Alfalfa broussonetia papyrifera silage and preparation method thereof

Abstract

The invention discloses alfalfa broussonetia papyrifera silage and a preparation method thereof. The alfalfa broussonetia papyrifera silage consists of 50-70% by mass of broussonetia papyrifera and 30-50% by mass of alfalfa. According to the alfalfa broussonetia papyrifera silage, broussonetia papyrifera and alfalfa are mixed for silage, the content of acid detergent fibers and neutral detergent fibers in broussonetia papyrifera silage and the content of lactic acid and acetic acid in the broussonetia papyrifera silage can be reduced, the abundance of beneficial bacteria in the broussonetia papyrifera silage is improved, the abundance of harmful bacteria in the broussonetia papyrifera silage is reduced, and therefore the quality of the broussonetia papyrifera silage is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to a silage, in particular to a silage of alfalfa and broussonetia papyrifera and a preparation method thereof. BACKGROUND

[0002] Alfalfa is a common forage used to make silage. Compared with hay, modulating alfalfa silage can effectively avoid the deterioration of forage quality caused by external factors such as rain, and can prevent the loss of protein caused by leaf shedding. At the same time, alfalfa can also improve the dry matter digestibility and palatability of forage after fermentation, and achieve the purpose of long-term preservation.

[0003] However, during the silage process, the protein of alfalfa is severely lost due to the decomposition of non-protein nitrogen (NPN) caused by the action of its own protease (Characterization of proteolysis in alfalfa and red clover. Crop science, 1995, 35(2): 537~541). According to research reports, the NPN in alfalfa after ensiling can reach 44%~87% of the total nitrogen in alfalfa (Dry matter level effects on alfalfa silage quality I. Nitrogen transformations. Transactions of the ASABE, 1987, 30(1): 7~14; A comparison of protein degradation during wilting and ensiling of six forage species. Canadian Journal of Plant Science, 1983, 63(4):903-912). NPN cannot be effectively utilized by livestock, so these lost proteins during the ensiling process can only be supplemented by increasing protein feed in the diet or increasing rapidly fermentable carbohydrates in the diet to maximize the use of NPN. However, this will inevitably increase the cost of the diet, and alfalfa will not be able to realize the nutritional characteristics of high-protein forage.

[0004] Broussonetia papyrifera, also known as paper mulberry, is a dicotyledonous plant of the Moraceae family. Broussonetia papyrifera contains high levels of crude protein (CP), crude fat, and phosphorus content, and has a high fresh grass yield, which has the potential to alleviate the shortage of protein feed in China. However, as a woody feed, broussonetia papyrifera has high cellulose and lignin content, which not only reduces the palatability of broussonetia papyrifera, but also makes it difficult for animals to absorb and utilize the nutritional components of broussonetia papyrifera, becoming an obstacle to broussonetia papyrifera as a high-quality protein feed.

[0005] It is to be noted that the information disclosed in the above BACKGROUND section is only for the purpose of facilitating the understanding of the background of the present disclosure and thus can include information that does not constitute the prior art known to those of ordinary skill in the art. SUMMARY

[0006] The present application aims to provide an alfalfa and broussonetia papyrifera silage and a preparation method thereof, which solves the problem that the existing broussonetia papyrifera silage has high cellulose and lignin, which is difficult to be absorbed and utilized by animals, can reduce the content of acid detergent fiber and neutral detergent fiber in the broussonetia papyrifera silage, and the content of lactic acid and acetic acid in the broussonetia papyrifera silage, and improve the abundance of beneficial bacteria and reduce the abundance of harmful bacteria in the broussonetia papyrifera silage, thereby improving the quality of the broussonetia papyrifera silage.

[0007] In order to achieve the above-mentioned purpose, the present application provides an alfalfa and broussonetia papyrifera silage, which is composed of 50-70% by mass of broussonetia papyrifera and 30-50% by mass of alfalfa.

[0008] Preferably, the silage is composed of 50% by mass of broussonetia papyrifera and 50% by mass of alfalfa.

[0009] Preferably, the silage is composed of 50-70% by mass of broussonetia papyrifera and 30-50% by mass of alfalfa and is ensiled for more than 14 days.

[0010] More preferably, the silage is composed of 50-70% by mass of broussonetia papyrifera and 30-50% by mass of alfalfa and is ensiled for more than 90 days.

[0011] The second object of the present application is to provide a preparation method of the alfalfa and broussonetia papyrifera silage, which comprises mixing 50-70% by mass of broussonetia papyrifera and 30-50% by mass of alfalfa, filling into a vacuum bag, sealing after vacuumizing, and storing at room temperature in the dark.

[0012] The third object of the present application is to provide a method for improving the quality of broussonetia papyrifera silage, which comprises mixing 50-70% by mass of broussonetia papyrifera and 30-50% by mass of alfalfa and ensiling for more than 14 days, and the improvement of the quality of the broussonetia papyrifera silage comprises any one or more than two of the following: (1) increasing the content of lactic acid or / and acetic acid in the broussonetia papyrifera silage; (2) reducing the content of acid detergent fiber or / and neutral detergent fiber in the broussonetia papyrifera silage; (3) increasing the abundance of beneficial bacteria or / and reducing the abundance of harmful bacteria in the broussonetia papyrifera silage.

[0013] Preferably, the ensiling time is more than 90 days.

[0014] Preferably, the abundance of beneficial bacteria in the bermuda silage is increased or / and the abundance of harmful bacteria is reduced, and the bermuda silage and alfalfa are mixed in a mass fraction of 50% and a mass fraction of 50%, and the silage is stored for more than 14 days.

[0015] Preferably, the beneficial bacteria comprise lactobacillus pentosus; or / and, the harmful bacteria comprise enterobacter ludwigii.

[0016] The bermuda silage and the preparation method thereof have the following advantages: The bermuda silage of the present application can reduce the content of acid detergent fiber and neutral detergent fiber in the bermuda silage, and the content of lactic acid and acetic acid in the bermuda silage, and increase the abundance of beneficial bacteria and reduce the abundance of harmful bacteria in the bermuda silage, thereby improving the quality of the bermuda silage. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The figure is the relative abundance of genus and species level microorganisms in different proportions of bermuda silage and bermuda mixed silage at different stages in experimental example 3 of the present application; (a) composition of microbial community at genus level; (b) composition of microbial community at species level.

[0018] Figure 2 The figure is the relative abundance of genus and species level microorganisms in different proportions of bermuda silage and bermuda mixed silage at different stages in experimental example 3 of the present application; (a) composition of microbial community at genus level; (b) composition of microbial community at species level. Figure 1 ; (a) fresh sample; (b) silage for 7 days; (c) silage for 14 days.

[0019] Figure 3 The figure is the relative abundance of genus and species level microorganisms in different proportions of bermuda silage and bermuda mixed silage at different stages in experimental example 3 of the present application; (a) composition of microbial community at genus level; (b) composition of microbial community at species level. Figure 2 ; (d) silage for 45 days; (e) silage for 90 days.

[0020] Figure 4 The figure is the statistical chart of topological parameters of bacterial co-occurrence network between different proportions of bermuda silage and bermuda mixed silage in experimental example 3 of the present application; (a) node number; (b) edge number; (c) betweenness; (d) homomorphism; (e) negative correlation / positive correlation.

[0021] Note: the fresh sample group is P0, P30, P50, P70, P100, and the symbol of each silage group represents each group (P0, P30, P50, P70, P100) + silage days (7, 14, 45, 90). DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0023] It should be noted that, in the embodiments, the specific conditions not specified are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased on the market.

[0024] In the present application, all the features defined in the form of a numerical range or a percentage range, such as numerical values, quantities, contents and concentrations, are only for the sake of brevity and convenience. Therefore, the description of the numerical range or the percentage range should be considered to have covered and specifically disclosed all possible sub-ranges and individual numerical values (including integers and fractions) within the range.

[0025] The features mentioned in the present application can be combined arbitrarily, as long as the combination of the features does not exist contradiction. All possible combinations should be considered as the scope disclosed in the specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless specifically stated, the disclosed features are only general examples of equivalent or similar features.

[0026] Experimental Example 1: Preparation of alfalfa and broussonetia papyrifera silage The broussonetia papyrifera with a height of about 100 cm was cut with a stubble of about 20 cm, and the alfalfa was harvested at the initial flowering stage. The alfalfa was dried to a moisture content of about 65%, and then was pressed, cut and shortened to 2-3 cm. The cut broussonetia papyrifera and alfalfa were mixed in different proportions (0:100, 30:70, 50:50, 70:30 and 100:0) to be marked as P0, P30, P50, P70 and P100 respectively. After being mixed uniformly, they were filled into polyethylene vacuum bags (500 g per bag), sealed after vacuumizing, and stored at room temperature in the dark. Samples were taken at 0, 7, 14, 45 and 90 d after silage, with 3 repeats at each time point.

[0027] Experimental Example 2: Analysis of fermentation characteristics and chemical components The fermentation characteristics and chemical components of the silages P0, P30, P50, P70 and P100 prepared in Experimental Example 1 were analyzed, as follows. 1. Determination of dry matter The dry matter (DM) was determined by oven drying at 65 ℃ until constant weight.

[0028] 2. Analysis of chemical components A 20 g subsample was mixed with 180 mL distilled water, homogenized for 50 s and filtered through 4 layers of medical gauze. The pH of the filtrate was immediately recorded with a pH meter. Subsequently, the filtrate was divided (10 mL) and acidified to pH 2, centrifuged at 10000 x g for 20 min at 4°C and filtered with a 0.22 µM dialyzer. Lactic acid (LA), acetic acid (AA) and butyric acid (BA) were determined by high-performance liquid chromatography; water-soluble carbohydrates (WSC) were determined by the anthrone-sulfuric acid method (Reference: The buffering constituents of herbage and of silage. Journal of the Science of Food and Agriculture, 1966, 17(6): 264-268).

[0029] Another 150 g subsample was freeze-dried for 72 h and ground through a 1 mm screen and used to determine neutral detergent fiber (aNDF), acid detergent fiber (ADF) and crude protein (CP). Neutral detergent fiber (aNDF), acid detergent fiber (ADF) and crude protein (CP) were determined according to Zhang Liying (Reference: “Feed analysis and feed quality detection technology”, Beijing: China Agricultural University Press, 2007). Ammoniacal nitrogen (NH3-N) was determined by the phenol-sodium hypochlorite colorimetric method. NPN, FAA-N and Peptide-N were determined according to Li Xujiao (Reference: “Alfalfa silage feed protein degradation mechanism and regulation research”, Beijing: China Agricultural University, 2018).

[0030] 3. Data processing The experiment used a 5 x 5 factorial design, including 5 treatments and 5 ensiling time points. The pH and organic acid data were analyzed using the general linear model procedure of SPSS software: Yij = μ + Ti + Dj + (T x D)ij + εij, where Yij represents the response variable, μ is the overall mean, Ti is the effect of different mixing ratio ensiling, Dj is the effect of ensiling time, (T x D)ij is the effect of the interaction between different mixing ratio ensiling and ensiling time, and εij is the error. One-way ANOVA was used to test the effects of fresh samples and 90-day ensiled chemical components. P < 0.05 indicates significant differences between treatments.

[0031] 4. Experimental results The nutritional components of the mixed fresh samples of different proportions of alfalfa and paper mulberry are shown in Table 1, and the DM contents of each treatment group have no significant difference. The CP content of alfalfa is significantly higher than that of paper mulberry, and the CP content gradually decreases with the increase of the addition proportion of paper mulberry (P<0.001). With the increase of the addition proportion of paper mulberry, the NDF content first decreases and then increases, and the NDF content of the P50 group is significantly lower than that of the other treatment groups (P<0.001); the ADF content has a gradually decreasing trend, and the ADF content of the P70 group is significantly lower than that of the other treatment groups (P<0.001). The WSC content of the P100 group is significantly higher than that of the other treatment groups (P<0.001).

[0032] Table 1 is the chemical composition of the mixed fresh samples of different proportions of alfalfa and paper mulberry

[0033] Item P0 P30 P50 P70 P100 p-value Dry matter DM / (%FM) 37.30±1.00 36.20±0.30 37.33±0.28 37.12±0.66 36.47±0.32 0.565 Crude protein CP / ((%DM) 20.87±0.23a 17.80±0.11b 14.94±0.10c 14.48±0.20c 9.53±0.21d <0.001 Neutral detergent fiber NDF / (%DM) 47.51±0.80a 47.55±0.38a 43.78±0.27c 45.35±0.30b 48.41±0.46a <0.001 Acid detergent fiber ADF / (%DM) 26.13±0.06a 24.53±1.20ab 25.96±0.43a 20.12±0.16c 23.40±0.23b <0.001 Soluble carbohydrates WSC / (%DM) 1.59±0.07d 2.12±0.6b 1.89±0.02c 1.64±0.07d 4.14±0.05a <0.001 Note: The same row and different lowercase letters represent significant difference (P<0.005), the same below.

[0034] With the fermentation, the CP and WSC contents of the silage gradually decrease because the nutrients are consumed by microorganisms. The nutritional components of the mixed silage of different proportions of alfalfa and paper mulberry after 90 days of fermentation are shown in Table 2, and the DM contents of each treatment group have no significant difference after 90 days of silage. With the increase of the addition proportion of paper mulberry, the change trend of the CP content of each treatment group is consistent with that before silage, and the CP content gradually decreases with the increase of the addition amount of paper mulberry (P<0.001). However, the reduction rate of the CP content of each treatment group shows a large difference, the CP content reduction rate of the P0 group is 10.21%, the P30 group is 4.49%, the P50 group is 4.28%, the P70 group is 8.83%, and the P100 group is 8.50%; the NDF contents of each group are reduced by 37.43%, 40.32%, 42.03%, 39.87% and 27.20% respectively compared with those before silage; the ADF contents of each group are reduced by 24.61%, 27.47%, 40.80%, 14.00% and 9.09% respectively compared with those before silage. After 90 days of silage, the WSC contents of each group are all below 1%, and the difference is not significant. After 90 days of silage, with the increase of the addition proportion of paper mulberry, the NH3-N, NPN and FAA-N contents gradually decrease, and the P0 group is the highest, which is significantly higher than the other treatment groups (P<0.001).

[0035] If a large amount of protein is decomposed into NPN during the silage of alfalfa, the amine substances produced will cause the feed intake of livestock to decrease, so the NPN component in the silage indicates that protein hydrolysis occurs during the fermentation process. From the above results, it can be seen that the amount of protein nitrogen converted into NPN gradually decreases with the increase of the addition proportion of paper mulberry, and the decrease of the NPN content of the present application may be related to the tannin in paper mulberry, which can bind plant proteins to protect protein from being converted into peptides and free amino acids.

[0036] NH3-N content is also an important indicator for measuring the degradation of silage protein, the lower the content, the less the protein degradation, the better the quality of silage fermentation. From the above results, the NH3-N content gradually decreases with the increase of proportion of paper mulberry.

[0037] The NDF and ADF contents of each group before ensiling are high, and the NDF and ADF contents of each group after ensiling decrease, which may be due to the increase of microbial abundance of degrading fiber, thereby enhancing the degradation of fiber material, and the NDF and ADF contents of the P50 group are significantly lower than those of the other groups, and the NDF and ADF contents of the P50 group decrease most obviously, by 42.03% and 40.80% respectively, indicating that mixed silage is beneficial to the degradation of fiber in the silage raw material, the P50 group has the highest degradation rate, the digestibility of forage grass is the highest, and the P50 group has the highest forage value.

[0038] Table 2 shows the chemical composition of alfalfa and paper mulberry mixed silage with different proportions after 90 days of ensiling Item P0 P30 P50 P70 P100 p-value Dry matter DM / (%FM) 36.86±0.76 36.51±0.04 36.57±0.33 36.27±0.23 36.65±1.00 0.967 Crude protein CP / ((%DM) 18.80±0.47a 17.00±0.06b 14.30±0.26c 13.20±0.37d 8.72±0.15e <0.001 Neutral detergent fiber NDF / (%DM) 29.71±0.16b 28.38±0.55bc 25.38±0.51d 27.27±0.72c 35.25±0.29a <0.001 Acid detergent fiber ADF / (%DM) 19.70±0.77b 17.79±0.52c 15.37±0.55d 17.30±0.30c 21.27±0.22a <0.001 Soluble carbohydrates WSC / (%DM) 0.30±0.01b 0.36±0.02ab 0.41±0.02a 0.36±0.04ab 0.30±0.04b 0.071 ammoniacal nitrogen NH3-N / (%TN) 15.91±0.25a 12.14±0.53b 10.64±1.74c 9.83±0.26cd 8.99±0.53d <0.001 Non-protein nitrogen NPN(%TN) 68.92±0.38a 65.71±1.58b 64.60±1.01b 58.39±0.87c 33.22±0.64d <0.001 Free amino acid nitrogen FAA-N(%TN) 37.12±0.03a 30.85±0.48b 26.92±1.53c 24.18±0.77d 23.26±0.04d <0.001 Peptide nitrogen Peptide-N(%TN) 15.88±0.26b 22.72±2.54a 27.04±2.04a 24.39±1.25a 0.98±0.31c <0.001 The fermentation characteristics of alfalfa and paper mulberry mixed silage with different proportions during the ensiling process are shown in Table 3. After 90 days of ensiling, the pH of each treatment group is greater than 4.5, and the pH of the alfalfa and paper mulberry mixed silage group is significantly lower than that of the alfalfa and paper mulberry single silage, and the pH of the alfalfa and paper mulberry mixed silage group is the lowest when the proportion of alfalfa and paper mulberry is 50% (P<0.001). The lactic acid and acetic acid contents of each treatment group gradually increase with the extension of ensiling time. After 90 days of ensiling, the lactic acid content of the P70 and P50 groups is significantly higher than that of the other groups, and the lactic acid content of the 100% paper mulberry treatment group is the lowest (P<0.001). After 90 days of ensiling, the acetic acid content of the 100% alfalfa silage group is the highest, and the acetic acid content gradually decreases with the increase of the addition amount of paper mulberry (P<0.001). The butyric acid content shows an opposite trend to the acetic acid content, and the butyric acid content of the 100% paper mulberry treatment group is significantly higher than that of the other groups (P<0.001). The lactic acid to acetic acid ratio increases with the increase of the addition proportion of paper mulberry, and the lactic acid to acetic acid ratio of the P0 and P30 treatment groups is lower than 2, and the lactic acid to acetic acid ratio of the P70 and P100 treatment groups is higher than 3.

[0039] Table 3 shows the fermentation quality of alfalfa and paper mulberry mixed silage at different stages

[0040] Note: T represents inoculation treatment; D represents ensiling time; TxD represents the interaction of inoculation treatment and ensiling time.

[0041] During the ensiling process, lactic acid bacteria convert WSC into organic acids, mainly LA, under anaerobic conditions, thereby lowering the pH to protect the feed from harmful microorganisms. The present invention found that the lactic acid content was the lowest when B. cumida was ensiled alone, and the lactic acid content was significantly higher when B. cumida was mixed with alfalfa. The acetic acid content gradually decreased with the increase of B. cumida addition, and the epiphytic microorganisms on the surface of B. cumida were mainly heterofermentative lactic acid bacteria. This indicates that when B. cumida is ensiled alone, not only is the fermentation quality poor, but the lower acetic acid content also affects the aerobic stability after opening.

[0042] The lactic acid to acetic acid ratio is usually used as a qualitative indicator to assess fermentation quality, and the lactic acid to acetic acid ratio of high-quality silage is about 2.5-3.0. The lactic acid to acetic acid ratio of P50 and P70 groups in the present invention is closer to 2.5-3.0, indicating that the quality of these two groups of silage is better. In addition, if BA is detected in the silage, it indicates that there are clostridia in the silage, which will exacerbate the loss of DM during ensiling. The BA content in P100 group was significantly higher than that in other groups throughout the entire ensiling process, indicating that ensiling B. cumida alone is not conducive to the preservation of nutrient content. From the comprehensive fermentation indicators, the pH of P50 group is lower, the LA content is higher, the lactic acid to acetic acid ratio is about 2.5-3.0, and the fermentation effect is better.

[0043] Experimental Example 3: Analysis of silage microbial community structure The microbial community structure of each silage P0, P30, P50, P70, P100 prepared in Experimental Example 1 was analyzed, as follows: 1. DNA extraction and amplification During the entire fermentation stage, 20 g of silage sample was evenly taken, frozen in liquid nitrogen, and stored at -80°C. The total DNA was extracted using a soil genomic DNA extraction kit (purchased from Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. The DNA concentration, purity, and quality were detected by Nano Drop ND-2000 spectrophotometer and 1% agarose gel electrophoresis. All DNA samples were stored at -20°C for further analysis.

[0044] PCR amplification used primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') to amplify the 16S rRNA gene of bacteria. Bacterial 16S amplification and sequencing data quality filtering, clustering, and analysis were performed. Through analysis of microbial community diversity, microbial community structure, and microbial network topology, the succession law of microorganisms during ensiling was explored.

[0045] 2. Experimental results 1) Microbial community species and relative abundance The composition of the microbial community at the genus level during the fermentation of silage in each treatment group is as follows: Figure 1 As shown in Figure a, before ensiling, the microbial composition of groups P0 and P30 was similar, mainly dominated by *Weissellella*, *Aerococcus*, and *Enterobacter*. The microbial composition of group P50 differed significantly from that of groups P0 and P30, mainly dominated by *Weissellella*, *Lactiplantibacillus*, and *Pediococcus*. Groups P70 and P100 were dominated by undesirable microorganisms; group P70 was dominated by *Escherichia-Shigella*, while group P100 was dominated by *Bacillus*. After ensiling was initiated, the microbial community succession in each treatment group underwent significant changes. The abundance of *Weissellella* significantly increased after 7 days of ensiling, and the *Weissellella*-dominated ensiling fermentation was maintained until 90 days of ensiling. Seven days after ensiling, the P0 group had no Aerococcus species compared to the other groups. The P30 and P50 groups had similar microbial compositions, dominated by Weissellella, Lactiplantibacillus, Pediococcus, and Aerococcus. Compared to the P30 and P50 groups, the P70 and P100 groups had higher abundance of Enterobacter and lower abundance of Lactiplantibacillus and Pediococcus. After 14 days of silage, compared with other groups, the P0 group had a higher abundance of *Lactiplantibacillus* and a lower abundance of *Aerococcus*. The P30 and P50 groups had similar microbial compositions, dominated by *Weissellella*, *Pediococcus*, and *Aerococcus*. The P70 and P100 groups had similar microbial compositions, dominated by *Weissellella*, *Enterobacter*, and *Aerococcus*. After 45 days of silage, the abundance of *Lactobacillus* in the P0 group increased significantly, while the microbial community succession in the other groups showed little change. After 90 days of silage, the abundance of *Lactiplantibacillus* in the P100 group increased.

[0046] The composition of the microbial community at the species level during the fermentation process of the silage of each treatment was as follows Figure 1As shown in Fig. b, before ensiling, the microbial composition of P0 group and P30 group was similar, mainly dominated by Weissellella cibaria and Aerococcus urinaeequi. The microbial composition of P50 group showed great difference compared with P0 and P30 groups, mainly dominated by Weissellella cibaria, Weissellella paramesenteroides, Pediococcus pentosaceus, Lactiplantibacillus pentosus and Weissellella hellenica. The microbial composition of P70 group was mainly dominated by Aerococcus urinaeequi, Weissellella cibaria, Weissellella paramesenteroides and Pediococcus pentosaceus. The microbial composition of P100 group was mainly dominated by streptococcus pneumoniae. After 7 days of ensiling, the microbial composition of P30 group and P50 group was similar, mainly dominated by Weissellella cibaria, Weissellella paramesenteroides, Pediococcus pentosaceus, Aerococcus urinaeequi and Lactiplantibacillus pentosus. Compared with P30 group and P50 group, Aerococcus urinaeequi was not found in P0 group. Compared with P30 group and P50 group, the abundance of Enterobacter ludwigii was higher in P70 group and P100 group, and the abundance of Lactococcus lactis was higher in P100 group. After 14 days of ensiling, the microbial composition of each group showed little difference. Compared with other groups, the abundance of Lactiplantibacillus pentosus was higher in P0 group, and the abundance of Pediococcus pentosaceus was lower in P70 group and P100 group. After 45 days of ensiling, the microbial composition of P30 group, P50 group and P70 group was similar. Compared with P30 group, P50 group and P70 group, Aerococcus urinaeequi was not found in P0 group, and the abundance of levilactobacillus brevis was higher.P100 group compared to the others had lower abundance of Pediococcus pentosaceus. After 90 days of ensiling, P30, P50 and P70 groups had similar microbiome composition, P0 group compared to the others had lower abundance of Aerococcus urinae equi and higher abundance of Weissellella hellenica. P100 group compared to the others had lower abundance of Pediococcus pentosaceus.

[0047] 2) Analysis of differences in bacterial composition between groups By linear discriminant analysis (LefSe) (see Figure 2 and Figure 3 ), before ensiling, Enterobacter was significantly present in P70 group, and Streptococcus pneumoniae was significantly more abundant in P100 group compared to the other treatments. After 7 days of ensiling, Lactobacillus was the most differentially abundant genus in P0 group, Enterobacter cloacae was significantly present in P70 group, and Enterobacter, Enterococcus were the most differentially abundant genera in P100 group. After 14 days of ensiling, Weissellella, Enterobacter and Enterococcus were the most differentially abundant genera in P100 group. After 45 days of ensiling, Enterococcus was the most differentially abundant genus in P70 group, Enterobacter was the most differentially abundant genus in P100 group, and Levilactobacillus and Lactiplantibacillus were the most differentially abundant genera in P0 group. After 90 days of ensiling, Enterobacter was the most differentially abundant genus in P70 group, Pediococcus was the most differentially abundant genus in P50 group, Leuconnostoc was the most differentially abundant genus in P100 group, and Weissellella was the most differentially abundant genus in P0 group.

[0048] Throughout the silage process, the abundance of Enterobacteriaceae increased with the increase in the proportion of paper mulberry added. In particular, the abundance of *Enterobacter ludwigii* was significantly higher in the P70 and P100 treatments than in other treatment groups, possibly related to their higher pH values. However, the presence of *Enterobacter ludwigii* negatively impacted the feed application of paper mulberry silage. Furthermore, *Pediococcus* was not detected when paper mulberry was ensiled alone. *Pediococcus* can rapidly convert pyruvate to LA via homofermentation using various substrates such as WSC, thereby lowering the pH value. Therefore, the high pH value in the P100 treatment group may be related to the absence of *Pediococcus*. After 45 days of silage, the P0 group showed a higher abundance of *Lactobacillus brevis*. *Lactobacillus brevis*, classified as a heterofermentative lactic acid bacterium, can convert lactic acid into acetic acid and propionic acid, explaining the significantly higher acetic acid content at 45 days of silage compared to other treatment groups. In the later stages of silage, the pH of the P50 group was significantly lower than other groups, while the LA content was significantly higher, indicating that the P50 group better promoted the growth of beneficial bacteria such as *Lactobacillus pentosus* and inhibited the growth of harmful bacteria. Microbiological results also confirmed this finding. Figure 1 Among the silage samples, *Lactobacillus pentosus* became the dominant bacteria in the P50 group after 90 days of silage, while *Enterobacter ludwigii* had extremely low abundance. Overall, the P50 group had the highest abundance of beneficial bacteria and the best silage effect.

[0049] 3) Bacterial co-occurrence network among groups The influence of bacterial co-occurrence networks on the complexity and stability of bacterial networks in silage with different proportions of mulberry and alfalfa mixtures was further investigated using bacterial co-occurrence networks. Different proportions of alfalfa and mulberry mixed silage resulted in bacterial co-occurrence networks exhibiting different symbiotic patterns. The complexity of the bacterial network was assessed using network topology parameters such as node number, edge number, betweenness, and assortativity; higher node and edge numbers, and lower betweenness and assortativity, indicated higher network complexity. The stability of the bacterial interaction network was assessed using negative / positive correlation (neg / pos); higher positive / negative correlation represented higher network stability. Figure 4 (of a~e).

[0050] The results showed that the proportion of Broussonetia papyrifera had a great influence on the bacterial co-occurrence network of alfalfa silage. Compared with pure alfalfa silage, the bacterial co-occurrence network complexity of P50 group was lower (lower node number, larger intermediate number and homophily), which may be due to the fact that the mixed silage group was dominated by lactic acid bacteria, which inhibited the growth of harmful microorganisms, so the interaction between bacteria was simpler.

[0051] The negative correlation between microorganisms in the network may weaken the competition relationship between bacterial communities, while the positive correlation can strengthen the competition relationship. Therefore, the greater the ratio of negative correlation to positive correlation, the weaker the competition between microbial communities, and the more stable the structure of microbial network. P30 group and P50 group reduced the stability (neg / pos ratio) of bacterial co-occurrence network, but P70 group increased the stability of bacterial co-occurrence network.

[0052] Although the content of the present application has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present application. After reading the above content, various modifications and alternatives of the present application will be apparent to those skilled in the art. Therefore, the protection scope of the present application should be defined by the appended claims.

Claims

1. A kind of alfalfa and mulberry silage, characterized in that, This silage consists of 50-70% paper mulberry and 30-50% alfalfa by mass.

2. The alfalfa and mulberry silage according to claim 1, characterized in that, The silage consists of 50% paper mulberry and 50% alfalfa by mass.

3. The alfalfa and mulberry silage according to claim 1, characterized in that, This silage is made of 50-70% paper mulberry and 30-50% alfalfa, which are ensiled for more than 14 days.

4. The alfalfa and mulberry silage according to claim 3, characterized in that, This silage is made of 50-70% paper mulberry and 30-50% alfalfa, and has been silaged for more than 90 days.

5. The method for preparing alfalfa and mulberry silage as described in any one of claims 1 to 4, characterized in that, The method includes: Mix 50-70% mulberry leaves and 30-50% alfalfa, fill the mixture into vacuum bags, seal them after vacuuming, and store them at room temperature away from light.

6. A method for improving the quality of paper mulberry silage, characterized in that, The method includes: Mix 50-70% paper mulberry and 30-50% alfalfa and ensilate for more than 14 days. The improvement of paper mulberry silage quality includes any one or more of the following: (1) Increase the lactic acid content and / or acetic acid content in paper mulberry silage; (2) Reduce the content of acid detergent fiber and / or neutral detergent fiber in paper mulberry silage; (3) Increase the abundance of beneficial bacteria and / or decrease the abundance of harmful bacteria in paper mulberry silage.

7. The method according to claim 6, characterized in that, The silage time is more than 90 days.

8. The method according to claim 6, characterized in that, To increase the abundance of beneficial bacteria and / or decrease the abundance of harmful bacteria in paper mulberry silage, mix 50% paper mulberry and 50% alfalfa by mass and ensile for more than 14 days.

9. The method according to claim 6, characterized in that, The beneficial bacteria include Lactobacillus pentosus; and / or the harmful bacteria include Enterobacter ludwig's enterobacter.

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