Culture method for improving solid content and group perfectness ratio of nostoc sphaeroids kutz and application of culture method
By using specific culture media and light control methods, the problems of low solid content and easy breakage in the artificial cultivation of Nostoc commune were solved, achieving high yield, high integrity rate and long shelf life.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-16
- Publication Date
- 2026-03-31
AI Technical Summary
In the existing technology, the low solid content and insufficient mechanical strength of the gelatinous sheath during the artificial cultivation of Nostoc commune make it prone to breakage and have a short shelf life, making it difficult to achieve high-yield and high-quality commercial production.
A specific culture medium formulation and light control method were used, including the addition of nutrients such as dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, and ferric ammonium citrate. The light wavelength was segmented and controlled by irradiating with 610-680nm red light to promote the synthesis of extracellular polysaccharides and enhance the strength of the gelatinous sheath.
It significantly increases the solids content and population integrity of *Gnaphalium affine*, extends shelf life, and improves growth stability and production efficiency.
Smart Images
Figure CN121759334A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microalgae cultivation technology, specifically to a cultivation method that significantly increases the dry matter content in a population of *Nostoc commune* through synergistic regulation of light and nutrients, and its application in improving the solid content, integrity rate, and shelf life of *Nostoc commune* populations. Background Technology
[0002] Nostoc commune is a freshwater cyanobacterium with extremely high nutritional value, rich in protein, polysaccharides, and various physiologically active substances, showing broad application prospects in the food, health product, and cosmetic fields. Nostoc commune is a multicellular organism, with a common gelatinous sheath surrounding the colony, easily forming a layered structure; wild Nostoc commune colonies can reach a diameter of 2 cm. Due to environmental changes and human factors, wild Nostoc commune resources are increasingly depleted, making artificial cultivation an inevitable choice.
[0003] Currently, the artificial cultivation of *Nostoc commune* (a type of algae) mostly uses conventional culture media such as BG-110. Under fluorescent light, the growth cycle is long (typically, it takes 40-100 days to grow a population of 0.5-1 mm in diameter to 5-8 mm at room temperature). As cultivation progresses, the particle size of the *Nostoc commune* population increases, but the internal solids (dry matter) content does not increase proportionally, resulting in excessively high water content and a decreased wet-to-dry ratio. This weakens the mechanical strength of the gelatinous sheath enveloping the algal filaments, making the population highly susceptible to breakage. Breakage not only directly reduces yield and product integrity, but the released organic debris also contaminates the culture medium, promoting the growth of other microorganisms and further inhibiting growth, creating a vicious cycle. Existing technologies alleviate this through frequent medium changes and cleaning, but these are costly and do not fundamentally solve the problem. Furthermore, *Nostoc commune* cultivated in natural conditions or using traditional methods generally has a low dry matter content (typically 0.9%-1.5%), resulting in a short shelf life for fresh products and hindering commercial distribution.
[0004] While existing technologies contain numerous studies on improving culture media or optimizing culture conditions for *Nostoc commune*, most focus on promoting biomass growth. There are few reports on specific technologies for targeted and efficient enhancement of intracellular dry matter (i.e., solids) accumulation, and the effects are limited. Therefore, developing a culture method that can fundamentally enhance the strength of the gelatinous sheath of *Nostoc commune* and increase solids content, thereby achieving a synergistic improvement in high yield, high integrity rate, and long shelf life, is a pressing technical challenge in this field. Summary of the Invention
[0005] This invention aims to solve at least one of the problems in the prior art by providing a cultivation method for improving the solid content and population integrity of *Nostoc commune*. This method, through the synergistic regulation of specific culture medium formulations and cultivation conditions (especially light), effectively promotes the synthesis and deposition of extracellular polysaccharides in *Nostoc commune*, strengthens the gelatinous sheath, thereby significantly increasing the solid content and population integrity, and extending the shelf life of fresh products.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: A cultivation method for improving the solid content and population integrity rate of *Gnaphalium affine* includes the following steps: S1. Preparation of culture medium: This culture medium contains specific concentrations of dipotassium hydrogen phosphate (15-60 mg / L), magnesium sulfate (20-60 mg / L), calcium chloride (15-50 mg / L), citric acid (4.0-8.0 mg / L), ferric ammonium citrate (5.0-9.0 mg / L), disodium ethylenediaminetetraacetate (1-2 mg / L), sodium carbonate (5-30 mg / L), and trace elements. Preferably, the culture medium further includes 50-2000 mg / L of sodium nitrate, more preferably 300 mg / L to 1500 mg / L.
[0007] Adjust the pH of the culture medium to ≥7.0 using sodium hydroxide or hydrochloric acid.
[0008] The preparation method of the *Ge Xian Mi* algae strain includes: providing a clean, spherical colony of *Ge Xian Mi*, homogenizing it to obtain filamentous algal slurry, and inoculating the filamentous algal slurry into BG-110 medium, and incubating it at a temperature of 15-25℃ and a light intensity of 20-1200 nm. The *Ge Xian Mi Zi* strain was obtained by culturing it for 10-15 days under the specified conditions.
[0009] S2. Inoculation: Inoculate the *Gnaphalium affine* seed or young *Gnaphalium affine* into the inoculation medium, preferably at a fresh weight density of 10-80 g / L.
[0010] S3. Aeration culture: At 10~32℃, introduce sterile air and irradiate with red light at a wavelength of 610~680nm (light intensity 20~1200 nm). The cells are cultured for 30-60 days. More preferably, a segmented light strategy is adopted: first, culture with a wavelength of 620-650 nm for 3-10 days to promote initial growth, then culture with a wavelength of 650-700 nm for 5-20 days to promote biomass accumulation, and finally culture with a wavelength of 640-680 nm for 5-15 days to enhance gelatin synthesis.
[0011] S4. Harvesting: After cultivation, collect the *Ge Xian Mi* population, wash it, and you can obtain a product with high solids content.
[0012] The beneficial effects of this invention are as follows: Significantly increases solids content: This invention, by adding a nitrogen source and red light irradiation, directionally regulates the carbon and nitrogen metabolism and extracellular polysaccharide synthesis of Nostoc commune, significantly increasing the dry matter content after 30 days of cultivation from approximately 1.2% to over 3.2%. With the extension of time, the dry matter content can be further increased, reaching a maximum of over 5.5%.
[0013] Enhanced population structure and integrity: High solids content means denser gelatinous sheaths and higher mechanical strength, resulting in smoother and rounder surfaces of the obtained Nostoc commune population, significantly reduced breakage rate, and significantly higher integrity rate than traditional methods.
[0014] Extended shelf life: The increased solids content directly increases intracellular solute concentration and antimicrobial substance content, as well as a denser gelatinous sheath, enhancing the fresh product's resistance to spoilage and extending its shelf life. Under refrigeration at 1-8℃, the shelf life can be extended to 60-120 days.
[0015] Promotes growth and stable production: A healthy population structure reduces culture medium contamination, creates a more stable growth environment, and is conducive to improving absolute growth rate and batch production stability. Attached Figure Description
[0016] Figure 1 Photographs of the *Gnaphalium affine* population after 30 days of culture (Comparative Example 10).
[0017] Figure 2 Photograph of the *Gnaphalium affine* population after 30 days of cultivation, Example 1 of this invention.
[0018] Figure 3 Schematic diagram comparing the changes in solid content of Nostoc commune during the cultivation process of Examples 1-9 and Comparative Examples 10-11.
[0019] Figure 4 Example 5 and Comparative Example 10: Schematic diagram comparing the changes in hardness of the Nostoc commune population during cultivation. Detailed Implementation
[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0021] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.
[0022] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.
[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.
[0024] Examples 1-9 and Comparative Examples 10-11 This invention provides a cultivation method for improving the solid content and population integrity rate of *Nostoc commune*, comprising the following steps: S1. Treatment of *Ge Xian Mi* seed source: The live *Ge Xian Mi* obtained from the paddy fields of Zouma Town, Hefeng County, Hubei Province after separation and purification was rinsed with sterile distilled water, then soaked in 75wt% ethanol solution for 20s to remove impurities and bacteria, and then washed 3 times with sterile BG-110 culture medium for later use.
[0025] Preparation of inoculation culture medium: This culture medium contains specific concentrations of dipotassium hydrogen phosphate (15-60 mg / L), magnesium sulfate (20-60 mg / L), calcium chloride (15-50 mg / L), citric acid (4.0-8.0 mg / L), ferric ammonium citrate (5.0-9.0 mg / L), disodium ethylenediaminetetraacetate (1-2 mg / L), sodium carbonate (5-30 mg / L), sodium nitrate (50-2000 mg / L), and trace elements. Further, the sodium nitrate content is preferably 300 mg / L to 1500 mg / L. Specifically, 11 culture media of Examples 1-9 and Comparative Examples 10-11 were prepared according to the contents of dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, disodium ethylenediaminetetraacetate, sodium carbonate, sodium nitrate, boric acid, manganese chloride, zinc sulfate, sodium molybdate, copper sulfate, and cobalt nitrate in Table 1, and the pH of the culture medium was adjusted to ≥7.0 with sodium hydroxide or hydrochloric acid.
[0026] S2. Inoculation: The cleaned and prepared Nostoc commune population is inoculated into the above culture medium at a fresh weight density of 10~80 g / L, and the pH is adjusted to 7.5 with 0.1mol / L HCl solution or NaOH solution.
[0027] S3. Cultivation of Nostoc commune: Cultivated at 10-32℃ for 75 days according to the conditions specified in Examples 1-9 and Comparative Examples 10 and 11 in Table 1, and the dry matter content and integrity rate were measured during the cultivation process. Figure 3 result.
[0028] Specifically, Examples 1-9 and Comparative Examples 10-11 all used a temperature of 20°C, with sterile air filtered through a 0.22μm filter membrane, and an airflow rate to culture flask volume ratio of 3:5. Examples 1-9 were irradiated with red light at a wavelength of 610-680nm (light intensity of 20-1200 nm). Comparative Example 10 was cultured under fluorescent light, and Comparative Example 11 was cultured under natural light for 60 days.
[0029] Examples 1-9 used the sodium nitrate-containing culture medium formulation of this invention and were irradiated with red light of a specific wavelength. The culture media of Comparative Examples 10 and 11 differed, particularly in that they did not contain sodium nitrate, and some other components were slightly different. The light sources also differed; Comparative Example 10 used fluorescent lighting, while Comparative Example 11 used natural light. All other conditions were the same, such as temperature, light intensity, and ventilation.
[0030] S4. Harvesting: After cultivation, collect the *Ge Xian Mi* population, wash it, and you can obtain a product with high solids content.
[0031] In Examples 8 and 9, the light exposure for the first 30 days employed a segmented strategy: first, cultivation at a wavelength of 620-650 nm for 3-10 days to promote initial growth; then, cultivation at a wavelength of 650-700 nm for 5-20 days to promote biomass accumulation; and finally, cultivation at a wavelength of 640-680 nm for 5-15 days to enhance gelatin synthesis. Specifically, in Example 8, the first stage involved cultivation at 640 nm with aeration for 8 days; the second stage involved cultivation at 665 nm with aeration for 15 days; and the third stage involved cultivation at 650 nm with aeration for 7 days. In Example 9, the first stage involved cultivation at 650 nm with aeration for 5 days; the second stage involved cultivation at 665 nm with aeration for 10 days; and the third stage involved cultivation at 680 nm with aeration for 15 days.
[0032] Table 1: Components of the inoculation medium (unless otherwise noted, unit: mg / L), culture conditions, and dry matter content after 30 days of culture.
[0033] Note: Boric acid to cobalt nitrate are trace elements. First, dissolve the boric acid to cobalt nitrate into a stock solution, and then add the stock solution to the culture medium at a concentration of 1 mL / L.
[0034] Results and Analysis: Dry matter content: As shown in the last row of Table 1, the dry matter content of *Gnaphalium affine* in Examples 1-9 of this invention after 30 days of cultivation was all higher than 3.2%, significantly higher than that of Comparative Example 10 (1.15%) and Comparative Example 11 (1.23%). Among them, under the general light strategy, Example 5 (sodium nitrate 1000 mg / L, red light 665nm) showed the best effect, reaching 4.28%. After changing the light strategy based on Example 5 (Examples 8 and 9), the dry matter content increased slightly, but it was not significant compared to Example 5, but it still showed significant improvement compared to Comparative Examples 10 and 11, indicating that the method of this invention is extremely effective in increasing the solid content.
[0035] Group morphology: After 30 days of cultivation, such as Figure 1 and Figure 2 As shown in the comparison, the *Gnaphalium affine* population cultivated by the method of this invention (…) Figure 2 The appearance is noticeably smoother and rounder, resulting in better commercial properties, while the surface of *Ge Xian Mi* cultivated under natural light is rough, uneven, and not smooth and round.
[0036] Dry matter content and properties: Refer to Figure 3 and Figure 4 The diagram illustrates that, during a 75-day cultivation period, the *Nostoc commune* cultivated using the method of this invention (as in Examples 1-7) consistently exhibited higher dry matter content (solids content) and population hardness compared to traditional methods (Comparative Examples 10 and 11). Furthermore, the difference widened over time during the first 60 days of cultivation, but ceased to increase after 60 days, indicating that this invention better maintains the structural strength and material accumulation advantages of the population within a certain cultivation period. The *Nostoc commune* obtained by this invention showed a significant increase in hardness compared to the comparative examples, but this increase ceased after day 45.
[0037] Mechanism Exploration: Dipotassium hydrogen phosphate provides phosphorus, magnesium sulfate provides magnesium and sulfur, calcium chloride provides calcium, ferric ammonium citrate, manganese chloride, boric acid, cobalt nitrate, and zinc chloride provide essential trace elements, sodium bicarbonate provides inorganic carbon, and sodium nitrate serves as a nitrogen source, providing the necessary nutrients for the growth of *Nostoc commune*. Specific wavelengths of red light (610-680 nm, optimal 665 nm) can be efficiently absorbed by *Nostoc commune* phycocyanin, promoting photosynthesis and carbon fixation. Combined with an optimized culture medium (especially suitable phosphorus, calcium, magnesium, and nitrogen sources), it can effectively guide the conversion of photosynthetic products towards the synthesis of extracellular polysaccharides (the main component of the gelatinous sheath), thereby enhancing the mechanical strength of the gelatinous sheath. During long-term cultivation, it effectively reduces the breakage rate of *Nostoc commune* populations, increases the integrity rate, and promotes the growth rate of *Nostoc commune* populations, thus increasing biomass while significantly increasing solids and strengthening the gelatinous sheath structure.
[0038] Performance testing The populations of *Gnaphalium affine* in Examples 1-9 and Comparative Examples 10-11 were tested after 30 days of culture, and the results are summarized in Table 2 below.
[0039] Table 2: Performance Test Results of Each Group of Nostoc commune Group Integrity rate (%) Shelf life of fresh products (1-8℃, days) Example 1 97.3 ≥90 Example 2 100 ≥90 Example 3 97.7 ≥95 Example 4 99.9 ≥100 Example 5 100 ≥120 Example 6 98.3 ≥100 Example 7 100.0 ≥100 Example 8 98.5 ≥120 Example 9 98.8 ≥120 Comparative Example 10 83.6 ≤30 Comparative Example 11 85.9 ≤35 For the integrity rate test, 100 mL of the *Gnaphalium affine* population cultured in each experimental group was randomly measured using a sampler as a sample for testing. The integrity rate was equal to the mass ratio of intact, unbroken *Gnaphalium affine* to broken *Gnaphalium affine*. In the fresh product shelf life test, the *Gnaphalium affine* obtained in the example with a storage temperature of 1-8℃ and a higher solids content also had a longer fresh product shelf life.
[0040] In summary, this invention, through innovative matching of culture medium components and light conditions, successfully solves the key technical problems of low solid content, easy breakage, and short shelf life in the artificial cultivation of Nostoc commune, providing an efficient and reliable method for the large-scale and commercial production of Nostoc commune.
[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention, such as replacing sodium nitrate with other nitrogen sources, should be included within the scope of protection of the present invention.
Claims
1. A cultivation method for improving the solid content and population integrity rate of *Gnaphalium affine*, characterized in that, Includes the following steps: Provide a culture medium with a pH value ≥ 7.0; Inoculate *Gnaphalium affine* strains or young *Gnaphalium affine* into the inoculation medium, and maintain the medium at a temperature of 10–32°C and a light intensity of 20–1200 ppm. Under conditions of light wavelength of 610~680nm, the plants were cultured with aeration for 30~60 days, and the population of *Gnaphalium affine* was collected. The inoculation culture medium comprises the following components at the following concentrations: dipotassium hydrogen phosphate 15-60 mg / L, magnesium sulfate 20-60 mg / L, calcium chloride 15-50 mg / L, citric acid 4-8 mg / L, ferric ammonium citrate 5-9 mg / L, disodium ethylenediaminetetraacetate 1-2 mg / L, sodium carbonate 5-30 mg / L, and trace elements.
2. The cultivation method according to claim 1, characterized in that, The light conditions for the cultivation process are divided into stages as follows: Phase 1: Cultured under aeration for 3-10 days at a light wavelength of 620-650nm; Second stage: Under conditions of light wavelength of 650~700nm, culture with ventilation for 5~20 days; The third stage: cultured under aeration for 5 to 15 days at a light wavelength of 640 to 680 nm.
3. The cultivation method according to claim 1, characterized in that, The *Spartina alterniflora* strain was inoculated into the inoculation medium at a fresh weight inoculation density of 10-80 g / L.
4. The cultivation method according to claim 1, characterized in that, The preparation method of the *Ge Xian Mi* algae strain includes: providing a clean, spherical colony of *Ge Xian Mi*, homogenizing it to obtain filamentous algal slurry, and inoculating the filamentous algal slurry into BG-110 medium, and incubating it at a temperature of 15-25℃ and a light intensity of 20-1200 nm. The *Ge Xian Mi Zi* strain was obtained by culturing it for 10-15 days under the specified conditions.
5. The cultivation method according to claim 4, characterized in that, The spherical population of *Ge Xian Mi* is derived from a wild *Ge Xian Mi* screening population or is obtained by the cultivation method described in any one of claims 1-3.
6. The cultivation method according to claim 1, characterized in that, The air introduced during the aeration culture process is sterile air filtered through a 0.22μm filter membrane.
7. The cultivation method according to claim 1, characterized in that, The trace elements include boric acid, manganese chloride, zinc sulfate, sodium molybdate, copper sulfate, and cobalt nitrate.
8. The cultivation method according to claim 7, characterized in that, The trace elements are added in the form of a trace element solution, which contains the following per liter: 2.86 g boric acid, 1.15 g manganese chloride, 0.13 g zinc sulfate, 0.33 g sodium molybdate, 0.05 g copper sulfate, and 0.03 g cobalt nitrate. The amount of the trace element solution added to the inoculation culture medium is 1 ml / L.
9. The cultivation method according to claim 1, characterized in that, The inoculation medium also includes 50 to 2000 mg / L of sodium nitrate, preferably 300 mg / L to 1500 mg / L.
10. The application of the cultivation method as described in any one of claims 1-9 in improving the shelf life of *Gnaphalium affine*, characterized in that... The shelf life of fresh Ge Xian Mi (a type of herb) can be extended by increasing the dry matter content of its population.