Heterotrophic euglena culture method for high-yield euglena polysaccharide

Abstract

The invention discloses an euglena heterotrophic culture method for high-yield euglena polysaccharide, which comprises the following steps: adding a culture medium into each stage of bioreactor, sterilizing the culture medium, cooling, adjusting the pH value, inoculating euglena algae species subjected to separation, activation and expanding culture, and starting heterotrophic culture. And adjusting the ammonia nitrogen concentration in the culture solution in real time according to the growth rates of the euglena cells in different stages. According to the invention, on the premise of not adding any allogenic material, the concentration of a nitrogen source in the culture solution is adjusted in real time, so that the cells rapidly reach a plateau phase to obtain high cell density, and the metabolic flow of algae cells in the plateau phase is transferred from cell division to euglena polysaccharide synthesis through nitrogen deficiency; therefore, the content of the euglena polysaccharide in the cells is increased to 65% or above.

Description

Technical Field

[0001] The present invention relates to the field of microalgae biotechnology, and in particular to a method for heterotrophic cultivation of Euglena for high-yield production of paramylon. Background Art

[0002] Euglena gracilis, also known as Euglena and Euglena viridis, is a single-celled eukaryote between animals and plants. The Euglena cell is about 60 μm long, and its body surface is covered with an elastic, striated pellicle. Euglena is suitable for survival in a relatively wide temperature and pH range, which are 20 - 35 °C and 2 - 6 respectively, and the optimal pH for Euglena growth is 3 - 4. Euglena contains rich nutritional components such as vitamins, mineral nutrients, amino acids, unsaturated fatty acids, chlorophyll, lutein, zeaxanthin, GABA and other 59 essential nutritional elements for the human body every day. Among them, the most important paramylon component is a unique component of the genus Euglena. Paramylon is a polysaccharide composed of linear β-1,3-glucan and is the energy storage substance of Euglena cells. Research shows that paramylon has activities such as improving human immunity, accelerating wound healing, alleviating atopic dermatitis and inhibiting influenza virus, and has application prospects in the food and medical fields.

[0003] Euglena can be cultured by photoautotrophy, mixotrophy and heterotrophy. During the photoautotrophic cultivation of Euglena, it is easily contaminated by natural enemy microorganisms, and both the biomass and the paramylon content in the cells are lower than those in mixotrophic and heterotrophic cultivation. Therefore, there are problems such as high production costs, low production efficiency and difficult product quality control in the photoautotrophic cultivation of Euglena. Mixotrophic cultivation requires a light and sterile environment, which has high requirements for the reactor and will greatly increase the cost. Therefore, it is difficult to industrialize the mixotrophic cultivation of Euglena. Heterotrophic cultivation is a controllable industrial production mode. Compared with photoautotrophic and mixotrophic cultivation, higher biomass and intracellular paramylon content can be obtained in the heterotrophic cultivation of Euglena. Therefore, the heterotrophic cultivation of Euglena is the best large-scale production method for paramylon.

[0004] Existing publicly available patent solutions for heterotrophic cultivation of Euglena to produce Euglena polysaccharide. For example, CN115125184A discloses a method for heterotrophic fermentation of Euglena. This method uses glucose as the main carbon source and corn steep liquor powder as the nitrogen source and secondary carbon source. Under the condition of adding plant hormones to promote growth and accumulation of Euglena polysaccharide, although the content of Euglena polysaccharide can reach 65 - 75%, the cell dry weight is only 13 - 18 g / L. CN114302950A discloses a method for heterotrophic cultivation of Euglena in a fermenter using one or more carbon sources and one or more nitrogen sources, and it is necessary to use acids (nitric acid, hydrochloric acid, sulfuric acid, phosphoric acid, and citric acid) or alkalis (sodium hydroxide, sodium carbonate, and sodium bicarbonate) to maintain the pH of the culture medium at 2 - 4. In this method, due to the excessive addition of acids and alkalis as the cultivation time prolongs, the salinity becomes too high, which affects the growth of Euglena. As a result, the cell dry weight of Euglena is only 26.25 g / L after 144 h of cultivation, and the average cell yield is only 0.179 g / L / h. CN104703611B discloses a method for heterotrophic cultivation of Euglena. This method uses glucose as the carbon source and ammonia water as the nitrogen source. Although it is not necessary to additionally supplement acids and alkalis to control the pH, it is necessary to add citric acid and citrate in the culture medium. Relying on the buffer system composed of these two substances, the pH is maintained at 2 - 5 during the cultivation of Euglena. Although the cell dry weight can finally reach more than 50 g / L, the content of Euglena polysaccharide is unstable, and the variation range is 30% - 70%. This is mainly because the buffer system composed of citric acid and citrate cannot stably control the pH of Euglena growth within the range of 3 - 4 (the optimal pH range for Euglena growth).

[0005] Although the above technologies can achieve heterotrophic cultivation of Euglena and production of Euglena polysaccharide, these cultivation methods have the following problems: (1) Although adding exogenous hormones can increase the content of Euglena polysaccharide, it does not significantly increase the cell density, and the addition of exogenous hormones will increase the production cost; (2) The growth of Euglena is affected by the salinity of the culture medium. Continuously supplementing acids and alkalis that are not absorbed during the growth of algal cells will make the salinity of the culture medium higher and higher, and the higher the salinity, the worse the cell growth; (3) Although Euglena can grow within the pH range of 2 - 6, the optimal pH for Euglena growth is 3 - 4. The higher the pH, the higher the probability of contamination during the cultivation process, and the unstable pH is not conducive to the growth of Euglena and the large - scale synthesis of Euglena polysaccharide; (4) In the traditional heterotrophic cultivation process of Euglena, the mechanical supplementation of nitrogen source does not adjust the feeding amount according to the growth of cells during the cultivation process, which easily leads to an excess of nutrients, and too high ammonia nitrogen concentration will cause cell poisoning and death. Therefore, it is necessary to design a method for heterotrophic cultivation of Euglena with high - yield Euglena polysaccharide. Summary of the Invention

[0006] In order to overcome the defects in the prior art, a method for heterotrophic cultivation of Euglena with high - yield Euglena polysaccharide is provided.

[0007] The present invention is achieved through the following solutions:

[0008] A method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena, the method comprising the following steps: adding a culture medium into bioreactors at all levels, sterilizing the culture medium, cooling it and adjusting the pH value, inoculating the separated, activated and expanded Euglena strain to start heterotrophic cultivation, and during the cultivation of Euglena in this method, the ammonia nitrogen concentration in the culture solution is adjusted in real time according to the growth rate of Euglena cells at different stages.

[0009] During the heterotrophic cultivation in bioreactors at all levels except shake flasks, the ammonia nitrogen concentration in the culture solution is adjusted in real time according to the growth rate of cells at different stages, so that the ammonia nitrogen concentration in the culture solution during the lag phase is 1.0 - 1.5 g / L, the ammonia nitrogen concentration in the culture solution during the logarithmic growth phase is 1.5 - 3.0 g / L, and the ammonia nitrogen concentration in the culture solution during the stationary phase is 0.1 - 1.0 g / L.

[0010] In the heterotrophic cultivation of Euglena, the nitrogen source is an ammonium salt solution or ammonia water; Euglena can adopt a batch culture mode or a fed-batch culture mode in bioreactors at all levels; the fed-batch culture is realized by adding a carbon source and automatically feeding a nitrogen source.

[0011] The heterotrophic cultivation of Euglena can be carried out in a bioreactor capable of heterotrophic cultivation, and the bioreactor capable of heterotrophic cultivation includes a shake flask, a mechanical stirring type, an air-lift type or a bubble column type; Euglena can adopt a batch culture and a fed-batch culture mode in a bioreactor capable of heterotrophic cultivation except a shake flask, and the fed-batch culture mode includes a fed-batch culture mode, a semi-continuous fed-batch culture mode and a continuous fed-batch culture mode; during the fed-batch culture process, the carbon source and the nitrogen source are added separately, and the fed-batch culture is realized by adding a carbon source and automatically feeding a nitrogen source.

[0012] In the fed-batch culture mode, after the glucose in the culture solution of bioreactors at all levels except shake flasks is exhausted or nearly exhausted, it is respectively added to 1 - 60 g / L. When the cell density of Euglena reaches more than 80 g / L and the polysaccharide content of Euglena reaches more than 65% of the cell dry weight, it is transferred to the next-level bioreactor or centrifuged for harvesting and spray-dried to obtain algal powder.

[0013] The culture medium used for culturing Euglena in bioreactors at all levels includes an initial culture medium and a fed-batch culture medium.

[0014] The glucose concentration in the initial culture medium and the culture medium after feeding is 1 - 60 g / L;

[0015] The culture medium used for the heterotrophic cultivation of Euglena also includes the following components: KH 2 PO 4 0.01 - 2.0 g / L, MgSO 4 ·7H 2O 0.1 - 2.0 g / L, CaCl 2 0.01 - 1 g / L, Fe solution, trace elements and water; wherein the composition of the Fe solution is FeSO 4 (NH 4 ) 2 SO 4 ·6H 2 O 1.0 - 20.0 g / L, EDTA 1.0 - 20.0 g / L; wherein the composition of the trace elements is H 3 BO 3 1.0 - 50.0 g / L, ZnSO 4 ·7H 2 O 5.0 - 50.0 g / L, MnSO 4 ·H 2 O 1.0 - 20.0 g / L, Na 2 MoO 4 ·2H 2 O 1.0 - 10.0 g / L, CuSO 4 ·5H 2 O 1.0 - 10.0 g / L, CoSO 4 ·7H 2 O 1.0 - 10.0 g / L;

[0016] Before the start of cultivation, the pH of the initial medium is adjusted to 3 - 4 with an acid, and the acids used include but are not limited to inorganic acids and organic acids such as sulfuric acid, hydrochloric acid, and nitric acid.

[0017] During the heterotrophic cultivation process in bioreactors at all levels except shake flasks, the temperature is controlled at 20 - 35 °C, the dissolved oxygen is not lower than 0.1% of the air saturation concentration, and the pH of the culture solution is automatically controlled at 3 - 4.

[0018] In the heterotrophic cultivation of the said Euglena, the Euglena strain used for the heterotrophic cultivation of Euglena in bioreactors at all levels is the cells heterotrophically cultivated in the previous - level bioreactor for 2 - 10 days.

[0019] In the heterotrophic cultivation of the said Euglena, the medium is sterilized by steam, maintained at 115 °C - 121 °C for 20 - 30 minutes. When the temperature drops to 20 - 35 °C, the separated, activated, and expanded Euglena strain is inoculated at 0.1% - 30% of the working volume to start heterotrophic cultivation.

[0020] The beneficial effects of the present invention are:

[0021] A heterotrophic cultivation method of Euglena for producing high-yield polysaccharide of Euglena can maintain the optimal pH required for the growth of Euglena during long-term heterotrophic cultivation without adding exogenous acid-base and buffer. The ammonia-nitrogen concentration in the culture solution is adjusted in real time according to the growth of cells at different culture stages, and the salinity of the culture solution is maintained stable during the cultivation of Euglena, avoiding the influence of increased salinity, too high ammonia-nitrogen concentration and too high or too low pH on cell growth and the synthesis of Euglena polysaccharide. Thus, Euglena can maintain a rapid growth state for a long time before reaching the plateau phase, and the cell density can quickly reach more than 80 g / L; maintaining a stable low pH can reduce the probability of contamination during heterotrophic cultivation; after Euglena grows to the plateau phase, by reducing the ammonia-nitrogen concentration, without adding any substances, the metabolic flux of algal cells can be transferred from cell division to the direction of Euglena polysaccharide synthesis through nitrogen deficiency, so that the content of Euglena polysaccharide in cells increases to more than 65%. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a process flow chart of the heterotrophic cultivation production process of Euglena of the present invention;

[0023] Figure 2 It is the curve of the heterotrophic cultivation process of Euglena in a 5L bioreactor;

[0024] Figure 3 It is the curve of the heterotrophic cultivation process of Euglena in a 50L bioreactor;

[0025] Figure 4 It is the curve of the heterotrophic cultivation process of Euglena in a 500L bioreactor;

[0026] Figure 5 It is the curve of the heterotrophic cultivation process of Euglena in a 10000L bioreactor. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The preferred embodiments of the present invention will be further described below with reference to the accompanying drawings:

[0028] As Figure 1 shown, a heterotrophic cultivation method of Euglena for producing high-yield polysaccharide of Euglena includes the following steps: adding the culture medium into each level of bioreactor, sterilizing the culture medium, cooling it and adjusting the pH value, and inoculating the separated, activated and expanded Euglena algal species to start heterotrophic cultivation. During the cultivation of Euglena in this method, the ammonia-nitrogen concentration in the culture solution is adjusted in real time according to the growth rate of Euglena cells at different stages.

[0029] During the heterotrophic culture process in bioreactors at all levels except shake flasks, the ammonia nitrogen concentration in the culture medium is adjusted in real time according to the growth rate of cells at different stages, so that the ammonia nitrogen concentration in the culture medium during the lag phase is 1.0 - 1.5 g / L, the ammonia nitrogen concentration in the logarithmic growth phase is 1.5 - 3.0 g / L, and the ammonia nitrogen concentration in the plateau phase is 0.1 - 1.0 g / L.

[0030] In the heterotrophic culture of the said Euglena, the nitrogen source is ammonium salt solution or ammonia water; Euglena can adopt batch culture mode or fed-batch culture mode in bioreactors at all levels; fed-batch culture is achieved by adding carbon source and automatically feeding nitrogen source.

[0031] The heterotrophic culture of the said Euglena can be carried out in a bioreactor capable of heterotrophic culture, and the bioreactor capable of heterotrophic culture includes shake flasks, mechanical stirring type, air-lift type or bubble column type; Euglena can adopt batch culture and fed-batch culture modes in bioreactors capable of heterotrophic culture except shake flasks, and the fed-batch culture mode includes fed-batch culture mode, semi-continuous fed-batch culture mode, continuous fed-batch culture mode; during the fed-batch culture process, carbon source and nitrogen source are added separately, and fed-batch culture is achieved by adding carbon source and automatically feeding nitrogen source.

[0032] In the fed-batch culture mode, after the glucose in the culture medium of bioreactors at all levels except shake flasks is exhausted or nearly exhausted, it is added to 1 - 60 g / L respectively. When the cell density of Euglena reaches more than 80 g / L and the content of Euglena polysaccharide reaches more than 65% of the cell dry weight, it is transferred to the next-level bioreactor or centrifuged for harvesting and spray-dried to obtain algal powder.

[0033] The culture medium used for culturing the said Euglena in bioreactors at all levels includes initial culture medium and feeding culture medium.

[0034] The glucose concentration in the initial culture medium and the culture medium after feeding is 1 - 60 g / L;

[0035] The culture medium used for the heterotrophic culture of the said Euglena also includes the following components: KH 2 PO 4 0.01 - 2.0 g / L, MgSO 4 ·7H 2 O 0.1 - 2.0 g / L, CaCl 2 0.01 - 1 g / L, Fe solution, trace elements and water; the composition of the said Fe solution is FeSO 4 (NH 4 ) 2 SO 4 ·6H 2 O 1.0 - 20.0 g / L, EDTA 1.0 - 20.0 g / L; the composition of trace elements is H 3 BO3 1.0 - 50.0 g / L, ZnSO 4 ·7H 2 O 5.0 - 50.0 g / L, MnSO 4 ·H 2 O 1.0 - 20.0 g / L, Na 2 MoO 4 ·2H 2 O 1.0 - 10.0 g / L, CuSO 4 ·5H 2 O 1.0 - 10.0 g / L, CoSO 4 ·7H 2 O 1.0 - 10.0 g / L;

[0036] Before the start of cultivation, the pH of the initial medium is adjusted to 3 - 4 with an acid. The acids used include, but are not limited to, inorganic acids and organic acids such as sulfuric acid, hydrochloric acid, and nitric acid.

[0037] During the heterotrophic cultivation process in bioreactors at all levels except shake flasks, the temperature is controlled at 20 - 35 °C, the dissolved oxygen is not lower than 0.1% of the air saturation concentration, and the pH of the culture solution is automatically controlled at 3 - 4.

[0038] In the heterotrophic cultivation of the said Euglena, the Euglena strain used for heterotrophic cultivation in bioreactors at all levels is the cells that have been heterotrophically cultivated in the previous - level bioreactor for 2 - 10 days.

[0039] In the heterotrophic cultivation of the said Euglena, the medium is sterilized by steam, maintained at 115 °C - 121 °C for 20 - 30 minutes. When the temperature drops to 20 - 35 °C, the separated, activated, and expanded Euglena strain is inoculated at 0.1% - 30% of the working volume to start heterotrophic cultivation.

[0040] The technical solution of this application proposes a production process for heterotrophic cultivation of Euglena that can adjust the ammonia - nitrogen concentration after feeding in real - time according to the growth of cells, stably maintain the salinity of the culture solution during the cultivation process, stably control the pH, and effectively induce Euglena cells to synthesize Euglena polysaccharide, providing technical support for the stable and efficient production of Euglena polysaccharide and laying a solid foundation for the large - scale production of Euglena polysaccharide.

[0041] Figure 1 This is the process flow chart for the heterotrophic cultivation production process of Euglena in the practical application of the present invention. As Figure 1As shown, add the appropriately prepared culture medium to each level of bioreactor, add acid to adjust the pH of the culture medium to 3 - 4, and the loading coefficient of the reactor is usually 0.6 - 0.8. Sterilize the culture medium by steam (115°C - 121°C, maintain for about 20 - 30 minutes). When the temperature drops to 20 - 35°C, inoculate the separated, activated, and expanded Euglena gracilis strain at 0.1% - 30% of the working volume to start heterotrophic culture. The Euglena gracilis strain used for heterotrophic culture in each level of bioreactor is the cells heterotrophically cultured in the previous level of bioreactor for 2 - 10 days. Among them, the shake flask seeds are cultured under the conditions of a temperature of 20 - 35°C and a rotation speed of 100 - 400 rpm.

[0042] In each level of bioreactor (except the shake flask), batch culture, fed-batch culture, semi-continuous feeding culture, and continuous feeding culture modes can be adopted, and the most commonly used one is the fed-batch culture mode. During the fed-batch culture process, automatically control the pH of the culture solution at 3 - 4 by feeding nitrogen source; when the carbon source in the culture medium is exhausted or the remaining concentration is relatively low, add concentrated carbon source, and then continue to culture until the carbon source is consumed again, and repeat the above feeding operation. Cycle in turn, and end this heterotrophic culture after feeding the carbon source 2 - 10 times.

[0043] During the culture process of Euglena gracilis cells in each level of bioreactor (except the shake flask), it is necessary to control the temperature at 20 - 35°C, preferably 25 - 32°C, and the dissolved oxygen is not less than 0.1% of the air saturation concentration. In a preferred embodiment, the dissolved oxygen is not less than 5% of the air saturation concentration.

[0044] At the end of heterotrophic culture, control the concentration of the carbon source to be basically consumed, preferably even zero. The term "basically consumed" used in this application means that the concentration of the nutrient component is below 0.1 g / L.

[0045] The methods for measuring the dry weight of Euglena gracilis cells and the content of Euglena gracilis polysaccharide involved in this application are as follows:

[0046] Determination of dry weight of algal cells: Take V milliliters of the culture solution during the Euglena gracilis culture process, centrifuge at 4000 rpm for 5 minutes, wash the centrifuged algal bodies 3 times with deionized water, transfer them to a weighing bottle {W1 (g)}, and dry them to a constant weight W2 (g) in an oven at 105°C. The dry weight Cx of the algal bodies can be calculated according to the following formula:

[0047] Cx (g / L) = (W2 - W1) / V / 1000.

[0048] Determination of Euglena polysaccharide in the culture medium: During the cultivation of Euglena, take V milliliters of the culture medium, centrifuge at 4000 rpm for 5 minutes, discard the supernatant, add a certain volume of SDS solution (2 g / L) to the algal cells, mix well and then ultrasonically vibrate until the solution turns completely white, centrifuge at 4000 rpm for 5 minutes, transfer the white precipitate part to a weighing bottle {W3 (g)} after discarding the supernatant, dry it in an oven at 105 °C until a constant weight of W4 (g) is obtained. Given the dry weight of the algal cells Cx, the content of Euglena polysaccharide P1 (%) during the cultivation process can be calculated according to the following formula:

[0049] P1 (%) = (W4 - W3) / V / 1000 / Cx.

[0050] The following further elaborates on the present application in combination with specific embodiments:

[0051] Example 1 (Heterotrophic cultivation process of Euglena in a 5L bioreactor)

[0052] Experimental group: The ammonia nitrogen concentration in the culture medium was adjusted in real time according to the cell growth rate during the cultivation process.

[0053] Control group: The ammonia nitrogen concentration in the culture medium was not adjusted during the cultivation process.

[0054] During the heterotrophic cultivation process, the temperature and dissolved oxygen were controlled as described in the specific implementation manner, and the concentrations of each component in the culture media of the experimental group and the control group were the same.

[0055] Experimental results: As Figure 2 shown, there were significant differences between the control group and the experimental group in the 5L bioreactor. When the ammonia nitrogen concentration in the culture medium was not adjusted during the cultivation process (control group), as the cultivation time extended, the salinity of the culture medium increased from 3.1 g / L to 8.5 g / L. When the dry weight increased to 50.53 g / L (cultivation for 123 h), the growth rate of the cells was significantly reduced due to the salinity, and they entered the growth plateau phase; the average growth rate of the cells between 0 - 123 h of cultivation was 0.411 g / L / h, and the content of Euglena polysaccharide at the end of the cultivation was 52.23%.

[0056] Compared with the control group, the ammonia nitrogen concentration in the culture medium was adjusted in real time according to the cell growth rate during the culture process (experimental group). During the culture process, the salinity of the culture medium fluctuated between 3.0 - 3.3 g / L. When the cell dry weight increased to 93.66 g / L (cultured for 148.5 h), due to the high viscosity of the culture medium and poor oxygen mass transfer, the cells entered the plateau phase, and the dry weight increased by 85.4% compared with the control group; the average cell growth rate between 0 - 148 h of culture was 0.631 g / L / h, which was 53.5% higher than that of the control group; after reducing the ammonia nitrogen concentration during the plateau phase, the content of Euglena polysaccharide increased to 67.46% at the end of the culture, which was 12.45% higher than before, indicating that during this process, the metabolic flux of algal cells shifted from cell division to the synthesis direction of Euglena polysaccharide; the content of Euglena polysaccharide increased by 29.2% compared with the control group, and the yield and productivity of Euglena polysaccharide increased by 120.0% and 123.45% respectively compared with the control group. It shows that compared with not adjusting the ammonia nitrogen concentration in the culture medium during the culture process, adjusting the ammonia nitrogen concentration in the culture medium in real time according to the cell growth rate during the culture process can greatly increase the cell dry weight and cell growth rate, and reducing the ammonia nitrogen concentration during the plateau phase can greatly increase the content, yield and productivity of Euglena polysaccharide.

[0057] Example 2 (Heterotrophic culture process of Euglena in a 50 L bioreactor)

[0058] Experimental group: The ammonia nitrogen concentration in the culture medium was adjusted in real time according to the cell growth rate during the culture process.

[0059] Control group: The ammonia nitrogen concentration in the culture medium was not adjusted during the culture process.

[0060] During the heterotrophic culture process, the temperature and dissolved oxygen were controlled as described in the specific implementation manner, and the concentrations of each component in the culture media of the experimental group and the control group were the same.

[0061] Experimental results: As Figure 3 shown, there were significant differences between the control group and the experimental group in the 50 L bioreactor. When the ammonia nitrogen concentration in the culture medium was not adjusted during the culture process (control group), as the culture time extended, the salinity of the culture medium increased from 3.2 g / L to 9.3 g / L, and when the dry weight increased to 52.13 g / L (cultured for 125 h), the growth rate of the cells was significantly reduced due to the influence of salinity and entered the growth plateau phase; the average cell growth rate between 0 - 125 h of culture was 0.417 g / L / h, and the content of Euglena polysaccharide at the end of the culture was 56.11%.

[0062] Compared with the control group, the ammonia-nitrogen concentration in the culture medium was adjusted in real time according to the cell growth rate during the cultivation process (experimental group). During the cultivation process, the salinity of the culture medium fluctuated between 3.1 - 3.4 g / L. When the cell dry weight increased to 101.86 g / L (cultivation for 144 h), due to the high viscosity of the culture medium and poor oxygen mass transfer, the cells entered the plateau phase, and the dry weight increased by 95.4% compared with the control group. The average cell growth rate during the cultivation from 0 - 144 h was 0.707 g / L / h, which was 69.6% higher than that of the control group. After reducing the ammonia-nitrogen concentration during the plateau phase, the content of Euglena polysaccharide increased to 68.34%, which was 18.48% higher than that before the plateau phase, indicating that during this process, the metabolic flux of algal cells shifted from cell division to the synthesis direction of Euglena polysaccharide. The content of Euglena polysaccharide increased by 21.8% compared with the control group, and the yield and productivity of Euglena polysaccharide increased by 123.40% and 120.83% respectively compared with the control group. This shows that compared with not adjusting the ammonia-nitrogen concentration in the culture medium during the cultivation process, adjusting the ammonia-nitrogen concentration in the culture medium in real time according to the cell growth rate during the cultivation process can significantly increase the cell dry weight and cell growth rate, and reducing the ammonia-nitrogen concentration during the plateau phase can significantly increase the content, yield and productivity of Euglena polysaccharide.

[0063] Example 3 (Heterotrophic cultivation process of Euglena in a 500 L bioreactor)

[0064] Experimental group: The ammonia-nitrogen concentration in the culture medium was adjusted in real time according to the cell growth rate during the cultivation process.

[0065] Control group: The ammonia-nitrogen concentration in the culture medium was not adjusted during the cultivation process.

[0066] During the heterotrophic cultivation process, the temperature and dissolved oxygen were controlled as described in the specific implementation manner, and the concentrations of each component in the culture media of the experimental group and the control group were the same.

[0067] Experimental results: As Figure 4 shown, there were significant differences between the control group and the experimental group in the 500 L bioreactor. When the ammonia-nitrogen concentration in the culture medium was not adjusted during the cultivation process (control group), as the cultivation time extended, the salinity of the culture medium increased from 3.0 g / L to 8.1 g / L, and when the dry weight increased to 32.29 g / L (cultivation for 68.85 h), the cells entered the growth plateau phase affected by the salinity. The average cell growth rate during the cultivation from 0 - 68.85 h was 0.469 g / L / h, and the content of Euglena polysaccharide at the end of the cultivation was 56.01%.

[0068] Compared with the control group, the ammonia nitrogen concentration in the culture medium was adjusted in real time according to the cell growth rate during the cultivation process (experimental group). During the cultivation process, the salinity of the culture medium fluctuated between 3.0 - 3.2 g / L. When the cell dry weight reached 91.73 g / L (cultivation for 90.43 h), due to the high viscosity of the culture medium and poor oxygen mass transfer, the cells entered the plateau phase, which was 184.1% higher than that of the control group; the average cell growth rate between 0 - 90.43 h of cultivation was 1.014 g / L / h, which was 122.0% higher than that of the control group; after reducing the ammonia nitrogen concentration during the plateau phase, the content of Euglena polysaccharide increased to 68.89%, indicating that during this process, the metabolic flux of algal cells shifted from cell division to the synthesis direction of Euglena polysaccharide; the content of Euglena polysaccharide was 23.0% higher than that of the control group, and the yield and productivity of Euglena polysaccharide were 249.2% and 140.90% higher than those of the control group respectively. It shows that compared with not adjusting the ammonia nitrogen concentration in the culture medium during the cultivation process, adjusting the ammonia nitrogen concentration in the culture medium in real time according to the cell growth rate during the cultivation process can greatly increase the cell dry weight and cell growth rate. After reducing the ammonia nitrogen concentration during the plateau phase, the content, yield and productivity of Euglena polysaccharide can be greatly increased.

[0069] Example 4 (Heterotrophic cultivation process of Euglena in a 10000 L bioreactor)

[0070] Experimental group: The ammonia nitrogen concentration in the culture medium was adjusted according to the cell growth rate during the cultivation process.

[0071] Control group: The ammonia nitrogen concentration in the culture medium was not adjusted during the cultivation process.

[0072] During the heterotrophic cultivation process, the temperature and dissolved oxygen were controlled as described in the specific implementation manner, and the concentrations of each component in the culture media of the experimental group and the control group were the same.

[0073] Experimental results: As Figure 5 shown, there were significant differences between the control group and the experimental group in the 10000 L bioreactor. When the ammonia nitrogen concentration in the culture medium was not adjusted during the cultivation process (control group), as the cultivation time extended, the salinity of the culture medium increased from 2.9 g / L to 8.4 g / L, and when the dry weight increased to 54.78 g / L (cultivation for 91.1 h), the cells entered the growth plateau phase affected by the salinity; the average cell growth rate between 0 - 91.1 h of cultivation was 0.601 g / L / h, and the content of Euglena polysaccharide at the end of cultivation was 58.11%.

[0074] Compared with the control group, the ammonia nitrogen concentration in the culture medium was adjusted in real time according to the cell growth rate during the culture process (experimental group). During the culture process, the salinity of the culture medium fluctuated between 2.9 - 3.3 g / L. When the cell dry weight reached 76.00 g / L (after 98.00 h of culture), due to the high viscosity of the culture medium and poor oxygen mass transfer, the cells entered the stationary phase, which was 38.7% higher than that of the control group; the average cell growth rate between 0 - 98.00 h of culture was 0.776 g / L / h, which was 29.1% higher than that of the control group; after reducing the ammonia nitrogen concentration during the stationary phase, the content of Euglena polysaccharide increased to 68.25%, which was 12.81% higher than that before nitrogen deficiency induction, indicating that during this process, the metabolic flux of algal cells shifted from cell division to the direction of Euglena polysaccharide synthesis; the content of Euglena polysaccharide was 17.45% higher than that of the control group, and the yield and productivity of Euglena polysaccharide were 68.64% and 38.80% higher than those of the control group respectively. It shows that compared with not adjusting the ammonia nitrogen concentration in the culture medium during the culture process, adjusting the ammonia nitrogen concentration in the culture medium in real time according to the cell growth rate during the culture process can greatly increase the cell dry weight and cell growth rate, and after reducing the ammonia nitrogen concentration during the stationary phase, it can greatly increase the content, yield and productivity of Euglena polysaccharide.

[0075] In summary, the method provided by this application does not require adding acid or base during the culture process, but automatically controls the pH of the culture medium by feeding nitrogen source, thus avoiding the continuous addition of acid and base during the culture process, which leads to an increasing salinity of the culture medium; in addition, according to the growth of cells during the culture process, the ammonia nitrogen concentration in the culture medium at different growth stages is adjusted in real time to ensure the stability of the salinity of the culture medium during the culture process, enabling the cells to quickly enter the stationary phase, and after reaching the stationary phase, by reducing the ammonia nitrogen concentration in the culture medium, the metabolic flux of algal cells is shifted from cell division to the direction of Euglena polysaccharide synthesis, thereby increasing the content of Euglena polysaccharide. This method can greatly increase the yield and productivity of Euglena cells and Euglena polysaccharide.

[0076] Although the technical solutions of the present invention have been described in detail and listed, it should be understood that for those skilled in the art, making modifications to the above embodiments or adopting equivalent alternative solutions are obvious to those skilled in the art. These modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena, the method comprising the following steps: adding a culture medium into bioreactors at all levels, sterilizing the culture medium, cooling it, adjusting the pH value, and inoculating the separated, activated and expanded Euglena strain to start heterotrophic cultivation. Characterized in that: During the cultivation of Euglena in this method, the ammonia nitrogen concentration in the culture solution is adjusted in real time according to the growth rate of Euglena cells at different stages.

2. The method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena according to claim 1, Characterized in that: During the heterotrophic cultivation in bioreactors at all levels except shake flasks, the ammonia nitrogen concentration in the culture solution is adjusted in real time according to the growth rate of cells at different stages, so that the ammonia nitrogen concentration in the culture solution during the lag phase is 1.0 - 1.5 g / L, the ammonia nitrogen concentration in the culture solution during the logarithmic growth phase is 1.5 - 3.0 g / L, and the ammonia nitrogen concentration in the culture solution during the plateau phase is 0.1 - 1.0 g / L.

3. The method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena according to claim 1, Characterized in that: In the heterotrophic cultivation of Euglena, the nitrogen source is an ammonium salt solution or ammonia water; Euglena can adopt a batch culture mode or a fed-batch culture mode in bioreactors at all levels; the fed-batch culture is realized by adding a carbon source and automatically feeding a nitrogen source.

4. The method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena according to claim 1, Characterized in that: The heterotrophic cultivation of Euglena can be carried out in a bioreactor capable of heterotrophic cultivation, and the bioreactor capable of heterotrophic cultivation includes a shake flask, a mechanical stirring type, an air-lift type or a bubble column type; Euglena can adopt a batch culture and a fed-batch culture mode in a bioreactor capable of heterotrophic cultivation except a shake flask, and the fed-batch culture mode includes a fed-batch culture mode, a semi-continuous fed-batch culture mode and a continuous fed-batch culture mode; during the fed-batch culture process, the carbon source and the nitrogen source are added separately, and the fed-batch culture is realized by adding a carbon source and automatically feeding a nitrogen source.

5. The method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena according to claim 1, Characterized in that: In the fed-batch culture mode, after the glucose in the culture solution of bioreactors at all levels except shake flasks is exhausted or nearly exhausted, it is respectively added to 1 - 60 g / L. When the cell density of Euglena reaches more than 80 g / L and the polysaccharide content of Euglena reaches more than 65% of the cell dry weight, it is transferred to the next-level bioreactor or centrifuged for harvesting and spray-dried to obtain algal powder.

6. The method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena according to claim 1, Characterized in that: The culture medium used for culturing Euglena in bioreactors at all levels includes an initial culture medium and a fed-batch culture medium.

7. The method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena according to claim 1, Characterized in that: The glucose concentration in the initial culture medium and the culture medium after feeding is 1 - 60 g / L; The medium used for heterotrophic cultivation of Euglena also includes the following components: KH 2 PO 4 0.01 - 2.0 g / L, MgSO 4 ·7H 2 O 0.1 - 2.0 g / L, CaCl 2 0.01 - 1 g / L, Fe solution, trace elements and water; wherein the composition of the Fe solution is FeSO 4 (NH 4 ) 2 SO 4 ·6H 2 O 1.0 - 20.0 g / L, EDTA 1.0 - 20.0 g / L; wherein the composition of the trace elements is H 3 BO 3 1.0 - 50.0 g / L, ZnSO 4 ·7H 2 O 5.0 - 50.0 g / L, MnSO 4 ·H 2 O 1.0 - 20.0 g / L, Na 2 MoO 4 ·2H 2 O 1.0 - 10.0 g / L, CuSO 4 ·5H 2 O 1.0 - 10.0 g / L, CoSO 4 ·7H 2 O 1.0 - 10.0 g / L; Before the start of cultivation, the pH of the initial culture medium is adjusted to 3 - 4 with an acid, and the acids used include but are not limited to inorganic acids and organic acids such as sulfuric acid, hydrochloric acid, nitric acid, etc.

8. The method for heterotrophic cultivation of Euglena for producing high-yield polysaccharide of Euglena according to claim 1, Characterized in that: During the heterotrophic culture process in bioreactors at all levels except shake flasks, control the temperature at 20-35°C, the dissolved oxygen not lower than 0.1% of the air saturation concentration, and automatically control the pH of the culture medium at 3-4.

9. A heterotrophic culture method of Euglena for high-yield production of Euglena polysaccharide according to claim 1, characterized in that: in the heterotrophic culture of Euglena, the Euglena strain used for the heterotrophic culture of Euglena in each bioreactor is the cells heterotrophically cultured in the previous-level bioreactor for 2-10 days.

10. A heterotrophic culture method of Euglena for high-yield production of Euglena polysaccharide according to claim 1, characterized in that: in the heterotrophic culture of Euglena, the culture medium is sterilized by steam, maintained at a temperature of 115°C - 121°C for 20 - 30 minutes, and when the temperature drops to 20 - 35°C, inoculate the separated, activated and expanded Euglena strain at 0.1% - 30% of the working volume to start heterotrophic culture.

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