CN106220727A - Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein - Google Patents
Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 87
- 102000008186 Collagen Human genes 0.000 title claims abstract description 62
- 108010035532 Collagen Proteins 0.000 title claims abstract description 62
- 239000002253 acid Substances 0.000 title claims abstract description 56
- 230000004044 response Effects 0.000 title claims abstract description 40
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 57
- 239000007788 liquid Substances 0.000 claims abstract description 56
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 42
- 238000001556 precipitation Methods 0.000 claims abstract description 42
- 239000012153 distilled water Substances 0.000 claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 22
- 239000011780 sodium chloride Substances 0.000 claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 18
- 241001482311 Trionychidae Species 0.000 claims abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000007710 freezing Methods 0.000 claims abstract description 11
- 230000008014 freezing Effects 0.000 claims abstract description 11
- 238000004108 freeze drying Methods 0.000 claims abstract description 8
- 239000012535 impurity Substances 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 36
- 238000000265 homogenisation Methods 0.000 claims description 27
- 238000003756 stirring Methods 0.000 claims description 21
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 238000004062 sedimentation Methods 0.000 claims description 9
- 238000000502 dialysis Methods 0.000 claims description 7
- 238000013019 agitation Methods 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 3
- 238000002386 leaching Methods 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- 229920001436 collagen Polymers 0.000 description 11
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 7
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 7
- 229960002591 hydroxyproline Drugs 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 238000000751 protein extraction Methods 0.000 description 5
- 230000009471 action Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 3
- 239000012490 blank solution Substances 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000005265 energy consumption Methods 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- 238000003809 water extraction Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000011344 liquid material Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- BBEJNBLSSWFDSN-UHFFFAOYSA-N methylamino benzoate Chemical compound CNOC(=O)C1=CC=CC=C1 BBEJNBLSSWFDSN-UHFFFAOYSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000003973 paint Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
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- 230000000630 rising effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The present invention relates to the extracting method of collagen protein.It is more particularly related to a kind of ultrasonic wave added acid extracting method utilizing response phase method to optimize collagen protein, including: carry out remove impurity process after taking fresh or freezing methods of soft-shell turtle skirt chopping；Remove miscellaneous after sample with solid-liquid ratio as 1:10～30 add 0.3～0.6mol/L acetic acid solution, it is placed in ultrasonic reactor carrying out batch (-type) supersound extraction, after the most ultrasonic first scheduled time, intermittently second scheduled time, the most repeatedly, supernatant is taken after Li Xin, in supernatant, add NaCl again, stand and filter after saltouing, after albumen precipitation redissolves with the acetic acid of same concentration, dialysing acetic acid and distilled water successively, last lyophilization obtains sample；First scheduled time was less than or equal to for second scheduled time.The invention provides a kind of extraction efficiency high, collagen protein dissolubility is good, raw material value and the highest collagen protein ultrasonic wave added acid extracting method of collagen protein utilization rate.
Description
Technical field
The present invention relates to the extracting method of a kind of collagen protein.It is more particularly related to one utilizes the response surface
Method optimizes the ultrasonic wave added acid extracting method of collagen protein.
Background technology
Collagen protein is the class formation albumen in animal tissue, has that viscosity is big, dissolubility is low and denaturation temperature is low etc.
Feature.Above feature limits its application to a certain extent.Its extracting method mainly have acid extraction method, alkali extraction method,
Enzyme extraction method and hot water extraction method etc..Long with soda acid extraction time, reagent dosage is big, produces substantial amounts of bazardous waste, extracts
Rate is low.Enzyme extraction method can keep the integrity of collagen structure, but extraction cost is high, and extraction time is longer, carries
Take rate relatively low.Although hot water extraction method's cost is the lowest, but extraction ratio is the lowest.
Summary of the invention
It is an object of the invention to solve at least the above, and the advantage that at least will be described later is provided.
It is a still further object of the present invention to provide a kind of extraction efficiency high, collagen protein dissolubility is good, raw material value
The collagen protein ultrasonic wave added acid extracting method the highest with collagen protein utilization rate.
In order to realize object of the present invention and further advantage, it is provided that one utilizes response phase method to optimize collagen protein
Ultrasonic wave added acid extracting method, comprise the following steps:
Step one, take the chopping of fresh or freezing methods of soft-shell turtle skirt after with solid-liquid ratio as 1:10～30 add mass fractions and be
1.0～the NaCl solution of 3.0%, at 20 DEG C, stir 6～12h, then high-speed homogenization 1～3min under 5000～10000rpm
After, collect primary sedimentation, with solid-liquid ratio as 1:10～30 add 0.3～0.6mol/L Na2CO3Solution, stirs 6 at 20 DEG C
～12h, then under 5000～10000rpm after high-speed homogenization 1～3min, collect secondary precipitation, with solid-liquid ratio as 1:20～30
Add the EDTA aqueous solution soaking remove impurity of 0.2～0.4mol/L, at 20 DEG C, stir 6～12h, then 5000～10000rpm
Lower high-speed homogenization 1～3min, collects three precipitations, uses distilled water cyclic washing, standby after fully draining, and adds and divides with quality
Number is the commensurability aqueous isopropanol that mass fraction is 5～20% of NaCl solution of 1.0～3.0%, in 20 DEG C soak 12～
30h, then high-speed homogenization 1～3min under 5000～10000rpm, collect four precipitations, then use distilled water cyclic washing,
Fully drain rear standby；
Step 2, take four times after fully draining in step one precipitations, with solid-liquid ratio as 1:10～30 add 0.3～
The acetic acid solution of 0.6mol/L, is placed in ultrasonic reactor carrying out batch (-type) supersound extraction, after the most ultrasonic first scheduled time,
Intermittently second scheduled time, the most repeatedly, ultrasonic power is 50～300W, and ultrasonic time is 50～70min, then 1000～
Take supernatant after being centrifuged 3～6min under 3000rmp, then in supernatant, add NaCl, until salinity is 0.9mol/L, stand
Saltout and filter after 18～30h, after albumen precipitation redissolves with the acetic acid of 0.3～0.6mol/L, successively to the acetic acid of 0.1mol/L with
Distilled water is dialysed, and last lyophilization obtains sample；
Wherein, described first scheduled time is less than or equal to described second scheduled time.
Preferably, described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, and described first
The scheduled time is 2s, and described second scheduled time is 2～6s.
Preferably, described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, described step
In one, fresh or freezing methods of soft-shell turtle skirt and mass fraction be the solid-liquid ratio of the NaCl solution of 2.0% be 1:20, mixing time
For 9h.
Preferably, described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, described step
In one, described primary sedimentation and the Na of 0.5mol/L2CO3The solid-liquid ratio of solution is 1:20, and mixing time is 9h.
Preferably, described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, described step
In one, described secondary precipitation is 1:25 with the feed liquid of the EDTA aqueous solution of 0.3mol/L, and mixing time is 10h.
Preferably, described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, described step
In one, described three precipitations are 1:20 with the solid-liquid ratio of the aqueous isopropanol that mass fraction is 10%, soak 24h in 20 DEG C.
Preferably, described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, described step
Stirring in one is continuous magnetic agitation.
The present invention at least includes following beneficial effect: ultrasonic wave added acid extracting method extract collagen protein have simple to operate,
The advantages such as the process time is short, albumen yield high and energy consumption is relatively low.The methods of soft-shell turtle skirt collagen protein yield that the method obtains is
19.8% (w/w), purity of protein is 59.6% (w/w), and classical acid lifting manipulation compares, and protein extracting ratio adds 48.5% (w/
W), purity improves 19.3% (w/w).Compared with classical acid lifting manipulation, ultrasonic wave added acid carries and significantly improves collagen protein
Dissolubility, after using ultrasound assisted extraction, the dissolubility of collagen protein improves 16.5%～22.7%.
Part is embodied by the further advantage of the present invention, target and feature by description below, and part also will be by this
Invention research and practice and be understood by the person skilled in the art.
Accompanying drawing explanation
Fig. 1 is the comparison diagram of the nitrogen solubility index of the method gained collagen protein of the method for the present invention and comparative example；
Fig. 2 is the ultrasonic time of present invention when fixing, and ultrasonic wave added acid collagen albumen is carried by ultrasonic power and liquid ratio
The response surface figure of the impact taken；
Fig. 3 is the liquid ratio of present invention when fixing, and ultrasonic wave added acid collagen albumen is carried by ultrasonic power and ultrasonic time
The response surface figure of the impact taken；
Fig. 4 is the ultrasonic power of present invention when fixing, and ultrasonic wave added acid collagen albumen is carried by solid-liquid ratio and ultrasonic time
The response surface figure of the impact taken.
Detailed description of the invention
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to description literary composition
Word can be implemented according to this.
Should be appreciated that used herein such as " have ", " comprising " and " including " term do not allot one or many
Other element individual or the existence of a combination thereof or interpolation.
The present invention provides a kind of ultrasonic wave added acid extracting method utilizing response phase method to optimize collagen protein, including following step
Rapid:
Step one, take the chopping of fresh or freezing methods of soft-shell turtle skirt after with solid-liquid ratio as 1:10～30 add mass fractions and be
1.0～the NaCl solution of 3.0%, at 20 DEG C, stir 6～12h, to remove water solublity and salting-in protein.Then 5000～
Under 10000rpm after high-speed homogenization 1～3min, collect primary sedimentation, with solid-liquid ratio as 1:10～30 add 0.3～0.6mol/L
Na2CO3Solution, stirs 6～12h, to remove noncollagen protein composition and pigment at 20 DEG C.Then 5000～
Under 10000rpm after high-speed homogenization 1～3min, collect secondary precipitation, with solid-liquid ratio as 1:20～30 add 0.2～0.4mol/L
EDTA aqueous solution soaking remove impurity, to remove mineral.6～12h are stirred, then height under 5000～10000rpm at 20 DEG C
Speed homogenate 1～3min, collects three precipitations, uses distilled water cyclic washing, standby after fully draining.Add and with mass fraction be
1.0～the commensurability aqueous isopropanol that mass fraction is 5～20% of NaCl solution of 3.0%, soak 12～30h in 20 DEG C, so
After under 5000～10000rpm high-speed homogenization 1～3min, collect four precipitations, to remove fat, then with distilled water repeatedly
Washing, standby after fully draining.
Step 2, take four times after fully draining in step one precipitations, with solid-liquid ratio as 1:10～30 add 0.3～
The acetic acid solution of 0.6mol/L, is placed in ultrasonic reactor carrying out batch (-type) supersound extraction, after the most ultrasonic first scheduled time,
Intermittently second scheduled time, wherein, described first scheduled time is less than or equal to described second scheduled time, repeatedly, the most ultrasonic
Power is 50～300W, and ultrasonic time is 50～70min.Carry out supersound extraction stage by stage so that extract more abundant, uniform,
Shorten the time of supersound extraction simultaneously.Then supernatant is taken after being centrifuged 3～6min under 1000～3000rmp, then to supernatant
Adding NaCl in liquid, until salinity is 0.9mol/L, standing saltouts filters after 18～30h, albumen precipitation with 0.3～
After the acetic acid of 0.6mol/L redissolves, acetic acid and the distilled water dialysis to 0.1mol/L successively, last lyophilization obtains sample.Logical
Cross dialysis and be further purified the acid collagen albumen that above step extraction obtains.
In the described ultrasonic wave added acid extracting method utilizing response phase method optimization collagen protein, described first scheduled time is
2s, described second scheduled time is 2～6s, so that the energy of ultrasound wave is fully discharged in mixture, and does not cause too high
Temperature.And ultrasonic time is short, the intermittent time is long, so that ultrasonic mixing is more uniform, and mixture is maintained at certain scope
In.
Described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, in described step one, fresh
Or the solid-liquid ratio of the NaCl solution that the methods of soft-shell turtle skirt of freezing and mass fraction are 2.0% is 1:20, mixing time is 9h.
Described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, in described step one, described
Primary sedimentation and the Na of 0.5mol/L2CO3The solid-liquid ratio of solution is 1:20, and mixing time is 9h.
Described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, in described step one, described
Secondary precipitation is 1:25 with the solid-liquid ratio of the EDTA aqueous solution of 0.3mol/L, and mixing time is 10h.
Described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, in described step one, described
Precipitating the solid-liquid ratio with the aqueous isopropanol that mass fraction is 10% for three times is 1:20, soaks 24h in 20 DEG C.
Described utilizes in the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, the stirring in described step one
It is continuous magnetic agitation.
Comparative example
The sour extracting method of a kind of collagen protein, comprises the following steps:
Step one, take the chopping of fresh or freezing methods of soft-shell turtle skirt after add mass fraction with solid-liquid ratio for 1:20 and be
The NaCl solution of 2.0%, continuous magnetic agitation 9h at 20 DEG C.Then, under 8000rpm after high-speed homogenization 2min, collect once
Precipitation, adds the Na of 0.5mol/L with solid-liquid ratio for 1:202CO3Solution, continuous magnetic agitation 9h at 20 DEG C.Then exist
Under 8000rpm after high-speed homogenization 2min, collect secondary precipitation, add the water-soluble immersion of EDTA of 0.3mol/L with solid-liquid ratio for 1:25
Bubble remove impurity.Continuous magnetic agitation 10h, then high-speed homogenization 2min under 8000rpm at 20 DEG C, collect three precipitations, with steaming
Distilled water cyclic washing, standby after fully draining.Adding mass fraction with solid-liquid ratio for 1:20 again is the aqueous isopropanol of 10%, in
20 DEG C are soaked 24h, then high-speed homogenization 2min under 8500rpm, collect four precipitations, then use distilled water cyclic washing, fill
Divide after draining standby.
Step 2, take four times after fully draining in step one precipitations, be the second that 1:20 adds 0.4mol/L with solid-liquid ratio
Acid solution, stirs.Then under 2000rmp, after centrifugal 5min, take supernatant, then in supernatant, add NaCl, until salt
Concentration is 0.9mol/L, filters, after the acetic acid of albumen precipitation 0.4mol/L redissolves, successively to 0.1mol/ after standing the 24h that saltouts
The acetic acid of L and distilled water dialysis, last lyophilization obtains sample.
Embodiment 1
A kind of ultrasonic wave added acid extracting method utilizing response phase method to optimize collagen protein, comprises the following steps:
Step one, take the chopping of fresh or freezing methods of soft-shell turtle skirt after add mass fraction with solid-liquid ratio for 1:20 and be
The NaCl solution of 2.0%, stirs 9h at 20 DEG C.Then, under 8000rpm after high-speed homogenization 2min, primary sedimentation is collected, with
Solid-liquid ratio is the Na that 1:20 adds 0.5mol/L2CO3Solution, stirs 9h at 20 DEG C.Then high-speed homogenization under 8000rpm
After 2min, collect secondary precipitation, add the EDTA aqueous solution soaking remove impurity of 0.3mol/L with solid-liquid ratio for 1:25.Stir at 20 DEG C
Mix 10h, then high-speed homogenization 2min under 8000rpm, collect three precipitations, use distilled water cyclic washing, fully drain standby
With.Adding mass fraction with solid-liquid ratio for 1:20 again is the aqueous isopropanol of 10%, soaks 24h in 20 DEG C, then at 8500rpm
Lower high-speed homogenization 2min, collects four precipitations, to remove fat, then uses distilled water cyclic washing, standby after fully draining.
Step 2, take four times after fully draining in step one precipitations, be the second that 1:20 adds 0.4mol/L with solid-liquid ratio
Acid solution, is placed in ultrasonic reactor carrying out batch (-type) supersound extraction, after every ultrasonic 2s, and intermittently 4s, the most repeatedly, ultrasonic merit
Rate is 200W, and ultrasonic time is 60min.Then under 2000rmp, after centrifugal 5min, take supernatant, then add in supernatant
NaCl, until salinity is 0.9mol/L, filters after standing the 24h that saltouts, after the acetic acid of albumen precipitation 0.4mol/L redissolves, depends on
The secondary acetic acid to 0.1mol/L and distilled water dialysis, last lyophilization obtains sample.
Response surface is the three dimensions of specific response value and corresponding factors composition, is mapped in formation etc. on two dimensional surface
High line, can reflect the reciprocal action of each factor and the impact on response value intuitively.The gradient of response surface curved surface can reflect this
The degree of strength that extraction rate of protein is affected by factor.Response surface design is relatively mild, and the impact of its tolerable treatment conditions is described.
The shape of contour map shows that the reciprocal action between variable is the most notable, and oval equal pitch contour shows that the reciprocal action between variable shows
Writing, circular equal pitch contour shows that reciprocal action is the most notable.Response surface curve is relatively steep, illustrates that the effect of response value is got over by this extracting parameter
Significantly, otherwise, certain extracting parameter is less to the effect of response value, shows as curve mild.
When ultrasonic time is fixed on level 0, ultrasonic power and liquid ratio are to ultrasonic wave added acid collagen protein extraction
Impact is shown in that response surface Fig. 1, ultrasonic power and liquid ratio are quadratic power effect to ultrasonic wave added acid collagen protein extraction yield, when
When liquid ratio is relatively low, improve ultrasonic power, decline after the rising that must take the lead in of ultrasonic wave added acid collagen albumen.Fig. 2 is fixative
When material ratio is for 20:1mL/g, ultrasonic power and the ultrasonic time impact on ultrasonic wave added acid collagen protein extraction, ultrasonic power
With ultrasonic time to ultrasonic wave added acid collagen protein extraction yield also in quadratic power effect, ultrasonic power is corresponding with ultrasonic time
Curved surface steeper, illustrate that the impact of the two factor relative result is notable, this is consistent with the result of variance analysis.Fig. 3 is solid
Determine ultrasonic power when being 200W, solid-liquid ratio and the ultrasonic time impact on ultrasonic wave added acid collagen protein extraction, extraction ratio with
The increase liquid material when ultrasonic time first raises, but increasing degree is inconspicuous.
The drafting of hydroxyproline standard curve
Accurately weigh hydroxyproline 100mg, with the dissolving with hydrochloric acid of 0.001mol/L, be configured to the hydroxyproline storage of 1mg/mL
Standby liquid, takes 1mL storing solution and is diluted to 100mL and is configured to the standard solution of 10 μ g/mL.Preparation 1mg is with the hydrochloric acid of 0.001mol/L
As blank solution, hydroxyproline standard solution and blank solution are separately added into toluene-sodium-sulfonchloramide solution 2mL, mixing, left at room temperature
20min, adds perchloric acid solution 2mL, mixing, left at room temperature 5min, adds methylaminobenzoate 2mL, mixes, 60 DEG C
Heating 20min carries out chromogenic reaction, is finally placed in cold water cooling.Return to zero with blank solution, measure the absorbance at 560nm, paint
Standard curve processed.
The mensuration of hydroxyproline content in sample
Testing sample in Example 1 about 10mg, in ampoule bottle, adds the hydrochloric acid of 1mL 6mol/L, uses alcohol blast burner
Hydrolyzing 3h after sealing at 130 DEG C, taking-up distilled water is settled to 10mL, filter paper filtering, takes 1mL by hydroxyproline standard curve
Assay method, with blank return to zero, measure testing sample absorbance.Hydroxyl in sample liquid is conversed by hydroxyproline standard curve
The concentration of proline.
The mensuration of dissolubility (nitrogen solubility index NSI)
Being dispersed in 20mL distilled water by collagen protein sample 0.4000g in embodiment 1, ambient temperatare is put
30min, with HCL solution or the pH value of 0.1mol/L NaOH solution regulation solution of 0.1mol/L, with homogenizer at 13400rpm
Lower homogenizing 1min, is settled to 25mL, and dispersion liquid, with the centrifugation 15min of 4000rpm, takes supernatant and collagen protein sample is used
Kjeldahl's method surveys nitrogen content, and formula is as follows:
NSI (%)=(total nitrogen in the nitrogen content/sample in supernatant) × 100
As it is shown in figure 1, curve above represents the molten finger of nitrogen utilizing the ultrasonic wave added acid of the present invention to carry collagen protein
With the change of pH, the curve being positioned at lower section, number represents that conventional acid proposes the nitrogen solubility index change with pH of collagen protein, it can be seen that
Ultrasonic wave added acid extracting method extract collagen protein have simple to operate, the process time is short, albumen yield is high and energy consumption is relatively low etc. excellent
Point.The methods of soft-shell turtle skirt collagen protein yield that the method obtains is 19.8% (w/w), and purity of protein is 59.6% (w/w), and passes
System acid lifting manipulation compares, and protein extracting ratio adds 48.5% (w/w), and purity improves 19.3% (w/w).With classical acid lifting manipulation phase
Ratio, ultrasonic wave added acid puies forward the dissolubility significantly improving collagen protein, after using ultrasound assisted extraction, the dissolving of collagen protein
Property improves 16.5%～22.7%.
Embodiment 2
A kind of ultrasonic wave added acid extracting method utilizing response phase method to optimize collagen protein, comprises the following steps:
Step one, take the chopping of fresh or freezing methods of soft-shell turtle skirt after add mass fraction with solid-liquid ratio for 1:10 and be
The NaCl solution of 1.0%, stirs 6h at 20 DEG C.Then, under 5000rpm after high-speed homogenization 1min, primary sedimentation is collected, with
Solid-liquid ratio is the Na that 1:10 adds 0.3mol/L2CO3Solution, stirs 6h at 20 DEG C.Then high-speed homogenization under 5000rpm
After 1min, collect secondary precipitation, add the EDTA aqueous solution soaking remove impurity of 0.2mol/L with solid-liquid ratio for 1:20.Stir at 20 DEG C
Mix 6h, then high-speed homogenization 1min under 5000rpm, collect three precipitations, use distilled water cyclic washing, fully drain standby
With.Adding mass fraction with solid-liquid ratio for 1:10 again is the aqueous isopropanol of 5%, soaks 12h in 20 DEG C, then at 5000rpm
Lower high-speed homogenization 1min, collects four precipitations, then uses distilled water cyclic washing, standby after fully draining.
Step 2, take four times after fully draining in step one precipitations, be the second that 1:10 adds 0.3mol/L with solid-liquid ratio
Acid solution, is placed in ultrasonic reactor carrying out batch (-type) supersound extraction, after every ultrasonic 2s, and intermittently 2s, the most repeatedly, ultrasonic merit
Rate is 50W, and ultrasonic time is 50min.Then under 1000rmp, after centrifugal 3min, take supernatant, then add in supernatant
NaCl, until salinity is 0.9mol/L, filters after standing the 18h that saltouts, after the acetic acid of albumen precipitation 0.3mol/L redissolves, depends on
The secondary acetic acid to 0.1mol/L and distilled water dialysis, last lyophilization obtains sample.
Embodiment 3
A kind of ultrasonic wave added acid extracting method utilizing response phase method to optimize collagen protein, comprises the following steps:
Step one, take the chopping of fresh or freezing methods of soft-shell turtle skirt after add mass fraction with solid-liquid ratio for 1:30 and be
The NaCl solution of 3.0%, stirs 2h at 20 DEG C.Then, under 10000rpm after high-speed homogenization 3min, primary sedimentation is collected, with
Solid-liquid ratio is the Na that 1:30 adds 0.6mol/L2CO3Solution, stirs 12h at 20 DEG C.Then high-speed homogenization under 10000rpm
After 3min, collect secondary precipitation, add the EDTA aqueous solution soaking remove impurity of 0.4mol/L with solid-liquid ratio for 1:30.Stir at 20 DEG C
Mix 12h, then high-speed homogenization 3min under 10000rpm, collect three precipitations, use distilled water cyclic washing, fully drain standby
With.Adding mass fraction with solid-liquid ratio for 1:30 again is the aqueous isopropanol of 20%, soaks 30h in 20 DEG C, then exists
High-speed homogenization 3min under 10000rpm, collects four precipitations, then uses distilled water cyclic washing, standby after fully draining.
Step 2, take four times after fully draining in step one precipitations, be the second that 1:30 adds 0.6mol/L with solid-liquid ratio
Acid solution, is placed in ultrasonic reactor carrying out batch (-type) supersound extraction, every ultrasonic 2s, intermittently 6s, the most repeatedly, and ultrasonic power
For 300W, ultrasonic time is 70min.Then under 3000rmp, after centrifugal 6min, take supernatant, then add in supernatant
NaCl, until salinity is 0.9mol/L, filters after standing the 30h that saltouts, after the acetic acid of albumen precipitation 0.6mol/L redissolves, depends on
The secondary acetic acid to 0.1mol/L and distilled water dialysis, last lyophilization obtains sample.
Although embodiment of the present invention are disclosed as above, but it is not restricted in description and embodiment listed
Using, it can be applied to various applicable the field of the invention completely, for those skilled in the art, and can be easily
Realizing other amendment, therefore under the general concept limited without departing substantially from claim and equivalency range, the present invention does not limit
In specific details with shown here as the legend with description.
Claims (7)
1. one kind utilizes the ultrasonic wave added acid extracting method that response phase method optimizes collagen protein, it is characterised in that comprise the following steps:
Step one, take the chopping of fresh or freezing methods of soft-shell turtle skirt after with solid-liquid ratio as 1:10～30 to add mass fractions be 1.0
～the NaCl solution of 3.0%, at 20 DEG C, stir 6～12h, then under 5000～10000rpm after high-speed homogenization 1～3min,
Collect primary sedimentation, with solid-liquid ratio as 1:10～30 add 0.3～0.6mol/L Na2CO3Solution, at 20 DEG C stir 6～
12h, then under 5000～10000rpm after high-speed homogenization 1～3min, collects secondary precipitation, with solid-liquid ratio as 1:20～30 add
Enter the EDTA aqueous solution soaking remove impurity of 0.2～0.4mol/L, at 20 DEG C, stir 6～12h, then under 5000～10000rpm
High-speed homogenization 1～3min, collects three precipitations, uses distilled water cyclic washing, standby after fully draining, and adds and mass fraction
It is the commensurability aqueous isopropanol that mass fraction is 5～20% of NaCl solution of 1.0～3.0%, soaks 12～30h in 20 DEG C,
Then high-speed homogenization 1～3min under 5000～10000rpm, collects four precipitations, then uses distilled water cyclic washing, fully
Drain rear standby；
Step 2, take four times after fully draining in step one precipitations, with solid-liquid ratio as 1:10～30 add 0.3～0.6mol/L
Acetic acid solution, be placed in ultrasonic reactor carrying out batch (-type) supersound extraction, after the most ultrasonic first scheduled time, intermittently second
The scheduled time, the most repeatedly, ultrasonic power is 50～300W, and ultrasonic time is 50～70min, then 1000～3000rmp
Under be centrifuged 3～6min after take supernatant, then in supernatant, add NaCl, until salinity is 0.9mol/L, stands and saltout 18
～filter after 30h, after albumen precipitation redissolves with the acetic acid of 0.3～0.6mol/L, acetic acid and the distilled water to 0.1mol/L successively
Dialysis, last lyophilization obtains sample；
Wherein, described first scheduled time is less than or equal to described second scheduled time.
Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein the most as claimed in claim 1, it is characterised in that
Described first scheduled time is 2s, and described second scheduled time is 2～6s.
Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein the most as claimed in claim 1, it is characterised in that
In described step one, fresh or freezing methods of soft-shell turtle skirt and mass fraction be the solid-liquid ratio of the NaCl solution of 2.0% be 1:20,
Mixing time is 9h.
Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein the most as claimed in claim 1, it is characterised in that
In described step one, described primary sedimentation and the Na of 0.5mol/L2CO3The solid-liquid ratio of solution is 1:20, and mixing time is 9h.
Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein the most as claimed in claim 1, it is characterised in that
In described step one, described secondary precipitation is 1:25 with the solid-liquid ratio of the EDTA aqueous solution of 0.3mol/L, and mixing time is 10h.
Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein the most as claimed in claim 3, it is characterised in that
In described step one, described three precipitations are 1:20 with the solid-liquid ratio of the aqueous isopropanol that mass fraction is 10%, in 20 DEG C of leachings
Bubble 24h.
Response phase method is utilized to optimize the ultrasonic wave added acid extracting method of collagen protein the most as claimed in claim 1, it is characterised in that
Stirring in described step one is continuous magnetic agitation.
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