Microbial protective agent in high-nutrient and high-activity compound fertilizer and preparation thereof

Abstract

The present application relates to a kind of high nutrient high activity chemical fertilizer biological compound fertilizer microbial protectant and preparation thereof, belong to biological extract technical field.The microbial protectant of the present application mainly includes fucoidan and tetrahydropyrimidine, other components also include at least one of burdock oligosaccharide, chitosan, amino acid, vegetable oil, fumaric acid, citric acid.The microbial protectant of the present application can protect microorganism in extreme environment, such as high salt environment, avoid the damage and death of microorganism, with wide application prospect, can be applied to the preparation of high activity microbial inoculant, and the preparation of high nutrient chemical fertilizer biological compound fertilizer.

Description

Technical Field

[0001] This invention relates to a microbial protectant in a high-nutrient, high-activity bio-compound fertilizer and its preparation, belonging to the field of bio-extractive technology. Background Technology

[0002] Microbial fertilizers, as a new type of fertilizer, have their own development advantages. Microbial fertilizers are inoculants prepared from one or more beneficial living microorganisms. Through their metabolic processes or metabolites, they improve plant growth conditions and promote plant growth. However, in the long-term practice of applying microbial fertilizers, it has been recognized that applying them alone in the field is difficult, has a slow effect, low farmer acceptance, and a short shelf life.

[0003] Biological compound fertilizers are made by combining specific beneficial microbial agents with traditional fertilizers. The organic combination of beneficial microbial agents and fertilizers can significantly improve nutrient utilization efficiency, enhance crop quality, improve soil, and increase crop resistance. However, maintaining the high activity of beneficial microorganisms while uniformly mixing high-salt-index fertilizers with live fertilizers to produce biological compound fertilizers remains a global challenge. In existing biological compound fertilizer preparation processes, one method of adding microorganisms is through granulation using a mixing and pelleting method with traditional fertilizers. However, the addition of microorganisms during granulation is susceptible to death due to factors such as high temperature, high salt, and dryness. Therefore, to ensure the effectiveness of biological compound fertilizers, it is crucial to address the issue of microbial activity. Patent document CN102617197A discloses a technology for surviving agricultural microbial agents in high-nutrient fertilizers. This involves adding 0.01‰ gibberellin crystal powder to agricultural microbial Bacillus bacterial solution to form mature spores, enhancing the reverse osmotic pressure of live bacteria, and reducing the damage caused by high salt content. Additionally, 10% humic acid is added to the bacterial solution adsorbent, utilizing microporous biological niches to isolate the harsh environment. This patent primarily utilizes the mature spores of Bacillus to resist adverse environments and survive long-term, as well as the microporous adsorption of humic acid. Patent document CN113880642A discloses a compound microbial fertilizer containing a microbial activity protectant composed of polyglutamic acid and trehalose. By adding polyglutamic acid and trehalose to the compound fertilizer as microbial activity protectants, damage to phosphate-solubilizing microorganisms during the preparation process is reduced, increasing the number of effective viable bacteria in the compound fertilizer. The protectant in this patent mainly protects the phosphate-solubilizing microorganisms in the compound microbial fertilizer, preventing significant damage and death.

[0004] To improve the effective activity of functional microorganisms in the preparation and storage of chemical fertilizer-biocompound fertilizers, it is crucial to study the effects of different microbial protectants and different formulation combinations on the number of viable bacteria. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a microbial protectant for high-nutrient, high-activity chemical fertilizer-biocompound fertilizer and its preparation.

[0006] Terminology Explanation:

[0007] The content of nitrogen, phosphorus, and potassium nutrients refers to the content of N, P2O5, and K2O.

[0008] The technical solution of the present invention is as follows:

[0009] A microbial protectant, mainly comprising fucoidan oligosaccharides and tetrahydropyrimidine.

[0010] According to a preferred embodiment of the present invention, the microbial protectant further includes at least one of burdock oligosaccharide, chitosan oligosaccharide, amino acids, vegetable oil, fumaric acid, and citric acid.

[0011] According to a preferred embodiment of the present invention, the mass fractions of each component in the microbial protectant are as follows: 65-85 parts of fucoidan, 4-6 parts of burdock oligosaccharide, 6-10 parts of chitosan oligosaccharide, 6-10 parts of amino acids, 5-10 parts of vegetable oil, 5-8 parts of tetrahydropyrimidine, 8-10 parts of fumaric acid, and 6-10 parts of citric acid.

[0012] According to a preferred embodiment of the present invention, the molecular weight of the fucoidan is below 2000 Da, the degree of polymerization is 2-20, and the 2-6 sugar content reaches more than 70%.

[0013] According to a preferred embodiment of the present invention, the degree of polymerization of the burdock oligosaccharide is 10-15.

[0014] According to a preferred embodiment of the present invention, the amino acid comprises one or more of glutamic acid, aspartic acid, and phenylalanine.

[0015] According to a preferred embodiment of the present invention, the types of vegetable oils include soybean oil and / or castor oil.

[0016] The brown algae oligosaccharides, burdock oligosaccharides, chitosan oligosaccharides, amino acids, vegetable oils, tetrahydropyrimidine, fumaric acid, and citric acid used in this invention can all be commercially available products.

[0017] The preparation method of the above-mentioned microbial protectant includes the following steps:

[0018] The raw materials of each component of the microbial protectant are quantitatively obtained, stirred and mixed evenly to obtain the final product.

[0019] According to a preferred embodiment of the present invention, a quantitative feeder is used to add the raw materials of each component of the microbial protectant into a mixer, and stirring is started. After stirring and mixing, the microbial protectant is obtained.

[0020] According to a preferred embodiment of the present invention, the product after being thoroughly mixed is packaged into bags using a metering and packaging machine.

[0021] A further preferred method is to store the packaged products in a dry and well-ventilated indoor environment.

[0022] The application of the above-mentioned microbial protectants in the preparation of microbial inoculant complexes.

[0023] The above-mentioned microbial protectant is mixed with microorganisms to prepare a microbial agent complex. The microorganisms in the microbial agent complex are effectively protected by the microbial protectant, which improves their survival rate in extreme environments and avoids damage and death.

[0024] The application of the above-mentioned microbial protectants in the preparation of chemical fertilizer and bio-compound fertilizer.

[0025] Applying the above-mentioned microbial protectants to chemical fertilizer-biocompound fertilizers can effectively protect the microorganisms in the fertilizer-biocompound fertilizers, prevent damage and death of microorganisms in the high-salt environment of chemical fertilizers, and effectively increase the nutrient content of inorganic fertilizers in the fertilizer-biocompound fertilizers.

[0026] A microbial agent complex containing the aforementioned microbial protectant.

[0027] According to a preferred embodiment of the present invention, the mass percentage of the microbial protectant in the microbial agent complex is 50% or more.

[0028] More preferably, the mass percentage of the microbial protectant in the composite microbial agent complex is 50-90%.

[0029] A chemical fertilizer-biocompound fertilizer containing the above-mentioned microbial protectant or the above-mentioned microbial agent compound.

[0030] According to a preferred embodiment of the present invention, the nitrogen, phosphorus, and potassium nutrient content of the fertilizer-biocompound fertilizer is ≥40%, and the number of viable microorganisms is ≥200 million CFU / g.

[0031] The beneficial effects of this invention are as follows:

[0032] 1. The microbial protectant of the present invention includes brown algae oligosaccharides, burdock oligosaccharides, chitosan oligosaccharides, amino acids, vegetable oils, tetrahydropyrimidine, fumaric acid, and citric acid. The microbial protectant of the present invention can protect microorganisms in extreme environments, such as high-salt environments, to avoid damage and death of microorganisms. It has broad application prospects and can be applied to the preparation of highly active microbial agents and the preparation of high-nutrient-content fertilizer-based compound fertilizers.

[0033] 2. After adding the above-mentioned microbial protectant to the microorganisms, the survival period of the microorganisms is extended by 6-12 months. After 12 months of storage, the number of viable bacteria in the microbial agent is 2×10⁻⁶. 8With a CFU / g or higher, the number of microorganisms in the soil exceeds 550,000 / g soil after 10 days of application, solving the problem that microbial fertilizers cannot be widely used in field crops.

[0034] 3. The number of live microorganisms in the chemical fertilizer-biocompound fertilizer produced by adding the protective agent of this invention is more than 200 million CFU / g, which is far higher than the standard of 020 million CFU / g specified in the compound microbial fertilizer standard "NY / T798-2015". This solves the problem of uniformly mixing high-salt-index chemical fertilizers with live beneficial microorganisms to produce chemical fertilizer-biocompound fertilizer while maintaining the high activity of beneficial microorganisms. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments, but is not limited thereto. Unless otherwise specified, all amounts of raw materials are parts by weight.

[0036] The components of the microbial protectant in this invention include one or more of the following: fucoidan, burdock oligosaccharide, chitosan oligosaccharide, amino acids, vegetable oil, tetrahydropyrimidine, fumaric acid, and citric acid.

[0037] Among them, brown algae oligosaccharides, burdock oligosaccharides, chitosan oligosaccharides, and amino acids maintain the natural structure of the original hydrated state of the microbial cell macromolecules in the chemical fertilizer-biocompound fertilizer through their polyhydroxy groups. At the same time, they form a vitrified matrix around the cells, which is similar to a physical barrier to resist the damage of high concentrations of salt to the cells, so that the cells suffer less loss of vitality and thus play a protective role.

[0038] The function of vegetable oil is to increase hydrophobicity and prevent microorganisms from coming into contact with fertilizers and water.

[0039] Tetrahydropyrimidine effectively protects the proteins in the spore coat by forming a "tetrahydropyrimidine hydrate," preventing cations from entering the spores and thus protecting the stability and activity of the spores in a high-concentration fertilizer environment.

[0040] The role of fumaric acid and citric acid is to enhance the rhizosphere chemotaxis and root surface film formation of beneficial microorganisms in chemical fertilizer and biological compound fertilizer, thereby increasing the colonization of microorganisms in the soil and increasing the rhizosphere colonization of microorganisms by 2.0 to 6.0 times.

[0041] The microbial protectant in this invention comprises the following components by weight: 65-85 parts of brown algae oligosaccharide, 4-6 parts of burdock oligosaccharide, 6-10 parts of chitosan oligosaccharide, 6-10 parts of amino acids, 5-10 parts of vegetable oil, 5-8 parts of tetrahydropyrimidine, 8-10 parts of fumaric acid, and 6-10 parts of citric acid.

[0042] In preferred technical solutions, the mass composition of fucoidan oligosaccharides can be 65, 68, 70, 72, 75, 77, 80, 83, or 85 parts. In preferred technical solutions, the mass composition of burdock oligosaccharides can be 4, 5, or 6 parts. In preferred technical solutions, the mass composition of chitosan oligosaccharides can be 6, 7, 8, 9, or 10 parts. In preferred technical solutions, the mass composition of amino acids can be 6, 7, 8, 9, or 10 parts. In preferred technical solutions, the mass composition of vegetable oil can be 5, 6, 7, 8, 9, or 10 parts. In preferred technical solutions, the mass composition of tetrahydropyrimidine can be 5, 6, 7, or 8 parts. In preferred technical solutions, the mass composition of fumaric acid can be 8, 9, or 10 parts. In preferred technical solutions, the mass composition of citric acid can be 6, 7, 8, 9, or 10 parts.

[0043] This invention allows for the adjustment of the composition and content of the microbial protectant according to the different types of microorganisms to be protected. For example, one microbial protectant has the following composition by weight: 75-85 parts of fucoidan, 5-8 parts of vegetable oil, 5-6 parts of tetrahydropyrimidine, and 8-10 parts of citric acid. This formulation is suitable for the protection of Bacillus subtilis (e.g., ACCC60364, ACCC19742, ACCC19743, ACCC60383).

[0044] For example, a microbial protectant has the following composition by weight: 65-75 parts of fucoidan, 4-6 parts of burdock oligosaccharide, 6-8 parts of vegetable oil, 5-8 parts of tetrahydropyrimidine, and 8-10 parts of fumaric acid. This formulation is suitable for the protection of Bacillus amyloliquefaciens (e.g., ACCC60428, ACCC10167, ACCC60382).

[0045] For example, a microbial protectant has the following composition by weight: 75-85 parts fucoidan, 6-8 parts chitosan oligosaccharide, 6-8 parts tetrahydropyrimidine, and 6-8 parts citric acid. This formulation is suitable for the protection of Bacillus mucilaginosa (e.g., CGMCC No. 17376, ACCC02983, ACCC10095, ACCC10090, ACCC0013), which has the function of degrading soil organic phosphorus, solubilizing potassium, and promoting the absorption of phosphorus and potassium by plants.

[0046] For example, a microbial protectant with the following composition by weight: 80-85 parts of fucoidan, 6-8 parts of chitosan oligosaccharide, 6-10 parts of amino acids, 6-8 parts of tetrahydropyrimidine, and 8-10 parts of fumaric acid. This formulation is suitable for the protection of Bacillus laterosporus (e.g., CGMCC No. 17377, ACCC 11079).

[0047] For example, a microbial protectant has the following composition by weight: 70-80 parts of fucoidan, 6-10 parts of chitosan oligosaccharide, 6-10 parts of vegetable oil, 6-8 parts of tetrahydropyrimidine, and 8-10 parts of fumaric acid. This formulation is suitable for the protection of mixed bacteria of Bacillus amyloliquefaciens (e.g., ACCC60428, ACCC10167, ACCC60382) and Bacillus laterosporus (e.g., CGMCC No.17377, ACCC11079).

[0048] For example, a microbial protectant has the following composition by weight: 75-85 parts fucoidan, 6-8 parts chitosan oligosaccharide, 5-8 parts vegetable oil, 5-8 parts tetrahydropyrimidine, and 6-10 parts citric acid. This formulation is suitable for the protection of mixed bacteria of Bacillus subtilis (e.g., ACCC60364, ACCC19742, ACCC19743, ACCC60383) and Bacillus mucilaginosa (e.g., CGMCCNo.17376, ACCC02983, ACCC10095, ACCC10090, ACCC0013).

[0049] The microbial protectant of this invention can also protect *Bacillus megaterium* JD2023Hb and *Bacillus subtilis* KC2023Hb. Furthermore, when *Bacillus megaterium* JD2023Hb and *Bacillus subtilis* KC2023Hb coexist, it can further enhance the tolerance of other microorganisms to high concentrations of inorganic fertilizers and improve the protective effect on other microorganisms. The preservation number of *Bacillus megaterium* JD2023Hb is CGMCC NO.29498, and the preservation number of *Bacillus subtilis* KC2023Hb is CGMCC NO.29499.

[0050] In the examples, the molecular weight of the brown algae oligosaccharides is below 2000 Da, the degree of polymerization is 2-20, and the 2-6 sugar content reaches more than 70%. They are obtained by fermenting brown algae, seaweed, and other marine plants. The preparation method can be found in patent document CN112342255A, or they can be obtained commercially.

[0051] In this example, the degree of polymerization of burdock oligosaccharides was 13, and they were extracted from secondary burdock roots using an enzymatic method. The dried burdock root sample was pulverized, passed through a 40-mesh sieve, and extracted with hot water at a solid-liquid ratio of 1:10 at 70°C for 90 minutes per extraction. The extract was then filtered, decolorized with activated charcoal, concentrated using a rotary evaporator, deproteinized using the papain-seveage method, precipitated with ethanol, centrifuged, washed multiple times, centrifuged again, and freeze-dried to obtain burdock oligosaccharide powder. It can also be commercially available.

[0052] The vegetable oil used in this example is soybean oil.

[0053] The chitosan oligosaccharide, amino acids, vegetable oil, tetrahydropyrimidine, fumaric acid, and citric acid used in this invention are all commercially available products.

[0054] Unless otherwise specified, all bacterial strains mentioned in the examples are publicly available strains and do not involve microbial preservation. All bacterial agents mentioned in the examples were processed by Shandong Lihaoyuan Bioengineering Co., Ltd.

[0055] Example 1:

[0056] A microbial protectant, with the following composition: 80 parts of brown algae oligosaccharide, 6 parts of vegetable oil, 5 parts of tetrahydropyrimidine, and 9 parts of citric acid.

[0057] The above-mentioned microbial protectant was prepared according to the following method:

[0058] Brown algae oligosaccharides, vegetable oil, tetrahydropyrimidine, and citric acid are added to a mixer using a quantitative feeder. The mixer is then turned on to mix the above raw materials evenly, thus obtaining a microbial protectant.

[0059] The above-mentioned microbial protectant was mixed evenly with Bacillus subtilis (ACCC60364) bacterial powder (200 billion CFU / g), with a mass ratio of microbial protectant to Bacillus subtilis bacterial powder of 7.5:1, to prepare a Bacillus subtilis inoculum containing the microbial protectant, wherein the viable number of Bacillus subtilis was 23.5 billion CFU / g. This Bacillus subtilis inoculum containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer, with a mass ratio of the inoculum to the NPK compound fertilizer of 1:28.4. After mixing evenly, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical value of the viable number of Bacillus subtilis in the obtained chemical fertilizer-biocompound fertilizer was 800 million CFU / g, and the NPK content was 51.7%. On the first day of production, the viable Bacillus subtilis count in the chemical-biological compound fertilizer was 730 million CFU / g. After 12 months of storage at room temperature, the viable Bacillus subtilis count in the fertilizer decreased to 635 million CFU / g. The application rate of the chemical-biological compound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus subtilis bacteria in the soil was 602,000 CFU / g soil.

[0060] Example 2:

[0061] A microbial protectant, identical to that in Example 1.

[0062] The above-mentioned microbial protectant was mixed evenly with Bacillus amyloliquefaciens (ACCC60428) bacterial powder (150 billion CFU / g), with a mass ratio of microbial protectant to Bacillus amyloliquefaciens bacterial powder of 5.56:1, to prepare a Bacillus amyloliquefaciens inoculum containing the microbial protectant, wherein the viable number of Bacillus amyloliquefaciens was 22.9 billion CFU / g. This Bacillus amyloliquefaciens inoculum containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer, with a mass ratio of the inoculum to the NPK compound fertilizer of 1:27.25. After mixing evenly, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical value of the viable number of Bacillus amyloliquefaciens in the obtained chemical fertilizer-biocompound fertilizer was 810 million CFU / g, and the NPK content was 51.6%. On the first day of production, the viable count of Bacillus amyloliquefaciens in the chemical-biological compound fertilizer was 635 million CFU / g. After 12 months of storage at room temperature, the viable count of Bacillus amyloliquefaciens in the fertilizer decreased to 580 million CFU / g. The application rate of the chemical-biological compound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus amyloliquefaciens in the soil was 585,000 CFU / g soil.

[0063] The above results indicate that although the microbial protectant shown in Example 1 can also be used to protect Bacillus amyloliquefaciens, its protective effect is far less than that for Bacillus subtilis.

[0064] Example 3:

[0065] A microbial protectant, identical to that in Example 1.

[0066] The above-mentioned microbial protectant, Bacillus megaterium JD2023Hb bacterial powder (100 billion CFU / g), Bacillus subtilis KC2023Hb bacterial powder (200 billion CFU / g), and Bacillus amyloliquefaciens (ACCC60428) bacterial powder (150 billion CFU / g) were mixed evenly. The mass ratio of the microbial protectant, Bacillus megaterium JD2023Hb bacterial powder, Bacillus subtilis KC2023Hb bacterial powder, and Bacillus amyloliquefaciens ACCC60428 bacterial powder was 3:0.1:0.1:0.54, thus preparing a mixed bacterial agent containing the microbial protectant, wherein the viable number of Bacillus amyloliquefaciens was 21.7 billion CFU / g. The above-mentioned mixed microbial inoculant containing microbial protectant was added to the nitrogen-phosphorus-potassium compound fertilizer at a mass ratio of 1:25.74. After thorough mixing, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical value of the viable Bacillus amyloliquefaciens count in the resulting chemical fertilizer-biocompound fertilizer was 810 million CFU / g, and the nitrogen, phosphorus, and potassium content was 51.4%. On the first day of production, the viable Bacillus amyloliquefaciens count in the chemical fertilizer-biocompound fertilizer was 726 million CFU / g. After 12 months of storage at room temperature, the viable Bacillus amyloliquefaciens count in the chemical fertilizer-biocompound fertilizer was 623 million CFU / g. The application rate of the chemical fertilizer-biocompound fertilizer was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus amyloliquefaciens in the soil was 603,000 CFU / g soil.

[0067] The above results indicate that, compared with Example 2, adding Bacillus megaterium JD2023Hb and Bacillus subtilis KC2023Hb bacterial powder to the microbial protectant can further improve the tolerance of Bacillus amyloliquefaciens to high concentrations of inorganic fertilizers and enhance the biological activity of Bacillus amyloliquefaciens.

[0068] Comparative Example 1:

[0069] A microbial protectant, which differs from Example 1 in that its composition is: 80 parts burdock oligosaccharide, 6 parts vegetable oil, 5 parts tetrahydropyrimidine, and 9 parts citric acid.

[0070] The above-mentioned microbial protectant was prepared according to the following method:

[0071] Burdock oligosaccharide, vegetable oil, tetrahydropyrimidine, and citric acid were added to a mixer using a quantitative feeder. The mixer was then turned on to mix the above raw materials evenly, thus obtaining a microbial protectant.

[0072] The above-mentioned microbial protectant was mixed evenly with Bacillus subtilis (ACCC60364) bacterial powder (200 billion CFU / g), with a mass ratio of microbial protectant to Bacillus subtilis bacterial powder of 7.5:1, to prepare a Bacillus subtilis inoculum containing the microbial protectant, wherein the viable number of Bacillus subtilis was 23.5 billion CFU / g. This Bacillus subtilis inoculum containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer, with a mass ratio of the inoculum to the NPK compound fertilizer of 1:28.4. After mixing evenly, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical value of the viable number of Bacillus subtilis in the obtained chemical fertilizer-biocompound fertilizer was 800 million CFU / g, and the NPK content was 51.7%. On the first day of production, the viable Bacillus subtilis count in the chemical-biocompound fertilizer was 680 million CFU / g. After 12 months of storage at room temperature, the viable Bacillus subtilis count in the fertilizer decreased to 603 million CFU / g. The application rate of the chemical-biocompound fertilizer was 100 kg / 667 m³. 2 Ten days later, the number of live Bacillus subtilis bacteria in the soil was 556,000 CFU / g soil.

[0073] The above results indicate that although replacing the brown algae oligosaccharide in the microbial protectant with burdock oligosaccharide can also protect microorganisms to a certain extent, the protective effect is inferior to that of Example 1. The composition of the microbial protectant in Example 1 is the optimal composition of the Bacillus subtilis protectant.

[0074] Comparative Example 2:

[0075] Without using microbial preservatives, Bacillus subtilis (ACCC60364) powder (200 billion CFU / g) was directly added to NPK compound fertilizer at a mass ratio of 1:249. After thorough mixing, the mixture was granulated to obtain a chemical-biological compound fertilizer. The theoretical value of the viable Bacillus subtilis count in the resulting chemical-biological compound fertilizer was 800 million CFU / g, with an NPK content of 53%. On the first day of production, the viable Bacillus subtilis count in the chemical-biological compound fertilizer was 520 million CFU / g. After 12 months of storage at room temperature, the viable Bacillus subtilis count in the chemical-biological compound fertilizer was 420 million CFU / g. The fertilizer was then applied to the soil at a rate of 100 kg / 667 m². 2 Ten days later, the number of live Bacillus subtilis bacteria in the soil was 354,000 CFU / g soil.

[0076] The above results indicate that without the use of microbial preservatives, a large number of microorganisms will die when mixed with inorganic fertilizers for granulation. Furthermore, the activity of microorganisms cannot be maintained during the storage of chemical fertilizer-biocompound fertilizers, and the number of viable microorganisms continues to decline.

[0077] Example 4:

[0078] A microbial protectant, with the following composition: 72 parts of fucoidan, 6 parts of burdock oligosaccharide, 8 parts of vegetable oil, 6 parts of tetrahydropyrimidine, and 8 parts of fumaric acid.

[0079] The above-mentioned microbial protectant was prepared according to the following method:

[0080] Brown algae oligosaccharides, burdock oligosaccharides, vegetable oil, tetrahydropyrimidine, and fumaric acid were added to a mixer using a quantitative feeder. The mixer was then turned on to mix the above raw materials evenly, thus obtaining a microbial protectant.

[0081] The above-mentioned microbial protectant was mixed evenly with Bacillus amyloliquefaciens (ACCC60428) bacterial powder (150 billion CFU / g), with a mass ratio of microbial protectant to Bacillus amyloliquefaciens of 5.56:1, to prepare a Bacillus amyloliquefaciens inoculum containing the microbial protectant, wherein the viable number of Bacillus amyloliquefaciens was 22.9 billion CFU / g. This Bacillus amyloliquefaciens inoculum containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer, with a mass ratio of the inoculum to the NPK compound fertilizer of 1:27.25. After mixing evenly, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical value of the viable number of Bacillus amyloliquefaciens in the obtained chemical fertilizer-biocompound fertilizer was 810 million CFU / g, and the NPK content was 51.6%. On the first day of production, the viable Bacillus amyloliquefaciens count in the chemical-biological compound fertilizer was 750 million CFU / g. After 12 months of storage at room temperature, the viable Bacillus amyloliquefaciens count in the fertilizer decreased to 689 million CFU / g. The application rate of the chemical-biological compound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus amyloliquefaciens in the soil was 623,000 CFU / g soil.

[0082] Example 5:

[0083] A microbial protectant, identical to that in Example 4.

[0084] The above-mentioned microbial protectant was mixed evenly with Bacillus subtilis (ACCC60364) bacterial powder (200 billion CFU / g), with a mass ratio of microbial protectant to Bacillus subtilis bacterial powder of 7.5:1, to prepare a Bacillus subtilis inoculum containing the microbial protectant, wherein the viable number of Bacillus subtilis was 23.5 billion CFU / g. This Bacillus subtilis inoculum containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer, with a mass ratio of the inoculum to the NPK compound fertilizer of 1:28.41. After mixing evenly, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical value of the viable number of Bacillus subtilis in the obtained chemical fertilizer-biocompound fertilizer was 800 million CFU / g, and the NPK content was 51.7%. On the first day of production, the number of viable Bacillus subtilis in the chemical-biocompound fertilizer was 626 million CFU / g. After 12 months of storage at room temperature, the number of viable Bacillus subtilis in the chemical-biocompound fertilizer was 573 million CFU / g. The application rate of the chemical-biocompound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of live Bacillus subtilis bacteria in the soil was 586,000 CFU / g soil.

[0085] The above results indicate that although the microbial protectant shown in Example 4 can also be used to protect Bacillus subtilis, its protective effect is not as good as that for Bacillus amyloliquefaciens.

[0086] Example 6:

[0087] A microbial protectant, identical to that in Example 4.

[0088] The above-mentioned microbial protectant, Bacillus megaterium JD2023Hb powder (100 billion CFU / g), Bacillus subtilis KC2023Hb powder (200 billion CFU / g), and Bacillus subtilis (ACCC60364) powder (200 billion CFU / g) were mixed evenly. The mass ratio of the microbial protectant, Bacillus megaterium JD2023Hb powder, Bacillus subtilis KC2023Hb powder, and Bacillus subtilis ACCC60364 powder was 3:0.1:0.1:0.4 to prepare a mixed microbial agent containing the microbial protectant, wherein the viable number of Bacillus subtilis ACCC60364 was 22.2 billion CFU / g. The above-mentioned mixed microbial inoculant containing microbial protectant was added to the nitrogen-phosphorus-potassium compound fertilizer at a mass ratio of 1:26.78. After thorough mixing, the mixture was granulated to obtain a chemical-fertilizer-biocompound fertilizer. The theoretical value of the viable Bacillus subtilis ACCC60364 count in the resulting chemical-fertilizer-biocompound fertilizer was 800 million CFU / g, and the nitrogen, phosphorus, and potassium content was 51.5%. On the first day of production, the viable Bacillus subtilis ACCC60364 count in the chemical-fertilizer-biocompound fertilizer was 724 million CFU / g. After 12 months of storage at room temperature, the viable Bacillus subtilis ACCC60364 count in the chemical-fertilizer-biocompound fertilizer was 682 million CFU / g. The application rate of the chemical-fertilizer-biocompound fertilizer was 100 kg / 667 m². 2 Ten days later, the viable count of Bacillus subtilis ACCC60364 was 618,000 CFU / g.

[0089] The above results indicate that, compared with Example 5, adding Bacillus megaterium JD2023Hb and Bacillus subtilis KC2023Hb bacterial powder to the microbial protectant can further improve the tolerance of Bacillus subtilis ACCC60364 to high concentrations of inorganic fertilizers and enhance the biological activity of Bacillus subtilis ACCC60364.

[0090] Comparative Example 3:

[0091] A microbial protectant, which differs from Example 4 in that its composition is: 72 parts of fucoidan, 6 parts of burdock oligosaccharide, 8 parts of vegetable oil, 6 parts of trehalose, and 8 parts of fumaric acid.

[0092] The above-mentioned microbial protectant was prepared according to the following method:

[0093] The following ingredients are added to a mixer using a quantitative feeder: fucoidan, burdock oligosaccharide, vegetable oil, trehalose, and fumaric acid. The mixer is then turned on to mix the ingredients evenly, thus obtaining the microbial protectant.

[0094] The above-mentioned microbial protectant was mixed evenly with Bacillus amyloliquefaciens (ACCC60428) bacterial powder (150 billion CFU / g), with a mass ratio of microbial protectant to Bacillus amyloliquefaciens bacterial powder of 5.56:1, to prepare a Bacillus amyloliquefaciens inoculum containing the microbial protectant, wherein the viable number of Bacillus amyloliquefaciens was 22.9 billion CFU / g. This Bacillus amyloliquefaciens inoculum containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer, with a mass ratio of the inoculum to the NPK compound fertilizer of 1:27.25. After mixing evenly, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical value of the viable number of Bacillus amyloliquefaciens in the obtained chemical fertilizer-biocompound fertilizer was 810 million CFU / g, and the NPK content was 51.6%. On the first day of production, the viable Bacillus amyloliquefaciens count in the chemical-biological compound fertilizer was 720 million CFU / g. After 12 months of storage at room temperature, the viable Bacillus amyloliquefaciens count in the fertilizer decreased to 635 million CFU / g. The application rate of the chemical-biological compound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus amyloliquefaciens in the soil was 563,000 CFU / g soil.

[0095] The above results indicate that, compared with Example 4, replacing tetrahydropyrimidine with trehalose in the microbial protectant, although both can stabilize the protein structure of the strain, the protective effect of the microbial protectant in Comparative Example 3 on Bacillus amyloliquefaciens is inferior to that in Example 4. The composition of the microbial protectant in Example 4 is the optimal composition for the protectant of Bacillus amyloliquefaciens.

[0096] Comparative Example 4:

[0097] Without using microbial preservatives, *Bacillus amyloliquefaciens* (ACCC60428) powder (150 billion CFU / g) was directly added to NPK compound fertilizer. The mass ratio of *Bacillus amyloliquefaciens* powder to NPK compound fertilizer was 1:184.9. After thorough mixing, the mixture was granulated to obtain a chemical-biological compound fertilizer. The theoretical value of the viable *Bacillus amyloliquefaciens* count in the resulting chemical-biological compound fertilizer was 810 million CFU / g, and the NPK content was 52.9%. On the first day of production, the viable *Bacillus amyloliquefaciens* count in the chemical-biological compound fertilizer was 590 million CFU / g. After 12 months of storage at room temperature, the viable *Bacillus amyloliquefaciens* count in the chemical-biological compound fertilizer was 460 million CFU / g. The application rate of the chemical-biological compound fertilizer was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus amyloliquefaciens in the soil was 364,000 CFU / g soil.

[0098] The above results indicate that without the use of microbial preservatives, a large number of microorganisms will die when mixed with inorganic fertilizers for granulation. Furthermore, the activity of microorganisms cannot be maintained during the storage of the fertilizer-biocompound fertilizer, and the number of viable microorganisms continues to decline.

[0099] Example 7:

[0100] A microbial protectant, with the following composition: 74 parts of fucoidan, 6 parts of chitosan oligosaccharide, 6 parts of vegetable oil, 6 parts of tetrahydropyrimidine, and 8 parts of fumaric acid.

[0101] The above-mentioned microbial protectant was prepared according to the following method:

[0102] A quantitative feeder is used to add brown algae oligosaccharides, chitosan oligosaccharides, vegetable oil, tetrahydropyrimidine, and fumaric acid into a mixer. The mixer is then turned on to mix the above raw materials evenly, thus obtaining a microbial protectant.

[0103] The above-mentioned microbial protectant was mixed evenly with *Bacillus amyloliquefaciens* (ACCC10167) powder (150 billion CFU / g) and *Bacillus laterosporus* powder (CGMCC No. 17377) (10 billion CFU / g). The mass ratio of the microbial protectant, *Bacillus amyloliquefaciens* powder, and *Bacillus laterosporus* powder was 3:0.4:2, resulting in a mixed microbial agent containing the microbial protectant. The total viable count of the mixed bacteria was 14.8 billion CFU / g. This mixed microbial agent containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer at a mass ratio of 1:17.52. After even mixing, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical viable count of *Bacillus amyloliquefaciens* in the obtained chemical fertilizer-biocompound fertilizer was 600 million CFU / g, the theoretical viable count of *Bacillus laterosporus* was 200 million CFU / g, and the nitrogen, phosphorus, and potassium content was 50.6%. On the first day of production, the viable count of *Bacillus amyloliquefaciens* in the chemical-biocompound fertilizer was 550 million CFU / g, and the viable count of *Bacillus laterosporus* was 180 million CFU / g. After 12 months of storage at room temperature, the viable count of *Bacillus amyloliquefaciens* in the chemical-biocompound fertilizer was 498 million CFU / g, and the viable count of *Bacillus laterosporus* was 155 million CFU / g. The application rate of the chemical-biocompound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus amyloliquefaciens in the soil was 409,000 CFU / g soil, and the number of viable Bacillus lateralis was 168,500 CFU / g soil.

[0104] Comparative Example 5:

[0105] A microbial protectant, which differs from Example 7 in that its composition is: 74 parts burdock oligosaccharide, 6 parts chitosan oligosaccharide, 6 parts vegetable oil, 6 parts tetrahydropyrimidine, and 8 parts fumaric acid.

[0106] The above-mentioned microbial protectant was prepared according to the following method:

[0107] Burdock oligosaccharide, chitosan oligosaccharide, vegetable oil, tetrahydropyrimidine, and fumaric acid were added to a mixer using a quantitative feeder. The mixer was then turned on to mix the above raw materials evenly, thus obtaining the microbial protectant.

[0108] The above-mentioned microbial protectant was mixed evenly with *Bacillus amyloliquefaciens* (ACCC10167) powder (150 billion CFU / g) and *Bacillus laterosporus* powder (CGMCC No. 17377) (10 billion CFU / g). The mass ratio of the microbial protectant, *Bacillus amyloliquefaciens* powder, and *Bacillus laterosporus* powder was 3:0.4:2, resulting in a mixed microbial agent containing the microbial protectant. The total viable count of the mixed bacteria was 14.8 billion CFU / g. This mixed microbial agent containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer at a mass ratio of 1:17.52. After even mixing, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical viable count of *Bacillus amyloliquefaciens* in the obtained chemical fertilizer-biocompound fertilizer was 600 million CFU / g, the theoretical viable count of *Bacillus laterosporus* was 200 million CFU / g, and the nitrogen, phosphorus, and potassium content was 50.6%. On the first day of production, the viable count of *Bacillus amyloliquefaciens* in the chemical-biocompound fertilizer was 510 million CFU / g, and the viable count of *Bacillus laterosporus* was 167 million CFU / g. After 12 months of storage at room temperature, the viable count of *Bacillus amyloliquefaciens* in the chemical-biocompound fertilizer was 463 million CFU / g, and the viable count of *Bacillus laterosporus* was 149 million CFU / g. The application rate of the chemical-biocompound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus amyloliquefaciens in the soil was 392,000 CFU / g soil, and the number of viable Bacillus lateralis was 163,500 CFU / g soil.

[0109] Comparative Example 6

[0110] Without using microbial preservatives, *Bacillus amyloliquefaciens* (ACCC10167) powder (150 billion CFU / g) and *Bacillus laterosporus* powder (CGMCC No. 17377) (10 billion CFU / g) were directly added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer. The mass ratio of the NPK compound fertilizer, *Bacillus amyloliquefaciens* powder, and *Bacillus laterosporus* powder was 97.6:0.4:2. After thorough mixing, the mixture was granulated to obtain a chemical-biological compound fertilizer. The theoretical value of the viable *Bacillus amyloliquefaciens* in the obtained chemical-biological compound fertilizer was 600 million CFU / g, the theoretical value of the viable *Bacillus laterosporus* powder was 200 million CFU / g, and the NPK content was 52.3%. On the first day of production, the viable count of *Bacillus amyloliquefaciens* in the chemical-biocompound fertilizer was 360 million CFU / g, and the viable count of *Bacillus laterosporus* was 120 million CFU / g. After 12 months of storage at room temperature, the viable count of *Bacillus amyloliquefaciens* in the chemical-biocompound fertilizer was 268 million CFU / g, and the viable count of *Bacillus laterosporus* was 55 million CFU / g. The application rate of the chemical-biocompound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of viable Bacillus amyloliquefaciens in the soil was 206,000 CFU / g soil, and the number of viable Bacillus lateralis was 53,500 CFU / g soil.

[0111] Example 8:

[0112] A microbial protectant, with the following composition: 75 parts of fucoidan, 7 parts of chitosan oligosaccharide, 6 parts of vegetable oil, 5 parts of tetrahydropyrimidine, and 7 parts of citric acid.

[0113] The above-mentioned microbial protectant was prepared according to the following method:

[0114] A quantitative feeder is used to add brown algae oligosaccharides, chitosan oligosaccharides, vegetable oil, tetrahydropyrimidine, and citric acid into a mixer. The mixer is then turned on to mix the above raw materials evenly, thus obtaining a microbial protectant.

[0115] The above-mentioned microbial protectant was mixed evenly with Bacillus subtilis (ACCC19742) powder (200 billion CFU / g) and Bacillus mucilaginosus (CGMCC No. 17376) powder (20 billion CFU / g). The mass ratio of the microbial protectant, Bacillus subtilis powder, and Bacillus mucilaginosus powder was 3:0.3:1, resulting in a mixed microbial agent containing the microbial protectant. The total viable count of the mixed bacteria was 18.6 billion CFU / g. This mixed microbial agent containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer at a mass ratio of 1:22.26. After even mixing, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical viable count of Bacillus subtilis in the obtained chemical fertilizer-biocompound fertilizer was 600 million CFU / g, the theoretical viable count of Bacillus mucilaginosus was 200 million CFU / g, and the nitrogen, phosphorus, and potassium content was 51.1%. On the first day of production, the viable count of Bacillus subtilis in the chemical-biocompound fertilizer was 560 million CFU / g, and the viable count of Bacillus spp. was 176 million CFU / g. After 12 months of storage at room temperature, the viable count of Bacillus subtilis in the chemical-biocompound fertilizer was 487 million CFU / g, and the viable count of Bacillus spp. was 150 million CFU / g. The application rate of the chemical-biocompound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of live Bacillus subtilis in the soil was 413,000 CFU / g soil, and the number of live Bacillus colloidis was 160,200 CFU / g soil.

[0116] Comparative Example 7

[0117] A microbial protectant, which differs from Example 8 in that its composition is: 75 parts of fucoidan, 7 parts of chitosan oligosaccharide, 6 parts of vegetable oil, 5 parts of trehalose, and 7 parts of citric acid.

[0118] The above-mentioned microbial protectant was prepared according to the following method:

[0119] A quantitative feeder is used to add fucoidan, chitosan oligosaccharide, vegetable oil, trehalose, and citric acid into a mixer. The mixer is then turned on to mix the above raw materials evenly, thus obtaining a microbial protectant.

[0120] The above-mentioned microbial protectant was mixed evenly with Bacillus subtilis (ACCC19742) powder (200 billion CFU / g) and Bacillus mucilaginosus (CGMCC No. 17376) powder (20 billion CFU / g). The mass ratio of the microbial protectant, Bacillus subtilis powder, and Bacillus mucilaginosus powder was 3:0.3:1, resulting in a mixed microbial agent containing the microbial protectant. The total viable count of the mixed bacteria was 18.6 billion CFU / g. This mixed microbial agent containing the microbial protectant was added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer at a mass ratio of 1:22.26. After even mixing, the mixture was granulated to obtain a chemical fertilizer-biocompound fertilizer. The theoretical viable count of Bacillus subtilis in the obtained chemical fertilizer-biocompound fertilizer was 600 million CFU / g, the theoretical viable count of Bacillus mucilaginosus was 200 million CFU / g, and the nitrogen, phosphorus, and potassium content was 51.1%. On the first day of production, the viable count of Bacillus subtilis in the chemical-biocompound fertilizer was 530 million CFU / g, and the viable count of Bacillus mucilaginosus was 165 million CFU / g. After 12 months of storage at room temperature, the viable count of Bacillus subtilis in the chemical-biocompound fertilizer was 465 million CFU / g, and the viable count of Bacillus mucilaginosus was 132 million CFU / g. The application rate of the chemical-biocompound fertilizer to the soil was 100 kg / 667 m². 2 Ten days later, the number of live Bacillus subtilis in the soil was 398,000 CFU / g soil, and the number of live Bacillus colloidis was 153,000 CFU / g soil.

[0121] Comparative Example 8

[0122] Without using microbial preservatives, Bacillus subtilis (ACCC19742) powder (200 billion CFU / g) and Bacillus mucilaginosus (CGMCC No. 17376) powder (20 billion CFU / g) were directly added to a nitrogen-phosphorus-potassium (NPK) compound fertilizer. The mass ratio of the NPK compound fertilizer, Bacillus subtilis powder, and Bacillus mucilaginosus powder was 98.7:0.3:1. After thorough mixing, the mixture was granulated to obtain a chemical-biological compound fertilizer. The theoretical value of the viable Bacillus subtilis count in the obtained chemical-biological compound fertilizer was 600 million CFU / g, the theoretical value of the viable Bacillus mucilaginosus count was 200 million CFU / g, and the NPK content was 52.8%. On the first day of production, the viable count of Bacillus subtilis in the chemical-biocompound fertilizer was 420 million CFU / g, and the viable count of Bacillus spp. was 102 million CFU / g. After 12 months of storage at room temperature, the viable count of Bacillus subtilis in the chemical-biocompound fertilizer was 289 million CFU / g, and the viable count of Bacillus spp. was 60 million CFU / g. The application rate of the chemical-biocompound fertilizer to the soil was 100 kg / 667 m². 2Ten days later, the number of live Bacillus subtilis in the soil was 210,500 CFU / g soil, and the number of live Bacillus spp. was 68,000 CFU / g soil.

Claims

1. A microbial protectant, characterized in that, It mainly includes alginate oligosaccharides and tetrahydropyrimidine; the mass fractions of each component in the microbial protectant are: alginate oligosaccharides 65-85 parts and tetrahydropyrimidine 5-8 parts.

2. The microbial protectant as described in claim 1, characterized in that, The microbial protectant also includes at least one of burdock oligosaccharide, chitosan oligosaccharide, amino acids, vegetable oil, fumaric acid, and citric acid.

3. The microbial protectant as described in claim 2, characterized in that, The mass fractions of each component in the microbial protectant are as follows: 65-85 parts of brown algae oligosaccharide, 4-6 parts of burdock oligosaccharide, 6-10 parts of chitosan oligosaccharide, 6-10 parts of amino acids, 5-10 parts of vegetable oil, 5-8 parts of tetrahydropyrimidine, 8-10 parts of fumaric acid, and 6-10 parts of citric acid.

4. The microbial protectant as described in claim 3, characterized in that, The molecular weight of the algae oligosaccharide is below 2000 Da, the degree of polymerization is 2-20, and the 2-6 sugar content reaches more than 70%.

5. The microbial protectant as described in claim 3, characterized in that, The degree of polymerization of the burdock oligosaccharide is 10-15.

6. The microbial protectant as described in claim 3, characterized in that, The amino acids include one or more of glutamic acid, aspartic acid, and phenylalanine.

7. The microbial protectant as described in claim 3, characterized in that, The types of vegetable oils include: soybean oil and / or castor oil.

8. The method for preparing the microbial protectant according to claim 1 or 2, characterized in that, Includes the following steps: The raw materials of each component of the microbial protectant are quantitatively obtained, stirred and mixed evenly to obtain the final product.

9. The preparation method according to claim 8, characterized in that, The raw materials of the microbial protectant are added to the mixer using a quantitative feeder, and the mixer is turned on. After mixing, the microbial protectant is obtained.

10. The use of the microbial protectant according to claim 1 or 2 in the preparation of microbial inoculant complexes.

11. The application of the microbial protectant according to claim 1 or 2 in the preparation of chemical fertilizer and bio-compound fertilizer.

12. A microbial agent complex containing the microbial protectant of claim 1 or 2.

13. The microbial inoculant complex as described in claim 12, characterized in that, The mass percentage of microbial protectant in the microbial agent complex is above 50%.

14. A chemical fertilizer-biocompound fertilizer containing the microbial protectant as described in claim 1 or 2 or the microbial inoculant complex as described in claim 12.

15. The chemical-biological compound fertilizer as described in claim 14, characterized in that, The chemical fertilizer-biocompound fertilizer contains ≥40% nitrogen, phosphorus, and potassium nutrients, and has a viable microbial count of over 200 million CFU / g.