Sterile production method of fresh dendrobium officinale protocorm

Abstract

The invention discloses a sterile production method of a fresh dendrobium officinale protocorm product, which comprises the following steps: taking dendrobium officinale seeds from capsules of dendrobium officinale, repeatedly washing the capsules with tap water until the capsules are free of foam, sterilizing, cutting the capsules open, and drying to obtain the dendrobium officinale protocorm product. Uniformly spreading the seeds on an M2 solid culture medium, keeping the illumination condition of 500Lx and the temperature of 25 + / -1 DEG C, and waiting for the seedlings to grow for 60-90 days; wherein the M2 solid culture medium comprises 1 / 2 MS macroelements without exogenous hormones, 35% of banana juice, 35% of composite carbon source and 2% of agar powder. The method has the beneficial effects that a technical route map for mass production by infinitely stacking sterilization and ventilation culture of the small glass containers of 10 liters and getting rid of the constraint of a shaking table is provided. The technical key point is to screen out that the growth rate which can reach 8.4 mg / g.d without depending on plant growth hormone (which is a rigid biological index).

Description

Technical Field

[0001] This invention relates to the field of Dendrobium officinale, specifically a method for the aseptic production of fresh protocorms. Background Technology

[0002] Our company's patent publication number is "ZL200910051663.5", which has been around for more than ten years. Over the years, we have tried various methods to update and iterate, innovate processes, and further improve quality and output, making it safer to consume. Therefore, we have improved this technical solution. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems by designing a sterile production method for fresh Dendrobium officinale protocorms.

[0004] The technical solution of the present invention to achieve the above objectives is as follows: a method for aseptic production of fresh Dendrobium officinale protocorms, (1) seed cultivation and inoculation. The seeds of Dendrobium officinale are obtained from the capsules of Dendrobium officinale. The capsules should be plump, mature but not cracked and free from pests and diseases. The capsules should be repeatedly rinsed with tap water until no foam is left, and then sterilized. The capsules are cut open and the seeds inside are evenly spread on M2 solid culture medium. The seedlings should be kept under a light of 500 Lx and a temperature of 25℃±1℃ for 60 to 90 days. The M2 solid culture medium is 1 / 2MS macro-elements without exogenous hormones + 35% banana juice + 35% compound carbon source + 2% agar powder. (2) Selection and subculture of protocorms The seedlings were transferred to M2 liquid medium and subcultured every 7 days. The temperature was 25℃±1℃ and the light intensity was natural indoors. After 45-60 days, protocorms were induced. The M2 liquid medium was 1 / 2MS macro-elements without exogenous hormones + 35% banana juice + 35% compound carbon source. (3) Hormone-free primary culture and screening The protocorms were inoculated into M3 liquid medium for primary liquid expansion culture. Subculture was performed every 10-15 days, with each subculture using a 1:2 ratio to expand the population. After 3-5 cycles, culture lines with normal morphology, green color, and similar size were selected and subcultured in M3 liquid medium every 10-15 days for 5-8 cycles to obtain mature cell lines. M2 liquid medium consisted of 1 / 2MS macronutrients without exogenous hormones, 35% banana juice, and 35% compound carbon source. Cultures were prepared in 500ml glass Erlenmeyer flasks on a shaker. (4) Hormone-free secondary culture The mature protocorms were inoculated into M3 liquid medium for secondary liquid expansion culture, and the conditioned medium from the primary culture was added. The culture was provided with 14-16 hours of light daily or natural sunlight for 10-15 days. The M3 liquid medium consisted of 1 / 2MS macro-elements without exogenous hormones, 35% banana juice, 30% sucrose, and 5% maltose. Culture was performed in 1000ml glass Erlenmeyer flasks on a shaker. (5) Hormone-free ultimate ventilation culture The glass barrel with 10L bubble aeration was used for culture, of which 5L was fresh M3 liquid culture medium and 1L was the conditioned culture medium used in step (4). The protocorms obtained in step (4) were selected for inoculation. The light intensity was maintained at 2000-2500Lx. The protocorms were cultured on a special culture rack at a 38-40 degree angle for 25-30 days. Fresh products were collected. (6) Processing of fresh products The above-mentioned sterile, fresh tubers can be directly delivered to the extraction and concentration manufacturer or consumed directly. Note: To maintain the freshness of the Dendrobium officinale tubers and ensure their nutritional content and bright appearance, a low-temperature preservation method is used for delivery.

[0005] Preferably, the sterilization in step (1) involves soaking in 50% ethanol for 3-5 seconds, then soaking in 20% "84" solution for 7-8 minutes, soaking in 0.1% mercuric chloride solution for 12-15 minutes, and finally rinsing several times with sterile water and then drying off.

[0006] Preferably, the conditioned medium in step (4) is the old culture medium after Dendrobium has been cultured in M2 liquid culture medium in step (3) for 10 to 15 days.

[0007] Preferably, the conditioned medium in step (5) is the old culture medium after Dendrobium has been cultured in M3 liquid culture medium in step (4) for 10 to 15 days.

[0008] Preferably, the specialized culture rack in step (5) occupies 1M of space per rack, has three layers, and six 10L containers in a row, with 18 containers placed on each rack.

[0009] Preferably, the 35% composite carbon source is composed of 25% sucrose, 5% maltose, and 5% sorbitol.

[0010] This invention discloses a method for the aseptic production of fresh Dendrobium officinale protocorms using the technical solution of this invention. It outlines a technical roadmap for mass production using infinitely stacked 10-liter glass containers with sterilization and aeration, freeing the constraints of shakers. The key technical point is the selection of protocorms that can achieve a growth rate of 8.4 mg / g·d (a rigid biological indicator) without relying on plant growth hormones. Selecting these protocorms with special growth functions is the core of this patented invention. The original Dendrobium officinale protocorms originate from Dendrobium officinale seeds. Seed-derived protocorms are numerous, have good synchronization, and the selection process is short and effective. The air sterilization filter uses a 0.45μm pore size to remove biological particles. The 10-liter glass containers are supplied with 1.5-2 liters of air per minute (the specific value depends on the condition of the Dendrobium officinale protocorms within the container), which must meet the oxygen requirements for growth. Attached Figure Description

[0011] Figure 1 This is a comparative chart showing the initial inoculation amount of Dendrobium officinale protocorms in various glass containers and the weight required after multiple generations; Detailed Implementation

[0012] Aseptic production method of fresh Dendrobium officinale corms (1) Seed culture and inoculation The seeds of Dendrobium officinale are obtained from the capsules of Dendrobium officinale. The capsules should be plump, mature but not cracked and free from pests and diseases. The capsules should be repeatedly rinsed with tap water until no foam is left, and then sterilized. The capsules are cut open and the seeds inside are evenly spread on M2 solid culture medium. The seedlings should be kept under a light of 500 Lx and a temperature of 25℃±1℃ for 60 to 90 days. The M2 solid culture medium is 1 / 2MS macro-elements without exogenous hormones + 35% banana juice + 35% compound carbon source + 2% agar powder. (2) Selection and subculture of protocorms The seedlings were transferred to M2 liquid medium and subcultured every 7 days. The temperature was 25℃±1℃ and the light intensity was natural indoors. After 45-60 days, protocorms were induced. The M2 liquid medium was 1 / 2MS macro-elements without exogenous hormones + 35% banana juice + 35% compound carbon source. (3) Hormone-free primary culture and screening The protocorms were inoculated into M3 liquid medium for primary liquid expansion culture. Subculture was performed every 10-15 days, with each subculture using a 1:2 ratio to expand the population. After 3-5 cycles, culture lines with normal morphology, green color, and similar size were selected and subcultured in M3 liquid medium every 10-15 days for 5-8 cycles to obtain mature cell lines. M2 liquid medium consisted of 1 / 2MS macronutrients without exogenous hormones, 35% banana juice, and 35% compound carbon source. Cultures were prepared in 500ml glass Erlenmeyer flasks on a shaker. (4) Hormone-free secondary culture The mature protocorms were inoculated into M3 liquid medium for secondary liquid expansion culture, and the conditioned medium from the primary culture was added. The culture was provided with 14-16 hours of light daily or natural sunlight for 10-15 days. The M3 liquid medium consisted of 1 / 2MS macro-elements without exogenous hormones, 35% banana juice, 30% sucrose, and 5% maltose. Culture was performed in 1000ml glass Erlenmeyer flasks on a shaker. (5) Hormone-free ultimate ventilation culture The glass barrel with 10L bubble aeration was used for culture, of which 5L was fresh M3 liquid culture medium and 1L was the conditioned culture medium used in step (4). The protocorms obtained in step (4) were selected for inoculation. The light intensity was maintained at 2000-2500Lx. The protocorms were cultured on a special culture rack at a 38-40 degree angle for 25-30 days. Fresh products were collected. (6) Processing of fresh products The above-mentioned sterile, fresh tubers can be directly delivered to the extraction and concentration manufacturer or consumed directly. Note: To maintain the freshness of the Dendrobium officinale tubers and ensure their nutritional content and bright appearance, a low-temperature preservation method is used for delivery.

[0013] Furthermore, the sterilization described in step (1) involves soaking in 50% ethanol for 3-5 seconds, then soaking in 20% "84" solution for 7-8 minutes, soaking in 0.1% mercuric chloride solution for 12-15 minutes, and finally rinsing several times with sterile water and then drying off.

[0014] Furthermore, the conditioned medium mentioned in step (4) is the old culture medium after Dendrobium has been cultured in the M2 liquid culture medium in step (3) for 10 to 15 days.

[0015] Furthermore, the conditioned medium mentioned in step (5) is the old culture medium after Dendrobium has been cultured in the M3 liquid culture medium in step (4) for 10 to 15 days.

[0016] Furthermore, the specialized culture rack described in step (5) occupies 1M of space per rack, has three layers, and six 10L containers in a row, with 18 containers placed on each rack.

[0017] Furthermore, the 35% composite carbon source consists of 25% sucrose, 5% maltose, and 5% sorbitol.

[0018] The above technical solutions only embody the preferred technical solutions of the present invention. Any modifications that may be made by those skilled in the art to certain parts thereof embody the principles of the present invention and fall within the protection scope of the present invention.

Claims

1. A method for aseptic production of fresh Dendrobium officinale protocorms, characterized in that: (1) Seed culture and inoculation The seeds of Dendrobium officinale are obtained from the capsules of Dendrobium officinale. The collected capsules should be plump, mature but not cracked and free from pests and diseases. The capsules are repeatedly rinsed with tap water until no foam is left, and then sterilized. The capsules are cut open and the seeds inside are evenly spread on M2 solid culture medium. The seedlings are kept under a light of 500 Lx and a temperature of 25℃±1℃ for 60 to 90 days. The M2 solid culture medium is 1 / 2MS macro-elements without exogenous hormones + 35% banana juice + 35% compound carbon source + 2% agar powder. (2) Selection and subculture of protocorms The seedlings were transferred to M2 liquid medium and subcultured every 7 days. The temperature was 25℃±1℃ and the light intensity was natural indoors. After 45-60 days, protocorms were induced. The M2 liquid medium was 1 / 2MS macro-elements without exogenous hormones + 35% banana juice + 35% compound carbon source. (3) Hormone-free primary culture and screening The protocorms were inoculated into M3 liquid medium for primary liquid expansion culture. Subculture was performed every 10-15 days, with each subculture using a 1:2 ratio to expand the population. After 3-5 cycles, culture lines with normal morphology, green color, and similar size were selected and subcultured in M3 liquid medium every 10-15 days for 5-8 cycles to obtain mature cell lines. M2 liquid medium consisted of 1 / 2MS macronutrients without exogenous hormones, 35% banana juice, and 35% compound carbon source. Cultures were prepared in 500ml glass Erlenmeyer flasks on a shaker. (4) Hormone-free secondary culture The mature protocorms were inoculated into M3 liquid medium for secondary liquid expansion culture, and the conditioned medium from the primary culture was added. The culture was provided with 14-16 hours of light daily or natural sunlight for 10-15 days. The M3 liquid medium consisted of 1 / 2MS macro-elements without exogenous hormones, 35% banana juice, 30% sucrose, and 5% maltose. Culture was performed in 1000ml glass Erlenmeyer flasks on a shaker. (5) Hormone-free ultimate ventilation culture The glass barrel with 10L bubble aeration was used for culture, of which 5L was fresh M3 liquid culture medium and 1L was the conditioned culture medium used in step (4). The protocorms obtained in step (4) were selected for inoculation. The light intensity was maintained at 2000-2500Lx. The protocorms were cultured on a special culture rack at a 38-40 degree angle for 25-30 days. Fresh products were collected. (6) Processing of fresh products The above-mentioned sterile original corms are directly delivered to the extraction and concentration liquid manufacturer or can be consumed directly. In order to maintain the freshness of the Dendrobium officinale original corms and ensure their nutritional components and bright appearance and color, a low-temperature preservation method is used for delivery.

2. The aseptic production method of fresh Dendrobium officinale protocorms according to claim 1, characterized in that, The sterilization process described in step (1) involves soaking in 50% ethanol for 3-5 seconds, then soaking in 20% "84" solution for 7-8 minutes, then soaking in 0.1% mercuric chloride solution for 12-15 minutes, and finally rinsing several times with sterile water and then drying off.

3. The aseptic production method of fresh Dendrobium officinale protocorms according to claim 1, characterized in that, The conditioned medium mentioned in step (4) is the old culture medium after Dendrobium has been cultured in the M2 liquid culture medium in step (3) for 10 to 15 days.

4. The aseptic production method of fresh Dendrobium officinale protocorms according to claim 1, characterized in that, The conditioned medium mentioned in step (5) is the old culture medium after Dendrobium has been cultured in the M3 liquid culture medium in step (4) for 10 to 15 days.

5. The aseptic production method of fresh Dendrobium officinale protocorms according to claim 1, characterized in that, The specialized culture rack described in step (5) occupies 1 meter of space per rack, has three layers, and six 10L containers in a row, with 18 containers placed on each rack.

6. The aseptic production method of fresh Dendrobium officinale protocorms according to claim 1, characterized in that, The 35% composite carbon source is composed of 25% sucrose, 5% maltose, and 5% sorbitol.

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