Carbon source fertilizer fermentation method for increasing content of polyphenol, flavone, small-molecule active peptide and organic carbon and application of carbon source fertilizer fermentation method for increasing content of polyphenol, flavone, small-molecule active peptide and organic carbon

Abstract

The invention discloses a carbon source fertilizer fermentation method for increasing the content of polyphenol, flavone, small molecule active peptide and organic carbon and application. According to the invention, soybeans and brown sugar are taken as main raw materials, a set of multi-strain, multi-stage and multi-layer fermentation method is developed, a specific fermentation flora is adopted, and the prepared carbon source fertilizer has the organic matter content of 180-210 g / L, the small molecule active peptide content of 95-115 g / L and the soluble small molecule organic carbon content of 102-136 g / L; moreover, the nutrient solution is rich in physiological activators such as flavone (about 530-650 mg / L), polyphenol (about 1200-1400 mg / L), polyglutamic acid (about 1200-2000 mg / L) and the like, and can comprehensively provide nutrients, better promote the growth of crops and improve the yield and quality. Meanwhile, the carbon source fertilizer prepared by the method can reduce the use of chemical fertilizers, has a remarkable effect, and provides more efficient liquid fertilizers rich in various active substances for agricultural production.

Description

Technical Field

[0001] This invention relates to the field of microbial and fermentation technology, and more specifically, to a method and application of carbon source fertilizer fermentation that increases the content of polyphenols, flavonoids, small molecule active peptides and organic carbon. Background Technology

[0002] Carbon is the most essential of the six macroelements for plants, a vital element for life. It accounts for 35% of plant dry matter and is a crucial yet often overlooked essential nutrient. If the soil cannot effectively supply crops with available carbon, crops will suffer from a chronic "carbon hunger."

[0003] Bio-Organic Carbon Fertilizer (BOCF) refers to a highly active organic carbon source fertilizer made from organic waste or industrial byproducts (such as straw, fruit pomace, molasses, etc.) through microbial fermentation or enzymatic hydrolysis. It has a high organic carbon content, providing a stable carbon source supply, and is rich in small-molecule active peptides, various organic acids, and other plant growth factors with biostimulatory effects. These natural small-molecule peptides are easily absorbed by plants and can be rapidly transported and transported through plant roots and leaves. When plants encounter stress conditions, they can provide amino acids and active substances, regulating plant immunity and achieving preventative and restorative functions. It also contains probiotics (such as Bacillus and Lactobacillus), promoting soil microecological balance and reducing the use of chemical fertilizers.

[0004] Existing fermentation methods for bio-organic carbon fertilizers typically employ aerobic-anaerobic coupled fermentation to improve humification efficiency; or use compound microbial agents (such as EM bacteria and cellulose-degrading bacteria) to enhance carbon conversion rates. The polyphenol and flavonoid content in bio-organic carbon fertilizers varies considerably depending on the source of raw materials (such as humic acid, seaweed fertilizer, and plant-derived organic fertilizer), fermentation process, and processing method. While existing fermentation methods can increase organic carbon content and conversion rates, they still suffer from low levels of active substances such as polyphenols, flavonoids, and small-molecule bioactive peptides. Organic matter and carbon sources alone are insufficient to provide comprehensive nutrition. Furthermore, polyphenols, flavonoids, and small-molecule bioactive peptides are crucial for plant development, improving crop yield and quality, promoting growth, enhancing crop resistance to stress, pests, and diseases, and improving the crop's ability to cope with extreme environments. For example, a method for preparing high-concentration organic carbonic acid fermentation broth, disclosed in existing research, uses water, sugar, molasses, and soybeans as raw materials, and involves three fermentations using different mixed strains. This method can increase the organic matter content to about 21%, but it also suffers from the problem of low content of active substances (such as flavonoids, polyphenols, small molecule carbon, and small molecule active peptides) in the fermentation products. Similarly, the content of small molecule peptides in existing BOCF product aqueous solutions is low, generally 0.5-2%. Therefore, there is still a lack of carbon source fertilizers rich in natural polyphenols, flavonoids, small molecule active peptides, and organic carbon, as well as their efficient preparation methods. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the problem that the content of polyphenols, flavonoids, small molecule active peptides and organic carbon in existing organic carbon source fertilizers is low. The present invention provides a carbon source fertilizer fermentation method and application that increases the content of polyphenols, flavonoids, small molecule active peptides and organic carbon.

[0006] The first objective of this invention is to provide a method for fermenting carbon source fertilizers that increases the content of polyphenols, flavonoids, small molecule active peptides, and organic carbon.

[0007] The second objective of this invention is to provide a carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides, and organic carbon.

[0008] The third objective of this invention is to provide applications for carbon source fertilizers.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for fermenting a carbon source fertilizer to increase the content of polyphenols, flavonoids, small molecule active peptides, and organic carbon, comprising the following steps: S1. Raw material preparation: Grind soybeans into a paste, boil them, mix them with brown sugar and purified water, and stir thoroughly to obtain a raw material mixture; the mass ratio of the raw materials is: soybeans: brown sugar: purified water = (1~2): (30~50): (20~40); S2. Inoculation with fermentation broth: Add the inoculum fermentation broth to the raw material mixture at an inoculation rate of 0.01~0.1% and stir evenly; the inoculum fermentation broth is prepared by fermentation of Bacillus subtilis, photosynthetic bacteria, yeast, Bacillus amyloliquefaciens, and lactic acid bacteria; the mass ratio of Bacillus subtilis:photosynthetic bacteria:yeast:Bacillus amyloliquefaciens:lactic acid bacteria = (1~2):(1~2):(1~2):(1~2):(1~2); S3. Multi-stage fermentation and aging: Aerobic fermentation is carried out. During the first month of fermentation, the mixture is stirred 1-2 times a day for 3-5 minutes. During the second and third months of fermentation, the mixture is stirred 1-2 times a month for 4-6 minutes. During the fourth and sixth months of fermentation, the mixture is allowed to stand for fermentation and then aged. After fermentation, the mixture is filtered and the filtrate is collected to obtain a carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon.

[0010] This invention uses soybeans, brown sugar, and water as main raw materials. Through a multi-strain, multi-stage, and multi-level fermentation and aging process, it increases the content of active substances such as polyphenols, flavonoids, small molecule active peptides, and organic carbon in carbon source fertilizer. By using specific fermentation bacteria combined with multi-stage fermentation treatment, a liquid carbon source fertilizer is prepared with an organic matter content of 180-210 g / L, a soluble small molecule organic carbon content of 102-136 g / L, and a small molecule active peptide (molecular weight <1000 Daltons) content of 95-115 g / L, including free amino acids and oligopeptides. It is also rich in physiologically active substances such as flavonoids (approximately 530-650 mg / L), polyphenols (approximately 1200-1400 mg / L), and polyglutamic acid (approximately 1200-2000 mg / L). The fertilizer is rich in nutrients and can provide more comprehensive nutrition. The content of active substances is higher than that of existing carbon source fertilizer products. The "multi-stage" method refers to the stirring process performed at different time stages during fermentation, while the "layered" method involves inoculating the fermented broth with pre-fermented microorganisms. This allows for the combined action of multiple fermenting bacteria and their metabolites, unlike existing methods that directly inoculate microorganisms before fermentation. This method utilizes specific microbial communities and metabolites to enhance the content and effectiveness of active ingredients in the carbon source fertilizer. Applying the carbon source fertilizer prepared by this invention to crop production promotes crop growth, increases yield and active ingredient content, and prevents soil nutrient loss. Its effects are superior to commercially available EM (Effective Microorganisms) products. This invention provides a novel, self-developed carbon source fertilizer rich in polyphenols, flavonoids, small-molecule active peptides, and organic carbon. It provides nutrition for various crops, and when used in combination with coenzymes and / or nutrient solutions, it enhances plant metabolic efficiency, nutrient absorption, and stress resistance, further improving its effectiveness. This results in better promotion of crop growth, increased yield and quality, and provides agricultural production with more high-efficiency liquid fertilizers rich in various active substances.

[0011] Preferably, the mass ratio of raw materials used in step S1 is: soybeans: brown sugar, purified water = (1~2): (40~50): (30~40).

[0012] More preferably, in step S1, the mass ratio of the raw materials is: soybeans: brown sugar, purified water = 2:40:30.

[0013] Preferably, in step S2, the mass ratio of Bacillus subtilis: photosynthetic bacteria: yeast: Bacillus amyloliquefaciens: lactic acid bacteria is 1:1:1:1:1.

[0014] Preferably, the preparation method of the bacterial fermentation broth in step S2 is as follows: the microorganisms are inoculated into the fermentation culture medium in a certain proportion and fermented at 25~35℃ for 2~4 days.

[0015] More preferably, the fermentation medium is LB medium.

[0016] Preferably, the OD of the fermentation broth in step S2 is... 600 =1.0±0.2.

[0017] Preferably, the inoculum amount of the microbial fermentation broth in step S2 is 0.05~0.1%.

[0018] More preferably, in step S2, the inoculation amount of the bacterial fermentation broth is 0.05% of the raw material mixture.

[0019] Preferably, the filtrate prepared by the method contains: organic matter content ≥18%, soluble small molecule organic carbon content ≥10%, flavonoid content ≥530 mg / L, small molecule active peptide content ≥9%, polyphenol content ≥1200 mg / L, and polyglutamic acid content ≥0.1%.

[0020] This invention provides a carbon source fertilizer, which is prepared by the above method.

[0021] This invention provides a product containing the above-mentioned carbon source fertilizer.

[0022] Preferably, the product also contains a nutrient solution or a coenzyme solution.

[0023] Preferably, the product contains ≥0.50 billion viable bacteria per mL.

[0024] More preferably, the nutrient solution is Genlidong nutrient solution or Vitality Source nutrient solution; the coenzyme solution is Tianzhongwang® coenzyme nutrient solution.

[0025] The present invention also provides the application of the above-mentioned carbon source fertilizer or product in promoting crop growth and improving crop yield and quality.

[0026] The present invention also provides the application of the above-mentioned carbon source fertilizer or product in reducing the heartburn rate of Chinese cabbage.

[0027] The present invention has the following beneficial effects: This invention utilizes soybeans and brown sugar as primary raw materials and develops a multi-stage fermentation method. Through multi-strain, multi-stage, and multi-level fermentation and aging, it can increase the content of polyphenols, flavonoids, small-molecule active peptides, and organic carbon in the fermentation products. Using a specific microbial fermentation broth, the prepared carbon source fertilizer has an organic matter content of 180–210 g / L, a soluble small-molecule organic carbon content of 102–136 g / L, and a small-molecule active peptide content of 95–115 g / L. It is also rich in physiologically active substances such as flavonoids (approximately 530–650 mg / L), polyphenols (approximately 1200–1400 mg / L), and polyglutamic acid (approximately 1200–2000 mg / L), providing comprehensive nutrition. When used in various crops, it can promote growth, increase yield and quality, and increase the content of active ingredients. Simultaneously, the carbon source fertilizer prepared using this method can significantly reduce the use of chemical fertilizers, providing agricultural production with more high-efficiency liquid fertilizers rich in various active substances. Attached Figure Description

[0028] Figure 1 Phenotypic diagrams of rice from different treatment groups.

[0029] Figure 2 Images of beets from different treatment groups.

[0030] Figure 3 This is a phenotypic diagram of the whole beet. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0033] Example 1: A fermentation method for carbon source fertilizer that increases the content of polyphenols, flavonoids, small molecule active peptides, and organic carbon. 1. Screening of bacterial strains The microorganisms used in this experiment were all commercially available common strains: Bacillus subtilis, photosynthetic bacteria, yeast, Bacillus amyloliquefaciens, lactic acid bacteria, Bifidobacterium, nitrifying bacteria, Bacillus licheniformis, and Streptomyces. Various fermentation broths were prepared by combining different strains for fermentation, and their fermentation effects were measured. The optimal fermentation combination was then selected. The specific fermentation combinations are as follows: A: Bacillus licheniformis + photosynthetic bacteria + yeast + Bacillus amyloliquefaciens + lactic acid bacteria + Streptomyces; B: Bacillus subtilis + photosynthetic bacteria + yeast + Bifidobacterium + lactic acid bacteria + nitrifying bacteria; C: Bacillus subtilis + photosynthetic bacteria + yeast + Bacillus licheniformis + lactic acid bacteria; D: Bacillus subtilis + photosynthetic bacteria + yeast + Bacillus amyloliquefaciens + lactic acid bacteria; E: Bacillus subtilis + photosynthetic bacteria + yeast + lactic acid bacteria; The different combinations of microorganisms mentioned above were inoculated into LB medium in equal proportions (e.g., a mass ratio of 1:1:1:1:1) and fermented in a reactor at room temperature (25-35°C) for 3 days. The resulting fermentation broth was then used for further processing. The OD value of the fermentation broth was measured. 600 =1.0±0.2. Subsequently, preliminary fermentation experiments were conducted using fermentation broths with different combinations of strains. Fermentation raw materials were prepared according to the mass ratio of soybeans, brown sugar, and water of 2:40:30. Then, 0.05% of the inoculum was added to the fermentation raw materials, stirred evenly, and fermentation was carried out. At one month, three months, and six months of fermentation, the same amount of fermentation product samples were taken to determine the content of polyphenols, flavonoids, and soluble small molecule organic carbon components.

[0034] The results are shown in Table 1. The table reveals significant differences in the content of polyphenols, flavonoids, small-molecule active peptides, and soluble small-molecule organic carbon after fermentation using different combinations of fermentation broths. Groups A and B, using a combination of six bacteria, showed varying effects with different strain combinations, but the differences between the two groups were not significant. In groups C and D, adjusting the types and numbers of strains showed that a combination of five strains was more effective than a combination of six, indicating a synergistic effect between different strains. Group D showed the best effect, significantly increasing the content of active substances. Group E, using a conventional combination of four carbon source fertilizer fermentation bacteria, showed slightly worse results than the previous groups. Finally, the fermentation broth prepared using a combination of Bacillus subtilis, photosynthetic bacteria, yeast, Bacillus amyloliquefaciens, and lactic acid bacteria yielded the best results and was used for subsequent experiments.

[0035] Table 1. Effects of different fermentation broth combinations

[0036] 2. Multi-stage fermentation and aging Take 20 kg of soybeans and soak them in filtered water for 8 hours, no more than 24 hours. After soaking, grind the soybeans into soy milk using a grinder and measure the soy milk concentration with a soy milk concentration meter; the concentration should be 10 degrees. Turn on the pump switch to pump the ground soy milk into a boiling tank and bring it to a boil. Take a one-ton fermentation tank, add 400-500 kg of brown sugar and 300-400 L of purified water, and use a stirrer to fully dissolve the brown sugar. After the brown sugar has dissolved, add 25-30 L of the boiled soy milk and stir thoroughly. Measure 2 L of the inoculum (using the fermentation combination D group of Bacillus subtilis, photosynthetic bacteria, yeast, Bacillus amyloliquefaciens, and lactic acid bacteria selected above) using a measuring cylinder, add it to the fermentation tank, continue stirring evenly, cover the tank, but do not completely seal it, and ferment under aerobic conditions.

[0037] From the start of fermentation, during the first month, stir each container twice a day, once in the morning and once in the afternoon, each time for 3-5 minutes. Stop stirring once a large number of bubbles begin to form. During the second and third months, stir once or twice a month, each time for at least 5 minutes. From the fourth to the sixth month, allow the fermentation to stand and undergo alcoholization without stirring. After the sixth month of fermentation, filter the fermentation liquid; the resulting filtrate can then be bottled to produce carbon source fertilizer.

[0038] 3. Determination of active ingredients The active ingredients of the carbon source fertilizer product prepared by the above fermentation were determined by ultraviolet-visible spectrophotometry.

[0039] The test results are shown in Table 2 below. The organic matter content of the prepared carbon source fertilizer is 180-210 g / L, the soluble small molecule organic carbon content is 102-136 g / L, and the small molecule active peptide content is 95-115 g / L (the fermentation process was tested and found to produce a large amount of small molecule active peptides (molecular weight <1000 Daltons), including free amino acids and oligopeptides, with a content as high as about 11%). It is also rich in physiologically active substances such as flavonoids (about 530-650 mg / L), polyphenols (about 1200-1400 mg / L), and polyglutamic acid (about 1200-2000 mg / L), and belongs to high-efficiency liquid fertilizer.

[0040] Table 2 Active components of carbon source fertilizer

[0041] 4. Comparison of different fermentation methods Different fermentation processes were used to compare their fermentation effects. The fermentation was carried out according to the following processes, with the same amount of fermentation raw materials and fermentation bacteria, the same fermentation environment conditions, and the same methods as above.

[0042] (1) Three-stage fermentation: In the first stage, Bacillus subtilis and photosynthetic bacteria are added in equal proportions for fermentation for 2 months; in the second stage, yeast and Bacillus amyloliquefaciens are introduced for fermentation for 1 month; in the third stage, lactic acid bacteria are introduced for fermentation for 1 month, and then alcoholic fermentation is carried out for 2 months. (2) Two-stage fermentation: In the first stage, Bacillus subtilis, photosynthetic bacteria and yeast are added in equal proportions for fermentation for 2 months; in the second stage, Bacillus amyloliquefaciens and lactic acid bacteria are introduced for fermentation for 2 months; finally, alcoholysis is carried out for 2 months. (3) Multi-level fermentation: The fermentation liquid of group D in Example 1 was directly added to the fermentation raw material for fermentation, fermented for 3 months, and then alcoholized for 3 months; (4) Multi-stage and multi-level fermentation: The fermentation liquid of group D in Example 1 is added directly to the fermentation source for fermentation. During the first month, each bucket is stirred twice a day, once in the morning and once in the afternoon, with each stirring time being 3 to 5 minutes. Stirring is stopped after a large number of bubbles are produced. During the second to third month after the start of fermentation, the bucket is stirred 1 to 2 times a month, with each stirring time being 5 minutes. During the fourth to sixth month after the start of fermentation, the bucket is left to ferment without stirring. (5) Multi-stage and multi-level fermentation: Bacillus subtilis, photosynthetic bacteria, yeast, Bacillus amyloliquefaciens and lactic acid bacteria are added to the fermentation raw materials in equal proportions for fermentation. During the first month, each bucket is stirred twice a day, once in the morning and once in the afternoon, with each stirring time being 3 to 5 minutes. Once a large number of bubbles are produced, stirring is stopped. During the second to third month after the start of fermentation, stirring is done 1 to 2 times a month, with each stirring time being 5 minutes. During the fourth to sixth month after the start of fermentation, the fermentation is allowed to stand and no stirring is required.

[0043] After fermentation, fermentation products obtained from different fermentation processes were collected, and the contents of polyphenols, flavonoids, small molecule active peptides, and soluble small molecule organic carbon were measured. The results are shown in Table 3 below. The results show that the content of components in carbon source fertilizers obtained from different fermentation processes is significantly different. The content of active components polyphenols, flavonoids, small molecule active peptides, and organic carbon after fermentation by using different strains in stages was lower. The effects of different strains at different times were significantly different. Although direct inoculation of the fermentation broth can increase the content of active components polyphenols, flavonoids, small molecule active peptides, and organic carbon, its effect is not as good as that of multi-stage, multi-level fermentation processes. Furthermore, direct inoculation of bacteria for fermentation will consume and utilize some of the active substances during the growth of the strains, which is not conducive to the generation of active substances.

[0044] Table 3. Content of active substances in different fermentation processes

[0045] Example 2: A carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon S1. Raw material preparation: Grind soybeans into a paste, boil it, mix it with brown sugar and purified water, stir thoroughly to obtain a raw material mixture for later use; The mass ratio of raw materials used is: soybeans: brown sugar, purified water = 2:40:30; S2. Inoculation with fermentation strains: Add 0.05% of the inoculum fermentation broth to the raw material mixture and stir evenly; The preparation method of the inoculum fermentation broth is as follows: Bacillus subtilis, photosynthetic bacteria, yeast, Bacillus amyloliquefaciens, and lactic acid bacteria are inoculated into LB fermentation medium at a mass ratio of 1:1:1:1:1 and fermented at 25~35℃ for 2~4 days to obtain the inoculum fermentation broth; S3. Multi-stage, multi-level fermentation and aging: Aerobic fermentation is carried out. During the first month of fermentation, the mixture is stirred 1-2 times a day for 3-5 minutes, and stirring is stopped after a large number of bubbles are produced. During the second and third months of fermentation, the mixture is stirred 1-2 times a month for 4-6 minutes. During the fourth and sixth months of fermentation, the mixture is allowed to stand for fermentation and aging. After the sixth month of fermentation, the fermentation liquid is filtered and the filtrate is collected to obtain a carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon.

[0046] The carbon source fertilizer produced contains: organic matter content ≥18%, small molecule active peptide content ≥9%, soluble small molecule organic carbon content ≥10%, flavonoid content ≥530 mg / L, polyphenol content ≥1200 mg / L, and polyglutamic acid content ≥0.1%.

[0047] Example 3: Application of carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon in Chinese cabbage 1. Materials and Methods (1) Overview of the experimental site: The experimental demonstration site is located in Chenyangou Village, Zhangbei County, Zhangjiakou City, Hebei Province. The soil type of the experimental site is chestnut calcareous soil. The terrain is flat, irrigation and drainage are convenient, and transportation is convenient. The previous crop was oats. The soil fertility level is moderate, and the soil pH value is about 8.16.

[0048] (2) Experimental material: Chinese cabbage, the variety of which is Four Seasons Emperor F1, is usually sold on the market 55-60 days after transplanting.

[0049] The experimental group used carbon source fertilizer prepared in Example 2, with an effective viable bacteria count ≥ 0.50 billion / mL. The control group used commercially available EM Original Dew® (purchased from Jiangxi Tianyi Biotechnology Development Co., Ltd.).

[0050] Conventional fertilization methods include: Sakefu compound fertilizer 15-15-15; urinary nitrogen, N≥27%; Stanley compound fertilizer 103.

[0051] (3) Experimental method: The experiment was conducted using a single-factor randomized block design with three treatments and three replicates. The specific treatments are as follows: Treatment 1 (T1): Conventional fertilization + 500 times dilution of carbon source fertilizer from Example 2 (each spraying amount is 0.1L / acre, diluted 500 times with water); Treatment 2 (T2): Conventional fertilization + "Tianyi EM Original Solution" ® "Water-soluble fertilizer 500 times dilution (each spraying amount is 0.1L / acre, diluted 500 times with water); Treatment 3 (CK): Conventional fertilization + the same amount of water as Treatment 1.

[0052] Conventional fertilization: Base fertilizer (broadcast application) 20 kg / mu of Sakafu compound fertilizer (15-15-15); Topdressing (applied with drip irrigation): 10 kg / mu of urinary nitrogen during the seedling stage and 15 kg / mu of urinary nitrogen during the rosette stage; Stanley compound fertilizer 10 kg / mu during the early heading stage and 15 kg / mu of Stanley compound fertilizer during the middle heading stage.

[0053] (3) Results determination: During the experiment, field management was carried out, and soil samples were collected before the experiment and on the day of crop harvest. These samples were sent to the company for testing (testing standards: organic matter GB9834-1988, total nitrogen GB7173-1987, available phosphorus GB12297-1990). During the maturity period of Chinese cabbage, 5 Chinese cabbage plants were selected from each plot, and their plant height, stem diameter, single plant weight, and total yield of the plot were measured and recorded. The number of Chinese cabbage plants and the number of plants with rotten tips in each plot were also calculated. Finally, during the maturity period of Chinese cabbage, 5 points were selected from each plot to collect Chinese cabbage samples. After mixing and processing, more than 1 kg of edible portion was taken and stored in a refrigerator at 0-4 degrees Celsius for the detection of soluble solids, protein, vitamin C, nitrates, etc., and the detection of crop pesticide residues.

[0054] 2. Results The yield measurement results are shown in Table 4 below. Different fertilizer treatments showed no significant differences in plant height, stem diameter, single plant weight, and yield per acre for Chinese cabbage, but there were significant differences in the heart-burning rate. Compared to the control (CK), the heart-burning rate of Chinese cabbage decreased after treatment T1, while treatment T2 significantly increased the heart-burning rate. This indicates that using the carbon source fertilizer in Example 2 does not cause heart-burning in Chinese cabbage and can reduce its heart-burning rate.

[0055] Among the different treatments, plant height was T1>CK>T2, while stem diameter, single plant weight, and yield per mu were T1>T2>CK. Moreover, compared with CK, the plant height, stem diameter, and single plant weight of Chinese cabbage in the T1 treatment increased by 4.10%, 2.98%, and 16.36%, respectively. The yield of Chinese cabbage under the T1 treatment increased by 950.36 kg per mu, with a yield increase of 16.21%, indicating that the carbon source fertilizer in Example 2 has a significant effect on increasing the yield of Chinese cabbage.

[0056] Table 4. Effects of different fertilizer treatments on Chinese cabbage yield

[0057] Note: According to the LSD test, in p When the value is ≤0.05, there is no significant difference in the numerical values ​​following the same lowercase letter.

[0058] The results are shown in Table 5 below. The table shows that different fertilizer treatments did not significantly affect the soluble sugar, vitamin C, and nitrate content of Chinese cabbage, but did significantly affect the total content of the 16 amino acids and the protein content. Compared to the control (CK), the total content of the 16 amino acids in the T1 treatment increased significantly by 78.13%, and the protein content increased significantly by 32.18%, with effects comparable to T2. Therefore, spraying the carbon source fertilizer of Example 2 can improve the quality of Chinese cabbage, enhance its physiological metabolic activities, and increase its resistance.

[0059] Table 5. Effects of different fertilizer treatments on the quality of Chinese cabbage

[0060] Meanwhile, the soluble sugar content of Chinese cabbage treated with T1 increased by 1.92%, and the vitamin C content increased by 13.27%. This indicates that spraying the carbon source fertilizer of Example 2 can increase the soluble sugar and vitamin C content of Chinese cabbage, thereby improving its quality. The nitrate content of each treatment was distributed around 130 mg / kg, which is within the range specified in the "Limits of Nitrate in Vegetable Seeds". Its soluble sugar and vitamin C increased by 1.92% and 13.27% respectively compared with the control, and the total amount of 16 amino acids and protein increased significantly by 78.13% and 32.18% respectively compared with the control. Spraying the carbon source fertilizer of Example 2 can improve the quality of Chinese cabbage, enhance its physiological metabolic activities, and improve its resistance.

[0061] The effects of different treatments on soil nutrients for Chinese cabbage are shown in Table 6 below. Neutral or slightly alkaline soils are beneficial for the growth of Chinese cabbage, and the soil pH in this experimental site was generally alkaline. Before the experiment, the soil had sufficient nitrogen, phosphorus, and potassium nutrients, and the organic matter content was at a moderate level (specifically determined according to the soil topsoil nutrient evaluation standards). After the experiment, the nitrogen, potassium, and organic matter content of each group of soil decreased, while phosphorus increased. All of these treatments provided good nutrition for the crop and had little impact on the soil's physicochemical properties.

[0062] Table 6. Soil nutrient content of Chinese cabbage under different treatments

[0063] Meanwhile, pesticide residue tests were conducted on Chinese cabbage from different treatment groups. The results showed that the detected pesticides in the Chinese cabbage under each treatment met the standards of the "National Food Safety Standard for Maximum Residue Limits of Pesticides in Food". Imidacloprid, carbofuran, carbendazim, and thiamethoxam were not detected.

[0064] In summary, the carbon source fertilizer of Example 2 showed that compared with the control, the plant height, stem diameter, single plant weight, and yield per mu of Chinese cabbage increased by 4.10%, 2.98%, 16.36%, and 16.21%, respectively. The rate of heartburn was also lower than both the control and treatment group 2, which significantly improved the commercial quality and yield of Chinese cabbage. Soluble sugar and vitamin C increased by 1.92% and 13.27% respectively compared with the control, while the total amount of 16 amino acids and protein increased significantly by 78.13% and 32.18% respectively, improving the nutritional quality and enhancing the physiological metabolic activity of Chinese cabbage, thus increasing its resistance. Therefore, the carbon source fertilizer prepared by this invention has a significant effect on improving the quality and efficiency of crops.

[0065] Example 4: Application of carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon in tea. 1. Materials and Methods (1) Overview of the experimental site: The experimental site was selected in the tea garden of Xiaxiao Village, Fubin Town, Raoping County, at Chuandu Tea Plantation (117.01670°E, 23.67845°N). The soil type of the experimental site is red soil of mountainous hills.

[0066] (2) Test materials: Fertilizers used in the experiment: The experimental group used the carbon source fertilizer prepared in Example 2; at the same time, coenzyme nutrient solution was used in conjunction with the carbon source fertilizer, and an experimental treatment group containing coenzyme was set up: Tianzhongwang® coenzyme nutrient solution was used, which was purchased from Cross-Strait Agricultural Technology Co., Ltd., registration number: Q / HXLA01-2020.

[0067] The control group was treated with commercially available Tianyi EM Original Dew® fertilizer, purchased from Jiangxi Tianyi Biotechnology Development Co., Ltd.; meanwhile, a plant growth hormone treatment group was set up, using gibberellin.

[0068] Experimental crop: tea, variety Lingtou Dancong (a national-level superior tea tree variety), with tree age of 20-25 years.

[0069] Conventional fertilization: 25 kg / mu of compound fertilizer.

[0070] (3) Experimental Design: The experiment was arranged as a single-factor randomized block design, with 5 treatments and 3 replicates. The experimental plot area was 64 m². 2 The length is 8 m and the width is 8 m. The specific treatments are as follows: Treatment 1 (T1): Conventional fertilization + 500 times dilution of carbon source fertilizer from Example 2 (without coenzyme); Treatment 2 (T2): Conventional fertilization + 500 times dilution of carbon source fertilizer from Example 2 (containing coenzyme); Treatment 3 (T3): Conventional fertilization + 500 times dilution of "Tianyi EM Original Dew" water-soluble fertilizer; Treatment 4 (T3): Conventional fertilization + "gibberellin"; Control (CK): Conventional fertilization + equal amount of water as treatment 1.

[0071] (4) Experimental Methods: After the tea garden was routinely fertilized, three sprayings were carried out at specific times to promote tea growth and improve quality. The first spraying was done 7-10 days before the tea trees sprouted to ensure abundant and uniform bud emergence; the second spraying was done when 60%-70% of the tea trees had sprouted to promote leaf growth; the third spraying was done after the tea trees sprouted to help the new buds grow evenly, the leaves thicken, and the internal contents become richer. During the spraying process, it was ensured that the water droplets remained on the leaf surface. Morphological and harvesting analyses were conducted after the tea leaves reached the stage of 1 bud and 3-4 leaves.

[0072] (5) Sample collection and yield determination: Fifteen mixed soil samples were collected at the test site before the first spraying. During the second spraying, control samples and three replicate soil samples from four different treatments were collected, for a total of 15 soil samples. All soil samples were air-dried and sent to Beijing PONY Testing Group Co., Ltd. for testing.

[0073] One week after the third spraying, tea leaves were harvested, weighed, and yielded. Harvesting was done by opening the leaves, calculating bud density and weighing the tea leaves within a 500×500mm square. Twenty new shoots were randomly selected to measure their length, leaf length, and leaf width. One hundred shoots with one bud and three leaves were selected and weighed to calculate the weight of 100 buds. Additionally, the thickness of the fourth new leaf on each shoot and the second older leaf below it were measured using a JC-YHD-2 leaf thickness gauge on three selected shoots. Dry-dried samples were prepared to measure the content of biochemical components, and the tea samples were processed into finished tea for sensory evaluation.

[0074] 2. Results The results of the effects on tea plant traits are shown in Table 7. The T4 treatment showed higher new shoot length, leaf length, leaf width, and leaf area compared to other treatments. However, the T4 treatment had the lowest thickness of the fourth new leaf, decreasing by 12.26% and 11.04% compared to the T1 and T2 treatments, respectively, with significant differences. The T2 treatment showed the best effect. This indicates that carbon source fertilizer, unlike gibberellin, provides sufficient nutrients and promotes robust tea plant growth, increasing leaf thickness with significant effects.

[0075] Table 7 Effects of different fertilizer treatments on tea plant traits

[0076] The results for tea yield are shown in Table 8. Treatment T1 showed the best bud density, with 90.31% of the buds having one bud and two to three leaves. Treatments T2 and T4 had the heaviest weight per 100 buds, followed by treatment T1. Treatments T1 and T4 had the highest weight of fresh tea leaves. The yield of fresh tea leaves in treatments T2 and T4 was significantly higher than other treatments. Compared to the control (CK), treatment T2 significantly increased the yield per acre for buds with three leaves, buds with four leaves, weight per 100 buds, and fresh tea leaf yield by 22.81%, 110.92%, 58.74%, 103.43%, and 67.03% respectively.

[0077] Table 8. Effects of different fertilizer treatments on tea yield and its components.

[0078] Spraying carbon source fertilizer can significantly promote the growth of tea trees, resulting in: thick stems, moderate internodes, thick and sturdy leaves, high bud density, and good overall growth. In contrast, tea treated with gibberellin has thin stems, large internodes, thin and curled leaves, and excessive vegetative growth.

[0079] The results of different treatments on the main biochemical components of tea are shown in Table 9. The water extract content of tea in each treatment group was higher than 42%, with T2 and T3 having the highest content. T2 and T3 also had higher tea polyphenol content, while CK had the lowest content. Among the water-soluble ash, T3 had the highest content, followed by T1 and T2. The crude fiber content of all treatment groups was within the required range, with T1 having the highest content. T1, T2, and T3 had relatively high phenol-to-amino acid ratios, resulting in a richer flavor, while CK and T4 had lower ratios, resulting in a relatively milder flavor.

[0080] Table 9. Effects of different fertilizer treatments on the main biochemical components of tea leaves.

[0081] Table 10 shows the effects of different fertilizer treatments on the main biochemical components of tea. The amino acid content of Dancong Oolong tea is typically 1.15%-2.96%, affecting aroma and taste. An amino acid content exceeding 2% can enhance the freshness and sweetness of the tea. This experiment found that treatment T4 contained high levels of amino acids, while other treatments showed little difference. Tea polysaccharides can alleviate bitterness; the higher the content, the richer the flavor. Treatment T1 had the highest tea polysaccharide content, while treatment T3 had the lowest. Tea ash is a residue composed of mineral elements and their oxides; the total ash content should not exceed 8%. In this experiment, treatments CK and T4 had higher total ash content. The caffeine content of Dancong Oolong tea ranges from 1.05%-5.33%; a moderate amount makes the tea soup fresh and refreshing, while excessive amounts bring bitterness. In this experiment, treatments T3 and T4 had higher caffeine content, while treatment T1 had the lowest. Theanine can alleviate bitterness and enhance sweetness. In this experiment, treatment T4 had the highest theanine content, while treatment T3 had the lowest. The total ash content of tea treated with carbon source fertilizer and coenzyme was reduced by 6.51%.

[0082] Table 10 Effects of different fertilizer treatments on the main biochemical components of tea leaves

[0083] In summary, the results show that carbon source fertilizer is rich in nutrients and promotes tea growth, resulting in robust growth, increased leaf thickness, and significant improvement in tea yield and active ingredient content. Furthermore, its combined use with coenzyme nutrient solution further enhances the effect, comparable to the commercially available EM Original Dew®, effectively promoting tea growth and improving its yield and quality.

[0084] Example 5: Application of carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon in rice. 1. Materials and Methods (1) Overview of the test site: The test soil was purple soil and red sandy soil developed from the parent material of the Suining Formation. The organic matter content was 9.24 g / kg, the water-soluble nitrogen content was 2.71 mg / kg, the available phosphorus was 3.02 mg / kg, the available potassium was 90.21 mg / kg, and the pH was 7.91.

[0085] (2) Test materials: Test crop: rice, variety Chuanxiangyou 6203. The test fertilizer was the carbon source fertilizer prepared in Example 2; coenzyme nutrient solution (main components: Ca+Mg≥100g / L), purchased from Cross-Strait Agricultural Technology Co., Ltd.; urea (N46%); compound fertilizer (N+P2O4+K2O≥45%).

[0086] (3) Experimental design: Treatment 1 (T1): Conventional fertilization + 300 times dilution of carbon source fertilizer from Example 2 (0.13 liters / acre per spray, diluted 300 times with water) + 800 times dilution of coenzyme nutrient solution (50 mL / acre per spray).

[0087] Treatment 2 (T2): Conventional fertilization + spraying with the same amount of water as Treatment 1 at the same time. Spray once each at the tillering stage, heading stage, and milk stage, for a total of 3 sprays.

[0088] Conventional fertilization: After the rice turns green, apply 50 kg of compound fertilizer (15-15-15) per mu, apply 15 kg of urea per mu during the tillering stage, and apply 25 kg of compound fertilizer per mu during the booting stage.

[0089] (4) Trial Management: Rice was managed according to local practices. Dry-seeding was carried out on April 2nd, transplanting on June 1st, fertilization on June 8th, pesticide spraying (fungicide and insecticide) on June 23rd, urea top dressing on June 28th, and compound fertilizer top dressing on August 2nd. Foliar fertilizer was sprayed at the tillering, booting, and milk stages in each treatment group. Harvesting took place on September 29th. After harvest, plant height, effective panicle number, total panicle number, panicle formation rate, and yield components were measured and statistically analyzed for each treatment group.

[0090] 2. Results The results of rice plant height and panicle formation rate are shown in Table 11. The plant height, effective panicle number and panicle formation rate of rice treated with carbon source fertilizer in Example 2 (treatment 1) were higher than those of conventional fertilization (treatment 2). Compared with the control, the effective panicle increased by 10.81%, the plant growth was better, and the theoretical yield increase effect was obvious.

[0091] Table 11 Effects on rice plant height and panicle formation rate

[0092] The results of the yield components of rice are shown in Table 12. Analysis of variance shows that there are significant differences in theoretical yield. Rice treated with carbon source fertilizer showed an 18.39% increase in yield. The yields of rice in different treatment groups were as follows: Figure 1 As shown, the use of carbon source fertilizer in Example 2 can significantly increase rice yield.

[0093] Table 12 Impact of factors affecting yield components of rice in Sichuan

[0094] Example 6: Application of carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon in sugar beets 1. Materials and Methods (1) Overview of the test site: COFCO Sugar Industry (Changji) Sugar Base in Yushugou Town, Changji City, Changji Prefecture, Xinjiang.

[0095] (2) Test materials: Experimental crop: sugar beet (variety: sv1555).

[0096] Fertilizers used in the test group: The carbon source fertilizer prepared in Example 2 was used in the test group. Coenzyme nutrient solution and root-power nutrient solution were used in conjunction with the carbon source fertilizer. Coenzyme nutrient solution was purchased from Cross-Strait Agricultural Technology Co., Ltd., registration number: ZLSRGD2021-00201; root-power nutrient solution was purchased from Cross-Strait Agricultural Technology Co., Ltd., fertilizer registration certificate number: microbial fertilizer (2021) approval number (10563).

[0097] Conventional fertilization: compound fertilizer (N+P2O5+K2O≥45%).

[0098] (3) Experimental design: Treatment 1 (60 mu demonstration area): Example 2: carbon source fertilizer + coenzyme nutrient solution + root-strengthening nutrient solution, the application method includes a combination of foliar spraying and ground irrigation.

[0099] Foliar spraying: Carbon source fertilizer was applied at concentrations of 150 mL / mu / time (300 times dilution), 150 mL / mu / time (300 times dilution), and 150 mL / mu / time (300 times dilution) at different growth stages of sugar beets; coenzyme nutrient solution was applied at concentrations of 60 mL / mu / time (800-1000 times dilution) at different growth stages of sugar beets; and a third application of Root Power Nutrient Solution at 70 mL / mu / time was added. A total of three applications were made during the seedling stage of sugar beets, using 50 L of water per mu per application.

[0100] Surface irrigation (drip irrigation): Used in conjunction with water and fertilizer during the daily field management of sugar beets, for a total of 6 times, using carbon source fertilizer at a concentration of 500 mL / mu / time and root-power nutrient solution at a concentration of 150 mL / mu / time.

[0101] Treatment 2 (control area 120 mu): conventional fertilization.

[0102] (4) Trial Management: The demonstration participation period was May 23rd. Foliar spraying was applied approximately every 7-15 days, with 3 applications and 6 applications of surface irrigation (drip irrigation) between May 23rd and August 12th. Foliar spraying: 3 applications, including 500 mL / mu / application of carbon source fertilizer, 60-70 mL / mu / application of coenzyme nutrient solution, and 150 mL / mu / application of root-strengthening nutrient solution, using 50 L / mu / application of water; spraying was done using a trailer truck. Surface irrigation (drip irrigation): This was integrated into routine field management of sugar beets, with 6 applications, using 500 mL / mu / application of carbon source fertilizer and 150 mL / mu / application of root-strengthening nutrient solution. At the end of the trial, yield measurements were conducted in both the experimental and control areas for acceptance.

[0103] 2. Results The theoretical yield results of sugar beets are shown in Table 13 below. The theoretical yield of sugar beets treated with carbon source fertilizer + coenzyme + root power (treatment 1) was higher than that of conventional fertilization (treatment 2), with a yield per mu (unit of land area) that increased by 33.4% compared to the control, and the difference was significant.

[0104] Table 13 Theoretical yield of sugar beets

[0105] The theoretical and experimental results of sugar beets are shown in Table 14 below. The experimental yield of sugar beets treated with carbon source fertilizer + coenzyme + root power (treatment 1) was higher than that of conventional fertilization (treatment 2), and the yield per mu increased by 37.8% compared with the control, which was significant.

[0106] Table 14 Measured yield of sugar beets

[0107] The sugar beet prepared using this invention during cultivation, such as Figure 2-3 As shown, according to statistical analysis, the theoretical yield of sugar beets increased by 33.4%, while the actual yield increased by 37.8%, far exceeding the theoretical yield increase. The carbon source fertilizer prepared by this invention, when used in combination with coenzymes and root power, has significant economic benefits and can be widely promoted and applied.

[0108] Example 7: Application of carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon in sugarcane 1. Materials and Methods (1) Overview of the test site: Luobai Township, Jiangzhou District, Chongzuo City, Guangxi Zhuang Autonomous Region.

[0109] (2) Test materials: Experimental crop: sugarcane, variety: Guitang 44.

[0110] Fertilizers used in the test group: The carbon source fertilizer prepared in Example 2 was used in the test group. At the same time, the carbon source fertilizer was used in combination with Vitality Source Nutrient Solution + Coenzyme Nutrient Solution + Root Power Nutrient Solution. The Vitality Source Nutrient Solution, Coenzyme Nutrient Solution and Root Power Nutrient Solution were all purchased from Cross-Strait Agricultural Technology Co., Ltd., with fertilizer registration certificate number: Microbial Fertilizer (2021) Approval No. (10563) and filing number: ZLSRGD2021-00201.

[0111] Conventional fertilization: compound fertilizer (N+P2O5+K2O≥45%).

[0112] (3) Experimental Design: This demonstration project used 28 mu (approximately 1.2 hectares) each for the demonstration and control areas. The agricultural machinery used was a drone (model 40). The water consumption per mu was 5-6 L. Specific treatments are as follows: Treatment 1 (T1): Conventional fertilization + Carbon source fertilizer stock solution from Example 2 (300 mL / mu per application) + Vitality source nutrient solution (300 mL / mu per application) + Coenzyme nutrient solution (60 mL / mu per application) + Root Power nutrient solution (120 mL / mu per application). Treatment 2 (T2): Conventional fertilization.

[0113] (4) Trial Management: The first foliar spray was applied when the sugarcane seedlings reached a leaf height of 50 cm, followed by a second spray 15 days later, and then sprayed every 15–30 days thereafter, for a total of 6 foliar sprays throughout the cycle. After harvest, the sugarcane stalk length, stalk diameter, cone weight, and yield of each treatment group were measured and recorded.

[0114] 2. Results The results of sugarcane stem length, stem diameter and weight are shown in Table 15 below. It shows that the sugarcane treated with carbon source fertilizer in Example 2 combined with coenzyme and root power (treatment 1) has higher stem length, stem diameter, stem weight and weight than the sugarcane treated with conventional fertilization (treatment 2). The plants have better growth and the yield increase is obvious.

[0115] Table 15 Effects of microbial fertilizer on sugarcane stem length, stem diameter, and stem thickness in Guangxi.

[0116] The sugarcane yield results are shown in Table 16 below. It shows that the sugarcane yield using the carbon source fertilizer of Example 2 combined with coenzyme and root power (treatment 1) is higher than that of conventional fertilization (treatment 2), and the yield per mu increased by 15.13% compared with the control, which is significant.

[0117] Table 16. Impact of Green Carbon Cultivation Management Technology on Sugarcane Yield in Guangxi

[0118] In summary, the results show that combining the carbon source fertilizer prepared by this invention with other fertilizer nutrient solutions can better improve sugarcane yield, increasing sugarcane yield by 15.13% after 3 months of cultivation.

[0119] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fermentation method for a carbon source fertilizer that increases the content of polyphenols, flavonoids, small molecule active peptides, and organic carbon, characterized in that, Includes the following method steps: S1. Raw material preparation: Grind soybeans into a paste, boil them, mix them with brown sugar and purified water, and stir thoroughly to obtain a raw material mixture; the mass ratio of the raw materials is: soybeans: brown sugar: purified water = (1~2): (30~50): (20~40); S2. Inoculation with fermentation broth: Add the inoculum fermentation broth to the raw material mixture at an inoculation rate of 0.01~0.1% and stir evenly; the inoculum fermentation broth is prepared by fermentation of Bacillus subtilis, photosynthetic bacteria, yeast, Bacillus amyloliquefaciens, and lactic acid bacteria; the mass ratio of Bacillus subtilis:photosynthetic bacteria:yeast:Bacillus amyloliquefaciens:lactic acid bacteria = (1~2):(1~2):(1~2):(1~2):(1~2); S3. Multi-stage fermentation and aging: Aerobic fermentation is carried out. During the first month of fermentation, the mixture is stirred 1-2 times a day for 3-5 minutes. During the second and third months of fermentation, the mixture is stirred 1-2 times a month for 4-6 minutes. During the fourth and sixth months of fermentation, the mixture is allowed to stand for fermentation and then aged. After fermentation, the mixture is filtered and the filtrate is collected to obtain a carbon source fertilizer rich in natural polyphenols, flavonoids, small molecule active peptides and organic carbon.

2. The method according to claim 1, characterized in that, Step S1 uses the following raw material mass ratio: soybeans: brown sugar: purified water = (1~2): (40~50): (30~40).

3. The method according to claim 1, characterized in that, OD of the fermentation broth in step S2 600 =1.0±0.

2.

4. The method according to claim 3, characterized in that, The preparation method of the bacterial fermentation broth in step S2 is as follows: the microorganisms are co-inoculated into the fermentation medium in proportion, and fermented at 25~35℃ for 2~4 days to obtain the broth.

5. The method according to claim 1, characterized in that, In step S2, the inoculum size of the fermentation broth is 0.05~0.1%.

6. The method according to claim 1, characterized in that, The carbon source fertilizer prepared by the method contains: organic matter content ≥18%, soluble small molecule organic carbon content ≥10%, small molecule active peptide content ≥9%, flavonoid content ≥530mg / L, polyphenol content ≥1200mg / L, and polyglutamic acid content ≥0.1%.

7. A carbon source fertilizer, characterized in that, It is prepared by the method described in any one of claims 1 to 6.

8. A product characterized in that, The fertilizer containing the carbon source as described in claim 7.

9. The product according to claim 8, characterized in that, The product also contains nutrient solution or coenzyme solution.

10. The use of the carbon source fertilizer of claim 7 or the product of claim 8 in promoting crop growth and improving crop yield and quality.

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