Separation method of endotoxin in rhamnolipid fermentation liquor

Abstract

The invention provides a method for separating endotoxin from rhamnolipid fermentation liquor, which comprises the following steps: 1) removing thalli containing the endotoxin from the rhamnolipid fermentation liquor through centrifugal separation to obtain clear liquor; (2) adding a surfactant into the clear liquid obtained in the step (1), adding calcium oxide at the same time, adjusting the pH value to be more than 10, separating endotoxin-containing cell debris through flocculation, and then centrifuging to obtain clear liquid; (3) adding acid into the clear liquid obtained in the step (2) to adjust the pH value to 2-3, and then performing centrifugal separation to obtain rhamnolipid precipitate; (4) adding alcohol into the rhamnolipid precipitate obtained in the step (3), and dissolving to form a solution; (5) adding an organic solvent into the solution obtained in the step (4), and extracting to obtain an extracting solution; and 6) removing the alcohol and the organic solvent from the extract obtained in the step 5), and then adjusting the pH value to 6-7 to obtain the rhamnolipid aqueous solution. The method is high in biological safety and easy for large-scale production. The endotoxin content of the product is lt; 0.1 EU / mg, which meets the medical standard.

Description

Technical Field

[0001] The present invention relates to the technical field related to the production of rhamnolipids, and specifically relates to a method for separating endotoxin in a rhamnolipid fermentation broth. Background Art

[0002] Rhamnolipids are biosurfactants produced by Gram-negative bacteria such as Pseudomonas aeruginosa or Pseudomonas putida under specific conditions. Due to their excellent surface activity, emulsifying properties, and non-toxic and biodegradable characteristics, rhamnolipids have broad application prospects in the fields of oil exploitation, medicine, food, daily chemicals, and environmental protection.

[0003] However, the rhamnolipid fermentation broth contains a large amount of endotoxin, which comes from the cell wall components of the rhamnolipid-producing bacteria and is the general term for the toxic substances present in the cells of Gram-negative bacteria, also called lipopolysaccharide. Specifically, it refers to the toxin released after the cell wall components of Gram-negative bacteria are lysed by the cells, and this component can cause serious adverse reactions such as human fever and shock, greatly limiting the application of rhamnolipids in fields with high safety requirements such as medicine and daily chemicals.

[0004] Currently, the methods for removing endotoxin from rhamnolipids mainly include two categories: chemical methods and physical methods. Chemical methods such as alkali lysis, acid lysis, oxidation methods, etc., although they can effectively remove endotoxin, will damage the structure of rhamnolipids and affect their biological activity. Physical methods such as ultrafiltration, ion exchange chromatography, etc., are complex in operation and high in cost, and it is difficult to achieve industrial production.

[0005] Since the above-mentioned existing technologies cannot well solve the problem of high endotoxin content in the rhamnolipid fermentation broth, restricting the application of rhamnolipids in fields with high safety requirements such as medicine and daily chemicals, therefore, there is an urgent need to develop a new method for removing endotoxin from rhamnolipids that is efficient, low-cost, and can be industrialized. Summary of the Invention

[0006] In order to overcome the above-mentioned drawbacks existing in the prior art, the purpose of the present invention is to provide a method for separating endotoxin in a rhamnolipid fermentation broth. This separation method can not only significantly reduce the endotoxin residue, but also does not damage the structure and activity of rhamnolipids, and has the advantages of high extraction efficiency, low cost, and industrialization. The separated rhamnolipids can meet the application requirements of fields with high safety requirements such as medicine and daily chemicals.

[0007] The present invention provides a method for separating endotoxin in a rhamnolipid fermentation broth, and the steps include:

[0008] 1) Centrifugal separation, the rhamnolipid fermentation broth is centrifugally separated to remove the endotoxin-containing bacteria, and a supernatant is obtained;

[0009] 2) Flocculation precipitation: Add a surfactant to the supernatant of step 1), and at the same time add calcium oxide and adjust the pH value to above 10. Separate the cell debris containing endotoxin by flocculation, and then centrifuge to obtain a supernatant;

[0010] 3) Acidification treatment: Add an acid to the supernatant of step 2) to adjust the pH value to 2 - 3, and then centrifuge to separate the rhamnolipid precipitate;

[0011] 4) Alcohol dissolution: Add alcohol to the rhamnolipid precipitate of step 3) to dissolve it to form a solution;

[0012] 5) Extraction and purification: Add an organic solvent to the solution of step 4) for extraction to obtain an extract;

[0013] 6) Refining: Remove the alcohol and organic solvent from the extract of step 5), and then adjust the pH value to 6 - 7 to obtain an aqueous rhamnolipid solution.

[0014] In one embodiment, for the rhamnolipid fermentation broth in step 1), the endotoxin content is 8000 - 10000 EU / mg, and it is detected by the limulus reagent test method;

[0015] Preferably, for the rhamnolipid fermentation broth, the dry weight content of rhamnolipid is 5 - 20 wt%.

[0016] In one embodiment, for the centrifugal separation in step 1), the temperature is 10 - 15 °C and the time is 5 - 10 min;

[0017] Furthermore, in some specific examples, for the centrifugal separation, the centrifugal force used is 5000 - 6000 G.

[0018] In one embodiment, for the surfactant in step 2), the addition amount is 0.5 - 1% of the supernatant mass;

[0019] Furthermore, in some specific examples, the surfactant is selected from one or more of sodium polyacrylate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium carboxymethyl cellulose, and sodium alginate.

[0020] In one embodiment, add calcium oxide to the supernatant of step 2) to adjust the pH value to above 10, preferably 10 - 12.

[0021] In one embodiment, for the centrifugation in step 2), the temperature is 10 - 15 °C and the time is 5 - 10 min;

[0022] Furthermore, in some specific examples, for the centrifugation, the centrifugal force used is 2500 - 3200 G.

[0023] In one embodiment, in the supernatant obtained by centrifugation in step 2), the endotoxin content therein needs to be controlled to be < 10 EU / mg;

[0024] When the endotoxin content in the supernatant is ≥ 10 EU / mg, steps 1) and 2) need to be repeated until the endotoxin content in the supernatant obtained by centrifugation is < 10 EU / mg.

[0025] In one embodiment, for the acid in step 3), the rate of change of pH value is controlled to be 0.05 - 0.5 pH / min during the addition to the supernatant;

[0026] Furthermore, in some specific examples, the temperature during the addition of the acid to the supernatant is controlled to be 4 - 10 °C;

[0027] Furthermore, in some specific examples, the acid used is an aqueous acid solution with a concentration of 20 - 30 wt%.

[0028] In one embodiment, the acid in step 3) is selected from one or more of sulfuric acid, phosphoric acid, and hydrochloric acid, preferably sulfuric acid.

[0029] In one embodiment, for the centrifugal separation in step 3), the temperature is 10 - 15 °C and the time is 5 - 10 min;

[0030] Furthermore, in some specific examples, the centrifugal force used for the centrifugal separation is 2500 - 3200 G.

[0031] In one embodiment, for the alcohol in step 4), its addition amount is controlled so that the light transmittance of the formed solution is ≥ 90%;

[0032] Furthermore, in some specific examples, after adding the alcohol, the solution is heated to 40 - 50 °C to make the light transmittance ≥ 98%;

[0033] Preferably, the heating rate used during the heating process of the solution is 1 - 2 °C / min.

[0034] In one embodiment, for the alcohol in step 4), a continuous or multiple feeding method is adopted, and the feeding rate is controlled to be ≤ 0.1 kg / h / kg solution.

[0035] In one embodiment, the alcohol in step 4) is selected from C1 - C4 alcohols, preferably one or more of ethanol, methanol, glycerol, and n-butanol.

[0036] In one embodiment, the organic solvent in step 5) is selected from non-polar organic solvents, preferably one or more of n-hexane, ethyl acetate, n-heptane, etc.;

[0037] Further, in some specific examples, the addition amount of the organic solvent is 1-1.5 times the mass of the solution.

[0038] In one embodiment, for the extraction in step 5), the temperature is 40-60 °C and the pressure is 0.1-0.5 Mpa.

[0039] In one embodiment, for the extraction in step 5), 8-12 stage countercurrent centrifugal extraction is adopted;

[0040] Further, in some specific examples, for the extraction, the residence time for each stage is 3-5 min and the centrifugal force is 2500-3200 G.

[0041] The rhamnolipid aqueous solution product obtained by the method of the present invention can reduce the endotoxin content to below 0.1 EU / mg, and the rhamnolipid recovery rate can reach over 95%.

[0042] Currently, rhamnolipid fermentation strains use Gram-negative bacteria such as Pseudomonas aeruginosa or Pseudomonas putida. The fermentation broth contains a large amount of endotoxins, which limits its application in the daily chemical and pharmaceutical fields. In the present invention, after the rhamnolipid fermentation broth removes the endotoxin-containing bacteria by centrifugation, calcium oxide and a surfactant are added to the centrifuged supernatant to flocculate the endotoxin-containing cell debris. After centrifugation, a rhamnolipid solution is obtained, and the endotoxin content can be reduced to <10 EU / mg.

[0043] After the rhamnolipid in the rhamnolipid solution obtained after the above centrifugation is acidified and precipitated, and then an alcohol is added for dissolution, the rhamnolipid micelles can be destroyed to form rhamnolipid single molecules, releasing the encapsulated endotoxins. After extraction and purification with an organic solvent, a rhamnolipid product with an endotoxin content of <0.1 EU / mg can be obtained.

[0044] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0045] The rhamnolipid product obtained by the method of the present invention can significantly reduce the endotoxin residue, with the endotoxin content <0.1 EU / mg, and the product meets the medical standards. Moreover, it has the advantages of high extraction efficiency, low cost, and easy realization of scale-up production. The separated rhamnolipid can meet the application requirements of fields with high safety such as medicine and daily chemicals. Specific Embodiments

[0046] Hereinafter, the content of the present invention will be described in detail.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The term "and / or" as used herein may include any and all combinations of one or more of the related listed items. It should be noted that the endpoints and any values disclosed in this specification for a range are not limited to that precise range or value, and such ranges or values should be understood to include values close to those ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint value of each range and a single point value, and between single point values may be combined with each other to obtain one or more new numerical ranges, and such numerical ranges should be regarded as specifically disclosed herein.

[0048] The present invention provides a method for separating endotoxin from rhamnolipid fermentation broth, including operations such as centrifugal separation, flocculation precipitation, acidification treatment, alcohol dissolution, extraction purification, and refining.

[0049] Specifically, the steps of the separation method include:

[0050] 1) Centrifugal separation: The rhamnolipid fermentation broth is centrifugally separated to remove the bacteria containing endotoxin, and a supernatant is obtained.

[0051] 2) Flocculation precipitation: A surfactant is added to the supernatant of step 1), and at the same time, calcium oxide is added and the pH value is adjusted to above 10. The cell debris containing endotoxin is separated by flocculation, and then centrifuged to obtain a supernatant.

[0052] 3) Acidification treatment: An acid is added to the supernatant of step 2) to adjust the pH value to 2 - 3, and then centrifugally separated to obtain a rhamnolipid precipitate.

[0053] 4) Alcohol dissolution: Alcohol is added to the rhamnolipid precipitate of step 3) for dissolution to form a solution.

[0054] 5) Extraction purification: An organic solvent is added to the solution of step 4) for extraction to obtain an extraction solution.

[0055] 6) Refining: The extraction solution of step 5) is de-alcoholized and the organic solvent is removed, and then the pH value is adjusted to 6 - 7 to obtain an aqueous rhamnolipid solution.

[0056] In the above separation method of the present invention, in step 1), there is no special requirement for the content of endotoxin in the raw material of the rhamnolipid fermentation broth to be treated. Any rhamnolipid fermentation broth can be treated by this separation method to further reduce its endotoxin content. Preferably, the endotoxin content in the rhamnolipid fermentation broth to be treated is 8000 EU / mg or more, including but not limited to 8000 EU / mg, 8300 EU / mg, 8500 EU / mg,

[0057] Ranges composed of 8800 EU / mg, 9000 EU / mg, 9300 EU / mg, 95000 EU / mg, 10000 EU / mg, 12000 EU / mg, 15000 EU / mg or any two of them, more preferably 8000 - 10000 EU / mg, are detected by the Limulus reagent test method;

[0058] There is no special requirement for the content of rhamnolipid in the rhamnolipid fermentation broth raw material, and it can be any rhamnolipid fermentation broth. According to the current preparation method, the dry weight content of rhamnolipid is usually 5 - 20 wt%, including but not limited to 5 wt%, 8 wt%, 10 wt%, 13 wt%, 15 wt%, 18 wt%, 20 wt% or ranges composed of any two of them.

[0059] Specifically, for the rhamnolipid fermentation broth in the present invention, there are no specific requirements for its source and type. For example, it can be obtained through commercial purchase, or can be self - made by any feasible fermentation method, including conventional methods in the art or improved methods based on conventional methods in the art. In specific applications, for example, it can be prepared by the methods disclosed in patents such as CN114196716A and CN117305092B. In specific applications, the relevant operations, process conditions actually adopted, and the devices used can all be selected conventionally in the art, and the present invention has no special requirements.

[0060] In step 1) of the above - mentioned separation method of the present invention, the rhamnolipid fermentation broth is separated by centrifugation, preferably the cells containing endotoxin are removed by disk centrifugation. The centrifugation separation is a conventional operation means in the field, with a temperature of 10 - 15 °C and a time of 5 - 10 min. Specifically, the temperature of the centrifugation separation includes but not limited to 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C or ranges composed of any two of them, and the time includes but not limited to 5 min, 6 min, 7 min, 8 min, 9 min, 10 min or ranges composed of any two of them;

[0061] The centrifugal force used for the centrifugation separation is 5000 - 6000 G, including but not limited to 5000 G, 5200 G, 5400 G, 5600 G, 5800 G, 6000 G or ranges composed of any two of them.

[0062] The precipitate after centrifugation separation is the cells containing endotoxin, and the supernatant is the rhamnolipid fermentation broth without cells. After detection by the Limulus reagent method, the endotoxin content in the supernatant can be reduced to 5000 - 10000 EU / mg.

[0063] In the above separation method of the present invention, in step 2), by adding calcium oxide to the supernatant after centrifugal separation to adjust the pH value to above 10 and adding a surfactant, cell debris containing endotoxin can be flocculated. After complete flocculation, centrifugal separation is carried out to obtain the supernatant, i.e., the rhamnolipid solution.

[0064] The surfactant therein is an auxiliary agent conventionally used in the field. Acting together with calcium oxide, it can completely sediment the cell debris in the fermentation broth. The present invention has no specific requirements for its type. For example, it can be one or more of sodium polyacrylate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium carboxymethyl cellulose, sodium alginate, etc.; the addition amount of the surfactant is 0.5-1% of the mass of the supernatant, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1% or the range composed of any two of them.

[0065] Adding calcium oxide to the supernatant after centrifugal separation, on the one hand, can act together with the surfactant to flocculate cell debris, and at the same time, calcium oxide has the function of adjusting the pH value of the supernatant. The pH value is adjusted to above 10 by the addition amount of calcium oxide, including but not limited to 10, 10.5, 11, 11.5, 12, 12.5, 13 or the range composed of any two of them. Theoretically, the higher the pH value, the better the flocculation effect. However, considering the conventional operation experience and economy, it is preferably adjusted to 10-12.

[0066] The centrifugation adopted therein is a conventional operation means in the field, with a temperature of 10-15 °C and a time of 5-10 min. Specifically, the temperature of centrifugal separation includes but not limited to 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C or the range composed of any two of them, and the time includes but not limited to 5 min, 6 min,

[0067] 7 min, 8 min, 9 min, 10 min or the range composed of any two of them;

[0068] The centrifugal force adopted in centrifugal separation is 2500-3200 G, including but not limited to 2500 G, 2600 G, 2700 G, 2800 G, 2900 G, 3000 G, 3100 G, 3200 G or the range composed of any two of them.

[0069] In the supernatant obtained by centrifugation in step 2), test the endotoxin content therein, and determine how to operate next according to whether the endotoxin content is <10 EU / mg:

[0070] When the endotoxin content in the supernatant is <10 EU / mg, perform the operation of step 3);

[0071] When the endotoxin content in the supernatant is ≥ 10 EU / mg, steps 1) and 2) need to be repeated until the endotoxin content in the supernatant obtained by centrifugation is < 10 EU / mg, and then the operation of step 3) is carried out.

[0072] In the above separation method of the present invention, in step 3), an appropriate amount of acid (such as sulfuric acid) is added to the supernatant, which can acidify the rhamnolipid component contained therein to form a precipitate, and then the rhamnolipid precipitate is obtained by centrifugation.

[0073] Among them, during the process of adding acid to the supernatant, the pH value change rate is controlled to be 0.05 - 0.5 pH / min, and the temperature is controlled to be 4 - 10 °C. Specifically, the pH value change rate temperature includes but is not limited to 0.05 pH / min, 0.1 pH / min, 0.2 pH / min, 0.3 pH / min, 0.4 pH / min, 0.5 pH / min or the range composed of any two of them, and the temperature includes but is not limited to 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C or the range composed of any two of them.

[0074] Preferably, the added acid is an aqueous acid solution with a concentration of 20 - 30 wt%, including but not limited to 20 wt%, 22 wt%, 24 wt%, 26 wt%, 28 wt%, 30 wt% or the range composed of any two of them.

[0075] Adjusting the system pH value within an appropriate range by adding an appropriate amount of acid is a conventional operation means in the chemical industry. The present invention has no special requirements for the type of acid added. For example, it can be one or more of sulfuric acid, phosphoric acid, and hydrochloric acid. However, considering wastewater treatment and equipment selection, sulfuric acid is preferably used.

[0076] Among them, centrifugal separation is a conventional operation means in the field, the temperature is 10 - 15 °C, and the time is 5 - 10 min. Specifically, the temperature of centrifugal separation includes but is not limited to 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C or the range composed of any two of them, and the time includes but is not limited to 5 min, 6 min,

[0077] 7 min, 8 min, 9 min, 10 min or the range composed of any two of them;

[0078] The centrifugal force used for centrifugal separation is 2500 - 3200 G, including but not limited to 2500 G, 2600 G, 2700 G, 2800 G, 2900 G, 3000 G, 3100 G, 3200 G or the range composed of any two of them.

[0079] In the above separation method of the present invention, in step 4), by adding an alcohol to the rhamnolipid precipitate for dissolution, the rhamnolipid micelles can be completely destroyed to form rhamnolipid single molecules, and at the same time, the encapsulated endotoxin is released.

[0080] Among them, when adding an alcohol to the rhamnolipid precipitate for dissolution, the light transmittance of the formed solution is measured simultaneously, and the addition of the alcohol is stopped when the light transmittance ≥ 90%, for example, including but not limited to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or the range composed of any two of them;

[0081] Then the solution is heated to 40 - 50 °C to make the light transmittance ≥ 98%;

[0082] Preferably, the heating rate used in the process of heating the solution is 1 - 2 °C / min, including but not limited to 1 °C / min, 1.2 °C / min, 1.4 °C / min, 1.6 °C / min, 1.8 °C / min, 2 °C / min or the range composed of any two of them.

[0083] Among them, when adding an alcohol for dissolution, the addition rate of the alcohol is controlled during this period. Preferably, a continuous or multiple feeding method is adopted, and the addition rate is controlled ≤ 0.1 kg / h / kg solution, including but not limited to 0.1 kg / h / kg,

[0084] 0.09 kg / h / kg, 0.08 kg / h / kg, 0.07 kg / h / kg, 0.06 kg / h / kg, 0.05 kg / h / kg, [[ID=1:16]]

[0085] 0.03 kg / h / kg, 0.01 kg / h / kg or the range composed of any two of them.

[0086] The alcohol added is used to dissolve the rhamnolipid precipitate and is selected from alcohols with C1 - C4, preferably one or more of ethanol, methanol, glycerol, and n-butanol.

[0087] In specific applications, an alcohol is added to the rhamnolipid precipitate for dissolution, the addition rate of the alcohol is controlled to be lower than 0.1 kg / h / kg solution, the addition of the alcohol is stopped when the light transmittance of the solution exceeds 90%, and the solution is slowly heated to 40 - 50 °C to make the light transmittance of the solution exceed 98%. At this time, the rhamnolipid micelles are completely destroyed and the encapsulated endotoxin is completely released.

[0088] The above separation method of the present invention further includes step 5) extraction and purification and step 6) refining operation, thereby obtaining a rhamnolipid product.

[0089] Step 5) The organic solvent used for extraction and purification is a non-polar organic solvent, preferably one or more of n-hexane, ethyl acetate, n-heptane, etc.; the addition amount of the organic solvent is 1-1.5 times the mass of the solution, including but not limited to 1 time, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times or the range composed of any two of them.

[0090] Among them, extraction is a conventional operation means in the field, the temperature is 40-60°C, and the pressure is 0.1-0.5 Mpa. Specifically, the extraction temperature includes but not limited to 40°C, 43°C, 45°C, 48°C, 50°C, 53°C, 55°C, 58°C, 60°C or the range composed of any two of them, and the pressure includes but not limited to 0.1 Mpa, 0.2 Mpa, 0.3 Mpa, 0.4 Mpa, 0.5 Mpa or the range composed of any two of them.

[0091] Preferably, countercurrent centrifugal extraction is adopted, and more preferably 8-12 stage countercurrent centrifugal extraction is adopted, such as 8 stages, 9 stages, 10 stages, 11 stages, 12 stages;

[0092] Among them, the residence time for each stage is 3-5 min, including but not limited to 3 min, 4 min, 5 min or the range composed of any two of them;

[0093] The centrifugal force used is 2500-3200 G, including but not limited to 2500 G, 2600 G, 2700 G, 2800 G, 2900 G, 3000 G, 3100 G, 3200 G or the range composed of any two of them.

[0094] The rhamnolipid aqueous solution product separated by the method of the present invention has an endotoxin content that can be reduced to less than 0.1 EU / mg, and the rhamnolipid recovery rate can reach more than 95%.

[0095] Step 6) The operation mode of refining is not specifically required in the present invention, and any achievable means in the field can be adopted. For example, the alcohol and organic solvent in the solution are removed by rectification at normal temperature and pressure, and then the pH value of the tower bottom liquid is adjusted to 6-7, such as 6, 6.2, 6.4, 6.6, 6.8, 7 or the range composed of any two of them, preferably 6.5-7, which is the rhamnolipid aqueous solution product.

[0096] Among them, the pH value of the tower bottom liquid is adjusted by conventional technical means. Preferably, the pH regulator can be sodium hydroxide, potassium hydroxide, etc.

[0097] In the separation method of the present invention, the clear liquid of step 2) is successively acidified and precipitated, and alcohol is dissolved to destroy the rhamnolipid micelles to release the encapsulated endotoxins, and then extracted and purified by organic solvents to further reduce the endotoxin content in the clear liquid from 10 EU / mg to below 0.1 EU / mg.

[0098] In order to facilitate the understanding of the present invention, the present invention will be further described below in conjunction with examples. It should be understood that the following examples are only for a better understanding of the present invention and do not mean that the present invention is limited to the following examples.

[0099] In the various examples and comparative examples of the present invention, the rhamnolipid fermentation broth used was prepared according to the method disclosed in patent CN114196716A. Other raw materials and reagents were purchased from commercial sources unless otherwise specified.

[0100] The main analytical methods used in the examples and comparative examples of the present invention are as follows:

[0101] Transmittance: absorbance value measured at 420nm wavelength.

[0102] Endotoxin content: Detected by Limulus amebocyte lysate test method, using the photometric method (interference test) of Method 2 (1143) of the General Rules of Part IV of the Pharmacopoeia of the People's Republic of China 2020 edition.

[0103] Rhamnolipid content: tested by anthrone sulfuric acid method.

[0104] Rhamnolipid recovery rate: (weight of rhamnolipid in rhamnolipid product / weight of rhamnolipid in fermentation broth)×100%.

[0105] Example 1

[0106] Method for separation of endotoxins in rhamnolipid fermentation broth:

[0107] 1) Pseudomonas aeruginosa rhamnolipid fermentation broth (endotoxin content 10,000 EU / mg, rhamnolipid dry weight content 20%) was centrifuged using a disc centrifuge at a centrifugal force of 5500 G for 8 minutes at a temperature of 12°C to separate the endotoxin-containing bacteria and obtain a rhamnolipid fermentation broth supernatant free of bacteria. The supernatant was tested using a Limulus amebocyte lysate assay, and the endotoxin content was 8,000 EU / mg.

[0108] 2) Calcium oxide was added to the supernatant to adjust the pH to 11, and sodium polyacrylate (0.8% by weight of the supernatant) was added to completely flocculate the endotoxin-containing cell debris. The supernatant was then centrifuged at 2800 g for 8 minutes at 13°C. The supernatant was tested for endotoxin content of 8 EU / mg using the Limulus amebocyte lysate assay.

[0109] 3) Add an aqueous sulfuric acid solution with a concentration of 25 wt% to the above centrifuged supernatant, control the pH change rate at 0.1 pH / min, the temperature at 6 °C, adjust the pH to 2.5, then perform centrifugal separation with a centrifugal force of 2800 G, a centrifugation time of 5 min, and a centrifugation temperature of 12 °C to obtain rhamnolipid precipitate.

[0110] 4) Add ethanol to the rhamnolipid precipitate for dissolution, control the alcohol addition rate at 0.08 kg / h / kg solution, stop adding alcohol when the light transmittance of the solution exceeds 92%, and then slowly heat it to 45 °C at a rate of 1 - 2 °C / min. At this time, the light transmittance exceeds 98.5%, stop heating to obtain a solution.

[0111] 5) Add an organic solvent to the solution and perform 10 - stage countercurrent centrifugal extraction. The organic solvent is a mixed solvent of n - hexane and ethyl acetate. The mass ratio of the solution to the solvent is 1:1.5, the centrifugal force is 2800 G, the residence time for each stage is 4 min, the extraction temperature is 45 °C, and the pressure is 0.5 MPa.

[0112] 6) Use normal - temperature and normal - pressure distillation to remove ethanol and organic solvents from the solution, and use sodium hydroxide to adjust the pH of the bottom liquid to 6.8 to obtain an aqueous rhamnolipid solution product. The endotoxin content is 0.03 EU / mg, meeting the medical standard; the rhamnolipid content is 32 wt%, and the rhamnolipid recovery rate is 96%.

[0113] Example 2

[0114] Method for separating endotoxin from rhamnolipid fermentation broth:

[0115] 1) Centrifuge the Pseudomonas putida rhamnolipid fermentation broth (endotoxin content 10000 EU / mg, rhamnolipid dry weight content 20%) by disk centrifugation with a centrifugal force of 6000 G, a centrifugation time of 7 min, and a centrifugation temperature of 15 °C to separate the bacteria containing endotoxin and obtain a cell - free rhamnolipid fermentation broth supernatant. The centrifuged supernatant is detected by the limulus reagent method, and the endotoxin content is 6000 EU / mg.

[0116] 2) Add calcium oxide to the above supernatant to adjust the pH to 10.5, and add 1% of sodium dodecyl sulfate based on the mass of the supernatant to completely flocculate the cell debris containing endotoxin, and then perform centrifugal separation with a centrifugal force of 3000 G, a centrifugation time of 6 min, and a centrifugation temperature of 10 °C. The centrifuged supernatant is detected by the limulus reagent method, and the endotoxin content is 8 EU / mg.

[0117] 3) Add an aqueous sulfuric acid solution with a concentration of 28 wt% to the above centrifuged supernatant, control the pH change rate at 0.1 pH / min, keep the temperature at 8 °C, adjust the pH to 2.2, then perform centrifugal separation with a centrifugal force of 3100 G, a centrifugation time of 10 min, and a centrifugation temperature of 14 °C to obtain rhamnolipid precipitate.

[0118] 4) Add methanol to the rhamnolipid precipitate for dissolution, control the alcohol addition rate at 0.06 kg / h / kg solution, stop adding alcohol when the light transmittance of the solution exceeds 95%, then slowly heat it up to 48 °C at a rate of 1 - 2 °C / min. At this time, the light transmittance exceeds 99%, stop heating to obtain a solution.

[0119] 5) Add an organic solvent to the solution and perform 8 - stage counter - current centrifugal extraction. The organic solvent is n - heptane, the mass ratio of the solution to the solvent is 1:1.5, the centrifugal force is 2900 G, the residence time for each stage is 5 min, the extraction temperature is 50 °C, and the pressure is 0.5 MPa.

[0120] 6) Use normal - temperature and normal - pressure distillation to remove methanol and the organic solvent from the solution, adjust the pH of the bottom liquid to 6.8 to obtain an aqueous rhamnolipid solution product. The endotoxin content is 0.02 EU / mg, meeting the medical standard, the rhamnolipid content is 31 wt%, and the rhamnolipid recovery rate is 95.5%.

[0121] Example 3

[0122] Method for separating endotoxin from rhamnolipid fermentation broth:

[0123] 1) Centrifuge the Pseudomonas aeruginosa rhamnolipid fermentation broth (endotoxin content 10000 EU / mg, rhamnolipid dry weight content 20%) by a disk centrifuge with a centrifugal force of 5800 G, a centrifugation time of 6 min, and a centrifugation temperature of 13 °C to separate the cells containing endotoxin and obtain a cell - free rhamnolipid fermentation broth supernatant. The centrifuged supernatant is detected by the limulus reagent method, and the endotoxin content is 5000 EU / mg.

[0124] 2) Add calcium oxide to the above supernatant to adjust the pH to 10, and add sodium dodecylbenzenesulfonate at 0.6% of the supernatant mass to completely flocculate the cell debris containing endotoxin, then perform centrifugal separation with a centrifugal force of 2600 G, a centrifugation time of 9 min, and a centrifugation temperature of 11 °C. The centrifuged supernatant is detected by the limulus reagent method, and the endotoxin content is 9 EU / mg.

[0125] 3) Add an aqueous sulfuric acid solution with a concentration of 22 wt% to the above centrifuged supernatant, control the pH change rate at 0.1 pH / min, keep the temperature at 5 °C, adjust the pH to 2.8, then perform centrifugal separation with a centrifugal force of 2800 G, a centrifugation time of 6 min, and a centrifugation temperature of 10 °C to obtain rhamnolipid precipitate.

[0126] 4) Ethanol was added to the rhamnolipid precipitate for dissolution, and the alcohol addition rate was controlled at 0.09 kg / h / kg solution. When the light transmittance of the solution exceeded 90%, the addition of alcohol was stopped, and then the temperature was slowly raised to 42 °C at a rate of 1 - 2 °C / min. At this time, the light transmittance exceeded 98%, and the heating was stopped to obtain a solution.

[0127] 5) An organic solvent was added to the solution, and 12 - stage countercurrent centrifugal extraction was carried out. The organic solvent was a mixed solvent of n - hexane and n - heptane. The mass ratio of the solution to the solvent was 1:1.5, the centrifugal force was 2700G, the residence time for each stage was 3 min, the extraction temperature was 55 °C, and the pressure was 0.5 MPa.

[0128] 6) Normal - temperature and normal - pressure rectification was used to remove ethanol and the organic solvent from the solution, and the pH of the bottom liquid was adjusted to 6.2 to obtain an aqueous rhamnolipid solution product. The endotoxin content was 0.04 EU / mg, meeting the medical standard. The rhamnolipid content was 31.5 wt%, and the rhamnolipid recovery rate was 97%.

[0129] Comparative Example 1

[0130] Referring to the separation method of Example 1, the difference was that: calcium oxide was not added in step 2), and other operations and conditions remained unchanged. An aqueous rhamnolipid solution product was obtained, in which the endotoxin content was 50 EU / mg, the rhamnolipid content was 31 wt%, and the rhamnolipid recovery rate was 94%.

[0131] Comparative Example 2

[0132] Referring to the separation method of Example 1, the difference was that: a surfactant was not added in step 2), and other operations and conditions remained unchanged. An aqueous rhamnolipid solution product was obtained, in which the endotoxin content was 200 EU / mg, the rhamnolipid content was 30 wt%, and the rhamnolipid recovery rate was 96%.

[0133] Comparative Example 3

[0134] Referring to the separation method of Example 1, the difference was that: the alcohol dissolution operation in step 4) was omitted, and the rhamnolipid precipitate in step 3) was directly used for the organic solvent extraction in step 5), and other operations and conditions remained unchanged. An aqueous rhamnolipid solution product was obtained, in which the endotoxin content was 6 EU / mg, the rhamnolipid content was 30 wt%, and the rhamnolipid recovery rate was 94%.

[0135] It is easy to understand that the above - mentioned examples are only for clear illustration and do not mean that the present invention is limited thereto. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for separating endotoxin from rhamnolipid fermentation broth, characterized in that the steps Comprising: 1) The rhamnolipid fermentation broth is centrifuged to remove the endotoxin-containing bacteria to obtain a supernatant; 2) A surfactant is added to the supernatant of step 1), and calcium oxide is added at the same time and the pH value is adjusted to above 10. The endotoxin-containing cell debris is separated by flocculation, and then centrifuged to obtain a supernatant; 3) An acid is added to the supernatant of step 2) to adjust the pH value to 2-3, and then centrifuged to separate the rhamnolipid precipitate; 4) An alcohol is added to the rhamnolipid precipitate of step 3) to dissolve it to form a solution; 5) An organic solvent is added to the solution of step 4) for extraction to obtain an extract; 6) The extract of step 5) is de-alcoholized and de-organic solventized, and then the pH value is adjusted to 6-7 to obtain an aqueous rhamnolipid solution.

2. The separation method according to claim 1, characterized in that, The rhamnolipid fermentation broth in step 1) has an endotoxin content of 8000-10000 EU / mg and is detected by the limulus reagent test method; preferably, the rhamnolipid fermentation broth has a rhamnolipid dry weight content of 5-20 wt%; and / or For the centrifugation separation in step 1), the temperature is 10-15 °C and the time is 5-10 min; preferably, the centrifugal force used for the centrifugation separation is 5000-6000 G.

3. The separation method according to claim 1 or 2, characterized in that The surfactant in step 2) is added in an amount of 0.5-1% of the mass of the supernatant; and / or The surfactant in step 2) is selected from one or more of sodium polyacrylate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, sodium carboxymethyl cellulose, and sodium alginate.

4. The separation method according to any one of claims 1-3, characterized in that, The pH value in step 2) is adjusted to 10-12; and / or For the centrifugation in step 2), the temperature is 10-15 °C and the time is 5-10 min; preferably, the centrifugal force used for the centrifugation is 2500-3200 G.

5. The separation method according to any one of claims 1 to 4, characterized in that, In the supernatant obtained by centrifugation in step 2), when the endotoxin content ≥ 10 EU / mg, steps 1) and 2) need to be repeated until the endotoxin content in the supernatant obtained by centrifugation is < 10 EU / mg.

6. The separation method according to any one of claims 1-5, characterized in that, For the acid in step 3), the rate of change of the pH value is controlled at 0.05-0.5 pH / min during the addition to the supernatant; and / or For the acid in step 3), the temperature during the addition to the supernatant is controlled at 4-10 °C; and / or For the acid in step 3), an acid aqueous solution with a concentration of 20-30 wt% is used; and / or The acid in step 3) is selected from one or more of sulfuric acid, phosphoric acid, and hydrochloric acid, preferably sulfuric acid.

7. The separation method according to any one of claims 1-6, characterized in that, For the centrifugation separation in step 3), the temperature is 10-15 °C and the time is 5-10 min; Preferably, the centrifugal force used for the centrifugation separation is 2500-3200 G.

8. The separation method according to any one of claims 1-7, characterized in that, For the alcohol in step 4), the addition amount is controlled so that the transmittance of the formed solution ≥ 90%; preferably, after adding the alcohol, the solution is heated to 40-50 °C to make the transmittance ≥ 98%; more preferably, the heating rate used during the heating process of the solution is 1-2 °C / min; and / or For the alcohol in step 4), a continuous or multiple feeding method is used, and the addition rate is controlled ≤ 0.1 kg / h / kg solution; and / or The alcohol in step 4) is selected from C1-C4 alcohols, preferably one or more of ethanol, methanol, glycerol, and n-butanol.

9. The separation method according to any one of claims 1-8, characterized in that, The organic solvent described in step 5) is selected from non-polar organic solvents, preferably one or more of n-hexane, ethyl acetate, and n-heptane; and / or The addition amount of the organic solvent described in step 5) is 1-1.5 times the mass of the solution.

10. The separation method according to any one of claims 1-9, characterized in that, For the extraction described in step 5), the temperature is 40-60°C and the pressure is 0.1-0.5 Mpa; Preferably, for the extraction, 8-12 stage countercurrent centrifugal extraction is used; more preferably, for the extraction, the residence time for each stage is 3-5 min and the centrifugal force is 2500-3200 G.