A cultivation method for increasing 1-deoxynojirimycin in mulberry leaves

Abstract

The present invention belongs to the field of plant cultivation technology and discloses a cultivation method for increasing 1-deoxynojirimycin in mulberry leaves. The present invention mixes base fertilizer into the cultivation medium and plants mulberry trees, applies Bacillus subtilis in the form of root irrigation during the mulberry growth period, and harvests mulberry leaves in a timely manner. The cultivation method of the present invention can promote the accumulation of DNJ in mulberry leaves and can reduce the level of nitrate nitrogen in the soil.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation, and in particular relates to a cultivation method for increasing 1-deoxynojirimycin in mulberry leaves. Background Art

[0002] 1-Deoxynojirimycin (DNJ) is a polyhydroxy alkaloid and a potent α-glucosidase inhibitor. DNJ and its derivatives can significantly lower fasting blood glucose levels, increase insulin sensitivity, and alleviate pancreatic β-cell damage, thereby effectively preventing and treating diabetes. Existing research has demonstrated that 1-deoxynojirimycin exhibits beneficial hypoglycemic and hypolipidemic properties, as well as antioxidant effects.

[0003] In 1976, Yoshiaki et al. in Japan first isolated and purified DNJ from mulberry root bark. DNJ is present in all organs of the mulberry tree, but its content varies depending on the variety, cultivation region, and the medicinal part of the tree. For mulberry leaves, factors such as leaf position, developmental stage, harvest time, and season all influence DNJ content. DNJ originates from plants and microorganisms. Besides mulberry, wild hyacinth, hyacinth, and Adenophora species also contain DNJ and its analogs, but their concentrations are lower than those in mulberry. DNJ produced by microbial fermentation is even lower, at around 0.01 mg / mL. DNJ in mulberry is primarily distributed in its leaves, roots, and branches, with mulberry leaves containing the highest DNJ content. Furthermore, mulberry leaves account for the largest proportion of the total dry matter in the mulberry tree. Therefore, mulberry leaves have become the primary source of natural DNJ.

[0004] Environmental stress, formulated fertilization, and the addition of exogenous elicitors can, to a certain extent, increase DNJ content in mulberry leaves. Increasing DNJ content in mulberry trees through cultural practices (formulated fertilization) is low-cost, quick, and easily scalable. Previous studies have shown that increasing nitrogen fertilizer application can increase DNJ content. However, excessive nitrogen fertilizer application can not only lead to excess nitrogen in the soil, reduced crop yields, decreased stress tolerance, and reduced crop quality, but can also cause soil nitrate and nitrite pollution. Summary of the Invention

[0005] In order to overcome the above problems, the purpose of the present invention is to provide a mulberry tree cultivation method that can greatly increase the DNJ content in mulberry leaves and reduce the nitrate nitrogen content in the soil.

[0006] The technical solutions of the present invention are as follows:

[0007] The invention provides a cultivation method for increasing 1-deoxynojirimycin in mulberry leaves, comprising the following steps: mixing base fertilizer into a cultivation medium and planting mulberry trees; applying Bacillus subtilis by way of root irrigation during the growth period of the mulberry trees; and harvesting mulberry leaves in due course.

[0008] As an embodiment, the base fertilizer is prepared by weighing 40 to 60 parts of mulberry branches, 20 to 30 parts of silkworm feces, 10 to 20 parts of dried cakes, and 2 to 3 parts of ammonium bicarbonate according to the weight of each component, mixing them evenly, adding water and quicklime, adjusting the pH to 6.0 to 7.0, and finally adding a composite bacterial agent for fermentation.

[0009] Preferably, the composite bacterial agent is a mixture of Bacillus amyloliquefaciens, Aspergillus oryzae, Trichoderma aculeatus, Candida utilis, Streptomyces tenuiflavus, and Lactobacillus acidophilus, and the number of viable bacteria is as follows: 5-6 parts of Bacillus amyloliquefaciens, 1-2 parts of Aspergillus oryzae, 1-2 parts of Trichoderma aculeatus, 1-2 parts of Candida utilis, 1 part of Streptomyces tenuiflavus, and 1 part of Lactobacillus acidophilus.

[0010] Preferably, the addition amount of the composite bacterial agent is 2‰ to 3‰ of the total mass of the material.

[0011] Preferably, the cultivation matrix is ​​garden soil, peat and perlite, and 55-65 parts of garden soil, 15-20 parts of peat, 8-12 parts of perlite and 8-12 parts of base fertilizer are mixed evenly by mass for planting.

[0012] Preferably, the total number of viable Bacillus subtilis bacteria per mulberry tree in a single irrigation is 0.5×10 10 ~2×10 10 CFU.

[0013] As an embodiment, Bacillus subtilis is applied once in July and once in September.

[0014] As an embodiment, the mulberry leaves are harvested 15 to 20 days after the application of Bacillus subtilis.

[0015] As an implementation method, fertilizer is applied once in mid-May, with 20 to 25 g of urea and 15-15-15N-P2O5-K2O compound fertilizer applied to each mulberry tree each time.

[0016] As an implementation method, mulberry seedlings of uniform size, seedling diameter greater than 5.0 mm, intact main roots, and root length greater than or equal to 100 mm are selected; the tender and weak parts at the top of the mulberry seedlings are cut off, leaving the seedlings 30 to 40 cm high; the thick heels are cut to 8 to 12 cm, and the mulberry seedlings are planted in a cultivation medium.

[0017] Compared with the prior art, the present invention has the following technical effects:

[0018] During the mulberry tree cultivation period, the invention applies Bacillus subtilis in the form of root irrigation, which can promote the accumulation of DNJ in mulberry leaves and reduce the levels of nitrate nitrogen and nitrite nitrogen in the soil.

[0019] The base fertilizer of the present invention contains mulberry branches, silkworm excrement and dried cake, which are obtained by adding a composite bacterial agent and fermenting. The mulberry branches have a high fiber content, and the silkworm excrement and dried cake have a high nutrient content. By mixing and fermenting the mulberry branches, silkworm excrement and dried cake, a relatively ideal carbon-nitrogen ratio and nutrient composition can be obtained. The use of the base fertilizer can efficiently improve the soil, effectively reduce the use of chemical fertilizers, and promote the synthesis of nitrogen dioxide by mulberry trees.

[0020] The composite microbial agent adopted by the present invention has a reasonable formula and is easy to use. It can efficiently ferment mulberry branches and silkworm excrement to produce nutrients such as various amino acids and growth hormones, and can inhibit the growth of some harmful bacteria in the silkworm excrement. The fermentation process will not cause secondary pollution. DETAILED DESCRIPTION

[0021] The present invention provides a cultivation method for increasing 1-deoxynojirimycin (DNJ) in mulberry leaves, comprising the following steps: mixing base fertilizer into a cultivation medium and planting mulberry trees; applying Bacillus subtilis by root irrigation during the mulberry tree's growth period; and harvesting the mulberry leaves at an appropriate time. The method can promote the accumulation of DNJ in mulberry leaves and reduce nitrate nitrogen levels in the soil.

[0022] The base fertilizer of the present invention is a base fertilizer specifically for mulberry cultivation, which is beneficial for promoting mulberry trees to synthesize DNJ and increasing the DNJ content in mulberry leaves. As an embodiment, the base fertilizer is prepared by weighing 40 to 60 parts of mulberry branches, 20 to 30 parts of silkworm excrement, 10 to 20 parts of dried cake, and 2 to 3 parts of ammonium bicarbonate according to the weight of each component, mixing them evenly, adding water and quicklime, adjusting the pH to 6.0 to 7.0, and finally adding a composite bacterial agent to ferment the resulting mixture. As a preferred embodiment, the weight of the base fertilizer components is 45 to 55 parts of mulberry branches, 24 to 27 parts of silkworm excrement, 13 to 18 parts of dried cake, and 2 to 3 parts of ammonium bicarbonate.

[0023] The present invention preferably comprises a mixture of Bacillus amyloliquefaciens, Aspergillus oryzae, Trichoderma aculeatus, Candida utilis, Streptomyces tenuifolius, and Lactobacillus acidophilus, wherein the above bacteria are prepared in the following proportions, calculated by viable cell count: 5-6 parts of Bacillus amyloliquefaciens, 1-2 parts of Aspergillus oryzae, 1-2 parts of Trichoderma aculeatus, 1-2 parts of Candida utilis, 1 part of Streptomyces tenuifolius, and 1 part of Lactobacillus acidophilus. The present invention does not particularly limit the source of the composite bacterial agent; the above bacterial agents can be purchased commercially and compounded, and the weight ratio of the compounded bacterial agents is determined based on the viable cell count content of the different bacterial agents. As an embodiment, commercially available Bacillus amyloliquefaciens (20 billion / g), Aspergillus oryzae (10 billion / g), Trichoderma acanthosporium (20 billion / g), Candida utilis (10 billion / g), Streptomyces tenuifolius (10 billion / g), and Lactobacillus acidophilus (10 billion / g) are compounded in a mass ratio of 3:2:1:1:1:1. In the present invention, the preferred amount of the composite bacterial agent added is 2‰ to 3‰ of the total mass of the material.

[0024] As one embodiment, the present invention employs composting as a fermentation method. When the compost temperature reaches above 60°C, the compost is promptly turned and fermentation is terminated until the raw material is odorless. The base fertilizer produced by the present invention can regulate the appropriate ratio of nitrogen forms and promote the synthesis of nitrogen nitrogen in mulberry trees. Experiments have shown that the base fertilizer can increase the nitrogen nitrogen content in mulberry leaves and reduce the nitrate nitrogen content in the soil.

[0025] As one embodiment, the present invention provides a cultivation medium for cultivating mulberry trees comprising a mixture of garden soil, peat, and perlite. A base fertilizer is mixed into the cultivation medium and the mulberry trees are cultivated together. Preferably, 55 to 65 parts garden soil, 15 to 20 parts peat, 8 to 12 parts perlite, and 8 to 12 parts base fertilizer are uniformly mixed by weight. The mixture of the cultivation medium and base fertilizer is placed in a cultivation container and set aside.

[0026] The present invention does not particularly limit the method of planting mulberry trees, and conventional methods can be used for planting. As an embodiment, mulberry seedlings are planted in mid-to-late February, and hybrid mulberry varieties with early germination, many stems, vigorous growth, resistance to felling, and late leaf drop are selected, such as Yuesang No. 11 and Guiyou No. 12. Mulberry seedlings with uniform size, seedling diameter greater than 5.0 mm, complete main roots, and root length greater than or equal to 100 mm are selected. The specific planting method is to cut off the tender and weak parts of the top of the mulberry seedlings, leaving a seedling height of 30 to 40 cm; cut the thick heels to about 8 to 12 cm, plant the mulberry seedlings in the cultivation medium, cover the soil and lift it lightly to allow the roots to stretch, then gently pat the soil with your hands to compact it, add a layer of loose soil, and water it enough to establish roots.

[0027] The mulberry trees of the present invention are managed with conventional water, fertilizer and pesticides during cultivation. As an embodiment, fertilizer is applied once in mid-May, with each mulberry tree being fertilized with 20 to 25 g of urea and compound fertilizer (N-P2O5-K2O: 15-15-15) each.

[0028] The present invention applies Bacillus subtilis by root irrigation during the mulberry tree growth period. Bacillus subtilis increases DNJ content by changing the soil microecology and regulating the genes related to DNJ synthesis in mulberry trees. As a preferred embodiment, Bacillus subtilis is applied once in July and once in September. During this period, mulberry trees have a vigorous metabolism and will synthesize a large amount of DNJ. At this time, root irrigation with Bacillus subtilis is more conducive to the accumulation of DNJ in mulberry leaves. Preferably, the total number of viable bacteria per mulberry tree in a single irrigation is 0.5×10 10 ~2×10 10 CFU, further 1×10 10 ~1.5×10 10CFU. The source of Bacillus subtilis is not particularly limited in the present invention and can be purchased commercially. Mulberry leaves are harvested 15-20 days after application of Bacillus subtilis. Root-feeding Bacillus subtilis can increase DNJ content in mulberry leaves and reduce soil nitrate nitrogen content. Experimental studies have shown that harvesting mulberry leaves during this period of time is most effective.

[0029] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments. The technical solutions of the present invention include but are not limited to the following embodiments.

[0030] Example 1

[0031] (1) Production of special base fertilizer

[0032] Mulberry branches are first crushed with a branch crusher and then pulverized with a multifunctional drug. Take 100kg of mulberry branches, 40kg of silkworm feces, 30kg of withered cakes, 4kg of ammonium bicarbonate, mix well, add water and an appropriate amount of quicklime, adjust pH to 7.0, add 400g of composite bacterial agent to carry out retting, and when the temperature reaches 60 DEG C, turn the heap in time until the raw material is odorless and finish fermentation. The composite bacterial agent is commercially available Bacillus amyloliquefaciens (20 billion / g), Aspergillus oryzae (10 billion / g), Trichoderma acanthoides (20 billion / g), Candida utilis (10 billion / g), Streptomyces tenuifolius (10 billion / g), Lactobacillus acidophilus (10 billion / g) and is composited in a mass ratio of 3:2:1:1:1:1.

[0033] (2) Preparation of cultivation medium

[0034] Mix 150 kg of garden soil, 50 kg of peat, 25 kg of perlite, and 25 kg of special base fertilizer to form the cultivation medium. Each seedling pot (inner diameter 32 cm, height 29 cm) is filled with approximately 15 kg of the cultivation medium and set aside.

[0035] (3) Planting

[0036] Plant Yuesang No. 11 mulberry seedlings on February 26th. Select seedlings of uniform size, with a seedling diameter greater than 5.0 mm, an intact taproot, and a root length greater than or equal to 100 mm. Prune the top of the seedling to remove the tender, weak parts, leaving it 30-40 cm tall; trim the thick base to about 10 cm. Plant the seedling in the substrate, cover with soil, and gently lift to allow the roots to stretch. Gently pat the soil with your hands to firm it, then add a layer of loose soil. Plant one seedling per pot and water thoroughly to establish roots.

[0037] (4) Fertilization

[0038] Fertilize once on May 16, applying 20g each of urea and compound fertilizer (N-P2O5-K2O: 15-15-15) to each mulberry tree.

[0039] (5) Application of Bacillus subtilis agent

[0040] On July 5 and September 8, Bacillus subtilis (20 billion / g, commercially available) was applied by root irrigation. Each mulberry tree was irrigated with 500 mL of Bacillus subtilis suspension (2×10 7 CFU / mL).

[0041] (6) Harvesting mulberry leaves

[0042] Mulberry leaves were harvested on July 20 and September 23 respectively.

[0043] Example 2

[0044] Same as Example 1, except that the concentration of the Bacillus subtilis suspension was 1×10 7 CFU / mL.

[0045] Example 3

[0046] Same as Example 1, except that the concentration of the Bacillus subtilis suspension was 4×10 7 CFU / mL.

[0047] Example 4

[0048] (1) Production of special base fertilizer

[0049] Mulberry branches are first crushed with a branch crusher and then pulverized with a multifunctional drug. 400kg of mulberry branches, 300kg of silkworm feces, 100kg of withered cakes, 20kg of ammonium bicarbonate are taken, mixed evenly, water and an appropriate amount of quicklime are added, pH is adjusted to 6.0, 1.5kg of composite bacterial agent is added for retting, and when the temperature reaches 60 DEG C, the heap is turned in time until the raw material is odorless and fermentation is terminated. The composite bacterial agent is commercially available Bacillus amyloliquefaciens (20 billion / g), Aspergillus oryzae (10 billion / g), Trichoderma acanthoides (20 billion / g), Candida utilis (10 billion / g), Streptomyces tenuifolius (10 billion / g), Lactobacillus acidophilus (10 billion / g) and is composited in a mass ratio of 3:2:1:1:1:1.

[0050] (2) Preparation of cultivation medium

[0051] Mix 160kg of garden soil, 45kg of peat, 30kg of perlite, and 30kg of special fertilizer to form the cultivation medium. Each pot should contain approximately 15kg of the cultivation medium and set aside.

[0052] (3) Planting

[0053] Plant Yuesang No. 11 mulberry seedlings on February 23rd. Select seedlings of uniform size, with a seedling diameter greater than 5.0 mm, intact taproots, and root lengths greater than or equal to 100 mm. Prune the tops of the seedlings, leaving them 30-40 cm tall; trim the thick base to about 12 cm. Plant the seedlings in the substrate, cover with soil, and gently lift to allow the roots to stretch. Gently pat the soil with your hands to firm it, then add a layer of loose soil. Plant one seedling per pot and water thoroughly to establish roots.

[0054] (4) Fertilization

[0055] Fertilize once on May 18, applying 20g each of urea and compound fertilizer (N-P2O5-K2O: 15-15-15) to each mulberry tree.

[0056] (5) Application of Bacillus subtilis agent

[0057] On July 10 and September 12, Bacillus subtilis (20 billion / g, commercially available) was applied by root irrigation. Each mulberry tree was irrigated with 500 mL of Bacillus subtilis suspension (2×10 7 CFU / mL).

[0058] (6) Harvesting mulberry leaves

[0059] Mulberry leaves were harvested on July 25 and September 27 respectively.

[0060] Example 5

[0061] (1) Production of special base fertilizer

[0062] Mulberry branches are first crushed with a branch crusher and then pulverized with a multifunctional drug. Take 600kg of mulberry branches, 200kg of silkworm feces, 200kg of withered cakes, 20kg of ammonium bicarbonate, mix well, add water and an appropriate amount of quicklime, adjust pH to 6.5, add 2kg of composite bacterial agent for retting, and turn the heap in time when the temperature reaches 60 DEG C, until the raw material is odorless and ends fermentation. The composite bacterial agent is commercially available Bacillus amyloliquefaciens (20 billion / g), Aspergillus oryzae (10 billion / g), Trichoderma acanthoides (20 billion / g), Candida utilis (10 billion / g), Streptomyces tenuifolius (10 billion / g), Lactobacillus acidophilus (10 billion / g) and is composited in a mass ratio of 3:1:1:2:1:1.

[0063] (2) Preparation of cultivation medium

[0064] Mix 140kg of garden soil, 40kg of peat, 20kg of perlite, and 20kg of special base fertilizer to form the cultivation medium. Each pot should contain approximately 15kg of the cultivation medium and set aside.

[0065] (3) Planting

[0066] Plant Yuesang No. 11 mulberry seedlings on February 23rd. Select seedlings of uniform size, with a seedling diameter greater than 5.0 mm, an intact taproot, and a root length greater than or equal to 100 mm. Prune the top of the seedling to remove the tender, weak parts, leaving it 30-40 cm tall; trim the thick base to about 10 cm. Plant the seedling in the substrate, cover it with soil, and gently lift it to allow the roots to stretch. Gently pat the soil with your hands to firm it, then add a layer of loose soil. Plant one seedling per pot and water it thoroughly to establish roots.

[0067] (4) Fertilization

[0068] Fertilize once on May 15th, applying 20g each of urea and compound fertilizer (N-P2O5-K2O: 15-15-15) to each mulberry tree.

[0069] (5) Application of Bacillus subtilis agent

[0070] On July 10 and September 12, Bacillus subtilis (20 billion / g, commercially available) was applied by root irrigation. Each mulberry tree was irrigated with 500 mL of Bacillus subtilis suspension (2×10 7 CFU / mL).

[0071] (6) Harvesting mulberry leaves

[0072] Mulberry leaves were harvested on July 25 and September 27 respectively.

[0073] Comparative Example 1

[0074] The same as Example 1, except that Bacillus subtilis was not used.

[0075] Comparative Example 2

[0076] The same as Example 1, except that no special base fertilizer is applied.

[0077] Comparative Example 3

[0078] The method is the same as Example 1, except that the base fertilizer preparation method is as follows: take 100 kg of mulberry branches, 40 kg of silkworm feces, 30 kg of dried cakes, and 4 kg of ammonium bicarbonate, mix them evenly, add water and an appropriate amount of quicklime, adjust the pH to 7.0, and add 400 g of compost fermentation agent (Beihai Qiangxing Biotechnology Co., Ltd.) for composting and fermentation.

[0079] Test example

[0080] The mulberry leaves obtained in Examples 1-3 and Comparative Examples 1-3 were dried at 70°C and pulverized, extracted with 0.05 mol / L hydrochloric acid, and the DNJ content was determined by pre-column derivatization followed by high-performance liquid chromatography (HPLC). On September 30, soil from each of the Examples and Comparative Examples was collected, air-dried, and the nitrate nitrogen (NO₃⁻N) content in the soil was determined by calcium chloride extraction followed by UV-visible spectrophotometry.

[0081] The test results are as follows:

[0082]

[0083] As shown in the table above, the mulberry cultivation method provided by the present invention significantly increases the DNJ content in mulberry leaves and reduces the nitrate nitrogen content in the soil. The DNJ content in the mulberry leaves of Example 1 was 15.34% to 48.80% higher than that of Comparative Example 1, 24.52% to 65.15% higher than that of Comparative Example 2, and 5.14% to 27.40% higher than that of Comparative Example 3. The nitrate nitrogen content in the soil of Example 1 was reduced by 13.14%, 20.65%, and 4.61% compared to Comparative Examples 1, 2, and 3, respectively.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for increasing 1-deoxynojirimycin in mulberry leaves, characterized in that: The following steps are involved: Mulberry trees were planted by mixing base fertilizer into the cultivation medium. During the growth period of the mulberry trees, Bacillus subtilis was applied by root irrigation. The total number of viable Bacillus subtilis bacteria per mulberry tree was 0.5×10 10 ~2×10 10 CFU, harvest mulberry leaves at the right time; The base fertilizer is prepared by weighing 40-60 parts of mulberry branches, 20-30 parts of silkworm feces, 10-20 parts of dried cakes, and 2-3 parts of ammonium bicarbonate according to the weight of each component, mixing them evenly, adding water and quicklime, adjusting the pH to 6.0-7.0, and finally adding a composite bacterial agent for fermentation; The composite bacterial agent comprises, in terms of viable bacteria count, 5-6 parts of Bacillus amyloliquefaciens, 1-2 parts of Aspergillus oryzae, 1-2 parts of Trichoderma acanthoides, 1-2 parts of Candida utilis, 1 part of Streptomyces tenuifolius and 1 part of Lactobacillus acidophilus.

2. The cultivation method according to claim 1, wherein The addition amount of the composite bacterial agent is 2‰ to 3‰ of the total mass of the material.

3. The cultivation method according to claim 1, wherein The cultivation matrix is ​​garden soil, peat and perlite. By weight, 55-65 parts of garden soil, 15-20 parts of peat, 8-12 parts of perlite and 8-12 parts of base fertilizer are evenly mixed for planting.

4. The cultivation method according to claim 1, wherein Bacillus subtilis was applied once in July and September respectively.

5. The cultivation method according to claim 1, wherein Mulberry leaves were harvested 15 to 20 days after application of Bacillus subtilis.

6. The cultivation method according to claim 1, wherein Fertilize once in mid-May, applying 20-25g each of urea and 15-15-15N-P2O5-K2O compound fertilizer to each mulberry tree.

7. The cultivation method according to claim 1, wherein Select mulberry seedlings of uniform size, seedling diameter greater than 5.0mm, complete main roots, and root length greater than or equal to 100mm; cut off the tender and weak parts at the top of the mulberry seedlings, leaving the seedlings 30-40cm high; cut the thick heels to 8-12cm, and plant the mulberry seedlings in the cultivation medium.

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