Method for enhancing production of chlorella protein through heterotrophic fermentation

Abstract

The invention relates to a method for enhancing chlorella protein production through heterotrophic fermentation. The method comprises a cell growth stage and a protein accumulation stage, in the cell growth stage, a culture medium with low-concentration ammonium nitrogen (high C / N ratio culture medium) as a nitrogen source is used for fed-batch culture, so that the chlorella cells reach high density; according to the method, protein accumulation is changed into high-concentration ammonium nitrogen (a low C / N ratio culture medium) which is used as a nitrogen source for fed-batch culture so as to promote accumulation of protein in cells, and an acetate solution is fed at the same time during the period so as to resist cytotoxicity of high-concentration ammonium to chlorella under a low C / N ratio induction culture condition; the efficient accumulation of protein in cells of the cell algae is promoted; meanwhile, the cell biomass is maintained not to be reduced or reduced slightly or even continuously increased, and finally, the technical effect of improving the quality (protein content) of a chlorella protein product and chlorella is achieved. The method is beneficial to reducing the production cost of the chlorella protein and improving the industrial economy.

Description

Technical Field

[0001] The invention belongs to the technical field of microalgae, and in particular relates to a method for strengthening the production of chlorella protein through heterotrophic fermentation. Background Art

[0002] Single-celled Chlorella from microalgae is considered to be an ideal high-quality protein source because of its fast growth rate, high protein content, and reasonable amino acid composition. At present, the commercial source of Chlorella biomass is mainly cultivated through traditional open raceway pools. This photoautotrophic cultivation mode is limited by factors such as seasons and cultivation environment conditions, and has defects such as low Chlorella biomass concentration, unstable product quality, high production cost, and low biomass production capacity. Heterotrophic cultivation using mature and controllable fermentation equipment can achieve stable and efficient production of Chlorella biomass, which has been proven to be a more economical and efficient way to produce Chlorella biomass. Protein content and protein yield are two important indicators for evaluating Chlorella protein production. The level of Chlorella protein content that can be achieved under heterotrophic fermentation determines the quality, price and application field of Chlorella protein. The protein content in commercial Chlorella products is generally required to be greater than 50%; and the level of protein yield determines the production cost and economy of Chlorella protein.

[0003] However, under heterotrophic culture, Chlorella cells usually tend to accumulate high levels of lipids or carbohydrates (>50%), while the protein content is low (<40%), which greatly limits the application of heterotrophic Chlorella biomass as a protein source in the field of food and feed. The results of existing literature show that the intracellular protein content of Chlorella can be increased by regulating the nitrogen nutrient supply during heterotrophic culture of Chlorella (increasing nitrogen concentration or reducing carbon-nitrogen ratio). Moreover, the type of nitrogen source in the culture medium also affects protein content and cell growth. Compared with nitrate nitrogen (NO3 - ) and urea nitrogen, the same molar concentration of ammonium nitrogen (NH4 +) is considered to be more conducive to the accumulation of intracellular protein in Chlorella because of its lowest energy consumption during utilization. For example, the prior art of Chinese patent application CN115161203B discloses the use of a two-stage culture mode to increase the protein yield of Chlorella under heterotrophic fermentation. This scheme divides the process of protein production by heterotrophic culture of Chlorella into two stages: biomass production and induced protein production. In the biomass production stage, ammonium chloride is used as the nitrogen source, and a high carbon and nitrogen (C / N=320-400:1) feed solution is used for feeding. When the cells reach the middle and late stages of logarithmic growth, ammonium chloride or urea is used as the nitrogen source, and the feed medium is switched to a low carbon and nitrogen ratio (C / N=3-12:1) feed medium to achieve protein induction. Experiments have shown that the protein content in the same period under low C / N ratio induction using ammonium chloride as the nitrogen source is higher than that when urea is used as the nitrogen source, but the cell biomass concentration loses part of the protein yield due to the toxic effect of high ammonium on cells under low C / N induction conditions. If urea is used instead of ammonium salt in the induction stage, the cell concentration will not drop significantly, which is conducive to maintaining the cell concentration, but the effect of increasing the intracellular protein content of Chlorella when urea is used as a nitrogen source is not as good as that of ammonium salt. Since protein yield = cell biomass (dry weight) × protein content, the final protein yield is directly related to the size of cell biomass and protein content. It can be seen that whether urea or ammonium is used as a nitrogen source in the induction protein production stage, it is impossible to maximize protein yield.

[0004] In order to solve technical problems such as the use of ammonium nitrogen easily leading to a decrease in cell biomass and the poor effect of urea on increasing cell protein content, the inventors believe that it is necessary to propose an improvement plan for the existing culture technology of Chlorella for protein production. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for strengthening the production of Chlorella protein by heterotrophic fermentation, which mainly alleviates the cytotoxicity of high ammonium to Chlorella cells by exogenously adding low-concentration acetic acid during the stage of inducing intracellular protein accumulation of Chlorella, thereby achieving the improvement of Chlorella protein content under high ammonium culture while maintaining the cell biomass without decreasing or decreasing slightly or even continuing to grow, thereby achieving the purpose of increasing the protein yield of Chlorella, solving the problem of high ammonium concentration in the prior art that resists cell growth and affects protein yield, which is beneficial to reducing the production cost of Chlorella protein and improving economy, and providing technical support for promoting the large-scale production of Chlorella as a cheap and high-quality protein source.

[0007] (II) Technical solution

[0008] In a first aspect, the present invention provides a method for enhancing the production of chlorella protein by heterotrophic fermentation, comprising: inoculating chlorella seed liquid into a basic culture medium of a fermentation tank for fermentation culture, during the entire fermentation process: the culture temperature is 22-30° C., the stirring speed and dissolved oxygen are coupled controlled during the culture process, the dissolved oxygen is controlled at 5-80%, and the pH of the fermentation culture liquid is 6.0-7.0; the fermentation process includes two stages: cell growth and protein accumulation;

[0009] The fermentation conditions in the cell growth phase are as follows: the glucose concentration in the fermentation medium is monitored, and when the glucose concentration drops below 5 g / L, a peristaltic pump is used to feed the growth feed medium for fed-batch culture, until the cell dry weight reaches 200 g / L or more, the cell growth phase ends and the protein accumulation phase begins immediately;

[0010] The fermentation conditions in the protein accumulation stage are as follows: the growth feed medium in the cell growth stage is switched to the induction feed medium for continued fed-batch culture, and an acetate solution is fed into the fermenter at the same time; during the culture process, the glucose concentration is monitored in real time, and the flow acceleration of the induction feed medium is adjusted in time according to the glucose concentration to maintain the glucose concentration in the culture solution at 1-20 g / L; at the same time, the acetate concentration in the culture solution in the fermenter is monitored, and the flow acceleration of the acetate solution is adjusted in time according to the acetate concentration to make the acetate concentration 1-50 mM; wherein the C / N of the induction feed medium is less than that of the growth feed medium;

[0011] Sampling was performed every 2-24 hours. When the cell dry weight remained unchanged or began to decrease after two consecutive sampling tests, the culture was terminated and the cells were harvested.

[0012] According to a preferred embodiment of the present invention, the basal culture medium is a modified Endo culture medium, and the growth feed culture medium and the induction feed culture medium are both concentrated modified Endo culture medium; the carbon source in the basal culture medium is glucose, and its concentration is 25-35 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 9-24; the carbon source in the growth feed culture medium is glucose, and its concentration is 720-780 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 320-400:1; the carbon source in the induction feed culture medium is glucose, and its concentration is 720-780 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 3-20:1.

[0013] According to a preferred embodiment of the present invention, the glucose content in the basal culture medium is 20 g / L, and the NH4Cl content is 1.5-4 g / L; the glucose content in the growth feed medium is 750 g / L, and the NH4Cl content is 3.34-4.18 g / L; the glucose content in the induction feed medium is 750 g / L, and the NH4Cl content is 66.875-445.8 g / L.

[0014] Preferably, during the protein accumulation stage, the acetate concentration in the fermentation broth is controlled at 5-50 mM, preferably 5-10 mM.

[0015] According to a preferred embodiment of the present invention, the acetate solution is acetic acid, acetate or a mixture of acetic acid and acetate, and the acetate is at least one of sodium acetate and potassium acetate; the acetate concentration in the acetate solution is 0.9-1.1M.

[0016] According to a preferred embodiment of the present invention, the Chlorella is a wild-type Chlorella sorokiniana or a Chlorella sorokiniana mutant with enhanced protein expression.

[0017] According to a preferred embodiment of the present invention, during the cell growth and protein accumulation stage, the fermentation temperature is maintained at 30° C., the stirring speed and dissolved oxygen are coupled controlled, and the dissolved oxygen is 20%.

[0018] According to a preferred embodiment of the present invention, during the cell growth and protein accumulation stage, ammonia water or 2-4 M NaOH solution is used to control the pH of the fermentation medium to 6.0-7.0, preferably 6.5±0.2.

[0019] According to a preferred embodiment of the present invention, a seed solution is prepared in a shaking flask, and the preparation process is as follows: in a sterile environment, the Chlorella sorokinensis cells activated by shaking flask are inoculated in a 1000mL shaking flask at a volume percentage of 2-10%, the volume of the culture solution in the shaking flask is 300-400mL, and shaking dark culture is performed, the culture temperature is 22-30°C, the rotation speed is 120-220rpm, and the culture time is 2-5 days; the carbon source in the shaking flask culture medium is glucose with a concentration of 30g / L, and the nitrogen source is KNO3 with a concentration of 2g / L.

[0020] In the present application, C / N is the molar ratio of C atoms to N atoms in the culture medium.

[0021] (III) Beneficial effects

[0022] (1) The present invention adopts a heterotrophic fermentation process to cultivate Chlorella to increase the protein yield of Chlorella. Ammonium nitrogen is used throughout the cultivation process, which is beneficial to the accumulation of intracellular protein of Chlorella. The fermentation process includes two stages: cell growth and protein accumulation. In the cell growth stage, a basal medium with a high C / N ratio is used for cultivation. During the period, a growth feed medium with a high C / N ratio is used for fed-batch culture to increase the cell dry weight in the culture solution to 200 g / L or more, thereby obtaining a high-density cell culture solution, which also provides a large amount of biomass base for obtaining protein yield. In the protein accumulation stage, an induction feed medium with a low C / N ratio is used for fed-batch culture. During the process, a low-concentration acetate solution is simultaneously fed, and the acetate content in the culture solution in the protein accumulation stage is controlled to be no more than 50 mM, so as to resist the cytotoxicity of high-concentration ammonium to Chlorella under the low C / N ratio induction culture conditions, promote the efficient accumulation of protein in the cell algae cells, and maintain the cell biomass without decreasing or decreasing or even continuing to increase, and finally achieve the purpose of significantly improving the protein yield of Chlorella and the quality of protein-containing Chlorella. The present invention is beneficial to reducing the production cost of Chlorella protein, improving economic efficiency, and providing technical support for promoting the large-scale production of Chlorella as a cheap and high-quality protein source.

[0023] (2) The present invention first discovered that low concentration of acetic acid (root) can reduce the toxicity of ammonium to Chlorella cells under high ammonium stress culture, so that Chlorella cells can significantly increase the intracellular protein content of Chlorella under high ammonium conditions while maintaining the cell biomass without decreasing or decreasing slightly or even continuing to grow, and finally achieve the simultaneous increase of protein content and yield. The scheme of the present invention is expected to be applied as a new method for alleviating ammonium toxicity in other microalgae high ammonium culture processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the dry weight (g / L) change curve of Chlorella during the culture period of 0-240h under the Chlorella fermentation culture mode of experimental group 1 and control groups 1-3.

[0025] Figure 2 This is the change curve of ammonium concentration (mM) in the culture medium during 0-240h of cultivation under the Chlorella fermentation culture mode of experimental group 1 and control groups 1-3.

[0026] Figure 3 This is the curve showing the change in protein content (%) of Chlorella during the culture period of 0-240h under the Chlorella fermentation culture mode of experimental group 1 and control groups 1-3.

[0027] Figure 4 This is the change curve of protein production (g / L) of Chlorella during the culture period of 0-240h under the Chlorella fermentation culture mode of experimental group 1 and control groups 1-3. DETAILED DESCRIPTION

[0028] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0029] The present invention provides a method for strengthening the production of chlorella protein by heterotrophic fermentation, which comprises: inoculating chlorella seed liquid into a basic culture medium of a fermentation tank for fermentation culture, during the whole fermentation process: the culture temperature is 22-30°C, the stirring speed and dissolved oxygen are coupled controlled during the culture process, the dissolved oxygen is controlled at 5-80%, and the pH of the fermentation culture liquid is 6.0-7.0; the fermentation process includes two stages: cell growth and protein accumulation;

[0030] The fermentation conditions in the cell growth phase are as follows: the glucose concentration in the fermentation medium is monitored, and when the glucose concentration drops below 5 g / L, a peristaltic pump is used to feed the growth feed medium for fed-batch culture, until the cell dry weight reaches 200 g / L or more, the cell growth phase ends and the protein accumulation phase begins immediately;

[0031] The fermentation conditions in the protein accumulation stage are as follows: the growth feed medium in the cell growth stage is switched to the induction feed medium for continued fed-batch culture, and an acetate solution is fed into the fermenter at the same time; during the culture process, the glucose concentration is monitored in real time, and the flow acceleration of the induction feed medium is adjusted in time according to the glucose concentration to maintain the glucose concentration in the culture solution at 1-20 g / L; at the same time, the acetate concentration in the culture solution in the fermenter is monitored, and the flow acceleration of the acetate solution is adjusted in time according to the acetate concentration to make the acetate concentration 1-50 mM; wherein the C / N of the induction feed medium is less than that of the growth feed medium;

[0032] Sampling was performed every 2-24 hours. When the cell dry weight remained unchanged or began to decrease after two consecutive sampling tests, the culture was terminated and the cells were harvested.

[0033] Among them, the culture conditions of the cell growth stage can refer to the prior art CN115161203B or CN110195019A. The main purpose of this stage is to obtain a large number of cell biomass bases to provide a biomass basis for the subsequent improvement of the protein quality of Chlorella. The culture conditions of this stage can be appropriately adjusted, for example, according to the ambient temperature, climate, Chlorella growth, initial inoculation concentration, etc., and there is no strict requirement. As long as the Chlorella cells with a dry weight of 200g / L or more in the logarithmic phase can be obtained in a short time, it is beneficial to shorten the culture cycle and improve production efficiency. If the initial inoculum is 10% (v / v) and the fermentation culture is carried out according to the culture conditions described in CN115161203B or the present invention, the dry weight of Chlorella can be basically reached within 120h. 200g / L or more.

[0034] After obtaining a high-density biomass culture solution, a fermentation culture is carried out in the protein accumulation stage. The main technical features of this process are: (1) using ammonium nitrogen and a culture solution with a low carbon-nitrogen ratio (C / N) for induced fed-batch culture, the energy consumption of the ammonium nitrogen utilization process is minimized, and the accumulation of intracellular protein of Chlorella is promoted; (2) during the induced fed-batch culture process, a low concentration of acetate is added (the acetate concentration of the culture solution is controlled at 1-50 mM) to resist the cytotoxicity of high concentration ammonium to Chlorella under low C / N ratio induced culture conditions, promote the efficient accumulation of protein in algal cells, and maintain the cell biomass without decreasing or decreasing or even continuing to grow, and finally achieve the technical effect of improving Chlorella protein products and Chlorella quality (protein content). Wherein, the acetate solution is acetic acid, acetate or a mixture of acetic acid and acetate, and the acetate is at least one of sodium acetate and potassium acetate; the total acetate concentration in the acetate solution is 0.9-1.1 M.

[0035] The basal medium is a modified Endo medium, and the growth feed medium and the induction feed medium are both concentrated modified Endo medium; the carbon source in the basal medium is glucose, the concentration of which is 25-35 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 9-24; the carbon source in the growth feed medium is glucose, the concentration of which is 720-780 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 320-400:1; the carbon source in the induction feed medium is glucose, the concentration of which is 720-780 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 3-20:1. The acetate provided in the acetate solution can also be used by Chlorella as a carbon source.

[0036] Preferably, the glucose content in the basal medium is 20 g / L, and the NH4Cl content is 1.5-4 g / L; the glucose content in the growth feed medium is 750 g / L, and the NH4Cl content is 3.34-4.18 g / L; the glucose content in the induction feed medium is 750 g / L, and the NH4Cl content is 66.875-445.8 g / L.

[0037] In addition to the carbon source and nitrogen source, the basal medium, growth feed medium and induction feed medium also contain a small amount of inorganic salts, and the inorganic salts are KH2PO4, MgSO4, trisodium citrate, FeSO4, EDTA mother solution and trace element mother solution. Among them, the basal medium is a modified Endo medium, and the growth feed medium and the induction feed medium are both concentrated modified Endo medium, so the inorganic salts contained therein are also several times the inorganic salts contained in the basal medium and are roughly consistent with the concentration multiple of the carbon source. However, those skilled in the art should know that inorganic salts do not have a decisive effect on the growth of Chlorella, and their addition amount and adjustment range are all conventional means, so the specific content values ​​of inorganic salts in each culture medium in the embodiment should not be regarded as limiting conditions of the scope of the present invention.

[0038] Further preferably, the compositions of the basal medium, the growth feed medium and the induction feed medium are as follows:

[0039] Basic culture medium: glucose 20g / L; NH4Cl 1.5-4g / L; KH2PO4 1.2g / L; MgSO4·7H2O 1.2g / L; trisodium citrate 0.2g / L; FeSO4 and EDTA mother solution 1mL / L; trace element mother solution 1mL / L, pH adjusted to 6.0. Among them, the composition of FeSO4 and EDTA mother solution is: FeSO4·7H2O 16g / L, EDTA 2.1g / L; the composition of trace element mother solution is: H3BO32.86g / L, ZnSO4·7H2O 0.222g / L, MnCl2·4H2O 1.81g / L, NaMoO4 0.021g / L, CuSO4·5H2O 0.07g / L.

[0040] Growth feed medium: glucose 750g / L; NH4Cl 3.34-4.18g / L; KH2PO4 60g / L; MgSO4·7H2O60g / L; trisodium citrate 5g / L; FeSO4 and EDTA mother solution 50mL / L; trace element mother solution 50mL / L. Among them, the composition of FeSO4 and EDTA mother solution is: FeSO4·7H2O16g / L, EDTA 2.1g / L; the composition of trace element mother solution is: H3BO3 2.86g / L, ZnSO4·7H2O0.222g / L, MnCl2·4H2O 1.81g / L, NaMoO4 0.021g / L, CuSO4·5H2O 0.07g / L.

[0041] Induction feed medium: glucose 750g / L; NH4Cl 66.875-445.8g / L; KH2PO4 60g / L; MgSO4·7H2O 60g / L; trisodium citrate 5g / L; FeSO4 and EDTA mother solution 50mL / L; trace element mother solution 50mL / L. Among them, the composition of FeSO4 and EDTA mother solution is: FeSO4·7H2O 16g / L, EDTA 2.1g / L; the composition of trace element mother solution is: H3BO3 2.86g / L, ZnSO4·7H2O 0.222g / L, MnCl2·4H2O 1.81g / L, NaMoO4 0.021g / L, CuSO4·5H2O 0.07g / L.

[0042] In order to illustrate the technical effect of the present invention, the following is described in combination with specific examples and control examples. It should be noted that the seed solution preparation method used in the examples and control examples is the same, and the preparation process is as follows: Under a sterile environment, the Sorokin Chlorella cells activated by shaking flasks are inoculated in a 1000mL shaking flask with a volume percentage of 10%, and the culture liquid in the shaking flask is 350mL. The shaking dark culture is carried out, the culture temperature is 30°C, the speed is 200rpm, and the culture time is 4 days. The composition of the shaking flask culture medium is: glucose 30g / L; KNO3 2g / L; KH2PO4 1.2g / L; MgSO4·7H2O 1.2g / L; trisodium citrate 0.2g / L; FeSO4 and EDTA mother solution 1mL / L; trace element mother solution 1mL / L, adjusted to pH 6.0. The composition of FeSO4 and EDTA mother solution is: FeSO4·7H2O 16g / L, EDTA 2.1g / L; the composition of trace element mother solution is: H3BO3 2.86g / L, ZnSO4·7H2O 0.222g / L, MnCl2·4H2O 1.81g / L, NaMoO4 0.021g / L, CuSO4·5H2O 0.07g / L. The activation process of Chlorella sorokinensis cells can refer to the prior art, such as CN110195019A, which performs two-stage seed activation and then inoculates into a shake bottle to prepare seed solution.

[0043] Example 1

[0044] The chlorella cultivation method of this embodiment is divided into two stages: cell growth and protein accumulation. The specific process is as follows: the seed liquid of wild-type Chlorella sorokinensis is inoculated into the basic culture medium of a 7.5L fermenter at 10% (v / v) for fermentation culture. The culture temperature is 30°C during the entire fermentation process. During the culture process, the stirring speed and dissolved oxygen are coupled controlled, and the dissolved oxygen is controlled at 20%. Ammonia water is used to control the pH of the culture solution at 6.5±0.2 during the entire fermentation period.

[0045] The composition of the basic culture medium is as follows: glucose 20 g / L; NH4Cl 2.8 g / L; the contents of the other components are as described above.

[0046] During the cell growth phase: monitor the glucose concentration in the fermentation medium. When the glucose concentration drops below 5 g / L, start using a peristaltic pump to feed the growth feed medium for fed-batch culture. After 120 hours of culture (when the cell dry weight reaches 200.0 g / L when sampled), the cell growth phase ends and the protein accumulation phase begins immediately.

[0047] The composition of the growth feed medium is: glucose 750 g / L; NH4Cl 3.4 g / L; the contents of the other components are as described above.

[0048] Protein accumulation stage: the growth feed medium in the cell growth stage is switched to the induction feed medium to continue the fed-batch culture, and at the same time, an acetate solution with a concentration of 1 M (the molar ratio of acetic acid to sodium acetate is 1:1) is fed into the fermenter; during the culture process, the glucose concentration is monitored in real time, and the flow acceleration of the induction feed medium is adjusted in time according to the glucose concentration to maintain the glucose concentration in the culture solution at 1-20 g / L; at the same time, the acetate concentration in the culture solution in the fermenter is monitored, and the flow acceleration of the acetate solution is adjusted in time according to the acetate concentration to make the acetate concentration 5-10 mM.

[0049] The composition of the induction feed medium is: glucose 750 g / L; NH4Cl 67.5 g / L; the contents of the other components are as described above.

[0050] Sampling was performed every 24 hours. When two consecutive sampling tests showed that the cell dry weight remained unchanged or began to decrease, the culture was terminated and harvested. The total culture period was 240 hours.

[0051] Comparative Example 1

[0052] The chlorella cultivation process of this control example is not divided into stages. The method is as follows: the seed liquid of wild-type Chlorella sorokinensis is inoculated into the basal culture medium of a 7.5L fermenter for fermentation and cultivation at 10% (v / v). The culture temperature is 30°C during the entire fermentation process. The stirring speed and dissolved oxygen are coupled and controlled during the culture process. The dissolved oxygen is controlled at 20%. Ammonia water is used to control the pH of the culture solution at 6.5±0.2 during the entire fermentation period. During the culture period, the glucose concentration in the fermentation culture solution is monitored. When the glucose concentration drops below 5g / L, a peristaltic pump is used to feed the growth feed medium for fed-batch culture, and the culture ends at 240h. The composition of the basal culture medium and the growth feed medium is referred to Example 1.

[0053] Comparative Example 2

[0054] The culturing process of Chlorella in this control example is the same as that in Example 1, except that no acetate solution is added in the "protein accumulation stage", the cell growth stage is cultured for 120 hours, and the total culture period is 240 hours.

[0055] Comparative Example 3

[0056] The chlorella cultivation process of this control example is the same as that of control example 2, except that 112.5 g / L urea is used as a nitrogen source to prepare the induction feed medium in the "protein accumulation stage" and the fed-batch culture is performed. The cell growth stage is cultured for 120 h, the total culture period is 240 h, and no acetate solution is fed in the "protein accumulation stage".

[0057] Example 2

[0058] The culturing process of Chlorella in this example is the same as that in Example 1, except that the acetate concentration of the culture solution in the protein accumulation stage is maintained at 40-50 mM by feeding a 1 M acetate solution (acetic acid and sodium acetate molar ratio 1:10) in the "protein accumulation stage". The cell growth stage is cultured for 120 hours, and the total culture period is 240 hours.

[0059] Example 3

[0060] The chlorella cultivation process of this example is the same as that of Example 1, except that the acetate concentration of the culture solution in the protein accumulation stage is maintained at 90-100 mM by feeding 1 M acetate solution (acetic acid and sodium acetate molar ratio 10:1) in the "protein accumulation stage". The cell growth stage is cultured for 120 hours, and the total culture period is 240 hours.

[0061] The production performance indicators such as cell dry weight (g / L), protein content (%) and protein yield (g / L) at the end of fermentation of Examples 1-3 and Control Examples 1-3 are compared as shown in Table 1:

[0062] Table 1: Groups

[0063]

[0064] The cell dry weight, ammonium concentration, protein content and protein yield of Example 1 and Comparative Examples 1-3 within 240 hours of fermentation culture were respectively plotted as follows. Figure 1-4 shown.

[0065] From Table 1 and Figure 1-4It can be seen that in Control Example 1, the cell dry weight was the largest at the end of fermentation, reaching 240 g / L, but the protein content was low at 36.7%, and the final protein yield was only 88.1 g / L. Although this culture mode can obtain a large amount of cell biomass, the quality of Chlorella is low, the protein content is low, and the economy is poor. However, Control Example 1 also shows that the use of low-concentration ammonium nitrogen as a nitrogen source (the C / N ratio of the growth feed medium is 393) as a feed medium for the entire process of fed-batch culture is conducive to maintaining the cell proliferation state and obtaining high-density cell biomass, but it is not conducive to the accumulation of protein in the cells.

[0066] From Table 1 and Figure 1-4 It can be seen that although the control example 2 was cultured in two stages, cell growth and protein accumulation, the induction feed medium with high concentration of ammonium chloride as the nitrogen source (C / N ratio of the induction feed medium was 19.8) was used for fed-batch culture during protein accumulation. The continuous supply of the induction feed medium with high concentration of ammonium nitrogen caused the accumulation of toxicity of ammonium nitrogen to the Chlorella cells. The cell biological dry weight decreased from 200g / L at 120h of fermentation to 120.5g / L at the end of 240h of culture. The cell dry weight (g / L) decreased by half during the period of 120-240h. It can be seen that ammonium nitrogen has obvious cytotoxicity to Chlorella cells, which leads to cell cessation of growth and even partial apoptosis. At the fermentation end of the control example 2, although the protein content (%) reached 58.5%, the protein yield was only 70.5g / L. However, the control example 2 also shows that high concentration of ammonium nitrogen is indeed conducive to improving the accumulation of protein in Chlorella cells.

[0067] From Table 1 and Figure 1-4 It can be seen that in Control Example 3, culture was carried out in two stages: cell growth and protein accumulation. In the protein accumulation, an induction feed medium with high concentration of urea as the nitrogen source (C / N ratio of the induction feed medium was 13.3) was used for fed-batch culture. At the end of fermentation (240h), the cell dry weight (g / L) was 196.2g / L, which was only 3.8g / L lower than 200g / L after 120h of fermentation, and the degree of decrease was not obvious. The protein content (%) was 50.6% at the end of fermentation, and the final protein yield was 99.28g / L. It can be seen that the use of urea to replace ammonium nitrogen in protein accumulation can reduce the toxicity of ammonium to Chlorella cells and maintain cell biomass, but the accumulation content of protein in the cells is not as good as the protein content in the cells when ammonium nitrogen is used in Control Example 2.

[0068] From Table 1 and Figure 1-4It can be seen that in Example 1, the culture was carried out in two stages, cell growth and protein accumulation. During protein accumulation, an induction feed medium with high concentration of ammonium nitrogen as the nitrogen source (C / N ratio of the induction feed medium was 19.8) was used for flow culture, and acetate was added to the fermenter to maintain the acetate concentration of the culture solution at 5-10mM to resist the toxicity of ammonium nitrogen to Chlorella cells. The cell dry weight at the end of fermentation for 240h was 212.0g / L, which was 12.0g / L more than that at 120h of fermentation. The protein content (%) at the end of fermentation was 57.6%, and the final protein yield was 122.1g / L. It can be seen that adding an appropriate amount of acetate when inducing protein accumulation using high concentration of ammonium nitrogen can maintain the double growth of cell dry weight and protein content, thereby significantly improving the quality and protein yield of Chlorella, and improving the economy of Chlorella protein production process.

[0069] Depend on Figure 2 As shown, during the 120-240h culture period of Control Example 1 and Example 1, the concentration of ammonium ions increased almost synchronously, which shows that compared with Control Example 1, the ammonium nitrogen itself did not decrease during the protein accumulation culture period in Example 1, but the use of acetate in Example 1 reduced the toxic effect of ammonium nitrogen on Chlorella cells.

[0070] As shown in Table 1, by comparing Example 1 with Example 2-3, it can be found that during the induction culture of protein accumulation, the acetate concentration in the fermentation broth needs to be controlled within a lower concentration range, otherwise it will also resist cell growth. For example, the acetate concentration of the culture broth in the protein accumulation stage of Example 3 is maintained at 90-100mM, and the cell dry weight (g / L) at the end of fermentation is only 125.4g / L. Although the protein content in the cells reaches 57.8%, the final protein yield is only 72.48g / L, and the protein yield is close to that of Reference Example 2, which is much lower than that of Example 1. In addition, the acetate concentration of the culture broth in the protein accumulation stage of Example 2 is maintained at 40-50mM, and the cell dry weight (g / L) at the end of fermentation 240h is 195.5g / L, and the cell dry weight is only 4.5g / L lower than 200g / L at 120h, and the degree of decline is not obvious. This shows that when the acetate concentration of the culture medium in the protein accumulation stage is maintained at 40-50 mM, it can also significantly resist the toxic effect of ammonium ions on Chlorella cells. At the end of the fermentation, the protein yield of Chlorella was 111.24 g / L, which was still higher than the urea group of Control Example 3 in all Examples and Control Examples, and the protein yield was second only to Example 1.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for enhancing the production of Chlorella protein by heterotrophic fermentation, characterized in that: It includes: The chlorella seed liquid is inoculated into the basic culture medium of the fermentation tank for fermentation culture. During the whole fermentation process: the culture temperature is 22-30°C, the stirring speed and dissolved oxygen are coupled controlled during the culture process, the dissolved oxygen is controlled at 5-80%, and the pH of the fermentation culture liquid is 6.0-7.0; the fermentation process includes two stages: cell growth and protein accumulation; The fermentation conditions in the cell growth phase are as follows: the glucose concentration in the fermentation medium is monitored, and when the glucose concentration drops below 5 g / L, a peristaltic pump is used to feed the growth feed medium for fed-batch culture, until the cell dry weight reaches 200 g / L or more, the cell growth phase ends and the protein accumulation phase begins immediately; The fermentation conditions in the protein accumulation stage are as follows: the growth feed medium in the cell growth stage is switched to the induction feed medium for continued fed-batch culture, and an acetate solution is fed into the fermenter at the same time; during the culture process, the glucose concentration is monitored in real time, and the flow acceleration of the induction feed medium is adjusted in time according to the glucose concentration to maintain the glucose concentration in the culture solution at 1-20 g / L; at the same time, the acetate concentration in the culture solution in the fermenter is monitored, and the flow acceleration of the acetate solution is adjusted in time according to the acetate concentration to make the acetate concentration 1-50 mM; wherein the C / N of the induction feed medium is less than that of the growth feed medium; Sampling was performed every 2-24 hours. When the cell dry weight remained unchanged or began to decrease after two consecutive sampling tests, the culture was terminated and the cells were harvested.

2. The method according to claim 1, characterized in that The basal culture medium is a modified Endo culture medium, and the growth feed culture medium and the induction feed culture medium are both concentrated modified Endo culture medium; the carbon source in the basal culture medium is glucose, and its concentration is 25-35 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 9-24; the carbon source in the growth feed culture medium is glucose, and its concentration is 720-780 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 320-400:1; the carbon source in the induction feed culture medium is glucose, and its concentration is 720-780 g / L, the nitrogen source is ammonium chloride, and the C / N ratio is 3-20:

1.

3. The method according to claim 2, characterized in that The glucose content in the basic culture medium is 20 g / L, and the NH4Cl content is 1.5-4 g / L; the glucose content in the growth feed culture medium is 750 g / L, and the NH4Cl content is 3.34-4.18 g / L; the glucose content in the induction feed culture medium is 750 g / L, and the NH4Cl content is 66.875-445.8 g / L.

4. The method according to claim 1, characterized in that During the protein accumulation phase, acetate solution was added to the fermenter to maintain the acetate concentration in the culture broth at 5-10 mM.

5. The method according to claim 1, characterized in that The acetate solution is acetic acid, acetate or a mixture of acetic acid and acetate, and the acetate is at least one of sodium acetate and potassium acetate; the acetate concentration in the acetate solution is 0.9-1.1M.

6. The method according to claim 1, characterized in that The chlorella is a wild-type Chlorella sorokiniana or a mutant of Chlorella sorokiniana with enhanced protein expression.

7. The method according to claim 1, characterized in that During the cell growth and protein accumulation phase, the fermentation temperature was maintained at 30°C, the stirring speed and dissolved oxygen were coupled controlled, and the dissolved oxygen was 20%.

8. The method according to claim 1, characterized in that: During the cell growth and protein accumulation stages, the pH of the fermentation broth was controlled at 6.0-7.0, 6.5±0.2 using aqueous ammonia or 2-4 M NaOH solution.

9. The method according to claim 1, characterized in that: The seed liquid is prepared in a shaking flask, and the preparation process is as follows: under a sterile environment, the Chlorella sorokinensis cells activated by shaking flask are inoculated in a 1000mL shaking flask at a volume percentage of 2-10%, the volume of the culture liquid in the shaking flask is 300-400mL, and shaking dark culture is carried out, the culture temperature is 22-30°C, the rotation speed is 120-220rpm, and the culture time is 2-5 days; the carbon source in the shaking flask culture medium is glucose with a concentration of 30g / L, and the nitrogen source is KNO3 with a concentration of 2g / L.

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