Xanthophyll-rich Chlorella sorojin as well as screening method and application thereof

Abstract

The invention relates to lutein-rich chlorella sorojin as well as a screening method and application thereof, and belongs to the technical field of algae. The chlorella sorojin is preserved in the China Center for Type Culture Collection on May 8, 2025, and the preservation number of the chlorella sorojin is CCTCC NO: M 2025999. The screened Chlorella sorojin rich in xanthophyll shows a significant growth rate in a BG11 culture medium, can effectively synthesize and accumulate xanthophyll in a metabolic process, and can be used as a xanthophyll source with great potential. The chlorella sorojin has important application potential in the fields of food, health care products and the like, and particularly can provide powerful support for sustainable development of related industries in the aspect of development of natural products.

Description

TECHNICAL FIELD

[0002] The application belongs to the technical field of algae, and particularly relates to a sororin chlorella rich in lutein as well as a screening method and application thereof. BACKGROUND

[0003] Lutein is an important natural carotenoid, which has the effects of antioxidation, anti-inflammation and retinal protection (blue light filtering and prevention of macular degeneration), and is widely used in the fields of food, health products, medicine and cosmetics. At present, about 95% of lutein in the world is obtained from marigold petal extraction, but marigold planting has problems such as long cycle, large labor, dependence on land and climate conditions, which limits the further development of natural lutein. As a natural lower plant containing lutein, chlorella has the advantages of fast growth, simple culture condition and all-year production, and is considered as a potential alternative source for lutein production. Screening of chlorella strains with high lutein yield is a prerequisite for efficient production, and has important scientific significance and commercial value. SUMMARY

[0004] A first object of the present application is to provide a sororin chlorella rich in lutein, which has a lutein content of 6.20 mg / g (dry weight) and a lutein yield of 26.41 mg / L under heterotrophic conditions, and a lutein content of 8.37 mg / g (dry weight) under autotrophic conditions.

[0005] A second object of the present application is to provide a screening method of the sororin chlorella rich in lutein.

[0006] A third object of the present application is to provide an application of the sororin chlorella rich in lutein.

[0007] In order to achieve the above objects, the technical scheme adopted by the present application is as follows: The sororin chlorella rich in lutein is preserved in China Center for Type Culture Collection on May 8, 2025, and has a preservation number of CCTCC NO: M 2025999.

[0008] The screening method of the sororin chlorella rich in lutein comprises the following steps: (1) water samples are taken, and the taken water samples are enriched and cultured in BG11 liquid medium, then diluted and spread, and then cultured until algal colonies appear, and single algal colonies are picked and purified; (2) the purified single algal colonies are transferred to BG11 liquid medium to obtain seed liquid; (3) the seed liquid is inoculated into BG11 liquid medium and cultured on a shaking table, and an advantageous algal strain is screened according to the lutein content and growth condition; (4) The dominant algae strain obtained in step (3) is identified in terms of species by using morphological identification and molecular biological identification.

[0009] Further, the enrichment culture condition in step (1) is that the temperature is 26-28 DEG C, the light intensity is 7000-9000 Lux, the humidity is 54-56 %, the light-dark cycle ratio is 12 h:12 h, and the time is 7-10 days.

[0010] Further, the dilution and coating step in step (1) is that the water sample after enrichment culture is gradiently diluted to obtain a diluent, and the diluents of various gradients are coated on the BG11 solid culture medium.

[0011] Further, the gradient dilution multiples are 10 -3 , 10 -4 , and 10 -5 .

[0012] Further, the culture condition for obtaining the seed liquid in step (2) is that the temperature is 26-28 DEG C, the rotation speed is 148-152 rpm, the light intensity is 24-26 %, and the culture time is 2-3 days.

[0013] Further, the inoculation amount of the seed liquid in step (3) accounts for 4-6 % of the volume of the BG11 liquid culture medium.

[0014] Further, the shaker culture condition in step (3) is that the temperature is 26-28 DEG C, the rotation speed is 148-152 rpm, the light intensity is 24-26 % or 0 %, and the culture time is 6-8 days.

[0015] Application of a lutein-rich Chlorella sorokiniana in the production of lutein.

[0016] The beneficial effects of the present application are: The lutein-rich Chlorella sorokiniana screened in the present application has a significant growth rate in the BG11 culture medium, can effectively synthesize and accumulate lutein in the metabolic process, and can be used as a very potential source of lutein. The Chlorella sorokiniana has important application potential in the fields of food and health care products, especially in the development of natural products, and can provide strong support for the sustainable development of related industries.

[0017] Compared with the conventional multiple plate streaking, the method of multiple dilution and coating in the present application can more efficiently obtain pure single algae. The Chlorella sorokiniana screened in the present application still has a high lutein content and yield under heterotrophic conditions, and the screened Chlorella sorokiniana has application potential in lutein production. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure for the dry weight, lutein content and yield of the advantageous algae strain in Example 1; Figure 2 Figure for the morphological structure of the lutein-rich Chlorella sorokiniana in Example 1 under an optical microscope 100X oil lens; Figure 3 Figure for the electropherogram of the lutein-rich Chlorella sorokiniana in Example 1; Figure 4 Figure for the phylogenetic tree of Chlorella sorokiniana CH7 constructed based on 18S rDNA gene sequence using the neighbor-joining method; Figure 5 Figure for the protein and polysaccharide content of the lutein-rich Chlorella sorokiniana in Example 2; Figure 6 Figure for the fatty acid types and content of the lutein-rich Chlorella sorokiniana in Example 2. DETAILED DESCRIPTION

[0019] The present application will be further described below in conjunction with the embodiments of the present application and the accompanying drawings.

[0020] The formula of the BG11 liquid culture medium is as follows: sodium nitrate 1.5 g / L, potassium phosphate dibasic 0.04 g / L, magnesium sulfate heptahydrate 0.075 g / L, calcium chloride dihydrate 0.036 g / L, citric acid 0.006 g / L, ferric ammonium citrate 0.006 g / L, ethylenediaminetetraacetic acid (EDTA) 0.001 g / L, sodium carbonate 0.02 g / L, boric acid 2.86 mg / L, manganese chloride monohydrate 1.81 mg / L, zinc sulfate heptahydrate 0.222 mg / L, copper sulfate pentahydrate 0.079 mg / L, sodium molybdate dihydrate 0.39 mg / L, cobalt nitrate hexahydrate 0.049 mg / L, pH 7.1±0.1.

[0021] The autotrophic culture is the BG11 liquid culture medium, and the light intensity in the simultaneous shaking culture is 25 %. The heterotrophic culture is the BG11 liquid culture medium with 20 g / L of glucose, and the light intensity in the simultaneous shaking culture is 0 %.

[0022] Example 1 The screening method of the lutein-rich Chlorella sorokiniana in Example 1 includes the following steps: (1) Collection and preservation of water samples: 25# plankton nets were used to collect water samples at different places in Chenghai Lake in Lijiang City, Yunnan Province, and the collected water samples were stored at 4 ℃ and enriched as soon as possible.

[0023] (2) Microalgae separation and purification: The water sample of step (1) was placed in a BG11 liquid medium and placed in a light incubator with a temperature of 28°C, a light intensity of 8000 Lux, a humidity of 55%, and a light-dark cycle ratio of 12 h:12 h for enrichment culture for 10 days. The water sample after enrichment culture was gradient diluted with sterile water to obtain a diluent, which was diluted by 10 -3 ,10 -4 ,10 -5 times, respectively. 100 μL of each gradient diluent was coated on a BG11 solid medium, followed by light culture. When algae appeared, single algae with different morphologies, sizes, and colors were picked and coated on a BG11 solid medium for dilution and coating. The process was repeated 1-2 times until the microalgae were purified. The preparation method of the BG11 solid medium was to add 1.2% agar powder to the BG11 liquid medium.

[0024] (3) Monoclonal algal culture: The purified single algae in step (2) were transferred to 20 mL of BG11 liquid medium and cultured at 28°C, 150 rpm, and 25% light intensity for 3 days as a seed liquid. The seed liquid was inoculated into 50 mL of BG11 liquid medium at a 5% inoculation amount, and autotrophic and heterotrophic culture was carried out in a 28°C, 150 rpm shaker to obtain an algal liquid. After 7 days of culture, 30 mL of algal liquid was collected, centrifuged at 12000 rpm at 4°C for 3 min, and the supernatant was removed to obtain a precipitate. The precipitate was resuspended with sterile water for 3 times to obtain algal mud, which was vacuum freeze-dried to obtain algal powder. The weight and dry weight were calculated.

[0025] (4) Lutein detection: 10 mg of algal powder was added with grinding beads, and a freeze grinding instrument was used for wall breaking treatment. After wall breaking, 1 mL of a mixed solution of methanol:dichloromethane at a volume ratio of 3:1 was added, and the mixture was fully vortexed and ultrasonicated at 40 kHz and 4°C in the dark. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter, and the lutein content was detected by high performance liquid chromatography.

[0026] The high performance liquid chromatography detection conditions were: Agilent ValueLab LC GP-C30 chromatographic column, ultraviolet detector, 450 nm, 25°C, sample size 10 μL, flow rate 1 mL / min, mobile phase methanol:dichloromethane:acetonitrile:water=67.5:22.5:9.5:0.5 (v / v) mixed solution, isocratic elution, detection time 15 min. According to the lutein content and growth condition, one dominant algal strain was selected, and the results are shown in Figure 1 .

[0027] (5) Algal strain identification: Figure 2The dominant algal strain was preliminarily judged as Chlorella under the morphology structure of optical microscope 100X oil lens, green color and circular shape, single cell. Further molecular identification was carried out, and the specific process was as follows: the dominant algal strain was picked and dropped into a 200 μL EP tube containing 20 μL sterile water, and was repeatedly blown to disperse the algal strain. The PCR tube was placed in a PCR instrument, and was heated at 100 ℃ for 20 min. After cooling, a DNA template was obtained. The 18S rDNA amplification forward primer was 18F: 5'-CCAACCTGGTTGATCCTGCCAGTA-3', the reverse primer was 18R: 5'-CCTTGTTACGACTTCACCTTCCTCT-3', and the PCR reaction condition was 95 ℃ pre-denaturation for 5 min, 95 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 2 min, 34 cycles, and a PCR amplification fragment was obtained. The amplification product was detected by 1.0 % agarose gel electrophoresis, and the result is shown in Figure 3 The PCR product was sequenced, the sequencing result was analyzed by Blast comparison in the GenBank database, and the software MEGA was used to construct a phylogenetic tree. Combined with morphological analysis, the microalgae was identified as Chlorella sorokiniana, and was named as Chlorella sorokiniana CH7.

[0028] Chlorella sorokiniana CH7 was preserved in the China Center for Type Culture Collection unit located in Wuhan, Hubei Province, Wuchang District, Luo Jia Mountain, Bajia Road on May 8, 2025, and the preservation number was CCTCC NO: M2025999.

[0029] Example 2 Chlorella sorokiniana CH7 in example 1 was cultured at 28 ℃ under 25 % light intensity for 7 days, and after multiple passages, the lutein content of Chlorella sorokiniana CH7 under autotrophic conditions reached 8.37 mg / g. Chlorella sorokiniana CH7 in example 1 was cultured under heterotrophic conditions at 28 ℃ in the dark for 7 days, and the lutein content was as high as 6.20 mg / g (dry weight). The protein content of Chlorella sorokiniana CH7 was determined according to the national standard GB / T33862-2017; the polysaccharide content of Chlorella sorokiniana CH7 was determined by the phenol-sulfuric acid method; the fatty acid types and content of Chlorella sorokiniana CH7 were extracted and detected by gas chromatography, and the test results are shown in Figure 5 , Figure 6 It can be seen from Figure 5 that the lutein-rich Chlorella sorokiniana in the application has a protein content of 52.59 % of the cell dry weight under autotrophic conditions, a polysaccharide content of 100.52 mg / g (dry weight), a protein content of 42.44 % of the cell dry weight under heterotrophic conditions, and a polysaccharide content as high as 145.26 mg / g.Figure 6 It can be seen that the total fatty acid content under heterotrophic conditions is significantly greater than that under autotrophic conditions, wherein C16:0, C16:1, C18:1 and C18:2 are the main fatty acid components. The Chlorella sorokiniana rich in lutein of the application has similar contents of C16:0 and C16:1 under heterotrophic conditions, which are 34.23 mg / g (dry weight) and 33.84 mg / g (dry weight) respectively, the content of C18:1 is 49.49 mg / g (dry weight), and the content of C18:2 is 58.11 mg / g (dry weight). C16:0, i.e. palmitic acid, can be rapidly metabolized by the liver into ketones to provide rapid energy for the brain. C16:1, i.e. palmitoleic acid, can protect the cardiovascular system, reduce cholesterol, and reduce the risk of cardiovascular disease. C18:1, i.e. oleic acid, can regulate blood lipids and has an anti-inflammatory effect, which helps to prevent atherosclerosis. C18:2, i.e. linoleic acid, can regulate the immune system and maintain skin health.

Claims

1. A strain of Chlorella sorokiniana enriched with lutein, characterized in that, The Chlorella sorokiniana is preserved in China Center for Type Culture Collection on May 8, 2025, and the preservation number is CCTCC NO: M 2025999.

2. A method of screening for a lutein-rich Chlorella sorokiniana according to claim 1, characterized by, The method comprises the following steps: (1) taking water samples, and then performing gradient dilution on the water samples after enrichment culture in BG11 liquid medium, and then performing dilution coating, and then culturing to obtain algae, and then picking single algae for purification; (2) culturing the purified single algae in BG11 liquid medium to obtain a seed liquid; (3) inoculating the seed liquid into BG11 liquid medium, and then performing shaking culture, and then screening a dominant algae strain according to the content of lutein and the growth condition; (4) performing species identification on the dominant algae strain obtained in step (3) by using morphological identification and molecular biological identification.

3. The method of screening for lutein-rich Chlorella sorokiniana according to claim 2, characterized in that, The enrichment culture in step (1) is performed under the following conditions: the temperature is 26-28 ℃, the light intensity is 7000-9000 Lux, the humidity is 54-56 %, the light / dark cycle ratio is 12 h:12 h, and the time is 7-10 d.

4. The method of screening of lutein-rich Chlorella sorokiniana according to claim 2, characterized in that, The dilution coating in step (1) is performed by: performing gradient dilution on the water samples after enrichment culture to obtain a diluent, and then coating the diluents of different gradients on BG11 solid medium.

5. The method of screening for lutein-rich Chlorella sorokiniana according to claim 4, characterized in that, The fold of the gradient dilution is 10, respectively -3 , 10 -4 , 10 -5 .

6. The method of screening of lutein-enriched Chlorella sorokiniana according to claim 2, characterized in that, The culture condition for obtaining the seed liquid in step (2) is: the temperature is 26-28 ℃, the rotation speed is 148-152 rpm, the light intensity is 24-26 %, and the culture time is 2-3 d.

7. The method of screening of lutein-enriched Chlorella sorokiniana according to claim 2, characterized in that, The inoculation amount of the seed liquid in step (3) accounts for 4-6 % of the volume of the BG11 liquid medium.

8. The method of screening of lutein-enriched Chlorella sorokiniana according to claim 2, characterized in that, The shaking culture in step (3) is performed under the following conditions: the temperature is 26-28 ℃, the rotation speed is 148-152 rpm, the light intensity is 24-26 % or 0 %, and the culture time is 6-8 d.

9. Use of the lutein-rich Chlorella sorokiniana in claim 1 in the production of lutein.

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