CN109908395A - A kind of sharkskin natural medical collagen protein sponge preparation method - Google Patents
A kind of sharkskin natural medical collagen protein sponge preparation method Download PDFInfo
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Abstract
The invention discloses a kind of sharkskin natural medical collagen protein sponge preparation methods, belong to technical field of biological medical material preparation, include the following steps: after sharkskin is removed watery blood, -20 DEG C of anxious jellies, casting skin layer and collagen layer are separated, remove foreign protein and lipid, then crushed with pulverizer；Will treated collagen layer, suspended with acetic acid solution, protease be added, is slowly stirred enzymatic hydrolysis, obtains unmodified collagen solution；The isolated supernatant of obtained collagen solution and precipitating, supernatant are saltoutd, precipitating is redissolved, and precipitating is dialysed after redissolving with dialysis membrane；Dialysis solution by is crosslinked, and freeze-drying obtains collagen protein sponge, packs to obtain medical collagen sponge.The present invention takes full advantage of the aquatic products processings waste such as sharkskin, and simple production process, strong operability is at low cost, and resulting collagen protein sponge purity is high can be widely applied to the fields such as biomaterial for medical purpose.
Description
Technical field
The present invention relates to a kind of collagen protein sponge preparation methods, more particularly to a kind of sharkskin natural medical collagen egg
Bai Haimian preparation method, belongs to technical field of biological medical material preparation.
Background technique
Collagen is that one kind is widely present in a kind of intracorporal fibrous structure protein of biology, poor according to molecular structure
It is different to be divided into the multiple types such as I, II, III, wherein Type I collagen albumen is most important existence form, structure feature in organism
The triple-helix structure formed by two α 1 and 2 peptide chain of α, due to collagen have good gel strength, emulsibility,
Low-viscosity, biocompatibility, water imbibition and moisture retention etc., it is widely used in food, medicine and biomaterial, change in recent years
The fields such as cosmetic, wherein collagen is bio-medical material derived from raw material, it has also become the most fast increasing of field of medical materials
One of long point, with collagen and the clinical application research of biomaterial, the market of collagen medical material will be into one
Step expands.
The source of collagen biomaterial is mainly to extract in ox heel string and ox-hide at this stage, since rabid ox disease, fowl are flowed
The problems such as sense, mouth tellurium epidemic disease, country carry out strict control, marine organisms collagen to the collagen product in animal tissue source
Due to its function is excellent, safety is good and it is cheap be easy to get, thus have a extensive future in the exploitation of technical field of biological material and conversion, but by
The more low factor of the thermal denaturation temperature of Yu Haiyang's biological collagen, limits its practical application, need through a variety of methods and
Technology is modified processing, just can apply to the production of medical material.
It had been reported that in recent years and prepares collagen in the skin and adjunct of grass carp, sturgeon, Tilapia mossambica etc., but do not had
There is the collagen in relevant report sharkskin source to extract, blue shark category Chondrichthyes Carcharhiniforme Carcharhinidae, the main quilt of Shark cartilage
As chondroitin sulfate is extracted, fin ray is used as preparing shark's fin high value added product, and sharkskin is the by-product in production process
Object is often used as feed processing.
Summary of the invention
The main object of the present invention is to provide for a kind of sharkskin natural medical collagen protein sponge preparation method, improves
The added value of sharkskin provides more reliable safer raw material sources for bio-medical material derived from collagen raw material.
The purpose of the present invention can reach by using following technical solution:
A kind of sharkskin natural medical collagen protein sponge preparation method, includes the following steps:
Step 1: the pretreatment of raw material
After sharkskin is removed watery blood, -22 DEG C to -18 DEG C freezings separate casting skin layer and collagen layer, remove removal of impurities egg
White and lipid, then crushed with pulverizer；
Step 2: enzymatic hydrolysis
It by step 1 treated collagen layer, is suspended with acetic acid solution, protease is added, is slowly stirred enzymatic hydrolysis, obtains
Unmodified collagen solution；
Step 3: the purifying of extract
The isolated supernatant of the collagen solution that step 2 is obtained and precipitating, supernatant are saltoutd, and precipitating is redissolved, and are sunk
It forms sediment and is dialysed after redissolving with dialysis membrane；
Step 4: the preparation of collagen protein sponge
It by the dialysis solution in step 3, is crosslinked, freeze-drying obtains collagen protein sponge, packs to obtain medical collagen sea
It is continuous.
Further, the step 1 to the overall process of step 4, is carried out under the conditions of being lower than 10 DEG C of temperature.
Further, in the step 1, the sharkskin of selection is the skin of blue shark or whale shark.
Further, in the step 1, fresh sharkskin is rinsed with clear water, after removing watery blood, -20 DEG C of anxious jellies,
The casting skin layer and collagen layer of sharkskin are separated, collagen layer is cut into small pieces, foreign protein is removed with NaOH, uses ethyl alcohol
Lipid is removed, adds the acetic acid solution of pre-cooling, then crushed with pulverizer.
Further, in the step 2, step 1 treated the collagen layer acetic acid solution of 0.5M is suspended, is pressed
Protease is added according to 1:1000, under conditions of being lower than 10 DEG C, is slowly stirred enzymatic hydrolysis 2-3 days, obtains unmodified collagen
Solution.
Further, in the step 3, the collagen solution that step 2 is obtained is placed in supercentrifuge high speed point
From obtaining supernatant and precipitating；
Isolated supernatant 10M NaOH solution tune pH to neutrality, is slowly added to 2.5MNaCl and saltouts；
It after isolated precipitating is centrifuged, is redissolved with 0.5M acetic acid solution, is then dialysed three days with dialysis membrane.
Further, in the step 3, in dialysis procedure, dialysis solution is taken, it is molten that silver nitrate is added dropwise in dialysis solution
Liquid can stop dialysing if that is, dialysis terminates without white precipitate, dialysis bag retention molecular weight 14000.
Further, in the step 4, the cross-linking method of selection is ultraviolet-crosslinkable and Co60Crosslinking is used in combination.
Further, in the step 4, by the dialysis solution in step 3, after ultraviolet-crosslinkable, it is put into freeze-drying
Freeze-drying, obtains collagen protein sponge, after collagen protein sponge is packed, uses Co in machine60After irradiation, medical collagen sea is obtained
It is continuous.
Further, it obtains medical collagen sponge and molecular weight test is carried out using PAGE gel electrophoresis, it is medical
Collagen protein sponge includes two kinds of α peptide chains and a β peptide chain, and electrophoretic migration position is between 100~120kD.
Advantageous effects of the invention: sharkskin natural medical collagen protein sponge preparation method provided by the invention,
The added value of sharkskin can be improved in simple process and low cost, mentions for bio-medical material derived from collagen raw material
For more reliable safer raw material sources；Sharkskin natural medical collagen molecules measurement obtained uses SDS-PAGE solidifying
Gel electrophoresis method, measuring shark collagen includes two kinds of α peptide chains (α 1 and α 2) and a β peptide chain (dimer of α peptide chain), the peptide
Chain composition characteristic is consistent with the peptide chain composition height of Type I collagen albumen, illustrates that extracting resulting collagen sample belongs to I type glue
Original compares the protein standard in electrophorogram it can be found that the electrophoretic migration position of 3 kinds of collagen α peptide chains is without bright
Significant difference is different, and between 100-120kD, it is preferable to extract quality.
Detailed description of the invention
Fig. 1 is the shark of a preferred embodiment of sharkskin natural medical collagen protein sponge preparation method according to the invention
Figure is separated by electrophoresis in the SDS-PAGE of collagen；
Fig. 2 is the cod of a preferred embodiment of sharkskin natural medical collagen protein sponge preparation method according to the invention
Figure is separated by electrophoresis in the SDS-PAGE of collagen.
Specific embodiment
To make the more clear and clear technical solution of the present invention of those skilled in the art, below with reference to examples and drawings
The present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Sharkskin natural medical collagen protein sponge preparation method provided in this embodiment, includes the following steps:
Step 1: the pretreatment of raw material
By fresh sharkskin, rinsed with clear water, after removing watery blood, -22 DEG C to -18 DEG C anxious jellies, by the casting skin of sharkskin
Layer separate with collagen layer, collagen layer is cut into small pieces, with NaOH removal foreign protein, with ethyl alcohol removal lipid, then plus
Enter the acetic acid solution of pre-cooling, then is crushed with pulverizer；
Step 2: enzymatic hydrolysis
It by step 1 treated collagen layer, is suspended with the acetic acid solution of 0.5M, protease is added according to 1:1000,
Under conditions of being lower than 10 DEG C, it is slowly stirred enzymatic hydrolysis 2-3 days, obtains unmodified collagen solution；
Step 3: the purifying of extract
The collagen solution that step 2 is obtained is placed in the separation of supercentrifuge high speed；Isolated supernatant is used
10M NaOH solution tune pH is slowly added to 2.5M NaCl and saltouts to neutrality；After isolated precipitating is centrifuged, use
0.5M acetic acid solution redissolves, and is then dialysed three days with dialysis membrane, in dialysis procedure, takes dialysis solution, drip in dialysis solution
Add silver nitrate solution, if that is, dialysis terminates without white precipitate, can stop dialysing, dialysis bag retention molecular weight 14000；
Step 4: the preparation of collagen protein sponge
By the dialysis solution in step 3, after ultraviolet-crosslinkable, it is put into freeze drier freeze-drying, obtains collagen
Sponge after packing collagen protein sponge, uses Co60After irradiation, medical collagen sponge is obtained.
Sharkskin natural medical collagen molecules measurement obtained uses PAGE gel electrophoresis, is detailed in Fig. 1
Shown, shark collagen includes two kinds of α peptide chains (α 1 and α 2) and a β peptide chain (dimer of α peptide chain), and peptide chain composition is special
Sign is consistent with the peptide chain composition height of Type I collagen albumen, illustrates that extracting resulting collagen sample belongs to Type I collagen.Control
Protein standard in electrophorogram it can be found that 3 kinds of collagen α peptide chains electrophoretic migration position no significant difference,
Between 100~120kD, it is preferable to extract quality.
In order to more clearly express above embodiment of the invention, following examples and reference examples are provided:
Embodiment 1:
It by the skin of blue shark, is rinsed with clear water, removes extra watery blood and meat etc., be put into -20 DEG C of anxious freeze and handle a week, take
What be can be convenient after out separates collagen layer and casting skin layer, then takes 15g fish-skin, after being cut into small pieces, adds 0.1M NaOH molten
Liquid 450ml handles 4hr, and removal foreign protein adds 10% ethanol solution 150ml after clear water rinsed clean, after, 16hr is handled,
After being cleaned up with clear water, add 0.5M acetic acid solution 200ml, impregnates after twenty minutes, blended with meat grinder, add 15mg pepsin
It handles within 10 DEG C of temperature, stirring enzymatic hydrolysis 72 hours, after centrifugation removes undissolved part, supernatant 10M NaOH solution tune
Then pH is saltoutd with 2.5M NaCl, is precipitated with 0.5M acetic acid, dialysed 72 hours or so, obtained unmodified natural to center
Then sharkskin collagen solution is used ultraviolet-crosslinkable 30 minutes, is freeze-dried to obtain medical collagen sponge, uses after packaging
Co60Irradiate to obtain sterile apyrogenic medical collagen sponge product.
Embodiment 2:
It by the skin of whale shark, is rinsed with clear water, removes extra watery blood and meat etc., be put into -20 DEG C of anxious freeze and handle a week, take
What be can be convenient after out separates collagen layer and casting skin layer, then takes 20g fish-skin, after being cut into small pieces, adds 0.1M NaOH molten
Liquid 600ml handles 4hr, and removal foreign protein adds 10% ethanol solution 200ml after clear water rinsed clean, after, 16hr is handled,
After being cleaned up with clear water, add 0.5M acetic acid solution 250ml, impregnates after twenty minutes, blended with meat grinder, add 20mg pepsin
It handles within 10 DEG C of temperature, stirring enzymatic hydrolysis 72 hours, after centrifugation removes undissolved part, supernatant 10M NaOH solution tune
Then pH is saltoutd with 2.5M NaCl, is precipitated with 0.5M acetic acid, dialysed 72 hours or so, obtained unmodified natural to center
Then sharkskin collagen solution is used ultraviolet-crosslinkable 30 minutes, is freeze-dried to obtain medical collagen sponge, uses after packaging
Co60Irradiate to obtain sterile apyrogenic medical collagen sponge product.
Embodiment 3:
It by the skin of whale shark, is rinsed with clear water, removes extra watery blood and meat etc., be put into -20 DEG C of anxious freeze and handle a week, take
What be can be convenient after out separates collagen layer and casting skin layer, then takes 25g fish-skin, after being cut into small pieces, adds 0.1M NaOH molten
Liquid 750ml handles 4hr, and removal foreign protein adds 10% ethanol solution 250ml after clear water rinsed clean, after, 16hr is handled,
After being cleaned up with clear water, add 0.5M acetic acid solution 300ml, impregnates after twenty minutes, blended with meat grinder, add 40mg pepsin
It handles within 10 DEG C of temperature, stirring enzymatic hydrolysis 72 hours, after centrifugation removes undissolved part, supernatant 10M NaOH solution tune
Then pH is saltoutd with 2.5M NaCl, is precipitated with 0.5M acetic acid, dialysed 72 hours or so, obtained unmodified natural to center
Then sharkskin collagen solution is used ultraviolet-crosslinkable 30 minutes, is freeze-dried to obtain medical collagen sponge, uses after packaging
Co60Irradiate to obtain sterile apyrogenic medical collagen sponge product.
Reference examples:
It by the skin of blue shark, is rinsed with clear water, removes extra watery blood and meat etc., be put into -20 DEG C of anxious freeze and handle a week, take
What be can be convenient after out separates collagen layer and casting skin layer, then takes 15g fish-skin, after being cut into small pieces, adds 0.1M NaOH molten
Liquid 450ml handles 4hr, and removal foreign protein adds 10% ethanol solution 150ml after clear water rinsed clean, after, 16hr is handled,
After being cleaned up with clear water, add 0.5M acetic acid solution 200ml, impregnates after twenty minutes, blended with meat grinder, add 15mg pepsin
It handles within 10 DEG C of temperature, stirring enzymatic hydrolysis 72 hours, after centrifugation removes undissolved part, supernatant 10M NaOH solution tune
Then pH is saltoutd with 2.5M NaCl, is precipitated with 0.5M acetic acid, dialysed 72 hours or so, obtained unmodified natural to center
Cod collagen solution.
According to above-described embodiment and the content of reference examples, its main feature is that the collagen-rich sharkskins such as blue shark are chosen,
Clear water rinsing removes remaining watery blood and impurity, then -20 DEG C of anxious jellies, in a cold or frozen state by collagen layer and casting skin layer point
It opens, is rinsed with clear water, remove remaining watery blood and meat, be cut into small pieces, remove foreign protein with NaOH, remove lipid with ethyl alcohol, then plus
Enter the acetic acid solution of pre-cooling, after pulverizer crushes, enzymatic hydrolysis, enzymolysis liquid is added NaCl and saltouts, then centrifugation obtains collagen and sinks
It forms sediment, precipitating is dissolved with acetic acid solution, the collagen solution for denaturation of dialysing naturally to be, after ultraviolet-crosslinkable, freeze-drying
White collagen protein sponge is obtained, uses Co after packaging60Irradiation, obtains medical collagen sponge.The present invention takes full advantage of shark
The aquatic products processings waste such as fish-skin, simple production process, strong operability is at low cost, resulting collagen protein sponge purity is high, can
It is widely used in the fields such as biomaterial for medical purpose.
In conclusion in the above-described embodiments, as depicted in figs. 1 and 2, sharkskin natural medical provided by the above embodiment
Collagen protein sponge preparation method, sharkskin natural medical collagen molecules measurement obtained use PAGE gel electric
Swimming method, measuring shark collagen includes two kinds of α peptide chains (α 1 and α 2) and a β peptide chain (dimer of α peptide chain), the peptide chain group
It is consistent with the peptide chain composition height of Type I collagen albumen at feature, illustrate that extracting resulting collagen sample belongs to Type I collagen,
The protein standard in electrophorogram is compareed it can be found that the electrophoretic migration position of 3 kinds of collagen α peptide chains is without obvious poor
It is different, between 100-120kD, it is preferable to extract quality.
The above, further embodiment only of the present invention, but scope of protection of the present invention is not limited thereto, and it is any
Within the scope of the present disclosure, according to the technique and scheme of the present invention and its design adds those familiar with the art
With equivalent substitution or change, protection scope of the present invention is belonged to.
Claims (10)
1. a kind of sharkskin natural medical collagen protein sponge preparation method, which comprises the steps of:
Step 1: the pretreatment of raw material
After sharkskin is removed watery blood, -22 DEG C to -18 DEG C freezings separate casting skin layer and collagen layer, removal foreign protein with
Lipid, then crushed with pulverizer；
Step 2: enzymatic hydrolysis
It by step 1 treated collagen layer, is suspended with acetic acid solution, protease is added, is slowly stirred enzymatic hydrolysis, obtains unchanged
The collagen solution of property；
Step 3: the purifying of extract
The isolated supernatant of the collagen solution that step 2 is obtained and precipitating, supernatant are saltoutd, and precipitating is redissolved, and precipitating is multiple
It is dialysed after molten with dialysis membrane；
Step 4: the preparation of collagen protein sponge
It by the dialysis solution in step 3, is crosslinked, freeze-drying obtains collagen protein sponge, packs to obtain medical collagen sponge.
2. a kind of sharkskin natural medical collagen protein sponge preparation method as described in claim 1, which is characterized in that described
Step 1 to the overall process of step 4, is carried out under the conditions of being lower than 10 DEG C of temperature.
3. a kind of sharkskin natural medical collagen protein sponge preparation method as described in claim 1, which is characterized in that described
In step 1, the sharkskin of selection is the skin of blue shark or whale shark.
4. a kind of sharkskin natural medical collagen protein sponge preparation method as described in claim 1, which is characterized in that described
In step 1, fresh sharkskin is rinsed with clear water, after removing watery blood, -20 DEG C of anxious jellies, by the casting skin layer and collagen of sharkskin
Albumin layer separation, collagen layer is cut into small pieces, and removes foreign protein with NaOH, is removed lipid with ethyl alcohol, is added pre-cooling
Acetic acid solution, then crushed with pulverizer.
5. a kind of sharkskin natural medical collagen protein sponge preparation method as described in claim 1, which is characterized in that described
In step 2, step 1 treated the collagen layer acetic acid solution of 0.5M is suspended, protease is added according to 1:1000,
Under conditions of 10 DEG C, it is slowly stirred enzymatic hydrolysis 2-3 days, obtains unmodified collagen solution.
6. a kind of sharkskin natural medical collagen protein sponge preparation method as described in claim 1, which is characterized in that described
In step 3, the collagen solution that step 2 is obtained is placed in the isolated supernatant of supercentrifuge high speed and precipitating；
Isolated supernatant 10M NaOH solution tune pH to neutrality, is slowly added to 2.5M NaCl and saltouts；
It after isolated precipitating is centrifuged, is redissolved with 0.5M acetic acid solution, is then dialysed three days with dialysis membrane.
7. a kind of sharkskin natural medical collagen protein sponge preparation method as claimed in claim 6, which is characterized in that described
In step 3, in dialysis procedure, dialysis solution is taken, silver nitrate solution is added dropwise in dialysis solution, if without white precipitate, i.e., thoroughly
Analysis terminates, and can stop dialysing, dialysis bag retention molecular weight 14000.
8. a kind of sharkskin natural medical collagen protein sponge preparation method as described in claim 1, which is characterized in that described
In step 4, the cross-linking method of selection is ultraviolet-crosslinkable and Co60Crosslinking is used in combination.
9. a kind of sharkskin natural medical collagen protein sponge preparation method as claimed in claim 8, which is characterized in that described
In step 4, by the dialysis solution in step 3, after ultraviolet-crosslinkable, it is put into freeze-drying in freeze drier, obtains collagen egg
Bai Haimian after packing collagen protein sponge, uses Co60After irradiation, medical collagen sponge is obtained.
10. a kind of sharkskin natural medical collagen protein sponge preparation method as claimed in claim 9, which is characterized in that
Medical collagen sponge carries out molecular weight test using PAGE gel electrophoresis, and medical collagen sponge includes two kinds
α peptide chain and a β peptide chain, electrophoretic migration position is between 100~120kD.
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