Trichocaulon yunnanense and preparation method of trichocaulon yunnanense

Abstract

The application belongs to the field of biological medicine manufacturing, and relates to a fine euglena protein and a preparation method thereof. The method comprises the following steps: S1, providing a fine euglena strain; S2, culturing the fine euglena strain in a modified CM culture medium at 25-32 DEG C; S3, adding protease and lipase to perform enzymolysis reaction; S4, adding a pH regulator to the enzymolysis solution or centrifuging the enzymolysis solution to obtain a protein precipitate; and S5, dissolving the protein precipitate with a phosphate buffer solution, and purifying the protein precipitate with a chromatographic column to obtain a solution of the fine euglena protein with a molecular weight of 55-70 kD. The application also relates to a composition containing the fine euglena protein and a pharmaceutical use of the fine euglena protein. The method provided by the application accumulates the fine euglena protein, and overcomes the defects of low extraction efficiency, non-green processing process and complex process in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing and relates to a thin Euglena protein with anti-inflammatory effects and its preparation method. Background Technology

[0002] In recent years, with the increasing health awareness of people and the growing demand for naturally derived bioactive ingredients, the development and utilization of microalgae resources has received increasing attention. Euglena slenderis (… Euglena gracilis As a single-celled microalga, it has the characteristics of fast growth, strong adaptability, and no cell wall. Moreover, its cells are rich in a variety of nutrients such as protein, polysaccharides, unsaturated fatty acids, vitamins and minerals. It is considered to be a functional food ingredient and source of bioactive substances with important application potential.

[0003] Existing studies have shown that the protein content of Euglena scabra can reach over 40% of its dry weight, with a relatively balanced amino acid composition, possessing good nutritional value and potential physiological activity. However, current research on Euglena scabra mainly focuses on its functional polysaccharide β-1,3-glucan as a feed additive or food supplement, while research on the isolation, purification, structural characterization, and bioactivity of its specific functional proteins is still limited. In particular, there are few reports on the anti-inflammatory effects of Euglena scabra proteins, and a lack of systematic preparation methods and efficacy verification limits its further application in health foods, pharmaceuticals, and cosmetics.

[0004] Inflammatory bowel disease (IBD) is a chronic disease characterized by persistent and excessive immune activation of the gut. Currently, most treatments for IBD focus on chemically synthesized anti-inflammatory drugs, but long-term use often leads to significant side effects or drug resistance. Therefore, identifying safe, effective, and low-toxicity anti-inflammatory components from natural resources for IBD intervention has become a current research hotspot. Euglena filamentosa, as a natural and safe biological resource, offers potential anti-inflammatory proteins that, if applied to IBD, could not only provide a new source of anti-inflammatory raw materials but also enhance the added value of Euglena filamentosa, aligning with the demands of green and sustainable industrial development.

[0005] However, existing methods for extracting and preparing Euglena fibrous protein (EGPro) mostly involve crude extraction or simple separation under highly alkaline conditions. These methods suffer from low extraction efficiency, low purity, easy loss of bioactivity, and unsustainable processing, making it difficult to obtain protein components with stable anti-inflammatory effects. For example, CN115197339B discloses a method for the combined extraction of Euglena fibrous protein and polysaccharides, but this method introduces large amounts of alkali and ethanol during the extraction process, resulting in a long extraction time and hindering the preservation of the protein's bioactivity. Similarly, CN114621313 B discloses an Euglena fibrous protein extract and its use in cosmetics, but this method introduces large amounts of ammonium sulfate during the extraction process, making the protein dialysis purification process time-consuming and limiting its large-scale industrial application. Furthermore, the lack of in-depth research on the structure of Euglena fibrous anti-inflammatory proteins and the unclear structure-activity relationship further restricts the development and application of related products.

[0006] Therefore, developing an efficient, mild, stable method suitable for large-scale preparation of Euglena filamentosa protein, and promoting its application in functional foods, pharmaceuticals, and cosmetics, is of great scientific significance and industrialization potential. Summary of the Invention

[0007] In view of this, the main objective of this invention is to develop Euglena filamentosa protein with anti-inflammatory effects and its efficient, stable and suitable method for large-scale preparation, so as to solve the problems of low extraction efficiency, low purity of Euglena filamentosa protein, easy loss of bioactivity, and environmentally unfriendly production and processing in the existing technology.

[0008] The objectives of this invention and the solutions to its technical problems can be achieved through the following technical solutions.

[0009] On one hand, the present invention provides a method for preparing slender Euglena protein, comprising the following steps: S1. Provides slender Euglena strains; S2. The *Euglena slenderis* strain was cultured at 25–32°C in a modified CM medium containing: calcium chloride dihydrate 0.1–0.5 g / L, magnesium sulfate heptahydrate 4.0–10.0 g / L, dipotassium hydrogen phosphate 2.0–8.0 g / L, ferrous sulfate 0.1–0.4 g / L, glucose 10.0–50.0 g / L, yeast extract 1.0–6.0 g / L, monosodium glutamate 2.0–10.0 g / L, complex amino acids 1.0–5.0 g / L, and vitamin B1. 12 0.001~0.01 g / L and vitamin H 0.001~0.01 g / L, pH 3.0~7.0, wherein the complex amino acids are glutamic acid, cysteine, methionine and trimethylglycine, in a mass ratio of 2:1:1:1; S3. Based on the weight of the Euglena culture medium, add 0.5% of protease and lipase to carry out enzymatic hydrolysis, and inactivate the enzymes after the reaction is completed. S4. Add a pH adjuster to the enzymatic hydrolysate, stir well, or centrifuge the hydrolysate and adjust the pH of the supernatant to 4.0-5.0 to obtain protein precipitation; and S5. Dissolve the protein precipitate with phosphate buffer, and purify the protein solution by chromatography to obtain a solution of slender Euglena protein with a molecular weight of 55-70 kD.

[0010] In an embodiment of the present invention, Euglena slenderis is Euglena slenderis with accession number GDMCC No. 66479.

[0011] In an embodiment of the present invention, in step S2, the modified CM culture medium preferably comprises 0.45 g / L calcium chloride dihydrate, 6.7 g / L magnesium sulfate heptahydrate, 7.0 g / L dipotassium hydrogen phosphate, 0.2 g / L ferrous sulfate, 40.0 g / L glucose, 5.0 g / L yeast extract, 5.0 g / L monosodium glutamate, 2.0 g / L complex amino acids, and vitamin B12. 12 0.005 g / L of vitamin H and 0.005 g / L of vitamin H, pH value 5.0.

[0012] In an embodiment of the present invention, the Euglena slenderis strain is cultured at 28°C in a modified CM medium. After 12 hours of culture, vitamin H is added to a concentration of 0.002-0.008 g / L, preferably 0.005 g / L. After 16 hours of culture, yeast extract is added to a concentration of 1-4 g / L, preferably 2 g / L. After 20 hours of culture, vitamin H and yeast extract are added to a concentration of 0.002-0.008 g / L (preferably 0.005 g / L) and 1-4 g / L (preferably 2 g / L), respectively. After 32 hours of culture, vitamin H is added to a concentration of 0.002-0.008 g / L, preferably 0.005 g / L. In an embodiment of the present invention, after culturing *Euglena spp.* for 36 hours, the biomass was 61.2 g / L, the protein content was 735 mg / g, and the protein yield was 1.25 g / (Lh). The accumulation of *Euglena spp.* protein could be promoted by adding vitamin H and yeast extract at different culture times.

[0013] In embodiments of the present invention, in step S3, the protease is one or a combination of two or more of neutral protease, alkaline protease, papain, and bromelain. In specific embodiments, for example, the protease is a mixture of neutral and alkaline protease, a mixture of neutral and papain, a mixture of neutral and bromelain, a mixture of alkaline and papain, a mixture of alkaline and bromelain, or a mixture of papain and bromelain. In specific embodiments, for example, the protease can be a mixture of neutral protease, papain, and bromelain, etc. In a preferred embodiment, the protease is a combination of neutral protease and bromelain in a 1:1 mass ratio.

[0014] In an embodiment of the present invention, in step S3, the mass ratio of lipase to protease is 1:1 to 1:5, for example 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:4.5, 1:4.6, 1:4.7, 1:5, etc., preferably 1:1 to 1:3, more preferably 1:2.

[0015] In an embodiment of the present invention, in step S3, the enzymatic hydrolysis temperature is 45-55°C and the enzymatic hydrolysis time is 60-150 minutes.

[0016] In an embodiment of the present invention, in step S4, the pH adjuster is selected from phosphoric acid, carbonic acid, or citric acid.

[0017] In an embodiment of the present invention, in step S5, the pH of the phosphate-buffered saline (PBS) is 7.4.

[0018] In an embodiment of the present invention, in step S5, the chromatography can be ion exchange chromatography or gel filtration chromatography, such as Superdex 75 column.

[0019] In a second aspect, the present invention provides a Euglena protein prepared by the above method, having a molecular weight of 55-70 kD and an essential amino acid content of 40.99%, wherein leucine accounts for 9.4%, valine accounts for 6.87%, and lysine accounts for 6.72%.

[0020] In a third aspect, the present invention provides an anti-inflammatory bowel disease composition comprising Euglena fibrous protein obtained by the method described in the first aspect or Euglena fibrous protein described in the second aspect.

[0021] In an embodiment of the present invention, inflammatory bowel disease can be gastroenteritis or inflammatory bowel disease.

[0022] In a fourth aspect, the present invention provides the use of the fibrous Euglena protein obtained by the method described in the first aspect or the fibrous Euglena protein described in the second aspect in the preparation of a medicament for treating inflammatory bowel disease.

[0023] In the implementation of the fourth option, inflammatory bowel disease can be gastroenteritis or inflammatory bowel disease.

[0024] This invention improves the accumulation of Euglena filamentosa protein by culturing it in a modified CM medium supplemented with vitamin H. The protein release rate is significantly increased by enzymatically hydrolyzing Euglena filamentosa using a combination of lipase and protease. Finally, after purification by ion exchange chromatography or gel filtration chromatography, the obtained Euglena filamentosa protein has a molecular weight concentrated in the range of 55-70 kDa, exhibiting good nutritional value and functional activity. This method successfully overcomes the shortcomings of existing technologies, such as low extraction efficiency, non-green processing, and complex processes. The prepared Euglena filamentosa protein can effectively alleviate inflammatory bowel disease, significantly reduce the number of intestinal neutrophils, and increase the number and height of intestinal villi, thus providing high-quality raw materials and reliable technical support for the development of novel microalgae-based anti-inflammatory functional foods and health products. Attached Figure Description

[0025] Figure 1 This is a bar chart showing the protein content of Euglena filamentosa during the non-metabolic and metabolic regulated culture processes of the algal strains of this invention.

[0026] Figure 2 The diagram (A) shows the scheme of supplementing yeast extract and vitamin H during the algal strain metabolism regulation culture process of the present invention, and the bar graph (B) shows the accumulation of the target product Euglena filamentosa protein.

[0027] Figure 3 This is a bar graph showing the effect of the Euglena fibrous protein (EGPro) of the present invention on the concentrations of inflammatory factors TNF-α (A) and IL-1β (B) secreted by RAW264.7 cells (* represents the difference compared with the model control group, ***, p<0.001).

[0028] Figure 4 This is a micrograph showing the effect of the fibrous Euglena protein (EGPro) of this invention on the morphology of RAW264.7 cells under lipopolysaccharide (LPS)-induced inflammatory state.

[0029] Figure 5 This is a pathological section image of a zebrafish gastrointestinal tract inflammation controlled by the euglena protein (EGPro) of this invention. Detailed Implementation

[0030] The preferred embodiments of the present invention will now be described in detail with reference to examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and intent, and all such modifications and substitutions fall within the scope of protection claimed in the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] Example 1: Cultivation of ZC-1007 Euglena based on protein accumulation in Euglena slenderis Original algal strain: Euglena slenderis ( Euglena gracilis Algal strain ZC-1007, with accession number GDMCC No.66479, is deposited at the Guangdong Provincial Center for Microbial Culture Collection and is used as the cultured algal strain.

[0033] Culture conditions: The original algal strain ZC-1007 was suspended in modified CM medium in the dark. The pH of the medium was adjusted to 5.0, and the culture temperature was set to 28℃. The modified CM medium contained 0.45 g / L calcium chloride dihydrate, 6.7 g / L magnesium sulfate heptahydrate, 7.0 g / L dipotassium hydrogen phosphate, 0.2 g / L ferrous sulfate, 40.0 g / L glucose, 5.0 g / L yeast extract, 5.0 g / L monosodium glutamate, 2.0 g / L complex amino acids, and vitamin B12. 12 0.005 g / L of sodium glutamate and 0.005 g / L of vitamin H. The complex amino acid is a mixture of glutamic acid, cysteine, methionine, and trimethylglycine in a ratio of 2:1:1:1. Glutamic acid and monosodium glutamate are combined to stabilize pH changes; methionine, as the initiating amino acid for protein translation, initiates the synthesis process and provides sulfur; cysteine ​​stabilizes protein structure by forming disulfide bonds and, as a glutathione precursor, alleviates oxidative stress and protects the synthesis system. Methionine and cysteine ​​work synergistically to improve the protein synthesis rate and ensure the correct folding and functional stability of the product. Since Euglena lacks a cell wall, trimethylglycine, as an osmolar regulator, helps cells maintain water balance in dehydrated or high-salt environments, which is beneficial for maintaining Euglena cell activity.

[0034] Example 2: Protein accumulation of Euglena ZC-1007 under metabolic regulation Culture conditions: Euglena ZC-1007 was inoculated into the above-mentioned modified CM medium and cultured in an environment with sufficient dissolved oxygen and a culture temperature of 28℃.

[0035] Metabolic Regulation: This invention promotes the accumulation of proteins in Euglena filamentosa by batch-adding yeast extract and vitamin H during the logarithmic growth phase (12-32 h) of ZC-1007. At 12 h of culture, vitamin H is added to a concentration of 0.005 g / L in the culture medium. At 16 h, yeast extract is added to a concentration of 2 g / L. At 20 h, vitamin H and yeast extract are added to concentrations of 0.005 g / L and 2 g / L, respectively. At 32 h, vitamin H is added to a concentration of 0.005 g / L, directly added to the culture tank according to its volume. Biomass Calculation Method: Biomass = Dry matter weight per unit volume of algal solution (g) / Unit volume (L) Protein yield calculation method: Protein yield = (Biomass dry matter weight * Dry matter protein content) / Culture time (h).

[0036] The results are as follows Figure 2 As shown. By Figure 2 It can be seen that after 36 hours of cultivation, the biomass of ZC-1007 cultured under metabolic regulation was 61.2 g / L, the protein content was 735 mg / g, and the protein yield of Euglena filamentosa was 1.25 g / (Lh). Compared with non-metabolic regulation culture, under the same culture period, the protein content of ZC-1007 cultured under metabolic regulation was significantly increased. After 36 hours of cultivation, the protein content of ZC-1007 cultured under metabolic regulation was 19.81% higher than that of non-metabolic regulation culture. Figure 1 ).

[0037] Example 3: Validation of the key component range of the modified CM culture medium This embodiment aims to verify the applicability of the range of values ​​for calcium chloride dihydrate, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, compound amino acids, and pH in the modified CM culture medium, and to confirm that the above parameters can achieve normal growth of Euglena filamentosa and efficient accumulation of target proteins within the specified range.

[0038] The experiment used the Euglena slendera strain with preservation number GDMCC No. 66479, and the culture conditions were uniform: culture temperature 28℃, total culture period 36 h. The metabolic regulation method was the same as in Example 2.

[0039] This experiment was conducted with a fixed baseline composition: glucose 40.0 g / L, yeast extract 5.0 g / L, monosodium glutamate 5.0 g / L, ferrous sulfate 0.2 g / L, and vitamin B12. 12 0.005 g / L, Vitamin H 0.005 g / L.

[0040] Ten experimental groups were set up by changing only the key component to be verified and the pH of the system. The specific formulations of each group are shown in the table below:

[0041] After 36 h of cultivation, the samples from each group were tested. All groups of Euglena filamentosa (1-10) showed good growth and normal cell proliferation and intracellular protein accumulation. The biomass of Euglena filamentosa ranged from 47.2 to 61.2 g / L, the algal protein content ranged from 462 to 735 mg / g, and the protein yield ranged from 0.61 to 1.25 g / (L·h). The results indicate that the following concentrations are suitable for the culture system of this invention: calcium chloride dihydrate (0.1-0.5 g / L), magnesium sulfate heptahydrate (4.0-10.0 g / L), dipotassium hydrogen phosphate (2.0-8.0 g / L), complex amino acids (1.0-5.0 g / L), and pH (3.0-7.0). These concentrations ensure stable cultivation and protein enrichment of Euglena filamentosa.

[0042] Comparative Example 4: Comparison with CN120818441A culture medium This comparative example uses Euglena slendera with accession number GDMCC No. 66479, cultured at 28℃ for 36 h, with the remaining culture environment and operating procedures being consistent with Example 2.

[0043] The culture medium was prepared according to Example CN120818441A: ammonium hydrogen phosphate 1.0 g / L, calcium chloride dihydrate 0.02 g / L, sodium citrate 0.8 g / L, boric acid 2.48 mg / L, ferric sulfate heptahydrate 3.0 mg / L, manganese chloride tetrahydrate 1.8 mg / L, cobalt sulfate heptahydrate 1.5 mg / L, zinc sulfate heptahydrate 0.4 mg / L, sodium molybdate dihydrate 0.2 mg / L, anhydrous copper sulfate 0.02 mg / L, glucose 20 g / L, yeast extract 4 g / L, potassium dihydrogen phosphate 12 g / L, magnesium sulfate 8 g / L, monosodium glutamate 8 g / L, vitamin B12 g / L. 12 0.005 g / L, Vitamin H 0.005 g / L, pH 6.0.

[0044] Results: Biomass was 38.7 g / L, protein content was 482 mg / g, and protein yield was 0.52 g / (L·h). It is evident that the protein yield of Example 2, at 1.25 g / (L·h), is 140% higher than that of the control medium CN120818441A, significantly superior to existing technologies.

[0045] Comparative Example 5: Rapeseed Meal Hydrolysate as a Substitute for Compound Amino Acids (Control) The experimental algal strains, culture environment, operating procedures, and detection methods were all consistent with those in Example 2 of this invention. The culture temperature was set at 28℃, and the dissolved oxygen in the culture medium was maintained throughout the entire process. The total culture period was 36 hours. The culture medium used in this experiment was based on the formulation of Example 2, except that the complex amino acid component was replaced with an equal mass of rapeseed meal hydrolysate, which had a nitrogen content of 2.5%. All other components and parameters remained unchanged.

[0046] Results: After 36 h of cultivation, the protein content in the algae was 591 mg / g, and the protein yield was 0.83 g / (L·h). Conclusion: The compound amino acids (glutamic acid, cysteine, methionine, and trimethylglycine) used in this invention increased the protein yield by 50.6% compared to rapeseed meal hydrolysate. The difference in effect between the two is significant, and the technical effect of this invention cannot be achieved simply by replacing components.

[0047] Example 6: Comparison of synergistic effects between different types of proteases and lipases The algal culture used in the experiment was obtained from Euglena slendera with preservation number GDMCC No. 66479, and cultured according to the conditions in Example 2. The lipase used was Lipozyme TL 100L (Novozymes), and it was combined with four different commercial proteases for combination experiments. The specific information of the proteases is as follows: neutral protease (Neutrase 0.8L, enzyme activity 0.8 AU / g, Novozymes (China) Biotechnology Co., Ltd.), alkaline protease (Alcalase 2.4L, enzyme activity 2.4 AU-A / g, Novozymes (China) Biotechnology Co., Ltd.), papain (Papain, enzyme activity 1.5 AU / g, Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.), and bromelain (Bromelain, enzyme activity 1.2 AU / g, Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.).

[0048] Standardized enzymatic hydrolysis parameters: the mass ratio of lipase to each individual protease was 1:1; the total enzyme content was 0.5% (w / w) based on the weight of the Euglena culture medium; and hydrolysis was performed at 50℃ for 120 minutes. Protein yield was used as the evaluation index, and each group was replicated three times. A control group using only the corresponding protease (without lipase) was also included.

[0049] Protein yield = (protein content in supernatant * volume of supernatant) / protein content in algae powder.

[0050] The results are shown in Table 1.

[0051] Table 1. Effects of different combinations of protease and lipase on protein yield

[0052] The yields of all lipase + protease combinations were significantly higher than those of the corresponding single enzyme combinations (p<0.01), indicating that lipases have a synergistic effect with the five proteases mentioned above. Among them, the combination of neutral protease and lipase had the highest yield, which was not significantly different from the yield of the combination of papain and lipase.

[0053] Example 7: Synergistic effect of two proteases combined with lipase To further improve protein yield, this example attempts to combine two proteases with lipase. Lipase accounts for 1 / 3 of the total enzyme mass, with the remaining 2 / 3 being a 1:1 mass mixture of the two proteases. The total enzyme content remains 0.5% (w / w Euglena culture medium), and other conditions are the same as in Example 6. The following combinations were used: neutral protease + alkaline protease; neutral protease + papain; neutral protease + bromelain; alkaline protease + papain; alkaline protease + bromelain; papain + bromelain.

[0054] The results are shown in Table 2.

[0055] Table 2. Protein yield with synergistic effect of two protease combinations and lipase

[0056] The above results indicate that the combination of lipase with neutral protease and papain can further increase the yield to 64.5%, which is superior to the combination of neutral protease alone (61.7%).

[0057] Comparative Example 8: Different proteases used alone (without lipase) To verify the synergistic necessity of lipase, a comparative experiment was conducted using only the five proteases mentioned above (without lipase). The conditions were the same as in Example 6, except that each protease was used individually, with a total enzyme amount of 0.5% (w / w Euglena broth). The yields are shown in the "Protein Yield Only" column of Table 1. The yields of all single enzymes were significantly lower than the corresponding lipase + protease combinations (p<0.01), demonstrating that the addition of lipase has a significant synergistic effect.

[0058] Example 9: Optimization of the mass ratio of lipase to protease (neutral protease: papain = 1:1) Based on Example 7, the total enzyme content was fixed at 0.5% (w / w Euglena culture medium), and the mass ratio of lipase to protease (neutral protease: papain = 1:1) was set to 1:1, 1:2, 1:3, 1:4, and 1:5. Other conditions were the same as in Example 3. Three replicates were performed for each group, and the protein yield was determined.

[0059] The results are shown in Table 3.

[0060] Table 3. Protein yield based on the mass ratio of lipase to protease (neutral protease: papain = 1:1)

[0061] The results showed that the protein yield reached its highest level (64.5%) when the ratio of lipase to protease was 1:2. Therefore, the preferred mass ratio is 1:1 to 1:3, and more preferably 1:2.

[0062] Example 10: Isolation and purification of Euglena gracilis protein (EGPro) Protein isolation from Euglena spp.: Add 0.5% (w / w) of total enzymes (based on the weight of the Euglena spp. culture medium), with a lipase:protease (neutral protease:papain = 1:1) mass ratio of 1:1. Hydrolyze at 50℃ for 120 minutes. After the reaction, inactivate the enzymes at 80℃ for 10 minutes. Add a small amount of 5% citric acid pH adjuster (pH 2.0) to the hydrolysate to adjust the pH to 4.5, stir thoroughly, and allow the protein to precipitate.

[0063] Euglena protein purification: Centrifuge the precipitated protein at 8000 rpm for 15 minutes, discard the supernatant, and retain the precipitate for later use; dissolve the protein precipitate with an appropriate amount of phosphate-buffered saline (PBS, pH 7.4), and add it to the Superdex gel filtration chromatography system. TM The protein was purified in a 75 Increase column (purchased from GlaxoSmithKline Life Sciences (Shanghai) Co., Ltd.) to obtain a solution of Euglena protein with a molecular weight of 55-70 kD.

[0064] Preparation of Euglena protein samples: Solutions of Euglena proteins with molecular weights of 55-70 kD were collected and concentrated to 1 / 10 of their original volume via rotary evaporation. The resulting lyophilized Euglena protein powder was then obtained. The obtained fine Euglena protein powder had a protein content of 92.3% and an essential amino acid content of 40.99%, exhibiting high purity and nutritional properties.

[0065] Method for calculating the protein content of Euglena: Protein content (%) = Nitrogen content in dry matter of Euglena protein (%) × 6.25 Method for calculating the proportion of essential amino acids in Euglena protein: The percentage of essential amino acids (%) = the content of essential amino acids in Euglena protein / the total amino acid content in Euglena protein × 100%.

[0066] The amino acid composition of the extracted Euglena protein is shown in Table 4.

[0067] Table 4. Amino acid composition analysis of extracted Euglena proteins

[0068] Example 11: Experiment on the inhibition of inflammatory cells by Euglena protein (EGPro) Experimental Design: An in vitro inflammation model was constructed using lipopolysaccharide (LPS)-induced RAW264.7 mouse mononuclear macrophage cell line (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.) to investigate the anti-inflammatory activity and mechanism of EGPro. The experiment included a control group (treated only with DMEM high-glucose medium (Hyclone) (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.), a model control group (1 μg / mL LPS), and low, medium, and high concentration groups of EGPro (treated with 10, 50, and 100 μg / mL and 1 μg / mL LPS respectively).

[0069] Intervention process: After the in vitro inflammation model was successfully established, cell morphology was observed under a microscope 24 h after treatment.

[0070] Results analysis: Effects of EGPro on the concentrations of inflammatory factors secreted by RAW264.7 cells: Compared with the model control group, the high-concentration EGPro group significantly reduced the secretion of the inflammatory cytokine TNF-α (p < 0.001); all three EGPro concentration groups significantly reduced the secretion of the inflammatory cytokine IL-1β (p < 0.001), indicating that EGPro has a certain anti-inflammatory effect. Figure 3 ).

[0071] Effects of EGPro on the morphology of RAW264.7 cells under LPS-induced inflammatory conditions: LPS-treated model cells exhibited an activated phenotype: they increased in size and developed pseudopodia, presenting a spindle shape. Figure 4 Compared to the model control group, some cells in the low-concentration (10 μg / mL) and medium-concentration (50 μg / mL) EGPro treatment groups showed some morphological recovery; the high-concentration (100 μg / mL) EGPro treatment group showed a significantly increased proportion of round, normal cells, with smaller and more rounded cell shapes. These results indicate that EGPro can reverse the morphological changes in RAW264.7 cells induced by LPS, demonstrating its significant anti-inflammatory activity.

[0072] Example 12: Experiment on the reduction of gastrointestinal inflammation by Euglena gracilis protein (EGPro) Experimental Design: Three-day-old wild-type AB strain zebrafish juveniles (provided by the fish breeding center of Beijing Huante Zhiyu Youjian Biotechnology Co., Ltd.) were divided into six groups: a normal control group (without trinitrobenzenesulfonic acid induction treatment), a model control group (treated with trinitrobenzenesulfonic acid), a positive control group (treated with mesalazine 500 µg / mL), and EGPro low, medium, and high concentration treatment groups (62.5, 125, and 250 µg / mL). A gastrointestinal mucosal injury model was established in the model control group, positive control group, and EGPro treatment groups using a trinitrobenzenesulfonic acid aqueous solution induction method (48 h, 28℃).

[0073] Intervention process: After successful establishment of the gastrointestinal mucosal injury model, the intervention was continued for each group. The positive control group (mesalazine 500 µg / mL) and the low, medium and high concentration EGPro treatment groups (62.5, 125, 250 μg / mL) were treated at 28℃ for 48 h.

[0074] Results analysis: Changes in the number of intestinal neutrophils (see Table 5): Starting 48 hours after intervention, EGPro significantly reduced the number of intestinal neutrophils, and EGPro had the effect of reducing gastrointestinal inflammation.

[0075] Table 5. Effects of different experimental groups on the number of intestinal neutrophils

[0076] Example 13: Experiment on the relief of colitis by Euglena protein (EGPro) Experimental Design: Three-day-old wild-type AB strain zebrafish juveniles (provided by the fish breeding center of Beijing Huante Zhiyu Youjian Biotechnology Co., Ltd.) were divided into six groups: a normal control group (without trinitrobenzenesulfonic acid induction treatment), a model control group (treated with trinitrobenzenesulfonic acid), a positive control group (treated with mesalazine 500 µg / mL), and EGPro low, medium, and high concentration treatment groups (62.5, 125, and 250 µg / mL). A gastrointestinal mucosal injury model was established in the model control group, positive control group, and EGPro treatment groups using a trinitrobenzenesulfonic acid aqueous solution induction method (48 h, 28℃).

[0077] Based on the pathological sections of the zebrafish gastrointestinal tract ( Figure 5 EGPro at a concentration of 250 µg / mL can alleviate intestinal distension and significantly increase the number and height of intestinal villi. Its effect is comparable to that of the positive control group (mesalazine 500 µg / mL), showing a relieving effect on colitis.

[0078] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Euglena slenderis ( Euglena gracilis A method for preparing protein, characterized in that, Includes the following steps: S1. Provides slender Euglena strains; S2. The *Euglena slenderis* strain was cultured at 25–32°C in a modified CM medium containing: calcium chloride dihydrate 0.1–0.5 g / L, magnesium sulfate heptahydrate 4.0–10.0 g / L, dipotassium hydrogen phosphate 2.0–8.0 g / L, ferrous sulfate 0.1–0.4 g / L, glucose 10.0–50.0 g / L, yeast extract 1.0–6.0 g / L, monosodium glutamate 2.0–10.0 g / L, complex amino acids 1.0–5.0 g / L, and vitamin B1. 12 0.001–0.01 g / L and vitamin H 0.001–0.01 g / L, pH 3.0–7.0, wherein the complex amino acid is glutamic acid, cysteine, methionine and trimethylglycine in a mass ratio of 2:1:1:1; S3. Based on the weight of the Euglena culture medium, add protease and lipase to a total enzyme content of 0.5% for enzymatic hydrolysis, and inactivate the enzymes after the reaction is completed. S4. Add a pH adjuster to the enzymatic hydrolysate, stir well, or centrifuge the enzymatic hydrolysate and adjust the pH of the supernatant to 4.0–5.0 to obtain protein precipitation; and S5. Dissolve the protein precipitate in phosphate-buffered saline (PBS, pH 7.4). After chromatographic purification, the protein solution yields a solution of slender Euglena protein with a molecular weight of 55–70 kD.

2. The preparation method according to claim 1, characterized in that, The *Euglena* species in question is *Euglena* species with accession number GDMCC No. 66479.

3. The preparation method according to claim 1, characterized in that, In step S2, the modified CM culture medium contains 0.45 g / L calcium chloride dihydrate, 6.7 g / L magnesium sulfate heptahydrate, 7.0 g / L dipotassium hydrogen phosphate, 0.2 g / L ferrous sulfate, 40.0 g / L glucose, 5.0 g / L yeast extract, 5.0 g / L monosodium glutamate, 2.0 g / L complex amino acids, and vitamin B12. 12 0.005 g / L of vitamin H and 0.005 g / L of vitamin H, pH value 5.

0.

4. The preparation method according to claim 1, characterized in that, Vitamin H is added after 12 hours of culture, with a concentration of 0.002–0.008 g / L, preferably 0.005 g / L, in the culture medium. After 16 hours of culture, yeast extract is added, with a concentration of 1–4 g / L, preferably 2 g / L, in the culture medium. After 20 hours of culture, vitamin H and yeast extract are added, with concentrations of 0.002–0.008 g / L (preferably 0.005 g / L) and 1–4 g / L (preferably 2 g / L), respectively, in the culture medium. After 32 hours of culture, vitamin H is added, with a concentration of 0.002–0.008 g / L, preferably 0.005 g / L, in the culture medium.

5. The preparation method according to claim 1, characterized in that, In step S3, the protease is one or a combination of two or more of the following: neutral protease, alkaline protease, papain, and bromelain.

6. The method according to claim 1, characterized in that, In step S3, the mass ratio of lipase to protease is 1:1 to 1:5, the hydrolysis temperature is 45 to 55°C, and the hydrolysis time is 60 to 150 minutes.

7. Slender Euglena protein, characterized in that... Prepared by the method described in claim 1, the molecular weight is 55-70 kD and the essential amino acid content is 40.99%.

8. An anti-inflammatory enteropathy composition comprising the fibrous Euglena protein of claim 7, wherein the inflammatory enteropathy is gastroenteritis or colitis.

9. Use of the fibrous Euglena protein of claim 7 in the preparation of a medicament for treating inflammatory bowel disease.

10. The use according to claim 1, characterized in that, The inflammatory bowel disease mentioned refers to gastroenteritis or colitis.

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