Method for producing bryophyllol by using dendrobium suspending protocorm
By continuously culturing Dendrobium protocorms with high Dendrobium content in a bioreactor and adding Indian piriformis culture, the problems of unstable Dendrobium content and high cost were solved, and efficient and stable production of Dendrobium was achieved.
Patent Information
- Application Number
- CN202211714635.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-12-30
- Publication Date
- 2025-11-11
- Estimated Expiration
- 2042-12-30
AI Technical Summary
The content of dendrobine varies greatly in dendrobium. Dendrobium is a precious Chinese medicine and grows slowly, making it difficult to cultivate. Currently, the extraction cost of dendrobine is high and is affected by the environment and climate, resulting in slow industrial development.
Dendrobium protocorms with high dendrobine content are automatically and continuously cultured in a bioreactor. Inactivated Indian piriformis culture is added to activate the dendrobine synthesis gene, and dendrobine is then produced in a factory setting using the bioreactor.
This method achieves high biomass yield and stable, controllable content of dendrobium, reduces labor costs, avoids seasonal and environmental influences, and improves the production efficiency of dendrobium.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for producing dendrobine from suspended protocorms of Dendrobium. Background Technology
[0002] Gigantol is a bibenzyl aromatic compound belonging to the genus Dendrobium (Dendrobium). Dendrodium sw Dendrobium contains relatively specific active substances in plants, possessing strong anti-inflammatory, anti-aging, and anti-tumor effects, making it one of the research hotspots of Dendrobium in recent years. Besides exhibiting tissue-specific and harvesting season-specific characteristics, the dendrobin content in Dendrobium varies considerably among different species. For example, the dendrobin content in Dendrobium huoshanense is relatively high among Dendrobium species, ranging from 0.1 to 0.2 mg / g, while in Dendrobium officinale it is mostly below 0.02 mg / g or undetectable, and differs from that in Dendrobium nobile (…). D. nobile ), Dendrobium nobile ( D. chrysotoxum Like other Dendrobium species included in the pharmacopoeia, it belongs to the Dendrobium species with low Dendrobium phenol content.
[0003] Dendrobium is a traditional and precious Chinese medicinal herb. It is a plant that metabolizes C3 and crassulacean acid, resulting in slow growth, high cultivation difficulty, and consistently high prices. Dendrobium phenols are present in Dendrobium at mg-level concentrations. Extraction of dendrobium phenols from Dendrobium is expensive and susceptible to environmental and climatic influences. Therefore, despite its excellent medicinal value, the development of related industries using Dendrobium phenols has been slow. This invention enables the industrial-scale production of Dendrobium protocorms, offering advantages such as high biomass yield, stable and controllable dendrobium phenol content, labor savings, and independence from seasonal and temporal variations. Summary of the Invention
[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for producing dendrobium phenols from suspended protocorms of Dendrobium. This method utilizes Dendrobium protocorms with high dendrobium phenol content for automated and continuous cultivation in a bioreactor to achieve industrialized production of dendrobium phenols. It features high biomass yield, stable and controllable dendrobium phenol content, labor savings, and is unaffected by time, space, or season. Furthermore, by adding inactivated Indian piriformis culture to the culture medium, the transcription level of genes related to dendrobium phenol synthesis can be effectively activated, thereby increasing dendrobium phenol yield.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] A method for producing dendrobine from suspended protocorms of Dendrobium includes the following steps:
[0007] (1) Induction of high dendrobium protocorms: In June-July, dendrobium stem segments or leaves with dendrobium content >0.1 mg / g in fresh stems and leaves of the current year were selected as explants. After sterilization, they were inoculated into induction medium for induction culture. After continuous induction for 40-60 days, the dendrobium suspension protocorms obtained were tested for dendrobium content. Protocorms with dendrobium content >0.2 mg / g were retained as dendrobium suspension protocorms with high dendrobium content.
[0008] (2) Subculture and proliferation of Dendrobium protocorms with high Dendrobium content: The Dendrobium suspension protocorms with high Dendrobium content obtained in step (1) were inoculated into a subculture and proliferation medium for continuous subculture. The subculture cycle was 30-45 days.
[0009] (3) Continuous culture in a bioreactor: The Dendrobium suspension protocorms obtained in step (2) are inoculated into a liquid culture medium at an inoculation amount of 10-20 g fresh weight / L and cultured in a bioreactor.
[0010] (4) Collection of protocorms and extraction of dendrobium phenols: After continuous culture for 20-30 days in step (3), the protocorms of Dendrobium suspension grow to the logarithmic phase. Collect 20-30% of the culture product every 1-2 days, replenish the culture medium and adjust the pH to 5.8. Then use the collected culture product to extract dendrobium phenols.
[0011] Further, the induction medium in step (1) is 1 / 2MS + BA 2.0-4.0 mg / L + 2,4-D 1.0-2.0 mg / L + sucrose 20 g / L + agar 5.5 g / L, pH 5.8.
[0012] Furthermore, the culture conditions in step (1) are 25 ℃, 16 h / d light, and light intensity of 2000 lx.
[0013] Further, the subculture culture medium in step (2) is 1 / 2MS + BA 0.5-1.0 mg / L + sucrose 20 g / L + agar 5.5 g / L, pH 5.8.
[0014] Furthermore, the conditions for subculture in step (2) are 25 ℃ and 16 h / d light.
[0015] Further, the liquid culture medium in step (3) is 1 / 2MS + BA 0.5-1.0 mg / L + sucrose 30-40 g / L + inactivated culture of Indian pyriformis 5-10 g / L, pH 5.8.
[0016] Further, the bioreactor in step (3) is set to the following conditions: temperature 25 °C, light intensity 16 h / d, light intensity 2000 lx, aeration rate of 0.1 L / min per L of culture medium, and stirring speed of 120 rpm.
[0017] The advantages of this invention are:
[0018] 1. The content of dendrobine varies greatly among different species of Dendrobium. This invention selects germplasm with high dendrobine content as explant material, which can effectively ensure the establishment of Dendrobium suspension protocorms with high dendrobine content.
[0019] 2. This invention utilizes a bioreactor to continuously cultivate Dendrobium suspension protocorms. Through continuous collection, the logarithmic growth phase of Dendrobium suspension protocorms can be maintained, thereby increasing the yield of Dendrobium suspension protocorms and reducing the production cost of Dendrobium phenols with reduced manual input.
[0020] 3. Indian piriformis is a symbiotic beneficial fungus, and its culture products can promote plant growth and the accumulation of secondary metabolites. This invention can effectively activate the transcription level of genes related to dendrobium synthesis and increase dendrobium yield by adding growth regulators and organic additives such as inactivated Indian piriformis culture to the culture medium. Attached Figure Description
[0021] Figure 1 A comparison chart of dendrobine content in the stems and leaves of different Dendrobium varieties;
[0022] Figure 2 A comparison of dendrobine content in suspension protocorms cultured for different durations after continuous culture with different amounts of inactivated Indian piriformis.
[0023] Figure 3 A diagram illustrating the effects of subculture of protocorms with high dendrobine content in 'Minghu No. 3';
[0024] Figure 4 The curve showing the change in dendrobine content during continuous cultivation of 'Minghu No. 3' protocorms with high dendrobine content. Detailed Implementation
[0025] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.
[0026] The inactivated culture of Indian pyriformis used was prepared by inactivating Indian pyriformis in a water bath at 100°C for 5 minutes.
[0027] Example 1
[0028] (1) Induction of Dendrobium protocorms with high dendrobine content: On June 20, leaves of Dendrobium germplasm "14L-3" with dendrobine content >0.1 mg / g in fresh stems and leaves of the current year were selected as explants. After disinfection with 75% alcohol for 30 s and sterilization with 0.1% mercuric chloride for 10 min, the plants were inoculated into an induction medium of 1 / 2 MS + BA 3.5 mg / L + 2,4-D 1.5 mg / L + sucrose 20 g / L + agar 5.5 g / L, pH 5.8, with an inoculation amount of 100 mg fresh weight / L. After continuous induction for 45 days at 25 ℃, 16 h / d light, and light intensity of 2000 lx, Dendrobium suspension protocorms were obtained. The dendrobine content was detected, and protocorms with dendrobine content >0.2 mg / g were retained as Dendrobium suspension protocorms with high dendrobine content.
[0029] (2) Subculture and proliferation of Dendrobium protocorms with high Dendrobium phenols: The Dendrobium suspension protocorms obtained in step (1) were inoculated into a subculture and proliferation medium of 1 / 2 MS + BA 0.8 mg / L + sucrose 20 g / L + agar 5.5 g / L, pH 5.8. The inoculation amount was 100 mg fresh weight / L. The subculture was carried out continuously at 25 ℃ and 16 h / d light conditions for 35 days.
[0030] (3) Continuous culture in bioreactor: The Dendrobium suspension protocorms obtained in step (2) were inoculated into a liquid culture medium of 1 / 2 MS + BA 0.8 mg / L + sucrose 40 g / L + Indian piriformis inactivated culture 5 g / L, pH 5.8. The inoculation amount was 20 g fresh weight / L. The bioreactor culture temperature was set at 25 ℃, light intensity was 16 h / d, light intensity was 2000 lx, aeration rate was 0.1 L / min per liter of culture medium, and stirring speed was 120 rpm.
[0031] (4) Collection of protocorms and extraction of dendrobium phenols: After continuous culture of dendrobium suspension protocorms for 25 days in step (3), the protocorms grow to the logarithmic phase. 30% of the culture product is collected every 2 days, and the culture medium is replenished and the pH is adjusted to 5.8. Then the collected culture product is dehydrated, dried, ground, extracted with methanol by ultrasonic method, centrifuged at 8000 rpm for 10 min, and the supernatant is dried under negative pressure at 70℃ to complete the extraction of dendrobium phenols.
[0032] Example 2:
[0033] (1) Induction of Dendrobium suspension protocorms with high dendrobine content: On July 20, leaves of Dendrobium germplasm "14L-3" with dendrobine content >0.1 mg / g in fresh stems and leaves of the current year were selected as explants. After disinfection with 75% alcohol for 30 s and sterilization with 0.1% mercuric chloride for 10 min, the plants were inoculated into an induction medium of 1 / 2 MS + BA 3 mg / L + 2,4-D 2.0 mg / L + sucrose 20 g / L + agar 5.5 g / L, pH 5.8, with an inoculation amount of 100 mg fresh weight / L. After continuous induction for 50 days at 25℃, 16 h / d light, and light intensity of 2000 lx, Dendrobium suspension protocorms were obtained. The dendrobine content was detected, and protocorms with dendrobine content >0.2 mg / g were retained as Dendrobium suspension protocorms with high dendrobine content.
[0034] (2) Subculture and proliferation of Dendrobium protocorms with high Dendrobium phenols: The Dendrobium suspension protocorms obtained in step (1) were inoculated into a subculture and proliferation medium of 1 / 2 MS + BA 1.0 mg / L + sucrose 20 g / L + agar 5.5 g / L, pH 5.8. The inoculation amount was 100 mg fresh weight / L. The subculture was carried out continuously at 25 ℃ and 16 h / d light conditions for 40 days.
[0035] (3) Continuous culture in bioreactor: The Dendrobium suspension protocorms obtained in step (2) were inoculated into a liquid culture medium of 1 / 2 MS + BA 1.0 mg / L + sucrose 35 g / L + Indian piriformis inactivated culture 5 g / L, pH 5.8. The inoculation amount was 20 g fresh weight / L. The bioreactor culture temperature was set at 25 ℃, light intensity was 16 h / d, light intensity was 2000 lx, aeration rate was 0.1 L / min per liter of culture medium, and stirring speed was 120 rpm.
[0036] (4) Collection of protocorms and extraction of dendrobium phenols: After continuous culture of dendrobium suspension protocorms for 20 days in step (3), the protocorms grow to the logarithmic phase. 30% of the culture product is collected every 2 days, and the culture medium is replenished and the pH is adjusted to 5.8. Then the collected culture product is dehydrated, dried, ground, extracted with methanol by ultrasonic method, centrifuged at 8000 rpm for 10 min, and the supernatant is dried under negative pressure at 70℃ to complete the crude extraction of dendrobium phenols.
[0037] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.
Claims
1. A method for producing dendrobine from suspended protocorms of Dendrobium, characterized in that: Includes the following steps: (1) Induction of high dendrobium protocorms: In June-July, leaves of Dendrobium germplasm "14L-3" with dendrobium content >0.1 mg / g in fresh stems and leaves of the current year were selected as explants. After sterilization, they were inoculated into induction medium for induction culture. After continuous induction for 40-60 days, the obtained Dendrobium suspension protocorms were tested for dendrobium content. Protocorms with dendrobium content >0.2 mg / g were retained as high dendrobium suspension protocorms. (2) Subculture and proliferation of Dendrobium protocorms with high dendrobine content: The Dendrobium suspension protocorms with high dendrobine content obtained in step (1) were inoculated into a subculture and proliferation medium for continuous subculture. The subculture cycle was 30-45 days. (3) Continuous culture in a bioreactor: The Dendrobium suspension protocorms obtained in step (2) are inoculated into a liquid culture medium at an inoculation amount of 10-20 g fresh weight / L and cultured in a bioreactor. (4) Collection of protocorms and extraction of dendrobium phenols: After continuous culture for 20-30 days in step (3), the protocorms of Dendrobium suspension grow to the logarithmic phase. Collect 20-30% of the culture product every 1-2 days, replenish the culture medium and adjust the pH to 5.
8. Then use the collected culture product to extract dendrobium phenols. The liquid culture medium in step (3) is 1 / 2MS + BA 0.5-1.0 mg / L + sucrose 30-40 g / L + inactivated culture of Indian piriformis 5-10 g / L, pH 5.
8.
2. The method for producing dendrobine from Dendrobium suspension protocorms according to claim 1, characterized in that: The induction medium in step (1) is 1 / 2MS + BA 2.0-4.0 mg / L + 2,4-D 1.0-2.0 mg / L + sucrose 20 g / L + agar 5.5 g / L, pH 5.
8.
3. The method for producing dendrobine from Dendrobium suspension protocorms according to claim 1, characterized in that: The culture conditions for step (1) are 25 ℃, 16 h / d light, and light intensity of 2000 lx.
4. The method for producing dendrobine from Dendrobium suspension protocorms according to claim 1, characterized in that: The subculture medium in step (2) is 1 / 2 MS + BA 0.5-1.0 mg / L + sucrose 20 g / L + agar 5.5 g / L, pH 5.
8.
5. A method for producing dendrobine from suspended protocorms of Dendrobium according to claim 1, characterized in that: The conditions for subculture in step (2) are 25 ℃ and 16 h / d light.
6. A method for producing dendrobine from Dendrobium suspension protocorms according to claim 1, characterized in that: The bioreactor settings in step (3) are: temperature 25 ℃, light irradiation 16 h / d, light intensity 2000 lx, aeration rate of 0.1 L / min, and stirring speed of 120 rpm.