A method for the continuous production of metabolites by plant cell suspension immobilization

Abstract

The application discloses a method for fixedly and continuously producing metabolites by plant suspension cells, and realizes fixed production of the plant suspension cells by fixing and culturing the high-metabolite-yield suspension cells in different fixed carriers, so that the ability of the plant suspension cells for continuously producing metabolites is effectively improved. Meanwhile, after the fixed culture, the continuous production culture is converted, and an optimized inducer is added into the culture medium, so that the efficiency of the fixed and continuous production of the metabolites by the plant suspension cells is remarkably improved, and efficient and continuous production of plant metabolites is realized.

Description

Technical Field

[0001] This invention belongs to the field of plant cell engineering, and specifically relates to a method for continuous production of metabolites by immobilizing plant suspension cells. Background Technology

[0002] Plants produce numerous metabolites, crucial for survival, reproduction, climate adaptability, symbiotic relationships, and pathogen resistance, and are widely used in pharmaceuticals, cosmeceuticals, and nutritional formulations. However, the direct, natural harvesting of plant metabolites is limited by seasonality, availability, species availability, and plant growth rate, leading to ecological damage. Furthermore, the stereochemical complexity of plant metabolites hinders attempts to produce them using chemical pathways; heterologous production, lacking fully characteristic secondary metabolic pathways, also results in differential compound expression. Plant cell suspension culture has emerged as a feasible technology for producing plant metabolites. Plant suspension cells not only produce a full spectrum of characteristic metabolites found in the parent plant but also exhibit stronger secondary metabolism compared to the parent plant, enabling the continuous production of metabolites.

[0003] Compared to suspension cells, plant cell immobilization offers advantages such as improved shear resistance, maintained cell viability, promoted synthesis and secretion of metabolites, reusability, increased production efficiency, and ease of post-production processing. Currently, most established immobilization technologies both domestically and internationally are embedding methods, where plant suspension cells are embedded in gel materials such as calcium alginate, agar, agarose, gelatin, carrageenan, and polyacrylamide. However, the media used in this method are prone to breakage, have poor strength, and cannot support cell growth. Adsorption immobilization, on the other hand, utilizes the inherent adsorption capacity of cells to actively or passively adsorb onto the surface of solid materials. The carriers used are porous and inert, exhibiting high stability and minimal impact on cell activity. The process is simple, the reaction conditions are mild, and the carriers can be reused repeatedly, enabling continuous plant cell production.

[0004] Therefore, this invention aims to study the effects of different immobilization carriers on plant suspension cells by using different suspension cells, in order to further establish an immobilization culture system and continuous production process related to plant suspension cells. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for continuous production of metabolites by immobilizing plant suspension cells, which addresses the shortcomings of the prior art.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for continuous production of metabolites by immobilizing plant suspension cells involves inoculating high-yield plant suspension cells into a liquid culture medium containing an immobilization carrier for cell immobilization, and then placing the immobilized cells into a liquid culture medium containing an inducer for continuous culture to produce metabolites.

[0008] The plant suspension cells mentioned above are grape suspension cells or hibiscus suspension cells.

[0009] The metabolites corresponding to the grape suspension cells are resveratrol and / or glycosides; the metabolites corresponding to the hibiscus suspension cells are anthocyanins.

[0010] The inoculation dosage is 6%-12% v / v;

[0011] Preferred inoculation amounts are 6% v / v, 8% v / v, and 12% v / v;

[0012] A more preferred inoculation amount is 12% v / v.

[0013] The immobilization carrier is a cotton towel with a concentration of 40-80g / L, a PE / PP composite short fiber with a concentration of 40-80g / L, a PE / PET composite short fiber with a concentration of 40-80g / L, a filter dust removal cloth with a concentration of 40-80g / L, or a polyethylene moisture-retaining blanket with a concentration of 40-80g / L.

[0014] Preferably, the immobilization carrier is a cotton towel with a concentration of 40-80 g / L;

[0015] More preferably, the immobilization carrier is a cotton towel with a concentration of 60 g / L.

[0016] The liquid culture medium further includes plant hormones, specifically, the hormones are 0.1-2 mg / L 2,4-D and 1-2 mg / L 6-BA, or the hormones are 0.1-2 mg / L 2,4-D and 0.2-1 mg / L KT;

[0017] Preferably, when using grape suspension cells to produce the metabolites resveratrol and / or glucosamine, the hormones are 0.2 mg / L 2,4-D and 1 mg / L 6-BA;

[0018] Preferably, when using roselle suspension cells to produce the metabolite anthocyanin, the hormones are 0.2 mg / L KT and 1 mg / L 2,4-D.

[0019] The liquid culture medium is B5 liquid culture medium.

[0020] The cell immobilization process involves two culture cycles, each lasting 6-8 days. After the first culture cycle, the immobilized carrier is separated from the solid and liquid, and then fresh liquid culture medium containing hormones is added. After the second culture cycle, the immobilized carrier is separated from the solid and liquid again.

[0021] Specifically, the cell immobilization culture conditions are as follows: under dark conditions, a rotation speed of 90-120 rpm and a culture temperature of 20-30℃, or under 2400 lux, 12h / d light conditions, a rotation speed of 90-120 rpm and a culture temperature of 20-30℃.

[0022] The inducer includes any one or a combination of several of the following: 5-15 mM dimethyl-β-cyclodextrin, 30-50 μM methyl jasmonate, 100-200 μM salicylic acid, 5-15 mM L-phenylalanine, 1-3 g / L 3-morpholinopropanesulfonic acid, 1-3 mg / L chitosan, 15-25 mg / L chitosan oligosaccharide, 1-3 mg / L chitin, 1-3 mg / L β-glucan, 30-50 mg / L acid-hydrolyzed casein, and 1-3 μM quercetin.

[0023] Preferably, the elicitor for the grape suspension cells is 40 μM methyl jasmonate, 10 mM dimethyl-β-cyclodextrin, 10 mM L-phenylalanine, and 3 g / L 3-morpholinopropanesulfonic acid.

[0024] Preferably, the elicitor for the roselle suspension cells is 40 μM methyl jasmonate.

[0025] The continuous culture involves a production cycle of 6-8 days, with fresh liquid culture medium containing inducers replacing the medium at the end of each production cycle.

[0026] Specifically, the continuous culture conditions are: darkness or 2400 lux, 12h / d light conditions, rotation speed of 90-120 rpm, culture temperature of 20-30℃, and a culture batch of 5-7 production cycles.

[0027] Beneficial effects:

[0028] (1) This invention establishes a plant suspension cell immobilization culture system by using different plant suspension cells and immobilization carriers, thereby realizing the continuous production of plant cells.

[0029] (2) This invention avoids the problems of low yield and short production time of suspended cells by immobilizing plant cells for continuous production, extends the production cycle of suspended cells, and effectively improves the productivity of metabolites.

[0030] (3) The present invention is simple to operate, low in cost, and its production is not limited by the plant itself, time, season and region. Attached Figure Description

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0032] Figure 1 Grape suspension cells were immobilized using different carriers. 1A shows the immobilized growth status of the cells on day 10 of immobilization culture. 1B shows the extracellular resveratrol yield of grape suspension cells immobilized using different carriers when the immobilization carrier concentration was 60 g / L. 1C shows the extracellular resveratrol yield of grape suspension cells immobilized using cotton towels with different concentrations.

[0033] Figure 2 The cell immobilization and growth status of hibiscus suspension cells immobilized in 60 g / L cotton towels on day 14.

[0034] Figure 3 The study used 60g / L cotton towels to immobilize "Kyoho" grape suspension cells for continuous production of resveratrol and glucosinolates. 3A shows the cell growth status during the second and fourth batches of immobilized production, 3B shows the extracellular resveratrol yield of different batches, and 3C shows the extracellular glucosinolate yield of different batches.

[0035] Figure 4 The chromatograms are for high performance liquid chromatography (HPLC) detection, where 4A is a 100 mg / L resveratrol standard and 4B is an immobilized extracellular resveratrol sample. Detailed Implementation

[0036] Based on the technical solution described in this invention, the embodiments provide detailed implementation methods and specific operating procedures. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the plant materials, reagents, and immobilization carriers are all commercially available unless otherwise specified.

[0037] The "Kyoho", "Xiahei", and "Domestic Red Globe" fruits used in this invention were purchased from supermarkets; the roselle seeds were purchased from Taobao (https: / / www.taobao.com); the B5 culture medium was from Qingdao Haibo Biotechnology, purchased from Wanqing Mall (http: / / www.wq-reagent.com); the immobilization vector was purchased from Alibaba's procurement platform (https: / / www.1688.com); and all plant hormones and inducers used were from Aladdin, purchased from Wanqing Mall (http: / / www.wq-reagent.com).

[0038] Example 1: Screening and culture of high-yield suspension cells

[0039] 1.1 Screening and culture of grape suspension cell lines for high resveratrol and glucosinolate production

[0040] Fruits of the "Kyoho," "Xiahei," and "Domestic Red Globe" varieties purchased from a supermarket were sterilized, and the grape skins were cut into approximately 1cm pieces. These were then inoculated into solid B5 medium containing 2 mg / L 6-BA and 0.5 mg / L 2,4-D, and induced under 25°C, 2400 lux, and 12 h / d light. After two weeks, "Domestic Red Globe" callus tissue was successfully induced, and after four weeks, "Kyoho" and "Xiahei" callus tissues were successfully induced. All three callus tissues exhibited red, green, yellow, and yellowish-brown colors. Resveratrol yield was extracted from the different colored callus tissues of "Kyoho," "Xiahei," and "Domestic Red Globe." The results showed that the yellow "Kyoho" callus tissue had the highest resveratrol content, while the yellowish-brown "Kyoho" callus tissue had the highest glucosinolate content.

[0041] Yellow and yellowish-brown, loosely textured callus tissue from the skin of "Kyoho" grapes with stable genetic characteristics was selected and inoculated at a rate of 6% v / v into liquid B5 medium containing 1-2 mg / L 6-BA and 0.2-1 mg / L 2,4-D. The medium was dispensed into 50 mL Erlenmeyer flasks (250 mL each) and cultured in the dark at 25°C with shaking at 110 rpm. "Kyoho" grape suspension cells were subcultured every 8 days. Before subculture, the suspension cells were shaken well, placed in a clean bench, and allowed to stand for several minutes. The supernatant was removed, and the middle portion of the cell culture was transferred to a shake flask containing fresh culture medium for further culture.

[0042] 1.2 Screening and Culture of High Anthocyanin-Producing Suspension Cell Lines for Roselle

[0043] After vernalizing hibiscus seeds in a refrigerator, the seeds were sterilized and inoculated into solid B5 medium. They were then cultured at 25°C under vertical light for 3-4 weeks, after which sterile hibiscus seedlings emerged. Leaves from these sterile seedlings were taken and cut into approximately 1cm sections. 2 Roselle callus tissue was successfully induced after inoculating the cells in solid B5 medium containing 2 mg / L 2,4-D and 0.2 mg / L KT, and inducing the cells at 25°C, 2400 lux, and 12 h / d light.

[0044] Select loosely textured callus tissue and inoculate it into liquid B5 medium at a 6% v / v inoculation rate. Dispense 50 mL of the medium into a 250 mL Erlenmeyer flask and incubate at 25°C, 2400 lux, 12 h / d light, and 120 rpm shaking. After 3-5 generations of suspension culture, when uniformly sized and shaped single cells appear, mix the cell supernatant with fresh solid medium to prepare a conditioned medium. Then, mix the selected single cells and small cell clusters with the conditioned medium and quickly pour the mixture into Petri dishes to form a thin layer. Incubate at 25°C under light. After approximately 25 days, small red cell clusters appear. Inoculate these red cell clusters into solid B5 medium containing 2 mg / L 2,4-D and 0.2 mg / L KT, and incubate at 25°C, 2400 lux, 12 h / d light to obtain anthocyanin-producing roselle callus tissue. Hibiscus callus tissue with loose texture and rapid growth rate producing anthocyanins was selected and inoculated into liquid B5 medium with hormone concentrations of 1-2 mg / L 2,4-D and 0.2-1 mg / L KT. The medium was placed in 250 mL Erlenmeyer flasks with a liquid volume of 50 mL and cultured at 25°C, 2400 lux, 12 h / d light, and 120 rpm shaking. Hibiscus suspension cells were subcultured every 6 days. Before subculturing, the suspension cells were shaken well, placed in a clean bench, allowed to stand for several minutes, the supernatant was removed, and the middle portion of the cell culture was transferred to a shake flask containing fresh culture medium for further culture.

[0045] Example 2: Screening of Suspension Cell Immobilization Materials

[0046] 2.1 Screening of Grape Suspension Cell Immobilization Materials

[0047] "Kyoho" grape suspension cells were inoculated at an 8% v / v rate into liquid B5 medium containing different immobilization carriers. The hormone concentrations were 1 mg / L 6-BA and 0.2 mg / L 2,4-D, respectively, in 50 mL Erlenmeyer flasks (250 mL each). The cells were cultured in the dark at 25°C and 110 rpm in a shaker. The immobilization carriers were: 40-80 g / L cotton towels, 60 g / L filter cloth, 60 g / L polyethylene moisture-retaining blankets, 60 g / L PE / PP composite short fibers, and 60 g / L PE / PET composite short fibers. Immobilized cells were cultured for 10 days, with samples taken every two days. 1 mL of the cell culture supernatant was extracted three times with ethyl acetate. The upper organic phase was collected, evaporated to dryness at 4°C, and dissolved in 1 mL of methanol. After complete dissolution, the sample was filtered through an organic filter membrane into a brown HPLC vial. High-performance liquid chromatography (HPLC) was performed using a 4.6 mm * 150 mm, 5 μm C18 column at 308 nm, 30°C, a flow rate of 0.6 mL / min, and a mobile phase of acetonitrile:0.2% phosphoric acid at a ratio of 30:70. Figure 4 ).

[0048] The results are as follows Figure 1 As shown in A, B, and C, *Gymnocalycium mihanovichii* grape suspension cells can be immobilized in the aforementioned carriers. Cotton towels and polyethylene moisturizing blankets showed the best immobilization effects (1A), with the highest extracellular resveratrol yield observed when suspension cells were immobilized using 60 g / L cotton towels (1B). Further investigation verified the effect of different cotton towel concentrations (40 g / L, 60 g / L, and 80 g / L) on resveratrol yield. The results (1C) showed that when the immobilization carrier was 60 g / L cotton towels, the highest extracellular resveratrol yield (2.83 mg / L) was observed on day 6 of immobilization, which was four times the yield of suspended extracellular resveratrol (0.70 mg / L).

[0049] Since glucosinolate is an oxidized dimer of resveratrol, in which two resveratrol units are oxidized and dimerized by peroxidase to produce glucosinolate, the yield of glucosinolate varies with the yield of resveratrol. Therefore, the extracellular glucosinolate yield is highest when the immobilization carrier is a cotton towel.

[0050] 2.2 Immobilization of Hibiscus Suspension Cells

[0051] Roselle suspension cells were inoculated at an inoculation rate of 8% v / v into B5 liquid medium containing 60 g / L cotton towels as immobilization carriers. The hormone concentrations were 1 mg / L 2,4-D and 0.2 mg / L KT. The medium was filled into 50 mL Erlenmeyer flasks at 25°C, under 2400 lux light for 12 h / d, and shaken at 120 rpm.

[0052] One week constitutes one culture cycle. After two weeks of immobilized culture, immobilized roselle cells were harvested, and anthocyanins were extracted. The anthocyanin content was determined using Giusti and Wrolstad's pH differential method. The results are as follows: Figure 2 As shown, the suspension cells and two parallel groups of immobilized cells in the non-high anthocyanin-producing cell line are yellow in color and do not produce red anthocyanins. In contrast, the suspension cells and two parallel groups of immobilized cells in the high anthocyanin-producing cell line both produce visible red anthocyanins. Specifically, the anthocyanin yields of the two parallel groups of immobilized cells in the high anthocyanin-producing cell line were 7.15 μg / g fresh weight and 16.68 μg / g fresh weight, respectively, while the anthocyanin yield of the suspension cells was 4.765 μg / g fresh weight. The anthocyanin yields of the two parallel groups of immobilized cells were 1.5 times and 3.5 times that of the suspension cells, respectively, indicating that immobilization significantly increased the anthocyanin yield of the cells.

[0053] Example 3: Screening of Suspension Cell Elicitors

[0054] 3.1 Screening of grape suspension cell inducers

[0055] Studies have shown that several inducers, including dimethyl-β-cyclodextrin, methyl jasmonate, salicylic acid, L-phenylalanine, chitosan, chitosan oligosaccharide, chitin, β-glucan, and acid-hydrolyzed casein, can increase the extracellular resveratrol production of grape suspension cells to varying degrees in grape suspension cell culture systems. Therefore, these inducers were screened in grape suspension cell systems.

[0056] "Kyoho" grape suspension cells were transferred at an inoculation rate of 12% v / v into liquid B5 medium containing 1 mg / L 6-BA and 0.2 mg / L 2,4-D. The medium was filled into 250 mL Erlenmeyer flasks with a volume of 50 mL and cultured in the dark at 25°C with shaking at 110 rpm. Using "Kyoho" grape suspension cells without inducers as the control group, the experimental group consisted of "Kyoho" grape suspension cells with a certain concentration of inducers: 5-15 mM dimethyl-β-cyclodextrin was added on the first day of suspension cell culture, or any one of the following was added on the fourth day of suspension culture: 30-50 μM methyl jasmonate, 100-200 μM salicylic acid, 5-15 mM L-phenylalanine, 1-3 mg / L chitosan, 15-25 mg / L chitosan oligosaccharide, 1-3 mg / L chitin, 1-3 mg / L β-glucan, or 30-50 mg / L acid-hydrolyzed casein. The suspension cells were cultured for 10 days, and the supernatant of the suspension cells was taken every two days to determine the yield of resveratrol and glucosamine in the supernatant of the suspension cell culture medium.

[0057] Tables 1 and 2 show the highest resveratrol and glucosinolate yields achievable by "Kyoho" grape suspension cells after 10 days of suspension cell culture under different types and concentrations of inducers. The results indicate that 10 mM dimethyl-β-cyclodextrin induced the highest extracellular resveratrol and glucosinolate yields, which were 250 times and 248.6 times that of the control group, respectively.

[0058] Table 1: Extracellular resveratrol production in grape suspension cells induced by different types and concentrations of elicitors

[0059]

[0060]

[0061] Table 2: Extracellular glucosinolate production in grape suspension cells induced by different types and concentrations of elicitors

[0062]

[0063] Martinez-Esteso et al. found that inducing grape suspension cells with a combination of cyclodextrin and methyl jasmonate could increase the yield of extracellular resveratrol. Therefore, based on the optimal concentrations of dimethyl-β-cyclodextrin and methyl jasmonate selected in Table 1, the two were combined to induce grape suspension cells. The culture conditions of the suspension cells remained unchanged, and the results are shown in Table 3. Table 3 shows the highest resveratrol and glucosamine yields achieved by "Kyoho" grape suspension cells after 10 days of culture under several inducer-induced conditions. The results showed that when 10 mM dimethyl-β-cyclodextrin and 40 μM methyl jasmonate were used for co-induction, the highest extracellular resveratrol yield was 234.52 mg / L, which was 418.8 times that of the control group, and the extracellular glucosamine yield was 174.87 mg / L, which was 485.8 times that of the control group. Compared with dimethyl-β-cyclodextrin induction, this further increased the yield of extracellular resveratrol and glucosamine.

[0064] L-Phenylalanine is a precursor for resveratrol biosynthesis. Tables 1 and 2 show that the optimal concentration for L-phenylalanine induction is 10 mM. Therefore, 10 mM L-phenylalanine was added as a third inducer to the aforementioned induction system of 10 mM dimethyl-β-cyclodextrin and 40 μM methyl jasmonate to co-induce grape suspension cells. The culture conditions for suspension cells remained unchanged. The results are shown in Table 3. When induced by the three inducers together, the extracellular resveratrol yield was 235.61 mg / L, and the extracellular glucose yield was 189.09 mg / L. Compared with the co-induction of 10 mM dimethyl-β-cyclodextrin and 40 μM methyl jasmonate, there was no significant increase in the extracellular resveratrol yield.

[0065] 3-Molarin propanesulfonic acid (MOPS) was used as a pH buffer to stabilize the pH in the culture system. Therefore, 1 g / L, 2 g / L, and 3 g / L of 3-Molarin propanesulfonic acid were added as a fourth inducer to the aforementioned induction system of 10 mM dimethyl-β-cyclodextrin, 40 μM methyl jasmonate, and 10 mM L-phenylalanine to co-induce grape suspension cells. The culture conditions for suspension cells remained unchanged. The results are shown in Table 3. When 10 mM dimethyl-β-cyclodextrin, 40 μM methyl jasmonate, 10 mM L-phenylalanine, and 3 g / L 3-Molarin propanesulfonic acid were co-induced, the extracellular resveratrol and glucosinolate yields were the highest, at 317.41 mg / L and 221.83 mg / L, respectively, which were 566.8 times and 616.2 times that of the control group, and significantly higher than those of 10 mM dimethyl-β-cyclodextrin, 40 μM methyl jasmonate, and 10 mM L-phenylalanine. The four inducer systems further increased the extracellular resveratrol production by 1.3-fold and 1.2-fold when co-induced with L-phenylalanine, respectively.

[0066] Since glucosinolate is an oxidized dimer of resveratrol, its yield varies with resveratrol yield. Therefore, the optimal type and concentration of inducers for inducing extracellular resveratrol production also apply to inducing extracellular glucosinolate production. Based on the above experimental results, four inducer combinations were selected: 10 mM dimethyl-β-cyclodextrin, 40 μM methyl jasmonate, 10 mM L-phenylalanine, and 3 g / L 3-morpholinopropanesulfonic acid. The immobilization continuous production experiment of Example 4.1 was then carried out.

[0067] Table 3: Co-induction of extracellular resveratrol and glucosinolate production in grape suspension cells by several inducers

[0068]

[0069] 3.2 Screening of Roselle Suspension Cell Elicitors

[0070] High-anthocyanin-producing suspension cells from hibiscus were inoculated at a rate of 12% v / v into liquid B5 medium containing 1 mg / L 2,4-D and 0.2 mg / L KT. The medium was placed in 250 mL Erlenmeyer flasks with a volume of 50 mL of liquid and cultured at 25°C, 2400 lux, 12 h / d light, and shaking at 120 rpm. The control group consisted of hibiscus suspension cells without inducers, while the experimental group consisted of hibiscus suspension cells with inducers. On the fourth day of suspension culture, one of the following was added to the hibiscus suspension cell system: 30-50 μM methyl jasmonate, 100-200 μM salicylic acid, 5-15 mM L-phenylalanine, or 1-3 μM quercetin. The suspension cells were cultured for 10 days, and samples were taken every two days to extract anthocyanins and determine their content.

[0071] Table 4 shows the highest anthocyanin yield achievable by hibiscus high-anthocyanin-producing suspension cells under different types and concentrations of inducers during 10 days of suspension cell culture. The results indicate that the suspension cells induced by 40 μM methyl jasmonate had the highest anthocyanin yield of 6.24 μg / g fresh weight, which is about twice that of the control group.

[0072] Table 4: Anthocyanin production in roselle suspension cells induced by different types and concentrations of elicitors

[0073]

[0074] Experiments revealed that roselle high-anthocyanin-producing suspension cells were prone to browning during production and were sensitive to elicitors. Therefore, only 40 μM methyl jasmonate was selected as an elicitor for the continuous production experiment of roselle suspension cell immobilization in Example 4.2.

[0075] Example 4: Immobilized Continuous Production Process

[0076] 4.1 Continuous Production of Extracellular Resveratrol and Grape Extract via Immobilized Grape Suspension Cells

[0077] "Kyoho" grape suspension cells were inoculated at a rate of 12% v / v into B5 liquid medium containing 1 mg / L 6-BA and 0.2 mg / L 2,4-D. 50 mL of the medium was placed in a 250 mL Erlenmeyer flask. The immobilization medium was a 60 g / L cotton towel. The culture was carried out at 25°C and 110 rpm in the dark. Each culture cycle lasted 6 days. At the end of each cycle, the immobilization medium was removed, and fresh B5 liquid medium was added. This process was repeated for two cycles. In the first cycle, the grape suspension cells were immobilized and adsorbed onto the cotton towel; in the second cycle, the cells were able to grow immobilized on the cotton towel.

[0078] After two culture cycles, a continuous production cycle was initiated, replacing the medium with fresh B5 liquid medium containing the inducer. Each production cycle lasted 8 days, and thereafter, the medium was replaced with fresh B5 liquid medium containing the inducer selected in Example 3.1: 40 μM methyl jasmonate, 10 mM dimethyl-β-cyclodextrin, 10 mM phenylalanine, and 3 g / L 3-morpholinopropanesulfonic acid. Samples were taken every two days to determine the extracellular resveratrol yield. The results are as follows... Figure 3 As shown in A and 3B, immobilized grape cells can be used for long-term continuous production. The extracellular resveratrol yield of immobilized cells with inducers was significantly higher than that without inducers. The average resveratrol yield per batch after adding inducers was 35.41 mg / L, 74.81 mg / L, 205.34 mg / L, 376.49 mg / L, 247.06 mg / L, 78.33 mg / L, and 24.44 mg / L, for a total of seven cycles. Although the yield was low in the early stages of continuous production due to cell growth and decreased later due to reduced cell activity, the resveratrol yield was significantly increased throughout the entire immobilized continuous production process, being 15.29, 27.95, 88.17, 205.49, 104.02, 39.13, and 15.97 times higher than that without inducers.

[0079] Since glucosinolate is an oxidized dimer of resveratrol, the conditions described above for the continuous production of extracellular resveratrol are also applicable to the continuous production of extracellular glucosinolate. Experimental results are as follows: Figure 3As shown in A and 3C, grape suspension cell immobilization can be carried out continuously for 7 cycles. The yield of extracellular glucosamine produced by continuous production with the addition of an inducer is significantly higher than that without an inducer. The average yield of glucosamine in each batch after adding the inducer is: 40.09 mg / L, 95.44 mg / L, 160.95 mg / L, 184.19 mg / L, 269.10 mg / L, 164.01 mg / L, and 53.84 mg / L. Although the glucosamine yield tends to stabilize in the early stage of continuous production and decreases in the later stage, the glucosamine yield is significantly increased throughout the entire immobilization continuous production process, being 23.08, 47.55, 92.15, 134.04, 151.07, 109.23, and 46.91 times that without an inducer.

[0080] 4.2 Continuous production of anthocyanins using hibiscus suspension cell immobilization

[0081] Roselle suspension cells producing high anthocyanins were inoculated at a rate of 12% v / v into B5 liquid medium containing 60 g / L cotton towels as immobilization carriers. The hormone concentrations were 1 mg / L 2,4-D and 0.2 mg / L KT. 50 mL of the medium was placed in 250 mL Erlenmeyer flasks and cultured at 25°C, 2400 lux, 12 h / d IPL, and 120 rpm shaking. Each culture cycle lasted 8 days. After each cycle, the immobilization carrier was retained, and the B5 liquid medium was replaced with fresh medium. This process was repeated for two cycles. In the first cycle, the roselle suspension cells were immobilized and adsorbed onto the cotton towel carrier; in the second cycle, the roselle cells were able to grow immobilized on the cotton towel.

[0082] After two culture cycles, a continuous production cycle was initiated, replacing the medium with fresh B5 liquid medium containing an inducer. Each production cycle lasted 6 days, and thereafter, the medium was replaced with fresh B5 liquid medium containing an inducer of 40 μM methyl jasmonate after each cycle. After each cycle, immobilized roselle cells were harvested, and anthocyanins were extracted. The anthocyanin content was determined using the pH differential method described by Giusti and Wrolstad. The results are shown in Table 5. Immobilization could produce continuously for 5 cycles. Compared with immobilization without an inducer, immobilization with an inducer increased the anthocyanin yield of suspension cells.

[0083] Table 5: Continuous anthocyanin production from hibiscus using suspension cell immobilization for high-yield anthocyanin production

[0084]

[0085] This invention demonstrates the ability of hibiscus and grape suspension cells to grow under immobilization using different immobilization carriers. Among them, 60 g / L cotton towel is the optimal choice for immobilization carrier. Immobilization increases the yield of metabolites of these two types of suspension cells. Furthermore, by first immobilizing and culturing plant suspension cells and then combining them with inducers for immobilization production, the continuous and efficient production of metabolites of these two plant suspension cells is achieved.

[0086] This invention provides a concept and method for the continuous production of metabolites using plant suspension cell immobilization. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for continuous production of metabolites using plant suspension cell immobilization, characterized in that, Plant suspension cells with high metabolite production were inoculated into a liquid culture medium containing an immobilization carrier for cell immobilization, and then the immobilized cells were placed in a liquid culture medium containing an inducer for continuous culture to produce metabolites. The plant suspension cells mentioned above are grape suspension cells or hibiscus suspension cells; The immobilization carrier is a cotton towel with a concentration of 40-80 g / L. The inducer includes any one or a combination of several of dimethyl-β-cyclodextrin, methyl jasmonate, and L-phenylalanine; The continuous culture is carried out in a production cycle of 6-8 days, and fresh liquid culture medium containing inducers is replaced at the end of each production cycle. The metabolites corresponding to the grape suspension cells are resveratrol and / or glucosinolate; the metabolites corresponding to the hibiscus suspension cells are anthocyanins. The inducers for grape suspension cells include any one or a combination of several of the following: 5-15 mM dimethyl-β-cyclodextrin, 30-50 μM methyl jasmonate, and 5-15 mM L-phenylalanine; the inducer for hibiscus suspension cells is 30-50 μM methyl jasmonate.

2. The method according to claim 1, characterized in that, The vaccination dose is 6%-12% v / v.

3. The method according to claim 2, characterized in that, The vaccinations mentioned above are administered at doses of 6% v / v, 8% v / v, and 12% v / v.

4. The method according to claim 1, characterized in that, The immobilization carrier is a cotton towel with a concentration of 60 g / L.

5. The method according to claim 1, characterized in that, The liquid culture medium is B5 liquid culture medium.

6. The method according to claim 1, characterized in that, The cell immobilization process involves two culture cycles, each lasting 6-8 days. After the first culture cycle, the immobilized carrier is separated from the solid and liquid, and then fresh liquid culture medium containing hormones is added. After the second culture cycle, the immobilized carrier is separated from the solid and liquid again.

7. The method according to claim 6, characterized in that, The cell immobilization is performed under the following conditions: in the dark, the rotation speed is 90-120 rpm and the culture temperature is 20-30 ℃; or under 2400 lux, 12 h / d light conditions, the rotation speed is 90-120 rpm and the culture temperature is 20-30 ℃.

8. The method according to claim 1, characterized in that, The inducers for the grape suspension cells are 40 μM methyl jasmonate, 10 mM dimethyl-β-cyclodextrin, and 10 mM L-phenylalanine, while the inducers for the roselle suspension cells are 40 μM methyl jasmonate.

9. The method according to claim 1, characterized in that, The continuous culture is carried out under the following conditions: darkness or 2400 lux, 12 h / d light conditions, rotation speed of 90-120 rpm, culture temperature of 20-30 ℃, and the culture batch consists of 5-7 production cycles.

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