Euglena gracilis strain producing high yield of beta-1,3-glucan and method for preparing the same

Abstract

The application belongs to the technical field of biological manufacturing, and relates to a kind of high-yield beta-1,3-glucan fine euglena (Euglena gracilis) strain ZC-1007 and its preparation method. Euglena gracilis ​ The preservation number of the strain ZC-1007 is GDMCC No.66479. The strain has high biomass density, fast growth rate and high yield of beta-1,3-glucan under light mixed culture conditions. The preparation method of the strain includes optimization of culture conditions and high-efficiency screening. Through specific culture medium and environmental control, the high-yield beta-1,3-glucan euglena strain ZC-1007 is obtained. The strain has good application prospect in the efficient preparation of functional component beta-1,3-glucan.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological manufacturing, and relates to a kind of high-yield β-1,3-glucan euglena strain and its preparation method. BACKGROUND

[0002] Euglena gracilis Euglena gracilis ) is a kind of unicellular green algae, with unique nutritional ingredients and biological active substances, wherein β-1,3-glucan is the highest content in euglena, has important function, it has been proved that β-1,3-glucan has many biological activities such as controlling sugar and reducing fat, improving immunity, regulating intestinal flora, anti-inflammatory and antioxidant, has important application prospect in the field of human and animal nutrition and health.

[0003] There are euglena and its culture method capable of producing β-1,3-glucan in the prior art. For example, CN202411005648.8 discloses a strain of euglena with high yield of β-1,3-glucan, but its β-1,3-glucan yield is 0.87 g / (L.h), which is still low, and is not conducive to scale industrial application. Therefore, developing a euglena strain with high yield of β-1,3-glucan is of great significance to promote the application of euglena in functional food industry. SUMMARY

[0004] Therefore, the main purpose of the present application is to provide a euglena gracilis strain with high efficiency of producing β-1,3-glucan and its preparation method, so as to effectively solve the problem of low yield in the prior art.

[0005] The purpose of the present application and the solution to its technical problems can be realized by the following technical scheme.

[0006] On the one hand, the present application provides a euglena gracilis strain with high yield of β-1,3-glucan ZC-1007, which has a preservation number of GDMCC No. 66479 and is preserved in Guangdong Microbial Culture Collection Center.

[0007] On the other hand, the present application provides a method for preparing a euglena gracilis strain with high yield of β-1,3-glucan, comprising the following steps:

[0008] S1. providing an original euglena strain;

[0009] S2. culturing the original strain in modified CM culture medium under constant light intensity of 30 μmol·m -2 ·s -1 for 18-36 h;

[0010] S3. Pick up the fast-growing single algae for purification treatment and subculture, carry out screening, and obtain the high-yield β-1,3-glucan euglena strain ZC-1007.

[0011] In an embodiment of the present application, the improved CM culture medium comprises ammonium hydrogen phosphate 0.5-2.0 g / L, calcium chloride dihydrate 0.01-0.05 g / L, sodium citrate 0.5-1.0 g / L, boric acid 2.0-4.0 mg / L, iron sulfate heptahydrate 1.0-4.0 mg / L, manganese chloride tetrahydrate 1.0-2.0 mg / L, cobalt sulfate heptahydrate 1.0-2.0 mg / L, zinc sulfate heptahydrate 0.1-0.5 mg / L, sodium molybdate dihydrate 0.1-0.4 mg / L, copper sulfate anhydrous 0.01-0.05 mg / L, glucose 10.0-50.0 g / L, yeast extract 1.0-6.0 g / L, potassium sulfate dihydrogen 0.5-4.0 g / L, magnesium sulfate anhydrous 8.0-15.0 g / L, monosodium glutamate 5.0-10.0 g / L, vitamin B12 0.001-0.01 g / L, and vitamin H 0.001-0.01 g / L, and the pH value is 3.5-7.0.

[0012] Compared with the prior art, the β-1,3-glucan yield of the euglena strain ZC-1007 of the present application is very high, after 72 hours of mixed culture and 12 hours of light-free induction, the biomass of the euglena strain ZC-1007 is as high as 121 g / L, the β-1,3-glucan content is as high as 750 mg / g of dry matter, and the β-1,3-glucan yield is as high as 1.08 g / (L.h), thereby having a broad application prospect in the fields of medicine, food, cosmetics, and feed, and providing reliable biological resources for green and pollution-free production of β-1,3-glucan.

[0013] The euglena strain ZC-1007 has been preserved in the Guangdong Microbial Culture Collection Center, located at the 5th Floor of the Experimental Building, Institute of Microbiology, Guangdong Academy of Sciences, 100 Middle Martyrs Road, Yuexiu District, Guangzhou, Guangdong, China, with a postal code of 510000, with a preservation number of GDMCC No. 66479, and a preservation date of June 6, 2025, and the taxonomic name of the strain is Euglena gracilis . BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a plate picture of the algal strain of the present application.

[0015] Figure 2 It is a microscope diagram of the algal strain of the present application.

[0016] Figure 3Monitoring data of the process of optimizing the mixed culture conditions of the high β-1,3-glucan-producing euglena of Example 3 in the present application.

[0017] Figure 4 A biomass accumulation curve and a target product β-1,3-glucan accumulation column chart during the culture process of the algal strain of the present application. Different lowercase letters indicate that there is a significant difference (P ≤ 0.01) between the products of GDMCC No. 63861 and ZC-1007 algal strains at different culture stages. DETAILED DESCRIPTION

[0018] The technical solutions of the present application will be described below in conjunction with specific embodiments, but those skilled in the art should understand that the embodiments described below are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present application.

[0019] In one aspect, the present application provides a high β-1,3-glucan-producing euglena strain ZC-1007, which has a preservation number of GDMCC No. 66479 and is preserved in the Guangdong Microbial Culture Collection Center.

[0020] In an embodiment of the present application, the culture characteristics of the algal strain ZC-1007 are smooth algal colony surface and neat edges on the plate, and the cell morphology under the microscope shows a shuttle shape with a cell size of about 35 μm, and the cell contents can be clearly observed as green oval particles.

[0021] In an embodiment of the present application, after mixed culture at 30°C for 84 hours, the biomass of the euglena strain ZC-1007 is as high as 121 g / L, the β-1,3-glucan content is as high as 750 mg / g of dry matter, and the β-1,3-glucan yield is as high as 1.08 g / (L.h).

[0022] In another aspect, the present application provides a method for preparing the euglena strain ZC-1007, comprising the following steps:

[0023] S1. Providing an original algal strain of euglena;

[0024] S2. Culturing the original algal strain in a modified CM culture medium under a constant light intensity of 30 µmol·m -2 ·s -1 for 24 hours, and culturing for 18-36 hours;

[0025] S3. Picking fast-growing single algae for purification treatment and subculture, screening, and obtaining the high β-1,3-glucan-producing euglena strain ZC-1007.

[0026] In an embodiment of the present invention, in step S2, the modified CM culture medium comprises: ammonium hydrogen phosphate 0.5–2.0 g / L, calcium chloride dihydrate 0.01–0.05 g / L, sodium citrate 0.5–1.0 g / L, boric acid 2.0–4.0 mg / L, ferric sulfate heptahydrate 1.0–4.0 mg / L, manganese chloride tetrahydrate 1.0–2.0 mg / L, cobalt sulfate heptahydrate 1.0–2.0 mg / L, zinc sulfate heptahydrate 0.1–0.5 mg / L, sodium molybdate dihydrate 0.1–0.4 mg / L, anhydrous copper sulfate 0.01–0.05 mg / L, glucose 10.0–50.0 g / L, yeast extract 1.0–6.0 g / L, potassium dihydrogen sulfate 0.5–4.0 g / L, anhydrous magnesium sulfate 8.0–15.0 g / L, monosodium glutamate 5.0–10.0 g / L, and vitamin B1. 12 0.001–0.01 g / L of ammonium diphosphate and vitamin H 0.001–0.01 g / L, with a pH of 3.5–7.0. In a specific embodiment of the present invention, the modified CM culture medium contains 1.0 g / L of ammonium hydrogen phosphate, 0.02 g / L of calcium chloride dihydrate, 0.8 g / L of sodium citrate, 2.48 mg / L of boric acid, 3.0 mg / L of ferric sulfate heptahydrate, 1.8 mg / L of manganese chloride tetrahydrate, 1.5 mg / L of cobalt sulfate heptahydrate, 0.4 mg / L of zinc sulfate heptahydrate, 0.2 mg / L of sodium molybdate dihydrate, 0.02 mg / L of anhydrous copper sulfate, 20.0 g / L of glucose, 4.0 g / L of yeast extract, 1.0 g / L of potassium dihydrogen phosphate, 12.0 g / L of magnesium sulfate, 8.0 g / L of monosodium glutamate, and vitamin B1. 12 0.005 g / L of vitamin H and 0.005 g / L of vitamin H, pH value is 6.0.

[0027] In the embodiment of the present invention, during the cultivation process in step S2, continuous feeding is required to maintain a carbon-nitrogen ratio of 18:1, ensuring sufficient carbon and nitrogen sources in the cultivation environment. The modified CM culture medium needs to be uniformly mixed and aerated to ensure the optimal growth environment for the algae.

[0028] In an embodiment of the present invention, the culture time in step S2 can be 18 to 36 hours. In a specific embodiment, the culture time can be 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours, with 24 hours being preferred.

[0029] In an embodiment of the present invention, the culture temperature in step S2 can be 25–35 °C. In a specific embodiment, the culture temperature can 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, or 35 °C, preferably 30 °C.

[0030] In an embodiment of the present invention, in step S3, the number of generations can be increased to 10.

[0031] 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.

[0032] 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.

[0033] Example 1: Screening and optimization of culture conditions for Euglena with high β-1,3-glucan production

[0034] Original algal strain provided: Euglena slenderis Euglena gracilis The original algal strain was selected from freshwater resources in Yantai, Shandong Province by Beijing Zaochen Biotechnology Co., Ltd. on October 20, 2024, and used as the base algal strain.

[0035] Culture conditions: The original algal 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℃. 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 phosphate, 12.0 g / L magnesium sulfate, 8.0 g / L monosodium glutamate, and vitamin B1. 12 0.005 g / L of nitrogen and phosphorus, 0.005 g / L of vitamin H, and pH 6.0. The appropriate amounts 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.

[0036] High-efficiency screening: single algae with rapid growth were picked for purification and subculture. Through multiple screenings, the euglena strain ZC-1007 with high β-1, 3-glucan yield was finally obtained. The single algae of the euglena strain ZC-1007 grew rapidly (see Figure 1 ), and the cell morphology was observed under a microscope to be green and in a shuttle shape (see Figure 2 ).

[0037] Example 2: Mixotrophic culture of euglena with high β-1, 3-glucan yield

[0038] Culture conditions: the euglena ZC-1007 was inoculated in the modified CM culture medium, the pH value of the culture medium was adjusted to 6.0, the culture temperature was set to 30°C, and the light intensity was 30 µmol·m -2 ·s -1 for 24 hours. The culture medium in the bioreactor was uniformly mixed and aerated to ensure the optimal growth environment of the algae strain. The formula of the modified CM culture medium is described in Example 1. The feed was supplemented during the culture to ensure sufficient carbon and nitrogen sources in the culture environment.

[0039] Mixotrophic culture: the mixotrophic culture was performed in the bioreactor, and the culture time was 84 hours. The biomass and β-1, 3-glucan content in the culture solution were determined as follows.

[0040] Biomass calculation method:

[0041] Biomass = dry matter weight (g) per unit volume (L)

[0042] β-1, 3-glucan content calculation method:

[0043] β-1, 3-glucan yield = (biomass dry matter weight * dry matter glucan content) / fermentation time (h).

[0044] 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 / (L.h).

[0045] Example 3: Optimization of mixotrophic culture conditions of euglena with high β-1, 3-glucan yield

[0046] Culture conditions: the euglena ZC-1007 was inoculated in the modified CM culture medium, the pH value of the culture medium was adjusted to 6.0, the culture temperature was set to 30°C, and the light intensity was 30 µmol·m -2 ·s -1Culture. The medium in the bioreactor was mixed evenly and aerated to ensure the optimal growth environment for the algae strain. The formula of the modified CM medium is described in Example 1. Continuous feeding was performed during the culture to maintain the carbon-nitrogen ratio at 18:1 and ensure sufficient carbon and nitrogen sources in the culture environment.

[0047] Mixed culture. The mixed culture was performed in the bioreactor for 72 hours. After the mixed culture, 12 hours of light-free induction was performed. The process monitoring data are shown in Figure 3 The biomass and β-1,3-glucan content in the culture solution were determined as follows.

[0048] Biomass calculation method:

[0049] Biomass = dry matter weight of algae solution per unit volume (g) / unit volume (L)

[0050] β-1,3-glucan content calculation method:

[0051] β-1,3-glucan yield = (biomass dry matter weight * dry matter glucan content) / fermentation time (h).

[0052] The results show that the biomass is 121 g / L, the β-1,3-glucan content is 750 mg / g, and the β-1,3-glucan yield is 1.08 g / (L.h). Compared with Example 2 without induction measures, the biomass increases by 23.47%, and the β-1,3-glucan yield increases by 38.46%.

[0053] Comparative Example 1: Comparison with E. gracilis strain GDMCC No. 63861

[0054] Experimental design: E. gracilis strain GDMCC No. 63861 and E. gracilis strain ZC-1007 of the application were inoculated in the modified CM medium (the formula of the modified CM medium is described in Example 1), the pH value of the medium was adjusted to 6.0, the culture temperature was set to 30°C, and the light intensity was 30 µmol·m -2 ·s -1 Culture. The medium in the bioreactor was mixed evenly and aerated to ensure the optimal growth environment for the algae strain. The mixed culture was performed in the bioreactor for 72 hours, and 12 hours of light-free induction was performed. The biomass and β-1,3-glucan content in the culture solution were determined.

[0055] Results analysis: the biomass of GDMCC No. 63861 and ZC-1007 after 84h culture was 93 g / L and 121 g / L respectively, the content of β-1, 3-glucan was 680 mg / g and 750 mg / g respectively, and the yield of β-1, 3-glucan was 0.75 g / (L.h) and 1.08 g / (L.h) respectively. Compared with GDMCC No. 63861, the yield of β-1, 3-glucan of ZC-1007 increased by 44.0%. During the culture process, the content of β-1, 3-glucan in the products produced by GDMCC No. 63861 and ZC-1007 was gradually increased with the extension of time, and after 84h culture, the content of β-1, 3-glucan in the products produced by ZC-1007 was 10.82% higher than that of GDMCC No. 63861, and the results are shown in Table 1. The biomass accumulation curve and the target product β-1, 3-glucan accumulation column chart of the algal strain of the application during the culture process are shown in Figure 4 Different small letters indicate that there is a significant difference (P ≤0.01) between the products produced by GDMCC No. 63861 and ZC-1007 algal strains at different culture stages.

[0056] Table 1.

[0057] Comparison of β-1, 3-glucan content between ZC-1007 and GDMCC No. 63861 during culture process

[0058]

[0059] The above shows and describes the basic principles and main features of the application and the advantages of the application. It should be understood by those skilled in the art that the application is not limited by the above examples, and the above examples and descriptions in the specification are only to illustrate the principles of the application, and various changes and improvements can be made to the application without departing from the spirit and scope of the application, and these changes and improvements all fall within the scope of the claimed application. The scope of protection of the application is defined by the appended claims and their equivalents.

Claims

1. Euglena slendera, a high producer of β-1,3-glucan ( Euglena gracilis Algal strain ZC-1007 is characterized by, The preservation number of the algal strain is GDMCC No. 66479.

2. The method for culturing the *Euglena slenderis* strain ZC-1007 as described in claim 1, characterized in that, The steps include: inoculating Euglena ZC-1007 into a modified CM medium, adjusting the pH of the medium to 6.0, setting the culture temperature to 30℃, and maintaining a constant light intensity of 30 µmol·m² for 24 hours. -2 ·s -1 The culture was carried out for 72 hours, followed by 12 hours of induction in the dark. Continuous feeding was performed during the culture process to maintain a carbon-to-nitrogen ratio of 18:

1. The modified CM medium contained 1.0 g / L ammonium diphosphate, 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 phosphate, 12.0 g / L magnesium sulfate, 8.0 g / L monosodium glutamate, and vitamin B1. 12 0.005 g / L of vitamin H and 0.005 g / L of vitamin H, pH value is 6.

0.

3. The method according to claim 2, characterized in that, After 12 hours of induction in the dark, the biomass of the Euglena strain 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).

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