A cultivation method for significantly increasing the content of dendrobium officinale mannans and application thereof

Abstract

This invention relates to the field of medicinal plant cultivation technology, specifically a cultivation method and application for significantly increasing the mannan content of Dendrobium officinale. The method includes: selecting a suitable cedar forest environment, fixing Dendrobium officinale seedlings using a soilless planting method, watering regularly, and spraying with a 200-fold diluted indigenous probiotic cultivation enzyme every 10 days, for a total of 10 sprays. The enzyme is fermented from native microorganisms in natural forest soil and plant materials, containing Bacillus amyloliquefaciens, yeast, active enzymes, and various minerals. This invention can significantly increase the yield of Dendrobium officinale and the content of active ingredients such as mannan and total amino acids, achieving green, efficient, and sustainable cultivation.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant cultivation technology, specifically a cultivation method and application for significantly increasing the mannan content of Dendrobium officinale. Background Technology

[0002] Dendrobium officinale, a perennial herb belonging to the genus Dendrobium in the Orchidaceae family, is a traditional and precious Chinese medicinal herb. It possesses various medicinal properties, including promoting saliva production and nourishing the stomach, nourishing yin and clearing heat, moistening the lungs and benefiting the kidneys, and improving eyesight and strengthening the lower back. Modern pharmacological studies have shown that Dendrobium officinale is rich in polysaccharides, mannans, amino acids, and various trace elements. Mannans are one of its important active ingredients, possessing multiple physiological functions such as enhancing immunity, anti-aging, and lowering blood sugar.

[0003] Currently, the main cultivation methods for Dendrobium officinale include greenhouse cultivation and semi-wild cultivation under forest cover. However, existing cultivation techniques generally suffer from the following problems: The yield is low; the yield of fresh product under conventional forest cultivation is only about 25 kg per mu, which is difficult to meet market demand. The content of active ingredients is unstable, especially the content of mannan, which is easily affected by the cultivation environment and management methods; Chemical pesticides are often used to control pests and diseases, which leads to pesticide residue risks and affects the quality and safety of medicinal materials. Traditional fertilization methods can easily lead to soil compaction and microbial imbalance, which can affect the long-term growth of Dendrobium officinale.

[0004] Existing technologies have attempted to promote plant growth using microbial inoculants, but these primarily target growth promotion and disease control, with limited effectiveness in enhancing specific active ingredients (such as mannan), and their compositions differ from those of this invention. Furthermore, conventional probiotic preparations often employ single or a few strains, resulting in limited functionality and insufficient adaptability to specific ecological conditions.

[0005] Therefore, developing a cultivation technique that can significantly increase the yield of Dendrobium officinale, especially the content of its medicinal components such as mannan, and is also green and environmentally friendly, is of great industrial significance. Summary of the Invention

[0006] The purpose of this invention is to provide a cultivation method that can significantly improve the yield and mannan content of Dendrobium officinale. This method achieves a synergistic improvement in the yield and quality of Dendrobium officinale by applying a specific composition of indigenous probiotic enzymes combined with an optimized understory bundled cultivation model.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A cultivation method and application for significantly increasing the mannan content of Dendrobium officinale includes the following steps: (1) Select a suitable understory cultivation environment: Select a cedar forest with a canopy density of 0.7-0.9, an altitude of 1500-1650m, a spacing of 3m×4m between trees, and a trunk diameter at breast height of 25-30cm. (2) Using the soilless planting method of binding: Bind the Dendrobium officinale seedlings to the trunk of the fir tree at a position more than 1.3m above the ground, with 25-30 seedlings per circle, a circle spacing of 30cm, and 4 circles per tree; (3) Water management: Spray water on Dendrobium officinale every 3 days to keep it moist; (4) Spraying with native probiotic enzymes: During the growth period of Dendrobium officinale, spray the diluted native probiotic planting enzyme solution once every 10 days at a dilution ratio of 1:200, and thoroughly wet the plants and the substrate. Spray a total of 10 times. The indigenous probiotic cultivation enzyme is fermented from indigenous microbial agents isolated and cultivated from specific natural forest soils and various plant raw materials. Its effective components include Bacillus amyloliquefaciens, Saccharomyces pp., active enzymes, and nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium.

[0008] Preferably, in the indigenous probiotic cultivation enzyme, the number of live Bacillus amyloliquefaciens is not less than 1×10^8 CFU / mL, and the number of live yeast is not less than 1×10^7 CFU / mL.

[0009] Preferably, the harvesting standard for Dendrobium officinale is a stem length ≥ 10cm.

[0010] This invention also claims protection for the use of the indigenous probiotic cultivation enzyme in increasing the yield of Dendrobium officinale and / or the content of mannan.

[0011] Furthermore, the present invention claims protection for Dendrobium officinale cultivated by the above method, wherein the mannan content is not less than 16.0%, the total amino acid content is not less than 28.0%, and the extract content is not less than 7.4%.

[0012] Compared with the prior art, the beneficial effects of the present invention are: Significantly increased yield: Through the systematic application of native probiotic enzymes, combined with the understory bundled cultivation model, the yield of fresh Dendrobium officinale per mu increased from the conventional 24.92 kg to 36.96 kg, an increase of 48.3%; The active ingredients have been significantly improved: the content of mannan has increased by 22.54%, the total amino acids by 5.44%, the extract by 6.62%, and the crude ash (fiber) by 14.4%, which significantly enhances the medicinal and nutritional value of Dendrobium officinale. Green and safe: The native probiotic enzymes are derived from natural forest soil, with no chemical additives. They can inhibit pathogens, decompose pesticide residues, reduce pesticide use, and meet the requirements for green planting of Chinese medicinal herbs. Eco-friendly: By improving the rhizosphere microbial community, promoting nutrient cycling, and enhancing plant resistance, sustainable cultivation can be achieved. Detailed Implementation

[0013] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0014] Example 1: Preparation of indigenous probiotic cultivation enzymes Microbial collection and isolation: In the original evergreen broad-leaved forests or mixed coniferous and broad-leaved forests (preferably areas untouched by human pollution) in Guizhou Province, 5-10 points were randomly selected to collect surface humus soil samples (0-20cm depth) and mix them evenly.

[0015] Enrichment and initial screening: Take 10g of mixed soil sample and add it to an Erlenmeyer flask containing 90mL of sterile physiological saline and glass beads. Shake for 30 minutes to prepare a soil suspension. Using a serial dilution method, spread the suspension onto beef extract peptone medium (for isolating bacteria) and potato dextrose agar medium (for isolating yeasts and fungi), and incubate at 28-30℃ for 48-72 hours.

[0016] Screening of functional strains: Screening for Bacillus amyloliquefaciens: Single colonies of bacteria with different morphologies were selected and preliminarily identified by Gram staining and spore staining. These colonies were then inoculated onto a medium with soluble starch as the sole carbon source and incubated at 30°C for 48 hours. Iodine solution was added, and the clear zone was observed. Colonies with a large ratio of the diameter of the hydrolysis zone to the colony diameter were selected. Further 16S rDNA sequencing confirmed that these were *Bacillus amyloliquefaciens*.

[0017] Yeast screening: Smooth, moist, milky white or light red colonies were selected and their typical yeast morphology was observed under a microscope. These colonies were then transferred to YPD liquid medium for further culture. Strains exhibiting abundant foam production and strong fermentation capabilities during growth were selected.

[0018] Compound fermentation: Culture medium preparation: By weight, take 40% wheat bran, 20% soybean meal, 15% corn flour, 15% rice husk powder, 5% brown sugar, 2% calcium carbonate, 1% potassium dihydrogen phosphate, 0.5% magnesium sulfate, and 0.5% zinc sulfate, and mix them evenly. Add sterile water at a material-to-water ratio of 1:1.2 and mix well.

[0019] Inoculation: The screened and purified Bacillus amyloliquefaciens liquid culture (viable count ≥1×10^9 CFU / mL) and yeast liquid culture (viable count ≥1×10^8 CFU / mL) were inoculated into the solid culture medium at a volume ratio of 3:1, and the total inoculation amount was 5% of the dry weight of the culture medium.

[0020] Fermentation: The inoculated material is placed in a breathable fermentation bag or tank and subjected to aerobic-facultative anaerobic solid-state fermentation at 30-35℃ and 70-80% relative humidity. During fermentation, the material is turned over every 24 hours for ventilation and heat dissipation. The fermentation cycle is 18-22 days. The fermentation endpoint is determined by observing changes in material temperature (first rising and then falling), the production of a strong yeast aroma, and the absence of mold.

[0021] Extraction and preparation: Add an equal weight of sterile water to the fermentation product, soak and extract for 12 hours, filter and collect the supernatant, which is the indigenous probiotic enzyme stock solution. Test its total effective viable bacteria count (Bacillus amyloliquefaciens ≥ 2.0 × 10^8 CFU / mL, yeast ≥ 5.0 × 10^7 CFU / mL), pH value between 4.0 and 5.5, and store in a sealed container in a cool place.

[0022] Example 2: Bundled cultivation and application of fungal agents under Dendrobium officinale forests Preparation of cultivation site: Site selection: Shicang Forest Farm, Jinsha County, Guizhou Province. A pure Chinese fir forest with uniform tree species, vigorous growth, and few pests and diseases was selected, with a measured stand canopy closure of 0.80.

[0023] Host tree selection: Mark cedar trees with a diameter at breast height of 25-30cm, rough bark and no severe peeling as host trees.

[0024] Seedlings and securing: Seedlings: Select Dendrobium officinale seedlings that have been tissue cultured and domesticated for more than one year, have well-developed root systems, thick stems, and are free from diseases and pests, with a seedling height of about 10-15cm.

[0025] Binding: On each selected cedar tree trunk, starting 1.3 meters above the ground, use environmentally friendly non-woven fabric strips or hemp rope as binding material. With the roots of the Dendrobium seedlings close to the bark, take 25-30 seedlings and wrap them around the trunk once, securing them with the fabric strips. The tightness should be moderate, avoiding damage to the seedlings and preventing slippage. Bind a second layer upwards at 30cm intervals, and so on, for a total of 4 layers per tree. All layers should be evenly distributed around the trunk.

[0026] Field management: Experimental design: An experimental group and a control group were set up, with 50 host trees in each group (i.e., 50 replicates). The planting and management conditions were kept consistent except for the application of fungicide.

[0027] Water management (same for both groups): Install a forest spraying system or use a handheld sprayer to spray water evenly on the Dendrobium officinale plants and attached tree trunks every morning or evening, every 3 days, to ensure that the bark and roots are moist.

[0028] Probiotic enzyme spraying (experimental group only): Starting from the time the seedlings resume growth after planting (approximately two weeks later), spray the leaves and the attached substrate every 10 days. Before use, accurately dilute the enzyme stock solution prepared in Example 1 with water at a volume ratio of 1:200. Spray evenly using a sprayer until the Dendrobium leaves and attached trunk are completely saturated and the liquid is just about to drip. Spray a total of 10 times during the entire growth cycle (approximately 100 days).

[0029] Control group treatment: At the same time that the experimental group was sprayed with diluted enzyme solution, the control group was sprayed with an equal amount of water.

[0030] Other management: Regularly remove vines and weeds around the tree trunks that severely obstruct sunlight. No chemical fertilizers or pesticides were applied to either group throughout the trial.

[0031] Harvesting and Sampling: Date: December 7, 2023 (approximately one year after planting).

[0032] Standard: Harvest all stems that are 10cm or longer using clean scissors.

[0033] Weighing: On-site weighing of each group of fresh produce was conducted using calibrated electronic scales, and the yield per acre was calculated (based on the actual planting spacing and host tree density).

[0034] Sample preparation: 1.0 kg of representative sample was randomly selected from each group of fresh products, quickly frozen with liquid nitrogen, and then stored in an ultra-low temperature freezer at -80℃ for subsequent component analysis.

[0035] Example 3: Yield and quality determination and data analysis Yield determination: As described in Example 2, yield was measured on-site. The average yield per mu (667 square meters) after conversion of total fresh weight in the experimental group was 36.96 kg, while the average yield per mu in the control group was 24.92 kg. The yield increase rate of the experimental group compared with the control group reached 48.3%, which was highly significant (p<0.01).

[0036] Third-party testing and analysis: The samples preserved in Example 2 were sent to a qualified third-party testing institution (such as the Food and Drug Inspection Institute of a certain province) for testing in accordance with the 2020 edition of the Chinese Pharmacopoeia and relevant national standards.

[0037] Detection methods: Mannan content was determined by high performance liquid chromatography (HPLC); total amino acid content was determined by an automatic amino acid analyzer; extract content was determined by hot water leaching; crude ash content was determined by ignition method; and crude polysaccharide content was determined by phenol-sulfuric acid method.

[0038] Results and Discussion: Yield and Composition Improvement: Data shows that the method of this invention (experimental group) not only achieved a significant leap in yield, but more importantly, made comprehensive progress in core medicinal components. The mannan content increased from 13.13% to 16.09%, a relative increase of 22.54%, which is the most valuable achievement of this method. Total amino acids, extractives, and crude ash (representing minerals and fiber) also showed significant increases, confirming the effectiveness of this method in improving the overall quality of Dendrobium officinale.

[0039] Analysis of changes in crude polysaccharides: The crude polysaccharide content in the experimental group (15.71 g / 100 g) was slightly lower than that in the control group (16.36 g / 100 g). However, considering the significant increase in mannan (a type of polysaccharide), this invention may have altered the composition and structure of Dendrobium officinale polysaccharides, making them more prone to synthesizing mannan-type polysaccharides with specific biological activities. Studies have shown that mannans with certain structures are more advantageous than total polysaccharides in regulating blood sugar and blood lipids; therefore, this change may endow the product with more precise health regulatory functions.

[0040] Mechanism correlation: The increased yield is attributed to the probiotics promoting nutrient supply and plant health; the specific enrichment of components such as mannan may be related to the regulation of related glycosyltransferase gene expression or metabolic flux allocation within Dendrobium by probiotic metabolites. This demonstrates the overall regulatory advantage of the "bacteria-tree-Dendrobium" system of this invention.

[0041] Example 4: Comparison with existing conventional greenhouse cultivation techniques To highlight the advantages of this invention, the same variety of Dendrobium officinale was selected and cultivated in a greenhouse using conventional pine bark substrate pot cultivation, compound fertilizer application, and necessary chemical pesticides as a control.

[0042] Results: The yield of fresh product under conventional greenhouse cultivation was approximately 35 kg per mu (comparable to that of this invention), but the mannan content was typically between 10% and 12%, significantly lower than the 16.09% of that produced by the forest-grown probiotic cultivation method of this invention. Furthermore, greenhouse products pose a potential risk of pesticide residues, and their flavor and taste are generally considered inferior to those of forest-grown biomimetic cultivation products.

[0043] Conclusion: This invention, while ensuring high yield, has unparalleled advantages over conventional technologies in terms of improving the content of core medicinal ingredients and product safety.

[0044] This invention provides a complete, specific, and reproducible method for cultivating Dendrobium officinale, especially clarifying the specific source, composition, preparation method, and key parameters for combining the indigenous probiotic enzyme with the forest-based bundled cultivation model (dilution ratio 1:200, 10 days / time, 10 times in total). This method achieves a predictable and significant improvement in the yield and quality of Dendrobium officinale, particularly the mannan content.

[0045] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A cultivation method for significantly increasing the mannan content of Dendrobium officinale, characterized in that, Includes the following steps: Select a cedar forest with a canopy closure of 0.7-0.9 as the cultivation site, at an altitude of 1500-1650m, with a tree spacing of 3m×4m and a trunk diameter at breast height of 25-30cm. The soilless planting method of binding is adopted: Dendrobium officinale seedlings are bound to the trunk of the fir tree at a height of more than 1.3m above the ground. 25-30 seedlings are used in each ring, the distance between adjacent rings is 30cm, and each tree is bound 4 times. Water management: Spray water on the Dendrobium officinale every 3 days; Spraying native probiotics for cultivation: During the growth period of Dendrobium officinale, spray a diluted solution of native probiotics for cultivation every 10 days at a dilution ratio of 1:

200. Ensure that the plants and the substrate attached to them are fully moistened when spraying. Spray a total of 10 times. The indigenous probiotic cultivation enzyme is prepared by fermenting indigenous microbial agents isolated and cultivated from natural forest soil with various plant raw materials. Its effective components include Bacillus amyloliquefaciens, yeast, active enzymes, and nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium.

2. The cultivation method according to claim 1, characterized in that, In the indigenous probiotic cultivation enzyme, the number of viable Bacillus amyloliquefaciens is not less than 1×10^8 CFU / mL, and the number of viable yeasts is not less than 1×10^7 CFU / mL.

3. The cultivation method according to claim 1, characterized in that, The active enzyme includes one or more of protease, amylase, cellulase and phytase.

4. The cultivation method according to claim 1, characterized in that, The plant-based raw materials include at least two of the following: wheat bran, soybean meal, straw, weeds, or fruit and vegetable residues.

5. The cultivation method according to claim 1, characterized in that, The harvesting standard for Dendrobium officinale is a stem length ≥10cm.

6. The application of an indigenous probiotic cultivation enzyme in increasing the yield and / or mannan content of Dendrobium officinale, characterized in that, The effective components of the indigenous probiotic cultivation enzyme include Bacillus amyloliquefaciens, yeast, active enzymes, and nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium, which are sprayed into the Dendrobium officinale cultivation system by the method described in any one of claims 1-5.

7. The application according to claim 6, characterized in that, The dilution ratio of the indigenous probiotic cultivation enzyme is 1:200, and the spraying frequency is once every 10 days, with a total of 10 spraying times.

8. A Dendrobium officinale cultivated by the method described in any one of claims 1-5, characterized in that, Its mannan content is not less than 16.0%, its total amino acid content is not less than 28.0%, and its extract content is not less than 7.4%.

9. The Dendrobium officinale according to claim 8, characterized in that, Its crude ash content is not less than 6.0%.

10. A Dendrobium officinale cultivation system, characterized in that, include: Cultivation medium: cedar forests with a canopy density of 0.7-0.9 and cedar trees with a diameter at breast height of 25-30 cm; Cultivation method: Soilless planting with binding, each tree is bound with 4 rings of Dendrobium officinale seedlings, with a ring spacing of 30cm; Management plan: Spray water once every 3 days, and spray with native probiotic cultivation enzyme diluted 1:200 once every 10 days, for a total of 10 sprays; The indigenous probiotic-grown enzyme contains Bacillus amyloliquefaciens, yeast, active enzymes, and mineral elements.