Summary of the invention
The objective of the invention is to propose the preparation method that a kind of collagen is modified bacteria cellulose composite membrane, the collagen that this method is prepared is modified bacteria cellulose composite membrane and is had excellent mechanical property and good permeable, ventilative or drainage, moisture sorption effect, simultaneously can promote wound healing, be used for guiding fields such as growing film, soft-tissue patch, short skin healing wound dressings, organization engineering skin and health care facial mask.
Technical scheme of the present invention is: prepares a kind of collagen and modifies bacteria cellulose composite membrane, be attached with collagen protein on the Nano grade fibrillar meshwork structure of bacteria cellulose, but even coated bacteria cellulosic fibre.In the surface of bacteria cellulose and network structure, form collagen layer by physics or chemical process, the physical method ultimate principle is collagen protein in the surface of bacteria cellulose prosperity and network structure by the physical adsorption deposition, and the ultimate principle of chemical reaction is aldehyde radical or carboxyl formation chemical bond in collagen protein residue and the functionalization bacteria cellulose film.
The present invention realizes the preparation of collagen modification bacteria cellulose composite membrane by the following method.
Its preparation method may further comprise the steps:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, 80 ~ 100 ℃ of following water-bath 60min or more than, wash out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 80 ~ 100 ℃ of following water-bath 30-60min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 3-5 time, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose (being designated as BC) behind the purifying, In Shade preservation afterwards;
Step 3, bacteria cellulose radical functino:
Select a series of oxidation systems that are applicable to cellulose materials such as TEMPO, Periodic acid or periodate, nitrogen peroxide etc. for use, hydroxyl oxygen on the bacteria cellulose is changed into carboxyl or aldehyde radical, with deionized water soaking flushing or remove residual oxidizer with ion exchange method repeatedly, obtain the bacteria cellulose film (being designated as OBC) of radical functino then;
The adjustment of step 4, bacteria cellulose water content and sterilization process:
Pressure with 20-1000N dewaters bacteria cellulose (BC and OBC), obtain the bacteria cellulose film of different moisture content, then bacteria cellulose film is passed through high temperature steaming, ultraviolet ray or Co60 radiation sterilization, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
Step 5, collagen are modified the recombining process of bacteria cellulose composite membrane:
Method 1 physics composite algorithm: the recombining process of collagen and BC
The collagen product is made into the solution of 0.01%-20% respectively, above-mentioned collagen solution is coated on BC film surface, the unit coating amount is 10-200 μ L/cm
2Or the collagen product is made into the solution of 0.01%-20% respectively, and above-mentioned BC film is dipped in 1s-24h in the collagen solution, make the physics composite collagen and modify bacteria cellulose composite membrane.
Method 2 chemically composited methods: the recombining process of collagen and OBC
The collagen product is made into the solution of 0.01%-20% respectively, above-mentioned collagen solution is coated on OBC film surface, the unit coating amount is 10-200 μ L/cm
2Or the collagen product is made into the solution of 0.01%-20% respectively, and above-mentioned OBC is dipped in 1s-24h in the collagen solution, make chemically composited collagen and modify bacteria cellulose composite membrane.
Aldehyde radical or carboxyl in the collagen modification bacteria cellulose composite membrane that method 2 makes in collagen protein residue and the functionalization bacteria cellulose film form chemical bond, reach stable Chemical bond.
Step 6, the collagen that will obtain by step 5 are modified bacteria cellulose composite membrane and be carried out drying treatment, obtain collagen and modify the compound dry film of bacteria cellulose; Perhaps will modify the bacteria cellulose composite membrane dehydration by the collagen that step 5 obtains, and adjust the collagen that obtains different water-contents again and modify the compound wet film of bacteria cellulose.
Described bacteria cellulose film is to belong in the bacteriums certain by genus acetobacter, Sarcina, Rhodopseudomonas, rhizobium, achromobacter, Alcaligenes, aerobacter, Azotobacter, Agrobacterium etc. nine to produce through fermentation, and the bacteria cellulose that produces has three-dimensional porous network structure.
The method of the drying treatment in the method one described step 5 comprises following 3 kinds of methods:
Normal temperature or 20 ℃ ~ 60 ℃ following freeze-day with constant temperature are handled under a, the ventilation state;
B, with bacteria cellulose drying treatment in-20 ℃ ~-60 ℃ freeze drier, or vacuum drying treatment;
C, the bacterial cellulose wet-coating chilling handled after, drying treatment under Vacuumdrier, thermostat container or normal temperature obtains the lower collagen of fragility and modifies bacteria cellulose composite membrane again.
Modify bacteria cellulose composite membrane by the collagen that the present invention prepares, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is the 0.1-20% of gross weight.
Compared with prior art, the present invention has following advantage and beneficial effect:
1, collagen that the present invention obtains is modified bacteria cellulose composite membrane, can promote the recovery of collagen content in the damaged skin;
2, the present invention utilizes unique texture and the character that bacteria cellulose has, and preparation contains the bacteria cellulose film of collagen, make bacteria cellulose obtain excellent biocompatibility and trauma repair ability, and the content of collagen is controlled;
3, the present invention adopts unique recombining process to prepare collagen and modifies bacteria cellulose composite membrane, permeable, the ventilative or drainage of existing collagen medicament existence, the problem of moisture sorption effect difference have been overcome, the novel method that provides collagen to use at burn trauma, cellular affinity is good, promotes the wound growth;
4, chemically composited legal system is equipped with in the collagen modification bacteria cellulose composite membrane of gained, and the functionalization group of the residue in the collagen protein in bacteria cellulose is combined by chemical bonds such as imine linkages, and it is compound more firm.Imine linkage can rupture under certain condition simultaneously, thereby makes collagen reach the purpose that control discharges;
5, preparation process of the present invention is simple, technical maturity, be suitable for the industrial production of the collagen modification bacteria cellulose composite membrane of different demands, can be used for guiding fields such as growing film, soft-tissue patch, short skin healing wound dressings, organization engineering skin and health care facial mask.
Embodiment
Below in conjunction with accompanying drawing, the preparation method who a kind of collagen of the present invention is modified bacteria cellulose composite membrane by specific examples is described further.
Embodiment one
The commercially available bacteria cellulose film clip that is made by acetobacter xylinum is become the bacteria cellulose sample of 10 * 10cm, and the preparation process that collagen is modified bacteria cellulose composite membrane is as follows:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, more than 90 ℃ of following water-bath 60min, washes out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 90 ℃ of following water-bath 30min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 5 times, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose behind the purifying, In Shade preservation afterwards.
The adjustment of step 3, bacteria cellulose water content and sterilization process:
After the bacteria cellulose pre-treatment is adjusted to neutrality, pressure with 200N dewaters bacteria cellulose, obtain the bacteria cellulose film of different moisture content, then bacteria cellulose film is sterilized by high temperature steaming, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
The recombining process of step 4, collagen and bacteria cellulose:
The collagen product is made into 1% solution, above-mentioned collagen solution is coated on the bacteria cellulose film surface, the unit coating amount is 20 μ L/cm
2
Step 5, the collagen that will obtain by step 4 are modified bacteria cellulose composite membrane 20 ℃ of freeze-day with constant temperature under ventilation state and are handled, and obtain collagen and modify the compound dry film of bacteria cellulose composite membrane.
Modify bacteria cellulose composite membrane by the collagen that a process for preparing, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is 1.2% of gross weight, and water content is 63.7% of original bulk.
Embodiment two
The commercially available bacteria cellulose film clip that is made by acetobacter xylinum is become the bacteria cellulose sample of 10 * 10cm, and the preparation process that collagen is modified bacteria cellulose composite membrane is as follows:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, more than 80 ℃ of following water-bath 60min, washes out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 80 ℃ of following water-bath 60min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 5 times, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose behind the purifying, In Shade preservation afterwards.
Step 3, bacteria cellulose radical functino:
Select the TEMPO oxidation system for use, the hydroxyl oxygen of C6 position on the bacteria cellulose is changed into carboxyl, with deionized water soaking flushing or remove residual oxidizer with ion exchange method repeatedly, obtain the bacteria cellulose film of radical functino then;
The adjustment of step 4, bacteria cellulose water content and sterilization process:
With the bacteria cellulose in the step 3, with the pressure of 500N bacteria cellulose is dewatered, obtain the bacteria cellulose film of different moisture content, then with bacteria cellulose film by disinfection by ultraviolet light, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
The recombining process of step 5, collagen and bacteria cellulose:
The collagen product is made into 5% solution respectively, above-mentioned collagen solution is coated on the bacteria cellulose film surface, the unit coating amount is 50 μ L/cm
2
Step 6, the collagen that will obtain by step 4 are modified bacteria cellulose composite membrane drying treatment in-50 ℃ freeze drier, obtain collagen and modify the compound dry film of bacteria cellulose composite membrane;
Modify bacteria cellulose composite membrane by the collagen that a process for preparing, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is 4.7% of gross weight, and water content is 35.4% of original bulk.
Embodiment three
The commercially available bacteria cellulose film clip that is made by acetobacter xylinum is become the bacteria cellulose sample of 10 * 10cm
Product, the preparation process that collagen is modified bacteria cellulose composite membrane is as follows:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, more than 85 ℃ of following water-bath 60min, washes out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 85 ℃ of following water-bath 45min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 5 times, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose behind the purifying, In Shade preservation afterwards.
The adjustment of step 3, bacteria cellulose water content and sterilization process:
After the bacteria cellulose pre-treatment is adjusted to neutrality, pressure with 50N dewaters bacteria cellulose, obtain the bacteria cellulose film of different moisture content, then bacteria cellulose film is passed through the Co60 radiation sterilization, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
The recombining process of step 4, collagen and bacteria cellulose:
The collagen product is made into 2% solution respectively, above-mentioned bacteria cellulose is dipped in 60s in the collagen solution.
Step 5, the collagen that will obtain by step 4 are modified the bacteria cellulose composite membrane dehydration, adjust the collagen that obtains different water-contents again and modify the bacteria cellulose composite membrane wet film.
Modify bacteria cellulose composite membrane by the collagen that a process for preparing, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is 2.7% of gross weight, and water content is 78.9% of original bulk.
Embodiment four
The commercially available bacteria cellulose film clip that is made by acetobacter xylinum is become the bacteria cellulose sample of 10 * 10cm, and the preparation process that collagen is modified bacteria cellulose composite membrane is as follows:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, more than 90 ℃ of following water-bath 60min, washes out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 80 ℃ of following water-bath 30min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 5 times, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose behind the purifying, In Shade preservation afterwards.
Step 3, bacteria cellulose radical functino:
Select the periodate oxidation system for use, the hydroxyl oxygen on bacteria cellulose C2, the C3 position is changed into aldehyde radical, with deionized water soaking flushing or remove residual oxidizer with ion exchange method repeatedly, obtain the bacteria cellulose film of radical functino then;
The adjustment of step 4, bacteria cellulose water content and sterilization process:
With the bacteria cellulose in the step 3, with the pressure of 500N bacteria cellulose is dewatered, obtain the bacteria cellulose film of different moisture content, then with bacteria cellulose film by disinfection by ultraviolet light, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
The recombining process of step 5, collagen and bacteria cellulose:
The collagen product is made into 6% solution respectively, above-mentioned bacteria cellulose is dipped in 1h in the collagen solution.
Step 6, the collagen that will obtain by step 4 are modified the bacteria cellulose composite membrane dehydration, adjust the collagen that obtains different water-contents again and modify the bacteria cellulose composite membrane wet film.
Modify bacteria cellulose composite membrane by the collagen that a process for preparing, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is 6.4% of gross weight, and water content is 45% of original bulk.
Embodiment five
The commercially available bacteria cellulose film clip that is made by acetobacter xylinum is become the bacteria cellulose sample of 10 * 10cm, and the preparation process that collagen is modified bacteria cellulose composite membrane is as follows:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, more than 95 ℃ of following water-bath 60min, washes out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 90 ℃ of following water-bath 30min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 4 times, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose behind the purifying, In Shade preservation afterwards.
The adjustment of step 3, bacteria cellulose water content and sterilization process:
After the bacteria cellulose pre-treatment is adjusted to neutrality, pressure with 800N dewaters bacteria cellulose, obtain the bacteria cellulose film of different moisture content, then bacteria cellulose film is sterilized by high temperature steaming, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
The recombining process of step 4, collagen and bacteria cellulose:
The collagen product is made into 15% solution respectively, above-mentioned collagen solution is coated on the bacteria cellulose film surface, the unit coating amount is 25 μ L/cm
2
After step 5, the collagen that will obtain by step 4 were modified the bacteria cellulose composite membrane chilling and handled, drying treatment in Vacuumdrier obtained collagen and modifies the compound dry film of bacteria cellulose composite membrane again.
Modify bacteria cellulose composite membrane by the collagen that a process for preparing, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is 7.2% of gross weight, and water content is 17.3% of original bulk.
Embodiment six
The commercially available bacteria cellulose film clip that is made by acetobacter xylinum is become the bacteria cellulose sample of 10 * 10cm, and the preparation process that collagen is modified bacteria cellulose composite membrane is as follows:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, more than 85 ℃ of following water-bath 60min, washes out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 100 ℃ of following water-bath 45min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 5 times, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose behind the purifying, In Shade preservation afterwards.
Step 3, bacteria cellulose radical functino:
Select the nitrogen peroxide oxidation system for use, the hydroxyl oxygen of C6 position on the bacteria cellulose is changed into carboxyl, with deionized water soaking flushing or remove residual oxidizer with ion exchange method repeatedly, obtain the bacteria cellulose film of radical functino then;
The adjustment of step 4, bacteria cellulose water content and sterilization process:
With the bacteria cellulose in the step 3, pressure with 100N dewaters bacteria cellulose, obtain the bacteria cellulose film of different moisture content, then bacteria cellulose film is passed through the Co60 radiation sterilization, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
The recombining process of step 5, collagen and bacteria cellulose:
The collagen product is made into 1% solution respectively, above-mentioned bacteria cellulose is dipped in 120s in the collagen solution.
Step 6, the collagen that will obtain by step 4 are modified bacteria cellulose composite membrane drying treatment in-45 ℃ freeze drier, obtain collagen and modify the compound dry film of bacteria cellulose composite membrane.
Modify bacteria cellulose composite membrane by the collagen that a process for preparing, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is 3.54% of gross weight, and water content is 70.2% of original bulk.
Embodiment seven
The commercially available bacteria cellulose film clip that is made by acetobacter xylinum is become the bacteria cellulose sample of 10 * 10cm, and the preparation process that collagen is modified bacteria cellulose composite membrane is as follows:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, more than 100 ℃ of following water-bath 60min, washes out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 95 ℃ of following water-bath 60min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 5 times, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose behind the purifying, In Shade preservation afterwards.
The adjustment of step 3, bacteria cellulose water content and sterilization process:
After the bacteria cellulose pre-treatment is adjusted to neutrality, pressure with 1000N dewaters bacteria cellulose, obtain the bacteria cellulose film of different moisture content, then bacteria cellulose film is passed through disinfection by ultraviolet light, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
The recombining process of step 4, collagen and bacteria cellulose:
The collagen product is made into 10% solution respectively, above-mentioned collagen solution is coated on the bacteria cellulose film surface, the unit coating amount is 100 μ L/cm
2
Step 5, the collagen that will obtain by step 4 are modified the bacteria cellulose composite membrane dehydration, adjust the collagen that obtains different water-contents again and modify the bacteria cellulose composite membrane wet film.
Modify bacteria cellulose composite membrane by the collagen that a process for preparing, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is 11.2% of gross weight, and water content is 12.8% of original bulk.
Embodiment eight
The commercially available bacteria cellulose film clip that is made by acetobacter xylinum is become the bacteria cellulose sample of 10 * 10cm, and the preparation process that collagen is modified bacteria cellulose composite membrane is as follows:
The pre-treatment of step 1, bacteria cellulose film:
Get bacteria cellulose and repeatedly wash with clear water, except striping surface medium and impurity.Again film is soaked in the NaOH solution of 0.1mol/L, more than 95 ℃ of following water-bath 60min, washes out thalline and residual substratum in the bacteria cellulose film.Bacteria cellulose film is soaked in the NaOH solution of 0.05mol/L, 95 ℃ of following water-bath 40min repeatedly wash with distilled water then again;
The purifying of step 2, bacteria cellulose film:
Bacteria cellulose film after the above-mentioned pre-treatment with distilled water immersion 4 times, is surveyed the pH value with the light press mold of pH test paper, and finally controlling the bacteria cellulose pH value is 7.2 ± 0.2, obtains the bacteria cellulose behind the purifying, In Shade preservation afterwards.
Step 3, bacteria cellulose radical functino:
Select the periodate oxidation system for use, the hydroxyl oxygen on bacteria cellulose C2, the C3 position is changed into aldehyde radical, with deionized water soaking flushing or remove residual oxidizer with ion exchange method repeatedly, obtain the bacteria cellulose film of radical functino then;
The adjustment of step 4, bacteria cellulose water content and sterilization process:
With the bacteria cellulose in the step 3, pressure with 150N dewaters bacteria cellulose, obtain the bacteria cellulose film of different moisture content, then bacteria cellulose film is passed through the Co60 radiation sterilization, reach the technology sterilization and require (processing requirement aseptic technique afterwards).
The recombining process of step 5, collagen and bacteria cellulose:
The collagen product is made into 7.5% solution respectively, above-mentioned bacteria cellulose is dipped in 600s in the collagen solution.
After step 6, the collagen that will obtain by step 4 were modified the bacteria cellulose composite membrane chilling and handled, drying treatment under Vacuumdrier, thermostat container or normal temperature obtained collagen and modifies the compound dry film of bacteria cellulose composite membrane again.
Modify bacteria cellulose composite membrane by the collagen that a process for preparing, be attached with collagen on the nano-scale fiber network structure of bacteria cellulose, the collagen content of described composite membrane is 6.3% of gross weight, and water content is 70.6% of original bulk.
More than embodiments of the invention are had been described in detail, but described content only is preferred embodiment of the present invention, can not be considered to for limiting practical range of the present invention.All equalizations of doing according to the present patent application scope change and improve etc., all should still belong within the patent covering scope of the present invention.