CN112029808B - Semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation - Google Patents
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Abstract
The invention discloses a semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation, which comprises the following steps: culturing seeds; a sophorolipid fermentation step; wherein, in the sophorolipid fermentation step, fermentation liquor is subjected to ultrasonic treatment to assist the sedimentation of sophorolipid. The semi-continuous fermentation method of the invention introduces ultrasonic waves in the sophorolipid fermentation process to accelerate the sedimentation rate of sophorolipid and reduce the loss of in-situ separation substrates and cells. The method can be helpful for the sedimentation of the sophorolipid in an environment with a low proportion of oil, not only can reduce the influence of the oil on the viscosity and the like of the fermentation liquor, but also can reduce the loss of the oil in the in-situ separation process and accelerate the separation of the sophorolipid.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to a semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation.
Background
Sophorolipids (SLs) are the most promising biosurfactants, have the characteristics of low toxicity, biodegradability and the like, and are widely applied to foods, cosmetics, pharmacy and petroleumAnd (4) industry. It has been reported that various microorganisms can synthesize sophorolipids, such as Candida, wickerhaniella, pichia, etc. Among them, candida bombicola is one of the strains which has the highest ability to synthesize sophorolipid at present and has been industrially used. The structure of sophorolipid is mainly composed of two hydrophilic groups composed of glucose and a hydrophobic group of fatty acid chain with 16-18 carbon, which form acid sophorolipid by beta-glycosidic bond, as shown in figure 1A. And the terminal carboxyl group of the acid type sophorolipid and the terminal hydroxyl group of the hydrophilic group 4' are subjected to esterification reaction under the action of lactonase to form lactone type sophorolipid, as shown in figure 1B. In addition, sophorolipids are also classified into nonacetylated, monoacetylated and bisacetylated products according to acetylation occurring at glucose C6' and C6 ″, and the structure and characteristics of sophorolipids are affected by the chain length, saturation degree and the like of fatty acids. Sophorolipids have been shown to be a mixture of more than 20 different structures. In the fermentation process, the sophorolipids with different structures have different properties, lactone sophorolipids have poor water solubility and are easy to form crystals in fermentation liquor, and acid sophorolipids have good water solubility. Therefore, the sophorolipid fermentation is a complex fermentation system containing solid crystals, oil, bacteria and other phases. Continuing with reference to FIGS. 1A and 1B, non-acetylated (R) 1 ,R 2 = OH), monoacetylation (R) 1 ＝-OH，R 2 ＝-OCH 3 ) And bis-acetylation (R) 1 ,R 2 ＝-OCH 3 )。
The biosurfactant has the advantages of environmental protection, good biocompatibility and the like, so that the biosurfactant has a very wide market. While the sophorolipid-producing strain is a nonpathogenic yeast and is safer than rhamnolipid (produced by Pseudomonas aeruginosa). However, sophorolipids are less competitive than chemical surfactants due to higher production costs. The sophorolipid and grease accumulated in the fermentation process can influence the rheological property and mass transfer mixing of the fermentation liquor, and the synthesis of sophorolipid at the later stage of fermentation is influenced. Therefore, semi-continuous fermentation becomes an important means for improving the yield of sophorolipid and reducing the cost. At present, semi-continuous fermentation is mainly based on the layering of sophorolipid and fermentation liquor, then sophorolipid is recovered, fermentation liquor supernatant is recycled for separation, and the method is the main method for improving the yield of sophorolipid and reducing the cost at present. Since the sedimentation of sophorolipid is the key point in the in-situ separation, how to accelerate the sedimentation rate of sophorolipid has important significance.
Therefore, it is highly desirable to provide a semi-continuous fermentation method based on ultrasonic-assisted sophorolipid precipitation, which accelerates the in-situ separation of sophorolipid by introducing ultrasonic assistance, reduces the loss of bacteria, and thus realizes efficient and rapid semi-continuous fermentation of sophorolipid based on in-situ separation.
Disclosure of Invention
The invention aims to provide a semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation, ultrasonic treatment is introduced in the sophorolipid fermentation process to separate sophorolipid, so that the sedimentation rate of sophorolipid can be accelerated in an environment with a low oil proportion, and meanwhile, the loss of in-situ separation substrates and cells is reduced.
In order to achieve the purpose, the invention adopts the following technical scheme.
The invention provides a semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation, which comprises the following steps: culturing seeds; a step of sophorolipid fermentation; wherein, in the fermentation step of the sophorolipid, the fermentation liquor is subjected to ultrasonic treatment to assist the sedimentation of the sophorolipid.
Further, the step of fermentation of sophorolipid comprises: inoculating the strain after seed culture to a fermentation medium, wherein the inoculation amount is 2.9% (OD) 600 80), the fermentation temperature was 25 ℃, the aeration was 0.5vvm, the initial rotation speed was 200rpm, the fermentation was carried out in a fermentor, wherein during the fermentation, the oil was fed into the fermentation broth at a rate of 3g/h, and the pH of the fermentation broth was maintained at 3.5.
Further, in the sophorolipid fermentation process, the ultrasonic treatment step includes: transferring the fermentation liquor to a separation device arranged on an ultrasonic platform, carrying out ultrasonic treatment on the fermentation liquor for 10min at the ultrasonic power of 100-300W to settle sophorolipid, separating out the settled sophorolipid, supplementing the supernatant to a fermentation tank, and continuing to carry out sophorolipid fermentation until the fermentation is finished.
Further, in the fermentation process of the sophorolipid, when the concentration of the sophorolipid product in the fermentation liquid reaches 100g/L, the fermentation liquid is subjected to ultrasonic treatment to separate the sophorolipid.
Further, in the sophorolipid fermentation process, the Dissolved Oxygen (DO) of the fermentation liquor is controlled by adjusting the rotating speed, the Dissolved Oxygen (DO) is maintained to be more than 40% within 0-36h, and the Dissolved Oxygen (DO) is maintained to be more than 25% after 36 h.
Further, in the ultrasonic treatment, the weight ratio (g/g) of the oil and fat to the sophorolipid in the fermentation solution is 0.10 to 0.20. Preferably, the weight ratio (g/g) of the oil and fat to the sophorolipid is 0.10 to 0.16.
Further, the grease is rapeseed oil.
Further, in the sophorolipid fermentation step, the concentration of glucose in the fermentation broth was maintained at 30 to 80g/L.
Further, the step of seed culture comprises: adding the strain into a seed culture medium, and shaking the flask for culturing for 48h under the conditions that the rotating speed is 200rpm and the temperature is 25 ℃.
Further, the seed medium comprises: 50g/L glucose, 1g/L KH 2 PO 4 4g/L of (NH) 4 ) 2 SO 4 0.5g/L MgSO 4 ·7H 2 O and 10g/L corn steep liquor. The seed culture medium is sterilized by high-temperature high-pressure steam at 115 ℃ for 30min.
Further, the fermentation medium comprises: 100g/L glucose, 1g/L KH 2 PO 4 4g/L of (NH) 4 ) 2 SO 4 0.5g/L MgSO 4 ·7H 2 O and 10g/L corn steep liquor. The fermentation medium is sterilized by high-temperature high-pressure steam at 115 ℃ for 30min.
In the present invention, the semi-continuous fermentation method used the C.bombicola ATCC 22214 strain for fermentation production of sophorolipid, the C.bombicola ATCC 22214 strain was purchased from Guangzhou city culture collection and 20% of the glycerol tubes were stored at-80 ℃.
The invention has the beneficial effects that:
the invention provides a semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation, wherein ultrasonic waves are introduced in the sophorolipid fermentation step to accelerate the sedimentation rate of sophorolipid and reduce the loss of in-situ separation substrates and cells. Ultrasonic wave not only can accelerate the gathering of sophorolipid granule, makes the tiny particle become bigger granule, and then accelerates to subside, and ultrasonic wave can remove the bubble fast moreover to reduce the bubble and rise the interference to sophorolipid settlement in-process. The semi-continuous fermentation method provided by the invention can be beneficial to the sedimentation of sophorolipid in an environment with a low oil proportion, not only can reduce the influence of oil on the viscosity of a fermentation solution and the like, but also can reduce the loss of oil in the in-situ separation process and accelerate the separation of sophorolipid.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1A is a chemical structure diagram of acid type sophorolipid.
FIG. 1B is a chemical structure diagram of lactone-type sophorolipid.
FIG. 2 is a schematic diagram of the apparatus for the fermentation step of sophorolipid in the semi-continuous fermentation method of the present invention.
FIG. 3A is a graph showing the effect of ultrasonic waves on the growth of the strain in test example 1.
FIG. 3B is a graph showing data on the effect of ultrasonic waves on sophorolipid production in test example 1.
FIG. 4A is a graph showing the sedimentation rate of sophorolipid assisted by ultrasonic waves at 100W ultrasonic power in test example 2.
FIG. 4B is a graph showing the sedimentation rate of sophorolipid assisted by ultrasonic waves at 200W in test example 2.
FIG. 4C is a graph showing the sedimentation rate of sophorolipid under ultrasonic power of 300W in test example 2.
FIG. 4D is a graph showing the maximum sedimentation rate under different power conditions (0 to 300W) in test example 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention. It is to be noted that the term "comprising" is used herein to mean "including but not limited to". The terms first, second, third and the like are used merely as labels, and do not impose numerical requirements or an order of establishment. Various embodiments of the invention may exist in a range of versions; it should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention; accordingly, the described range descriptions should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, it is contemplated that the description of a range from 1 to 6 has specifically disclosed sub-ranges such as, for example, from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within a range such as, for example, 1, 2, 3, 4, 5, and 6, as applicable regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any number (fractional or integer) recited within the indicated range. The sizes and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Conversely, unless otherwise indicated, various sizes are intended to indicate both the recited value and a range that is functionally equivalent to the recited value. For example, a disclosed size of "10 microns" refers to "about 10 microns".
The embodiment of the invention provides a semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation, which comprises the following steps: culturing seeds; a sophorolipid fermentation step; wherein, in the fermentation step of the sophorolipid, the fermentation liquor is subjected to ultrasonic treatment to assist the sedimentation of the sophorolipid.
In this example, the semi-continuous fermentation process used C.bombicola ATCC 22214 strain for the production of sophorolipid by fermentation, C.bombicola ATCC 22214 was purchased from Guangzhou, inc. and 20% of the glycerol tubes were stored at-80 ℃.
In this embodiment, the fermentation of sophorolipid comprises: inoculating the strain after seed culture to a fermentation medium, wherein the inoculation amount is 2.9% (OD) 600 80), the fermentation temperature is 25 ℃, the aeration is 0.5vvm, the initial rotating speed is 200rpm, and the fermentation is carried out in a fermentation tank, wherein in the fermentation process, grease is fed into the fermentation liquor at the speed of 3g/h, and the pH value of the fermentation liquor is maintained at 3.5.
In this embodiment, referring to fig. 2, in the fermentation process of sophorolipid, the ultrasonic treatment step includes: transferring the fermentation liquid in the fermentation tank to a separation device arranged on an ultrasonic platform, carrying out ultrasonic treatment on the fermentation liquid for 3-10 min at the ultrasonic power of 100-300W to settle the sophorolipid, separating the settled sophorolipid to obtain crude sophorolipid, then replenishing the supernatant in the separation device to the fermentation tank, and continuing to carry out sophorolipid fermentation until the fermentation is finished.
In some embodiments, during the fermentation of sophorolipid, the fermentation broth is sonicated after the concentration of sophorolipid product in the fermentation broth reaches 100g/L to isolate sophorolipid.
In some embodiments, during the fermentation of the sophorolipid, the Dissolved Oxygen (DO) in the fermentation liquid is controlled by adjusting the rotating speed, the Dissolved Oxygen (DO) is maintained above 40% within 0-36h, and the Dissolved Oxygen (DO) is maintained above 25% after 36 h.
In some embodiments, in the ultrasonic treatment, the concentration or yield of the sophorolipid in the fermentation tank can be monitored on line, and the weight ratio (Oil/SLs) of the Oil to the sophorolipid in the fermentation liquid is controlled to be 0.10 to 0.20g/g. For example, the weight ratio of the oil to the sophorolipid may be 0.10, 0.12, 0.14, 0.16, 0.18 or 0.20g/g.
In some embodiments, the grease may be rapeseed oil.
In some embodiments, the concentration of glucose in the fermentation broth is maintained between 30 and 80g/L during the fermentation.
In some embodiments, the step of seed culturing comprises: adding the strain into a seed culture medium, and shaking the flask for culturing for 48h under the conditions that the rotating speed is 200rpm and the temperature is 25 ℃.
In some embodiments, the seed medium comprises: 50g/L glucose, 1g/L KH 2 PO 4 4g/L of (NH) 4 ) 2 SO 4 0.5g/L MgSO 4 ·7H 2 O and 10g/L corn steep liquor. The seed culture medium is sterilized by high-temperature high-pressure steam at 115 ℃ for 30min.
In some embodiments, the fermentation medium comprises: 100g/L glucose, 1g/L KH 2 PO 4 4g/L of (NH) 4 ) 2 SO 4 0.5g/L MgSO 4 ·7H 2 O and 10g/L corn steep liquor. The fermentation medium is sterilized by high-temperature high-pressure steam at 115 ℃ for 30min.
In the invention, the sophorolipid content is gradually increased along with the increase of the fermentation period, and the sophorolipid is settled under a certain oil proportion. The average sedimentation rate is shown in formula (1); it was further found that the sophorolipid sedimentation height is proportional to the sophorolipid concentration, as shown in formula (2); the final calculation formula for the sedimentation rate of sophorolipid is shown in formula (3):
(1)
(2)
(3)
wherein in the formula
Represents the average sedimentation velocity (cm/s), H, of sophorolipid 1 Represents the height (cm) of the fermentation broth in a 10mL settling tube, H 2 Represents the sophorolipid height (cm) in a 10mL settling tube, t represents the settling time(s), c represents the sophorolipid concentration (g/L), and a and b represent the fitting coefficients, respectively.
Example 1:
the present example provides a semi-continuous fermentation method based on ultrasonic-assisted sophorolipid precipitation, which comprises a step of seed culture and a step of sophorolipid fermentation, as shown below.
In this example, seed culture: in a 1L baffled shake flask, 200mL of medium was added and incubated for 48h on a shaker at 200rpm and 25 ℃ respectively.
In this example, sophorolipid fermentation: in a 5L fermenter, the initial volume was 2.5L and the inoculum size was 2.9% (OD) 600 80), fermenting; the fermentation temperature is 25 ℃, the ventilation volume is 0.5vvm, and the initial rotating speed is 200rpm; the Dissolved Oxygen (DO) is maintained above 40% by adjusting the rotation speed for 0-36h, and is maintained above 25% after 36 h. The pH was maintained at 3.5 during the culture by 4M NaOH adjustment. The fed-batch fermentation period was 168h, rapeseed oil (3 g/h) was fed to the fermentation broth, and the glucose concentration was maintained at 30g/L to 80g/L.
In the semi-continuous fermentation process of sophorolipid in this example, the ratio of oil (rapeseed oil) to sophorolipid is controlled to be 0.10 (g/g), so as to realize rational sedimentation of sophorolipid, please refer to fig. 2: when the concentration of the sophorolipid product in the fermentation tank reaches 100g/L, rapidly pumping the fermentation liquor in the fermentation tank into a separation device from a port a through a peristaltic pump, simultaneously placing the separation device into an ultrasonic machine, wherein the ultrasonic power is 100W, the ultrasonic time is 10min, separating sophorolipid from a port c after 1min of pause, and replenishing the supernatant into the fermentation tank from a port b again for continuous fermentation; repeating the above sophorolipid separation step until the sophorolipid concentration in the fermenter is below 60 g/L.
In this embodiment, in the ultrasonic treatment, the concentration or yield of sophorolipid in the fermentation tank can be monitored on line, so as to better regulate and control the ratio of oil and fat to sophorolipid.
Comparative example 1:
comparative example 1 differs from example 1 only in that: the ultrasonic power in comparative example 1 was 200W, and the other conditions were exactly the same as in example 1.
Comparative example 2:
comparative example 2 differs from example 1 only in that: the ultrasonic power in comparative example 2 was 300W, and the other conditions were exactly the same as in example 1.
Test example 1:
this test example examined the growth of the strain and the production of sophorolipid in the semi-continuous fermentation method of the present invention to observe the effect of ultrasonic waves on the growth and production of the strain.
The method comprises the following steps: taking a proper amount of fermentation liquor obtained in the example 1, the comparative example 1 and the comparative example 2, carrying out ultrasonic treatment at different ultrasonic powers (such as 100W, 200W and 300W) and ultrasonic times (such as 0min, 10min, 20 min, 30min, 40 min, 50 min and 60 min), diluting the treated fermentation liquor to a proper concentration, coating a flat plate, and observing the growth condition of the strain. Meanwhile, the fermentation liquor after ultrasonic treatment is centrifuged, and thalli are inoculated into a culture medium only containing glucose and grease for synthesizing sophorolipid, and the production conditions of ultrasonic strains and non-ultrasonic strains are compared. The results are shown in detail in FIGS. 3A and 3B.
FIG. 3A is a graph of data on the effect of ultrasound on strain growth.
FIG. 3B is a data plot of the effect of ultrasound on sophorolipid production.
As can be seen from FIG. 3, the growth and production of the strain were not affected after the strain was subjected to different ultrasonic powers and ultrasonic times during the fermentation process.
Test example 2:
(1) in this test example, sophorolipid precipitation in the semicontinuous fermentation method was subjected to rate measurement, and the precipitation rate was observed and test data was recorded.
The method comprises the following steps: the same semi-continuous fermentation method as in example 1 was used, in this test example, ultrasonic waves were used to assist the sedimentation of sophorolipid under the condition that the mass ratio (g/g) of oil (rapeseed oil) to sophorolipid was 0.10 to 0.22, and the sedimentation rate of sophorolipid was examined; wherein the power of the ultrasonic wave is 100W, 200W and 300W respectively, and the ultrasonic treatment time is 0s, 10s, 30s, 60s, 120s and 180s respectively. The experimental data were recorded and shown in detail in fig. 4A, 4B and 4C.
FIG. 4A is a graph of ultrasonic-assisted sophorolipid sedimentation rate at 100W ultrasonic power.
FIG. 4B is a graph of ultrasonic-assisted sophorolipid sedimentation rate at 200W ultrasonic power.
FIG. 4C is a graph of ultrasonic-assisted sophorolipid sedimentation rate at 300W ultrasonic power.
As can be seen from fig. 4A, 4B and 4C, the sedimentation rate of sophorolipid increases with the increase of the ultrasonic treatment time; on the other hand, along with the increase of the ultrasonic power, the sedimentation rate of sophorolipid is also obviously increased. However, the sedimentation rate of sophorolipid has a maximum value under different oil sophorolipid proportions, and the maximum value is not influenced by the ultrasonic time and power. In addition, compared with the method without ultrasonic treatment, the method can obviously improve the sedimentation rate of sophorolipid after ultrasonic treatment; can realize high-efficiency sophorolipid separation under the condition of lower proportion of oil sophorolipid, and further can reduce the loss of cells, glucose and grease.
(2) In order to understand the maximum sedimentation rate under the assistance of ultrasonic waves at different powers in the semi-continuous fermentation method, the ultrasonic waves were used to assist the sedimentation of sophorolipid at a mass ratio (g/g) of oil and fat (rapeseed oil) to sophorolipid of 0.10-0.22, wherein the ultrasonic wave powers were 0W, 100W, 200W, and 300W, respectively, and the other conditions were the same as those in the semi-continuous fermentation method of example 1. The test data was recorded and shown in detail in figure 4D.
FIG. 4D is a graph of maximum sedimentation rate at different powers (0-300W).
As can be seen from fig. 4D, in comparison with the ultrasonic power of 0W (i.e. blank control), the ultrasonic wave has different effects on the sophorolipid sedimentation rate of different oil sophorolipid ratios, the oil ratio is from high to low, and the ultrasonic wave increases the sedimentation rate by 46.9% to 485.41%. Therefore, ultrasonic waves can effectively assist the sedimentation of sophorolipid, and the sedimentation rate of sophorolipid can be higher under the condition of lower oil content.
(3) Statistics were carried out on the average loss rate and separation efficiency of cells, glucose and lipids in the semi-continuous fermentation method for ultrasonic-assisted sophorolipid precipitation described above, and the average loss rate and separation efficiency of cells, glucose and lipids in the semi-continuous fermentation method for precipitating sophorolipid without ultrasonic treatment (blank control test) are shown in table 1.
TABLE 1
Wherein the content of the first and second substances,
as can be seen from the data in table 1, the ultrasonic-assisted sedimentation reduces the specific losses of cells, glucose and oil by 68.2%, 16.2% and 65.5%, respectively, improves the in-situ separation efficiency by 25.7%, has a significant effect of ultrasonic-assisted sophorolipid sedimentation in the sophorolipid fermentation step, can improve the sedimentation rate of sophorolipid under the condition of a low oil-to-fat ratio, i.e., can improve the sophorolipid separation efficiency, and can also reduce the cell loss, glucose loss and oil loss in the fermentation broth. Therefore, the ultrasonic-assisted in-situ separation can realize efficient sophorolipid separation and lower loss rate in a complex sophorolipid fermentation system.
In summary, although the sophorolipid can be settled by adjusting the proportion of oil and fat in the fermentation liquid, a part of cells, glucose and oil and fat are usually carried out along with the separation of sophorolipid in the in-situ separation process. For example, when the proportion of the oil sophorolipid is controlled to be 1:4, although sophorolipid can settle faster, more oil is lost; and the oil-fat ratio is less than 3: at 20, although the loss of grease can be reduced, the settling rate is also slowed. However, according to FIG. 4, for example, when the ratio of the oil and fat to the sophorolipid is 0.10g/g, the sedimentation rate reaches 0.0165cm/s at the maximum. Therefore, the ultrasonic wave is introduced in the sophorolipid fermentation process, so that the sedimentation rate of sophorolipid can be increased under the condition of low oil proportion, the loss of in-situ separation substrate and cells is reduced, and the beneficial effect is obvious.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation is characterized by comprising the following steps:
culturing seeds;
a step of sophorolipid fermentation;
wherein the step of seed culture employs a strain of C.bombicola deposited as ATCC No. 22214;
the sophorolipid fermentation step comprises the following steps:
inoculating the strain subjected to seed culture into a fermentation medium, wherein the inoculation amount is 2.9%, the fermentation temperature is 25 ℃, the ventilation amount is 0.5vvm, the initial rotation speed is 200rpm, and fermenting is carried out in a fermentation tank, wherein in the fermentation process, grease is fed into the fermentation liquor at the speed of 3g/h, and the pH of the fermentation liquor is maintained to be 3.5;
the fermentation medium comprises: 100g/L glucose, 1g/L KH 2 PO 4 4g/L of (NH) 4 ) 2 SO 4 0.5g/L MgSO 4 ·7H 2 O and 10g/L corn steep liquor; sterilizing the fermentation culture medium by high-temperature high-pressure steam at 115 ℃ for 30min;
in the fermentation process of the sophorolipid, when the concentration of the sophorolipid product in the fermentation liquid reaches 100g/L, carrying out ultrasonic treatment on the fermentation liquid to assist the sophorolipid to settle and separate the sophorolipid; at the beginning of the ultrasonic treatment, the weight ratio of the oil and fat to the sophorolipid in the fermentation liquid is (0.10-0.20 g) to 1g;
the step of ultrasonic treatment comprises: and transferring the fermentation liquor to a separation device arranged on an ultrasonic platform, carrying out ultrasonic treatment on the fermentation liquor for 3-10 min at the ultrasonic power of 100-300W to settle the sophorolipid, separating out the settled sophorolipid, supplementing the supernatant to a fermentation tank, and continuing to carry out sophorolipid fermentation until the fermentation is finished.
2. The method of claim 1, wherein the dissolved oxygen in the fermentation broth is controlled by adjusting the rotation speed during the fermentation of sophorolipid, wherein the dissolved oxygen is maintained at 40% or more for 0-36h, and the dissolved oxygen is maintained at 25% or more after 36 h.
3. The semi-continuous fermentation method based on ultrasonic-assisted sophorolipid precipitation as claimed in claim 1, wherein the concentration of glucose in the fermentation broth is maintained at 30 to 80g/L in the sophorolipid fermentation step.
4. The semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation according to claim 1, wherein the step of seed culture comprises: adding the strain into a seed culture medium, and shaking the flask for culturing for 48h under the conditions that the rotating speed is 200rpm and the temperature is 25 ℃.
5. The semi-continuous fermentation method based on ultrasonic-assisted sophorolipid sedimentation according to claim 4, wherein the seed culture medium comprises: 50g/L glucose, 1g/L KH 2 PO 4 4g/L of (NH) 4 ) 2 SO 4 0.5g/L MgSO 4 ·7H 2 O and 10g/L corn steep liquor; the seed culture medium is sterilized by high-temperature high-pressure steam at 115 ℃ for 30min.
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