A method for preparing sophorolipids with an open fermentation process

Abstract

The present application relates to the field of microbial fermentation, and discloses a method for preparing sophorolipids by using an open fermentation process, which comprises the following steps: placing a fermentation medium in a fermentation tank, inoculating Candida utilis at a 10% (v / v) inoculation amount to perform fermentation, wherein the 10% (v / v) inoculation amount is the volume percentage of seed liquid to fermentation liquid, the fermentation temperature is kept at 25-30 DEG C, the rotation speed is 300-800 rpm, and the aeration amount is 0.4-1.0 vvm; after the fermentation starts, 120-150 g / L of plant oil and 120-180 g / L of glucose are added in batches; and during the whole fermentation cycle, no sterilization operation is performed on the fermentation medium, the fermentation tank and the supplementary materials. The present application has high fermentation efficiency and simplified fermentation operation, and can effectively solve the problems of increased production cost and possible deterioration of the fermentation medium caused by sterilization operation.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, and specifically relates to a method for preparing sophorolipids using an open fermentation process. Background Technology

[0002] Sophorolipids, a type of glycolipid nonionic biosurfactant, are generally obtained through batch or continuous microbial fermentation. Compared with traditional chemically synthesized surfactants, sophorolipids meet the current development needs of green chemistry, with advantages including, but not limited to: 1. Superior surface interfacial activity and emulsifying properties; 2. Green and safe production process; 3. Raw materials are generally oil-soluble carbon sources such as vegetable oils and water-soluble carbon sources such as glucose, both of which are renewable resources, effectively reducing fossil fuel consumption and carbon dioxide emissions; 4. Sophorolipids are low in toxicity, easily biodegradable, and have good environmental compatibility; 5. Excellent resistance to high salt and high temperatures. Currently, sophorolipids, as a representative of green and efficient biosurfactants, are gradually gaining widespread application in industries such as daily chemicals, environmental remediation, food and pharmaceuticals, and oil extraction. However, the production cost of sophorolipids is higher than that of nonionic chemically synthesized surfactants with similar performance, which restricts the large-scale application of sophorolipid biosurfactants in various industries.

[0003] Generally, batch or continuous fermentation of sophorolipids requires sterilization of the fermentation substrate, culture medium, and fermentation tank using dry or wet heat methods to ensure that the fermentation medium and equipment are in a sterile state and to avoid interference from other microorganisms. Sterilization not only consumes a large amount of heat energy, increases fermentation time, and significantly increases the cost of sophorolipid fermentation, but also the high temperature and pressure during sterilization may cause the culture medium, such as glucose, to deteriorate, increasing the risk of fermentation failure.

[0004] Sophorolipids, as a secondary metabolite of *Candida*, maintain a stable pH of around 3.0 in the late logarithmic phase of fermentation. This acidic environment is unfavorable for the survival of most other microorganisms. Secondly, in the early stages of sophorolipid fermentation, increasing the concentration of readily available water-soluble carbon sources such as glucose and fructose enhances the growth rate of *Candida*. Glucose content is typically as high as 150 g / L. This high sugar content significantly increases the osmotic pressure of the culture medium, leading to growth inhibition of microbial cells due to water loss in the high osmotic pressure environment. However, *Candida*, found in chicory nectar and bumblebee honey, possesses a natural advantage in tolerating high sugar levels. Furthermore, sophorolipid biosurfactants themselves have good antibacterial properties, effectively inhibiting the growth of bacteria such as *Escherichia coli*, *Staphylococcus aureus*, *Bacillus subtilis*, and *Pseudomonas aeruginosa*. Finally, high concentrations of vegetable oils and oleic acid, as fermentation substrates, also have some bactericidal effects, but these can be utilized and metabolized by yeast. Therefore, the aforementioned characteristics of Candida albicans and its fermentation substrate make it possible to produce it through open fermentation.

[0005] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0006] To address or at least alleviate one or more of the above problems, a method for preparing sophorolipids using an open fermentation process is provided. This method offers high fermentation efficiency and simplified fermentation operations, effectively solving the problems of increased production costs due to sterilization operations and potential deterioration of the fermentation medium.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a method for preparing sophorolipids using an open fermentation process is provided, comprising the following steps: Fermentation culture: Place fermentation medium in a fermenter and inoculate with Candida albicans at an inoculation rate of 10% (v / v). The 10% (v / v) inoculation rate is the percentage of seed liquid volume to fermentation liquid volume. Maintain the fermentation temperature at 25℃~30℃, the rotation speed at 300~800 rpm, and the aeration rate at 0.4~1.0 vvm. Feeding: After fermentation begins, add 120-150 g / L of vegetable oil and 120-180 g / L of glucose in batches; during the complete fermentation cycle, the fermentation medium, the fermenter, and the feeding are not sterilized.

[0008] Preferably, the method further includes preparing a seed solution: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 25-30 ℃ and 160-220 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed culture that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued for 24 h under the conditions of (1) to obtain the secondary seed culture.

[0009] Preferably, the seed culture medium comprises: 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose, with water as the solvent, and sterilized at 115°C for 30 min.

[0010] Preferably, the fermentation medium comprises: 2.5 g / L yeast powder, 5 g / L sodium citrate, 4 g / L magnesium sulfate, 2 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.1 g / L sodium chloride, 0.1 g / L anhydrous calcium chloride, and water as the solvent.

[0011] Preferably, the hydrophobic carbon source is vegetable oil, and the hydrophilic carbon source is glucose.

[0012] Preferably, the vegetable oil is at least one of soybean oil, peanut oil, rapeseed oil, corn oil, olive oil, cottonseed oil, and oleic acid.

[0013] Preferably, a first portion of glucose solution is added during the fermentation culture, and a second portion of glucose solution is added during the feeding process, wherein the amounts of the first portion and the second portion are equal.

[0014] Preferably, the feeding is carried out in four stages, at 16h, 40h, 64h and 88h after the start of fermentation.

[0015] Preferably, it also includes semi-continuous fermentation: when the batch fermentation reaches the peak yield, 20% of the fermentation broth is removed, and an equal amount of fresh fermentation medium is added, along with 30g of glucose and 24g of vegetable oil, marking this as the first cycle. The fermentation broth is transferred every 24 hours, and the cycle number is incremented by one. This process is repeated until the average lipid production rate within the cycle is lower than the batch fermentation lipid production rate, at which point fermentation ends. The average lipid production rate of the batch fermentation refers to the total lipid production of the entire batch fermentation process divided by the total fermentation time.

[0016] Preferably, the method further includes the steps of extraction, centrifugation and separation from the fermentation broth to obtain sophorolipids.

[0017] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art: This invention omits the sterilization step before fermentation, avoiding culture medium deterioration under high temperature and pressure, improving fermentation efficiency, and significantly reducing the production cost of sophorolipids. The inoculum size is increased from 5% (v / v) to 10% (v / v). The increased number of strains, combined with the natural acid and osmotic tolerance of Candida albicans, prevents contamination by other microorganisms during fermentation, ensuring a high yield of sophorolipids. These two advantages will promote the industrial production and commercial application of sophorolipids.

[0018] This invention utilizes the unique biological characteristics of Candida albicans and its fermentation system to construct a natural barrier against contaminating microorganisms, making it possible to produce sophorolipids through open fermentation. This completely simplifies the fermentation equipment and operating procedures. By adopting an open fermentation process, the production cost of sophorolipids is significantly reduced. At the same time, the simplified process lowers the operating threshold and labor costs, thereby significantly reducing the unit production cost of sophorolipids and effectively improving the efficiency of sophorolipid fermentation production.

[0019] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0020] The accompanying drawings, as part of this invention, are provided to further illustrate the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation thereof. Clearly, the drawings described below are merely some embodiments; those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0021] In the attached diagram: Figure 1 This is a schematic diagram of the process for preparing sophorolipids using an open fermentation process in this specific embodiment. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0023] In this invention, references to numerical ranges, such as numerical values, percentages, proportions, or ratios, unless otherwise stated, should be understood as meaning that the range is considered continuous, and its disclosure includes not only the two explicitly stated endpoint values, i.e., the minimum and maximum values, but also all intermediate values ​​between these two endpoints. When the range is explicitly limited to containing only integers, it is considered to directly and individually enumerate every integer within the range, including the two endpoint integers. Any numerical range disclosed in this invention encompasses any and all sub-intervals within it. If multiple ranges describing the same feature are provided in this invention, these ranges can be combined with each other.

[0024] Please see Figure 1 , Figure 1 This is a flowchart illustrating the open fermentation process for preparing sophorolipids, where gray areas represent areas that do not require sterilization, and red areas represent areas that require sterilization or filtration. This invention provides a method for preparing sophorolipids using an open fermentation process, comprising the following steps: Fermentation culture: Place fermentation medium in a fermenter, add 120~180g / L glucose solution, and inoculate with Candida albicans at an inoculation rate of 10% (v / v). The 10% (v / v) inoculation rate is the percentage of seed liquid volume to fermentation liquid volume. Maintain the fermentation temperature at 25℃~30℃, the rotation speed at 300rpm~800rpm, and the aeration rate at 0.4~1.0vvm. The gas introduced into the fermenter passes through a 0.22 μm filter membrane to remove airborne bacteria, etc. Feeding: After fermentation begins, add 120-150 g / L of hydrophobic carbon source and 120-180 g / L of hydrophilic carbon source in batches; wherein, during fermentation culture and feeding, the fermentation culture medium, the fermenter and the feeding are not sterilized.

[0025] In some embodiments, the preparation of seed solution is also included: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 25-30 ℃ and 160-220 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed culture that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued for 24 h under the conditions of (1) to obtain the secondary seed culture.

[0026] In some embodiments, the seed culture medium comprises: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, water as the solvent, and sterilized at 115°C for 30 min.

[0027] Specifically, the seed culture medium is prepared by adding 10g of yeast powder, 20g of peptone, and 20g of glucose to every 1L of tap water and sterilizing at 115℃ for 30min.

[0028] In some embodiments, the fermentation medium comprises: 2.5 g / L yeast extract, 5 g / L sodium citrate, 4 g / L magnesium sulfate, 2 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.1 g / L sodium chloride, 0.1 g / L anhydrous calcium chloride, and water as the solvent.

[0029] Specifically, the fermentation medium is as follows: 2.5g of yeast powder, 5.0g of sodium citrate, 4.0g of magnesium sulfate, 2.0g of ammonium sulfate, 1.0g of potassium dihydrogen phosphate, 0.1g of sodium chloride, and 0.1g of anhydrous calcium chloride are added to every 1L of tap water, without sterilization.

[0030] In some implementations, the hydrophobic carbon source is vegetable oil, and the hydrophilic carbon source is glucose.

[0031] In some embodiments, the vegetable oil is at least one of soybean oil, peanut oil, rapeseed oil, corn oil, olive oil, cottonseed oil, and oleic acid.

[0032] Specifically, the vegetable oil is at least one of soybean oil, oleic acid, or waste cooking oil.

[0033] In some implementations, a first portion of glucose solution is added during fermentation culture, and a second portion of glucose solution is added during feeding, with the amounts of the first and second portions being equal.

[0034] Specifically, the first part is half of the total glucose addition, and the second part is the other half. The total glucose addition refers to the total amount of glucose added throughout the entire fermentation process, including initial addition and feed addition. The initial addition and feed addition are equal.

[0035] In some implementations, feeding is carried out in four stages, at 16h, 40h, 64h and 88h after the start of fermentation.

[0036] In some implementations, the amount of glucose added each time is 30g, 37.5g, 45g, 60g, or any range or value between two of these values.

[0037] In some embodiments, semi-continuous fermentation is also included: when the batch fermentation reaches the peak yield, 20% of the fermentation broth is removed, and an equal amount of fresh fermentation medium is added, along with 30g of glucose and 24g of vegetable oil, marking the first cycle. The fermentation broth is transferred every 24 hours, and the cycle number is incremented by one. This process is repeated until the average lipid production rate within the cycle is lower than the batch fermentation lipid production rate, at which point fermentation ends. The average lipid production rate of the batch fermentation refers to the total lipid production of the entire batch fermentation process divided by the total fermentation time.

[0038] In some implementations, 60g of glucose and 48g of vegetable oil are fed each cycle.

[0039] It should be noted that "peak yield" refers to the time point when the concentration of sophorolipids reaches its highest value, which can be determined by periodic sampling and testing; "lipid production rate" refers to the rate of sophorolipid formation (g / h) within each cycle; and "average lipid production rate of batch fermentation" refers to the total lipid production of the entire batch fermentation process divided by the total fermentation time.

[0040] Example 1: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 30 ℃ and 180 rpm to obtain the first-stage seed culture.

[0041] (2) In 50 ml of sterilized seed culture medium, 2.5 ml of primary seed liquid was inoculated at a 5% inoculation rate and cultured at 30℃ and 180 rpm for 24 h to obtain secondary seed liquid, which is the seed fermentation broth used for fermentation. (3) Add 2L of fermentation medium to a 5L fermenter, and immediately add half of the total amount of 120g / L glucose. Add the secondary seed liquid to the fermenter at an inoculation rate of 10% (v / v) for fermentation. Control the temperature at 30℃, the aeration rate at 1.0vvm, the rotation speed at 400rpm, and the fermentation time at about 150h. (4) Add 120g / L soybean oil in batches at 16, 40, 64 and 88 hours after the start of fermentation, with 60g added each time; add the remaining glucose in batches at 120g / L at 16, 40, 64 and 88 hours after the start of fermentation, with 30g added each time. (5) Sophorolipids were separated from the fermentation broth by extraction, centrifugation and other operations, with a fermentation yield of 160 g / L.

[0042] Example 2: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 30 ℃ and 180 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed liquid that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued at 30℃ and 180 rpm for 24 h to obtain the secondary seed liquid, which is the seed fermentation liquid used for fermentation. (3) Add 2L of fermentation medium to a 5L fermenter, and immediately add half of the total amount of 150g / L glucose. Add the secondary seed liquid to the fermenter at an inoculation rate of 10% (v / v) for fermentation. Control the temperature at 30℃, the aeration rate at 1.0vvm, the rotation speed at 400rpm, and the fermentation time at about 150h. (4) Add 120 g / L of oleic acid in batches at 16, 40, 64 and 88 h after the start of fermentation, with 60 g added each time; add the remaining glucose in batches at 150 g / L at 16, 40, 64 and 88 h after the start of fermentation, with 45 g added each time. (5) When the yield of sophorolipid is the highest (144h), semi-continuous open fermentation is started. 400ml of 20% (v / v) (volume percentage of total fermentation liquid) fermentation liquid is removed from the fermentation system, and 400ml of 20% (v / v) (volume percentage of total fermentation liquid) fresh fermentation medium is added. At the same time, 60g of glucose and 48g of vegetable oil are added respectively. This is recorded as the first cycle. (6) At 168h, 400ml of 20% (v / v) fermentation broth was removed from the fermentation system and 400ml of 20% (v / v) fresh fermentation medium was added. At the same time, 60g of glucose and 48g of oleic acid were added respectively. This was recorded as the second cycle. (7) Transfer the fermentation broth every 24 hours and increment the cycle number by one, as shown in (6), until the average lipid production rate in that cycle is lower than the batch fermentation lipid production rate (1.86 g / h), which is considered the end of fermentation. (8) Fermentation was completed in the 386th hour, i.e. the tenth cycle, with a lipid production rate of 2.06 g / h, which is 11% higher than the traditional batch fermentation yield. The unit cost of sophorolipid was 187 yuan / kg, which is 17% lower than the intermittent process cost of 226 yuan / kg.

[0043] Example 3: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 30 ℃ and 180 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed liquid that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued at 30℃ and 180 rpm for 24 h to obtain the secondary seed liquid, which is the seed fermentation liquid used for fermentation. (3) Add 2L of fermentation medium to a 5L fermenter, and immediately add half of the total amount of 150g / L glucose. Add the secondary seed liquid to the fermenter at an inoculation rate of 10% (v / v) for fermentation. Control the temperature at 30℃, the aeration rate at 1.0vvm, the rotation speed at 400rpm, and the fermentation time at about 144h. (4) Add 120 g / L of oleic acid in batches at 16, 40, 64 and 88 h after the start of fermentation, with 60 g added each time; add the remaining glucose in batches at 150 g / L at 16, 40, 64 and 88 h after the start of fermentation, with 45 g added each time. (5) Sophorolipids were separated from the fermentation broth by extraction, centrifugation and other operations, with a fermentation yield of 134 g / L.

[0044] Example 4: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 30 ℃ and 180 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed liquid that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued at 30℃ and 180 rpm for 24 h to obtain the secondary seed liquid, which is the seed fermentation liquid used for fermentation. (3) Add 2L of fermentation medium to a 5L fermenter, and immediately add half of the total amount of 150g / L glucose. Add the secondary seed liquid to the fermenter at an inoculation rate of 10% (v / v) for fermentation. Control the temperature at 30℃, the aeration rate at 1.0vvm, the rotation speed at 450rpm, and the fermentation time at about 144h. (4) Add 120g / L of vegetable oil in batches at 16, 40, 64 and 88 hours after the start of fermentation, with 60g added each time; add the remaining glucose in batches at 150g / L at 16, 40, 64 and 88 hours after the start of fermentation, with 45g added each time. (5) Sophorolipids were separated from the fermentation broth by extraction, centrifugation and other operations, with a fermentation yield of 130 g / L.

[0045] Example 5: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 30 ℃ and 180 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed liquid that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued at 30℃ and 180 rpm for 24 h to obtain the secondary seed liquid, which is the seed fermentation liquid used for fermentation. (3) Add 2L of fermentation medium to a 5L fermenter, and immediately add half of the total amount of 150g / L glucose. Add the secondary seed liquid to the fermenter at an inoculation rate of 10% (v / v) for fermentation. Control the temperature at 30℃, the aeration rate at 1.0vvm, the rotation speed at 350rpm, and the fermentation time at about 144h. (4) Add 120g / L of vegetable oil in batches at 16, 40, 64 and 88 hours after the start of fermentation, with 60g added each time; add the remaining glucose in batches at 150g / L at 16, 40, 64 and 88 hours after the start of fermentation, with 45g added each time. (5) Sophorolipids were separated from the fermentation broth by extraction, centrifugation and other operations, with a fermentation yield of 90 g / L.

[0046] Example 6: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 30 ℃ and 180 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed liquid that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued at 30℃ and 180 rpm for 24 h to obtain the secondary seed liquid, which is the seed fermentation liquid used for fermentation. (3) Add 2L of fermentation medium to a 5L fermenter, and after sterilization, cool to 30℃. Immediately add half of the total amount of 150g / L glucose. Add the secondary seed liquid to the fermenter at an inoculation rate of 5% (v / v) for fermentation. Control the temperature at 30℃, the aeration rate at 1.0vvm, the rotation speed at 400rpm, and the fermentation time at about 160h. (4) Add 120g / L of vegetable oil in batches at 16, 40, 64 and 88 hours after the start of fermentation, with 60g added each time; add the remaining glucose in batches at 150g / L at 16, 40, 64 and 88 hours after the start of fermentation, with 37.5g added each time. (5) Sophorolipids were separated from the fermentation broth by extraction, centrifugation and other operations, with a fermentation yield of 144.5 g / L.

[0047] Example 7: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 30 ℃ and 180 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed liquid that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued at 30℃ and 180 rpm for 24 h to obtain the secondary seed liquid, which is the seed fermentation liquid used for fermentation. (3) Add 2L of fermentation medium to a 5L fermenter, and immediately add half of the total amount of glucose (180g / L). Add the secondary seed liquid to the fermenter at an inoculation rate of 10% (v / v) for fermentation. Control the temperature at 30℃, the aeration rate at 1.0vvm, the rotation speed at 400rpm, and the fermentation time at about 144h. (4) Add 120g / L of vegetable oil in batches at 16, 40, 64 and 88 hours after the start of fermentation, with 60g added each time; add the remaining glucose in batches at 150g / L at 16, 40, 64 and 88 hours after the start of fermentation, with 45g added each time. (5) Sophorolipids were separated from the fermentation broth by extraction, centrifugation and other operations, with a fermentation yield of 125.8 g / L.

[0048] Example 8: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 30 ℃ and 180 rpm to obtain the first-stage seed culture. (2) In 50 ml of sterilized seed culture medium, 2.5 ml of the primary seed liquid that has been shaken and cultured was inoculated at a 5% inoculation rate. The culture was continued at 30℃ and 180 rpm for 24 h to obtain the secondary seed liquid, which is the seed fermentation liquid used for fermentation. (3) Add 2L of fermentation medium to a 5L fermenter, and immediately add half of the total amount of 150g / L glucose. Add the secondary seed liquid to the fermenter at an inoculation rate of 10% (v / v) for fermentation. Control the temperature at 30℃, the aeration rate at 1.0vvm, the rotation speed at 400rpm, and the fermentation time at about 144h. (4) Add 120g / L of kitchen waste oil in batches at 16, 40, 64 and 88 hours after the start of fermentation, with 60g added each time; add the remaining glucose in batches at 150g / L at 16, 40, 64 and 88 hours after the start of fermentation, with 37.5g added each time. (5) Sophorolipids were separated from the fermentation broth by extraction, centrifugation and other operations, with a fermentation yield of 132.3 g / L.

[0049] To demonstrate the effectiveness and operability of open fermentation for sophorolipids, Example 6 was added to compare the yield difference between open fermentation and strict fermentation with Example 3.

[0050] The fermentation conditions in Example 3 and Example 6 were the same except for whether sterilization was included and the inoculum size.

[0051] The inoculum size of 10% (v / v) in Example 3 is higher than that of 5% (v / v) in Example 6. This is an optimized result that enables Candida albicans to have a survival advantage in open fermentation.

[0052] In Example 6, strict aseptic principles were followed; all equipment, instruments, culture media, and feed were sterilized before use. Except for Example 6, in the other examples, the fermenters, culture media, and feed were not sterilized during the fermentation process.

[0053] The comparison of sophorolipid yields in Examples 3 and 6 shows that the yield of sophorolipids under open fermentation conditions is essentially the same as that under strict fermentation conditions.

[0054] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. The implementation schemes in the above embodiments can also be further combined or replaced. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing sophorolipids using an open fermentation process, characterized in that, Includes the following steps: Fermentation culture: Fermentation medium is placed in a fermenter, and Candida albicans is inoculated at an inoculum rate of 10% (v / v) for fermentation. The 10% (v / v) inoculum rate is the percentage of the seed liquid volume to the total fermentation liquid volume. The total fermentation liquid volume is the sum of the fermentation medium volume and the seed liquid volume. The fermentation temperature is maintained at 25℃~30℃, the rotation speed is 300rpm~800rpm, and the aeration rate is 0.4~1.0vvm. Feeding: After fermentation begins, add 120-150 g / L of hydrophobic carbon source and 120-180 g / L of hydrophilic carbon source in batches; wherein, during fermentation culture and feeding, the fermentation culture medium, the fermenter and the feeding are not sterilized.

2. The method according to claim 1, characterized in that, It also includes the preparation of seed liquid: (1) Inoculate one loopful of Candida albicans into 5 ml of sterilized seed culture medium and culture it in a shaking incubator for 24 h at 25-30 ℃ and 160-220 rpm to obtain the first-stage seed culture. (2) In a sterilized 50ml seed culture medium, 2.5ml of primary seed liquid was inoculated at a 5% inoculation rate. The culture was continued for 24h under the conditions of (1) to obtain secondary seed liquid, which is the seed fermentation liquid used for fermentation.

3. The method according to claim 2, characterized in that, The seed culture medium contains: 10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose, and the remainder is water, and is sterilized at 115°C for 30 min.

4. The method according to claim 1, characterized in that, The fermentation medium comprises: 2.5 g / L yeast extract, 5 g / L sodium citrate, 4 g / L magnesium sulfate, 2 g / L ammonium sulfate, 1 g / L potassium dihydrogen phosphate, 0.1 g / L sodium chloride, 0.1 g / L anhydrous calcium chloride, and water as the solvent.

5. The method according to claim 1, characterized in that, The hydrophobic carbon source is vegetable oil, and the hydrophilic carbon source is glucose.

6. The method according to claim 5, characterized in that, The vegetable oil is at least one of soybean oil, peanut oil, rapeseed oil, corn oil, olive oil, cottonseed oil, and oleic acid.

7. The method according to claim 5, characterized in that, A first portion of glucose is added during the fermentation culture, and a second portion of glucose is added during the feeding. The amounts of the first and second portions are the same.

8. The method according to claim 1, characterized in that, The feeding was carried out in four stages, at 16h, 40h, 64h and 88h after the start of fermentation.

9. The method according to claim 6, characterized in that, It also includes semi-continuous fermentation: when the batch fermentation reaches the peak yield, 20% of the fermentation broth is removed, and an equal amount of fresh fermentation medium is added, along with 30g of glucose and 24g of vegetable oil. This is recorded as the first cycle. Fermentation broth is transferred every 24 hours, and the cycle number is incremented by one. This operation is repeated until the average lipid production rate within the cycle is lower than the batch fermentation lipid production rate, at which point fermentation ends. The average lipid production rate of the batch fermentation refers to the total lipid production of the entire batch fermentation process divided by the total fermentation time.

10. The method according to claim 1, characterized in that, It also includes the steps of extraction, centrifugation and separation from the fermentation broth to obtain sophorolipids.

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