A method for culturing the protocorm of Dendrobium officinale, which is an ideal protein

Abstract

The present invention belongs to the technical field of medicinal plant cell culture, and specifically relates to a method for culturing Dendrobium officinale protocorms, which is an ideal protein. The method comprises the following steps: (1) inducing Dendrobium officinale protocorms, (2) multiplying and screening the Dendrobium officinale protocorms, (3) liquid suspension culture and screening of the Dendrobium officinale protocorms, and (4) culturing the Dendrobium officinale protocorms in a bioreactor. The Dendrobium officinale protocorms obtained by the present invention contain essential amino acids accounting for approximately 40% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids is greater than 60%. The composition of the Dendrobium officinale protocorms meets the ideal protein standards proposed by FAO / WHO (Food and Agriculture Organization of the United Nations and World Health Organization).

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal plant cell culture, and in particular relates to a method for culturing a protocorm of Dendrobium officinale containing an ideal protein. Background Art

[0002] Protein is the material basis for all life activities and plays a crucial role in regulating human physiological and biochemical metabolism. Plant protein is an important sustainable source of protein. Using plant protein to supplement or partially replace animal protein has become a new development trend. This is primarily due to increasing consumer demand for protein and concerns about the health and environmental impacts of excessive meat consumption. According to the reference standards provided by the FAO / WHO (Food and Agriculture Organization of the United Nations and World Health Organization), ideal protein is defined when essential amino acids account for approximately 40% of the total amino acids and the ratio of essential to non-essential amino acids is greater than 60%. Ideal protein is the protein with the most appropriate amino acid ratio recommended by the FAO / WHO for human needs, meaning it has the highest absorption and utilization rate in the human body. This evaluation standard is based on the amino acid ratio required by the human body, rather than the absolute content of one or more amino acids.

[0003] Dendrobium officinale Kimura et Migo is a traditional and precious Chinese medicinal material. Dendrobium officinale is rich in a variety of amino acids, including arginine, aspartic acid, lysine, etc. These amino acids are the basic components of proteins and are also an important material basis for their medicinal effects. The 2020 edition of the "Chinese Pharmacopoeia" records that Dendrobium officinale has the effects of nourishing the stomach and producing body fluid, nourishing yin and clearing heat. It is used to treat body fluid damage caused by febrile diseases, dry mouth and thirst, insufficient stomach yin, poor appetite and dry retching, persistent heat after illness, yin deficiency and fire, bone steaming and fatigue, blurred vision, and weakness of muscles and bones. As a Chinese medicinal material that can be used as both medicine and food, Dendrobium officinale has not only medicinal value, but also nutritional value that cannot be ignored.

[0004] Due to the special growth environment and extremely difficult reproduction of Dendrobium officinale, coupled with its extremely high economic value, irrational development and utilization have led to a decreasing number of resources. It has now been included in the "National List of Key Protected Wild Plants" and cannot meet market demand. From the current research progress, the artificial large-scale cultivation of Dendrobium officinale still has the following problems: (1) High production cost of tissue culture seedlings. In the rapid propagation process of Dendrobium officinale, the number of transfer cultures is high, and the labor and raw material costs are high. At the same time, the survival rate of transplanted seedlings is low. (2) High requirements for cultivation conditions and management measures. The substrate, light, temperature, humidity, ventilation and other conditions need to meet or approach the requirements of the process regulations. At the same time, targeted fertilization and pest control should be carried out according to the growth and development of the seedlings. (3) The level of standardization needs to be improved. Dendrobium officinale is mainly used as a raw material for functional foods and medicines. It is necessary to establish and implement standardized standards in all aspects such as tissue culture, cultivation, processing, and inspection to improve the level of standardized production. In particular, the cultivation of medicinal plants often uses large amounts of pesticides, resulting in serious pesticide residue and heavy metal residue problems.

[0005] Using plant cell culture technology to obtain plant tissue cultures and replace the original plant with plant tissue cultures is an effective way to address the shortage of wild Dendrobium officinale resources. Protocorm culture of Dendrobium officinale has the advantages of a short production cycle, a controllable production process, and uniform product quality. It can be developed and utilized as an alternative resource to wild Dendrobium officinale to address its limited supply. The use of plant cell culture technology to cultivate protocorms of Dendrobium officinale has long been studied both domestically and internationally, with numerous achievements in laboratory and pilot experiments. However, many challenges remain in the process of industrialization, primarily due to process limitations such as slow cell growth, low yield, and unstable yield. In particular, the transition from shake flask culture to commercial production using bioreactors is extremely difficult. Difficulties in scaling up culture in reactors are a major technical barrier and limiting factor to industrialization. Therefore, developing a method for scalable protocorm culture using bioreactors is of great significance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for culturing Dendrobium officinale protocorms which is an ideal protein with a short culture cycle, high yield, no hormone residue, high safety and is suitable for industrial production.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A method for culturing the protocorm of Dendrobium officinale, which is an ideal protein, comprises the following steps:

[0009] (1) Induction of Dendrobium officinale protocorms: Explants of Dendrobium officinale from the wild were cleaned and sterilized, and then inoculated on protocorm induction medium. The protocorms of Dendrobium officinale were induced under light conditions.

[0010] (2) Proliferation culture and screening of Dendrobium officinale protocorms: The protocorms obtained in step (1) are inoculated into a proliferation medium and continuously subcultured under light conditions, and protocorms with stable and uniform cell morphology and short proliferation cycles are screened to establish a cell line;

[0011] (3) Liquid suspension culture and screening of Dendrobium officinale protocorm: The protocorm cell line obtained by screening in step (2) is transferred into a liquid culture medium for suspension culture in a shake flask, and a Dendrobium officinale protocorm suspension cell culture line containing the ideal protein is screened and established, which is the seed cell used for production;

[0012] (4) Cultivation of Dendrobium officinale protocorms in a bioreactor: The seed cells from step (3) are transferred into a bioreactor for production cultivation to obtain protocorms.

[0013] Furthermore, step (2) is to inoculate the protocorms obtained in step (1) into a proliferation medium for continuous subculture, and in each subculture, select dedifferentiated protocorms with loose morphology, uniform size, and obvious new proliferating cells from the harvested protocorms as seeds for subculture; the screening process is continued for at least 10 generations until the protocorms obtained are in a completely dedifferentiated state, have loose morphology, uniform size, stable proliferation cycle, and bright green color, thereby establishing a protocorm solid culture cell line;

[0014] Furthermore, step (3) is to transfer the protocorm cell line obtained by screening in step (2) into a liquid culture medium for suspension culture in a shake flask, and in each subculture, select the protocorms that are completely dedifferentiated and remove the protocorms that are differentiated; select the protocorms with obvious new proliferating cells and good dispersion, and remove the hard protocorms and protocorms in the late proliferation stage; select the protocorms with bright green color and remove the protocorms with brown cells; the screening process is continued for at least 10 generations until the protocorms obtained are all completely dedifferentiated, uniform cell clusters with strong proliferation ability, good dispersion, and bright green color, thereby establishing a suspension cell culture system;

[0015] The wild Dendrobium officinale explant in step (1) is a 2-3 year old young stem of Dendrobium officinale with at least one stem node;

[0016] In step (1), the protocorm induction medium is MS basal medium, and the final concentration of the medium is 0.1-5 mg·L -1 NAA1-naphthaleneacetic acid, 0.1-5 mg·L -1 6-BA (6-benzylaminopurine), 10-50 g·L -1 Sucrose and 5-15 g·L -1 Agar; the pH value of the culture medium is 5.4-6.2;

[0017] Lighting conditions: The light source consists of white light or a mixture of white light and blue light, or white light and red light, and the lighting time is 8 to 16 hours a day -1 , the light intensity is 10~100μmol·m -2 ·s -1 ;

[0018] The induction temperature is 15-30°C.

[0019] In step (2), the proliferation medium is N6 basal medium, and the final concentration of the added medium is 0.1-5 mg·L -1 NAA, 20-150 g·L -1 Potato extract, 10-50 g·L -1 Sucrose and 5-15 g·L -1 Agar; the pH value of the culture medium is 5.4-6.2;

[0020] Potato extract is obtained by peeling and weighing potatoes, cutting them into thin slices, heating them at 100°C for 10 minutes, and filtering to remove solids.

[0021] The seeding density of the continuous subculture proliferation culture in step (2) is 50 to 200 g·L -1 ;

[0022] Lighting conditions: The light source is white light or a mixture of white light and blue light, or white light and red light, and the lighting time is 8 to 16 hours a day. -1 , the light intensity is 10~100μmol·m -2 ·s -1 ;

[0023] The culture temperature is 15-30°C;

[0024] Subculture once every 2 to 6 weeks.

[0025] In step (3), the liquid culture medium is N6 basal culture medium, and the final concentration of the added medium is 0.1-5 mg·L -1 NAA, 20-150 g·L -1 Potato extract, 10-50 g·L -1 Sucrose; the pH value of the culture medium is 5.4-6.2.

[0026] Potato extract is obtained by peeling and weighing potatoes, cutting them into thin slices, heating them at 100°C for 10 minutes, and filtering to remove solids.

[0027] The suspension culture in the shake flask in step (3) is carried out by rotary shaking on a shaker with a speed of 80 to 150 r / min. -1 Lighting conditions: The light source consists of white light or a mixture of white light and blue light, or white light and red light, and the lighting time is 8 to 16 hours a day. -1, the light intensity is 10~100μmol·m -2 ·s -1 Temperature: 15-30°C; inoculation density: 50-200 g·L -1 ; Subculture once every 2 to 6 weeks.

[0028] The culture medium used in the protocorm bioreactor culture in step (4) is N6 basal medium, with a final concentration of 0.1 to 5 mg·L -1 NAA, 0.1-3 g·L -1 Potato flour, 10-50 g / L -1 Sucrose; the pH value of the culture medium is 5.4-6.2.

[0029] The protocorm bioreactor culture in step (4) is a bubble reactor culture; the ventilation volume is 0.02-0.2Nm 3 ·h -1 Lighting conditions: The light source consists of white light or a mixture of white light and blue light, or white light and red light, and the lighting time is 8 to 16 hours a day. -1 , the light intensity is 10~100μmol·m -2 ·s -1 Temperature: 15-30°C; inoculation density: 50-200 g·L -1 ; The culture period is 2 to 4 weeks.

[0030] The protocorm obtained in the above step (4) can also be vacuum-frozen and vacuum-dried in sequence, and then crushed and filtered to prepare freeze-dried powder of Dendrobium officinale protocorm.

[0031] Furthermore, the protocorms were washed with pure water to remove the culture medium, and after removing the water through a 40-60 mesh filter, the protocorms were spread in a freeze-drying tray with a thickness of 2 cm, and after vacuum freezing, vacuum drying was performed, and the vacuum degree was maintained at 1-10 Pa.

[0032] Vacuum freezing is as follows: the protocorm is placed in a vacuum freeze dryer, the temperature of the protocorm is lowered to -50°C, and maintained for 5 hours;

[0033] The vacuum drying process is as follows: the temperature of the original corm is maintained at -45℃~-40℃ for 4 hours; then the temperature of the original corm is raised and maintained at -40℃~-20℃ for 5 hours; then the temperature of the original corm is raised and maintained at -20℃~0℃ for 6 hours; then the temperature of the original corm is raised and maintained at 0℃~40℃ for 8 hours; finally the temperature of the original corm is raised and maintained at 45℃ for 5 hours.

[0034] The ideal protein cultivated by the method comprises about 40% essential amino acids of the total amino acids in the protocorm of Dendrobium officinale, and the ratio of essential amino acids to non-essential amino acids is higher than 60%, which meets the ideal protein standards proposed by FAO / WHO (Food and Agriculture Organization of the United Nations and World Health Organization).

[0035] The beneficial effects of the present invention are as follows:

[0036] 1. The Dendrobium officinale protocorm culture method of the present invention has a short culture cycle and high yield. The bioreactor culture cycle is 2 to 4 weeks and the yield is 25 to 35 g / L (dry weight).

[0037] 2. The essential amino acid content of the Dendrobium officinale protocorm obtained by the present invention accounts for about 40% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids is higher than 60%. Its composition meets the ideal protein standards proposed by FAO / WHO (Food and Agriculture Organization of the United Nations and World Health Organization).

[0038] 3. The present invention adopts bioreactor culture, the whole process is controllable, the technology is stable, and it is suitable for industrial production.

[0039] 4. The method of the present invention is simple in process, the obtained protocorm has no hormone residue, is highly safe, and can provide a new source of amino acids for food and clinical medicine.

[0040] 5. The method of the present invention is not restricted by season and region and can be produced continuously. DETAILED DESCRIPTION

[0041] Example 1

[0042] 1. Induction of Protocorms from Explants

[0043] Take 2-3 year old young stems of wild-cultivated Dendrobium officinale and rinse them with ultrapure water. On a clean bench, immerse the stem segments in 75% alcohol and cut them into 5 cm segments, each with 2-3 nodes. Soak for 30 seconds and rinse three times with sterile water. Then soak in a sodium hypochlorite solution for 8 minutes and rinse three times with sterile water. Use sterile filter paper to absorb the surface moisture of the stem segments, cut off the wounds at both ends, and cut them into small segments with one node. Inoculate them onto protocorm induction medium and induce the production of Dendrobium officinale protocorms under light conditions.

[0044] The protocorm induction medium was MS basal medium, with a final concentration of 0.5 mg·L -1 NAA (1-naphthylacetic acid), 1 mg·L -1 6-BA (6-benzylaminopurine), 30 g·L -1 Sucrose and 8g·L -1Agar; pH value of the culture medium is 5.8. The culture bottle used is 240mL, containing 40mL of solid culture medium.

[0045] Lighting conditions: white light, illumination time 12h·d -1 , the light intensity was 30 μmol·m -2 ·s -1 ; The induction temperature was 25℃.

[0046] 2. Screening and Establishment of Protocorm Solid Culture Cell Lines

[0047] The induced protocorms were inoculated into proliferation medium and continuously subcultured. During each subculture, dedifferentiated protocorms with loose morphology, uniform size, and obvious new proliferating cells were selected from the harvested protocorms as seeds for subculture. The screening process lasted for 15 generations until the protocorms obtained were in a completely dedifferentiated state with loose morphology, uniform size, stable proliferation cycle, and bright green color, thus establishing a protocorm solid culture cell line.

[0048] The proliferation medium was N6 basal medium, and the final concentration of the added medium was 0.5 mg·L -1 NAA, 100 g L -1 Potato extract, 30g·L -1 Sucrose and 8g·L -1 Agar; medium pH 5.8; potato extract is obtained by peeling, weighing, thinly slicing potatoes, heating at 100°C for 10 minutes, and filtering to remove solids. The culture flask used is 240 mL and contains 40 mL of solid medium.

[0049] The inoculation density of the continuous subculture was 100 g·L -1 ; Culture under illumination, temperature is 25℃, subculture once every 4 weeks. Light conditions: light source is white light, illumination time is 12h·d -1 , the light intensity was 30 μmol·m -2 ·s -1 .

[0050] 3. Establishment of a suspension cell culture system of Dendrobium officinale protocorm

[0051] The protocorm cell line is transferred into liquid culture medium for suspension culture in shake flasks. During each subculture, completely dedifferentiated protocorms are selected and differentiated protocorms are removed; protocorms with obvious new proliferating cells and good dispersion are selected, and hard protocorms and protocorms in the late proliferation stage are removed; protocorms with bright green color are selected and protocorms with brown cells are removed; the screening process continues for 20 generations until the protocorms obtained are all completely dedifferentiated, uniform cell clusters with strong proliferation ability, good dispersion, and bright green color, and a suspension cell culture system is established, which is the seed cell used for production.

[0052] The liquid medium used for the suspension culture of protocorm cell lines in shake flasks was N6 basal medium, with a final concentration of 0.5 mg·L -1 NAA, 100 g L -1 Potato extract, 30g·L -1 Sucrose; the medium pH is 5.8. A 100 mL shake flask is used, containing 20 mL of liquid medium, and sealed with double-layer aluminum foil.

[0053] The suspension culture of protocorm cell lines was carried out in a shaker with a rotational shaking speed of 100 r·min. -1 ; Illumination culture; Temperature: 25°C; Inoculation density: 100 g·L -1 ; Subculture once every 4 weeks. Lighting conditions: Light source composition is white light, lighting time is 12h·d -1 , the light intensity was 30 μmol·m -2 ·s -1 .

[0054] 4. Bioreactor Culture of Dendrobium officinale Protocorm

[0055] The protocorm seed cells of Dendrobium officinale that have completed growth and culture are transferred into a bioreactor for production culture to obtain the protocorm.

[0056] The culture medium used in the protocorm bioreactor culture was N6 basal medium, with a final concentration of 0.5 mg·L - 1 NAA, 2 g L -1 Potato flour, 30g·L -1 Sucrose; pH value of the culture medium was 5.8.

[0057] The bioreactor culture was a bubble reactor with a ventilation volume of 0.06 Nm 3 ·h -1 ; Illumination culture; Temperature: 25°C; Inoculation density: 100 g·L -1 Lighting conditions: The light source is white light, and the lighting time is 12h·d -1 , the light intensity was 40 μmol·m-2 ·s -1 .

[0058] On the 14th day (336 h) of protocorm growth, the cultured protocorms were harvested.

[0059] 5. Preparation of Freeze-dried Powder of Dendrobium officinale Protocorm

[0060] The protocorms harvested after production and cultivation are successively subjected to vacuum freezing and vacuum drying, and then crushed and filtered by an ultrafine grinder to prepare freeze-dried powder of the Dendrobium officinale protocorms.

[0061] The vacuum freeze-drying of the harvested protocorms specifically refers to washing the protocorms with pure water to remove the culture medium, removing the moisture through a 40-60 mesh filter, spreading the protocorms in a 2 cm thickness on a freeze-drying tray, vacuum freezing, and vacuum drying, with the vacuum degree maintained at 1-10 Pa.

[0062] Vacuum freezing was performed by placing the protocorms into a vacuum freeze dryer, lowering the temperature of the protocorms to -50°C, and maintaining it for 5 h;

[0063] The vacuum drying process is as follows: the temperature of the protocorm is maintained at -45℃~-40℃ for 4 hours; then the temperature of the protocorm is raised and maintained at -40℃~-20℃ for 5 hours; then the temperature of the protocorm is raised and maintained at -20℃~0℃ for 6 hours; then the temperature of the protocorm is raised and maintained at 0℃~40℃ for 8 hours; finally the temperature of the protocorm is raised and maintained at 45℃ for 5 hours.

[0064] The yield of the protocorm of Dendrobium officinale was 32 g·L -1 (Dry weight). Determination of amino acid and 1-naphthylacetic acid content in freeze-dried powder of Dendrobium officinale protocorm

[0065] The method for determining the amino acid content in freeze-dried powder of Dendrobium officinale protocorm is high performance liquid chromatography; the method for determining the 1-naphthacetic acid content refers to SN / T 2228-2008, determination of 31 acidic herbicide residues in imported and exported foods (gas chromatography-mass spectrometry).

[0066] In this embodiment, the detection result of the 1-naphthylacetic acid content was not detected; the detection result of the amino acids was that the essential amino acid content accounted for 40.12% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids was 67.01%, as shown in Table 1 and Table 2.

[0067] Example 2

[0068] The difference from Example 1 is that the medium used for the suspension culture of the protocorm cell line in the shake flask is N6 basal medium, and the final concentration of the medium is 2 mg·L -1 NAA, 80g·L -1Potato extract, 30g·L -1 sucrose; the pH value of the culture medium was 5.8, and other conditions were the same as in Example 1.

[0069] According to the measurement, the yield of the protocorm of Dendrobium officinale in this embodiment is 30g·L -1 (dry weight).

[0070] In this embodiment, the detection result of the 1-naphthylacetic acid content was not detected; the detection result of the amino acids was that the essential amino acids accounted for 41.25% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids was 70.19%.

[0071] Example 3

[0072] The difference from Example 1 is that the protocorm cell line suspension culture was cultured under illumination, the light source was a mixture of white light and red light, and the illumination time was 16 h·d. -1 , the light intensity was 40 μmol·m -2 ·s -1 , other conditions are the same as in Example 1.

[0073] The yield of the protocorm of Dendrobium officinale was 33 g·L -1 (dry weight).

[0074] In this embodiment, the detection result of the 1-naphthylacetic acid content was not detected; the detection result of the amino acids was that the essential amino acid content accounted for 40.05% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids was 66.81%.

[0075] Example 4

[0076] The difference from Example 1 is that the culture medium used in the protocorm bioreactor culture is N6 basal medium, and the final concentration of 1 mg·L -1 NAA, 0.5 g·L -1 Potato flour, 30g·L -1 Sucrose; pH value of the culture medium was 5.8.

[0077] The bioreactor culture was a bubble reactor with a ventilation volume of 0.1 Nm 3 ·h -1 ; Illumination culture; the light source is white light, and the illumination time is 12h·d -1 , the light intensity was 30 μmol·m -2 ·s -1 The other conditions are the same as those in Example 1.

[0078] On the 21st day (504h) of protocorm growth, the cultured protocorms were harvested. According to the measurement, the yield of the protocorms of Dendrobium officinale in this embodiment was 35g·L-1 (dry weight).

[0079] In this embodiment, the detection result of the 1-naphthylacetic acid content was not detected; the detection result of the amino acids was that the essential amino acids accounted for 42.10% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids was 72.71%.

[0080] Comparative Example 1

[0081] The culture method and preparation method of Dendrobium officinale protocorm are the same as those in Example 1, except that the culture medium used in the protocorm bioreactor culture is N6 basal medium, and the final concentration of 0.5 mg·L -1 NAA, 100 g L -1 Potato extract, 30g·L -1 Sucrose; the pH value of the culture medium was 5.8. The same batch of samples as in Example 1 was tested according to the method in Example 1.

[0082] The yield of the protocorm of Dendrobium officinale in this comparative example was determined to be 35 g·L -1 (dry weight).

[0083] In this comparative example, the detection result of 1-naphthylacetic acid content was not detected; the detection result of amino acids was that the essential amino acid content accounted for 34.87% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids was 53.55%, as shown in Table 1 and Table 2.

[0084] Comparative Example 2

[0085] A certain brand of commercially available freeze-dried Dendrobium officinale powder (Dendrobium officinale is cultivated in the wild) and the freeze-dried Dendrobium officinale protocorm powder in Example 1 were tested in the same batch as the sample in Example 1 according to the method in Example 1. The commercially available freeze-dried Dendrobium officinale powder was tested in accordance with the "Hubei Province Traditional Chinese Medicine Preparation Specifications", product batch number 20191206.

[0086] The test result of 1-naphthaleneacetic acid content in the commercially available freeze-dried powder of Dendrobium officinale was not detected; the test result of amino acids showed that the essential amino acid content accounted for 37.01% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids was 58.74%, as shown in Tables 1 and 2 for details.

[0087] Table 1: Amino acid composition and content analysis of freeze-dried powder of Dendrobium officinale protocorm and freeze-dried powder of Dendrobium officinale (commercially available)

[0088]

[0089] Table 2: Amino acid ratio analysis of freeze-dried powder of Dendrobium officinale protocorm and freeze-dried powder of Dendrobium officinale (commercially available)

[0090]

[0091]

[0092] As can be seen from the above examples, the protocorms of Dendrobium officinale obtained by the method of the embodiment of the present invention do not contain hormones (1-naphthylacetic acid), which is safer. In addition, the essential amino acid content in the protocorms of Dendrobium officinale obtained by the method of the embodiment of the present invention is about 40% of the total amino acids, and the ratio of essential amino acids to non-essential amino acids is higher than 60%. Its composition meets the ideal protein standard proposed by FAO / WHO (Food and Agriculture Organization of the United Nations and World Health Organization). At the same time, as can be seen from Comparative Example 1, when the culture conditions are changed, the essential amino acid content and the ratio of essential amino acids to non-essential amino acids in the protocorms of Dendrobium officinale change significantly. Therefore, the present invention can obtain the protocorms of Dendrobium officinale that meet the ideal protein standard by controlling specific culture conditions and the combination of various culture conditions. As can be seen from Comparative Example 2, the protein quality of the protocorms of Dendrobium officinale obtained by the method of the embodiment of the present invention is significantly better than that of existing commercially available products.

Claims

1. A method for culturing the protocorm of Dendrobium officinale, which is an ideal protein, characterized by: The following steps are included: (1) Induction of Dendrobium officinale protocorms: Explants of Dendrobium officinale from the wild were cleaned and sterilized, and then inoculated on protocorm induction medium. The protocorms of Dendrobium officinale were induced under light conditions. (2) Proliferation culture and screening of Dendrobium officinale protocorms: The protocorms obtained in step (1) are inoculated into a proliferation culture medium and continuously subcultured under light conditions, and protocorms with stable and uniform cell morphology and short proliferation cycles are screened to establish cell lines; (3) Liquid suspension culture and screening of Dendrobium officinale protocorm: The protocorm cell line obtained by screening in step (2) is transferred into liquid culture medium for suspension culture in shake flasks, and a suspension cell culture line is screened and established, which is the seed cell used for production; (4) Cultivation of Dendrobium officinale protocorm in bioreactor: The seed cells in step (3) are transferred into a bioreactor for production culture to obtain protocorms; wherein the culture medium used for the protocorm bioreactor culture is N6 basal medium + a final concentration of 0.1-5 mg·L -1 NAA + 0.1~3g·L -1 Potato flour +10~50g·L -1 Sucrose; medium pH value is 5.4-6.2; The explant of wild Dendrobium officinale in step (1) is a 2-3 year old young stem of Dendrobium officinale with at least one stem node; Step (1) The protocorm induction medium is MS basal medium + final concentration of 0.1-5 mg·L -1 NAA + 0.1~5mg·L -1 6-BA+10~50g·L -1 Sucrose +5-15g·L -1 Agar; the pH value of the culture medium is 5.4-6.2; The proliferation medium in step (2) is N6 basal medium + final concentration of 0.1-5 mg·L -1 NAA+20~150g·L -1 Potato extract + 10-50g·L -1 Sucrose +5-15g·L -1 Agar; the pH value of the culture medium is 5.4-6.2; The liquid culture medium in step (3) is N6 basal culture medium + final concentration of 0.1-5 mg·L -1 NAA+20~150g·L -1 Potato extract + 10-50g·L -1 Sucrose; the pH value of the culture medium is 5.4-6.

2.

2. The culture method according to claim 1, wherein: Step (2) is to inoculate the protocorms obtained in step (1) into a proliferation culture medium for continuous subculture. During each subculture, dedifferentiated protocorms with loose morphology, uniform size, and obvious new proliferating cells are selected from the harvested protocorms as seeds for subculture. The screening process lasts for at least 10 generations until the protocorms obtained are in a completely dedifferentiated state, have loose morphology, uniform size, stable proliferation cycle, and bright green color, thereby establishing a protocorm solid culture cell line.

3. The culture method according to claim 1, wherein: Step (3) is to transfer the protocorm cell line obtained by screening in step (2) into a liquid culture medium for shake flask suspension culture. During each subculture, completely dedifferentiated protocorms are selected and differentiated protocorms are removed; protocorms with obvious newly proliferated cells and good dispersion are selected, and hard protocorms or protocorms in the late stage of proliferation are removed; Select protocorms with bright green color and remove protocorms with brown cells; the screening process continues for at least 10 generations until the protocorms obtained are all completely dedifferentiated, uniformly shaped cell clusters with strong proliferation ability, good dispersibility, and bright green color, and a suspension cell culture system is established.

4. The culture method according to claim 1, wherein: Step (1) Illumination conditions: The light source is white light or a mixture of white light and blue light, or white light and red light, and the illumination time is 8 to 16 hours per day. -1 , the light intensity is 10~100μmol·m -2 ·s -1 ; The induction temperature is 15-30°C.

5. The culture method according to claim 1, wherein: The inoculation density of the continuous subculture proliferation culture in step (2) is 50-200 g·L -1 ; Lighting conditions in step (2): The light source is white light or a mixture of white light and blue light, or white light and red light, and the illumination time is 8 to 16 hours per day. -1 , the light intensity is 10~100μmol·m -2 ·s -1 ; The culture temperature is 15-30℃; subculture once every 2-6 weeks.

6. The culture method according to claim 1, wherein: The suspension culture in the shake flask in step (3) is carried out by rotary shaking culture on a shaker with a speed of 80 to 150 r / min. -1 Lighting conditions: The light source consists of white light or a mixture of white light and blue light, or white light and red light, and the lighting time is 8 to 16 hours a day. -1 , the light intensity is 10~100μmol·m -2 ·s -1 Temperature: 15-30°C; inoculation density: 50-200 g·L -1 ; Subculture once every 2 to 6 weeks.

7. The culture method according to claim 1, wherein: The bioreactor in step (4) is a bubble reactor; the ventilation volume is 0.02 to 0.2 Nm 3 ·h -1 Lighting conditions: The light source consists of white light or a mixture of white light and blue light, or white light and red light, and the lighting time is 8 to 16 hours a day. -1 , the light intensity is 10~100μmol·m -2 ·s -1 Temperature: 15-30°C; inoculation density: 50-200 g·L -1 ; The culture period is 2 to 4 weeks.

8. The culture method according to claim 1, wherein: The protocorm obtained in step (4) can be vacuum-frozen and vacuum-dried in sequence, and then crushed and filtered to prepare freeze-dried powder of Dendrobium officinale protocorm.

9. The culture method according to claim 8, wherein: Vacuum freezing is as follows: vacuum degree is maintained at 1-10 Pa, temperature is -50°C, and maintained for 5 hours; The vacuum drying is carried out by maintaining the vacuum degree at 1-10 Pa and the temperature of the original corm at -45℃~-40℃ for 4 hours; then the temperature of the original corm is raised and maintained at -40℃~-20℃ for 5 hours; then the temperature of the original corm is raised and maintained at -20℃~0℃ for 6 hours; then the temperature of the original corm is raised and maintained at 0℃~40℃ for 8 hours; finally the temperature of the original corm is raised and maintained at 45℃ for 5 hours.

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