A kind of preparation method of medical collagen protein powder
One, technical field
The present invention relates to a kind of preparation method of medical collagen protein powder, belong to the preparation field of biomedical material.
Two, background technology
Collagen protein is a kind of extracellular matrix, belongs to structural protein, accounts for 1/3 of mammalian proteins matter total amount, is the main component that constitutes reticular tissue such as skin, ligament, cartilage, tendon or organ.The collagen protein of confirming has 26 types at present, wherein with maximum (the Pace James M of I type content, Corrado Marcella, MisseroCaterina.Identification, characterization and expression analysis of a newfibrillar collagen gen.Matrix Biology, 2003,1 (22): 3-14).The special quaternary structure that collagen protein had has determined its unique physical and chemical properties and good biocompatibility, degradability, reduced immunogenicity and performances such as cell adapted property, cell proliferation and promotion blood clotting (but to defend China, Wang Kunyu, Ceng Rui, Deng, the medical use of collagen and development prospect thereof, " biomedical engineering and clinical " .2004,8 (1): 45-48).
In recent years at home and abroad, aspects such as the development and utilization (Zhou Wenchang that the research of collagen protein mainly concentrates on the separating technology of the discovery of novel collagen and performance study, collagen protein and improves the chemically modified of the purity of collagen goods, collagen protein, crosslinked and composite collagen material, but defend China, Liao Longli, Deng. collagen protein and the application of matrix material in medical science that forms with superpolymer thereof. leather science and engineering, 2004,2 (14): 30-34).Along with to the understanding of collagen with the many biological functions headed by the proliferative cell, make the collagen base biological medical material especially noticeable, at medical field, remove the surgery dressing membrane, styptic sponge, operating sutures, outside the hemostatic agent, the foreign study person mainly concentrates on the preparation of research and utilization collagen protein has the planning function to human body soft tissue collagen injection liquid, collagen base delivery system, skin, bone, cardiovascular, oesophagus, the equivalent material of tracheae etc., external at present research and the patent report (Chen Jingtao that has more collagen base medical material, Xu Zheng, Gu Qisheng. the latest developments of collagen protein research and development. " Shanghai biomedical engineering " magazine, 2004,25 (2): 52-55,47), and domestic feasibility study and clinical application also be in the starting stage (Mu Changdao. the research of the extraction of collagen protein and biomembrane material thereof. the post-doctor of Sichuan University paper, 2003).
The preparation of medical collagen has vast market prospect.Report according to statistics, collagen protein be only with regard to the application of medical material aspect, sold in 2003 to surpass 4,000,000,000 U.S. dollars, and also annual with 12% rate increase.The special performance of collagen protein with and wide market application prospect will impel people that collagen protein and goods thereof are carried out extensively dark people's research, collagen protein will promote the well-being of mankind more widely.
Medical collagen production gordian technique comprises extraction, purifying, modification of the choosing of raw material, collagen protein etc.The history of collagen protein technology of preparing is longer, as far back as nineteen twenty-nine just have patented technology with phosphoric acid dissolving animal bone tissue extraction collagen (Xu Xinyu. the extraction of collagen, modification, crosslinked and use. dialysis and artificial organs, 2004,3 (15): 38-47).Up to the present, the collagen preparation is still to be extracted as the master from flesh tissue.According to lot of documents both domestic and external report, up to the present be applied to the preparation of coml collagen and only be type i collagen, and have only the price people of the type i collagen that from the skin of animal or heel string, extracts just can accept.The collagen of other type is preparation under study for action only, all is unsuitable for mass production owing to cost an arm and a leg.
Because collagen is water insoluble, it is very difficult it being separated from biological tissue and extract.Particularly importantly, need to keep well the triple helix structure of collagen in leaching process, the reason excess processes does not make collagen further be decomposed into gelatin and loses the physical strength that collagen possesses, and this difficulty that just makes collagen extract is bigger.
In sum, prior art has the following disadvantages:
1. only considered that with ox-hide, ox tendon and mouse tail tendon be raw material, its resource-constrained, and have the risk of carrying crazy heifer disease virus;
2. singlely take the in the majority of Enzymatic Extraction, extraction process complexity, cycle are long;
3. productive rate is low, generally at (dry weight basis) below 16%;
4. production cost height.
Three, summary of the invention
The objective of the invention is to provide a kind of preparation method of medical collagen pulvis at the deficiencies in the prior art, be characterized in that with purified pigskin cheap and easy to get be raw material, after pre-treatment, with purified pigskin freeze-drying, pulverizing, adopt again prozyme collagen treatment agent E extract, refining and dry, obtain the medical collagen pulvis.
Purpose of the present invention is realized that by following technical measures wherein said starting material umber is parts by weight except that specifying.
The preparation method of medical collagen contains following steps:
(1) pre-treatment: get purified pigskin 100 weight parts, put into rotary drum, the adding temperature is 32-40 ℃ a deionized water 300-600 weight part, and sodium-chlor 3-10 weight part rotates 20-150min; Control dried waste liquid, the adding temperature is 32-40 ℃ a deionized water 300-600 weight part, and peregal 1-5 weight part rotates 20-150min; Control dried waste liquid, add deionized water 300-600 weight part, rotate 30-40min; Control dried waste liquid, add deionized water 300-600 weight part, rotate 30-40min;
(2) cut apart: take out pretreated skin base from rotary drum, centrifuge dewatering is divided into the fritter of 2-6 * 2-6cm then;
(3) freeze-drying: little skin bit is put into Freeze Drying Equipment, and the cooling parameter is decided to be the 2 ℃/min that descends, and reduces to-34 ℃ until condenser temperature, is evacuated to 45-150mbar and keeps 24h, and the 8-15h that slowly heats up then makes condenser temperature rise to 15-30 ℃, finishes freeze-drying process;
(4) pulverize: on the onal machine little skin bit is ground into the cuticle grain, its granularity requirements is: 0.5-1.0 * 0.5-1.0mm;
(5) pickling: the dry cuticle grain of getting 100 weight parts is put in the rotary drum, Tutofusin tris-the hydrochloride that adds the 1000-5000 weight part, pH is 7.4 damping fluid, regulate the interior temperature of drum with ice cube and reach 4-15 ℃, intermittently rotate, rotation 3min/30min * 2-9 time, rotate 10min then continuously, intermittently rotate again, rotation 3min/30min * 1-6 time, leave standstill 30min, with acidproof vacuum pump sucking-off rotary drum clear liquid at the middle and upper levels, in rotary drum, add HAc again, regulate pH=1.8-2.8, temperature is 4-15 ℃ in the rotary drum, leaves standstill to soak 1-4h;
(6) enzymic digestion: adjusting also keeps the interior temperature of rotary drum to reach 4-15 ℃, adds the prozyme collagen treatment agent E of 0.5-4.0 weight part, rotates 4-12h continuously;
(7) once saltout: with acidproof vacuum pump the enzymic digestion liquid pump is gone into to carry out centrifugation in the whizzer, and with acidproof vacuum pump supernatant liquor is pumped in the rotary drum, the pH value of adjusting clear liquid with NaOH solution in the rotation, slowly adds (NH to 7.2-8.2
4)
2SO
4, make its concentration reach 0.8-1.8mol/L, leave standstill 6-14h;
(8) acid dissolving: go into to carry out centrifugation in the whizzer with the acidproof vacuum pump liquid pump of will saltouing, get the throw out in the whizzer, abandon supernatant liquor, and with in the 0.1-1.2MHAc solution that has prepared in advance in the immigration of the throw out in the whizzer rotary drum, rotate 10-40min, obtain rough collagen solution;
(9) secondary is saltoutd: with rough collagen solution acid for adjusting pH is 1.8-2.8, and centrifugation in whizzer pumps into this clear liquid in another rotary drum with acidproof vacuum pump, in the rotation, slowly adds (NH in the clear liquid in rotary drum
4)
2SO
4, make its concentration reach 0.8-1.8mol/L, then, the pH value of adjusting clear liquid with NaOH solution leaves standstill 8-18h to 6.8-7.4;
(10) acid dissolving: pump supernatant liquor with acidproof vacuum pump, abandon it, leave and take throw out; In the rotation, slowly add 0.2-0.8M HAc solution 100-300 weight part, rotate 10-40min from drum gudgeon;
(11) classification: with resulting collagen solution doubly, remove particulate, adopt the gel-filtration chromatography to carry out classification then by the millipore filtration of 0.2 μ m with deionized water dilution 1-5;
(12) dialysis: pour the collagen solution of different fractions into the 36mm dialysis tubing respectively, dialysis molecular weight 10kD is earlier to the potassium primary phosphate of 0.04mol/L and 0.02mol/L or sodium dihydrogen phosphate dialysis 2 days, again to distill water dialysis 3 days;
(13) spraying drying: respectively the collagen solution of different fractions is dissolved into the dilute solution of concentration less than 0.5wt% with 0.2-0.8M HAc solution, is lower than spraying drying under 60 ℃ the condition in temperature, the packing of weighing back hermetically drying is preserved.
The proportioning of prozyme collagen treatment agent E is: 3305 aspartic proteases, 3.2 weight parts of 50,000 units, stomach en-0.8 weight part of 0.4 ten thousand units, papoid 1.2 weight parts of 0.4 ten thousand units, wilkinite 1.8 weight parts, wheat bran 5.5 weight parts.
Millipore filtration is the millipore filtration that mixed cellulose ester is made.
Rotary drum is wooden rotary drum of suspension type or stainless steel rotary drum.
Dialysis tubing is that man-made fiber is made.
Adopt method of the present invention to handle the medical collagen protein powder that obtains, its salient features is as follows:
1. outward appearance: white powder, the impurity that no naked eyes are visible;
2. moisture :≤10% (m/m);
3. heavy metal content :≤10 μ g/g (m/m);
4. water regain: should be not less than 2.0 times of powder material own wt;
5. ash content :≤1% (m/m);
6. sterility test: should be aseptic, aseptic validity period should be no less than 1 year;
7. purity: 〉=90% (m/m);
8.pH value: 5.0-7.0
9. amino acid analysis: should not contain halfcystine and tryptophane;
10. oxyproline analysis: content should be not less than 8% (m/m) of total protein content;
11. biology performance:
Collagen protein powder should carry out biological assessment according to the requirement of GB/T 16886.1, should not discharge the material that human body is had undesirable action.
(1) cell toxicity test: cell-cytotoxic reaction is not more than 1 grade.
(2) sensitization test: no sensitivity response.
(3) intradermoreaction test: primary cutaneous stimulation index (PII) is 0.0.
(4) muscle implantation test: implanting 4 all back tissue reactions and negative control does not have significant difference.
Adopt method of the present invention to handle the medical collagen protein powder that obtains, be used for:
1. prepare the bio-medical mould material;
2. prepare the medical fibre material;
3. medicament slow release material;
4. hemostatic material.
The present invention compared with prior art has the following advantages:
1. directly utilize pigskin cheap and easy to get, wide material sources to be the feedstock production medical collagen, overcome and adopted beef tendon, mouse tail tendon to prepare the problem of the scarcity of resources of medical collagen, also can avoid adopting ox-hide may carry the risk of crazy heifer disease virus for the feedstock production medical collagen;
2. adopt prozyme collagen treatment agent that pigskin is carried out enzymic digestion, speed of response is fast, and the productive rate height can access the collagen protein of expecting molecular weight and distribution thereof, has improved the controllability of digestive process;
3. whole process is carried out under lower temperature, has kept the biological activity of collagen protein to greatest extent;
4. simple to operate, processing condition are content with very little, and running cost is low, and are with short production cycle;
5. leaching process does not have " three wastes " generation, and it is environmentally friendly that production process belongs to.
Four, embodiment
Below by embodiment the present invention is carried out concrete description; be necessary to be pointed out that at this; present embodiment only is used for the present invention is further specified; and can not be interpreted as restriction to protection scope of the present invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of foregoing invention.
Embodiment 1
(1) pre-treatment: get purified pigskin 100 weight parts, put into rotary drum, the adding temperature is 32 ℃ deionized water 300 weight parts, and sodium-chlor 4 weight parts rotate 60min; Control dried waste liquid, the adding temperature is 32 ℃ deionized water 300 weight parts, and peregal 1 weight part rotates 60min; Control dried waste liquid, add deionized water 300 weight parts, rotate 30min; Control dried waste liquid, add deionized water 300 weight parts, rotate 30min;
(2) cut apart: take out pretreated skin base from rotary drum, centrifuge dewatering is divided into the fritter of 2-6 * 2-6cm then;
(3) freeze-drying: little skin bit is put into Freeze Drying Equipment, and the cooling parameter is decided to be the 2 ℃/min that descends, and reduces to-34 ℃ until condenser temperature, is evacuated to 45-150mbar and keeps 24h, and the 8h that slowly heats up then makes condenser temperature rise to 15 ℃, finishes freeze-drying process;
(4) pulverize: on the onal machine little skin bit is ground into the cuticle grain, its granularity requirements is: 0.5-1.0 * 0.5-1.0mm;
(5) pickling: the dry cuticle grain of getting 100 weight parts is put in the rotary drum, Tutofusin tris-the hydrochloride that adds 2000 weight parts, pH is 7.4 damping fluid, regulate the interior temperature of drum with ice cube and reach 6 ℃, intermittently rotate, rotate 3min/30min * 4 time, rotate 10min then continuously, intermittently rotate again, rotate 3min/30min * 2 time, leave standstill 30min, with acidproof vacuum pump sucking-off rotary drum clear liquid at the middle and upper levels, in rotary drum, add HAc again, regulate pH=2.2, temperature is 6 ℃ in the rotary drum, leaves standstill to soak 2h;
(6) enzymic digestion: adjusting also keeps the interior temperature of rotary drum to reach 6 ℃, adds the prozyme collagen treatment agent E of 1.0 weight parts, rotates 8h continuously;
(7) once saltout: with acidproof vacuum pump the enzymic digestion liquid pump is gone into to carry out centrifugation in the whizzer, and supernatant liquor is pumped in the rotary drum,, in the rotation, slowly add (NH with the pH value to 7.2 of NaOH solution adjustment clear liquid with acidproof vacuum pump
4)
2SO
4, make its concentration reach 1.0mol/L, leave standstill 6h;
(8) acid dissolving: go into to carry out centrifugation in the whizzer with the acidproof vacuum pump liquid pump of will saltouing, get the throw out in the whizzer, abandon supernatant liquor, and with in the 0.6MHAc solution that has prepared in advance in the immigration of the throw out in the whizzer rotary drum, rotate 20min, obtain rough collagen solution;
(9) secondary is saltoutd: with rough collagen solution acid for adjusting pH is 2.2, and centrifugation in whizzer pumps into this clear liquid in another rotary drum with acidproof vacuum pump, in the rotation, slowly adds (NH in the clear liquid in rotary drum
4)
2SO
4, make its concentration reach 1.0mol/L, then, the pH value to 7.0 with NaOH solution adjustment clear liquid leaves standstill 10h;
(10) acid dissolving: pump supernatant liquor with acidproof vacuum pump, abandon it, leave and take throw out; In the rotation, slowly add 0.2M HAc solution 100 weight parts, rotate 20min from drum gudgeon;
(11) classification: with 1 times of resulting collagen solution dilution, remove particulate, adopt the gel-filtration chromatography to carry out classification then by the millipore filtration of 0.2 μ m;
(12) dialysis: pour the collagen solution of different fractions into the 36mm dialysis tubing respectively, dialysis molecular weight 10kD is earlier to the potassium primary phosphate of 0.04mol/L and 0.02mol/L or sodium dihydrogen phosphate dialysis 2 days, again to distill water dialysis 3 days;
(13) spraying drying: respectively the collagen solution of different fractions is dissolved into the dilute solution of concentration less than 0.5wt% with 0.2M HAc solution, is lower than spraying drying under 60 ℃ the condition in temperature, the packing of weighing back hermetically drying is preserved.
Embodiment 2
(1) pre-treatment: get purified pigskin 100 weight parts, put into rotary drum, the adding temperature is 36 ℃ deionized water 450 weight parts, and sodium-chlor 7 weight parts rotate 120min; Control dried waste liquid, add temperature again and be 36 ℃ deionized water 450 weight parts, peregal 3 weight parts rotate 120min; Control dried waste liquid, add deionized water 450 weight parts, rotate 35min; Control dried waste liquid, add deionized water 450 weight parts, rotate 35min;
(2) cut apart: take out pretreated skin base from rotary drum, centrifuge dewatering is divided into the fritter of 2-6 * 2-6cm then;
(3) freeze-drying: little skin bit is put into Freeze Drying Equipment, and the cooling parameter is decided to be the 2 ℃/min that descends, and reduces to-34 ℃ until condenser temperature, is evacuated to 45-150mbar and keeps 24h, and the 12h that slowly heats up then makes condenser temperature rise to 22 ℃, promptly finishes freeze-drying process;
(4) pulverize: on the onal machine little skin bit is ground into the cuticle grain, its granularity requirements is: 0.5-1.0 * 0.5-1.0mm;
(5) pickling: the dry cuticle grain of getting 100 weight parts is put in the rotary drum, Tutofusin tris-the hydrochloride that adds 3500 weight parts, pH is 7.4 damping fluid, regulate the interior temperature of drum with ice cube and reach 11 ℃, intermittently rotate, rotate 3min/30min * 6 time, rotate 10min then continuously, intermittently rotate again, rotate 3min/30min * 4 time, leave standstill 30min, with acidproof vacuum pump sucking-off rotary drum clear liquid at the middle and upper levels, in rotary drum, add HAc again, regulate pH=2.4, temperature is 11 ℃ in the rotary drum, leaves standstill to soak 3h;
(6) enzymic digestion: adjusting also keeps the interior temperature of rotary drum to reach 11 ℃, adds the prozyme collagen treatment agent E of 3.0 weight parts, rotates 10h continuously;
(7) once saltout: with acidproof vacuum pump the enzymic digestion liquid pump is gone into to carry out centrifugation in the whizzer, and supernatant liquor is pumped in the rotary drum,, in the rotation, slowly add (NH with the pH value to 7.6 of 6MNaOH solution adjustment clear liquid with acidproof vacuum pump
4)
2SO
4, make its concentration reach 1.4mol/L, leave standstill 10h;
(8) acid dissolving: go into to carry out centrifugation in the whizzer with the acidproof vacuum pump liquid pump of will saltouing, get the throw out in the whizzer, abandon supernatant liquor, and with in the 0.9MHAc solution that has prepared in advance in the immigration of the throw out in the whizzer rotary drum, rotate 30min, obtain rough collagen solution;
(9) secondary is saltoutd: with rough collagen solution acid for adjusting pH is 2.5, and centrifugation in whizzer pumps into this clear liquid in another rotary drum with acidproof vacuum pump, in the rotation, slowly adds (NH in the clear liquid in rotary drum
4)
2SO
4, make its concentration reach 1.4mol/L, then, the pH value to 7.2 with NaOH solution adjustment clear liquid leaves standstill 14h;
(10) acid dissolving: pump supernatant liquor with acidproof vacuum pump, abandon it, leave and take throw out; In the rotation, slowly add 0.5M HAc solution 200 weight parts, rotate 30min from drum gudgeon;
(11) classification: with 3 times of resulting collagen solution dilutions, remove particulate, adopt the gel-filtration chromatography to carry out classification then by the millipore filtration of 0.2 μ m;
(12) dialysis: pour the collagen solution of different fractions into the 36mm dialysis tubing respectively, dialysis molecular weight 10kD is earlier to the potassium primary phosphate of 0.04mol/L and 0.02mol/L or sodium dihydrogen phosphate dialysis 2 days, again to distill water dialysis 3 days;
(13) spraying drying: respectively the collagen solution of different fractions is dissolved into the dilute solution of concentration less than 0.5wt% with 0.5M HAC solution, is lower than spraying drying under 60 ℃ the condition in temperature, the packing of weighing back hermetically drying is preserved.
Embodiment 3
(1) pre-treatment: get purified pigskin 100 weight parts, put into rotary drum, the adding temperature is 40 ℃ deionized water 600 weight parts, and sodium-chlor 10 weight parts rotate 150min; Control dried waste liquid, add temperature again and be 40 ℃ deionized water 600 weight parts, peregal 5 weight parts rotate 150min; Control dried waste liquid, add deionized water 600 weight parts, rotate 40min; Control dried waste liquid, add deionized water 600 weight parts, rotate 40min;
(2) cut apart: take out pretreated skin base from rotary drum, centrifuge dewatering is divided into the fritter of 2-6 * 2-6cm then;
(3) freeze-drying: little skin bit is put into Freeze Drying Equipment, and the cooling parameter is decided to be the 2 ℃/min that descends, and reduces to-34 ℃ until condenser temperature, is evacuated to 45-150mbar and keeps 24h, and the 8-15h that slowly heats up then makes condenser temperature rise to 30 ℃, promptly finishes freeze-drying process;
(4) pulverize: on the onal machine little skin bit is ground into the cuticle grain, its granularity requirements is: 0.5-1.0 * 0.5-1.0mm;
(5) pickling: the dry cuticle grain of getting 100 weight parts is put in the rotary drum, Tutofusin tris-the hydrochloride that adds 5000 weight parts, pH is 7.4 damping fluid, regulate the interior temperature of drum with ice cube and reach 15 ℃, intermittently rotate, rotate 3min/30min * 9 time, rotate 10min then continuously, intermittently rotate again, rotate 3min/30min * 6 time, leave standstill 30min, with the careful sucking-off rotary drum of acidproof vacuum pump clear liquid at the middle and upper levels, in rotary drum, add HAc again, regulate pH=2.8, temperature is 15 ℃ in the rotary drum, leaves standstill to soak 4h;
(6) enzymic digestion: adjusting also keeps the interior temperature of rotary drum to reach 15 ℃, adds the prozyme collagen treatment agent E of 4.0 weight parts, rotates 12h continuously;
(7) once saltout: with acidproof vacuum pump the enzymic digestion liquid pump is gone into to carry out centrifugation in the whizzer, and supernatant liquor is pumped in the rotary drum,, in the rotation, slowly add (NH with the pH value to 8.2 of NaOH solution adjustment clear liquid with acidproof vacuum pump
4)
2SO
4, make its concentration reach 1.8mol/L, leave standstill 14h;
(8) acid dissolving: go into to carry out centrifugation in the whizzer with the acidproof vacuum pump liquid pump of will saltouing, get the throw out in the whizzer, abandon supernatant liquor, and with in the 1.2MHAc solution that has prepared in advance in the immigration of the throw out in the whizzer rotary drum, rotate 40min, obtain rough collagen solution;
(9) secondary is saltoutd: it is 2.8 that rough collagen solution is transferred pH with acid, and centrifugation in whizzer pumps into this clear liquid in another rotary drum with acidproof vacuum pump, in the rotation, slowly adds (NH in the clear liquid in rotary drum
4)
2SO
4, make its concentration reach 1.8mol/L, then, the pH value to 7.4 with NaOH solution adjustment clear liquid leaves standstill 18h;
(10) acid dissolving: pump supernatant liquor with acidproof vacuum pump, abandon it, leave and take throw out; In the rotation, slowly add 0.8M HAc solution 300 weight parts, rotate 40min from drum gudgeon;
(11) classification: resulting collagen solution is suitably diluted, remove particulate, adopt the gel-filtration chromatography to carry out classification then by the millipore filtration of 0.2 μ m;
(12) dialysis: pour the collagen solution of different fractions into the 36mm dialysis tubing respectively, dialysis molecular weight 10kD is earlier to the potassium primary phosphate of 0.04mol/L and 0.02mol/L or sodium dihydrogen phosphate dialysis 2 days, again to distill water dialysis 3 days;
(13) spraying drying: respectively the collagen solution of different fractions is dissolved into the dilute solution of concentration less than 0.5wt% with 0.8M HAC solution, is lower than spraying drying under 60 ℃ the condition in temperature, the packing of weighing back hermetically drying is preserved.
Annotate: the used collagen treatment agent E of the present invention is that Chengdu enlightening Australia collagen Hitek Ltd produces, and millipore filtration is that Haining holy magnificent filter material factory produces, and dialysis tubing and other chemical reagent are that market is bought.