-2% carry out enzymolysis 1.5h, by reaction solution 95
After 15min is lived in enzyme deactivation under DEG C water bath condition, ultrafiltration is carried out by the ultrafiltration membrane of molecule interception 3kDa-10kDa, filters out small molecule
Target peptide, and collect the macromolecule substrate of retention;The macromolecule substrate being collected into is repeated into enzymolysis process 1-2 times, until precipitation
Middle protein content is less than 5%;
(3) small molecule target peptide is dried
The small molecule target peptide that will be filtered out in step (2), it is freeze-dried to remove moisture removal, wheat glutamine peptide is made
Powder;
(4) prepared by goldbeater's skin nutrition peptide microballoon
Seaweed is added in wheat glutamine peptide powder in step (3) by the sodium alginate aqueous solution of configuration quality concentration 2%
In acid sodium aqueous solution, after stirring evenly, 1% chitosan solution of mass concentration and 2% calcium chloride water of mass concentration is added, mixes
After even, 1-2h is incubated, freeze-dried or spray drying process removes moisture removal, and wheat glutamine peptide microballoon is made, i.e. goldbeater's skin is sought
Support peptide microballoon.
In above-mentioned technical proposal, the raw material of the hydrolyzed wheat protein powder is Gluten or is prepared by wheat flour extraction small
Wheat protein hydrolysate, protein content >=80%.
In above-mentioned technical proposal, sodium alginate and wheat glutamine peptide powder mass ratio are 1 in step (4):2-3.
The beneficial effects of the present invention are:
(1) the hydrolyzed wheat protein powder used in the present invention is provided by Wuhan Ke Tai biochemical technologies limited liability company,
It is that a kind of protein content is high, the hydrolyzed wheat protein powder of good water solubility avoids hydrolyzed wheat protein powder and meets water conglomeration, enzymolysis
The problem of inefficiency.
(2) when there are the glutamine peptide of high concentration, some glutamine peptide is unable to fully be absorbed and utilized, this hair
Bright goldbeater's skin nutrition peptide microballoon reduces glutamine and enters body by controlling glutamine from the rate of release in the microballoon
Absorption rate, to play more preferably enteral nutrition effect.The present invention hydrolyzes egg using hydrolyzed wheat protein as raw material, using wheat
White enzymolysis is organically combined with microsphere preparation technology, is effectively made to realize wheat glutamine peptide and goldbeater's skin nutrition peptide microballoon
It is standby, it is confirmed through animal experiment, compared to the oligomeric wheat peptide of high glutamine, which seeks with enteron aisle outstanding
Support effect.
(3) in the present invention, goldbeater's skin nutrition peptide microballoon decomposites glutamine and chitosan under hydrochloric acid in gastric juice effect, chitosan into
After entering enteron aisle, the profitable strain that can be proliferated in enteron aisle inhibits pernicious bacteria, promotes intestines peristalsis, removes the stool of big enteral
And toxin, adjust function of intestinal canal.Chitosan and glutamine can improve animal mucosal structure simultaneously, improve digestive organs work(
Can, increase the intestinal villus length of ileum, duodenum etc., improve intestinal environment, promote the growth and development of intestinal villus, increases intestines
Absorption of the road to nutriment.
(4) goldbeater's skin nutrition peptide microballoon produced by the present invention have goldbeater's skin nutritive effect outstanding, can be used as clinical application or
The dispensing of food, health products, feed has widened hydrolyzed wheat protein application range, for improving hydrolyzed wheat protein added value
It has important practical significance.
Description of the drawings
Fig. 1 goldbeater's skin nutrition peptide microballoon In-vitro release curves.
Specific implementation mode
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail:
Experimental method in following embodiments is unless otherwise specified conventional method.Original as used in the following examples
Material, reagent etc. are commercially available products unless otherwise specified.The hydrolyzed wheat protein powder that the present invention uses, by Thailand of Wuhan section
Biochemical technology limited liability company provides, and the hydrolyzed wheat protein powder of Wuhan Chortle Bio-chem. Technology Co., Ltd.'s production is with flour
For raw material extraction Soluble plant (wheat) albumen, be one kind with water existing for polypeptide, small molecular protein and amino acid form
Dissolubility wheat gluten is a kind of with high protein content (80% or more), and solubility height (95% or more) is simultaneously containing very high paddy ammonia
The protein of amide (30% or so) and high sanitary standard (food-grade), low ash content (being less than 1.2%).
Compound protease Protamex (1.5Au/g) used in the present invention is provided by Novozymes Company of Denmark, basic protein
Enzyme (Alcalase 2.4L) is provided by Novozymes Company of Denmark.
Embodiment 1:Preparation prepared by goldbeater's skin nutrition peptide microballoon
1, a kind of method preparing goldbeater's skin nutrition peptide microballoon using hydrolyzed wheat protein, includes the following steps:
(1) hydrolyzed wheat protein feed liquid is configured
10g hydrolyzed wheat protein raw materials are added water into a concentration of 5%-10% of albumen quality, adjustment suspension temperature is 60
DEG C, and using the method for magnetic agitation, 30-45min is persistently stirred, and unit for uniform suspension is formed, it is spare;
(2) stepwise discretization
The pH value of suspension is 7.0 ± 0.5 in regulating step (1), maintains 50 ± 2 DEG C of temperature, adds suspension quality
After the Protamex compound proteases of 1.5-2.5% carry out enzymolysis 1h, by reaction solution, enzyme deactivation is lived under 95 DEG C of water bath conditions
15min, it is 8.5 to adjust pH value, temperature 60 C, and the Alcalase alkali proteases of addition reaction solution quality 1-2% are digested
1.5h, by reaction solution, enzyme deactivation is lived after 15min under 95 DEG C of water bath conditions, by the ultrafiltration membrane of molecule interception 3kDa-10kDa into
Row ultrafiltration filters out small molecule target peptide, and collects the macromolecule substrate of retention;The macromolecule substrate being collected into is repeated to digest
Journey 1-2 times, until protein content is less than 5% in precipitation;
(3) small molecule target peptide is dried
The small molecule target peptide that will be filtered out in step (2), it is freeze-dried to remove moisture removal, obtain 5.8g wheat glutamy
Amine Gly-His-Lys;
(4) prepared by goldbeater's skin nutrition peptide microballoon
Sodium alginate is added in small molecule target peptide in step (3) by the sodium alginate aqueous solution of configuration quality concentration 2%
In aqueous solution, wherein sodium alginate and wheat glutamine peptide powder mass ratio are 1:After stirring evenly, mass concentration is added in 2-3
2% calcium chloride water of 1% chitosan solution and mass concentration after mixing, incubates 1-2h, and spray drying process removes moisture removal, makes
Obtain 2.1g wheat glutamine peptide microballoons, i.e. goldbeater's skin nutrition peptide microballoon.
Further, hydrolyzed wheat protein raw material is Gluten or extracts the hydrolyzed wheat protein prepared by wheat flour,
Quality index meets albumen (butt) >=80%, ash content (butt)≤2.0, crude fat (butt)≤2.0, and starch (butt)≤
15%.
In the present invention, goldbeater's skin nutrition peptide microballoon decomposites glutamine and chitosan under hydrochloric acid in gastric juice effect, and chitosan enters
After enteron aisle, the profitable strain that can be proliferated in enteron aisle, inhibit pernicious bacteria, promote intestines peristalsis, remove big enteral stool and
Toxin adjusts function of intestinal canal.Chitosan and glutamine can improve animal mucosal structure simultaneously, improve digestive organs function,
Increase the intestinal villus length of ileum, duodenum etc., improve intestinal environment, promote the growth and development of intestinal villus, increases enteron aisle
Absorption to nutriment.
2, goldbeater's skin nutrition peptide microsphere encapsulation rate and release performance test
Granulometry is carried out to goldbeater's skin nutrition peptide microballoon with the laser granulometry of Zetasizer Nano ZS90, it is few
Amount sample (about 10mg), which is dissolved in 10ml deionized waters, to be measured, and ultrasonic disperse 5min measures the average grain diameter of powder 1.5
±0.2um。
Goldbeater's skin nutrition peptide microsphere encapsulation rate and carrying drug ratio:0.8g goldbeater's skin nutrition peptide microballoons are taken to be placed in 200mL0.5mol/L's
60 DEG C of reflux 8h of HCl solution, wait for that microballoon is completely dissolved, and after 3000 times of dilutions, are sought to goldbeater's skin using high performance gel filtration chromatography
The content for supporting peptide microballoon glutamine is detected;Simultaneously by wheat glutamine peptide powder 3 × 106After diluting again, using height
Effect gel filtration chromatography is detected the content of the glutamine in wheat glutamine peptide powder;Goldbeater's skin nutrition peptide microballoon
Encapsulation rate (%)=(quality of the quality of microballoon glutamine/input wheat glutamine peptide glutamine) × 100%;
The drugloading rate (%) of goldbeater's skin nutrition peptide microballoon=(quality of the quality of microballoon glutamine/goldbeater's skin nutrition peptide microballoon) ×
100%, it is shown in Table 1.
The performance of 1 goldbeater's skin nutrition peptide microballoon of table
Goldbeater's skin nutrition peptide microballoon gross mass | 2.10 |
Microvesicle glutamine content (ugmL-1) | 0.94 |
Total quality containing glutamine (g) of microvesicle | 1.48 |
Drugloading rate (%) | 70.5 |
Wheat glutamine peptide glutamine content (ugmL-1) | 1.14 |
Wheat glutamine peptide glutamine gross mass (g) | 2.28g |
Encapsulation rate (%) | 64.9 |
Wheat glutamine peptide glutamine ratio (%) | 39.3 |
Goldbeater's skin nutrition peptide microballoon release in vitro:Goldbeater's skin nutrition peptide microballoon is put into bag filter, it is molten that phosphate-buffered is added
Liquid (PBS, pH=6.5), is clamped with dialysis clamp, is placed in PBS, while 37 DEG C of temperature controlled water baths.Respectively 1,2,4,6,8,
10,12h is sampled from external solution, while supplementing PBS.Using high performance gel filtration chromatography to goldbeater's skin nutrition peptide microsphere outside
Release different periods (1,2,4,6,8,10,12h) content of external solution glutamine is detected the content of glutamine
It being detected, and calculates glutamine Cumulative release amount and cumulative percentage release, result of calculation is shown in Table 2, using the time as horizontal axis,
Glutamine cumulative percentage release is mapped, as shown in Figure 1.Calculation formula is:The paddy ammonia at cumulative percentage release=certain moment
Glutamine amount × 100% for amide Cumulative release amount/always contain, is shown in Table 2.
2 goldbeater's skin nutrition peptide microballoon release in vitro of table
As shown in Table 2, microballoon discharges 76.6% within 12h, has good slow release effect, can be by controlling glutamy
Amine reduces the absorption rate that glutamine enters body, to play more preferably enteral nutrition from the rate of release in the microballoon
Effect.
The content of glutamine is detected using high performance gel filtration chromatography in the present embodiment, chromatographic condition is such as
Under:HLPC chromatograph models:LC-20A;Chromatographic column:NH 4.6 × 250mm of ¢, chromatographic column number:312316;Mobile phase:Acetonitrile/phosphorus
Acid dihydride potassium=30/70 (V/V), wherein a concentration of 0.05mol/L of potassium dihydrogen phosphate:6.8g potassium dihydrogen phosphates are taken, are added water to
1000mL is 4.0 with phosphoric acid regulating ph value;Detection wavelength:UV215nm;Flow velocity:1.0ml/min;Column temperature:35℃.
Embodiment 2:Effect study of the goldbeater's skin nutrition peptide microballoon to young baby's goldbeater's skin
Experiment packet:A groups:Hydrolyzed wheat protein powder;B groups:(small molecular target peptide of the present invention is through cold for small molecule target peptide
Dry or spray drying process is lyophilized and removes moisture removal, small molecule target Gly-His-Lys are made);C groups:Goldbeater's skin nutrition peptide microballoon;D groups:0.9% life
Manage brine
(1) Effect study of the goldbeater's skin nutrition peptide microballoon to piglet goldbeater's skin
Piglet similar in 12 30 ages in days, weight is taken, 4 groups are equally divided into.Experiment sets A groups:Feed hydrolyzed wheat protein
Powder (3g/kg/d);B groups:Feed wheat glutamine peptide powder (3g/kg/d);C groups:Feed goldbeater's skin nutrition peptide microballoon (3g/kg/
d);D groups:0.9% physiological saline 3ml is fed, since the same day, each group removes basal feed feeding, and arbitrarily taking the photograph water, normally feeding is outer,
The empirically 1 time/d of dosage feeding of each group design.After carrying out feeding in one week, piglet sacrificed by exsanguination, surgical method will be tested
Abdominal cavity is opened, Duodenal Tissues are taken out, after normal saline flushing is clean, is put into 10% neutral formalin fixed, conventional dehydration,
Paraffin embedding, the variation of light microscopic observation Duodenal Mucosa structure, the results are shown in Table 3 after slice HE dyeing.
The variation of 3 piglet duodenal mucosa structure of table
As shown in Table 3, compared to the normal feeding of D groups, A groups, B groups and C groups have rush to the Duodenal Mucosa of piglet
It is acted on into growth and development, compared to A group hydrolyzed wheat proteins powder, B group wheat glutamine peptide powder, C group goldbeater's skin nutrition peptide microballoons
It is stronger to the facilitation of puppy intestinal mucosa growth and development, intestinal villus length and Crypt depth shoals, intestinal villus length/hidden
Nest depth ratio becomes larger, so that the production performance of piglet increases.
(2) Effect study of the goldbeater's skin nutrition peptide microballoon to puppy goldbeater's skin
24 weight 3.0 ± 0.5kg puppies are taken, 4 groups are equally divided into.Experiment sets A groups:Feed hydrolyzed wheat protein powder
(2g/kg/d);B groups:Feed wheat glutamine peptide powder (2g/kg/d);C groups:Feed goldbeater's skin nutrition peptide microballoon (2g/kg/d);D
Group:0.9% physiological saline 2ml is fed, since the same day, each group removes basal feed feeding, and arbitrarily taking the photograph water, normally feeding is outer, by real
Test the 1 time/d of dosage feeding of each group design.After carrying out feeding in one week, by Experimental puppies sacrificed by exsanguination, surgical method is opened
Duodenum, ileal tissue are taken out in abdominal cavity, after normal saline flushing is clean, is put into 10% neutral formalin and fixes, conventional de-
Water, paraffin embedding, the small intestinal villous height of light microscopic observation duodenum and ileum, the results are shown in Table 4 after slice HE dyeing.
The villi height of table 4 puppy duodenum and ileum
Duodenal intestinal villus length/um | Intestinal villus length/um of ileum | |
A groups | 557.9±4.3 | 427.6±4.3 |
B groups | 587.5±3.5 | 496.5±3.5 |
C groups | 645.7±2.4 | 539.4±2.4 |
D groups | 443.7±9.1 | 355.1±9.1 |
As shown in Table 4, compared to the normal feeding of D groups, A groups, B groups and C groups have the duodenum of puppy and ileum
Enhancing development acts on, and compared to A group hydrolyzed wheat proteins powder, B group wheat glutamine peptide powder, C group goldbeater's skin nutrition peptides are micro-
Ball is stronger to the facilitation of puppy intestinal mucosa growth and development, and in addition intestinal villus length is more uniform in C groups, the digestion of intestinal mucosa
Function is more sound.
The study show that there are significant promotion intestines to young baby according to the goldbeater's skin nutrition peptide microballoon prepared by the inventive method
The effect of mucous membrane growth and development has ideal enteral nutrition effect.
Goldbeater's skin nutrition peptide microballoon produced by the present invention has goldbeater's skin nutritive effect outstanding, can be used as clinical application or food
The dispensing of product, health products, feed has widened hydrolyzed wheat protein application range, for improving hydrolyzed wheat protein added value tool
There is important realistic meaning.
Finally illustrate, the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to compared with
Good embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the right of invention.
Claims (3)
1. a kind of method preparing goldbeater's skin nutrition peptide microballoon using hydrolyzed wheat protein, it is characterised in that:Include the following steps:
(1) hydrolyzed wheat protein feed liquid is configured
Hydrolyzed wheat protein raw material is added water into a concentration of 5%-10% of albumen quality, adjustment suspension temperature is 60 DEG C, and is adopted
With the method for magnetic agitation, 30-45min is persistently stirred, forms unit for uniform suspension, it is spare;
(2) stepwise discretization
The pH value of suspension is 7.0 in regulating step (1), maintains temperature 50 C, addition suspension quality 1.5-2.5%'s
After Protamex compound proteases carry out enzymolysis 1h, by reaction solution, enzyme deactivation work 15min, adjusting pH value are under 95 DEG C of water bath conditions
8.5, the Alcalase alkali proteases of temperature 60 C, addition reaction solution quality 1-2% carry out enzymolysis 1.5h, by reaction solution 95
After 15min is lived in enzyme deactivation under DEG C water bath condition, ultrafiltration is carried out by the ultrafiltration membrane of molecule interception 3kDa-10kDa, filters out small molecule
Target peptide, and collect the macromolecule substrate of retention;The macromolecule substrate being collected into is repeated into enzymolysis process 1-2 times, until precipitation
Middle protein content is less than 5%;
(3) small molecule target peptide is dried
The small molecule target peptide that will be filtered out in step (2), it is freeze-dried to remove moisture removal, wheat glutamine peptide powder is made;
(4) prepared by goldbeater's skin nutrition peptide microballoon
Sodium alginate is added in wheat glutamine peptide powder in step (3) by the sodium alginate aqueous solution of configuration quality concentration 2%
In aqueous solution, after stirring evenly, 1% chitosan solution of mass concentration and 2% calcium chloride water of mass concentration, mixing is added
Afterwards, 1-2h is incubated, freeze-dried or spray drying process removes moisture removal, wheat glutamine peptide microballoon, i.e. goldbeater's skin nutrition is made
Peptide microballoon.
2. the method according to claim 1 for preparing goldbeater's skin nutrition peptide microballoon using hydrolyzed wheat protein, which is characterized in that
The raw material of the hydrolyzed wheat protein powder is Gluten or the hydrolyzed wheat protein by wheat flour extraction preparation, protein content
>=80%.
3. the method according to claim 1 for preparing goldbeater's skin nutrition peptide microballoon using hydrolyzed wheat protein, which is characterized in that
Sodium alginate and wheat glutamine peptide powder mass ratio are 1 in step (4):2-3.
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Application publication date: 20180918 |