Method for preparing sophorolipids by using candida strain

Abstract

Disclosed in the present application is a method for preparing sophorolipids by using a Candida strain. The method comprises inoculating a Candida seed liquid into a fermentation medium for fermentation, initiating the addition of a feed medium after about 48 h to 52 h of fermentation, and terminating the feeding after about 144 h to 152 h of fermentation, wherein the feed medium contains about 60 g / L to about 100 g / L glucose, about 60 g / L to about 100 g / L rapeseed oil, and about 6 g / L to about 10 g / L yeast powder.

Description

Method for preparing sophorolipids using Candida albicans

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. CN2025103613462, filed on March 26, 2025, entitled “Method for preparing sophorolipids using Candida albicans”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of fermentation engineering, and specifically to a method for preparing sophorolipids using Candida albicans. Background Technology

[0004] Sophorolipids are biosurfactants produced by yeast fermentation. Structurally, they are classified into lactone and acid types, with different configurations exhibiting different properties. Lactone-type sophorolipids possess excellent bioactivity and low toxicity, making them widely used in the pharmaceutical field; acid-type sophorolipids exhibit good surface activity and solubility, finding wide application in the daily chemical industry. Compared to traditional chemically synthesized surfactants, sophorolipid biosurfactants not only possess solubilizing, emulsifying, wetting, and surface tension-reducing properties, but also exhibit non-toxicity, biodegradability, and environmental friendliness, demonstrating significant potential for future applications in replacing traditional chemical surfactants.

[0005] Since different configurations of sophorolipids have different applications, the main problem limiting their application is how to flexibly prepare products with different acid-to-protein ratios to meet diverse downstream demands. High production costs are also a limiting factor. Therefore, screening high-yield strains, reducing raw material costs, optimizing fermentation conditions to increase yield, and optimizing separation processes to improve product yield and purity are all key areas of current sophorolipid research. Some methods have yielded products with a single dominant configuration by separating sophorolipid mixtures. For example, patent CN201410617794.6 reports the separation of lactone-type products from sophorolipid mixtures through crystallization and extraction, but this does not involve the recovery of acid-type products, resulting in a low overall yield. The separation process of bioactive surfactants produced through fermentation generally generates a large amount of waste, such as wastewater and waste microbial cells. Effectively recycling these wastes is an effective way to reduce production costs, but there are currently no reports on the recycling of waste from sophorolipid production. Other techniques have been developed to separate and purify sophorolipids and rhamnolipids using resin adsorption separation, organic solvent extraction, and column chromatography. Patent application CN109678914A uses ion exchange resin and macroporous adsorption resin to treat sophorolipid solutions containing oil components. In the process of treating ion exchange resin and 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 bacterial residues and protein and other pollutants, ion exchange resin and macroporous adsorption resin also have problems such as limited service life, high cost and complex process treatment, and a large amount of waste is discharged.

[0006] Therefore, there is an urgent need in this field to develop novel methods for preparing sophorolipids. Summary of the Invention

[0007] Therefore, it is necessary to provide at least one method for preparing sophorolipids using Candida albicans.

[0008] In a first aspect of this application, a method for preparing sophorolipids is provided, the method comprising:

[0009] The seed culture of Candida albicans was inoculated into a fermentation medium for fermentation: the fermentation medium contained about 60 g / L to about 100 g / L glucose, about 60 g / L to about 100 g / L rapeseed oil, about 6 g / L to about 10 g / L yeast extract and about 1 g / L to about 3 g / L dipotassium hydrogen phosphate, the fermentation pH was about 3.0 to about 4.0, the fermentation temperature was about 30°C to about 33°C, and the relative dissolved oxygen in the fermentation broth was maintained at about 28% to about 32%.

[0010] Feeding: Feeding medium is added starting from about 48 to about 52 hours after fermentation and is stopped after about 144 to about 152 hours of fermentation. The feeding medium contains about 60 g / L to about 100 g / L of glucose, about 60 g / L to about 100 g / L of rapeseed oil and about 6 g / L to about 10 g / L of yeast extract.

[0011] In some embodiments, the Candida albicans includes engineered strain ATCC22214.

[0012] In some embodiments, the feeding medium is added continuously at a rate of about 20 g / (L·h) to about 25 g / (L·h).

[0013] In some embodiments, the method further includes seed culture; the seed culture includes primary seed culture and secondary seed culture.

[0014] In some embodiments, the seed culture media for primary and secondary seed cultures each independently contain about 2 g / L to about 5 g / L glucose, about 8 g / L to about 10 g / L yeast extract, and about 2 g / L to about 3 g / L ammonium sulfate. In some embodiments, the pH of the seed culture media for primary and secondary seed cultures each independently is about 3.0 to about 4.0.

[0015] In some embodiments, the conditions for primary and secondary seed culture each independently include: a temperature of about 30°C to about 33°C; and a time of about 24 hours to about 48 hours. In some embodiments, the temperature is about 30°C to about 31°C; and in some embodiments, the time is about 30 hours to about 36 hours.

[0016] In some embodiments, the OD of the primary seed culture obtained from primary seed culture is... 600 The value is greater than 5. In some embodiments, the OD of the primary seed culture obtained from primary seed culture is... 600 The value is approximately 5.1 to approximately 5.5.

[0017] In some embodiments, the OD of the secondary seed culture obtained from the secondary seed culture is... 600 The value is greater than 5. In some embodiments, the OD value of the secondary seed culture obtained from secondary seed culture is greater than 5. 600 The value is approximately 5.1 to approximately 5.5.

[0018] In some embodiments, the inoculation amount of the primary seed solution is 2% (v / v) to about 8% (v / v), for example, about 3% (v / v) to about 5% (v / v); "% (v / v)" is the volume percentage of the seed solution to be inoculated into the seed culture medium.

[0019] In some embodiments, the inoculation amount of the secondary seed liquid is about 3 wt% to about 8 wt%, for example, about 4 wt% to about 5 wt%; "wt%" is the percentage of the secondary seed liquid by mass of the fermentation medium to be inoculated.

[0020] In some embodiments, the fermentation medium occupies about 30% to about 50% of the fermentation vessel volume. In some embodiments, the fermentation medium occupies about 35% to about 40% of the fermentation vessel volume.

[0021] In some embodiments, the method further includes separating and purifying the fermentation broth obtained from fermentation;

[0022] The separation and purification process includes:

[0023] a. Add ethyl acetate to the fermentation broth, stir, and let stand until complete phase separation, then collect and retain the supernatant liquid;

[0024] b. Add an alkane solvent to the upper liquid, stir, allow to stand until complete phase separation, collect and retain the lower liquid; and,

[0025] c. Solvent removal is performed on the lower liquid layer to prepare high-purity lactone-type sophorolipids; the removal method may optionally include vacuum evaporation.

[0026] In some implementations, step a satisfies one or more of the conditions shown in C1) to C4):

[0027] C1) The amount of ethyl acetate added is about 0.5 to about 1.5 times the volume of the fermentation broth, optionally about 1 times;

[0028] C2) Stirring and settling are carried out independently at approximately 20°C to approximately 40°C;

[0029] C3) The stirring speed is from about 150 rpm to about 250 rpm, and the time is from about 10 min to about 20 min;

[0030] C4) The settling time is approximately 2 hours to approximately 6 hours.

[0031] In some implementations, step b satisfies one or more of the conditions shown in D1) to D5):

[0032] D1) The amount of alkane solvent added is approximately 0.5 to approximately 1 times the amount of the lower liquid layer;

[0033] D2) Stirring and settling are carried out independently at approximately 20°C to approximately 40°C;

[0034] D3) The alkane solvent is selected from any one of n-hexane, n-heptane, and n-octane;

[0035] D4) The stirring speed is from about 100 rpm to about 200 rpm, and the time is from about 30 min to about 60 min;

[0036] D5) The settling time is approximately 10 hours to approximately 20 hours.

[0037] In some embodiments, the separation and purification further includes a side-processing step;

[0038] The side-processing step includes spray drying the lower liquid obtained after complete phase separation in step a to obtain a powdered solid, thus preparing a crude sophorolipid product; the spray drying temperature can optionally be from about 110°C to about 120°C.

[0039] In some embodiments, the method further includes preparing products with different acid ratios based on high-purity lactone-type sophorolipids, including:

[0040] The high-purity lactone-type sophorolipid was mixed with alkali and water in a certain proportion and then stirred and matured to prepare products with different acid ratios.

[0041] In some embodiments, the stirring speed is from about 150 rpm to about 250 rpm, and the stirring time is from about 5 h to about 10 h.

[0042] In some embodiments, the alkaline solution comprises an aqueous solution of NaOH.

[0043] In some embodiments, the mass concentration of the NaOH aqueous solution is optionally from about 5 wt% to about 20 wt%.

[0044] The method for preparing sophorolipids containing this application is simple and easy to operate, greatly simplifying the process of producing sophorolipid products with different configurations. This significantly improves the feasibility of producing products with different configurations using the same production equipment, and reduces production costs to a certain extent. The final product has a high proportion of single-configuration products.

[0045] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. 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 illustrating this application.

[0047] Figure 1 shows the production process of sophorolipid in one embodiment of this application. Detailed Implementation

[0048] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0050] 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 other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0051] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0052] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0053] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0054] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0055] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0056] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0057] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0058] In this application, the term "first" in "first aspect" is used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should it be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first" serves only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0059] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every 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 ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0060] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0061] The method for preparing sophorolipids provided in this application can yield a product mainly composed of lactones. A high-purity product can be obtained through a two-stage extraction process. Subsequently, alkali solution can be added to adjust the product according to downstream needs, resulting in liquid products with different acid ratios for use in the home care field. Furthermore, the separated bacterial wastewater can be spray-dried to obtain a powdered solid, which can be used as a low-end product in the agrochemical industry.

[0062] The preparation method is simple to operate, has a short process, and is environmentally friendly. It can obtain high-purity sophorolipids with different configurations. Moreover, the production route is short, efficient, and energy-saving. In some embodiments, the fermentation and separation processes are flexible, which can obtain products with different acid ratios in high yield. The production route can fully utilize sophorolipids, with no separation loss, low amount of waste, and high recovery rate.

[0063] In a first aspect of this application, a method for preparing sophorolipids is provided, the method comprising:

[0064] The seed culture of Candida albicans was inoculated into a fermentation medium for fermentation: the fermentation medium contained about 60 g / L to about 100 g / L glucose, about 60 g / L to about 100 g / L rapeseed oil, about 6 g / L to about 10 g / L yeast extract and about 1 g / L to about 3 g / L dipotassium hydrogen phosphate, the fermentation pH was about 3.0 to about 4.0, the fermentation temperature was about 30°C to about 33°C, and the relative dissolved oxygen in the fermentation broth was maintained at about 28% to about 32%.

[0065] Feeding: Feeding medium is added starting from about 48 to about 52 hours after fermentation and is stopped after about 144 to about 152 hours of fermentation. The feeding medium contains about 60 g / L to about 100 g / L of glucose, about 60 g / L to about 100 g / L of rapeseed oil and about 6 g / L to about 10 g / L of yeast extract.

[0066] In some embodiments, the concentration of glucose in the fermentation medium is about 60 g / L, about 61 g / L, about 62 g / L, about 63 g / L, about 64 g / L, about 65 g / L, about 66 g / L, about 67 g / L, about 68 g / L, about 69 g / L, about 70 g / L, about 71 g / L, about 72 g / L, about 73 g / L, about 74 g / L, about 75 g / L, about 76 g / L, about 77 g / L, about 78 g / L, about 79 g / L, about 8 g / L, about 8 g / L. 0 g / L, approximately 81 g / L, approximately 82 g / L, approximately 83 g / L, approximately 84 g / L, approximately 85 g / L, approximately 86 g / L, approximately 87 g / L, approximately 88 g / L, approximately 89 g / L, approximately 90 g / L, approximately 91 g / L, approximately 92 g / L, approximately 93 g / L, approximately 94 g / L, approximately 95 g / L, approximately 96 g / L, approximately 97 g / L, approximately 98 g / L, approximately 99 g / L, approximately 100 g / L, or any range or value between two values.

[0067] In some embodiments, the concentration of rapeseed oil in the fermentation medium is about 60 g / L, about 61 g / L, about 62 g / L, about 63 g / L, about 64 g / L, about 65 g / L, about 66 g / L, about 67 g / L, about 68 g / L, about 69 g / L, about 70 g / L, about 71 g / L, about 72 g / L, about 73 g / L, about 74 g / L, about 75 g / L, about 76 g / L, about 77 g / L, about 78 g / L, about 79 g / L, about 8 g / L. 0 g / L, approximately 81 g / L, approximately 82 g / L, approximately 83 g / L, approximately 84 g / L, approximately 85 g / L, approximately 86 g / L, approximately 87 g / L, approximately 88 g / L, approximately 89 g / L, approximately 90 g / L, approximately 91 g / L, approximately 92 g / L, approximately 93 g / L, approximately 94 g / L, approximately 95 g / L, approximately 96 g / L, approximately 97 g / L, approximately 98 g / L, approximately 99 g / L, approximately 100 g / L, or any range or value between two values.

[0068] In some embodiments, the concentration of yeast extract in the fermentation medium is about 6 g / L, about 6.5 g / L, about 7 g / L, about 7.5 g / L, about 8 g / L, about 8.5 g / L, about 9 g / L, about 9.5 g / L, about 10 g / L, or any range or value between two values.

[0069] In some embodiments, the concentration of dipotassium hydrogen phosphate in the fermentation medium is about 1 g / L, about 1.1 g / L, about 1.2 g / L, about 1.3 g / L, about 1.4 g / L, about 1.5 g / L, about 1.6 g / L, about 1.7 g / L, about 1.8 g / L, about 1.9 g / L, about 2 g / L, about 2.1 g / L, about 2.2 g / L, about 2.3 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.7 g / L, about 2.8 g / L, about 2.9 g / L, about 3 g / L, or any range or value between two values.

[0070] The pH value during fermentation (which may be simply referred to as "fermentation pH") may, for example, be about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, or a range or value between any two values. This pH may be the pH of the fermentation medium or the pH value of the fermentation system (e.g., at the start of fermentation).

[0071] Fermentation temperature can be 30℃, 30.5℃, 31℃, 31.5℃, 32℃, 32.5℃, 33℃, or any range or value between two of these values.

[0072] In some embodiments, the relative dissolved oxygen in the fermentation broth is maintained at about 28%, about 28.5%, about 29%, about 29.5%, about 30%, about 30.5%, about 31%, about 31.5%, about 32%, or any range or value between two values.

[0073] Unless otherwise specified, the terms "fermentation broth," "fermentation effluent," and "bioreactor flow" in this application may be used interchangeably to refer to the liquid phase that retains microorganisms, feed material (such as fermentation medium), and fermentation products, which may be contained in one or more bioreactors (such as fermenters). It should be understood that the fermentation broth is not limited to a single point in time (such as the end of fermentation), but is continuously present. After fermentation begins, the aforementioned liquid phase in the bioreactor at any point in time during the fermentation process can be referred to as fermentation broth.

[0074] Unless otherwise specified, the term "relative dissolved oxygen" in this application refers to the ratio of the actual dissolved oxygen concentration to the theoretical saturated dissolved oxygen concentration under those conditions in a fermentation system, usually expressed as a percentage (%). For example, relative dissolved oxygen (%) = (actual dissolved oxygen concentration / theoretical saturated dissolved oxygen concentration) × 100%.

[0075] In some embodiments, the feed medium comprises about 60 g / L to about 100 g / L of glucose, about 60 g / L to about 100 g / L of rapeseed oil, and about 6 g / L to about 10 g / L of yeast extract.

[0076] In some embodiments, the glucose concentration in the fed culture medium is about 60 g / L, about 61 g / L, about 62 g / L, about 63 g / L, about 64 g / L, about 65 g / L, about 66 g / L, about 67 g / L, about 68 g / L, about 69 g / L, about 70 g / L, about 71 g / L, about 72 g / L, about 73 g / L, about 74 g / L, about 75 g / L, about 76 g / L, about 77 g / L, about 78 g / L, about 79 g / L, about 8 g / L, about 8 g / L. 0 g / L, approximately 81 g / L, approximately 82 g / L, approximately 83 g / L, approximately 84 g / L, approximately 85 g / L, approximately 86 g / L, approximately 87 g / L, approximately 88 g / L, approximately 89 g / L, approximately 90 g / L, approximately 91 g / L, approximately 92 g / L, approximately 93 g / L, approximately 94 g / L, approximately 95 g / L, approximately 96 g / L, approximately 97 g / L, approximately 98 g / L, approximately 99 g / L, approximately 100 g / L, or any range or value between two values.

[0077] In some embodiments, the concentration of rapeseed oil in the fed culture medium is about 60 g / L, about 61 g / L, about 62 g / L, about 63 g / L, about 64 g / L, about 65 g / L, about 66 g / L, about 67 g / L, about 68 g / L, about 69 g / L, about 70 g / L, about 71 g / L, about 72 g / L, about 73 g / L, about 74 g / L, about 75 g / L, about 76 g / L, about 77 g / L, about 78 g / L, about 79 g / L, about 8 g / L, and about 8 g / L. 0 g / L, approximately 81 g / L, approximately 82 g / L, approximately 83 g / L, approximately 84 g / L, approximately 85 g / L, approximately 86 g / L, approximately 87 g / L, approximately 88 g / L, approximately 89 g / L, approximately 90 g / L, approximately 91 g / L, approximately 92 g / L, approximately 93 g / L, approximately 94 g / L, approximately 95 g / L, approximately 96 g / L, approximately 97 g / L, approximately 98 g / L, approximately 99 g / L, approximately 100 g / L, or any range or value between two values.

[0078] In some embodiments, the concentration of yeast extract in the fed culture medium is about 6 g / L, about 6.5 g / L, about 7 g / L, about 7.5 g / L, about 8 g / L, about 8.5 g / L, about 9 g / L, about 9.5 g / L, about 10 g / L, or any range or value between two values.

[0079] The feeding medium can be added approximately 48 to 52 hours after fermentation. For example, it can be added approximately 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, or 52 hours after fermentation (or any range or value between any two values).

[0080] Feeding can be stopped approximately 144 to approximately 152 hours after the start of fermentation. For example, feeding can be stopped approximately 144 hours, approximately 145 hours, approximately 146 hours, approximately 147 hours, approximately 148 hours, approximately 149 hours, approximately 150 hours, approximately 151 hours, or approximately 152 hours after fermentation (or any range or value between any two of these values).

[0081] The fermentation time can be from about 168 hours to about 170 hours, for example, it can be about 168 hours, about 168.5 hours, about 169 hours, about 169.5 hours, about 170 hours, or any range or value between two values.

[0082] In some embodiments, the Candida albicans used in the method includes the engineered Candida albicans strain ATCC22214. In some embodiments, the Candida albicans is the engineered Candida albicans strain ATCC22214. The engineered Candida albicans strain ATCC22214 can be a conventionally available engineered Candida albicans strain ATCC22214 in the art, for example, purchased from the Guangdong Institute of Microbiology.

[0083] In some embodiments, the feeding medium is added continuously at a rate of about 20 g / (L·h) to about 25 g / (L·h).

[0084] The method of adding feed culture medium in this application can be the conventional method of adding feed culture medium in the art, such as continuous addition.

[0085] In some implementations, the feeding rate is from about 20 g / (L·h) to about 25 g / (L·h). While not wishing to be limited by any theory, it has been found that feeding rates below about 20 g / (L·h) may reduce sophorolipid production and potentially lead to fermentation abnormalities; while feeding rates above about 25 g / (L·h) may result in incomplete consumption of raw materials, excessive residues, and conversion into other impurities, leading to an increase in the impurity content of the fermentation broth.

[0086] In some embodiments, the method for preparing sophorolipids further includes seed culture. Exemplarily, seed culture includes primary seed culture and secondary seed culture.

[0087] In some embodiments, seed culture includes inoculating Candida albicans (such as engineered Candida albicans ATCC22214) into a primary seed culture medium, fermenting it to obtain a primary seed liquid, and then inoculating the primary seed liquid into a secondary seed culture medium for further fermentation to form a bacterial suspension, thus obtaining a secondary seed liquid.

[0088] In some embodiments, the primary culture medium for primary seed culture contains about 2 g / L to about 5 g / L glucose, about 8 g / L to about 10 g / L yeast extract, and about 2 g / L to about 3 g / L ammonium sulfate.

[0089] In some embodiments, the secondary culture medium for secondary seed culture contains about 2 g / L to about 5 g / L glucose, about 8 g / L to about 10 g / L yeast extract, and about 2 g / L to about 3 g / L ammonium sulfate.

[0090] In some embodiments, the concentration of glucose in the primary or secondary seed culture medium is about 2 g / L, about 2.5 g / L, about 3 g / L, about 3.5 g / L, about 4 g / L, about 4.5 g / L, about 5 g / L, or any range or value between two values.

[0091] In some embodiments, the concentration of yeast extract in the primary or secondary seed culture medium is about 8 g / L, about 8.5 g / L, about 9 g / L, about 9.5 g / L, about 10 g / L, or any range or value between two of these values.

[0092] In some embodiments, the concentration of ammonium sulfate in the primary or secondary seed culture medium is about 2.1 g / L, about 2.2 g / L, about 2.3 g / L, about 2.4 g / L, about 2.5 g / L, about 2.6 g / L, about 2.7 g / L, about 2.8 g / L, about 2.9 g / L, about 3.0 g / L, or any range or value between two of these values.

[0093] In some embodiments, the pH of the primary seed culture medium and the secondary seed culture medium are each independently about 3.0 to about 4.0, for example about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, or any range or value between two values.

[0094] In some embodiments, the temperatures for primary and secondary seed cultures are each independently set at approximately 30°C to approximately 33°C, for example, approximately 30°C, approximately 30.5°C, approximately 31°C, approximately 31.5°C, approximately 32°C, approximately 32.5°C, approximately 33°C, or any range or value between two such values. In some embodiments, the temperatures for primary and secondary seed cultures are each independently set at approximately 30°C to approximately 31°C. While not wishing to be limited by any theory, it is believed that excessively low or high temperatures may lead to slow cell growth and metabolism, potentially affecting the rate of sophorolipid production.

[0095] In some embodiments, the primary seed culture and secondary seed culture times are each independently from approximately 24 hours to approximately 48 hours. Exemplarily, they can each be independently approximately 24 hours, approximately 25 hours, approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 31 hours, approximately 32 hours, approximately 33 hours, approximately 34 hours, approximately 35 hours, approximately 36 hours, approximately 37 hours, approximately 38 hours, approximately 39 hours, approximately 40 hours, approximately 41 hours, approximately 42 hours, approximately 43 hours, approximately 44 hours, approximately 45 hours, approximately 46 hours, approximately 47 hours, approximately 48 hours, or any range or value between two values. In some embodiments, the primary seed culture and secondary seed culture times are each independently from 30 hours to 36 hours.

[0096] In some embodiments, the OD of the primary seed culture obtained from primary seed culture is... 600 The value is greater than 5. In some embodiments, the OD of the primary seed culture obtained from primary seed culture is... 600 The value is not higher than 6. In some embodiments, the OD value of the primary seed culture obtained from primary seed culture is... 600 The value is approximately 5.1 to approximately 5.5. For example, the OD of the primary seed solution... 600 The value is approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, or a range or value between any two values.

[0097] In some embodiments, the OD of the secondary seed culture obtained from the secondary seed culture is... 600 The value is greater than 5. In some embodiments, the OD value of the secondary seed culture obtained from secondary seed culture is greater than 5. 600 The value is approximately 5.1 to approximately 5.5. In some embodiments, the OD value of the secondary seed culture obtained from secondary seed culture is... 600 The value is not higher than 6. In some embodiments, the OD value of the secondary seed culture obtained from secondary seed culture is... 600 The value is approximately 5.1 to approximately 5.5. For example, the OD of the secondary seed solution... 600 The value is approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, or a range or value between any two values.

[0098] Unless otherwise specified, the term "OD" in this application is an abbreviation for optical density. The energy difference before and after light passes through a analyte is the energy absorbed by the analyte. At a specific wavelength, there is a quantitative relationship between the concentration of the same analyte and the energy absorbed. 600 "OD value" refers to the optical density measured at a wavelength of 600 nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is commonly used to indicate bacterial cell density. Detecting OD values ​​to indicate bacterial concentration is a routine practice in this field, and OD values ​​and bacterial concentration have a linear relationship.

[0099] In some embodiments, the inoculation volume of the primary seed culture is from about 2% (v / v) to about 8% (v / v); "% (v / v)" is the volume percentage of the seed culture medium (e.g., secondary seed medium) to which it is to be inoculated. Exemplarily, the inoculation volume of the primary seed culture is about 2% (v / v), about 3% (v / v), about 4% (v / v), about 5% (v / v), about 6% (v / v), about 7% (v / v), about 8% (v / v), or a range or value between any two values. In some embodiments, the inoculation volume of the primary seed culture is from about 3% (v / v) to about 5% (v / v).

[0100] In some embodiments, the inoculation amount of the secondary seed culture is from about 3 wt% to about 8 wt%; "wt%" is the percentage by mass of the secondary seed culture to be inoculated into the fermentation medium. In some embodiments, the inoculation amount of the secondary seed culture is about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, or any range or value between any two values, for example, from about 4 wt% to about 5 wt%.

[0101] In some embodiments, the fermentation medium comprises about 30% to about 50% of the volume of the fermentation vessel (e.g., fermenters of various volumes, such as 5L fermenters, 7.5L fermenters, etc.). In some embodiments, the fermentation medium comprises about 35% to about 40% of the volume of the fermentation vessel, for example, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, or any range or value between two of these values.

[0102] In some embodiments, the method for preparing sophorolipids further includes separating and purifying the fermentation broth obtained from fermentation;

[0103] Separation and purification include:

[0104] a. Add ethyl acetate to the fermentation broth obtained from fermentation, stir, let stand until complete phase separation, collect and retain the supernatant liquid;

[0105] b. Add an alkane solvent to the resulting upper layer, stir, and allow to stand until complete phase separation, then collect and retain the lower layer; and,

[0106] c. Solvent removal is performed on the resulting lower liquid layer to prepare high-purity lactone-type sophorolipid.

[0107] In some embodiments, the amount of ethyl acetate added in step a is about 0.5 to about 1.5 times the volume of the fermentation broth. Exemplarily, it can be about 1 times.

[0108] In some embodiments, the stirring and settling in step a are each performed independently at approximately 20°C to approximately 40°C. Exemplarily, these are, for example, approximately 20°C, approximately 21°C, approximately 22°C, approximately 23°C, approximately 24°C, approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C, approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C, approximately 35°C, approximately 36°C, approximately 37°C, approximately 38°C, approximately 39°C, approximately 40°C, or any range or value between two such values. In some embodiments, both stirring and settling are performed at approximately 25°C.

[0109] I do not wish to be limited by any theory, but I believe that the solubility of the fermentation broth in ethyl acetate may decrease below about 20°C, while side reactions may occur above about 40°C.

[0110] In some embodiments, the stirring speed in step a is from about 150 rpm to about 250 rpm, and the time is from about 10 min to about 20 min. Exemplarily, the stirring speed may be about 150 rpm, about 155 rpm, about 160 rpm, about 165 rpm, about 170 rpm, about 175 rpm, about 180 rpm, about 185 rpm, about 190 rpm, about 195 rpm, about 200 rpm, about 205 rpm, about 210 rpm, about 215 rpm, about 220 rpm, about 225 rpm, about 230 rpm, about 235 rpm, about 240 rpm, about 245 rpm, about 250 rpm, or any range or value between two such values. For example, the stirring time may be approximately 10 min, approximately 11 min, approximately 12 min, approximately 13 min, approximately 14 min, 15 min, approximately 16 min, approximately 17 min, approximately 18 min, approximately 19 min, approximately 20 min, or any range or value between two of these values. In some embodiments, the stirring speed in step a is approximately 200 rpm, and the time is approximately 15 min.

[0111] In some implementations, the settling time in step a is approximately 2 hours to approximately 6 hours. Exemplarily, the settling time is approximately 2 hours, approximately 2.5 hours, approximately 3 hours, approximately 3.5 hours, approximately 4 hours, approximately 4.5 hours, approximately 5 hours, approximately 5.5 hours, approximately 6 hours, or any range or value between two values.

[0112] In some implementations, the settling time in step a is about 4 hours, and the settling temperature is about 25°C.

[0113] In some embodiments, the amount of alkane solvent added in step b is about 0.5 to about 1 times the amount of the lower liquid. Exemplarily, for example, it is about 0.5 times, about 0.6 times, about 0.7 times, about 0.8 times, about 0.9 times, about 1 time, or any range or value between two such values. In some embodiments, the amount of alkane solvent added in step b is about 0.8 times the amount of the lower liquid.

[0114] In some embodiments, the stirring and settling in step b are each carried out independently at approximately 20°C to approximately 40°C. Exemplary values ​​include, for example, approximately 20°C, approximately 21°C, approximately 22°C, approximately 23°C, 24°C, approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C, approximately 30°C, approximately 31°C, approximately 32°C, approximately 33°C, approximately 34°C, approximately 35°C, approximately 36°C, approximately 37°C, approximately 38°C, approximately 39°C, 40°C, or any range or value between two such values. While not intended to be theoretically limited, it is believed that the solubility of the fermentation broth in ethyl acetate may decrease below approximately 20°C, while side reactions may occur above approximately 40°C. In some embodiments, both stirring and settling are carried out at approximately 25°C.

[0115] In some embodiments, the alkane solvent in step b is selected from one or more of n-hexane, n-heptane, and n-octane. For example, n-hexane, n-heptane, or n-octane is used for extraction.

[0116] In some embodiments, the stirring speed in step b is from about 100 rpm to about 200 rpm, and the time is from about 30 min to about 60 min.

[0117] In some embodiments, the stirring speed in step b is approximately 100 rpm, approximately 110 rpm, approximately 120 rpm, approximately 130 rpm, approximately 140 rpm, approximately 150 rpm, approximately 155 rpm, approximately 160 rpm, approximately 165 rpm, approximately 170 rpm, approximately 175 rpm, approximately 180 rpm, approximately 185 rpm, approximately 190 rpm, approximately 195 rpm, approximately 200 rpm, or any range or value between two of these values.

[0118] In some embodiments, the stirring time in step b is approximately 30 min, approximately 35 min, approximately 40 min, approximately 45 min, approximately 50 min, approximately 55 min, approximately 60 min, or any range or value between two of these values. In some embodiments, the stirring time in step b is approximately 40 min.

[0119] In some implementations, the stirring speed and time are approximately 150 rpm and approximately 40 min, respectively.

[0120] In some embodiments, the settling time in step b is approximately 10 hours to approximately 20 hours. Exemplarily, the settling time can be approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, approximately 18 hours, approximately 19 hours, approximately 20 hours, or any range or value between two such values. In some embodiments, the settling time in step b is approximately 15 hours, and the settling temperature is approximately 25°C.

[0121] In some implementations, the removal method in step c may include vacuum evaporation.

[0122] In some embodiments, the prepared sophorolipid has a purity greater than 98%, a yield greater than 95%, and a lactone content greater than 90%.

[0123] The method for determining sophorolipid content is a conventional method used in this field. For example, the sulfuric acid-anthrone method.

[0124] In some implementations, the separation and purification process also includes a side-processing step;

[0125] The side-processing steps include spray drying the lower liquid obtained after complete phase separation in step a to obtain a powdered solid, thus preparing the crude sophorolipid product.

[0126] In some embodiments, the spray drying temperature is from about 110°C to about 120°C. For example, about 110°C, about 111°C, about 112°C, about 113°C, about 114°C, about 115°C, about 116°C, about 117°C, about 118°C, about 119°C, about 120°C, or any range or value between two such values. In some embodiments, the spray drying temperature is about 115°C. In some embodiments, the effective content of sophorolipids in the solid powder obtained after spray drying is from about 19% to about 40%.

[0127] In some embodiments, the method further includes preparing products with different acid ratios based on high-purity lactone-type sophorolipids, including:

[0128] High-purity lactone-type sophorolipids were mixed with alkali and water in a certain proportion and then stirred and matured to prepare products with different acid ratios.

[0129] Optionally, the mixture may be allowed to stand after stirring and maturation. For example, it may be allowed to stand at about 20°C to about 40°C.

[0130] In some embodiments, high-purity lactone-type sophorolipids are mixed with an alkaline solution, and then different masses of water are added to prepare products with different acid ratios. For example, see Table 1 below for mixing instructions.

[0131] Table 1

[0132] *: "A" represents the grade, indicating acidic sophorolipid.

[0133] In some embodiments, the stirring speed is from about 150 rpm to about 250 rpm, and the stirring time is from about 5 hours to about 10 hours. For example, the stirring speed can be about 150 rpm, about 155 rpm, about 160 rpm, about 165 rpm, about 170 rpm, about 175 rpm, about 180 rpm, about 185 rpm, about 190 rpm, about 195 rpm, about 200 rpm, about 205 rpm, about 210 rpm, about 215 rpm, about 220 rpm, about 225 rpm, about 230 rpm, about 235 rpm, about 240 rpm, about 245 rpm, about 250 rpm, or any range or value between two values. For example, the stirring time can be about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, or any range or value between two values. In some embodiments, the stirring speed is about 200 rpm, and the stirring time is about 8 hours. The stirring temperature is usually not strictly limited, for example, it can be around 30°C.

[0134] In some embodiments, the alkaline solution comprises an aqueous NaOH solution; the mass concentration of the aqueous NaOH solution is optionally from about 5 wt% to about 20 wt%. Exemplarily, for example, it is about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, or any range or value between two values.

[0135] High-purity sophorolipid products were obtained through simple two-stage extraction. Products with different acid ratios were obtained by adjusting the alkaline hydrolysis process. Wastewater generated during separation was recycled to prepare low-end sophorolipid products. This enabled the preparation of products of different specifications using the same batch of fermentation broth, meeting the application needs of different fields, with no separation loss throughout the process.

[0136] The following are some examples.

[0137] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0138] The fermentation equipment used in the following examples and comparative examples is as follows: Everson 7.5L fermenter, constant temperature shaker (Shanghai Hetian), and constant temperature biological incubator (Shanghai Keheng).

[0139] The separation equipment is as follows: 10L glass reactor (with stirring), Japanese RIKEN rotary evaporator N-1100D-W, 3L three-necked flask, 1L flask, and Shanghai Jinghong water bath.

[0140] Main raw material sources

[0141] The reagents used, their sources, and purities are as follows: Anhydrous ethanol (analytical grade), purchased from Comio; 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; dipotassium hydrogen phosphate (analytical grade), purchased from Sinopharm; ethyl acetate (analytical grade), purchased from Sinopharm; Candida bombicola engineered strain ATCC22214, purchased from Guangdong Institute of Microbiology; n-hexane (analytical grade), n-heptane (analytical grade), n-octane (analytical grade), purchased from Comio; ethyl acetate (analytical grade), purchased from Comio; sodium hydroxide (analytical grade), purchased from Sinopharm.

[0142] Main testing methods

[0143] The method for determining the content of sophorolipids is the sulfuric acid-anthrone method, and the specific operation method is as follows:

[0144] (1) Preparation of anthrone reagent

[0145] Accurately weigh 0.1g of anthrone, add 100mL of concentrated sulfuric acid, mix well, and this is the anthrone reagent.

[0146] (2) Prepare a 1 mmol / mL anhydrous glucose solution

[0147] Weigh a certain mass of anhydrous glucose and dry it to constant weight in a drying oven at 105℃. Weigh 0.1800g of the constant-weight anhydrous glucose and dilute it to volume in a 100mL volumetric flask, then shake well. Its concentration is 10mmol / L. Then take 0.5, 1, 2, 3, and 4mL respectively and dilute to 100mL, with concentrations of 0.5, 0.1, 0.2, 0.3, and 0.4mmol / L respectively. 1) Preparation of anthrone solution: Dissolve 0.2g of anthrone in 100mL of 80% sulfuric acid, protect from light, and prepare fresh before use; do not store.

[0148] (3) Anthrone method for plotting glucose standard curve

[0149] Measure 1 mL of anthrone reagent and standard glucose solution into stoppered test tubes, respectively, and quickly place them in an ice-water bath to cool. 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 used in the experiment was 1 mL of distilled water.

[0150] The absorbance was determined using the anthrone method, and a standard curve was plotted. y = ax + b

[0151] Where y is the glucose content and x is the absorbance.

[0152] (4) Determination of sophorolipid content in fermentation broth

[0153] Add 1 mL of ethyl acetate to 0.5 mL of fermentation broth, mix well, let stand for 5 min, centrifuge at 1000 rpm for 10 min, and dilute the supernatant solution 50-100 times. Determine the total sugar content in the ethyl acetate layer using the anthrone method. Subtract the glucose content from the supernatant to obtain the lactone-type sophorolipid content. Add 1 mL of ethanol to 0.5 mL of fermentation broth, mix well, centrifuge, and dilute the liquid 50-100 times. Determine the total sugar concentration in the supernatant using the anthrone method. Subtract the residual glucose from the supernatant to calculate the total sophorolipid yield. Subtract the lactone content to obtain the acidic sophorolipid content.

[0154] The final formula for calculating the sophorolipid content is:

[0155] Sophorolipid content = (total sugar content in the reaction solution - glucose content) × dilution factor × 1.91.

[0156] The ratio of acidic to lactone-type sophorolipids was determined by dissolving them in different solvents and measuring the content of acidic and lactone-type sophorolipids.

[0157] Example 1

[0158] Prepare the culture medium according to the following proportions.

[0159] Seed culture medium composition: glucose 2g / L, yeast extract 8g / L, ammonium sulfate 2g / L.

[0160] Fermentation medium composition: glucose 60 g / L, rapeseed oil 60 g / L, yeast extract 6 g / L, dipotassium hydrogen phosphate 1 g / L. Initial medium volume was 30% (v / v).

[0161] The supplemental culture medium consists of 60 g / L glucose, 60 g / L rapeseed oil, and 6 g / L yeast powder.

[0162] Candida albicans ATCC22214 was inoculated into seed culture medium and fermented for 24 hours to obtain primary seed culture. The fermentation conditions were 30℃ and pH 3.0. Then, the primary seed culture (OD) was... 600(5.3) Inoculate the seed culture medium again at an inoculation rate of 2% (v / v) and continue fermentation culture for 24 hours to obtain secondary seed liquid.

[0163] Secondary seed solution (OD) 600 The inoculum (6.0%) was inoculated into the fermentation medium at a rate of 3 wt%, and fermentation was carried out under the following conditions: temperature 30℃, pH 3.0, dissolved oxygen relative concentration 28%. Feeding was started after 48 hours of fermentation at a rate of 20 g / (L·h), and feeding was stopped after 144 hours. Fermentation was stopped after 168 hours. 5 L of fermentation broth was obtained. Analysis showed that the yield of sophorolipids was 280 g / L, with a lactone content of 85%.

[0164] The obtained fermentation broth was separated and purified according to the following separation process:

[0165] 5 L of fermentation broth was added to 2.5 L of ethyl acetate, stirred for 10 min, allowed to stand for 2 h, and then the phases were separated. The upper layer, approximately 3.2 L, was obtained. 1.6 L of n-hexane was added, the mixture was stirred for 30 min, allowed to stand for 10 h, and the phases were separated again. The lower layer, approximately 2.1 L, was collected. The solvent was removed by vacuum evaporation to obtain 1.4 kg of concentrate, which was high-purity sophorolipid with a purity of 95%.

[0166] Product blending:

[0167] Preparation of 50% Product A: Take 1000g of concentrated solution, add 300g of 10wt% NaOH aqueous solution and 700g of water, mix well, stir for 8 hours to mature, and obtain 2000g of aqueous solution product. Product yield is 95%. Testing shows that the acidic form of sophorolipids accounts for 51%, and the sophorolipid content is 95%.

[0168] The lower aqueous phase obtained from the first extraction totaled 4.1 L. After spray drying at 110 °C, 200 g of powder product was obtained. Analysis showed that the sophorolipid content was 35%. Sophorolipids accounted for 5%.

[0169] The production process of sophorolipids can be seen in Figure 1.

[0170] Example 2

[0171] Compared with Example 1, the following changes are made:

[0172] Seed culture medium composition: glucose 5g / L, yeast powder 10g / L, ammonium sulfate 3g / L.

[0173] Fermentation medium composition: glucose 100g / L, rapeseed oil 100g / L, yeast powder 10g / L, dipotassium hydrogen phosphate 3g / L.

[0174] The supplemental culture medium consists of 100 g / L glucose, 100 g / L rapeseed oil, and 10 g / L yeast powder.

[0175] Candida albicans ATCC22214 was inoculated into seed culture medium and fermented for 48 h to obtain primary seed culture. The fermentation conditions were 33℃ and pH 4.0. Then, the primary seed culture (OD) was... 600 (5.5) At an inoculum rate of 8% (v / v), the seed culture was inoculated again and fermented for another 48 hours to obtain the secondary seed culture (OD). 600 (5.4).

[0176] The secondary seed culture was inoculated into the fermentation medium at an inoculum rate of 8 wt%, and fermentation was carried out under the following conditions: temperature 33℃, pH 4.0, and dissolved oxygen relative concentration 32%. Feeding was started after 52 h of fermentation at a rate of 25 g / (L·h), and stopped after 152 h. Fermentation was stopped after 170 h. 5 L of fermentation broth was obtained, and analysis showed that the yield of sophorolipids was 320 g / L, with a lactone content of 82%.

[0177] The obtained fermentation broth was separated and purified according to the following separation process:

[0178] 3 L of fermentation broth was added to 4.5 L of ethyl acetate, stirred for 20 min, allowed to stand for 6 h, and the phases were separated. The upper layer, approximately 4 L, was separated, and 4 L of heptahexane was added. The mixture was stirred for 60 min, allowed to stand for 20 h, and the phases were separated again. The lower layer, approximately 3 L, was collected. The solvent was removed by vacuum evaporation to obtain 972 g of high-purity sophorolipid with a purity of 96.8%.

[0179] Product blending:

[0180] Take 500g of concentrated solution, 400g of 10wt% NaOH aqueous solution, and 100g of water, mix them evenly, stir for 10h to mature, and obtain 1000g of aqueous solution product with an acid form ratio of 90%, with a product yield of 98%.

[0181] The lower aqueous phase obtained from the first extraction totaled 3.1 L. After spray drying at 110 °C, 100 g of powder product was obtained. Analysis showed that the sophorolipid content was 19.2%, representing 2% of the total product.

[0182] Example 3

[0183] Seed culture medium composition: glucose 3.5 g / L, yeast extract 9 g / L, ammonium sulfate 2.5 g / L.

[0184] Fermentation medium composition: glucose 70g / L, rapeseed oil 90g / L, yeast powder 8.5g / L, dipotassium hydrogen phosphate 2g / L.

[0185] The supplemental culture medium consists of: 70 g / L glucose, 90 g / L rapeseed oil, and 8.5 g / L yeast powder.

[0186] Fermentation process:

[0187] Candida albicans ATCC22214 was inoculated into seed culture medium and fermented for 36 hours to obtain primary seed culture. The fermentation conditions were 31℃, pH 3.5, and 220 rpm. The primary seed culture was then divided according to (OD...) 600 At an inoculum size of 5.2)6% (v / v), the seed culture medium was inoculated again, and fermentation was continued for 36 hours to obtain the secondary seed culture (OD). 600 (5.3).

[0188] The secondary seed culture was inoculated into the fermentation medium at a 6 wt% inoculum, and fermentation was carried out under the following conditions: temperature 31℃, pH 3.5, agitator speed 220 rpm, and dissolved oxygen relative concentration 30%. After 50 h of fermentation, feeding was started at a rate of 22 g / (L·h), and feeding was stopped after 150 h. Fermentation was then stopped after 170 h. 4.5 L of fermentation broth was obtained. Analysis showed that the yield of sophorolipids was 300 g / L, with a lactone content of 79%.

[0189] Separation process:

[0190] Add 4L of ethyl acetate to 4L of fermentation broth, stir at room temperature for 15 minutes, let stand for 4 hours, and separate the phases. Collect about 5L of the upper liquid, add 4L of n-octane, stir for 40 minutes, let stand for 15 hours, 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 sophorolipid with a purity of 97%.

[0191] Product blending:

[0192] 70% A: Take 1000g of concentrated solution, 600g of 10% NaOH aqueous solution, and 400g of water, mix them evenly, stir at room temperature for 8 hours to mature, and obtain 2000g of aqueous solution product with an acidity ratio of 70%, with a product yield of 95%.

[0193] The lower aqueous phase obtained from the first extraction totaled 3L. After spray drying at 120℃, 150g of powder product was obtained. Analysis showed that the sophorolipid content was 40%, and the sophorolipid percentage was 5%.

[0194] Comparative Example 1

[0195] Prepare the culture medium according to the following proportions.

[0196] Seed culture medium: glucose 5g / L, yeast extract 10g / L, ammonium sulfate 3g / L.

[0197] Fermentation medium and fed medium: glucose 50g / L, rapeseed oil 50g / L, ammonium sulfate 5g / L, yeast powder 5g / L, potassium dihydrogen phosphate 1g / L.

[0198] The seed culture medium was dispensed into two 1L shake flasks, each containing 20% ​​of the liquid volume. The fermentation culture medium was dispensed into a 7.5L fermenter in 3L. The feed culture medium was prepared in 1L and dispensed into a 2L feed bottle. All culture media were sterilized at high temperature before use.

[0199] 1) Seed culture: Preserved Candida albicans was inoculated into sterilized seed culture medium and activated at 30℃ and 200 rpm for 40 h to obtain primary seed culture. Then, the primary seed culture was inoculated into fresh sterilized seed culture medium at a volume ratio of 6% and cultured at 30℃ and 200 rpm for 40 h to form a bacterial suspension. OD was then measured. 600 The concentration is 7.1, which indicates it is a secondary seed solution.

[0200] 2) Fermentation Culture: The secondary seed culture was inoculated into a 7.5L fermenter at a mass ratio of 5% and fermentation began at a temperature of 30℃, a pH of 3.5, a rotation speed of 200 rpm, and a dissolved oxygen relative concentration of 30%. Feeding was started after 24 hours of fermentation at a rate of 20 g / L / H and stopped at the end of the fermentation period. Fermentation was stopped after 96 hours. 4.1L of fermentation broth was obtained, with a sophorolipid yield of 158 g / L, of which 65% was acidic and the remainder was lactone.

[0201] Comparative Example 2

[0202] Seed culture medium: glucose 5g / L, yeast extract 10g / L, ammonium sulfate 3g / L.

[0203] Fermentation medium and fed medium: glucose 50g / L, rapeseed oil 50g / L, ammonium sulfate 5g / L, yeast powder 5g / L, potassium dihydrogen phosphate 1g / L.

[0204] The seed culture medium was dispensed into two 1L shake flasks, each containing 20% ​​of the liquid volume. The fermentation culture medium was dispensed into a 7.5L fermenter in 3L. The feed culture medium was prepared in 1L and dispensed into a 2L feed bottle. All culture media were sterilized at high temperature before use.

[0205] 1) Seed culture: Preserved Candida albicans was inoculated into sterilized seed culture medium and activated at 30℃ and 200 rpm for 40 h to obtain primary seed culture. Then, the primary seed culture was inoculated into fresh sterilized seed culture medium at a volume ratio of 6% and cultured at 30℃ and 200 rpm for 40 h to form a bacterial suspension. OD was then measured. 600 The concentration is 7.1, which indicates it is a secondary seed culture.

[0206] 2) Fermentation Culture: The secondary seed culture was inoculated into a 7.5L fermenter at a mass ratio of 5% and fermentation began at a temperature of 30℃, a pH of 3.5, a rotation speed of 200 rpm, and a dissolved oxygen relative concentration of 15%. Feeding was started after 24 hours of fermentation at a rate of 20 g / L / H and stopped at the end of the fermentation period. Fermentation was stopped after 142 hours. 5.4L of fermentation broth was obtained, with a sophorolipid yield of 112 g / L, of which 50% was acidic and the remainder was lactone.

[0207] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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.

[0208] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method of preparing a sophorolipid, wherein, The method includes: The seed culture of Candida albicans was inoculated into a fermentation medium for fermentation: the fermentation medium contained about 60 g / L to about 100 g / L glucose, about 60 g / L to about 100 g / L rapeseed oil, about 6 g / L to about 10 g / L yeast extract and about 1 g / L to about 3 g / L dipotassium hydrogen phosphate, the fermentation pH was about 3.0 to about 4.0, the fermentation temperature was about 30°C to about 33°C, and the relative dissolved oxygen in the fermentation broth was maintained at about 28% to about 32%. Feeding: Feeding medium is added starting from about 48 to about 52 hours after fermentation and is stopped after about 144 to about 152 hours of fermentation. The feeding medium contains about 60 g / L to about 100 g / L of glucose, about 60 g / L to about 100 g / L of rapeseed oil and about 6 g / L to about 10 g / L of yeast extract.

2. The method of claim 1, wherein, The Candida yeast includes the engineered strain ATCC22214.

3. The method of claim 1, wherein, The feeding medium was added continuously at a rate of approximately 20 g / (L·h) to approximately 25 g / (L·h).

4. The method of any one of claims 1-3, wherein, The method further includes seed culture; the seed culture includes primary seed culture and secondary seed culture. Primary seed culture and secondary seed culture meet one or more of the conditions shown in A1) to A2) below: A1) Seed culture media for primary and secondary seed cultures each independently contain about 2 g / L to about 5 g / L glucose, about 8 g / L to about 10 g / L yeast extract and about 2 g / L to about 3 g / L ammonium sulfate, optionally with a pH of about 3.0 to about 4.0; A2) The conditions for primary seed culture and secondary seed culture are each independent and include: The temperature is from about 30°C to about 33°C, or alternatively from about 30°C to about 31°C; the time is from about 24 hours to about 48 hours, or alternatively from about 30 hours to about 36 hours.

5. The method of claim 4, wherein, Seed culture solutions obtained from seed culture meet one or more of the conditions shown in B1) to B4) below: B1 ) OD of the primary seed broth obtained from the primary seed culture 600 greater than 5, optionally from about 5.1 to about 5.5; B2) the OD of the secondary seed broth obtained from the secondary seed culture 600 greater than 5, optionally from about 5.1 to about 5.5; B3) The inoculation amount of the primary seed solution is about 2% (v / v) to about 8% (v / v), optionally about 3% (v / v) to about 5% (v / v); "% (v / v)" is the volume percentage of the seed solution to be inoculated into the seed culture medium; B4) The inoculation amount of the secondary seed liquid is about 3 wt% to about 8 wt%, optionally about 4 wt% to about 5 wt%; "wt%" is the percentage of the secondary seed liquid by mass of the fermentation medium to be inoculated.

6. The method according to any one of claims 1 to 5, wherein, The fermentation medium occupies approximately 30% to 50% of the fermentation vessel volume.

7. The method of claim 6, wherein, The fermentation medium occupies approximately 35% to 40% of the fermentation vessel volume.

8. The method according to any one of claims 1 to 7, wherein, The method also includes separating and purifying the fermentation broth obtained from fermentation; The separation and purification process includes: a. Add ethyl acetate to the fermentation broth, stir, and let stand until complete phase separation, then collect and retain the supernatant liquid; b. Add an alkane solvent to the upper liquid, stir, allow to stand until complete phase separation, collect and retain the lower liquid; and, c. Solvent removal is performed on the lower liquid layer to prepare high-purity lactone-type sophorolipids; the removal method may optionally include vacuum evaporation.

9. The method of claim 8, wherein, Step a satisfies one or more of the following conditions C1) to C4): C1) The amount of ethyl acetate added is about 0.5 to about 1.5 times the volume of the fermentation broth, optionally about 1 times; C2) Stirring and settling are carried out independently at approximately 20°C to approximately 40°C; C3) The stirring speed is from about 150 rpm to about 250 rpm, and the time is from about 10 min to about 20 min; C4) The settling time is approximately 2 hours to approximately 6 hours; Step b satisfies one or more of the conditions shown in D1) to D5): D1) The amount of alkane solvent added is approximately 0.5 to approximately 1 times the amount of the lower liquid layer; D2) Stirring and settling are carried out independently at approximately 20°C to approximately 40°C; D3) The alkane solvent is selected from any one or more of n-hexane, n-heptane, and n-octane; D4) The stirring speed is from about 100 rpm to about 200 rpm, and the time is from about 30 min to about 60 min; D5) The settling time is approximately 10 hours to approximately 20 hours.

10. The method of claim 8 or 9, wherein, The separation and purification process also includes a side-processing step; The side-processing step includes spray drying the lower liquid obtained after complete phase separation in step a to obtain a powdered solid, thus preparing the crude sophorolipid product.

11. The method of claim 10, wherein, The spray drying temperature is approximately 110°C to approximately 120°C.

12. The method according to any one of claims 8 to 11, wherein, The method also includes preparing products with different acid ratios based on high-purity lactone-type sophorolipids, including: The high-purity lactone-type sophorolipid was mixed with alkali and water in a certain proportion and then stirred and matured to prepare products with different acid ratios.

13. The method of claim 12, wherein, The stirring speed is from approximately 150 rpm to approximately 250 rpm, and the stirring time is from approximately 5 hours to approximately 10 hours.

14. The method of claim 12, wherein, The alkaline solution includes an aqueous solution of NaOH.

15. The method of claim 14, wherein, The mass concentration of the NaOH aqueous solution is from about 5 wt% to about 20 wt%.

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