CN107177654A - A kind of leech polypeptide powder and application - Google Patents
A kind of leech polypeptide powder and application Download PDFInfo
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- CN107177654A CN107177654A CN201710412958.5A CN201710412958A CN107177654A CN 107177654 A CN107177654 A CN 107177654A CN 201710412958 A CN201710412958 A CN 201710412958A CN 107177654 A CN107177654 A CN 107177654A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/26—Iron; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/30—Zinc; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/42—Phosphorus; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
- C12G3/00—Preparation of other alcoholic beverages
- C12G3/04—Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
The invention discloses a kind of leech polypeptide powder, prepared by following steps：Pretreatment：Natural leech is taken, sodium chloride solution immersion is added；Defibrination：Homogenate, filters off impurity, obtains slurries；It is modified：Slurries are handled with reference to microwave, modified solution is obtained；Hydrolysis：Papain is added in modified solution, regulation pH is 4.5 6, modified solution is hydrolyzed processing with reference to microwave, be then cooled to 4~10 DEG C, constant temperature is placed；Handled with reference to microwave, stop reaction, obtain hydrating solution；Inactivation：By hydrating solution heat inactivation；Filtering：Filter to take filtrate；Dry：Filtrate is dried, leech polypeptide powder is obtained；The leech polypeptide powder is obtained in the way of microwave radiation technology combined filtering, the efficient composition in the high income of polypeptide, and the polypeptide that can be obtained；Invention additionally discloses the application of leech polypeptide powder, for leech polypeptide powder is used to prepare wine；The wine is easy to absorb, the effect with lipid-loweringing.
Description
Technical field
The present invention relates to a kind of leech polypeptide powder and application, belong to extractive technique field.
Background technology
Leech is with a long history as traditional Chinese medicine simply, the traditional Chinese medical science think leech have blood-breaking, by becoming silted up, stimulating the menstrual flow the effects such as.Through grinding
Study carefully and show, Chinese traditional medicine leech can be such that 6- ketone-PGF12 in blood in human body in substantially increases, and TXA2 is decreased obviously, there is significant drop blood
Fat is acted on, and Aorta atheromatous plague is substantially declined, visible collagen fiber hyperplasia in patch, and cholesterol crystal is reduced.
Atherosclerosis, a variety of cardiovascular and cerebrovascular diseases of preventing and treating, which are prevented and treated, for Chinese traditional medicine leech provides certain theoretical foundation.Also send out simultaneously
Now thrombophlebitis, hepatic sclerosis, respiratory disease are treated with Chinese traditional medicine leech all achieve satisfied effect.
So people gradually extract for leech to utility therein, the thing with anticoagulation is isolated
After matter-hirudin, extensive and deep pharmaceutical research has been carried out to it both at home and abroad.Hirudin (hirudin) is leech
(Leech) the most notable and most studied a kind of composition of activity in various active composition and its in salivary gland has been extracted,
The small protein (polypeptide) that it is made up of 65-66 amino acid.Hirudin has extremely strong inhibitory action to fibrin ferment,
It is to find the natural specific inhibitor of most strong fibrin ferment so far.
The content of the invention
In order to overcome the deficiencies in the prior art, first purpose of the invention is to provide a kind of leech polypeptide powder,
The leech polypeptide powder is obtained in the way of microwave radiation technology combined filtering, the high income of polypeptide, and the polypeptide that can be obtained
In efficient composition, the effect with logical blood vessel, reducing blood lipid, and be easier to be absorbed by the body.
Second object of the present invention is to provide a kind of application of leech polypeptide powder, for by above-mentioned leech albumen
Polypeptide powder is used to prepare wine, and leech polypeptide powder auxiliary improves the palatability of wine, and the wine is easy to absorb, with low antigenicity,
Effect with lipid-loweringing.
Realizing first purpose of the present invention can be reached by adopting the following technical scheme that：A kind of leech polypeptide
Powder, is prepared by following steps：
Pretreatment：Natural leech is taken, sodium chloride solution immersion 5-15min is added；Then sodium chloride solution is removed；
Defibrination：Pure water is added in pretreated natural leech and pH is adjusted to 5.5-6.5；Homogenate, filters off impurity, obtains
To slurries；
It is modified：Under conditions of temperature is 65~85 DEG C, processing 5-10min, then 60 DEG C are carried out to slurries with reference to microwave
Under conditions of be incubated 20-40min, obtain modified solution；
Hydrolysis：Papain is added in modified solution, regulation pH is 4.5-6, is 8000-9500MHz with reference to frequency
Microwave modified solution is hydrolyzed processing 10-20min, be then cooled to 4~10 DEG C, constant temperature places 10-20min；With reference to
Frequency is that 4000-6000MHz microwaves carry out processing 5-15min, stops reaction, obtains hydrating solution；
Inactivation：Hydrating solution is heated to 95~115 DEG C, 15~25min is handled, obtains inactivating hydrolyzate；
Filtering：Inactivation hydrolyzate is cooled to 20~30 DEG C, ultra filtration leaching filtrate；
Dry：Filtrate is dried, leech polypeptide powder is obtained.
Further, in pre-treatment step, the concentration of sodium chloride solution is 11-20g/L.
Further, in refining step, pH is adjusted by acetum.
Further, in modification procedure, the frequency of microwave is 0.7-1.2GHz.
Further, in hydrolysing step, the addition of papain is 6000-18000U/g modified solutions.
Further, in filtration step, filtrate is taken using the ultrafiltration membrance filter of 8000-11000 molecular weight.
Further, in filtration step, the pressure that ultra-filtration process applies is 0.1-0.2MPa.
Further, in drying steps, under -35~-20 DEG C of vacuum condition, filtrate is dried.
Realize that second object of the present invention can reach by adopting the following technical scheme that：A kind of leech polypeptide powder
Application, by leech polypeptide powder as described above be used for prepare wine；Wine includes the following active ingredient by densimeter：
Further, wine includes the following active ingredient by densimeter：
Compared with prior art, the beneficial effects of the present invention are：
1st, leech polypeptide powder of the invention is obtained in the way of microwave radiation technology combined filtering, and the high income of polypeptide is obtained
Hirudin and other peptide fragment, amino acid and small molecules for being easily absorbed by the human body are taken, the effect with logical blood vessel, reducing blood lipid is more held
Easily absorbed；
2nd, leech polypeptide powder of the invention is with microwave radiation technology preparation technology, in protein macromolecule in the presence of microwave
Many peptide bonds or amino acid side chain produce relevant, orderly vibrations, greatly improve enzymolysis efficiency；In hydrolysing step, pass through
The temperature setting of heating-cooling-heating, is able to maintain that the balance of microwave energy, and the peptide fragment to be obtained in albumen is not destroyed, tool
It is improved the active effect of hydrolase；
3rd, the wine prepared in the present invention by leech polypeptide powder has good palatability, and the wine is easy to absorb, tool
There is low antigenicity, the effect with lipid-loweringing.
Embodiment
Below, with reference to embodiment, the present invention is described further：
A kind of leech polypeptide powder, is prepared by following steps：
Pretreatment：Natural leech is taken, it is that 11-20g/L sodium chloride solutions soak 5-15min to add concentration；Then dechlorination is removed
Change sodium solution；
Defibrination：Pure water is added in pretreated natural leech and pH to 5.5-6.5 is adjusted by acetum；It is even
Slurry, filters off impurity, obtains slurries；
It is modified：Temperature be 65~85 DEG C under conditions of, with reference to frequency for 0.7-1.2GHz microwave to slurries at
5-10min is managed, then 20-40min is incubated under conditions of 60 DEG C, obtains modified solution；
Hydrolysis：Papain is added in modified solution, the addition of papain is modified for 6000-18000U/g
Solution, regulation pH is 4.5-6, modified solution is hydrolyzed processing 10- with reference to frequency for 8000-9500MHz microwave
20min, is then cooled to 4~10 DEG C, constant temperature places 10-20min；It is that 4000-6000MHz microwaves carry out processing 5- with reference to frequency
15min, stops reaction, obtains hydrating solution；Hydrolysis first adds papain, is hydrolyzed with the microwave radiation technology of upper frequency, should
Protein macromolecule many peptide bonds or amino acid side chain is caused to produce relevant, orderly vibrations under frequency, and solution heats up, and promotes water
Efficiency is solved, is then significantly cooled, then is handled by the microwave of lower frequency, can be delayed with reference to the microwave treatment of discontinuous
Microwave energy is rushed, the balance of microwave energy is maintained, the peptide fragment to be obtained in albumen is not destroyed, extraction efficiency is greatly improved；
Inactivation：Hydrating solution is heated to 95~115 DEG C, 15~25min is handled, obtains inactivating hydrolyzate；
Filtering：Inactivation hydrolyzate is cooled to 20~30 DEG C, filter is taken using the ultrafiltration membrance filter of 8000-11000 molecular weight
Liquid；The pressure that ultra-filtration process applies is 0.1-0.2MPa；It can be removed under the molecular weight and not hydrolyze high molecular weight protein and peptide fragment, obtained
Take hirudin and other peptide fragment, amino acid and small molecules for being easily absorbed by the human body；
Dry：Under -35~-20 DEG C of vacuum condition, filtrate is dried, leech polypeptide powder is obtained.
A kind of application of leech polypeptide powder, above-mentioned leech polypeptide powder is used to prepare wine；Wine is included by dense
Spend the following active ingredient of meter：
Under above alcohol concentration, the leech polypeptide powder of extraction has a preferable activity, leech polypeptide powder with
Under other compositions and the synergy of alcohol, the wine has promoting blood circulation and removing blood stasis, anticoagulant, antithrombotic, improves blood circulation and drop blood
Effect of fat.
Embodiment 1：
A kind of leech polypeptide powder, is prepared by following steps：
Pretreatment：Natural leech is taken, it is that 18g/L sodium chloride solutions soak 15min to add concentration；Then sodium chloride is removed molten
Liquid；
Defibrination：Pure water is added in pretreated natural leech and pH to 6 is adjusted by acetum；Homogenate, is filtered off
Impurity, obtains slurries；
It is modified：Under conditions of temperature is 70 DEG C, processing 10min is carried out to slurries for 0.8GHz microwave with reference to frequency,
Then 30min is incubated under conditions of 60 DEG C, obtain modified solution；
Hydrolysis：Papain is added in modified solution, the addition of papain is 15000U/g modified solutions,
It is 5 to adjust pH, and processing 18min is hydrolyzed to modified solution for 8500MHz microwave with reference to frequency, 9 DEG C, perseverance are then cooled to
Temperature places 15min；It is that 5800MHz microwaves carry out processing 8min with reference to frequency, stops reaction, obtain hydrating solution；
Inactivation：Hydrating solution is heated to 115 DEG C, 20min is handled, obtains inactivating hydrolyzate；
Filtering：Inactivation hydrolyzate is cooled to 30 DEG C, filtrate is taken using the ultrafiltration membrance filter of 10000 molecular weight；Ultra filtration
The pressure that journey applies is 0.2MPa；
Dry：Under -25 DEG C of vacuum condition, filtrate is dried, leech polypeptide powder is obtained.
Embodiment 2：
The characteristics of the present embodiment, is：
It is modified：Under conditions of temperature is 80 DEG C, processing 10min is carried out to slurries for 1.1GHz microwave with reference to frequency,
Then 30min is incubated under conditions of 60 DEG C, obtain modified solution.
Pretreatment, defibrination, hydrolysis, inactivation, filtering and drying steps and embodiment 1 are identical.
Embodiment 3：
The characteristics of the present embodiment, is：
Hydrolysis：Papain is added in modified solution, the addition of papain is modified for 6000-18000U/g
Solution, regulation pH is 6, and processing 12min is hydrolyzed to modified solution for 9500MHz microwave with reference to frequency, 5 are then cooled to
DEG C, constant temperature places 15min；It is that 4500MHz microwaves carry out processing 13min with reference to frequency, stops reaction, obtain hydrating solution.
Pretreatment, defibrination, hydrolysis, inactivation, filtering and drying steps and embodiment 1 are identical.
Comparative example 1：
Comparative example 1 is modified using routine techniques to albumen：
It is modified：It is under conditions of 70 DEG C, processing 10min to be carried out to slurries, is then incubated under conditions of 60 DEG C in temperature
30min, obtains modified solution；
Remaining step and parameter are same as Example 1.
Comparative example 2：
Handled in the hydrolysing step of comparative example 2 not through cooling and uninterrupted microwave：
Hydrolysis：Papain is added in modified solution, the addition of papain is modified for 6000-18000U/g
Solution, regulation pH is 5, and processing 26min is hydrolyzed to modified solution for 8500MHz microwave with reference to frequency, stops reaction, obtains
To hydrating solution；
Remaining step and parameter are same as Example 1.
Comparative example 3：
Albumen is hydrolyzed not through cooling step using routine techniques for comparative example 3：
Hydrolysis：Papain is added in modified solution, the addition of papain is modified for 6000-18000U/g
Solution, regulation pH is 5, modified solution is hydrolyzed processing 18min with reference to frequency for 8500MHz microwave；It is with reference to frequency
5800MHz microwaves carry out processing 8min, stop reaction, obtain hydrating solution；
Remaining step and parameter are same as Example 1.
Determine embodiment 1-3 and comparative example 1-3 peptide contents and yield is detected, as a result as shown in Table 1：
The peptide content of form 1 and yield are detected
It can be obtained from form 1, the mode of microwave radiation technology hydrolysis can significantly improve peptide content ratio and yield, wherein comparative example
2 eliminate uninterrupted microwave and cooling step in hydrolysing step, although peptide content ratio and yield ratio comparative example 1,3 are high but right
It is still relatively low in embodiment 1-3, illustrate that extraction of the uninterrupted microwave combination cooling to leech polypeptide powder plays the role of positive.
Embodiment 4：
The leech polypeptide powder that embodiment 1 is obtained, for preparing wine, wine include by densimeter it is following effectively into
Point：
Leech polypeptide powder, zinc gluconate (Zn are added in rice wine2+Source), calcium carbonate Ca2+Source), sulphur
Sour magnesium (Mg2+Source), ferric phosphate (Fe3+And PO4 3-Source), soak more than 3 months, leech polypeptide is obtained after filtering
Wine.Obtained wine is detected, as shown in Table 2, nature examination is as shown in Table 3 for organoleptic detection：
The organoleptic detection of the embodiment 4 of form 2
| Project | Sensory results |
| Color and luster | Oolemma rose |
| Flavour and fragrance | With fragranced, slight bitter, sweet flavour；Rice wine fragrance, free from extraneous odour |
| Local flavor | Entrance is soft and fine and smooth, stingless larynx is felt, do not have suspension |
| Tissue morphology | Clear, there is a small amount of sediment after being long placed in |
The nature examination of form 3
For those skilled in the art, technical scheme that can be as described above and design, make other each
It is kind corresponding to change and deform, and all these change and deformation should all belong to the protection model of the claims in the present invention
Within enclosing.
Claims (10)
1. a kind of leech polypeptide powder, it is characterised in that prepared by following steps：
Pretreatment：Natural leech is taken, sodium chloride solution immersion 5-15min is added；Then sodium chloride solution is removed；
Defibrination：Pure water is added in pretreated natural leech and pH is adjusted to 5.5-6.5；Homogenate, filters off impurity, is starched
Liquid；
It is modified：Under conditions of temperature is 65~85 DEG C, with reference to microwave slurries are carried out with processing 5-10min, then 60 DEG C bar
20-40min is incubated under part, modified solution is obtained；
Hydrolysis：Papain is added in modified solution, regulation pH is 4.5-6, is the micro- of 8000-9500MHz with reference to frequency
Ripple modified solution is hydrolyzed processing 10-20min, is then cooled to 4~10 DEG C, constant temperature places 10-20min；With reference to frequency
Processing 5-15min is carried out for 4000-6000MHz microwaves, stops reaction, obtains hydrating solution；
Inactivation：Hydrating solution is heated to 95~115 DEG C, 15~25min is handled, obtains inactivating hydrolyzate；
Filtering：Inactivation hydrolyzate is cooled to 20~30 DEG C, ultra filtration leaching filtrate；
Dry：Filtrate is dried, leech polypeptide powder is obtained.
2. leech polypeptide powder as claimed in claim 1, it is characterised in that：In pre-treatment step, the sodium chloride solution
Concentration be 11-20g/L.
3. leech polypeptide powder as claimed in claim 1, it is characterised in that：In refining step, adjusted by acetum
pH。
4. leech polypeptide powder as claimed in claim 1, it is characterised in that：In modification procedure, the frequency of microwave is 0.7-
1.2GHz。
5. leech polypeptide powder as claimed in claim 1, it is characterised in that：In hydrolysing step, the addition of papain
Measure as 6000-18000U/g modified solutions.
6. leech polypeptide powder as claimed in claim 1, it is characterised in that：In filtration step, 8000-11000 points are used
The ultrafiltration membrance filter of son amount takes filtrate.
7. leech polypeptide powder as claimed in claim 1, it is characterised in that：In filtration step, the pressure that ultra-filtration process applies
Power is 0.1-0.2MPa.
8. leech polypeptide powder as claimed in claim 1, it is characterised in that：In drying steps, -35~-20 DEG C of vacuum
Under the conditions of, filtrate is dried.
9. a kind of application of leech polypeptide powder, it is characterised in that：Leech polypeptide powder as claimed in claim 1 is used
In preparing wine；The wine includes the following active ingredient by densimeter：
10. the application of leech polypeptide powder as claimed in claim 9, it is characterised in that：The wine is included by densimeter
Following active ingredient：
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| CN201710412958.5A CN107177654A (en) | 2017-06-05 | 2017-06-05 | A kind of leech polypeptide powder and application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710412958.5A CN107177654A (en) | 2017-06-05 | 2017-06-05 | A kind of leech polypeptide powder and application |
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Family
ID=59835676
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5194589A (en) * | 1986-09-18 | 1993-03-16 | Pennsylvania Hospital | Leech protein having anticoagulant and antimetastatic properties |
| CN101759775A (en) * | 2008-11-13 | 2010-06-30 | 北京和润创新医药科技发展有限公司 | Leech active small peptide and preparation method and application thereof |
| CN105385739A (en) * | 2015-12-09 | 2016-03-09 | 梁尚文 | Method for extracting protein peptide from poecilobdella manillensis |
| CN105999247A (en) * | 2016-06-14 | 2016-10-12 | 唐校岩 | Medicinal hirudin and raw-leech liquor and preparation method and application thereof |
| CN106434816A (en) * | 2016-12-05 | 2017-02-22 | 广州蛭肽素医药科技有限公司 | Method of using leech to prepare hypolipidemic peptide |
-
2017
- 2017-06-05 CN CN201710412958.5A patent/CN107177654A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5194589A (en) * | 1986-09-18 | 1993-03-16 | Pennsylvania Hospital | Leech protein having anticoagulant and antimetastatic properties |
| CN101759775A (en) * | 2008-11-13 | 2010-06-30 | 北京和润创新医药科技发展有限公司 | Leech active small peptide and preparation method and application thereof |
| CN105385739A (en) * | 2015-12-09 | 2016-03-09 | 梁尚文 | Method for extracting protein peptide from poecilobdella manillensis |
| CN105999247A (en) * | 2016-06-14 | 2016-10-12 | 唐校岩 | Medicinal hirudin and raw-leech liquor and preparation method and application thereof |
| CN106434816A (en) * | 2016-12-05 | 2017-02-22 | 广州蛭肽素医药科技有限公司 | Method of using leech to prepare hypolipidemic peptide |
Non-Patent Citations (2)
| Title |
|---|
| ISOLDA P.BASKOVA ET AL.: ""Arterial antithrombotic effect of piyav1t, the novel pharmacological preparation from the medicinal leech, and of its components, prostanoids and enzyme destabilase"", 《THROMBOSIS RESEARCH》 * |
| 姜海波 等: "水蛭素抑制凝血作用探讨", 《河北中医》 * |
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