Preparation method of brown sugar fermentation product for improving saline-alkaline resistance of crops as well as product and application of brown sugar fermentation product

Abstract

The invention provides a preparation method of a brown sugar fermentation product capable of improving saline-alkaline resistance of crops as well as a product and application of the brown sugar fermentation product. The method comprises the following steps: mixing brown sugar, fermentation enzyme and a buffer solution, inoculating zymophyte to perform two-stage fermentation, performing solid-liquid separation, and collecting liquid to obtain the brown sugar fermentation product, the fermentation enzyme comprises any one or a combination of at least two of cellulase, amylase or protease; the zymophyte comprises any one of bacillus subtilis, bacillus licheniformis or bacillus amyloliquefaciens or a combination of at least two of the bacillus subtilis, the bacillus licheniformis and the bacillus amyloliquefaciens. The brown sugar fermentation product prepared by the method disclosed by the invention can obviously improve the biomass of crops, improve photosynthesis and moisture state, maintain salt ion balance and relieve oxidative stress induced by salt and alkali.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural biological stress resistance and soil improvement, and particularly relates to a preparation method of a brown sugar fermentation product for improving salt and alkali tolerance of crops, the product and application thereof. BACKGROUND

[0002] Land salinization has become a global environmental problem, which seriously restricts agricultural development. According to the statistics of the United Nations Educational, Scientific and Cultural Organization and the Food and Agriculture Organization, the global area of saline-alkali land is about 954 million hectares, of which the salinization and secondary salinization land in China is about 99 million hectares, accounting for about 10.4% of the global saline-alkali land and about 10% of the cultivated land area in China. According to statistics, soil salinization causes an economic loss of about 10 billion yuan per year in China, and the grain yield reduction caused by abiotic stress is more than 20%, which has become an important limiting factor for the sustainable development of agriculture in China. Therefore, reasonable development and utilization of saline-alkali land is the key to improving the growth environment of crops and promoting high yield and high efficiency of characteristic crops. Studies have shown that excessive soil salt will damage the integrity of plant cell membranes, inhibit plant growth, cause ion toxicity and nutrient deficiency, and osmotic stress and ion toxicity caused by salt and alkali stress often lead to oxidative damage in plants.

[0003] CN102643136A discloses a nutrient agent for improving salt and alkali resistance of plants, which is composed of the following raw materials in a certain proportion by weight: water-retaining agent 5-10 parts, indole-3-butyric acid 0.02-0.04 parts, gibberellin 0.01-0.03 parts, polyaspartic acid 0.03-0.05 parts, fully decomposed crop straw 20-30 parts, fully decomposed cattle or pig manure 30-40 parts, yeast 0.06-0.15 parts, quicklime 1-10 parts, ferrous sulfate 10-15 parts, copper sulfate 0.6-3 parts, manganese sulfate 3-6 parts, potassium dihydrogen phosphate 0.8-3 parts. It can reduce the impact of salt damage to a certain extent, but the introduction of more than ten components such as indole-3-butyric acid, gibberellin and quicklime makes the composition too complex, the implementation cost is relatively high, and soil nutrient imbalance is easily caused.

[0004] CN1784494A discloses a method and composition for improving stress tolerance of plants. Arabidopsis MYB8 transcription factor (also known as HOS10) is involved in regulating the stress response of plants to cold, drought, osmotic stress, plant hormone abscisic acid and salinity, and by adding a DNA molecule to the plants, the cold resistance, drought resistance and salt resistance of plant cells are increased. Although some progress has been made in existing breeding techniques, such as improving the salt resistance of crops by exogenous DNA introduction technology, other measures still need to be combined to improve the salt resistance effect.

[0005] Therefore, it is urgent to use pure natural ingredients to alleviate the damage of soil salt and alkali stress to plants and improve the suitability of saline-alkali land for crop planting. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing brown sugar fermentation products that improve the salt and alkali tolerance of crops, as well as the products and applications thereof. The brown sugar fermentation products obtained by the method of this invention can significantly increase the biomass of crops, improve photosynthesis and water status, maintain salt ion balance, and reduce salt and alkali induced oxidative stress.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing a brown sugar fermentation product that enhances the salt and alkali tolerance of crops. The method includes: mixing brown sugar, fermentation enzyme and buffer solution, inoculating fermentation bacteria for two-stage fermentation, separating solid and liquid, collecting liquid, and obtaining the brown sugar fermentation product.

[0009] The fermentation enzyme includes any one or a combination of at least two of cellulase, amylase, or protease.

[0010] The fermentation bacteria include any one or a combination of at least two of Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens.

[0011] The two-stage fermentation includes a first-stage fermentation and a second-stage fermentation.

[0012] The fermentation temperature in the first stage is 25-30℃, for example, 25℃, 25.5℃, 26℃, 26.5℃, 27℃, 27.5℃, 28℃, 28.5℃, 29℃, 29.5℃, or 30℃; the time is 10-15 h, for example, 10 h, 10.5 h, 11 h, 11.5 h, 12 h, 12.5 h, 13 h, 13.5 h, 14 h, 14.5 h, or 15 h; the pH value is 5.5-6.0, for example, 5.5, 5.55, 5.6, 5.65, 5.7, 5.75, 5.8, 5.85, 5.9, 5.95, or 6.0; the viable count of the inoculated fermentation bacteria is (1-3)×10⁻⁶. 7 CFU / mL, the specific point values ​​in (1-3) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0013] The fermentation temperature in the second stage is 30-35℃, for example, 30℃, 30.5℃, 31℃, 31.5℃, 32℃, 32.5℃, 33℃, 33.5℃, 34℃, 34.5℃, or 35℃; the time is 12-60 h, for example, 12 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 50 h, 55 h, or 60 h; the pH value is 6.0-6.5, for example, 6.0, 6.05, 6.1, 6.15, 6.2, 6.25, 6.3, 6.35, 6.4, 6.45, or 6.5; the viable count of the inoculated fermentation bacteria is (3-5)×10⁻⁶. 7 For example, the specific point values ​​in (3-5) can be 3, 3.1, 3.2, 3.5, 3.7, 4, 4.3, 4.5, 4.8 or 5, etc.

[0014] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0015] The brown sugar fermentation product obtained by this invention through a specific two-stage fermentation process is rich in flavonoids, polysaccharides, organic acids, and various secondary metabolites. It has a significant growth-promoting effect, which can enhance the biomass accumulation of plants, promote coordinated growth of the aboveground parts and roots, and simultaneously enhance the water and salt uptake regulation function of the roots. It increases the bound water content of plants, enhances the cell osmotic regulation capacity, and prevents dehydration or plasmolysis under salt stress. It effectively promotes potassium ion absorption and inhibits sodium ion accumulation, regulates ion transport and distribution, and improves salt ion homeostasis. It reduces salt- and alkali-induced oxidative stress, enhances the plant's energy absorption and heat dissipation, and improves the photosynthetic stability of plants under salt stress.

[0016] This invention achieves the aforementioned salt and alkali resistance properties by controlling the range of four parameters: temperature, time, pH value, and viable cell count of fermentation bacteria, so that the first-stage fermentation and the second-stage fermentation can be synergistically combined to reach the optimal fermentation process.

[0017] Preferably, the initial concentration of brown sugar in the fermentation system is 5-10 g / L, for example, it can be 5 g / L, 5.5 g / L, 6 g / L, 6.5 g / L, 7 g / L, 7.5 g / L, 8 g / L, 8.5 g / L, 9 g / L, 9.5 g / L or 10 g / L, etc.

[0018] Preferably, the initial concentration of the fermentation enzyme in the fermentation system is 0.5-1.2 g / L, for example, it can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L or 1.2 g / L, etc.

[0019] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0020] Preferably, the fermentation enzymes include cellulase, amylase, and protease.

[0021] Cellulase can degrade cellulose components in fermentation substrates, amylase can promote the hydrolysis of polysaccharides, and protease can improve nitrogen source utilization. A combination of cellulase, amylase, and protease is preferred for brown sugar fermentation. These three enzymes have a synergistic effect, better assisting fermentation, improving the fermentation process, increasing the content of soluble metabolites in brown sugar fermentation products, and further enhancing the salt and alkali tolerance of crops.

[0022] Preferably, the mass ratio of cellulase, amylase and protease is (1-3):(2-5):(1-3).

[0023] The specific point values ​​in the first (1-3) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0024] The specific point values ​​in (2-5) can be 2, 2.1, 2.2, 2.5, 2.7, 3, 3.3, 3.5, 3.8, 4, 4.5 or 5, etc.

[0025] The specific point values ​​in the second (1-3) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0026] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0027] Preferably, the fermentation bacteria include Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens.

[0028] A combination of Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens was selected for two-stage fermentation. The three bacteria have a synergistic effect, and the two-stage fermentation is rationally regulated under their action. The effective components and their content of the fermentation products in the first stage are within a reasonable range, which is fully adapted to the second stage fermentation, improves the fermentation process, significantly increases the content of soluble metabolites in the final product, and further enhances the salt and alkali tolerance of crops.

[0029] Preferably, the ratio of viable bacteria of Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens is (1-5):(1-5):(1-5).

[0030] The specific point values ​​in the first (1-5) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8, 3, 3.5, 4, 4.5 or 5, etc.

[0031] The specific point values ​​in the second (1-5) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8, 3, 3.5, 4, 4.5 or 5, etc.

[0032] The specific point values ​​in the third (1-5) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8, 3, 3.5, 4, 4.5 or 5, etc.

[0033] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0034] Preferably, the buffer solution includes any one or a combination of at least two of PBS buffer, HEPES buffer, or MOPS buffer.

[0035] Preferably, the collected liquid further includes a concentration and drying step.

[0036] In a second aspect, the present invention provides a brown sugar fermentation product prepared by the method described in the first aspect.

[0037] Thirdly, the present invention provides the application of the brown sugar fermentation product as described in the second aspect in improving the salt and alkali tolerance of crops.

[0038] Fourthly, the present invention provides an organic preparation for improving the salt and alkali tolerance of crops, the organic preparation comprising the brown sugar fermentation product and stabilizing agent described in the second aspect;

[0039] The stabilizing agent includes any one or a combination of at least two of xanthan gum, sodium carboxymethyl cellulose, sodium alginate, or gum arabic.

[0040] The aforementioned stabilizing agents can enhance the adhesion, slow-release ability, and stability of brown sugar fermentation products in the rhizosphere environment, and maintain the activity of the active ingredients.

[0041] Preferably, the stabilizing agent includes xanthan gum, sodium carboxymethyl cellulose, sodium alginate, and gum arabic.

[0042] The combination of the above four stabilizing agents has better suspension stability and activity retention.

[0043] Preferably, the mass ratio of xanthan gum, sodium carboxymethyl cellulose, sodium alginate and gum arabic is (1-3):(1-3):(1-2):(1-2).

[0044] The specific point values ​​in the first (1-3) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0045] The specific point values ​​in the second (1-3) can be 1, 1.1, 1.2, 1.5, 1.7, 2, 2.3, 2.5, 2.8 or 3, etc.

[0046] The specific point value in the first (1-2) can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2, etc.

[0047] The specific point value in the second (1-2) can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2, etc.

[0048] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0049] Preferably, the mass ratio of the brown sugar fermentation product to the stabilizing agent is (10-30):(0.1-0.5).

[0050] The specific point values ​​in (10-30) can be 10, 11, 12, 15, 17, 20, 23, 25, 28 or 30, etc.

[0051] The specific point values ​​in (0.1-0.5) can be 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5, etc.

[0052] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0053] Preferably, the amount of brown sugar fermentation product applied is 0.5-4.5 g / L, for example, it can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.2 g / L, 1.5 g / L, 1.7 g / L, 2 g / L, 2.3 g / L, 2.5 g / L, 2.8 g / L, 3 g / L, 3.5 g / L, 4 g / L or 4.5 g / L, etc.

[0054] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0055] Preferably, the crop includes any one of corn, soybeans, rice, wheat, or sugarcane.

[0056] Preferably, the crop is corn.

[0057] The organic preparation was first applied to the rhizosphere one week after corn emergence, with 25-35 mL applied per plant; thereafter, it was applied every 2-4 days for 2-4 consecutive applications. During the application process, the soil moisture was maintained at 70-80% of field capacity to ensure a stable rhizosphere environment.

[0058] Preferably, the organic formulation further includes agriculturally acceptable functional adjuvants.

[0059] Preferably, the agriculturally acceptable functional adjuvant includes any one or a combination of at least two of solvents, emulsifiers, dispersants, wetting agents, binders, defoamers, diluents, thickeners, or antifreeze agents.

[0060] Preferably, the dosage form of the organic preparation includes any one of powder, suspension, microemulsion, ointment, plaster or smear.

[0061] Preferably, the organic agent is applied by any one or a combination of at least two of the following methods: foliar spraying, soil mixing, root irrigation, seed soaking, trench application, or hole application.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The brown sugar fermentation product obtained by this invention through a specific two-stage fermentation process is rich in flavonoids, polysaccharides, organic acids, and various secondary metabolites. It has a significant growth-promoting effect, which can enhance the biomass accumulation of plants, promote coordinated growth of the aboveground parts and roots, and simultaneously enhance the water and salt uptake regulation function of the roots. It increases the bound water content of plants, enhances the cell osmotic regulation capacity, and prevents dehydration or plasmolysis under salt stress. It effectively promotes potassium ion absorption and inhibits sodium ion accumulation, regulates ion transport and distribution, and improves salt ion homeostasis. It reduces salt- and alkali-induced oxidative stress, enhances the plant's energy absorption and heat dissipation, and improves the photosynthetic stability of plants under salt stress. Detailed Implementation

[0064] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0065] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0066] The sources of materials used in the following specific embodiments are as follows:

[0067] Cellulase was obtained from Novozymes (China) Biotechnology Co., Ltd.; amylase from Shanghai Yuanye Biotechnology Co., Ltd.; protease from Beijing Solarbio Technology Co., Ltd.; Bacillus subtilis from China Center for Type Culture Collection (CCTCC), accession number CCTCC M 2018456; Bacillus licheniformis from China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC 1.6852; and Bacillus amyloliquefaciens from China Industrial Microbiological Culture Collection Center (CICC), accession number CICC 10250.

[0068] Example 1

[0069] This embodiment provides a method for preparing brown sugar fermentation products, the method comprising:

[0070] Mix 7.5 g brown sugar, 0.2 g cellulase, 0.4 g amylase, 0.2 g protease, and 1 L PBS buffer, and stir until completely dissolved. Inoculate with Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, with initial viable counts of 0.6 × 10⁻⁶ cells / mL for each species in the first stage of fermentation. 7 CFU / mL, 0.5×10 7 CFU / mL and 0.9×10 7 The concentration was maintained at CFU / mL, pH 5.7, and cultured at 27°C for 13 h. Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens were then inoculated again, with initial viable counts of 2 × 10⁻⁶ CFU / mL for each species in the second stage fermentation system. 7 CFU / mL, 1×10 7 CFU / mL and 1×10 7 The mixture was incubated at 32°C for 36 h at a concentration of CFU / mL and a pH of 6.3. The solution was then filtered, collected, concentrated, and dried to obtain the brown sugar fermentation product.

[0071] Example 2

[0072] This embodiment provides a method for preparing brown sugar fermentation products, the method comprising:

[0073] Mix 10 g brown sugar, 0.3 g cellulase, 0.5 g amylase, 0.3 g protease, and 1 L PBS buffer, and stir until completely dissolved. Inoculate with Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, with an initial viable count of 1 × 10⁻⁶ cells / mL for each species in the first stage of fermentation. 7 CFU / mL, 1×10 7 CFU / mL and 1×10 7The concentration was maintained at CFU / mL, pH 5.5, and cultured at 25°C for 15 h. Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens were then inoculated again, with initial viable counts of 1×10⁻⁶ for each species in the second stage fermentation system. 7 CFU / mL, 3×10 7 CFU / mL and 1×10 7 The mixture was incubated at CFU / mL, with the pH maintained at 6.0, at 30°C for 60 h. Afterwards, the mixture was filtered, the liquid collected, concentrated, and dried to obtain the brown sugar fermentation product.

[0074] Example 3

[0075] This embodiment provides a method for preparing brown sugar fermentation products, the method comprising:

[0076] Mix 5 g brown sugar, 0.1 g cellulase, 0.2 g amylase, 0.2 g protease, and 1 L PBS buffer, and stir until completely dissolved. Inoculate with Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, with an initial viable count of 0.4 × 10⁻⁶ cells / mL for each species in the first stage of fermentation. 7 CFU / mL, 0.3×10 7 CFU / mL and 0.3×10 7 The concentration was maintained at CFU / mL, pH 6.0, and cultured at 30°C for 10 h. Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens were then inoculated again, with initial viable counts of 1 × 10⁻⁶ CFU / mL for each species in the second stage fermentation system. 7 CFU / mL, 1×10 7 CFU / mL and 1×10 7 The mixture was incubated at 35°C for 12 h at a concentration of CFU / mL and a pH of 6.5. The liquid was then filtered, collected, concentrated, and dried to obtain the brown sugar fermentation product.

[0077] Example 4

[0078] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that: Bacillus subtilis was not inoculated in the first stage of fermentation, and the reduced number of viable bacteria was proportionally allocated to Bacillus licheniformis and Bacillus amyloliquefaciens; Bacillus subtilis was not inoculated in the second stage of fermentation, and the reduced number of viable bacteria was proportionally allocated to Bacillus licheniformis and Bacillus amyloliquefaciens. All other steps and raw materials remain unchanged.

[0079] Example 5

[0080] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that: in the first stage of fermentation, Bacillus licheniformis was not inoculated, and the reduced number of viable bacteria was proportionally allocated to Bacillus subtilis and Bacillus amyloliquefaciens; in the second stage of fermentation, Bacillus licheniformis was not inoculated, and the reduced number of viable bacteria was proportionally allocated to Bacillus subtilis and Bacillus amyloliquefaciens. All other steps and raw materials remain unchanged.

[0081] Example 6

[0082] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that: in the first stage of fermentation, Bacillus amyloliquefaciens was not inoculated, and the reduced number of viable bacteria was proportionally allocated to Bacillus subtilis and Bacillus licheniformis; in the second stage of fermentation, Bacillus amyloliquefaciens was not inoculated, and the reduced number of viable bacteria was proportionally allocated to Bacillus subtilis and Bacillus licheniformis. All other steps and raw materials remain unchanged.

[0083] Example 7

[0084] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that cellulase is not added, and its mass is proportionally allocated to amylase and protease. All other steps and raw materials remain unchanged.

[0085] Example 8

[0086] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that no amylase is added. Instead, the reduced mass of amylase is proportionally allocated to cellulase and protease. All other steps and raw materials remain unchanged.

[0087] Example 9

[0088] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that no protease is added. Instead, the reduced mass is proportionally allocated to cellulase and amylase. All other steps and raw materials remain unchanged.

[0089] Comparative Example 1

[0090] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that "cultivating at 27°C for 13 h" in the first stage of fermentation is replaced with "cultivating at 22°C for 18 h", and "cultivating at 32°C for 36 h" in the second stage of fermentation is replaced with "cultivating at 37°C for 10 h". All other steps and raw materials remain unchanged.

[0091] Comparative Example 2

[0092] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that the "cultivation at 27°C for 13 h" in the first stage of fermentation is replaced with "cultivation at 33°C for 8 h", and the "cultivation at 32°C for 36 h" in the second stage of fermentation is replaced with "cultivation at 28°C for 62 h". All other steps and raw materials remain unchanged.

[0093] Comparative Example 3

[0094] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that "maintaining pH value of 5.7" in the first stage of fermentation is replaced with "maintaining pH value of 5.2", and "maintaining pH value of 6.3" in the second stage of fermentation is replaced with "maintaining pH value of 6.7". All other steps and raw materials remain unchanged.

[0095] Comparative Example 4

[0096] This embodiment provides a method for preparing brown sugar fermentation products. The only difference between this method and Example 1 is that "maintaining pH value of 5.7" in the first stage of fermentation is replaced with "maintaining pH value of 6.2", and "maintaining pH value of 6.3" in the second stage of fermentation is replaced with "maintaining pH value of 5.8". All other steps and raw materials remain unchanged.

[0097] Comparative Example 5

[0098] This embodiment provides a method for preparing brown sugar fermentation products, which differs from Example 1 only in that: in the first stage of fermentation, Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens are inoculated, with the initial viable counts of these bacteria in the first stage fermentation system being 0.6 × 10⁻⁶. 7 CFU / mL, 0.5×10 7 CFU / mL and 0.9×10 7 Replace "CFU / mL" with "Inoculated with Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, with initial viable counts of 0.2 × 10⁻⁶ CFU / mL in the first stage fermentation system." 7 CFU / mL, 0.1×10 7 CFU / mL and 0.2×10 7 "CFU / mL"; During the second stage of fermentation, "Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens were inoculated again, with initial viable counts of 2 × 10⁻⁶ CFU / mL for each." 7 CFU / mL, 1×10 7 CFU / mL and 1×10 7 Replace "CFU / mL" with "Re-inoculated with Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, with initial viable counts of 1.5 × 10⁻⁶ CFU / mL in the second stage fermentation system."7 CFU / mL, 1.5×10 7 CFU / mL and 2.5×10 7 "CFU / mL", all other steps and raw materials remain unchanged.

[0099] Comparative Example 6

[0100] This embodiment provides a method for preparing brown sugar fermentation products, which differs from Example 1 only in that: in the first stage of fermentation, Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens are inoculated, with the initial viable counts of these bacteria in the first stage fermentation system being 0.6 × 10⁻⁶. 7 CFU / mL, 0.5×10 7 CFU / mL and 0.9×10 7 Replace "CFU / mL" with "Inoculated with Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, with an initial viable count of 1×10⁻⁶ CFU / mL in the first stage fermentation system." 7 CFU / mL, 1×10 7 CFU / mL and 1.5×10 7 "CFU / mL"; During the second stage of fermentation, "Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens were inoculated again, with initial viable counts of 2 × 10⁻⁶ CFU / mL for each." 7 CFU / mL, 1×10 7 CFU / mL and 1×10 7 Replace "CFU / mL" with "Re-inoculated with Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, with initial viable counts of 0.9 × 10⁻⁶ CFU / mL in the second stage fermentation system." 7 CFU / mL, 0.6×10 7 CFU / mL and 1×10 7 "CFU / mL", all other steps and raw materials remain unchanged.

[0101] Comparative Example 7

[0102] This comparative example provides a method for preparing a brown sugar fermentation product, the method comprising:

[0103] Mix 7.5 g brown sugar, 0.2 g cellulase, 0.4 g amylase, 0.2 g protease, and 1 L PBS buffer, and stir until completely dissolved. Inoculate with Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens, with an initial viable count of 2 × 10⁻⁶ cells / mL for each species in the first stage of fermentation. 7 CFU / mL, 1×10 7 CFU / mL and 1×10 7The concentration was maintained at CFU / mL, pH 6.3, and cultured at 32℃ for 36 h. Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens were then inoculated again, with initial viable counts of 0.6 × 10⁻⁶ for each species in the second stage fermentation system. 7 CFU / mL, 0.5×10 7 CFU / mL and 0.9×10 7 The mixture was incubated at 27°C for 13 h at a concentration of CFU / mL and a pH of 5.7. The liquid was then filtered, collected, concentrated, and dried to obtain the brown sugar fermentation product.

[0104] Comparative Example 8

[0105] This comparative example provides a method for preparing a brown sugar fermentation product, the method comprising:

[0106] Mix 7.5 g brown sugar, 0.2 g cellulase, 0.4 g amylase, 0.2 g protease, and 1 L PBS buffer, and stir until completely dissolved. Inoculate with *Bacillus subtilis*, *Bacillus licheniformis*, and *Bacillus amyloliquefaciens*, with initial viable counts of 2.6 × 10⁻⁶ cells / mL, respectively. 7 CFU / mL, 1.5×10 7 CFU / mL and 1.9×10 7 The mixture was incubated at 29.5°C for 49 h at a concentration of CFU / mL and a pH of 6. The liquid was then filtered, collected, concentrated, and dried to obtain the brown sugar fermentation product.

[0107] Test case

[0108] Dissolve 15 g of the brown sugar fermentation product provided in Examples 1-9 or Comparative Examples 1-8 in 10 L of PBS buffer, then add 0.1 g xanthan gum, 0.1 g sodium carboxymethyl cellulose, 0.1 g sodium alginate and 0.1 g gum arabic and mix well to prepare an organic preparation.

[0109] The test was conducted in a greenhouse at the Beijing Academy of Agricultural and Forestry Sciences, using a potted saline-alkali soil method. A 300 g sample of saline-alkali soil (sampled from Huanghua, Hebei Province, with a pH of 7.8 and an EC value of 1.5 m) was taken. SAfter passing through a 2 mm sieve, the seeds were placed in flowerpots. Two fresh-eating corn seeds of the variety "Nongke Nuo 336" were sown in each pot, covered with 1 cm of soil, thoroughly watered, and then covered with a thin film to promote germination. After emergence, the film was removed and the plants were shaded for two days to allow them to recover. One week after emergence, organic fertilizer was applied to the root zone, 30 mL per plant, applied every 3 days for a total of 3 applications. The application time was fixed at 9:00-10:00 AM, maintaining soil moisture content at 70-80% of field capacity and ensuring stable salinity and water conditions. An equal amount of water was used as a blank control group. Relevant indicators were measured on the 3rd day after the last application. All indicators were measured three times independently and repeatedly, and the Duncan multiple comparison method (p<0.05) was used to analyze significant differences.

[0110] As shown in Table 1, Examples 1-3 demonstrate that after treatment with an organic agent containing brown sugar fermentation products, the aboveground and root dry weight of maize seedlings under salt stress significantly increased, while the root-to-shoot ratio showed no significant difference. This indicates that the aboveground and root systems grew in a coordinated manner, and the water and salt absorption regulation functions of the roots were simultaneously enhanced. The brown sugar fermentation products provided by this invention can effectively alleviate growth inhibition under salt stress and improve biomass accumulation. Examples 4-6 show that Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens have a synergistic effect, with the three bacteria synergistically regulating the two-stage fermentation process and affecting the effect of brown sugar fermentation products on improving plant biomass accumulation. Examples 7-9 show that cellulase, amylase, and protease have a synergistic effect, with the three enzymes synergistically assisting the two-stage fermentation process and affecting the effect of brown sugar fermentation products on improving plant biomass accumulation. Comparative Examples 1-6 show that the temperature, time, pH value, and viable cell count range of the two-stage fermentation all affect the effect of brown sugar fermentation products on improving plant biomass accumulation. As can be seen from Comparative Examples 7-8, the first and second stages of fermentation are complementary. If the order of the two stages is interchanged or they are combined into a single stage of fermentation, the effect of brown sugar fermentation products on plant biomass accumulation will be affected.

[0111] Table 1

[0112]

[0113] As shown in Table 2, Examples 1-3 demonstrate that treatment of corn seedlings under salt stress with an organic agent containing brown sugar fermentation products resulted in a decrease in free water content and an increase in bound water content in the leaves. The increase in bound water helps maintain enzyme activity and cellular metabolic stability, preventing dehydration or plasmolysis under salt stress. Potassium ions play a crucial role in maintaining osmotic balance, activating enzyme activity, and stabilizing membrane structure. The brown sugar fermentation products provided by this invention effectively enhance the physiological adaptability of plants under salt stress by regulating ion transport and distribution, improving salt ion homeostasis, and enhancing cellular osmotic regulation. Examples 4-6 show that Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens have a synergistic effect, with the three bacteria synergistically regulating the two-stage fermentation process and influencing the regulation of plant water status and ion homeostasis by the brown sugar fermentation products. Examples 7-9 demonstrate that cellulase, amylase, and protease have a synergistic effect, with the three enzymes synergistically assisting the two-stage fermentation process and influencing the regulation of plant water status and ion homeostasis by the brown sugar fermentation products. Comparative Examples 1-6 show that the temperature, time, pH value, and viable cell count of the fermentation bacteria in both stages of fermentation all affect the regulation of plant water status and ion homeostasis by the brown sugar fermentation products. Comparative Examples 7-8 show that the first and second stages of fermentation are complementary; interchange or merging them into a single stage of fermentation will affect the regulation of plant water status and ion homeostasis by the brown sugar fermentation products.

[0114] Table 2

[0115]

[0116] As shown in Table 3, Examples 1-3 demonstrate that treatment with organic agents containing brown sugar fermentation products significantly increased the hydroxyl radical scavenging rate, SOD enzyme activity, and CAT enzyme activity in maize seedlings under salt stress. The brown sugar fermentation products provided by this invention effectively enhance the antioxidant defense system of plants under salt stress and improve reactive oxygen species scavenging capacity. Examples 4-6 show that Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens have a synergistic effect, with the three bacteria synergistically regulating the two-stage fermentation process and influencing the regulation of the plant's antioxidant defense system by the brown sugar fermentation products. Examples 7-9 show that cellulase, amylase, and protease have a synergistic effect, with the three enzymes synergistically assisting the two-stage fermentation process and influencing the regulation of the plant's antioxidant defense system by the brown sugar fermentation products. Comparative Examples 1-6 show that the temperature, time, pH value, and viable cell count range of the two-stage fermentation all affect the regulation of the plant's antioxidant defense system by the brown sugar fermentation products. As can be seen from Comparative Examples 7-8, the first and second stages of fermentation are complementary. If the order of the two stages is interchanged or they are combined into a single stage of fermentation, it will affect the regulation of the plant's antioxidant defense system by the brown sugar fermentation products.

[0117] Table 3

[0118]

[0119] As shown in Table 4, as illustrated in Examples 1-3, treatment of maize seedlings under salt stress with an organic agent containing brown sugar fermentation products significantly reduced the H2O2 and MDA content in the leaves, indicating that membrane lipid peroxidation was inhibited; the SPAD value significantly increased, indicating an increase in chlorophyll content and enhanced photosynthetic activity. The brown sugar fermentation product provided by this invention can effectively alleviate salt-induced oxidative stress and improve the stability of the photosynthetic system, demonstrating a good salt tolerance protection effect from a physiological perspective. As shown in Table 5, treatment of maize seedlings under salt stress with an organic agent containing brown sugar fermentation products affected the fluorescence parameters and photochemical efficiency of the leaves, with a significant increase in Fv / Fo and Fv / Fm, indicating improved PSII electron transport efficiency and reduced damage to light energy capture and conversion under salt stress. The brown sugar fermentation product provided by this invention can effectively protect the integrity of the photosystem structure, maintain the stability of the electron transport chain, and thus maintain photosynthetic efficiency and energy utilization levels. As shown in Table 6, treatment of maize seedlings under salt stress with an organic agent containing brown sugar fermentation products affected parameters related to light energy absorption, transmission, and utilization in the leaves. Both ABS / RC and DIo / RC significantly increased, indicating enhanced light energy absorption and dissipation capacity per unit reaction center. TRo / RC increased, while ETo / RC decreased, suggesting that some absorbed energy was used for defense reactions rather than electron transport. The brown sugar fermentation products provided by this invention can effectively optimize and regulate light energy distribution, enhance energy absorption and heat dissipation, reduce the risk of photoinhibition, and maintain energy balance, thereby ensuring the stable and sustainable operation of the photosystem under salt stress.

[0120] Examples 4-6 show that *Bacillus subtilis*, *Bacillus licheniformis*, and *Bacillus amyloliquefaciens* have a synergistic effect, co-regulating the two-stage fermentation process and influencing the regulation of oxidative stress and photosynthesis in plants by the brown sugar fermentation products. Examples 7-9 show that cellulase, amylase, and protease have a synergistic effect, co-assisting the two-stage fermentation process and influencing the regulation of oxidative stress and photosynthesis in plants by the brown sugar fermentation products. Comparative Examples 1-6 show that the temperature, time, pH, and viable cell count ranges of the two-stage fermentation all affect the regulation of oxidative stress and photosynthesis in plants by the brown sugar fermentation products. Comparative Examples 7-8 show that the first and second stage fermentation processes are complementary; interchange or merging them into a single stage of fermentation will affect the regulation of oxidative stress and photosynthesis in plants by the brown sugar fermentation products.

[0121] Table 4

[0122]

[0123] Table 5

[0124]

[0125] Table 6

[0126]

[0127] This invention illustrates, through the above embodiments, a method for preparing a brown sugar fermentation product that enhances the salt and alkali tolerance of crops, as well as the product and its application. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the product, addition of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0128] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0129] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing a brown sugar fermentation product that enhances the salt and alkali tolerance of crops, characterized in that, The method includes: mixing brown sugar, fermentation enzyme and buffer solution, inoculating fermentation bacteria to carry out two-stage fermentation, separating solid and liquid, collecting liquid, and obtaining the brown sugar fermentation product. The fermentation enzyme includes any one or a combination of at least two of cellulase, amylase, or protease. The fermentation bacteria include any one or a combination of at least two of Bacillus subtilis, Bacillus licheniformis, or Bacillus amyloliquefaciens. The two-stage fermentation includes a first-stage fermentation and a second-stage fermentation; the first-stage fermentation is carried out at a temperature of 25-30℃ for 10-15 hours, with a pH of 5.5-6.0, and the viable count of the inoculated fermentation bacteria is (1-3)×10⁻⁶. 7 CFU / mL; the second stage fermentation temperature is 30-35℃, the time is 12-60 h, the pH value is 6.0-6.5, and the viable count of the inoculated fermentation bacteria is (3-5)×10⁻⁶. 7 CFU / mL.

2. The method according to claim 1, characterized in that, The initial concentration of brown sugar in the fermentation system is 5-10 g / L.

3. The method according to claim 1, characterized in that, The initial concentration of the fermentation enzyme in the fermentation system is 0.5-1.2 g / L.

4. The method according to claim 1, characterized in that, The fermentation enzymes include cellulase, amylase, and protease; The mass ratio of cellulase, amylase and protease is (1-3):(2-5):(1-3).

5. The method according to claim 1, characterized in that, The fermentation bacteria include Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens; The ratio of viable Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens is (1-5):(1-5):(1-5).

6. The method according to claim 1, characterized in that, The buffer solution includes any one or a combination of at least two of PBS buffer, HEPES buffer, or MOPS buffer. The process of collecting the liquid also includes a concentration and drying step.

7. The brown sugar fermentation product prepared by the method according to any one of claims 1-6.

8. The application of the brown sugar fermentation product according to claim 7 in improving the salt and alkali tolerance of crops.

9. An organic preparation for enhancing the salt and alkali tolerance of crops, characterized in that, The organic preparation includes the brown sugar fermentation product and stabilizing agent as described in claim 7; The stabilizing agent includes any one or a combination of at least two of xanthan gum, sodium carboxymethyl cellulose, sodium alginate, or gum arabic.

10. The organic preparation according to claim 9, characterized in that, The mass ratio of the brown sugar fermentation product to the stabilizing agent is (10-30):(0.1-0.5); The application rate of the brown sugar fermentation product is 0.5-4.5 g / L; The crops include any one of corn, soybeans, rice, wheat, or sugarcane.

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