CN110468174A - A kind of preparation method of leech active oligopeptides - Google Patents
A kind of preparation method of leech active oligopeptides Download PDFInfo
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Abstract
This application discloses a kind of preparation methods of leech active oligopeptides, comprising: is digested using alkali protease and trypsase to leech, obtains the leech active oligopeptides；Wherein, the molecular weight of the leech active oligopeptides is lower than 500Da.Method described herein solves leech polypeptide method for preparing extractive in the prior art and is difficult to ensure the technical issues of polypeptide molecular weight is smaller and molecular weight distribution is concentrated.
Description
Technical field
This application involves a kind of preparation methods of leech active oligopeptides, belong to the preparation field of biological peptide.
Background technique
Leech is first recorded in Shennong's Herbal: " main by extravesated blood, hemostasis is closed the moon, the accumulation of blood-breaking lump in the abdomen, s.m.p, dredging water passages." tool
There is blood-breaking promoting menstruation, by the function of stagnation resolvation disease.Modern pharmacology thinks, leech has the effects that anticoagulant, thrombolysis, antitumor.Its is anticoagulant
The active constituent of thrombolysis is mainly protein, component polypeptides.The hirudin secreted in leech saliva is presently considered to strongest
Anticoagulating active substance.But extract that hirudin yield is low, cost is excessively high from saliva, it is difficult to mass production.Therefore, it sells in the market
Various natural hirudin prices it is also very expensive, purity, activity are difficult to ensure.Therefore there is an urgent need to one kind can reduce into
Originally, the extracting method of simple possible, to improve leech class anticoagulant active substance in the application of medicine and healthcare field.
Leech is as Chinese medicine, and traditionally use is beaten the powder mode of taking after mixing it with water and taken, and then enters after gastrointestinal tract is degraded internal
Anticoagulant and thrombolytic activity is played, therefore also centainly containing the functional domain segment of anticoagulant active in leech body.Enzyme process is used at present
The active peptide extracted in leech body is largely used, and has the activity in many documents or patent report Enzymatic Extraction leech
Peptide.In view of the natural barrier of human body alimentary canal, the only peptide of small molecule can just be absorbed by the body utilization.At present in these reports
Since leech raw material is different, the technique of use is different, and obtained polypeptide products have very big difference.Or focus on high activity peptide
Extraction, and have ignored the size of molecular weight；Or focus on obtaining oligopeptides, and have ignored active reservation.
Summary of the invention
According to the one aspect of the application, a kind of preparation method of leech active oligopeptides is provided, this method solve existing
The technology for having leech polypeptide method for preparing extractive in technology to be difficult to ensure that polypeptide molecular weight is smaller and molecular weight distribution is concentrated is asked
Topic, leech preparation method of oligopeptide products therefrom antithrombin activity lower problem and leech have stench problem.
The method that Controlled-enzymatic Hydrolysis is taken in the preparation method of leech active oligopeptides described herein, passes through preferred protease
Type leech is digested, obtaining existing high activity again has the oligopeptides of small-molecular-weight.As drug, health care product or
When skin care item, not only guarantee there is higher bioavilability, but also guarantees its activity and stability with higher.
The preparation method of the leech active oligopeptides characterized by comprising
Leech is digested using alkali protease and trypsase, obtains the leech active oligopeptides；
Wherein, the molecular weight of the leech active oligopeptides is lower than 500Da.
Optionally, the leech oligopeptides has the antithrombin activity of 480U/g or more.
Optionally, the leech oligopeptides has the antithrombin activity of 520U/g or more.
Optionally, the leech oligopeptides has the antithrombin activity of 560U/g or more.
Optionally, the colourless nothing of leech active oligopeptides is stench.
Optionally, the condition of the enzymatic hydrolysis are as follows:
The pH of enzymatic hydrolysis is 7-10；
The temperature of enzymatic hydrolysis is 50-80 DEG C；
The time of enzymatic hydrolysis is 1-6 hours.
Optionally, the pH upper limit digested in the condition of the enzymatic hydrolysis is selected from 7.5,8,8.5,9 or 10；Lower limit be selected from 7,7.5,
8,8.5 or 9.
Optionally, the temperature upper limit digested in the condition of the enzymatic hydrolysis is selected from 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C or 80
℃；Lower limit is selected from 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C or 75 DEG C.
Optionally, the time upper limit of the enzymatic hydrolysis is selected from 2 hours, 3 hours, 4 hours, 5 hours or 6 hours；Lower limit is selected from 1
Hour, 2 hours, 3 hours, 4 hours or 5 hours.
Optionally, the leech digested is leech freeze-dried powder.
Optionally, the preparation method of the leech freeze-dried powder includes: that leech living body or its frozen goods thaw, and stirring obtains
To homogenate；The homogenate is freeze-dried to obtain freeze-dried powder.
Optionally, the leech freeze-dried powder carries out enzymolysis processing after being added to the water dissolution.
Optionally, the leech freeze-dried powder carries out enzymolysis processing after 10-30 times of (weight) water dissolution is added.
As one of specific embodiment, the preparation method of the leech active oligopeptides includes: to take freeze-dried powder suitable
After adding 5-30 times of water dissolution, alkali protease is added by the 1%-10% of freeze-dried powder weight, in pH7-10, temperature 50-80 in amount
DEG C, it reacts 1-6 hours, adds 1%-10% trypsase enzyme, in pH7-10, reaction 1-5 hours of 50-80 DEG C of temperature, thereafter
Active carbon and white bole is added in high-temperature inactivation, centrifugation, supernatant, after carrying out deodorant decoloring reaction, after filtering, takes supernatant, adjusts
PH is saved to neutrality, is freeze-dried the leech oligopeptides to get low molecule high activity.
Optionally, the additional amount of the alkali protease is the 0.8%-10% of leech weight；
The additional amount of the trypsase is the 1%-10% of leech weight；
Wherein, the weight of the leech is subject to the weight of leech freeze-dried powder.
Optionally, the additional amount of the alkali protease is the 1%-10% of leech weight.
Optionally, the additional amount of the alkali protease be leech weight the upper limit be selected from 0.9%, 1%, 1.1%,
1.4%, 1.7%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%,
8.5%, 9%, 9.5% or 10%；Lower limit be selected from 0.8%, 0.9%, 1%, 1.1%, 1.4%, 1.7%, 2%, 2.5%,
3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9% or 9.5%.
Optionally, the additional amount of the trypsase be leech weight the upper limit be selected from 1.1%, 1.4%, 1.7%, 2%,
2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or
10%；Lower limit be selected from 1%, 1.1%, 1.4%, 1.7%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%,
6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9% or 9.5%.
Optionally, the preparation method of the leech active oligopeptides includes:
Enzymatic hydrolysis I is carried out to leech using alkali protease first, trypsase is then added and carries out enzymolysis II, is obtained described
Leech active oligopeptides.
Optionally, the condition of the enzymatic hydrolysis I are as follows:
It is 7-10 in pH, temperature is 50-80 DEG C, and reacting is to be digested under conditions of 1-6 hours；
The condition of the enzymolysis II are as follows:
It is 7-10 in pH, temperature is 50-80 DEG C, and reacting is to be digested under conditions of 1-5 hours.
Optionally, the pH upper limit digested in the condition of the enzymatic hydrolysis I is selected from 7.5,8,8.5,9 or 10；Lower limit be selected from 7,
7.5,8,8.5 or 9.
Optionally, in the condition of the enzymatic hydrolysis I temperature upper limit that digests be selected from 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C or
80℃；Lower limit is selected from 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C or 75 DEG C.
Optionally, the time upper limit of the enzymatic hydrolysis I is selected from 2 hours, 3 hours, 4 hours, 5 hours or 6 hours；Lower limit is selected from
1 hour, 2 hours, 3 hours, 4 hours or 5 hours.
Optionally, the pH upper limit digested in the condition of the enzymolysis II is selected from 7.5,8,8.5,9,9.5 or 10；Lower limit is selected from
7,7.5,8,8.5,9 or 9.5.
Optionally, the temperature upper limit digested in the condition of the enzymolysis II be selected from 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C or
80℃；Lower limit is selected from 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C or 75 DEG C.
Optionally, the time upper limit of the enzymolysis II is selected from 2 hours, 3 hours, 4 hours or 5 hours；It is small that lower limit is selected from 1
When, 2 hours, 3 hours or 4 hours.
Optionally, inactivation treatment is carried out after the enzymatic hydrolysis.
Optionally, the condition of the inactivation treatment are as follows: 80~100 DEG C of 10~40min of processing.
Optionally, deodorant decolorization is carried out after the enzymatic hydrolysis；
The deodorant decolorization includes: to carry out deodorant decoloration using active carbon and white bole.
Optionally, the condition of the deodorant decolorization are as follows:
Temperature is 40-70 DEG C；
Time is 0.3-3 hours.
Optionally, the additional amount of the active carbon is the 0.5-10% of enzymolysis liquid weight；
The additional amount of the white bole is the 0.5-10% of enzymolysis liquid weight.
The enzymolysis liquid is enzymolysis liquid to be processed after digesting.
Optionally, it is inactivated after enzymatic hydrolysis, centrifugal treating, obtained supernatant is enzymolysis liquid to be processed.
Optionally, temperature upper limit is selected from 50 DEG C, 60 DEG C or 70 DEG C during the deodorant decolorization；Lower limit is selected from 40
DEG C, 50 DEG C or 60 DEG C.
Optionally, during the deodorant decolorization time upper limit be selected from 0.5 hour, 1 hour, 1.5 hours, 2 hours,
2.5 hours or 3 hours；Lower limit is selected from 0.3 hour, 0.5 hour, 1 hour, 1.5 hours, 2 hours or 2.5 hours.
Optionally, the additional amount of the active carbon be enzymolysis liquid weight the percentage upper limit be selected from 1%, 2%, 2.5%,
3%, 5%, 6%, 8% or 10%；Lower limit is selected from 0.5%, 1%, 2%, 2.5%, 3%, 5%, 6% or 8%.
Optionally, the additional amount of the white bole be enzymolysis liquid weight the percentage upper limit be selected from 1%, 2%, 2.5%,
3%, 5%, 6%, 8% or 10%；Lower limit is selected from 0.5%, 1%, 2%, 2.5%, 3%, 5%, 6% or 8%.
Optionally, after carrying out the deodorant decolorization, filtering takes supernatant, adjusts pH to neutrality, freeze-drying obtains
To the leech active oligopeptides.
Optionally, the leech digested passes through homogenized.
Optionally, the preparation method of the leech active oligopeptides includes:
(1) leech homogenate is freeze-dried, obtains freeze-dried powder；
(2) freeze-dried powder described in step (1) is taken, alkali protease enzymatic hydrolysis is added after being dissolved in water, tryptose is then added
Enzyme enzymatic hydrolysis, inactivation, centrifugation obtain supernatant；
(3) active carbon and white bole are added in supernatant described in step (2), deodorant decoloration takes after filtering
Clear liquid adjusts pH to neutrality, and freeze-drying obtains the leech active oligopeptides.
Optionally, the leech active oligopeptides that the method is prepared is in the drug of anticoagulant and thrombolytic, health product raw material, makeup
Application in product raw material.
As one of specific embodiment, using leech as raw material in the preparation method of the leech active oligopeptides,
The oligopeptides with high anticoagulation is obtained after homogenate, enzymatic hydrolysis, decoloration deodorization.
As one of specific embodiment, the preparation method of the leech active oligopeptides includes:
After leech living body or frozen goods are thawed, it is put into stirring in refiner and is made even to paste and without blocky meat
Slurries.Freeze-dried powder is obtained after homogenate is freeze-dried, and is sealed spare.It takes freeze-dried powder appropriate, after adding 5-30 times of water dissolution, presses
Alkali protease is added in the 0.8%-10% of freeze-dried powder weight, in 50-80 DEG C of pH7-10, temperature, reacts 1-6 hours, adds
1%-10% trypsase enzyme, in pH7-10, reaction 1-5 hours of 50-80 DEG C of temperature, high-temperature inactivation, centrifugation, supernatant add thereafter
Enter active carbon and white bole, after carrying out deodorant decoloring reaction, after filtering, take supernatant, adjusts pH to neutrality, be freeze-dried, i.e.,
Obtain the leech oligopeptides of low molecule high activity.
As one of specific embodiment, the preparation method of the leech active oligopeptides includes:
By leech living body, it is put into stirring in refiner and homogenate is made to paste and without blocky meat.Homogenate is freezed
Freeze-dried powder is obtained after drying, is sealed spare.It takes freeze-dried powder appropriate, after adding 20 times of water dissolutions, adds by the 1.7% of freeze-dried powder weight
Enter alkali protease, is reacted 2 hours under the conditions of pH9, temperature are 55 degree, 2.5% trypsase enzyme is added, in pH8,60 DEG C
Between react 3 hours, 85 DEG C of inactivation 15min thereafter, centrifugation, it is 3 that supernatant, which adjusts pH, and 5% active carbon and 2.5% white pottery is added
Soil carries out deodorant decoloring reaction 1h under 60 DEG C of water-baths, after filtering, take supernatant, adjusts pH to neutrality, is freeze-dried to get low
The leech oligopeptides of molecule high activity.
The beneficial effect that the application can generate includes:
(1) complex enzyme zymohydrolysis technique is used in combination in the preparation method of herein described leech active oligopeptides and carries out leech widow
Peptide extracts, and controls temperature, pH value, and the time while ratio of enzyme bottom, guarantees that the molecular weight of gained peptide is small and range is stablized, anticoagulant
The feature that blood is active and polypeptide retention rate is high.And in existing extraction process, or focus on the extraction of high activity peptide, and have ignored
The size of molecular weight；Or focus on obtaining oligopeptides, and have ignored active reservation.
(2) compared with prior art, this law solve that polypeptide molecular weight in existing scheme is larger and molecular weight distribution not
The problems in collection and the in the prior art lower problem of leech preparation method of oligopeptide products therefrom antithrombin activity.
(3) in the preparation method of the application, two kinds of protease used under alkaline condition is selected, the pH item used is reacted
Part is close, and preparation process is simple, easy to produce；
(4) the application leech active oligopeptides obtained can be used as the drug of anticoagulant and thrombolytic, health product raw material, can also be with
It is used as cosmetic material.
Detailed description of the invention
Fig. 1 is tyrosine solution standard curve in embodiment 7；
Fig. 2 is 8 Plays albumen high-efficient liquid phase chromatogram of embodiment；
Fig. 3 is in embodiment 8 according to retention time and the resulting standard curve of molecular weight logarithm；
Fig. 4 is papain enzymolysis product HPLC figure in embodiment 8；
Fig. 5 is 8 neutral and alkali protease hydrolyzed product HPLC of embodiment figure；
Fig. 6 is trypsin digestion product HPLC chromatogram in embodiment 8；
Product HPLC schemes after Fig. 7 alkali protease and trypsin digestion；
Product HPLC schemes after Fig. 8 alkali protease and papain enzymolysis；
Fig. 9 is active carbon decolorizing effect figure under condition of different pH in embodiment 12；Wherein, figure acceptance of the bid note is respectively active carbon
Additive amount and pH, from left to right successively are as follows: pH3,3%；PH3,1%；PH4,3%；PH4,1%；PH5,1%；PH5,3%；
Figure 10 is white bole decolorizing effect figure in embodiment 12；From left to right successively are as follows: enzymolysis liquid stoste；White bole 4%；
White bole 6%；White bole 8%；
Figure 11 is that decolorizing effect figure is used in combination in active carbon and white bole in embodiment 12；From left to right successively are as follows: stoste；
5% active carbon, 2.5% white bole；5% active carbon；3% active carbon, 2.5% white bole；1% active carbon, 2.5% white bole.
Specific embodiment
The application is described in detail below with reference to embodiment, but the application is not limited to these embodiments.
Unless otherwise instructed, the raw material in embodiments herein is bought by commercial sources, in which:
Leech is provided by Guangxi Fu Xinyi group, and identified kind is hiruto.Casein (lot number: 9000719, Beijing
Baeyer enlightening Bioisystech Co., Ltd), alkali protease (Solarbio, lot number: 718I022), papain
(Solarbio, lot number: 518F027), trypsase (Solarbio, lot number: 310P041), forint phenol (Solarbio, lot number:
120I043).Trichloroacetic acid (aladdin, lot number: E1808060), natrium carbonicum calcinatum (Macklin, lot number: M33318011),
NaOH (pure, the lot number of analysis: 1503041, Xilong Chemical Co., Ltd), HCl (pure, the lot number of analysis: 20170426, Beijing
Chemical plant), Aprotinin (lot number: N26F9W54634, Shanghai Yuan Ye Science and Technology Ltd.), bacitracin (lot number: S19A681, on
Hai Yuanye Science and Technology Ltd.), aminoacetic acid-aminoacetic acid-aminoacetic acid (enjy-tech, lot number: M10101.5), aminoacetic acid-second ammonia
Acid-Tyr-Arg (enjy-tech, lot number: M10102.5), acetonitrile (Fisher Chemical, lot number: 184265),
Methanol (Fisher Chemical, lot number: 164786), trishydroxymethylaminomethane (Tris) (lot number: 77861, Beijing Baeyer
Enlightening Bioisystech Co., Ltd), and trifluoroacetic acid (lot number: 20180112, Tianjin good fortune morning chemical reagent factory), HPLC water is super
Pure water, remaining is deionized water with water.Fibrinogen (Sigma F8630, lot number: AP0036), TT assay kit (batch
Number: 121197, Shanghai Sun Bio-Tech Co., Ltd.)；Active carbon (is purchased from Beijing Chemical Plant, lot number 20180525)；White pottery
Soil (is purchased from Macklin, lot number: M153618079).
Analysis method is as follows in embodiments herein:
It is surveyed using TU-1901 double beam ultraviolet-visible spectrophotometer (Beijing Puxi General Instrument Co., Ltd)
Determine proteinase activity and leech oligopeptides percent of decolourization；LG-PABER type platelet aggregation coagulation factor analyzer (Beijing generation Supreme Being science
Instrument company) measurement thrombin time (thrombin activity)；(thermo fisher scientific is public for high performance liquid chromatograph
Department) and liquid chromatogram gel chromatographic columns (TOSOH Co., Ltd, English name: TSKgel G2000SWXL, product number:
0008540, column specification: 7.8*300) for measuring the molecular weight of leech oligopeptides.
In embodiments herein:
PH is adjusted by 1mol/LNaOH aqueous solution or HCl.
The preparation of leech homogenate and its freeze-dried powder
By leech (hiruto) living body or -80 DEG C of frozen goods, stir about 30min in refiner is put into after defrosting, until paste
And without blocky meat, freeze-drying for 24 hours after (temperature: -45 DEG C, pressure: 7.9pa) freeze-dried powder, be sealed spare.
Embodiment 1
Leech living body is put into stirring in refiner, homogenate is made to paste and without blocky meat.Homogenate is freezed
Freeze-dried powder is obtained after drying, is sealed spare.Freeze-dried powder 5g is taken, adds and is dissolved in 100mL water, is added by the 1.7% of freeze-dried powder weight
Enter alkali protease, reacted 2 hours under the conditions of pH is 9, temperature is 55 DEG C, then pancreas egg is added by the 2.5% of freeze-dried powder weight
White enzyme reacts 3 hours under conditions of pH is 8, temperature is 60 DEG C, and 85 DEG C of inactivation 15min, centrifugation, supernatant adjust pH thereafter
It is 3,5wt% active carbon and 2.5wt% white bole (being 100% calculating with supernatant quality) is added, is taken off under 60 DEG C of water-baths
Raw meat decoloring reaction 1h after filtering, takes supernatant, adjusts pH to neutrality, freeze-drying is to get molecular weight 500 hereinafter, anticoagulating active
The leech active oligopeptides of 500U/g or more.
Embodiment 2
Leech living body is put into stirring in refiner, homogenate is made to paste and without blocky meat.Homogenate is freezed
Freeze-dried powder is obtained after drying, is sealed spare.Freeze-dried powder 5g is taken, adds and is dissolved in 100mL water, is added by the 1.1% of freeze-dried powder weight
Enter alkali protease, reacted 1 hour under the conditions of pH is 9, temperature is 55 degree, then pancreas egg is added by the 2.5% of freeze-dried powder weight
White enzyme reacts 4 hours under conditions of pH is 8, temperature is 60 DEG C, and 85 DEG C of inactivation 15min, centrifugation, supernatant adjust pH thereafter
It is 3,3wt% active carbon and 2.5wt% white bole (being 100% calculating with supernatant quality) is added, is taken off under 60 DEG C of water-baths
Raw meat decoloring reaction 1h after filtering, takes supernatant, adjusts pH to neutrality, freeze-drying is to get molecular weight 500 hereinafter, anticoagulating active
The leech active oligopeptides of 500U/g or more.
Embodiment 3
Leech living body is put into stirring in refiner, homogenate is made to paste and without blocky meat.Homogenate is freezed
Freeze-dried powder is obtained after drying, is sealed spare.Freeze-dried powder 5g is taken, adds and is dissolved in 100mL water, is added by the 1.4% of freeze-dried powder weight
Enter alkali protease, reacted 2 hours under the conditions of pH is 8.5, temperature is 60 degree, then pancreas is added by the 2.5% of freeze-dried powder weight
Protease reacts 2 hours under conditions of pH is 8, temperature is 60 DEG C, and 85 DEG C of inactivation 15min, centrifugation, supernatant are adjusted thereafter
PH is 3, and 5wt% active carbon and 2wt% white bole (being 100% calculating with supernatant quality) is added, is taken off under 60 DEG C of water-baths
Raw meat decoloring reaction 1h after filtering, takes supernatant, adjusts pH to neutrality, freeze-drying is to get molecular weight 500 hereinafter, anticoagulating active
The leech active oligopeptides of 500U/g or more.
Embodiment 4
Leech living body is put into stirring in refiner, homogenate is made to paste and without blocky meat.Homogenate is freezed
Freeze-dried powder is obtained after drying, is sealed spare.Freeze-dried powder 5g is taken, adds and is dissolved in 100mL water, is added by the 1.1% of freeze-dried powder weight
Enter alkali protease, reacted 2 hours under the conditions of pH is 9.5, temperature is 65 degree, then pancreas is added by the 2.5% of freeze-dried powder weight
Protease reacts 2 hours under conditions of pH is 8, temperature is 60 DEG C, and 85 DEG C of inactivation 15min, centrifugation, supernatant are adjusted thereafter
PH is 3, and 5wt% active carbon and 3wt% white bole (being 100% calculating with supernatant quality) is added, is taken off under 60 DEG C of water-baths
Raw meat decoloring reaction 1h after filtering, takes supernatant, adjusts pH to neutrality, freeze-drying is to get molecular weight 500 hereinafter, anticoagulating active
The leech active oligopeptides of 500U/g or more.
Embodiment 5
Leech living body is put into stirring in refiner, homogenate is made to paste and without blocky meat.Homogenate is freezed
Freeze-dried powder is obtained after drying, is sealed spare.Freeze-dried powder 5g is taken, adds and is dissolved in 100mL water, is added by the 1.7% of freeze-dried powder weight
Enter alkali protease, reacted 2 hours under the conditions of pH is 9, temperature is 60 degree, then 2.5% press freeze-dried powder weight addition pancreas egg
White enzyme reacts 3 hours under conditions of pH is 8, temperature is 60 DEG C, and 85 DEG C of inactivation 15min, centrifugation, supernatant adjust pH thereafter
It is 3,3wt% active carbon and 6wt% white bole (being 100% calculating with supernatant quality) is added, carries out deodorant under 60 DEG C of water-baths
Decoloring reaction 1h after filtering, takes supernatant, is freeze-dried the leech oligopeptides to get low molecule high activity.
Embodiment 6
Leech living body is put into stirring in refiner, homogenate is made to paste and without blocky meat.Homogenate is freezed
Freeze-dried powder is obtained after drying, is sealed spare.Freeze-dried powder 5g is taken, adds and is dissolved in 100mL water, is added by the 1.7% of freeze-dried powder weight
Enter alkali protease and 2.5% trypsase, is reacted under the conditions of pH is 8, temperature is 60 degree 3 hours, 85 DEG C of inactivations thereafter
15min, centrifugation, it is 3 that supernatant, which adjusts pH, and 5wt% active carbon and 2.5wt% white bole is added (with supernatant quality for 100%
Calculate), deodorant decoloring reaction 1h is carried out under 60 DEG C of water-baths, after filtering, takes supernatant, adjusts pH to neutrality, freeze-drying to get
Molecular weight 500 hereinafter, anticoagulating active 500U/g or more leech active oligopeptides.
The measurement of 7. enzyme activity standard curve making of embodiment and a variety of enzyme activities
For the not same problem of the enzyme activity convenient for unified different manufacturers, vitality test, later period are carried out to the hydrolase of buying
It can feed intake by enzyme activity.The enzyme activity of alkali protease, trypsase, papain has been investigated in experiment.
(7-1) experimental method
(7-1-1) enzyme activity standard curve making
The drafting of tyrosine standard curve: 10 colorimetric cylinder numbers are taken, are divided to two groups in parallel, every group is accurately added 1mL respectively
Concentration is the standard tyrosine solution of 20,40,60,80 and 100 μ g/mL, then is separately added into 5mL 0.4mol/L Na2CO3Solution
With 0.5mLFolin- phenol reagent, rapid oscillation is mixed, and sets the 20min that develops the color in 40 DEG C of waters bath with thermostatic control, and taking out with deionized water is sky
White control measures each pipe light absorption value at 660nm.Using tyrosine concentration as abscissa, light absorption value is ordinate, draws out junket ammonia
Acidity scale directrix curve.
(7-1-2) enzyme solution preparation to be measured
0.1g alkali protease, 0.1g trypsase are taken respectively, and 0.1g papain is settled to pH7.4PBS solution
50mL。
(7-1-3) measures enzyme activity
3 colorimetric cylinder numbers are taken, the good protein enzyme solution 1mL (step (7-1-2)) of addition beforehand dilution in every pipe, No. 1
Pipe is blank control (operation repetitive), and after liquid of protease is added in No. 1 pipe, 0.4mol/L solution of trichloroacetic acid is added immediately
2mL makes enzyme not in contact with living to substrate slip.1mL2wt% caseic aqueous solution is added in another two test tubes, mixes rapidly, immediately
It is put into 40 DEG C of waters bath with thermostatic control accurate timing constant temperature 10min, 0.4mol/L solution of trichloroacetic acid 2mL is rapidly added, to terminate enzyme
Reaction.1m L2wt% caseic aqueous solution is added into No. 1 pipe simultaneously, shakes up.After 3 colorimetric cylinders stand 10min at room temperature
4000r/min is centrifuged 15min, takes each pipe supernatant 1m L to be moved into the colorimetric cylinder of another 3 numbers respectively, 5mL is respectively added
0.4mol/L Na2CO3Solution and 0.5mL Folin- phenol reagent, rapid oscillation mix, set in 40 DEG C of waters bath with thermostatic control and develop the color
20min takes out and measures each pipe light absorption value at 660nm.The calculating of enzyme activity is according to formula:
Prolease activity
In formula, U-proteolytic enzyme unit of activity number (U/g or U/mL)；A-sample parallel test average absorbance
Value；K-extinction constant, the amount (μ g) of the tyrosine when absorbance is 1；N-enzyme solution extension rate；4-reaction reagents
Volume (mL).
(7-2) experimental result and analysis
Tyrosine standard curve is drawn according to (7-1-1) aforesaid operations method, shown in result figure 1.
As shown in Figure 1, tyrosine solution calibration curve formula is y=0.0067x+0.0079, R2=0.9991；It is linear good
Good, being computed K value is 148.07.
To the albumen of the protease (papain, trypsase and alkali protease) of 3 kinds of selected separate sources
The results are shown in Table 1 for enzyme activity determination.
The energy value and optimum reaction conditions of 1 three kinds of protease of table
| Protease | Absorbance | Energy value U/g |
| Alkali protease | 0.597 | 1.77x105 |
| Trypsase | 0.537 | 1.59x105 |
| Papain | 0.195 | 5.8x105 |
The measurement of 8 polypeptide molecular weight of embodiment
Different hydrolases is different to the restriction enzyme site of protein, therefore the polypeptide molecular weight for having digested acquisition is also different.
(8-1) experimental method
(8-1-1) list enzyme enzymatic hydrolysis
After taking 3 groups of centrifuge tubes that the dissolution of leech freeze-dried powder 0.50g, 10mL water is added, it is optimum that pH is adjusted to each enzyme respectively
PH, respectively 8.5,8.0,7.0.Be separately added into alkali protease, trypsase, papain (enzyme amount of addition with
2000U/g conversion), in addition to leech freeze-dried powder is not added, remaining operation is identical for each group blank control.6h is reacted at 60 DEG C, 85 DEG C go out
15min living, adjusts pH to 7.0,10000r/min to be centrifuged 10min, takes supernatant.
The molecular weight determination of (8-1-2) leech enzymatic product
High performance liquid chromatograph condition: high performance liquid chromatograph；Liquid-phase chromatographic analysis column (TOSOH Co., Ltd, English name
Claim: TSKgel G2000SWXL, product number: 0008540, column specification: 7.8*300)；Wavelength: 220nm；Flow velocity: 0.5mL/
min；Column temperature: 30 DEG C；Sample volume: 10 μ L.
Mobile phase preparation method are as follows: acetonitrile: water: trifluoroacetic acid=45:55:0.1 (volume ratio), wavelength: 220nm, flow velocity:
0.5mL/min, column temperature: 30 DEG C, sample volume: 10 μ L.
Enzymolysis liquid is crossed into 0.45 μm of miillpore filter, as test solution.Take Aprotinin (6500Da), bacitracin
(1450Da), aminoacetic acid-aminoacetic acid-aminoacetic acid (189Da), aminoacetic acid-aminoacetic acid-Tyr-Arg (451Da) in right amount,
With 0.45 μm of miillpore filter is crossed after flowing phased soln, as standard solution.
(8-2) experimental result and analysis
(8-2-1) standard protein high-efficient liquid phase chromatogram and standard curve
Aprotinin (6500Da), bacitracin (1450Da), aminoacetic acid-aminoacetic acid-Tyr-Arg (451Da), second ammonia
Acid-aminoacetic acid-aminoacetic acid (189Da) high-efficient liquid phase chromatogram is as shown in Figure 2.According to retention time and molecular weight logarithm institute
The standard curve obtained is as shown in Figure 3.
The average molecular of elution time (T) and standard items of this experiment according to the standard items of different molecular weight on a column
The logarithm of quality (Mr) is in some linear, and principle can (in formula, logMr be that standard items are opposite with formula logMr=AT+B
The logarithm of molecular mass, T are elution time, and B is the intercept of standard curve, and A is the slope of standard curve).
Experimental data is substituted into formula, obtaining standard items appearance time-molecular mass standard curve equation is
Y=-0.2561x+7.3798 (R2=0.9922) (formula 1)
(8-2-2) leech enzymatic product high-efficient liquid phase chromatogram and molecular weight distribution
It is digested using papain, alkali protease and trypsase according to method under (8-1-1) item, enzymatic hydrolysis produces
Object carries out gel chromatography analysis, and obtained HPLC is as shown in Fig. 4, Fig. 5, Fig. 6.Wherein, Fig. 4 is papain in embodiment 8
Enzymolysis product HPLC figure；Fig. 5 is 8 neutral and alkali protease hydrolyzed product HPLC of embodiment figure；Fig. 6 is trypsase in embodiment 8
Enzymolysis product HPLC chromatogram.
According to gel chromatography elution order: the big compound of molecular weight first flows out chromatographic column, from papain, alkaline egg
It is big that the HPLC chromatogram of white enzyme and trypsase can be seen that the peptides retention time obtained after alkali protease enzymatic hydrolysis
Part concentrates between 14-20min, i.e. molecular weight is concentrated mainly between 17-20min between 189-6500, i.e. molecule
Amount is below 3000.The appearance time of papain and trypsase product is more early, and the appearance time of most of product exists
Before 14min, i.e., molecular weight is greater than 6500.
(8-3) conclusion
According to the above analysis, the active peptide for using alkali protease that can obtain low molecular weight as hydrolase is selected.
9 complex enzyme zymohydrolysis of embodiment
Leech can obtain the peptide fragment of relatively small molecular weight through alkali protease enzymatic hydrolysis, to investigate alkali protease+tryptose
The peptide that more small-molecular-weight whether can be obtained after enzyme, alkali protease+papain combination, has carried out complex enzyme zymohydrolysis experiment.
(9-1) experimental method
After taking 2 groups of centrifuge tubes that the dissolution of leech freeze-dried powder 0.50g, 10mL water is added, pH is adjusted to 8.5, basic protein is added
Enzyme (enzyme amount of addition is converted with 2000U/g), in addition to leech freeze-dried powder is not added, remaining operation is identical for each group blank control.60℃
After lower reaction 4h, pH is adjusted to 8.0,7.0 respectively, is separately added into trypsase, (enzyme amount of addition is with 2000U/ for papain
G conversion), 4h, 85 DEG C of inactivation 15min are reacted at 60 DEG C, are adjusted pH to 7.0,10000r/min centrifugation 10min, are taken supernatant.
(9-2) experimental result and analysis
By alkali protease+trypsase, alkali protease+papain complex enzyme for hydrolyzing product by (8-1-2) method into
Row gel chromatography analysis measurement molecular weight, resulting HPLC chromatogram are as shown in Figure 7, Figure 8.Wherein, Fig. 7 alkali protease and pancreas
Product HPLC schemes after protease hydrolyzed；Product HPLC schemes after Fig. 8 alkali protease and papain enzymolysis.
Fig. 7, Fig. 8 are compared with Fig. 5, after alkali protease enzymatic hydrolysis, trypsase or papain are added, when component retains
Between move back, show that resulting bioactive peptide molecule amount reduces, two kinds of complex enzymes combinations can get the lower active peptide of molecular weight, this
Outside in terms of appearance time, the time of all components appearance is more concentrated, and shows more to concentrate using complex enzyme hydrolysis molecular weight of product.
As can be seen from the above: alkali protease respectively and after trypsase and papain combination, can be obtained
The active peptide that the peptide fragment and molecular weight of more small-molecular-weight are more concentrated.
The optimization of 10 alkali protease enzymatic hydrolysis condition of embodiment
Hydrolysis temperature, pH, enzyme concentration, substrate dosage etc. all can be to the activity influences of enzymolysis product in investigation enzymolysis process.
For the peptides for obtaining most highly active, the enzymatic hydrolysis condition of alkali protease is carried out using the anticoagulating active of gained enzymolysis liquid as index excellent
Change.
(10-1) experimental method
(10-1-1) activity determination method (thrombin time method)
It takes 50 μ L of 0.5wt% aqueous fibrinogen solution in test cup, is separately added into 50 μ L of solution to be measured, test pearl 1
, after 37 DEG C of incubation 5min, the 50 μ L of TT reagent that 37 DEG C of pre-temperatures are added is taken out immediately, records setting time, while with reagent sky
It is white to record its thrombin time (TT) for reference.(10-1-2) experiment condition single factor exploration
In experiment of single factor, each factor takes 5 parallel pipes.Take 0.5g leech freeze-dried powder+10mL water (empty in parallel pipe
For white control group in addition to leech freeze-dried powder is not added, remaining operation is identical), the pH of reaction system is adjusted to 1mol/L Na OH solution
8.5,5.65mg alkali protease is added in each tube, is put into 60 DEG C of water-baths and reacts 4h, 85 DEG C of water-baths inactivate 15min,
The pH of liquid in 5 parallel pipes of each factor is adjusted to 7,10000r/min centrifugation 10min, supernatant is taken, dilutes 15 times, measurement
TT value finally judges its activity height with different leech sample TT values.
Enzymolysis process optimization is tested, and in experiment of single factor, each factor takes 5 parallel pipes, only changes experiment of single factor item
Part, other experiment conditions are constant.In pH gradient experiment, pH gradient is set are as follows: 7.5,8,8.5,9,9.5；In temperature gradient experiment,
Set temperature gradient are as follows: 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C；In enzyme amount gradient experiment, enzyme amount gradient is set are as follows: 0.8%,
1.1%, 1.4%, 1.7%, 2.0%, 2.5%, that is, the alkali protease being added is between 1416U/g-4425U/g；Time ladder
In degree experiment, setting time gradient: 1h, 2h, 3h, 4h, 5h.
Wherein, enzyme amount is to weigh by the percentage of leech freeze-dried powder weight in the present embodiment.
(10-1-3) optimum condition orthogonal design is investigated and verifying
According to single factor exploration as a result, choosing pH is 8.5,9,9.5,55 DEG C of reaction temperature, 60 DEG C, 65 DEG C, enzyme concentration
1.1%, 1.4%, 1.7%, reaction time 1h, 2h, 3h carry out orthogonal experiment, determine optimum reaction condition.
9 50mL centrifuge tubes are taken, 0.5g leech freeze-dried powder is weighed, is separately added into 10mL water, liquid in pipe is adjusted to respectively
Different pH are separately added into different amounts of alkali protease, digest different time, 85 DEG C of inactivation 15min at different temperatures respectively
Afterwards, it adjusts pH to 7,10000r/min to be centrifuged 10min, takes supernatant, dilute 15 times, measure its TT value by 10-1-1 method.Each pipe
In experiment condition see the table below 2:
Table 2
| Serial number | Temperature/DEG C | pH | Time/h | Enzyme amount/% |
| 1 | 55 | 8.5 | 1 | 1.1 |
| 2 | 55 | 9 | 2 | 1.7 |
| 3 | 55 | 9.5 | 3 | 1.4 |
| 4 | 60 | 8.5 | 2 | 1.4 |
| 5 | 60 | 9 | 3 | 1.1 |
| 6 | 60 | 9.5 | 1 | 1.7 |
| 7 | 65 | 8.5 | 3 | 1.7 |
| 8 | 65 | 9 | 1 | 1.4 |
| 9 | 65 | 9.5 | 2 | 1.1 |
(10-2) result and analysis
(10-2-1) single factor exploration result
Each factor is investigated when by method under (10-1-2) item to leech enzymatic, and the results are shown in Table 3.
Leech enzymatic liquid obtained by 3 experiment of single factor of table and its blank control TT measurement result
From 3 interpretation of result of table, for reacting pH, optimal activity is shown for 9 or so in pH；For temperature condition, In
60 DEG C or so show optimum activity；For enzyme concentration condition, optimal activity is shown 1.4% or so；When for reaction
Between, reach optimum activity in 2h or more.
(10-2-2) orthogonal experiment investigates result
According to single factor exploration as a result, choosing pH is 8.5,9,9.5,55 DEG C of reaction temperature, 60 DEG C, 65 DEG C, enzyme concentration
1.1%, 1.4%, 1.7%, reaction time 1h, 2h, 3h carry out orthogonal experiment, and according to orthogonal table, it is real to carry out enzymatic hydrolysis
It tests, TT determination of activity is carried out to the enzymolysis product under the conditions of each, as a result as follows:
4 difference way crossover study TT measurement result (n=4) of table
This experiment carries out process optimization to the enzymatic hydrolysis condition of leech freeze-dried powder enzymolysis liquid, according to experiment of single factor and orthogonal examination
Test acquired results, the peak enzymolysis-ability condition of alkali protease are as follows: pH=9, enzyme amount: 1.7% alkali protease, reaction temperature: 55
DEG C, the reaction time: 2h.
Alkali protease peak enzymolysis-ability condition is used again, digests leech freeze-dried powder in the same way, and measure its TT value,
(sample :+700 μ L water of 50 μ L sample measures after 15 times of dilution), as a result are as follows: 36.8,37.1,36.3,37.9.
According to above-mentioned analysis, leech enzymatic experiment pH7.5-9.5, -65 DEG C of temperature 45 C, enzyme concentration 0.8%-2.5%,
In reaction time 2h-6h section, compared to the blank group, very high antithrombin activity is shown.Knot obtained by orthogonal test
Fruit, the peak enzymolysis-ability condition of alkali protease are as follows: pH=9, enzyme amount: 1.7% alkali protease, reaction temperature: 55 DEG C, when reaction
Between: 2h.
11 complex enzyme condition optimizing of embodiment
Alkali protease+trypsase is chosen in experiment, two methods of alkali protease+papain carry out leech compound
Enzyme enzymatic hydrolysis.After the completion of alkali protease enzymatic hydrolysis, enzymolysis liquid is separately adjusted to angularly trypsase and the most suitable pH of papain, point
Not Jia Ru 2000U/g trypsase and papain, select trypsase and the optimum pH of papain and temperature condition respectively,
That is trypsase pH8.0, temperature 60 C；Papain pH7.0, temperature 60 C investigate different enzymes under the conditions of above-mentioned pH and temperature
The time is solved on the active influence of final product.
(11-1) experimental method
Four centrifuge tubes are taken, precision weighs four parts of leech homogenate freeze-dried powder 0.50g, is separately added into 10mL deionized water
(for blank in addition to leech freeze-dried powder is not added, remaining operation is identical), is adjusted to alkali protease optimum pH 9, alkali is added in each pipe
Property protease 1.7% (0.0085g) after magnetic agitation 2h at 55 DEG C, 85 DEG C of inactivation 15min, is adjusted to trypsase optimum pH
8.0 (papains 7.0), be separately added into trypsase 0.0126g (papain 0.0034g, the enzyme amount of addition with
2000U/g conversion), distinguish 1h, 2h, 3h, 4h, 85 DEG C of inactivation 15min of magnetic agitation at 60 DEG C, adjusts pH to 7.0,10000r/min
It is centrifuged 10min, supernatant is taken, obtains mother liquor, it is spare.Mother liquor is taken to dilute 15 times, measurement TT activity.
(11-2) experimental result
According to (11-1) method, two methods of alkali protease+trypsase, alkali protease+papain is respectively adopted
Complex enzyme zymohydrolysis is carried out to leech, the thrombin time (TT) of enzymolysis product is as shown in table 5 under the conditions of different enzymolysis times.
5 alkali protease of table and complex enzyme zymohydrolysis product TT are active (s)
As can be seen from the results in the table that alkali protease enzymolysis product activity is higher, after trypsase and papain is added,
Activity is declined, and after especially papain is added, activity decline is serious.Comprehensively consider molecular weight and active double factor, adopts
Preferable activity and lower molecular weight can be obtained by being shared with alkali protease and trypsase.
According to above-mentioned analysis, preferable activity and lower point can be obtained by being shared using alkali protease and trypsase
Son amount.The enzymatic hydrolysis condition of complex enzyme are as follows: weigh leech homogenate freeze-dried powder 0.50g, 10mL deionized water is added, is adjusted to alkaline egg
Alkali protease 1.7% is added in white enzyme optimum pH 9 in each pipe, at 55 DEG C after magnetic agitation 2h, 85 DEG C of inactivation 15min, adjusts
To pH value 8.0, it is added trypsase 0.0126g (2000U/g), at 60 DEG C of difference after magnetic agitation 3h, 85 DEG C of inactivation 15min,
It adjusts pH to 7.0,10000r/min to be centrifuged 10min, takes supernatant to get leech active peptidase hydrolyzed liquor.
Enzymatic hydrolysis leech is combined compared to alkali protease and papain, alkali protease and trypsase combine enzymatic hydrolysis leech
Obtaining active oligopeptide, not only molecular weight is low, but also activity is higher.
12 decoloration process of embodiment
Leech alkali protease and trypsin digestion liquid are brown, have bad smell, therefore carry out decolorization to it.
Enzymolysis liquid described in the present embodiment is the resulting enzymolysis liquid of embodiment 1 (supernatant obtained after centrifugation).
(12-1) experimental method
Active carbon decoloring is applied alone in (12-1-1)
Take 10ml enzymolysis liquid, be adjusted to different pH (3,4,5) respectively, be separately added into different carbon content actives (1wt%, 3wt%,
5wt%) (being 100% calculating with enzymolysis liquid quality), 1h is placed in 60 DEG C of water-baths, and 10000r/min is centrifuged 10min, takes supernatant,
Observe its color.
(12-1-2) is applied alone white bole to decolourize
10ml enzymolysis liquid is taken, respectively plus 30min, 10000r/min are placed in 0.4g, 0.6g, 0.8g white bole, 50 DEG C of water-baths
It is centrifuged 10min, supernatant is taken, observes its color.
(12-1-3) is simultaneously with white bole and active carbon decoloring
10ml enzymolysis liquid is taken, adjusts pH to 3,5 respectively, respectively plus 1wt%, 3wt%, 5wt% active carbon are (with enzymolysis liquid quality
Calculated for 100%), 1h are placed in addition 2.5wt% white bole (being calculated with enzymolysis liquid quality for 100%), 60 DEG C of water-baths,
10000r/min is centrifuged 10min, observes its color.
(12-1-4) percent of decolourization and the measurement of peptide retention rate and calculating
(1) percent of decolourization
Ultraviolet full wavelength scanner is carried out to leech enzymatic liquid, discovery has suction at 575nm in visible absorption wave-length coverage
It receives, therefore wavelength is scheduled on measurement decoloration front and back absorbance A at 575nm, calculate percent of decolourization.Percent of decolourization=(before decolourizing after A- decoloration
A)/decolourize preceding A.
(2) peptide retention rate
Using bovine serum albumin(BSA) as standard protein, assay is carried out to leech oligopeptides using Coomassie Brilliant Blue, it is accurate
10.00mg bovine serum albumin(BSA) (BSA) is weighed, water is added to be settled to 10mL, is shaken up female to get the BSA that mass concentration is 1.0g/L
Liquid, it is spare.Precision weighs 10.00mg Coomassie brilliant G-250, and 95% ethyl alcohol 5mL dissolution is added, adds 85wt% phosphoric acid
10mL adds water to be settled to 100mL, shakes up, filtering, obtains the Coomassie Brillant Blue solution that mass concentration is 0.10g/L, spare.Take BSA
Mother liquor is diluted with water to a series of mass concentrations (0.05,0.1,0.2,0.4,0.6,0.8,1.0g/L), takes various concentration
Each 50 μ L of BSA titer, is separately added into 4mL Coomassie Brillant Blue solution, shakes up, take out immediately after being protected from light 5min, chooses
595nm, surveying absorbance, (blank control: 50 μ L water add 4mL Coomassie Brillant Blue solution, and 5min is kept in dark place after shaking up, and survey extinction
Degree).Finally obtaining equation of linear regression is y=0.38393x+0.04651 (R2=0.9980, n=7).
Leech enzymatic liquid absorbance is measured at wavelength 595nm according to the above method, substitutes into regression equation, calculates peptide content,
Calculate peptide retention rate.Peptide content before peptide content/decoloration after peptide retention rate=decoloration.
(12-2) experimental result and analysis
(12-2-1) singly adds active carbon
Single when being decolourized using active carbon, the decolorizing effect under condition of different pH, as a result such as Fig. 9 institute has been investigated in experiment
Show.As pH is reduced, the decolorizing effect of active carbon improves, and has preferable decolorizing effect when pH3, and with active carbon additional amount
Increase, decolorizing effect enhancing, but cannot decolourize completely.
(12-2-2) singly adds white bole
When single plus white bole, the sepia of leech enzymatic liquid can not be sloughed, as shown in Figure 10.
(12-2-3) active carbon and white bole are used in combination
Under conditions of pH is 3, while active carbon (1wt%-5wt%) and white bole (2.5wt%), decolorizing effect is added
Shown in Figure 11.It can be seen from figure 11 that decolorizing effect is better than active carbon is applied alone after active carbon and white bole share, and with
The additional amount of active carbon increases to 5wt% from 1wt%, and decolorizing effect is more preferable, and colourless transparent solution is presented after decoloration, at this time de-
Color rate is 80%, and peptide retention rate is 92%.
According to above-mentioned test, active carbon and white bole are used in mixed way with preferable decolorizing effect, and optimum condition is that pH is
3, activated carbon addition 5wt%, white bole additive amount 2.5wt%.Enzymolysis liquid is handled using the method, deodorization effect is bright
It is aobvious, enzymolysis liquid lighter, while the retention rate of active peptide can achieve 92%.
Embodiment 2 carries out deodorant decoloration using active carbon and white bole in other enzymolysis process into embodiment 11, obtains
Result similar to the above: deodorization effect is obvious, enzymolysis liquid lighter, while the retention rate of active peptide is high.
13 leech oligopeptides anticoagulating active of embodiment and molecular weight determination
The measurement of (13-1) anticoagulating active
After carrying out enzymatic hydrolysis decoloration to leech by above-mentioned steps, freeze-drying.Take freeze-dried powder Tris-HCl (pH=7.4) slow
Fliud flushing is configured to the solution of 0.01g/mL, measures 100 μ L, sets in small test tube, be added containing 0.5wt% (ox) fibrinogen (with
Coagulum meter) 200 μ L of water buffer, shake up, set in water-bath (37 DEG C ± 0.5 DEG C) temperature leaching 5 minutes, be added dropwise in every 1mL containing 40
The thrombin solution (being added dropwise for every 1 minute 1 time, 5 μ L, gently shakes up when being added dropwise every time) of unit records consumption blood coagulation to solidifying
The volume of enzyme solutions, is calculated as follows:
U=C1V1/C2V2
The every 1g unit containing thrombin activity of U, U/g in formula；
C1The concentration (u/mL) of-thrombin solution；C2The concentration (g/mL) of-test solution；V1- consumption fibrin ferment is molten
The volume (μ L) of liquid；V2The additional amount (μ L) of-test solution,；
Take three batch leech enzymatic liquid (supernatant after being digested in embodiment 1, digest in embodiment 2 after supernatant, implement
Supernatant after being digested in example 3) freeze-dried powder, the antithrombin activity of three batches is respectively 520U/g, 520U/g, 560U/ after measured
g。
(13-2) molecular weight determination
According to Fig. 7, alkali protease and trypsin digestion product appearance time and formula 1 are calculated, molecule in enzymolysis product
The oligopeptides of 500 or less (i.e. after appearance time 18.3min) of amount accounts for 90% of gross product or more, thus using alkali protease and
Trypsase can obtain the active peptide below of molecular weight 500.
The analysis of 14 data of embodiment:
1. molecular weight compares
Using alkali protease, trypsase, papain, alkali protease+trypsase complex enzyme, basic protein
Enzyme+papain complex enzyme respectively digests leech, and enzymolysis product carries out exclusion chromatography analysis, according to retention time
Molecular weight information is measured, obtained result is as shown in Fig. 4,5,6,7,8, and after comparing with standard protein retention time, discovery is used
When single enzyme digests, the molecular weight that trypsase and papain obtain is most of after 6500 or more, alkali protease enzymatic hydrolysis
The molecular weight of leech active peptide is concentrated mainly on 6500 or less；Using alkali protease+trypsase complex enzyme, alkali protease
After+trypsase complex enzyme is digested, active peptide obtained molecular weight is mainly below 500.Moreover, from Fig. 4,5,6,7,8
As a result it is found that product appearance time is more concentrated after using alkali protease+trypsase complex enzyme hydrolysis, i.e., molecular weight is concentrated.
2. activity comparison
By method under (11-1) item, using alkali protease+trypsase, alkali protease+papain combined-enzyme method pair
Leech is digested, and is digested first using alkali protease, trypsase and papain is then respectively adding, when digesting different
Between.After enzymolysis liquid decoloration, freeze-drying.Enzymolysis liquid freeze-dried powder is measured into antithrombin activity, the results are shown in Table 6.
6 alkali protease of table and complex enzyme zymohydrolysis product antithrombin activity (U/g)
As can be seen from Table 6, using alkali protease+trypsase than alkali protease+papain combined-enzyme method
With higher anticoagulating active.
The above is only several embodiments of the application, not does any type of limitation to the application, although this Shen
Please disclosed as above with preferred embodiment, however not to limit the application, any person skilled in the art is not taking off
In the range of technical scheme, a little variation or modification are made using the technology contents of the disclosure above and is equal to
Case study on implementation is imitated, is belonged in technical proposal scope.
Claims (10)
1. a kind of preparation method of leech active oligopeptides characterized by comprising
Leech is digested using alkali protease and trypsase, obtains the leech active oligopeptides；
Wherein, the molecular weight of the leech active oligopeptides is lower than 500Da.
2. the method according to claim 1, wherein the condition of the enzymatic hydrolysis are as follows:
The pH of enzymatic hydrolysis is 7-10；
The temperature of enzymatic hydrolysis is 50-80 DEG C；
The time of enzymatic hydrolysis is 1-6 hours.
3. the method according to claim 1, wherein the additional amount of the alkali protease is leech weight
0.8%-10%；
The additional amount of the trypsase is the 1%-10% of leech weight；
Wherein, the weight of the leech is subject to the weight of leech freeze-dried powder.
4. the method according to claim 1, wherein the preparation method of the leech active oligopeptides includes:
Enzymatic hydrolysis I is carried out to leech using alkali protease first, trypsase is then added and carries out enzymolysis II, obtains the leech
Active oligopeptide.
5. according to the method described in claim 4, it is characterized in that, the condition of the enzymatic hydrolysis I are as follows:
It is 7-10 in pH, temperature is 50-80 DEG C, and reacting is to be digested under conditions of 1-6 hours；
The condition of the enzymolysis II are as follows:
It is 7-10 in pH, temperature is 50-80 DEG C, and reacting is to be digested under conditions of 1-5 hours.
6. the method according to claim 1, wherein carrying out deodorant decolorization after the enzymatic hydrolysis；
The deodorant decolorization includes: to carry out deodorant decoloration using active carbon and white bole.
7. according to the method described in claim 6, it is characterized in that, the condition of the deodorant decolorization are as follows:
Temperature is 40 DEG C -70 DEG C；
Time is 0.3-3 hours；
The additional amount of the active carbon is the 0.5-10% of enzymolysis liquid weight；
The additional amount of the white bole is the 0.5-10% of enzymolysis liquid weight.
8. the method according to claim 1, wherein the leech digested passes through homogenized.
9. the method according to claim 1, wherein the preparation method of the leech active oligopeptides includes:
(1) leech homogenate is freeze-dried, obtains freeze-dried powder；
(2) freeze-dried powder described in step (1) is taken, alkali protease enzymatic hydrolysis is added after being dissolved in water, trypsase enzyme is then added
Solution, inactivation, centrifugation obtain supernatant；
(3) active carbon and white bole being added in supernatant described in step (2), deodorant decoloration takes supernatant after filtering,
PH is adjusted to neutrality, freeze-drying obtains the leech active oligopeptides.
10. method according to any one of claims 1 to 9, which is characterized in that the leech active that the method is prepared
Application of the oligopeptides in the drug of anticoagulant and thrombolytic, health product raw material, cosmetic material.
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| CN113155943A (en) * | 2021-01-27 | 2021-07-23 | 中国科学院生态环境研究中心 | Method for biodegradation of trypsin-induced carbon black particles and analysis of products thereof |
| CN113155943B (en) * | 2021-01-27 | 2022-06-17 | 中国科学院生态环境研究中心 | Method for biodegradation of trypsin-induced carbon black particles and analysis of products thereof |
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