A space desert alga obtained by space breeding and its applications

Abstract

The present invention discloses a desert alga improved by space breeding and its applications. Through induced mutations in the space environment and subsequent screening on the ground, a desert alga has been obtained. This desert alga exhibits a rapid growth rate and excellent outdoor adaptability. At the same time, it can efficiently accumulate two bioactive substances, polysaccharides and flavonoids, which have important commercial and health values. Therefore, this desert alga shows good application potential in the production of high-quality desert alga powder, polysaccharides, flavonoids, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and more particularly, to a space desert alga obtained by space breeding and its applications. Background Art

[0002] Desert algae are a type of algae that can survive in extremely arid and highly saline environments, possessing excellent drought tolerance, salt tolerance, and ultraviolet resistance. These algae can synthesize various bioactive substances with commercial value, such as polysaccharides and flavonoids. Polysaccharides are macromolecular compounds composed of multiple monosaccharide molecules and have functions such as regulating immune function and reducing blood sugar. Flavonoids are a class of natural compounds with various effects such as antioxidant, anti-inflammatory, and anti-cancer properties. However, in the natural state, the growth rate of desert algae is slow and the biomass is small, which limits their commercial production. The technical bottlenecks in the deep processing of desert algae are as follows: 1) There is a lack of high-performance engineered desert alga strains and desert algae with high polysaccharide and flavonoid contents; 2) The slow growth rate of desert algae increases the raw material cost of desert algae.

[0003] Space breeding technology provides a method for cultivating new crop varieties through genetic variation under special space environments, which helps to improve the growth rate and yield of crops. Microalgae space breeding is a relatively efficient artificial breeding method that utilizes the mutagenic effects of factors such as cosmic radiation, microgravity, and complex electromagnetic environments on microalgae to cause genetic variation in microalgae cells, obtaining a large number of mutant strains at once, and then screening out alga strains with fast growth rates, high biomass, stable genetic traits, and development value from the mutagenized strains. Compared with traditional breeding, the greatest advantage of space mutagenesis breeding is the high mutation probability, wide mutation range, and short breeding cycle, which can create high-quality germplasm resources in a relatively short time. However, there is currently no research on space breeding of desert algae to increase the content of their bioactive substances.

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

[0005] The object of the present invention is to provide a space desert alga obtained by space breeding and its applications. This desert alga not only has a fast growth rate and high biomass, but also can grow well under strong outdoor adaptability conditions, and can efficiently accumulate algal polysaccharides and flavonoids.

[0006] In a first aspect, the present invention provides a strain of desert alga named Hassallia sp. P06, with a deposit number of CCTCC M 20242054, deposited on September 23, 2024, at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China.

[0007] The desert algae in the present invention are a type of cyanobacteria belonging to the genus Hassallia sp., which grow in extremely arid and high-salt environments. Some studies have found that desert algae contain rich bioactive substances. On this basis, Hassallia sp. P06 was obtained through induction and screening in the space environment. Experiments have proved that Hassallia sp. P06 is significantly superior to wild-type desert algae in terms of growth rate and bioactive substance accumulation. Therefore, Hassallia sp. P06 provided by the present invention has good commercial development potential and broad application prospects.

[0008] Second, the present invention also provides the application of the above-mentioned desert algae in the production of algal polysaccharides.

[0009] Third, the present invention also provides the application of the above-mentioned desert algae in the production of flavonoids.

[0010] Fourth, the present invention also provides the application of the above-mentioned desert algae in the production of desert algae powder.

[0011] Fifth, the present invention also provides the application of the above-mentioned desert algae in the preparation of cosmetics, food, drugs, and feeds.

[0012] Hassallia sp. P06 obtained by the present invention through induction and screening in the space environment can be applied to the production of related algal polysaccharides, flavonoids, and algal powder. The produced algal polysaccharides and flavonoids can be further applied to the preparation of products such as cosmetics, food, and drugs, and the algal polysaccharides, flavonoids, and algal powder can be further applied to the preparation of products such as feeds.

[0013] Seventh, the present invention also provides an algal bioproduce, which contains the above-mentioned desert algae.

[0014] Eighth, the present invention also provides a method for producing algal polysaccharides, which produces bioactive components by culturing the above-mentioned desert algae.

[0015] Ninth, the present invention also provides a method for producing flavonoids, which produces flavonoids by culturing the above-mentioned desert algae.

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

[0017] Through mutation induced by the space environment and screening on the ground, a strain of desert alga Hassallia sp. P06 was obtained. This strain of desert alga exhibits a rapid growth rate and excellent outdoor adaptability. At the same time, it can efficiently accumulate two bioactive substances, polysaccharides and flavonoids, which have important commercial and health values. Therefore, Hassallia sp. P06 shows good application potential in the production of high-quality desert alga powder, polysaccharides, flavonoids, etc. 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 will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

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

[0020] Figure 2 is the screening of the Hassallia sp. P06 strain of the desert alga;

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

[0022] Figure 4 is the comparison of the flavonoid content between the Hassallia sp. P06 strain of the desert alga and the wild-type desert alga strain. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0024] The features and performance of the present invention will be further described in detail below with reference to the embodiments.

[0025] Embodiment 1

[0026] This embodiment is the breeding and trait evaluation of the space desert alga 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] Culture medium preparation: To promote the growth of P06 strain and obtain high-value bioactive substances, a culture medium with a specific ratio is used in this example. The specific formula is as follows:

[0037] In each liter of the culture medium, add sodium nitrate (NaNO 3 ): 1.5 g, dipotassium hydrogen phosphate (K 2 HPO 4 ): 0.04 g, magnesium sulfate (MgSO 4 ·7H 2 O): 0.075 g, calcium chloride (CaCl 2 ·2H 2 O): 0.036 g, sodium carbonate (Na 2 CO 3 ): 0.02 g, boric acid (H 3 BO 3 ): 2.86 mg, manganese chloride (MnCl 2 ·4H 2 O): 1.86 mg, sodium molybdate (Na 2 MoO 4 ·2H 2 O): 0.22 mg, copper sulfate (CuSO 4 ·5H 2 O): 0.012 mg, zinc sulfate (ZnSO 4 ·7H 2 O): 0.022 mg and cobalt chloride (CoCl 2 ·6H 2 O): 0.013 mg. After fully mixing, perform high-temperature sterilization to ensure a sterile state.

[0038] Cultivation conditions and harvesting:

[0039] Cultivation object: The space desert alga P06 strain and the original wild-type desert alga without space mutagenesis are used as a control group, and an equal amount is inoculated into the above-prepared culture medium.

[0040] Cultivation environment: Set the light intensity to 2500 Lux and cultivate for 15 days under the condition of a constant temperature of 28 °C. Then, detect various bioactive substances of the obtained space desert alga P06 strain and the original wild-type desert alga without space mutagenesis under the same conditions.

[0041] Detection of polysaccharide content: Under the action of concentrated sulfuric acid, polysaccharides are hydrolyzed to generate monosaccharides, and then rapidly dehydrated to form furfural derivatives, which then condense with phenol to form an orange-yellow compound, and the color is stable. At a wavelength of 488 nm and within a certain concentration range, its absorbance is linearly proportional to the polysaccharide content. Therefore, the absorbance can be measured using a spectrophotometer.

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

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

[0044] (2) Determination of polysaccharide content in algae: 1) Accurately weigh 5 g of algal powder, dissolve it in 30 mL of water, and mix well; 2) Ultrasonic power is 650 W, ultrasonic for 30 min to disrupt the cell structure, and then place it in a water bath at 85 °C for extraction for 2 h; 3) Centrifuge at 5000 r / min for 10 min, and the supernatant is the algal polysaccharide solution; 4) Add 4 times the volume of 95% ethanol, and precipitate at 4 °C for 24 h; 5) Centrifuge at 5000 r / min for 10 min, collect the precipitate, and dissolve the precipitate with an appropriate amount of water to obtain the crude polysaccharide extract; 6) According to the absorbance value of the measured sample extract, use the standard curve to calculate the content of polysaccharide in algae. The detection results of polysaccharide in algae are as Figure 3 shown.

[0045] Detection of flavonoid content: Use the NaNO 2 -Al(NO 3 ) 3 colorimetric method for ultraviolet spectrophotometric detection of content. The principle is that flavonoids form a chelate with Al(NO 2 ) 3 in the presence of NaNO 3 under weak alkaline conditions. At this time, adding NaOH solution shows orange-red color and has a stable characteristic absorption peak at 510 nm.

[0046] (1) Preparation of standard curve: Accurately weigh 5 mg of rutin standard substance, make up the volume to 50 mL with deionized water to prepare a 0.2 mg / mL standard solution. Take 0, 1, 2, 3, 4, 5, 6 mL of rutin standard solution into 25 mL brown volumetric flasks respectively, add 70% ethanol aqueous solution to make up the volume to 10 mL for each, then add 1 mL of 5% NaNO 2 solution, shake well and let stand for 6 min, add 10% Al(NO 3 )3 Take 1 mL of the solution, shake well and let stand for 6 min. Add 10 mL of 10% NaOH solution, shake well and let stand for 15 min. Make up the volume to 25 mL with distilled water. According to the ultraviolet-visible spectrophotometry, perform colorimetric determination at a wavelength of 510 nm, using the reagent as the blank reference.

[0047] (2) Determination of flavonoid content in desert algae: 1) Accurately weigh 5 g of algal powder and dissolve it in 25 mL of absolute ethanol, and mix well; 2) Ultrasonic power is 650 W, ultrasonic for 30 min to disrupt the cell structure; 3) Centrifuge at 4500 r / min for 15 min, and the supernatant is the flavonoid extract of desert algae; 4) Take 1 mL of the extract, make up the volume to 10 mL with 70% ethanol aqueous solution, and add 5% NaNO 2 Take 1 mL of the solution, shake well and let stand for 6 min. Add 1 mL of 10% Al(NO 3 ) 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. Make up the volume to 25 mL with distilled water; 5) According to the ultraviolet-visible spectrophotometry, measure the absorbance at a wavelength of 510 nm, record and analyze the values. The detection results of flavonoids in desert algae are as Figure 4 shown.

[0048] Result comparison: The P06 strain showed significant growth advantages. The harvested biomass concentration reached 0.793 g / L, and at the same time, there were high levels of secondary metabolites, including a polysaccharide content of 16.308 g / 100 g and a 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: Further, the P06 strain was subjected to 21 rounds of continuous subculture. 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 those of existing algal strains.

[0050] This space desert algae P06 strain 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 are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within 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. The use according to any one of claims 2 to 4, 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.

6. The use according to claim 5, 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.

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

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

9. 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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