Method for preparing acid sophorolipid

Abstract

The invention discloses a method for preparing acid sophorolipid, and belongs to the technical field of biological fermentation. According to the method, candida is used as a fermentation strain, a composite carbon source culture medium containing a water-soluble carbon source and an oil-soluble carbon source is adopted, and the content of residual oil is monitored in real time in the fermentation process; when the residual oil content exceeds a threshold value of 5g / L, the emulsifying agent of 0.1 g / L-0. 5g / L is supplemented, and the stirring rotating speed is increased by 20-50rpm, so that the emulsification of an oil phase and the utilization of the thalli on the oil-soluble carbon source are promoted. By accurately controlling the residual oil content in the fermentation process, the proportion of the acidic sophorolipid and the carbon source conversion rate are remarkably increased, the proportion of the acidic sophorolipid is increased to 60% from 30% of a traditional process, the carbon source conversion rate is increased to 65% from 40%, the problems that the increasing means of the proportion of the acidic sophorolipid is single, and the carbon source is wasted are effectively solved, and the method is suitable for industrial production. The method has the advantages of simple process, low cost, environmental protection and the like.

Description

Technical Field

[0001] This application relates to the field of biosurfactant production technology, specifically to a method for preparing acidic sophorolipids. Background Technology

[0002] Sophorolipids are a class of glycolipid biosurfactants produced by yeast metabolism. They have excellent properties such as being non-toxic, non-irritating, and readily biodegradable. They have shown promising application prospects in many fields such as medicine, cosmetics, food processing, and environmental protection, and are considered to be one of the most promising biosurfactants to replace chemical surfactants.

[0003] Sophorolipids can be structurally classified into two types: lactone type (LSL) and acid type (ASL). Lactone type sophorolipids excel in reducing surface tension and antibacterial activity, while acid type sophorolipids have superior foam-forming ability and solubility, making them urgently needed in specific application areas such as high-end detergents and foam stabilizers.

[0004] Currently, industrial production of sophorolipids all employs fermentation processes. The ratio of acidic to lactone-type sophorolipids is significantly influenced by strain characteristics and fermentation conditions, and the proportion of acidic sophorolipids is generally low (mostly 30%-40%), making it difficult to meet the demands of high-value-added applications. Traditional fermentation processes often neglect the impact of residual oil content on the composition of sophorolipids, resulting in difficulty in increasing the proportion of acidic sophorolipids in the product. Furthermore, excessively high residual oil content reduces carbon source conversion efficiency, leading to resource waste and increased production costs.

[0005] Therefore, developing a fermentation method that can effectively control residual oil content and increase the proportion of acidic sophorolipids is of great practical significance and application value. Summary of the Invention

[0006] Based on this, this application provides at least one method for preparing acidic sophorolipids.

[0007] One aspect of this application provides a method for preparing acidic sophorolipids, the method comprising:

[0008] Fermentation culture: The seed culture of Candida albicans was inoculated into the fermentation medium for fermentation. The fermentation temperature was 25℃~32℃, and the total dissolved oxygen relative value of the fermentation broth was maintained at 20%~60%.

[0009] Supplementing water-soluble and oil-soluble carbon sources: When the initial sugar concentration in the fermentation broth is less than or equal to 15 g / L, supplementing water-soluble and oil-soluble carbon sources, and controlling the residual sugar concentration to be 10 g / L~15 g / L;

[0010] Add emulsifier: When the residual oil content in the fermentation broth is greater than or equal to 5 g / L, add emulsifier and increase the stirring speed to continue fermentation.

[0011] The fermentation medium contains water-soluble carbon source, nitrogen source, inorganic salts and water.

[0012] In some embodiments, the Candida species includes one or more of Candida bombicola, Candida bombicola, and Wickerhamiella versatilis.

[0013] In some embodiments, the *Candida globosa* is *Candida globosa* with accession number CGMCC 1576.

[0014] In some embodiments, the bumblebee Candida is the bumblebee Candida species with accession number ATCC 22214.

[0015] In some embodiments, the Candida albicans strain is not an acidic strain.

[0016] In some embodiments, the fermentation medium conforms to one or more of the items shown in A1) to A4) below:

[0017] A1) The water-soluble carbon source includes sugar;

[0018] A2) The oil-soluble carbon source includes at least one of soybean oil, rapeseed oil, peanut oil, palm oil, corn oil, and sunflower oil;

[0019] A3) The nitrogen source selected includes at least one of yeast extract, peptone, urea, corn steep liquor, ammonium sulfate, and ammonium chloride;

[0020] A4) The inorganic salts include at least one of potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate heptahydrate, zinc chloride, ferrous sulfate, copper sulfate, and manganese sulfate.

[0021] In some implementations, the sugar in A1 is, for example, glucose, molasses, etc.

[0022] In some embodiments, the fermentation medium comprises: 20 g / L to 40 g / L glucose, 5 g / L to 20 g / L corn steep liquor, 1 g / L to 10 g / L ammonium chloride, 0.1 g / L to 10 g / L dipotassium hydrogen phosphate, 0.1 g / L to 10 g / L potassium dihydrogen phosphate, 0.1 g / L to 5 g / L magnesium sulfate heptahydrate, 0.01 g / L to 5 g / L ferrous sulfate, 0.01 g / L to 1 g / L manganese sulfate, 0.1 g / L to 1 g / L zinc chloride, and 0.1 g / L to 5 g / L copper sulfate.

[0023] In some implementations, the fermentation rotation speed is 100 rpm to 600 rpm.

[0024] In some embodiments, during the fermentation culture step, the inoculation amount of the seed liquid is 5% (v / v) to 10% (v / v).

[0025] In some embodiments, the method for preparing the seed solution includes:

[0026] Candida albicans was inoculated into an activation medium and cultured at 28℃~32℃ and 180 rpm~220 rpm for 18h~24h to prepare a seed culture.

[0027] In some implementations, the step of adding emulsifier conforms to one or more of the items shown in B1) to B4) below:

[0028] B1) The amount of the emulsifier used is 0.1 g / L to 0.5 g / L;

[0029] B2) The stirring speed can be increased by 20 rpm to 50 rpm;

[0030] B3) The emulsifier includes at least one of Tween-80 and Span-80;

[0031] B4) Regularly monitor the residual oil content in the fermentation broth.

[0032] In some implementations, the detection cycle for residual oil content in B4 is 6h to 24h.

[0033] In some implementations, the fermentation time is 160h to 240h.

[0034] In some embodiments, the method further includes a product extraction step.

[0035] In some embodiments, the product extraction steps include: after fermentation, collecting the fermentation broth, separating and purifying sophorolipids by ethyl acetate extraction, analyzing the content of acidic sophorolipids and lactone-type sophorolipids, and calculating the proportion of acidic sophorolipids.

[0036] The method for preparing acidic sophorolipids provided in one embodiment of this application has at least the following advantages:

[0037] The proportion of acidic sophorolipids has been significantly increased: by controlling the residual oil content, the proportion of acidic sophorolipids can be increased from 30% in the traditional process to 60%, which meets the market demand for products with a high proportion of acidic sophorolipids and expands the application fields of sophorolipids.

[0038] Improved carbon source conversion rate: By monitoring and controlling the residual oil content during fermentation, the residual oil content in the fermentation broth was effectively reduced, carbon source waste was reduced, and the carbon source conversion rate was increased from the original 40% to 65%.

[0039] Simple process and low cost: By monitoring the residual oil content in real time and taking simple control measures, no complicated equipment or additional process steps are required, making it easy to apply in industrial applications. Attached Figure Description

[0040] 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.

[0041] Figure 1 This is a schematic diagram comparing the proportion of acidic sophorolipids in an embodiment of this application with that of a comparative example. Detailed Implementation

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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."

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Unless otherwise specified, the term "residual oil" in this application refers to carbon sources (e.g., oil-soluble carbon sources) that are not fully utilized by microorganisms during fermentation, and its components include, for example, fatty acids.

[0056] 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.

[0057] 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 the same 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%.

[0058] 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. 620 "OD value" refers to the optical density measured at a wavelength of 620 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.

[0059] One aspect of this application provides a method for preparing acidic sophorolipids.

[0060] The inventors of this application made an accidental discovery during the experiment that, in the process of preparing acidic sophorolipids by fermentation of microorganisms (such as non-acidic strains), controlling the residual oil content in the fermentation broth can affect the proportion of acidic sophorolipids in the final prepared sophorolipids, hence this application is filed.

[0061] In some embodiments, the method for preparing acidic sophorolipids includes the following steps:

[0062] S100. Fermentation culture: The seed culture of Candida albicans is inoculated into the fermentation medium for fermentation. The fermentation temperature is 25℃~32℃, and the total dissolved oxygen relative value of the fermentation broth is maintained at 20%~60%.

[0063] S200. Supplement water-soluble and oil-soluble carbon sources: When the initial sugar concentration in the fermentation broth is less than or equal to 15 g / L, supplement water-soluble and oil-soluble carbon sources, and control the residual sugar concentration to 10 g / L~15 g / L;

[0064] S300. Add emulsifier: When the residual oil content in the fermentation broth is greater than or equal to 5 g / L, add emulsifier and increase the stirring speed to continue fermentation.

[0065] The fermentation medium contains water-soluble carbon source, nitrogen source, inorganic salts and water.

[0066] The Candida species mentioned in this application may be the Candida species commonly used in the art to prepare acidic sophorolipids, such as Candida globulus, Candida bumblebee, and Candida wilkens.

[0067] In some embodiments, step S100 may use a seed culture of at least one Candida species, such as Candida globosum, Candida bumblebee, or Candida wilk.

[0068] In some embodiments, step S100 may use seed cultures of two species of Candida, such as simultaneously inoculating seed cultures of Candida globosa and Candida bumblebee, or simultaneously inoculating seed cultures of Candida spp. and Candida bumblebee, or simultaneously inoculating seed cultures of Candida spp. and Candida globosa.

[0069] In some embodiments, the *Candida globosa* is *Candida globosa* with accession number CGMCC 1576.

[0070] In some embodiments, the bumblebee Candida is the bumblebee Candida species with accession number ATCC 22214.

[0071] In some embodiments, the fermentation temperature in step S100 is 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, or any range or value between two values.

[0072] We do not wish to be limited by any theory, but we have found that when the fermentation temperature is outside the above range, the fermentation yield of sophorolipids decreases by 30% to 40%.

[0073] In some implementations, the relative value of dissolved oxygen in step S100 is 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or any range or value between two values.

[0074] We do not wish to be limited by any theory, but we have found that when the relative dissolved oxygen value is not within the above range, the production of sophorolipids decreases by 40% to 60%.

[0075] In some embodiments, the Candida albicans strain is not an acidic strain.

[0076] Unless otherwise specified, the term "acidic strain" in this application refers to a class of selected or genetically modified yeast strains whose core characteristic is that they primarily produce acidic sophorolipids, rather than lactone-type sophorolipids.

[0077] In some embodiments, the water-soluble carbon source in the fermentation medium in step S100 includes sugar.

[0078] In some embodiments, the sugar is, for example, glucose, molasses, etc.

[0079] For example, the water-soluble carbon source includes at least one of glucose and molasses, such as glucose only, molasses only, or both glucose and molasses.

[0080] In some embodiments, the oil-soluble carbon source in the fermentation medium in step S100 includes at least one of soybean oil, rapeseed oil, peanut oil, palm oil, corn oil, and sunflower oil. For example, rapeseed oil may be used.

[0081] In some embodiments, the nitrogen source in the fermentation medium in step S100 is selected from at least one of yeast extract, peptone, urea, corn steep liquor, ammonium sulfate, and ammonium chloride. Exemplarily, it may include corn steep liquor, ammonium sulfate, and / or ammonium chloride.

[0082] In some embodiments, the inorganic salts in the fermentation medium in step S100 include at least one of potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate heptahydrate, zinc chloride, ferrous sulfate, copper sulfate, and manganese sulfate.

[0083] In some embodiments, the fermentation medium in step S100 comprises: 20 g / L to 40 g / L glucose, 5 g / L to 20 g / L corn steep liquor, 1 g / L to 10 g / L ammonium chloride, 0.1 g / L to 10 g / L dipotassium hydrogen phosphate, 0.1 g / L to 10 g / L potassium dihydrogen phosphate, 0.1 g / L to 5 g / L magnesium sulfate heptahydrate, 0.01 g / L to 5 g / L ferrous sulfate, 0.01 g / L to 1 g / L manganese sulfate, 0.1 g / L to 1 g / L zinc chloride, and 0.1 g / L to 5 g / L copper sulfate.

[0084] In some embodiments, the concentration of glucose in the fermentation medium may be, for example, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, or any range or value between two such values.

[0085] In some embodiments, the concentration of corn steep liquor in the fermentation medium may be, for example, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, or any range or value between two such values.

[0086] In some embodiments, the concentration of ammonium chloride in the fermentation medium may be, for example, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any range or value between two values.

[0087] In some embodiments, the concentration of dipotassium hydrogen phosphate in the fermentation medium may be, for example, 0.1 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any range or value between two values.

[0088] In some embodiments, the concentration of potassium dihydrogen phosphate in the fermentation medium may be, for example, 0.1 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, or any range or value between two values.

[0089] In some embodiments, the concentration of magnesium sulfate heptahydrate in the fermentation medium may be, for example, 0.1 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 1.5 g / L, 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 any range or value between two values.

[0090] In some embodiments, the concentration of ferrous sulfate in the fermentation medium may be, for example, 0.01 g / L, 0.03 g / L, 0.05 g / L, 0.07 g / L, 0.09 g / L, 0.1 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 1.5 g / L, 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 any range or value between two values.

[0091] In some embodiments, the concentration of manganese sulfate in the fermentation medium may be, for example, 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, or any range or value between two values.

[0092] In some embodiments, the concentration of zinc chloride in the fermentation medium may be, for example, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, or any range or value between two values.

[0093] In some embodiments, the concentration of copper sulfate in the fermentation medium may be, for example, 0.1 g / L, 0.5 g / L, 0.8 g / L, 1 g / L, 1.5 g / L, 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 any range or value between two values.

[0094] In some embodiments, the fermentation medium in step S100 comprises: 30 g / L glucose, 5 g / L corn steep liquor, 4 g / L ammonium chloride, 2 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 2 g / L ferrous sulfate, 1 g / L manganese sulfate, 1 g / L zinc chloride and 0.5 g / L copper sulfate.

[0095] In some embodiments, the fermentation rotation speed in step S100 is 100 rpm to 600 rpm. For example, it may be 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, or any range or value between two values.

[0096] In some embodiments, in step S100, the inoculation amount of the seed solution is 5% (v / v) to 10% (v / v). For example, it may be 5% (v / v), 6% (v / v), 7% (v / v), 8% (v / v), 9% (v / v), 10% (v / v), or any range or value between two values.

[0097] In some embodiments, in step S200, when the concentration of the initial sugar in the fermentation broth is below 15 g / L, such as 15 g / L, 14 g / L, 13 g / L, 12 g / L, 11 g / L, 10 g / L, 9 g / L, 8 g / L, 7 g / L, 6 g / L, 5 g / L, 4 g / L, 3 g / L, etc., water-soluble carbon source and oil-soluble carbon source are added, and the residual sugar concentration is controlled to be 10 g / L~15 g / L.

[0098] In some embodiments, in step S200, the water-soluble carbon source and oil-soluble carbon source added are glucose and rapeseed oil, respectively. For example, the flow rate ratio of the two is 1:1 during addition.

[0099] In some embodiments, the concentration of the water-soluble carbon source added in step S200 is about 600 g / L, for example, 500 g / L to 700 g / L.

[0100] I do not wish to be limited by any theory, but I believe that if the concentration of water-soluble carbon source is too high, it will easily crystallize, while if the concentration is too low, it will result in a large volume of additional material.

[0101] In some implementations, the monitoring period for the initial sugar concentration in the fermentation broth is 6h to 24h, exemplarily 12h.

[0102] The water-soluble carbon source in this step is, for example, the same water-soluble carbon source contained in the fermentation medium in step S100, such as glucose.

[0103] In some embodiments, the method for preparing acidic sophorolipids further includes the step of preparing seed liquid S000.

[0104] For example, step S000 includes:

[0105] Candida albicans was inoculated into an activation medium and cultured at 28℃~32℃ and 180rpm~220rpm for 18 h~24 h to prepare a seed culture.

[0106] In some embodiments, the temperature for preparing the seed solution in step S000 is 28°C, 29°C, 30°C, 31°C, 32°C, or any range or value between two of these values.

[0107] In some implementations, the rotational speed in step S000 is 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, or any range or value between two values.

[0108] In some implementations, the incubation time in step S000 is 18h, 19h, 20h, 21h, 22h, 23h, 24h, or any range or value between two values.

[0109] In some embodiments, the amount of emulsifier used in step S300 is 0.1 g / L to 0.5 g / L. Exemplarily, such as 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, or any range or value between two values.

[0110] While not wanting to be limited by any theory, it is believed that when the amount of emulsifier is higher than 0.5 g / L, the emulsification effect will not be significantly enhanced, and excessive amounts may affect the separation of subsequent products; while when it is lower than 0.1 g / L, its emulsification effect is not obvious.

[0111] In some implementations, the increase in stirring speed in step S300 is 20 rpm to 50 rpm; for example, 20 rpm, 25 rpm, 30 rpm, 35 rpm, 40 rpm, 45 rpm, 50 rpm, or any range or value between two values.

[0112] In some embodiments, the emulsifier in step S300 may be a conventional emulsifier in the art, for example, Tween-80, Span-80, etc.

[0113] In some implementations, in step S300, the residual oil content in the fermentation broth is monitored periodically.

[0114] In some implementations, the detection cycle for residual oil content is 6h to 24h. For example, the residual oil content is detected every 6h, every 8h, every 10h, every 12h, every 14h, every 16h, every 18h, every 20h, every 22h, every 24h, or any value between two of these two.

[0115] In some implementations, the fermentation time is 160h to 240h. Examples include 160h, 168h, 170h, 180h, 190h, 200h, 210h, 220h, 230h, 240h, or any value between two of these.

[0116] In some embodiments, the method for preparing acidic sophorolipids further includes a product extraction step S400.

[0117] In some embodiments, step S400 includes: after fermentation, collecting the fermentation broth, separating and purifying sophorolipids by ethyl acetate extraction, analyzing the content of acidic sophorolipids and lactone-type sophorolipids, and calculating the proportion of acidic sophorolipids.

[0118] In some embodiments, a fermentation method is provided to increase the proportion of acidic sophorolipids by controlling the residual oil content during fermentation, in order to solve the problems of limited existing technologies and waste of carbon sources.

[0119] In some embodiments, the fermentation method for increasing the proportion of acidic sophorolipids by controlling the residual oil content includes the following steps:

[0120] (1) Activation of strain: Candida albicans is inoculated into activation medium and cultured at 28-32℃ and 180-220r / min for 18-24h to obtain seed liquid; the Candida albicans is, for example, Candida globulus.

[0121] (2) Culture medium preparation: Prepare fermentation culture medium, which is composed of water-soluble carbon source, nitrogen source, inorganic salt and water; the water-soluble carbon source is preferably glucose; the oil-soluble carbon source is preferably rapeseed oil.

[0122] (3) Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 5%-10%, control the fermentation temperature at 25-32℃ and dissolved oxygen at 20-60%, and monitor the residual oil content in the fermentation liquid regularly. When the residual oil content exceeds 5g / L, add 0.1-0.5g / L emulsifier and increase the stirring speed by 20-50rpm to promote oil phase emulsification and the utilization of oil by the cells. Terminate fermentation after 168h. The emulsifier is preferably Tween-80.

[0123] (4) Product extraction: After fermentation, the fermentation broth was collected and the sophorolipids were separated and purified by ethyl acetate extraction. The contents of acidic sophorolipids and lactone sophorolipids were analyzed and the proportion of acidic sophorolipids was calculated.

[0124] The following are some examples.

[0125] 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.

[0126] The main sources of the raw materials used in the following examples and comparative examples are as follows:

[0127] The main raw materials are shown in Table 1.

[0128] Table 1

[0129]

[0130] (1) Residual oil detection:

[0131] 1) Take 1-2g of fermentation broth and place it in a 15mL centrifuge tube. Weigh the fermentation broth sample as m1.

[0132] 2) Then add 3 mL of n-hexane and 1 mL of acetonitrile, and vortex until homogeneous;

[0133] 3) Then centrifuge at 5000 r / min for 10 min. Transfer the supernatant after centrifugation to a weighing pan with a pre-weighed weight m2 using a pipette. Add 3 mL of n-hexane to the lower fermentation broth and repeat the extraction twice. Combine the three extracts, evaporate to dryness, and weigh m3. Calculate the residual oil content (g / kg). Residual oil content = (m3-m2) / m1, unit g / kg.

[0134] (2) Residual sugar detection: The fermentation broth sample was centrifuged, the supernatant was diluted 50 times with water, and the sample was detected using the SBA-40C biosensor;

[0135] (3) Sophorolipid yield detection: The method for detecting sophorolipid content is the sulfuric acid-anthrone method, and the specific operation method is as follows:

[0136] 1) Preparation of anthrone reagent

[0137] Accurately weigh 0.1 g of anthrone, add 100 mL of concentrated sulfuric acid, mix well, and this is the anthrone reagent.

[0138] 2) Prepare a 1 mmol / mL anhydrous glucose solution.

[0139] Weigh a certain mass of anhydrous glucose and dry it to constant weight in a drying oven at 105℃. Weigh 0.1800 g of the constant-weight anhydrous glucose and dilute it to 100 mL in a volumetric flask, then shake well. Its concentration is 10 mmol / L. Then take 0.5, 1, 2, 3, and 4 mL of the diluted glucose solution and dilute it to 100 mL, with concentrations of 0.5, 0.1, 0.2, 0.3, and 0.4 mmol / L, respectively.

[0140] 3) Plotting a glucose standard curve using the anthrone method

[0141] 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.

[0142] The absorbance was determined using the anthrone method, and a standard curve was plotted.

[0143] y = ax + b; where y is the glucose content and x is the absorbance.

[0144] 4) Determination of sophorolipid content in fermentation broth

[0145] 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 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.

[0146] The final formula for calculating the sophorolipid content is:

[0147] Sophorolipid content = (total sugar content in reaction solution - glucose content) × dilution factor × 1.91;

[0148] The percentage of acidic sophorolipids = acidic sophorolipid content / total sophorolipids × 100.

[0149] (4) Calculate carbon source conversion rate = sophorolipid production / (glucose consumption mass + vegetable oil consumption mass), unit g / g.

[0150] Example 1

[0151] (1) Seed culture: The glycerol tube strain stored at low temperature was inoculated into 50 mL of YPD liquid medium and cultured in a constant temperature shaker at 28℃ and 180 rpm for 24 h. The inoculation amount of the strain was 1% (volume ratio: v / v).

[0152] The yeast strain mentioned above is wild-type Candida bombicola (S. bombicola) CGMCC 1576, which has been disclosed in Chinese Patent ZL200610042190 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 28, 2006, with accession number CGMCC No. 1576.

[0153] The YPD culture medium mentioned above consists of: 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone.

[0154] (2) Fermentation medium preparation: glucose 30 g / L, corn steep liquor 5 g / L, ammonium chloride 4 g / L, dipotassium hydrogen phosphate 2 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate 2 g / L, manganese sulfate 1 g / L, zinc chloride 1 g / L, copper sulfate 0.5 g / L. The concentration of the added glucose solution was 600 g / L. When adding glucose, rapeseed oil was added at the same time.

[0155] (3) Fermentation culture: Prepare a 5L fermenter, and inoculate the mature seeds cultivated in step (1) into a 3L sterile fermentation medium at an inoculation rate of 5% (v / v). The fermentation temperature is 28℃, the fermentation pH is 4, the air volume is controlled at 3L / min, and the rotation speed is 100~600rpm. The dissolved oxygen is maintained at 40% by adjusting the stirring speed and the aeration rate.

[0156] During fermentation, the residual sugar and residual oil content were measured every 12 hours. When the residual sugar concentration was consumed to 15 g / L, glucose and rapeseed oil were added. The mass ratio of added sugar to added oil was 1:1.

[0157] When the residual oil content exceeds 5 g / L, add 0.3 g / L of Tween-80 and increase the stirring speed by 30 rpm to maintain the residual oil concentration below 5 g / L during fermentation.

[0158] (4) Product extraction: Fermentation was terminated after 168 h of fermentation. The fermentation broth was collected and purified by ethyl acetate extraction. The residual oil content was 2 g / L, the proportion of acidic sophorolipids was 55%, the carbon source conversion rate was 64%, and the total amount of sophorolipids was 207 g / L.

[0159] Example 2

[0160] (1) Seed culture: The glycerol tube strain stored at low temperature was inoculated into 50 mL of YPD liquid medium and cultured in a constant temperature shaker at 30℃ and 180 rpm for 24 h. The inoculation amount of the strain was 1% (volume ratio).

[0161] The yeast strain mentioned above is wild-type Candida bombicola (S. bombicola) CGMCC 1576, which has been disclosed in Chinese Patent ZL200610042190 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 28, 2006, with accession number CGMCC No. 1576.

[0162] The YPD culture medium mentioned above consists of: 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone.

[0163] (2) Fermentation medium preparation: glucose 20 g / L, corn steep liquor 10 g / L, ammonium chloride 4 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 5 g / L, ferrous sulfate 2 g / L, manganese sulfate 1 g / L, zinc chloride 1 g / L, copper sulfate 0.5 g / L. The concentration of the added glucose solution was 600 g / L. When adding glucose, rapeseed oil was added at the same time.

[0164] (3) Fermentation culture: Prepare a 5L fermenter. Inoculate the matured seeds from step (1) into a 3L sterile fermentation medium at an inoculation rate of 8% (v / v). The fermentation temperature is 30℃, the fermentation pH is 4, the air flow rate is controlled at 3L / min, and the stirring speed is 100~600rpm. The dissolved oxygen is maintained at 40% by adjusting the stirring speed and aeration rate. During the fermentation process, the residual sugar content and residual oil content are measured every 12 hours. When the residual sugar concentration is consumed to 15g / L, glucose and rapeseed oil are added. The mass ratio of added sugar to added oil is 1:1. When the residual oil content exceeds 5g / L, 0.1g / L of Tween-80 is added, and the stirring speed is increased by 40rpm. During the fermentation process, the residual oil concentration is maintained below 5g / L.

[0165] (4) Product extraction: Fermentation was terminated after 168 h of fermentation. The fermentation broth was collected and purified by ethyl acetate extraction. The residual oil content was 4 g / L, the proportion of acidic sophorolipids was 50%, the carbon source conversion rate was 50%, and the total amount of sophorolipids was 190 g / L.

[0166] Example 3

[0167] (1) Seed culture: The glycerol tube strain stored at low temperature was inoculated into 50 mL of YPD liquid medium and cultured in a constant temperature shaker at 30℃ and 180 rpm for 24 h. The inoculation amount of the strain was 1% (volume ratio).

[0168] The yeast strain mentioned above is wild-type Candida bombicola (S. bombicola) CGMCC 1576, which has been disclosed in Chinese Patent ZL200610042190 and was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 28, 2006, with accession number CGMCC No. 1576.

[0169] The YPD culture medium mentioned above consists of: 20 g / L glucose, 10 g / L yeast extract, and 20 g / L peptone.

[0170] (2) Fermentation medium preparation: glucose 20 g / L, corn steep liquor 10 g / L, ammonium chloride 4 g / L, dipotassium hydrogen phosphate 3 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 5 g / L, ferrous sulfate 2 g / L, manganese sulfate 1 g / L, zinc chloride 1 g / L, copper sulfate 0.5 g / L. The concentration of the added glucose solution was 600 g / L. When adding glucose, rapeseed oil was added at the same time.

[0171] (3) Fermentation culture: Prepare a 5L fermenter. Inoculate the matured seeds from step (1) into a 3L sterile fermentation medium at an inoculation rate of 8% (v / v). The fermentation temperature is 30℃, the fermentation pH is 4, the air flow rate is controlled at 3L / min, and the stirring speed is 100~600rpm. The dissolved oxygen is maintained at 40% by adjusting the stirring speed and aeration rate. During the fermentation process, the residual sugar and residual oil content are measured every 12 hours. When the residual sugar concentration is consumed to 10g / L, glucose and rapeseed oil are added. The mass ratio of added sugar to added oil is 1:1. When the residual oil content exceeds 5g / L, 0.5g / L of Tween-80 is added, and the stirring speed is increased by 50rpm. During the fermentation process, the residual oil concentration is maintained below 5g / L.

[0172] Fermentation was terminated after 168 hours, and the fermentation broth was collected. Sophorolipids were separated and purified by ethyl acetate extraction. The residual oil content was 1 g / L, the proportion of acidic sophorolipids was 60%, the carbon source conversion rate was 65%, and the total amount of sophorolipids was 210 g / L.

[0173] Example 4

[0174] The operating steps are the same as in Example 1, except that the emulsifier is 0.1 g / L Span-80.

[0175] Fermentation was terminated after 168 hours, and the fermentation broth was collected. Sophorolipids were separated and purified by ethyl acetate extraction. The residual oil content was 5 g / L, the proportion of acidic sophorolipids was 52%, the carbon source conversion rate was 49%, and the total amount of sophorolipids was 186 g / L.

[0176] Example 5

[0177] The operating steps are the same as in Example 2, except that the emulsifier is 0.3 g / L Span-80.

[0178] Fermentation was terminated after 168 hours, and the fermentation broth was collected. Sophorolipids were separated and purified by ethyl acetate extraction. The residual oil content was 3 g / L, the proportion of acidic sophorolipids was 57%, the carbon source conversion rate was 63%, and the total amount of sophorolipids was 205 g / L.

[0179] Example 6

[0180] The operation steps are the same as in Example 3, except that the emulsifier is 0.5 g / L Tween, and the fermentation strain used is Candida bumblebee, strain number ATCC22214, purchased from Guangdong Institute of Microbiology.

[0181] Fermentation was terminated after 168 hours, and the fermentation broth was collected. Sophorolipids were separated and purified by ethyl acetate extraction. The residual oil content was 1 g / L, the proportion of acidic sophorolipids was 60%, the carbon source conversion rate was 64.6%, and the total amount of sophorolipids was 208 g / L.

[0182] Comparative Example 1

[0183] The operation steps are the same as in Example 1, except that the residual oil content is not controlled. When the residual oil content is higher than 5g / L, no emulsifier is added. At the end of fermentation, the residual oil content is relatively high.

[0184] Fermentation was terminated after 168 hours, and the fermentation broth was collected. Sophorolipids were separated and purified by ethyl acetate extraction. The residual oil content was determined to be 15 g / L, the proportion of acidic sophorolipids was 30%, the carbon source conversion rate was 40%, and the total amount of sophorolipids was 220 g / L.

[0185] Comparative Example 2

[0186] The operation steps are the same as in Example 1, except that the residual oil content is not controlled. When the residual oil content is higher than 5g / L, no emulsifier is added. At the end of fermentation, the residual oil content is relatively high.

[0187] Fermentation was terminated after 168 hours, and the fermentation broth was collected. Sophorolipids were separated and purified by ethyl acetate extraction. The residual oil content was 10 g / L, the proportion of acidic sophorolipids was 40%, the carbon source conversion rate was 42%, and the total amount of sophorolipids was 210 g / L.

[0188] For the effects of different residual oil contents in the fermentation system on the proportion of acidic sophorolipids and carbon source conversion rate in the fermentation products in the above examples and comparative examples, please refer to [link to relevant documentation]. Figure 1 .

[0189] Comparative Example 3

[0190] The operating steps were the same as in Example 1, except that the strain used was an acidic strain. The original strain of this strain was deposited at the China General Microbiological Culture Collection Center (CGMCC No. 1576). It was obtained by genetic modification, knocking out the lactonease gene, and screening. During the fermentation process, different residual oil concentrations were controlled, and the experimental results are shown in Table 2 below.

[0191] Table 2. Proportion of acidic sophorolipids at different residual oil concentrations controlled by acidic strains

[0192]

[0193] Table 2 shows that during the fermentation of acidic sophoroliposide using acidic strains, the residual oil concentration does not affect the acidic content of the final sophoroliposide.

[0194] The comparison results of the above embodiments and comparative examples clearly show that the fermentation method of the present invention, by controlling the residual oil content, has significant advantages in reducing residual oil content, improving carbon source conversion rate, and increasing the proportion of acidic sophorolipids.

[0195] 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.

[0196] 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 for preparing acidic sophorolipids, characterized in that, The method includes: Fermentation culture: The seed culture of Candida albicans was inoculated into the fermentation medium for fermentation. The fermentation temperature was 25℃~32℃, and the total dissolved oxygen relative value of the fermentation broth was maintained at 20%~60%. Supplementing water-soluble and oil-soluble carbon sources: When the initial sugar concentration in the fermentation broth is less than or equal to 15 g / L, supplementing water-soluble and oil-soluble carbon sources, and controlling the residual sugar concentration to be 10 g / L~15 g / L; Add emulsifier: When the residual oil content in the fermentation broth is greater than or equal to 5 g / L, add emulsifier and increase the stirring speed to continue fermentation. The fermentation medium contains water-soluble carbon source, nitrogen source, inorganic salts and water.

2. The method for preparing acidic sophorolipids according to claim 1, characterized in that, The Candida species include one or more of the following: *S. bombicola*, *Candida bombicola*, and *Wickerhamiella versatilis*. Optionally, the *Candida globosa* species is *Candida globosa* with accession number CGMCC 1576; Optionally, the bumblebee Candida is the bumblebee Candida species with accession number ATCC 22214; Optionally, the Candida albicans strain is not an acidic strain.

3. The method for preparing acidic sophorolipids according to claim 1, characterized in that, The fermentation medium conforms to one or more of the items shown in A1) to A4) below: A1) The water-soluble carbon source includes sugar; the sugar may optionally include at least one of glucose and molasses; A2) The oil-soluble carbon source includes at least one of soybean oil, rapeseed oil, peanut oil, palm oil, corn oil, and sunflower oil; A3) The nitrogen source selected includes at least one of yeast extract, peptone, urea, corn steep liquor, ammonium sulfate, and ammonium chloride; A4) The inorganic salts include at least one of potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate heptahydrate, zinc chloride, ferrous sulfate, copper sulfate, and manganese sulfate.

4. The method for preparing acidic sophorolipids according to claim 3, characterized in that, The fermentation medium contains: 20 g / L to 40 g / L glucose, 5 g / L to 20 g / L corn steep liquor, 1 g / L to 10 g / L ammonium chloride, 0.1 g / L to 10 g / L dipotassium hydrogen phosphate, 0.1 g / L to 10 g / L potassium dihydrogen phosphate, 0.1 g / L to 5 g / L magnesium sulfate heptahydrate, 0.01 g / L to 5 g / L ferrous sulfate, 0.01 g / L to 1 g / L manganese sulfate, 0.1 g / L to 1 g / L zinc chloride, and 0.1 g / L to 5 g / L copper sulfate.

5. The method for preparing acidic sophorolipids according to claim 1, characterized in that, The fermentation speed is 100 rpm to 600 rpm.

6. The method for preparing acidic sophorolipids according to claim 1, characterized in that, In the fermentation culture step, the inoculation amount of the seed liquid is 5% (v / v) to 10% (v / v). Optionally, the method for preparing the seed liquid includes: Candida albicans was inoculated into an activation medium and cultured at 28℃~32℃ and 180 rpm~220 rpm for 18 h~24 h to prepare a seed culture.

7. The method for preparing acidic sophorolipids according to claim 1, characterized in that, The step of adding emulsifier conforms to one or more of the items shown in B1) to B4) below: B1) The amount of the emulsifier used is 0.1 g / L to 0.5 g / L; B2) The stirring speed can be increased by 20 rpm to 50 rpm; B3) The emulsifier includes at least one of Tween-80 and Span-80; B4) Regularly monitor the residual oil content in the fermentation broth.

8. The method for preparing acidic sophorolipids according to claim 7, characterized in that, In B4, the detection cycle for residual oil content is 6h~24h.

9. The method for preparing acidic sophorolipids according to any one of claims 1 to 8, characterized in that, The fermentation time is 160h~240h.

10. The method for preparing acidic sophorolipids according to any one of claims 1 to 8, characterized in that, The method also includes a product extraction step; Optionally, the product extraction steps include: after fermentation, collecting the fermentation broth, separating and purifying sophorolipids by ethyl acetate extraction, analyzing the content of acidic sophorolipids and lactone-type sophorolipids, and calculating the proportion of acidic sophorolipids.

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