A root chicory stem and leaf mixed corn stalk silage feed and a preparation method thereof

Abstract

The present application belongs to the technical field of green agriculture organic waste silage, and particularly relates to a kind of root chicory stem and leaf and corn straw mixed silage and a preparation method thereof. The preparation method steps are as follows: after mixing different proportions of root chicory stem and leaf and corn straw, plant lactobacillus, short lactobacillus and lactobacillus buchneri are used as silage fermentation bacteria to prepare silage by silage fermentation, and in vitro rumen degradation test is also carried out. The present application overcomes the shortcomings of the prior art, and improves the mixing silage efficiency and quality by adding different proportions of mixing silage and different lactobacillus.

Description

Technical Field

[0001] This invention belongs to the field of green agricultural organic waste silage technology, specifically relating to a mixed silage of root chicory stems and leaves with corn stalks and its preparation method. Background Technology

[0002] With the large-scale and intensive development of the livestock industry, the shortage of feed resources, especially feed grains, is becoming increasingly serious. Developing grain-saving livestock farming and developing new unconventional feed resources are effective ways to alleviate the feed shortage. Although a huge amount of green agricultural organic waste and low-value crop straw are generated every year, they are limited by processing technology and storage conditions, and the feed conversion rate is low.

[0003] In recent years, with the popularization of the concept of food and medicine sharing the same origin, root chicory has been widely introduced and cultivated due to its rich content of active ingredients such as inulin. However, in the industrial harvesting process of root chicory, a large amount of by-products—root chicory stems and leaves—are generated. Although these stems and leaves have advantages such as being tender and juicy, palatable, and high in crude protein, their dry matter content is extremely low (about 16%), making them highly susceptible to mold and rot under conventional storage conditions. Directly discarding them not only causes serious waste of resources and non-point source pollution but also increases farmers' processing costs. Although corn stalks have a huge yield, their high degree of lignification, poor palatability, and low digestibility make them unsuitable for direct feeding to ruminants, limiting their efficient utilization in animal husbandry.

[0004] Currently, the industry mostly uses physical sun-drying or single-raw-material silage to process feed. However, physical sun-drying is greatly affected by weather, and high-moisture materials suffer significant nutrient loss and are prone to bacterial growth during the sun-drying process. High-moisture root chicory stems and leaves are difficult to compact when ensiled alone, and there is a lot of exudate, which easily leads to the proliferation of harmful microorganisms such as Clostridium difficile, resulting in spoilage and foul odor. Simple corn stalks have insufficient sugar content, resulting in insufficient lactic acid fermentation and unstable silage quality. Although existing technologies use feed-grade root chicory stems and leaves and silage corn as raw materials for mixed silage, they rely on the special feed varieties and the high sugar content of whole corn plants. For mixed silage of two agricultural wastes, namely root chicory by-product stems and leaves and grain corn stalks, the moisture content of the silage raw materials is unsuitable, and the corn stalks are lacking in fermentable sugars and have a coarse fiber structure. Therefore, it is difficult to directly apply the existing technologies for separate silage, and there are problems such as difficulty in initiating fermentation, easy mold growth, and low nutrient retention. Summary of the Invention

[0005] The purpose of this invention is to provide a mixed silage of root chicory stems and leaves and corn stalks and its preparation method, thereby overcoming the shortcomings of the prior art. By mixing silage raw materials with compound microbial agents in different proportions and anaerobic fermenting them, mixed silage is obtained, which improves the fermentation quality of mixed silage and prolongs the aerobic stability of silage, while retaining the nutrients of the raw materials.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: On the one hand, the present invention provides a method for preparing silage of root chicory stems and leaves mixed with corn stalks, comprising the following steps: spraying silage fermentation bacteria into the silage raw material composed of root chicory stems and leaves and corn stalks, and carrying out silage fermentation under sealed anaerobic conditions; Silage fermentation bacteria are Lactobacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus brevis ( Lactobacillus brevis ) and Lactobacillus brunelli ( Lentilactobacillus buchneri )composition; Lactobacillus brevis has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 20, 2023. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 27664.

[0007] On the other hand, the present invention provides a method for preparing a mixed silage of root chicory stems and leaves and corn stalks, thereby obtaining a mixed silage of root chicory stems and leaves and corn stalks.

[0008] The beneficial effects of this invention are: (1) This invention uses root chicory stems and leaves and corn stalks as raw materials, and adds a specific composition of silage fermentation bacteria (composed of Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus buchneri) for anaerobic fermentation. The resulting silage has the characteristics of high lactic acid and acetic acid content, low pH value, large number of lactic acid bacteria, no mold, and low moisture content. It can be used as ruminant feed, and can develop green agricultural organic waste resources, realize the utilization of feed, turn waste into treasure, save feed costs, and improve the economic benefits of farmers.

[0009] (2) The present invention has the characteristics of simple and feasible preparation method and large-scale production. It can be used for the development of silage from unconventional feed resources, and is also suitable for the development of feed to reduce rumen methane emissions in ruminants. The above-mentioned technical effects and excellent results have been fully verified by multiple experimental indicators such as silage quality, nutrients, in vitro rumen degradation rate, and methane emissions, and have significant practical value. Attached Figure Description

[0010] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0011] Figure 1 To investigate the effects of different lactic acid bacteria starter cultures on the aerobic stability of a mixture of root chicory stems and leaves with corn straw in silage; Figure 2The relative abundance of microbial communities at the genus level in the silage of root chicory stems and leaves mixed with corn stalks; Figure 3 The figure shows the comparative analysis of the microbial community at the genus level in the mixed silage of root chicory stems and leaves and corn straw after the addition of compound microbial agents. In the figure, a is the abundance ratio of microbial genus, b is the difference in the ratio of microbial genus, c is the comprehensive effect size, and d is the difference multiple. Figure 4 A heatmap showing the differential clustering of metabolites from the silage of root chicory stems and leaves mixed with corn stalks after the addition of compound microbial agents; Figure 5 Correlation analysis of microbial community and metabolites in mixed silage of root chicory stems and leaves and corn straw with the addition of compound microbial agents. Detailed Implementation

[0012] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0013] Among them, Lactobacillus plantarum ( Lactiplantibacillus plantarum This is a commercially available product, product number BNCC191046, purchased from Beina Innovation Biotechnology Co., Ltd.

[0014] Lactobacillus brevis ( Lactobacillus brevis DN-1 has been sent to the China General Microbiological Culture Collection Center (CGMCC) on June 20, 2023. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 27664.

[0015] Lactobacillus bruneri ( Lentilactobacillus buchneri (This refers to a commercially available product, trade number NCIMB-40788, purchased from Raman Company; or using a product published in The effects of a heterofermentative lactic acid bacterial inoculant containing...) Lentilactobacillus hilgardii and Lentilactobacillus buchneri Lactobacillus buchneri with or without chitinases on theensiling, aerobic stability, and in vitro ruminal fermentation of whole-cropcorn silages, Niu et al.

[0016] To address the shortcomings of separate ensiling of root chicory stems and leaves and corn stalks, which are prone to spoilage due to high moisture content and poor fermentation due to low sugar content, existing mixed silage methods cannot adapt to the extreme differences in moisture and sugar content between root chicory and grain straw. This results in large moisture gradients after mixing, making it difficult to precisely control the fermentation environment, which easily leads to localized mold, secondary fermentation, and uneven nutrient distribution. This invention systematically investigates the effects of mixing fresh root chicory stems and leaves with corn stalks in different proportions on silage quality and in vitro rumen fermentation characteristics. The aim is to overcome the technical barriers to the co-fermentation of inferior by-products and provide a scientific basis for fully utilizing locally sourced agricultural by-product feed resources, achieving loss reduction and efficiency improvement, and alleviating the shortage of forage for ruminants in the region.

[0017] On the one hand, the present invention provides a method for preparing silage of root chicory stems and leaves mixed with corn stalks, comprising the following steps: spraying silage fermentation bacteria into the silage raw material composed of root chicory stems and leaves and corn stalks, and carrying out silage fermentation under sealed anaerobic conditions; Silage fermentation bacteria are Lactobacillus plantarum ( Lactiplantibacillus plantarum ) 、 Lactobacillus brevis ( Lactobacillus brevis ) and Lactobacillus brunelli ( Lentilactobacillus buchneri )composition; Lactobacillus brevis has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 20, 2023. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 27664.

[0018] A synergistic fermentation system was constructed by precisely compounding specific functional bacterial groups (Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus bruneri) to achieve rapid acidification and stable fermentation of a mixture of root chicory stems and leaves and corn stalks under a sealed anaerobic environment. Lactobacillus plantarum, as a homofermenting bacterium, rapidly consumes water-soluble sugars to produce large amounts of lactic acid, quickly lowering the pH value to inhibit the growth of harmful microorganisms such as Clostridium perfringens; Lactobacillus brevis synergistically enhances acid resistance and improves flavor; while Lactobacillus bruneri produces acetic acid through heterofermentation, effectively inhibiting mold growth and preventing secondary fermentation after opening the fermentation pit.

[0019] In some other embodiments, the mass ratio of root chicory stems and leaves to corn stalks is (80-95):(5-20). For example, the mass ratio of root chicory stems and leaves to corn stalks is any one of 95:5, 90:10, 85:15, and 80:20. Within this range, root chicory stems and leaves are the main component, providing ample lactic acid fermentation substrate (water-soluble sugars) and a high-quality protein source, compensating for the nutritional deficiencies of corn stalks alone. The addition of corn stalks plays a crucial role in "physical support" and "drying regulation," and its coarse, hard fiber structure effectively improves the packing porosity of the material, significantly increasing the packing and compaction density.

[0020] In some other embodiments, the mass ratio of root chicory stems and leaves to corn stalks is 90:10. This ratio ensures that the silage substrate contains sufficient fermentation substrates such as soluble sugars and proteins, driving the rapid proliferation of lactic acid bacteria and lowering the pH value for efficient preservation. The coarse and hard fibrous skeleton of corn stalks forms a stable physical support structure in the pile, effectively solving the problem of "difficulty in compaction and air residue" caused by the soft texture of high-moisture root chicory stems and leaves when ensiled alone, and significantly reducing the risk of growth of harmful bacteria such as Clostridium perfringens.

[0021] In some other embodiments, the length of the root chicory stems and leaves is <2cm; the length of the corn stalks is 0.5-2cm. Specifically, the fresh root chicory stems and leaves are cut short to <2cm, and the corn stalks are air-dried and shredded. This ensures uniform inoculation and rapid acid production in the early stages of fermentation, effectively prevents compaction and oxygen deficiency or localized mold growth caused by excessively fine materials, and optimizes the fermentation microenvironment and nutrient distribution of the mixed silage.

[0022] In some other embodiments, 1 mL of silage fermentation bacteria is sprayed onto each gram of silage raw material; the activity of the silage fermentation bacteria is ≥2 ×10⁻⁶. 6 CFU / g. Preferably, the activities of *Lactobacillus plantarum*, *Lactobacillus brevis*, and *Lactobacillus brunelli* are 1×10⁻⁶ CFU / g. 6 CFU / g, 0.5×10 6 CFU / g and 0.5×10 6 CFU / g.

[0023] In some other embodiments, the ratio of Lactobacillus plantarum, Lactobacillus brunelli, and Lactobacillus brevis is (1-2):1:1.

[0024] In some other embodiments, the ratio of Lactobacillus plantarum, Lactobacillus brunelli, and Lactobacillus brevis is 2:1:1.

[0025] In some other implementations, the silage fermentation temperature is 20-35°C and the time is 30-70 days.

[0026] On the other hand, the present invention provides a method for preparing a mixed silage of root chicory stems and leaves and corn stalks, thereby obtaining a mixed silage of root chicory stems and leaves and corn stalks.

[0027] In some other embodiments, the dry matter content of the silage made from a mixture of root chicory stems and leaves and corn stalks is 25-30 wt%, the pH is 3.5-3.8, the lactic acid content is >26 g / kg DM, the acetic acid content is >6 g / kg DM, the stem and leaf structure is well maintained, and the silage is yellowish-green, has a strong sour taste, and is aromatic.

[0028] Example 1 This embodiment screens the silage fermentation quality of different proportions of root chicory stems and leaves mixed with corn straw, specifically including the following steps: A single-factor experimental design was used. Fresh root chicory stems and leaves were chopped to <2cm and mixed with air-dried and shredded corn stalks at different mass ratios of 100:0 (Group I), 95:5 (Group II), 90:10 (Group III), 85:15 (Group IV), and 80:20 (Group V). The mixtures were then thoroughly mixed with *Lactobacillus plantarum* (L. plantarum). Lactiplantibacillus plantarum The content is 2×10 6 After 60 days (d) of sealed anaerobic silage fermentation at room temperature (CFU / g), samples were taken from the bags to evaluate the sensory quality of the silage (the sensory quality of silage was graded according to the internationally accepted sensory evaluation method based on color, odor and texture established by the German National Agricultural Association (DLG)). The fermentation quality of the silage was analyzed, and then each group of silage was used as a substrate for in vitro rumen fermentation experiments.

[0029] Root chicory stems and leaves (Belgian root chicory) are fresh stems and leaves harvested from the chicory planting base in Tongliao City, Inner Mongolia Autonomous Region, and the air-dried corn stalks are from shredded corn stalks in Tongliao City, Inner Mongolia Autonomous Region. Their nutritional levels are shown in Table 1.

[0030] Table 1. Nutrient levels (dry matter basis) of root chicory stems and leaves mixed with corn stalks before silage.

[0031] Performance testing: (1) Evaluation of sensory quality: The sensory quality evaluation results of silage made from different proportions of root chicory stems and leaves mixed with corn stalks are shown in Table 2.

[0032] Table 2 Sensory quality of silage made from different proportions of root chicory stems and leaves mixed with corn stalks

[0033] As shown in Table 2, Group I silage made from single-stem chicory stems and leaves was yellowish-brown and dull in color, with damaged stem and leaf structure, a small amount of exudate, and a sour taste with a hint of butyric acid; Group II silage was yellowish-brown in color, with slightly damaged stem and leaf structure, a sour aroma, and a mild butyric acid taste; Group III silage was yellowish-green in color, with intact stem and leaf structure, a strong sour taste, and an aromatic taste; Group IV silage was yellowish-green in color, with intact stem and leaf structure, and an aromatic sour taste; Group V silage was yellowish-green in color, with intact stem and leaf structure, and a weak aromatic sour taste.

[0034] (2) Fermentation quality: Table 3 shows the silage fermentation quality of different proportions of root chicory stems and leaves mixed with corn straw.

[0035] Table 3. Silage fermentation quality of different proportions of root chicory stems and leaves mixed with corn stalks

[0036] Table 3 shows that after 60 days of mixed silage fermentation, the pH of each group ranged from 3.66 to 3.76. Group III had a significantly higher lactic acid content than the other groups. P <0.05%, with group I having the lowest lactic acid content. Acetic acid and propionic acid contents gradually decreased with the decrease in the proportion of root chicory stems and leaves; group I had the highest butyric acid content, while the butyric acid content of groups III, IV, and V did not differ significantly ( P >0.05. The ammonia nitrogen / total nitrogen ratio in group I was significantly higher than that in the other groups ( P <0.05).

[0037] (3) Overall score: The comprehensive scores of silage made from different proportions of root chicory stems and leaves mixed with corn stalks are shown in Table 4.

[0038] Table 4. Overall scores for silage made from different proportions of root chicory stems and leaves mixed with corn stalks

[0039] As shown in Table 4, all groups achieved an excellent overall score. Group I had the highest butyric acid / total acid ratio and the lowest lactic acid / total acid ratio, resulting in the lowest overall score. Group III had the highest overall score of 95.

[0040] (4) Effects of in vitro rumen fermentation characteristics: The effects of different proportions of root chicory stems and leaves mixed with corn straw on the in vitro rumen fermentation characteristics are shown in Table 5.

[0041] Table 5. Effects of different proportions of root chicory stems and leaves mixed with corn straw for silage on in vitro fermentation gas production and gas production parameters.

[0042] Table 5 shows that as the proportion of stems and leaves in root chicory decreased, the gas production and gas production rate gradually decreased, while the gas production per gram of substrate degradation gradually increased. With prolonged fermentation time, the gas production per gram of substrate degradation gradually increased, reaching a peak at 12 h and then gradually decreasing. As the proportion of stems and leaves in root chicory decreased, methane (CH4) production and gas production rate gradually decreased, but the CH4 production per gram of substrate degradation gradually increased. The CH4 production per gram of substrate degradation at 6 h was significantly lower in groups I, II, and III than in groups IV and V. P <0.05%, the CH4 production per gram of substrate at 12 and 24 h in Group I was significantly lower than that in other groups ( P <0.05), the CH4 yield per gram of substrate degraded in groups I and II after 48 h was significantly lower than that in groups III, IV, and V, and group III was significantly lower than that in groups IV and V. P <0.05).

[0043] (5) Effects on in vitro rumen fermentation characteristics: The effects of different proportions of root chicory stems and leaves mixed with corn straw on the in vitro rumen fermentation characteristics are shown in Table 6.

[0044] Table 6. Comprehensive evaluation of the silage effect of different proportions of root chicory stems and leaves mixed with corn straw.

[0045] Table 6 shows that membership function analysis was performed on the nine indicators of each group of silage. Positive indicators included CP, WSC, lactic acid, and gas production, while negative indicators included pH, ammonia nitrogen / total nitrogen, acetic acid, and propionic acid content. The comprehensive value was ranked according to the average membership function value; the higher the average value, the higher the comprehensive value. The comprehensive ranking of the groups was: Group III (0.68) > Group II (0.63) > Group I (0.55) > Group IV (0.51) > Group V (0.36). Based on the comprehensive evaluation of 11 indicators, Group III (90:10 ratio of root chicory stems and leaves to corn stalks) showed the best silage performance.

[0046] Example 2 This embodiment optimizes the fermentation agent used for ensiling root chicory stems and leaves mixed with corn stalks, specifically including the following steps: Five different lactic acid bacteria treatment groups were set up: (1) no additive control group (labeled CK); (2) Lactobacillus plantarum group (labeled LP); (3) Lactobacillus brevis group (labeled LB1); (4) Lactobacillus bruneri group (labeled LB2); (5) a mixed group of Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus bruneri in a ratio of 2:1:1 (labeled LPB). The activity of the added bacteria was 2 × 10⁻⁶. 6CFU / g. The additive was dissolved in 10 mL of distilled water and sprayed evenly onto the continuously mixed silage material using a micro-sprayer. The control group was sprayed with an equal amount of sterile water. Chicory stems and leaves were thoroughly mixed with corn stalks at a mass ratio of 90:10, and then sealed for anaerobic silage fermentation for 60 days. Samples were then taken after opening the bags. The nutritional level, fermentation quality, and aerobic stability of the silage were first analyzed, followed by the detection of microbial structure and metabolites in the control and probiotic groups.

[0047] After 60 days of ensiling, 15.0 g samples were taken and genomic DNA was extracted from the microbial community using the EZNA® Soil DNA Kit (Omega Bio-tek, USA). The V3-V4 variable region of the 16S rRNA gene was amplified by PCR using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3'(SEQ ID NO: 1)) and 806R (5'-GGACTACHVGGGTWTCTAAT-3'(SEQ ID NO: 2)) carrying barcode sequences. The PCR products were purified and quantified after recovery using a 2% agarose gel. Libraries were constructed from the purified PCR products (amplicones) using the NEXTFLEX® Rapid DNA-Seq Kit and sequenced on the Illumina Nextseq2000 platform (Shanghai Meiji Bio-tek Technology Co., Ltd.). After sequencing, a standard curve was established based on the theoretical copy number and the actual sequence number of the spike-in internal reference, and the number of reads of each ASV in the sample was converted into the absolute copy number of the 16S rRNA gene, thereby obtaining information on the absolute abundance of the microbial community.

[0048] After mixing, the silage samples were rapidly aliquoted into 15 mL centrifuge tubes, flash-frozen in liquid nitrogen, and then stored at low temperature on dry ice before being sent to the Megibio platform for LC-MS non-targeted metabolomics analysis. Following Megibio's standard procedures, 100 mg of silage sample was placed in a 2 mL centrifuge tube, and a 6 mm diameter grinding bead and 800 μL of internal standard solution (80% methanol-water) were added to extract metabolites. The mixture was incubated at -20℃ for 30 min, centrifuged for 15 min (4℃, 13,000 g), and the supernatant was transferred for analysis. 20 µL of supernatant from each sample was collected, mixed, and used as a quality control (QC) sample. A QC sample was inserted for every five samples to verify the repeatability of the analytical process. After LC-MS, raw data were imported into the metabolomics software Progenesis QI v3.0 (Waters) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Variables with RSD > 30% in QC samples were removed, generating a data matrix containing information such as retention time, mass-to-charge ratio, and peak intensity. The software was used for characteristic peak search and identification, matching MS and MS / MS mass spectrometry information with metabolic databases to obtain metabolite information, which was then uploaded to the Meiji Cloud platform (cloud.majorbio.com) for analysis. After standardization and preprocessing, raw data were annotated with metabolites. PLS-DA analysis was performed using MetaX software to obtain the VIP values ​​of metabolites. Statistical analysis was performed on differentially expressed metabolites between groups, calculating the fold differences. Differentially expressed metabolites were screened using VIP > 1.0, P < 0.05, and FC > 1.5 / FC < 0.667. Further KEGG pathway enrichment analysis and Fisher's test were performed using SciPyStats to identify biological pathways related to the experimental treatment.

[0049] Performance testing: (1) Effects of nutrients: The effects of different Lactobacillus fermentation agents on the nutrients (dry matter basis) of mixed silage of root chicory stems and leaves with corn straw are shown in Table 7.

[0050] Table 7. Effects of different types of lactic acid bacteria additives on the nutrient content of mixed silage of root chicory stems and leaves with corn straw.

[0051] As shown in Table 7, after 60 days of silage fermentation, different Lactobacillus treatments had a significant impact on all nutritional indicators of the mixed silage of root chicory stems and leaves with corn straw. P <0.05%, the DM content in the LP, LB1, LB2, and LPB groups was significantly higher than that in the CK group ( P <0.05%, with the DM content in the LB2 and LPB groups being significantly higher than that in the LP and LB1 groups ( P<0.05), there was no significant difference between the LP group and the LB1 group (P>0.05). The CK group had the highest NDF and ADF levels, significantly higher than the LP, LB1, LB2, and LPB groups. P <0.05%. There were no significant differences in CP content among the LP, LB1, LB2, and LPB groups. P >0.05), and all were significantly higher than the CK group ( P <0.05. The WSC content in the CK group was significantly higher than that in the other four groups ( P <0.05), and the WSC content was lowest in the LPB group.

[0052] (2) Effects on fermentation quality: The effects of different lactic acid bacteria starter cultures on the fermentation quality of mixed silage of root chicory stems and leaves with corn straw are shown in Table 8.

[0053] Table 8. Effects of different Lactobacillus starter cultures on the fermentation quality of silage made from a mixture of root chicory stems and leaves and corn straw.

[0054] As shown in Table 8, after 60 days of mixed silage fermentation, there was no significant difference in pH among the groups. P >0.05), and all were less than 4. Lactic acid, acetic acid, and propionic acid levels were all increased in the LP, LB1, LB2, and LPB groups and significantly higher than in the CK group ( P <0.05%, the LPB group had the highest acetic acid content. Butyric acid was not detected in the LP, LB1, and LB2 groups. The ammonia nitrogen / total nitrogen ratio in the CK group was significantly higher than that in the LP, LB1, LB2, and LPB groups. P <0.05).

[0055] (3) Effects on aerobic stability: The effects of different lactic acid bacteria starter cultures on the aerobic stability of mixed silage of root chicory stems and leaves with corn stalks, such as Figure 1 As shown. By Figure 1 It can be seen that after 60 days of mixed silage fermentation, the aerobic stability of the compound microbial agent group was significantly higher than that of the other groups. P <0.05. The aerobic stability of the CK group was significantly lower than that of the LP, LB1, and LB2 groups ( P> 0.05). Therefore, adding compound microbial agents can improve the aerobic stability of silage.

[0056] (4) Effects on microorganisms: The effects of adding compound microbial agents on the microorganisms in the mixed silage of root chicory stems and leaves and corn straw are as follows: Figure 2 As shown.

[0057] Depend on Figure 2 It can be seen that, in terms of relative abundance at the genus level, the core dominant genera in the CK group include *Lactobacillus* (…). Levilactobacillus ) accounted for 41.76%, and Weissella spp. (Weissella ) accounted for 21.36%, Pediococcus spp. ( Pediococcus ) accounted for 17.20%, Lactobacillus plantarum ( Lactiplantibacillus 6.25% were lactic acid bacteria, accounting for approximately 86.57% of the total. Minor genera included unclassified genera of Enterobacteriaceae. Enterobacteriaceae ), Enterococcus spp. Enterococcus The relative abundance of *Lactobacillus plantarum* was 13.43%. The dominant genus in the LPB group was *Lactobacillus plantarum*. Lactiplantibacillus ) accounted for 64.88%, holding an absolute dominant position. Among other lactic acid bacteria genera, *Lactobacillus* genus (… Levilactobacillus ), Weissella spp. Weissella ), Pediococcus ( Pediococcus The relative abundance of these genera decreased significantly, accounting for only 12.53% in total. The relative abundance of other genera totaled 22.59%.

[0058] (5) Comparative analysis of species differences in the effects of adding compound microbial agents on the silage of root chicory stems and leaves mixed with corn straw The differences in microbial community structure between the CK and LPB groups were analyzed at the genus level using the Wilcoxon rank-sum test. Figure 3 As shown, the differences in bacterial composition between the two groups are visually illustrated. In the LPB group, *Lactobacillus plantarum* (…) Lactiplantibacillus ) is the dominant genus, accounting for an absolute majority, and its relative abundance is significantly higher than that of the CK group ( P <0.05%. Lactobacillus spp. in the CK group ( Levilactobacillus ), Weissella spp. Weissella ), Pediococcus ( Pediococcus ), Enterococcus spp. Enterococcus The relative abundance was significantly higher than that of the LPB group. P <0.05%. In both groups, *Trichophyton* spp. ( Lachnotalea Clostridium ( Clostridium ), genus Rombutz ( Romboutsia Agrobacterium spp. Agrobacterium ), Zurich bacillus ( Turicibacter Overall abundance was low, but the Log2FC of the LPB group was relatively large, indicating that although the fold change was significant, the contribution of absolute abundance was limited. Figure 3 (a) The scatter plot shows the abundance difference between the two groups and their 95% confidence intervals. Points to the left of 0 indicate that the genus is more abundant in the LPB group, and points to the right of 0 indicate that the genus is more abundant in the CK group. Confidence intervals that do not cross 0 indicate that the direction of the difference is relatively stable. Figure 3 (b) Combined Effect Size (Combine ES) Figure 3 c) and the difference factor (Log2FC) Figure 3d) further illustrates the direction and intensity of the difference.

[0059] (6) Effect of adding compound microbial agents on the metabolites of mixed silage of root chicory stems and leaves with corn straw Cluster analysis of the top 50 differentially metabolites between the two groups was performed, and the results are as follows: Figure 4 As shown, the CK group and the LPB group clustered independently into two classes, with the six replicates within each group clustering closely together (CK-1~CK-6 and LPB-1~LPB-6), and the samples between groups were clearly separated. Differential metabolites (metabolites 1-50, specific metabolites are shown in Table 9) could be further divided into multiple subclusters (subclusters 1-10), and different subclusters showed opposite accumulation trends in the two groups: some metabolites were generally upregulated in the LB2 group but decreased in the CK group, while others were the opposite. This further validated the metabolic differences between the groups.

[0060] Table 9 Metabolites in Silage

[0061] (7) Correlation analysis of microbial community and metabolites in mixed silage of root chicory stems and leaves and corn straw after the addition of compound microbial agents like Figure 5 As shown, based on Pearson's correlation heatmap, the relationship between dominant microbial genera (genera 1-10) and differential metabolites (products 1-20) was analyzed. Specific genera and metabolite names are shown in Table 10. Among the differential metabolites screened between the CK and LB2 groups, apatides, radish phenol A, 2-fucosylated lactose, tuliposide B, D-galactosyl-(1→3)-D-galactosyl-(1→3)-L-arabinose, pallidolol 3-glucoside, naringin 4'-glucoside, glycyrrhizin-4'-[3-acetylapigenyl-(1->2)-glucoside], carnoside A, and α-trisaccharide were associated with *Lactobacillus plantarum* genus (…). Lactiplantibacillus ) showed a significant positive correlation ( P <0.05, compared with Lactobacillus spp. ( Levilactobacillus ) 、 Weissella spp. Weissella ), Pediococcus ( Pediococcus ), Enterococcus ( Enterococcus ) showed a significant negative correlation ( P <0.05%. Metabolites such as lycorine, chrysoeriol, and aflatoxin Q1, and *Lactobacillus* spp. (…) Levilactobacillus ), Weissella spp. Weissella), Pediococcus ( Pediococcus ), Enterococcus spp. Enterococcus It showed a significant positive correlation with (P<0.05) and with Lactobacillus plantarum ( Lactiplantibacillus ) showed a significant negative correlation ( P <0.05). Therefore, compound microbial agents can regulate the microorganisms and metabolites in the mixed silage of root chicory stems and leaves and corn stalks, improve the fermentation quality of silage and preserve its nutritional value, and reduce the production of harmful bacteria and metabolites.

[0062] Table 10 Names of Microbial Genus and Metabolites

[0063] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing silage of root chicory stems and leaves mixed with corn stalks, characterized in that, Includes the following steps: Spray silage fermentation bacteria onto the silage material composed of root chicory stems and leaves and corn stalks, and carry out silage fermentation under sealed anaerobic conditions; The silage fermentation bacteria are Lactobacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus brevis ( Lactobacillus brevis ) and Lactobacillus brunelli ( Lentilactobacillus buchneri )composition; The Lactobacillus brevis has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on June 20, 2023. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 27664. Lactobacillus plantarum ( Lactiplantibacillus plantarum This is a commercially available product, product number BNCC191046, purchased from Beina Innovation Biotechnology Co., Ltd. Lactobacillus bruneri ( Lentilactobacillus buchneri This is a commercially available product, product number NCIMB-40788, purchased from Raman Company; The mass ratio of root chicory stems and leaves to corn stalks is (80-95):(5-20); Spray 0.5-1.5 mL of silage fermentation bacteria per gram of silage raw material; the activity of the silage fermentation bacteria is ≥2×10⁻⁶. 6 CFU / g; The ratio of Lactobacillus plantarum, Lactobacillus brunelli, and Lactobacillus brevis is 2:1:

1.

2. The method for preparing silage of root chicory stems and leaves mixed with corn stalks as described in claim 1, characterized in that, The mass ratio of root chicory stems and leaves to corn stalks is 90:

10.

3. The method for preparing silage of root chicory stems and leaves mixed with corn stalks as described in claim 1, characterized in that, The length of the root chicory stems and leaves is <2cm; the length of the corn stalks is 0.5-2cm.

4. The method for preparing silage of root chicory stems and leaves mixed with corn stalks as described in claim 1, characterized in that, The silage fermentation temperature is 20-35℃, and the time is 30-70 days.

5. A silage prepared by the method of preparing root chicory stems and leaves mixed with corn stalks according to any one of claims 1-4.

6. The silage of root chicory stems and leaves mixed with corn stalks as described in claim 5, characterized in that, The dry matter content of the silage made from a mixture of root chicory stems and leaves and corn stalks is 25-30 wt%, and the pH is 3.5-3.8.

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