Method for preparing sophorolipids using candida

Abstract

The invention discloses a method for preparing sophorolipid by using candida mycoderma bacteria. The method comprises the following steps: inoculating a seed solution of candida mycoderma bacteria into a fermentation culture medium for fermentation, supplementing materials and the like. According to the method, a lactone-type-based product can be obtained, a high-purity product can be further obtained through a two-stage extraction process, alkali liquor is added according to downstream requirements subsequently for blending, and liquid products with different acid type proportions are obtained and used in the field of home care; in addition, the bacterium-containing wastewater obtained through separation can be subjected to spray drying to obtain a powdery solid, and the powdery solid can be used as a low-end product in the agrochemical direction.

Description

Technical Field

[0001] The present application relates to the field of fermentation engineering, and in particular to a method for preparing sophorolipids using Candida albicans. Background Art

[0002] Sophorolipids are a type of biosurfactant produced by yeast fermentation. They are structurally divided into lactone type and acid type, and products of different configurations have different properties. Lactone type sophorolipids have good biological activity and low toxicity, and are widely used in the medical field; acid type sophorolipids have good surface activity and solubility, and are widely used in the daily chemical field. Compared with traditional chemical synthetic surfactants, sophorolipid biosurfactants not only have the functions of solubilization, emulsification, wetting, and reducing surface tension, but also have the characteristics of non-toxicity, biodegradability, and environmental friendliness. They have great application potential in replacing traditional chemical surfactants in the future.

[0003] Since products with different configurations have different application fields, how to flexibly prepare products with different acid ratio configurations to meet different downstream needs has become the main problem that currently limits the application of sophorolipids. In addition, the high production cost is also one of the limiting factors. Therefore, screening high-yield strains, reducing raw material costs, optimizing fermentation conditions to increase production, and optimizing separation processes to increase product yield and purity are all the focus of current sophorolipid research. There are products with a single configuration as the main component obtained by separating a sophorolipid mixture. For example, patent CN201410617794.6 reported the separation of lactone products from a sophorolipid mixture by separation means such as crystallization and extraction, but did not involve the recovery of acid products, resulting in a low overall yield. Fermented biological surfaces generally produce a large amount of three wastes, such as waste water, waste bacteria, etc., during the separation process. How to effectively recycle these three wastes is also an effective way to reduce production costs, but there is no report on the recycling of the three wastes of sophorolipids. There are also technologies for separating and purifying sophorolipids and rhamnolipids by resin adsorption separation, organic solvent extraction, and column chromatography. Patent application CN109678914A uses ion exchange resin and macroporous adsorption resin to treat a sophorolipid solution containing oil components. In the process of treating the ion exchange resin and the macroporous adsorption resin, a large amount of reagents such as ethanol, ethyl acetate, hydrochloric acid and sodium hydroxide are used. At the same time, due to the presence of pollutants such as bacterial residues and proteins, the ion exchange resin and the macroporous adsorption resin also have the problems of limited service life, high cost and complex process treatment, and a large amount of three wastes are discharged.

[0004] Therefore, there is an urgent need in the art to develop a new method for preparing sophorolipids. Summary of the invention

[0005] Based on this, it is necessary to provide at least a method for preparing sophorolipids using Candida albicans.

[0006] In a first aspect of the present application, a method for preparing a sophorolipid is provided, the method comprising:

[0007] The seed liquid of Candida is inoculated into a fermentation medium for fermentation: the fermentation medium comprises 60 g / L-100 g / L glucose, 60 g / L-100 g / L rapeseed oil, 6 g / L-10 g / L yeast powder and 1 g / L-3 g / L dipotassium hydrogen phosphate, the fermentation pH is 3.0-4.0; the fermentation temperature is 30°C-33°C, and the relative value of dissolved oxygen in the fermentation liquid is maintained at 28%-32%;

[0008] Feeding: Feed medium was added after 48 h~52 h of fermentation and was stopped after 144 h~152 h of fermentation. The feed medium contained 60 g / L~100 g / L glucose, 60 g / L~100 g / L rapeseed oil and 6 g / L~10 g / L yeast powder.

[0009] In some embodiments, the Candida species includes engineered bacteria ATCC22214.

[0010] In some embodiments, the feed medium is added continuously at a rate of 20 g / (L·h) to 25 g / (L·h).

[0011] In some embodiments, the method further comprises seed culture; the seed culture comprises primary seed culture and secondary seed culture.

[0012] In some embodiments, the seed culture medium for primary seed culture and secondary seed culture each independently comprises 2 g / L to 5 g / L glucose, 8 g / L to 10 g / L yeast powder, and 2 g / L to 3 g / L ammonium sulfate. In some embodiments, the pH of the seed culture medium for primary seed culture and secondary seed culture is each independently 3.0 to 4.0.

[0013] In some embodiments, the conditions for primary seed culture and secondary seed culture each independently include: temperature of 30°C to 33°C; time of 24 h to 48 h. In some embodiments, the temperature is 30°C to 31°C. In some embodiments, the time is 30 h to 36 h.

[0014] In some embodiments, the OD of the primary seed solution obtained by primary seed culture is 600 The value is greater than 5. In some embodiments, the OD of the primary seed solution obtained by primary seed culture is 600 The value is 5.1~5.5.

[0015] In some embodiments, the OD of the secondary seed solution obtained by the secondary seed culture is 600 The value is greater than 5. In some embodiments, the OD of the secondary seed solution obtained by the secondary seed culture is 600 The value is 5.1~5.5.

[0016] Unless otherwise specified, the term "OD" in this application is the abbreviation of optical density. The energy difference before and after light passes through the object is the energy absorbed by the object. At a specific wavelength, there is a quantitative relationship between the concentration of the same object and the absorbed energy. 600 " is the optical density value measured when the wavelength is set to 600nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate bacterial cell density. Detecting OD values ​​to indicate bacterial concentration is a common method used in this field. The OD value and bacterial concentration are linearly related.

[0017] In some embodiments, the inoculation amount of the primary seed solution is 2% (v / v) to 8% (v / v), for example, 3% (v / v) to 5% (v / v); "% (v / v)" is the volume percentage of the seed solution in the seed culture medium to be inoculated.

[0018] In some embodiments, the inoculation amount of the secondary seed liquid is 3 wt%~8 wt%, for example, 4 wt%~5wt%; "wt%" is the mass percentage of the secondary seed liquid to the fermentation medium to be inoculated.

[0019] In some embodiments, the fermentation medium accounts for 30% to 50% of the volume of the fermentation vessel. In some embodiments, the fermentation medium accounts for 35% to 40% of the volume of the fermentation vessel.

[0020] In some embodiments, the method further comprises separating and purifying the fermentation broth obtained by fermentation;

[0021] The separation and purification comprises:

[0022] a. Add ethyl acetate to the fermentation broth, stir, let stand until the phases are completely separated, collect and retain the upper liquid;

[0023] b. adding an alkane solvent to the upper liquid, stirring and standing until the phases are completely separated, collecting and retaining the lower liquid; and,

[0024] c. removing the solvent from the lower liquid to prepare a highly pure lactone-type sophorolipid; the removal method may optionally include evaporation under reduced pressure.

[0025] In some embodiments, step a satisfies one or more of the following conditions C1) to C4):

[0026] C1) the amount of ethyl acetate added is 0.5 to 1.5 times the volume of the fermentation broth, and optionally 1 times;

[0027] C2) stirring and standing are performed independently at 20°C to 40°C;

[0028] C3) The stirring speed is 150 rpm~250 rpm, and the time is 10 min~20 min;

[0029] C4) The standing time is 2 h~6 h.

[0030] In some embodiments, step b satisfies one or more of the following conditions D1) to D5):

[0031] D1) The amount of alkane solvent added is 0.5 to 1 times of the lower liquid;

[0032] D2) stirring and standing are performed independently at 20℃~40℃;

[0033] D3) the alkane solvent is selected from any one of n-hexane, n-heptane and n-octane;

[0034] D4) The stirring speed is 100 rpm~200 rpm, and the time is 30 min~60 min;

[0035] D5) The standing time is 10 h~20 h.

[0036] In some embodiments, the separation and purification further comprises a by-treatment step;

[0037] The side treatment step comprises spray drying the lower layer liquid obtained after complete phase separation in step a to obtain a powdery solid to prepare a crude sophorolipid product; the spray drying temperature can be optionally 110°C to 120°C.

[0038] In some embodiments, the method further comprises preparing products with different acid-type ratios based on high-purity lactone-type sophorolipids, comprising:

[0039] The high-purity lactone-type sophorolipid is mixed with alkali solution and water in proportion, and stirred and matured to prepare products with different acid-type proportions.

[0040] In some embodiments, the stirring speed is 150 rpm to 250 rpm, and the stirring time is 5 h to 10 h.

[0041] In some embodiments, the alkali solution includes a NaOH aqueous solution; the mass concentration of the NaOH aqueous solution can optionally be 5 wt% to 20 wt%.

[0042] The method for preparing the sophorolipids of the present application is simple in process and easy to operate, which greatly simplifies the process difficulty of producing sophorolipid products with different configurations, greatly improves the feasibility of producing products with different configurations with the same production equipment, and reduces the production cost to a certain extent; the proportion of single-configuration products in the final product is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0044] Figure 1 This is a sophorolipid production process in one embodiment of the present application. DETAILED DESCRIPTION

[0045] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0047] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0048] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0049] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0050] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0051] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0052] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0053] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0054] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0055] In the present application, the term "first" in the "first aspect" is used for descriptive purposes only and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first" only serves the purpose of non-exhaustive enumeration and description, and it should be understood that it does not constitute a closed limitation on quantity.

[0056] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0057] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0058] The method for preparing the sophorolipid provided in the present application can obtain a lactone-based product. A high-purity product can be further obtained through a two-stage extraction process, and then alkali solution is added for blending according to downstream demand to obtain a liquid product with different acid ratios for use in the field of home care. In addition, the separated bacterial wastewater can be spray-dried to obtain a powdered solid, which can be used as a low-end product in the agricultural chemical industry.

[0059] The preparation method has simple process operation, short flow, and environmentally friendly process, and can obtain high-purity sophorolipids with different configurations; and the production route is short, efficient, and energy-saving; in some embodiments, the fermentation and separation processes are flexible, and products with different acid ratios can be obtained with high yields. The production route can fully utilize the sophorolipids without separation losses, with low amounts of three wastes and high recovery rates.

[0060] In a first aspect of the present application, a method for preparing a sophorolipid is provided, the method comprising:

[0061] The seed liquid of Candida is inoculated into a fermentation medium for fermentation: the fermentation medium contains 60 g / L~100 g / L glucose, 60 g / L~100 g / L rapeseed oil, 6 g / L~10 g / L yeast powder and 1 g / L~3 g / L dipotassium hydrogen phosphate, the fermentation pH is 3.0~4.0; the fermentation temperature is 30℃~33℃, and the relative value of dissolved oxygen in the fermentation liquid is maintained at 28%~32%;

[0062] Feeding: Feed medium was added after 48 h~52 h of fermentation and was stopped after 144 h~152 h of fermentation. The feed medium contained 60 g / L~100 g / L glucose, 60 g / L~100 g / L rapeseed oil and 6 g / L~10 g / L yeast powder.

[0063] In some embodiments, the concentration of glucose in the fermentation medium is 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 76 g / L, 77 g / L, 78 g / L, 79 g / L, 80 g / L, 81 g / L, 82 g / L, 83 g / L, 84 g / L, 85 g / L, 86 g / L, 87 g / L, 88 g / L, 89 g / L, 90 g / L, 91 g / L, 92 g / L, 93 g / L, 94 g / L, 95 g / L, 96 g / L, 97 g / L, 98 g / L, 99 g / L, 100 g / L, or a range or value between any two values.

[0064] In some embodiments, the concentration of rapeseed oil in the fermentation medium is 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 76 g / L, 77 g / L, 78 g / L, 79 g / L, 80 g / L, 81 g / L, 82 g / L, 83 g / L, 84 g / L, 85 g / L, 86 g / L, 87 g / L, 88 g / L, 89 g / L, 90 g / L, 91 g / L, 92 g / L, 93 g / L, 94 g / L, 95 g / L, 96 g / L, 97 g / L, 98 g / L, 99 g / L, 100 g / L, or a range or value between any two values.

[0065] In some embodiments, the concentration of yeast powder in the fermentation medium is 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, or a range or value between any two values.

[0066] In some embodiments, the concentration of potassium hydrogen phosphate in the fermentation medium is 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3 g / L, or a range or value between any two values.

[0067] The pH value during fermentation (hereinafter referred to as "fermentation pH") may be illustratively 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, or a range or value between any two values. The pH may be the pH of the fermentation medium or the pH of the fermentation system (e.g., at the start of fermentation).

[0068] The fermentation temperature may be 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, or a range or value between any two values.

[0069] In some embodiments, the relative value of dissolved oxygen in the fermentation broth is maintained at 28%, 28.5%, 29%, 29.5%, 30%, 30.5%, 31%, 31.5%, 32%, or a range or value between any two values.

[0070] Unless otherwise specified, the terms "fermentation broth", "fermentation effluent", "bioreactor flow" and the like in this application may be used interchangeably, meaning a liquid phase retaining microorganisms, feed materials (such as fermentation medium, etc.) and fermentation products, which may be contained in one or more bioreactors (such as fermenters, etc.). It should be understood that the fermentation broth is not limited to a certain time point (such as the end of fermentation), but exists continuously. After the start of fermentation, at any time point during the fermentation process, the above-mentioned liquid phase in the bioreactor can be referred to as fermentation broth.

[0071] Unless otherwise specified, the term "relative dissolved oxygen value" in this application refers to the ratio of the actual dissolved oxygen concentration to the theoretical saturated dissolved oxygen concentration under the conditions in the fermentation system, usually expressed as a percentage (%). For example, relative dissolved oxygen value (%) = (actual dissolved oxygen concentration / theoretical saturated dissolved oxygen concentration) × 100%.

[0072] In some embodiments, the feed medium comprises 60 g / L-100 g / L glucose, 60 g / L-100 g / L rapeseed oil, and 6 g / L-10 g / L yeast powder.

[0073] In some embodiments, the concentration of glucose in the feed medium is 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 76 g / L, 77 g / L, 78 g / L, 79 g / L, 80 g / L, 81 g / L, 82 g / L, 83 g / L, 84 g / L, 85 g / L, 86 g / L, 87 g / L, 88 g / L, 89 g / L, 90 g / L, 91 g / L, 92 g / L, 93 g / L, 94 g / L, 95 g / L, 96 g / L, 97 g / L, 98 g / L, 99 g / L, 100 g / L, or a range or value between any two values.

[0074] In some embodiments, the concentration of rapeseed oil in the feed medium is 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / L, 65 g / L, 66 g / L, 67 g / L, 68 g / L, 69 g / L, 70 g / L, 71 g / L, 72 g / L, 73 g / L, 74 g / L, 75 g / L, 76 g / L, 77 g / L, 78 g / L, 79 g / L, 80 g / L, 81 g / L, 82 g / L, 83 g / L, 84 g / L, 85 g / L, 86 g / L, 87 g / L, 88 g / L, 89 g / L, 90 g / L, 91 g / L, 92 g / L, 93 g / L, 94 g / L, 95 g / L, 96 g / L, 97 g / L, 98 g / L, 99 g / L, 100 g / L, or a range or value between any two values.

[0075] In some embodiments, the concentration of yeast powder in the feed medium is 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, or a range or value between any two values.

[0076] The feed medium may be added after 48 to 52 hours of fermentation. For example, it may be added after 48 hours, 48.5 hours, 49 hours, 49.5 hours, 50 hours, 50.5 hours, 51 hours, 51.5 hours or 52 hours (or a range or value between any two values) of fermentation.

[0077] The timing of stopping feeding can be selected 144 h to 152 h after the start of fermentation. Exemplarily, feeding can be stopped after 144 h, 145 h, 146 h, 147 h, 148 h, 149 h, 150 h, 151 h or 152 h of fermentation (or a range or value between any two values).

[0078] The fermentation time may be 168 h to 170 h. For example, it may be 168 h, 168.5 h, 169 h, 169.5 h, 170 h, or a range or value between any two values.

[0079] In some embodiments, the Candida used in the method includes Candida engineered bacteria ATCC 22214. In some embodiments, the Candida is Candida engineered bacteria ATCC 22214. Candida engineered bacteria ATCC 22214 can be conventional Candida engineered bacteria ATCC 22214 in the art, for example, purchased from Guangdong Institute of Microbiology.

[0080] In some embodiments, the feed medium is added continuously at a rate of 20 g / (L·h) to 25 g / (L·h).

[0081] The method of adding the feed medium in the present application can be a conventional method of adding the feed medium in the art, such as continuous addition.

[0082] In some embodiments, the feeding rate is 20 g / (L·h) to 25 g / (L·h). Without wishing to be bound by any theory, it is found that when the feeding rate is lower than 20 g / (L·h), the yield of sophorolipids may decrease, which may lead to abnormal fermentation; when the feeding rate is higher than 25 g / (L·h), incomplete consumption of raw materials may occur, and the residue may be too high, which may be converted into other impurities, resulting in an increase in the impurity content in the fermentation broth.

[0083] In some embodiments, the method for preparing sophorolipids further comprises seed culture. Exemplarily, the seed culture comprises primary seed culture and secondary seed culture.

[0084] In some embodiments, seed culture includes inoculating Candida (such as engineered Candida ATCC22214) into a primary seed culture medium, fermenting and culturing to obtain a primary seed solution, and then inoculating the primary seed solution into a secondary seed culture medium, continuing fermentation and culturing to form a bacterial suspension, thereby obtaining a secondary seed solution.

[0085] In some embodiments, the primary culture medium for primary seed culture comprises 2 g / L to 5 g / L glucose, 8 g / L to 10 g / L yeast powder, and 2 g / L to 3 g / L ammonium sulfate.

[0086] In some embodiments, the secondary culture medium for secondary seed culture comprises 2 g / L to 5 g / L glucose, 8 g / L to 10 g / L yeast powder, and 2 g / L to 3 g / L ammonium sulfate.

[0087] In some embodiments, the concentration of glucose in the primary seed culture medium or the secondary seed culture medium is 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, or a range or value between any two values.

[0088] In some embodiments, the concentration of yeast powder in the primary seed culture medium or the secondary seed culture medium is 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L, 10 g / L, or a range or value between any two values.

[0089] In some embodiments, the concentration of ammonium sulfate in the primary seed culture medium or the secondary seed culture medium is 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, or a range or value between any two values.

[0090] In some embodiments, the pH of the primary seed culture medium and the secondary seed culture medium are each independently 3.0 to 4.0, for example, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, or a range or value between any two values.

[0091] In some embodiments, the temperature of the primary seed culture and the secondary seed culture is independently 30°C to 33°C, for example, 30°C, 30.5°C, 31°C, 31.5°C, 32°C, 32.5°C, 33°C, or a range or value between any two values. In some embodiments, the temperature of the primary seed culture and the secondary seed culture is independently 30°C to 31°C. Without wishing to be bound by any theory, it is believed that too low or too high a temperature may result in slow bacterial growth and metabolism, which may affect the production rate of sophorolipids.

[0092] In some embodiments, the time for primary seed culture and secondary seed culture is independently 24 h to 48 h. For example, they can be independently 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, or a range or value between any two values. In some embodiments, the time for primary seed culture and secondary seed culture is independently 30 h to 36 h.

[0093] In some embodiments, the OD of the primary seed solution obtained by primary seed culture is 600 The value is greater than 5. In some embodiments, the OD of the primary seed solution obtained by primary seed culture is 600 The value is not higher than 6. In some embodiments, the OD of the primary seed solution obtained by primary seed culture is 600 The value is 5.1 to 5.5. For example, the OD of the first seed solution 600 The value is 5.1, 5.2, 5.3, 5.4, 5.5, or a range or value between any two values.

[0094] In some embodiments, the OD of the secondary seed solution obtained by the secondary seed culture is 600 The value is greater than 5. In some embodiments, the OD of the secondary seed solution obtained by the secondary seed culture is 600 In some embodiments, the OD of the secondary seed solution obtained by the secondary seed culture is 5.1 to 5.5. 600 The value is not higher than 6. In some embodiments, the OD of the secondary seed solution obtained by the secondary seed culture is 600 The value is 5.1 to 5.5. For example, the OD of the secondary seed solution is 600 The value is 5.1, 5.2, 5.3, 5.4, 5.5, or a range or value between any two values.

[0095] In some embodiments, the inoculation amount of the primary seed solution is 2% (v / v) to 8% (v / v); "% (v / v)" is the volume percentage of the seed solution in the seed culture medium (e.g., secondary seed culture medium) to be inoculated. Exemplarily, the inoculation amount of the primary seed solution is 2% (v / v), 3% (v / v), 4% (v / v), 5% (v / v), 6% (v / v), 7% (v / v), 8% (v / v), or a range or value between any two values. In some embodiments, the inoculation amount of the primary seed solution is 3% (v / v) to 5% (v / v).

[0096] In some embodiments, the inoculation amount of the secondary seed solution is 3 wt% to 8 wt%; "wt%" is the mass percentage of the secondary seed solution to the fermentation medium to be inoculated. In some embodiments, the inoculation amount of the secondary seed solution is 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, or a range or value between any two values, for example, 4 wt% to 5 wt%.

[0097] In some embodiments, the fermentation medium accounts for 30% to 50% of the volume of the fermentation container (e.g., fermentors of various volumes, such as 5 L fermentors, 7.5 L fermentors, etc.). In some embodiments, the fermentation medium accounts for 35% to 40% of the volume of the fermentation container, such as 35%, 36%, 37%, 38%, 39%, 40%, or any range or value between two values.

[0098] In some embodiments, the method for preparing sophorolipids further comprises separating and purifying the fermentation broth obtained by fermentation;

[0099] Separation and purification include:

[0100] a. Add ethyl acetate to the fermentation broth obtained by fermentation, stir and allow to stand until complete phase separation, collect and retain the upper liquid;

[0101] b. adding an alkane solvent to the obtained upper liquid, stirring, standing until the phases are completely separated, collecting and retaining the lower liquid; and,

[0102] c. removing the solvent from the obtained lower liquid to prepare a high-purity lactone-type sophorolipid.

[0103] In some embodiments, the amount of ethyl acetate added in step a is 0.5 to 1.5 times the volume of the fermentation broth. Exemplarily, it can be 1 times.

[0104] In some embodiments, the stirring and standing in step a are each independently carried out at 20°C to 40°C. For example, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or a range or value between any two values. In some embodiments, the stirring and standing are both carried out at 25°C.

[0105] Without wishing to be bound by any theory, it is believed that the solubility of the fermentation broth in ethyl acetate may be poor below 20°C, while side reactions may occur above 40°C.

[0106] In some embodiments, the rotating speed of stirring in step a is 150 rpm ~ 250 rpm, and the time is 10 min ~ 20 min. Exemplarily, the rotating speed of stirring can be 150 rpm, 155 rpm, 160 rpm, 165 rpm, 170 rpm, 175 rpm, 180 rpm, 185 rpm, 190 rpm, 195 rpm, 200 rpm, 205 rpm, 210 rpm, 215 rpm, 220 rpm, 225rpm, 230 rpm, 235 rpm, 240 rpm, 245 rpm, 250 rpm, or the range or numerical value between any two numerical values. Exemplarily, the time of stirring can be 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17min, 18 min, 19 min, 20 min, or the range or numerical value between any two numerical values. In some embodiments, the stirring speed in step a is 200 rpm and the stirring time is 15 min.

[0107] In some embodiments, the standing time in step a is 2 h to 6 h. Exemplarily, the standing time is 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 6 h, or a range or value between any two values.

[0108] In some embodiments, the standing time in step a is 4 h and the standing temperature is 25°C.

[0109] In some embodiments, the amount of the alkane solvent added in step b is 0.5 to 1 times the amount of the lower layer liquid. For example, it is 0.5, 0.6, 0.7, 0.8, 0.9, 1, or a range or value between any two values. In some embodiments, the amount of the alkane solvent added in step b is 0.8 times the amount of the lower layer liquid.

[0110] In some embodiments, stirring and standing in step b are each independently carried out at 20°C to 40°C. Exemplarily, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or a range or value between any two values. It is not intended to be limited by any theory, but it is believed that the dissolution effect of the fermentation broth in ethyl acetate may deteriorate when it is lower than 20°C, and there is the possibility of side reactions above 40°C. In some embodiments, stirring and standing are both carried out at 25°C.

[0111] In some embodiments, the alkane solvent in step b is selected from any one or more of n-hexane, n-heptane and n-octane. For example, n-hexane, n-heptane or n-octane is used for extraction.

[0112] In some embodiments, the stirring speed in step b is 100 rpm to 200 rpm, and the time is 30 min to 60 min.

[0113] In some embodiments, the stirring speed in step b is 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 155 rpm, 160 rpm, 165 rpm, 170 rpm, 175 rpm, 180 rpm, 185 rpm, 190 rpm, 195 rpm, 200 rpm, or a range or value between any two values.

[0114] In some embodiments, the stirring time in step b is 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or a range or value between any two values. In some embodiments, the stirring time in step b is 40 min.

[0115] In some embodiments, the stirring speed and time are 150 rpm and 40 min, respectively.

[0116] In some embodiments, the standing time in step b is 10 h to 20 h. Exemplarily, the standing time may be 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, or a range or value between any two values. In some embodiments, the standing time in step b is 15 h, and the standing temperature is 25 ° C.

[0117] In some embodiments, the removal method in step c may include evaporation under reduced pressure.

[0118] In some embodiments, the purity of the prepared sophorolipid is greater than 98%, the yield is greater than 95%, and the lactone type accounts for more than 90%.

[0119] The method for determining the content of sophorolipids may be a conventional method for determining the content of sophorolipids in the art, such as the sulfuric acid-anthrone method.

[0120] In some embodiments, the separation and purification further comprises a by-treatment step;

[0121] The side treatment step comprises spray drying the lower layer liquid obtained after complete phase separation in step a to obtain a powdery solid to prepare a crude sophorolipid product.

[0122] In some embodiments, the spray drying temperature is 110°C to 120°C. For example, 110°C, 111°C, 112°C, 113°C, 114°C, 115°C, 116°C, 117°C, 118°C, 119°C, 120°C, or a range or value between any two values. In some embodiments, the spray drying temperature is 115°C. In some embodiments, the effective content of sophorolipids in the solid powder obtained after spray drying is 19% to 40%.

[0123] In some embodiments, the method further comprises preparing products with different acid-type ratios based on high-purity lactone-type sophorolipids, comprising:

[0124] After mixing high-purity lactone-type sophorolipids with alkali solution and water in proportion, stirring and ripening, products with different acid-type proportions are prepared.

[0125] Optionally, after stirring and aging, the mixture is allowed to stand. For example, the mixture may be allowed to stand at 20°C to 40°C.

[0126] In some embodiments, high-purity lactone-type sophorolipids are mixed with alkali solution, and then different qualities of water are added to prepare products with different acid ratios. For example, the mixture can be prepared as shown in Table 1 below.

[0127] Table 1

[0128]

[0129] : "A" represents the brand number, indicating acid-type sophorolipid.

[0130] In some embodiments, the speed of stirring is 150 rpm ~ 250 rpm, and the duration of stirring is 5 h ~ 10 h. Exemplarily, the rotating speed of stirring can be 150 rpm, 155 rpm, 160 rpm, 165 rpm, 170 rpm, 175 rpm, 180 rpm, 185 rpm, 190 rpm, 195 rpm, 200 rpm, 205 rpm, 210 rpm, 215 rpm, 220 rpm, 225 rpm, 230rpm, 235 rpm, 240 rpm, 245 rpm, 250 rpm, or the range or numerical value between any two numerical values. Exemplarily, the time of stirring can be 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, or the range or numerical value between any two numerical values. In some embodiments, the speed of stirring is 200 rpm, and the duration of stirring is 8 h. The stirring temperature is generally not strictly limited and may be, for example, 30°C.

[0131] In some embodiments, the alkali solution includes a NaOH aqueous solution; the mass concentration of the NaOH aqueous solution is optionally 5 wt% to 20 wt%. For example, it is 5 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, or a range or value between any two values.

[0132] High-purity sophorolipid products are obtained through simple two-stage extraction, and products with different acid ratios are obtained by adjusting the alkaline hydrolysis process. The wastewater generated by separation is recycled to prepare low-end sophorolipid products. This allows the use of the same batch of fermentation broth to prepare products of different specifications to meet application needs in different fields, with no separation loss throughout the process.

[0133] Some examples are provided below.

[0134] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0135] The fermentation equipment used in the following examples and comparative examples are as follows: Iverson 7.5L fermenter, constant temperature shaker (Shanghai Hetian), constant temperature biological incubator (Shanghai Keheng).

[0136] The separation equipment was as follows: 10L glass reactor (with stirring), Japan Rika rotary evaporator N-1100D-W, 3L three-necked flask, 1L eggplant-shaped bottle, and Shanghai Jinghong water bath.

[0137] Main raw material sources

[0138] The reagents, sources and purity used are as follows: anhydrous ethanol (analytical grade), purchased from Komiou; rapeseed oil (superior grade), purchased from COFCO; glucose (analytical grade), purchased from Sinopharm; yeast powder (analytical grade), purchased from Angel Yeast; concentrated sulfuric acid, 98%, analytical grade, purchased from Sinopharm; ammonium sulfate (analytical grade), purchased from Sinopharm; potassium dihydrogen phosphate (analytical grade), purchased from Sinopharm; ethyl acetate (analytical grade), purchased from Sinopharm; Candida bombicola engineered bacteria ATCC22214, purchased from Guangdong Institute of Microbiology. n-Hexane (analytical grade), n-heptane (analytical grade), n-octane (analytical grade), purchased from Komiou; ethyl acetate (analytical grade), purchased from Komiou. Sodium hydroxide (analytical grade), purchased from Sinopharm.

[0139] Main test methods

[0140] The method for detecting the content of sophorolipids is the sulfuric acid-anthrone method, and the specific operation method is as follows:

[0141] (1) Preparation of anthrone reagent

[0142] Accurately weigh 0.1 g of anthrone, add 100 mL of concentrated sulfuric acid, and mix well to obtain the anthrone reagent.

[0143] (2) Prepare 1 mmol / mL anhydrous glucose solution

[0144] Weigh a certain mass of anhydrous glucose, dry it in a 105℃ oven to constant weight, weigh 0.1800 g of anhydrous glucose of constant weight, dilute it to a 100 mL volumetric flask, and shake it well. Its concentration is 10 mmol / L. Then take 0.5, 1, 2, 3, and 4 mL respectively and dilute it to 100 mL, and its concentrations are 0.5, 0.1, 0.2, 0.3, and 0.4 mmol / L respectively. 1) Prepare anthrone solution: Take 0.2g of anthrone and dissolve it in 100mL of 80% sulfuric acid by volume, keep it away from light and set aside for use. Prepare it immediately before use and do not store it.

[0145] (3) Draw the glucose standard curve using the anthrone method

[0146] Measure 1 mL of anthrone reagent and standard glucose solution respectively and place them in stoppered test tubes, quickly cool them in an ice water bath, then heat them in a boiling water bath for 10 min, cool them in an ice bath, and measure the OD value at a wavelength of 620 nm. The blank control for the experiment is 1 mL of distilled water.

[0147] The absorbance was determined by the anthrone method and a standard curve was drawn.

[0148]

[0149] Among them, y is the glucose content and x is the absorbance.

[0150] (4) Determination of sophorolipid content in fermentation broth

[0151] Take 0.5 mL of fermentation broth and add 1 mL of ethyl acetate, mix well and let stand for 5 min, centrifuge at 1000 rpm for 10 min, take the upper solution and dilute it 50-100 times, use the anthrone method to determine the total sugar content in the ethyl acetate layer, and subtract the glucose content in the supernatant to obtain the lactone-type sophorolipid content. Take 0.5 mL of fermentation broth and add 1 mL of ethanol, mix well and centrifuge, take the liquid and dilute it 50-100 times, use the anthrone method to determine the total sugar concentration in the supernatant, and subtract the residual glucose in the supernatant to convert it to the total sophorolipid yield. Subtract the lactone content to obtain the acid content.

[0152] The calculation formula of the final sophorolipid content is:

[0153] Sophorolipid content = (total sugar content in the reaction solution - glucose content) × dilution factor × 1.91.

[0154] The ratio of acid-type and lactone-type sophorolipids is obtained by measuring the acid-type and lactone-type contents by dissolving in different solvents.

[0155] Example 1

[0156] Prepare the culture medium according to the following ratios

[0157] Seed culture medium composition: glucose 2g / L, yeast powder 8g / L, ammonium sulfate 2g / L.

[0158] The fermentation medium composition: glucose 60g / L, rapeseed oil 60g / L, yeast powder 6g / L, dipotassium hydrogen phosphate 1g / L. The initial medium liquid volume is 30% (v / v).

[0159] Composition of feed medium: glucose 60 g / L, rapeseed oil 60 g / L, yeast powder 6 g / L.

[0160] Candida ATCC22214 was inoculated into the seed culture medium and fermented for 24 h to obtain the first-level seed solution. The fermentation conditions were 30°C and pH 3.0. The first-level seed solution (OD 600 5.3) was inoculated into the seed culture medium again at a 2% (v / v) inoculation rate and the fermentation culture was continued for 24 h to obtain the secondary seed solution.

[0161] Secondary seed solution (OD 600 The fermentation medium was inoculated with 3 wt% of the inoculum (6.0) and fermented under the following conditions: temperature 30°C, pH 3.0, and relative dissolved oxygen value 28%. Feeding was started after 48 h of fermentation at a rate of 20 g / (L·h). Feeding was stopped after 144 h of fermentation and fermentation was stopped after 168 h of fermentation. 5 L of fermentation broth was obtained. After testing, the yield of sophorolipids was 280 g / L and the lactone ratio was 85%.

[0162] The obtained fermentation broth is separated and purified according to the following separation process:

[0163] Take 5L of fermentation broth and add 2.5L of ethyl acetate, stir for 10 min, let stand for 2 h, and separate the phases. Separate and obtain about 3.2L of the upper layer, add 1.6L of n-hexane, stir for 30 min, let stand for 10 h, separate the phases, and collect about 2.1L of the lower layer liquid. Evaporate under reduced pressure to remove the solvent to obtain 1.4kg of concentrate, which is a high-purity sophorolipid with a purity of 95%.

[0164] Product formulation:

[0165] Preparation of 50%A product: Take 1000g of concentrated solution, add 300g of 10 wt% NaOH aqueous solution and 700g of water, mix well, stir for 8 hours and mature to obtain 2000g of aqueous solution product. The product yield is 95%. After testing, the proportion of sophorolipid acid type is 51%. Sophorolipid accounts for 95%.

[0166] The lower aqueous phase obtained from the first extraction was 4.1 L in total, and after spray drying at 110°C, 200 g of powder product was obtained. After testing, the sophorolipid content was 35%. Sophorolipids accounted for 5%.

[0167] The production process of sophorolipids can be found in Figure 1 .

[0168] Example 2

[0169] Compared with Example 1, the following changes are made:

[0170] Seed culture medium composition: glucose 5g / L, yeast powder 10g / L, ammonium sulfate 3g / L.

[0171] Fermentation medium composition: glucose 100g / L, rapeseed oil 100g / L, yeast powder 10g / L, dipotassium hydrogen phosphate 3g / L.

[0172] Composition of feed medium: glucose 100 g / L, rapeseed oil 100 g / L, yeast powder 10 g / L.

[0173] Candida ATCC22214 was inoculated into the seed culture medium and fermented for 48 h to obtain the first-level seed solution. The fermentation conditions were 33 °C and pH 4.0. The first-level seed solution (OD 600 The seed medium was inoculated with 8% (v / v) of 5.5% of the inoculum and fermented for 48 h to obtain the secondary seed solution (OD 600 is 5.4).

[0174] The secondary seed liquid was inoculated into the fermentation medium at an inoculation rate of 8 wt%, and fermentation was carried out under the following fermentation conditions: temperature 33°C, pH 4.0, and relative dissolved oxygen value 32%. Feeding began after 52 h of fermentation, with a feeding rate of 25 g / (L·h), and feeding was stopped after 152 h of fermentation, and fermentation was stopped after 170 h of fermentation. 5L of fermentation broth was obtained, and the yield of sophorolipids was 320 g / L and the lactone ratio was 82%.

[0175] The obtained fermentation broth is separated and purified according to the following separation process:

[0176] Take 3L of fermentation broth and add 4.5L of ethyl acetate, stir for 20 min, let stand for 6 h, and separate the phases. Separate and obtain about 4L of the upper layer, add 4L of heptane, stir for 60 min, let stand for 20 h, separate the phases, and collect about 3L of the lower layer liquid. Evaporate under reduced pressure to remove the solvent and obtain 972g of high-purity sophorolipids with a purity of 96.8%.

[0177] Product formulation:

[0178] Take 500 g of the concentrated solution, 400 g of 10 wt% NaOH aqueous solution, and 100 g of water, mix them evenly, stir and mature for 10 h, and obtain 1000 g of an aqueous solution product with an acid type ratio of 90%, and the product yield is 98%.

[0179] The lower aqueous phase obtained from the first extraction was 3.1 L in total, and after spray drying at 110°C, 100 g of powder product was obtained. After testing, the sophorolipid content was 19.2%, and the sophorolipid accounted for 2%.

[0180] Example 3

[0181] Seed culture medium composition: glucose 3.5g / L, yeast powder 9g / L, ammonium sulfate 2.5g / L.

[0182] Fermentation medium composition: glucose 70g / L, rapeseed oil 90g / L, yeast powder 8.5g / L, dipotassium hydrogen phosphate 2g / L.

[0183] Composition of feed medium: glucose 70 g / L, rapeseed oil 90 g / L, yeast powder 8.5 g / L.

[0184] Fermentation process:

[0185] Candida ATCC22214 was inoculated into the seed culture medium and fermented for 36 h to obtain the first-level seed solution. The fermentation conditions were temperature 31 °C, pH 3.5, and rotation speed 220 rpm. The first-level seed solution was then 600 The inoculum volume was 5.2) and 6% (v / v) was inoculated into the seed medium again, and the fermentation culture was continued for 36 h to obtain the secondary seed solution (OD 600 is 5.3).

[0186] The secondary seed liquid was inoculated into the fermentation medium at a 6 wt% inoculation rate, and fermentation was carried out under the following fermentation conditions: temperature 31°C, pH 3.5, rotation speed 220rpm, and relative dissolved oxygen value 30%. After 50 h of fermentation, feeding began at a feeding rate of 22 g / (L·h), and feeding was stopped after 150 h of fermentation, and fermentation was stopped after 170 h of fermentation. 4.5 L of fermentation liquid was obtained, and the yield of sophorolipids was 300 g / L and the lactone type ratio was 79%.

[0187] Separation process:

[0188] Take 4L of fermentation broth and add 4L of ethyl acetate, stir at room temperature for 15 min, let stand for 4 h, and separate the phases. Collect about 5L of the upper liquid, add 4L of n-octane, stir for 40 min, let stand for 15 h, separate the phases, collect about 3.2L of the lower liquid, evaporate under reduced pressure to remove the solvent, and obtain about 1175g of high-purity sophorolipids with a purity of 97%.

[0189] Product formulation:

[0190] 70%A: Take 1000g of the concentrate, 600g of 10% NaOH aqueous solution, and 400g of water, mix them evenly, stir and mature them at room temperature for 8 h, and obtain 2000g of an aqueous solution product with an acid ratio of 70%, and the product yield is 95%.

[0191] The lower aqueous phase obtained from the first extraction was 3 L in total, and after spray drying at 120°C, 150 g of powder product was obtained. After testing, the sophorolipid content was 40%, and the sophorolipid accounted for 5%.

[0192] Comparative Example 1

[0193] Prepare the culture medium according to the following ratios

[0194] Seed culture medium: glucose 5g / L, yeast powder 10g / L, ammonium sulfate 3g / L.

[0195] Fermentation medium and feed medium: glucose 50 g / L, rapeseed oil 50 g / L, ammonium sulfate 5 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 1 g / L.

[0196] The seed culture medium was divided into two 1L shake flasks with a liquid volume of 20%, the fermentation culture medium was divided into 3L and placed in a 7.5L fermenter, and the feed culture medium was prepared and divided into 1L and placed in a 2L feed bottle. After all the culture media were prepared, they were sterilized at high temperature and set aside for use.

[0197] 1) Seed culture: Inoculate Candida into sterilized seed culture medium, activate at 30°C and 200 rpm for 40 h to obtain primary seed solution, then inoculate the primary seed solution at a volume ratio of 6% into new sterilized seed culture medium, incubate at 30°C and 200 rpm for 40 h to form a bacterial suspension, and measure OD 600 is 7.1, which is the secondary seed solution;

[0198] 2) Fermentation culture: The secondary seed culture solution was inoculated into a 7.5L fermentation tank at a mass ratio of 5%, and fermentation was started at a fermentation temperature of 30°C, a fermentation pH of 3.5, a rotation speed of 200rpm, and a relative dissolved oxygen value of 30%. Feeding began after 24 hours of fermentation at a feeding rate of 20g / L / H, and stopped at the end of the fermentation. The fermentation was stopped after 96 hours. 4.1L of fermentation liquid was obtained, and the yield of sophorolipids was 158g / L, of which the acid type accounted for 65% and the rest was the lactone type.

[0199] Comparative Example 2

[0200] Seed culture medium: glucose 5g / L, yeast powder 10g / L, ammonium sulfate 3g / L.

[0201] Fermentation medium and feed medium: glucose 50 g / L, rapeseed oil 50 g / L, ammonium sulfate 5 g / L, yeast powder 5 g / L, potassium dihydrogen phosphate 1 g / L.

[0202] The seed culture medium was divided into two 1L shake flasks with a liquid volume of 20%, the fermentation culture medium was divided into 3L and placed in a 7.5L fermenter, and the feed culture medium was prepared and divided into 1L and placed in a 2L feed bottle. After all the culture media were prepared, they were sterilized at high temperature and set aside for use.

[0203] 1) Seed culture: Inoculate Candida into sterilized seed culture medium, activate at 30°C and 200 rpm for 40 h to obtain primary seed solution, then inoculate the primary seed solution at a volume ratio of 6% into new sterilized seed culture medium, incubate at 30°C and 200 rpm for 40 h to form a bacterial suspension, and measure OD600 is 7.1, which is the secondary seed solution;

[0204] 2) Fermentation culture: The secondary seed culture solution was inoculated into a 7.5L fermentation tank at a mass ratio of 5%, and fermentation was started at a fermentation temperature of 30°C, a fermentation pH of 3.5, a rotation speed of 200rpm, and a relative dissolved oxygen value of 15%. Feeding began after 24 hours of fermentation at a feeding rate of 20g / L / H, and stopped at the end of the fermentation. The fermentation was stopped after 142 hours. 5.4L of fermentation liquid was obtained, and the yield of sophorolipids was 112g / L, of which the acid type accounted for 50% and the rest was the lactone type.

[0205] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0206] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A method for preparing a sophorolipid, characterized in that: The method comprises: The seed liquid of Candida is inoculated into a fermentation medium for fermentation: the fermentation medium comprises 60 g / L-100 g / L glucose, 60 g / L-100 g / L rapeseed oil, 6 g / L-10 g / L yeast powder and 1 g / L-3 g / L dipotassium hydrogen phosphate, the fermentation pH is 3.0-4.0; the fermentation temperature is 30°C-33°C, and the relative value of dissolved oxygen in the fermentation liquid is maintained at 28%-32%; Feeding: Feed medium was added after 48 h~52 h of fermentation and was stopped after 144 h~152 h of fermentation. The feed medium contained 60 g / L~100 g / L glucose, 60 g / L~100 g / L rapeseed oil and 6 g / L~10 g / L yeast powder.

2. The method according to claim 1, characterized in that The Candida species include the engineered bacteria ATCC22214.

3. The method according to claim 1, characterized in that The feeding medium is added continuously at a rate of 20 g / (L·h)~25 g / (L·h).

4. The method according to any one of claims 1 to 3, characterized in that: The method further comprises seed cultivation; the seed cultivation comprises primary seed cultivation and secondary seed cultivation; The primary seed culture and the secondary seed culture meet one or more of the following conditions A1) to A2): A1) the seed culture medium for the primary seed culture and the secondary seed culture each independently comprises 2 g / L-5 g / L glucose, 8 g / L-10 g / L yeast powder and 2 g / L-3 g / L ammonium sulfate; A2) The conditions for the primary seed culture and the secondary seed culture independently include: a temperature of 30°C to 33°C; and a time of 24 h to 48 h.

5. The method according to claim 4, characterized in that A1) The pH of the primary seed culture medium and the secondary seed culture medium are independently 3.0-4.

0.

6. The method according to claim 4, characterized in that A2) The medium temperature is 30℃~31℃.

7. The method according to claim 4, characterized in that A2) The time is 30 h~36 h.

8. The method according to claim 4, characterized in that The seed culture solution obtained by seed culture meets one or more of the following conditions B1) to B4): B1) OD of the primary seed solution obtained from primary seed culture 600 The value is greater than 5; B2) OD of the secondary seed solution obtained from the secondary seed culture 600 The value is greater than 5; B3) the inoculation amount of the primary seed solution is 2% (v / v) to 8% (v / v), "% (v / v)" is the volume percentage of the seed solution to the seed culture medium to be inoculated; B4) The inoculation amount of the secondary seed liquid is 3 wt%~8 wt%, "wt%" is the mass percentage of the secondary seed liquid to the fermentation medium to be inoculated.

9. The method according to claim 8, characterized in that OD of the first-stage seed solution obtained from the first-stage seed culture 600 The OD of the secondary seed solution obtained by secondary seed culture 600 The values ​​are each independently 5.1 to 5.

5.

10. The method according to claim 8, characterized in that The inoculation amount of the primary seed solution is 3% (v / v) to 5% (v / v).

11. The method according to claim 8, characterized in that The inoculation amount of the secondary seed solution is 4 wt%~5 wt%.

12. The method according to any one of claims 1 to 3, characterized in that: The fermentation medium accounts for 30%~50% of the volume of the fermentation vessel.

13. The method according to claim 12, characterized in that The fermentation medium accounts for 35%~40% of the volume of the fermentation vessel.

14. The method according to any one of claims 1 to 3, characterized in that: The method further comprises separating and purifying the fermentation liquid obtained by fermentation; The separation and purification comprises: a. Add ethyl acetate to the fermentation broth, stir, let stand until the phases are completely separated, collect and retain the upper liquid; b. adding an alkane solvent to the upper liquid, stirring and standing until the phases are completely separated, collecting and retaining the lower liquid; and, c. removing the solvent from the lower layer of liquid to prepare a high-purity lactone-type sophorolipid.

15. The method according to claim 14, characterized in that In step c, the removal method includes evaporation under reduced pressure.

16. The method according to claim 14, characterized in that Step a satisfies one or more of the following conditions C1) to C4): C1) the amount of ethyl acetate added is 0.5 to 1.5 times the volume of the fermentation broth; C2) stirring and standing are performed independently at 20°C to 40°C; C3) The stirring speed is 150 rpm~250 rpm, and the time is 10 min~20 min; C4) The standing time is 2 h~6 h; Step b satisfies one or more of the following conditions D1) to D5): D1) The amount of alkane solvent added is 0.5 to 1 times of the lower liquid; D2) stirring and standing are performed independently at 20℃~40℃; D3) the alkane solvent is selected from any one or more of n-hexane, n-heptane and n-octane; D4) The stirring speed is 100 rpm~200 rpm, and the time is 30 min~60 min; D5) The standing time is 10 h~20 h.

17. The method according to claim 16, characterized in that C1) The amount of ethyl acetate added is 1 times the volume of the fermentation broth.

18. The method of claim 14, wherein: The separation and purification also includes a side treatment step; The side treatment step comprises spray drying the lower layer liquid obtained after complete phase separation in step a to obtain a powdery solid to prepare a crude sophorolipid product.

19. The method according to claim 18, characterized in that The spray drying temperature is 110°C~120°C.

20. The method of claim 14, wherein: The method also includes preparing products with different acid-type ratios based on high-purity lactone-type sophorolipids, including: The high-purity lactone-type sophorolipid is mixed with alkali solution and water in proportion, and stirred and matured to prepare products with different acid-type proportions.

21. The method of claim 20, wherein: The stirring speed is 150 rpm~250 rpm, and the stirring time is 5 h~10 h.

22. The method of claim 20, wherein: The alkali solution includes a NaOH aqueous solution.

23. The method of claim 22, wherein: The mass concentration of the NaOH aqueous solution is 5 wt%~20wt%.

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