Organic fertilizer obtained by fermenting wine lees and preparation method thereof

Abstract

The invention discloses an organic fertilizer obtained by fermenting distiller's grains and a preparation method thereof, and belongs to the technical field of distiller's grains processing and fermentation fertilizer preparation. The method comprises the following steps: adding corn flour, peptone, glucose and agar to sterile water to mix to obtain a corn solid culture medium; inoculating Trichoderma harzianum into the corn solid culture medium to culture to obtain a spore plate; washing the spore plate with sterile water to obtain a spore suspension; adding corn flour, magnesium sulfate heptahydrate, ammonium sulfate and brown sugar to sterile water to mix to obtain a corn liquid culture medium; inoculating the spore suspension into the corn liquid culture medium to culture to obtain Trichoderma harzianum seed liquid; mixing white wine distiller's grains with monohydrated citric acid to obtain a modified white wine distiller's grains solution; mixing and fermenting the Trichoderma harzianum seed liquid, the modified white wine distiller's grains solution and wheat straw, and drying to obtain the organic fertilizer. The invention improves the activity of Trichoderma harzianum in the organic fertilizer, thereby improving the disease resistance of the organic fertilizer.

Description

Technical Field

[0001] The invention belongs to the technical field of distiller's grains processing and fermentation fertilizer preparation, and particularly relates to an organic fertilizer obtained by fermenting distiller's grains and a preparation method thereof. Background Art

[0002] Liquor lees are the main by-product of liquor brewing, accounting for about 65-80% of the raw materials for liquor production. It is composed of the residue left after wheat, barley, corn and sorghum are fermented by saccharification and fermentation agents such as Daqu, Xiaoqu or Fuqu. In my country, the output of liquor brewing is large and the market demand is high. About 20 million tons of liquor lees can be produced each year. Due to the high acidity and easy corruption of liquor lees, the redevelopment of liquor lees is of great significance to environmental protection and resource utilization. Liquor lees are rich in crude protein, cellulose, amino acids and various trace elements, making the comprehensive utilization of liquor lees possible. At present, liquor lees are used to make fertilizers mainly through natural composting. The fertilizers produced by this method are easy to make crops sick.

[0003] Patent CN102653477B discloses a method for preparing a distiller's grains type bio-organic fertilizer, comprising the following steps: (1) Preparation of Aspergillus fumigatus seed culture solution: a. Preparation of seed culture medium; b. Seed resurrection; c. Seed activation; d. Seed expansion culture; (2) Preparation of bio-organic fertilizer: Mixing the distiller's grains and water at a mass ratio of 1:1 to 1:2 to obtain a culture medium matrix, inoculating 2 to 6% of the Aspergillus fumigatus seed culture solution at a liquid-solid ratio of ml / g, aerating 15 to 30%, and culturing at 42 to 48°C for 4 to 7 days. Aspergillus fumigatus is an environmental pathogenic filamentous fungus that can cause fatal diseases in immunocompromised people, and may cause harm to personnel both in the preparation process of distiller's grains type bio-organic fertilizer and in the use process of distiller's grains type bio-organic fertilizer.

[0004] Patent CN114774288A discloses Trichoderma harzianum and its application and microbial agent for promoting strawberry growth and development against anthracnose. Trichoderma harzianum CM-26 is classified and named as Trichoderma harzianum, and its deposit number is CGMCC NO.40010. The microbial agent comprises the fermentation liquid of the Trichoderma harzianum, and may further comprise potassium alginate and / or raspberry leaf extract. The microbial agent prepared by the Trichoderma harzianum shows significant efficacy in promoting strawberry growth, promoting photosynthesis and preventing and controlling strawberry anthracnose.

[0005] Molds play an important role in the field of biological fermentation. There are many types of molds, and not all molds are suitable for the fermentation production of organic fertilizers from liquor lees. Trichoderma has a more prominent application in the field of plant disease resistance because most Trichoderma can produce a variety of biologically active substances that have antagonistic effects on plant pathogenic fungi and bacteria, such as chitinase, β-1,3-glucanase, cellulase and protease to decompose the cell wall of plant pathogenic fungi or secrete extracellular enzymes such as glucosidase. Trichoderma does not produce drug resistance, has no safe interval for drug use, and has no pesticide residues. Compared with other chemical pesticides and biological pesticides, it reduces the adverse effects on the land environment and human body. The cultivation of Trichoderma usually requires multiple separate fermentation amplifications to control the reproduction iteration rate of Trichoderma, so that various nutrients will not be completely absorbed in the first few iterations of Trichoderma, resulting in a sudden drop in the number of live cells of Trichoderma in the subsequent period. However, for large-scale industrial production of organic fertilizers, multiple separate fermentation amplifications are too cumbersome and difficult to implement.

[0006] Therefore, studying an organic fertilizer prepared by fermentation of distiller's grains with disease resistance has broad application prospects. Summary of the invention

[0007] According to the deficiencies in the prior art, the present invention obtains a corn solid culture medium that is conducive to the resurrection of Trichoderma harzianum by a suitable ratio of corn flour, peptone, glucose, agar and sterile water, and resurrects Trichoderma harzianum. Then, a corn liquid culture medium that is conducive to the amplification of Trichoderma harzianum is obtained by a suitable ratio of corn flour, magnesium sulfate heptahydrate, ammonium sulfate, brown sugar and sterile water, and Trichoderma harzianum is amplified. Then, the liquor lees containing a large amount of protein, amino acids and cellulose are modified, and the protein, amino acids, cellulose and hydrated citric acid are cross-linked, so as to achieve the encapsulation of the liquor lees nutrients. Compared with the unencapsulated nutrients, the encapsulated nutrients can achieve slow decomposition and then regulate the fermentation metabolism of Trichoderma harzianum. On the one hand, it can achieve the effect of controlling the reproduction iteration rate of Trichoderma harzianum, and realize one-time continuous fermentation; on the other hand, it can better retain the nutrients in the liquor lees organic fertilizer that are not too much absorbed by Trichoderma harzianum and cause the problem of abnormal fermentation. Finally, the Trichoderma harzianum seed liquid, the modified liquor lees solution and the wheat straw are evenly mixed and spread on a glass plate for fermentation. The hollow tubular structure of the wheat straw and the spreading on the glass plate can increase the contact area between the aerobic Trichoderma harzianum and oxygen, thereby improving the survival rate of Trichoderma harzianum and the expression of anti-disease substances, solving the technical problems raised in the background technology. Specifically, the technical solution of the present invention includes the following contents:

[0008] Add corn flour, peptone, glucose and agar into sterile water and mix, then heat to 50°C and stir until completely dissolved, then pour into a culture dish, and solidify by natural cooling to obtain a corn solid culture medium;

[0009] inoculating Trichoderma harzianum into the corn solid culture medium to obtain a spore plate;

[0010] Washing the spore plate with sterile water to obtain a spore suspension;

[0011] Add corn flour, magnesium sulfate heptahydrate, ammonium sulfate and brown sugar into sterile water and mix, then heat to 50°C and stir until completely dissolved, then pour into a conical flask to obtain a corn liquid culture medium;

[0012] inoculating the spore suspension into the corn liquid culture medium to obtain Trichoderma harzianum seed solution;

[0013] Mixing liquor lees and water in a mass ratio of 1:1 to obtain a liquor lees solution, adding monohydrated citric acid and sodium hydroxide to the liquor lees solution, adjusting the pH to 7.0-8.0, heating to 50° C., stirring and reacting for 1-1.5 hours to obtain a modified liquor lees solution;

[0014] The organic fertilizer is obtained by mixing the Trichoderma harzianum seed solution, the modified liquor lees solution and wheat straw and fermenting them.

[0015] Furthermore, the mass ratio of corn flour: peptone: glucose: agar: sterile water in the corn solid culture medium is 10:8:1:12:1000. The use of corn flour and peptone is more convenient to prepare and has a better effect on the revival of Trichoderma harzianum than conventional potato glucose solid culture medium.

[0016] Furthermore, the spore plate is a plate containing Trichoderma harzianum obtained by inoculating Trichoderma harzianum into the corn solid culture medium and culturing it for one day under an environment of temperature 28° C. and humidity 80%.

[0017] Furthermore, the process of washing the spore plate with sterile water includes washing the spore plate twice in succession with 5 ml of sterile water.

[0018] Furthermore, the mass ratio of corn flour: magnesium sulfate heptahydrate: ammonium sulfate: brown sugar: sterile water in the corn liquid culture medium is 10: 1: 2: 4: 1000. The use of brown sugar has the effect of having a better amplification effect on Trichoderma harzianum than conventional culture medium using glucose.

[0019] Furthermore, the process of inoculating the spore suspension into the corn liquid culture medium for cultivation is to inoculate the spore suspension into the corn liquid culture medium and culture it under shaking at a temperature of 24° C. and a humidity of 65% for 5 to 8 days.

[0020] Furthermore, the usage of the citric acid monohydrate is 0.5 mol of citric acid monohydrate per 1000 g of the white wine lees solution.

[0021] Furthermore, the mass ratio of the Trichoderma harzianum seed solution: the modified white wine lees solution and wheat straw is 1:20:10.

[0022] Furthermore, the process of drying after the mixed fermentation of the Trichoderma harzianum seed solution, the modified liquor lees solution and the wheat straw includes evenly mixing the Trichoderma harzianum seed solution, the modified liquor lees solution and the wheat straw, spreading them on a glass plate, fermenting them in an environment at a temperature of 28° C. for 20 to 25 days and then drying them. The use of wheat straw is beneficial to the fermentation of Trichoderma harzianum, and the hollow tubular structure of the wheat straw is beneficial to ventilation, which enhances the aerobic fermentation of Trichoderma harzianum, improves the activity of Trichoderma harzianum in the organic fertilizer, and thus improves the disease resistance of the organic fertilizer.

[0023] Furthermore, the mass ratio of the Trichoderma harzianum seed solution: the modified white wine lees solution: wheat straw is 1:20:10.

[0024] Furthermore, the water content needs to be maintained above 30% during the mixed fermentation process.

[0025] The invention discloses an organic fertilizer prepared by a method for preparing organic fertilizer obtained by fermenting wine lees.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention improves the effect and efficiency of the resuscitation by using corn flour and peptone, which are more effective for the resurrection culture of Trichoderma harzianum, as solid culture medium. And brown sugar, which has a promoting effect on the amplification culture of Trichoderma harzianum, is added to the corn flour liquid culture medium to improve the amplification effect and efficiency. Finally, the Trichoderma harzianum seed liquid, the modified white wine lees solution and the wheat straw are evenly mixed and spread on a glass plate for fermentation. The hollow tubular structure of the wheat straw spread on the glass plate can increase the contact area between the aerobic Trichoderma harzianum and oxygen, thereby improving the viability of Trichoderma harzianum and the expression ability of disease-resistant substances.

[0028] (2) The present invention modifies the liquor lees containing a large amount of protein, amino acids and cellulose, and allows citric acid to undergo cross-linking reactions with hydroxyl groups and amino groups respectively in an alkaline environment, so that the protein, amino acids and cellulose are cross-linked to form a cage-like or claw-like encapsulation structure, and other substances are encapsulated therein. This encapsulated cross-linked structure prevents the nutrients in the liquor lees from being quickly absorbed by Trichoderma harzianum, and needs to be gradually decomposed into small molecules and released during the fermentation process to regulate the fermentation metabolism of Trichoderma harzianum. On the one hand, it can control the reproduction iteration rate of Trichoderma harzianum by limiting the release of nutrients required for the reproduction of Trichoderma harzianum, thereby achieving the purpose of one-time continuous fermentation; on the other hand, it can better retain the nutrients required by plants in the liquor lees organic fertilizer so that they will not be excessively absorbed by Trichoderma harzianum and cause fermentation abnormalities.

[0029] (3) Through the results of the number of live bacteria of organic fertilizers, the content of nutrients required by plants, and the anti-disease efficacy of Example 6 and Examples 1 to 5, it can be found that when the white wine lees solution is modified, if the cross-linking reaction time is too long, the number of protein, amino acid and cellulose cross-linked encapsulation structures will be too large and difficult to decompose, resulting in the inability of Trichoderma harzianum to obtain sufficient nutrients for reproduction in the early stage of iteration, which reduces the number of live bacteria of Trichoderma harzianum, and in the later stage of iteration, it is unable to obtain sufficient nutrients for expressing metabolites with anti-disease efficacy, which deteriorates the anti-disease effect of the organic fertilizer, affects the further fermentation of the white wine lees solution, and reduces the content of nutrients required by plants. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below through the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the raw materials and reagents used in the present invention are commercially available or can be prepared by known methods.

[0032] The strain number is CICC 13056 and was purchased from the China Industrial Microbiology Culture Collection Center.

[0033] The Botrytis cinerea strain numbered CGMCC 34581 was purchased from this general microbial strain collection management center.

[0034] Preparation Example 1:

[0035] The preparation of corn solid culture medium includes the following steps:

[0036] Add 1000ml of sterile water to a beaker, add 10g of corn flour, 8g of peptone, 1g of glucose and 12g of agar to 1000ml of sterile water respectively, stir well and pour into a wide-mouth flask, then heat the wide-mouth flask with an alcohol stove, heat to 50℃ and stir until completely dissolved, pour into a conical flask for wet heat sterilization, after wet heat sterilization, wait until it cools to 50-55℃ and then pour into a 90mm plate and let cool to obtain corn solid culture medium.

[0037] Preparation Example 2:

[0038] The preparation of corn liquid culture medium includes the following steps:

[0039] Add 1000 ml of sterile water to a beaker, add 10 g of corn flour, 1 g of magnesium sulfate heptahydrate, 2 g of ammonium sulfate and 4 g of brown sugar to 1000 ml of sterile water respectively, stir evenly and pour into a wide-mouth flask, then heat the wide-mouth flask with an alcohol stove, heat to 50°C and stir until completely dissolved, then pour into a conical flask for wet heat sterilization to obtain corn liquid culture medium.

[0040] Embodiment 1:

[0041] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0042] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator and set at a temperature of 28° C. and a humidity of 80% for culturing for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquids were mixed twice to obtain a spore suspension; the spore suspension was inoculated into the corn liquid culture medium conical flask of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / min, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for culturing for 5 days. Add 1000ml sterile water and 1000g liquor lees to a beaker, stir evenly and add 105.1g monohydrated citric acid, stir evenly again and add appropriate amount of sodium hydroxide, adjust pH to 7.0-8.0 and pour into a heating container and heat to 50°C, continue to react for 1.5h to obtain a modified liquor lees solution. Take 100g of the Trichoderma harzianum seed solution obtained after cultivation, add it to 2000g of the modified liquor lees solution, then add 1000g of granular straw, stir well and spread it on a 10cm thick glass plate, then place the glass plate in a sunny constant temperature greenhouse, set the temperature to 28°C, detect the water content in the glass plate every day, ensure that the water content is above 30% by watering, and dry it after fermentation for 20 days to obtain organic fertilizer.

[0043] Embodiment 2:

[0044] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0045] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator and set at a temperature of 28° C. and a humidity of 80% for culturing for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquid was mixed twice to obtain a spore suspension; the spore suspension was inoculated into the corn liquid culture medium conical flask of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / m, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for culturing for 8 days. Add 1000ml sterile water and 1000g liquor lees to a beaker, stir evenly and add 105.1g monohydrated citric acid, stir evenly again and add appropriate amount of sodium hydroxide, adjust pH to 7.0-8.0 and pour into a heating container and heat to 50°C, continue to react for 1.5h to obtain a modified liquor lees solution. Take 100g of the Trichoderma harzianum seed solution obtained after cultivation, add it to 2000g of the modified liquor lees solution, then add 1000g of granular straw, stir well and spread it on a 10cm thick glass plate, then place the glass plate in a sunny constant temperature greenhouse, set the temperature to 28°C, detect the water content in the glass plate every day, ensure that the water content is above 30% by watering, and dry it after fermentation for 20 days to obtain organic fertilizer.

[0046] Embodiment 3:

[0047] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0048] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator and set at a temperature of 28° C. and a humidity of 80% for culturing for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquids were mixed twice to obtain a spore suspension; the spore suspension was inoculated into the corn liquid culture medium conical flask of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / m, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for culturing for 5 days. Add 1000ml sterile water and 1000g liquor lees to a beaker, stir evenly and add 105.1g monohydrated citric acid, stir evenly again and add appropriate amount of sodium hydroxide, adjust pH to 7.0-8.0 and pour into a heating container and heat to 50°C, continue to react for 1.5h to obtain a modified liquor lees solution. Take 100g of the Trichoderma harzianum seed solution obtained after cultivation, add it to 2000g of the modified liquor lees solution, then add 1000g of granular wheat straw, stir well and spread it on a 10cm thick glass plate, then place the glass plate in a sunny constant temperature greenhouse, set the temperature to 28°C, detect the water content in the glass plate every day, ensure that the water content is above 30% by watering, and dry it after fermentation for 25 days to obtain organic fertilizer.

[0049] Embodiment 4:

[0050] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0051] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator and set at a temperature of 28° C. and a humidity of 80% for culturing for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquid was mixed twice to obtain a spore suspension; the spore suspension was inoculated into the corn liquid culture medium conical flask of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / m, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for culturing for 8 days. Add 1000ml sterile water and 1000g liquor lees to a beaker, stir evenly and add 105.1g monohydrated citric acid, stir evenly again and add appropriate amount of sodium hydroxide, adjust pH to 7.0-8.0 and pour into a heating container and heat to 50°C, continue to react for 1.5h to obtain a modified liquor lees solution. Take 100g of the Trichoderma harzianum seed solution obtained after cultivation, add it to 2000g of the modified liquor lees solution, then add 1000g of granular wheat straw, stir well and spread it on a 10cm thick glass plate, then place the glass plate in a sunny constant temperature greenhouse, set the temperature to 28°C, detect the water content in the glass plate every day, ensure that the water content is above 30% by watering, and dry it after fermentation for 25 days to obtain organic fertilizer.

[0052] Embodiment 5:

[0053] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0054] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator and set at a temperature of 28° C. and a humidity of 80% for culturing for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquid was mixed twice to obtain a spore suspension; the spore suspension was inoculated into the corn liquid culture medium conical flask of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / m, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for culturing for 8 days. Add 1000ml sterile water and 1000g liquor lees to a beaker, stir evenly and add 105.1g monohydrated citric acid, stir evenly again and add appropriate amount of sodium hydroxide, adjust pH to 7.0-8.0 and pour into a heating container and heat to 50°C, continue to react for 1h to obtain a modified liquor lees solution. Take 100g of the Trichoderma harzianum seed solution obtained after cultivation, add it to 2000g of the modified liquor lees solution, then add 1000g of granular wheat straw, stir well and spread it on a 10cm thick glass plate, then place the glass plate in a sunny constant temperature greenhouse, set the temperature to 28°C, detect the water content in the glass plate every day, ensure that the water content is above 30% by watering, and dry it after fermentation for 25 days to obtain organic fertilizer.

[0055] Embodiment 6:

[0056] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0057] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator and set at a temperature of 28° C. and a humidity of 80% for culturing for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquid was mixed twice to obtain a spore suspension; the spore suspension was inoculated into the corn liquid culture medium conical flask of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / m, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for culturing for 8 days. Add 1000ml sterile water and 1000g liquor lees to a beaker, stir evenly, add 105.1g citric acid monohydrate, stir evenly again, add an appropriate amount of sodium hydroxide, adjust the pH to 7.0-8.0, pour into a heating container and heat to 50°C, continue to react for 2h, and obtain a modified liquor lees solution. Add 1000ml sterile water and 1000g liquor lees to a beaker, stir evenly, add 105.1g citric acid monohydrate, stir evenly again, add an appropriate amount of sodium hydroxide, adjust the pH to 7.0-8.0, pour into a heating container and heat to 50°C, continue to react for 2h, and obtain a modified liquor lees solution. 100 g of the Trichoderma harzianum seed liquid obtained after cultivation was added to 2000 g of the modified liquor lees solution, and then 1000 g of granular wheat straw was added. After being fully stirred, the mixture was spread on a 10 cm thick glass plate, and then the glass plate was placed in a constant temperature greenhouse with sufficient sunlight, the temperature was set to 28° C. The water content in the glass plate was tested every day, and the water content was ensured to be above 30% by watering. After fermentation for 25 days, the organic fertilizer was obtained by drying.

[0058] Comparative Example 1:

[0059] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0060] The corn solid culture medium plates in Preparation Example 1 in Example 4 were replaced with commonly used PDA culture medium plates, and the other conditions remained the same as in Example 4.

[0061] Comparative Example 2:

[0062] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0063] The corn liquid culture medium of Preparation Example 2 in Example 4 was replaced with a commonly used corn liquid culture medium without brown sugar, and the other conditions remained the same as in Example 4.

[0064] Comparative Example 3:

[0065] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0066] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator and set at a temperature of 28° C. and a humidity of 80% for culturing for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquids were mixed twice to obtain a spore suspension; the spore suspension was inoculated into the corn liquid culture medium conical flask of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / m, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for culturing for 5 days. Add 1000ml sterile water and 1000g white wine lees in a beaker, stir evenly and add 105.1g monohydrated citric acid, stir evenly again and add appropriate amount of sodium hydroxide, adjust pH to 7.0-8.0 and pour into a heating container and heat to 50°C, continue to react for 1.5h to obtain a modified white wine lees solution. Take 100g of the harzianum seed solution obtained after cultivation, add it to 3000g of the modified white wine lees solution, stir well and spread it on a 10cm thick glass plate, then place the glass plate in a sunny constant temperature greenhouse, set the temperature to 28°C, detect the water content in the glass plate every day, ensure that the water content is above 30% by watering, and dry it after fermentation for 20 days to obtain organic fertilizer.

[0067] Comparative Example 4:

[0068] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0069] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator and set at a temperature of 28° C. and a humidity of 80% for culturing for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquids were mixed twice to obtain a spore suspension; the spore suspension was inoculated into a conical flask of corn liquid culture medium of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / m, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for culturing for 5 days. Add 1000ml sterile water and 1000g white wine lees in a beaker, stir evenly and add 105.1g monohydrated citric acid, stir evenly again and add appropriate amount of sodium hydroxide, adjust pH to 7.0-8.0 and pour into a heating container and heat to 50°C, continue to react for 1.5h to obtain a modified white wine lees solution. Take 100g of the harzianum seed solution obtained after cultivation, add it to 2000g of the modified white wine lees solution, then add 1000g of granular wheat straw, stir well and pour into a stainless steel barrel, then place the stainless steel barrel in a sunny constant temperature greenhouse, set the temperature to 28°C, detect the water content in the glass plate every day, ensure that the water content is above 30% by watering, and dry it after fermentation for 20 days to obtain organic fertilizer.

[0070] Comparative Example 5:

[0071] A method for preparing organic fertilizer obtained by fermenting wine lees, specifically comprising the following steps:

[0072] The Trichoderma harzianum strain numbered CICC 13056 was inoculated into the corn solid culture medium plate of Preparation Example 1, and then placed in a mold incubator with the temperature set at 28° C. and the humidity set at 80% for 1 day; 5 ml of sterile water was added to the incubated plate as a rinse liquid, and after shaking for 2 minutes, the rinse liquid was poured into a test tube, and the operation was repeated once, and the rinse liquids were mixed twice to obtain a spore suspension; the spore suspension was inoculated into a conical flask of the corn liquid culture medium of Preparation Example 2, and then the conical flask was sealed and placed on an oscillator, and placed together in a mold incubator, the oscillation frequency was adjusted to 30 r / m, and the temperature of the mold incubator was set at 24° C. and the humidity was set at 65% for 8 days. Add 1000 ml of sterile water, 1000 g of white wine lees and 1000 g of granular wheat straw into a beaker, stir well and spread on a 10 cm thick glass plate, then place the glass plate in a constant temperature greenhouse with plenty of sunlight, set the temperature to 28°C, check the water content in the glass plate every day, ensure that the water content is above 30% by watering, and dry after fermentation for 25 days to obtain organic fertilizer.

[0073] The organic fertilizers obtained in Examples 1 to 6 and Comparative Examples 1 to 5 were fully mixed with water and then allowed to stand. The upper layer of liquid was centrifuged, and the supernatant after centrifugation was diluted. After dilution, a blood cell count was performed to obtain the number of viable Trichoderma harzianum as shown in the following table:

[0074] Sources Trichoderma harzianum viable count cfu / g Example 1 <![CDATA[4.8×10 7 ]]> Example 2 <![CDATA[7.1×10 7 ]]> Example 3 <![CDATA[1.2×10 8 ]]> Example 4 <![CDATA[3.8×10 8 ]]> Example 5 <![CDATA[6.3×10 7 ]]> Example 6 <![CDATA[8.6×10 4 ]]> Comparative Example 1 <![CDATA[2.2×10 6 ]]> Comparative Example 2 <![CDATA[4.5×10 5 ]]> Comparative Example 3 <![CDATA[2.1×10 5 ]]> Comparative Example 4 <![CDATA[6.7×10 5 <!-- 7 -->]]> Comparative Example 5 <![CDATA[1.9×10 5 ]]>

[0075] The organic fertilizers obtained in Examples 1 to 6 and Comparative Examples 1 to 5 were allowed to stand, and the content of organic matter (%) was obtained by potassium dichromate volumetric method; the content of phosphorus (%) was obtained by vanadium molybdenum yellow colorimetry; the content of potassium (%) was obtained by flame photometry; the content of nitrogen (%) was obtained by sulfuric acid-hydrogen peroxide digestion method, and the content of nutrients required by plants was obtained as shown in the following table:

[0076] Sources Organic matter / % phosphorus / % Potassium / % nitrogen / % Example 1 82.9 5.12 1.24 4.98 Example 2 83.4 5.04 1.36 5.17 Example 3 82.7 5.18 1.28 4.98 Example 4 80.4 5.43 1.35 5.23 Example 5 82.3 5.42 1.31 5.16 Example 6 90.7 2.96 0.34 3.87 Comparative Example 1 83.3 5.36 1.31 5.07 Comparative Example 2 81.7 5.12 1.19 4.84 Comparative Example 3 86.5 4.21 0.82 4.21 Comparative Example 4 87.3 4.32 0.95 4.34 Comparative Example 5 78.7 5.51 1.39 5.22

[0077] Eleven tomato plots of the same area were isolated in the same soil environment, and the same batch of tomato seeds were planted respectively, and it was ensured that there were 50 well-growing tomato vines on each tomato plot. Until the 60th day, all tomato vines were inoculated with Botrytis cinerea with strain number CGMCC 34581, and the organic fertilizers obtained in Examples 1 to 6 and Comparative Examples 1 to 5 were applied to the 11 tomato plots respectively, 10 g of fertilizer was applied to each tomato vine, and the fertilizers were applied three times in succession, each time with an interval of 10 days, and the tomato vines were observed to see whether they were infected with the disease. The results of the disease resistance are shown in Table 1:

[0078]

[0079]

[0080] (1) It can be seen from Comparative Example 1 that although the use of PDA medium for the revival of Trichoderma harzianum has little effect on the nutrients in the organic fertilizer, it will significantly affect the number of live bacteria of Trichoderma harzianum, resulting in a decrease in the disease resistance effect. This may be because the number of Trichoderma harzianum is reduced after the revival. Although the nutrients in the liquor lees are sufficient for the reproduction and fermentation of Trichoderma harzianum, it is not fully fermented within a limited number of days.

[0081] (2) It can be seen from Comparative Example 2 that the use of corn liquid culture medium without brown sugar for the amplification of Trichoderma harzianum has an impact on the nutritional components in the organic fertilizer and the number of viable bacteria of Trichoderma harzianum, and the disease resistance effect is also greatly affected. This may be because the number of viable bacteria or the working expression efficiency of Trichoderma harzianum after amplification cannot meet the requirements of the fermentation of the modified white wine lees solution.

[0082] (3) It can be seen from Comparative Example 3 that the lack of greater ventilation brought by the porous structure of wheat straw caused Trichoderma harzianum to carry out more anaerobic fermentation, resulting in a decrease in the reproduction rate of Trichoderma harzianum and a decrease in the number of viable bacteria. However, since the viability of Trichoderma harzianum may not have decreased too much, Trichoderma harzianum is still metabolizing and expressing disease-resistant substances, resulting in the actual decrease in the level of disease-resistant effect being not completely consistent with the decrease in the number of viable bacteria.

[0083] (4) It can be seen from Comparative Example 4 that flat paving can bring a larger oxygen contact area, allowing Trichoderma harzianum to carry out more aerobic fermentation. The overall impact is similar to that of Comparative Example 3, but the degree of impact is not as high as that of Comparative Example 3, which further illustrates that oxygen has a great influence on the reproduction and expression of Trichoderma harzianum during the fermentation process.

[0084] (5) It can be seen from Comparative Example 5 that the number of live bacteria of Trichoderma harzianum is severely reduced, but the content of nutrients is on the high side. This shows that the nutrients required for the reproduction and expression of Trichoderma harzianum are not completely consistent with the nutrients required by plants. Since the white wine lees have not been modified, Trichoderma harzianum consumes a large amount of required nutrients in the initial iteration, resulting in insufficient nutrition in subsequent iterations and a large number of Trichoderma harzianum deaths. However, the cellulase produced by the metabolism of Trichoderma harzianum is still further enzymolyzing the white wine lees to release the nutrients required by plants.

[0085] The above-described embodiments provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected.

Claims

1. A method for preparing organic fertilizer obtained by fermentation of distiller's grains, characterized in that: The preparation method comprises the following steps: Add corn flour, peptone, glucose and agar into sterile water and mix, then heat to 50°C and stir until completely dissolved, then pour into a culture dish, and solidify by natural cooling to obtain a corn solid culture medium; inoculating Trichoderma harzianum into the corn solid culture medium to obtain a spore plate; Washing the spore plate with sterile water to obtain a spore suspension; Add corn flour, magnesium sulfate heptahydrate, ammonium sulfate and brown sugar into sterile water and mix, then heat to 50°C and stir until completely dissolved, then pour into a conical flask to obtain a corn liquid culture medium; inoculating the spore suspension into the corn liquid culture medium to obtain a Trichoderma harzianum seed solution; The liquor lees and water are mixed in a mass ratio of 1:1 to obtain a liquor lees solution, monohydrated citric acid is added to the liquor lees solution and stirred evenly, sodium hydroxide is added to adjust the pH to 7.0-8.0, and the mixture is heated to 50° C. and stirred for reaction for 1-1.5 hours to obtain a modified liquor lees solution; The organic fertilizer is obtained by mixing the Trichoderma harzianum seed solution, the modified liquor lees solution and wheat straw for fermentation and then drying; The usage amount of the citric acid monohydrate is 0.5 mol of citric acid monohydrate per 1000 g of the liquor lees solution; The process of drying after mixed fermentation of the Trichoderma harzianum seed solution, the modified white wine lees solution and wheat straw comprises mixing the Trichoderma harzianum seed solution, the modified white wine lees solution and wheat straw evenly, spreading them on a glass plate, fermenting them in an environment at a temperature of 28° C. for 20 to 25 days and then drying them.

2. The method for preparing an organic fertilizer obtained by fermentation of wine lees according to claim 1, characterized in that: The mass ratio of corn flour: peptone: glucose: agar: sterile water in the corn solid culture medium is 10:8:1:12:1000.

3. The method for preparing an organic fertilizer obtained by fermentation of wine lees according to claim 1, characterized in that: The process of washing the spore plate with sterile water includes washing the spore plate twice in succession with 5 ml of sterile water.

4. The method for preparing an organic fertilizer obtained by fermentation of wine lees according to claim 1, characterized in that: The mass ratio of corn flour: magnesium sulfate heptahydrate: ammonium sulfate: brown sugar: sterile water in the corn liquid culture medium is 10:1:2:4:1000.

5. The method for preparing an organic fertilizer obtained by fermentation of wine lees according to claim 1, characterized in that: The mass ratio of the Trichoderma harzianum seed solution: the modified liquor lees solution and the wheat straw is 1:20:

10.

6. The method for preparing an organic fertilizer obtained by fermentation of wine lees according to claim 1, characterized in that: The water content needs to be maintained above 30% during the mixed fermentation process.

7. An organic fertilizer prepared by the method for preparing an organic fertilizer obtained by fermentation of wine lees according to any one of claims 1 to 6.

Images