阅读说明：本技术 一种去除发酵法PHAs中内毒素的方法 (Method for removing endotoxin in PHAs by fermentation method ) 是由 沈宏伟 余柳松 吕金艳 司徒卫 林枫 于 2021-01-04 设计创作，主要内容包括：本发明涉及医用材料技术领域,尤其涉及一种去除发酵法PHAs中内毒素的方法。微生物发酵法生产的PHAs中含有内毒素,含量值超过限量标准,使其难以作为医疗器械产品的原料使用。采用化学降解法能有效去除内毒素,但很可能造成PHAs的降解,使其分子量降低从而失去应用价值。针对上述问题,本发明提供一种去除发酵法PHAs中内毒素的方法,主要利用内毒素脂质A中β-1,6糖苷键在酸性条件下容易水解,酸性体系中加入甘油等小分子醇可以保护PHAs的酯键不被破坏的特点,不仅成功去除了PHAs中大量的内毒素,还使得PHAs几乎不被降解,其分子量基本不变,取得了显著的效果,此方法简单、高效,具有较高的商业价值。(The invention relates to the technical field of medical materials, in particular to a method for removing endotoxin in PHAs by a fermentation method. PHAs produced by microbial fermentation contain endotoxin, and the content value exceeds the limit standard, so that the PHAs are difficult to be used as raw materials of medical appliance products. Endotoxin can be effectively removed by adopting a chemical degradation method, but the degradation of PHAs is likely to be caused, so that the molecular weight of PHAs is reduced, and the application value is lost. Aiming at the problems, the invention provides a method for removing endotoxin in fermentation-process PHAs, which mainly utilizes the characteristic that beta-1, 6 glycosidic bonds in endotoxin lipid A are easy to hydrolyze under an acidic condition, and micromolecular alcohol such as glycerol is added into an acidic system to protect ester bonds of the PHAs from being damaged, thereby successfully removing a large amount of endotoxin in the PHAs, ensuring that the PHAs are hardly degraded, ensuring that the molecular weight of the PHAs is basically unchanged, obtaining remarkable effects, and having simple and efficient method and higher commercial value.)

1. A method for removing endotoxin in PHAs by fermentation method is characterized in that: the method comprises the following steps:

(1) adding acid into medical pure water, and adjusting the pH =0-3 of the solution;

(2) adding small molecular alcohol into the solution to make the mass volume concentration of the small molecular alcohol in the solution be 0.001-0.01g/cm³Stirring to obtain a uniform solution;

(3) adding 1 weight part of PHAs into 3-50 weight parts of the uniform solution obtained in the step (2), uniformly stirring, treating at 5-70 ℃ for 30min-12h, then centrifugally separating to remove supernatant and collect bottom precipitate, washing the obtained precipitate with pyrogen-free medical water for 3 times, fully stirring for 30min each time, finally centrifugally separating to collect bottom precipitate, freeze-drying for 24h, and then vacuum-drying at 40 ℃ under-0.1 MPa for 24h to obtain PHAs solid.

2. The method of claim 1 for removing endotoxins from fermentative PHAs, wherein: the acid is hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid or butyric acid.

3. The method of claim 1 for removing endotoxins from fermentative PHAs, wherein: the small molecular alcohol is glycerol, propylene glycol, butanediol or hexanediol.

4. The method of claim 1 for removing endotoxins from fermentative PHAs, wherein: the monomer of the PHAs comprises PHB, PHBV, P (HDD-HO-HTDE), P (3HB-4HB), P (HO-HD-HDD), P (HB-HHx), P (3HB-4HB-3HHx), P (HHx-HO) or P (HD-HDD-HO-HHx).

5. The method of claim 1 for removing endotoxins from fermentative PHAs, wherein: the rotating speed of the centrifuge in the step (3) is 3000 and 12000 rpm.

Technical Field

The invention relates to the technical field of medical materials, in particular to a method for removing endotoxin in PHAs by a fermentation method.

Background

Polyhydroxyalkanoates (PHAs) are a kind of natural high molecular polyesters synthesized by microorganisms, and have become one of the important candidate materials for current biomedical materials due to their good physical properties such as thermoplasticity, piezoelectricity, optical activity and mechanical properties, and good biological and environmental properties such as biodegradability, biocompatibility, biorenewability, surface modifiability and nontoxicity of degradation products.

At present, most of the methods for producing PHAs are microbial fermentation methods, and the strains producing PHAs are gram-negative bacteria. Gram-negative bacteria contain an antigenic complex of proteins, polysaccharides and lipids (i.e., bacterial endotoxins) which are released when the cell is disrupted or lysed. Endotoxin can cause various clinical reactions in human body, and if PHAs containing a large amount of endotoxin is used in medical health, particularly medical tissue engineering, the PHAs can cause fever, headache, nausea, vomiting and shock of human body, and even death in severe cases. Therefore, there are strict requirements for the materials of medical devices used in clinical applications at home and abroad, for example: the U.S. Food and Drug Administration (FDA) requires that the endotoxin content of medical devices should not exceed 20 EU/g.

Since PHAs are intracellular products, they must be isolated and purified by breaking cell walls or changing cell permeability. If gram-negative bacteria are used for fermentation production of PHAs, a large amount of endotoxin is released to pollute PHAs products while wall breaking is carried out on the products, and the application of the products in the aspect of medical tissue engineering is further influenced. Therefore, removal of endotoxin must be performed before the PHAs material is clinically used to meet the requirements specified by the FDA.

At present, the chemical degradation method is a commonly used method for removing endotoxin in PHAs, and the chemical degradation method means that endotoxin is degraded BY strong acid, strong base or strong oxidizer to remove endotoxin (Campbell D H, Cherkin A. the present Destricton OF Pyrogens BY Hydrogen PEROXIDE [ J ] Science,1945,102(2656): 535) 536), but the method is likely to cause degradation OF PHAs, so that the molecular weight is reduced and the application value is lost.

Disclosure of Invention

Aiming at the problems in the prior art, the technical problems to be solved by the invention are as follows: PHAs produced by microbial fermentation contain endotoxin, and the content value exceeds the limit standard, so that the PHAs are difficult to be used as raw materials of medical appliance products. Endotoxin can be effectively removed by adopting a chemical degradation method, but the degradation of PHAs is likely to be caused, so that the molecular weight of PHAs is reduced, and the application value is lost.

The technical scheme adopted by the invention for solving the technical problems is as follows: the invention provides a method for removing endotoxin in fermentation PHAs, which comprises the following steps:

(1) adding acid into medical pure water, and adjusting the pH value of the solution to be 0-3;

(2) adding micromolecular alcohol into the solution to ensure that the mass volume concentration of the micromolecular alcohol in the solution is 0.001-0.01g/cm3, and stirring to obtain uniform solution;

(3) adding 1 weight part of PHAs into 3-50 weight parts of the uniform solution obtained in the step (2), uniformly stirring, treating at 5-70 ℃ for 30min-12h, then centrifugally separating to remove supernatant and collect bottom precipitate, washing the obtained precipitate with pyrogen-free medical water for 3 times, fully stirring for 30min each time, finally centrifugally separating to collect bottom precipitate, freeze-drying for 24h, and then vacuum-drying at 40 ℃ under-0.1 MPa for 24h to obtain PHAs solid.

Specifically, the acid is hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, or butyric acid.

Specifically, the small molecular alcohol is glycerol, propylene glycol, butanediol or hexanediol.

Specifically, the monomer of the PHAs includes PHB, PHBV, P (HDD-HO-HTDE), P (3HB-4HB), P (HO-HD-HDD), P (HB-HHx), P (3HB-4HB-3HHx), P (HHx-HO), or P (HD-HDD-HO-HHx).

Specifically, the rotation speed of the centrifuge in the step (3) is 3000-12000 rpm.

The invention has the beneficial effects that:

(1) the method for removing endotoxin in PHAs is simple and efficient, can effectively remove the endotoxin in PHAs, and can prevent PHAs from being degraded;

(2) the invention mainly utilizes the characteristics that beta-1, 6 glycosidic bonds in endotoxin lipid A are easy to hydrolyze under acidic conditions, and micromolecular alcohols such as glycerol are added into an acidic system to protect ester bonds of PHAs from being damaged, thereby successfully removing a large amount of endotoxin in PHAs, ensuring that PHAs are hardly degraded, ensuring that the molecular weight of PHAs is basically unchanged and obtaining remarkable effect.

Detailed Description

The present invention will now be described in further detail with reference to examples.

The acid used to adjust the pH of the solution used in the following examples of the invention is hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid or butyric acid.

The rotation speed of the centrifuge in the step (3) of the following example of the present invention is 3000-12000 rpm.

Example 1

(1) Adding acid into 100mL of medical pure water, and adjusting the pH value of the solution to be 2;

(2) adding 0.5mL of glycerol into the solution, and stirring to obtain a uniform solution;

(3) and (3) taking 150g of the uniform solution obtained in the step (2), adding 10g of PHB with the endotoxin content of 15EU/g and the molecular weight of 60.6kDa, uniformly stirring, treating at 37 ℃ for 5h, then carrying out centrifugal separation to remove supernatant, collecting bottom precipitate, washing the obtained precipitate with pyrogen-free medical water for 3 times, fully stirring for 30min each time, finally carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24h, and carrying out vacuum drying at 40 ℃ and under-0.1 MPa for 24h to obtain the PHB solid.

Example 2

(1) Adding acid into 100mL of medical pure water, and adjusting the pH value of the solution to be 0;

(2) adding 0.1mL of butanediol into the solution, and stirring to obtain a uniform solution;

(3) and (3) adding 10g of PHBV with endotoxin content of 8EU/g and molecular weight of 620kDa into 30g of the uniform solution obtained in the step (2), uniformly stirring, treating at 5 ℃ for 12h, then carrying out centrifugal separation to remove supernatant and collect bottom precipitate, washing the obtained precipitate for 3 times by using non-heat source medical water, fully stirring for 30min each time, finally carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24h, and carrying out vacuum drying at 40 ℃ and under-0.1 MPa for 24h to obtain the PHBV solid.

Example 3

(1) Adding acid into 100mL of medical pure water, and adjusting the pH value of the solution to be 3;

(2) adding 0.2mL of propylene glycol into the solution, and stirring to obtain a uniform solution;

(3) and (3) adding 10g of P (3HB-4HB) with the endotoxin content of 11EU/g and the molecular weight of 580kDa into 500g of the uniform solution obtained in the step (2), uniformly stirring, treating at 70 ℃ for 1h, then carrying out centrifugal separation to remove supernatant and collect bottom precipitate, washing the obtained precipitate with pyrogen-free medical water for 3 times, fully stirring for 30min each time, finally carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24h, and carrying out vacuum drying at 40 ℃ and under-0.1 MPa for 24h to obtain the P (3HB-4HB) solid.

Example 4

(1) Adding acid into 100mL of medical pure water, and adjusting the pH value of the solution to be 2;

(2) adding 0.3mL of glycerol into the solution, and stirring to obtain a uniform solution;

(3) and (3) taking 300g of the uniform solution obtained in the step (2), adding 10g of P (HD-HDD) with the endotoxin content of 4EU/g and the molecular weight of 550kDa, uniformly stirring, treating at 50 ℃ for 3 hours, then carrying out centrifugal separation to remove supernatant and collect bottom precipitate, washing the obtained precipitate with pyrogen-free medical water for 3 times, fully stirring for 30min each time, finally carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24 hours, and carrying out vacuum drying at 40 ℃ and under-0.1 MPa for 24 hours to obtain the P (HD-HDD) solid.

Example 5

(1) Adding acid into 100mL of medical pure water, and adjusting the pH value of the solution to be 2.5;

(2) adding 0.6mL of butanediol into the solution, and stirring to obtain a uniform solution;

(3) and (3) adding 10g of P (HHx-HO) with the endotoxin content of 7.3EU/g and the molecular weight of 650kDa into 100g of the uniform solution obtained in the step (2), uniformly stirring, treating at 50 ℃ for 3h, then carrying out centrifugal separation to remove supernatant and collect bottom precipitate, washing the obtained precipitate with pyrogen-free medical water for 3 times, fully stirring for 30min each time, finally, carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24h, and then carrying out vacuum drying at 40 ℃ and under-0.1 MPa for 24h to obtain the P (HHx-HO) solid.

Example 6

(1) Adding acid into 100mL of medical pure water, and adjusting the pH value of the solution to be 2;

(2) adding 0.5mL of hexanediol into the solution, and stirring to obtain a uniform solution;

(3) and (3) taking 150g of the uniform solution obtained in the step (2), adding 10g of P (HB-HHx) with the endotoxin content of 15EU/g and the molecular weight of 60.6kDa, uniformly stirring, treating at 37 ℃ for 5h, then carrying out centrifugal separation to remove supernate and collect bottom precipitate, washing the obtained precipitate with pyrogen-free medical water for 3 times, fully stirring for 30min each time, finally carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24h, and then carrying out vacuum drying at 40 ℃ and under-0.1 MPa for 24h to obtain the P (HB-HHx) solid.

Comparative example 1 differs from example 1 in that: the endotoxin of PHB in comparative example 1 was removed according to the following procedure:

(1) adding acid into 100mL of medical pure water, and adjusting the pH value of the solution to be 2;

(2) and (2) taking 150g of the solution obtained in the step (1), adding 10g of PHB with the endotoxin content of 15EU/g and the molecular weight of 606kDa, uniformly stirring, treating at 37 ℃ for 5h, then carrying out centrifugal separation to remove supernatant, collecting bottom precipitate, washing the obtained precipitate for 3 times with non-heat source medical water, fully stirring for 30min each time, finally carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24h, and carrying out vacuum drying at 40 ℃ under-0.1 MPa for 24h to obtain the PHB solid.

Comparative example 2 differs from example 1 in that: the endotoxin of PHB in comparative example 2 was removed according to the following procedure:

(1) adding 0.5mL of glycerol into 100mL of medical pure water, and stirring to obtain a uniform solution;

(3) and (3) taking 150g of the uniform solution obtained in the step (2), adding 10g of PHB with the endotoxin content of 15EU/g and the molecular weight of 606kDa, uniformly stirring, treating at 37 ℃ for 5h, then carrying out centrifugal separation to remove supernatant, collecting bottom precipitate, washing the obtained precipitate for 3 times with non-heat source medical water, fully stirring for 30min each time, finally carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24h, and carrying out vacuum drying at 40 ℃ under-0.1 MPa for 24h to obtain the PHB solid.

Comparative example 3 differs from example 1 in that: the endotoxin of PHB in comparative example 3 was removed according to the following procedure:

(1) adding sodium bicarbonate into 100mL of medical pure water, and adjusting the pH value of the solution to be 10;

(2) adding 0.5mL of glycerol into the solution, and stirring to obtain a uniform solution;

(3) and (3) taking 150g of the uniform solution obtained in the step (2), adding 10g of PHB with the endotoxin content of 15EU/g and the molecular weight of 606kDa, uniformly stirring, treating at 37 ℃ for 5h, then carrying out centrifugal separation to remove supernatant, collecting bottom precipitate, washing the obtained precipitate for 3 times with non-heat source medical water, fully stirring for 30min each time, finally carrying out centrifugal separation to collect the bottom precipitate, carrying out freeze drying for 24h, and carrying out vacuum drying at 40 ℃ under-0.1 MPa for 24h to obtain the PHB solid.

Performance evaluation:

the purified PHAs of examples 1-6 and comparative examples 1-3 were assayed for endotoxin content according to the endotoxin test method (Limulus reagent gel method) described in "Chinese pharmacopoeia"; the molecular weight of PHAs purified in examples 1-6 and comparative examples 1-3 was determined by GPC. Specific results are shown in table 1:

TABLE 1

Test item	Endotoxin (EU/g)	Molecular weight (kDa)
			Example 1	0.015	560
Example 2	0.030	580
			Example 3	0.026	535
Example 4	0.011	508
			Example 5	0.023	600
Example 6	0.035	591
			Comparative example 1	0.012	120
Comparative example 2	14.4	603
			Comparative example 3	0.019	30

In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.