A method for preparing mulberry leaf protein feed and the resulting product

Abstract

This invention provides a method for preparing mulberry leaf protein feed, which involves fermenting the following raw materials in parts by weight: 30-35 parts mulberry leaves; 5-10 parts fiber regulator; 2-3 parts nitrogen source; 1-4 parts modified Mandel nutrient solution; and 50-60 parts distilled water. The fiber regulator is selected from one or two of wheat bran and corn flour; the nitrogen source is selected from one or more of urea, ammonium sulfate, and ammonia water; and the fermentation strain is one or more of Aspergillus oryzae, Hansenula polymorpha, Bacillus subtilis, and Bacillus licheniformis. This invention's method for preparing mulberry protein feed, through microbial fermentation of mulberry leaves combined with appropriate raw material ratios and optimized nutrient solution, yields a high-protein, easily absorbed, and low-cost mulberry protein feed.

Description

Technical Field

[0001] This invention belongs to the technical field of animal feed, specifically relating to a method for preparing a high-protein feed mulberry and the resulting product. Background Technology

[0002] In recent years, my country's livestock industry has developed rapidly, leading to a continuous increase in demand for high-quality protein feed. This significant growth in livestock production necessitates providing animals with more protein sources. However, existing protein feed resources mainly rely on soybean meal, which is relatively scarce. To better meet the needs of livestock development, the Ministry of Agriculture and Rural Affairs formulated the "Three-Year Action Plan for Reducing and Replacing Soybean Meal in Feed" in April 2023. This plan encourages cattle and sheep farmers to reduce their use of concentrate feed through measures such as "improving efficiency, increasing supply, and adjusting structure." With the rapid development of livestock, traditional protein feeds can no longer meet the growing demand. Therefore, it is urgent to develop new protein feed resources, especially high-quality fermented protein feeds, to alleviate the shortage of protein feed resources.

[0003] Mulberry trees are deciduous perennial woody plants with strong resistance to adverse conditions and are widely cultivated in my country. Mulberry leaves are a plant resource rich in protein, vitamins, and minerals, with broad application potential. Mulberry leaves have a high protein content and are rich in saturated fatty acids; the crude protein content in the dry matter of mulberry leaves reaches 15%–30%, and the amino acid composition is balanced, making them particularly suitable for animal feed. Currently, mulberry leaves are gradually being used in the breeding of various livestock, poultry, and aquatic animals, demonstrating special feed value. However, directly using mulberry leaves as feed has the following problems: high crude fiber content, reducing the digestibility and absorption efficiency of animals; high content of anti-nutritional factors, affecting feed quality and digestibility; low protein utilization rate, especially for ruminants and non-ruminants; poor palatability, leading to insufficient feed intake. Further research is needed on technologies to reduce the crude fiber content of mulberry leaves, increase the true protein content, and reduce the anti-nutritional factor tannins. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a method for preparing mulberry leaf protein feed with mulberry leaves as the main raw material. Through scientific formulation and fermentation processes, the protein utilization rate and palatability of mulberry leaves are effectively improved.

[0005] A second objective of this invention is to propose the addition of feed prepared by the aforementioned method.

[0006] The technical solution for achieving the above-mentioned objective of this invention is as follows:

[0007] A method for preparing mulberry leaf protein feed involves fermenting the following raw materials in parts by weight: 30-35 parts mulberry leaves; 5-10 parts fiber regulator; 2-3 parts nitrogen source; 1-4 parts modified Mandel nutrient solution; and 50-60 parts distilled water. The fiber regulator is selected from one or two of wheat bran and corn flour, and the nitrogen source is selected from one or more of urea, ammonium sulfate, and ammonia water.

[0008] The fermentation strain is Aspergillus oryzae, and one or more of the following: Hansenula polymorpha, Bacillus subtilis, and Bacillus licheniformis.

[0009] The mulberry leaves contain 70-85 wt% moisture, 8.0-11.0% true protein and 12.0-15.0% crude fiber on a dry matter basis.

[0010] Furthermore, the fiber regulator is wheat bran and corn flour in a mass ratio of 4:4 to 6; the nitrogen source is urea and ammonium sulfate in a mass ratio of 1:1 to 2.

[0011] The chemical composition of the modified Mandel nutrient solution is as follows: MgSO4·7H2O: 0.3wt%; CaCl2: 0.45wt%; KH2PO4: 2wt%; FeSO4·7H2O: 0.005wt%; CoCl2: 0.002wt%; MnSO4·H2O: 0.0016wt%; ZnSO4·H2O: 0.0014wt%, with the balance being water.

[0012] A preferred embodiment of the present invention is that the fermentation strain is a mixture of Aspergillus oryzae, Hansenula polysaccharide, and a third strain, wherein the third strain is Bacillus licheniformis or Bacillus subtilis, and the fermentation strain is inoculated in the form of a bacterial suspension, wherein the volume ratio of the bacterial suspension of Aspergillus oryzae, Hansenula polysaccharide, and the third strain is 2-3:1-4:1.

[0013] More preferably, the preservation number of the *Hansenula polymorpha* is CGMCC No. 34449.

[0014] The method for preparing the mulberry leaf protein feed includes the following steps:

[0015] 1) Raw material preparation: Wash and dry mulberry leaves to 8-12% moisture content, then crush them to a particle size of less than 1 mm; crush the fiber regulator and sieve it for later use; mix mulberry leaf powder, wheat bran and corn flour evenly in proportion; dissolve the nitrogen source in distilled water, add the modified nutrient solution, stir evenly and then mix it thoroughly with the mixed matrix;

[0016] 2) Fermentation: Inoculate with a suspension of fermentation bacteria at 8-12% of the total substrate weight; ferment at 25-30℃ for 48-72 hours, stirring once every 10-15 hours during the fermentation process;

[0017] 3) Drying and pelleting: The fermented feed is dried at 62-65℃ until the moisture content is less than 10%; after drying, it is pressed into pelleted protein feed by a feed pellet mill.

[0018] In step 1), after the fiber conditioner is pulverized, it can be passed through a 20-mesh to 10-mesh sieve, for example, through a 1 mm sieve.

[0019] The *Aspergillus oryzae* strain was inoculated using a bacterial suspension. The preparation method of the bacterial suspension was as follows: the *Aspergillus oryzae* strain was transferred to potato dextrose agar plates in a petri dish and cultured at 24–28°C for 5–7 days; then, the spores were eluted with a modified Mandel nutrient solution and diluted to a spore count of 10-1. 7 CFU / mL.

[0020] The third bacterial strain was inoculated as a bacterial suspension. The bacterial suspension was prepared by adding 1 wt% peptone, 0.3 wt% beef extract, and 0.5 wt% NaCl to distilled water, adjusting the pH to 6.0–6.5, sterilizing, and cooling to obtain a nutrient broth liquid culture medium. *Bacillus licheniformis* was inoculated into the nutrient broth liquid culture medium and cultured at 35–38°C for 10–15 hours. The third bacterial strain was either *Bacillus licheniformis* or *Bacillus subtilis*.

[0021] The *Hansenula polymorpha* strain was inoculated using a bacterial suspension. The bacterial suspension was prepared by adding 1 wt% peptone, 0.5 wt% potato starch, 0.5 wt% NaCl, and 1.5% glucose to distilled water, adjusting the pH to 6.0–6.5, sterilizing, and cooling to obtain potato glucose liquid culture medium. The *Hansenula polymorpha* strain was then inoculated into the potato glucose liquid culture medium and cultured at 26–28°C for 10–14 hours.

[0022] pH can be adjusted using 0.1 mol / L HCl, 0.1 mol / L NaOH, or other pH adjusters known in the art.

[0023] Furthermore, the total inoculation amount of Aspergillus oryzae, Hansenula juncetamol, and the third strain is 10%, and the inoculation is carried out according to the ratio of Aspergillus oryzae, Hansenula juncetamol, and Bacillus licheniformis 2:3:1, that is, 10 mL of the mixed bacterial suspension of Aspergillus oryzae, Hansenula juncetamol, and the third strain is inoculated into every 100 g of fermentation medium. For example, 3.3 mL of Aspergillus oryzae spore suspension, 5 mL of Hansenula juncetamol bacterial suspension, and 1.67 mL of Bacillus licheniformis bacterial suspension are inoculated into every 100 g of fermentation medium.

[0024] The viable cell counts in the three bacterial suspensions were the same, ranging from 0.5 to 5 × 10⁶. 7 CFU / mL, for example, the viable count in all three bacterial suspensions is 10. 7 CFU / mL.

[0025] The feed prepared by the method described in this invention.

[0026] The method for preparing mulberry leaf protein feed proposed in this invention involves microbial fermentation of mulberry leaves, combined with appropriate raw material ratios and optimized nutrient solutions, to obtain a high-protein, easily absorbed, and low-cost mulberry protein feed.

[0027] The beneficial effects of this invention are as follows:

[0028] 1. Low cost: Utilizing mulberry leaves to prepare protein feed is both economical and environmentally friendly. Reduced waste: Fermentation converts non-protein nitrogen in mulberry leaves into usable microbial protein, avoiding resource waste. It can replace some expensive protein feeds (such as soybean meal and fishmeal). Compared to traditional feeds, fermented mulberry leaf feed is: lower in cost; more nutritionally balanced; and has a wider range of applications, suitable for ruminants, monogastric animals, and even poultry.

[0029] 2. Improve the protein content and quality of mulberry leaves: Enzymes secreted by microorganisms can partially degrade the insoluble protein in mulberry leaves, converting it into small peptides and free amino acids that are more easily digested and absorbed, thereby improving the nutritional value of the protein.

[0030] 3. Reducing anti-nutritional factors (such as tannins): Tannins in mulberry leaves can affect the utilization of proteins and minerals. During aerobic fermentation, the enzyme activity of microorganisms (such as tanninase) can break down tannins, reducing their inhibitory effect on nutrient absorption.

[0031] 4. Improving the palatability of mulberry leaves: Due to their high tannin and fiber content, direct feeding of mulberry leaves may reduce palatability. The role of fermentation: Aerobic fermentation can significantly reduce the bitterness and astringency of mulberry leaves, while generating aromatic substances (such as short-chain fatty acids and esters), improving the flavor of the feed.

[0032] 5. Enhanced Probiotic Effects: Probiotics used during fermentation (such as yeast and Bacillus licheniformis) proliferate significantly in the feed. After feeding: Probiotics can regulate the animal's intestinal microbiota, promote the growth of beneficial bacteria, and inhibit the growth of harmful bacteria. This improves the animal's immunity and reduces the incidence of intestinal diseases.

[0033] 6. Low energy consumption, no pollution, low investment, and low operating costs; fermentation effect is good at 25℃, and room temperature can meet the requirements during the mulberry leaf harvesting season; no waste residue or wastewater is discharged during the entire fermentation process, reducing environmental pollution. The preparation process does not require high temperature and high pressure, has low energy consumption, and no wastewater or waste residue discharge, making it suitable for large-scale promotion.

[0034] 7. Improves the palatability and digestibility of feed, and enhances its nutritional value; This invention uses Aspergillus oryzae, Hansenula polysaccharide, and Bacillus licheniformis to ferment mulberry leaves. All three bacteria are microorganisms permitted for use as feed additives, thus ensuring safety. Aspergillus oryzae, Hansenula polysaccharide, and Bacillus licheniformis can secrete a variety of enzyme systems. When these three proliferate in large quantities, they can effectively convert the crude fiber in mulberry leaves into a protein feed rich in various amino acids, vitamins, enzymes, bioactive substances, and growth regulators, with a true protein content of 14.0–15.4% and a crude protein content of 26.5–28.1%. Attached Figure Description

[0035] Figure 1 This is a flowchart of the preparation process for mulberry leaf protein feed.

[0036] Figure 2 The morphology of Hansenula polymorpha was observed in the yeast.

[0037] Figure 3 The growth of *Hansenula polymorpha* strain on tanninase identification medium was studied. Detailed Implementation

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0039] The mulberry leaves used in this embodiment were sourced from the Shichao Nursery Planting Farmers Professional Cooperative in Suiyang District, Shangqiu City, and the mulberry variety was Gui Sang You 12. The moisture content of the mulberry leaves used was 70–85 wt%, and on a dry matter basis, the true protein content was 9.0–10.5%, the crude fiber content was 12.0%–15.0%, and the tannin content was 1.2%–2.0%.

[0040] In the examples, the strains of *Aspergillus oryzae* (CGMCC No. 3.13905), *Bacillus licheniformis* (CGMCC No. 1.8791), *Saccharomycees cerevisiae* (CGMCC No. 2.3880), and *Bacillus subtilis* (CGMCC No. 1.12939) were all purchased from the China General Microbiological Culture Collection Center (CGMCC).

[0041] Hanseniaspora pseudoguilliermondii was isolated from a fermented total mixed ration (TMR) feed in our laboratory. Its accession number is CGMCC No. 34449, the deposit date is May 7, 2025, and the deposit location is China General Microbiological Culture Collection Center.

[0042] Unless otherwise specified, all methods used in this instruction manual are existing techniques in the field. All raw materials used are commercially available.

[0043] Example 1

[0044] This embodiment describes the isolation and identification of *Hansenula polymorpha*.

[0045] The *Hansenula polymorpha* strain used in this experiment (CGMCC No. 34449) was isolated from a fermented total mixed diet (TMR) (composed of corn, alfalfa, and cottonseed meal) from China Agricultural University. After enrichment culture, yeast colonies were initially isolated on YPD agar plates using the dilution plating method.

[0046] Observation under an optical microscope (1000× oil immersion): The cells are typically elliptical (approximately 2–5 × 4–10 μm in size), reproduce by unipolar budding, and show no pseudohyphae formation (consistent with the characteristics of *Hansenula polymorpha*). See [link to relevant documentation]. Figure 2 After culturing for 72 hours on a dedicated identification medium containing 1% tannic acid, a distinct clear hydrolysis zone appeared around the colonies, confirming that this strain possesses a highly efficient tanninase secretion capacity, capable of hydrolyzing tannic acid to produce gallic acid. See also... Figure 3 .

[0047] This strain has the following outstanding characteristics: acid resistance and fermentation adaptability: it grows stably in an environment of pH 3.0–6.0; polyphenol degradation ability: it efficiently degrades anti-nutritional factors (tannins, gossypol) in cottonseed meal and alfalfa; feed improvement potential: by hydrolyzing plant polyphenols, it improves TMR protein utilization and reduces the metabolic burden on ruminants.

[0048] Example 2

[0049] This embodiment provides a method for preparing mulberry leaf protein feed; the process is described in [link to process description]. Figure 1 The steps include:

[0050] 1) Preparation of fermentation raw materials:

[0051] First, the raw materials are pre-treated: mulberry leaves are crushed and passed through a 1mm sieve for later use; fiber regulators (wheat bran, cornmeal) are crushed and passed through a 1mm sieve for later use.

[0052] By weight, the fermentation ingredients consist of 34 parts mulberry leaves, 5 parts cornmeal, 5 parts wheat bran, 1 part ammonium sulfate, 1 part urea, 1 part modified Mandel nutrient solution, and 53 parts distilled water; mix thoroughly and set aside.

[0053] 2) Solid-state fermentation: A 10% inoculum of *Hansenula polymorpha* (CGMCC No. 34449) was added to the fermentation feed, stirred thoroughly, and fermented at 30°C for 72 hours. The mixture was stirred every 12 hours during fermentation to ensure uniform fermentation.

[0054] The *Hansenula polymorpha* strain was inoculated using a bacterial suspension. The suspension was prepared as follows: 1 wt% peptone, 0.5 wt% potato starch, 0.5 wt% NaCl, and 1.5% glucose were added to distilled water. The pH was adjusted to 6.0–6.5 with 0.1 mol / L HCl and 0.1 mol / L NaOH. After sterilization and cooling, a potato glucose liquid culture medium was prepared. *Hansenula polymorpha* was inoculated into the potato glucose liquid culture medium and cultured at 28°C and 150 rpm for 12 hours on a shaker. The medium was then diluted to a spore count of 10-1. 7 A suspension of cells / mL;

[0055] 3) Drying: Dry the fermented feed at 65℃ until the moisture content is less than 10%; after drying, press it into granular protein feed using a feed pellet mill.

[0056] 1-4 parts of Mandel nutrient solution can be added to the fermentation raw materials. The present invention proposes an improved chemical composition for the Mandel nutrient solution as follows:

[0057] MgSO4·7H2O: 0.3wt%; CaCl2: 0.45wt%; KH2PO4: 2wt%; FeSO4·7H2O: 0.005wt%; CoCl2: 0.002wt%; MnSO4·H2O: 0.0016wt%; ZnSO4·H2O: 0.0014wt%. The above chemical components were dissolved in distilled water, brought to a final volume of 100mL, and stored at 4℃ for later use.

[0058] In the conventional Mandel nutrient solution, the CaCl2 content is 0.3 wt%. Through exploration and comparison in the culture experiments of Hansenula polysaccharide, the inventors chose to increase the CaCl2 content to 0.45 wt%, which is more conducive to the growth of the strain.

[0059] The protein feed product obtained in this embodiment has a true protein content of 14.0% and a crude protein content of 27.6%.

[0060] Comparative Example 1:

[0061] The preparation method of mulberry leaf protein feed provided in this example is the same as that in Example 2, wherein the inoculated Hansenula polymorpha was commercially available (Shanghai Xuanke Biotechnology Co., Ltd.).

[0062] The protein feed product obtained in this example has a true protein content of 11.0% and a crude protein content of 26.1%.

[0063] Example 3

[0064] This embodiment provides a method for preparing mulberry leaf protein feed; the process is described in [link to process description]. Figure 1 The steps include:

[0065] 1) Raw material pretreatment: Crush mulberry leaves and pass them through a 1mm sieve for later use; crush fiber regulators (wheat bran, cornmeal) and pass them through a 1mm sieve for later use. Mix mulberry leaf powder, wheat bran, and cornmeal to prepare fermentation raw materials.

[0066] 2) Cultivation of fermentation strains: Preparation of Aspergillus oryzae spore suspension, Hansenula polymorpha suspension, and Bacillus licheniformis suspension:

[0067] One loopful of *Aspergillus oryzae* strain was transferred to potato dextrose agar plates and incubated at 25°C for 5-7 days. 15 mL of modified Mendel's salt solution was added to each petri dish, and the spores were gently scraped off with a spreader. The mixture was filtered through four layers of sterile gauze into a sterile Erlenmeyer flask. Sterile glass beads were added to disperse the spores, and the solution was diluted appropriately. The spore count was then performed using a hemocytometer to ensure a count of 10-1. 7 cells / mL suspension

[0068] The *Hansenula polymorpha* was inoculated using a bacterial suspension, and the preparation method of the bacterial suspension was the same as in Example 2.

[0069] The *Bacillus licheniformis* was inoculated as a bacterial suspension. The bacterial suspension was prepared by adding 1 wt% peptone, 0.3 wt% beef extract, and 0.5 wt% NaCl to distilled water, adjusting the pH to 6.0–6.5 with 0.1 mol / L HCl and 0.1 mol / L NaOH, and then sterilizing and cooling to obtain a nutrient broth liquid culture medium. One loopful of *Bacillus licheniformis* was transferred into the nutrient broth liquid culture medium and cultured at 37°C and 150 rpm for 12 hours on a shaker. The culture was then diluted to a spore count of 10-1. 7 A suspension of CFU / mL.

[0070] 3) Preparation of fermentation raw materials:

[0071] By weight, the fermentation ingredients include 30 parts mulberry leaves, 4 parts cornmeal, 4 parts wheat bran, 1 part ammonium sulfate, 1 part urea, 1 part modified Mandel nutrient solution, and 59 parts distilled water; mix thoroughly and set aside.

[0072] 4) Solid-state fermentation: Inoculate the fermentation raw materials with 3.3% Aspergillus oryzae spore suspension, 5% Hansenula polymorpha spore suspension and 1.67% Bacillus licheniformis spore suspension, stir evenly and ferment at 25°C for 72 hours.

[0073] 5) Drying: Dry the fermented material at 65℃ to obtain the protein feed product.

[0074] The protein feed product obtained in this embodiment has a true protein content of 15.4% and a crude protein content of 27.0%. The tannin content in the fermentation product is 1.2%.

[0075] Example 4

[0076] The raw material pretreatment and preparation of the fermentation strain suspension in this embodiment are the same as in Example 3. The Bacillus subtilis is inoculated as a bacterial suspension. The preparation method of the bacterial suspension is as follows: 1 wt% peptone, 0.3 wt% beef extract, and 0.5 wt% NaCl are added to distilled water, and the pH is adjusted to 6.0–6.5 with 0.1 mol / L HCl and 0.1 mol / L NaOH. After sterilization and cooling, a nutrient broth liquid culture medium is obtained. Bacillus subtilis is inoculated into the nutrient broth liquid culture medium and cultured at 35–38°C for 10–15 hours.

[0077] 1) Preparation of fermentation raw materials:

[0078] By weight, the fermentation ingredients consist of 38 parts mulberry leaves, 5 parts cornmeal, 3 parts wheat bran, 1 part ammonium sulfate, 1 part urea, 1 part modified Mandel nutrient solution, and 51 parts distilled water; mix thoroughly and set aside.

[0079] 2) Solid-state fermentation: 6.6% Aspergillus oryzae spore suspension, 3.3% Hansenula polymorpha suspension and 3.3% Bacillus subtilis suspension were inoculated into the fermentation raw materials, stirred evenly and then fermented in a solid state at 28°C for 60 hours.

[0080] 3) Drying: Dry the fermented material at 65℃ to obtain the protein feed product.

[0081] In the protein feed product of Example 4, the true protein content was 14.8%, and the crude protein content was 27.1%.

[0082] Example 5

[0083] The raw material pretreatment and the preparation of the fermentation strain suspension in this embodiment are the same as in Embodiments 3 and 4.

[0084] 1) Preparation of fermentation raw materials:

[0085] By weight, the fermentation ingredients consist of 34 parts mulberry leaves, 5 parts cornmeal, 5 parts wheat bran, 1 part ammonium sulfate, 1 part urea, 1 part modified Mendel nutrient solution, and 53 parts distilled water; mix thoroughly and set aside.

[0086] 2) Solid-state fermentation: 5% Aspergillus oryzae spore suspension, 3.3% Hansenula polymorpha spore suspension and 5% Bacillus subtilis spore suspension were inoculated into the fermentation raw materials, stirred evenly and fermented at 30℃ for 48 hours.

[0087] 3) Drying: Dry the fermented material at 65℃ to obtain the protein feed product.

[0088] In the protein feed product of Example 5, the true protein content is 14.5%, and the crude protein content is 26.5%.

[0089] Example 6

[0090] The raw material pretreatment and the preparation of the fermentation strain suspension in this embodiment are the same as in Example 3.

[0091] 3) Preparation of fermentation raw materials:

[0092] By weight, the fermentation ingredients consist of 32 parts mulberry leaves, 4 parts cornmeal, 4 parts wheat bran, 1 part ammonium sulfate, 1 part urea, 1 part modified Mandel nutrient solution, and 59 parts distilled water; mix thoroughly and set aside.

[0093] 4) Solid-state fermentation: Inoculate the fermentation raw materials with 5% inoculum of Hansenula polymorpha and 5% inoculum of Bacillus licheniformis, stir evenly, and ferment at 25°C for 72 hours.

[0094] 5) Drying: Dry the fermented material at 65℃ to obtain the protein feed product.

[0095] In the protein feed product of Example 6, the true protein content is 14.5%, and the crude protein content is 28.0%.

[0096] Example 7

[0097] The raw material pretreatment and fermentation strain cultivation in this embodiment are the same as in Example 3.

[0098] 1) Preparation of fermentation raw materials:

[0099] By weight, the fermentation ingredients consist of 38 parts mulberry leaves, 5 parts cornmeal, 3 parts wheat bran, 1 part ammonium sulfate, 1 part urea, 1 part modified Mandel nutrient solution, and 51 parts distilled water; mix thoroughly and set aside.

[0100] 2) Solid-state fermentation: 5% of Aspergillus oryzae spore suspension and 5% of Hansenula polymorpha suspension were inoculated into the fermentation raw materials, stirred evenly, and then solid-state fermented at 28°C for 60 hours.

[0101] 3) Drying: Dry the fermented material at 65℃ to obtain the protein feed product.

[0102] In the protein feed product of Example 7, the true protein content was 14.0%, and the crude protein content was 28.1%.

[0103] Comparative Example 2

[0104] The raw material pretreatment in this example is the same as in Example 3.

[0105] 1) Preparation of fermentation raw materials:

[0106] By weight, the fermentation ingredients include 30 parts mulberry leaves, 4 parts cornmeal, 4 parts wheat bran, 1 part ammonium sulfate, 1 part urea, 1 part modified Mendel nutrient solution, and 51 parts distilled water; mix thoroughly and set aside.

[0107] 2) Solid-state fermentation: Inoculate the fermentation raw materials with 10% of the inoculum of brewer's yeast suspension, stir evenly, and then ferment in a solid-state environment at 28°C for 72 hours.

[0108] The *Saccharomyces cerevisiae* was inoculated as a bacterial suspension. The bacterial suspension was prepared by adding 1 wt% peptone, 0.5 wt% potato extract, 0.5 wt% NaCl, and 1.5% glucose to distilled water. The pH was adjusted to 6.0–6.5 with 0.1 mol / L HCl and 0.1 mol / L NaOH. After sterilization and cooling, a potato glucose liquid culture medium was prepared. The *Saccharomyces cerevisiae* was inoculated into the potato glucose liquid culture medium and cultured at 28°C and 150 rpm for 12 hours on a shaker. The medium was then diluted to a spore count of 10-1. 7 A suspension of cells / mL;

[0109] 3) Drying: Dry the fermented material at 65℃ to obtain the protein feed product.

[0110] In this comparative protein feed product, the true protein content is 12.9%, and the crude protein content is 31.9%.

[0111] Comparative Example 3

[0112] The raw material pretreatment and the preparation of the fermentation strain suspension in this comparative example are the same as in Example 3.

[0113] 1) Preparation of fermentation raw materials:

[0114] By weight, the fermentation ingredients include 30 parts mulberry leaves, 4 parts cornmeal, 4 parts wheat bran, 1 part ammonium sulfate, 1 part urea, 1 part modified Mandel nutrient solution, and 51 parts distilled water; mix thoroughly and set aside.

[0115] 2) Solid-state fermentation: Inoculate the fermentation raw material with a 10% inoculum of Bacillus licheniformis suspension, stir evenly, and then ferment in a solid-state environment at 28°C for 72 hours.

[0116] 3) Drying: Dry the fermented material at 65℃ to obtain the protein feed product.

[0117] In this comparative protein feed product, the true protein content is 11.1%, and the crude protein content is 30.1%.

[0118] Compared with examples using other bacterial suspensions, the addition of *Hansenula polymorpha* significantly increased the protein content in the fermentation products.

[0119] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from its spirit and essence should fall within the protection scope of the present invention.

Claims

1. A method for preparing mulberry leaf protein feed, characterized in that, The following raw materials are fermented in parts by weight: 30-35 parts mulberry leaves; 5-10 parts fiber regulator; 2-3 parts nitrogen source; 1-4 parts modified Mandel nutrient solution; 50-60 parts distilled water; the fiber regulator is wheat bran and corn flour; the nitrogen source is selected from one or more of urea, ammonium sulfate, and ammonia water; the modified Mandel nutrient solution has the following composition: MgSO4·7H2O: 0.3wt%; CaCl2: 0.45wt%; KH2PO4: 2wt%; FeSO4·7H2O: 0.005wt%; CoCl2: 0.002wt%; MnSO4·H2O: 0.0016wt%; ZnSO4·H2O: 0.0014wt%, with the remainder being water; The fermentation strain is a mixture of Aspergillus oryzae, Hansenula spp. guaranae, and a third strain, wherein the third strain is Bacillus licheniformis or Bacillus subtilis. The fermentation strain is inoculated in the form of a bacterial suspension, wherein the volume ratio of the bacterial suspension of Aspergillus oryzae, Hansenula spp. guaranae, and the third strain is 2-3:1-4:1; the preservation number of Hansenula spp. guaranae is CGMCC No. 34449.

2. The method for preparing mulberry leaf protein feed according to claim 1, characterized in that, The mulberry leaves have a moisture content of 70-85 wt%, a true protein content of 8.0-11.0% and a crude fiber content of 12.0%-15.0% on a dry matter basis.

3. The method for preparing mulberry leaf protein feed according to claim 1, characterized in that, The fiber regulator is wheat bran and corn flour in a mass ratio of 4:4 to 6; the nitrogen source is urea and ammonium sulfate in a mass ratio of 1:1 to 2.

4. The method for preparing mulberry leaf protein feed according to any one of claims 1 to 3, characterized in that, Including the following steps: 1) Raw material preparation: Wash and dry mulberry leaves to 8-12% moisture content, then crush them to a particle size of less than 1 mm; crush the fiber regulator and sieve it for later use; mix mulberry leaf powder, wheat bran and corn flour evenly according to the ratio; dissolve the nitrogen source in distilled water, add the modified nutrient solution, stir evenly and then mix it thoroughly with the mixed matrix; 2) Fermentation: Inoculate with a suspension of fermentation bacteria at 8-12% of the total substrate weight; ferment at 25-30 ℃ for 48-72 hours, stirring once every 10-15 hours during the fermentation process; 3) Drying and pelleting: The fermented feed is dried at 62-65 ℃ until the moisture content is less than 10%; after drying, it is pressed into pelleted protein feed by a feed pellet mill.

5. The method for preparing mulberry leaf protein feed according to claim 4, characterized in that, The *Aspergillus oryzae* strain was inoculated as a spore suspension. The spore suspension was prepared by transferring the *Aspergillus oryzae* strain onto potato dextrose agar plates and culturing at 24–28 °C for 5–7 days; then eluting the spores with a modified Mandel nutrient solution and diluting to a spore count of 0.5–5 × 10⁻⁶. 7 CFU / mL; The third bacterial strain was inoculated as a bacterial suspension. The bacterial suspension was prepared by adding 1 wt% peptone, 0.3 wt% beef extract, and 0.5 wt% NaCl to distilled water, adjusting the pH to 6.0-6.5, sterilizing and cooling to obtain a nutrient broth liquid culture medium; the bacterial strain was inoculated into the nutrient broth liquid culture medium and cultured at 35-38℃ for 10-15 hours; the third bacterial strain was Bacillus licheniformis or Bacillus subtilis.

6. The method for preparing mulberry leaf protein feed according to claim 4, characterized in that, The *Hansenula polymorpha* strain was inoculated using a bacterial suspension. The bacterial suspension was prepared by adding 1 wt% peptone, 0.5 wt% potato starch, 0.5 wt% NaCl, and 1.5% glucose to distilled water, adjusting the pH to 6.0–6.5, sterilizing, and cooling to obtain potato glucose liquid culture medium. The *Hansenula polymorpha* strain was inoculated into the potato glucose liquid culture medium and cultured at 26–28 °C for 10–14 hours.

7. The feed prepared by the method according to any one of claims 1 to 6.

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