A method for preparing a schizochytrium spore

Abstract

The application provides a preparation method of a schizochytrium sp. protoplast. The preparation method of the schizochytrium sp. protoplast comprises the following steps: fermenting and culturing schizochytrium to obtain a fermentation liquor; a first fermentation stage: the temperature is 26-28 DEG C, and the culturing time is 36-40 h; a second fermentation stage: the temperature is 30-32 DEG C; and no sugar is additionally supplemented in the process of the fermentation culture. A large amount of protoplasts can be obtained by controlling the culturing conditions in the fermentation culture stage.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, more particularly, to a preparation method of Schizochytrium protoplast. BACKGROUND

[0002] Schizochytrium, also known as Schizochytrium aggregatum, belongs to a kind of marine fungi of Thraustochytriales, is single-cell, spherical, and the diameter is about 15 μm. Because it can accumulate high safety and high purity oil in cells, it is a representative strain for producing omega-3 PUFAs. As a non-model organism, the genetic characteristics and genomics of Schizochytrium are less studied. Because there is no stable additional plasmid, when performing genetic modification, the target gene needs to be integrated into the host chromosome for homologous recombination. The methods for introducing the target gene into the host include calcium ion method, protoplast method and electroporation method. Among them, the protoplast refers to a viable cell surrounded by a plasma membrane after removing the cell wall, which can take in genetic material such as cell nucleus, organelle genome or exogenous DNA fragment, and is a very good transient expression system. Through the preparation of protoplast, the genetic transformation of Schizochytrium can be facilitated, and technical support is provided for obtaining high-yield omega-3 PUFAs by using protoplast fusion technology.

[0003] So far, the successful transformation of cells has been realized by using protoplast as the material in many algal substances. However, the cell wall structure of Schizochytrium is tight and tough, and contains a large amount of oil wrapped by liposomes in the inside, and the preparation scheme and technology of protoplast are rarely reported. In the prior art, it is shown that the protoplast can be prepared by adding additional enzymes, for example, in the patent CN104694402A, the protoplasts of Schizochytrium and Aspergillus niger are prepared by the method of protoplast fusion, the protoplasts of the two parents are fused, and after further screening and culture, a Schizochytrium strain capable of producing DHA by using starch as a carbon source is successfully obtained. However, it can be seen that a large number of required enzymes are needed for wall breaking pretreatment, and different enzyme hydrolysis reaction conditions need to be designed, including temperature, pH, enzyme hydrolysis time, etc. The method is complicated and time-consuming. SUMMARY

[0004] The first purpose of the present application is to provide a preparation method of Schizochytrium protoplast. The present application is found in a very accidental research that a large amount of protoplast can be obtained by controlling the culture conditions during the fermentation culture stage. The protoplast obtained by using the preparation method of the present application is almost indistinguishable from the protoplast obtained by using the external enzyme hydrolysis method to break the wall.

[0005] The preparation method of Schizochytrium protoplast provided by the present application comprises the following steps: fermenting and culturing a Schizochytrium strain to obtain a fermentation broth;

[0006] The fermentation culture comprises:

[0007] The first fermentation stage is at a temperature of 26-28℃, and the culture time is 36-40h;

[0008] The second fermentation stage is at a temperature of 30-32℃;

[0009] During the fermentation culture, no additional sugar is added.

[0010] The present application can obtain a large number of Schizochytrium sp. protoplasts by precisely controlling the fermentation parameters in the fermentation culture.

[0011] The method provided by the present application is suitable for Schizochytrium, especially CABIO-A-2-IV, with a preservation number of CCTCC M2021565. The preservation unit is: China Center for Type Culture Collection; the preservation address is: Wuhan University, Wuhan, China, with a postcode of 430072; and the preservation date is May 19, 2021. The strain has been disclosed in Chinese patent application CN202111476958.4.

[0012] In a preferred embodiment of the present application, in the first fermentation stage, the aeration amount is 1.5-2vvm, and the pH is controlled at 6.5±0.5; in the second fermentation stage, the aeration amount is 0.8-1vvm, and the pH is controlled at 8.5±0.5. By controlling the above parameters within the above range, the preparation effect can be better.

[0013] In addition to controlling the aeration amount and the pH value, in another preferred embodiment of the present application, by controlling the rotation speed in the first fermentation stage and the second fermentation stage, the purpose of further optimizing the preparation effect can also be achieved. That is, the rotation speed in the first fermentation stage is controlled at 200-250rpm, and the rotation speed in the second fermentation stage is controlled at 150-180rpm. In the present application, under the condition of ensuring the temperature control of the first fermentation stage and the second fermentation stage in the core invention point and not adding sugar, controlling the aeration amount, the pH value in the two fermentation stages and controlling the rotation speed of the two fermentation stages are two schemes for optimizing the preparation effect. The former is suitable for fermentation tank preparation with parameters easy to control, and the latter is especially suitable for shake flask preparation examples in which the aeration amount and the pH are difficult to control. Meanwhile, in the preparation examples of the fermentation tank, the two methods can be combined without reducing the implementation effect.

[0014] In the present application, the fermentation medium can use the fermentation medium commonly used in the art, and different from the conventional fermentation culture, the present application does not need to supplement sugar in the fermentation process. In the present application, the first fermentation stage and the second fermentation stage are to distinguish the parameter control stage, and the first fermentation stage and the second fermentation stage are carried out in the same fermentation medium. In a preferred embodiment of the present application, the fermentation medium includes 5-10% carbon source, 1-5% sodium glutamate, 0.5-2% organic nitrogen source, 0.1-1% KH2PO4, 0.1-1% MgSO4 and 1-5% Na2SO4; the pH is natural. Among them, the organic carbon source can include but is not limited to one or more of glucose, fructose, glycerol, and in a specific embodiment, the organic carbon source is selected from glucose. The organic nitrogen source includes but is not limited to one or more of yeast extract, yeast extract, yeast powder, and peptone, and in a specific embodiment, the organic nitrogen source is selected from yeast extract.

[0015] In a preferred embodiment of the present application, the total fermentation time (i.e. including the culture time of the first fermentation stage and the culture time of the second fermentation stage) is preferably 90-98h.

[0016] In the embodiments of the present application, when preparing the Schizochytrium sp. protoplast using the method of the present application, the step of activation and / or seed expansion culture can be added before the fermentation culture according to the fermentation scale. In a preferred embodiment of the present application, the OD before the fermentation culture is preferably 12-17. That is, in a preferred embodiment, the Schizochytrium sp. strain can be inoculated into the activation and / or seed expansion culture medium for culture, so that the OD is 12-17, and then the fermentation culture is carried out to obtain the fermentation broth. The seed expansion culture can also include primary culture, secondary culture, etc., so that the initial bacterial concentration matches the fermentation scale and meets the needs of the present application when the fermentation culture is carried out. In the present application, the activation and / or seed expansion culture medium can be selected from the commonly used culture medium in the art.

[0017] In the embodiments of the present application, the activation and / or seed expansion culture medium can include:

[0018] 2-6% glucose, 2-8% sodium glutamate, 0.6-1.5% yeast extract, 0.5-1% KH2PO4, 0.5-1.2% MgSO4, 0.5-3% NaCl, 0.01-0.1% CaCl2, pH natural. In the present application, the culture medium (including the fermentation medium, the activation and / or seed expansion culture medium) is usually a liquid medium, i.e. in addition to the effective ingredients, the rest is water. The skilled person in the art can also add agar to the activation and / or seed expansion culture medium to make a solid culture medium according to the needs. Among them, the temperature of the activation and / or seed liquid culture is preferably 28-32°C.

[0019] In a preferred embodiment of the present application, the step of centrifuging the fermentation broth obtained from the fermentation culture, taking the lower layer, washing with buffer HPBS, and diluting and resuspending to obtain a suspension is further included.

[0020] In the buffer HPBS, the pH is preferably 5.8, and the buffer HPBS is a high-osmotic buffer solution, which refers to the addition of 0.7 mol / L KCl to 0.2 mol / L PBS.

[0021] In the present application, unless otherwise specified, "%" is mass percent.

[0022] Another object of the present application is to provide the Schizochytrium sp. protoplast obtained by the above preparation method.

[0023] The present application is based on the accidental discovery that a large number of cells of Schizochytrium sp. present a protoplast-like state (i.e., cells without obvious cell wall wrapping) at the late fermentation stage. By comparing the protoplasts obtained by the exogenous enzymatic method, there is almost no obvious difference. Through this phenomenon, it is speculated that, on the one hand, the cells are in a state of nutrient deficiency (the main components of the fungal cell wall are chitin, cellulose, glucan, and mannan, which are all polysaccharides of monosaccharides, and can also be used as nutrients by cells in the absence of nutrients), and on the other hand, part of the cells reach the senescence stage and undergo autolysis, resulting in the release of enzymes in the cells. Under the dual action of these two aspects, the cell wall is lysed, thereby forming protoplasts. At the same time, the present application surprisingly found that these protoplasts can maintain a certain activity. Based on this innovative discovery, the applicant found that a large number of protoplasts can be obtained by controlling the fermentation culture conditions of Schizochytrium sp., and the obtained protoplasts are almost indistinguishable from the protoplasts obtained by the exogenous enzymatic method. The method provided by the present application can replace the traditional and tedious exogenous enzyme method. At the same time, the protoplasts obtained by the method provided by the present application are washed, resuspended, and regenerated, and the survival rate is more than half. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The figure is the state of Schizochytrium sp. at different fermentation stages (left: early stage, right: late stage) under a microscope (water sheet) in Example 1.

[0025] Figure 2 The figure is the bromophenol blue staining state of Schizochytrium sp. at different fermentation stages (left: early stage, right: late stage) under a microscope (fixed sheet) in Example 1. DETAILED DESCRIPTION

[0026] The specific embodiments of the present application will be further described in detail below in conjunction with examples. The following examples are used to illustrate the present application, but are not intended to limit the scope of the present application.

[0027] Example 1

[0028] (1) The preservation number of Schizochytrium sp. CABIO-A-2-IV is CCTCC M 2021565.

[0029] (2) The strain is inoculated into a primary activation medium and cultured at a temperature of 28°C and a shaking speed of 220 r / min for 40 h to obtain a primary activation liquid.

[0030] (3) The primary activation liquid obtained in step (2) is inoculated into a secondary activation medium at an inoculation amount of 2.5%, and cultured at a temperature of 28°C and a speed of 220 r / min for 30 h to obtain a secondary activation liquid.

[0031] (4) Preparation of protoplasts in a fermentation tank: the secondary activation liquid obtained in step (3) is inoculated into a fermentation medium at a liquid loading amount of 60% and an inoculation amount of 5%, and cultured at a temperature of 28°C for 40 h, with a ventilation amount of 2 vvm and pH controlled at 6.5±0.5. After 40 h, the temperature is raised to 30°C, the ventilation amount is reduced to 1 vvm, and pH is controlled at 8.5±0.5. The stirring speed is maintained at 200 rpm, and the total fermentation culture time is 96 h to obtain a fermentation liquid containing protoplasts.

[0032] The activation medium (the primary activation medium and the secondary activation medium) comprises glucose 4%, sodium glutamate 3%, yeast extract 0.6%, KH2PO4 0.6%, MgSO4 0.8%, NaCl 2%, CaCl2 0.03%, and agar 2%, with a natural pH.

[0033] The fermentation medium comprises glucose 8%, sodium glutamate 2%, yeast extract 0.5%, KH2PO4 0.3%, MgSO4 0.4%, and Na2SO4 3%, with a natural pH.

[0034] (5) The fermentation liquid obtained in step (4) is centrifuged (at 4000 rpm), and the supernatant is removed, washed twice with a hypertonic buffer HPBS (pH=5.8, 0.2 mol / L PBS added with 0.7 mol / L KCl), and resuspended with HPBS to obtain a suspension.

[0035] Effect detection of preparation of protoplasts:

[0036] Figure 1 The state of Schizochytrium sp. under a microscope (water sheet) in different fermentation periods in this example is shown, in which the left picture is the early stage (40 h of fermentation), and the right picture is the later stage (end of fermentation). As can be seen from the picture, one clear cell can be seen in the early stage, and the cell boundary is blurred in the later stage.

[0037] The protoplasts appear in the late stage of culture, when the nutrients are consumed, a small amount of cells die and lyse, at this time the supernatant is no longer clear after centrifugation of the fermentation broth, and the absorbance value OD600 of the prepared suspension begins to decrease, which is used as the standard for obtaining the protoplasts. In the test, the OD600 of the prepared suspension was detected every 6h until 72h of fermentation, and the change in the absorbance value of the suspension was used as the basis for judging the amount of protoplasts obtained, and the final fermentation time was determined to be 96h. The culture time in the following examples is also determined by this method.

[0038] The suspension after the OD600 begins to decrease is subjected to microscopic examination, and the results show that there are a large number of protoplasts with transparent color around the cells relative to the untreated Schizochytrium cells, and no obvious cell wall is observed, as shown in Figure 1 .

[0039] After staining with bromophenol blue, the staining state is observed. Among them, Figure 2 The bromophenol blue staining state (fixed piece) of the Schizochytrium under a microscope at different fermentation periods in this example is shown, wherein the left picture is the early stage (40h of fermentation), and the right picture is the late stage (end of fermentation). It can be seen from the picture that the cells can be effectively stained in the early stage due to the integrity of the cell wall, and the cells cannot be effectively stained in the late stage, indicating that the cell wall is broken in the late stage.

[0040] The protoplasts are counted using a hemocytometer, and the protoplast release rate is calculated to be 90%; the wall breaking condition and the protoplast state are observed, and the protoplast viability is detected after staining with trypan blue (dead cells are stained blue): the protoplast viability is 85%.

[0041] Example 2

[0042] (1) The preservation number of the Schizochytrium sp. CABIO-A-2-IV described in the present application is CCTCC M 2021565.

[0043] (2) The strain is inoculated into a primary activation medium for culture, the culture temperature is 28°C, the shaking speed of the shaking table is 220r / min, and the culture is performed for 40h to obtain a primary activation liquid.

[0044] (3) The primary activation liquid obtained in step (2) is inoculated into a secondary activation medium, the inoculation amount is 2.5%, the culture temperature is 28°C, the speed is 220r / min, and the culture is performed for 30h to obtain a secondary activation liquid.

[0045] (4) Shake flask fermentation to prepare protoplasts: the secondary activation liquid obtained in step (3) was inoculated into fermentation medium, the liquid loading amount was 30%, the inoculation amount was 5%, the culture temperature was 28°C before 40h, the rotation speed was 250rpm, the temperature was increased to 30°C after 40h, the rotation speed was adjusted to 180rpm, the total fermentation culture time was 92h, and the fermentation liquid containing protoplasts was obtained.

[0046] The activation medium (primary and secondary activation medium) comprises: glucose 4%, sodium glutamate 3%, yeast extract 0.6%, KH2PO4 0.6%, MgSO4 0.8%, NaCl 2%, CaCl2 0.03%, agar 2%, and the pH is natural.

[0047] The fermentation medium comprises: glucose 8%, sodium glutamate 2%, yeast extract 0.5%, KH2PO4 0.3%, MgSO4 0.4%, Na2SO4 3%, and the pH is natural.

[0048] (5) The fermentation liquid obtained in step (4) was centrifuged (4000rpm), the supernatant was removed, the protoplasts were washed twice with high-osmotic buffer HPBS (pH=5.8, 0.2mol / L PBS added with 0.7mol / L KCl), and the protoplasts were resuspended and diluted with HPBS to obtain a suspension.

[0049] After bromophenol blue staining, the protoplasts were counted using a hemocytometer, the protoplast release rate was calculated to be 93%; the protoplast state and the wall breaking condition were observed, and the protoplast viability was detected after trypan blue staining; the protoplast production rate was 65%.

[0050] Example 3

[0051] The method provided in this example is basically the same as that in example 1, and the only difference is that:

[0052] (4) Fermentor preparation of protoplasts: the secondary activation liquid obtained in step (3) was inoculated into fermentation medium, the liquid loading amount was 60%, the inoculation amount was 5%, the culture temperature was 26°C before 40h, the aeration amount was 1.5vvm, the pH was controlled at 6.5±0.5, the temperature was increased to 30°C after 36h, the aeration amount was reduced to 0.8vvm, the pH was controlled at 8.5±0.5, the stirring speed was maintained at 200rpm, the total fermentation culture time was 98h, and the fermentation liquid containing protoplasts was obtained.

[0053] After bromophenol blue staining, the protoplasts were counted using a hemocytometer, the protoplast release rate was calculated to be 92%; the protoplast state and the wall breaking condition were observed, and the protoplast viability was detected after trypan blue staining; the protoplast viability was 83%.

[0054] Example 4

[0055] The method provided by this embodiment is basically the same as that provided by embodiment 1, and the difference is only that:

[0056] (4) Preparation of protoplasts in a fermenter: the secondary activation solution obtained in step (3) is inoculated into a fermentation medium, the liquid loading amount is 60%, the inoculation amount is 5%, the culture temperature is 28°C, the aeration amount is 1.5vvm, the pH is controlled at 6.5±0.5, the temperature is increased to 32°C after 36h, the aeration amount is reduced to 0.8vvm, the pH is controlled at 8.5±0.5, the stirring speed is maintained at 200rpm, the total fermentation culture time is 90h, and a fermentation solution containing protoplasts is obtained.

[0057] After bromophenol blue staining, the protoplasts are counted using a hemocytometer, and the protoplast release rate is calculated to be 93%; the wall breaking condition and the protoplast state are observed, and after trypan blue staining, the protoplast viability is detected; the protoplast viability is 76%.

[0058] Comparative Example 1

[0059] The preparation method of the protoplasts of the Schizochytrium provided in this comparative example is the same as that in embodiment 1, and the difference is only that:

[0060] (4) Preparation of protoplasts in a fermenter: the secondary activation solution obtained in step (3) is inoculated into a fermentation medium, the liquid loading amount is 60%, the inoculation amount is 5%, the culture temperature is 30°C, the pH is maintained at 7-8 during the culture, the aeration amount is 2vvm, the stirring speed is maintained at 200rpm, 5% of glucose is supplemented after 48h of fermentation, and the total fermentation culture time is 120h, which is the conventional Schizochytrium culture time.

[0061] Result: After bromophenol blue staining, the staining state is observed, the protoplasts are counted using a hemocytometer, and the protoplast release rate is calculated to be 20%; since the release rate is low, the subsequent viability detection is not performed.

[0062] Comparative Example 2

[0063] The preparation method of the protoplasts of the Schizochytrium provided in this comparative example is the same as that in embodiment 1, and the difference is only that:

[0064] (4) 5% of glucose is supplemented after 48h of fermentation, and the total fermentation culture time is 120h, which is the conventional Schizochytrium culture time.

[0065] Result: After bromophenol blue staining, the staining state is observed, the protoplasts are counted using a hemocytometer, and the protoplast release rate is calculated to be 35%; since the release rate is low, the subsequent viability detection is not performed.

[0066] Comparative Example 3

[0067] The preparation method of the Schizochytrium sp. protoplast provided by the present comparative example is the same as that of Example 1, except that the Schizochytrium sp. with the preservation number CCTCCM2019990 is used.

[0068] Results: After staining with bromophenol blue, the staining state was observed, the protoplasts were counted using a hemocytometer, and the protoplast release rate was calculated to be 80%; the cell wall breaking and the protoplast state were observed, and the protoplast viability was detected after staining with trypan blue: the protoplast productivity was 35% of the protoplast viability, indicating that more cells were actually autolyzed at the end of fermentation.

[0069] Comparative Example 4

[0070] The present comparative example is basically the same as the method provided by Example 1, except that:

[0071] (4) Preparation of protoplasts in a fermenter: the secondary activation solution obtained in step (3) was inoculated into a fermentation medium at an inoculation amount of 5%, and the fermentation was carried out at 34°C for 40 h, with the pH maintained at 7-8 and the aeration amount of 1.5 vvm. The total fermentation time was 78 h, and the end point determination method was the same as that of Example 1, to obtain a fermentation broth.

[0072] The protoplasts were counted using a hemocytometer, and the protoplast release rate was calculated to be 85%; the protoplast viability was detected after staining with trypan blue: the protoplast productivity was 25% of the protoplast viability, indicating that more cells were actually autolyzed at the end of fermentation under this method.

[0073] As can be seen from the above examples and comparative examples, a large number of Schizochytrium sp. protoplasts can be obtained by precisely controlling the fermentation culture conditions of Schizochytrium sp.

[0074] Finally, the method of the present application is only a preferred embodiment, and is not intended to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a Schizochytrium sp. protoplast, characterized by, The method comprises the following steps: fermenting Schizochytrium sp. to obtain a fermentation liquor; The fermentation culture comprises: The first fermentation stage is at a temperature of 26-28℃ for 36-40 hours; The second fermentation stage is at a temperature of 30-32℃; During the fermentation culture, no additional sugar is added; The Schizochytrium sp. is Schizochytrium sp. CABIO-A-2-IV, with a preservation number of CCTCC M 2021565; When the fermentation culture is carried out in a fermenter, in the first fermentation stage, the aeration rate is 1.5-2vvm, and the pH is controlled at 6.5±0.5; in the second fermentation stage, the aeration rate is 0.8-1vvm, and the pH is controlled at 8.5±0.5; When the fermentation culture is carried out in a shake flask, in the first fermentation stage, the rotation speed is 200-250rpm; in the second fermentation stage, the rotation speed is 150-180rpm.

2. The production method according to claim 1, characterized by, The culture medium of the fermentation culture comprises 5-10% of a carbon source, 1-5% of sodium glutamate, 0.5-2% of an organic nitrogen source, 0.1-1% of KH2PO4, 0.1-1% of MgSO4 and 1-5% of Na2SO4, and the pH is natural.

3. The preparation method according to claim 1, characterized in that, The total culture time of the fermentation stage is 90-98 hours.

4. The method of claim 1, wherein, Before the fermentation culture, the Schizochytrium sp. strain is activated and / or seed culture is enlarged; the culture medium of the activation and / or seed culture comprises: 2-6% of glucose, 2-8% of sodium glutamate, 0.6-1.5% of yeast extract, 0.5-1% of KH2PO4, 0.5-1.2% of MgSO4, 0.5-3% of NaCl, 0.01-0.1% of CaCl2, and the pH is natural.

5. The preparation method according to claim 4, characterized in that, The temperature of the activation and / or seed culture is 28-32℃.

6. The method of claim 1, wherein, The method further comprises the following steps: centrifuging the fermentation liquor, taking the lower layer, washing and diluting the resuspended sample with buffer HPBS to obtain a suspension.

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