Microbial preparation suitable for saline-alkali soil and preparation method and application thereof

Abstract

The invention belongs to the technical field of microbial preparations, and particularly relates to a microbial preparation suitable for saline-alkali soil and a preparation method and application thereof. The microbial agent comprises the following components in parts by weight: 4-25 parts of bacillus subtilis, 4-30 parts of saccharomyces cerevisiae, 40-85 parts of a synergistic carrier and 5-20 parts of a protective carrier, the synergistic carrier comprises one or more than two selected from a substance group consisting of yeast metabolites, mineral humic acid and amino acid powder; the protective carrier comprises one or more than two selected from a substance group consisting of maltodextrin, trehalose, corn starch and attapulgite. According to the invention, the bacillus subtilis and the saccharomyces cerevisiae cooperate with each other, and the synergistic carrier and the protective carrier are combined, so that the obtained microbial preparation has an excellent effect of improving saline-alkali soil, and also has the effects of promoting crop growth and improving saline-alkali resistance of crops. The microbial agent is simple in preparation process, easy to operate, stable in quality, low in cost and more suitable for industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial preparations, and in particular relates to a microbial preparation suitable for saline-alkali soil and a preparation method and application thereof. Background Art

[0002] Saline-alkali soil is a general term for saline soil and alkaline soil. Soil salinization, also known as soil salinization, refers to the geological process and phenomenon in which the salt content of the soil surface increases continuously under natural and human influences and exceeds a certain limit. Saline-alkali soil affects the absorption of nutrients by crops, reduces the effectiveness of soil nutrients, and deteriorates the physical and biological properties of the soil. Studies have shown that excessive soil salt content will seriously affect the yield of crops.

[0003] The current biological improvement technology of saline-alkali soil is to use the life activities of microorganisms to increase the content of nitrogen, available phosphorus and available potassium in the soil, convert some substances in the soil that cannot be directly used by plants into nutrients that can be absorbed and utilized by them, inhibit plant pathogens by adjusting the material level of crop contents, improve soil fertility, reduce the salt content and pH of saline-alkali soil, and achieve the purpose of improving saline-alkali soil conditions. At present, strains with growth-promoting and disease-resistant properties have been made into microbial fertilizers or microbial pesticides, but they are mainly used in conventional soils, and their application in saline-alkali soils is not ideal. Therefore, it is urgent to develop a microbial preparation suitable for saline-alkali land and with excellent performance. Summary of the invention

[0004] The technical problem solved by the present invention is that the effect of applying microbial fertilizers in saline-alkali soil in the prior art is not ideal.

[0005] In view of the above technical problems, the present invention provides a microbial preparation suitable for saline-alkali soil and a preparation method and application thereof.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a microbial preparation suitable for saline-alkali soil, which comprises, by weight: 4 to 25 parts of Bacillus subtilis, 4 to 30 parts of Saccharomyces cerevisiae, 40 to 85 parts of a synergistic carrier and 5 to 20 parts of a protective carrier; wherein the synergistic carrier comprises one or more of a substance group consisting of yeast metabolites, mineral humic acid and amino acid powder; and the protective carrier comprises one or more of a substance group consisting of maltodextrin, trehalose, corn starch and attapulgite.

[0008] Preferably, the microbial preparation comprises 4 to 20 parts by weight of Bacillus subtilis; more preferably, the microbial preparation comprises 4 to 13 parts by weight of Bacillus subtilis; and even more preferably, the microbial preparation comprises 4 to 6 parts by weight of Bacillus subtilis. And / or

[0009] Preferably, the microbial preparation comprises 4 to 20 parts of Saccharomyces cerevisiae by weight; more preferably, the microbial preparation comprises 4 to 6 parts of Saccharomyces cerevisiae. And / or

[0010] Preferably, the microbial preparation comprises 60 to 85 parts of the synergistic carrier by weight; more preferably, the microbial preparation comprises 75 to 85 parts of the synergistic carrier; and even more preferably, the microbial preparation comprises 78 to 82 parts of the synergistic carrier. And / or

[0011] Preferably, the microbial preparation comprises 5 to 15 parts of protective carriers by weight; more preferably, the microbial preparation comprises 5 to 12 parts of protective carriers; and even more preferably, the microbial preparation comprises 8 to 11 parts of protective carriers. And / or

[0012] Preferably, the Bacillus subtilis is Bacillus subtilis subsp. subtilis tc-4, which was deposited in China Center for Type Culture Collection (CCTCC) on April 17, 2023, with a deposit number of CCTCC NO: M 2023541. And / or

[0013] Preferably, the brewer's yeast is brewer's yeast YY (Saccharomyces cerevisiae high wine yeast YY), which was deposited in the China Center for Type Culture Collection (CCTCC) on February 1, 2021, with a deposit number of CCTCCNO: M 2021171; or the brewer's yeast is brewer's yeast d8.8 (Saccharomyces cerevisiae d8.8), which was deposited in the China Center for Type Culture Collection (CCTCC) on March 29, 2017, with a deposit number of CCTCC NO: M2017148; more preferably, the brewer's yeast is brewer's yeast YY (Saccharomyces cerevisiae high wine yeast YY), which was deposited in the China Center for Type Culture Collection (CCTCC) on February 1, 2021, with a deposit number of CCTCC NO: M 2021171.

[0014] and / or

[0015] Preferably, the synergistic carrier includes yeast metabolites; more preferably, the yeast metabolites are metabolites obtained by fermenting molasses to extract yeast; more preferably, the molasses includes beet molasses; and / or, more preferably, the content of fulvic acid in the yeast metabolites is 15% to 30% based on the dry weight of the yeast metabolites; more preferably, the content of betaine in the yeast metabolites is 6% to 15% based on the dry weight of the yeast metabolites. And / or

[0016] Preferably, the protective carrier comprises one or more selected from the group consisting of trehalose, corn starch and attapulgite; more preferably, the protective carrier comprises corn starch and / or attapulgite.

[0017] Preferably, the effective viable count of Bacillus subtilis in the microbial preparation is 4 billion to 25 billion CFU / g; more preferably, the effective viable count of Bacillus subtilis in the microbial preparation is 4 billion to 20 billion CFU / g. And / or

[0018] Preferably, the effective viable count of Saccharomyces cerevisiae in the microbial preparation is 800 million to 6 billion CFU / g; more preferably, the effective viable count of Saccharomyces cerevisiae in the microbial preparation is 800 million to 4 billion CFU / g.

[0019] and / or

[0020] Preferably, the Bacillus subtilis is Bacillus subtilis powder, and the preparation method thereof comprises the following steps:

[0021] Step 1: Activation of Bacillus subtilis;

[0022] Step 2: inoculating the activated Bacillus subtilis into a culture medium for culturing to obtain a seed solution; and

[0023] Step 3: inoculating the seed liquid into a fermentation medium for culturing to obtain a fermentation liquid, and drying to obtain Bacillus subtilis powder;

[0024] More preferably, in step 1, based on the mass of the culture medium used for activation, the culture medium used for activation comprises 0.5% to 1.5% peptone, 0.1% to 0.5% beef extract, 0.3% to 0.7% sodium chloride and 1.5% to 2% agar, and the remainder is water; further preferably, the culture medium used for activation comprises 1% peptone, 0.3% beef extract, 0.5% sodium chloride and 1.5% to 2% agar, and the remainder is water; and / or

[0025] More preferably, in step 1, the culture temperature is 35-39° C.; and / or the culture time is 21-27 h; and / or

[0026] More preferably, the cultured Bacillus subtilis is further activated;

[0027] More preferably, the activation medium comprises, by weight of the activation medium, 0.5% to 1.5% peptone, 0.1% to 0.5% beef extract, 0.3% to 0.7% sodium chloride and 1.5% to 2% agar, with the remainder being water; most preferably, the activation medium comprises 1% peptone, 0.3% beef extract, 0.5% sodium chloride and 1.5% to 2% agar, with the remainder being water; and / or

[0028] Preferably, the temperature of the further activation culture is 35-39° C.; and / or, the culture time is 21-27 h; and / or,

[0029] More preferably, in step 2, based on the total mass of the culture medium, the culture medium includes 0.5% to 1.5% peptone, 0.1% to 0.5% beef powder, 0.3% to 0.7% sodium chloride and 0.05% to 0.15% glucose, and the rest is water; further preferably, the culture medium includes 1% peptone, 0.3% beef powder, 0.5% sodium chloride and 0.1% glucose, and the rest is water; and / or, the culture temperature is 35 to 39° C.; and / or, the culture time is 10 to 15 h; and / or, the rotation speed is 100 to 200 rpm; and / or

[0030] More preferably, in step 3, based on the total mass of the culture medium, the culture medium includes 3% to 5% corn flour, 3% to 5% soybean meal, 0.05% to 0.15% magnesium sulfate and 0.05% to 0.15% calcium chloride, and the rest is water; more preferably, the culture medium includes 4% corn flour, 4% soybean meal, 0.1% magnesium sulfate and 0.1% calcium chloride, and the rest is water; and / or, the culture temperature is 35 to 39°C; and / or, the culture time is 15 to 20h; and / or, the ventilation volume is 20 to 50L / min; and / or, the tank pressure is 0.2 to 0.5 standard atmospheres. And / or preferably, the brewer's yeast is brewer's yeast powder, and its preparation method comprises the following steps:

[0031] Step 1: Activation of Saccharomyces cerevisiae;

[0032] Step 2: inoculating the activated Saccharomyces cerevisiae into a culture medium for culturing to obtain a seed solution;

[0033] Step 3: inoculating the seed liquid into a fermentation medium for culturing to obtain a fermentation liquid, and separating and drying to obtain brewer's yeast powder;

[0034] More preferably, in step 1, based on the total mass of the culture medium used for activation, the culture medium used for activation comprises 0.5% to 1.5% peptone, 1.5% to 2.5% glucose, 0.1% to 1% yeast extract powder and 1.1 to 1.7% agar, and the remainder is water; more preferably, the culture medium used for activation comprises 1% peptone, 2% glucose, 0.5% yeast extract powder and 1.4% agar, and the remainder is water; and / or, the culture temperature is 28 to 32° C.; and / or, the culture time is 21 to 27 h; and / or,

[0035] More preferably, in step 2, the activated Saccharomyces cerevisiae is inoculated into the culture medium used for the primary seed solution for culturing to obtain the primary seed solution; then, the primary seed solution is inoculated into the culture medium used for the secondary seed solution for culturing to obtain the secondary seed solution (also referred to as seed solution); more preferably, the culture conditions of the primary seed solution are: the culture temperature is 28 to 32° C.; and / or, the culture time is 12 to 18 hours; and / or, more preferably, the composition of the culture medium used for the primary seed solution is: 90 to 110 g of sucrose per liter of water, 18 to 22 g of yeast extract powder, and 10 to 25 g of sucrose per liter of water. g, 0.5-1.5 g magnesium sulfate and 0.5-1.5 g zinc sulfate; more preferably, the composition of the culture medium used for the primary seed solution is: 100 g sucrose, 20 g yeast extract powder, 1 g magnesium sulfate and 1 g zinc sulfate per liter of water; and / or, more preferably, the culture conditions of the secondary seed solution are: the inoculation amount of the primary seed solution is 1%-3% based on the volume of the culture medium; and / or, the culture temperature is 28-32°C; and / or, the culture time is 12-18h; and / or, the culture medium used for the secondary seed solution includes beet molasses; and / or,

[0036] More preferably, in step 3, the inoculation amount of the seed solution is 1% to 3% based on the volume of the culture medium; and / or the culture medium includes beet molasses; and / or the culture temperature is 28 to 32° C.; and / or the culture time is 12 to 18 hours.

[0037] In a second aspect, the present invention provides a method for preparing a microbial preparation suitable for saline-alkali soil, comprising the following steps:

[0038] The microbial preparation is obtained by mixing the synergistic carrier, Bacillus subtilis, Saccharomyces cerevisiae and the protective carrier.

[0039] Preferably, a microbial preparation suitable for saline-alkali soil is prepared by the above-mentioned method for preparing the microbial preparation suitable for saline-alkali soil.

[0040] In a third aspect, the present invention provides an application of a microbial preparation suitable for saline-alkali soil in improving saline-alkali soil.

[0041] Preferably, the application amount of the microbial preparation suitable for saline-alkali soil is 10-20 kg / hm2 ; and / or

[0042] Preferably, the microbial preparation is used by drip irrigation or flushing, and is suitable for the seedling growth period of crops. And / or

[0043] Preferably, improving saline-alkali soil includes improving soil fertility, reducing soil alkalinity and / or reducing soil conductivity.

[0044] In a fourth aspect, the present invention provides an application of a microbial preparation suitable for saline-alkali soil in promoting the growth of crops and / or improving the salt-alkali tolerance of crops.

[0045] Preferably, the crop is a leguminous crop. And / or

[0046] Preferably, the application amount of the microbial preparation suitable for saline-alkali soil is 10-20 kg / hm 2 . and / or

[0047] Preferably, the microbial preparation is used by drip irrigation or flushing, and is suitable for the seedling growth period of crops.

[0048] In a fifth aspect, the present invention provides a composition for improving saline-alkali soil, wherein the composition for improving saline-alkali soil comprises the above-mentioned microbial preparation suitable for saline-alkali soil.

[0049] Preferably, improving saline-alkali soil includes improving soil fertility, reducing soil alkalinity and / or reducing soil conductivity.

[0050] In a sixth aspect, the present invention provides a composition for promoting the growth of crops and / or improving the salt-alkali tolerance of crops, wherein the composition for promoting the growth of crops and / or improving the salt-alkali tolerance of crops comprises the above-mentioned microbial preparation suitable for saline-alkali soil.

[0051] Beneficial Effects of the Invention

[0052] (1) The present invention uses Bacillus subtilis and Saccharomyces cerevisiae to cooperate with each other and combines a synergistic carrier and a protective carrier to make the obtained microbial preparation have an excellent effect of improving saline-alkali soil, which is specifically reflected in improving soil fertility, reducing soil alkalinity and reducing soil conductivity.

[0053] (2) The microbial preparation provided by the present invention also has the function of promoting the growth of crops and improving the salt-alkali resistance of crops. It is particularly suitable for bean crops and has a more excellent effect in bean crops.

[0054] (3) In the present invention, in particular, a microbial preparation is obtained by combining Bacillus subtilis subsp. subtilis tc-4 and Saccharomyces cerevisiae YY with a protective carrier and a synergistic carrier, and by regulating the content ratio of the above four components, the components synergize with each other, so that it has a better effect of improving saline-alkali soil, and also has a better growth-promoting effect and stress resistance effect on crops.

[0055] (4) The microbial preparation provided by the present invention is safe for crops and the environment, reduces the input of chemical substances, and reduces environmental pressure.

[0056] (5) The preparation process of the microbial agent provided by the present invention is simple and easy to operate, and has low cost, and is more suitable for industrial production.

[0057] Strain deposit information

[0058] In the present invention, Bacillus subtilis subsp. subtilistc-4 was deposited in China Center for Type Culture Collection (CCTCC) on April 17, 2023, with a deposit number of CCTCC NO: M 2023541, and a deposit address: Wuhan University, Wuhan, China, zip code: 430072; telephone: (027) 68754052. The Bacillus subtilis subsp. subtilis tc-4 is disclosed in Chinese patent application number 202311624023.5.

[0059] In the present invention, the brewer's yeast Saccharomyces cerevisiae high-wine yeast YY (Saccharomyces cerevisiae high-wine yeast YY) was deposited in the China Center for Type Culture Collection (CCTCC) on February 1, 2021, with a deposit number of CCTCC NO: M2021171, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027) 68754052. The brewer's yeast Saccharomyces cerevisiae high-wine yeast YY is disclosed in the Chinese patent application number 202110342874.5.

[0060] In the present invention, the brewer's yeast d8.8 (Saccharomyces cerevisiae d8.8) was deposited in the China Center for Type Culture Collection (CCTCC) on March 29, 2017, with a deposit number of CCTCCNO: M2017148, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027) 68754052. The brewer's yeast d8.8 is disclosed in the Chinese patent application number 201710519421.9. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a graph evaluating the growth-promoting effect of the microbial preparation prepared in the example.

[0062] Figure 2 This is a diagram for evaluating the stress resistance of the microbial preparation prepared in the example. DETAILED DESCRIPTION

[0063] As mentioned above, the object of the present invention is to provide a microbial preparation suitable for saline-alkali soil and a preparation method and application thereof.

[0064] In the present invention, the first purpose is to provide a microbial preparation suitable for saline-alkali soil, which includes, by weight: 4 to 25 parts of Bacillus subtilis, 4 to 30 parts of Saccharomyces cerevisiae, 40 to 85 parts of a synergistic carrier and 5 to 20 parts of a protective carrier; wherein the synergistic carrier includes one or more of a substance group consisting of yeast metabolites, mineral humic acid and amino acid powder; the protective carrier includes one or more of a substance group consisting of maltodextrin, trehalose, corn starch and attapulgite.

[0065] Preferably, the microbial preparation includes 4 to 25 parts of Bacillus subtilis, for example, it may contain 4 to 22 parts, 4 to 21 parts, 4 to 15 parts or 4 to 6 parts of Bacillus subtilis. The Bacillus subtilis is preferably Bacillus subtilis subsp. subtilis tc-4. For example, in some embodiments, the microbial preparation includes 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 parts, or contains Bacillus subtilis within a numerical range consisting of any two of the above specific numerical values ​​as endpoints, preferably Bacillus subtilis powder, more preferably Bacillus subtilis subsp. subtilis tc-4 powder (which is powder prepared by Bacillus subtilis subsp. subtilis tc-4); and / or,

[0066] Preferably, the microbial preparation includes 4 to 30 parts of Saccharomyces cerevisiae, for example, it may contain 4 to 25 parts, 4 to 22 parts, 4 to 21 parts, 4 to 15 parts or 4 to 6 parts of Saccharomyces cerevisiae. The Saccharomyces cerevisiae is preferably Saccharomyces cerevisiae high wine yeast YY. For example, in some embodiments, the microbial preparation includes 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 parts, or contains Saccharomyces cerevisiae within a numerical range consisting of any two of the above specific values ​​as endpoints, preferably Saccharomyces cerevisiae powder, more preferably Saccharomyces cerevisiae high wine yeast YY powder (which is powder prepared by Saccharomyces cerevisiae high wine yeast YY); and / or,

[0067] Preferably, the microbial preparation comprises 40 to 85 parts of a synergistic carrier, for example, 50 to 85 parts, 55 to 85 parts, 70 to 85 parts, 75 to 85 parts or 77 to 83 parts of a synergistic carrier. The synergistic carrier comprises one or more selected from the group consisting of yeast metabolites, mineral humic acid and amino acid powder; preferably yeast metabolites. For example, in some embodiments, the microbial preparation comprises 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85 parts, or contains a synergistic carrier within a numerical range consisting of any two of the above specific values ​​as endpoints, preferably a yeast metabolite; and / or,

[0068] Preferably, the microbial preparation includes 5 to 20 parts of a protective carrier, for example, it may contain 5 to 18 parts, 5 to 16 parts, 5 to 13 parts, 5 to 11 parts or 7 to 13 parts of a protective carrier. The protective carrier includes one or more selected from the group consisting of maltodextrin, trehalose, corn starch and attapulgite; preferably corn starch and / or attapulgite. For example, in some embodiments, the microbial preparation includes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 parts, or contains a protective carrier within a numerical range consisting of any two of the above specific values ​​as endpoints, preferably corn starch and / or attapulgite.

[0069] In the present invention, the synergistic carrier refers to a type of carrier that can promote the effect of the microbial agent, reduce the amount of microbial agent added to a certain extent, and work synergistically with other components. The synergistic carrier includes one or more of yeast metabolites, mineral humic acid and amino acid powder; preferably, the synergistic carrier is a yeast metabolite. The yeast metabolite is a metabolite obtained by fermenting molasses as a raw material to extract yeast; preferably, the molasses includes beet molasses. Yeast metabolites are most preferred because yeast metabolites contain stress resistance factors such as betaine, and also contain soil maintenance factor components such as fulvic acid, which synergize with microbial agents to improve soil environment, enhance plant resistance, and increase production and quality. More preferably, yeast metabolites contain 15% to 30% fulvic acid and / or 6% to 15% betaine.

[0070] In the present invention, the protective carrier refers to a type of carrier that can improve the survival rate of live bacteria in microbial preparations and extend the shelf life of the microbial preparations. In some specific embodiments, the protective carrier is selected from one or more of maltodextrin, trehalose, corn starch and attapulgite. Maltodextrin, trehalose, corn starch and / or attapulgite are selected as synergistic carriers because they can effectively improve soil alkalinity, optimize the physical and chemical properties of the soil, and reduce the loss of soil moisture. Among them, preferably, the protective carrier is corn starch and / or attapulgite because it is low in cost and has a more significant protective effect on live bacteria in microbial preparations.

[0071] In the present invention, the Bacillus subtilis contains dormant spores, which can help the bacteria survive adverse environments, produce growth-promoting substances IAA, promote plant growth, produce stress-resistant substances, and enhance the resistance of plants to adverse environments. The brewer's yeast contains a large amount of trehalose, glucan, manno-oligosaccharide, growth-promoting substance IAA, stress-resistant substances salicylic acid and jasmonic acid and other substances, which enhance the stress resistance of the yeast body itself and the applied plants. Bacillus subtilis and brewer's yeast have strong stress resistance themselves and can survive in saline-alkali environments. They jointly produce growth-promoting and stress-resistant substances. The synergistic effect of the two bacteria improves the growth performance of plants in saline-alkali environments, and can produce polysaccharides to improve soil structure.

[0072] In the present invention, a microbial preparation is obtained by combining Bacillus subtilis subsp. subtilis tc-4 and Saccharomyces cerevisiae YY with a protective carrier and a synergistic carrier, and by adjusting the content ratio of the above four components, the components are synergistically enhanced, so that it has an excellent effect of improving saline-alkali soil, and has an excellent growth-promoting effect and stress resistance effect on crops, and the content of each component and / or the type of the component within the preferred range has a better effect, and within a more preferred range, the degree of improvement of saline-alkali soil and the growth-promoting and stress-resistant effect on crops can be further improved. Preferably, the microbial preparation of the present invention is particularly suitable for legume crops, and has a more excellent effect when applied to legume crops.

[0073] The second object of the present invention is to provide a method for preparing a microbial preparation, which comprises the following steps: sequentially adding a synergistic carrier, Bacillus subtilis powder, Saccharomyces cerevisiae powder and a protective carrier, mixing them in a mixing bin for 5 to 8 minutes, and obtaining a microbial preparation.

[0074] Preferably, the method for preparing the Bacillus subtilis powder comprises the following steps:

[0075] Step 1: inoculating Bacillus subtilis in a culture medium comprising 0.5% to 1.5% peptone, 0.1% to 0.5% beef extract, 0.3% to 0.7% sodium chloride, 1.5% to 2% agar and water, and culturing at a temperature of 35 to 39° C. for 21 to 27 hours; inoculating the obtained single colony in a culture medium comprising 0.5% to 1.5% peptone, 0.1% to 0.5% beef extract, 0.3% to 0.7% sodium chloride, 1.5% to 2% agar and water, and continuing to culture at a temperature of 35 to 39° C. for 21 to 27 hours to obtain activated Bacillus subtilis;

[0076] Step 2: inoculating a single colony of the activated Bacillus subtilis into a culture medium comprising 0.5% to 1.5% peptone, 0.1% to 0.5% beef powder, 0.3% to 0.7% sodium chloride, 0.05% to 0.15% glucose and water, and culturing for 10 to 15 hours at a temperature of 35 to 39° C. and a rotation speed of 100 to 200 rpm to obtain a seed solution;

[0077] Step 3: Inoculate the entire inoculum of the seed solution into a culture medium comprising 3% to 5% corn flour, 3% to 5% soybean meal, 0.05% to 0.15% magnesium sulfate, 0.05% to 0.15% calcium chloride and water, and culture for 15 to 20 hours at a temperature of 35 to 39° C., a ventilation volume of 20 to 50 L / min and a tank pressure of 0.2 to 0.5 standard atmospheric pressure to obtain a fermentation liquid. The fermentation liquid is spray-dried to obtain Bacillus subtilis powder.

[0078] And / or, preferably, the method for preparing the brewer's yeast powder comprises the following steps:

[0079] Step 1: inoculating brewer's yeast into a culture medium comprising 0.5% to 1.5% peptone, 1.5% to 2.5% glucose, 0.1% to 1% yeast extract powder, 1.1% to 1.7% agar and water, and culturing at a temperature of 28 to 32° C. for 21 to 27 hours;

[0080] Step 2: inoculating the activated brewer's yeast into a culture medium for a primary seed solution containing 90-110 g of sucrose, 18-22 g of yeast extract powder, 0.5-1.5 g of magnesium sulfate and 0.5-1.5 g of zinc sulfate per liter of water for culturing at a temperature of 28-32° C. for 12-18 hours to obtain a primary seed solution; then, inoculating the primary seed solution at an inoculum amount of 1% to 3% by volume of the culture medium into a culture medium for a secondary seed solution containing beet molasses for culturing at a temperature of 28-32° C. for 12-18 hours to obtain a secondary seed solution (also referred to as seed solution);

[0081] Step 3: Based on the volume of the culture medium, the seed liquid is inoculated at a volume percentage of 1% to 3% into a fermentation medium containing beet molasses for cultivation at a temperature of 28 to 32° C. for 12 to 18 hours to obtain a fermentation liquid, which is separated and drum squeezed to obtain brewer's yeast powder.

[0082] The third purpose of the present invention is to provide a microbial preparation suitable for saline-alkali soil for use in improving saline-alkali soil, promoting crop growth and / or improving the salt-alkali tolerance of leguminous crops. The microbial preparation is used for drip irrigation or flushing, and is suitable for the seedling growth period of crops, with an application rate of 10-20 kg / hm 2 .

[0083] Unless otherwise specified, the various reagents / instruments used in the examples and comparative examples of the present invention are conventional commercially available products. The experimental material information used in the present invention is shown in the following table:

[0084] Table 1 Experimental materials and vendors

[0085]

[0086] In the present invention, the NA plate is the purchased nutrient agar NA, which contains 1% peptone, 0.3% beef extract, 0.5% sodium chloride, 1.5% agar, and the rest is water. The NB medium is the purchased nutrient broth NB, which contains 1% peptone, 0.3% beef powder, 0.5% sodium chloride, 0.1% glucose, and the rest is water. The YPD plate is the purchased YPD agar (yeast extract powder peptone glucose medium), which contains 1% peptone, 2% glucose, 0.5% yeast extract powder and 1.4% agar, and the rest is water.

[0087] The present invention does not impose any limitation on the source of beet molasses, and any commercially available or homemade beet molasses can be used in the present invention. Preferably, the present inventors have found through research that as long as the betaine content of the commercially available or homemade beet molasses is ≥3wt%, it can be used in the present invention; more preferably, the betaine content in the beet molasses is 3wt% to 8wt%. In some specific embodiments, the beet molasses comes from COFCO Sugar.

[0088] In the present invention, yeast metabolites include yeast polysaccharide (calculated as β-glucan+mannan) ≥2.5%, fulvic acid ≥15%, betaine ≥6%, secondary elements (Ca+Mg) ≥1%, total nutrients (N, P2O5 and K2O) ≥10%, organic matter content ≥15%, and water insoluble matter ≤5%.

[0089] In some specific embodiments, the present invention does not impose any limitation on the source of yeast metabolites, and any commercially available or homemade yeast metabolites can be used in the present invention. Specifically, the inventors have found through research that as long as the betaine content of the commercially available or homemade yeast metabolites is ≥6% and the fulvic acid content is ≥15%, they can be used in the present invention.

[0090] Preferably, based on the weight of the yeast metabolites, the yeast metabolites include 24% to 26% crude protein, 50% to 55% crude ash, 3% to 5% total nitrogen, 15% to 28% organic matter, 15% to 30% fulvic acid, and / or 6% to 15% betaine.

[0091] More preferably, the yeast metabolites further include calcium 4% to 5.5%, magnesium 0.1% to 1%, sodium 7% to 8%, β-glucan 1% to 2%, mannan 2% to 3%, humic acid 2% to 3%, potassium 13 to 15%, phosphorus 0.1% to 1%, sulfur 3% to 4%, chloride ion 15 to 18%, and / or hydrolyzed amino acids 9% to 13%.

[0092] In the present invention, the detection components of yeast metabolites purchased from Yili Fubang New Agriculture Co., Ltd. are shown in Table 2 below:

[0093] Table 2 Detection components of yeast metabolites

[0094]

[0095]

[0096]

[0097] In the present invention, the method for detecting mannan comprises the following steps:

[0098] Sample treatment: Accurately weigh 400 mg (accurate to 0.1 mg) of sample and place it in a 20 mL heat-resistant glass test tube with a screw cap, add 6.0 mL of hydrochloric acid (37%), carefully cover the vial and mix it with a vortex mixer to obtain a uniform suspension. Place the vial in a 30°C water bath for 45 minutes, and oscillate and mix it with a vortex mixer every 15 minutes. Then transfer the suspension quantitatively to a 200 mL Dewar flask, wash the test tube several times with about 100 mL to 120 mL of water, and add the washing liquid to the Dewar flask. Place the Dewar flask in an autoclave and treat it at 121°C for 60 minutes. After taking it out, cool it, adjust the solution to pH 6-7 with sodium hydroxide solution, and then make it dilute to 200 mL. Filter it with a cellulose acetate membrane with a pore size of 0.45 microns for use.

[0099] Chromatographic conditions: Use pure water as the mobile phase, the flow rate is 0.5mL / min, the column temperature is 80℃, and the sample is injected after the instrument baseline is stable.

[0100] Drawing of standard curve: Pipette 1, 2, 3, 4, 5 mL of mannose standard solution (1 g / L, weigh 0.2500 g of mannose, dilute to 250 mL with pure water) into a 10 mL volumetric flask, dilute to scale with high-purity water, and obtain a standard series of mannose of 100, 200, 300, 400, 500 mg / L. Accurately inject 20 μL under the above chromatographic conditions, obtain the regression equation between the chromatographic peak area and the mass concentration of the standard, and draw the standard curve.

[0101] Determination of samples and reference substances: Under the same chromatographic conditions, inject the treated samples into the chromatograph and record the retention time and peak area of ​​each chromatographic peak. Use the retention time of the sugar standard chromatographic peak for qualitative analysis and the peak area of ​​the sugar standard chromatographic peak for quantitative analysis.

[0102] Result calculation: The content of mannan is calculated according to the following formula:

[0103]

[0104] Where:

[0105] X---the content of mannan in the sample, %;

[0106] A---The mannose content of the sample solution obtained from the standard curve based on the peak area of ​​the sample solution, mg / L;

[0107] m---Weigh the mass of the sample, g;

[0108] 0.2---The volume of the sample after treatment with the reference substance, L;

[0109] 0.9---The coefficient for converting mannose to mannan.

[0110] In the present invention, the yeast extract has a total nitrogen content of ≥9.0%, an amino nitrogen content of ≥3.0%, an ash content of ≤15%, a sodium chloride content of ≤2%, and a moisture content of ≤6%. In some specific embodiments, the present invention does not impose any limitation on the source of the yeast extract, and any commercially available or homemade yeast extract can be used in the present invention. Specifically, the inventors have found through research that as long as the commercially available or homemade yeast extract has a total nitrogen content of ≥9.0% and / or an amino nitrogen content of ≥3.0%, it can be used in the present invention. Preferably, the yeast extract has a total nitrogen content of 9% to 15%, and / or an amino nitrogen content of 3% to 5%.

[0111] Preferably, the yeast extract comprises 41000-42000 mg / kg of trace elements based on the weight of the yeast extract. More preferably, the trace elements comprise 32760-32770 mg / kg of potassium, 5836-5846 mg / kg of sodium, 533-543 mg / kg of calcium and 2029-2039 mg / kg of magnesium. Even more preferably, the trace elements comprise 32767.33 mg / kg of potassium, 5841.92 mg / kg of sodium, 538.71 mg / kg of calcium and 2034.06 mg / kg of magnesium.

[0112] And / or, preferably, based on the weight of the yeast extract powder, the yeast extract powder includes 5000-6000 ppm of vitamins; more preferably, the yeast extract powder includes 2-3 ppm of vitamin B1 (detection method: GB5009.84-2016 National Food Safety Standard Determination of Vitamin B1 in Foods - First Method - High Performance Liquid Chromatography), 38-44 ppm of vitamin B2 (detection method: GB5009.85-2016 National Food Safety Standard Determination of Vitamin B2 in Foods - First Method - High Performance Liquid Chromatography), and 78-84 ppm of vitamin B5 (detection method: GB 5009.210-2023 National Food Safety Standard Determination of Pantothenic Acid in Foods Method 3 Microbiological Method), Vitamin B6 content is 10-13ppm (Detection method: GB5009.154-2023 National Food Safety Standard Determination of Vitamin B6 in Foods Method 3 High Performance Liquid Chromatography-Fluorescence Detection Method), Vitamin B7 content is 4-7ppm (Detection method: GB 5009.259-2023 National Food Safety Standard Determination of Biotin in Foods Method 2 Microbiological Method), Vitamin B9 content is 23-27ppm (Detection method: GB 5009.211-2022 National Food Safety Standard Determination of Folic Acid in Foods), Vitamin B12 content is 0.15-0.25μg / 100g (Detection method: GB The content of choline is 3010-3025 ppm (detection method: GB 5413.20-2022 National Food Safety Standard Determination of Choline in Infant Food and Dairy Products Method 3 Liquid Chromatography-Tandem Mass Spectrometry), the content of inositol is 2010-2020 ppm (detection method: GB 5009.270-2023 National Food Safety Standard Determination of Inositol in Food Method 1 Gas Chromatography), and / or the content of niacin is 310-325 ppm (detection method: GB 5009.89-2023 National Food Safety Standard Determination of Nicotinic Acid and Nicotinamide in Food Method 1 High Performance Liquid Chromatography). More preferably, the yeast extract powder comprises: 2.6 ppm of vitamin B1, 41.6 ppm of vitamin B2, 81.3 ppm of vitamin B5, 11.5 ppm of vitamin B6, 5.79 ppm of vitamin B7, 25.1 ppm of vitamin B9, 0.21 μg / 100 g of vitamin B12, 3017 ppm of choline, 2016 ppm of inositol, and / or 318 ppm of niacin.

[0113] And / or, preferably, based on the weight of the yeast extract, the yeast extract comprises 57% to 61% of hydrolyzed amino acids. More preferably, the yeast extract comprises 59.16% of hydrolyzed amino acids.

[0114] In the present invention, the content of total available nutrients in the soil is the total content of hydrolyzable nitrogen, available phosphorus and available potassium, wherein the determination of the hydrolyzable nitrogen content refers to the determination of hydrolyzable nitrogen in the determination of forest soil nitrogen in the forestry industry standard LY / T1228-2015 of the People's Republic of China; the determination of the available phosphorus content refers to the agricultural industry standard NY / T1121.7-2014 of the People's Republic of China, Part 7: Determination of available phosphorus in soil; the determination of the available potassium content refers to the agricultural industry standard NY / T889-2004 of the People's Republic of China, Determination of available potassium and slow-release potassium in soil. The determination of the organic matter content in the soil refers to the agricultural industry standard NY / T 1121.6-2006 of the People's Republic of China, Part 6: Determination of soil organic matter. The conductivity in the soil refers to the electrode method for the determination of soil conductivity in the national environmental protection standard HJ 802-2017 of the People's Republic of China. The soluble salt concentration in the soil is tested using a conductivity meter. The soil pH value refers to the agricultural industry standard of the People's Republic of China NY / T 1121.2-2006 Soil Testing Part 2: Determination of Soil pH.

[0115] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0116] Example 1

[0117] (1) A formulation of a microbial preparation suitable for saline-alkali soil

[0118] A microbial preparation suitable for saline-alkali soil comprises, by weight, 80 parts of yeast metabolites, 5 parts of Bacillus subtilis powder, 5 parts of Saccharomyces cerevisiae powder and 10 parts of corn starch, wherein the content of fulvic acid in the yeast metabolites is 26.12%, and the content of betaine is 11.9%.

[0119] Wherein, the Bacillus subtilis in the Bacillus subtilis powder is Bacillus subtilis subsp. subtilis tc-4, which was deposited in the China Center for Type Culture Collection (CCTCC) on April 17, 2023, with a deposit number of CCTCC NO: M 2023541, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027) 68754052. The Bacillus subtilis subsp. subtilis tc-4 is disclosed in the Chinese patent application number 202311624023.5.

[0120] The preparation method of the Bacillus subtilis powder comprises the following steps:

[0121] Step 1: Use an inoculation loop to dip into the Bacillus subtilis subsp. subtilis tc-4 bacterial solution stored in glycerol at -80°C, streak culture on NA plate (i.e., purchased nutrient agar NA), culture at 37°C for 24 hours, and obtain a single colony. Pick a single colony and continue to streak culture on the NA plate, culture at 37°C for 24 hours to obtain activated Bacillus subtilis tc-4.

[0122] Step 2: Pick a complete single colony with an inoculation loop and inoculate it into a conical flask containing 200 mL of NB culture medium. Place it in a shaker and culture it at 37°C for 12 hours at a speed of 160 r / min to obtain seed solution.

[0123] Step 3: First, 15L of liquid culture medium is loaded into a 30L fermentation tank. The preparation method of the liquid culture medium includes: adding 4% corn flour, 4% soybean meal, 0.1% magnesium sulfate and 0.1% calcium chloride based on the total mass of the culture medium, and then adding tap water to 15L, adjusting the pH to 7, and sterilizing at high temperature and high pressure for 30min at a temperature of 121°C. Then, all 200mL of seed liquid obtained in step 2 is transferred into a 30L fermentation tank for fermentation and culture. The culture conditions are: ventilation volume 20-50L / min, tank pressure 0.2-0.5 standard atmospheric pressure, and ventilation fermentation at a temperature of 37°C for 17-19h. Finally, the fermented liquid is spray-dried to obtain Bacillus subtilis powder.

[0124] The Saccharomyces cerevisiae in the Saccharomyces cerevisiae powder is Saccharomyces cerevisiae YY, which was deposited in the China Center for Type Culture Collection (CCTCC) on February 1, 2021, with a deposit number of CCTCC NO: M 2021171, and a deposit address of: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027) 68754052. The Saccharomyces cerevisiae YY is disclosed in the Chinese patent application number 202110342874.5.

[0125] The preparation method of the saccharomyces cerevisiae powder comprises the following steps:

[0126] Step 1: Use an inoculating loop to dip into the bacterial liquid of Saccharomyces cerevisiae YY (i.e., Saccharomyces cerevisiae YY) frozen in glycerol at -80°C, streak it on YPD agar medium, and obtain a single colony after culturing at 30°C for 24 hours.

[0127] Step 2: Pick a complete single colony with an inoculation loop, inoculate it into the culture medium used for the primary seed solution for culturing at 30°C for 15 hours to obtain the primary seed solution of yeast. The preparation method of the culture medium used for the primary seed solution includes: dissolving 100g of sucrose, 20g of yeast extract powder, 1g of magnesium sulfate and 1g of zinc sulfate in 1L of water, and adjusting the pH to 4.8 with concentrated sulfuric acid.

[0128] The yeast primary seed liquid is inoculated into the medium used for the secondary seed liquid at an inoculation rate of 1% to 3% based on the volume of the medium, and the culture is cultured at a temperature of 30° C. for 15 hours, the culture liquid is separated by a separator, and the precipitate is washed with water to obtain the yeast seed liquid (also called the secondary seed liquid). The preparation method of the medium used for the secondary seed liquid includes: taking 8L of beet molasses and adjusting the pH to 5.5 with concentrated sulfuric acid.

[0129] Step 3: Based on the volume of the culture medium, the yeast seed liquid is inoculated into the fermentation tank at an inoculation rate of 1% to 3%, and fermented at a temperature of 30°C for 15 hours to obtain a yeast liquid. The preparation method of the culture medium in the fermentation tank includes the following steps: beet molasses is taken, and the pH is adjusted to 5.5 with concentrated sulfuric acid. The yeast liquid is separated by a separator, and the precipitate is washed with water, and then squeezed and dried by a rotary drum to obtain brewer's yeast powder.

[0130] (2) A method for preparing a microbial preparation suitable for saline-alkali soil

[0131] A method for preparing a microbial preparation suitable for saline-alkali soil comprises the following steps:

[0132] The yeast metabolites, Bacillus subtilis powder, Saccharomyces cerevisiae powder and corn starch are added into a mixing bin and mixed for 5 to 8 minutes to obtain a microbial preparation.

[0133] Example 2

[0134] (1) A formulation of a microbial preparation suitable for saline-alkali soil

[0135] A microbial preparation suitable for saline-alkali soil comprises, by weight, 40 parts of yeast metabolites, 20 parts of Bacillus subtilis powder, 13 parts of Saccharomyces cerevisiae powder, 5 parts of corn starch and 10 parts of attapulgite, wherein the content of fulvic acid in the yeast metabolites is 26.12%, and the content of betaine is 11.9%.

[0136] Wherein, the Bacillus subtilis in the Bacillus subtilis powder is Bacillus subtilis subsp. subtilis tc-4. The preparation method of the Bacillus subtilis powder is the same as the preparation method of the Bacillus subtilis powder in Example 1.

[0137] The Saccharomyces cerevisiae powder is Saccharomyces cerevisiae YY. The preparation method of the Saccharomyces cerevisiae powder is the same as the preparation method of the Saccharomyces cerevisiae powder in Example 1.

[0138] (2) A method for preparing a microbial preparation suitable for saline-alkali soil

[0139] A method for preparing a microbial preparation suitable for saline-alkali soil comprises the following steps:

[0140] The yeast metabolites, Bacillus subtilis powder, Saccharomyces cerevisiae powder, corn starch and attapulgite are added into a mixing bin and mixed for 5 to 8 minutes to obtain a microbial preparation.

[0141] Example 3

[0142] (1) A formulation of a microbial preparation suitable for saline-alkali soil

[0143] A microbial preparation suitable for saline-alkali soil comprises, by weight, 60 parts of yeast metabolites, 13 parts of Bacillus subtilis powder, 20 parts of Saccharomyces cerevisiae powder and 5 parts of corn starch, wherein the content of fulvic acid in the yeast metabolites is 26.12%, and the content of betaine is 11.9%.

[0144] Wherein, the Bacillus subtilis in the Bacillus subtilis powder is Bacillus subtilis subsp. subtilis tc-4. The preparation method of the Bacillus subtilis powder is the same as the preparation method of the Bacillus subtilis powder in Example 1.

[0145] The Saccharomyces cerevisiae powder is Saccharomyces cerevisiae YY. The preparation method of the Saccharomyces cerevisiae powder is the same as the preparation method of the Saccharomyces cerevisiae powder in Example 1.

[0146] (2) A method for preparing a microbial preparation suitable for saline-alkali soil

[0147] A method for preparing a microbial preparation suitable for saline-alkali soil comprises the following steps:

[0148] The yeast metabolites, Bacillus subtilis powder, Saccharomyces cerevisiae powder and corn starch are added into a mixing bin and mixed for 5 to 8 minutes to obtain a microbial preparation.

[0149] Example 4

[0150] (1) A formulation of a microbial preparation suitable for saline-alkali soil

[0151] A microbial preparation suitable for saline-alkali soil comprises, by weight, 80 parts of yeast metabolites, 25 parts of Bacillus subtilis powder, 30 parts of Saccharomyces cerevisiae powder and 10 parts of corn starch, wherein the content of fulvic acid in the yeast metabolites is 26.12%, and the content of betaine is 11.9%.

[0152] Wherein, the Bacillus subtilis in the Bacillus subtilis powder is Bacillus subtilis subsp. subtilis tc-4. The preparation method of the Bacillus subtilis powder is the same as the preparation method of the Bacillus subtilis powder in Example 1.

[0153] The Saccharomyces cerevisiae powder is Saccharomyces cerevisiae YY. The preparation method of the Saccharomyces cerevisiae powder is the same as the preparation method of the Saccharomyces cerevisiae powder in Example 1.

[0154] (2) A method for preparing a microbial preparation suitable for saline-alkali soil

[0155] A method for preparing a microbial preparation suitable for saline-alkali soil comprises the following steps:

[0156] The yeast metabolites, Bacillus subtilis powder, Saccharomyces cerevisiae powder and corn starch are added into a mixing bin and mixed for 5 to 8 minutes to obtain a microbial preparation.

[0157] Example 5

[0158] (1) A formulation of a microbial preparation suitable for saline-alkali soil

[0159] A microbial preparation suitable for saline-alkali soil comprises, by weight, 80 parts of yeast metabolites, 5 parts of Bacillus subtilis powder, 5 parts of Saccharomyces cerevisiae powder and 10 parts of maltodextrin, wherein the content of fulvic acid in the yeast metabolites is 26.12%, and the content of betaine is 11.9%.

[0160] Wherein, the Bacillus subtilis in the Bacillus subtilis powder is Bacillus subtilis subsp. subtilis tc-4. The preparation method of the Bacillus subtilis powder is the same as the preparation method of the Bacillus subtilis powder in Example 1.

[0161] The Saccharomyces cerevisiae powder is Saccharomyces cerevisiae YY. The preparation method of the Saccharomyces cerevisiae powder is the same as the preparation method of the Saccharomyces cerevisiae powder in Example 1.

[0162] (2) A method for preparing a microbial preparation suitable for saline-alkali soil

[0163] A method for preparing a microbial preparation suitable for saline-alkali soil comprises the following steps:

[0164] The yeast metabolites, Bacillus subtilis powder, Saccharomyces cerevisiae powder and corn starch are added into a mixing bin and mixed for 5 to 8 minutes to obtain a microbial preparation.

[0165] Example 6

[0166] Compared with Example 1, the Bacillus subtilis powder prepared from Bacillus subtilis subsp. subtilis tc-4 was replaced with commercially available Bacillus subtilis powder (powdered, trade name: Bacillus subtilis, sold by Shandong Weilan Biotechnology Co., Ltd.). The details are as follows:

[0167] (1) A formulation of a microbial preparation suitable for saline-alkali soil

[0168] A microbial preparation suitable for saline-alkali soil comprises, by weight, 80 parts of yeast metabolites, 5 parts of Bacillus subtilis powder (commercially available), 5 parts of Saccharomyces cerevisiae powder and 10 parts of corn starch, wherein the content of fulvic acid in the yeast metabolites is 26.12%, and the content of betaine is 11.9%.

[0169] Wherein, the Bacillus subtilis powder is commercially available from Shandong Weilan Biotechnology Co., Ltd.

[0170] The Saccharomyces cerevisiae powder is Saccharomyces cerevisiae YY. The preparation method of the Saccharomyces cerevisiae powder is the same as the preparation method of the Saccharomyces cerevisiae powder in Example 1.

[0171] (2) A method for preparing a microbial preparation suitable for saline-alkali soil

[0172] A method for preparing a microbial preparation suitable for saline-alkali soil comprises the following steps:

[0173] The yeast metabolites, Bacillus subtilis powder (commercially available), Saccharomyces cerevisiae powder and corn starch were added to a mixing bin and mixed for 5 to 8 minutes to obtain a microbial preparation.

[0174] Example 7

[0175] Compared with Example 4, the Saccharomyces cerevisiae high wine yeast YY (Saccharomyces cerevisiae high wine yeast YY) in the Saccharomyces cerevisiae powder was replaced with Saccharomyces cerevisiae d8.8, which was deposited in the China Center for Type Culture Collection (CCTCC) on March 29, 2017, with a deposit number of CCTCC NO: M2017148, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072, telephone: (027) 68754052. The Saccharomyces cerevisiae d8.8 is disclosed in the Chinese patent application number 201710519421.9. The details are as follows:

[0176] (1) A formulation of a microbial preparation suitable for saline-alkali soil

[0177] A microbial preparation suitable for saline-alkali soil comprises, by weight, 80 parts of yeast metabolites, 25 parts of Bacillus subtilis powder, 30 parts of Saccharomyces cerevisiae powder and 10 parts of corn starch, wherein the content of fulvic acid in the yeast metabolites is 26.12% and the content of betaine is 11.9%.

[0178] Wherein, the Bacillus subtilis in the Bacillus subtilis powder is Bacillus subtilis subsp. subtilis tc-4. The preparation method of the Bacillus subtilis powder is the same as the preparation method of the Bacillus subtilis powder in Example 1.

[0179] Wherein, the Saccharomyces cerevisiae in the Saccharomyces cerevisiae powder is Saccharomyces cerevisiae d8.8.

[0180] The preparation method of the saccharomyces cerevisiae powder comprises the following steps:

[0181] Step 1: Use an inoculation loop to dip into the glycerol-free suspension of Saccharomyces cerevisiae d8.8 stored at -80°C, streak it on YPD agar, and culture it at 30°C for 24 hours to obtain a single colony.

[0182] Step 2: Pick a complete single colony with an inoculation loop, inoculate it into the culture medium used for the primary seed solution for culturing at 30°C for 15 hours to obtain the primary seed solution of yeast. The preparation method of the culture medium used for the primary seed solution includes: dissolving 100g of sucrose, 20g of yeast extract powder, 1g of magnesium sulfate and 1g of zinc sulfate in 1L of water, and adjusting the pH to 4.8 with concentrated sulfuric acid.

[0183] The yeast primary seed liquid is inoculated into the medium used for the secondary seed liquid at an inoculation rate of 1% to 3% based on the volume of the medium, and the culture is cultured at a temperature of 30° C. for 15 hours, the culture liquid is separated by a separator, and the precipitate is washed with water to obtain the yeast seed liquid (also called the secondary seed liquid). The preparation method of the medium used for the secondary seed liquid includes: taking 8L of beet molasses and adjusting the pH to 5.5 with concentrated sulfuric acid.

[0184] Step 3: Based on the volume of the culture medium, the yeast seed liquid is inoculated into the fermentation tank at an inoculation rate of 1% to 3%, and fermented at a temperature of 30°C for 15 hours to obtain a yeast liquid. The preparation method of the culture medium in the fermentation tank includes the following steps: taking beet molasses, adjusting the pH to 5.5 with concentrated sulfuric acid. The yeast liquid is separated by a separator, the precipitate is washed with water, and then squeezed and dried by a rotary drum to obtain the brewer's yeast powder.

[0185] (2) A method for preparing a microbial preparation suitable for saline-alkali soil

[0186] A method for preparing a microbial preparation suitable for saline-alkali soil comprises the following steps:

[0187] The yeast metabolites, Bacillus subtilis powder, Saccharomyces cerevisiae powder (strain: Saccharomyces cerevisiae d8.8) and corn starch are added into a mixing bin and mixed for 5 to 8 minutes to obtain a microbial preparation.

[0188] Application example 1: Stability test

[0189] The evaluation of stability includes the following steps: storing the microbial preparation prepared in the example at room temperature indoors, using a woven bag plus an inner film bag for storage, with the inner film bag and the woven bag being the inner film bag and the woven bag, sampling and measuring the data stored for 12 months, testing once a month, and determining the effective viable count by referring to the plate count method in the agricultural industry standard of the People's Republic of China NY / T 2321-2013 Microbial Fertilizer Product Inspection Procedure, calculating the survival rate and coefficient of variation (i.e., CV value) stored for one year, and the calculation method is as follows:

[0190]

[0191] Wherein, X is the survival rate, in %, A is the effective viable bacteria count of the prepared microbial preparation after 12 months of storage, in 100 million CFU / g; B is the initial effective viable bacteria count of the prepared microbial preparation (the initial effective viable bacteria count refers to the microbial preparation with a storage time of 0 months), in 100 million CFU / g.

[0192]

[0193] Wherein, CV is the coefficient of variation, in %, SD is the standard deviation of the effective viable count for 12 months; Mean is the monthly average value of the effective viable count for 12 months.

[0194] Table 3 Stability index results of the microbial preparations prepared in Example

[0195]

[0196] As shown in Table 3 above, in the microbial preparations prepared in Examples 1-7, the survival rate of Bacillus subtilis reached more than 75%, and the CV value was within 15%; the survival rate of Saccharomyces cerevisiae reached more than 59%, and the CV value was within 15.1%. Among them, in the microbial preparations prepared in Examples 4-7, the survival rate of Bacillus subtilis was 75.0% to 79.9%, the CV value was 10.9% to 13.7%, and the survival rate of Saccharomyces cerevisiae was 59.5% to 72.9%, and the CV value was 11.8% to 15.1%, indicating that the microbial preparations prepared in Examples 4-7 have good stability. In the microbial preparations prepared in Examples 1-3, the survival rate of Bacillus subtilis is 85.0% to 88.3%, the CV value is 8.9% to 9.6% (less than 10%), the survival rate of Saccharomyces cerevisiae is 73.6% to 84.0%, the CV value is 9.3% to 9.8% (less than 10%), and the effective viable count data of Bacillus subtilis and Saccharomyces cerevisiae fluctuate less, indicating that the stability of the microbial preparations obtained is better. The above data show that each raw material component and its content within the preferred range can achieve a more excellent technical effect.

[0197] Application example 2: Fertility experiment

[0198] Experimental samples: samples of microbial preparations prepared in Examples 1-7.

[0199] Experimental method: The alkaline nutrient classification of the experimental plot in Xili Village, Zhanhua District, Binzhou City, Shandong Province, is classified as medium-low, with severe alkalinity and slight salinization. The crop planted is soybean, and the variety is Zhonghuang 13.

[0200] The experimental plot was divided into eight plots of equal size, each with an area of ​​30m 2, base fertilizer is conventional fertilizer (compound fertilizer and organic fertilizer). In the early stage of soybean planting (July to August), the 7 experimental samples prepared in Examples 1-7 were applied to the roots twice at an application rate of 1000 g / mu, and soil samples were taken after soybean harvest for physical and chemical property analysis.

[0201] Table 4 Result data of soil salinity and alkali physical and chemical properties

[0202]

[0203] In Table 4, CK represents the control group, that is, only conventional fertilization is applied without adding the microbial preparation of the present invention, and the other conditions are the same as those in the embodiment.

[0204] As can be seen from Table 4 above, compared with CK, after adding the microbial preparation of the present invention, the content of total nutrients and organic matter are increased, and the conductivity, soluble salt content and pH value are also decreased, which shows that the microbial preparation of the present invention has the effect of improving soil fertility and improving soil alkalinity. Among them, in Examples 4-7, the total nutrients increased by 5.90% to 10.69%, the organic matter increased by 2.06% to 7.94%, the conductivity decreased by 0.61% to 2.03%, the soluble salt content decreased by 3.22% to 9.68%, and the pH value decreased by 2.76% to 6.28%. The above data show that the microbial preparations prepared in Examples 4-7 have the effect of improving soil fertility and improving saline-alkali soil. In Examples 1-3, the total nutrients increased by 16.01% to 21.30%, the organic matter increased by 8.35% to 12.37%, the conductivity decreased by 1.09% to 22.29%, the soluble salt content decreased by 6.45% to 15.81%, and the pH value decreased by 7.72% to 10.80%. The above data show that the microbial preparations prepared in Examples 1-3 have a better effect of improving soil fertility and improving soil salinity. The above data show that each raw material component and its content within the preferred range can achieve a better technical effect.

[0205] Application example 3: Growth promotion experiment

[0206] Experimental samples: samples of microbial preparations prepared in Examples 1-7.

[0207] Experimental method: potting method was used, and root irrigation was performed when soybean seedlings were 2 cm tall. Control group CK was set up, and the control group was treated with clean water. The 7 treatment groups were treated with the microbial preparation samples prepared in Example 1, and three replicates were set up for each treatment group. Weigh 2 g of the microbial preparation experimental sample prepared in Example, dilute 500 times, take 200 mL of the experimental sample solution from each pot for root irrigation, repeat the treatment after an interval of 7 days, and count the relevant growth data after 14 days. The specific data are shown in Table 5 and Figure 1 shown.

[0208] Table 5 Results of soybean growth promotion experiment

[0209] project Soybean stem length / cm CK 9.23±0.64 Example 1 13.13±0.95 Example 2 12.05±1.12 Example 3 12.53±1.08 Example 4 11.53±0.62 Example 5 10.40±0.9 Example 6 9.60±1.12 Example 7 10.00±1.05

[0210] Figure 1 Table 5 shows the soybean stem length results after applying the microbial preparations prepared in Examples 1-7. Figure 1 As shown in Table 5, the application of the microbial preparations prepared in the embodiments of the present invention is beneficial to increasing the stem length of soybeans. Among them, the stem length of soybeans in the Example 1-3 group is 12.05-13.13 cm, which is 30.55%-42.25% longer than that in the blank control group CK, which is greater than 30%; the stem length of soybeans in the Example 4-7 group is 9.60-11.53 cm, which is 4.00%-24.92% longer than that in the blank control group CK. The above data show that the Example 1-3 group has a more significant growth-promoting effect on the stem length of soybeans than the Example 4-7 group; this shows that each raw material component and its content within the preferred range can achieve a better technical effect.

[0211] Application example 4: Stress resistance test

[0212] Experimental samples: samples of microbial preparations prepared in Examples 1-7.

[0213] Experimental method: The potting method was used to conduct root irrigation experiments at the soybean seedling stage. Two control groups were set up, namely the clear water control group CK1 and the salt water control group CK2. The seven treatment groups were treated with the microbial preparation samples prepared in Examples 1-7, respectively. Each treatment group was set up with three replicates. Except for the clear water control group CK1, the salt water control group CK2 and the groups in Examples 1-7 were treated with 200 mL of 0.6% salt water for root irrigation once when the soybean seedlings were 2 cm tall. Weigh 2 g of the experimental sample of the microbial preparation prepared in the example, dilute it 500 times, take 200 mL of the experimental sample solution from each pot for root irrigation, repeat the treatment after an interval of 7 days, and count the growth data for 14 days. The specific data are shown in Tables 6 and Figure 2 shown.

[0214] Table 6 Results of soybean stress resistance experiment

[0215] project Soybean stem length / cm CK1 8.25±0.50 CK2 4.07±0.76 Example 1 9.60±0.49 Example 2 8.28±0.22 Example 3 8.58±0.48 Example 4 7.70±0.24 Example 5 8.00±0.26 Example 6 7.70±0.79 Example 7 7.60±0.17

[0216] Figure 2 Table 6 shows the stem length of soybeans after application of the microbial preparations prepared in Examples 1-7 in simulated saline-alkali soil. Figure 2As shown in Table 6, the application of the microbial preparations prepared in the embodiments of the present invention is beneficial to improving the stem length of soybeans grown in saline-alkali soil. Among them, the stem length of soybeans in Example 1-3 groups is 8.28-9.60 cm, which is 103.44%-135.87% higher than that of the salt water control group CK2, and is consistent with the growth of the clear water control group CK1. The Example 1 group is better than the clear water control group CK1. The stem length of soybeans in Example 4-7 groups is 7.60-8.00 cm, which is 86.73%-96.56% higher than that of the salt water control group CK2, and is comparable to the growth of the clear water control group CK1. The above data show that the Example 1-3 groups have a more significant growth-promoting effect on soybeans grown in saline-alkali soil than the Example 4-7 groups, and a better stress resistance effect; this shows that each raw material component and its content within the preferred range can achieve a better technical effect.

[0217] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A microbial preparation suitable for saline-alkali soil, characterized in that: In parts by weight, it comprises: 4 to 25 parts of Bacillus subtilis, 4 to 30 parts of Saccharomyces cerevisiae, 40 to 85 parts of synergistic carriers and 5 to 20 parts of protective carriers; The synergistic carrier includes one or more than two selected from the group consisting of yeast metabolites, mineral humic acid and amino acid powder; the protective carrier includes one or more than two selected from the group consisting of maltodextrin, trehalose, corn starch and attapulgite.

2. The microbial preparation suitable for saline-alkali soil according to claim 1, wherein: In parts by weight, the microbial preparation comprises 4 to 20 parts of Bacillus subtilis; preferably, the microbial preparation comprises 4 to 13 parts of Bacillus subtilis; more preferably, the microbial preparation comprises 4 to 6 parts of Bacillus subtilis; and / or, in parts by weight, the microbial preparation comprises 4 to 20 parts of Saccharomyces cerevisiae; preferably, the microbial preparation comprises 4 to 6 parts of Saccharomyces cerevisiae; And / or, by weight, the microbial preparation comprises 60 to 85 parts of the synergistic carrier; preferably, the microbial preparation comprises 75 to 85 parts of the synergistic carrier; more preferably, the microbial preparation comprises 78 to 82 parts of the synergistic carrier; And / or, based on weight, the microbial preparation includes 5 to 15 parts of a protective carrier; preferably, the microbial preparation includes 5 to 12 parts of a protective carrier; more preferably, the microbial preparation includes 8 to 11 parts of a protective carrier.

3. The microbial preparation suitable for saline-alkali soil according to claim 1 or 2, wherein: The Bacillus subtilis is Bacillus subtilis subsp. subtilis tc-4, which was deposited in the China Center for Type Culture Collection (CCTCC) on April 17, 2023, with a deposit number of CCTCC NO: M 2023541; And / or, the brewer's yeast is brewer's yeast high wine yeast YY (Saccharomyces cerevisiae high wine yeast YY), which was deposited in China Center for Type Culture Collection (CCTCC) on February 1, 2021, with a deposit number of CCTCC NO: M2021171; or the brewer's yeast is brewer's yeast d8.8 (Saccharomyces cerevisiae d8.8), which was deposited in China Center for Type Culture Collection (CCTCC) on March 29, 2017, with a deposit number of CCTCC NO: M2017148; preferably, the brewer's yeast is brewer's yeast high wine yeast YY (Saccharomyces cerevisiae high wine yeast YY), which was deposited in China Center for Type Culture Collection (CCTCC) on February 1, 2021, with a deposit number of CCTCC NO: M 2021171.

4. The microbial preparation suitable for saline-alkali soil according to any one of claims 1 to 3, wherein: The synergistic carrier includes yeast metabolites; Preferably, the yeast metabolite is a metabolite produced by extracting yeast by fermentation using molasses as raw material; More preferably, the molasses comprises beet molasses; And / or, more preferably, based on the dry weight of the yeast metabolites, the content of fulvic acid in the yeast metabolites is 15% to 30%; further preferably, based on the dry weight of the yeast metabolites, the content of betaine in the yeast metabolites is 6% to 15%.

5. The microbial preparation suitable for saline-alkali soil according to any one of claims 1 to 4, wherein: The protective carrier includes one or more than two selected from the group consisting of trehalose, corn starch and attapulgite; preferably, the protective carrier includes corn starch and / or attapulgite.

6. The microbial preparation suitable for saline-alkali soil according to any one of claims 1 to 5, wherein: The effective viable bacteria count of Bacillus subtilis in the microbial preparation is 4 billion to 25 billion CFU / g; preferably, the effective viable bacteria count of Bacillus subtilis in the microbial preparation is 4 billion to 20 billion CFU / g; And / or, the effective viable count of Saccharomyces cerevisiae in the microbial preparation is 800 million to 6 billion CFU / g; preferably, the effective viable count of Saccharomyces cerevisiae in the microbial preparation is 800 million to 4 billion CFU / g.

7. The microbial preparation suitable for saline-alkali soil according to any one of claims 1 to 6, wherein: The Bacillus subtilis is Bacillus subtilis powder, and its preparation method comprises the following steps: Step 1: Activation of Bacillus subtilis; Step 2: inoculating the activated Bacillus subtilis into a culture medium for culturing to obtain a seed solution; and Step 3: inoculating the seed liquid into a fermentation medium for culturing to obtain a fermentation liquid, and drying to obtain Bacillus subtilis powder; Preferably, in step 1, based on the mass of the culture medium used for activation, the culture medium used for activation comprises 0.5%-1.5% peptone, 0.1%-0.5% beef extract, 0.3%-0.7% sodium chloride and 1.5%-2% agar, and the rest is water; and / or, the culture temperature is 35-39°C; and / or, the culture time is 21-27h; more preferably, the cultured Bacillus subtilis is further activated; further preferably, based on the mass of the culture medium used for activation, the culture medium used for activation comprises 0.5%-1.5% peptone, 0.1%-0.5% beef extract, 0.3%-0.7% sodium chloride and 1.5%-2% agar, and the rest is water; and / or, the culture temperature is 35-39°C; and / or, the culture time is 21-27h; And / or, preferably, in step 2, based on the total mass of the culture medium, the culture medium includes 0.5% to 1.5% peptone, 0.1% to 0.5% beef powder, 0.3% to 0.7% sodium chloride and 0.05% to 0.15% glucose, and the rest is water; and / or, the culture temperature is 35 to 39° C.; and / or, the culture time is 10 to 15 hours; and / or, the rotation speed is 100 to 200 rpm; And / or, preferably, in step 3, based on the total mass of the culture medium, the culture medium includes 3% to 5% corn flour, 3% to 5% soybean meal, 0.05% to 0.15% magnesium sulfate and 0.05% to 0.15% calcium chloride, and the rest is water; and / or, the culture temperature is 35 to 39°C; and / or, the culture time is 15 to 20h; and / or, the ventilation volume is 20 to 50L / min; and / or, the tank pressure is 0.2 to 0.5 standard atmospheres.

8. The microbial preparation suitable for saline-alkali soil according to any one of claims 1 to 7, wherein: The brewer's yeast is brewer's yeast powder, and the preparation method thereof comprises the following steps: Step 1: Activation of Saccharomyces cerevisiae; Step 2: inoculating the activated Saccharomyces cerevisiae into a culture medium for culturing to obtain a seed solution; and Step 3: inoculating the seed liquid into a fermentation medium for culturing to obtain a fermentation liquid, and separating and drying to obtain brewer's yeast powder; Preferably, in step 1, the activation medium comprises, based on the mass of the activation medium, 0.5% to 1.5% peptone, 1.5% to 2.5% glucose, 0.1% to 1% yeast extract powder and 1.1 to 1.7% agar, and the remainder is water; and / or the culture temperature is 28 to 32° C.; and / or the culture time is 21 to 27 hours; And / or, preferably, in step 2, the activated Saccharomyces cerevisiae is inoculated into the culture medium used for the primary seed solution for culturing to obtain the primary seed solution; Then, the primary seed liquid is inoculated into the culture medium used for the secondary seed liquid for culturing to obtain the secondary seed liquid; more preferably, the culture conditions of the primary seed liquid are: the culture temperature is 28-32° C.; and / or, the culture time is 12-18 hours; and / or, more preferably, the composition of the culture medium used for the primary seed liquid is: 90-110 g of sucrose, 18-22 g of yeast extract powder, 0.5-1.5 g of magnesium sulfate and 0.5-1.5 g of zinc sulfate per liter of water; and / or, more preferably, the culture conditions for preparing the secondary seed liquid are: the inoculation amount of the primary seed liquid is 1%-3% based on the volume of the culture medium; and / or, the culture temperature is 28-32° C.; and / or, the culture time is 12-18 hours; and / or, the culture medium used for the secondary seed liquid includes beet molasses; And / or, preferably, in step 3, the inoculation amount of the seed solution is 1% to 3% based on the volume of the culture medium; and / or, the culture medium includes beet molasses; and / or, the culture temperature is 28 to 32° C.; and / or, the culture time is 12 to 18 hours.

9. A method for preparing the microbial preparation suitable for saline-alkali soil according to any one of claims 1 to 8, characterized in that: The steps include: The microbial preparation is obtained by mixing the synergistic carrier, Bacillus subtilis, Saccharomyces cerevisiae and the protective carrier.

10. A microbial preparation suitable for saline-alkali soil, characterized in that: The microbial preparation is prepared by the method for preparing the microbial preparation suitable for saline-alkali soil as described in claim 9.

11. An application of a microbial preparation suitable for saline-alkali soil in improving saline-alkali soil, characterized in that: The microbial preparation applicable to saline-alkali soil is the microbial preparation applicable to saline-alkali soil according to any one of claims 1 to 8 or the microbial preparation applicable to saline-alkali soil according to claim 10; Preferably, improving saline-alkali soil includes improving soil fertility, reducing soil alkalinity and / or reducing soil conductivity.

12. The use in improving saline-alkali soil according to claim 11, wherein: The application amount of the microbial preparation suitable for saline-alkali soil is 10-20 kg / hm 2 ; Preferably, the microbial preparation is used by drip irrigation or flushing, and is suitable for the seedling growth period of crops.

13. Use of a microbial preparation suitable for saline-alkali soil in promoting crop growth and / or improving the salt-alkali tolerance of crops, characterized in that: The microbial preparation suitable for saline-alkali soil is the microbial preparation suitable for saline-alkali soil according to any one of claims 1 to 8 or the microbial preparation suitable for saline-alkali soil prepared by the method according to claim 10; Preferably, the crops are leguminous crops; More preferably, the leguminous crop is soybean.

14. The use of claim 13 in promoting the growth of crops and / or improving the salt-alkali tolerance of crops, wherein: The application amount of the microbial preparation suitable for saline-alkali soil is 10-20 kg / hm 2 ; Preferably, the microbial preparation is used by drip irrigation or flushing, and is suitable for the seedling growth period of crops.

15. A composition for improving saline-alkali soil, characterized in that: The composition for improving saline-alkali soil comprises the microbial preparation applicable to saline-alkali soil according to any one of claims 1 to 8 or the microbial preparation applicable to saline-alkali soil according to claim 10; Preferably, improving saline-alkali soil includes improving soil fertility, reducing soil alkalinity and / or reducing soil conductivity.

16. A composition for promoting the growth of crops and / or improving the salt-alkali tolerance of crops, characterized in that: The composition for promoting crop growth and / or improving the salt-alkali tolerance of crops comprises the microbial preparation applicable to saline-alkali soil according to any one of claims 1 to 8 or the microbial preparation applicable to saline-alkali soil according to claim 10.

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