Bacillus amyloliquefaciens strain RSD8 and application thereof

Abstract

The application discloses a bacillus amyloliquefaciens RSD8 and application thereof, and belongs to the field of microorganisms.The bacillus amyloliquefaciens RSD8 is preserved in the Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO: 68841.The bacillus amyloliquefaciens RSD8 is isolated and screened from whole corn stalks, and has the abilities of acid tolerance, cellulose degradation and acid production.In the silage fermentation taking whole corn stalks as main raw materials, compared with natural fermentation, the addition of the bacillus amyloliquefaciens RSD8 inoculum can not only reduce the pH value and the content of ammonia nitrogen, increase the content of lactic acid and acetic acid in the feed, promote the reduction of the content of acid detergent fiber and neutral detergent fiber, but also reduce the consumption of nutrients (dry matter, soluble carbohydrate and crude protein) during the silage fermentation, thereby providing a strain resource application reference for improving the quality of the silage feed.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of amylolytic Bacillus RSD8 and its applications. Background Technology

[0002] my country has a vast corn planting area and abundant straw resources, but a large amount of straw is burned or returned to the field, resulting in resource waste and environmental pollution. Silage is an important way to utilize straw as feed, preserving the nutritional characteristics of green fodder through microbial fermentation under anaerobic conditions. However, the complex structure of lignocellulose in whole corn stalks and its difficulty in degradation directly lead to excessively high fiber content and low utilization rate of soluble sugars, severely restricting the improvement of silage quality.

[0003] Bacteria of the genus *Paenibacillus* have attracted attention for their ability to secrete various cell wall-degrading enzymes (such as cellulase, xylanase, and pectinase) in the degradation of plant residues and the resource utilization of agricultural waste. Previous studies have shown that strains such as *Paenibacillus polymyxa* and *Paenibacillus glucanolyticus* can be used in silage fermentation to improve silage quality by degrading lignocellulose. *Paenibacillus amylolyticus* is a known cellulase-producing strain: strain ZJJ-9 has a cellulase activity of 34.26 U / mL; patent CN110257293B discloses that strain KY15 has cellulose-degrading ability and can be used to promote plant growth; other studies have reported that strain 27C64 has low cellulase activity and cannot effectively utilize cellulose. However, none of the above studies have addressed the application of *Paenibacillus amylolyticus* in silage fermentation, nor have they addressed the acid tolerance and acid-producing capacity of this strain.

[0004] Applying cellulosic strains with cellulose-degrading capabilities to silage fermentation requires not only lignocellulose-degrading activity but also the ability to maintain activity and function in the acidic environment created by the rapid pH drop during fermentation. Not all cellulase-producing strains can meet this requirement. Therefore, for the resource utilization and feed application of whole-plant corn stalks, screening for functional strains with acid tolerance, cellulose degradation, and acid-producing capabilities, and applying them to whole-plant corn silage fermentation, is of great significance for improving silage quality and promoting the resource utilization of straw. Summary of the Invention

[0005] The technical problem to be solved by this invention is: to address the problem of difficult degradation of lignocellulose in traditional silage fermentation and the problem of improving the quality of silage fermentation, to isolate and screen strains with acid tolerance, cellulose degradation and acid production capabilities, and to apply them to silage fermentation using whole corn stalks as raw materials. One objective of this invention is to provide a Bacillus strain with acid tolerance, cellulose degradation and acid production capabilities, and another objective is to provide the application of this strain.

[0006] The technical solution of the present invention is: a strain of Paenibacillus amylolyticus RSD8, abbreviated as RSD8, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on August 17, 2026, with the accession number GDMCC NO: 68841. The address of the depository is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0007] Microbial inoculants containing Paenibacillus amylolyticus RSD8.

[0008] Application of Paenibacillus amylolyticus RSD8 or microbial agents containing this strain in the silage fermentation of whole-plant corn stalks.

[0009] Furthermore, the treatment is silage fermentation.

[0010] Furthermore, the silage fermentation refers to cutting fresh whole corn plants into 2-5 cm segments, inoculating them with the aforementioned Paenibacillus amylolyticus RSD8 or other microbial agents, and then carrying out silage fermentation.

[0011] Furthermore, the silage fermentation method is as follows: using fresh whole-plant corn stalks cut into 2-5 cm pieces as raw material, controlling the moisture content to 65%-75%, and fermenting at a rate of 1 × 10⁻⁶ cm / s. 6 The above-mentioned microbial agent was added at an application rate of CFU / g, thoroughly mixed, compacted, and vacuum-sealed. It was then fermented in the dark at 25 ± 2 ℃ for 30 days.

[0012] Compared with the prior art, the present invention has the following unexpected technical effects:

[0013] This invention is the first to discover the extreme acid tolerance and acid-producing capacity of Paenibacillus amylolyticus: while this strain has been reported to have cellulose degradation capabilities in the prior art, its acid tolerance and acid-producing capacity have never been discussed. During silage fermentation, the pH value rapidly drops below 4.0, placing special demands on the strain's acid tolerance; this invention is the first to confirm that this strain possesses this crucial characteristic.

[0014] The cellulase activity of the strain of this invention is significantly superior to that of known strains: compared with the reported cellulase activity of Paenibacillus mylolyticus ZJJ-9 (34.26 U / mL), the cellulase activity of the strain RSD8 of this invention reaches 46.58 U / mL, which is 36% higher. This significant advantage in enzyme activity cannot be reasonably predicted from the prior art.

[0015] This invention marks the first successful application of this strain to silage fermentation: Paenibacillus amylolyticus was applied for the first time to whole-plant corn stalk silage fermentation, achieving multi-dimensional improvements in silage quality, including a decrease in pH to 3.43, a significant increase in lactic acid and acetic acid content, a significant decrease in ammonia nitrogen content, a significant decrease in acid detergent fiber and neutral detergent fiber content, and a significant increase in crude protein and soluble carbohydrate content.

[0016] The preservation information is as follows:

[0017] Paenibacillus amylolyticus RSD8, or RSD8 for short, was deposited on August 17, 2026, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 68841), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Attached Figure Description

[0018] Figure 1 Photographs showing the colony morphology of Paenibacillus amylolyticus RSD8 on tryptic soybean agar (TSA medium);

[0019] Figure 2 A phylogenetic tree of Paenibacillus mylolyticus RSD8 constructed based on 16S rRNA sequence results;

[0020] Figure 3 The initial screening results for cellulase production by Paenibacillus amylolyticus RSD8;

[0021] Figure 4 The initial screening results for acid production by Paenibacillus amylolyticus RSD8;

[0022] Figure 5 The appearance changes of whole corn stalks before and after silage fermentation; Note: A: silage raw material; B: CK treatment group; C: treatment group with added RSD8 microbial agent;

[0023] Figure 6 pH changes before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0024] Figure 7 The change in ammonia nitrogen content before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0025] Figure 8 The change in lactic acid content before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0026] Figure 9 The change in acetic acid content before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0027] Figure 10 The change in dry matter content before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0028] Figure 11 The change in crude protein content before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0029] Figure 12 The change in soluble carbohydrate content before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0030] Figure 13 The change in acid detergent fiber content before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0031] Figure 14The change in neutral detergent fiber content before and after silage fermentation; Note: FM: silage raw material; CK: CK treatment group; RSD8: treatment group with added RSD8 microbial agent;

[0032] Figure 15 Analysis of bacterial microbial community α-diversity before and after silage fermentation;

[0033] Figure 16 The composition of the bacterial microbial community before and after silage fermentation; Note: A: Phylum level; B: Genus level. Detailed Implementation

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0035] In this application:

[0036] Tryptic soybean agar medium (TSA medium): 15 g tryptic peptone, 5 g soybean peptone, 5 g NaCl, 20 g agar, 1000 mL distilled water, pH 7.2;

[0037] Tryptic soybean peptone liquid medium (TSB medium): 17 g tryptic peptone, 3 g soybean peptone, 5 g NaCl, 2.5 g K2HPO4, 2.5 g glucose, 20 g agar, 1000 mL distilled water, pH 7.2;

[0038] Cellulase primary screening medium: CMC-Na 2.0 g, KH2PO4 0.5 g, Na2SO4 1.0 g, MgSO4·7H2O 0.25 g, Congo red 1 g, agar 20 g, distilled water 1000 mL, pH 7.0;

[0039] Cellulase rescreening medium: glucose 5 g, yeast powder 10 g, KH2PO4 0.5 g, MgSO4·7H2O 0.3 g, (NH4)2SO4 1 g, CaCl2 1 g, NaCl 1 g, distilled water 1000 mL, pH 7.2;

[0040] Primary screening medium for acid production: 10.0 g tryptone, 5 g glucose, 2.0 g soluble starch, 0.04 g bromocresol purple, 20 g agar, 1000 mL distilled water, pH 7.0;

[0041] Acid production screening medium: 20 g glucose, 5.0 g peptone, 5.0 g NaCl, 0.5 g yeast extract, 0.5 g KH2PO4, 0.05 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 1000 mL distilled water, pH 7.0.

[0042] Example 1: Isolation and identification of Paenibacillus amylolyticus RSD8

[0043] 1.1 Isolation of Paenibacillus amylolyticus RSD8

[0044] Weigh 10 g of fresh corn stalks into a 250 mL sterile Erlenmeyer flask containing 90 mL of sterile water. Place the flask in a shaker, set the temperature to 30 ℃, and shake at 180 r / min for 30 min to obtain 10 g of fresh corn stalks. -1 The bacterial suspensions were serially diluted by placing them in a constant-temperature metal bath at 80 °C for 10 min. The high-temperature-treated bacterial suspensions were then serially diluted using sterile physiological saline, with 100 μL of each 10x dilution being taken from each serially diluted suspension. -1 10 -2 10 -3 The bacterial suspension was spread onto TSA medium and incubated at 30 °C for 48 h. Single colonies were picked and purified on TSA medium. The purified strains were placed in 40% (V / V) glycerol cryovials and stored at -80 °C.

[0045] 1.2 Identification of Paenibacillus amylolyticus RSD8

[0046] 1.2.1 Observation of strain morphology

[0047] Single colonies of strain RSD8 were picked and inoculated onto TSA medium. After incubation in a 30 ℃ incubator for 48 h, the colony morphology of the strain was observed.

[0048] 1.2.2 DNA extraction and PCR amplification of the strain

[0049] (1) Reagents: Chelex 100 (Beijing Solarbio Science & Technology Co., Ltd.), Mix (Sangon Biotech Co., Ltd.).

[0050] (2) Primers

[0051] Forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0052] Reverse primer 1492R: 5'-TACGACTTAACCCCAATCGC-3'.

[0053] (3) Bacterial DNA extraction

[0054] Pick a single colony using a disposable inoculation loop and place it into a 2 mL EP tube. Add 200 μL of 5% Chelex 100 solution, incubate in a metal bath at 100 °C for 15 min, vortex thoroughly to mix, and cool. Centrifuge at 12000 r / min for 5 min, and use the supernatant as a DNA template. Store at -20 °C.

[0055] (4) Bacterial 16S rRNA gene amplification

[0056] The purified bacterial DNA template was amplified using universal primers 27F and 1492R for bacterial 16S rRNA. The 16S rRNA gene amplification conditions were as follows: 1 μL DNA template (50 ng / μL), 12.5 μL 2× Taq PCR mix, 1 μL each of forward and reverse primers (1.0 μmol / L), ddH2O added to a final volume of 25 μL, incubated at 95 ℃ for 5 min, 95 ℃ for 30 s, 55 ℃ for 45 s, 72 ℃ for 1.5 min, for 35 cycles, followed by 72 ℃ for 10 min. After PCR amplification and band detection using 1.5% agarose gel electrophoresis, the products were sent to Sangon Biotech Co., Ltd. for sequencing.

[0057] 1.2.3 16S rRNA gene sequence analysis and phylogenetic tree construction

[0058] The sequence obtained after sequencing (SEQ ID No. 1) was uploaded to the EzBioCloud database for comparison. The 16S rRNA gene sequence of the published standard strain of the same genus with the highest similarity was selected as the reference sequence. The sequence of the Bacillus subtilis (NCIB 3610) standard strain was used as the outgroup. Multiple sequence alignment analysis was performed using Clustal X software. A phylogenetic tree was constructed using MEGA 11 software based on the Neighbor-joining algorithm to determine the taxonomic position of the strain.

[0059] 1.3 Experimental Results

[0060] Amylolyticus RSD8 was isolated from fresh corn stalks. After incubation on TSA medium at 30 ℃ for 48 h, the colonies were round, 2-3 mm in diameter, slightly raised, with rough edges and an uneven surface, and light yellow in color. They were easy to pick up. The growth conditions were aerobic. Figure 1 The 16S rRNA sequence of strain RSD8 was determined, and homology was compared with the EzBioCloud database. Phylogenetic analysis was performed using MEGA 11 software. The nucleotide sequence homology of the 16S rRNA of this strain with that of Paenibacillus amylolyticus was 100%. Based on the combined results of morphological characteristics and 16S rRNA sequence homology analysis, strain RSD8 was identified as Paenibacillus amylolyticus. Figure 2 ).

[0061] Example 2: Functional determination of the acid tolerance, cellulose degradation and acid production capacity of Paenibacillus amylolyticus RSD8

[0062] 2.1 Determination of acid tolerance

[0063] The pH of the PBS solution was adjusted to 1, 2, 3, and 7 using 10% HCl solution, resulting in four gradients (pH=7 served as the control group). The solutions were then filtered through a 0.22 µm sterile filter membrane before use. After culturing the bacterial strain in TSB medium for 16 h, the culture was centrifuged at 10000 rpm for 5 min at 4 ℃. The bacterial culture was inoculated at a 2% inoculum into PBS solutions at different pH values, incubated for 4 h, and the absorbance was measured at 600 nm, denoted as A1. A 10% inoculum was added to pH=7 PBS buffer as a control, incubated for 4 h, and the absorbance was measured at 600 nm, denoted as A0. Each experiment was performed in triplicate. The acid tolerance rate was calculated using the following formula:

[0064] Acid tolerance rate (%) = A1 / A0 × 100%

[0065] In the formula: A1: absorbance value of the experimental group;

[0066] A0: Absorbance value of the control group;

[0067] 100%: Unit conversion factor;

[0068] 2.2 Determination of cellulose degradation capacity

[0069] 2.2.1 Initial screening of cellulose degradation ability

[0070] After inoculating the strain in TSB medium and culturing for 16 h, 200 µL of the bacterial suspension was evenly spread on TSA medium and incubated at 30 ℃ for 24 h. The strain and medium were then used to prepare a bacterial cake using a 5 mm diameter punch and inoculated onto cellulase screening medium. The cake was incubated at 30 ℃ for 48 h, and the presence or absence of a transparent hydrolysis zone around the bacterial cells was observed. The diameter of the hydrolysis zone (D) and the diameter of the colony (d) were measured, and the ratio (D / d) was calculated. A higher D / d value indicates a stronger ability of the strain to degrade cellulose. Each experiment was repeated in triplicate.

[0071] 2.2.2 Cellulase Activity Assay

[0072] (1) Drawing the standard curve

[0073] Prepare a 100 μg / mL glucose standard solution, dilute it to obtain glucose solutions with concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL. Take 2 mL of each diluted solution, add 1.5 mL of DNS reagent, shake well, boil in a water bath for 5 min, cool immediately, and bring the volume to 25 mL. After shaking well, measure the OD value at 540 nm. Plot a glucose standard curve with absorbance as the ordinate and glucose content as the abscissa.

[0074] (2) Cellulase activity assay.

[0075] After inoculating the bacterial strain in TSB medium and culturing for 16 h, the bacterial solution was inoculated into cellulase screening medium at a ratio of 3%, and cultured at 30 ℃ with shaking at 180 r / min for 5 days. The supernatant was obtained by centrifugation at 9000 r / min for 10 min, which is the crude enzyme solution. 0.5 mL of the crude enzyme solution was taken, and 1.5 mL of 1% CMC-Na solution was added. The mixture was incubated in a 45 ℃ water bath for 20 min, and immediately 1.5 mL of DNS chromogenic reagent was added. The mixture was then boiled in a water bath for 5 min, cooled, and brought to a final volume of 25 mL. The OD value was measured at 540 nm and compared with the standard curve. The glucose content (m1) was calculated from the OD value. Simultaneously, 0.5 mL of the crude enzyme solution was taken, and 1.5 mL of distilled water and 1.5 mL of DNS chromogenic reagent were added. The mixture was boiled in a water bath for 5 min, cooled, and brought to a final volume of 25 mL. Each experiment was repeated in triplicate. The OD value was measured at 540 nm and compared with the standard curve. The glucose content (m0) was calculated from the OD value. The enzyme activity was calculated using the following formula:

[0076] Cellulase activity (U / mL) = (m1 - m0) × 2 × 1000 / 20

[0077] In the formula: m1: glucose content of crude enzyme solution;

[0078] m0: Glucose content in the control group;

[0079] 2: Divide the crude enzyme solution into multiples;

[0080] 1000: Unit conversion factor;

[0081] 20: Reaction time 20 min.

[0082] 2.3 Determination of acid production capacity

[0083] 2.3.1 Initial screening of acid production capacity

[0084] After culturing the strain in TSB medium for 16 h, centrifuging at 10000 r / min at 4 ℃ for 5 min, washing twice with 2 mL PBS solution, and resuspending to obtain a bacterial suspension. The bacterial suspension was inoculated into an acid-producing screening medium by spot inoculation and cultured at 37 ℃ for 24 h. The presence of yellow color around the colonies was used to determine whether the strain had acid-producing ability.

[0085] 2.3.2 Determination of acid production capacity

[0086] After inoculating the strain in TSB medium and culturing for 16 h, the bacterial solution was inoculated into acid production screening medium at a ratio of 3%, and cultured at 30 ℃ and 180 r / min for 2 days with shaking. Each group of experiments was repeated in 3 replicates. After the culture was completed, the pH value of the supernatant was measured to evaluate the acid production capacity of the strain.

[0087] 2.4 Experimental Results

[0088] Tests showed that *Paenibacillus amylolyticus* RSD8 exhibits tolerance to acidic environments, surviving at extreme acidic conditions of pH 1.0, pH 2.0, and pH 3.0 with a survival rate exceeding 75% (Table 1). It also demonstrates the ability to produce cellulase and acid. Figure 3 and 4 (Table 2). The cellulase activity was 46.58 U / mL, and the acid-producing capacity was good. After 24 h of culture, the pH value reached 3.98 (Table 2). The results show that Paenibacillus amylolyticus RSD8 has good tolerance to acidic environment, cellulase production and acid production capacity, and has the potential for further development and application.

[0089] Table 1. Acid tolerance of Paenibacillus amylolyticus RSD8

[0090]

[0091] Table 2. Cellulase and acid production capacity of Paenibacillus amylolyticus RSD8

[0092]

[0093] Example 3 Preparation of microbial inoculants

[0094] This embodiment provides a microbial inoculant, the preparation method of which is as follows:

[0095] Paenibacillus amylolyticus RSD8 plates were streaked onto TSA medium. Pure strains were picked and placed in sterile physiological saline. The bacterial suspension concentration was adjusted to 10⁻⁶ using the McFarland turbidimetric method. 9 The bacterial suspension was inoculated into TSB medium at a concentration of CFU / mL at a rate of 3%, and cultured at 37 ℃ with shaking at 180 r / min for 16 h to obtain the microbial inoculum, which had an effective viable count of 1.0 × 10⁻⁶. 8 CFU / g.

[0096] Example 4: Application of Paenibacillus amylolyticus RSD8 in whole-plant corn silage fermentation

[0097] 4.1 Silage Preparation

[0098] After harvesting fresh whole-plant corn stalks, cut them into 2-5 cm pieces. Control the moisture content of the corn stalks to 65%-75% by sun-drying or spraying with a suitable amount of water. Weigh 1 kg of whole-plant corn stalk pieces and mix according to 1 × 10⁻⁶ cm. 6 The microbial inoculant described in Example 3 was added at a dosage of CFU / g fresh corn and sprayed evenly onto the silage material. The silage fermentation time was set to 30 days. Two treatment groups were set up in the experiment: (1) no microbial inoculant was added, only an equal amount of sterile water was added as a blank control group (CK group); (2) microbial inoculant containing Paenibacillus amylolyticus RSD8 was added (RSD8 group). After the bacterial solution and silage material were thoroughly mixed, they were put into polyethylene bags, compacted, and vacuum sealed to make bagged silage. Each treatment was repeated 3 times. Fermentation was carried out at 25 ± 2 ℃ in the dark for 30 days. After the fermentation was completed, the bags were opened immediately to determine the fermentation quality.

[0099] 4.2 Sensory evaluation

[0100] The silage was scored using the German Agricultural Association (DLG) scoring criteria after fermentation at each time point. Based on a comprehensive score of various indicators, the silage was divided into four grades: excellent, good, average, and spoiled. A score >16 indicates excellent, 9-16 indicates good, 5-9 indicates average, and 5-9 indicates spoiled. The scoring indicators are shown in Table 3.

[0101] Table 3 Sensory Evaluation Standards for Silage

[0102]

[0103] 4.3 Silage Fermentation Quality Analysis

[0104] Weigh 10 g of fresh silage sample, add 90 mL of distilled water, and extract overnight at 4 ℃. Centrifuge the extract at 10000 r / min, 4 ℃ for 5 min to obtain the supernatant, and measure the pH using a pH meter. Determine the ammonia nitrogen content using the phenol-hypochlorous acid colorimetric method. Determine the lactic acid and acetic acid content of the sample using high-performance liquid chromatography (HPLC). Dry the fresh silage sample to constant weight and determine the dry matter content. Grind the dried sample through a 40-mesh sieve for the determination of relevant nutrients. Determine the crude protein content using the Kjeldahl method; determine the soluble carbohydrate content using the anthrone-sulfuric acid colorimetric method; and determine the content of neutral detergent fiber and acid detergent fiber using a fully automated fiber analyzer.

[0105] 4.4 Microbial Community Analysis

[0106] Weigh 1 g of fresh silage sample and extract total DNA using a DNA extraction kit (MP Biomedicals, CA, USA). DNA purity and concentration were assessed by 1% agarose gel electrophoresis. The V3-V4 region of the DNA was amplified using primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The 25 μL amplification reaction mixture consisted of: 5 μL buffer, 2 μL 2.5 mm dNTPs, 1 μL each of primers 338F and 806R, 0.25 μL DNA polymerase, 1 μL DNA template, and 14.75 μL ddH2O. PCR amplification program: 94 ℃ pre-denaturation for 3 min; denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 40 s, for a total of 35 cycles; final extension at 72 ℃ for 10 min. The amplified products were detected by 1% agarose gel electrophoresis. The qualified DNA was sent to Shanghai Paisenno Biotechnology Co., Ltd., and paired-end sequencing was performed using the Illumina NovaSeq sequencing platform.

[0107] 4.5 Experimental Results

[0108] 4.5.1 Sensory evaluation results

[0109] After one month of silage fermentation, the silage from each treatment group was as follows: Figure 5 As shown, in terms of odor, the CK group had a slight butyric acid odor, while the RSD8 group had no soyric odor and instead had an aromatic fruity smell. In terms of structure, all treatment groups maintained good stem and leaf structure. In terms of color, all treatment groups showed slight discoloration, turning yellowish-brown. The odor, structure, and color of the samples were scored using the German Agricultural Society (DLG) scoring method. The results showed that the silage of the CK group (15 points) was initially rated as good by sensory evaluation, while the silage of the RSD8 group (19 points) was initially rated as excellent by sensory evaluation.

[0110] 4.5.2 pH value determination of silage

[0111] pH value is an important indicator for evaluating the quality of silage fermentation. Most harmful microorganisms cannot survive in an acidic environment, and a lower pH value can effectively inhibit harmful strains and reduce nutrient loss. A pH value below 4.2 is considered one of the standards for high-quality silage. The pH values ​​of the silage raw materials and each treatment group after one month of silage fermentation are shown below. Figure 6 As shown, the pH value of the CK group in this study was 4.22, while the pH value of RSD8 (3.43) was significantly lower than that of the CK group (P<0.05) and reached below 4.2, indicating that the addition of RSD8 microbial agent can significantly improve the quality of silage.

[0112] 4.5.3 Determination of Ammonia Nitrogen Content in Silage

[0113] Ammonia nitrogen content is an indicator of the degree of protein decomposition in silage. However, animals' ability to absorb and utilize ammonia nitrogen is far lower than their ability to absorb and utilize protein. Higher ammonia nitrogen content indicates that more protein is broken down into ammonia nitrogen, resulting in poorer fermentation quality and lower nutritional value of the silage. The ammonia nitrogen content of the silage raw materials and each treatment group after one month of silage fermentation is shown below. Figure 7 As shown, compared with the CK group, the addition of RSD8 microbial agent significantly reduced the content of ammonia nitrogen (P<0.05), indicating that the bacterial treatment effectively inhibited the decomposition of protein during silage fermentation and improved the quality of silage.

[0114] 4.5.4 Determination of Organic Acid Content in Silage

[0115] Lactic acid and acetic acid are closely related to the fermentation quality of silage; higher levels of these substances result in better silage quality. The lactic acid and acetic acid contents of the silage raw materials and each treatment group after one month of silage fermentation are shown below. Figure 8 and Figure 9 As shown, compared with the CK group, the addition of RSD8 microbial agent significantly increased the content of lactic acid and acetic acid (P<0.05), and the microbial treatment effectively improved the quality of silage.

[0116] 4.5.5 Determination of Dry Matter Content in Silage

[0117] During silage fermentation, nutrients in the silage are continuously consumed through plant respiration and microbial activity, leading to a decrease in dry matter (DM) content. The silage raw materials and the DM content of each treatment group after one month of silage fermentation are shown below. Figure 10 As shown, the dry matter content of the RSD8 group was significantly higher than that of the CK group (P<0.05). This means that although the RSD8 exogenous microbial agent was added, it not only did not cause excessive consumption of the dry matter content of the silage, but also reduced the dry matter loss during the fermentation process and improved the quality of the silage.

[0118] 4.5.6 Determination of crude protein content in silage

[0119] The crude protein content of the silage raw materials and each treatment group after one month of silage fermentation is as follows: Figure 11 As shown, although the protein content inevitably decreases significantly due to the vigorous activity of microorganisms during silage fermentation, which decomposes proteins into ammonia nitrogen, the addition of RSD8 microbial agent significantly increased the crude protein content of silage compared to the control group (P<0.05), thus preserving the protein content of the silage raw materials to a greater extent and effectively improving the quality of the silage.

[0120] 4.5.7 Determination of soluble carbohydrate content in silage

[0121] Soluble carbohydrates are also an important energy source for microbial activity during silage fermentation; intense microbial activity leads to the rapid consumption of soluble carbohydrates. The soluble carbohydrate content of the silage raw materials and each treatment group after one month of silage fermentation is shown below. Figure 12 As shown, compared with the CK group, the addition of RSD8 microbial agent significantly increased the soluble carbohydrate content of silage (P<0.05), effectively improving the quality of silage.

[0122] 4.5.8 Determination of acid detergent fiber and neutral detergent fiber content in silage

[0123] Excessive levels of acid detergent fiber (ADF) and neutral detergent fiber (NDF) in feed can negatively impact animal feed intake and digestibility, while lower ADF and NDF levels can improve the palatability of silage. The ADF and NDF contents of the silage raw materials and the silage fermentation group after one month are shown below. Figure 13 and Figure 14 As shown, compared with the CK group, the addition of RSD8 microbial agent significantly reduced the content of acid detergent fiber and neutral detergent fiber in silage (P<0.05), and promoted the improvement of palatability of the feed.

[0124] 4.5.9 Analysis of Microbial Diversity in Silage

[0125] Alpha diversity of microbial communities in silage raw materials and each treatment group after one month of silage fermentation was analyzed, and the results are as follows: Figure 15 As shown, the Chao1 index and Simpson index were used to characterize species richness and diversity, respectively. Compared with silage raw materials, silage fermentation significantly reduced the Chao1 and Simpson indices in both the CK and RSD8 groups (P<0.05). This is because the lower pH environment during silage fermentation inhibits the growth of most microorganisms. Furthermore, the Chao1 and Simpson indices in the RSD8 group were significantly lower than those in the CK group, indicating that the addition of RSD8 microbial agent significantly reduced the richness and diversity of the microbial community in silage (P<0.05).

[0126] 4.5.10 Analysis of the microbial community composition of silage

[0127] Analysis of bacterial community composition in silage raw materials and samples from the CK and RSD8 groups after one month of silage fermentation revealed that at the phylum level ( Figure 16A) The dominant bacterial phyla in silage feed mainly consist of Proteobacteria, Bacteroidetes, and Actinobacteriota. After one month of silage fermentation, the dominant bacterial phyla in the silage changed significantly. For both the CK and RSD8 groups, Firmicutes and Proteobacteria were the dominant phyla, differing only in relative abundance. The abundance of Firmicutes in the RSD8 group was higher than that in the CK group, while the abundance of Proteobacteria was lower. Further in-depth analysis of the bacterial community composition at the genus level was conducted. Figure 16 B), the silage raw materials are mainly composed of bacteria such as Chryseobacterium, Deinococcus, Sphingobacterium, and Stenotrophomonas. After one month of silage fermentation, the composition of the bacterial microbial community changed significantly. The CK group was mainly composed of bacteria such as Levilactobacillus, Lactiplantibacillus, Klebsiella, and Weissella. The RSD8 group was mainly composed of bacteria such as Lactiplantibacillus and Levilactobacillus. In addition, compared with the CK group, RSD8 significantly reduced the relative abundance of Klebsiella (P<0.05), inhibiting the growth of harmful microbial genera in the silage.

[0128] 4.6 Technical Effect Analysis

[0129] The results of this embodiment demonstrate that the strain of the present invention produced significant technical effects in the silage fermentation of whole-plant corn straw:

[0130] (1) pH value significantly reduced: After adding RSD8 microbial agent, the pH value of silage dropped to 3.43, which was significantly lower than 4.22 in the CK group (P<0.05), reaching the standard of high-quality silage (pH<4.2).

[0131] (2) The ammonia nitrogen content was significantly reduced: Compared with the CK group, the ammonia nitrogen content of the RSD8 group was significantly reduced (P<0.05), indicating that the addition of RSD8 inoculant effectively inhibited the decomposition of protein during silage;

[0132] (3) The content of organic acids increased significantly: The content of lactic acid and acetic acid in the RSD8 group was significantly higher than that in the CK group (P<0.05). The increase in organic acid content directly reflects the improvement in the quality of silage fermentation.

[0133] (4) The fiber content was significantly reduced: the content of acid detergent fiber and neutral detergent fiber in the RSD8 group was significantly lower than that in the CK group (P<0.05), indicating that the strain effectively degraded the fiber components in the straw by secreting cellulase with an enzyme activity of 46.58 U / mL.

[0134] (5) Improved nutrient retention: The dry matter, crude protein and soluble carbohydrate content of the RSD8 group were significantly higher than those of the CK group (P<0.05), indicating that the addition of RSD8 microbial agent reduced the consumption of nutrients during silage.

[0135] (6) Optimization of microbial community structure: The RSD8 group significantly reduced the relative abundance of potentially harmful bacteria such as Klebsiella (P<0.05).

Claims

1. A strain of Paenibacillus amylolyticus RSD8 is deposited at Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO: 68841.

2. A microbial agent containing Paenibacillus amylolyticus RSD8 as described in claim 1.

3. The application of Paenibacillus amylolyticus RSD8 as described in claim 1 or the microbial agent as described in claim 2 in the silage fermentation of whole-plant corn stalks.

4. The application according to claim 3, characterized in that, The whole-plant corn stalk silage fermentation refers to cutting fresh whole-plant corn stalks into 2-5 cm segments, inoculating them with Paenibacillus amylolyticus RSD8 as described in claim 1 or the microbial agent as described in claim 2, and then carrying out silage fermentation.

5. The application according to claim 4, characterized in that, The method for preparing whole-plant corn silage through fermentation is as follows: Adjust the moisture content of the whole-plant corn stalk segments as described in claim 4 to 65%-75%, and add the *Paenibacillus amylolyticus* RSD8 as described in claim 1 or the microbial inoculant as described in claim 2 at a ratio of 1 × 10⁻⁶. 6 Add CFU / g, mix thoroughly, compact, and vacuum seal. Ferment at 25 ± 2 ℃ in the dark for 30 days.

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