CN118272470A

CN118272470A - Method for rapidly accumulating Euglena polysaccharide and efficiently and stably producing Euglena polysaccharide

Info

Publication number: CN118272470A
Application number: CN202211725124.7A
Authority: CN
Inventors: 李元广; 万民熙; 王伟良
Original assignee: Yunnan Baoshan Zeyuan Algae Industry Health Technology Co ltd
Current assignee: Yunnan Baoshan Zeyuan Algae Industry Health Technology Co ltd
Priority date: 2022-12-30
Filing date: 2022-12-30
Publication date: 2024-07-02

Abstract

The invention discloses a method for rapidly accumulating Euglena polysaccharide and efficiently and stably producing Euglena polysaccharide, which comprises the following steps: inoculating euglena species into a reactor for heterotrophic culture; the carbon-nitrogen mass ratio of the culture medium required by heterotrophic culture of the euglena is 1.5:1-2:1; the euglena can adopt a single batch culture mode or a feed supplement culture mode in each level of bioreactor, and the culture medium is starved for 2-8 hours after the nitrogen in the culture medium is exhausted in the feed supplement culture mode, and then the feed supplement is carried out; the carbon source in the culture medium used for heterotrophic culture of euglena is glucose, and the nitrogen source is glutamic acid. The invention is beneficial to the quick accumulation of the euglena polysaccharide by the euglena cells and obviously improves the growth rate of the euglena cells.

Description

Method for rapidly accumulating Euglena polysaccharide and efficiently and stably producing Euglena polysaccharide

Technical Field

The invention relates to the technical field of microalgae biology, in particular to a method for rapidly accumulating and efficiently and stably producing euglena polysaccharide.

Background

Euglena (Euglenagracilis), also known as euglena, is a single-cell eukaryotic organism that is intermediate between animals and plants. The euglena mainly live in ditches, ponds or slow flows rich in organic substances, and can be greatly propagated in warm seasons to make water green. The body is green and fusiform, about 60 μm long, with a large round nucleus in the middle of the body later. The body surface is covered with an elastic surface film with twills. The temperature range of the euglena suitable for survival is wider, the suitable temperature for propagation is 20-35 ℃, and the euglena grows most vigorously in the three seasons of spring, summer and autumn, especially 6-9 months.

Euglena contains 59 kinds of nutrient elements necessary for human body such as vitamins, mineral nutrients, amino acids, unsaturated fatty acids, chlorophyll, lutein, zeaxanthin, GABA, etc. The most important of which is the characteristic component of Euglena. The Euglena polysaccharide is a polysaccharide body composed of linear beta-1, 3-glucan, and is used for efficiently storing sugar produced by Euglena in photosynthesis. It is as nondigestible as dietary fiber, and after degradation of its interior, it is found that there are countless pinholes in the complex structure of the spiral winding. Because of the characteristic of no cell wall of the euglena, compared with spirulina and chlorella, the euglena has high efficient absorption rate of 93.1 percent, and simultaneously contains all amino acids required by human bodies similar to breast milk.

The main nutrition mode of the euglena is the same as most microalgae according to the difference of the carbon source and the illumination utilized by growth, and can be divided into three types of photoautotrophic, heterotrophic and mixed culture (also called as concurrently culture). The euglena mainly utilizes light energy to carry out photosynthesis under the condition of light through chlorophyll. Under the condition of no light, the euglena can absorb organic substances dissolved in water through the body surface.

The ratio of the growth condition of the euglena cells to the polysaccharide content of the euglena under different culture modes is shown in table 1. Heterotrophic culture is a way of nutrition of microalgae under complete light-shielding conditions with organic carbon as carbon. Compared with photoautotrophic culture and mixotrophic culture, the cells can be purely cultured under heterotrophic conditions and are not affected by protozoa, bacteria, fungi, algae and other organisms, and can be maintained in vegetative cell stage for a long time, so that higher cell density and faster cell growth rate can be obtained. The Euglena can be used as inorganic nitrogen source (ammonium sulfate, etc.) and organic nitrogen source (glutamic acid, glutamate, corn steep liquor, etc.) during heterotrophic culture. Glutamic acid can be used as an ideal nitrogen source in heterotrophic culture of Euglena. The euglena is suitable for growing in an environment with the pH value of 3-3.5, and the pH value of the culture medium containing glutamic acid is just between 3-3.5, so that the glutamic acid is used as a nitrogen source in the heterotrophic culture of the euglena, the low pH condition of the culture medium is ensured to be suitable for the rapid growth of the euglena on the premise of not adding acid, and meanwhile, the risk of bacteria contamination in the heterotrophic culture process of the euglena is greatly reduced. The molecular formula (C ₅H₉O₄ N) of glutamic acid simultaneously contains C, N elements, in the heterotrophic culture process of euglena, glutamic acid is converted into alpha-ketoglutarate and NH ₄ ⁺ by using self-synthesized glutamate dehydrogenase, the alpha-ketoglutarate participates in the carbon metabolism process (tricarboxylic acid circulation) of euglena cells, and NH ₄ ⁺ can be taken as a nitrogen source to be directly absorbed by the euglena cells to participate in nitrogen metabolism synthesis of protein. Therefore, the glutamic acid can be used as a carbon source and a nitrogen source in the heterotrophic culture process of the euglena.

Under normal conditions, the consumption rate of glucose is faster than the consumption rate of glutamic acid in the heterotrophic culture process of the euglena, and partial glucose is directly synthesized into euglena polysaccharide after being absorbed by the euglena cells, so that the euglena polysaccharide content of the euglena cells reaches a higher level (the euglena polysaccharide content is more than 60%) at the initial stage of culture, and the euglena polysaccharide can be rapidly accumulated and simultaneously the rapid growth of the cells can be ensured. However, there are cases where the consumption rate of glutamic acid is too high in production, resulting in a lower content of euglena polysaccharide and a slower growth rate. There are two reasons why this occurs: ① The nitrogen metabolism of the euglena cells is enhanced. The glutamic acid is taken as a nitrogen source to be quickly absorbed by cells for synthesizing protein, and simultaneously alpha-ketoglutarate formed by conversion of the glutamic acid is also involved in a cell carbon metabolism path, so that the speed of glucose absorbed by the euglena cells is greatly reduced, the cell nitrogen metabolism effect is enhanced, the capacity of the euglena cells for synthesizing euglena polysaccharide is reduced, the intracellular protein content is overhigh (about 40% of the euglena polysaccharide and about 40% of the protein content), and the cell growth is not facilitated; ② The carbon nitrogen ratio is small. The low glucose concentration and/or the high glutamic acid concentration in the initial culture medium or the fed culture medium can lead to low carbon nitrogen ratio, increase the glutamic acid absorption rate of the euglena cells, and further influence the synthesis of euglena polysaccharide.

On the one hand, the production cost is increased (the price of glutamic acid is far higher than that of glucose) if the consumption rate of glutamic acid is kept too fast for a long time in the heterotrophic culture process of euglena; on the other hand, the pH value of the algae liquid in the reactor after inoculation or feed supplement is rapidly increased (NH ₄ ⁺ is accumulated in a large amount), the risk of bacteria infection in the heterotrophic process is greatly increased, and the added acid can increase the salinity of the algae liquid, which is not beneficial to the growth of cells, and simultaneously the cost is increased; in addition, the content of the euglena polysaccharide (the content of the euglena polysaccharide is the most important index for evaluating the quality of the algae cells) and the cell growth rate in the algae cells can be reduced, the stability of the euglena production is reduced, and the long-term development of enterprises is not facilitated. Therefore, a method for rapidly accumulating euglena polysaccharide and efficiently and stably producing euglena polysaccharide is needed to be designed.

Disclosure of Invention

In order to overcome the defects in the prior art, the method for rapidly accumulating the euglena polysaccharide and efficiently and stably producing the euglena polysaccharide is provided.

The invention is realized by the following scheme:

a method for rapidly accumulating and efficiently and stably producing Euglena polysaccharide by Euglena comprises the following steps: inoculating euglena species into a reactor for heterotrophic culture, wherein the carbon-nitrogen mass ratio of a culture medium required by the euglena heterotrophic culture is 1.5:1-2:1;

The euglena can adopt a single batch culture mode or a feed supplement culture mode in each level of bioreactor, and the culture medium is starved for 2-8 hours after the nitrogen in the culture medium is exhausted in the feed supplement culture mode, and then the feed supplement is carried out;

The carbon source in the culture medium used for heterotrophic culture of euglena is glucose, and the nitrogen source is glutamic acid.

The culture medium used for heterotrophic culture of the euglena comprises an initial culture medium and a fed culture medium, wherein the mass ratio of carbon to nitrogen of the initial culture medium to that of the fed culture medium are 1.5:1-2:1.

The concentration of glucose is 10-50 g/L, and the concentration of glutamic acid is 1-10 g/L.

The culture medium for heterotrophic culture of euglena also comprises the following components: KH ₂PO₄0.01～2.0g/L、MgSO₄·7H₂O0.1～2.0g/L、CaCl₂ 0.01.01-1 g/L, fe solution, trace elements and water; wherein the composition of the Fe solution is FeSO ₄(NH₄)₂SO₄·6H₂ O1.0-20.0 g/L, EDTA 1.0.0-20.0 g/L; wherein the trace elements comprise H₃BO₃1.0～50.0g/L、ZnSO₄·7H₂O5.0～50.0g/L、MnSO₄·H₂O1.0～20.0g/L、Na₂MoO₄·2H₂O1.0～10.0g/L、CuSO₄·5H₂O1.0～10.0g/L、CoSO₄·7H₂O1.0～10.0g/L.

The heterotrophic culture of euglena can be carried out in a shake flask, a mechanical stirring type, an airlift type or a bubbling type bioreactor capable of heterotrophic culture;

the euglena may be cultured in a single batch and fed-batch mode in a bioreactor other than shake flask, including fed-batch, semi-continuous, continuous fed-batch modes.

The euglena seeds inoculated into 10-50L secondary bioreactor for heterotrophic culture are euglena cells which are cultivated in shake flask of primary bioreactor for 2-10 days.

When heterotrophic culture of euglena is carried out, the dissolved oxygen of each level of bioreactor except the shake flask is controlled to be more than 1%.

Before inoculating the bioreactor except shake flask, the culture medium is first filled into the bioreactor, and after inoculating algae cells, fed-batch culture is performed, and corresponding materials are fed into the corresponding bioreactor along with continuous consumption of nutrients.

Under the fed-batch culture mode, the semi-continuous fed-batch culture mode and the continuous fed-batch culture mode, the bioreactors except the shake flask are starved for 2-8 hours after the glutamic acid is exhausted and fed, and glucose can be fed simultaneously with the glutamic acid or can be fed immediately after the glucose is exhausted.

Under the feed supplement culture mode, the glutamic acid and the glucose of each level of bioreactor except the shake flask are respectively supplemented to the initial concentration after being exhausted, the feed supplement steps are continuously repeated after the shake flask is exhausted again, and when the density of the euglena cells reaches more than 50g/L and the content of the euglena polysaccharide reaches more than 60% of the dry weight of the cells, the euglena cells are transplanted to the next level of bioreactor or centrifugally harvested and spray-dried to obtain the algae powder.

The beneficial effects of the invention are as follows:

1. According to the method for rapidly accumulating the euglena polysaccharide and efficiently and stably producing the euglena polysaccharide, the carbon-nitrogen mass ratio in the culture medium is set to be 1.5:1-2:1 after initial euglena or feeding, and meanwhile, in a fed-batch culture mode, the euglena polysaccharide is rapidly accumulated and rapidly grown in the heterotrophic culture process by starving for 2-8 hours after glutamic acid is exhausted and then feeding is performed, so that the problem of excessively high consumption rate of the glutamic acid in the heterotrophic culture process of the euglena can be inhibited, the increase of production cost is avoided, the risk of bacteria infection in the heterotrophic process is greatly reduced, and the rapid accumulation and growth of the euglena polysaccharide in the heterotrophic culture process of the euglena cell are facilitated, so that the growth rate of the euglena polysaccharide is rapidly accumulated and the growth rate of the euglena cell is remarkably improved.

2. The invention can solve the problems that the nitrogen metabolism of the euglena cells is enhanced, and the consumption rate of glutamic acid is too high due to smaller carbon nitrogen in an initial or feed medium, so that the content of euglena polysaccharide in cells is low and the growth rate is slow, and the heterotrophic culture process of the euglena is more efficient and stable, thereby obtaining the euglena powder with high yield and high euglena polysaccharide content.

3. The invention improves the heterotrophic culture process of the euglena, ensures that the euglena production is more efficient and stable, and is beneficial to the development and popularization of euglena enterprises.

Drawings

FIG. 1 is a process flow diagram of a heterotrophic culture production process of euglena of the invention;

FIG. 2 is a schematic diagram showing the effect of nitrogen starvation on the heterotrophic culture process of Euglena in a 10000L bioreactor after glutamate depletion;

FIG. 3 is a graph showing the effect of different initial and fed media carbon-nitrogen ratios on the heterotrophic culture process of Euglena in a 10000L bioreactor.

Detailed Description

Preferred embodiments of the present invention will be further described with reference to the accompanying drawings:

The culture medium used for heterotrophic culture of the euglena comprises an initial culture medium and a fed culture medium, wherein the mass ratio of carbon to nitrogen is 1.5:1-2:1.

The concentration of glucose is 10-50 g/L, and the concentration of glutamic acid is 1-10 g/L;

Heterotrophic culture can be performed in shake flasks, mechanically stirred, airlift or bubble heterotrophic culture bioreactors;

Fig. 1 is a process flow chart of a heterotrophic culture production process of euglena in practical application, and as can be seen from fig. 1, the method mainly comprises the following steps:

A first part: preparing heterotrophic shake flask seeds of Euglena.

A second part: and (3) when the carbon-nitrogen mass ratio in the culture medium after initial or feeding is less than 1.5:1, the euglena is cultivated in different stages of reactors in a heterotrophic mode.

Third section: and when the nitrogen metabolism of the euglena cells is enhanced, namely the protein is rapidly accumulated, the euglena is heterotrophically cultured in each stage of reactor.

Fourth part: the carbon-nitrogen mass ratio in the culture medium is 1.5:1-2:1 after initial or fed-batch culture, and meanwhile, in the fed-batch culture mode, the culture process of the euglena in heterotrophic culture in each stage of reactors is carried out after 2-8h of starvation after glutamic acid is exhausted.

As shown in FIG. 1, in the first part, the algae species used for heterotrophic culture of Euglena in the secondary reactor (10-50L) are Euglena cells cultivated for 2-10 days in the shake flask of the primary reactor.

Adding the corresponding prepared culture medium into each level of biological reactors, regulating the pH of the culture medium to 2.0-7.0 by conventional means such as acid or alkali (in the application, the pH of the culture medium is about 3 without adding acid or alkali due to the existence of glutamic acid), adding water to a working volume, generally the loading coefficient is 0.6-0.8, then steam sterilizing (115-121 ℃ for about 20-30 minutes), and when the temperature is reduced to 20-40 ℃, inoculating the euglena after separation, activation and culture expansion according to 0.1-30% of the working volume to start heterotrophic culture.

As shown in figure 1, the algae species used for heterotrophically culturing the euglena in the secondary bioreactor is cells which are heterotrophically cultured for 2-10 days in a shake flask of the primary bioreactor, and the shake flask is cultured at the temperature of 20-35 ℃ and the rotating speed of 100-400 rpm. The second part to the fourth part are all in fed-batch culture mode in each level of bioreactor (except shake flask).

After the inoculation is completed and heterotrophic culture is started, when the nitrogen source, the organic carbon source or the phosphorus source in the culture medium is consumed (usually 24-72 hours), the culture medium needs to be fed with a carbon source (such as glucose), a nitrogen source (such as glutamic acid) or a phosphorus source (such as KH ₂PO₄), and the fed nutritional ingredients are the corresponding culture mediums after concentration respectively, so that the euglena is promoted to continue growing. Wherein, 0.1-5 ml/L of concentrated H ₂SO₄ can be added into the nitrogen source feed liquid to adjust the pH value and the solubility of the nitrogen source in the reactor. The material can be fed every 5-24 hours, the feeding concentration of the carbon source can be 5-30 g/L, and the feeding concentration of the nitrogen source solution can be 1-10 g/L. And then continuing to culture until the nitrogen source or the carbon source is consumed again, and repeating the feeding operation. And (3) sequentially circulating, and ending the heterotrophic culture after feeding for 2-10 times.

In the culture process, proper culture conditions are controlled to enable euglena to grow normally. Generally, the temperature is controlled to be 20-40 ℃, and the optimal temperature is 25-32 ℃, the dissolved oxygen is not lower than 1% of air saturation concentration, and the pH value after feeding is not higher than 7.0. In a preferred embodiment, the dissolved oxygen is not less than 5% in air saturation concentration and the post-feed pH is not greater than 5.0. In other preferred embodiments, the dissolved oxygen is not less than 10% of the air saturation concentration and the post-feed pH is not greater than 4.0.

During the culture process, the pH does not need to be automatically controlled, generally, as the culture is carried out, the pH can slowly rise as nutrients are continuously consumed until the nutrients are consumed, the pH can rise to approximately 8.0, and at the moment, the pH of the culture solution can be quickly reduced to the initial level through the feed.

At the end of the heterotrophic culture, the concentration of the nutrient components such as carbon, nitrogen and/or phosphorus is controlled to be substantially consumed, preferably even zero. As used herein, the term "substantially depleted" means that the concentration of the nutrient components is below 0.01 mM.

The application relates to a method for measuring the dry weight of euglena cells, the content of euglena polysaccharide and the content of protein, which comprises the following steps:

determination of algal cell dry weight: during the culture of euglena, culture solution V ml is taken, centrifuged at 4000rpm for 5 minutes, the centrifuged algae is washed with deionized water for 3 times, transferred into a weighing bottle { W1 (g) }, and dried in an oven at 105 ℃ to constant weight W2 (g). The algal dry weight Cx can be calculated according to the following formula:

Cx(g/L)＝(W2-W1)/V/1000。

Determination of Euglena polysaccharide during cultivation: taking culture solution V milliliter in the process of euglena culture, centrifuging at 4000rpm for 5 minutes, adding a certain volume of SDS solution (2 g/L) into the euglena after supernatant is discarded, carrying out ultrasonic vibration until the solution becomes completely white after uniform mixing, centrifuging at 4000rpm for 5 minutes, transferring a white precipitate part into a weighing bottle { W3 (g) }, drying in an oven at 105 ℃ until the weight of the euglena W4 (g) is constant, and calculating the known dry weight Cx of the euglena, wherein the euglena polysaccharide content P1 (%) in the process of culture can be calculated according to the following formula:

P1(％)＝(W4-W3)/V/1000/Cx。

Determination of Euglena polysaccharide in algae powder: about 0.1g of algae powder (m) and a centrifuge tube are taken, a certain volume of SDS solution (2 g/L) is added, after uniform mixing, ultrasonic vibration is carried out until the solution becomes white completely, centrifugation is carried out at 4000rpm for 5 minutes, after supernatant is discarded, the white precipitate is transferred into a weighing bottle { W5 (g) }, and the mixture is dried to constant weight W6 (g) in an oven at 105 ℃. The content P2 (%) of euglena polysaccharide in the algae powder can be calculated according to the following formula:

P2(％)＝(W6-W5)/1000/m

Determining a sampling volume according to an estimated cell biomass value of the algae liquid (for example, the algae density is about 65g/L, then 3ml of algae liquid is required to be measured, about 0.2g of dry algae) is required to be transferred to a centrifuge tube, centrifugation is carried out at 4000rpm for 10 minutes, supernatant is discarded, water is used for washing, secondary centrifugation is carried out, supernatant is discarded, the obtained algae mud is transferred to a digestion tube, 0.2g of copper sulfate, 3g of potassium sulfate and 10ml of concentrated sulfuric acid are added for digestion at 200 ℃ for 5 minutes, the temperature is raised to 350 ℃ for digestion for 10 minutes, finally the temperature is raised to 440 ℃ for digestion for 3 hours, and the algae mud is transferred to a Kjeldahl nitrogen determination instrument for distillation. And (3) titrating the sample by using hydrochloric acid after distillation, and calculating the protein content according to a formula by using the volume of the hydrochloric acid. The formula is as follows:

X＝[(V1-V2)×c×0.0140×6.25]/m

Wherein X is the content of protein in the sample, and the unit is; v1 is the volume of hydrochloric acid titration liquid consumed by a sample, and the unit is milliliter (mL); v2 is the volume of the hydrochloric acid standard titration solution consumed by the reagent blank, and the unit is milliliter (mL); c is the concentration of the hydrochloric acid solution, and the unit is mol per liter (mol/L); m is the mass of the sample in grams (g)

Table 1 comparison of the culture conditions reported in the literature for Euglena with the present invention

Document [1] is ：GrimmP,RisseJM,CholewaD,etal.Applicabilityof Euglenagracilisforbiorefineriesdemonstratedbytheproductionofα-tocopherolandparamylonfollowedbyanaerobicdigestion[J].Journal ofBiotechnology,2015.

Document [2] is: boz ˇ idar AFriehsK,LotzM,etal.Productionof paramylon,aβ-1,3-glucan,byheterotrophicgrowthofEuglenagracilis onpotatoliquorinfed-batchandrepeated-batchmodeofcultivation[J].EngineeringinLifeSciences,2011

The application is further illustrated by the following examples:

Example 1 (influence of Nitrogen starvation after glutamic acid depletion on the heterotrophic culture process of Euglena in 10000L bioreactor)

Adding heterotrophic culture medium and deionized water to 10000L bioreactor, sterilizing, and inoculating Euglena when temperature is reduced to 30+ -1deg.C, and starting fed-batch heterotrophic culture.

Experimental group: the initial medium was nitrogen starved for 5h after glutamate depletion and was supplemented with glutamate. The temperature in the culture process is 30+/-1 ℃, and the dissolved oxygen is controlled to be more than 1%.

Control group: the initial medium was depleted of glutamate and immediately after nitrogen starvation was supplemented with glutamate. The temperature in the culture process is 30+/-1 ℃, and the dissolved oxygen is controlled to be more than 1%.

Heterotrophic and feed media as described in the detailed description, the experimental and control media had the same concentrations of each component.

Experimental results: as shown in FIG. 2, after the initial culture medium is starved for 5 hours of nitrogen after the glutamic acid is exhausted, the glutamic acid is supplemented, the content of the euglena polysaccharide in the euglena cells reaches 61.26 percent within 14.43 hours of culture, in the fed-batch heterotrophic culture process (after 14.43 hours of culture), the content of the euglena polysaccharide is always above 60 percent, the protein content is about 23 percent, and the cell density reaches 63.785g/L within 91.33 hours of culture; the initial culture medium is filled with glutamic acid immediately after the glutamic acid is exhausted and nitrogen starvation, the content of the euglena polysaccharide in the cells is lower than 60% in the whole fed-batch culture process, the content of the euglena polysaccharide is gradually reduced along with the extension of the culture time, the content of the euglena polysaccharide is only 42.78% when the culture is carried out for 112.06h, the protein content is gradually increased, the final concentration reaches 41.7%, and the final cell density is only 29.67g/L. During heterotrophic culture of Euglena, the initial culture medium is starved for 5h of nitrogen after glutamic acid is exhausted, which is beneficial to inhibiting nitrogen metabolism, so that the Euglena polysaccharide in cells can reach more than 60% in a very short time, and is beneficial to cell growth.

Example 2 (Effect of different initial and fed Medium carbon-to-carbon ratios on the heterotrophic culture of Euglena in a 10000L bioreactor)

Experimental group: the initial carbon-nitrogen ratio of the culture medium is 1.5:1. The temperature in the culture process is 30+/-1 ℃, and the dissolved oxygen is controlled to be more than 1%.

Control group: the initial carbon-nitrogen ratio of the culture medium is 2.5:1. The temperature in the culture process is 30+/-1 ℃, and the dissolved oxygen is controlled to be more than 1%.

Heterotrophic and feed media as described in the embodiments, the concentrations of the other components in the experimental and control media are the same, except for the initial carbon to nitrogen ratio.

Experimental results: as shown in FIG. 3, when the carbon-nitrogen ratio in the initial and fed culture media is 1.5:1, the content of the euglena polysaccharide in the euglena cells reaches 64.82% within 17.58h of culture, and during the fed-batch heterotrophic culture (after 17.58h of culture), the content of the euglena polysaccharide always keeps above 60%, the protein content is about 23%, and the cell density reaches 43.27g/L within 50.51h of culture; and when the carbon-nitrogen ratio in the culture medium after initial and fed-batch is 2.5:1, the content of the euglena polysaccharide in the cells is lower than 60% in the whole fed-batch culture process, and the content of the euglena polysaccharide is gradually reduced along with the extension of the culture time, the content of the euglena polysaccharide is only 42.18% in 90.18h of culture, the content of protein is gradually increased, the final reaches 40.4%, and the final cell density is only 28.67g/L. The carbon nitrogen ratio in the culture medium after initial and feeding is smaller (2.5:1), which is more beneficial to the synthesis of protein by algae cells; when the carbon and nitrogen in the culture medium after the initial and the feed supplement are 1.5:1, the method is more favorable for the rapid synthesis and growth of the euglena polysaccharide by the cells.

In conclusion, the method provided by the application can solve the problems that the consumption rate of glutamic acid is too high due to the enhancement of nitrogen metabolism of the euglena cells and the low consumption rate of carbon nitrogen in an initial or feed medium in production, so that the content of euglena polysaccharide in cells is low and the growth rate is slow, and the heterotrophic culture process of euglena is more efficient and stable, so that the euglena powder with high yield and high euglena polysaccharide content is obtained.

While the invention has been described and illustrated in considerable detail, it should be understood that modifications and equivalents to the above-described embodiments will become apparent to those skilled in the art, and that such modifications and improvements may be made without departing from the spirit of the invention.

Claims

1. A method for rapidly accumulating and efficiently and stably producing euglena polysaccharide is characterized by comprising the following steps: inoculating euglena into a reactor for heterotrophic culture, wherein the carbon-nitrogen mass ratio of a culture medium required by the heterotrophic culture is 1.5:1-2:1;

2. The method for rapidly accumulating and efficiently stabilizing the production of euglena polysaccharide according to claim 1, which is characterized in that: the culture medium used for heterotrophic culture of the euglena comprises an initial culture medium and a fed culture medium, wherein the carbon-nitrogen ratio of the initial culture medium to the fed culture medium is 1.5:1-2:1.

3. The method for rapidly accumulating and efficiently stabilizing the production of euglena polysaccharide according to claim 1, which is characterized in that: on the premise that the carbon-nitrogen ratio in an initial culture medium or a fed culture medium is 1.5:1-2:1, the glucose concentration is 10-50 g/L, and the glutamic acid concentration is 1-10 g/L;

4. The method for rapidly accumulating and efficiently stabilizing the production of euglena polysaccharide according to claim 1, which is characterized in that: heterotrophic culture can be performed in shake flasks, mechanically stirred, airlift or bubble heterotrophic culture bioreactors;

5. The method for rapidly accumulating and efficiently stabilizing the production of euglena polysaccharide according to claim 4, wherein the method comprises the following steps: the euglena seeds inoculated into 10-50L secondary bioreactor for heterotrophic culture are euglena cells which are cultivated in shake flask of primary bioreactor for 2-10 days.

6. The method for rapidly accumulating and efficiently stabilizing the production of euglena polysaccharide according to claim 4, wherein the method comprises the following steps: when heterotrophic culture of euglena is carried out, the dissolved oxygen of each level of bioreactor except the shake flask is controlled to be more than 1%.

7. The method for rapidly accumulating and efficiently stabilizing the production of euglena polysaccharide according to claim 4, wherein the method comprises the following steps: before inoculating the bioreactor except shake flask, the culture medium is first filled into the bioreactor, and after inoculating algae cells, fed-batch culture is performed, and corresponding materials are fed into the corresponding bioreactor along with continuous consumption of nutrients.

8. The method for rapidly accumulating and efficiently stabilizing the production of euglena polysaccharide according to claim 7, wherein the method comprises the following steps: under the fed-batch culture mode, the semi-continuous fed-batch culture mode and the continuous fed-batch culture mode, the bioreactors except the shake flask are starved for 2-8 hours after the glutamic acid is exhausted and fed, and glucose can be fed simultaneously with the glutamic acid or can be fed immediately after the glucose is exhausted.

9. The method for rapidly accumulating and efficiently stabilizing the production of euglena polysaccharide according to claim 7, wherein the method comprises the following steps: under the feed supplement culture mode, the glutamic acid and the glucose of each level of bioreactor except the shake flask are respectively supplemented to the initial concentration after being exhausted, the feed supplement steps are continuously repeated after the shake flask is exhausted again, and when the density of the euglena cells reaches more than 50g/L and the content of the euglena polysaccharide reaches more than 60% of the dry weight of the cells, the euglena cells are transplanted to the next level of bioreactor or centrifugally harvested and spray-dried to obtain the algae powder.