CN108998489A - A kind of preparation method of Wood frog antibiotic peptides - Google Patents
A kind of preparation method of Wood frog antibiotic peptides Download PDFInfo
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- CN108998489A CN108998489A CN201810909476.5A CN201810909476A CN108998489A CN 108998489 A CN108998489 A CN 108998489A CN 201810909476 A CN201810909476 A CN 201810909476A CN 108998489 A CN108998489 A CN 108998489A
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Abstract
The present invention discloses a kind of preparation method of Wood frog antibiotic peptides, this method comprises: (1) digests；(2) it deploys；(3) emulsifier is prepared；(4) mixing and high speed shear；(5) it is freeze-dried.The Wood frog antibiotic peptides powder hygroscopicity as made from this method is low, brew is good, has good fungistatic effect, and the preparation method is suitable for industrialized production.
Description
Technical field
The invention belongs to technical field of biological extraction, more particularly, to a kind of preparation method of Wood frog antibiotic peptides.
Background technique
The skin and internal organ of amphibian animal have extensive pharmacological activity and clinical efficacy, and many amphibian animals are in
State belongs to traditional Chinese medicine and national drug and is widely applied, such as Dongbei forest-frog (oviductus ranae), Chinese toad, Bombina maxima.?
Report pharmacological activity has broad spectrum antimicrobial effect, antitumor, office's local anaesthesia, analgesia, immunological regulation, Cardiovascular System
Deng.
Wood frog (Rana chensinensis), also known as breathes out assorted toad, wood frog's fallopian tube etc., is Amphibia, Anura, Ranidae, wood frog
Belong to, is China's endemic species, main producing region is distributed in the Northeast of China, especially based on Jilin.30-40 age in 20th century, Japan
Scholar's Kubo Tian Qingguang, longevity Tian Wenci youth etc. have carried out fundamental analysis to the chemical component of oviductus ranae, and test result shows: albumen
Matter accounts for 50% or so of wood frog oil component, additionally containing a certain amount of fat, carbohydrate, nitrogen phosphate and sulfur etc..
There is long history in China to the application of wood frog class drug, but main to its active constituent and pharmacological Quality Research
The small organic molecules such as alkaloid are concentrated on, it is also less to its bioactive peptide object Quality Research.Dongbei forest-frog is distributed mainly on China
Northeast Changbaishan area, be the national folk drug of one of the characteristic resources animal in China and characteristic.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of Wood frog antibiotic peptides, Wood frog antibiotic peptides powder made from this method
With certain antibacterial activity, it is suitable for industrialized production.
In order to achieve the above objectives, the present invention adopts the following technical solutions:
The present invention provides a kind of preparation method of Wood frog antibiotic peptides, method includes the following steps:
(1) it digests: using oviductus ranae as primary raw material, Controlled-enzymatic Hydrolysis being carried out using conbined enzymolysis, face light yellow complexion, water is made
The enzymolysis liquid that Xie Du is 40%, the polypeptide molecular weight of hydrolyzable moiety is 1000-7000 dalton；
(2) it deploys: taking maltodextrin, beta-cyclodextrin, sepg whey albumen powder WPI95, concentrated whey protein powder respectively
WPC80 is placed in clean dried container, under conditions of being sufficiently stirred, is slowly added into certain mass made from step (1)
In enzymolysis liquid, magnetic agitation is to being completely dissolved；
(3) it prepares emulsifier: with the deionized water dissolving monoglyceride of a small amount of room temperature, being tuned into scattered paste shape, heating water bath to cream
Change 70-85 DEG C of temperature, oscillator acutely shakes, and is allowed to dissolution and forms white " milky " liquid, and water bath with thermostatic control heat preservation is spare；
(4) mixing and high speed shear: under conditions of magnetic agitation 700-1000r/min, to step (2) allotment obtained
And dissolve complete material liquid and carry out heating water bath, after rising to 70-85 DEG C of emulsifying temperature, it is slowly added to cream made from step (3)
The content control of agent, emulsifier is sufficiently mixed in 0.01-0.03%, magnetic agitation 8-10min, then again in 7000rpm item
Emulsification feed liquid is made in high speed shear 5min under part, magnetic agitation 5min；This step high speed shear treatment is to promote emulsification abundant
Effective means；
(5) it is freeze-dried: step (4) emulsification feed liquid obtained being cooled to room temperature, is put into -80 DEG C of ultralow temperature ice later
Case precooling 5-7h, then using the dry powder processed of full-automatic vacuum freeze drying machine, main -52 DEG C, 20h of freezing, it is rear freeze 4h to get
Wood frog antibiotic peptides powder.
Further, in step (2), 5-25% maltodextrin, 1-10% beta-cyclodextrin, 5-20% separation cream are weighed respectively
Albumin powder WPI95,5-20% concentrated whey protein powder WPC80.
Further, in step (3) and step (4), emulsifying temperature is 80 DEG C.
Further, in step (4), emulsifier content 0.02%.
Further, ultra low temperature freezer precooling 6h in step (5).
Following antibacterial activity test experience proves that Wood frog antibiotic peptides produced by the present invention have good antibacterial activity.
Antibacterial activity detection: using cylinder plate method, and culture medium is plain agar culture medium.It is injected separately into the culture of heating and melting
Base 20m1 is used as bottom in plate, makes its uniform feeding in ware bottom, after solidification, after separately taking the appropriate heating and melting of culture medium,
Bacteria suspension to be measured is added in plate respectively, shakes up, it is made uniformly to spread out cloth on bottom, as bacterium layer.After cooling, in plate
Equidistant Oxford cup 6 for being uniformly put into oneself and sterilizing.The testing compound solution of 0.3mg/ml concentration is added in first steel bowl
Sample liquid is added using doubling dilution in 0.1m1, remaining Oxford cup, and inhibition zone size is observed in 37 DEG C of cultures.Inhibition zone 10mm with
On conduct minimal inhibitory concentration.
Above-mentioned test in triplicate, is averaged.The bacteriostatic experiment result such as Tables 1 and 2 of two antibacterial peptides.
1 dybowskii-1CDYa bacteriostatic activity of table
* the staphylococcus aureus of methicillin-resistant
2 dybowskii-2CDYa bacteriostatic activity of table
* the staphylococcus aureus of methicillin-resistant
Beneficial effects of the present invention: the present invention uses Freeze Drying Technique powder, using oviductus ranae enzymatic hydrolysate as primary raw material,
After oviductus ranae dry product is made as enzymolysis liquid, on the basis of oviductus ranae enzymatic hydrolysate, by rational allocation and emulsifying process, freezing is utilized
The Wood frog antibiotic peptides powder that dry technology is processed as, and hygroscopicity is low, brew is good is suitable for industrialized production, and obtained
Wood frog antibiotic peptides have good fungistatic effect.
Specific embodiment
In order to illustrate more clearly of the present invention, below with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, this should not be limited with this
The protection scope of invention.
Embodiment 1
(1) it digests: using oviductus ranae as primary raw material, Controlled-enzymatic Hydrolysis, obtained enzymolysis liquid being carried out using conbined enzymolysis
Face light yellow complexion, fishy smell is light, slightly tart flavour and bitter taste, the polypeptide molecular weight of degree of hydrolysis 40%, hydrolyzable moiety is mainly distributed on
Between 1000~7000 dalton；
(2) it deploys: weighing 25% maltodextrin, 5% beta-cyclodextrin, 5% sepg whey albumen powder WPI95,20% respectively
Concentrated whey protein powder WPC80 is placed in clean dried container, under conditions of being sufficiently stirred, is slowly added into step (1) system
Certain mass enzymolysis liquid in, magnetic agitation is to being completely dissolved；
(3) it prepares emulsifier: with the deionized water dissolving monoglyceride of a small amount of room temperature, being tuned into scattered paste shape, heating water bath to cream
Change temperature 70 C, oscillator acutely shakes, and is allowed to dissolution and forms white " milky " liquid, and water bath with thermostatic control heat preservation is spare；
(4) mixing and high speed shear: under conditions of magnetic agitation 700r/min, allotment obtained to step (2) is simultaneously molten
It solves complete material liquid and carries out heating water bath, after rising to 70 DEG C of emulsifying temperature, be slowly added to emulsifier made from step (3), cream
The content of agent is 0.01%, and magnetic agitation 8min is sufficiently mixed, then with the well-mixed feed liquid of emulsifier in 7000rpm
Under the conditions of high speed shear 5min, magnetic agitation 5min, be made emulsification feed liquid；High speed shear processing is to promote emulsification adequately effective
Means；
(5) it is freeze-dried: step (4) emulsification feed liquid obtained being cooled to room temperature, is put into -80 DEG C of ultralow temperature ice later
Case precooling 5h, then using the dry powder processed of full-automatic vacuum freeze drying machine, main -52 DEG C, 20h of freezing, the rear 4h that freezes is to get woods
Frog antibacterial Gly-His-Lys.
Embodiment 2
(1) it digests: using oviductus ranae as primary raw material, Controlled-enzymatic Hydrolysis, obtained enzymolysis liquid being carried out using conbined enzymolysis
Face light yellow complexion, fishy smell is light, slightly tart flavour and bitter taste, the polypeptide molecular weight of degree of hydrolysis 40%, hydrolyzable moiety is mainly distributed on
Between 1000~7000 dalton；
(2) it deploys: weighing 5% maltodextrin, 10% beta-cyclodextrin, 20% sepg whey albumen powder WPI95,5% respectively
Concentrated whey protein powder WPC80 is placed in clean dried container, under conditions of being sufficiently stirred, is slowly added into step (1) system
Certain mass enzymolysis liquid in, magnetic agitation is to being completely dissolved；
(3) it prepares emulsifier: with the deionized water dissolving monoglyceride of a small amount of room temperature, being tuned into scattered paste shape, heating water bath to cream
Change 80 DEG C of temperature, oscillator acutely shakes, and is allowed to dissolution and forms white " milky " liquid, and water bath with thermostatic control heat preservation is spare；
(4) mixing and high speed shear: under conditions of magnetic agitation 800r/min, allotment obtained to step (2) is simultaneously molten
It solves complete material liquid and carries out heating water bath, after rising to 80 DEG C of emulsifying temperature, be slowly added to emulsifier made from step (3), cream
The content of agent is 0.03%, and magnetic agitation 8min is sufficiently mixed, then with the well-mixed feed liquid of emulsifier in 7000rpm
Under the conditions of high speed shear 5min, magnetic agitation 5min, be made emulsification feed liquid；High speed shear processing is to promote emulsification adequately effective
Means；
(5) it is freeze-dried: step (4) emulsification feed liquid obtained being cooled to room temperature, is put into -80 DEG C of ultralow temperature ice later
Case precooling 7h, then using the dry powder processed of full-automatic vacuum freeze drying machine, main -52 DEG C, 20h of freezing, the rear 4h that freezes is to get woods
Frog antibacterial Gly-His-Lys.
Embodiment 3
(1) it digests: using oviductus ranae as primary raw material, Controlled-enzymatic Hydrolysis, obtained enzymolysis liquid being carried out using conbined enzymolysis
Face light yellow complexion, fishy smell is light, slightly tart flavour and bitter taste, the polypeptide molecular weight of degree of hydrolysis 40%, hydrolyzable moiety is mainly distributed on
Between 1000~7000 dalton；
(2) it deploys: weighing 20% maltodextrin, 1% beta-cyclodextrin, 15% sepg whey albumen powder WPI95,15% respectively
Concentrated whey protein powder WPC80 is placed in clean dried container, under conditions of being sufficiently stirred, is slowly added into step (1) system
Certain mass enzymolysis liquid in, magnetic agitation is to being completely dissolved；
(3) it prepares emulsifier: with the deionized water dissolving monoglyceride of a small amount of room temperature, being tuned into scattered paste shape, heating water bath to cream
Change 85 DEG C of temperature, oscillator acutely shakes, and is allowed to dissolution and forms white " milky " liquid, and water bath with thermostatic control heat preservation is spare；
(4) mixing and high speed shear: under conditions of magnetic agitation 1000r/min, allotment obtained to step (2) is simultaneously molten
It solves complete material liquid and carries out heating water bath, after rising to 85 DEG C of emulsifying temperature, be slowly added to emulsifier made from step (3), cream
The content of agent is 0.02%, and magnetic agitation 10min is sufficiently mixed, then with the well-mixed feed liquid of emulsifier in 7000rpm
Under the conditions of high speed shear 5min, magnetic agitation 5min, be made emulsification feed liquid；High speed shear processing is to promote emulsification adequately effective
Means；
(5) it is freeze-dried: step (4) emulsification feed liquid obtained being cooled to room temperature, is put into -80 DEG C of ultralow temperature ice later
Case precooling 6h, then using the dry powder processed of full-automatic vacuum freeze drying machine, main -52 DEG C, 20h of freezing, the rear 4h that freezes is to get woods
Frog antibacterial Gly-His-Lys.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention may be used also on the basis of the above description for those of ordinary skill in the art
To make other variations or changes in different ways, all embodiments can not be exhaustive here, it is all to belong to this hair
The obvious changes or variations that bright technical solution is extended out are still in the scope of protection of the present invention.
Claims (5)
1. a kind of preparation method of Wood frog antibiotic peptides, which is characterized in that method includes the following steps:
(1) it digests: using oviductus ranae as raw material, using conbined enzymolysis that degree of hydrolysis is made as the peptide molecule of 40%, hydrolyzable moiety
Amount is the enzymolysis liquid of 1000-7000 dalton；
(2) it deploys: taking maltodextrin, beta-cyclodextrin, sepg whey albumen powder WPI95, concentrated whey protein powder WPC80 respectively,
Under agitation, it is added in enzymolysis liquid made from step (1), stirring is to being completely dissolved；
(3) it prepares emulsifier: using deionized water dissolving monoglyceride, for heating water bath to 70-85 DEG C of emulsifying temperature, concussion is allowed to molten
Solution forms white " milky " liquid, water bath with thermostatic control heat preservation；
(4) mixing and high speed shear: under conditions of magnetic agitation 700-1000r/min, to material liquid made from step (2) into
After rising to 70-85 DEG C of emulsifying temperature, emulsifier made from step (3), emulsifier content 0.01- is added in row heating water bath
0.03%, magnetic agitation 8-10min, then high speed shear 5min under the conditions of 7000rpm, magnetic agitation 5min again, are made cream
Material liquid；
(5) it is freeze-dried: step (4) emulsification feed liquid obtained being cooled to room temperature, later -80 DEG C of precooling 5-7h, re-dry
Powder processed, main -52 DEG C of freezing, 20h freezes 4h afterwards, obtains Wood frog antibiotic peptides powder.
2. the method according to claim 1, wherein weighing 5-25% maltodextrin, 1- respectively in step (2)
10% beta-cyclodextrin, 5-20% sepg whey albumen powder WPI95,5-20% concentrated whey protein powder WPC80.
3. the method according to claim 1, wherein the emulsifying temperature is 80 in step (3) and step (4)
℃。
4. the method according to claim 1, wherein in step (4), the emulsifier content 0.02%.
5. the method according to claim 1, wherein precooling 6h in step (5).
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| CN201810909476.5A CN108998489A (en) | 2018-08-10 | 2018-08-10 | A kind of preparation method of Wood frog antibiotic peptides |
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| CN201810909476.5A CN108998489A (en) | 2018-08-10 | 2018-08-10 | A kind of preparation method of Wood frog antibiotic peptides |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042138A (en) * | 2019-04-10 | 2019-07-23 | 常熟理工学院 | The preparation method and its separation method and purposes of a kind of oviductus ranae anti-oxidation peptide component |
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| CN103343153A (en) * | 2013-05-25 | 2013-10-09 | 吉林农业大学 | Method for preparing forest frog oil peptides by enzymatic hydrolysis and forest frog oil peptides |
| CN104744566A (en) * | 2015-04-08 | 2015-07-01 | 中国人民解放军军事医学科学院军事兽医研究所 | Antibacterial peptide separated from skin secretions of wood frog and preparation method of antibacterial peptide |
| CN105724901A (en) * | 2014-12-11 | 2016-07-06 | 黑龙江众生生物工程有限公司 | Preparation method of oviductus ranae polypeptide |
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| CN103343153A (en) * | 2013-05-25 | 2013-10-09 | 吉林农业大学 | Method for preparing forest frog oil peptides by enzymatic hydrolysis and forest frog oil peptides |
| CN105724901A (en) * | 2014-12-11 | 2016-07-06 | 黑龙江众生生物工程有限公司 | Preparation method of oviductus ranae polypeptide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110042138A (en) * | 2019-04-10 | 2019-07-23 | 常熟理工学院 | The preparation method and its separation method and purposes of a kind of oviductus ranae anti-oxidation peptide component |
| CN110042138B (en) * | 2019-04-10 | 2020-12-29 | 常熟理工学院 | Preparation method of rana japonica oil antioxidant peptide component, separation method and application thereof |
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Application publication date: 20181214 |