Method for obtaining high yield of anthocyanins based on stable sedum lineare stem cells

Abstract

The application discloses a method for obtaining high-yield anthocyanins based on stable sedum aizoon stem cells. First, ferric iron phenylalanine chelate is added to a traditional liquid culture medium to promote the synthesis of anthocyanins and the growth of the sedum aizoon stem cells. Then, the accumulation of anthocyanins in the sedum aizoon stem cells is improved by culturing under specific light stimulation conditions, a stable seed system of the sedum aizoon stem cells is established, and the seed system is expanded for production. Finally, the sedum aizoon stem cells are co-cultured with lactobacillus plantarum to promote the release and extraction of anthocyanins. The lactobacillus plantarum is selected and added to the system at the end of the culture, and the lactic acid bacteria can produce hydrolytic enzymes, dextran and acid, and have the characteristics of acting on the plant cell wall, so that the anthocyanins can be released. The growth of the lactic acid bacteria reduces the pH of the system, the amount of HCl used in extraction can be reduced, and the extraction efficiency is improved. Anthocyanins can be extracted from the cells, and can also be collected from the liquid culture system to achieve the effect of double-system extraction.

Description

Technical Field

[0001] The invention relates to the field of stem cell culture technology, and more specifically, to a method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells. Background Technology

[0002] Anthocyanins are a class of water-soluble natural flavonoid plant pigments that can be rapidly absorbed by the body. They possess strong antioxidant capabilities, inhibit inflammatory responses, and enhance immunity, making them one of the most effective natural antioxidants for scavenging excess free radicals in the body. As our understanding of the functions of anthocyanins deepens, research on anthocyanins in various natural plants has become a hot topic in the field of natural product research. Based on cell totipotency, plant cells possess both the original characteristics of the plant and anthocyanins can be extracted from stem cells. Currently, the anthocyanin extraction rate from field-cultivated Sedum aizoon dry plants is only 0.2 mg / g. Existing techniques typically use traditional culture media to culture Sedum aizoon stem cells. Although the anthocyanin extraction rate from cultured Sedum aizoon stem cells is somewhat higher than that from field-cultivated plants, it is still not ideal.

[0003] Sedum sarcodactylis ( Sedum aizoon Sedum aizoon (L.) is a perennial herb belonging to the genus Sedum in the family Crassulaceae, also known as Fei Cai, Tu San Qi, etc. The roots and whole plant are used medicinally, with the roots harvested in spring and autumn. Sedum aizoon is recorded in books such as the *Dictionary of Traditional Chinese Medicine* and *Chinese Materia Medica*. It is a commonly used medicine among the Miao ethnic group, often used for hemostasis, analgesia, stopping hematemesis, hemoptysis due to injury, epistaxis, hemoptysis in pulmonary diseases, and cough suppression. Sedum aizoon contains abundant active ingredients, mainly polysaccharides, flavonoids, and alkaloids, including oleanolic acid, β-sitosterol, ursolic acid, and arbutin.

[0004] Oleanolic acid can reduce liver damage, promote hepatocyte regeneration, and inhibit liver fibrosis. β-Sitosterol can lower plasma cholesterol and relieve cough. Ursolic acid has sedative, anti-inflammatory, antibacterial, anti-diabetic, and anti-ulcer biological effects. Ursolic acid has significant antioxidant properties and is widely used as a pharmaceutical and cosmetic ingredient. Arbutin, by inhibiting the activity of tyrosinase, an enzyme that produces melanin, can reduce melanin production. Through its binding to tyrosinase, it accelerates the decomposition and excretion of melanin, thereby reducing skin pigmentation, removing age spots and freckles, and can be used for skin whitening.

[0005] As the superior value of Sedum aizoon is increasingly recognized, the demand for it continues to rise, and current production cannot adequately meet market demand. Therefore, there is an urgent need to provide a method that can improve the stability of Sedum aizoon stem cells and rapidly increase the yield of anthocyanins to address the key issues that those skilled in the art need to resolve. Summary of the Invention

[0006] This invention provides a method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells, aiming to solve the problem of producing a natural plant extract with antioxidant properties.

[0007] To achieve the above objectives, this invention provides a method for obtaining high-yield anthocyanins based on stable Sedum aizoon stem cells. This invention utilizes the medicinal plant Sedum aizoon to induce purplish-red callus tissue, which is then domesticated in a solid culture medium and subcultured in a liquid culture medium. The liquid culture medium is supplemented with 30 mg / L phenylalanine-chelated iron to promote anthocyanin synthesis and cell growth in the Sedum aizoon stem cells. The system is then subjected to specific light stimulation at an intensity of 800-1600 lx, with a red-to-blue light ratio of 2-6:3-5, to induce high anthocyanin content and stable genetic characteristics in the cells. Furthermore, the action of lactic acid bacteria in generating hydrolytic enzymes opens cell permeability, releasing intracellular anthocyanins into the liquid culture medium, enabling cells to synthesize more anthocyanins. During lactic acid bacteria growth, glucose is converted into dextran, which in turn enriches anthocyanins, forming stable anthocyanin-dextran conjugates. The acid produced by the lactic acid bacteria lowers the pH of the system, allowing for a reduction in the amount of HCl used during extraction and improving extraction efficiency. This method can obtain anthocyanins in high quantities, improve the extraction rate of Sedum sarmentosum stem cell production system, and establish a complete production system to obtain anthocyanins efficiently and in high yield.

[0008] A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: The leaves of sterile Sedum aizoon seedlings were cut into pieces and inoculated into an induction medium for induction culture to induce purplish-red callus tissue; the induction medium was a solid medium composed of 20 g / L MS, 2.0 mg / L 2,4-D, 1.0 mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in 1 L of water.

[0009] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was peeled off from the leaves and inoculated into a proliferation medium for subculture to obtain Sedum sarmentosum stem cells; the proliferation medium was a solid medium composed of 20 g / L MS, 1.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in 1 L of water.

[0010] S3. Screening of Sedum sarmentosum stem cells: Using an ultraviolet spectrophotometer to determine the anthocyanin content of cells, Sedum sarmentosum stem cells with an anthocyanin content of 6.5 mg / g dry weight, loose and fragile, and high viability were screened.

[0011] S4. Establishment of the Sedum aizoon stem cell suspension culture system: The Sedum aizoon stem cells screened in step S3 were seeded into liquid culture medium, and 30 mg / L was added to the liquid culture medium for suspension culture to obtain a suspension culture system; the liquid culture medium consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, and 3% sucrose.

[0012] S5. Specific light stimulation enhances the accumulation of anthocyanins in Sedum sarmentosum stem cells: The suspension culture system obtained in step S4 is cultured in a shaker to obtain an initial liquid culture system, from which stable Sedum sarmentosum stem cell seeds are cultured.

[0013] S6. Promoting anthocyanin release from cells by co-culturing *Lactobacillus plantarum* and *Sedum aizoon* stem cells: After repeating the shaker culture of step S5 for one cycle, *Lactobacillus plantarum* was added to the liquid culture system obtained in step S5. The light intensity was 800-1600 lx, the red-blue light intensity ratio was 2-6:3-5, the photoperiod was 8-12 h / d, and the photoculture was carried out for 3.5-4.5 days to obtain the liquid culture system in the final stage.

[0014] S7. Extraction of anthocyanins from Sedum sarmentosum stem cells and liquid culture system: The obtained liquid culture system at the end of the period was filtered to obtain filtrate and Sedum sarmentosum stem cells. The filtrate was filtered and evaporated to dryness to obtain crude anthocyanin extract. The powder obtained by drying, grinding and sieving Sedum sarmentosum stem cells was extracted with an extractant to obtain crude anthocyanin extract.

[0015] In the preferred embodiment, the size of the cut leaf wound in step S1 is 0.8~1.2cm and the number of wounds is 3~5; in the induction culture, 6~10 leaves are inoculated in 50mL of induction medium and cultured in the dark at a temperature of 22~28℃ for 40~45 days, and the inoculation container is a 300mL tissue culture bottle.

[0016] In the preferred embodiment, the peeling tool in step S2 is tweezers; the subculture involves inoculating 0.8-1.2g of purplish-red callus tissue into every 50mL of proliferation medium, and culturing in the dark at a temperature of 22-28℃, with a subculture cycle of 19-21 days and 3-5 subculture cycles.

[0017] In a preferred embodiment, after the loose and fragile callus tissue described in step S3 is inoculated into a liquid culture medium, it can be dispersed into Sedum sarmentosum stem cells of 0.8~1.2 mm; the high viability refers to the Sedum sarmentosum stem cells that can achieve a 5-fold increase in 10 days and an 11.6-fold increase in 20 days.

[0018] In the preferred embodiment, in step S4, 0.8-1.2g of Sedum aizoon stem cells are inoculated into each 100mL of liquid culture medium for suspension culture, and the culture is carried out in the dark for 18-22 days at a temperature of 22-28℃ with a shaking speed of 100-120rpm; the amount of phenylalanine chelated iron added is 30mg / L; and the pH of the suspension culture system is 5.8-6.0.

[0019] In the preferred embodiment, the preparation process of phenylalanine chelated iron in step S4 is as follows: Take 0.95~1.05g of phenylalanine and ferrous chloride, dissolve them in 100mL of water, place them in a three-necked flask under 300mL liquid nitrogen conditions, add 0.45~0.55g of iron powder, adjust the pH to 3.5~5.0 with sodium hydroxide, stir, and reflux the mixture. After reacting for 1h, centrifuge the mixture at 8000rpm for 5min, take the supernatant for alcohol precipitation, filter it with a vacuum pump to obtain brown filter residue, wash the filter residue with ethanol 5~6 times, and dry it in an oven at 60℃ for 18~20h to obtain phenylalanine chelated iron.

[0020] In the preferred embodiment, the shaker culture in steps S5-S6 lasts for 19-21 days per cycle, divided into two stages: the first stage is dark culture for 9-11 days, and the second stage is light culture for 9-11 days. The light intensity for the light culture is 800-1600 lx, the red-blue light intensity ratio is 2-6:3-5, and the light cycle is 8-12 h / d. The culture temperature in steps S5-S6 is 22-28℃, and the rotation speed is 100-120 rpm. After the shaker culture in step S6, 1 mL / 100 mL of 10... 6 cfu / mL Lactobacillus plantarum

[0021] In the preferred embodiment, the extractant in step S7 is a mixed solution of 0.5% hydrochloric acid and 50% ethanol, extracted at a material-to-liquid ratio of 1:40. The extraction is performed by ultrasonication at 40°C for 30 minutes. The filtration is carried out using a vacuum pump. The drying is performed in an oven at 38-42°C. The screening is done using a No. 3 sieve.

[0022] An optimal method for improving the stability of Sedum aizoon stem cells and obtaining anthocyanins in high yield is characterized by the following steps: S1. Induction of Sedum aizoon callus: Take 8 leaves from sterile Sedum aizoon seedlings, cut a 1cm wound on each leaf, place them in 50mL of induction medium, and incubate in the dark at 25℃ for 40 days to obtain purplish-red callus.

[0023] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus tissue induced in step S1 was peeled off from the leaves with tweezers, and 1g was placed in 50mL of proliferation medium and cultured in the dark at 25℃. One cycle was 20 days, and the culture was repeated for 4 cycles to obtain Sedum sarmentosum stem cells.

[0024] S3. Screening of Sedum sarmentosum stem cells: Using an ultraviolet spectrophotometer to determine the anthocyanin content of cells, Sedum sarmentosum stem cells with a dry weight anthocyanin content of 6.5 mg / g, which can be dispersed into 0.8~1.2 mm after inoculation into liquid culture medium, and whose viability can increase by 5 times in 10 days and by 11.6 times in 20 days were selected.

[0025] S4. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of Sedum aizoon stem cells selected in step S3 was picked up with tweezers and seeded into the liquid culture medium. 30 mg / L of phenylalanine chelated iron was added to the liquid culture medium, the pH was adjusted to 5.8, and the culture was carried out in the dark at 25 °C with a shaking speed of 110 rpm for 20 days to obtain the suspension culture system.

[0026] S5. Specific light stimulation enhances the growth of Sedum aizoon stem cells and the accumulation of anthocyanin content: The obtained suspension culture system was cultured in shake flasks at a speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity of the light culture was 800 lx, the light cycle was 12 h / d, and the red-blue light intensity ratio was 6:3, thus obtaining an initial liquid culture system and cultivating stable Sedum aizoon stem cell seeds.

[0027] S6. Promoting anthocyanin release from cells through co-culture with *Lactobacillus plantarum* and *Sedum aizoon* stem cells: Repeat the shaker culture described in step S6 with the initial liquid culture system at 110 rpm and 25°C. After one culture cycle, add 1 mL of 10... 6 Lactobacillus plantarum at cfu / mL was cultured in shake flasks at 25℃, 110 rpm, 800 lx light intensity, and 12 h / d light cycle for 4 days to obtain the final liquid culture system.

[0028] S7. Extraction of anthocyanins from Sedum sarmentosum stem cells and liquid culture system: The obtained liquid culture system at the end of the period was filtered to obtain filtrate and Sedum sarmentosum stem cells; the filtrate was filtered and evaporated to obtain crude anthocyanin extract; the powder obtained by drying, grinding and sieving the Sedum sarmentosum stem cells was extracted at a material-to-liquid ratio of 1:40, the extract was a mixed solution of 0.5% hydrochloric acid and 50% ethanol solution, ultrasonicated at 40℃ for 30 min, filtered and evaporated to obtain crude anthocyanin extract.

[0029] The beneficial effects of this invention are as follows: First, by adding phenylalanine to chelate iron, cell yield and anthocyanin content are increased. Second, specific light stimulation further increases intracellular anthocyanin content. Finally, by adding *Lactobacillus plantarum*, cell permeability is improved, allowing for better release of intracellular substances. Simultaneously, utilizing the acid-producing properties of microorganisms reduces the need for organic reagents during extraction.

[0030] This invention induces purplish-red Sedum aizoon callus under the original characteristics of Sedum aizoon. Because Sedum aizoon contains abundant active substances, and based on cell totipotency, plant cells possess both the original characteristics of the plant and anthocyanins can be extracted from suspended stem cells. Phenylalanine chelated iron is a novel amino acid chelate. This invention, by adding phenylalanine chelated iron to the liquid culture medium, allows phenylalanine to act as a precursor, promoting the formation of anthocyanin precursors; Fe... 2+ It can promote the production of anthocyanin synthase and simultaneously adsorb anthocyanins, achieving a dual effect of enhancing both anthocyanin precursor and anthocyanin synthase synthesis. This process is beneficial for anthocyanin accumulation and cell growth. Through chelation of Fe... 2+ The addition of precursor substances and exogenous light stimulation promote the growth of Sedum aizoon stem cells and the accumulation of anthocyanin content, effectively reducing the browning rate of the system to achieve optimal production conditions and lay the foundation for the production of cell-based foods. The anthocyanin yield of cells cultured in this medium is twice that of the original culture medium compared to traditional culture media, and 64 times that of wild plants. This invention utilizes the principles of plant cell engineering to induce callus tissue in Sedum aizoon, resulting in purplish-red callus tissue with a high anthocyanin content. Exogenous stimulation of cells can significantly increase the growth of suspension stem cells and the accumulation of anthocyanins within the cells.

[0031] This invention utilizes the strong division and rapid growth characteristics of stem cells, reducing the cumbersome preparation process of cultured cell seeds. Simultaneously, leveraging the excellent properties of lactic acid bacteria, it reduces the use of organic reagents. Co-culturing Sedum aizoon stem cells in the later stages of growth facilitates the release of intracellular anthocyanins into the extracellular space, as well as the enrichment and extraction of anthocyanins, effectively increasing the release amount and extraction rate of anthocyanins. This method achieves a higher anthocyanin yield in a short period, replacing field-grown Sedum aizoon. Cells can multiply rapidly in a short time, reaching a 13-fold increase in 20 days, with a shorter growth cycle than field-grown plants and faster growth than traditional callus tissue. The culture obtained by this invention shows an 83% increase in anthocyanin yield compared to field-grown plants.

[0032] The anthocyanins obtained in this invention are natural pigments biosynthesized from Sedum sarmentosum stem cells. They are flavonoids. As a natural edible pigment, anthocyanins can be directly and stably absorbed by tissues. Compared with synthetic pigments, they have the advantages of being non-toxic, harmless, and highly safe. At the same time, anthocyanins also have antioxidant, anti-glycation, and anti-mutagenic activities. They have physiological functions such as lowering blood pressure, lowering blood lipids, protecting liver function, and improving vision. They can be used in the food and pharmaceutical industries, and can meet people's demand for natural pigments.

[0033] Anthocyanin synthesis and cell growth status are related to environmental conditions. The relationship between anthocyanin synthesis and cells in Sedum aizoon is intermediate, falling between growth-coupled and non-growth-coupled types. Therefore, finding a balance between biomass requirements and anthocyanin accumulation is crucial. The temperature range and light duration culture of this invention promote cell growth, division, and anthocyanin accumulation. Furthermore, regulating light stimulation of Sedum aizoon suspension stem cells can more effectively address the browning rate of Sedum aizoon stem cells.

[0034] Plants need light to absorb nutrients, utilizing its energy for photosynthesis to obtain the necessary nutrients for growth and development; plant cells are no exception. Different wavelengths of light have specific effects on the active substances within plant cells. Low-wavelength ultraviolet light (280-315nm) has little impact on plant growth. Ultraviolet light (315-400nm) primarily affects photoperiod and can have a bactericidal effect, but prolonged stimulation of cells is not recommended. Blue light (400-520nm) is second only to red light in its importance for photosynthesis; chlorophyll and carotenoids absorb the most blue light, making it essential for plant growth. Orange light (520-610nm) has little effect on plants. Red light (610-720nm) is beneficial for carbohydrate synthesis, significantly impacts photosynthesis and photoperiod, accelerates growth, and plays a role in seed germination, seedling growth, and anthocyanin synthesis. Plants have low absorption rates of infrared radiation in the 730-740nm range, which can stimulate cell elongation. Therefore, this invention uses a 6:3 red-blue light ratio to stimulate Sedum aizoon stem cells to achieve optimal growth and anthocyanin accumulation.

[0035] Lactobacillus plantarum was added to the system at the end of the culture phase. Lactic acid bacteria produce hydrolytic enzymes, glucans, and produce acid, which act on the plant cell wall, releasing anthocyanins. The glucans enrich the anthocyanins. The growth of lactic acid bacteria lowers the pH of the system, allowing for a reduction in the amount of HCl used during extraction, thus improving extraction efficiency. This allows for extraction from cells and collection from the liquid, achieving a dual-system extraction effect. This invention achieves high-efficiency production, significantly reducing production time, saving cultivation costs, and lowering energy consumption. Furthermore, using natural medicinal plants to produce the target product is safer and more reliable. The anthocyanin extract enriched by glucan greatly improves the stability of anthocyanins, making it suitable for food processing, pharmaceuticals, health products, and packaging materials. It can enhance immunity and has anti-aging properties, fully utilizing its excellent performance. The extraction rate of Sedum aizoon stem cells in this invention reaches 2.55%, which is 5 times that of blueberries and 25 times that of black mulberries. Current research on anthocyanin production through suspension cells is relatively limited, allowing for the expansion of research and application to more species. Attached Figure Description

[0036] Picture 1 This is an overview diagram of the innovative points and main processes of this invention. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1 (Establishment of Suspension Cell Lines) A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in water.

[0039] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in water.

[0040] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0041] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ to obtain phenylalanine chelated iron.

[0042] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 30 mg / L phenylalanine chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, and 3% sucrose dissolved in water.

[0043] S6. Extraction of anthocyanins from Sedum aizoon stem cells: The suspension culture system obtained in step S5 was filtered to collect Sedum sarmentosum stem cells, or the filtered Sedum sarmentosum stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected (the latter is beneficial for the preservation of anthocyanins in the cells). The collected powder was sieved through a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol solution as the extractant. The mixture was sonicated at 40°C for 30 minutes, filtered, and evaporated to dryness to obtain crude anthocyanin extract.

[0044] Example 2 (Establishment of suspension cell line + light culture - light intensity ratio of 2:5) A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in water.

[0045] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0046] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0047] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ for 18 hours to obtain phenylalanine chelated iron.

[0048] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 30 mg / L phenylalanine chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, and 3% sucrose dissolved in water.

[0049] S6. Specific light stimulation increases the growth rate of Sedum aizoon stem cells and the accumulation of anthocyanin content: The suspension culture system obtained in step S5 was cultured in shake flasks at a rotation speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity for the light culture was 800 lx, and the light cycle was 12 h / d (the light duration could be adjusted according to the state of the Sedum sarmentosum stem cells, and the same treatment was performed in the examples or comparatives below). The ratio of red to blue light intensity was 2:5 to obtain a liquid culture system, which cultured stable Sedum sarmentosum stem cell seeds.

[0050] S7. Extraction of anthocyanins from Sedum aizoon stem cells: The liquid culture system obtained in step S6 was filtered to collect Sedum sarmentosum stem cells, or the filtered Sedum sarmentosum stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected (the latter is beneficial for the preservation of anthocyanins in the cells). The collected powder was sieved using a No. 3 sieve, and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol solution as the extractant. The mixture was sonicated at 40°C for 30 minutes, filtered, and evaporated to dryness to obtain crude anthocyanin extract.

[0051] Example 3 (Establishment of suspension cell line + light culture - light intensity ratio of 5:1) A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in water.

[0052] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in water.

[0053] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0054] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ to obtain phenylalanine chelated iron.

[0055] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 3 mg of phenylalanine-chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, and 3% sucrose dissolved in water.

[0056] S6. Specific light stimulation increases the growth rate of Sedum aizoon stem cells and the accumulation of anthocyanin content: The suspension culture system obtained in step S5 was cultured in shake flasks at a rotation speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity of the light culture was 800 lx, the light cycle was 12 h / d, and the red-blue light intensity ratio was 5:1, thus obtaining a liquid culture system and culturing stable Sedum sarmentosum stem cell seeds.

[0057] S7. Extraction of anthocyanins from Sedum aizoon stem cells: The liquid culture system was filtered to collect Sedum aizoon stem cells, or the filtered Sedum aizoon stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected (the latter is beneficial for the preservation of intracellular anthocyanins). The collected powder was sieved using a No. 3 sieve, and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol solution as the extractant. The mixture was sonicated at 40°C for 30 minutes, filtered, and evaporated to dryness to obtain crude anthocyanin extract.

[0058] Example 4 (Establishment of suspension cell line + light culture - light intensity ratio of 5:2) A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in water.

[0059] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in water.

[0060] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0061] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ to obtain phenylalanine chelated iron.

[0062] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 30 mg / L phenylalanine chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, and 3% sucrose.

[0063] S6. Specific light stimulation increases the growth rate of Sedum aizoon stem cells and the accumulation of anthocyanin content: The suspension culture system obtained in step S5 was cultured in shake flasks at a rotation speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity of the light culture was 800 lx, the light cycle was 12 h / d, and the red-blue light intensity ratio was 5:2, thus obtaining a liquid culture system and culturing stable Sedum sarmentosum stem cell seeds.

[0064] S7. Extraction of anthocyanins from Sedum aizoon stem cells: The liquid culture system was filtered to collect Sedum aizoon stem cells, or the filtered Sedum aizoon stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected (the latter is beneficial for the preservation of intracellular anthocyanins). The collected powder was sieved using a No. 3 sieve, and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol solution as the extractant. The mixture was sonicated at 40°C for 30 minutes, filtered, and evaporated to dryness to obtain crude anthocyanin extract.

[0065] Example 5 (Establishment of suspension cell line + light culture - light intensity ratio of 6:3) A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in water.

[0066] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0067] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0068] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ to obtain phenylalanine chelated iron.

[0069] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 30 mg / L phenylalanine chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0070] S6. Specific light stimulation increases the growth rate of Sedum aizoon stem cells and the accumulation of anthocyanin content: The suspension culture system obtained in step S5 was cultured in shake flasks at a rotation speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity of the light culture was 800 lx, the light cycle was 12 h / d, and the red-blue light intensity ratio was 6:3, thus obtaining a liquid culture system and culturing stable Sedum sarmentosum stem cell seeds.

[0071] S7. Extraction of anthocyanins from Sedum aizoon stem cells: The liquid culture system was filtered to collect Sedum aizoon stem cells, or the filtered Sedum aizoon stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected (the latter is beneficial for the preservation of intracellular anthocyanins). The collected powder was sieved using a No. 3 sieve, and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol solution as the extractant. The mixture was sonicated at 40°C for 30 minutes, filtered, and evaporated to dryness to obtain crude anthocyanin extract.

[0072] Example 6 (Establishment of suspension cell line + light culture with red to blue light intensity ratio of 6:3 + co-culture of Lactobacillus plantarum) A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0073] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0074] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0075] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ to obtain phenylalanine chelated iron.

[0076] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 30 mg / L phenylalanine chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0077] S6. Specific light stimulation increases the growth rate of Sedum aizoon stem cells and the accumulation of anthocyanin content: The suspension culture system obtained in step S5 was cultured in shake flasks at a rotation speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity of the light culture was 800 lx, the light cycle was 12 h / d, and the red-blue light intensity ratio was 6:3, thus obtaining the initial liquid culture system and culturing stable Sedum sarmentosum stem cell seeds.

[0078] S7. Promoting anthocyanin release from cells by co-culturing Lactobacillus plantarum and Sedum aizoon stem cells: The initial liquid culture system obtained in step S6 was repeated with the shaker culture described in step S6 at a rotation speed of 110 rpm and a temperature of 25°C. After one culture cycle, 1 mL of 10... 6 Lactobacillus plantarum at cfu / mL was cultured in shake flasks for 4 days under constant temperature, rotation speed, light intensity, and red-blue light intensity ratio.

[0079] S8. Extraction of anthocyanins from Sedum aizoon stem cells: The liquid culture system obtained in step S7 was filtered to obtain filtrate and Sedum aizoon stem cells. The filtrate was collected and evaporated to dryness to obtain crude anthocyanin extract (this step reduced the amount of HCl and ethanol used). The Sedum aizoon stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected. The collected powder was sieved through a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixed solution of 0.5% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to dryness to obtain crude anthocyanin extract.

[0080] Comparative Example 1 (Field cultivation - normal growth - sunlight) Sedum aizoon plants grown in the field (at 15-25℃ under natural light, with a growth cycle of 60-90 days) were dried in an oven at 40℃ until constant weight, then pulverized into powder and collected. The collected powder was sieved through a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixed solution of 0.8% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40℃ for 30 minutes, followed by filtration and evaporation to obtain crude anthocyanin extract.

[0081] Comparative Example 2 (Ordinary callus tissue + dark culture) A method for obtaining high-yield anthocyanins based on Sedum aizoon callus includes the following steps: S1. Induction of Sedum aizoon callus: Vigorous, healthy young leaves were selected as material. The leaves were cut into 1cm pieces, and eight leaves were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0082] S2. Accumulation of anthocyanins in the callus tissue of Sedum aizoon: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1 g of the detached callus was inoculated into 50 mL of proliferation medium and cultured in the dark at 25°C for 20 days as one culture cycle. The proliferation medium used was a solid medium composed of 20 g / L MS, 1.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0083] S3. Establishment of a suspension culture system for Sedum aizoon callus: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* callus tissue cultured in step S2 was inoculated into the liquid culture medium using forceps, and the pH was adjusted to 5.8. The culture was incubated in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0084] S4. The amount of callus growth and anthocyanin accumulation in Sedum aizoon: The callus tissue obtained in step S3 was cultured in the dark at a temperature of 25℃ for 20 days per cycle to obtain Sedum sarmentosum callus tissue induced from young leaves.

[0085] S5. Extraction of anthocyanins from Sedum aizoon callus: The liquid culture system obtained in step S4 was filtered, and the Sedum aizoon callus tissue was collected. Alternatively, the filtered Sedum aizoon callus tissue was dried in an oven at 40°C until constant weight, pulverized into powder, and collected. The collected powder was sieved through a No. 3 sieve, and extracted at a material-to-liquid ratio of 1:40 using a mixed solution of 0.8% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to obtain crude anthocyanin extract.

[0086] Comparative Example 3 (Stem cells cultured in conventional culture medium (without added precursor substances) - dark culture) The traditional method for culturing Sedum aizoon stem cells to promote anthocyanin synthesis includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0087] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0088] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0089] S4. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps, and the pH was adjusted to 5.8. The culture was incubated in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0090] S5. Extraction of anthocyanins from Sedum aizoon stem cells: The liquid culture system obtained in step S4 was filtered to collect Sedum sarmentosum stem cells, or the filtered Sedum sarmentosum stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected. The collected powder was sieved through a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol solution as the extractant. The mixture was sonicated at 40°C for 30 minutes, filtered, and evaporated to dryness to obtain crude anthocyanin extract.

[0091] Comparative Example 4 (Ordinary callus tissue + full-spectrum culture) A method for obtaining high-yield anthocyanins based on Sedum aizoon callus includes the following steps: S1. Induction of callus tissue from young leaves of Sedum aizoon: Vigorous, healthy young leaves were selected as material. The leaves were cut into 1cm pieces, and eight leaves were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0092] S2. Accumulation of anthocyanins in the callus tissue of Sedum aizoon: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1 g of the detached callus was inoculated into 50 mL of proliferation medium and cultured at 25℃ for 20 days as one culture cycle. The proliferation medium used was a solid medium composed of 20 g / L MS, 1.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0093] S3. Establishment of a suspension culture system for Sedum aizoon callus: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* callus tissue cultured in step S2 was inoculated into the liquid culture medium using forceps, and the pH was adjusted to 5.8. The culture was incubated in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0094] S4. Specific light stimulation increases the growth of Sedum aizoon callus and the accumulation of anthocyanin content: The callus tissue obtained in step S3 was cultured at a temperature of 25℃ for 20 days per cycle. Each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light culture was conducted under full-spectrum conditions with a light intensity of 800 lx and a light cycle of 12 h / d, resulting in a liquid culture system containing Sedum sarmentosum callus tissue directly induced from young leaves.

[0095] S5. Extraction of anthocyanins from Sedum aizoon callus: The Sedum aizoon callus tissue directly induced from the young leaves obtained in step S4 was removed with tweezers and placed in a petri dish. It was dried in an oven at 40°C until constant weight, then pulverized into powder and collected (the latter is beneficial for the preservation of intracellular anthocyanins). The collected powder was sieved using a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to obtain crude anthocyanin extract.

[0096] Comparative Example 5 (Ordinary callus tissue + red-blue light 2:5 culture) A method for obtaining high-yield anthocyanins based on Sedum aizoon callus includes the following steps: S1. Induction of callus tissue from young leaves of Sedum aizoon: Vigorous, healthy young leaves were selected as material. The leaves were cut into 1cm pieces, and eight leaves were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0097] S2. Accumulation of anthocyanins in the callus tissue of Sedum aizoon: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1 g of the detached callus was inoculated into 50 mL of proliferation medium and cultured at 25℃ for 20 days as one culture cycle. The proliferation medium used was a solid medium composed of 20 g / L MS, 1.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0098] S3. Establishment of a suspension culture system for Sedum aizoon callus: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* callus tissue cultured in step S2 was inoculated into the liquid culture medium using forceps, and the pH was adjusted to 5.8. The culture was incubated in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0099] S4. Specific light stimulation increases the growth of Sedum aizoon callus and the accumulation of anthocyanin content: The callus tissue obtained in step S3 was cultured at a temperature of 25℃ for 20 days per cycle. Each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity for the light culture was 800 lx, the light cycle was 12 h / d, and the red-blue light ratio was 2:5. This yielded a liquid culture system containing Sedum sarmentosum callus tissue directly induced from young leaves.

[0100] S5. Extraction of anthocyanins from Sedum aizoon callus: The Sedum aizoon callus tissue directly induced from the young leaves obtained in step S4 was removed with tweezers and placed in a petri dish. It was dried in an oven at 40°C until constant weight, then pulverized into powder and collected (the latter is beneficial for the preservation of intracellular anthocyanins). The collected powder was sieved using a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to obtain crude anthocyanin extract.

[0101] Comparative Example 6 (Ordinary callus tissue + red-blue light 5:1 culture) A method for obtaining high-yield anthocyanins based on Sedum aizoon callus includes the following steps: S1. Induction of callus tissue from young leaves of Sedum aizoon: Vigorous, healthy young leaves were selected as material. The leaves were cut into 1cm pieces, and eight leaves were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0102] S2. Accumulation of anthocyanins in the callus tissue of Sedum aizoon: The purplish-red callus induced in step S1 was gently detached from the leaf using tweezers. 1 g of the detached callus was inoculated into 50 mL of proliferation medium and cultured at 25°C for 20 days as one culture cycle. The proliferation medium used was a solid medium composed of 20 g / L MS, 1.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0103] S3. Establishment of a suspension culture system for Sedum aizoon callus: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* callus tissue cultured in step S2 was inoculated into the liquid culture medium using forceps, and the pH was adjusted to 5.8. The culture was incubated in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0104] S4. Specific light stimulation increases the growth of Sedum aizoon callus and the accumulation of anthocyanin content: The callus tissue obtained in step S3 was cultured at a temperature of 25℃ for 20 days per cycle. Each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity for the light culture was 800 lx, the light cycle was 12 h / d, and the ratio of red to blue light intensity was 5:1. This yielded a liquid culture system containing Sedum sarmentosum callus tissue directly induced from young leaves.

[0105] S5. Extraction of anthocyanins from Sedum aizoon callus: The Sedum aizoon callus tissue directly induced from the young leaves obtained in step S4 was removed with tweezers and placed in a petri dish. It was dried in an oven at 40°C until constant weight, then pulverized into powder and collected (the latter is beneficial for the preservation of intracellular anthocyanins). The collected powder was sieved using a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to obtain crude anthocyanin extract.

[0106] Comparative Example 7 (Ordinary callus tissue + red-blue light 5:2 culture) A method for obtaining high-yield anthocyanins based on Sedum aizoon callus includes the following steps: S1. Induction of callus tissue from young leaves of Sedum aizoon: Vigorous, healthy young leaves were selected as material. The leaves were cut into 1cm pieces, and eight leaves were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0107] S2. Accumulation of anthocyanins in the callus tissue of Sedum aizoon: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1 g of the detached callus was inoculated into 50 mL of proliferation medium and cultured at 25℃ for 20 days as one culture cycle. The proliferation medium used was a solid medium composed of 20 g / L MS, 1.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0108] S3. Establishment of a suspension culture system for Sedum aizoon callus: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* callus tissue cultured in step S2 was inoculated into the liquid culture medium using forceps, and the pH was adjusted to 5.8. The culture was incubated in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0109] S4. Specific light stimulation increases the growth of Sedum aizoon callus and the accumulation of anthocyanin content: The callus tissue obtained in step S2 was cultured at a temperature of 25℃ for 20 days per cycle. Each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity for the light culture was 800 lx, the light cycle was 12 h / d, and the ratio of red to blue light intensity was 5:2. This yielded a liquid culture system containing Sedum sarmentosum callus tissue directly induced from young leaves.

[0110] S5. Extraction of anthocyanins from Sedum aizoon callus: The Sedum aizoon callus tissue directly induced from the young leaves obtained in step S4 was removed with tweezers and placed in a petri dish. It was dried in an oven at 40°C until constant weight, then pulverized into powder and collected (the latter is beneficial for the preservation of intracellular anthocyanins). The collected powder was sieved using a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to obtain crude anthocyanin extract.

[0111] Comparative Example 8 (Ordinary callus tissue + red-blue light 6:3 culture) A method for obtaining high-yield anthocyanins based on Sedum aizoon callus includes the following steps: S1. Induction of callus tissue from young leaves of Sedum aizoon: Vigorous, healthy young leaves were selected as material. The leaves were cut into 1cm pieces, and eight leaves were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0112] S2. Accumulation of anthocyanins in the callus tissue of Sedum aizoon: The purplish-red callus induced in step S1 was gently detached from the leaf using tweezers. 1 g of the detached callus was inoculated into 50 mL of proliferation medium and cultured at 25°C for 20 days as one culture cycle. The proliferation medium used was a solid medium composed of 20 g / L MS, 1.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0113] S3. Establishment of a suspension culture system for Sedum aizoon callus: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* callus tissue cultured in step S2 was inoculated into the liquid culture medium using forceps, and the pH was adjusted to 5.8. The culture was incubated in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0114] S4. Specific light stimulation increases the growth of Sedum aizoon callus and the accumulation of anthocyanin content: The callus tissue obtained in step S3 was cultured at a temperature of 25℃ for 20 days per cycle. Each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity for the light culture was 800 lx, the light cycle was 12 h / d, and the ratio of red to blue light intensity was 6:3. This yielded a liquid culture system containing Sedum sarmentosum callus tissue directly induced from young leaves.

[0115] S5. Extraction of anthocyanins from Sedum aizoon callus: The Sedum aizoon callus tissue directly induced from the young leaves obtained in step S4 was removed with tweezers and placed in a petri dish. It was dried in an oven at 40°C until constant weight, then pulverized into powder and collected (the latter is beneficial for the preservation of intracellular anthocyanins). The collected powder was sieved using a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to obtain crude anthocyanin extract.

[0116] Comparative Example 9 (Establishment of suspension cell lines + light culture - full spectrum) A method for obtaining high-yield anthocyanins based on Sedum aizoon callus includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0117] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0118] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0119] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ for 18 hours to obtain phenylalanine chelated iron.

[0120] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 30 mg / L phenylalanine chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0121] S6. Specific light stimulation increases the growth rate of Sedum aizoon stem cells and the accumulation of anthocyanin content: The suspension culture system obtained in step S5 was cultured in shake flasks at a rotation speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light culture was carried out under full-spectrum conditions with a light intensity of 800 lx and a light cycle of 12 h / d, to obtain the final liquid culture system.

[0122] S7. Extraction of anthocyanins from Sedum aizoon stem cells: The liquid culture system obtained in step S6 was filtered to collect Sedum sarmentosum stem cells, or the filtered Sedum sarmentosum stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected (the latter is beneficial for the preservation of anthocyanins in the cells). The collected powder was sieved using a No. 3 sieve, and extracted at a material-to-liquid ratio of 1:40 using a mixture of 0.8% hydrochloric acid and 50% ethanol solution as the extractant. The mixture was sonicated at 40°C for 30 minutes, filtered, and evaporated to dryness to obtain crude anthocyanin extract.

[0123] Comparative Example 10 (Precursor material added to suspension cell culture + light culture with red to blue light intensity ratio of 6:3 + organic solvent extraction) A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0124] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0125] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0126] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ to obtain phenylalanine chelated iron.

[0127] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 3 mg of phenylalanine-chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0128] S6. Specific light stimulation increases the growth rate of Sedum aizoon stem cells and the accumulation of anthocyanin content: The suspension culture system obtained in step S5 was cultured in shake flasks at a rotation speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity of the light culture was 800 lx, the light cycle was 12 h / d, and the red-blue light intensity ratio was 6:3, resulting in the final liquid culture system.

[0129] S7. Extraction of anthocyanins from Sedum aizoon stem cells: The liquid culture system obtained in step S6 was filtered to collect Sedum sarmentosum stem cells, or the filtered Sedum sarmentosum stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected (the latter is beneficial for the preservation of anthocyanins in the cells). The collected powder was sieved using a No. 3 sieve, and extracted at a material-to-liquid ratio of 1:40 using an 80% ethanol solution as the extractant. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to obtain crude anthocyanin extract.

[0130] Comparative Example 11 (Precursor-added suspension cell culture + light culture with red to blue light intensity ratio of 6:3 + yeast co-culture) A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells includes the following steps: S1. Induction of Sedum aizoon callus: Sterile Sedum seedlings with vigorous growth were selected as the material. Leaves of the sterile seedlings were cut into 1cm pieces, and eight pieces were inoculated into 50mL of induction medium and cultured in the dark at 25℃ for 40 days to induce purplish-red callus tissue. The induction medium used was a solid medium composed of 20g / L MS, 2mg / L 2,4-D, 1mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0131] S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was gently detached from the leaf using forceps. 1g of the detached callus was inoculated into 50mL of proliferation medium and cultured in the dark at 25℃. Four subculture cycles were performed, each lasting 20 days, to obtain Sedum sarmentosum stem cells (Sedum sarmentosum embryogenic callus). The proliferation medium used was a solid medium composed of 20g / L MS, 1.5mg / L 2,4-D, 0.5mg / L 6-BA, 3% sucrose, 0.6% agar, and water.

[0132] S3, Screening of Sedum aizoon stem cells: Using a UV spectrophotometer to determine the anthocyanin content of cells, we screened out Sedum sarmentosum stem cells with high anthocyanin content (dry weight content reaching 6.5 mg / g), loose and fragile (after inoculation into liquid culture medium, they can be dispersed into stem cells of 0.8~1.2 mm), and vigorous growth (high vitality) (the growth rate can reach 5 times in 10 days and 11.6 times in 20 days) for later use.

[0133] S4. Preparation of phenylalanine chelated iron: Weigh 1g of phenylalanine and 1g of ferrous chloride, dissolve them in 100mL of water, and place the solution in a three-necked flask under liquid nitrogen conditions. Add 0.5g of iron powder and adjust the pH to 4.0 with sodium hydroxide. Stir to promote the reaction and reflux. After reacting for 1 hour, centrifuge the mixture. Utilizing the difference in solubility of the amino acid chelate and its free components in ethanol (ethanol is a product of the reaction), precipitate the supernatant with alcohol, then filter it using a vacuum pump to obtain a brown filter residue. Wash the filter residue 5-6 times with ethanol and dry it in an oven at 60℃ to obtain phenylalanine chelated iron.

[0134] S5. Establishment of the Sedum aizoon stem cell suspension culture system: 100 mL of liquid culture medium was placed in a 250 mL shake flask. 1 g of *Sedum aizoon* stem cells selected in step S3 were seeded into the liquid culture medium using forceps. 3 mg of phenylalanine-chelated iron was added to the liquid culture medium, and the pH was adjusted to 5.8. The culture was carried out in the dark at 25°C with a shaking speed of 110 rpm for 20 days to obtain a suspension culture system. The liquid culture medium used in the process consisted of 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and water.

[0135] S6. Specific light stimulation increases the growth rate of Sedum aizoon stem cells and the accumulation of anthocyanin content: The suspension culture system obtained in step S5 was cultured in shake flasks at a rotation speed of 110 rpm and a temperature of 25°C. Each shake flask culture cycle was 20 days, and each cycle was divided into two stages: the first stage was dark culture for 10 days; the second stage was light culture for 10 days. The light intensity of the light culture was 800 lx, the light cycle was 12 h / d, and the red-blue light intensity ratio was 6:3, thus obtaining the initial liquid culture system.

[0136] S7. Promoting anthocyanin release from cells by co-culturing yeast with Sedum aizoon stem cells: The initial liquid culture system was repeatedly cultured on a shaker as described in step S6 at a speed of 110 rpm and a temperature of 25°C. After one culture cycle, 1 mL of 10... 6 Yeast cells at cfu / mL were cultured in shake flasks for 4 days under constant temperature, rotation speed, light intensity, and red-blue light intensity ratio to obtain the final liquid culture system.

[0137] S8. Extraction of anthocyanins from Sedum aizoon stem cells: The obtained liquid culture system at the end of the treatment period was filtered to obtain filtrate and Sedum aizoon stem cells. The filtrate was collected and evaporated to dryness to obtain crude anthocyanin extract (this step reduced the amount of HCl and ethanol used). The Sedum aizoon stem cells were dried in an oven at 40°C to constant weight, pulverized into powder, and collected. The collected powder was sieved through a No. 3 sieve and extracted at a material-to-liquid ratio of 1:40 using a mixed solution of 0.5% hydrochloric acid and 50% ethanol. The extraction was performed by sonication at 40°C for 30 minutes, followed by filtration and evaporation to dryness to obtain crude anthocyanin extract.

[0138] Table 1. Comparison of anthocyanin content and stable cell growth in Sedum aizoon cells.

[0139] As shown in Table 1, higher cell fresh weight and growth rate indicate stable cell growth, meaning better cell stability. The percentage increase in anthocyanin content in Table 1 is relative to Comparative Example 1.

[0140] Comparative Example 1: Field-grown, normally growing Sedum aizoon plants had an anthocyanin content (dry weight) of 0.2 mg / g, a cell fresh weight of 2g, and a 1-fold increase in growth over 20 days, indicating low anthocyanin content. Comparative Example 2: Ordinary callus tissue (using young leaves as explants to induce cell dedifferentiation and proliferation) showed a 9% increase in anthocyanin content compared to Comparative Example 1, with a cell fresh weight of 2.9g and a 1.9-fold increase in growth over 20 days. While the anthocyanin content was increased, cell growth was slow. Comparative Example 3: Sedum aizoon stem cells were selected from leaves of vigorous, healthy, sterile Sedum aizoon seedlings. Under ordinary callus culture conditions, the induced purplish-red callus tissue was screened and then suspended in a traditional liquid culture medium. The anthocyanin content increased by 31.5%, a significant increase compared to field-grown Sedum aizoon plants, with a cell fresh weight of 12.6g and an 11.6-fold increase in growth over 20 days, indicating rapid cell growth. The culture conditions for Example 1 and stem cells (Comparative Example 3) were the same. Precursor material (phenylalanine chelated iron) was added to the traditional liquid culture medium for suspension culture. The anthocyanin content was 64% higher than that of field-grown plants, the fresh cell weight was 13.9g, and the cell growth rate was 12.9 times after 20 days. The anthocyanin content was the highest among the cases in Table 1, and the cell growth was also the most stable.

[0141] Table 2 Comparison of anthocyanin content under different light conditions

[0142] Table 2 compares the anthocyanin content under different light conditions. The *Sedum aizoon* stem cells used in this invention and ordinary callus tissue (prepared from young leaves) were cultured under the same conditions using the culture systems described in Examples 1-5 and Comparative Example 9 of this invention. Anthocyanin synthesis was compared under different light stimuli and in field cultivation. Plants need light to absorb nutrients and utilize light energy for photosynthesis to obtain the necessary nutrients for growth and development; plant cells are no exception. Different wavelengths of light have specific effects on the active substances within plant cells. Using different wavelengths of light for culture is beneficial for the transformation of these active substances.

[0143] The selected Sedum aizoon suspension stem cells were seeded in liquid culture medium containing 20 g / L MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, 3% sucrose, and 30 mg / L phenylalanine chelated iron, and cultured in a shaker at 110 rpm and 25°C. After 10 days of culture, the Sedum aizoon stem cell culture system was placed under a light intensity of 800 lx with a red-blue light intensity ratio of 2~6:3~5, for 12 hours per day. This effectively improved the growth rate and anthocyanin accumulation of Sedum aizoon stem cells, while also effectively addressing the browning problem of Sedum aizoon stem cells, reducing the browning rate of the system, and enabling the cells to serve as stable stem cell seeds for seeding in larger culture systems.

[0144] Sedum sarcodactylon stem cells cultured for 20 days were filtered and collected. Fresh Sedum sarcodactylon stem cells were obtained and can be directly used for anthocyanin extraction. Alternatively, the filtered Sedum sarcodactylon stem cells can be dried in an oven at 40℃ to constant weight, pulverized, and collected; this method is beneficial for the preservation of intracellular anthocyanins. The absorbance was measured at 530 nm, and the anthocyanin content was calculated, leading to the following conclusions: As shown in Table 2, Examples 1-5 respectively enhanced anthocyanin accumulation in Sedum sarmentosum stem cells by applying different light stimuli; Comparative Examples 4-8 respectively enhanced anthocyanin accumulation in ordinary callus tissue prepared from young leaves by applying different light stimuli; Comparative Example 9 enhanced anthocyanin accumulation in Sedum sarmentosum stem cells by applying full-spectrum light stimuli. Specific light types are shown in Table 2. The percentage increase in anthocyanin content (dry weight) in Table 2 is based on normally growing Sedum sarmentosum plants cultivated in the field.

[0145] Table 2 shows that, compared to field-grown Sedum sarmentosum plants, the anthocyanin content of Comparative Example 2 and Example 1 was increased, with Example 1 showing a 62% increase and Comparative Example 1 a 9% increase. When the anthocyanin synthesis of callus prepared from young leaves was stimulated under a full-spectrum light intensity ratio of 2:5 (red to blue), the anthocyanin content remained unchanged compared to the dark culture conditions of Comparative Example 2. The anthocyanin content of callus prepared from young leaves in Comparative Examples 5-8 was significantly higher than that in Comparative Example 2. Specifically, the anthocyanin content of callus prepared from young leaves in Comparative Example 8 was more than 20% higher than that of field-grown Sedum sarmentosum plants in Comparative Example 1. This indicates that a 6:3 red to blue light ratio has an effect on anthocyanin synthesis.

[0146] In Table 2, the liquid culture system of cells cultured under any light conditions with added precursor substance (phenylalanine chelated iron) promoted anthocyanin synthesis, and the anthocyanin content was increased compared to the dark culture conditions in Example 1. The increase in anthocyanin content in Examples 2 and 3 was less significant, while the increases in anthocyanin content in Examples 4 and 5 and Comparative Example 9 were more pronounced. Compared to the normally growing Sedum aizoon plants in field cultivation in Comparative Example 1, the anthocyanin content in Examples 4 and 5 and Comparative Example 9 increased by more than 75%, indicating that a red-blue light intensity ratio of 2-6:3-5 has a significant effect on anthocyanin synthesis. Among these, the effect was most significant when the red-blue light intensity ratio was 6:3 in Example 5.

[0147] Under the same light conditions, compared with cell culture supplemented with precursor (phenylalanine chelated iron), the anthocyanin content (dry weight) of callus prepared from young leaves was significantly higher in the latter than that of callus prepared from young leaves. Specifically, under a red-blue light intensity ratio of 6:3, the anthocyanin content (dry weight) in the callus prepared from young leaves in Comparative Example 8 reached 4.2 mg / g, which was more than 20% higher than that of normally grown Sedum sarmentosum stem cell plants in field cultivation. The anthocyanin content (dry weight) in Example 5 was the highest among the cases in Table 2, reaching 16.8 mg / g, which was 83% higher than that of normally grown Sedum sarmentosum stem cell plants in field cultivation, and significantly higher than the anthocyanin content (dry weight) in the callus prepared from young leaves in Comparative Example 8.

[0148] In summary, among the cases in Table 2, suspension culture of the liquid culture system with added phenylalanine chelated iron under a red-to-blue light intensity ratio of 6:3 showed the best effect in promoting anthocyanin synthesis. Therefore, Example 5 is the most effective among the cases in Table 2. Furthermore, stable and heritable Sedum aizoon stem cell seeds could be obtained after light stimulation.

[0149] Table 3 Effect of different extraction conditions on anthocyanin yield

[0150] Table 3 shows the effect of different extraction conditions on anthocyanin yield. Lactic acid bacteria can generate hydrolytic enzymes, opening cell permeability and releasing intracellular anthocyanins into the liquid culture medium, thus increasing intracellular anthocyanin synthesis. During growth, lactic acid bacteria utilize glucose to convert it into dextran, which in turn enriches anthocyanins, forming stable anthocyanin-dextran coupling compounds. The acid produced during lactic acid bacteria growth lowers the pH of the system, allowing for a reduction in HCl usage and improved extraction efficiency. Co-culturing with lactic acid bacteria can improve anthocyanin extraction rate, reduce the use of organic reagents, and decrease environmental pollution. 1 mL of 10% HCl was added to every 100 mL of a 20-day-old Sedum aizoon suspension cell culture system. 6After co-culturing *Lactobacillus plantarum* at CFU / mL for 4 days, anthocyanins were extracted and collected from the culture liquid and cells. During extraction, the lactic acid bacteria can lower the pH of the culture system, reducing the amount of HCl used, thus increasing extraction efficiency and reducing environmental pollution. This method can obtain anthocyanins in high yields, improving the extraction rate for *Sedum aizoon* stem cell production systems. The anthocyanin extraction rate using the lactic acid bacteria co-culture method can reach 2.55%, which is 1.83 times higher than that of traditional organic extraction. Simultaneously, the acid-producing characteristics of lactic acid bacteria can reduce the use of organic reagents. This technology can establish a complete production system for anthocyanin extraction, achieving high efficiency and high yield. Evaluation was conducted per 100 mL of liquid volume, and the results are shown in Table 3.

[0151] As shown in Table 3, under different extraction conditions, Comparative Example 10 used organic solvents as the extraction agent to extract anthocyanins from Sedum aizoon stem cells, with an extraction yield of 14 mg / g and an extraction rate of only 1.4%. Example 5 used HCl + alcohol as the extraction agent to extract anthocyanins from Sedum aizoon stem cells, with an extraction yield of 16.8 mg / g. Although the extraction rate was improved compared to organic solvent extraction, it was only 1.68%. Comparative Example 11 involved Sedum aizoon stem cells co-cultured with yeast and then using HCl + alcohol as the extraction agent in a liquid culture system to extract anthocyanins, with an extraction yield of 12.7 mg / g. The extraction rate was not only not higher than that of HCl + alcohol extraction alone, but actually lower. Example 6 involved Sedum aizoon stem cells co-cultured with lactic acid bacteria and then using HCl + alcohol extraction in a liquid culture system to extract anthocyanins, with an extraction yield of 25.5 mg / g. This was significantly improved compared to organic solvent extraction and HCl + alcohol extraction, with an extraction rate as high as 2.55%.

[0152] Based on the data from Examples 1 and 5-6 of the present invention in Tables 1-3, the following conclusions are drawn: In Example 1 of this invention, the anthocyanin content was 12.6 mg / g. In Example 5, based on the suspension culture system established in Example 1, the suspension culture system was cultured under light stimulation with a red-to-blue light intensity ratio of 6:3, resulting in a significant increase in anthocyanin content compared to Example 1, reaching 16.8 mg / g. Furthermore, in Example 6, based on the final culture system of Example 5, Lactobacillus plantarum was used to co-culture with Sedum aizoon stem cells to promote the release of anthocyanins from Sedum aizoon stem cells. After culture, anthocyanins could be extracted from the Sedum aizoon stem cells and the cultured liquid culture system, with an anthocyanin extraction yield as high as 25.5% and an extraction rate of 2.55%, which was significantly higher than the anthocyanin content in Example 5.

[0153] In summary, Example 6 of this invention firstly, phenylalanine chelated iron was added to the liquid culture medium to promote the synthesis of anthocyanins in Sedum sarmentosum stem cells and cell growth; then, under a light intensity ratio of 6:3, stimulation was used to enhance the accumulation of anthocyanins in Sedum sarmentosum stem cells and cell stability; finally, co-culture with Lactobacillus plantarum and Sedum sarmentosum stem cells promoted the release and extraction of anthocyanins, resulting in the highest anthocyanin content.

[0154] The various embodiments are described in a progressive manner, with each embodiment focusing on the differences from other implementation cases. Similar or identical parts between the various implementation cases can be referred to each other.

[0155] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0156] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells, characterized in that, Includes the following steps: S1. Induction of Sedum aizoon callus: The leaves of sterile Sedum aizoon seedlings were cut into pieces and inoculated into an induction medium for induction culture to induce purplish-red callus tissue; the induction medium was a solid medium composed of MS, 2.0 mg / L 2,4-D, 1.0 mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in 1L of water. S2. Accumulation of anthocyanins in Sedum sarmentosum stem cells: The purplish-red callus induced in step S1 was peeled off from the leaves and inoculated into a proliferation medium for subculture to obtain Sedum sarmentosum stem cells; the proliferation medium was a solid medium composed of MS, 1.5 mg / L 2,4-D, 0.5 mg / L 6-BA, 3% sucrose, and 0.6% agar dissolved in 1 L of water; S3. Screening of Sedum sarmentosum stem cells: The anthocyanin content of cells was determined by ultraviolet spectrophotometer, and Sedum sarmentosum stem cells with an anthocyanin dry weight content of 6.5 mg / g were screened. S4. Establishment of the Sedum aizoon stem cell suspension culture system: The Sedum aizoon stem cells screened in step S3 were seeded into liquid culture medium, and 30 mg / L phenylalanine chelated iron was added to the liquid culture medium. The suspension culture system was obtained by suspension culture. The liquid culture medium consisted of MS, 1.0 mg / L 2,4-D, 0.3 mg / L 6-BA, 0.3 mg / L NAA, and 3% sucrose. S5. Light stimulation enhances the growth of Sedum sarmentosum stem cells and the accumulation of anthocyanin content. The suspension culture system obtained in step S4 is cultured in shake flasks at a rotation speed of 100-120 rpm and a temperature of 22-28°C. Each shake flask culture cycle is 19-21 days, and each cycle is divided into two stages: the first stage is dark culture for 9-11 days; the second stage is light culture for 9-11 days. The light intensity for light culture is 800-1600 lx, the light cycle is 8-12 h / d, and the ratio of red light to blue light intensity is 2-6:3-5, thus obtaining an initial liquid culture system and cultivating stable Sedum sarmentosum stem cell seeds. S6. Promoting anthocyanin release by co-culturing *Lactobacillus plantarum* and *Sedum aizoon* stem cells: After repeating the shake-flask culture cycle described in step S5 for one cycle in the initial liquid culture system, add 1 mL of a 10% concentration of [unspecified ingredient] to every 100 mL of the culture system. 6 The final liquid culture system was obtained by using CFU / mL Lactobacillus plantarum bacterial culture, with a light intensity of 800~1600 lx, a red light to blue light intensity ratio of 2~6:3~5, a photoperiod of 8~12 h / d, a photoculture period of 3.5~4.5 days, a culture temperature of 22~28℃, and a rotation speed of 100~120 rpm. S7. Extraction of anthocyanins from Sedum sarmentosum stem cells and liquid culture system: The liquid culture system at the end stage is filtered to obtain filtrate and Sedum sarmentosum stem cells. The filtrate is filtered and evaporated to dryness to obtain crude anthocyanin extract. The powder obtained by drying, grinding and sieving Sedum sarmentosum stem cells is extracted with an extractant to obtain crude anthocyanin extract.

2. The method for obtaining high-yield anthocyanins based on stable Sedum aizoon stem cells according to claim 1, characterized in that, Step S1: Cut the leaves into small pieces with wounds of 0.8-1.2 cm in size and 3-5 wounds in total. In the induction culture step, 6-10 leaves are inoculated into each 50 mL induction culture medium and cultured in the dark at a temperature of 22-28℃ for 40-45 days. The inoculation container is a 300 mL tissue culture bottle.

3. The method for obtaining high-yield anthocyanins based on stable Sedum aizoon stem cells according to claim 1, characterized in that, Step S2, the peeling tool is tweezers; in the subculture step, 0.8~1.2g of purplish-red callus tissue is inoculated into every 50mL of proliferation medium, and cultured in the dark at a temperature of 22~28℃, with a subculture cycle of 19~21 days and 3~5 subculture cycles.

4. The method for obtaining high-yield anthocyanins based on stable Sedum aizoon stem cells according to claim 1, characterized in that, In the suspension culture step, 0.8~1.2g of Sedum aizoon stem cells are inoculated into every 100mL of liquid culture medium and cultured in the dark for 18~22 days at a temperature of 22~28℃ with a shaking speed of 100~120rpm; the pH of the suspension culture system is 5.8~6.

0.

5. The method for obtaining high-yield anthocyanins based on stable Sedum aizoon stem cells according to claim 1, characterized in that, The preparation process of the phenylalanine chelated iron is as follows: Take 0.95~1.05g of phenylalanine and ferrous chloride, dissolve them in 100mL of water, place them in a three-necked flask under 300mL liquid nitrogen conditions, add 0.45~0.55g of iron powder, adjust the pH to 3.5~5.0 with sodium hydroxide, stir, and reflux the mixture. After reacting for 1h, centrifuge the mixture at 8000rpm for 5min, take the supernatant for alcohol precipitation, filter it with a vacuum pump to obtain brown filter residue, wash the filter residue with ethanol 5~6 times, and dry it in an oven at 60℃ for 18~20h to obtain phenylalanine chelated iron.

6. The method for obtaining high-yield anthocyanins based on stable Sedum sarmentosum stem cells according to claim 1, characterized in that, The extractant in step S7 is a mixed solution of 0.5% hydrochloric acid and 50% ethanol, extracted at a material-to-liquid ratio of 1:

40. The extraction is performed by ultrasonication at 40°C for 30 minutes. The filtration is carried out using a vacuum pump. The drying is performed in an oven at 38-42°C. The screening is done using a No. 3 sieve.

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