Corn steep liquor fermentation and its application in preparing water-soluble nitrogen fertilizer

Abstract

The application provides corn syrup fermentation and application thereof in preparation of water-soluble nitrogen fertilizer, and belongs to the technical field of water-soluble fertilizer.The preparation method of the Rhizopus kind koji provided by the application comprises the following steps: mixing Rhizopus spore suspension and culture medium, and carrying out fermentation culture at 28 DEG C to 40 DEG C for 48 h to 108 h; the culture medium is composed of dry materials and liquid materials, the dry materials are composed of soybean meal and bran, and the liquid materials are composed of glucose mother liquor, corn syrup and water.The preparation method of the Rhizopus kind koji can improve the neutral protease activity in the Rhizopus kind koji, so as to improve the subsequent fermentation efficiency of corn syrup and improve the functional activity of the fermentation product obtained by corn syrup fermentation.The corn syrup fermentation provided by the application can improve the small peptide content in the corn syrup fermentation product.The corn syrup fermentation provided by the application can replace nitrogen fertilizer to promote the growth and development of soybean.

Description

Technical Field

[0001] This invention belongs to the field of water-soluble fertilizer technology, specifically relating to a corn steep liquor fermentation product and its application in the preparation of water-soluble nitrogen fertilizer. Background Technology

[0002] Corn steep liquor is one of the main byproducts of wet corn starch production, produced in huge quantities and rich in nutrients such as protein and carbohydrates. With the increase in corn processing volume in recent years, the output of this byproduct has also doubled year after year. Currently, the main uses of corn steep liquor include its use as a nitrogen source for fermentation, but the utilization rate remains limited, resulting in resource waste and a lack of resource-based and rational utilization methods. How to effectively utilize the nutrients in corn steep liquor has become a technical challenge for corn processing enterprises.

[0003] Mucor, a common microbial species in fermented foods, can secrete various hydrolytic enzymes, such as proteases and cellulases, with high hydrolysis efficiency. However, there are currently no reports on using Mucor koji to ferment corn steep liquor. Water-soluble fertilizers are fast-acting, high-concentration fertilizers that can be completely dissolved in water, directly absorbed by plants, and are quick to take effect with low dosage. Therefore, preparing corn steep liquor into water-soluble fertilizers has broad application prospects; however, there are currently no records in this field on how to prepare corn steep liquor into water-soluble fertilizers. Summary of the Invention

[0004] In view of this, one of the objectives of the present invention is to provide a high-yield mucor koji and its preparation method.

[0005] The second objective of this invention is to provide a corn steep liquor fermentation product that can be used as a water-soluble nitrogen fertilizer.

[0006] The third objective of this invention is to provide a method for promoting soybean growth and development by using corn steep liquor fermentation to replace nitrogen fertilizer.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a high-yield neutral protease-producing Rhizopus koji, comprising the following steps: mixing Rhizopus spore suspension with a culture medium and fermenting at 28℃~40℃ for 48h~108h; the culture medium consists of dry material and liquid material, the dry material consists of soybean meal and wheat bran, and the liquid material consists of glucose mother liquor, corn steep liquor and water; the weight-to-volume ratio of the dry material and the liquid material is 20g:10~30mL; the weight ratio of the soybean meal and wheat bran is (1:19)~(4:16); the volume ratio of the corn steep liquor, glucose mother liquor and water is (0:2:13)~(3:2:10).

[0008] Preferably, the concentration of the Mucor spore suspension is 4 × 10⁻⁶. 6Cells / mL ~5×10 6 The amount of the Rhizopus spore suspension added is based on the weight of the dry material, which is 0.8 mL to 2.4 mL of Rhizopus spore suspension per 20 g of dry material; the Rhizopus spore suspension is Rhizopus wutongqiaoensis YY-25, with the preservation number CGMCC No.12109.

[0009] The present invention also provides a Mucor spawn with high yield of neutral protease, which is prepared by the above preparation method.

[0010] The present invention also provides the application of the above-mentioned Mucor koji in the preparation of corn steep liquor fermentation products that can be used as water-soluble nitrogen fertilizer.

[0011] The present invention also provides a corn steep liquor ferment that can be used as a water-soluble nitrogen fertilizer, which is prepared by the following method: corn steep liquor is mixed with sodium hydroxide, calcium hydroxide is added and mixed, then the above-mentioned Rhizopus inoculum is added, fermented, and the supernatant is taken to obtain the corn steep liquor ferment.

[0012] Preferably, the mass ratio of Rhizopus inoculum to corn steep liquor is 0.5~2.5:100.

[0013] Preferably, the mass ratio of sodium hydroxide to corn steep liquor is 1.79:100, and the mass ratio of calcium hydroxide to corn steep liquor is 0.29:100.

[0014] Preferably, the fermentation temperature is 45℃~65℃, and the fermentation time is 0~6h.

[0015] The present invention also provides the application of the above-mentioned corn steep liquor fermentation product in the preparation of water-soluble nitrogen fertilizer.

[0016] The present invention also provides a method for promoting soybean growth and development, wherein the above-mentioned corn steep liquor fermentation product is applied as nitrogen fertilizer when applying base fertilizer.

[0017] The beneficial effects of this invention are: The method for preparing *Mucor* koji provided by this invention can improve the activity of neutral protease in the *Mucor* koji, thereby improving the fermentation efficiency of corn steep liquor and the functional activity of the fermentation products obtained from corn steep liquor fermentation. Using the *Mucor* koji provided by this invention to ferment corn steep liquor can increase the content of small peptides in the corn steep liquor fermentation product. The corn steep liquor fermentation product provided by this invention can replace nitrogen fertilizer to promote the growth and development of soybeans. Attached Figure Description

[0018] Figure 1 The effect of different soybean meal addition amounts in dry feed on the activity of neutral protease in Rhizopus koji.

[0019] Figure 2The effect of the amount of liquid material added (moisture content) on the activity of neutral protease in Mucor spores;

[0020] Figure 3 The effect of glucose mother liquor addition on the activity of neutral protease in Mucor spores;

[0021] Figure 4 The effect of corn steep liquor addition on the activity of neutral protease in Mucor koji.

[0022] Figure 5 The effect of inoculum size of Mucor spore suspension (bacterial suspension) on the activity of neutral protease in Mucor spores;

[0023] Figure 6 The effect of fermentation time on the activity of neutral protease in Mucor spores;

[0024] Figure 7 The effect of fermentation temperature on the activity of neutral protease in Mucor koji;

[0025] Figure 8 The effect of the amount of Rhizopus inoculum added on the content of small peptides in corn steep liquor fermentation product;

[0026] Figure 9 The effect of fermentation temperature on the content of small peptides in corn steep liquor fermentation products;

[0027] Figure 10 The effect of fermentation time on the content of small peptides in corn steep liquor fermentation product;

[0028] Figure 11 Soybean plant height in different treatment groups;

[0029] Figure 12 The thickness of soybean stems in different treatment groups;

[0030] Figure 13 The chlorophyll content of soybean leaves in different treatment groups;

[0031] Figure 14 Leaf area of ​​soybean leaves in different treatment groups;

[0032] Figure 15 Fresh weight of soybean leaves for different treatment groups;

[0033] Figure 16 Dry weight of soybean leaves in different treatment groups;

[0034] Figure 17 Fresh weight of soybean stems in different treatment groups;

[0035] Figure 18 The dry weight of soybean stems for different treatment groups;

[0036] In the above figures, the letters on the bars mean that, among any two groups, different letters indicate significant differences (p < 0.05). Biological Preservation Information

[0037] The *Mucor* species used in this invention is *Mucor wutongqiaoensis* YY-25, which was deposited on March 28, 2016, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 12109, and taxonomically named *Mucor wutongqiaoensis*. Mucor wutungkiao . Detailed Implementation

[0038] This invention provides a method for preparing a high-yield neutral protease-producing Rhizopus koji, comprising the following steps: mixing Rhizopus spore suspension with a culture medium and fermenting at 28℃~40℃ for 48h~108h; the culture medium consists of dry material and liquid material, the dry material consists of soybean meal and wheat bran, and the liquid material consists of glucose mother liquor, corn steep liquor and water; the weight-to-volume ratio of the dry material and the liquid material is 20g:10~30mL; the weight ratio of soybean meal and wheat bran is (1:19)~(4:16); the volume ratio of corn steep liquor, glucose mother liquor and water is (0:2:13)~(3:2:10).

[0039] In this invention, the preferred method for preparing the *Mucor* spore suspension includes the following steps: injecting sterile water into a *Mucor* slant, scraping *Mucor* spores and hyphae using an inoculation loop, pouring the mixture containing hyphae and spores into a conical flask containing glass beads, and shaking it in a shaker at 28°C and 180 r / min. In this invention, the shaking time is preferably 1 hour, and the concentration of the *Mucor* spore suspension is preferably 4 × 10⁻⁶. 6 Cells / mL ~5×10 6 The strain of *Rhizopus* spores in the suspension is preferably *Rhizopus wutongqiaoensis* YY-25, with accession number CGMCC No. 12109.

[0040] In this invention, the amount of *Mucor spore* suspension added is based on the weight of the dry material, preferably 0.8 mL to 2.4 mL per 20 g of dry material, more preferably 1.0 mL to 2.0 mL per 20 g of dry material. In this invention, the weight-to-volume ratio of the dry material to the liquid material is preferably 20 g: 12-20 mL, more preferably 20 g: 15-18 mL; the weight ratio of soybean meal to wheat bran is preferably (2:18) to (3:17). In the culture medium provided by this invention, the amount of soybean meal is significantly reduced, further reducing production costs; the volume ratio of corn steep liquor, glucose mother liquor, and water is preferably (1:2:12) to (2:2:11). This invention does not have specific limitations on the specific sources of the raw materials in the culture medium; conventional commercially available products in the art can be used. In this invention, after the culture medium is prepared, it is preferably sterilized before adding the *Mucor spore* suspension for fermentation. The sterilization conditions are preferably sterilization at 121°C for 20 min. After sterilization, while still hot, shake the clumps of culture medium in the container to break them up. After cooling to room temperature, add the Mucor spore suspension. In the Mucor spawn preparation method of the present invention, the fermentation method is preferably shaker fermentation, the shaking speed of the shaker is preferably 10 r / min, and during the fermentation process, it is preferable to shake the flask once every 24 hours.

[0041] The present invention also provides a Mucor spawn with high yield of neutral protease, which is prepared by the above preparation method.

[0042] The present invention also provides the application of the above-mentioned Mucor koji in the preparation of corn steep liquor fermentation products that can be used as water-soluble nitrogen fertilizer.

[0043] The present invention also provides a corn steep liquor ferment that can be used as a water-soluble nitrogen fertilizer, which is prepared by the following method: corn steep liquor is mixed with sodium hydroxide, calcium hydroxide is added and mixed, then the above-mentioned Rhizopus inoculum is added, fermented, and the supernatant is taken to obtain the corn steep liquor ferment.

[0044] In this invention, the preferred mass ratio of sodium hydroxide to corn steep liquor is 1.79:100. After adding sodium hydroxide to the corn steep liquor, stirring is preferably required for 20 minutes, during which the pH is adjusted to maintain at 5.5-6.0. Then, calcium hydroxide is added, with a preferred mass ratio of calcium hydroxide to corn steep liquor of 0.29:100. After adding calcium hydroxide, stirring is preferably required for 40 minutes, during which the pH is still adjusted to maintain at 5.5-6.0. The addition of sodium hydroxide and calcium hydroxide to the corn steep liquor in this invention serves two purposes: firstly, to adjust the pH of the system, and secondly, to flocculate impurities in the corn steep liquor. In this invention, the preferred mass ratio of *Rhizopus* koji to corn steep liquor is 0.5-2.5:100, more preferably 1.0-2.0:100; the preferred fermentation temperature is 45℃-65℃, more preferably 50℃-60℃; and the preferred fermentation time is 0h-6h, more preferably 2h-5h, and even more preferably 3h-4h. In this invention, when preparing corn steep liquor fermentation product, the fermentation method is preferably stirred fermentation. After fermentation, it is preferably filtered and centrifuged. The filtration is preferably carried out using four layers of gauze, and the centrifugation is preferably carried out at 4000 r / min for 20 min.

[0045] This invention also provides the application of the above-mentioned corn steep liquor fermentation product in the preparation of water-soluble nitrogen fertilizer. The corn steep liquor fermentation product provided by this invention can be used as a fast-acting, high-concentration fertilizer. When used as a water-soluble nitrogen fertilizer, it can be completely dissolved in water and directly absorbed by plants, with rapid effects and low dosage.

[0046] This invention also provides a method for promoting soybean growth and development, wherein the aforementioned corn steep liquor fermentation product is applied as nitrogen fertilizer when applying base fertilizer. In this invention, the soybean is preferably Heihe 35 variety, and the preferred indicators for soybean growth and development include leaf chlorophyll content, leaf area, leaf fresh weight and dry weight, and stem fresh weight and dry weight.

[0047] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Unless otherwise specified, the following embodiments are all conventional methods.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] The corn steep liquor used in the following examples was purchased from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd.; the glucose mother liquor was purchased from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd.

[0051] Example 1 A method for preparing a Mucor koji with high neutral protease production, comprising the following steps: Preparation of *Rhizopus wutongqiaoensis* YY-25 spore suspension: Sterile water was added to the *Rhizopus wutongqiaoensis* YY-25 slant agar (the slant agar medium was PDA medium). *Rhizopus* spores and hyphae were scraped off using an inoculation loop. The mixture containing hyphae and spores was poured into an Erlenmeyer flask containing glass beads and placed in a shaker at 28℃ and 180 r / min until a concentration of 5 × 10⁻⁶ was obtained. 6 A suspension of Mucor spores per mL.

[0052] Culture medium preparation: Weigh 2g of soybean meal and 18g of wheat bran (passed through a 30-mesh sieve) into an Erlenmeyer flask as dry material; measure 2mL of glucose stock solution, 2mL of corn steep liquor, and 11mL of tap water into a 50mL beaker and stir well to obtain liquid material; pour the liquid material into the Erlenmeyer flask, stir the liquid material and dry material evenly with a glass rod, and sterilize at 121℃ for 20min. After sterilization, while still hot, shake to break up any clumps of culture medium in the Erlenmeyer flask, and allow it to cool to room temperature before use.

[0053] The concentration is 5×10 6 1.2 mL of a Mucor spore suspension per mL was mixed with the culture medium prepared above. The conical flask was placed in a shaker at 30 °C and 10 r / min. The flask was shaken once every 24 hours and fermented for 72 hours to obtain Mucor starter.

[0054] Example 2 A high-yield mucor koji of neutral protease was prepared by the following method: Preparation of *Rhizopus wutongqiaoensis* YY-25 spore suspension: Sterile water was injected into the slant of *Rhizopus wutongqiaoensis* YY-25. *Rhizopus* spores and hyphae were scraped using an inoculation loop. The mixture containing hyphae and spores was poured into an Erlenmeyer flask containing glass beads and placed in a shaker at 28℃ and 180 r / min until a concentration of 4 × 10⁻⁶ was obtained. 6 A suspension of Mucor spores per mL.

[0055] Culture medium preparation: Weigh 3g of soybean meal and 17g of wheat bran (passed through a 30-mesh sieve) into an Erlenmeyer flask as dry material; measure 2mL of glucose stock solution, 3mL of corn steep liquor, and 10mL of tap water into a 50mL beaker and stir well to obtain liquid material; pour the liquid material into the Erlenmeyer flask, stir the liquid material and dry material evenly with a glass rod, and sterilize at 121℃ for 20min. After sterilization, while still hot, shake to break up any clumps of culture medium in the Erlenmeyer flask, and use after cooling to room temperature.

[0056] The concentration is 4×10 6 Mix 1.5 mL of a Mucor spore suspension per mL with the culture medium prepared above, place the conical flask in a shaker at 28 °C and 10 r / min, shake the flask once every 24 h, and ferment for 92 h to obtain Mucor starter.

[0057] Experimental Example 1 The fermentation conditions of Mucor koji were optimized using single-factor experiments with neutral protease activity as an indicator, as detailed below: (1) The dry material in the culture medium is 20g, and the liquid material is 20mL. The dry material consists of soybean meal and wheat bran that have passed through a 30-mesh sieve. The mass ratio of soybean meal to wheat bran is set to 1:19, 2:18, 3:17, 4:16, and 5:15, respectively (each group is labeled as having 1g, 2g, 3g, 4g, and 5g of soybean meal added). The liquid material consists of water, with a concentration of 5×10⁻⁶ in 1.2mL. 6 A suspension of *Mucor* spores per mL was inoculated into culture media with different mass ratios (soybean meal and wheat bran), shaken well, and incubated at 28°C for 72 h in a shaker at 10 rpm. All other steps were the same as in Example 1. After incubation, neutral protease activity was measured (neutral protease activity was determined according to national standard GB / T 23527-2009) to determine the optimal mass ratio of soybean meal to wheat bran for *Mucor* koji fermentation.

[0058] The results are as follows Figure 1 As shown, the optimal reaction conditions for subsequent experiments were set at 2g soybean meal and 18g wheat bran.

[0059] (2) The dry material in the culture medium is 20g, and the liquid material (water content) is set at 5mL, 10mL, 15mL, 20mL, 25mL, and 30mL respectively. The dry material consists of soybean meal and wheat bran that have passed through a 30-mesh sieve. The mass ratio of soybean meal to wheat bran in the dry material is 2:18. The liquid material consists of water. 1.2mL of the solution with a concentration of 5×10 6 A suspension of *Mucor* spores per mL was inoculated into culture media with different moisture contents, shaken well, and incubated at 28°C for 72 h in a shaker at 10 rpm. All other steps were the same as in Example 1. After incubation, neutral protease activity was measured (neutral protease activity was determined according to national standard GB / T 23527-2009) to determine the optimal moisture content of the *Mucor* spore fermentation medium.

[0060] The results are as follows Figure 2 As shown, a water content of 15 mL was used as the optimal reaction condition for subsequent experiments.

[0061] (3) The dry material in the culture medium is 20g, and the liquid material is 15mL. The liquid material consists of glucose mother liquor and water. The volume ratio of glucose mother liquor to water is set as follows: 0:15, 1:14, 2:13, 3:12, 4:11, 5:10, 6:9, 7:8 (each group is labeled as the group with glucose mother liquor added at 0mL, 1mL, 2mL, 3mL, 4mL, 5mL, 6mL, and 7mL). The dry material consists of soybean meal and wheat bran that have passed through a 30-mesh sieve. The mass ratio of soybean meal to wheat bran in the dry material is 2:18. 1.2mL of 5×106 A suspension of *Mucor* spores per mL was inoculated into culture media containing different glucose stock solutions, shaken well, and incubated at 28°C for 72 h in a shaker at 10 rpm. All other steps were the same as in Example 1. After incubation, neutral protease activity was measured (neutral protease activity was determined according to national standard GB / T 23527-2009) to determine the optimal amount of glucose stock solution added to the *Mucor* spore fermentation medium.

[0062] The results are as follows Figure 3 As shown, the optimal fermentation conditions for subsequent experiments were determined by adding 2 mL of glucose mother liquor (corresponding to a glucose mother liquor to water volume ratio of 2:13).

[0063] (4) The dry material in the culture medium is 20g, and the liquid material is 15mL. The liquid material consists of corn steep liquor, glucose mother liquor, and water. The volume ratio of corn steep liquor: glucose mother liquor: tap water is set as 0:2:13, 1:2:12, 2:2:11, 3:2:10, 4:2:9, and 5:2:8, respectively (each group is labeled as the group with corn steep liquor added at 0mL, 1mL, 2mL, 3mL, 4mL, and 5mL, respectively). The dry material consists of soybean meal and wheat bran that have passed through a 30-mesh sieve. The mass ratio of soybean meal to wheat bran is 2:18. 1.2mL of a 5×10⁻⁶ solution is added. 6 A suspension of *Mucor* spores per mL was inoculated into culture media with different amounts of corn steep liquor, shaken well, and incubated at 28°C for 72 h in a shaker at 10 rpm. All other steps were the same as in Example 1. After incubation, neutral protease activity was measured (neutral protease activity was determined according to national standard GB / T 23527-2009) to determine the optimal amount of corn steep liquor for *Mucor* koji fermentation.

[0064] The results are as follows Figure 4 As shown, the optimal fermentation conditions for subsequent experiments were determined by adding 2 mL of corn steep liquor (corresponding to a group with a volume ratio of corn steep liquor, glucose mother liquor, and water of 2:2:11).

[0065] (5) The dry material in the culture medium is 20g, and the liquid material is 15mL. The liquid material consists of corn steep liquor, glucose mother liquor, and water. The volume ratio of corn steep liquor: glucose mother liquor: tap water is 2:2:11. The dry material consists of soybean meal and wheat bran that have passed through a 30-mesh sieve. The mass ratio of soybean meal to wheat bran is 2:18. 0.4mL, 0.8mL, 1.2mL, 1.6mL, 2.0mL, and 2.4mL of the liquid material, with a concentration of 5×10⁻⁶, are added respectively. 6A spore suspension of *Mucor* spores per mL was inoculated into the culture medium (the inoculation amount of *Mucor* spore suspension was based on 20 g of dry material, and the volumetric percentages corresponding to the inoculation amounts for the above groups were 2%, 4%, 6%, 8%, 10%, and 12%, respectively). The mixture was shaken well and placed in a shaker at 10 r / min, and incubated at 28°C for 72 h. All other steps were the same as in Example 1. After incubation, the neutral protease activity was measured (neutral protease activity was determined according to national standard GB / T 23527-2009) to determine the optimal inoculation amount of *Mucor* spore suspension for the *Mucor* koji fermentation medium.

[0066] The results are as follows Figure 5 As shown, the optimal fermentation conditions for subsequent experiments were determined by using a Mucor spore suspension (bacterial suspension) inoculum of 6% (1.2 mL).

[0067] (6) The dry material in the culture medium is 20g, and the liquid material is 15mL. The liquid material consists of corn steep liquor, glucose mother liquor, and water. The volume ratio of corn steep liquor: glucose mother liquor: tap water is 2:2:11. The dry material consists of soybean meal and wheat bran that have passed through a 30-mesh sieve. The mass ratio of soybean meal to wheat bran is 2:18. 1.2mL of a 5×10⁻⁶ solution is added. 6 A suspension of *Mucor* spores per mL was inoculated into the culture medium, shaken well, and placed in a shaker at 10 rpm. The mixture was then incubated at 28°C for 48 h, 60 h, 72 h, 84 h, 96 h, 108 h, and 120 h, respectively. All other steps were the same as in Example 1. After incubation, neutral protease activity was measured (neutral protease activity was determined according to national standard GB / T 23527-2009) to determine the optimal fermentation time for *Mucor* spore fermentation medium.

[0068] The results are as follows Figure 6 As shown, a fermentation time of 72 hours was used as the optimal fermentation condition for subsequent experiments.

[0069] (7) The dry material in the culture medium is 20g, and the liquid material is 15mL. The liquid material consists of corn steep liquor, glucose mother liquor, and water. The volume ratio of corn steep liquor: glucose mother liquor: tap water is 2:2:11. The dry material consists of soybean meal and wheat bran that have passed through a 30-mesh sieve. The mass ratio of soybean meal to wheat bran is 2:18. 1.2mL of a 5×10⁻⁶ solution is added. 6 A suspension of *Mucor* spores per mL was inoculated into the culture medium, shaken well, and placed in a shaker at 10 rpm. The mixture was then incubated at 26°C, 28°C, 30°C, 32°C, and 40°C for 72 h each. All other steps were the same as in Example 1. After incubation, neutral protease activity was measured (neutral protease activity was determined according to national standard GB / T 23527-2009) to determine the optimal fermentation temperature for *Mucor* koji.

[0070] The results are as follows Figure 7 As shown, a fermentation temperature of 30℃ was selected as the optimal fermentation condition for subsequent experiments.

[0071] The optimal fermentation conditions were determined through response surface methodology: 20g of dry material and 15mL of liquid material. The liquid material consisted of corn steep liquor, glucose mother liquor, and water, with a volume ratio of corn steep liquor: glucose mother liquor: tap water of 2:2:11. The dry material consisted of soybean meal and wheat bran that had passed through a 30-mesh sieve, with a mass ratio of soybean meal to wheat bran of 2:18. The inoculum amount of Mucor spore suspension was 1.2mL (based on the weight of 20g of dry material, the volume percentage of the inoculum amount was 6%). The fermentation time was 72h, and the fermentation temperature was 30℃.

[0072] Example 3 A corn steep liquor ferment that can be used as a water-soluble nitrogen fertilizer is prepared by the following method: 100g of corn steep liquor was placed in a beaker and heated to 55℃ in a magnetically stirred pot. Sodium hydroxide was added to the corn steep liquor at a mass ratio of 1.79:100, and the mixture was stirred for 20 minutes, maintaining the pH at 5.5-6.0. Then, calcium hydroxide was added to the corn steep liquor at a mass ratio of 0.29:100, and the mixture was stirred for 40 minutes, again maintaining the pH at 5.5-6.0. After the reaction was complete, 1.5g of the *Mucor* inoculum prepared in Example 1 was added, and the mixture was kept at a constant temperature of 55℃ and stirred for 4 hours for fermentation. After fermentation, the mixture was filtered through four layers of gauze and then centrifuged at 4000 rpm for 20 minutes. The supernatant was taken as the fermented corn steep liquor.

[0073] Example 4 A corn steep liquor ferment that can be used as a water-soluble nitrogen fertilizer is prepared by the following method: 100g of corn steep liquor was placed in a beaker and heated to 60℃ in a magnetically stirred pot. Sodium hydroxide was added to the corn steep liquor at a mass ratio of 1.79:100, and the mixture was stirred for 20 minutes, maintaining the pH at 5.5-6.0. Then, calcium hydroxide was added to the corn steep liquor at a mass ratio of 0.29:100, and the mixture was stirred for 40 minutes, again maintaining the pH at 5.5-6.0. After the reaction was complete, 2.0g of the *Mucor* inoculum prepared in Example 1 was added, and the mixture was kept at a constant temperature of 60℃ and stirred for 5 hours for fermentation. After fermentation, the mixture was filtered through four layers of gauze, then centrifuged at 4000 rpm for 20 minutes. The supernatant was taken as the fermented corn steep liquor.

[0074] Experimental Example 2 The method for preparing corn steep liquor fermentation product was optimized using single-factor methods, as follows: (1) The difference from Example 3 is that the mass ratio of the *Rhizopus* koji prepared in Example 1 to corn steep liquor was adjusted to 0:100, 0.5:100, 1:100, 1.5:100, 2:100, 2.5:100, and 3:100 (each group was labeled as having 0g, 0.5g, 1g, 1.5g, 2g, 2.5g, and 3g of *Rhizopus* koji added, respectively), and the fermentation temperature was 50℃. All other aspects were the same as in Example 3. After fermentation, the content of small peptides in the corn steep liquor fermented with different inoculum amounts of *Rhizopus* koji was determined. The content of small peptides was determined using the Folin-Ciocalteu method.

[0075] The results are as follows Figure 8 As shown, the optimal fermentation conditions for subsequent experiments were set at 1.5g of Rhizopus koji (the mass ratio of Rhizopus koji to corn steep liquor was 1.5:100).

[0076] (2) The difference from Example 3 is that the fermentation temperature was set to 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, and 65℃ respectively, while the rest were the same as in Example 3. After fermentation, the content of small peptides in the corn steep liquor fermented at different fermentation temperatures was measured. The content of small peptides was determined by the Folin-Ciocalteu method.

[0077] The results are as follows Figure 9 As shown, a fermentation temperature of 55℃ was used as the optimal fermentation condition for subsequent experiments.

[0078] (3) The difference from Example 3 is that the fermentation time was set to 0h, 1h, 2h, 3h, 4h, 5h, and 6h respectively, while the rest were the same as in Example 3. After fermentation, the content of small peptides in corn steep liquor fermented at different fermentation times was measured. The content of small peptides was determined by the Folin-Ciocalteu method.

[0079] The results are as follows Figure 10 As shown, the increase after 4 hours of fermentation is relatively small, but if the fermentation time is extended by 2 hours, the production cost will increase. Therefore, a fermentation time of 4 hours is adopted as the optimal fermentation condition for subsequent experiments.

[0080] The optimal fermentation conditions were determined through response surface methodology: 1.5g of Rhizopus koji (the mass ratio of Rhizopus koji to corn steep liquor was 1.5:100), a fermentation temperature of 55℃, and a fermentation time of 4h.

[0081] Example 5 A method for promoting soybean growth and development involves applying the corn steep liquor fermented in Example 3 as nitrogen fertilizer when applying base fertilizer.

[0082] Experimental Example 3 The following is an experiment on using corn steep liquor fermented at different fermentation times as nitrogen fertilizer to regulate soybean seedling growth: The inorganic nitrogen fertilizer used in this experiment was urea, the phosphate fertilizer was calcium phosphate, the potassium fertilizer was potassium chloride, and the organic nitrogen fertilizer was corn steep liquor fermented at different fermentation times. The soybean variety used was Heihe 35. The study investigated the effects of different treatment groups of basal fertilizer on the growth and development of soybean seedlings.

[0083] The pot experiment consisted of 6 treatment groups (blank control group, fertilizer group, and 4 corn steep liquor fermentation groups with different fermentation times). Each treatment was divided into 3 pots, with 3 replicates. Each pot contained 8 seeds and 2 kg of soil. Fertilizer was mixed with the soil and then placed into the pot. The blank control group (CK) received no fertilizer, while the fertilizer group (CF) received urea, calcium phosphate, and potassium chloride.

[0084] The four corn steep liquor fermentation groups with different fermentation times are as follows: FCSL-0h: Corn steep liquor fermented for 0h (the difference from Example 3 is that the fermentation time is 0h, and the rest is the same as Example 3) is used to replace the urea in the CF group. The missing phosphorus and potassium fertilizers are supplemented by chemical fertilizers to ensure that the nitrogen, phosphorus and potassium content is consistent with the CF group. FCSL-2h: Corn steep liquor fermented for 2 hours (the difference from Example 3 is that the fermentation time is 2 hours, and the rest is the same as Example 3) is used to replace the urea in the CF group. The missing phosphorus and potassium fertilizers are supplemented by chemical fertilizers to ensure that the nitrogen, phosphorus and potassium content is consistent with the CF group. FCSL-3h: Corn steep liquor fermented for 3 hours (the difference from Example 3 is that the fermentation time is 3 hours, and the rest is the same as Example 3) is used to replace the urea in the CF group. The missing phosphorus and potassium fertilizers are supplemented by chemical fertilizers to ensure that the nitrogen, phosphorus and potassium content is consistent with the CF group. FCSL-4h: Corn steep liquor fermented for 4 hours (obtained in Example 3) was used to replace the urea in the CF group. The missing phosphorus and potassium fertilizers were supplemented by chemical fertilizers to ensure that the nitrogen, phosphorus and potassium content was consistent with that of the CF group.

[0085] The specific experimental plan is shown in Table 1 below: Table 1. Fertilizer application rates for different treatment groups

[0086] Note: 1. CK is the blank control group, CF is the fertilizer group, and FCSL-0h~FCSL-4h are corn steep liquor fermentation fertilizer groups with different fermentation times.

[0087] 2. Urea contains 46% N, calcium phosphate contains 36% P2O5, and potassium chloride contains 60% K2O. The fermented corn steep liquor from 0 hours contains 2.8745% N, 0.0257% P, and 1.9106% K; from 2 hours it contains 2.9200% N, 0.0287% P, and 1.9180% K; from 3 hours it contains 2.7010% N, 0.0314% P, and 2.1820% K; and from 4 hours it contains 2.8970% N, 0.0391% P, and 2.0615% K. The fermented corn steep liquor can replace urea, and the missing phosphate and potassium fertilizers can be supplemented by chemical fertilizers.

[0088] After the soybeans have grown their first compound leaves, plant height and stem diameter are measured every 10 days. After 60 days of planting, soybean agronomic indicators (chlorophyll content, leaf area, leaf fresh weight and stem fresh weight, leaf dry weight and stem dry weight) are measured.

[0089] Plant height: Measured using a measuring tape with an accuracy of 0.1cm, the measurement point is the vertical distance from above the first compound leaf of the soybean plant to the growing point; Stem diameter: The base of the first compound leaf of the soybean plant was measured using a vernier caliper with an accuracy of 0.01 mm; Chlorophyll content: The relative chlorophyll content (SPAD value) of each soybean leaf was measured using a chlorophyll meter (TYS-4N), and the chlorophyll content of the leaves in each treatment group was recorded. Leaf area: The leaf area of ​​soybean plants was measured using a portable leaf area meter YMJ-B, and the leaf area of ​​each treatment group was recorded. Leaf and stem fresh weight: After the experiment, the leaf and stem weights were directly measured and recorded for each treatment group. Leaf dry weight and stem dry weight: After the experiment, the leaves and stems were blanched at 105℃ for 30 minutes and then placed in an 80℃ oven for 48 hours. The leaf dry weight and stem dry weight of each treatment group were weighed and recorded.

[0090] The results are shown below: Soybean plant height in different treatment groups, such as Figure 11 As shown, the CK group had the lowest plant height, indicating that appropriate application of basal fertilizer can promote soybean growth. The soybean plant height in the corn steep liquor treatment group was significantly different from that in the CF group, indicating that corn steep liquor can replace nitrogen fertilizer to promote soybean growth.

[0091] Soybean stems in different treatment groups were as thick as Figure 12 As shown, the CK group had the lowest stem diameter, indicating that appropriate application of base fertilizer can promote soybean growth. The soybean stem diameter of the treatment group using corn steep liquor fermentation was significantly different from that of the CF group, indicating that corn steep liquor fermentation can replace nitrogen fertilizer to promote soybean growth.

[0092] Soybean leaf chlorophyll content in different treatment groups, such as Figure 13 As shown, the CK treatment group had the lowest chlorophyll content in its leaves, indicating that appropriate application of base fertilizer would increase the chlorophyll content of soybean leaves. The FCSL-4h treatment group had the highest chlorophyll content in its soybean leaves, showing a significant advantage.

[0093] Soybean leaf area in different treatment groups as follows Figure 14 As shown, the CK treatment group had the lowest leaf area, indicating that appropriate application of base fertilizer will increase the leaf area of ​​soybean leaves. The FCSL-3h treatment group had the largest leaf area of ​​soybean leaves, showing a significant advantage.

[0094] Fresh weight of soybean leaves in different treatment groups as follows Figure 15 As shown, the CK treatment group had the lightest leaf fresh weight, indicating that appropriate application of base fertilizer will increase the fresh weight of soybean leaves. The FCSL-3h and FCSL-4h treatment groups showed stable effects and significant advantages in leaf fresh weight.

[0095] Soybean leaf dry weight in different treatment groups as follows Figure 16 As shown, the CK treatment group had the lightest leaf dry weight, indicating that appropriate application of base fertilizer will increase the dry weight of soybean leaves. The FCSL-4h treatment group showed stable effects and a significant advantage in leaf dry weight, indicating that the FCSL-4h treatment group had the highest accumulation of dry matter in its leaves.

[0096] Fresh weight of soybean stems in different treatment groups as follows Figure 17 As shown, the CK treatment group had the lightest stem fresh weight, indicating that appropriate application of base fertilizer will increase the fresh weight of soybean stems. The FCSL-3h treatment group showed stable effects and a significant advantage in stem fresh weight.

[0097] Soybean stem dry weight in different treatment groups as follows Figure 18 As shown, the CK treatment group had the lightest stem dry weight, indicating that appropriate application of base fertilizer will increase the dry weight of soybean stems. The FCSL-3h treatment group showed stable results and a significant advantage in stem dry weight, indicating that the FCSL-3h treatment group had a lot of cellulose accumulation in the stems.

[0098] The results above indicate that the corn steep liquor fermentation group can exert a similar regulatory effect as the CF group, and both can effectively promote soybean growth, suggesting that corn steep liquor fermentation can replace nitrogen fertilizer to achieve the same function.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a high-yield *Mucor* koji mold, characterized in that, The process includes the following steps: mixing the Mucor spore suspension with the culture medium and fermenting at 28℃~40℃ for 48h~108h; the culture medium consists of dry material and liquid material, the dry material consists of soybean meal and wheat bran, and the liquid material consists of glucose mother liquor, corn steep liquor and water; the weight-to-volume ratio of the dry material and liquid material is 20g:10~30mL; the weight ratio of soybean meal and wheat bran is (1:19)~(4:16); the volume ratio of corn steep liquor, glucose mother liquor and water is (0:2:13)~(3:2:10).

2. The preparation method according to claim 1, characterized in that, The concentration of the Mucor spore suspension was 4 × 10⁻⁶. 6 Cells / mL ~5×10 6 The amount of the Rhizopus spore suspension added is based on the weight of the dry material, which is 0.8~2.4mL of Rhizopus spore suspension added per 20g of dry material; the Rhizopus spore suspension is Rhizopus wutongqiaoensis YY-25, with the preservation number CGMCC No.12109.

3. A Mucor spawn with high yield of neutral protease, characterized in that, It is prepared by the preparation method described in claim 1 or 2.

4. The use of the Mucor koji of claim 3 in the preparation of corn steep liquor fermentation products that can be used as water-soluble nitrogen fertilizer.

5. A corn steep liquor fermentation product that can be used as a water-soluble nitrogen fertilizer, characterized in that, It is prepared by the following method: corn steep liquor is mixed with sodium hydroxide, calcium hydroxide is added and mixed, then the Rhizopus koji of claim 3 is added, fermented, and the supernatant is taken to obtain corn steep liquor fermentation product.

6. The corn steep liquor fermentation product according to claim 5, characterized in that, The mass ratio of Rhizopus koji to corn steep liquor is 0.5~2.5:

100.

7. The corn steep liquor fermentation product according to claim 5, characterized in that, The mass ratio of sodium hydroxide to corn steep liquor is 1.79:100, and the mass ratio of calcium hydroxide to corn steep liquor is 0.29:

100.

8. The corn steep liquor fermentation product according to claim 5, characterized in that, The fermentation temperature is 45℃~65℃, and the fermentation time is 0~6h.

9. The use of the corn steep liquor fermentation product according to any one of claims 5 to 8 in the preparation of water-soluble nitrogen fertilizer.

10. A method for promoting soybean growth and development, characterized in that, When applying base fertilizer, the corn steep liquor fermentation product as described in any one of claims 5 to 8 shall be applied as nitrogen fertilizer.

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