Composite soaking agent for promoting synchronous seedling emergence of Chinese yam tubers as well as preparation method and application of composite soaking agent

Abstract

The invention provides a composite soaking agent for promoting synchronous seedling emergence of Chinese yam tubers as well as a preparation method and application of the composite soaking agent. The composite soaking agent is prepared from the following raw materials in parts by weight: 0.05 to 0.2 part of melatonin nano-liposome, 0.5 to 2.0 parts of sodium lignin sulfonate, 2 to 5 parts of garlic extracting solution, 5 to 10 parts of abscisic acid degrading enzyme activating agent, 3 to 8 parts of mitochondria activating agent, 2 to 6 parts of pyruvate kinase activating agent and 800 to 900 parts of purified water. The compound soaking agent solves the heterogeneity problem of tuber germination from multiple aspects through a five-in-one synergistic network of hormone regulation, energy enhancement, metabolism activation, oxidation resistance and bacterium resistance, and promotes tuber full seedling, so that integrated management of water, fertilizer and illumination in the later period is facilitated, the yield is increased, and the economic benefit is increased. A technical solution of a multi-target and multi-component synergistic mechanism is provided for high-quality and high-yield Chinese yam, and the method has remarkable application and popularization values.

Description

Technical Field

[0001] This invention relates to the field of agricultural planting technology, specifically to a compound soaking agent for promoting the synchronous emergence of yam tubers, its preparation method, and its application. Background Technology

[0002] Yam possesses both nutritional and economic value. Its fleshy tubers are rich in mucoprotein, dietary fiber, and various trace elements, making it a nourishing food with medicinal properties, known for its spleen-strengthening, stomach-benefiting, lung-moistening, and cough-relieving effects. It is also a raw material for processing yam powder, diosgenin, and other products, with stable market demand. Currently, yam cultivation mainly employs three methods: tuber cutting propagation, bulbil propagation, and stem cuttings, with tuber cultivation being the most common and widely used. However, due to differences in bud vigor in different parts of the tuber, coupled with uneven nutrient consumption during storage, germination times often vary, with the same batch of tubers potentially germinating at different rates by more than twenty days. This asynchronous germination not only increases the difficulty of field management but also affects the unified control of light, temperature, water, and fertilizer, ultimately potentially leading to inconsistent plant growth stages and yield fluctuations. Summary of the Invention

[0003] In view of this, the present invention proposes a compound soaking agent for promoting synchronous emergence of yam tubers, its preparation method and application, in order to solve the above problems.

[0004] The technical solution of this invention is implemented as follows:

[0005] A compound soaking agent for promoting synchronous emergence of yam tubers comprises the following raw materials in parts by weight: 0.05-0.2 parts melatonin nanoliposomes, 0.5-2.0 parts sodium lignin sulfonate, 2-5 parts garlic extract, 5-10 parts abscisic acid degrading enzyme activator, 3-8 parts mitochondrial activator, 2-6 parts pyruvate kinase activator, and 800-900 parts purified water.

[0006] Furthermore, melatonin nanoliposomes were prepared by the following method: soybean lecithin, cholesterol, melatonin, and vitamin E were mixed in a mass ratio of (15-20):(3-4):(1.0-1.5):(0.4-0.6) to obtain a mixture; the mixture was added to a chloroform-ethanol solution in a mass-volume ratio of 1:(8-12) to obtain a solution; the solution was then rotary evaporated at 40-45℃ and 80-120 rpm for 40-60 min to obtain a lipid membrane; subsequently, the solution was added to the lipid membrane in a volume ratio of 1: Add PBS buffer preheated to 40-45℃ to 8-12, stir in a water bath at 40-45℃ for 25-35 minutes to obtain a crude emulsion; then transfer it to an ultrasonic treatment device and treat it in pulse mode at an ultrasonic power of 200-400W for 10 minutes, working in pulse mode for 3 seconds, pausing for 2 seconds, and repeating this cycle; then treat it in a high-pressure homogenizer for 3-5 times at 700-900 bar, cool it to 3-5℃ after homogenization, and then filter it through a 0.22μm filter membrane to obtain melatonin nanoliposomes.

[0007] Furthermore, the garlic extract is prepared by the following method: Fresh garlic cloves are peeled and sprayed with cold water at 3-5℃, then quickly frozen in liquid nitrogen at -15 to -20℃ for 4-6 minutes, then crushed, and then 0.1-0.2% of a compound enzyme by weight of garlic is added and enzymatically hydrolyzed at 34-36℃ for 2 hours. After the enzymatic hydrolysis is completed, the temperature is raised to 85-90℃ to inactivate the enzyme for 3-5 minutes, and then centrifuged at 4000-6000 rpm for 10-15 minutes. The supernatant is collected to obtain the garlic extract; wherein the compound enzyme consists of alliinase and cellulase in a mass ratio of 1:1.5-2.

[0008] Furthermore, the abscisic acid degrading enzyme activator is composed of soybean extract and Bacillus amyloliquefaciens fermentation broth in a volume ratio of (2.5-3.5):(1.5-2.5).

[0009] Furthermore, the soybean extract is prepared by the following method: Dry soybeans are pulverized and passed through a 40-60 mesh sieve. Petroleum ether is added at a solid-liquid ratio of 1:4-6 (g / mL), and the mixture is extracted using a Soxhlet extract at 55-65℃ for 3.5-4.5 hours. Subsequently, the mixture is placed in a fume hood and evaporated at 40-45℃ for 3-5 hours to obtain defatted soybean powder. 65-75 wt% ethanol is added to the defatted soybean powder at a solid-liquid ratio of 1:8-12 (g / mL), and the mixture is extracted using a water bath at 55-65℃ and an irradiation rate of 150-250 rpm. Extract by stirring at a speed of 1 pm for 1.8-2.2 h. After extraction, centrifuge at 4000-6000 rpm for 8-12 min and collect the supernatant. Repeat the extraction once with the lower precipitate and combine the two supernatants. Then, rotary evaporate to 15-20% of the original volume at 40-45℃ and -0.08 to -0.10 MPa. Add 0.04-0.06% vitamin C of the evaporated volume and stir at 3-5℃ for 20-30 min to obtain soybean extract.

[0010] Furthermore, the Bacillus amyloliquefaciens fermentation broth was prepared by the following method: Bacillus amyloliquefaciens strain was streaked onto an agar slant and incubated at 35-39℃ for 22-26 hours to form single colonies; these single colonies were then inoculated into 50 mL of seed culture medium and incubated at 35-39℃ with shaking at 150-250 rpm for 11-13 hours. Subsequently, 5% of this inoculum was inoculated into 500 mL of seed culture medium and incubated at 35-39℃ with shaking at 150-250 rpm. The culture medium was shaken at rpm for 7-9 hours; then, it was inoculated into the fermentation tank at an 8% inoculum and cultured at 33-35℃ and 300-500 rpm for 36-40 hours with a fermentation tank aeration rate of 1.0-1.2 vvm. When the residual sugar was <5 g / L, concentrated corn steep liquor was added at a flow rate of 40-50 mL / h. After fermentation, the mixture was centrifuged at 5000-7000 rpm for 15-20 minutes at 3-5℃, and the supernatant was collected. The supernatant was filtered through a 100 kDa filter membrane to obtain the Bacillus amyloliquefaciens fermentation broth. The slant culture medium includes the following components: 8-12 g / L peptone, 4-6 g / L beef extract, 4-6 g / L sodium chloride, and 15-25 g / L agar. The seed culture medium includes the following components: 8-12 g / L peptone, 4-6 g / L yeast extract, 15-25 g / L glucose, and 0.4-0.6 g / L magnesium sulfate heptahydrate. The fermentation culture medium includes the following components: 25-35 g / L starch, 15-25 g / L soybean meal, 8-12 g / L corn steep liquor, 0.8-1.0 g / L potassium dihydrogen phosphate, and 1.5-2.5 g / L calcium carbonate.

[0011] Furthermore, the mitochondrial activator is composed of α-lipoic acid and betaine in a mass ratio of (0.2-0.4):(1.2-1.8).

[0012] Furthermore, the pyruvate kinase activator is composed of magnesium citrate, berberine, and echinacea extract in a mass ratio of (0.4-0.6):(0.04-0.06):(1.5-2.5).

[0013] Furthermore, the echinacea extract was prepared by the following method: Echinacea roots were taken, washed, and dried at 40-45℃ until the moisture content was ≤8%. The roots were then pulverized to 40-60 mesh. 65-75 wt% ethanol was added at a material-to-liquid ratio of 1:8-12 (g / mL). Extraction was carried out under ultrasonic power of 200-400W and 38-42℃ for 40-60 minutes. After extraction, the extract was filtered out, and the residue was extracted again. The two extracts were combined and then rotary evaporated at 40℃ to 15-20% of the original volume to obtain a crude extract. The crude extract was then subjected to molecular distillation at 75-85℃ and 0.1-0.2 Pa to collect the light fraction, thus obtaining the echinacea extract.

[0014] Furthermore, a method for preparing a compound soaking agent to promote synchronous emergence of yam tubers includes the following steps:

[0015] S1. Add sodium lignosulfonate to purified water and stir until homogeneous to obtain a premixed solution;

[0016] S2. Add melatonin nanoliposomes, garlic extract, mitochondrial activator, abscisic acid degradation enzyme activator, and pyruvate kinase activator to the premix in sequence, stir well to obtain a mixture;

[0017] S3. Place the mixture into a homogenizer and homogenize it 2-3 times to obtain the composite soaking agent.

[0018] Furthermore, the stirring speed for S1 is 150-250 rpm and the stirring time is 8-12 min; the stirring speed for S2 is 250-350 rpm and the stirring time is 10-20 min; and the homogenization pressure for S3 is 500-700 bar.

[0019] Furthermore, a compound soaking agent is provided to promote the synchronous emergence of yam tubers, which is applied to soak yam tubers before planting.

[0020] Compared with existing technologies, the beneficial effects of this invention are as follows: The composite soaking agent of this invention solves the heterogeneity problem of tuber germination from multiple levels through a synergistic network of "hormone regulation - energy enhancement - metabolic activation - antioxidation - antibacterial". Melatonin nanoliposomes can scavenge free radicals and induce the activity of superoxide dismutase and catalase, reducing oxidative damage to tubers. Melatonin nanoliposomes can also inhibit abscisic acid synthesis and promote gibberellin accumulation, breaking tuber dormancy and promoting synchronous germination. Its nanoliposome form improves melatonin bioavailability, stabilizes mitochondrial membrane potential, and reduces energy dissipation, thereby significantly shortening germination time and enhancing seedling resistance. Sodium lignin sulfonate reduces the surface tension of the soaking solution, promoting the uniform dispersion and penetration of other components on the tuber epidermis; simultaneously, through osmotic regulation, it softens the tuber epidermis, reducing the mechanical resistance of the radicle / embryo breaking through the epidermis, and reducing germination delay differences by optimizing the seed potato imbibition process. Garlic extract, used in combination with melatonin, enhances the antioxidant system and improves germination uniformity. Its active ingredients, such as allicin and thiosulfinates, can effectively kill pathogens on the tuber surface and reduce seedling mortality caused by diseases. The abscisic acid degrading enzyme activator contains Bacillus amyloliquefaciens, which secretes ABA-8′-hydroxylase to convert abscisic acid into inactive products. Isoflavones in its soybean extract activate the metabolic activity of Bacillus amyloliquefaciens, accelerating abscisic acid degradation, reducing abscisic acid concentration, relieving its inhibition of germination, and promoting gibberellin accumulation, significantly shortening emergence time and reducing germination time differences. The mitochondrial activator contains α-lipoic acid, which catalyzes pyruvate metabolism and scavenges reactive oxygen species, improving mitochondrial ATP synthesis efficiency; betaine promotes tuber water absorption through osmotic regulation, softens the epidermis, and acts as a methyl donor to regulate hormone synthesis and activity, coordinating energy distribution and cell division / elongation. The pyruvate kinase activator contains magnesium citrate to supplement the Mg2+ required for pyruvate production. + Cofactors stabilize enzyme structure; berberine activates the AMPK pathway, upregulates pyruvate gene expression, and increases enzyme protein synthesis; echinacea extract scavenges reactive oxygen species produced by glycolysis, regulates osmotic pressure to promote enzyme-substrate contact, and inhibits abscisic acid synthesis, thus relieving its inhibition on pyruvate activity. Detailed Implementation

[0021] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0022] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0023] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0024] Example 1

[0025] A compound soaking agent for promoting synchronous emergence of yam tubers comprises the following raw materials in parts by weight: 0.05 parts melatonin nanoliposomes, 0.5 parts sodium lignin sulfonate, 2 parts garlic extract, 5 parts abscisic acid degrading enzyme activator, 3 parts mitochondrial activator, 2 parts pyruvate kinase activator, and 800 parts purified water. The abscisic acid degrading enzyme activator is composed of soybean extract and Bacillus amyloliquefaciens fermentation broth in a volume ratio of 2.5:1.5; the mitochondrial activator is composed of α-lipoic acid and betaine in a mass ratio of 0.2:1.2; and the pyruvate kinase activator is composed of magnesium citrate, berberine, and echinacea extract in a mass ratio of 0.4:0.04:1.5.

[0026] The melatonin nanoliposomes were prepared by the following method: soybean lecithin, cholesterol, melatonin, and vitamin E were mixed in a mass ratio of 15:3:1.0:0.4 to obtain a mixture; the mixture was added to a chloroform-ethanol solution in a mass-volume ratio of 1:8 to obtain a mixture solution; the mixture solution was evaporated at 80 rpm for 60 min at 40 °C to obtain a lipid membrane; then PBS buffer preheated to 40 °C was added to the lipid membrane in a volume ratio of 1:8, and the mixture was stirred in a 40 °C water bath for 35 min to obtain a crude emulsion; then the mixture was transferred to an ultrasonic processing device and processed in pulse mode at an ultrasonic power of 200 W for 15 min, with a 3 s pulse mode operation followed by a 2 s pause, and this cycle was repeated; then the mixture was processed in a high-pressure homogenizer at 700 bar for 5 cycles; after homogenization, the mixture was cooled to 3 °C and then filtered through a 0.22 μm filter membrane to obtain the melatonin nanoliposomes.

[0027] The garlic extract was prepared by the following method: fresh garlic was peeled to obtain garlic cloves, which were then sprayed with cold water at 3°C, and then quick-frozen in liquid nitrogen at -15°C for 6 minutes. After crushing, 0.1% of the garlic mass of the compound enzyme was added and enzymatically hydrolyzed at 34°C for 2 hours. After the enzymatic hydrolysis was completed, the temperature was raised to 85°C to inactivate the enzyme for 5 minutes. Then, the mixture was centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected to obtain the garlic extract. The compound enzyme consisted of alliinase and cellulase in a mass ratio of 1:1.5.

[0028] The soybean extract was prepared by the following method: dried soybeans were pulverized, passed through a 40-mesh sieve, and petroleum ether was added at a ratio of 1:4 (g / mL) to the liquid. The mixture was extracted using a Soxhlet extractor at 55°C for 4.5 hours, and then evaporated at 40°C for 5 hours in a fume hood to obtain defatted soybean powder. 75wt% ethanol was added to the defatted soybean powder at a ratio of 1:8 (g / mL) to the liquid. The mixture was extracted by stirring at 150 rpm for 2.2 hours in a water bath at 55°C. After extraction, the mixture was centrifuged at 4000 rpm for 12 minutes, and the supernatant was collected. The lower precipitate was extracted once more, and the two supernatants were combined. The mixture was then rotary evaporated at 40°C and -0.08 MPa to 20% of its original volume. 0.04% vitamin C (by volume after evaporation) was added, and the mixture was stirred at 3°C ​​for 20 minutes to obtain the soybean extract.

[0029] The Bacillus amyloliquefaciens fermentation broth was prepared by the following method: Bacillus amyloliquefaciens strain was streaked onto an agar slant and incubated at 35℃ for 26 h to form single colonies. These single colonies were then inoculated into 50 mL of seed culture medium and incubated at 35℃ with shaking at 150 rpm for 13 h. Subsequently, a 5% inoculum was added to 500 mL of seed culture medium and incubated at 35℃ with shaking at 150 rpm for 9 h. Then, an 8% inoculum was added to the fermentation medium in a fermenter and incubated at 33℃ with shaking at 300 rpm for 40 h. The fermenter aeration rate was 1.0 vvm. When the residual sugar was <5 g / L, concentrated corn steep liquor was added at a flow rate of 40 mL / h. After fermentation, the mixture was centrifuged at 5000 rpm for 20 min at 3℃, and the supernatant was collected. The supernatant was filtered through a 100 kDa filter membrane to obtain the Bacillus amyloliquefaciens fermentation broth. The slant culture medium includes the following components: 8 g / L peptone, 4 g / L beef extract, 4 g / L sodium chloride, and 15 g / L agar; the seed culture medium includes the following components: 8 g / L peptone, 4 g / L yeast extract, 15 g / L glucose, and 0.4 g / L magnesium sulfate heptahydrate; and the fermentation culture medium includes the following components: 25 g / L starch, 15 g / L soybean meal, 8 g / L corn steep liquor, 0.8 g / L potassium dihydrogen phosphate, and 1.5 g / L calcium carbonate.

[0030] The echinacea extract was prepared by the following method: Echinacea roots were taken, washed, and dried at 40℃ to a moisture content of 8%. The roots were then pulverized to 40 mesh. 75wt% ethanol was added at a material-to-liquid ratio of 1:8 (g / mL), and extracted for 60 min under ultrasonic power of 200W and 38℃. After extraction, the extract was filtered out, and the residue was extracted again. The two extracts were combined, and then rotary evaporated at 40℃ to 20% of the original volume to obtain a crude extract. The crude extract was subjected to molecular distillation at 75℃ and 0.1 Pa, and the light fraction was collected to obtain the echinacea extract.

[0031] Example 2

[0032] A compound soaking agent for promoting synchronous emergence of yam tubers comprises the following raw materials in parts by weight: 0.2 parts melatonin nanoliposomes, 2.0 parts sodium lignin sulfonate, 5 parts garlic extract, 10 parts abscisic acid degrading enzyme activator, 8 parts mitochondrial activator, 6 parts pyruvate kinase activator, and 900 parts purified water. The abscisic acid degrading enzyme activator is composed of soybean extract and Bacillus amyloliquefaciens fermentation broth in a volume ratio of 3.5:2.5; the mitochondrial activator is composed of α-lipoic acid and betaine in a mass ratio of 0.4:1.8; and the pyruvate kinase activator is composed of magnesium citrate, berberine, and echinacea extract in a mass ratio of 0.6:0.06:2.5.

[0033] The melatonin nanoliposomes were prepared by the following method: soybean lecithin, cholesterol, melatonin, and vitamin E were mixed in a mass ratio of 20:4:1.5:0.6 to obtain a mixture; the mixture was added to a chloroform-ethanol solution at a mass-volume ratio of 1:12 to obtain a mixture solution; the mixture solution was evaporated at 120 rpm for 40 min at 45 °C to obtain a lipid membrane; then PBS buffer preheated to 45 °C was added to the lipid membrane at a volume ratio of 1:12, and the mixture was stirred in a 45 °C water bath for 25 min to obtain a crude emulsion; then the mixture was transferred to an ultrasonic processing device and processed in pulse mode at an ultrasonic power of 400 W for 10 min, with a 3 s pulse mode operation followed by a 2 s pause, and this cycle was repeated; then the mixture was processed in a high-pressure homogenizer at 900 bar for 3 times; after homogenization, the mixture was cooled to 5 °C and then filtered through a 0.22 μm filter membrane to obtain the melatonin nanoliposomes.

[0034] The garlic extract was prepared by the following method: fresh garlic was peeled to obtain garlic cloves, which were then sprayed with 5°C cold water and then quick-frozen in liquid nitrogen at -20°C for 4 minutes. After crushing, 0.2% of the garlic mass of the compound enzyme was added and enzymatically hydrolyzed at 36°C for 2 hours. After the enzymatic hydrolysis was completed, the temperature was raised to 90°C to inactivate the enzyme for 3 minutes. Then, the mixture was centrifuged at 6000 rpm for 10 minutes, and the supernatant was collected to obtain the garlic extract. The compound enzyme consisted of alliinase and cellulase in a mass ratio of 1:2.

[0035] The soybean extract was prepared by the following method: dried soybeans were pulverized, passed through a 60-mesh sieve, and petroleum ether was added at a ratio of 1:6 (g / mL) to the liquid. The mixture was extracted using a Soxhlet extractor at 65°C for 3.5 hours, and then evaporated at 45°C for 3 hours in a fume hood to obtain defatted soybean powder. 65 wt% ethanol was added to the defatted soybean powder at a ratio of 1:12 (g / mL) to the liquid. The mixture was extracted by stirring at 250 rpm for 1.8 hours in a water bath at 65°C. After extraction, the mixture was centrifuged at 6000 rpm for 8 minutes, and the supernatant was collected. The lower precipitate was extracted once more, and the two supernatants were combined. The mixture was then rotary evaporated at 45°C and -0.10 MPa to 15% of its original volume. 0.06% of vitamin C (by volume after evaporation) was added, and the mixture was stirred at 5°C for 20 minutes to obtain the soybean extract.

[0036] The Bacillus amyloliquefaciens fermentation broth was prepared by the following method: Bacillus amyloliquefaciens was streaked onto an agar slant and incubated at 39℃ for 22 h to form single colonies. These single colonies were then inoculated into 50 mL of seed culture medium and incubated at 39℃ with shaking at 250 rpm for 11 h. Subsequently, a 5% inoculum was added to 500 mL of seed culture medium and incubated at 39℃ with shaking at 250 rpm for 7 h. Then, an 8% inoculum was added to the fermentation medium in a fermenter and incubated at 33℃ with shaking at 500 rpm for 36 h. The fermenter aeration rate was 1.2 vvm. When the residual sugar was <5 g / L, concentrated corn steep liquor was added at a flow rate of 50 mL / h. After fermentation, the mixture was centrifuged at 7000 rpm for 15 min at 5℃, and the supernatant was collected. The supernatant was filtered through a 100 kDa filter membrane to obtain the Bacillus amyloliquefaciens fermentation broth. The slant culture medium includes the following components: 12 g / L peptone, 6 g / L beef extract, 6 g / L sodium chloride, and 25 g / L agar; the seed culture medium includes the following components: 12 g / L peptone, 6 g / L yeast extract, 25 g / L glucose, and 0.6 g / L magnesium sulfate heptahydrate; and the fermentation culture medium includes the following components: 35 g / L starch, 25 g / L soybean meal, 12 g / L corn steep liquor, 1.0 g / L potassium dihydrogen phosphate, and 2.5 g / L calcium carbonate.

[0037] The echinacea extract was prepared by the following method: Echinacea roots were taken, washed, and dried at 45℃ to a moisture content of 6%. The roots were then pulverized to 60 mesh. 65wt% ethanol was added at a material-to-liquid ratio of 1:12 (g / mL), and extracted for 40 min under ultrasonic power of 400W and 42℃. After extraction, the extract was filtered out, and the residue was extracted again. The two extracts were combined, and then rotary evaporated at 40℃ to 15% of the original volume to obtain a crude extract. The crude extract was then subjected to molecular distillation at 85℃ and 0.2 Pa to collect the light fraction, thus obtaining the echinacea extract.

[0038] Example 3

[0039] A compound soaking agent for promoting synchronous emergence of yam tubers comprises the following raw materials in parts by weight: 0.12 parts melatonin nanoliposomes, 1.2 parts sodium lignin sulfonate, 3.5 parts garlic extract, 7.5 parts abscisic acid degrading enzyme activator, 5.5 parts mitochondrial activator, 4 parts pyruvate kinase activator, and 850 parts purified water. The abscisic acid degrading enzyme activator is composed of soybean extract and Bacillus amyloliquefaciens fermentation broth in a volume ratio of 3.0:2.0; the mitochondrial activator is composed of α-lipoic acid and betaine in a mass ratio of 0.3:1.5; and the pyruvate kinase activator is composed of magnesium citrate, berberine, and echinacea extract in a mass ratio of 0.5:0.05:2.0.

[0040] The melatonin nanoliposomes were prepared by the following method: soybean lecithin, cholesterol, melatonin, and vitamin E were mixed in a mass ratio of 17.5:3.5:1.3:0.5 to obtain a mixture; the mixture was added to a chloroform-ethanol solution at a mass-volume ratio of 1:10 to obtain a mixture solution; the mixture solution was evaporated at 100 rpm for 50 min at 42.5℃ to obtain a lipid membrane; then PBS buffer preheated to 42.5℃ was added to the lipid membrane at a volume ratio of 1:10, and the mixture was stirred in a water bath at 42.5℃ for 30 min to obtain a crude emulsion; then the mixture was transferred to an ultrasonic treatment device and treated in pulse mode at an ultrasonic power of 300W for 12.5 min, with a 3 s pulse mode operation followed by a 2 s pause, and this cycle was repeated; then the mixture was cyclically treated in a high-pressure homogenizer at 800 bar for 4 times; after homogenization, the mixture was cooled to 4℃ and then filtered through a 0.22 μm filter membrane to obtain the melatonin nanoliposomes.

[0041] The garlic extract was prepared by the following method: fresh garlic was peeled to obtain garlic cloves, which were then sprayed with cold water at 4°C, and then quick-frozen in liquid nitrogen at -18°C for 5 minutes. After crushing, 0.2% of the garlic mass of the compound enzyme was added and enzymatically hydrolyzed at 35°C for 2 hours. After the enzymatic hydrolysis was completed, the temperature was raised to 88°C to inactivate the enzyme for 4 minutes. Then, the mixture was centrifuged at 5000 rpm for 12.5 minutes, and the supernatant was collected to obtain the garlic extract. The compound enzyme consisted of alliinase and cellulase in a mass ratio of 1:1.8.

[0042] The soybean extract was prepared by the following method: dried soybeans were pulverized, passed through a 50-mesh sieve, and petroleum ether was added at a ratio of 1:5 (g / mL) to the liquid. The mixture was extracted using a Soxhlet extractor at 60℃ for 4.0 h, and then evaporated at 42.5℃ for 4 h in a fume hood to obtain defatted soybean powder. 70wt% ethanol was added to the defatted soybean powder at a ratio of 1:10 (g / mL) to the liquid. The mixture was extracted by stirring at 200 rpm for 2.0 h in a water bath at 60℃. After extraction, the mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected. The lower precipitate was extracted once more. The two supernatants were combined and then rotary evaporated at 42.5℃ and -0.09 MPa to 18% of the original volume. 0.05% of the evaporated volume of vitamin C was added, and the mixture was stirred at 4℃ for 25 min to obtain the soybean extract.

[0043] The Bacillus amyloliquefaciens fermentation broth was prepared by the following method: Bacillus amyloliquefaciens strain was streaked onto an agar slant and incubated at 37°C for 24 hours to form single colonies. These single colonies were then inoculated into 50 mL of seed culture medium and incubated at 37°C with shaking at 200 rpm for 12 hours. Subsequently, a 5% inoculum was added to 500 mL of seed culture medium and incubated at 37°C with shaking at 200 rpm for 8 hours. Then, an 8% inoculum was added to the fermentation medium in a fermenter and incubated at 34°C with shaking at 400 rpm for 38 hours. The fermenter aeration rate was 1.1 vvm. When the residual sugar was <5 g / L, concentrated corn steep liquor was added at a flow rate of 45 mL / h. After fermentation, the mixture was centrifuged at 6000 rpm for 18 minutes at 4°C, and the supernatant was collected. The supernatant was filtered through a 100 kDa filter membrane to obtain the Bacillus amyloliquefaciens fermentation broth. The slant culture medium includes the following components: 10 g / L peptone, 5 g / L beef extract, 5 g / L sodium chloride, and 20 g / L agar; the seed culture medium includes the following components: 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, and 0.5 g / L magnesium sulfate heptahydrate; and the fermentation culture medium includes the following components: 30 g / L starch, 20 g / L soybean meal, 10 g / L corn steep liquor, 0.9 g / L potassium dihydrogen phosphate, and 2.0 g / L calcium carbonate.

[0044] The echinacea extract was prepared by the following method: Echinacea roots were washed, dried at 42.5℃ to a moisture content of 5%, and then pulverized to 50 mesh. 70wt% ethanol was added at a material-to-liquid ratio of 1:10 (g / mL), and extracted for 50 min under ultrasonic power of 300W and 40℃. After extraction, the extract was filtered out, and the residue was extracted again. The two extracts were combined, and then rotary evaporated at 40℃ to 18% of the original volume to obtain a crude extract. The crude extract was then subjected to molecular distillation at 80℃ and 0.2 Pa to collect the light fraction, thus obtaining the echinacea extract.

[0045] The compound soaking agent for promoting synchronous emergence of yam tubers described in Examples 1-3 is prepared by the following method, specifically including the following steps:

[0046] S1. Add sodium lignosulfonate to purified water and stir at 200 rpm for 10 min to obtain a premixed solution.

[0047] S2. Melatonin nanoliposomes, garlic extract, mitochondrial activator, abscisic acid degradation enzyme activator, and pyruvate kinase activator are added sequentially to the premix and stirred at 300 rpm for 15 min to obtain the mixture.

[0048] S3. Place the mixture into a homogenizer and homogenize it three times at a homogenization pressure of 600 bar to obtain the composite soaking agent.

[0049] Example 4

[0050] Compared with Example 3, the difference in this embodiment is that the compound soaking agent for promoting synchronous emergence of yam tubers is prepared by the following method, specifically including the following steps:

[0051] S1. Add sodium lignosulfonate to purified water and stir at 150 rpm for 12 min to obtain a premixed solution;

[0052] S2. Melatonin nanoliposomes, garlic extract, mitochondrial activator, abscisic acid degradation enzyme activator, and pyruvate kinase activator are added sequentially to the premix and stirred at 250 rpm for 20 min to obtain the mixture.

[0053] S3. Place the mixture into a homogenizer and homogenize it three times at a homogenization pressure of 500 bar to obtain the composite soaking agent.

[0054] Example 5

[0055] Compared with Example 3, the difference in this embodiment is that the compound soaking agent for promoting synchronous emergence of yam tubers is prepared by the following method, specifically including the following steps:

[0056] S1. Add sodium lignosulfonate to purified water and stir at 250 rpm for 8 min to obtain a premixed solution;

[0057] S2. Melatonin nanoliposomes, garlic extract, mitochondrial activator, abscisic acid degradation enzyme activator, and pyruvate kinase activator are added sequentially to the premix and stirred at 350 rpm for 10 min to obtain the mixture.

[0058] S3. Place the mixture into a homogenizer and homogenize it twice at a homogenization pressure of 700 bar to obtain the composite soaking agent.

[0059] Comparative Example 1

[0060] Compared with Example 3, this comparative example does not contain melatonin nanoliposomes in its raw materials. The composite soaking agent for promoting synchronous emergence of yam tubers is prepared by the following method, specifically including the following steps:

[0061] S1. Add sodium lignosulfonate to purified water and stir at 200 rpm for 10 min to obtain a premixed solution.

[0062] S2. Add garlic extract, mitochondrial activator, abscisic acid degradation enzyme activator and pyruvate kinase activator to the premix in sequence, and stir at 300 rpm for 15 min to obtain the mixture.

[0063] S3. Place the mixture into a homogenizer and homogenize it three times at a homogenization pressure of 600 bar to obtain the composite soaking agent.

[0064] Comparative Example 2

[0065] Compared with Example 3, this comparative example differs in that the raw materials do not contain mitochondrial activators. The compound soaking agent for promoting synchronous emergence of yam tubers is prepared by the following method, specifically including the following steps:

[0066] S1. Add sodium lignosulfonate to purified water and stir at 200 rpm for 10 min to obtain a premixed solution.

[0067] S2. Melatonin nanoliposomes, garlic extract, abscisic acid degrading enzyme activator, and pyruvate kinase activator are added sequentially to the premixed solution and stirred at 300 rpm for 15 min to obtain the mixture.

[0068] S3. Place the mixture into a homogenizer and homogenize it three times at a homogenization pressure of 600 bar to obtain the composite soaking agent.

[0069] Comparative Example 3

[0070] Compared with Example 3, this comparative example differs in that the raw materials do not contain abscisic acid degrading enzyme activator. The compound soaking agent for promoting synchronous emergence of yam tubers is prepared by the following method, specifically including the following steps:

[0071] S1. Add sodium lignosulfonate to purified water and stir at 200 rpm for 10 min to obtain a premixed solution.

[0072] S2. Add melatonin nanoliposomes, garlic extract, mitochondrial activator and pyruvate kinase activator to the premix in sequence, and stir at 300 rpm for 15 min to obtain the mixture.

[0073] S3. Place the mixture into a homogenizer and homogenize it three times at a homogenization pressure of 600 bar to obtain the composite soaking agent.

[0074] Comparative Example 4

[0075] Compared with Example 3, this comparative example differs in that the raw materials do not contain pyruvate kinase activator. The compound soaking agent for promoting synchronous emergence of yam tubers is prepared by the following method, specifically including the following steps:

[0076] S1. Add sodium lignosulfonate to purified water and stir at 200 rpm for 10 min to obtain a premixed solution.

[0077] S2. Add melatonin nanoliposomes, garlic extract, mitochondrial activator, and abscisic acid degradation enzyme activator to the premix in sequence, and stir at 300 rpm for 15 min to obtain the mixture.

[0078] S3. Place the mixture into a homogenizer and homogenize it three times at a homogenization pressure of 600 bar to obtain the composite soaking agent.

[0079] field trials

[0080] Take pre-prepared yam tubers as planting materials, cut them into equal sizes according to planting requirements, and randomly divide them into 10 portions, each containing 1000 stem segments. Soak each portion simultaneously in an equal volume of the soaking solution prepared in Examples 1-5 and Comparative Examples 1-4, diluted 10 times. A blank control group is also set up, soaked in an equal volume of purified water. Soaking time is 24 hours for each portion. After soaking, apply wood ash to the cut surfaces. Plant the soaked stem segments according to equal plant spacing, row spacing, fertilizer application, and watering amount, with each group planting 1 acre. Observe daily after sowing, record the emergence date of each plant, and calculate the standard deviation of emergence time for all plants in the group, rounding the result to one decimal place. A smaller standard deviation indicates more concentrated emergence. On the 30th day after planting, measure the height of each seedling, calculate the standard deviation, rounding the result to one decimal place. A smaller standard deviation indicates less variation in seedling height. Then, follow uniform water and fertilizer management until harvest. Harvest uniformly, and count the yam yield of each group, rounding the result to one decimal place. The results are all recorded in Table 1.

[0081] Table 1

[0082]

[0083]

[0084] As can be seen from Table 1, after soaking in the compound soaking agent of the present invention, the difference in the emergence time of yam tubers is small, the difference in seedling height is also small, and the yield per mu of yam is also higher, with Example 3 showing better results.

[0085] Comparing Comparative Example 1 with Example 3, melatonin nanoliposomes can enter tuber cells via endocytosis or lipid fusion, improving the uniformity of melatonin distribution within the tuber. After entering the tuber, melatonin nanoliposomes can regulate the balance between abscisic acid and gibberellin, making the "germination signal threshold" of different tubers more consistent and shortening the differences in germination initiation time. Melatonin promotes coleoptile cell division and elongation by regulating cyclin, auxin, and brassinolide signals, resulting in more synchronized seedling growth. Furthermore, melatonin can activate α-amylase and β-amylase activity, accelerating starch hydrolysis, while upregulating the expression of sucrose synthase and sucrose phosphate synthase genes, promoting sugar transport to the embryo and providing energy for cell division and radicle / embryo elongation. Therefore, soaking in a composite soaking agent containing melatonin nanoliposomes promotes uniform seedling emergence and germination growth.

[0086] Comparing Comparative Example 2 and Example 3, the α-lipoic acid in the mitochondrial activator of this invention, by regenerating antioxidants such as vitamin C, vitamin E, and glutathione, eliminates reactive oxygen species leaking from the mitochondrial electron transport chain, reduces mitochondrial membrane lipid peroxidation and DNA damage, maintains mitochondrial membrane potential stability, and has a significant repair effect on tubers that have aged during storage and whose mitochondrial function has declined. Betaine enters the cytoplasm and mitochondria, lowers the cell water potential, promotes tuber water absorption, softens the epidermis, reduces the mechanical resistance of the radicle / embryo to break through the epidermis, and accelerates germination initiation. In addition, α-lipoic acid eliminates free radicals, and betaine increases the activity of superoxide dismutase and catalase, jointly reducing mitochondrial oxidative damage and avoiding seedling delay caused by individual differences in stress resistance. Betaine regulates the balance of endogenous hormones, and α-lipoic acid maintains the stability of hormone signaling pathways through antioxidant effects, reducing differences in seedling height caused by local metabolic imbalances. Therefore, soaking in the compound soaking agent containing the mitochondrial activator can promote uniform seedling emergence and germination growth of tubers.

[0087] Comparing Comparative Example 3 with Example 3, the Bacillus amyloliquefaciens fermentation broth in the abscisic acid degrading enzyme activator of the present invention, through the secretion of abscisic acid degrading enzymes by Bacillus amyloliquefaciens, can decompose abscisic acid into inactive products. Isoflavones in soybean extract can activate the metabolic activity of Bacillus amyloliquefaciens, further accelerating abscisic acid degradation and relieving its inhibitory effect on tuber germination. After the abscisic acid concentration decreases, the difference in germination delay between different individuals is significantly reduced. Furthermore, the nitrogen, phosphorus, and potassium metabolites in soybean extract provide balanced nutrition to the roots, enabling seedling roots to absorb water and nutrients synchronously, reducing differences in seedling height caused by nutrient competition. Moreover, the Bacillus amyloliquefaciens in the fermentation broth can also competitively suppress soil-borne pathogens, reducing seedling mortality caused by diseases. In addition, the hydrolytic enzymes in the Bacillus amyloliquefaciens fermentation broth can decompose the cork layer and cuticle of the tuber epidermis, reducing the resistance of the radicle / embryo to breaking through the epidermis. The amino acids and organic acids in soybean extract can penetrate and soften the epidermal cell walls, making the softening degree of different tubers more uniform and reducing the differences in germination initiation time. Furthermore, the disease-resistant proteins and antioxidants in the Bacillus amyloliquefaciens fermentation broth can enhance the stress resistance of tubers and seedlings, reducing the interference of diseases, drought, and other stresses on growth. Because the stress resistance of seedlings from different tubers tends to be more uniform, growth fluctuations decrease, and differences in seedling height are further reduced. Therefore, soaking in a compound soaking agent containing abscisic acid degrading enzyme activator can promote uniform seedling emergence and germination growth of tubers.

[0088] By comparing Comparative Example 4 with Example 3, since pyruvate is Mg-dependent... 2+ The metalloenzyme, magnesium citrate in the pyruvate kinase activator of the composite soaking agent of this invention, contains magnesium. 2+ By binding to the enzyme's active site, Mg maintains its three-dimensional conformational stability, promoting substrate binding and catalytic reactions. During tuber storage, Mg... 2+ It may be lost due to metabolic activities, leading to a decrease in pyruvate activity. Magnesium citrate releases Mg... 2+Berberine directly replenishes the cofactors required for pyruvate, restoring its catalytic activity. Berberine regulates energy metabolism by activating the adenosine monophosphate-activated protein kinase pathway. Echinacea extract, rich in polysaccharides and phenolic compounds, prevents pyruvate oxidative inactivation by scavenging reactive oxygen species leaked during glycolysis; the polysaccharides soften the tuber epidermis by absorbing water and swelling, reducing cytoplasmic osmotic pressure, which facilitates the penetration and absorption of the compound soaking agent, resulting in more uniform softening of the epidermis across different tubers and reducing differences in germination initiation time. The phenolic compounds indirectly alleviate the inhibition of pyruvate activity by abscisic acid by inhibiting abscisic acid synthesis or promoting gibberellin synthesis. Simultaneously, the AMPK pathway activated by berberine antagonizes the inhibition of glycolysis by abscisic acid, ensuring that the pyruvate activity of different tubers is no longer affected by differences in abscisic acid regulation, leading to more uniform germination signals. Therefore, the pyruvate kinase activator composed of magnesium citrate, berberine, and echinacea extract, through the Mg2+ of magnesium citrate... 2+ The synergistic effects of berberine's AMPK signal regulation and echinacea extract's antioxidant and metabolic support, along with the enhanced PK activity, synchronized energy supply, and optimized hormone balance, have solved the heterogeneity problem in yam tuber germination and growth, ultimately reducing differences in emergence time and seedling height.

[0089] Comparing Comparative Example 5 with Example 3, it is evident that the compound soaking agent of the present invention has a significant effect on uniform yam tuber emergence. After soaking in the compound soaking agent of the present invention, the differences in emergence time and seedling height are significantly reduced. Due to the small differences between seedlings, the yield per acre of yam is also higher under uniform water and fertilizer management.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound soaking agent for promoting synchronous emergence of yam tubers, characterized in that, The ingredients include the following parts by weight: 0.05-0.2 parts melatonin nanoliposomes, 0.5-2.0 parts sodium lignosulfonate, 2-5 parts garlic extract, 5-10 parts abscisic acid degrading enzyme activator, 3-8 parts mitochondrial activator, 2-6 parts pyruvate kinase activator, and 800-900 parts purified water.

2. The compound soaking agent for promoting synchronous emergence of yam tubers as described in claim 1, characterized in that, The melatonin nanoliposomes were prepared by the following method: soybean lecithin, cholesterol, melatonin, and vitamin E were mixed in a mass ratio of (15-20):(3-4):(1.0-1.5):(0.4-0.6) to obtain a mixture; the mixture was added to a chloroform-ethanol solution in a mass-volume ratio of 1:(8-12) to obtain a liquid mixture; the liquid mixture was rotary evaporated at 80-120 rpm for 40-60 min at 40-45℃ to obtain a lipid membrane; subsequently, the lipid membrane was injected with a liquid mixture in a volume ratio of 1:8- 12. Add PBS buffer preheated to 40-45℃, stir in a water bath at 40-45℃ for 25-35 min to obtain crude emulsion; then transfer to an ultrasonic treatment device, treat in pulse mode at ultrasonic power of 200-400W for 10 min, work in pulse mode for 3s, pause for 2s, and repeat this cycle; then treat in a high pressure homogenizer for 3-5 times at 700-900 bar, cool to 3-5℃ after homogenization, and then filter through a 0.22μm filter membrane to obtain melatonin nanoliposomes.

3. The compound soaking agent for promoting synchronous emergence of yam tubers as described in claim 1, characterized in that, The garlic extract is prepared by the following method: Fresh garlic cloves are peeled and sprayed with cold water at 3-5℃, then quickly frozen in liquid nitrogen at -15 to -20℃ for 4-6 minutes, then crushed, and then 0.1-0.2% of a compound enzyme by weight of garlic is added and enzymatically hydrolyzed at 34-36℃ for 2 hours. After enzymatic hydrolysis, the temperature is raised to 85-90℃ to inactivate the enzyme for 3-5 minutes, and then centrifuged at 4000-6000 rpm for 10-15 minutes. The supernatant is collected to obtain the garlic extract. The compound enzyme consists of alliinase and cellulase in a mass ratio of 1:1.5-2.

4. The compound soaking agent for promoting synchronous emergence of yam tubers as described in claim 1, characterized in that, The abscisic acid degrading enzyme activator is composed of soybean extract and Bacillus amyloliquefaciens fermentation broth in a volume ratio of (2.5-3.5):(1.5-2.5).

5. The compound soaking agent for promoting synchronous emergence of yam tubers as described in claim 1, characterized in that, The mitochondrial activator consists of α-lipoic acid and betaine in a mass ratio of (0.2-0.4):(1.2-1.8).

6. The compound soaking agent for promoting synchronous emergence of yam tubers as described in claim 1, characterized in that, The pyruvate kinase activator is composed of magnesium citrate, berberine, and echinacea extract in a mass ratio of (0.4-0.6):(0.04-0.06):(1.5-2.5).

7. A method for preparing a compound soaking agent for promoting synchronous emergence of yam tubers as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Add sodium lignosulfonate to purified water and stir until homogeneous to obtain a premixed solution; S2. Add melatonin nanoliposomes, garlic extract, mitochondrial activator, abscisic acid degradation enzyme activator, and pyruvate kinase activator to the premix in sequence, stir well to obtain a mixture; S3. Place the mixture into a homogenizer and homogenize it 2-3 times to obtain the composite soaking agent.

8. The method for preparing a compound soaking agent for promoting synchronous emergence of yam tubers as described in claim 7, characterized in that, The stirring speed of S1 is 150-250 rpm and the stirring time is 8-12 min; the stirring speed of S2 is 250-350 rpm and the stirring time is 10-20 min; and the homogenization pressure of S3 is 500-700 bar.

9. A compound soaking agent for promoting synchronous emergence of yam tubers as described in any one of claims 1-6, characterized in that, The compound soaking agent is used to soak yam tubers before planting.

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