Space desert algae bred in space and application of space desert algae

Abstract

The invention discloses a strain of desert algae improved by space breeding and application of the desert algae. According to the invention, a strain of desert algae is obtained through variation induced by a space environment and ground screening, and the strain of desert algae shows high growth rate and excellent outdoor adaptability, and can efficiently accumulate two bioactive substances, namely algal polysaccharides and flavones, which have important commercial and health values. Therefore, the strain of the desert algae shows better application potential in the aspects of producing high-quality desert algae powder, polysaccharide, flavone and the like.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a space desert algae bred in space and applications thereof. Background Art

[0002] Desert algae is a type of algae that can survive in extremely arid and high-salt environments, and has excellent drought resistance, salt resistance, and UV resistance. This algae can synthesize a variety of commercially valuable bioactive substances, such as polysaccharides and flavonoids. Polysaccharides are macromolecular compounds composed of multiple monosaccharide molecules, which have the functions of regulating immune function and lowering blood sugar. Flavonoids are a class of natural compounds with multiple effects such as antioxidant, anti-inflammatory, and anti-cancer. However, the slow growth rate and small biomass of desert algae in the natural state limit its commercial production. The technical bottlenecks of deep processing of desert algae are: 1) the lack of high-performance engineered desert algae strains, and the lack of desert algae that produces high polysaccharide and flavonoid content; 2) the slow growth rate of desert algae increases the cost of desert algae raw materials.

[0003] Space breeding technology provides a method to cultivate new crop varieties through genetic variation in the special environment of space, which helps to increase the growth rate and yield of crops. Space breeding of microalgae is a relatively efficient artificial breeding method. It uses the mutagenic effects of cosmic radiation, microgravity and complex electromagnetic environment on microalgae to cause genetic variation in microalgae cells, obtain large mutant strains at one time, and then screen out algae strains with fast growth rate, high biomass, stable genetic traits and development value from the mutant strains. Compared with traditional breeding, the biggest advantage of space mutagenesis breeding is the high probability of variation, wide range of variation and short breeding cycle, which can create high-quality germplasm resources in a relatively short period of time. However, there is currently no research on space breeding of desert algae to increase the content of its bioactive substances.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a space desert algae bred in space and its application. The desert algae not only grows fast and has a large biomass, but can also grow well under strong adaptability conditions outdoors, and can efficiently accumulate algae polysaccharides and flavonoids.

[0006] In a first aspect, the present invention provides a desert algae, which is named Hassallia sp. P06, with a preservation number of CCTCC M 20242054, a preservation time of September 23, 2024, a preservation unit of China Center for Type Culture Collection (CCTCC), and a preservation address of Wuhan University, Wuhan, China.

[0007] The desert algae in the present invention is a cyanobacteria belonging to the genus Hassallia sp., which grows in an extremely arid and high-salt environment. Studies have found that desert algae contain rich biologically active substances. On this basis, the present invention obtains Hassallia sp. P06 through space environment induction and screening. Experiments have shown that Hassallia sp. P06 is significantly superior to wild-type desert algae in growth rate and accumulation of biologically active substances. Therefore, the Hassallia sp. P06 provided by the present invention has good commercial development potential and broad application prospects.

[0008] In a second aspect, the present invention also provides the use of the above-mentioned desert algae in the production of algae polysaccharides.

[0009] In a third aspect, the present invention also provides the use of the above-mentioned desert algae in the production of flavonoid compounds.

[0010] In a fourth aspect, the present invention also provides the use of the above-mentioned desert algae in the production of desert algae powder.

[0011] In a fifth aspect, the present invention also provides the use of the above-mentioned desert algae in the preparation of cosmetics, food, medicine and feed.

[0012] The Hassallia sp. P06 obtained by induction and screening in the space environment of the present invention can be applied to the production of related algae polysaccharides, flavonoid compounds and algae powder, and the produced algae polysaccharides and flavonoid compounds can be further applied to the preparation of cosmetics, food, medicines and other products, and the algae polysaccharides, flavonoid compounds and algae powder can be further applied to the preparation of feed and other products.

[0013] In a seventh aspect, the present invention also provides an algae biological product, which comprises the above-mentioned desert algae.

[0014] In an eighth aspect, the present invention also provides a method for producing algae polysaccharides, which produces biologically active ingredients by culturing the above-mentioned desert algae.

[0015] In a ninth aspect, the present invention also provides a method for producing flavonoid compounds, which produces flavonoid compounds by culturing the above-mentioned desert algae.

[0016] The present invention has the following beneficial effects:

[0017] The present invention obtains a desert algae Hassalliasp.P06 through mutation induced by space environment and ground screening. The desert algae shows a fast growth rate and excellent outdoor adaptability, and can efficiently accumulate algae polysaccharides and flavonoids, two biologically active substances with important commercial and health values. Therefore, the desert algae Hassalliasp.P06 shows good application potential in producing high-quality desert algae powder, polysaccharides and flavonoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 It is the cell morphology of desert algae;

[0020] Figure 2 For the screening of desert algae Hassallia sp.P06 strain;

[0021] Figure 3 The comparison of the polysaccharide content between the desert algae Hassallia sp.P06 strain and the wild type desert algae strain;

[0022] Figure 4 This is a comparison of the flavonoid content in the desert algae Hassallia sp.P06 strain and the wild-type desert algae strain. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0024] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0025] Example 1

[0026] This example is the breeding and trait evaluation of space desert algae P06 strain

[0027] 1) Space launch and initial screening: First, the desert algae (Hassalli asp.) strain that has been carried into the space environment is rejuvenated and amplified in a 50 mL Erlenmeyer flask until it reaches an appropriate cell density, and then the initial screening process is carried out.

[0028] 2) Isolation of single-cell mutants: Single algae filaments were picked from the algae culture and inoculated into 96-well plates under sterile conditions. Each well contained an independent mutant and was cultured under appropriate light conditions for about 15 days. During this period, the color changes of each well were observed, and the optical density (OD750) at a wavelength of 750nm was measured using a microplate reader to evaluate the growth of the algae.

[0029] 3) Propagation and evaluation of mutant strains: Based on the 96-well plate culture results, algal strains with significantly accelerated growth rates were selected and transferred to 50 mL Erlenmeyer flasks for further propagation. Subsequently, a comprehensive performance evaluation of these candidate mutant strains was performed using 500 mL Erlenmeyer flasks.

[0030] 4) Trait selection and determination: Through detailed analysis of the mutant strains in the flask culture stage, including measurement of biomass concentration, polysaccharides and flavonoids, a mutant strain with excellent comprehensive traits was finally screened out and named Hassallia sp. P06, hereinafter referred to as P06 strain.

[0031] The biomass acquisition and detection method is as follows: after culturing the space desert algae P06 strain and the original wild desert algae without space mutation, add them to the centrifuge tube and centrifuge at 4000r / min for 10 minutes, discard the supernatant and wash with distilled water, repeat twice. Then, put the washed algae into an 85℃ oven to dry to constant weight, and measure twice to get the average value to obtain accurate biomass, so as to effectively evaluate the growth and biomass accumulation of desert algae.

[0032] like Figure 1 As shown in the figure, the morphological characteristics of the P06 strain are as follows: the strain presents a typical branching shape, with the diameter of the algal filaments ranging from 5-10 μm, forming regular branches and 200-500 μm in length, without forming heterocysts and posterior wall spores, and the gelatinous sheath covering the cells is extremely thin or almost invisible.

[0033] Figure 2 This is a comparison chart of the biomass detection results of different algae strains during the screening process. It can be seen from the figure that the biomass concentration of the P06 strain is the highest.

[0034] Example 2

[0035] This example is the acquisition and detection of biologically active substances of the space desert algae P06 strain

[0036] Medium preparation: In order to promote the growth of the P06 strain and obtain high-value bioactive substances, this example uses a culture medium with a specific ratio. The specific formula is as follows:

[0037] In each liter of culture medium, add sodium nitrate (NaNO3): 1.5g, dipotassium hydrogen phosphate (K2HPO4): 0.04g, magnesium sulfate (MgSO4·7H2O): 0.075g, calcium chloride (CaCl2·2H2O): 0.036g, sodium carbonate (Na2CO3): 0.02g, boric acid (H3BO3): 2.86mg, manganese chloride (MnCl2·4H2O): 1.86mg, sodium molybdate (Na2MoO4·2H2O): 0.22mg, copper sulfate (CuSO4·5H2O): 0.012mg, zinc sulfate (ZnSO4·7H2O): 0.022mg and cobalt chloride (CoCl2·6H2O): 0.013mg, mix thoroughly and then sterilize at high temperature to ensure sterility.

[0038] Culture conditions and harvest:

[0039] Culture objects: The space desert algae P06 strain and the original wild-type desert algae without space mutation were used as the control group, and inoculated into the above-prepared culture medium in equal amounts.

[0040] Culture environment: The light intensity was set at 2500 Lux and the culture was carried out at a constant temperature of 28°C for 15 days. The obtained space desert algae P06 strain was then tested for various bioactive substances under the same conditions as the original wild-type desert algae that had not been induced by space mutation.

[0041] Polysaccharide content detection: Under the action of concentrated sulfuric acid, polysaccharides are hydrolyzed into monosaccharides, and quickly dehydrated to form aldehyde derivatives, which are then condensed with phenol to form orange-yellow compounds with stable color. At a wavelength of 488nm and within a certain concentration range, its absorbance is linearly proportional to the polysaccharide content, so its absorbance can be measured using a spectrophotometer.

[0042] (1) Establishment of standard curve (phenol-sulfuric acid method): Accurately weigh 10 mg of anhydrous glucose standard substance and place it in a 100-mL volumetric flask. Add appropriate amount of distilled water to dissolve it, dilute to the scale, and shake well. At this time, the glucose concentration is 0.1 mg / mL.

[0043] Take 0, 100 μL, 200 μL, 400 μL, 600 μL, 800 μL and 1000 μL of the above-mentioned reference solution, respectively place them in stoppered test tubes, dilute to 1 mL, continue to add 1 mL of 5% phenol solution respectively, shake well, quickly add 5 mL of concentrated sulfuric acid (add slowly), shake well, keep warm in a boiling water bath for 10-15 min, take out, cool to room temperature, use the corresponding reagent as a blank control, measure the absorbance at a wavelength of 488 nm according to the UV-Vis spectrophotometry, and draw a standard curve with the absorbance value as the ordinate and the glucose concentration as the abscissa.

[0044] (2) Determination of algae polysaccharide content: 1) Accurately weigh 5g of algae powder, dissolve in 30mL of water, and mix thoroughly; 2) Ultrasonic power 650W, ultrasonic for 30min, ultrasonic destroy the cell structure, and then place in 85℃ water bath for 2h; 3) Centrifuge at 5,000r / min for 10min, the supernatant is the algae polysaccharide solution; 4) Add 4 times the volume of 95% ethanol, precipitate at 4℃ for 24h; 5) Centrifuge at 5,000r / min for 10min, collect the precipitate, and then dissolve the precipitate with an appropriate amount of water to obtain a crude polysaccharide extract; 6) According to the absorbance value of the sample extract, use the standard curve to calculate the algae polysaccharide content. The detection results of algae polysaccharides are as follows: Figure 3 shown.

[0045] Flavonoid content detection: The content is detected by UV spectrophotometry using the NaNO2-Al(NO3)3 colorimetric method of flavonoid compounds. The principle is that flavonoids form an integration with Al(NO3)3 in the presence of weak alkalinity and NaNO2. At this time, adding NaOH solution will show orange-red color, and there is a stable characteristic absorption peak at 510nm.

[0046] (1) Preparation of standard curve: Accurately weigh 5 mg of rutin standard, dilute to 50 mL with deionized water, and prepare a 0.2 mg / mL standard solution. Take 0, 1, 2, 3, 4, 5, and 6 mL of rutin standard solution into a 25 mL brown volumetric flask, add 70% ethanol aqueous solution to each volume to 10 mL, then add 1 mL of 5% NaNO2 solution, shake well and let stand for 6 min, add 1 mL of 10% Al(NO3)3 solution, shake well and let stand for 6 min, add 10 mL of 10% NaOH solution, shake well and let stand for 15 min, and distilled water to 25 mL. According to the UV-Vis spectrophotometry, colorimetric determination is performed at a wavelength of 510 nm, and the reagent is used as a blank reference.

[0047] (2) Determination of flavonoids content in desert algae: 1) Accurately weigh 5g of algae powder, dissolve in 25mL of anhydrous ethanol, and mix thoroughly; 2) Ultrasonic power 650W, ultrasonic for 30min, ultrasonic destroys cell structure; 3) Centrifuge at 4500r / min for 15min, the supernatant is the flavonoids extract of desert algae; 4) Take 1mL of the extract, dilute to 10ml with 70% ethanol aqueous solution, add 1mL of 5% NaNO2 solution, shake well and let stand for 6min, add 1mL of 10% Al(NO3)3 solution, shake well and let stand for 6min, add 10mL of 10% NaOH solution, shake well and let stand for 15min, dilute to 25mL with distilled water; 5) According to the UV-visible spectrophotometry, measure the absorbance at a wavelength of 510nm, record and analyze the values. The detection results of desert algae flavonoids are as follows Figure 4 shown.

[0048] Comparison of results: The P06 strain showed a significant growth advantage, with a harvested biomass concentration of 0.793 g / L, accompanied by high levels of secondary metabolites, including polysaccharide content of 16.308 g / 100 g and flavonoid content of 0.0428 g / 100 g. In contrast, the wild-type desert algae in the control group had a harvested biomass concentration of 0.672 g / L, a polysaccharide content of 9.516 g / 100 g, and a flavonoid content of 0.0237 g / 100 g.

[0049] Verification of genetic stability: Furthermore, the P06 strain was subjected to 21 rounds of continuous subculture, and the results showed that its characteristics of high biomass yield, high polysaccharide and high flavonoid content were stably inherited, and all indicators were significantly better than existing algae strains.

[0050] The P06 strain of space desert algae not only achieved efficient production of biomass, but also ensured the stable accumulation of high-value components, showing its great potential in the field of biological resource development. In particular, after multiple rounds of subculture, the excellent traits of the P06 strain remained unchanged, proving its reliability and sustainability in practical applications.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A desert algae, characterized in that: The name of the desert algae is Hassallia sp. P06, and the preservation number is: CCTCC M 20242054.

2. Use of the desert algae as claimed in claim 1 in the production of desert algae polysaccharides.

3. Use of the desert algae as claimed in claim 1 in producing flavonoids.

4. Use of the desert algae as claimed in claim 1 in producing desert algae powder.

5. Use of the desert algae as claimed in claim 1 in the preparation of cosmetics, food and feed.

6. The use according to any one of claims 2 to 5, characterized in that: The method for culturing the desert algae comprises: inoculating the desert algae into a culture medium, and culturing for 5-20 days under the conditions of a light intensity of 1500-5000 Lux and a constant temperature of 20-45° C.

7. The use according to claim 6, characterized in that: The components of the culture medium include: 1.00-5.00 g / L sodium nitrate, 0.01-0.1 g / L potassium dihydrogen phosphate, 0.05-0.1 g / L magnesium sulfate, 0.01-0.1 g / L calcium chloride, 0.01-0.10 g / L sodium carbonate, 1.00-5.00 mg / L boric acid, 1.00-5.00 mg / L manganese chloride, 0.1-1.0 mg / L sodium molybdate, 0.01-0.10 mg / L copper sulfate, 0.01-0.10 mg / L zinc sulfate and 0.01-0.10 mg / L cobalt chloride.

8. An algae biological product, characterized in that: Comprising the desert algae described in claim 1.

9. A method for producing algae polysaccharides, characterized in that: Algal polysaccharide is produced by culturing the desert algae according to claim 1.

10. A method for producing algae flavonoid compounds, characterized in that: The algae flavonoids are produced by culturing the desert algae according to claim 1.

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