CN118255973A

CN118255973A - Polyhydroxyalkanoate with low endotoxin and preparation method and application thereof

Info

Publication number: CN118255973A
Application number: CN202410270726.0A
Authority: CN
Inventors: 杨历帆; 杨宏宇; 杨继帅; 刘兴甜; 高紫君
Original assignee: Beijing Micro Structure Factory Biotechnology Co ltd
Current assignee: Beijing Micro Structure Factory Biotechnology Co ltd
Filing date: 2024-03-11
Publication date: 2024-06-28

Abstract

The invention relates to the technical field of new materials, and provides a polyhydroxyalkanoate with low endotoxin, a preparation method and application thereof, wherein the method comprises the following steps: performing enzymolysis on thalli derived from fermentation liquor, wherein the thalli contains polyhydroxyalkanoate in cells; and (3) performing enzymolysis to obtain an extracting solution containing polyhydroxyalkanoate, wherein the extracting solution also contains endotoxin, and treating the extracting solution by using a solution containing a polyoxyethylene surfactant. The endotoxin content of the polyhydroxyalkanoate final product prepared by the method is below 0.5EU/mL, the weight average molecular weight of the polyhydroxyalkanoate is above 100kDa, and the application of the polyhydroxyalkanoate in cosmetics and medical equipment is widened.

Description

Polyhydroxyalkanoate with low endotoxin and preparation method and application thereof

Technical Field

The invention relates to the technical field of new materials, relates to a method for reducing endotoxin content in polyhydroxyalkanoate products, and in particular relates to polyhydroxyalkanoate with low endotoxin content, and a preparation method and application thereof.

Background

Polyhydroxyalkanoate (Polyhydroxyalkanoate, PHA) is a novel bio-friendly material obtained by fermentation, gradually appears in public vision, can be used in a plurality of fields such as medical instruments, disposable packaging materials, textile fibers and the like, and is a green and environment-friendly polymer material with the most potential accepted due to excellent biodegradability and biocompatibility. Along with the rising of polyhydroxyalkanoates, a plurality of companies already put into production at present, but if the polyhydroxyalkanoates are to be realized to replace traditional plastics completely, a plurality of defects exist, wherein the most important reason is that compared with the traditional chemical synthesis method, the biological fermentation extraction method has the problems of partial cell and protein residues, lower purity, higher endotoxin, deeper color after heating and melting and the like in the purification process, thus the final product is limited in the fields of foods, cosmetics, medicines and the like, the market share of the polyhydroxyalkanoates is reduced, and the popularization difficulty of the polyhydroxyalkanoates is increased.

Wherein, endotoxin has the effect of causing a series of inflammatory reactions of the organism, and can cause fever, headache, nausea, vomiting and shock of the human body, and even death phenomenon can occur in severe cases. The U.S. Food and Drug Administration (FDA) requires that the endotoxin content of medical devices should not exceed 0.5EU/mL, and therefore it is critical to produce polyhydroxyalkanoate products with lower endotoxin content. Endotoxins are not proteins and are therefore very thermostable. The biological activity of the material is destroyed even if the material is heated at a high temperature of 100 ℃ for 1 hour and is not destroyed, and the biological activity of the material is destroyed only when the material is heated at 160 ℃ for 2 to 4 hours or boiled for 30 minutes by heating with strong alkali, strong acid or strong oxidant. Endotoxin is a component of the cell wall of gram-negative bacteria, called lipopolysaccharide. Although endotoxins are tightly embedded in the cell wall, they are constantly released into the surrounding environment, not only due to cell disintegration and death, but also during normal cell growth and division. Therefore, the production of the low endotoxin polyhydroxyalkanoate product is not only to control the fermentation process to avoid bacterial contamination, but also to prevent endotoxin in vessels, tanks and the environment from contaminating the product during the extraction process.

The current industry for extracting polyhydroxyalkanoates is mainly divided into two types, namely an organic extraction method and an aqueous extraction method. The organic extraction method is to use a halide extraction solvent such as chloroform, methylene dichloride and the like to dissolve and purify polyhydroxyalkanoate, but the method has the defects of difficult solvent recovery, dangerous production environment, high volatility of the organic solvent at high temperature, complicated operation for industrial production and high extraction cost due to the need of using special organic solvent extraction equipment. The water phase extraction method is to break cells by an enzymolysis wall breaking or physical wall breaking method to release the polyhydroxyalkanoate in the cells, and then remove impurities by washing for many times, but the polyhydroxyalkanoate product obtained by the method has higher endotoxin. Although endotoxin can be effectively removed by chemical degradation, it causes a decrease in molecular weight. Endotoxin can be effectively removed by adopting high-temperature methods, but the higher temperature can melt polyhydroxyalkanoate and reduce the molecular weight.

Thus, there is a need to propose a method for producing endotoxin in polyhydroxyalkanoate products by a fermentation process for removal which is effective in removing endotoxin with little reduction in molecular weight. Patent application CN113121807a discloses a method for removing endotoxin in fermenting PHAs, which comprises the following specific implementation steps: (1) Fully and uniformly mixing 1 part of PHAs with 5-100 parts of solvent (0.01-0.5 organic acid or inorganic acid: 100 chlorinated hydrocarbon), centrifuging, collecting sediment I and supernatant I, washing the sediment I with clean water without heat source to remove the solvent on the surface, and drying. (2) Adding methanol or ethanol into the supernatant I to make the ratio of the methanol or the ethanol reach 25-30%, centrifuging, collecting a precipitate II and a supernatant II, washing the precipitate II with clear water without a heat source to remove the solvent on the surface, and drying. (3) Continuously adding methanol or ethanol into the supernatant II to make the ratio of the methanol or the ethanol reach 45-50%, centrifuging, collecting sediment III and supernatant III, washing the sediment III with clean water without heat source to remove the solvent on the surface, and drying. (4) Continuously adding methanol or ethanol into the supernatant III to make the proportion of the methanol or the ethanol reach 45-50%, centrifuging, collecting a precipitate IV, washing the precipitate IV with clean water without a heat source to remove the solvent on the surface, and drying. The obtained precipitate I, II, III and IV are low endotoxin PHAs products with different molecular weights. The method has the following defects: (1) Organic solvents such as chlorinated hydrocarbons are volatile, specialized organic solvent equipment is required in production, and the solvents are harmful to the environment and difficult to recover. (2) The polyhydroxyalkanoate with low molecular weight can be dissolved at normal temperature after being added into a solvent, but the polyhydroxyalkanoate with high molecular weight is not easy to be dissolved in the solvent, and is usually heated to more than 100 ℃, but the polyhydroxyalkanoate is degraded greatly due to long-time heating, so that the application value is lost.

Disclosure of Invention

The invention provides a polyhydroxyalkanoate with low endotoxin, a preparation method and application thereof, which are used for solving the defects of large molecular weight reduction range, large use of a large amount of organic solvents and limited application range existing in the process of removing endotoxin in the production of polyhydroxyalkanoate products by a fermentation method in the prior art.

In a first aspect, the present invention provides a method for reducing endotoxin content in polyhydroxyalkanoate products comprising: performing enzymolysis on thalli derived from fermentation liquor, wherein the thalli contains polyhydroxyalkanoate in cells; and (3) performing enzymolysis to obtain an extracting solution containing polyhydroxyalkanoate, wherein the extracting solution also contains endotoxin, and treating the extracting solution by using a polyoxyethylene surfactant.

Two different polar groups exist in the molecular structure of polyoxyethylene surfactants: hydrophilic group and hydrophobic group, in actual use, hydrophilic group can make the surfactant dissolve in water to smoothly realize the fusion with the solution in water, hydrophobic group can make the surfactant dissolve specific material and wrap up it, be convenient for directly wash the removal with water. It comprises the following steps: anionic surfactants, cationic surfactants, polyoxyethylene nonionic surfactants, and specialty surfactants.

In experiments, it was found that the polyoxyethylene surfactant plays a role in reducing the endotoxin content of the polyhydroxyalkanoate product. The effect of the nonionic surfactant is more remarkable.

Polyhydroxyalkanoates referred to in the present invention may be classified into homopolymers and copolymers according to the monomer composition. Depending on the number of carbon atoms of the monomer, it may be a short chain polyhydroxyalkanoate (i.e., a hydroxy fatty acid of C3-C5 as a monomer) or a medium long chain polyhydroxyalkanoate (i.e., a hydroxy fatty acid of C6-C18 as a monomer), but is not limited thereto.

The polyhydroxyalkanoate may be a homopolymer including, but not limited to, polyhydroxypropionate (PHP), polyhydroxybutyrate (PHB), polyhydroxyoctanoate (PHO), polyhydroxyvalerate (PHV), and the like, for example, poly-3-hydroxybutyrate (P3 HB), poly-4-hydroxybutyrate (P4 HB), poly-3-hydroxypropionate (P3 HP), or poly-3-hydroxyvalerate (P3 HV), and the like.

The polyhydroxyalkanoate may be a copolymer, for example, a copolymer of 3-hydroxybutyric acid and 4-hydroxybutyric acid (P34 HB), a copolymer of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (PHBHHx), a copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (PHBV), a copolymer of 3-hydroxyoctanoic acid and 3-hydroxyhexanoic acid (P3 HO3 HHx), a copolymer of 3-hydroxybutyric acid, 3-hydroxyvaleric acid and 3-hydroxyhexanoic acid (PHBVHHx), a copolymer of 3-hydroxybutyric acid co-4-hydroxybutyric acid and 3-hydroxyvaleric acid (P3 HB-co-4HB-co-3 HV), and the like.

According to the method for reducing the endotoxin content in the polyhydroxyalkanoate product provided by the invention, the polyoxyethylene type surfactant comprises a polyoxyethylene nonionic surfactant;

preferably, the polyoxyethylene type surfactant comprises polyoxyethylene-8-octylphenyl ether and/or glycerol polyoxyethylene ether.

Wherein, the structural formula of the glycerol polyoxyethylene ether is as follows:

Which is a commonly used surfactant.

The polyoxyethylene-8-octyl phenyl ether is also called triton, is used as a surfactant for rinsing tissue samples and the like, and is prepared into a water solution with specific concentration for the first time, and the polyoxyethylene-8-octyl phenyl ether and/or glycerol polyoxyethylene ether are found to be capable of remarkably reducing the endotoxin content in a polyhydroxyalkanoate product under a small amount of addition, so that the polyoxyethylene-8-octyl phenyl ether and/or glycerol polyoxyethylene ether plays a key role in the marketization application of polyhydroxyalkanoate in cosmetics and medical appliances.

The method for reducing the endotoxin content in the polyhydroxyalkanoate product provided by the invention comprises the following steps: treating the extract with an aqueous solution of a polyoxyethylene surfactant having a mass-to-volume concentration of 0.1-2%. If the concentration is less than 0.1%, the surfactant is too little, the endotoxin removal is insufficient, if the concentration is more than 2%, the surfactant is excessively used, firstly, the use amount of the reagent is increased, so that the cost is increased, secondly, the surfactant needs to be washed and removed by using a large amount of water, and if the addition amount is excessively large, the subsequent washing water is excessively used, so that the cost of water, labor, equipment and the like is increased.

According to the method for reducing the endotoxin content in the polyhydroxyalkanoate product provided by the invention, polyhydroxyalkanoate in the extracting solution accounts for 5-30% of the volume of the solution containing the polyoxyethylene surfactant.

In a second aspect, the present invention also provides a process for the preparation of low endotoxin polyhydroxyalkanoates using a method for reducing endotoxin content in polyhydroxyalkanoate products as described above.

According to the preparation method of the polyhydroxyalkanoate with low endotoxin, provided by the invention, the extracting solution is subjected to solid-liquid separation to obtain polyhydroxyalkanoate precipitate, the content of endotoxin in the polyhydroxyalkanoate precipitate is more than 5EU/g, and the polyhydroxyalkanoate precipitate is treated by using a solution containing a polyoxyethylene surfactant;

Preferably, the polyhydroxyalkanoate precipitate accounts for 5-30% of the volume of the solution containing the polyoxyethylene surfactant; if the ratio is too high, the fluidity of the system after mixing the two becomes poor, and the efficiency is lowered.

In general, the mass content of polyhydroxyalkanoate in the polyhydroxyalkanoate precipitate is not less than 93%, preferably not less than 98%;

preferably, the polyhydroxyalkanoate in the polyhydroxyalkanoate precipitate has a weight average molecular weight of 100kDa or more.

The preparation method of the low endotoxin polyhydroxyalkanoate provided by the invention comprises the following steps:

Mixing a solution containing a polyoxyethylene surfactant with the polyhydroxyalkanoate precipitate, and then treating the mixture at the temperature of 30-50 ℃ for 20 min-6 h to obtain a feed liquid;

And (3) carrying out solid-liquid separation, washing for 1-5 times and drying on the feed liquid to obtain the polyhydroxyalkanoate final product.

Preferably, the treatment at 30-50 ℃ for 20 min-6 h comprises: and (5) standing or fully mixing the mixed system in a stirring state.

Preferably, the drying is preferably performed using a vacuum freeze dryer.

The obtained polyhydroxyalkanoate end product can be directly used as the polyhydroxyalkanoate with low endotoxin in the invention.

separating from the fermentation broth to obtain thalli, re-suspending the thalli, breaking wall and cracking to obtain a cracking solution;

Carrying out solid-liquid separation on the pyrolysis liquid, and carrying out enzymolysis on the obtained precipitate after resuspension;

After the enzymolysis is finished, carrying out solid-liquid separation, and washing the obtained precipitate for a plurality of times by adopting a mode of adding water, carrying out solid-liquid separation and discarding supernatant;

and drying after the washing is finished to obtain polyhydroxyalkanoate precipitate.

As a raw material for producing the polyhydroxyalkanoate, a halophilic fermentation broth may be used, wherein halophilic bacteria include halophilic bacteria and/or halophilic archaea, preferably, halophilic bacteria, more preferably, bacteria of the genus Halomonas (Halomonas) and their derivative strains or combinations thereof, and more preferably, any species under the genus Monomonas, for example, Halomonas bluephagenesis、Halomonas campaniensis、Halomonas aydingkolgenesis、Halomonas aerodenitrificans、Halomonas halocynthiae. Further preferably Halomonas bluephagenesis TD, accession number CGMCC No.4353, available from university of Qinghua.

In order to reduce the decrease of the molecular weight of the polyhydroxyalkanoate, when the polyhydroxyalkanoate product is prepared by adopting an aqueous phase extraction method, technological parameters with small influence on the molecular weight of the polyhydroxyalkanoate are adopted, generally, when the polyhydroxyalkanoate is extracted by adopting conventional enzymolysis liquid, the decrease degree of the molecular weight of the polyhydroxyalkanoate in the obtained polyhydroxyalkanoate product is less than 5%, the purity of the product is more than 98%, wherein the endotoxin content is more than 5EU/g, preferably 5EU/g to 100EU/g.

Preferably, more than one surfactant selected from fatty alcohol polyoxyethylene ether, fatty alcohol polyoxyethylene ether sulfate and fatty alcohol polyoxyethylene ether carboxylate is added during the wall breaking and cracking;

the enzymolysis adopts a compound enzyme preparation comprising lysozyme, nucleic acid degrading enzyme, dextranase, mannanase, snailase and protease;

The extraction method of the present invention includes, but is not limited to, the extraction method of polyhydroxyalkanoate described in CN 115786411B.

In a third aspect, the present invention also provides a polyhydroxyalkanoate end product obtained by the method of preparing a low endotoxin polyhydroxyalkanoate as described above; preferably, the endotoxin content in the polyhydroxyalkanoate final product is below 0.5 EU/mL; further preferably, the final polyhydroxyalkanoate product has an endotoxin content of 0.05EU/g or less, and the polyhydroxyalkanoate has a weight average molecular weight of 100kDa or more, preferably 800kDa or more.

In a fourth aspect, the invention also provides the use of the polyhydroxyalkanoate end product as described above in cosmetics, medical devices.

According to the low-endotoxin polyhydroxyalkanoate, the preparation method and the application thereof, the polyoxyethylene surfactant is used as the active ingredient of the detergent, and the polyhydroxyalkanoate product containing endotoxin is washed, so that a large amount of endotoxin can be removed, and the color reaction of the polyhydroxyalkanoate in the granulating and application processes can be reduced, so that the polyhydroxyalkanoate product has wider application scene. The polyoxyethylene surfactant has small addition amount and lower cost, is harmless to the environment and human body in industrial production, does not need to use a specific instrument, and avoids using volatile substances such as chlorinated hydrocarbon and the like which are harmful to the human body. More importantly, the molecular weight of the polyhydroxyalkanoate is not degraded in the washing process, so that the performance of the polyhydroxyalkanoate is ensured.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.

In the embodiment of the invention, all used appliances are required to be cleaned or treated at high temperature to remove endotoxin and other pollution sources, and all the appliances comprise centrifugal cups, glass beakers and the like, and the cleaning method comprises the following steps: soaking for 1h by adopting NaOH solution (or 30% hydrogen peroxide) with the mass concentration of 1-10%, cleaning, and drying at 60 ℃ for later use; the high temperature treatment includes: the device is put into an environment with the temperature of 180-250 ℃ for 30-60 minutes of dry heat treatment.

In embodiments of the present invention, the instrument surface, table top, and all steps are sterilized and all are performed in an ultra clean bench or production clean work area.

In the embodiment of the invention, the pure water without endotoxin is prepared by using an ultrapure water machine. Specifically: after most of pyrogens are filtered out by RO membrane, the water in the water purifier is sterilized by ultraviolet lamp with 253.7nm, so that the pyrogens with strong activity lose biological activity, and finally, an ultrafiltration membrane capable of filtering out residual pyrogens and protein with molecular weight more than 5000 is used, so that the water quality of the final effluent can reach the water quality standard without bacteria and pyrogens.

The normal temperature in the embodiment of the invention is 10-40 ℃, and the corresponding effect of the invention can be realized within the temperature range, preferably 25 ℃.

Example 1

A method for reducing endotoxin content in polyhydroxyalkanoate product, comprising the steps of:

(1) Adding polyoxyethylene-8-octyl phenyl ether into 1L pure water without endotoxin to make the mass concentration of the polyoxyethylene-8-octyl phenyl ether be 0.1%, and uniformly mixing for later use.

(2) 200G of polyhydroxyalkanoate product having endotoxin of 30EU/g and molecular weight of 1264kDa, which was PHB product obtained by using the aqueous phase extraction method of patent CN115786411B, was taken, and the purity thereof was 98.45%.

Adding PHB product into the solution obtained in the step (1), stirring uniformly, stirring continuously at normal temperature for 2h, centrifuging, removing supernatant, collecting precipitate, washing precipitate with endotoxin-free pure water for 3 times, and collecting precipitate after centrifuging. The precipitate was dried in a vacuum freeze-dryer and taken out, and the endotoxin content and the polyhydroxyalkanoate molecular weight were measured, and the results are shown in Table 1.

Example 2

(1) Adding glycerol polyoxyethylene ether into 1L pure water without endotoxin to make the mass concentration of the glycerol polyoxyethylene ether be 0.5%, and uniformly mixing for later use.

(2) 200G of polyhydroxyalkanoate product with endotoxin of 45EU/g and molecular weight of 1057kDa, which is PHB product obtained by using the aqueous phase extraction method of patent CN115786411B, is taken, and the purity of the polyhydroxyalkanoate product is 97.56%.

Example 3

A preparation method of polyhydroxyalkanoate comprises the following steps:

(1) Taking 300g of fresh PHB fermentation liquor, wherein the molecular weight of PHB in the fermentation liquor is 1128kDa;

Centrifuging the fermentation liquor, collecting precipitate, adding purified water into the precipitate to re-suspend the precipitate, and performing combined treatment for 4 hours by using a high-pressure homogenizer and a compound enzyme preparation to obtain enzymolysis liquor; wherein the complex enzyme preparation is referred to in example 1 of CN 115786411B.

(2) Adopting pure water to centrifugally wash the enzymolysis liquid obtained in the step (1) for 3 times, and then collecting precipitate; the endotoxin in the precipitate was 143EU/g, the molecular weight of PHB was 1021kDa, and the purity of PHB was 97.37%.

(3) Adding 22g of the precipitate collected in the step (2) into 150mL of polyoxyethylene-8-octyl phenyl ether water solution (prepared by pure water without endotoxin) with the mass concentration of 0.1%, stirring uniformly, continuously stirring for 2 hours at normal temperature, centrifuging, removing the supernatant, collecting the precipitate, washing the precipitate with pure water without endotoxin for 3 times, and collecting the precipitate after centrifuging. The precipitate was dried in a vacuum freeze-dryer and taken out, and the endotoxin content and the polyhydroxyalkanoate molecular weight were measured, and the results are shown in Table 1.

Example 4

Substantially the same as in example 3, the only difference is that: the mass concentration of the polyoxyethylene-8-octyl phenyl ether aqueous solution is reduced to 0.05 percent.

Example 5

Substantially the same as in example 3, the only difference is that: the mass concentration of the polyoxyethylene-8-octyl phenyl ether aqueous solution is improved to 1.5%.

Example 6

Substantially the same as in example 3, the only difference is that: the mass concentration of the polyoxyethylene-8-octyl phenyl ether aqueous solution is improved to 2 percent.

Example 7

Substantially the same as in example 3, except that: 300g of fresh P34HB fermentation broth was taken, the molecular weight of P34HB in this broth being 1038kDa.

Example 8

Substantially the same as in example 3, except that: 300g of fresh PHBHHx fermentation broth was taken, and the molecular weight of PHBHHx in the fermentation broth was 868kDa.

Example 9

Substantially the same as in example 3, except that: 300g of fresh P3HB-co-4HB-co-3HV broth having a molecular weight of 955kDa was taken.

The aqueous phase extraction procedure of examples 7 to 9 was carried out with reference to CN115786411B, the endotoxin content of the obtained precipitate was higher than 5EU/g before carrying out step (3) of example 3, and the endotoxin content and polyhydroxyalkanoate molecular weight thereof were measured after carrying out step (3) of example 3, and the results are shown in table 1.

Comparative example 1

(1) 10g of polyhydroxyalkanoate product with a molecular weight of 784kDa and an endotoxin content of 55EU/g was placed in 1L of chloroform solution, heated to 80℃and maintained for 2 hours, until the precipitate was completely dissolved.

(2) Slowly adding ethanol into the feed liquid obtained in the step (1) until the liquid becomes turbid, and stopping after all polyhydroxy fatty acid esters are precipitated by the ethanol.

(3) And (3) centrifuging the feed liquid obtained in the step (2) by using a centrifuge, and collecting the precipitate.

(4) Washing the precipitate collected in the step (3) with pure water without endotoxin for 3 times, and collecting the precipitate after centrifugation. The precipitate was dried in a vacuum freeze-dryer and taken out, and the endotoxin content and the polyhydroxyalkanoate molecular weight were measured, and the results are shown in Table 1.

TABLE 1

In the present invention, a limulus reagent is used for measuring endotoxin.

It is seen from examples 3 to 6 that when the mass concentration of the polyoxyethylene-8-octylphenyl ether aqueous solution is too small, it is difficult to reduce the endotoxin content of the resulting polyhydroxyalkanoate precipitate, and if the mass concentration of the polyoxyethylene-8-octylphenyl ether aqueous solution is further increased to more than 2%, the endotoxin content of the resulting polyhydroxyalkanoate precipitate can be reduced, but the number of pure water washing in step (3) is required to be 4 to 5 times to remove the surfactant in water. This results in an increase in the number of washes, increasing water costs and labor costs.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for reducing endotoxin content in polyhydroxyalkanoate products comprising: performing enzymolysis on thalli derived from fermentation liquor, wherein the thalli contains polyhydroxyalkanoate in cells; and (3) performing enzymolysis to obtain an extracting solution containing polyhydroxyalkanoate, wherein the extracting solution also contains endotoxin, and treating the extracting solution by using a polyoxyethylene surfactant.

2. The method of reducing endotoxin content in a polyhydroxyalkanoate product of claim 1, wherein said polyoxyethylene surfactant comprises a polyoxyethylene nonionic surfactant;

3. A method for reducing endotoxin content in polyhydroxyalkanoate product as claimed in claim 1 or claim 2, comprising: treating the extract with an aqueous solution of a polyoxyethylene surfactant having a mass-to-volume concentration of 0.1-2%.

4. A method for reducing endotoxin content in a polyhydroxyalkanoate product as claimed in any one of claims 1 to 3, wherein the mass of polyhydroxyalkanoate in the extract is 5 to 30% by volume of the polyoxyethylene surfactant-containing solution.

5. A method for preparing a low endotoxin polyhydroxyalkanoate comprising: a method for reducing endotoxin content in polyhydroxyalkanoate product as claimed in any one of claims 1 to 4.

6. The method for producing a polyhydroxyalkanoate having a low endotoxin content as claimed in claim 5, wherein the polyhydroxyalkanoate precipitate is obtained by subjecting the extract to solid-liquid separation, wherein the content of endotoxin in the polyhydroxyalkanoate precipitate is 5EU/g or more, and wherein the polyhydroxyalkanoate precipitate is treated with a solution containing a polyoxyethylene type surfactant.

7. The method for producing a polyhydroxyalkanoate having low endotoxin as claimed in claim 6, comprising:

8. The method for producing a polyhydroxyalkanoate having low endotoxin as claimed in claim 6 or 7, comprising:

9. A polyhydroxyalkanoate end product obtained by the method for producing a polyhydroxyalkanoate of low endotoxin as claimed in any one of claims 5 to 8; preferably, the final polyhydroxyalkanoate product has an endotoxin content of 0.5EU/mL or less and a weight average molecular weight of 100kDa or more.

10. Use of the polyhydroxyalkanoate end product of claim 9 in cosmetics and medical devices.