Mulberry leaf-vinasse fermentation composition

Abstract

The invention discloses a mulberry leaf-vinasse fermentation composition which comprises the following raw materials: vinasse, mulberry leaves and a complex microbial inoculant, the complex microbial inoculants comprise saccharomyces cerevisiae, bacillus subtilis and lactobacillus plantarum. The mulberry leaf-vinasse fermentation composition is obtained by performing anaerobic solid state fermentation on the raw materials, namely crushing vinasse and mulberry leaves to 20-50 meshes, uniformly mixing, adjusting the water content of the raw materials to be 50-70wt%, inoculating a complex microbial inoculant, uniformly stirring, filling into a breathing bag, sealing at 37 + / -0.5 DEG C, and fermenting for 4-6 days, thereby obtaining the mulberry leaf-vinasse fermentation composition. According to the mulberry leaf-vinasse fermentation composition, the mulberry leaves and the vinasse are mixed, then the complex microbial inoculant is inoculated for solid-state fermentation, the pH value of the prepared mulberry leaf-vinasse fermentation composition is remarkably reduced, meanwhile, the lactic acid content is remarkably increased, the antinutritional factor content is reduced, true protein is well preserved, the antioxidant activity is more reserved, and the fermentation effect of the mulberry leaves and the vinasse is improved; the feeding value of the mulberry leaves and the vinasse is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and more specifically, relates to a mulberry leaf-vinasse fermentation composition. Background Art

[0002] With the rapid growth of the global population, the demand for foods like meat, eggs, and dairy products continues to surge, posing a significant risk of feed shortages for the rapidly expanding livestock industry. Corn and soybeans are not only primary feed ingredients but are also consumed directly by humans, leading livestock companies to face persistently high feed costs. To address this critical issue, developing new feeds tailored to local conditions is one effective measure.

[0003] Distillers' grains (DG) are a byproduct of Chinese liquor production. After fermentation and distillation, DG has a high water content and acidity, making it susceptible to spoilage if not properly handled. Although rich in protein, starch, bioactive factors, cellulose, and high levels of amino acids, adding DG directly to the diet can inhibit digestion and even cause toxicity in non-ruminant animals, making it a poor substitute for conventional feed.

[0004] Mulberry (Morus alba), a plant of the genus Morus in the Moraceae family, has been cultivated in China for over 5,000 years. The annual biomass yield of mulberry leaves (ML) is approximately 250-300 kg / km². Due to its high crude protein content, mulberry leaves are considered a high-quality feed ingredient. Phytochemicals contained in mulberry leaves, such as phenols (flavonoids, chlorogenic acid) and alkaloids (1-deoxynojirimycin, buckwheat alkaloids), have antibacterial, anti-inflammatory, anthelmintic, and antioxidant activities. However, fresh mulberry leaves have a high water content, making them difficult to preserve long-term. They also contain anti-nutritional factors such as tannins. Excessive addition can interfere with the animal's metabolic absorption of feed nutrients, impacting their health and the yield and quality of livestock and poultry products, limiting their use in feed.

[0005] Solid-state fermentation (SSF) is a process that uses porous solid matrices to cultivate microorganisms. It can convert low-value by-products into high-value products such as spores, probiotics, biofungicides, and biopesticides. This technology can also degrade cellulose and is therefore widely used to improve the quality of by-products. Common fermentation bacteria include lactic acid bacteria, Bacillus, and yeast. For example, solid-state fermentation of rice bran by Bacillus subtilis and Saccharomyces cerevisiae can reduce the fiber and anti-nutritional factor content, thereby increasing the nutritional value and antioxidant capacity; while fermentation of rice by Lactobacillus plantarum can reduce the lignocellulose content and increase the lactic acid content. Although solid-state fermentation is an effective method for the resource utilization of distiller's grains, its high moisture and acidity can affect the fermentation process. Summary of the Invention

[0006] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0007] In order to achieve these purposes and other advantages of the present invention, a mulberry leaf-vintner's grains fermentation composition is provided, which comprises the following raw materials: vintner's grains, mulberry leaves and a composite bacterial agent;

[0008] The composite bacterial agent comprises saccharomyces cerevisiae, bacillus subtilis and lactobacillus plantarum.

[0009] Preferably, the dry mass ratio of the vinasse and mulberry leaves is 1:0.5-2; and the dosage of the composite bacterial agent is 5-15wt% of the total mass of the vinasse and mulberry leaves.

[0010] Preferably, the mulberry leaf-vinasse fermentation composition is obtained by anaerobic solid-state fermentation of the raw materials.

[0011] Preferably, in the composite bacterial agent, Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum are composed in a mass ratio of 1-3:1-3:1-3.

[0012] Preferably, the composite bacterial agent further comprises Lactobacillus brevis.

[0013] Preferably, in the composite bacterial agent, Saccharomyces cerevisiae, Bacillus subtilis, Lactobacillus plantarum and Lactobacillus brevis are composed in a mass ratio of 1-3:1-3:1-3:1-3:1-3.

[0014] Preferably, the composite bacterial agent is Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus brevis.

[0015] Preferably, in the composite bacterial agent, Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus brevis are composed in a mass ratio of 1-3:1-3:1-3.

[0016] Preferably, the specific steps of the anaerobic solid-state fermentation are: crushing the vinasse and mulberry leaves into 20-50 mesh and then mixing them evenly, adjusting the moisture content of the raw materials to 50-70wt%, then inoculating the composite bacterial agent and stirring evenly, putting them into a breathing bag, sealing them and fermenting them at 37°C±0.5°C for 4-6 days to obtain a mulberry leaf-vinasse fermentation composition.

[0017] Preferably, the raw materials of the mulberry leaf-vintner's grains fermentation composition further include: additives; wherein the amount of the additives is 5 to 15 wt% of the total mass of the vintner's grains and mulberry leaves.

[0018] Preferably, the preparation method of the additive comprises the following steps:

[0019] S1. Grind cinnamon bark through a 40-60 mesh sieve, add 5-10 times the mass of 75wt% ethanol, soak for 20-50 min, then place in an ultrasonic instrument for ultrasonic extraction for 20-60 min, separate the extract and solid matter, add the solid matter again into 5-10 times the mass of 75wt% ethanol, ultrasonically extract for 20-60 min, and filter and collect the extract; combine the two extracts, concentrate to 1 / 5 of the original volume by vacuum rotary evaporation at 45°C, and freeze-dry to obtain a cinnamon bark extract; wherein the ultrasonic extraction temperature is 35-50°C, the ultrasonic power is 200-500W, and the ultrasonic frequency is 25-40kHz;

[0020] S2. Add Bletilla striata polysaccharide and isomaltulose into distilled water, ultrasonicate for 30 to 90 minutes, then add cinnamon extract, continue ultrasonicate for 30 to 90 minutes, and freeze-dry to obtain an additive; wherein the mass ratio of Bletilla striata polysaccharide, cinnamon extract, and isomaltulose is 1:0.5 to 1:0.05 to 0.2, and the mass volume ratio of Bletilla striata polysaccharide and distilled water is 1 g:3 to 8 mL; the power of the ultrasound is 200 to 500 W, and the frequency is 40 to 70 kHz.

[0021] Preferably, the specific steps of the anaerobic solid-state fermentation are: adding the additive to 5 to 10 times the mass of distilled water, stirring evenly to obtain a mixed solution; crushing the lees and mulberry leaves into 20 to 50 mesh and mixing evenly, adding the mixed solution, stirring evenly, adjusting the water content of the raw materials to 50 to 70wt%, then inoculating the composite bacterial agent and stirring evenly, placing the mixture into a breathing bag, sealing the mixture and fermenting it at 37°C ± 0.5°C for 4 to 6 days to obtain a mulberry leaf-lees fermentation composition.

[0022] A use of the mulberry leaf-vinasse fermentation composition as described above in preparing animal feed.

[0023] The present invention has at least the following beneficial effects: the present invention mixes mulberry leaves with distiller's grains, and then inoculates a composite bacterial agent for solid-state fermentation. The pH value of the prepared mulberry leaf-distiller's grains fermentation composition is significantly reduced (4.07-4.16), while the lactic acid content is significantly increased, the anti-nutritional factor content is reduced, the true protein is well preserved, and the antioxidant activity is more retained. This solves the problems of high anti-nutritional factor content in mulberry leaves and high acidity in distiller's grains, improves the fermentation effect of mulberry leaves and distiller's grains, effectively increases the feeding value of mulberry leaves and distiller's grains, and provides a new idea for solving the problem of feed resource shortage.

[0024] The present invention adopts Lactobacillus brevis and / or Lactobacillus plantarum to form a composite microbial agent in combination with Bacillus subtilis and Saccharomyces cerevisiae. By inoculating the three composite microbial agents, the microbial community structure in the solid-state fermentation system is changed, the relative abundance of lactic acid bacteria is significantly increased after fermentation, the growth of harmful microorganisms is effectively controlled, and the solid-state fermentation quality is improved. Fermentation using the composite microbial agent of the present invention can effectively improve the physical and chemical properties of the raw materials and enhance their feeding value, providing a theoretical basis for the application of mulberry leaves and distiller's grains as feed. Among them, the microbial compounding scheme of group B (Saccharomyces cerevisiae:Bacillus subtilis:Lactobacillus plantarum=1:1:1) is the optimal exogenous microorganism for mulberry leaves and distiller's grains.

[0025] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The anti-nutritional factor content and degradation rate of the compositions of Examples 1-3 and Comparative Examples 1-6 before and after fermentation, wherein ac are the tannin content and degradation rate of Comparative Examples 1-3, Comparative Examples 4-6, and Example 1-3, respectively, and df are the phytic acid content and degradation rate of Comparative Examples 1-3, Comparative Examples 4-6, and Example 1-3, respectively; in the figure, different lowercase letters indicate significant differences in anti-nutritional factor content (p<0.05), different uppercase letters indicate significant differences in degradation rate (p<0.05), and no letters indicate no significant differences;

[0027] Figure 2 The antioxidant capacity changes of the compositions of Examples 1-3 and Comparative Examples 1-6 before and after fermentation, wherein ac are the DPPH scavenging activities and retention rates of Comparative Examples 1-3, Comparative Examples 4-6, and Examples 1-3, respectively, and df are the ABTS scavenging activities and retention rates of Comparative Examples 1-3, Comparative Examples 4-6, and Examples 1-3, respectively; different lowercase letters in the figure indicate significant differences in antioxidant capacity (p<0.05), different uppercase letters indicate significant differences in retention rates (p<0.05), and no letters indicate no significant differences;

[0028] Figure 3 The relative abundance of microorganisms before and after fermentation of the compositions of Examples 1-3 and Comparative Examples 1-6, a is a heat map at the phylum level, b is a histogram at the genus level; in the figure, S is the mulberry leaf group, J is the distiller's grains group, and JS is the mulberry leaf and distiller's grains mixed group;

[0029] Figure 4 The α diversity of the compositions of Examples 1-3 and Comparative Examples 1-6 before and after fermentation, a represents Chao, b represents ACE, c represents Simpson, and d represents Shannon; in the figure, S represents the mulberry leaf group, J represents the distiller's grains group, and JS represents the mulberry leaf and distiller's grains mixed group;

[0030] Figure 5 a is the Venn diagram of different groups at the genus level, Figure 5 b Non-metric multidimensional scaling analysis of different groups, Figure 5 c, d Linear discriminant analysis effect size (LEfSe) results of bacterial composition; in the figure, S is the mulberry leaf group, J is the distiller's grains group, and JS is the mulberry leaf and distiller's grains mixed group;

[0031] Figure 6 This is a heat map of the correlation between the microbial genus level and physical and chemical properties; in the figure, S is the mulberry leaf group, J is the distiller's grains group, and JS is the mulberry leaf and distiller's grains mixed group. DETAILED DESCRIPTION

[0032] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0033] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0034] In the following examples, the yeast Saccharomyces cerevisiae used was CICC 1202, purchased from the China Industrial Culture Collection of Microorganisms; Bacillus subtilis was CCTCC NO: M2024804 (Chinese patent CN2024107543110); Lactobacillus brevis was CGMCC1.2028, purchased from the China General Culture Collection of Microorganisms; Lactobacillus plantarum was CGMCC 1.12974, purchased from China General Microbial Culture Collection Center; pre-screening was carried out in YPD medium (Saccharomyces cerevisiae), LB medium (Bacillus subtilis), and MRS medium (Lactobacillus brevis and Lactobacillus plantarum), respectively. The initial pH of the culture medium was 7.0, 6.5, and 5.7, respectively. Saccharomyces cerevisiae and Bacillus subtilis were cultured at 37°C, 110 rpm, and shaking for 24 h, while Lactobacillus plantarum and Lactobacillus brevis were cultured statically at 37°C for 24 h.

[0035] Example 1

[0036] A mulberry leaf-vintner's grains fermentation composition comprises the following raw materials: vintner's grains, mulberry leaves, and a composite bacterial agent; wherein, based on dry weight, the mass ratio of the vintner's grains to the mulberry leaves is 1:1, and the amount of the composite bacterial agent is 10 wt% of the total mass of the vintner's grains and the mulberry leaves;

[0037] The composite bacterial agent is composed of Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus brevis in a mass ratio of 1:1:1;

[0038] The preparation method of the mulberry leaf-vintner's grains fermentation composition comprises the following steps: grinding vintner's grains and mulberry leaves into 30 meshes and then uniformly mixing them; adjusting the water content of the raw materials to 60wt%, inoculating a composite bacterial agent and uniformly stirring; maintaining a natural pH; and placing the mixture into breathing bags (23cm×30cm, Wenzhou Wangting Packaging Co., Ltd., Zhejiang, China) with 200g of fermentation material in each bag. The mixture is sealed and fermented at 37°C±0.5°C for 5 days to obtain the mulberry leaf-vintner's grains fermentation composition.

[0039] Example 2

[0040] This embodiment is basically the same as Example 1, except that the composite bacterial agent of this embodiment is composed of Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus plantarum in a mass ratio of 1:1:1.

[0041] Example 3

[0042] This embodiment is basically the same as Example 1, except that the composite bacterial agent of this embodiment is composed of Saccharomyces cerevisiae, Bacillus subtilis, Lactobacillus brevis, and Lactobacillus plantarum in a mass ratio of 1:1:1:1.

[0043] Example 4

[0044] A mulberry leaf-vintner's grain fermentation composition comprises the following raw materials: vintner's grains, mulberry leaves, a composite bacterial agent, and additives; wherein, based on dry weight, the mass ratio of the vintner's grains to the mulberry leaves is 1:1, the amount of the composite bacterial agent is 10wt% of the total mass of the vintner's grains and the mulberry leaves, and the amount of the additive is 10wt% of the total mass of the vintner's grains and the mulberry leaves;

[0045] The composite bacterial agent is composed of Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1:1;

[0046] The preparation method of the additive is:

[0047] S1. Cinnamon bark was crushed and passed through a 50-mesh sieve, added to 8 times the mass of 75wt% ethanol, soaked for 30 min, and then placed in an ultrasonic instrument for ultrasonic extraction for 30 min, the extract and solid matter were separated, the solid matter was again added to 8 times the mass of 75wt% ethanol, ultrasonically extracted for 30 min, and the extract was collected by filtration; the two extracts were combined, concentrated to 1 / 5 of the original volume by vacuum rotary evaporation at 45°C, and freeze-dried to obtain a cinnamon bark extract; wherein the ultrasonic extraction temperature was 45°C, the ultrasonic power was 300W, and the ultrasonic frequency was 35kHz;

[0048] S2. Add Bletilla striata polysaccharide and isomaltulose to distilled water, ultrasonicate for 1 hour, then add cinnamon extract, continue ultrasonicate for 1 hour, and freeze-dry to obtain an additive; wherein the mass ratio of Bletilla striata polysaccharide, cinnamon extract, and isomaltulose is 1:0.6:0.1, and the mass volume ratio of Bletilla striata polysaccharide and distilled water is 1 g:5 mL; the ultrasonic power is 300 W and the frequency is 55 kHz;

[0049] The preparation method of the mulberry leaf-vintner's grains fermentation composition comprises the following steps: adding an additive to 8 times the mass of distilled water, stirring uniformly to obtain a mixed solution; crushing the vintner's grains and mulberry leaves into 30 meshes, mixing uniformly, adding the mixed solution, stirring uniformly, adjusting the water content of the raw materials to 60wt%, inoculating a composite bacterial agent and stirring uniformly, maintaining a natural pH value, and filling the mixture into a breathing bag (23cm×30cm, Wenzhou Wangting Packaging Co., Ltd., Zhejiang, China), filling 200g of fermentation material into each bag, sealing the bag, and fermenting at 37°C±0.5°C for 5 days to obtain the mulberry leaf-vintner's grains fermentation composition.

[0050] In this embodiment, additives are added as raw materials based on embodiment 2.

[0051] Example 5

[0052] A mulberry leaf-vintner's grain fermentation composition comprises the following raw materials: vintner's grains, mulberry leaves, a composite bacterial agent, and additives; wherein, based on dry weight, the mass ratio of the vintner's grains to the mulberry leaves is 1:1, the amount of the composite bacterial agent is 10wt% of the total mass of the vintner's grains and the mulberry leaves, and the amount of the additive is 10wt% of the total mass of the vintner's grains and the mulberry leaves;

[0053] The composite bacterial agent is composed of Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1:1;

[0054] The preparation method of the additive comprises: adding Bletilla striata polysaccharide and isomaltulose to distilled water, ultrasonicating for 2 hours, and freeze-drying to obtain the additive; wherein the mass ratio of Bletilla striata polysaccharide to isomaltulose is 1:0.1, and the mass volume ratio of Bletilla striata polysaccharide to distilled water is 1 g:5 mL; the ultrasonic power is 300 W and the frequency is 55 kHz;

[0055] The preparation method of the mulberry leaf-vintner's grains fermentation composition comprises the following steps: adding an additive to 8 times the mass of distilled water, stirring uniformly to obtain a mixed solution; crushing the vintner's grains and mulberry leaves into 30 meshes, mixing uniformly, adding the mixed solution, stirring uniformly, adjusting the water content of the raw materials to 60wt%, inoculating a composite bacterial agent and stirring uniformly, maintaining a natural pH value, and filling the mixture into a breathing bag (23cm×30cm, Wenzhou Wangting Packaging Co., Ltd., Zhejiang, China), filling 200g of fermentation material into each bag, sealing the bag, and fermenting at 37°C±0.5°C for 5 days to obtain the mulberry leaf-vintner's grains fermentation composition.

[0056] Compared with Example 4, this example only changes the preparation method of the additive, that is, cinnamon extract is not used in the additive.

[0057] Example 6

[0058] A mulberry leaf-vintner's grain fermentation composition comprises the following raw materials: vintner's grains, mulberry leaves, a composite bacterial agent, and additives; wherein, based on dry weight, the mass ratio of the vintner's grains to the mulberry leaves is 1:1, the amount of the composite bacterial agent is 10wt% of the total mass of the vintner's grains and the mulberry leaves, and the amount of the additive is 10wt% of the total mass of the vintner's grains and the mulberry leaves;

[0059] The composite bacterial agent is composed of Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1:1;

[0060] The preparation method of the additive is:

[0061] S1. Cinnamon bark was crushed and passed through a 50-mesh sieve, added to 8 times the mass of 75wt% ethanol, soaked for 30 min, and then placed in an ultrasonic instrument for ultrasonic extraction for 30 min, the extract and solid matter were separated, the solid matter was again added to 8 times the mass of 75wt% ethanol, ultrasonically extracted for 30 min, and the extract was collected by filtration; the two extracts were combined, concentrated to 1 / 5 of the original volume by vacuum rotary evaporation at 45°C, and freeze-dried to obtain a cinnamon bark extract; wherein the ultrasonic extraction temperature was 45°C, the ultrasonic power was 300W, and the ultrasonic frequency was 35kHz;

[0062] S2. Add Bletilla striata polysaccharide to distilled water, sonicate for 1 hour, then add cinnamon extract, continue sonicating for 1 hour, and freeze-dry to obtain an additive; wherein the mass ratio of Bletilla striata polysaccharide to cinnamon extract is 1:0.6, and the mass volume ratio of Bletilla striata polysaccharide to distilled water is 1 g:5 mL; the ultrasonic power is 300 W and the frequency is 55 kHz;

[0063] The preparation method of the mulberry leaf-vintner's grains fermentation composition comprises the following steps: adding an additive to 8 times the mass of distilled water, stirring uniformly to obtain a mixed solution; crushing the vintner's grains and mulberry leaves into 30 meshes, mixing uniformly, adding the mixed solution, stirring uniformly, adjusting the water content of the raw materials to 60wt%, inoculating a composite bacterial agent and stirring uniformly, maintaining a natural pH value, and filling the mixture into a breathing bag (23cm×30cm, Wenzhou Wangting Packaging Co., Ltd., Zhejiang, China), filling 200g of fermentation material into each bag, sealing the bag, and fermenting at 37°C±0.5°C for 5 days to obtain the mulberry leaf-vintner's grains fermentation composition.

[0064] Compared with Example 4, this example only changed the preparation method of the additive, that is, no isomaltulose was used in the additive.

[0065] Example 7

[0066] A mulberry leaf-vintner's grain fermentation composition comprises the following raw materials: vintner's grains, mulberry leaves, a composite bacterial agent, and additives; wherein, based on dry weight, the mass ratio of the vintner's grains to the mulberry leaves is 1:1, the amount of the composite bacterial agent is 10wt% of the total mass of the vintner's grains and the mulberry leaves, and the amount of the additive is 10wt% of the total mass of the vintner's grains and the mulberry leaves;

[0067] The composite bacterial agent is composed of Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum in a mass ratio of 1:1:1;

[0068] The preparation method of cinnamon extract comprises the following steps: grinding cinnamon, passing it through a 50-mesh sieve, adding it to 8 times the mass of 75wt% ethanol, soaking it for 30 minutes, then placing it in an ultrasonic instrument for ultrasonic extraction for 30 minutes, separating the extract and solid matter, adding the solid matter again to 8 times the mass of 75wt% ethanol, ultrasonically extracting it for 30 minutes, filtering and collecting the extract; combining the two extracts, concentrating them to 1 / 5 of their original volume by vacuum rotary evaporation at 45°C, and freeze-drying them to obtain the cinnamon extract; wherein the ultrasonic extraction temperature is 45°C, the ultrasonic power is 300W, and the ultrasonic frequency is 35kHz;

[0069] The preparation method of a mulberry leaf-vintner's lees fermentation composition comprises the following steps: mixing bletilla striata polysaccharide, cinnamon bark extract, and isomaltulose in a mass ratio of 1:0.6:0.1 to obtain an additive, adding the additive into 8 times the mass of distilled water, and stirring the mixture to obtain a mixed solution; crushing the vintner's lees and mulberry leaves into 30 mesh sizes, mixing the mixture to obtain a mixed solution, stirring the mixture to obtain a mixed solution, adjusting the water content of the raw materials to 60wt%, inoculating a composite bacterial agent, stirring the mixture to obtain a mixed solution, maintaining a natural pH value, and placing the mixture into a breathing bag (23cm×30cm, Wenzhou Wangting Packaging Co., Ltd., Zhejiang, China), filling each bag with 200g of fermentation material, sealing the bag, and fermenting the mixture at 37°C±0.5°C for 5 days to obtain the mulberry leaf-vintner's lees fermentation composition.

[0070] Compared with Example 4, this example directly adds Bletilla striata polysaccharide, cinnamon extract and isomaltulose.

[0071] Comparative Example 1

[0072] This comparative example is basically the same as Example 1, except that the raw materials of this comparative example do not contain distiller's grains, but only contain mulberry leaves and a composite bacterial agent, and a mulberry leaf fermentation composition is obtained.

[0073] Comparative Example 2

[0074] This comparative example is basically the same as Example 2, except that the raw materials of this comparative example do not contain distiller's grains, but only contain mulberry leaves and a composite bacterial agent, and a mulberry leaf fermentation composition is obtained.

[0075] Comparative Example 3

[0076] This comparative example is basically the same as Example 3, except that the raw materials of this comparative example do not contain distiller's grains, but only contain mulberry leaves and a composite bacterial agent, and a mulberry leaf fermentation composition is obtained.

[0077] Comparative Example 4

[0078] This comparative example is basically the same as Example 1, except that the raw materials of this comparative example do not contain mulberry leaves, but only contain distiller's grains and a composite bacterial agent to obtain a distiller's grains fermentation composition.

[0079] Comparative Example 5

[0080] This comparative example is basically the same as Example 2, except that the raw materials of this comparative example do not contain mulberry leaves, but only contain distiller's grains and a composite bacterial agent to obtain a distiller's grains fermentation composition.

[0081] Comparative Example 6

[0082] This comparative example is basically the same as Example 3, except that the raw materials of this comparative example do not contain mulberry leaves, but only contain distiller's grains and a composite bacterial agent to obtain a distiller's grains fermentation composition.

[0083] The raw materials of the fermentation compositions of Examples 1-3 and Comparative Examples 1-6 are shown in Table 1-3.

[0084] Table 1 Mulberry leaf fermentation composition

[0085]

[0086] Table 2 Wine lees fermentation composition

[0087]

[0088] Table 3 Mulberry leaf-vinasse fermentation composition

[0089]

[0090] (1) Analysis of chemical composition and anti-nutritional factors

[0091] 1) Conventional index determination:

[0092] Dry matter (DM): measured after drying in an oven at 105°C to constant weight;

[0093] pH value: measured using a glass electrode pH meter (PHS-3C, Shanghai Yidian Scientific Instrument Co., Ltd., Shanghai, China);

[0094] True protein (TP): determined using a Kjeldahl nitrogen analyzer according to the AOAC method (Chi, Z., Deng, M., Tian, H., Liu, D., Li, Y., Liu, G., Sun, B., Guo, Y., 2022. Effects of Mulberry Leaves and Pennisetum Hybrid Mix-Silage on Fermentation Parameters and Bacterial Community. Fermentation 8, 197.);

[0095] Soluble sugar (WSC): measured using the anthrone colorimetric method (Murphy, RP, 1958. A method for the extraction of plant samples and the determination of total soluble carbohydrates. J. Sci. Food Agric. 9, 714-717.);

[0096] Neutral detergent fiber (NDF): Determined according to the method of Van Soest et al. (Van Soest, PJ, Robertson, JB, Lewis, BA, 1991. Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. J. Dairy Sci. 74, 3583-3597).

[0097] Ammoniacal nitrogen (AN) was determined by the phenol-hypochlorite colorimetric method (Ke, WC, Ding, WR, Xu, DM, Ding, LM, Zhang, P., Li, FD, Guo, XS, 2017. Effects of addition of malic or citric acids on fermentation quality and chemical characteristics of alfalfasilage. J. Dairy Sci. 100, 8958-8966.).

[0098] 2) Antioxidant capacity determination:

[0099] ABTS scavenging ability and DPPH scavenging ability were evaluated according to the method of Kim et al. (Kim, D.-O., Lee, KW, Lee, HJ, Lee, CY, 2002. Vitamin C Equivalent Antioxidant Capacity (VCEAC) of Phenolic Phytochemicals. J. Agric. Food Chem. 50, 3713-3717).

[0100] 3) Determination of anti-nutritional factors:

[0101] Phytic acid content: determined by the method of Su et al. (Su, W., Jiang, Z., Hao, L., Li, W., Gong, T., Zhang, Y., Du, S., Wang, C., Lu, Z., Jin, M., Wang, Y., 2021. Variations of Soybean Meal and Corn Mixed Substrates in Physicochemical Characteristics and Microbiota During Two-Stage Solid-State Fermentation. Front. Microbiol. 12, 688839.);

[0102] Tannin content: determined by Folin colorimetry (Deshpande, SS, Cheryan, M., Salunkhe, DK, Luh, BS, 1986. Tannin analysis of food products. CR CCrit. Rev. Food Sci. Nutr. 24, 401-449).

[0103] 4) Organic acid analysis (gas chromatography):

[0104] The following organic acids were determined using a RP-FFAP column (30 m × 0.25 mm × 0.25 μm) and a flame ionization detector (FID):

[0105] Lactic acid: injection volume 1 μL, column temperature 110°C (maintain for 5 min), carrier gas N2, H2 flow rate 40 mL / min;

[0106] Acetic acid, propionic acid, butyric acid, pentanoic acid, and isovaleric acid: injection volume 1 μL, column temperature initially 70°C, increased at 10°C / min to 120°C and held for 3 min, then increased at 15°C / min to 180°C and held for 3 min, carrier gas N2, H2 flow rate 40 mL / min.

[0107] (2) Bacterial quantitative qPCR analysis

[0108] Quantitative PCR was performed using the same 16S rRNA primers used for sequencing. Amplification was performed using the ABI 7300Fast Real-Time PCR System (Applied Biosystems, USA) with the following protocol: initial denaturation at 95°C for 3 minutes; amplification cycles (40 cycles): 95°C for 5 seconds, 58°C for 30 seconds, and 72°C for 1 minute;

[0109] The plasmid DNA with known copy number was diluted ten-fold (10 3 -10 7 A standard curve was established using 16S rRNA gene amplification and standard curve analysis, and the copy number was automatically analyzed using the SDS software (version 2.4) provided with the qPCR system.

[0110] (3) Bacterial community analysis

[0111] DNA extraction and amplification

[0112] use Total microbial genomic DNA was extracted using a soil DNA extraction kit (Omega Bio-tek, USA) according to the manufacturer's instructions. DNA quality and concentration were determined by 1.0% agarose gel electrophoresis and a NanoDrop2000 spectrophotometer (Thermo Scientific, USA) and stored at −80°C until use. Amplification was performed using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') targeting the V3-V4 hypervariable region of the bacterial 16S rRNA gene in a T100 PCR instrument (BIO-RAD, USA). The PCR reaction system (20 μL) consisted of 4 μL 5× Fast Pfu buffer, 2 μL 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL Fast Pfu polymerase, and 10 ng of template DNA, with ddH2O added to the volume. Amplification program: initial denaturation at 95°C for 3 minutes; 29 cycles (95°C for 30 seconds, 53°C for 30 seconds, 72°C for 45 seconds); final extension at 72°C for 10 minutes; storage at 4°C;

[0113] Product purification and sequencing

[0114] PCR products were recovered by 2% agarose gel electrophoresis and purified using a PCR purification kit (YuHua, Shanghai, China) according to the manufacturer's instructions. They were quantified using Qubit 4.0 (Thermo Fisher Scientific, USA). Equimolar amounts of the purified products were mixed and subjected to paired-end sequencing on an Illumina Nextseq 2000 platform (Illumina, USA) according to the standard protocol of Shanghai Meiji Biopharmaceutical Technology Co., Ltd.

[0115] Bioinformatics analysis

[0116] The original FASTQ files were split using a Perl script, quality-controlled using fastp (v0.19.6), and spliced using FLASH (v1.2.7). Data analysis was performed on the MajorBio Cloud Platform (https: / / cloud.majorbio.com). Based on OTU (operational taxonomic unit) information, rarefaction curves and alpha diversity indices (including the number of observed OTUs, Chao1 richness index, Shannon diversity index, and Good's coverage) were calculated using Mothur v1.30.1 software. Nonmetric multidimensional scaling (NMDS) analysis based on the Bray-Curtis distance matrix was performed using the Vegan v2.5-3 software package to assess the similarity of microbial communities between samples. The linear discriminant analysis effect size (LEfSe) method (http: / / huttenhower.sph.harvard.edu / LEfSe) was used to identify significantly different bacterial taxa at the phylum to genus level (LDA values > 0, P < 0.05) between groups. Finally, heat map analysis revealed correlations between species and physicochemical parameters.

[0117] (4) Statistical analysis

[0118] The data were analyzed using the Duncan multiple comparison test (P < 0.05) using R language. The results are expressed as mean ± standard deviation, and the graphs were drawn using Origin 2018.

[0119] The changes in chemical composition of the compositions of Examples 1-3 and Comparative Examples 1-6 before and after fermentation are shown in Table 4, the changes in organic acid content are shown in Table 5, and the changes in the number of lactic acid bacteria are shown in Table 6. In the table, group CK is a group not inoculated with a bacterial agent; RM is a raw material (before fermentation); treatment groups: Group A is inoculated with Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus brevis; Group B is inoculated with Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus plantarum; Group C is inoculated with Saccharomyces cerevisiae, Bacillus subtilis, Lactobacillus brevis, and Lactobacillus plantarum; "-" indicates absence or not detected; "ND" indicates not detected.

[0120] Silage is easy to succeed when the soluble sugar (WSC) content of the raw material exceeds 5%. The WSC content of the mulberry leaves used in the present invention is 9%, and the WSC content is still 6% after mixing with the lees. However, the WSC content of the mulberry leaves was significantly reduced after inoculation with the composite bacterial agent. After inoculation with the lees, it was reduced compared with the raw material, but there was no significant difference compared with the CK group. The WSC content of mulberry leaves after mixed fermentation with lees was significantly higher than that of CK, and groups B and C still had higher content. The neutral detergent fiber (NDF) content of the three raw materials after fermentation was not significantly different from that of the raw materials, and increased to varying degrees after fermentation with the inoculation of bacterial agents. Previous studies have found that inoculation with lactic acid bacteria will reduce the WSC content and produce lactic acid, and will not affect the NDF content. The strains used in the present invention do not show the ability to degrade cellulose. Since the cellulose is not degraded, the WSC content is not replenished, so after the dry matter is lost, the proportion of cellulose increases.

[0121] pH is a key indicator for silage production, and a pH < 4.2 is generally considered successful. In this study, the reference standard for evaluating successful silage is a pH below 4.2. The pH of all mulberry leaf-treated groups decreased significantly after fermentation, with Group B having the lowest pH. Table 5 shows that compared to the CK group, the mulberry leaf-treated groups showed significantly lower AA (acetic acid) and PA (propionic acid) contents, while significantly higher LA (lactic acid) content. Group B had the lowest AA and PA contents, while having the highest LA content. In the distiller's grains, the pH remained unchanged, and the organic acid contents in the grains remained unchanged. After mixed fermentation of mulberry leaves and distiller's grains, the pH of all treated groups decreased significantly compared to the CK group, with Group B having the lowest pH. The CK group showed a significant increase in AA and PA contents after fermentation. Compared to the CK group, the treated groups showed significantly lower AA and PA contents, while significantly higher LA contents. Group B had the lowest AA and PA contents. The high lactic acid content may be the cause of the pH decrease. The lack of significant pH change in the distiller's grains may be due to the low initial pH, which makes it difficult for most bacterial species to colonize the grains. Except for the lees group, inoculation of exogenous bacteria could significantly reduce the production of AA and PA after fermentation.

[0122] After inoculation, the true protein (TP) content of the mulberry leaf and distiller's grains and mulberry leaf mixed fermentations did not significantly decrease compared to the raw material. Within the distiller's grains, the TP content of the CK group was significantly lower compared to the raw material, while there were no significant differences in TP content among groups A, B, and C. The results indicate that after inoculation with exogenous bacteria, most protein was retained, likely due to the rapid decrease in pH after fermentation, which inhibited protein degradation by microorganisms such as Enterobacteriaceae. With the exception of mulberry leaf group A, ammonia nitrogen (AN) content increased significantly after fermentation compared to the raw material. However, AN content was lower in the CK group after inoculation with exogenous bacteria. AN is a key indicator of silage quality, and an increase in AN content is generally associated with proteolysis and amino acid deamination. During the fermentation process, AN increases with microbial activity. The AN content after inoculation with exogenous bacteria was lower than in the CK group, indicating that microbial activities such as proteolysis were inhibited by inoculation with exogenous bacteria, which is beneficial for improving fermentation quality.

[0123] qPCR analysis showed that Lactobacillus was not found in the mulberry leaf raw material and the mixed raw material of lees and mulberry leaves, but only in the lees, containing 8.68×10 2 copies. After mulberry leaves were inoculated with exogenous bacteria for fermentation, the number of lactic acid bacteria copies increased significantly, and group B had the highest number of gene copies. After mixed fermentation of distiller's grains and mulberry leaves, group CK had the highest number of copies, while groups A, B, and C were significantly lower than group CK. The number of lactic acid bacteria increased after mixing distiller's grains with mulberry leaves, indicating that the addition of mulberry leaves is beneficial to the survival of lactic acid bacteria. In addition, the order of magnitude of the number of lactic acid bacteria gene copies in groups A, B, and C after mixing mulberry leaves with distiller's grains and mulberry leaves was different. Correlation analysis showed that the relative abundance of L. brevis in mulberry leaves was negatively correlated with the total copy number. The reduction in the total number of bacteria in groups A and C may be due to the addition of L. brevis. During the mixed fermentation of distiller's grains and mulberry leaves, the relative abundance of L. plantarum was negatively correlated with the total number of bacteria. Therefore, the total number of bacteria in distiller's grains and mulberry leaf groups B and C also decreased.

[0124] The anti-nutritional factor content and degradation rate of the compositions of Examples 1-3 and Comparative Examples 1-6 before and after fermentation are as follows: Figure 1 As shown in the figure, ac are the tannin content and degradation rate of mulberry leaf group, distiller's grains group, and mulberry leaf and distiller's grains mixed group, respectively; df are the phytic acid content and degradation rate of mulberry leaf group, distiller's grains group, and mulberry leaf and distiller's grains mixed group, respectively. Phytic acid is a heat-resistant anti-nutritional factor that can complex with calcium 2+ 、Cu 3+ Such as divalent and trivalent metal cations, thereby inhibiting the utilization of minerals. Figure 1As shown in Figures d, e, and f, the phytic acid content was significantly reduced after fermentation. After fermentation, except for the phytic acid content in mulberry leaf group A, which was higher than that in the other mulberry leaf fermentation groups, there was no significant difference in phytic acid content between the mixed fermentation of distiller's grains and distiller's grains and mulberry leaves. Studies have shown that the decomposition of phytic acid may be related to a decrease in pH, and a lower pH can enhance the activity of phytase. In this study, the Pearson correlation coefficient between pH and phytic acid degradation rate was -0.497 (p < 0.05), indicating that a decrease in pH promotes the decomposition of phytic acid.

[0125] Tannins play a dual role in animal nutrition. Studies have shown that appropriate amounts of tannins can promote amino acid absorption in the small intestine, thereby improving animal growth performance. However, feeding tannin-rich feeds can reduce animal feed intake and digestibility, thereby affecting animal growth. In this study, the tannin content of the three raw materials after fermentation was significantly reduced, but there were differences among the different bacterial strain treatment groups. Group A of mulberry leaves had the lowest content and was significantly lower than Group CK and Group B. After fermentation of distiller's grains, there was no significant difference in tannin content among the treatment groups. In the mixed distiller's grains and mulberry leaf group, the tannin content of Group B was significantly higher than that of Group CK. There was no obvious pattern in the changes in tannin content of different raw materials after fermentation. Lactobacillus has been identified as producing tannin-degrading enzymes from fermented foods, and the addition of lactobacillus can degrade tannins.

[0126] The antioxidant capacity of the compositions of Examples 1-3 and Comparative Examples 1-6 before and after fermentation is as follows: Figure 2As shown, ac represents the DPPH scavenging activity and retention rate of the mulberry leaf group, distiller's grains group, and a mixture of mulberry leaf and distiller's grains group, respectively; df represents the ABTS scavenging activity and retention rate of the mulberry leaf group, distiller's grains group, and a mixture of mulberry leaf and distiller's grains group, respectively. There was no significant difference in DPPH scavenging activity among the fermented mulberry leaf, distiller's grains, and distiller's grains-mulberry leaf mixture, but the activity was significantly lower than that of the raw material. The DPPH retention rate was lowest in the fermented mulberry leaf, while it increased in the fermented mulberry leaf and distiller's grains mixture, indicating that mixed fermentation of mulberry leaf and distiller's grains can enhance the retention of DPPH free radical scavenging activity. The ABTS scavenging activity of mulberry leaf after fermentation was significantly lower than that of the raw material, and there was no significant difference between Groups A, B, and C and Group CK. After distiller's grains fermentation, the ABTS scavenging activity of Groups CK, B, and C was significantly higher than that of the raw material distiller's grains. After distiller's grains-mulberry leaf mixture, the ABTS scavenging activity of Groups C and CK was higher than that of the raw material, and the ABTS scavenging activity of Group CK was significantly higher than that of Groups A and B. Antioxidant activity is related to the content of polyphenols, vitamins, and amino acids. Adding exogenous microorganisms can alter pH to maintain antioxidant activity. DPPH and ABTS free radical scavenging activity are both methods for evaluating total antioxidant activity. However, previous studies have shown divergent trends in DPPH and ABTS scavenging activity, and different assay methods have shown different correlations with the levels of substances such as polyphenols and ascorbic acid. The DPPH and ABTS results may be due to differences in amino acid or peptide content. In the mixed fermentation of distiller's grains and mulberry leaves, the antioxidant activity retention rate was improved compared to the mulberry leaf group, and the antioxidant activity was improved compared to the distiller's grains group.

[0127] Table 4

[0128]

[0129] Note: Different lowercase letters indicate significant differences in the proportions of different strains on different substrates (p<0.05), and no letters indicate no significant differences, the same below.

[0130] Table 5

[0131]

[0132] Table 6

[0133]

[0134] Table 7 shows the dry matter recovery rate DMR, anti-nutritional factor degradation rate, true protein content and antioxidant activity retention rate of the mulberry leaf-vintner's lees fermentation composition of Example 4-7. It can be seen that Example 4 uses Bletilla striata polysaccharide, isomaltulose and cinnamon extract to prepare the additive, which is beneficial to the growth and metabolism of Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum; at the same time, the prepared additive has good dispersibility and is evenly dispersed in the pores of vintner's lees and mulberry leaves, avoiding the porous structure of the fermentation substrate that will increase the oxygen content and cause aerobic bacteria to reproduce, so that the inoculated composite bacterial agent rapidly proliferates and enters an anaerobic metabolic state, further improving the solid-state fermentation effect. Compared with Example 2, the mulberry leaf-vintner's lees fermentation composition obtained in Example 4-7 has an improved dry matter recovery rate, an improved tannin and phytic acid degradation rate, an increased true protein content, and an improved antioxidant activity retention rate.

[0135] Table 7

[0136]

[0137] Solid-state fermentation is carried out by a complex variety of microorganisms. It is crucial to understand the changes in microbial communities under the fermentation treatment of mulberry leaves, lees, mixed raw materials and compound bacterial agents. Figure 3 In Figure a, the heatmap indicates that higher relative abundance indicates redder colors, while lower relative abundance indicates bluer colors. Proteobacteria were the predominant phylum in all three raw materials. The abundance of Firmicutes increased in the distiller's grains treated with a microbial inoculation after fermentation. In the mulberry leaf group, Proteobacteria were the dominant phylum at the phylum level in the CK, followed by Firmicutes. After inoculation, the relative abundance of Firmicutes increased significantly in treatments A, B, and C, becoming the predominant phylum, while the abundance of Proteobacteria decreased significantly. Proteobacteria were the dominant phylum in the mixed distiller's grains and mulberry leaves. After fermentation, Firmicutes became the predominant phylum in the CK, with the relative abundance of Firmicutes in the inoculation group significantly higher than in the CK. Increased abundance of Firmicutes is beneficial for fermentation, as Firmicutes includes numerous genera important for fermentation and capable of breaking down macromolecules such as starch and cellulose. Several studies on silage have shown that Proteobacteria often initially dominate the raw material but are gradually replaced by Firmicutes after ensilage, a finding similar to that observed in this study. The above results show that inoculation of microbial agents can significantly change the microbial community, inhibit the growth of Proteobacteria, and mixing with wine lees can improve the fermentation effect of mulberry leaves.

[0138] like Figure 3As shown in (b), at the genus level, Methylobacterium Methylorubrum and Shingomonas were primarily enriched in the mulberry leaf raw material. After fermentation, the relative abundances of Enterobacter, unclassified_o_Enterobacterales, Franconibacter, and Enterococcus significantly increased in the CK and A groups. Compared with the CK group, the relative abundance of Lactobacillus increased significantly in treatments A, B, and C, while the relative abundance of Enterobacter decreased significantly. Lactobacillus became the dominant genus in treatments B and C, accounting for over 96% of the total bacterial load, while the relative abundance of Lactobacillus in group A was only 32.76%. Acetobacter, consistently the dominant strain in distiller's grains, decreased significantly in treatments A, B, and C after fermentation compared with the CK group. Bacillus increased in treatments A, B, and C. In a mixture of distiller's grains and mulberry leaves, the dominant strains were Acetobacter, Methylobacterium Methylorubrum, and Shingomonas. After fermentation, the relative abundance of Lactobacillus increased significantly in all groups, while the relative abundance of Acetobacter decreased significantly. The increased relative abundance of Lactobacillus in the CK group may be due to the presence of a small amount of Lactobacillus in the raw material. The pH decreases after mixing mulberry leaves with distiller's grains, creating an environment favorable for Lactobacillus proliferation, leading to its dominance in the CK. In the treated group, the addition of exogenous bacteria may have further increased the abundance of Lactobacillus. Lactobacillus is a key strain in feed fermentation, secreting lactic acid, lowering pH, and inhibiting some undesirable bacteria, thereby improving fermentation quality. Enterobacter is generally considered an undesirable microorganism in silage because it metabolizes protein into NH3-N, reducing the nutritional value of the feed, and competes with Lactobacillus for limited nutrients. Enterobacteriaceae can also convert lactic acid produced by lactic acid bacteria into AA. Since AA is less acidic than lactic acid, it causes the pH to rise, thereby disrupting the growth environment of lactic acid bacteria. The relative abundance of Shingomonas and Methylobacterium in mulberry leaf raw materials decreased significantly after fermentation. This may be due to competition between Shingomonas and Methylobacterium and lactic acid bacteria, thus inhibiting their proliferation.Lactobacillus has a relatively low abundance in wine lees. The acetic acid produced by Acetobacter can exert a certain antibacterial effect, but too high acetic acid concentration may affect the growth of lactic acid bacteria and lead to a reduction in the number of beneficial bacteria.

[0139] Figure 4 Figure 2 shows the trends in bacterial community diversity under different treatments, including (a) Chao, (b) ACE, (c) Simpson, and (d) Shannon. Chao, ACE, and Shannon were significantly higher in the mulberry leaf sample than in the other samples. In the mixed distiller's grains and mulberry leaf sample, there were no significant differences in diversity indices between the CK and treated groups. When distiller's grains were fermented alone, no significant differences in diversity were observed between the groups. Figure 5 The results of Venn in a indicate that fermentation led to a decrease in the number of bacterial OTUs in the mulberry leaf and mulberry leaf + lees groups. However, the number of unique bacterial OTUs increased in the lees group. Figure 5 In b, the results of the non-metric multidimensional scaling (NMDS) plot showed that the raw material of the wine lees and the samples after fermentation were highly clustered. This may be because the acidity of the wine lees was high and the exogenous bacteria could not successfully colonize, resulting in the inoculation of the microorganisms that could not significantly affect the community structure of the wine lees. When mulberry leaves were fermented alone, the raw material, CK, and inoculation of the microorganisms were clearly divided into 5 groups in the NMDS plot. After the wine lees were mixed with mulberry leaves, the treatment group and the CK group were significantly dispersed, indicating that the bacterial population composition changed after the inoculation of exogenous bacteria. The linear discriminant analysis effect size (LEfSe) analysis showed that ( Figure 5 c, d) In the mulberry leaf CK group, the relative abundance of Gammaproteobacteria (LDA > 5.5) increased. Inoculation with the mulberry leaf B group increased the abundance of O_Lactobacillales. In the distiller's grains B group, the relative abundance of g_Bacillus increased. These results demonstrate that inoculation with the inoculum significantly altered the bacterial community structure within the sample.

[0140] Microbial communities are complex multispecies combinations, and studying the correlation between bacterial communities and sample physicochemical properties can provide a deeper understanding of the key bacteria that affect sample quality. Figure 6Among the samples, Lactobacillus was significantly negatively correlated with pH, WSC, Tannin, Phytic acid, and DPPH, and significantly positively correlated with LA. Acetobacter was negatively correlated with TP and pH, and positively correlated with NDF, aceric acid, butyric acid, pentanoic acid, and isovaleric acid. Methylobacterium Methyloubrum and Sphingomonas were significantly negatively correlated with NDF, aceric acid, pentanoic acid, and isovaleric acid, and significantly positively correlated with WSC, NDF, Tannin, Phytic acid, and DPPH. These results suggest that AN production may be primarily due to increased Acetobacter abundance, and that an increase in Acetobacter can lead to a loss of protein nutrients.

[0141] As food resources become increasingly scarce, the development of new feed resources is particularly important. Mulberry leaves and distiller's grains are potential nutrient-rich feed raw materials, but their feeding value is limited by the high content of anti-nutritional factors in mulberry leaves and the high acidity of distiller's grains. The present invention compounds mulberry leaves and distiller's grains, comprehensively utilizing the advantages of mulberry leaves and distiller's grains and improving their shortcomings. The present invention also discloses the effects of a composite bacterial agent composed of Lactobacillus brevis, Lactobacillus plantarum, Bacillus subtilis and Saccharomyces cerevisiae on the solid-state fermentation quality and microbial community of mulberry leaves, distiller's grains and a mixed group of mulberry leaves and distiller's grains. The study found that after solid-state fermentation:

[0142] 1) After the mulberry leaves and distiller's grains were mixed and inoculated with the composite bacterial agent, the pH value of the mulberry leaf-distiller's grains fermentation composition was significantly reduced (≤4.16), and a large amount of lactic acid was produced, with a maximum of 83.43 mg / g (Example 2);

[0143] 2) The true protein in the fermentation composition is well preserved (retention rate >99%);

[0144] 3) The content of anti-nutritional factors (phytic acid, tannin) in the fermentation composition is significantly reduced;

[0145] 4) Compared with the fermentation alone in the comparative example, the antioxidant activity and antioxidant activity retention rate were improved by fermenting the mixture of mulberry leaves and distiller's grains in the embodiment, with the DPPH scavenging ability retention rate being 52.66% (Example 3) and the ABTS scavenging ability retention rate being 114.35% (Example 3);

[0146] 5) Microbial community analysis showed that the relative abundance of lactic acid bacteria increased significantly after fermentation and became the dominant genus; correlation analysis revealed that lactic acid bacteria had a significant impact on multiple indicators;

[0147] 6) Example 4: An additive is prepared with Bletilla striata polysaccharide, isomaltulose and cinnamon extract, and mixed with mulberry leaves and vinasse, which is beneficial to the growth and metabolism of Bacillus subtilis, Saccharomyces cerevisiae and Lactobacillus plantarum, and causes the inoculated composite bacterial agent to proliferate rapidly and enter an anaerobic metabolic state, further improving the solid-state fermentation effect. Compared with Example 2, the mulberry leaf-vinasse fermentation composition obtained in Example 4 has an improved dry matter recovery rate, an improved tannin and phytic acid degradation rate, an increased true protein content, and an improved antioxidant activity retention rate.

[0148] The results showed that fermentation with the composite microbial agent of the present invention can effectively improve the physical and chemical properties of the raw materials and enhance their feed value, providing a new approach to solving the problem of feed resource shortage and providing a theoretical basis for the feed application of these two materials, mulberry leaves and distiller's grains. In summary, the microbial compound scheme of Group B (Saccharomyces cerevisiae: Bacillus subtilis: Lactobacillus plantarum = 1:1:1) is the optimal exogenous microorganism for the mulberry leaves and mulberry leaf distiller's grains combination. This invention provides new insights into the comprehensive utilization of mulberry leaves and distiller's grains, laying a theoretical foundation for further research.

[0149] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A mulberry leaf-vinasse fermentation composition, characterized in that: It includes the following raw materials: Wine lees, mulberry leaves and compound microbial agents; The composite bacterial agent comprises saccharomyces cerevisiae, bacillus subtilis and lactobacillus plantarum.

2. The mulberry leaf-vinasse fermentation composition according to claim 1, characterized in that: The dry mass ratio of the vinasse and mulberry leaves is 1:0.5-2; the dosage of the composite bacterial agent is 5-15wt% of the total mass of the vinasse and mulberry leaves.

3. The mulberry leaf-vinasse fermentation composition according to claim 1, wherein: The mulberry leaf-vintner's lees fermentation composition is obtained by anaerobic solid-state fermentation of the raw materials. The specific steps of the anaerobic solid-state fermentation are: crushing the vintner's lees and mulberry leaves into 20-50 meshes and then mixing them evenly, adjusting the water content of the raw materials to 50-70wt%, then inoculating a composite bacterial agent and stirring evenly, putting them into a breathing bag, sealing them and fermenting them at 37°C±0.5°C for 4-6 days to obtain the mulberry leaf-vintner's lees fermentation composition.

4. The mulberry leaf-vinasse fermentation composition according to claim 1, wherein: In the composite bacterial agent, saccharomyces cerevisiae, bacillus subtilis and lactobacillus plantarum are composed in a mass ratio of 1-3:1-3:1-3.

5. The mulberry leaf-vinasse fermentation composition according to claim 1, wherein: The composite bacterial agent also includes Lactobacillus brevis.

6. The mulberry leaf-vinasse fermentation composition according to claim 5, characterized in that: In the composite bacterial agent, saccharomyces cerevisiae, bacillus subtilis, lactobacillus plantarum and lactobacillus brevis are composed in a mass ratio of 1-3:1-3:1-3:1-3:1-3.

7. The mulberry leaf-vinasse fermentation composition according to claim 1, wherein: The composite bacterial agent comprises saccharomyces cerevisiae, bacillus subtilis and lactobacillus brevis.

8. The mulberry leaf-vinasse fermentation composition according to claim 7, characterized in that: In the composite bacterial agent, saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus brevis are composed in a mass ratio of 1-3:1-3:1-3.

9. The mulberry leaf-vinasse fermentation composition according to claim 1, wherein: The raw materials of the mulberry leaf-vinasse fermentation composition further include: additives; The amount of the additive is 5 to 15 wt% of the total mass of the lees and mulberry leaves; The preparation method of the additive comprises the following steps: S1. Grind cinnamon bark through a 40-60 mesh sieve, add 5-10 times the mass of 75wt% ethanol, soak for 20-50 min, then place in an ultrasonic instrument for ultrasonic extraction for 20-60 min, separate the extract and solid matter, add the solid matter again into 5-10 times the mass of 75wt% ethanol, ultrasonically extract for 20-60 min, and filter and collect the extract; combine the two extracts, concentrate to 1 / 5 of the original volume by vacuum rotary evaporation at 45°C, and freeze-dry to obtain a cinnamon bark extract; wherein the ultrasonic extraction temperature is 35-50°C, the ultrasonic power is 200-500W, and the ultrasonic frequency is 25-40kHz; S2. Add Bletilla striata polysaccharide and isomaltulose into distilled water, ultrasonicate for 30 to 90 minutes, then add cinnamon extract, continue ultrasonicate for 30 to 90 minutes, and freeze-dry to obtain an additive; wherein the mass ratio of Bletilla striata polysaccharide, cinnamon extract, and isomaltulose is 1:0.5 to 1:0.05 to 0.2, and the mass volume ratio of Bletilla striata polysaccharide and distilled water is 1 g:3 to 8 mL; the power of the ultrasound is 200 to 500 W, and the frequency is 40 to 70 kHz.

10. Use of the mulberry leaf-vinasse fermentation composition according to any one of claims 1 to 9 in preparing animal feed.

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