CN105136699A - Protease collagen hydrolysis activity determination method based on undenatured dyed hide powder substrate, and uses thereof - Google Patents
Protease collagen hydrolysis activity determination method based on undenatured dyed hide powder substrate, and uses thereof Download PDFInfo
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Abstract
The invention provides a determination method for collagen hydrolysis activity of proteases based on a substrate of undenatured dyed hide powder. The determination method comprises the following steps: (1) preparing the dyed hide powder: an intermediated reticular layer of the animal hide is processed to get the hide powder, then the hide powder is dispersed in a NaCl solution to form a hide powder suspension, and the impurities in the hide powder fully enters the liquid phase through oscillation, a low temperature active dye solution is added according to a proportion of 1 mass part of the hide powder to 0.15-4.0 mass parts of low temperature active dye, and the low temperature active dye is covalently bound with collagen of the hide powder by oscillating the reaction liquid, a fixing agent solution is added according to a ratio of 1 mass parts of the hide powder to 0.05-1.2 mass parts of a fixing agent, and after fully oscillation, filtering, rinsing, dehydration, drying and crushing, the final product is obtained; (2) a standard curve of the dyed hide powder is measured; (3) to-be-measured reaction liquid and a blank contrast solution are prepared; and (4) absorbance is measured and enzyme activity is calculated. The method can be used for measuring collagen protease hydrolysis activity under acidic, neutral, and alkaline conditions.
Description
Technical field
The invention belongs to prolease activity and measure field, relate to the proteinase collagen hydrolysate vigour-testing method based on non-sex change dyeing skin foundation cream thing and application thereof.
Background technology
Collagen is the major structural protein forming Animal Skin, the structure of collagen is wound in distinctive triple helix by three α peptide chains, polypeptied chain sequence is repeated to form by Gly-x-y sequence, Gly residue is piled up along the central shaft of triple helix, the y residue of the x residue of the Gly on one chain and second strand and the 3rd strand is adjacent, the C=O of the N-H of each Gly residue and the x residue of adjacent chain forms hydrogen bond, and also participate in the formation of interchain hydrogen bond due to the hydroxyl of Hyp residue, triple helix is stablized further and is strengthened, due to above feature, collagen is not easily by general protease hydrolytic, but can be degraded by the enzyme with collagen enzyme activity.
Proteinase is applied in the industries such as food, medicine and process hides more and more.In food industry, hydrolysis of animal micromicro obtains collagen polypeptide, and this hydrolysate has good trophic function, physicochemical property and physiological function; At medical industry, the Hydrolyzed Collagen obtained by collagen effectively can promote the healing of trauma skin, improves bone strength, suppress blood pressure rising etc.; In leather industry, Collagenase is widely used in immersion, depilation, the operation such as softening.Therefore, set up proteinase collagen hydrolysate vigor testing methods, be conducive to the performance understanding collagen hydrolysate enzyme, and reasonably apply according to its performance.
The casein that the assay method of prolease activity has to dissolve is as the LVU method of substrate, tryptic conversion method of multiplicity, folin's methods, the Anson method being substrate with the haemocyanin of sex change, the prolease activity assay method being substrate with succinyl-alanyl-alanyl-prolyl-leucyl-paranitroanilinum (Suc-Ala-Ala-Pro-Leu-pNA).But because casein, methemalbumin, Suc-Ala-Ala-Pro-Leu-pNA substrate to differ comparatively large with the character of collagen, thus the prolease activity that records of above method there is no actual directive significance to proteinase in the application in collagen process etc.Analysis of amino acids rule is this principle of the distinctive amino acid of collagen according to hydroxyproline, by mensuration proteinase, the hydroxyproline content in gained hydrolysate after collagen effect is calculated to the collagen hydrolysate vigor of proteinase, although the method can overcome substrate otherness, also exist and measure cumbersome, the cycle that records longer deficiency.
For the mensuration of the collagen hydrolysate vigor of proteinase, dyeing skin powder also can be adopted as substrate, this substrate is hydrolyzed under the catalytic action of proteinase, the enzymolysis product discharged is with dye molecule, make hydrolyzate have color and be easy to detect, as Azocoll, Congored and and the dyeing skin pruinescence such as CommassieBrilliantBlue be used as the vigor that substrate measures proteinase.But these dyestuffs mainly contain the acidity of the hydrophilic radical such as sulfonic group and carboxyl or direct dyestuff, they are mainly by the sulfonic group on dye molecule, carboxyl combines with Van der Waals force and hydrogen bond with collagen, this adhesion is more weak, under acid or alkaline conditions, dye molecule on dyeing skin foundation cream thing easily comes off or the color of dyestuff easily has greatly changed, cause the light absorption characteristics of dyeing skin foundation cream thing hydrolyzate unstable, thus the method is only suitable for the vigor measuring proteinase in neutral conditions usually, be not suitable for measuring prolease activity under the pH value condition of wide spectrum.Because the kind of proteinase is more, according to the Optimun pH of proteinase, proteinase can be divided into acidity, neutrality and alkali protease, and the method is difficult to the collagen hydrolysate vigor of Accurate Determining acidity and alkali protease, causes its application to be limited by very large.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, there is provided a kind of proteinase collagen hydrolysate vigour-testing method based on non-sex change dyeing skin foundation cream thing, to widen the pH value scope of application of the existing prolease activity assay method based on dyeing skin foundation cream thing.
The proteinase collagen hydrolysate vigour-testing method based on non-sex change dyeing skin foundation cream thing of foregoing invention object can be realized, comprise the following steps:
(1) preparation dyeing skin powder: the skin powder produced by Animal Skin central web layer being dispersed in concentration is that to form skin powder concentration in the NaCl solution of 0.005 ~ 0.2g/mL be the suspending liquid of 0.05 ~ 0.1g/mL, the impurity of Pi Fenzhong is made fully to enter liquid phase in 10 ~ 45 DEG C of vibrations, then 0.15 ~ 4.0 mass parts low temperature active dyestuff is used according to every mass parts skin powder, the amount of 0.05 ~ 1.2 mass parts colouring stabilizer, add low temperature active dye solution, the collagen generation covalent bond of low temperature active dyestuff and Pi Fenzhong is made in 10 ~ 45 DEG C of vibrations, add fixer solution again, filter after 10 ~ 45 DEG C fully vibration, NH is added in gained solid phase skin powder
4cl solution is to liquid phase in neutral, and gained skin powder is washed with water to eluate after filtration is afterwards colourless, and the Pi Fenzai after washing is through dehydration, dry, pulverizing obtained dyeing skin powder,
(2) dyeing skin powder typical curve is measured: the dyeing skin powder taking some different quality parts, the damping fluid of equivalent and the protein enzyme solution of equivalent is added respectively to each dyeing Pi Fenzhong, in 20 ~ 50 DEG C of vibrations to each dyeing skin powder complete hydrolysis, the some parts of hydrolyzates that the concentration in gradient obtaining dyeing skin powder hydrolysate changes, the absorbance of each part hydrolyzate is measured with determined wavelength, with the concentration of the skin powder that dyes be horizontal ordinate, absorbance for ordinate, drawing standard curve;
(3) reactant liquor to be measured and blank liquid is prepared
1 ~ 20mL damping fluid is added in 0.5 ~ 50mg dyes skin, be heated to measure temperature, add the protein enzyme solution 0.1 ~ 5mL doubly formed by proteinase dilute with water N, enzyme digestion reaction Tmin is carried out in mensuration temperature under vibration, the time chien shih dyeing skin powder of controlled enzymatic hydrolysis reaction is not totally consumed, centrifugal immediately after enzyme digestion reaction, gained supernatant is reactant liquor to be measured;
Add 1 ~ 20mL damping fluid to 0.5 ~ 50mg dyeing Pi Fenzhong, be heated to measure temperature, in mensuration temperature oscillation Tmin, add the protein enzyme solution 0.1 ~ 5mL doubly formed by proteinase dilute with water N, centrifugal immediately, gained supernatant is blank liquid;
(4) record absorbance and calculate enzyme activity: getting blank liquid and reactant liquor to be measured measures absorbance under determined wavelength, being denoted as A respectively
0and A, the collagen hydrolysate vigor of meter proteinase is calculated according to formula (1),
In formula (1), U is the collagen hydrolysate vigor of proteinase, A
0the absorbance of blank liquid and reactant liquor to be measured is respectively, V with A
1for the cumulative volume of reaction system after enzyme digestion reaction, mL, V
2for the addition of dilution N protein enzyme solution doubly, mL, T are the time of enzyme digestion reaction, and min, K are the slope of typical curve, and N is the extension rate of proteinase.
In technique scheme, described low temperature active dyestuff is X-type reactive dye, preferentially adopts Reactive Brilliant Blue X-BR, reactive brilliant red x-3b, reactive brilliant yellow X-7R or reactive brilliant orange X-GN.
In technique scheme, described proteinase is acid protease, neutral proteinase or alkali protease.
In technique scheme, described colouring stabilizer is Na
2cO
3, Na
2hCO
3, NaOH, at least one in alkaline agent.
In technique scheme, the concentration of described low temperature active dyestuff is 0.015 ~ 0.2g/mL, and the concentration of fixer solution is 0.01 ~ 0.2g/mL.
In technique scheme, the pH value of described damping fluid is 1 ~ 14, preferential adopt Bloomsbury smooth-Robinson, Robert damping fluid, sodium carbonate-bicarbonate damping fluid, sodium dihydrogen phosphate-sodium hydrate buffer solution or Tris-HCl damping fluid.
In technique scheme, the defining method of described determined wavelength is: to dyeing Pi Fenzhong with damping fluid and protein enzyme solution, in 20 ~ 50 DEG C of vibrations to dyeing skin powder complete hydrolysis, the hydrolyzate damping fluid of the dyeing skin powder obtained is diluted and measures its absorption curves in 400 ~ 800nm wavelength coverage, using maximum absorption wavelength as determined wavelength.
In technique scheme, the described skin powder produced by Animal Skin central web layer of step (1) and dyeing skin powder were the skin powder of 40 ~ 200 mesh sieves.
In technique scheme, the Animal Skin for the preparation of dyeing skin powder is preferably ox-hide, pigskin or sheepskin.
In technique scheme, step (1) after adding fixer solution, preferably 10 ~ 45 DEG C vibration 30 ~ 60min.
In technique scheme, enzyme activity is defined as: at mensuration temperature and pH value condition, and the amount that the dyeing skin powder of catalysis 1 μ g per minute is hydrolyzed into solution desirable proteins enzyme is a unit of activity.
Present invention also offers a kind of application of said method, said method is applicable to the collagen hydrolysate vigor measuring proteinase or protease preparation in process hides, food or field of medicaments.
Compared with prior art, the present invention has following beneficial effect:
1. the invention provides a kind of new method measuring proteinase collagen hydrolysate vigor, the method adopts low temperature active dyestuff to dye to skin powder, covalent bond is defined between the collagen of Pi Fenzhong and low temperature active dyestuff, because covalently bound form compares hydrogen bond, the combination of the forms such as Van der Waals force is more firm, even if the dye molecule therefore dyeed in skin powder is also not easy to come off under acidity and alkali condition, in addition the optical absorption characteristics of dyestuff has no significant change when potential of hydrogen is different, thus the method for the invention is applicable to measure enzyme activity under wider pH value condition, to acidity, neutral and alkali protease all uses.
2. because the method for the invention adopts low temperature active dyestuff to dye to skin powder at 10 ~ 45 DEG C, skin powder generation thermal denaturation can be avoided in the dyeing of this temperature, the collagen hydrolysate vigor of proteinase is measured using unmodified dyeing skin powder as substrate, reaction system is close to practical application, and the enzyme activity result thus adopting the method for the invention to measure is stronger to the directive significance be actually used in.
3. the hydrolyzate that the dyeing skin pruinescence protease hydrolytic adopted in the method for the invention is formed can produce absorption in visible-range, the concentration of hydrolysate is easy to detect, the method of the invention has advantage simple to operate, quick, sensitive, with low cost, is easy to apply.
Accompanying drawing explanation
Fig. 1 be dyeing skin powder hydrolyzate in embodiment 1 when pH value is 3 ~ 11 in the absorption curves of 400 ~ 800nm wavelength coverage, in Fig. 1, the curve that the absorbance at 600nm place is descending is record under pH value is the condition of 3,5,9,11 and 7 successively, and OD indicates absorbance.
Embodiment
Below by embodiment, the present invention is specifically described; what be necessary to herein means out is; embodiment is only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.
In following each embodiment, described PY enzyme is a kind of alkali protease, purchased from Chengdu Da Wei company limited; Described LT enzyme is a kind of acid protease, purchased from Shandong Longke Enzyme Co., Ltd.; The instrument adopted mainly contains: FA2004 type analysis balance, pHS-3C type pH meter, LG16-W type high speed micro hydro-extractor, UV2501PC ultraviolet-visible spectrophotometer, MS-100 type constant temperature blending instrument.
Embodiment 1
Based on the PY enzyme collagen hydro vigor testing methods of non-sex change dyeing skin foundation cream thing, step is as follows:
(1) reagent prepares
Preparation 0.1mol/L Bloomsbury smooth-Robinson, Robert damping fluid (B-R damping fluid): take 6.183g boric acid and be dissolved in appropriate distilled water, add 11.5294g phosphoric acid and 6.005g acetic acid, be settled to 1L with behind NaOH solution adjust ph to 3 ~ 12 of 4mol/L with distilled water.
The NaCl solution of preparation 0.005g/mL: take 0.5gNaCl distilled water and be settled to 100mL.
The Reactive Brilliant Blue X-BR solution of preparation 0.015g/mL: take 1.5g Reactive Brilliant Blue X-BR distilled water and be settled to 100mL.
The Na of preparation 0.01g/mL
2cO
3solution: take 1gNa
2cO
3100mL is settled to distilled water.
The NH of preparation 0.05g/mL
4cl solution: take 5gNH
4cl distilled water is settled to 100mL.
(2) preparation dyeing skin powder: ox-hide, after immersion, degreasing, is got central web layer and fully washed, then with acetone dehydration, natural drying, pulverizing, cross 40 mesh sieves after obtained skin powder; Take this skin powder 5g and be placed in conical flask, add NaCl solution 100mL, vibration to ox-hide is dispersed in NaCl solution and forms skin powder suspending liquid, the impurity of Pi Fenzhong is made to enter liquid phase in 35 DEG C of vibration 2h, add Reactive Brilliant Blue X-BR solution 50mL, make the collagen generation covalent bond of Reactive Brilliant Blue X-BR and Pi Fenzhong in 35 DEG C of vibration 60min, add Na
2cO
3solution 50mL, then in 35 DEG C of vibration 60min, filter, in gained solid phase, add NH
4cl solution regulates the pH value of liquid phase to being about 7, and spending deionized water skin powder is colorless state to eluate, is dewatered by the skin powder acetone after washing, natural drying, grinding, crosses 50 mesh sieves obtained dyeing skin powder;
(3) determined wavelength is determined: take 50mg dyeing skin powder and be placed in conical flask, add 4mLB-R damping fluid and 1mLPY enzyme solutions, in 40 DEG C of vibrations to dyeing skin powder complete hydrolysis in constant temperature blending instrument, obtain the hydrolyzate of dyeing skin powder, get this hydrolyzate 5 parts, adjust ph to 3 respectively, 5, 7, 9, 11 obtain 5 samples, then in 400 ~ 800nm wavelength coverage, detect the absorption curves of each sample respectively, result as shown in Figure 1, using maximum absorption wavelength 600nm as determined wavelength, be it can also be seen that by Fig. 1, the Reactive Brilliant Blue X-BR molecule that the present embodiment adopts is at wider pH value range all energy stable existences, its optical absorption characteristics does not have significant change.
(4) measure dyeing skin powder typical curve: accurately take 0,10,20,30,40, the 50mg skin powder that dyes is placed in conical flask, add B-R damping fluid and the 1mLPY enzyme solutions of 4mLpH=9 respectively, in 40 DEG C of vibrations to dyeing skin powder complete hydrolysis in constant temperature blending instrument, obtain the concentration in gradient change hydrolyzate of 6 parts of dyeing skin powder hydrolysates, record the absorbance of each hydrolyzate at 600nm wavelength place, then with the skin powder concentration that dyes for horizontal ordinate, absorbance is the typical curve that ordinate draws dyeing skin powder, slope K=0.435 of this typical curve.
(5) reactant liquor to be measured and blank liquid is prepared
Reactant liquor to be measured: PY enzyme distilled water diluting 30000 is doubly made PY enzyme solutions.Take 5 ± 0.1mg and dye skin powder as substrate, add the B-R damping fluid of 1mLpH=9, PY enzyme solutions described in 0.2mL is added after being heated to 40 DEG C, in constant temperature blending instrument at oscillating condition in 40 DEG C of enzyme digestion reaction 30min, the skin powder that now dyes is totally consumed, centrifugal immediately, gained supernatant is reactant liquor to be measured, and after enzyme digestion reaction, the cumulative volume of reaction system is 1.2mL;
Blank liquid: take 5 ± 0.1mg and dye skin powder as substrate, add the B-R damping fluid of 1mLpH=9, be heated to 40 DEG C, in 40 DEG C of vibration 30min in constant temperature blending instrument, then the PY enzyme solutions of 0.2mL dilute with water 15000 times is added, centrifugal immediately, gained supernatant is blank liquid.
(6) measure absorbance calculate enzyme activity: get blank liquid and reactant liquor to be measured measures absorbance at 600nm wavelength place, be denoted as A respectively
0and A; The collagen hydrolysate vigor of meter PY enzyme is calculated according to formula (1), the collagen hydrolysate vigor of PY enzyme is defined as: temperature be 40 DEG C, under the condition of pH=9.0, the amount that the dyeing skin powder of catalysis 1 μ g per minute is hydrolyzed into PY enzyme needed for solution is a unit of activity U.
In formula (1), U is the collagen hydrolysate vigor of proteinase, A
0the absorbance of blank liquid and reactant liquor to be measured is respectively, V with A
1for the cumulative volume of reaction system after enzyme digestion reaction, mL, V
2for the addition of dilution N protein enzyme solution doubly, mL, T are the time of enzyme digestion reaction, and min, K are the slope of typical curve, and N is the extension rate of proteinase.A=0.358 (mean value measured for 5 times), A
0=0.088, V
1=1.2mL, V
2=0.2mL, T=30min, K=0.435, N=30000, bring formula (1) into, then:
Embodiment 2
Based on the LT enzyme collagen hydro vigor testing methods of non-sex change dyeing skin foundation cream thing, step is as follows:
Reagent prepares identical with embodiment 1 with the process of preparation dyeing skin powder, and determined wavelength is 600nm.
(1) measure dyeing skin powder typical curve: accurately take 0,10,20,30,40, the 50mg skin powder that dyes is placed in conical flask, add B-R damping fluid and the 1mLLT enzyme solutions of 4mLpH=4 respectively, in 40 DEG C of vibrations to dyeing skin powder complete hydrolysis in constant temperature blending instrument, obtain the concentration in gradient change hydrolyzate of 6 parts of dyeing skin powder hydrolysates, record the absorbance of each hydrolyzate at 600nm wavelength place, then with the skin powder concentration that dyes for horizontal ordinate, absorbance is the typical curve that ordinate draws dyeing skin powder, slope K=0.435 of this typical curve.
(2) reactant liquor to be measured and blank liquid is prepared
Reactant liquor to be measured: LT enzyme distilled water diluting 200 is doubly made LT enzyme solutions.Take 5 ± 0.1mg and dye skin powder as substrate, add the B-R damping fluid of 1mLpH=4, LT enzyme solutions described in 0.2mL is added after being heated to 40 DEG C, in constant temperature blending instrument under oscillating condition in 40 DEG C of enzyme digestion reaction 30min, the skin powder that now dyes is totally consumed, centrifugal immediately, gained supernatant is reactant liquor to be measured, and after enzyme digestion reaction, the cumulative volume of reaction system is 1.2mL;
Blank liquid: take 5 ± 0.1mg and dye skin powder as substrate, add the B-R damping fluid of 1mLpH=4, be heated to 40 DEG C, in 40 DEG C of vibration 30min in constant temperature blending instrument, then the LT enzyme solutions of 0.2mL dilute with water 15000 times is added, centrifugal immediately, gained supernatant is blank liquid.
(3) measure absorbance calculate enzyme activity: get blank liquid and reactant liquor to be measured measures absorbance at 600nm wavelength place, be denoted as A respectively
0and A; The collagen hydrolysate vigor of meter LT enzyme is calculated according to formula (1), the collagen hydrolysate vigor of LT enzyme is defined as: temperature be 40 DEG C, under the condition of pH=4.0, the amount that the dyeing skin powder of catalysis 1 μ g per minute is hydrolyzed into PY enzyme needed for solution is a unit of activity U.
In formula (1), U is the collagen hydrolysate vigor of proteinase, A
0the absorbance of blank liquid and reactant liquor to be measured is respectively, V with A
1for the cumulative volume of reaction system after enzyme digestion reaction, mL, V
2for the addition of dilution N protein enzyme solution doubly, mL, T are the time of enzyme digestion reaction, and min, K are the slope of typical curve, and N is the extension rate of proteinase.A=0.408 (mean value measured for 5 times), A
0=0.093, V
1=1.2mL, V
2=0.2mL, T=30min, K=0.435, N=200, bring formula (1) into, then:
Embodiment 3
Operation based on the PY enzyme collagen hydro vigor testing methods of non-sex change dyeing skin foundation cream thing is substantially the same manner as Example 1, and difference is the sodium carbonate-bicarbonate damping fluid adopting pH=9, and the preparation method of dyeing skin powder is as follows:
Ox-hide, after immersion, degreasing, is got central web layer and is fully washed, then with acetone dehydration, and obtained skin powder after natural drying, pulverizing, excessively 100 mesh sieves; Take this skin powder 5g and be placed in conical flask, adding concentration is 0.1g/mLNaCl solution 50mL, vibration to ox-hide is dispersed in NaCl solution and forms skin powder suspending liquid, the impurity of Pi Fenzhong is made to enter liquid phase in 45 DEG C of vibration 1h, add the reactive brilliant red x-3b solution 100mL that concentration is 0.2g/mL, make the collagen generation covalent bond of reactive brilliant red x-3b and Pi Fenzhong in 45 DEG C of vibration 60min, add the Na that concentration is 0.2g/mL
2hCO
3solution 30mL, then in 45 DEG C of vibration 60min, filter, in gained solid phase, add the NH that concentration is 0.05g/mL
4cl solution regulates the pH value of liquid phase to being about 7, and spending deionized water skin powder is colorless state to eluate, is dewatered by the skin powder acetone after washing, natural drying, grinding, crosses 200 mesh sieves obtained dyeing skin powder.
Embodiment 4
Operation based on the PY enzyme collagen hydro vigor testing methods of non-sex change dyeing skin foundation cream thing is substantially the same manner as Example 1, and difference is the Tris-HCl damping fluid adopting pH=9, and the preparation method of dyeing skin powder is as follows:
Ox-hide, after immersion, degreasing, is got central web layer and is fully washed, then with acetone dehydration, and obtained skin powder after natural drying, pulverizing, excessively 80 mesh sieves; Take this skin powder 5g and be placed in conical flask, adding concentration is 0.2g/mLNaCl solution 100mL, vibration to ox-hide is dispersed in NaCl solution and forms skin powder suspending liquid, the impurity of Pi Fenzhong is made to enter liquid phase in 10 DEG C of vibration 2h, add the reactive brilliant yellow X-7R solution 30mL that concentration is 0.15g/mL, the collagen generation covalent bond of reactive brilliant yellow X-7R and Pi Fenzhong is made in 10 DEG C of vibration 30min, add the NaOH solution 10mL that concentration is 0.1g/mL, again in 10 DEG C of vibration 40min, filter, in gained solid phase, add the NH that concentration is 0.05g/mL
4cl solution regulates the pH value of liquid phase to being about 7, and spending deionized water skin powder is colorless state to eluate, is dewatered by the skin powder acetone after washing, natural drying, grinding, crosses 100 mesh sieves obtained dyeing skin powder.
Embodiment 5
Operation based on the PY enzyme collagen hydro vigor testing methods of non-sex change dyeing skin foundation cream thing is substantially the same manner as Example 1, and difference is the Tris-HCl damping fluid adopting pH=9, and the preparation method of dyeing skin powder is as follows:
Ox-hide, after immersion, degreasing, is got central web layer and is fully washed, then with acetone dehydration, and obtained skin powder after natural drying, pulverizing, excessively 100 mesh sieves; Take this skin powder 5g and be placed in conical flask, adding concentration is 0.01g/mLNaCl solution 100mL, vibration to ox-hide is dispersed in NaCl solution and forms skin powder suspending liquid, the impurity of Pi Fenzhong is made to enter liquid phase in 35 DEG C of vibration 2h, add the reactive brilliant orange X-GN solution 50mL that concentration is 0.05g/mL, the collagen generation covalent bond of reactive brilliant orange X-GN and Pi Fenzhong is made in 10 DEG C of vibration 60min, add the NaOH solution 30mL that concentration is 0.05g/mL, again in 35 DEG C of vibration 30min, filter, in gained solid phase, add the NH that concentration is 0.05g/mL
4cl solution adjust ph is to being about 7, and spending deionized water skin powder is colorless state to eluate, is dewatered by the skin powder acetone after washing, natural drying, grinding, crosses 200 mesh sieves obtained dyeing skin powder.
Claims (10)
1., based on a proteinase collagen hydrolysate vigour-testing method for non-sex change dyeing skin foundation cream thing, it is characterized in that comprising the following steps:
(1) preparation dyeing skin powder: the skin powder produced by Animal Skin central web layer being dispersed in concentration is that to form skin powder concentration in the NaCl solution of 0.005 ~ 0.2g/mL be the suspending liquid of 0.05 ~ 0.1g/mL, the impurity of Pi Fenzhong is made fully to enter liquid phase in 10 ~ 45 DEG C of vibrations, then 0.15 ~ 4.0 mass parts low temperature active dyestuff is used according to every mass parts skin powder, the amount of 0.05 ~ 1.2 mass parts colouring stabilizer, add low temperature active dye solution, the collagen generation covalent bond of low temperature active dyestuff and Pi Fenzhong is made in 10 ~ 45 DEG C of vibrations, add fixer solution again, filter after 10 ~ 45 DEG C fully vibration, NH is added in gained solid phase skin powder
4cl solution is to liquid phase in neutral, and gained skin powder is washed with water to eluate after filtration is afterwards colourless, and the Pi Fenzai after washing is through dehydration, dry, pulverizing obtained dyeing skin powder,
(2) dyeing skin powder typical curve is measured: the dyeing skin powder taking some different quality parts, the damping fluid of equivalent and the protein enzyme solution of equivalent is added respectively to each dyeing Pi Fenzhong, in 20 ~ 50 DEG C of vibrations to each dyeing skin powder complete hydrolysis, the some parts of hydrolyzates that the concentration in gradient obtaining dyeing skin powder hydrolysate changes, the absorbance of each part hydrolyzate is measured with determined wavelength, with the concentration of the skin powder that dyes be horizontal ordinate, absorbance for ordinate, drawing standard curve;
(3) reactant liquor to be measured and blank liquid is prepared
1 ~ 20mL damping fluid is added in 0.5 ~ 50mg dyes skin, be heated to measure temperature, add the protein enzyme solution 0.1 ~ 5mL doubly formed by proteinase dilute with water N, enzyme digestion reaction Tmin is carried out in mensuration temperature under vibration, the time chien shih dyeing skin powder of controlled enzymatic hydrolysis reaction is not totally consumed, centrifugal immediately after enzyme digestion reaction, gained supernatant is reactant liquor to be measured;
Add 1 ~ 20mL damping fluid to 0.5 ~ 50mg dyeing Pi Fenzhong, be heated to measure temperature, in mensuration temperature oscillation Tmin, add the protein enzyme solution 0.1 ~ 5mL doubly formed by proteinase dilute with water N, centrifugal immediately, gained supernatant is blank liquid;
(4) record absorbance and calculate enzyme activity: getting blank liquid and reactant liquor to be measured measures absorbance under determined wavelength, being denoted as A respectively
0and A, the collagen hydrolysate vigor of meter proteinase is calculated according to formula (1),
In formula (1), U is the collagen hydrolysate vigor of proteinase, A
0the absorbance of blank liquid and reactant liquor to be measured is respectively, V with A
1for the cumulative volume of reaction system after enzyme digestion reaction, mL, V
2for the addition of dilution N protein enzyme solution doubly, mL, T are the time of enzyme digestion reaction, and min, K are the slope of typical curve, and N is the extension rate of proteinase.
2., according to claim 1 based on the proteinase collagen hydrolysate vigour-testing method of non-sex change dyeing skin foundation cream thing, it is characterized in that described low temperature active dyestuff is X-type reactive dye.
3., according to claim 2 based on the proteinase collagen hydrolysate vigour-testing method of non-sex change dyeing skin foundation cream thing, it is characterized in that described X-type reactive dye are Reactive Brilliant Blue X-BR, reactive brilliant red x-3b, reactive brilliant yellow X-7R or reactive brilliant orange X-GN.
4., according to claim 1 based on the proteinase collagen hydrolysate vigour-testing method of non-sex change dyeing skin foundation cream thing, it is characterized in that described proteinase is acid protease, neutral proteinase or alkali protease.
5., according to claim 1 based on the proteinase collagen hydrolysate vigour-testing method of non-sex change dyeing skin foundation cream thing, it is characterized in that described colouring stabilizer is Na
2cO
3, Na
2hCO
3, NaOH, at least one in alkaline agent.
6., according to claim 1 based on the proteinase collagen hydrolysate vigour-testing method of non-sex change dyeing skin foundation cream thing, it is characterized in that the concentration of described low temperature active dyestuff is 0.015 ~ 0.2g/mL, the concentration of fixer solution is 0.01 ~ 0.2g/mL.
7., according to claim 1 based on the proteinase collagen hydrolysate vigour-testing method of non-sex change dyeing skin foundation cream thing, it is characterized in that the pH value of described damping fluid is 1 ~ 14.
8. according to claim 6 based on the proteinase collagen hydrolysate vigour-testing method of non-sex change dyeing skin foundation cream thing, it is characterized in that described damping fluid be Bloomsbury smooth-Robinson, Robert damping fluid, sodium carbonate-bicarbonate damping fluid, sodium dihydrogen phosphate-sodium hydrate buffer solution or Tris-HCl damping fluid.
9. according to the proteinase collagen hydrolysate vigour-testing method of the skin foundation cream thing that dyes based on non-sex change one of claim 1 to 8 Suo Shu, it is characterized in that the defining method of described determined wavelength is: to dyeing Pi Fenzhong with damping fluid and protein enzyme solution, in 20 ~ 50 DEG C of vibrations to dyeing skin powder complete hydrolysis, the hydrolyzate damping fluid of the dyeing skin powder obtained is diluted and measures its absorption curves in 400 ~ 800nm wavelength coverage, using maximum absorption wavelength as determined wavelength.
10. the described proteinase collagen hydrolysate vigour-testing method based on non-sex change dyeing skin foundation cream thing of one of claim 1 to 9, is applicable to the collagen hydrolysate vigor measuring proteinase or protease preparation in process hides, food or field of medicaments.
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| CN108588175A (en) * | 2018-03-27 | 2018-09-28 | 四川大学 | Assay method and its application of the protease to elastic protein fiber catalyzing hydrolysis vigor |
| CN108845133A (en) * | 2018-03-27 | 2018-11-20 | 四川大学 | A kind of catalyzing hydrolysis method of evaluating performance of protease to chrome tanning collagen fabric |
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