A compound biological preparation for broussonetia papyrifera silage fermentation and application thereof

Abstract

The application provides a composite biological preparation for fermentation of broussonetia papyrifera silage and application thereof, and belongs to the technical field of animal feed. The composite biological preparation for fermentation of broussonetia papyrifera silage comprises lactic acid bacteria and tannase; the ratio of the viable cell count of the lactic acid bacteria to the enzyme activity of the tannase is (1-10) x 10 5 CFU: 0.1 U; and the ratio of the viable cell count of lactobacillus plantarum to that of paracaseolyticum in the lactic acid bacteria is (1-3):(0.8-1.2). The composite biological preparation is applied to preparation of broussonetia papyrifera silage, can effectively improve the nutritional quality, flavonoids, total phenol and lactic acid content of the feed, simultaneously reduces the tannin, acetic acid and ammonia nitrogen content, and reduces the pH value, thereby providing a new means for improving the meat quality of livestock, and having important significance for utilization and popularization of broussonetia papyrifera.

Description

Technical Field

[0001] This invention belongs to the field of animal feed technology, specifically relating to a compound biological agent for fermenting paper mulberry silage and its application. Background Technology

[0002] Paper mulberry (Broussonetia papyrifera) is a deciduous tree belonging to the genus Broussonetia in the family Moraceae. It grows rapidly and is highly adaptable, making it widely used as a feed ingredient for livestock and poultry in southern my country. In the hot and humid climate of southern my country, high-protein forage grasses such as alfalfa are difficult to cultivate and utilize. Paper mulberry trees with a height of 0.8-1.2m have a total harvest protein content of >18%, and the protein content of the leaves is as high as 20-30%. The rumen degradation rate of paper mulberry protein reaches over 90%, making it a suitable protein feed substitute for alfalfa in the diets of cattle, sheep, and other rumen-producing animals, thus addressing the protein feed shortage in southern my country. Furthermore, paper mulberry is rich in amino acids, vitamins, carbohydrates, minerals, and other nutrients, as well as flavonoids and lignans, which have antioxidant and antibacterial effects, significantly improving pork quality. Paper mulberry has low environmental requirements, simple cultivation and management techniques, and improves the ecological environment. Developing paper mulberry feed can compensate for the shortage of grain feed, resolve the conflict between humans and livestock for food, and offer significant economic and ecological benefits.

[0003] Freshly harvested mulberry leaves are characterized by high water content, low soluble carbohydrate content, high protein content, and strong buffering capacity. Direct ensiling and fermentation results in a high final pH value, leading to the proliferation of Clostridium difficile and the production of harmful substances such as ammonia and butyric acid, thus reducing the quality of the mulberry silage. Furthermore, the tannins in mulberry silage are anti-nutritional factors that inhibit protein degradation in the rumen, reduce protein utilization, and negatively impact livestock production performance. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a compound biological agent for fermenting paper mulberry silage, which can not only effectively reduce the tannin content and the proportion of ammonia nitrogen, but also increase the content of nutrients and bioactive components such as starch, total phenols, and flavonoids in paper mulberry silage, and also increase the lactic acid content, thereby reducing the pH value of the feed.

[0005] This invention provides a compound biological agent for fermenting paper mulberry silage, comprising lactic acid bacteria and tannins; The ratio of viable lactic acid bacteria count to tannin enzyme activity is (1~10)×10⁻¹⁰. 5 CFU: 0.1U; The lactic acid bacteria include Lactobacillus plantarum and Lactobacillus paracasei; The viable count ratio of *Lactobacillus plantarum* to *Lactobacillus paracasei* is (1~3):(0.8~1.2).

[0006] Preferably, the ratio of viable lactic acid bacteria count to tannin enzyme activity is 5 × 10⁻⁶. 5 CFU: 0.1U.

[0007] Preferably, the ratio of viable bacteria of *Lactobacillus plantarum* to *Lactobacillus paracasei* is 2:1.

[0008] This invention provides the application of the compound biological agent in the preparation of paper mulberry silage.

[0009] Preferably, the application includes at least one of the following: Improve the nutritional quality of paper mulberry silage; Reduce the tannin content in paper mulberry silage; Increase the flavonoid and total phenol content in paper mulberry silage; Increase the lactic acid content in paper mulberry silage; This helps reduce the pH, acetic acid, and ammonia nitrogen content in paper mulberry silage.

[0010] Preferably, the raw materials for preparing paper mulberry silage include one or more of paper mulberry leaves, paper mulberry roots, paper mulberry bark, and paper mulberry branches.

[0011] Preferably, the dry matter content of the whole paper mulberry tree is 27% to 34%.

[0012] Preferably, in the preparation of the paper mulberry silage, the amount of lactic acid bacteria added to the compound biological agent is (1~10)×10. 5 CFU / g raw material; The amount of tanninase added is 80~120U / kg of raw material.

[0013] Preferably, the fermentation temperature of the paper mulberry silage is 15~35℃ during preparation.

[0014] Preferably, the fermentation time of the paper mulberry silage is 1 year.

[0015] This invention provides a compound biological agent for fermenting paper mulberry silage, comprising lactic acid bacteria and tannin enzymes; the ratio of viable count of the lactic acid bacteria to enzyme activity of the tannin enzymes is (1~10)×10⁻¹⁰. 5CFU: 0.1U; the viable count ratio of *Lactobacillus plantarum* and *Lactobacillus paracasei* is (1~3):(0.8~1.2). Experiments have shown that using the aforementioned compound biological agent to prepare mulberry silage can effectively reduce the tannin content, an anti-nutritional factor in mulberry, by more than 20%. The compound biological agent can also significantly increase the content of flavonoids and total phenols in mulberry silage, thereby improving the antioxidant activity and antibacterial effect of the feed, and improving the quality of the fed animals. The compound biological agent can also significantly improve the nutritional quality of mulberry silage; for example, it significantly increases starch content compared to the blank control group, while the crude protein reduction rate is not significant, and significantly reduces aNDF content compared to other additives such as corn cob, rice husk powder, and molasses. The compound biological agent can also significantly improve the fermentation quality of mulberry silage, significantly reduce the pH value of the feed, reduce the acetic acid content in the silage, and reduce the ammonia nitrogen content in the mulberry silage. Although individual tannin enzymes also effectively reduce tannin content in fermented products, their effects on active ingredient production, nutritional quality, and fermentation quality are inferior to the aforementioned compound biological agent. The dosage and cost of using tannin enzymes during fermentation are significantly lower than those of the compound cellulase group; therefore, the compound biological agent can improve nutritional quality while reducing costs. It is evident that the compound biological agent provides an ideal fermentation preparation for producing silage with low levels of harmful substances, high nutritional value, and excellent fermentation quality. Attached Figure Description

[0016] Figure 1 The pH values ​​of the paper mulberry silage in groups 1 to 12 were measured. Figure 2 The results of acetic acid determination in mulberry silage from groups 1 to 12; Figure 3 The results of lactic acid determination in mulberry silage from groups 1 to 12; Figure 4 The results of the determination of the ammonia nitrogen / total nitrogen ratio in the silage of Broussonetia papyrifera in groups 1 to 12; Figure 5 The results show the starch content determination of Broussonetia papyrifera silage in groups 1 to 12; Figure 6 The results show the crude protein content of mulberry silage in groups 1 to 12. Figure 7 The results of the determination of aNDF content in mulberry silage in groups 1 to 12; Figure 8 The results of flavonoid content determination in mulberry silage from groups 1 to 12; Figure 9 The results of the determination of total phenol content in mulberry silage from groups 1 to 12; Figure 10The results show the determination of tannin content in mulberry silage from groups 1 to 12. Detailed Implementation

[0017] This invention provides a compound biological agent for fermenting paper mulberry silage, comprising lactic acid bacteria and tannin enzymes; the ratio of viable count of the lactic acid bacteria to enzyme activity of the tannin enzymes is (1~10)×10⁻¹⁰. 5 CFU: 0.1U; The live count ratio of Lactobacillus plantarum and Lactobacillus paracasei is (1~3):(0.8~1.2).

[0018] In this invention, the ratio of viable count of lactic acid bacteria to enzyme activity of tannins is preferably 5 × 10⁻⁶. 5 CFU: 0.1U. The preferred viable count ratio of *Lactobacillus plantarum* to *Lactobacillus paracasei* is 2:1. This invention does not impose any particular limitation on the strains of *Lactobacillus plantarum* and *Lactobacillus paracasei*; any strains of *Lactobacillus plantarum* and *Lactobacillus paracasei* well-known in the art can be used. In this embodiment, the *Lactobacillus plantarum* and *Lactobacillus paracasei* were purchased from the China Center for Type Culture Collection. The tanninase was purchased from Zhong Sheng Biotechnology Co., Ltd., with a purity of 97%.

[0019] In this invention, the compound biological agent effectively improves the fermentation quality of mulberry silage. This improvement includes reducing ammonia nitrogen content, lowering the pH value of the silage, increasing lactic acid (LA), and decreasing acetic acid (AA) content. Lowering ammonia nitrogen content helps reduce the content of harmful substances in the feed, while simultaneously reducing protein degradation and retaining a higher protein content. Lowering the pH value also helps reduce the growth of unwanted microorganisms and improve feed quality. Increasing lactic acid content helps lower the pH value of the feed.

[0020] In this invention, the compound biological agent can effectively improve the nutritional quality of mulberry silage. This improvement preferably includes increasing dry matter content and starch content. The tanninase group and the compound biological agent group showed comparable effectiveness in increasing starch content, second only to the *Lactobacillus plantarum* + compound cellulase group. However, because the amount of tanninase used and the cost were significantly lower than the compound cellulase group, group 9 achieved the effect of improving nutritional quality while reducing costs, second only to the *Lactobacillus plantarum* and cellulase combination. Furthermore, this invention analyzed the effects of compound biological agent treatment on the crude protein and structural carbohydrate content in mulberry silage. Structural carbohydrates included neutral detergent fiber (aNDF, treated with amylase) and acid detergent fiber (ADF). The results showed that crude protein decreased to some extent in all treatment groups, but the reduction in crude protein in the compound biological agent group did not exceed 10%. The increase in acid detergent fiber in the compound biological agent group was not significant, but the decrease in neutral detergent fiber was significant.

[0021] In this invention, the compound biological agent can affect the number of microorganisms in mulberry silage. The microorganisms preferably include lactic acid bacteria, yeast, and mold. Because the compound biological agent can significantly reduce the pH value in the feed, the growth of lactic acid bacteria is inhibited. Simultaneously, the compound biological agent results in no significant difference in the number of yeasts in the feed compared to the blank control group. The compound biological agent also ensures that no mold is detected in the feed, meeting feed safety requirements.

[0022] In this invention, the compound biological agent can effectively increase the content of active ingredients and anti-nutritional factors. The active ingredients preferably include flavonoids and total phenols. The anti-nutritional factors include tannins. The compound biological agent can effectively increase the flavonoid content, exceeding that of the individual lactic acid bacteria compound group (LAB) and tannin enzyme group. The compound biological agent can effectively reduce the tannin content, with better results than the individual lactic acid bacteria compound group (LAB) and comparable to the individual tannin enzyme group. Regarding the total phenol content, the compound biological agent can significantly increase the total phenol content, showing a significant difference compared to the control group. Although the increase is not as great as that of the individual lactic acid bacteria compound group (LAB), it is better than that of the individual tannin enzyme group.

[0023] In summary, the compound biological agent protected by this invention exhibits excellent performance in reducing tannin content, ammonia nitrogen content, and pH value, as well as increasing flavonoid content, total phenols, lactic acid, and starch content. Therefore, using the compound biological agent as a fermentation agent to prepare mulberry silage can effectively improve the quality of the feed, thereby improving the quality of the animals being fed.

[0024] This invention provides the application of the compound biological agent in the preparation of paper mulberry silage.

[0025] In this invention, the compound biological agent preferably has at least one of the following applications: Improve the nutritional quality of paper mulberry silage; Reduce the tannin content in paper mulberry silage; Increase the flavonoid and total phenol content in paper mulberry silage; Increase the lactic acid content in paper mulberry silage; This helps reduce the pH, acetic acid, and ammonia nitrogen content in paper mulberry silage.

[0026] In this invention, the raw materials for preparing paper mulberry silage preferably include one or more of the following: paper mulberry leaves, paper mulberry roots, paper mulberry bark, and paper mulberry branches. In this embodiment, the whole paper mulberry plant is used as the raw material to prepare paper mulberry silage. The dry matter content of the whole paper mulberry plant is preferably 27% to 34%, more preferably 28% to 32%, and most preferably 30%.

[0027] In this invention, the preferred amount of lactic acid bacteria added during the preparation of the paper mulberry silage is (1~10)×10⁻⁶. 5 CFU / g of raw material, preferably 3~8×10 5 The optimal CFU / g raw material ratio is 5×10⁻⁶. 5 CFU / g raw material. The preferred amount of tanninase added is 80~120U / kg raw material, more preferably 90~110U / kg raw material, and most preferably 100U / kg raw material. The preferred mass percentage of the added tanninase is 0.2%.

[0028] In this invention, the fermentation temperature of the paper mulberry silage is preferably 15-35℃, more preferably 20-30℃, and most preferably 25℃. The fermentation time of the paper mulberry silage is preferably one year.

[0029] The following detailed description, in conjunction with embodiments, illustrates a compound biological agent for fermenting mulberry silage provided by the present invention and its application, but these should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 Preparation of a compound biological agent (LAB+DE) for fermenting paper mulberry silage and a method for making paper mulberry silage. 1. Activation of Lactobacillus plantarum and Lactobacillus paracasei The viable counts of *Lactobacillus plantarum* and *Lactobacillus paracasei* lyophilized powders were 2.67 × 10⁻⁶. 10 CFU / g and 1.33×10 10 CFU / g, respectively, according to the inoculation amount of 3.33×10 8 CFU / ml and 1.67×10 8 The CFU / ml ratio was inoculated into 1 ml of sterile skim milk powder solution and activated at room temperature for 2 h to obtain Lactobacillus plantarum and Lactobacillus paracasei bacterial suspensions.

[0031] The viable cell counts of *Lactobacillus plantarum* and *Lactobacillus paracasei* cultures were determined separately, and the viable cell concentration of *Lactobacillus plantarum* culture was found to be 3.33 × 10⁻⁶. 8 The CFU / ml concentration of *Lactobacillus paracasei* in the culture was 1.67 × 10⁻⁶. 8 CFU / ml. Then, according to the live count ratio of *Lactobacillus plantarum* and *Lactobacillus paracasei* at 2:1, take 1 ml of *Lactobacillus plantarum* bacterial suspension and 1 ml of *Lactobacillus paracasei* bacterial suspension respectively, mix them, and add distilled water to 20 ml to obtain a total concentration of 5 × 10⁻⁶ CFU / ml. 8 The CFU mixed bacterial solution corresponds to 1 kg of silage raw material.

[0032] 2. Processing of paper mulberry silage raw materials The paper mulberry silage raw material is properly processed to ensure that its dry matter content is between 27% and 34%. The harvested paper mulberry silage raw material is evenly cut into small pieces of 2-3 cm with a chaff cutter and thoroughly mixed. It is then divided into three portions of 1 kg each using the quartering method.

[0033] 3. Fermentation Based on a total vaccination volume of 5×10 5 The mixed bacterial solution was sprayed onto the mulberry silage material at a concentration of CFU / g, and tanninase was added at a concentration of 0.2%. After thorough homogenization, each 1 kg portion was divided into three equal parts, placed into silage bags, vacuum-sealed, and stored as three replicates for each treatment. The storage time was one year at room temperature. The storage temperature was 15~35℃.

[0034] Comparative Example 1 Preparation of a microbial preparation (LAB) for fermenting paper mulberry silage and a method for making paper mulberry silage. Based on Example 1, the addition of tanninase was omitted before ensiling, while other operations remained the same.

[0035] Comparative Example 2 Preparation of a compound enzyme preparation (E) for fermenting paper mulberry silage and a method for making paper mulberry silage. Paper mulberry silage was prepared according to the method in Example 1. Cellulase, xylanase, glucanase, and pectinase were accurately weighed at addition rates of 10 U / g, 120 U / g, 40 U / g, and 20 U / g of the raw material, respectively, and then dissolved thoroughly in 18 ml of distilled water. The enzyme activities of cellulase, xylanase, glucanase, and pectinase were 230,000 U / g, 300,000 U / g, 1,000,000 U / g, and 30,000 U / g, respectively. All four enzymes were in pure enzyme form and purchased from Guangdong Yiduoli Biotechnology Co., Ltd.

[0036] Before silage fermentation, the enzyme solution is sprayed onto the raw materials and thoroughly mixed. Then, mulberry silage is prepared according to the fermentation method of Example 1.

[0037] Comparative Example 3 A method for producing mulberry silage using tanninase (DE) Paper mulberry silage was prepared according to the method in Example 1. Tanninase was sprayed onto the paper mulberry silage raw material at an addition rate of 0.2%, and after thorough and uniform mixing, paper mulberry silage was prepared according to the fermentation method in Example 1.

[0038] Comparative Example 4 A method for producing paper mulberry silage using rice husk powder (DKF). Paper mulberry silage was prepared according to the method in Example 1. Rice husk powder was added to the paper mulberry silage at a dosage of 5%, and the mixture was thoroughly stirred. Then, paper mulberry silage was prepared according to the fermentation method in Example 1.

[0039] Comparative Example 5 A method for producing paper mulberry silage using corn cobs (YMX) Paper mulberry silage was prepared according to the method in Example 1. Corn cobs were added to the paper mulberry silage at a dosage of 5%, and the mixture was thoroughly stirred. Then, the paper mulberry silage was prepared using the fermentation method described in Example 1.

[0040] Comparative Example 6 A method for producing paper mulberry silage using molasses (M) Paper mulberry silage was prepared according to the method in Example 1. Molasses was added to the paper mulberry silage at a dosage of 1% and stirred thoroughly. Then, paper mulberry silage was prepared according to the fermentation method in Example 1.

[0041] Comparative Example 7 A method for producing mulberry silage using compound microbial agents and compound enzyme preparations (LAB+E) Paper mulberry silage was prepared according to the method of Example 1. A compound microbial culture was inoculated into the paper mulberry silage according to the method of Comparative Example 1, and a compound enzyme preparation was added according to the method of Comparative Example 2. Then, paper mulberry silage was prepared according to the fermentation method of Example 1.

[0042] Comparative Example 8 A method for producing mulberry silage using compound microbial agents and tannin enzymes (LAB+DE) Paper mulberry silage was prepared according to the method of Example 1. A compound microbial culture was inoculated into the paper mulberry silage according to the method of Comparative Example 1, and tanninase was added according to the method of Comparative Example 3. Then, paper mulberry silage was prepared according to the fermentation method of Example 1.

[0043] Comparative Example 9 A method for producing mulberry silage using compound microbial inoculants and rice husk powder (LAB+DKF) Paper mulberry silage was prepared according to the method of Example 1. Compound microbial bacteria were inoculated into the paper mulberry silage according to the method of Comparative Example 1, and rice husk powder was added according to the method of Comparative Example 4. Then, paper mulberry silage was prepared according to the fermentation method of Example 1.

[0044] Comparative Example 10 A method for producing mulberry silage using compound microbial agents and corn cobs (LAB+YMX) Paper mulberry silage was prepared according to the method of Example 1. Compound microbial bacteria were inoculated into the paper mulberry silage according to the method of Comparative Example 1, and corn cobs were added according to the method of Comparative Example 5. Then, paper mulberry silage was prepared according to the fermentation method of Example 1.

[0045] Comparative Example 11 A method for producing paper mulberry silage using compound microbial agents and molasses (LAB+M) Paper mulberry silage was prepared according to the method of Example 1. A compound microbial culture was inoculated into the paper mulberry silage according to the method of Comparative Example 1, and molasses was added according to the method of Comparative Example 6. Then, paper mulberry silage was prepared by fermentation according to the method of Example 1.

[0046] Comparative Example 12 A method for producing mulberry silage based on natural fermentation Paper mulberry silage was prepared according to the method in Example 1. 2 ml of sterilized skim milk powder and 18 ml of distilled water were added to the paper mulberry silage, and the mixture was thoroughly mixed. Then, the paper mulberry silage was prepared according to the fermentation method in Example 1.

[0047] Example 2 The paper mulberry silage prepared in Example 1 and Comparative Examples 1-12 were numbered and grouped, and comprehensively evaluated in terms of nutritional quality, fermentation quality, microbial quantity and bioactive components, and anti-nutritional factor content. 1. Detection of fermentation quality of paper mulberry silage After opening, each batch of paper mulberry silage was mixed evenly, and 20g of silage was taken using the five-point sampling method, 180ml of distilled water was added, and the mixture was shaken evenly by hand and then placed in a 4℃ refrigerator for 24h for extraction. The extract was then filtered through four layers of gauze and qualitative filter paper to obtain the extract.

[0048] The pH value of silage was measured using a Mettler pH meter.

[0049] The extract was filtered through a 0.22 μm disposable aqueous filter, and the filtrate was analyzed by high-performance liquid chromatography (HPLC) to determine lactic acid (LA) and acetic acid (AA), respectively. The chromatographic conditions were as follows: HPLC system: Agilent 1260 (Agilent Technologies); detector: variable wavelength detector (VWD); column: Shodex RSpak KC-G (6.0 mm × 50 mm) + Shodex RSpak KC-811S-DVB gel C (8.0 mm × 30 cm, Shimadzu Corporation); injection volume: 5 µL; mobile phase: 3 mM HClO4; flow rate: 1 mL / min. -1 Column temperature: 60℃; Detection wavelength: 210nm; Running time: 20min.

[0050] The ratio of ammonia nitrogen to total nitrogen was determined using the phenol-sodium hypochlorite method.

[0051] The results are shown in Table 1 and... Figures 1-4 .

[0052] Table 1. Effects of different biological agents on the fermentation quality of Broussonetia papyrifera silage.

[0053] As shown in Table 1, compared with the blank control group 1, all groups significantly reduced the pH value of the silage. The pH reduction rates in groups 2 to 12 were 6.93%, 9.78%, 8.70%, 7.07%, 8.22%, 8.36%, 10.46%, 10.94%, 7.54%, 8.76%, and 9.38%, respectively. Groups 8 and 9 showed the best results, with pH reduction rates exceeding 10%.

[0054] The lactic acid content of groups 2, 3, 4, 6, 7, 9, 10, 11, and 12 increased by 14.31%, 17.56%, 29.88%, 5.58%, 24.41%, 15.39%, 14.12%, 3.23%, and 29.61%, respectively. The highest increases were observed in groups 4 and 12, followed by groups 7 and 3.

[0055] Compared with the blank control group 1, all groups from 2 to 12 reduced the acetic acid content of silage. Groups 6, 12, 10, 5, 7 and 11 reduced the content by more than 20%, with groups 7 and 11 showing the best results, reducing the acetic acid content by 29.26% and 28.86%, respectively.

[0056] Compared with the blank control group 1, all groups 2 through 12 significantly reduced the ammonia nitrogen / total nitrogen ratio in silage, with reductions of 39.21%, 31.14%, 34.37%, 37.29%, 36.08%, 49.18%, 36.10%, 40.68%, 36.05%, 35.04%, and 40.68%, respectively. Group 7 showed the best effect, followed by groups 12 and 9. Group 9 had the lowest pH value of 4.37, and its ammonia nitrogen / total nitrogen ratio was not significantly different from that of the best-performing group 7.

[0057] 2. Testing the nutritional quality of paper mulberry silage The remaining approximately 250g of sample was blanched at 105℃ for 30 minutes and then dried at 65℃ to constant weight. The dry matter content (DM) was then calculated.

[0058] The samples were pulverized (through a 1mm sieve) and sealed in self-sealing bags for nutritional quality determination. The total nitrogen content was determined using a FORS Kjeldahl nitrogen analyzer, and the crude protein content of the silage was calculated as total nitrogen × 6.25.

[0059] Neutral detergent fiber (aNDF, treated with amylase) and acid detergent fiber (ADF) were tested using Ankom200i automated fiber analyzer from Ankom Corporation.

[0060] The content of soluble sugars was determined by the anthrone colorimetric method, and the content of starch was determined by the perchloric acid hydrolysis anthrone colorimetric method.

[0061] The contents of tannins, flavonoids, and total phenols were detected using kits, namely the Tannin Content Detection Kit (BC1395-100 tubes / 96 samples), the Plant Flavonoid Content Detection Kit (BC1335-100 tubes / 48 samples), and the Plant Total Phenolic (TP) Content Detection Kit (BC1345-100 tubes / 48 samples), all of which were from Solarbio and purchased from Nanjing Xinkenida Biotechnology Co., Ltd.

[0062] The effects of different fermentation agents on the dry matter, soluble sugar, and starch content of paper mulberry silage are shown in Table 2 and 3. Figure 5 and Figure 6 .

[0063] Table 2. Effects of different biological agents on the dry matter and non-structural carbohydrate content of Broussonetia papyrifera silage.

[0064] Compared with the blank control group, the dry matter content of groups 5, 6, 10 and 11 increased due to the addition of air-dried rice husk powder and corn cob. However, except for group 11, which was significantly higher than the blank control group, the other treatment groups were not significantly different from group 1.

[0065] In terms of soluble sugar content, the remaining soluble sugar in the mulberry silage was less than 1%. The remaining soluble sugar content in group 6 was significantly lower than that in the blank control group. The content of compound additives in groups 10, 11, and 12 was significantly lower than that in the blank control group. This is because the use of lactic acid bacteria consumed more soluble sugar. The soluble sugar content in other groups was lower than that in the blank control group, but none of them were significant.

[0066] Compared with the blank control group (group 1), the starch content of groups 2, 3, 4, 5, 7, 8, 9, and 12 increased by 26.05%, 21.94%, 34.87%, 5.33%, 25.24%, 44.39%, 30.13%, and 17.65%, respectively. Group 8 showed the best effect, followed by groups 4 and 9, but there was no significant difference between them and group 8. The dosage and cost of tanninase were significantly lower than those of the compound cellulase group; therefore, group 9's ability to improve nutritional quality while reducing costs was second only to the combination of *Lactobacillus plantarum* and cellulase.

[0067] The effects of different biological agents on crude protein and structural carbohydrates in mulberry silage are shown in Table 3 and 4. Figure 7 .

[0068] Table 3. Effects of different biological agents on the crude protein and structural carbohydrate content of paper mulberry silage.

[0069] Compared with the blank control group (group 1), the crude protein content of all treatment groups decreased to some extent. Among them, the crude protein reduction of groups 2, 12, 8, 9, 4, 3 and 7 did not exceed 10%, and the smallest reduction was in group 2, with a crude protein reduction of 1.83%.

[0070] ADF levels increased in all treatment groups, with groups 8 and 9 showing the lowest ADF levels, except for the control group. Compared to the blank control group 1, aNDF levels in groups 2 and 3 decreased by 2.03% and 0.4%, respectively, while aNDF levels in groups 8 and 9 decreased by 8.54% and 10.04%, respectively.

[0071] In terms of crude protein content, groups 5, 6, 10, and 11 showed the most significant decreases, while aNDF and ADF also increased to some extent, indicating that the addition of corn cob and rice husk powder had a certain negative impact on mulberry silage. Meanwhile, groups 8 and 9, with a crude protein content reduction of no more than 10%, resulted in a decrease in aNDF content of more than 8%, which was significantly better than the use of rice husk powder and corn cob.

[0072] The contents of lactic acid bacteria (MRS medium, incubated at 37℃ for 48 h), yeast, and mold in mulberry silage were determined by plate counting method (Bengal red medium, incubated at 28℃ for 3-7 days). The results are shown in Table 4.

[0073] Table 4. Effects of different biological agents on the number of microorganisms in Broussonetia papyrifera silage.

[0074] After one year of silage fermentation, the anaerobic fermentation process has stabilized. Lactic acid bacteria are significantly inhibited by low pH levels, with groups 8, 9, and 12 showing the lowest lactic acid bacteria counts. Groups 8 and 9, due to their low pH, have significantly fewer lactic acid bacteria than the control group. The mulberry silage contains relatively few yeasts, with group 12 having the lowest count and group 6 the highest. Mold was mostly undetectable, with only minor, repeated detections in groups 1, 6, 10, and 11. This may be due to exogenous mold introduced from corn cobs and rice husk powder that was not completely removed after fermentation.

[0075] The results of the determination of bioactive components and anti-nutritional factors in paper mulberry silage are shown in Table 5 and 2016. Figures 8-10 .

[0076] Table 5. Effects of different biological agents on the content of bioactive components and antinutritional factors in Broussonetia papyrifera silage.

[0077] Compared with the blank control group 1, all groups increased the flavonoid content in the mulberry silage, and the increase was more than 200%. Group 5 had the best effect, and there was no significant difference among the other treatment groups.

[0078] Compared with the blank control group 1, all groups increased the total phenol content. Group 2 showed the best effect, with a 50.25% increase in total phenol content. Groups 12 and 9 showed the next best effects, with increases of 38.02% and 34.47% in total phenol content, respectively.

[0079] Compared with the blank control group 1, all groups reduced tannin content. Groups 8, 9, 4, 7, 11 and 10 reduced tannin content by more than 20%, with group 10 showing the best effect, with a reduction of 28.92%.

[0080] The increase in flavonoid content in groups 5 and 6, and the decrease in tannin content in groups 10 and 11, were mainly due to the increase in flavonoid content and the decrease in tannin content caused by the exogenous addition of rice husk powder and corn cob. However, this also led to a decrease in crude protein content and an increase in fiber content.

[0081] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a compound biological agent for fermenting paper mulberry silage in the preparation of paper mulberry silage, wherein the compound biological agent has the following application: improving the nutritional quality of paper mulberry silage; wherein the nutritional quality of paper mulberry silage is rich in starch; The compound biological agent is composed of lactic acid bacteria and tannin enzymes; The ratio of viable lactic acid bacteria count to tannin enzyme activity is (1~10)×10⁻¹⁰. 5 CFU: 0.1U; The lactic acid bacteria include Lactobacillus plantarum and Lactobacillus paracasei; The viable count ratio of *Lactobacillus plantarum* to *Lactobacillus paracasei* is (1~3):(0.8~1.2). When preparing the paper mulberry silage, the amount of lactic acid bacteria added is (1~10)×10. 5 CFU / g raw material; The amount of tanninase added is 80~120U / kg of raw material; The added tannin enzyme has a mass percentage of 0.2%.

2. The application according to claim 1, characterized in that, The ratio of viable lactic acid bacteria count to tannin enzyme activity is 5 × 10⁻⁶. 5 CFU: 0.1U.

3. The application according to claim 1, characterized in that, The ratio of viable Lactobacillus plantarum to Lactobacillus paracasei is 2:

1.

4. The application according to claim 1, characterized in that, The raw materials for preparing paper mulberry silage include one or more of the following: paper mulberry leaves, paper mulberry roots, paper mulberry bark, and paper mulberry branches.

5. The application according to claim 4, characterized in that, The dry matter content of the whole paper mulberry tree is 27% to 34%.

6. The application according to claim 1, characterized in that, When preparing the paper mulberry silage, the fermentation temperature of the paper mulberry silage is 15~35℃.

7. The application according to any one of claims 3 to 6, characterized in that, The fermentation time for the paper mulberry silage is 1 year.

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