Euglena gracilis strain with high yield of beta-1, 3-glucan and preparation method of euglena gracilis strain

Abstract

The invention belongs to the technical field of biological manufacturing, and relates to an Euglena gracilis algal strain ZC-1007 with high yield of beta-1, 3-glucan and a preparation method of the Euglena gracilis algal strain ZC-1007. The preservation number of the algal strain ZC-1007 is GDMCC (China General Microbiological Culture Collection Center) No.66479. The algal strain is high in biomass density, high in growth speed and high in beta-1, 3-glucan yield under the light polyculture condition. The preparation method of the euglena strain comprises the steps of optimizing culture conditions and efficiently screening, and obtaining the euglena strain ZC-1007 with high yield of beta-1, 3-glucan through a specific culture medium and environment control. The strain has a good application prospect in the aspect of efficiently preparing a functional component beta-1, 3-glucan.

Description

Technical Field

[0001] The present invention relates to the technical field of biomanufacturing, and in particular to a high-yield beta-1,3-glucan Euglena gracilis strain and a preparation method thereof. Background Art

[0002] Euglena gracilis ( Euglena gracilis ) is a single-cell green algae with unique nutrients and bioactive substances. Among them, β-1,3-glucan is the substance with the highest content in naked algae and has important functional effects. It has been proven that β-1,3-glucan has multiple biological activities such as controlling blood sugar and lipids, improving immunity, regulating intestinal flora, anti-inflammation and anti-oxidation, etc., and has important application prospects in the field of nutrition and health of humans and animals.

[0003] Prior art already exists for Euglena that can produce β-1,3-glucan and methods for cultivating them. For example, CN202411005648.8 discloses a high-yield Euglena strain of β-1,3-glucan. However, its β-1,3-glucan yield is 0.87 g / L / h, which is still relatively low and unsuitable for large-scale industrial applications. Therefore, developing a high-yield Euglena strain of β-1,3-glucan is of great significance for promoting the application of Euglena in the functional food industry. Summary of the Invention

[0004] In view of this, the main purpose of the present invention is to provide a Euglena gracilis strain that efficiently produces β-1,3-glucan and a preparation method thereof, so as to effectively solve the problem of low yield in the prior art.

[0005] The purpose of the present invention and the solution to its technical problems can be achieved through the following technical solutions.

[0006] In one aspect, the present invention provides a high-yield β-1,3-glucan Euglena gracilis strain ZC-1007, which has a deposit number of GDMCC No. 66479 and is deposited in the Guangdong Provincial Microbial Culture Collection Center.

[0007] In another aspect, the present invention provides a method for preparing a high-yielding β-1,3-glucan Euglena strain, comprising the following steps: S1. Provide the original strain of Euglena gracilis; S2. The original algae strain was cultured in modified CM medium under a constant light intensity of 30 μmol·m -2 ·s -1 Culture for 18-36 hours; S3. Fast-growing algae were selected for purification and subculture, and screening was performed to obtain the gracilis algae strain ZC-1007, which produces high β-1,3-glucan.

[0008] In an embodiment of the present invention, the improved CM medium comprises 0.5-2.0 g / L of ammonium hydrogen phosphate, 0.01-0.05 g / L of calcium chloride dihydrate, 0.5-1.0 g / L of sodium citrate, 2.0-4.0 mg / L of boric acid, 1.0-4.0 mg / L of ferric sulfate heptahydrate, 1.0-2.0 mg / L of manganese chloride tetrahydrate, 1.0-2.0 mg / L of cobalt sulfate heptahydrate, 0.1-0.5 mg / L of zinc sulfate heptahydrate, 0.1-0.4 mg / L of sodium molybdate dihydrate, 0.01-0.05 mg / L of anhydrous copper sulfate, 10.0-50.0 g / L of glucose, 1.0-6.0 g / L of yeast extract, 0.5-4.0 g / L of potassium dihydrogen sulfate, 8.0-15.0 g / L of anhydrous magnesium sulfate, 5.0-10.0 g / L of monosodium glutamate, and vitamin B12. 0.001~0.01g / L and vitamin H 0.001~0.01g / L, pH value is 3.5~7.0.

[0009] Compared with the prior art, the β-1,3-glucan yield of the gracilis algae strain ZC-1007 of the present invention is very high. After 72 hours of mixed culture and 12 hours of light-proof induction, the biomass of the gracilis algae strain ZC-1007 is as high as 121 g / L, the β-1,3-glucan content is as high as 750 mg / g dry matter, and the β-1,3-glucan yield is as high as 1.08 g / (Lh). Therefore, it has broad application prospects in the fields of medicine, food, cosmetics, and feed, and provides a reliable biological resource for the green and pollution-free production of β-1,3-glucan.

[0010] The strain ZC-1007 of Euglena gracilis is now deposited in Guangdong Microbial Culture Collection Center, address: 5th Floor, Dayuan Experimental Building, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, China, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code 510000, with the deposit number GDMCC No. 66479 and the deposit date June 6, 2025. The taxonomic name of this strain is Euglena gracilis . BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a flat plate picture of the algae strain of the present invention.

[0012] Figure 2 Schematic diagram of the algae strain of the present invention under microscope.

[0013] Figure 3 These are the monitoring data for the process of optimizing the polyculture conditions of Euglena for high-yield β-1,3-glucan in Example 3 of the present invention.

[0014] Figure 4The following are a graph showing biomass accumulation during the cultivation of the present algae strain and a bar graph showing the accumulation of the target product, β-1,3-glucan. Different lowercase letters indicate significant differences (P ≤ 0.01) in the products produced by GDMCC No. 63861 and ZC-1007 strains at different stages of cultivation. DETAILED DESCRIPTION

[0015] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments. However, it should be understood by those skilled in the art that the embodiments described below are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0016] In one aspect, the present invention provides a high-yield β-1,3-glucan Euglena gracilis strain ZC-1007, which has a deposit number of GDMCC No. 66479 and is deposited in the Guangdong Provincial Microbial Culture Collection Center.

[0017] In the embodiment of the present invention, the culture characteristics of the algae strain ZC-1007 are that the algae colony surface on the plate is smooth and has neat edges, the cell morphology is spindle-shaped under a microscope, the cell size is about 35 μm, and the cell contents can be clearly observed as green oval particles.

[0018] In an embodiment of the present invention, after 84 hours of mixed culture at 30°C, the biomass of the Euglena gracilis strain ZC-1007 was as high as 121 g / L, the β-1,3-glucan content was as high as 750 mg / g dry matter, and the β-1,3-glucan yield was as high as 1.08 g / (Lh).

[0019] In another aspect, the present invention provides a method for preparing the Euglena gracilis strain ZC-1007, comprising the following steps: S1. Provide the original strain of Euglena gracilis; S2. The original algae strain was cultured in modified CM medium under a constant light intensity of 30 µmol·m -2 ·s -1 Culture for 18-36 hours; S3. Fast-growing algae were selected for purification and subculture, and screening was performed to obtain the gracilis algae strain ZC-1007, which produces high β-1,3-glucan.

[0020] In an embodiment of the present invention, in step S2, the improved CM medium comprises 0.5-2.0 g / L of ammonium hydrogen phosphate, 0.01-0.05 g / L of calcium chloride dihydrate, 0.5-1.0 g / L of sodium citrate, 2.0-4.0 mg / L of boric acid, 1.0-4.0 mg / L of ferric sulfate heptahydrate, 1.0-2.0 mg / L of manganese chloride tetrahydrate, 1.0-2.0 mg / L of cobalt sulfate heptahydrate, 0.1-0.5 mg / L of zinc sulfate heptahydrate, 0.1-0.4 mg / L of sodium molybdate dihydrate, 0.01-0.05 mg / L of anhydrous copper sulfate, 10.0-50.0 g / L of glucose, 1.0-6.0 g / L of yeast extract, 0.5-4.0 g / L of potassium dihydrogen sulfate, 8.0-15.0 g / L of anhydrous magnesium sulfate, 5.0-10.0 g / L of monosodium glutamate, and vitamin B12. 12 0.001-0.01 g / L and vitamin H 0.001-0.01 g / L, pH value is 3.5-7.0. In a specific embodiment of the present invention, the improved CM medium contains 1.0 g / L ammonium hydrogen phosphate, 0.02 g / L calcium chloride dihydrate, 0.8 g / L sodium citrate, 2.48 mg / L boric acid, 3.0 mg / L ferric sulfate heptahydrate, 1.8 mg / L manganese chloride tetrahydrate, 1.5 mg / L cobalt sulfate heptahydrate, 0.4 mg / L zinc sulfate heptahydrate, 0.2 mg / L sodium molybdate dihydrate, 0.02 mg / L anhydrous copper sulfate, 20.0 g / L glucose, 4.0 g / L yeast extract, 1.0 g / L potassium dihydrogen sulfate, 12.0 g / L magnesium sulfate, 8.0 g / L monosodium glutamate, vitamin B 12 0.005 g / L and vitamin H 0.005 g / L, pH value is 6.0.

[0021] In an embodiment of the present invention, during the culture process of step S2, continuous feeding is required to maintain a carbon-nitrogen ratio of 18:1 to ensure that the culture environment has sufficient carbon and nitrogen sources. The modified CM culture medium needs to be evenly mixed and aerated to ensure the optimal growth environment for the algae strain.

[0022] In an embodiment of the present invention, in step S2, the incubation time may be 18 to 36 hours. In a specific embodiment, the incubation time may be 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours, preferably 24 hours.

[0023] In an embodiment of the present invention, in step S2, the culture temperature may be 25-35° C. In a specific embodiment, the culture temperature may be 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 30.5° C., 31° C., 31.5° C., 32° C., 32.5° C., 33° C., 33.5° C., 34° C., 34.5° C., 35° C., preferably 30° C.

[0024] In an embodiment of the present invention, in step S3, the cells may be passaged 10 times.

[0025] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention, and all such modifications and substitutions fall within the scope of the claims of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0027] Example 1: Screening of Euglena with High β-1,3-Glucan Production and Optimization of Culture Conditions Original algae strain provided: Euglena gracilis Euglena gracilis The original algae strain was screened by Beijing Zaochen Biotechnology Co., Ltd. from the freshwater resources in Yantai, Shandong on October 20, 2024, and was used as the basic algae strain.

[0028] Culture conditions: The original algae strain was suspended in modified CM medium in the dark, the pH of the medium was adjusted to 6.0, and the culture temperature was set at 30°C. The modified CM medium contained 1.0 g / L ammonium hydrogen phosphate, 0.02 g / L calcium chloride dihydrate, 0.8 g / L sodium citrate, 2.48 mg / L boric acid, 3.0 mg / L ferric sulfate heptahydrate, 1.8 mg / L manganese chloride tetrahydrate, 1.5 mg / L cobalt sulfate heptahydrate, 0.4 mg / L zinc sulfate heptahydrate, 0.2 mg / L sodium molybdate dihydrate, 0.02 mg / L anhydrous copper sulfate, 20.0 g / L glucose, 4.0 g / L yeast extract, 1.0 g / L potassium dihydrogen sulfate, 12.0 g / L magnesium sulfate, 8.0 g / L monosodium glutamate, and vitamin B12. 12 0.005 g / L of nitrate and 0.005 g / L of vitamin H, with a pH of 6.0. The appropriate amount of nitrogen, phosphorus, carbon, and trace elements in the modified CM medium can promote the rapid growth of Euglena and the accumulation of β-1,3-glucan.

[0029] Efficient screening: Fast-growing single algae were selected for purification and subculture. After multiple screenings, the slender Euglena strain ZC-1007 with high β-1,3-glucan production was finally obtained. The slender Euglena strain grows rapidly on flat plates (see Figure 1 ), observed under a microscope, the cell morphology is spindle-shaped green (see Figure 2 ).

[0030] Example 2: Polyculture of Euglena with High β-1,3-Glucan Production Culture conditions: Euglena ZC-1007 was inoculated into modified CM medium. The pH value of the medium was adjusted to 6.0, the culture temperature was set at 30°C, and the light intensity was kept constant for 24 hours at 30 µmol·m -2 ·s -1 Cultivation. The culture medium in the bioreactor is uniformly mixed and aerated to ensure an optimal growth environment for the algae strains. The formulation of the modified CM medium is as described in Example 1. Feeding during cultivation ensures that the culture environment has sufficient carbon and nitrogen sources.

[0031] Mixed Culture: Mixed culture was carried out in a bioreactor for 84 hours, and the biomass and β-1,3-glucan content in the culture medium were determined as follows.

[0032] Biomass calculation method: Biomass = dry matter weight per unit volume of algae (g) / unit volume (L) Calculation method of β-1,3-glucan content: β-1,3-glucan yield = (biomass dry matter weight * dry matter glucan content) / fermentation time (h).

[0033] The results showed that the biomass was 98 g / L, the β-1,3-glucan content was 670 mg / g, and the β-1,3-glucan yield was 0.78 g / (Lh).

[0034] Example 3: Optimization of polyculture conditions for high-yield β-1,3-glucan Euglena Culture conditions: Euglena ZC-1007 was inoculated into modified CM medium. The pH value of the medium was adjusted to 6.0, the culture temperature was set at 30°C, and the light intensity was 30 µmol·m -2 ·s -1 Cultivate. The culture medium in the bioreactor is uniformly mixed and aerated to ensure an optimal growth environment for the algae strains. The formulation of the modified CM medium is as described in Example 1. Continuous feeding is performed during the culture process to maintain a carbon-nitrogen ratio of 18:1 to ensure sufficient carbon and nitrogen sources in the culture environment.

[0035] Polyculture: Polyculture was carried out in a bioreactor for 72 hours. After the polyculture was completed, the induction was carried out in the dark for 12 hours. The process monitoring data is shown in Figure 3 , the biomass and β-1,3-glucan content in the culture broth were determined as follows.

[0036] Biomass calculation method: Biomass = dry matter weight per unit volume of algae (g) / unit volume (L) Calculation method of β-1,3-glucan content: β-1,3-glucan yield = (biomass dry matter weight * dry matter glucan content) / fermentation time (h).

[0037] The results showed that the biomass was 121 g / L, the β-1,3-glucan content was 750 mg / g, and the β-1,3-glucan yield was 1.08 g / (Lh). Compared with Example 2 without induction measures, the biomass increased by 23.47% and the β-1,3-glucan yield increased by 38.46%.

[0038] Comparative Example 1: Comparison with Euglena gracilis strain GDMCC No.63861

[0039] Experimental design: The strain GDMCC No. 63861 and the strain ZC-1007 of the present invention were inoculated into a modified CM medium (the formula of the modified CM medium is as described in Example 1). The pH value of the medium was adjusted to 6.0, the culture temperature was set at 30°C, and a constant light intensity of 30 μmol·m was maintained for 24 hours. -2 ·s -1 Cultivation. The culture medium in the bioreactor was uniformly mixed and aerated to ensure an optimal growth environment for the algae. Polyculture was performed in the bioreactor for 72 hours, followed by 12 hours of induction in the dark. Biomass and β-1,3-glucan content in the culture medium were measured.

[0040] Results: After 84 hours of cultivation, the biomass of GDMCC No. 63861 and ZC-1007 were 93 g / L and 121 g / L, respectively. The β-1,3-glucan contents were 680 mg / g and 750 mg / g, respectively. The β-1,3-glucan yields were 0.75 g / L / h and 1.08 g / L / h, respectively. ZC-1007 exhibited a 44.0% increase in β-1,3-glucan yield compared to GDMCC No. 63861. During the cultivation process, the β-1,3-glucan content in the products produced by both GDMCC No. 63861 and ZC-1007 gradually increased over time. After 84 hours of cultivation, the β-1,3-glucan content in the product produced by ZC-1007 was 10.82% higher than that of GDMCC No. 63861. The results are shown in Table 1. The biomass accumulation curve during the cultivation of the algae strain of the present invention and the target product β-1,3-glucan accumulation bar graph are shown in Figure 4 Different lowercase letters indicate significant differences (P ≤ 0.01) between the products produced by GDMCC No. 63861 and ZC-1007 algae strains at different culture stages.

[0041] Table 1. Comparison of β-1,3-glucan content during the culture of ZC-1007 and GDMCC No.63861

[0042] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. High-yield β-1,3-glucan Euglena gracilis ( Euglena gracilis ) algae strain ZC-1007, characterized in that The deposit number of the algae strain is GDMCC No. 66479.

2. A method for preparing the Euglena gracilis strain ZC-1007 according to claim 1, characterized in that: The steps include: S1. Provide the original strain of Euglena gracilis; S2. The original algae strain was cultured in a modified CM medium under a constant light intensity of 30 μmol·m -2 ·s -1 Culture for 18-36 hours; S3. Selecting fast-growing single algae for purification and subculture, and screening to obtain a gracilis algae strain with high β-1,3-glucan production.

3. The method according to claim 2, characterized in that In step S2, the modified CM medium contains 0.5-2.0 g / L of ammonium hydrogen phosphate, 0.01-0.05 g / L of calcium chloride dihydrate, 0.5-1.0 g / L of sodium citrate, 2.0-4.0 mg / L of boric acid, 1.0-4.0 mg / L of ferric sulfate heptahydrate, 1.0-2.0 mg / L of manganese chloride tetrahydrate, 1.0-2.0 mg / L of cobalt sulfate heptahydrate, 0.1-0.5 mg / L of zinc sulfate heptahydrate, 0.1-0.4 mg / L of sodium molybdate dihydrate, 0.01-0.05 mg / L of anhydrous copper sulfate, 10.0-50.0 g / L of glucose, 1.0-6.0 g / L of yeast extract, 0.5-4.0 g / L of potassium dihydrogen sulfate, 8.0-15.0 g / L of anhydrous magnesium sulfate, 5.0-10.0 g / L of monosodium glutamate, and vitamin B12. 12 0.001~0.01g / L and vitamin H 0.001~0.01g / L, pH value is 3.5~7.

0.

4. The method according to claim 3, wherein The modified CM medium contains 1.0 g / L ammonium hydrogen phosphate, 0.02 g / L calcium chloride dihydrate, 0.8 g / L sodium citrate, 2.48 mg / L boric acid, 3.0 mg / L ferric sulfate heptahydrate, 1.8 mg / L manganese chloride tetrahydrate, 1.5 mg / L cobalt sulfate heptahydrate, 0.4 mg / L zinc sulfate heptahydrate, 0.2 mg / L sodium molybdate dihydrate, 0.02 mg / L anhydrous copper sulfate, 20.0 g / L glucose, 4.0 g / L yeast extract, 1.0 g / L potassium dihydrogen sulfate, 12.0 g / L magnesium sulfate, 8.0 g / L monosodium glutamate, and vitamin B12. 12 0.005 g / L and vitamin H 0.005 g / L, pH value is 6.

0.

5. The method according to claim 2, wherein In step S2, the culture temperature may be 25-35°C.

6. The method according to claim 2, characterized in that In step S3, the cells can be passaged 10 times.

7. The method according to claim 2, wherein After the Euglena strain is polycultured at 30° C. for 84 hours, the biomass is 100-121 g / L, the β-1,3-glucan content is 650-750 mg / g, and the β-1,3-glucan yield is 0.9-1.08 g / (Lh).

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