CN101983725B - Inactivation method of pathogenic microorganisms of chitosan - Google Patents
Inactivation method of pathogenic microorganisms of chitosan Download PDFInfo
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- CN101983725B CN101983725B CN 201010547705 CN201010547705A CN101983725B CN 101983725 B CN101983725 B CN 101983725B CN 201010547705 CN201010547705 CN 201010547705 CN 201010547705 A CN201010547705 A CN 201010547705A CN 101983725 B CN101983725 B CN 101983725B
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Abstract
An inactivation method of the pathogenic microorganisms of chitosan belongs to the technical field of the biological material processing. The method comprises the following steps: performing primary inactivation to the pathogenic microorganisms of chitosan with NaOH solution, then drying chitosan, introducing nitrogen, sealing, and performing 10-50kGy of gamma-ray irradiation at -20 to 30 DEG C to inactivate possible pathogenic microorganisms in chitosan and ensure the safety of chitosan used as the biological material. Compared with the existing inactivation method of the pathogenic microorganisms, by using the method of the invention, the pathogenic microorganisms such as bacteria and viruses can be effectively killed, the quality indexes of chitosan such as deacetylation degree and crystallization degree can not change and the reduction of the molecular weight is obviously lower than that of other inactivation methods. Therefore, the quality and function of the biological material can be maintained while the pathogenic microorganisms brought by the possible pollution can be effectively killed.
Description
Technical field
The present invention relates to chitosan, relate in particular to a kind of inactivation method of pathogenic microorganisms of chitosan, belong to the biomaterial processing technique field.
Background technology
Chitosan chemistry poly-(Isosorbide-5-Nitrae)-2-amino by name-2-deoxidation-callose mainly makes through deacetylated by extensively being present in the chitin that extracts in arthropod shell and Mycophyta cell wall, is a kind of natural aminopolysaccharide.Because it has good biocompatibility, biological degradability, chitosan is widely used in fields such as biomaterial, medicine, food.But chitosan derives from animal body, may be by the various microbiological contaminations such as from virus to the antibacterial; Simultaneously chitosan also may because of the impact of the factors such as instrument and vessel, reagent, production environment, be subject to the pollution of the pathogenic microorganisms such as antibacterial, virus in processing, storage.If these pollutant are propagated to the people, will cause serious health problem.These pollutant also may reduce the material that destruction is even polluted.
That the Method for Inactivation of Bacteria of existing biomaterial comprises is xeothermic, damp and hot, filtration etc.; Virus inactivating method comprises S/D method (organic solvent/surfactant method), Filtration, heating, oxirane etc.They can be referring to patent CN1225020A, patent application 200910209501.X etc. in the application of biomaterial sterilization etc.
But these methods have limitation to the deactivation of chitosan pathogenic microorganism: the S/D method is only effective to enveloped virus; Heating is to heat-resisting viral weak effect, and experiment finds that heating can cause the degraded of chitosan product: after 170 ℃ of 2 hours dry heat sterilizations, chitosan is become brown by off-white color; After 120 ℃ of 4 hours dry heat sterilizations, chitosan becomes brown color, and molecular weight is reduced to initial 27.6%; After 121 ℃ of moist heat sterilizations, chitosan becomes yellow by off-white color, and molecular weight is reduced to initial 56.8%.Alkylating agent may cause the reactions such as degraded of material.Filtration is also inapplicable to full-bodied product such as chitosan.
Gamma-radiation is a kind of electromagnetic radiation that is interacted and produced by subatomic particle, and it all has killing action to various microorganisms, includes peplos and nonenveloped virus and all genotype materials.Compare with traditional antibacterial, virus inactivating method, gamma-radiation irradiation has its unique advantage: article are heated up, be applicable to avoid the sterilization of hot article, except some plastics, living cells and preparation thereof, many medical articles are available its sterilization that carries out disinfection all.2, penetration power is strong.Each position of radiolucent photographed object product generally is not subjected to the restriction of article packing, form.3, the photographed object product can not produce artificial radioactivity, non-residual toxicity after irradiation, and method is easy.4, economize energy, be particularly suited for heavy industrialization and process.Due to above-mentioned advantage, gamma-radiation irradiation is used also more and more in biomaterial to the deactivation of antibacterial and virus, as patent CN1665388A, CN101757651A, CN1628860A.
Generally, the gamma-radiation irradiation of 20～50kGy dosage almost can all antibacterial and the virus of deactivation.But the report such as Fang Yuee adopts 2.5～20kGy gamma-radiation irradiation chitosan film deactivation gold Portugal bacterium and bacillus subtilis, and irradiation has caused that the chitosan molecule amount reduces simultaneously, but concrete numerical value is not reported.The dosage that the irradiation dose that inactivation of virus needs needs greater than sterilization.Irradiation dose is larger, and is also larger to the damage of biological macromolecule material.After the chitosan solution 2-10kGy irradiation such as Choi, molecular weight sharply descends, and during the above irradiation of 100kGy, solution becomes brown.The chitosan solids such as Gryczka molecular weight and molecular weight and crystallinity after 6.3～300kGy irradiation change, and after 14.4kGy irradiation, molecular weight namely is reduced to 30% of initial molecular weight, and after 300kGy irradiation, molecular weight is only 2.9% of initial molecular weight.The chitosan solution irradiation post crystallization degree such as Kang reduce.The quality of the variation meeting appreciable impact chitosan of these molecular weight, degree of crystallinity etc. limits it as the application of biomaterial.
As seen, when the key that chitosan is carried out the pathogenic microorganism deactivation was thoroughly to carry out deactivation, it was very important reducing to greatest extent the biological activity of material and the impact of structure to the greatest extent.Therefore, seek a kind of all viruses of energy deactivation, on less, the easy to operate method of chitosan mass impact, will help to improve the safety of chitosan product simultaneously, and guarantee that its quality is suitable as biomaterial and uses.
NaOH solution can the deactivation pathogenic microorganism, but because a lot of biomaterials do not tolerate highly basic, its application is rarely found.Chitosan is stable to NaOH, but because chitosan is insoluble to alkali, the NaOH of aqueous phase is difficult to the pathogenic microorganism that deactivation is wrapped in chitosan solid particle inside, be subjected to the restriction of such two-phase system to be difficult to the complete inactivation pathogenic microorganism, so the method has significant limitation to pathogenic microorganism deactivation in chitosan.
In sum, chitosan derives from animal body, relates to the risk of virus and infectious agent etc.The Method for Inactivation of Bacteria of existing biomaterial is not suitable for chitosan.Gamma-radiation is the method for pathogenic microorganism in a kind of effective deactivation chitosan, but the pathogen in the complete inactivation chitosan needs higher irradiation dose, and the degraded to chitosan during high dose is comparatively remarkable, and its quality is had certain influence.The restriction that in NaOH deactivation chitosan, pathogenic microorganism is subject to two-phase system has very large limitation.Therefore, seek a kind of all viruses of energy deactivation, simultaneously on less, the easy to operate method of chitosan mass impact, the safety that will help to improve the chitosan product.
Summary of the invention
The present invention be for overcome PI pathogenic microorganism inactivation technology in the pathogenic microorganism deactivation of chitosan defective and provide a kind of when guaranteeing effectively to kill the pathogenic microorganism that may pollute, keep the inactivation method of pathogenic microorganisms of function and the character of chitosan.
Purpose of the present invention can be achieved through the following technical solutions:
Adopt NaOH solution to carry out preliminary deactivation to pathogenic microorganism in chitosan, then that chitosan is dry, fill nitrogen and sealing, temperature control irradiation.
[0013]The NaOH solution of mass concentration 4.0%～40% is adopted in described preliminary sterilization, and NaOH solution and chitosan dosage mass ratio are 20～50 ︰ 1, and temperature is room temperature to 100 ℃, tentatively sterilizes to total plate count less than 1000cfu/g; Then filter, be washed to neutrality.
Described drying is to include but not limited to the modes such as constant pressure and dry, drying under reduced pressure, lyophilization, makes the chitosan water content less than 10%.
Described nitrogen-filled seal is to get a certain amount of chitosan to suitable container, and inflated with nitrogen and sealing immediately to reduce the dividing potential drop of oxygen, reduce the active oxygen that irradiation produces, and reduce irradiation to the degraded of chitosan.
Chitosan after described temperature control irradiation refers to seal is under ℃ condition of temperature-20～30, with the gamma-radiation irradiation of 10～50kGy.
The residual titre of infectious virus in chitosan after temperature control irradiation in chitosan≤0.5 lg TCID
50Clump count is reduced to≤10cfu/g.
Beneficial effect of the present invention: compared with prior art, the present invention reduces the biological load of chitosan by the preliminary deactivation pathogenic microorganism of NaOH solution; Then can realize aseptic virus-freely by the gamma-radiation irradiation of 10～50kGy, avoid high dosage irradiation.Therefore the deacetylation of chitosan, degree of crystallinity etc. remain unchanged before and after deactivation, and molecular weight reduces amplitude significantly lower than other inactivation method of pathogenic microorganisms; Also avoided other deactivation modes to cause degradation of chitosan or be difficult to the shortcomings such as complete inactivation.When guaranteeing effectively to kill the pathogenic microorganism that may pollute, keep the quality and performance of chitosan, after deactivation, the chitosan product is applicable to use as biomaterial.
The specific embodiment
Embodiment 1
Get 1 part, chitosan sample, add successively approximately 10
7Cfu/g Bacillus pumilus spore (Spores of Bacillus pumilus), approximately 10
7Cfu/g bacillus subtilis spore (Spores of Bacillus subtilis) and approximately 10
6TCID
50The pig parvoviral of/g (PPV), the packing of 1g/ bottle.Add 4.0%NaOH solution, the NaOH solution usage is 1,80 ℃ of insulation of 20 ︰ with the chitosan ratio, makes and sterilizes to total plate count less than 1000cfu/g.Filter, be washed to neutrality, ampoule bottle packing, lyophilization, dry rear moisture 9.6%.Inflated with nitrogen, sealing immediately.-20 ℃ are carried out 50kGy gamma-radiation irradiation.Adopt Reed-muench TCID after irradiation
50Method is measured virus titer and is reduced (total reduction factor, TRF) situation, and measures the situation that clump count reduces.
Experimental result shows: 50kGy gamma-radiation irradiation after 80 ℃ of preliminary deactivations of 4.0%NaOH solution, the residual titre of PPV≤0.5 lg TCID
50, clump count is reduced to≤10cfu/g.Simultaneously deactivation front and rear casing polysaccharide degree of crystallinity is respectively 36.4%, 36.7%, deacetylation is 84.2%, and molecular weight is reduced to 76.6% of initial molecular weight.
Embodiment 2
The preparation of chitosan is with embodiment 1.
Be that 15%, NaOH solution is that 1,60 ℃ of 30 ︰ sterilizes to total plate count less than 1000cfu/g with the chitosan dosage ratio with the NaOH concentration of polymer solution.Filter, be washed to neutrality.Drying under reduced pressure is to moisture 8.8 %.The ampoule bottle packing, inflated with nitrogen, sealing.Room temperature is carried out accumulated dose 25kGy gamma-radiation irradiation, the residual titre of PPV after irradiation≤0.5 lg TCID
50, clump count is reduced to≤10cfu/g.After predose, chitosan degree of crystallinity is respectively 36.4,36.7%, and deacetylation is respectively 84.2% and 84.6%, and molecular weight is reduced to 77.8 % of initial molecular weight.
Embodiment 3
The preparation of chitosan is with embodiment 1.
Be that 40%, NaOH solution is 50 ︰ 1 with the chitosan dosage ratio with the NaOH concentration of polymer solution, temperature is room temperature, sterilizes to total plate count less than 1000cfu/g.Filter, be washed to neutrality.Drying under reduced pressure, dry rear moisture 8.9 %.The ampoule bottle packing, inflated with nitrogen, sealing.30 ℃ are carried out accumulated dose 10kGy gamma-radiation irradiation, the residual titre of PPV after irradiation≤0.5 lg TCID
50, clump count is reduced to≤10cfu/g.After predose, chitosan degree of crystallinity is respectively 36.4,36.2%, and deacetylation is respectively 84.2% and 85.0%, and molecular weight is reduced to 80.1% of initial molecular weight.
Embodiment 4
The preparation of chitosan is with embodiment 1.
Be that 30%, NaOH solution is 40 ︰ 1 with the chitosan dosage ratio with the NaOH concentration of polymer solution, temperature is 100 ℃, sterilizes to total plate count less than 1000cfu/g.Filter, be washed to neutrality.Constant pressure and dry, dry rear moisture 9.2 %.The ampoule bottle packing, inflated with nitrogen, sealing.-10 ℃ are carried out accumulated dose 30kGy gamma-radiation irradiation, the residual titre of PPV after irradiation≤0.5 lg TCID
50, clump count is reduced to≤10cfu/g.After predose, chitosan degree of crystallinity is respectively 36.5,36.3%, and deacetylation is respectively 84.1% and 84.7%, and molecular weight is reduced to 78.2% of initial molecular weight.
Claims (1)
1. the inactivation method of pathogenic microorganisms of a chitosan, is characterized in that adopting NaOH solution to carry out preliminary deactivation to pathogenic microorganism in chitosan, and is then that chitosan is dry, fills nitrogen and sealing, temperature control irradiation; Step is:
(1) adopt NaOH solution to carry out preliminary deactivation to pathogenic microorganism in chitosan: described NaOH concentration of polymer solution is 4.0%～40%, NaOH solution is 20～50 ︰ 1 with the chitosan mass ratio, temperature is room temperature to 100 ℃, preliminary deactivation to total plate count less than 1000cfu/g; Then filter, be washed to neutrality;
(2) described drying: adopt constant pressure and dry, drying under reduced pressure or lyophilization, make the chitosan water content less than 10%;
(3) described nitrogen-filled seal: get a certain amount of chitosan to suitable container, inflated with nitrogen and sealing immediately to reduce the dividing potential drop of oxygen, reduce the active oxygen that irradiation produces, and reduce irradiation to the degraded of chitosan;
(4) described temperature control irradiation: the chitosan after sealing is under ℃ condition of temperature-20～30, with the gamma-radiation irradiation of 10～50kGy;
After temperature control irradiation, the residual titre of infectious virus in chitosan≤0.5 lg TCID
50Clump count is reduced to≤10cfu/g.
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| CN103341187B (en) * | 2012-06-28 | 2016-06-22 | 四川远大蜀阳药业股份有限公司 | A kind of method of terminal inactivation of pathogenic microorganism |
| US10213521B2 (en) * | 2015-06-11 | 2019-02-26 | Medtronic Xomed, Inc. | Useful polysaccharide after radiation sterilization |
| CN108570117A (en) * | 2017-03-13 | 2018-09-25 | 青岛博益特生物材料股份有限公司 | A kind of chitosan virus removal technique and its application |
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| CN101366954A (en) * | 2008-09-19 | 2009-02-18 | 乐普（北京）医疗器械股份有限公司 | Biocidal treatment method for biological coating medical device |
| WO2009132225A2 (en) * | 2008-04-24 | 2009-10-29 | Medtronic, Inc. | Rehydratable polysaccharide particles and sponge |
| CN101664566A (en) * | 2009-09-18 | 2010-03-10 | 浙江大学 | Method for modifying three-dimensional chitosan bars by Co60-gamma-ray radiation |
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| WO2009132225A2 (en) * | 2008-04-24 | 2009-10-29 | Medtronic, Inc. | Rehydratable polysaccharide particles and sponge |
| CN101366954A (en) * | 2008-09-19 | 2009-02-18 | 乐普（北京）医疗器械股份有限公司 | Biocidal treatment method for biological coating medical device |
| CN101664566A (en) * | 2009-09-18 | 2010-03-10 | 浙江大学 | Method for modifying three-dimensional chitosan bars by Co60-gamma-ray radiation |
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