CN105861608A - Method for preparing tortoise protein polypeptides - Google Patents
Method for preparing tortoise protein polypeptides Download PDFInfo
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- CN105861608A CN105861608A CN201610373189.8A CN201610373189A CN105861608A CN 105861608 A CN105861608 A CN 105861608A CN 201610373189 A CN201610373189 A CN 201610373189A CN 105861608 A CN105861608 A CN 105861608A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
Abstract
The invention discloses a preparation method and application of protein polypeptides in tortoise plastron. The preparation comprises the following steps: pulverizing tortoise plastron, passing through a 50-mesh screen, mixing a decalcifying agent solution with the tortoise fine powder, sufficiently reacting, adding a NaOH solution, and sufficiently reacting to obtain a reaction solution; centrifuging the reaction solution, maintaining the precipitate, and cleaning the precipitate to obtain the decalcified tortoise fine powder; mixing double distilled water with the decalcified tortoise fine powder, and boiling to obtain a tortoise protein leaching solution; and adding proteinase into the tortoise protein leaching solution, and sufficiently reacting to obtain the tortoise protein polypeptides. The method saves the time and labor, and can perform industrial production simply, quickly and efficiently. The pilot plant test proves that the technological parameters can be transferred to industrialization.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of method preparing Testudinis protein polypeptide.
Background technology
Carapax et Plastrum Testudinis, i.e. Carapax Et Plastrum Testudinis, for carapace and the sternite of Testudinidae animal Testudinis, have replenishing kidney and strengthening bone, nourishing YIN for suppressing the hyperactive YANG,
The effects such as controlling nocturnal emission with astringent drugs hemostasis, replenishing blood for nourishing heart, for one of conventional famous and precious tonic herb.Ancient times recipe such as Bushen Pills,
The Chinese patent medicines such as full deer Da Bu Wan, fixed female ball all contain Carapax Et Plastrum Testudinis, and are to be ground into fine powder to be used as medicine.When the Warring states
The Classic of Mountains and Rivers in generation to " Dragon Lord book on Chinese herbal medicine warp " to " draft detailed outline " arrives " pharmacopeia " in modern times again and all records
Edible and the medicinal efficacy of Testudinis.Carapax Et Plastrum Testudinis is rich in several amino acids, protein, calcium and multiple beneficial trace unit
Element, is therefore also commonly used in functional food and medicated diet.
Biologically active polypeptide refers to regulate the vital movement of living organism or have some physiological activity
The general name of the small molecule active polypeptide of (such as hormonal action, immunomodulating, antioxidation, antitumor etc.),
These materials are widely present in animal, plant and microorganism.Therefore, biologically active polypeptide be screening of medicaments,
Prepare the natural resources treasure-house of vaccine and food additive.
The structure of biologically active peptide can be from simple dipeptides to the polypeptide of relatively macromole.By 2 to 10 amino
The linear peptides that acid is formed by peptide bond is referred to as oligopeptide or little peptide；By peptide linkage get up more than 10 aminoacid
Polymer then be referred to as polypeptide.In recent years, scientist it has been investigated that, protein is through digestive tract enzymatic hydrolysis
Rear mainly with absorbance in intestinal of the form of little peptide or polypeptide higher than protein and aminoacid, and polypeptide tool
There is promotion lipid metabolism, reduce cholesterol, promotion mineral absorption and promote the physiological functions such as immunity of organism, tool
There are preferable acid, heat stability and water solublity and good physiologically active.Therefore, use zymolysis technique by Carapax Et Plastrum Testudinis
Proteolytic enzyme becomes polypeptide, can be effectively improved the utilization rate of Carapax Et Plastrum Testudinis protein, increases Carapax Et Plastrum Testudinis value-added content of product, opens
Send deep processing Carapax Et Plastrum Testudinis protein product.
Biological polypeptide mainly has four kinds of sources, respectively proteolysis polypeptide, fermentable metabolising polypeptide,
Natural biological polypeptide and chemically synthesized polypeptide.Wherein enzymatic isolation method not only yield is big, low cost, course of reaction not
Harmful substance being produced, meeting the food development trend of " natural, nutrition, safety ", so becoming current
Main polypeptide generates method.
Summary of the invention
It is an object of the invention to propose a kind of method simply, efficiently preparing Carapax et Plastrum Testudinis protein polypeptide from Carapax et Plastrum Testudinis.
In order to realize this purpose, the technical solution used in the present invention is: the preparation of a kind of Testudinis protein polypeptide
Method, comprises the steps: that taking dry Carapax et Plastrum Testudinis grinding and sieving obtains Testudinis fine powder；It is 1 according to solid-to-liquid ratio:
Decalcifying agent solution is mixed with described Testudinis fine powder and fully reacts by 2～1:10, is subsequently added into NaOH solution, fills
Reactant liquor is obtained after dividing reaction；Precipitation is retained, after precipitation obtains decalcification after cleaning up after centrifugal for reactant liquor
Testudinis fine powder；Being that distilled water is mixed by 1:5～1:20 with the Testudinis fine powder after decalcification according to solid-to-liquid ratio, 1h～6h is boiled in boiling
After obtain Testudinis albumen dissolution fluid；And according to the enzyme amount of 5000U～30000U in described Testudinis albumen dissolution fluid
Add protease, fully obtain Testudinis protein polypeptide after reaction.
Further, decalcifying agent is HCl or EDTA.
Further, decalcifying agent is EDTA, and the concentration of described EDTA is 0.1～0.75mol/L.
Further, decalcifying agent solution shakes after mixing with described Testudinis fine powder so that it is fully reacts, shakes
The time of shaking is 1～6H.
Further, after decalcifying agent solution fully react with described Testudinis fine powder, add NaOH shake,
It is made fully to react, the concentration of NaOH is 1mol/L, and the shaking time is 6～24H.
Further, reactant liquor being centrifuged the rotating speed used is 12000rpm.
Further, the method obtaining the Testudinis fine powder after decalcification after precipitation being cleaned up is to use deionized water
Clean precipitation 2 times.
Further, the Testudinis fine powder after decalcification adds deionized water according to solid-to-liquid ratio 1:15, and boiling boils 6 hours.
Further, protease be trypsin, pepsin, papain, alkaline protease and in
One in property protease.
Further, protease is neutral protease, and enzyme amount is 10000～30000U, enzymolysis time be 6～
24h。
After present invention decalcifying agent carries out pre-treatment to Testudinis powder, Testudinis protein extraction rate processes than non-decalcification and adds
6.8 times, after process is boiled in decalcification, Testudinis protein extraction rate adds 34.3 times than untreated.Through decalcification, boiling
After boiling, being directly added into protease hydrolyzed Testudinis albumen dissolution fluid, degree of hydrolysis reaches 13.62%, and protein polypeptide obtains
Rate is 9.64%, and the method is the most efficient, it is possible to quickly realize industrialization.
Accompanying drawing explanation
Fig. 1 is that the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method is molten with the albumen of other treatment conditions Tortoise Shells
Go out rate comparison diagram；
Fig. 2 is the decalcification efficiency comparative figure of the different decalcifying agents of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method；
Fig. 3 is the decalcification efficiency pair of the different EDTA concentration of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Than figure；
Fig. 4 is the decalcification efficiency comparative figure of the different solid ratio of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method；
Fig. 5 is that the calcium deposit of the different shaking times of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method is containing the most right
Than figure；
Fig. 6 is the Testudinis amyloid proteins dissolution of the different times of boiling of boiling of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Rate comparison diagram；
Fig. 7 is that monkey is boiled in the decalcification Testudinis powder boiling of the different solid ratio of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Albumen stripping quantity comparison diagram；
Fig. 8 is the different protease hydrolysis Carapax Et Plastrum Testudinis protein liquids of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Degree of hydrolysis comparison diagram；
Fig. 9 is the enzymolysis degree of hydrolysis contrast of the different enzyme dosages of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Figure；
Figure 10 is the enzymolysis degree of hydrolysis pair of the different enzymolysis times of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Than figure；
Figure 11 is the enzymolysis degree of hydrolysis pair of the different concentration of substrate of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Than figure；
Figure 12 is the enzymolysis degree of hydrolysis contrast of the different pH value of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Figure；
Figure 13 is the enzymolysis degree of hydrolysis pair of the condition of different temperatures of the present invention a kind of Carapax Et Plastrum Testudinis protein polypeptide preparation method
Than figure.
Detailed description of the invention
Following example are merely to illustrate the present invention, and are not limited to the scope of the present invention.Without departing substantially from this
In the case of spirit and essence, the amendment that the inventive method, step or condition are made or replacement, all
Belong to the scope of the present invention.
If not specializing, the routine that technological means used in embodiment is well known to those skilled in the art
Means.
Embodiment 1
A kind of method preparing Testudinis protein polypeptide, comprises the steps:
(1) take dry Tortoise Shell and be broken into coarse powder, obtain Testudinis fine powder through 50 mesh sieves；
(2) weigh the 5g Testudinis fine powder of gained in step (1), add the EDTA of the 0.75mol/L of 10ml
Mix, shake 1h, add 1mol/L NaOH and continue shaking 6h, obtain reactant liquor；
(3) use the reactant liquor of 12000rpm centrifugation step (2) gained at normal temperatures, remove supernatant, use
Deionized water cleans after precipitating 2 times and is centrifuged the Testudinis fine powder after going supernatant to obtain decalcification；
(4) in step (3), the decalcification Testudinis fine powder of gained adds 25ml distilled water, and boiling is boiled 1 hour,
Testudinis albumen dissolution fluid；
(5) according to the enzyme amount of 20000U in the Testudinis albumen dissolution fluid of step (4) gained, Trypsin is added
Enzyme enzymolysis Testudinis collagen solution 6h, it is thus achieved that Testudinis protein polypeptide.
Embodiment 2
A kind of method preparing Testudinis protein polypeptide, comprises the steps:
(1) take dry Tortoise Shell and be broken into coarse powder, obtain Testudinis fine powder through 50 mesh sieves；
(2) weigh the 5g Testudinis fine powder of gained in step (1), add the EDTA of the 0.5M of 50mol/L
Mix, shake 1h, add 1mol/L NaOH and continue shaking 24h, obtain reactant liquor；
(3) use the reactant liquor of 12000rpm centrifugation step (2) gained at normal temperatures, remove supernatant, use
Deionized water cleans after precipitating 2 times and is centrifuged the Testudinis fine powder after going supernatant to obtain decalcification；
(4) in step (3), the decalcification Testudinis fine powder of gained adds 30ml distilled water, and boiling is boiled 2 hours,
Testudinis albumen dissolution fluid；
(5) according to the enzyme amount of 5000U in the Testudinis albumen dissolution fluid of step (4) gained, pepsin is added
Enzyme enzymolysis Testudinis collagen solution 24h, it is thus achieved that Testudinis protein polypeptide.
Embodiment 3
A kind of method preparing Testudinis protein polypeptide, comprises the steps:
(1) take dry Tortoise Shell and be broken into coarse powder, obtain Testudinis fine powder through 50 mesh sieves；
(2) weigh the 5g Testudinis fine powder of gained in step (1), add the EDTA of the 0.3mol/L of 40ml
Mix, shake 1h, add 1mol/L NaOH and continue shaking 15h, obtain reactant liquor；
(3) use the reactant liquor of 12000rpm centrifugation step (2) gained at normal temperatures, remove supernatant, use
Deionized water cleans after precipitating 2 times and is centrifuged the Testudinis fine powder after going supernatant to obtain decalcification；
(4) in step (3), the decalcification Testudinis fine powder of gained adds 100ml distilled water, and boiling is boiled 3 hours,
Obtain Testudinis albumen dissolution fluid；
(5) according to the enzyme amount of 100000U in the Testudinis albumen dissolution fluid of step (4) gained, Fructus Chaenomelis is added
Protease hydrolyzed Testudinis collagen solution 15h, it is thus achieved that Testudinis protein polypeptide.
Embodiment 4
A kind of method preparing Testudinis protein polypeptide, comprises the steps:
(1) take dry Tortoise Shell and be broken into coarse powder, obtain Testudinis fine powder through 50 mesh sieves；
(2) weighing the 5g Testudinis fine powder of gained in step (1), the HCL of the 1mol/L adding 30ml enters
Row mixing, shakes 3h, adds 1mol/L NaOH and continues shaking 12h, obtains reactant liquor；
(3) use the reactant liquor of 12000rpm centrifugation step (2) gained at normal temperatures, remove supernatant, use
Deionized water cleans after precipitating 2 times and is centrifuged the Testudinis fine powder after going supernatant to obtain decalcification；
(4) in step (3), the decalcification Testudinis fine powder of gained adds 50ml distilled water, and boiling is boiled 4 hours,
Testudinis albumen dissolution fluid；
(5) according to the enzyme amount of 30000U in the Testudinis albumen dissolution fluid of step (4) gained, alkalescence egg is added
White enzyme enzymolysis Testudinis collagen solution 12h, it is thus achieved that Testudinis protein polypeptide.
Embodiment 5
A kind of method preparing Testudinis protein polypeptide, comprises the steps:
(1) take dry Tortoise Shell and be broken into coarse powder, obtain Testudinis fine powder through 50 mesh sieves；
(2) weigh the 5g Testudinis fine powder of gained in step (1), add the HCL of the 0.5mol/L of 30ml
Mix, shake 1h, add 1mol/L NaOH and continue shaking 20h, obtain reactant liquor；
(3) use the reactant liquor of 12000rpm centrifugation step (2) gained at normal temperatures, remove supernatant, use
Deionized water cleans after precipitating 2 times and is centrifuged the Testudinis fine powder after going supernatant to obtain decalcification；
(4) in step (3), the decalcification Testudinis fine powder of gained adds 80ml distilled water, and boiling is boiled 5 hours,
Testudinis albumen dissolution fluid；
(5) according to the enzyme amount of 25000U in the Testudinis albumen dissolution fluid of step (4) gained, neutral egg is added
White enzyme enzymolysis Testudinis collagen solution 24h, it is thus achieved that Testudinis protein polypeptide.
Embodiment 6
A kind of method preparing Testudinis protein polypeptide, comprises the steps:
(1) take dry Tortoise Shell and be broken into coarse powder, obtain Testudinis fine powder through 50 mesh sieves；
(2) weigh the 5g Testudinis fine powder of gained in step (1), add the EDTA of the 0.1mol/L of 30ml
Mix, shake 3h, add 1mol/L NaOH and continue shaking 15h, obtain reactant liquor；
(3) use the reactant liquor of 12000rpm centrifugation step (2) gained at normal temperatures, remove supernatant, use
Deionized water cleans after precipitating 2 times and is centrifuged the Testudinis fine powder after going supernatant to obtain decalcification；
(4) in step (3), the decalcification Testudinis fine powder of gained adds 60ml distilled water, and boiling is boiled 6 hours,
Testudinis albumen dissolution fluid；
(5) according to the enzyme amount of 15000U in the Testudinis albumen dissolution fluid of step (4) gained, pepsin is added
Enzyme enzymolysis Testudinis collagen solution 10h, it is thus achieved that Testudinis protein polypeptide.
The Testudinis amyloid proteins dissolution rate of embodiment 7 different pre-treatments condition
Experiment condition is as follows:
Experimental result is shown in Fig. 1.
The different decalcifying agent impact on Testudinis powder decalcification efficiency of embodiment 8
Respectively with the EDTA solution of 0.25mol/L and 0.5mol/L of 5 times of quality, 0.5mol/L and
The hydrochloric acid solution of 1.0mol/L shakes 6h to 5g Testudinis fine powder (crossing 50 mesh) respectively, then with the 1 of 50mL
After the NaOH solution of mol/L soaks shaking 1d, centrifugal, take supernatant, adjust pH value to 3.8, claim
Precipitation quality, determines the decalcification effect of different decalcifying agent.Result is shown in Fig. 2.
The impact on Testudinis powder decalcification efficiency of the embodiment 9 decalcifying agent variable concentrations
Weigh 5g Testudinis fine powder (crossing 50 mesh), be the EDTA solution that 1:6 adds variable concentrations according to solid-to-liquid ratio
(0.125,0.25,0.5,0.75), shakes 6h, then soaks by the NaOH solution of the 1mol/L of 50mL
After bubble shaking 1d, centrifugal.Take supernatant, adjust pH value to 3.8, claim precipitation quality, determine different de-
The decalcification effect of calcium preparation concentration.Result is shown in Fig. 3.
The impact of embodiment 10 different solid comparison Testudinis powder decalcification efficiency
Weigh 5g Testudinis fine powder (crossing 50 mesh), be that 1:2,1:4,1:6,1:8,1:10 add in fact according to solid-to-liquid ratio
Execute the solution of the optimal EDTA concentration (0.5M) of example 9 gained, shake 6h, then with the 1mol/L of 50mL
NaOH solution soak shaking 1d after, centrifuging and taking supernatant, adjust pH value to 3.8, claim precipitation quality,
Determine the decalcification effect of different solid ratio.Result is shown in Fig. 4.
The different decalcification time impact on Testudinis powder decalcification efficiency of embodiment 11
Weigh 5g Testudinis fine powder (cross 50 mesh), add 0.5M EDTA 30mL according to 1:6 solid-to-liquid ratio, shaking 6,
12,18,24,48h, then after soaking shaking 1d by the NaOH solution of the 1mol/L of 50mL, from
The heart takes supernatant, adjusts pH value to 3.8, claims precipitation quality, determines the decalcification effect of different decalcification time.
Result is shown in Fig. 5.
The boiling that embodiment 12 is different boils the time to the impact of Testudinis protein extraction rate
Weighing 5g Testudinis fine powder (crossing 50 mesh), the optimal conditions obtained according to embodiment 1-5 carries out Testudinis fine powder and takes off
Calcium, is centrifuged after removing supernatant, and deposit sample adds distilled water according to 1:10 solid-to-liquid ratio, steaming and decocting respectively in boiling water bath
1,2,3,4,5,6h, measure total protein content (TCA method) in supernatant, determine optimal digestion time.
Result is shown in Fig. 6.
Embodiment 13 different solid comparison Testudinis protein extraction rate affects
Weighing 5g Testudinis fine powder (crossing 50 mesh), the optimal conditions obtained according to embodiment 1-5 carries out Testudinis fine powder
Decalcification, is centrifuged after removing supernatant, and deposit sample adds distilled water according to 1:5,1:10,1:15,1:20 solid-to-liquid ratio,
According to steaming and decocting 6h in the result boiling water bath of embodiment 6, measure total protein content (TCA method) in supernatant, really
Fixed optimal solid-to-liquid ratio.Result is shown in Fig. 7.
The different protease impact on Testudinis albumen dissolution fluid degree of hydrolysis of embodiment 14
Obtaining Testudinis albumen dissolution fluid according to the experiment condition of embodiment 7-13 experiment gained, regulation protein concentration is
1mg/mL, at pepsin, trypsin, neutral protease, papain, alkaline protease
Under the conditions of Shi, it is added separately in the centrifuge tube containing 12mL Testudinis albumen dissolution fluid according to 10000U enzyme amount,
Enzymolysis 12h, formol titration measures degree of hydrolysis, determines the suitableeest enzyme.Result is shown in Fig. 8.
The different neutral protein enzyme dosage impact on Testudinis albumen dissolution fluid degree of hydrolysis of embodiment 15
Obtaining Testudinis albumen dissolution fluid according to the experiment condition of embodiment 7-13 experiment gained, regulation pH is 7, egg
White concentration is 1mg/mL, under the conditions of 48 DEG C, according to 5000,10000,15000,20000,25000,
The enzyme amount of 30000U is added separately in the centrifuge tube containing 12mL Testudinis albumen dissolution fluid, enzymolysis 12h, first
Aldehyde titration measuring degree of hydrolysis, determines optimal enzyme dosage.Result is shown in Fig. 9.
Carapax Et Plastrum Testudinis protein liquid degree of hydrolysis is affected by the different enzymolysis time of embodiment 16
Obtaining Testudinis albumen dissolution fluid according to the experiment condition of embodiment 7-13 experiment gained, regulation pH is 7, egg
White concentration is 1mg/mL, under the conditions of 48 DEG C, is added separately to containing 12mL according to the enzyme dosage of 20000U
In the centrifuge tube of Testudinis albumen dissolution fluid, respectively enzymolysis 1,2,3,4,5,6,9,12,15h, formaldehyde drips
Method of determining measures degree of hydrolysis, determines optimal enzymolysis time.Result is shown in Figure 10.
Carapax Et Plastrum Testudinis protein liquid degree of hydrolysis is affected by the different concentration of substrate of embodiment 17
Obtaining Testudinis albumen dissolution fluid according to the experiment condition of embodiment 7-13 experiment gained, regulation pH is 7, takes
Appropriate Testudinis albumen dissolution fluid is diluted to 0.5,1,2,3mg/mL concentration, respectively take 12mL, 48 DEG C of conditions
Under, according to the enzyme dosage enzymolysis 12h of 20000U, formol titration measures degree of hydrolysis, determines optimal enzymolysis
Concentration of substrate.Result is shown in Figure 11.
Carapax Et Plastrum Testudinis protein liquid degree of hydrolysis is affected by the different pH value of embodiment 18
Obtaining Testudinis albumen dissolution fluid according to the experiment condition of embodiment 7-13 experiment gained, regulation protein concentration is
1mg/mL, takes 12mL Testudinis albumen dissolution fluid, and regulation pH is 6.6,6.7,6.8,6.9,7.0,7.1 respectively,
Under the conditions of 48 DEG C, according to the enzyme dosage of 20000U be added separately to containing 12mL Testudinis albumen dissolution fluid from
In heart pipe, enzymolysis 12h, formol titration measures degree of hydrolysis, determines the pH value of optimal enzymolysis.Result is shown in figure
12。
Carapax Et Plastrum Testudinis protein liquid degree of hydrolysis is affected by embodiment 19 different temperatures
Obtaining Testudinis albumen dissolution fluid according to the experiment condition of embodiment 7-13 experiment gained, regulation pH is 6.8,
Protein concentration is 1mg/mL, respectively takes 12mL, under the conditions of 46,47,48,49,50 DEG C, according to 20000U
Enzyme dosage enzymolysis 12h, formol titration measure degree of hydrolysis, determine the temperature conditions of optimal enzymolysis.Result is shown in
Figure 13.
Embodiment 20 different enzyme amount, enzymolysis time, the Orthogonal experiment results of hydrolysis temperature
Testudinis albumen dissolution fluid is obtained, according to embodiment 9-13 according to the experiment condition of embodiment 7-13 experiment gained
Single factor test condition obtain optimal enzyme dosage (20000U), enzymolysis time (12h), hydrolysis temperature (48
DEG C) 3 factors of condition setting carry out orthogonal test, regulate pH6.8, protein concentration 1mg/mL, respectively take
12mL tests, and the results are shown in Table 1.
Table 1 different enzyme amount, enzymolysis time, the Orthogonal experiment results of hydrolysis temperature
Table 1 different enzyme amount, enzymolysis time, the Orthogonal experiment results of hydrolysis temperature
Embodiment 21 orthogonal test the result
Testudinis albumen dissolution fluid is obtained, according to embodiment 14 according to the experiment condition of embodiment 7-13 experiment gained
The orthogonal experiments obtained, selects optimal enzyme dosage (25000U), enzymolysis time (10h), enzymolysis temperature
Degree (49 DEG C) is verified, regulates pH6.8, protein concentration 1mg/mL, respectively takes 12mL and test,
The results are shown in Table 2.
Table 2 orthogonal test the result
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed,
But therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that, for this area
Those of ordinary skill for, without departing from the inventive concept of the premise, it is also possible to make some deformation and
Improving, these broadly fall into protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be with appended
Claim is as the criterion.
Claims (10)
1. the preparation method of a Testudinis protein polypeptide, it is characterised in that comprise the steps:
Take dry Carapax et Plastrum Testudinis grinding and sieving and obtain Testudinis fine powder；
It is that decalcifying agent solution is mixed with described Testudinis fine powder and fills by 1:2～1:10 according to solid-to-liquid ratio
Divide reaction, be subsequently added into NaOH solution, fully obtain reactant liquor after reaction；
Precipitation is retained, after described precipitation obtains decalcification after cleaning up after centrifugal for described reactant liquor
Testudinis fine powder；
It is that distilled water is mixed by 1:5～1:20 with the Testudinis fine powder after described decalcification according to solid-to-liquid ratio,
Boiling obtains Testudinis albumen dissolution fluid after boiling 1h～6h；And
In described Testudinis albumen dissolution fluid, protease is added according to the enzyme amount of 5000U～30000U,
Fully obtain Testudinis protein polypeptide after reaction.
The method of preparation Testudinis protein polypeptide the most according to claim 1, it is characterised in that
Described decalcifying agent is HCl or EDTA.
The method of preparation Testudinis protein polypeptide the most according to claim 2, it is characterised in that
Described decalcifying agent is EDTA, and the concentration of described EDTA is 0.1～0.75mol/L.
The method of preparation Testudinis protein polypeptide the most according to claim 1, it is characterised in that
Described decalcifying agent solution shakes after mixing with described Testudinis fine powder so that it is fully react, shaking
Time is 1～6H.
The preparation method of Testudinis protein polypeptide the most according to claim 4, it is characterised in that
After described decalcifying agent solution fully reacts with described Testudinis fine powder, add NaOH shake, make
It fully reacts, the concentration of described NaOH is 1mol/L, and the shaking time is 6～24H.
The preparation method of Testudinis protein polypeptide the most according to claim 1, it is characterised in that
Described is 12000rpm by centrifugal for the reactant liquor rotating speed used.
The preparation method of Testudinis protein polypeptide the most according to claim 1, it is characterised in that
Described precipitation is cleaned up after obtain decalcification after the method for Testudinis fine powder be to use deionized water
Clean precipitation 2 times.
The preparation method of Testudinis protein polypeptide the most according to claim 1, it is characterised in that
Testudinis fine powder after described decalcification adds deionized water according to solid-to-liquid ratio 1:15, and boiling boils 6 hours.
The preparation method of Testudinis protein polypeptide the most according to claim 1, it is characterised in that
Described protease is trypsin, pepsin, papain, alkaline protease and neutrality
One in protease.
The preparation method of Testudinis protein polypeptide the most according to claim 6, it is characterised in that
Described protease is neutral protease, and enzyme amount is 10000～30000U, enzymolysis time be 6～
24h。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107095800A (en) * | 2017-05-04 | 2017-08-29 | 安徽万甲宴食品科技有限公司 | Composition and preparation method containing turtle peptide and the facial mask containing said composition |
| CN107373016A (en) * | 2017-08-21 | 2017-11-24 | 张帆 | A kind of preparation method of tortoise polypeptide |
| CN116870102A (en) * | 2023-07-27 | 2023-10-13 | 北京力晟鸿吉信息科技发展有限公司 | Traditional Chinese medicine composition for reducing blood sugar and application thereof |
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| CN116870102A (en) * | 2023-07-27 | 2023-10-13 | 北京力晟鸿吉信息科技发展有限公司 | Traditional Chinese medicine composition for reducing blood sugar and application thereof |
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