Xanthomonas campestris pv. campestris strain CCRCGPMS 001 and its use

Abstract

The application discloses a strain of Drysillus zoogleoides CCRCGPMR002 and an application thereof, and belongs to the field of microorganisms. The strain is preserved in the Guangdong Microbial Culture Collection Center, and has a preservation number of GDMCC NO: 65222. The strain of the application is separated from bamboo leaves of bamboo which is the main food of giant pandas. The strain has good growth advantages, and has the abilities of producing acid, resisting acid, resisting bile salt and resisting artificial gastric juice. The strain can promote the decomposition of cellulose and lignocellulose, has a good inhibiting effect on pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Salmonella, and has moderate to high sensitivity to commonly used antibiotics in clinics. The application of the CCRCGPMR002 strain in the preparation of silage can accelerate the silage fermentation and the conversion of substances, reduce the pH value and the content of ammonia nitrogen, promote the decomposition of cellulose, reduce the loss of dry matter and nutrients, and improve the quality of the silage.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of Zurich Sciccibacter erturicensis and its applications. Background Technology

[0002] Silage is a forage storage method that utilizes microbial fermentation under anaerobic conditions to preserve the nutritional properties of green fodder. Its core is homolactic fermentation dominated by lactic acid bacteria, which lowers the pH value and inhibits putrefactive bacteria through acid production, thus achieving long-term preservation of the feed. However, the complex structure formed by lignin, cellulose, and hemicellulose intertwined in the cell walls of gramineous forage crops constitutes a physical barrier that hinders microbial enzymatic degradation. Therefore, adding exogenous microbial strains capable of degrading lignocellulose to break down structural polysaccharides into fermentable sugars is a key strategy for improving the nutritional value of silage.

[0003] The bamboo, the staple food of giant pandas (common species include *Phyllostachys nigra*, *Phyllostachys pubescens*, and *Phyllostachys aurea*), is home to a large number of epiphytic and endophytic microorganisms on its surface and inside. These microorganisms, which have long been colonized in the high-fiber, high-lignin bamboo leaf tissue, may have formed a unique lignocellulose-degrading enzyme system during the co-evolution with the host plant. *Siccibacterturicensis* is a facultative anaerobic bacterium that can grow under both aerobic and anaerobic conditions and carry out metabolic activities. Silage fermentation, however, takes place under anaerobic conditions, which allows this strain derived from bamboo leaves to naturally adapt to the oxygen conditions of the silage environment.

[0004] The currently disclosed strains of *Siccibacter turicensis* include the type strain DSM18397 isolated from fruit powder and the T9 strain used in cantaloupe cultivation, neither of which is related to silage. In 2020, Dhakal et al. isolated this strain from kangaroo feces. Although they observed signs of cellulose degradation and identified the bcs operon gene using CMC plate hydrolysis zones, its cellulase activity could not be detected in the culture supernatant, and the detection of hemicellulase and lignin-degrading enzymes was not involved, nor were any practical applications discussed. Currently, there are no reports of *Siccibacter turicensis* isolated from bamboo leaves, the staple food of giant pandas, or its application in silage, nor are there any reports of quantitative data on the simultaneous activity of hemicellulase, cellulase, and lignin-degrading enzymes in this strain. Summary of the Invention

[0005] The technical problem to be solved by this invention is: in order to isolate and screen lignocellulose-degrading bacteria with probiotic functions and apply them to the preparation of corn silage, one of the objectives of this invention is to provide a lignocellulose-degrading bacterium with probiotic functions, and another objective is to provide the application of this strain.

[0006] The technical solution of the present invention is: Siccibacter turicensis CCRCGPMR002, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 27, 2024, with accession number GDMCC NO: 65222. The address of the depository is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0007] Microbial inoculum containing Siccibacter turicensis CCRCGPMR002.

[0008] The above-mentioned application of Zürich Siccibacter turicensis CCRCGPMR002 or microbial inoculants in the preparation of silage.

[0009] Furthermore, the silage is corn silage.

[0010] Further, the preparation involves inoculating whole-plant corn at the waxy maturity stage with the aforementioned Siccibacter turicensis CCRCGPMR002 or microbial inoculant for silage fermentation.

[0011] Further, the preparation includes the following steps:

[0012] (1) Naturally sun-dry fresh whole corn plants at the waxy ripening stage until they wilt to a moisture content of 60%~65%, then chop them into pieces of 2~3 cm;

[0013] (2) Spray the wilted corn treated in step (1) evenly with the bacterial solution of *Siccibacter turicensis* CCRCGPMR002 as described in claim 1, with an inoculation amount of not less than 1×10⁻⁶. 6 CFU / g, vacuum packaged and ensiled for 30-90 days, fermentation temperature 25-35℃.

[0014] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0015] This invention uses bamboo leaves, the staple food of giant pandas, as the isolation source to screen and obtain a strain of *Siccibacter turicensis*, CCRCGPMR002. This strain is the first to quantitatively demonstrate the simultaneous presence of three enzymes: hemicellulase, cellulase, and lignin-degrading enzyme. Simultaneously, this strain exhibits excellent acid production capacity, acid resistance, bile salt resistance, resistance to artificial intestinal fluid (survival rate 84.30%), and resistance to artificial gastric fluid, among other beneficial characteristics. It also shows antibacterial effects against pathogens such as *Staphylococcus aureus*, enteropathogenic *Escherichia coli*, and *Salmonella enteritidis*, without producing hemolysis, and is sensitive to commonly used clinical antibiotics. When the microbial agent prepared using this strain was applied to the fermentation of corn silage, the lactic acid content increased by 34.9%, the soluble carbohydrate (WSC) content increased by 154.3%, the pH value decreased from 4.89 to 4.54, the ammonia nitrogen content decreased from 2.58 g / kg to 2.29 g / kg, and the dry matter and crude protein content were significantly improved, resulting in a comprehensive improvement in silage quality.

[0016] The combined presence of the aforementioned multiple beneficial and functional properties, along with the significant quantitative change in silage effect, constitutes the unexpected technical effect of this invention compared to the prior art. Specifically: (1) In the prior art, the cellulase activity of the kangaroo-derived S. zurich S. xerase strain is clearly described as too weak to be measured, while the three enzyme activities of the strain of this invention are clearly measurable and the values ​​are significant; (2) The prior art does not involve the hemicellulase and lignin-degrading enzyme activities of this strain at all, while this invention is the first to quantitatively confirm the existence of these two enzyme activities; (3) The prior art does not involve any practical application of this strain, while this invention is the first to successfully apply it to silage and achieve a significant improvement in fermentation effect. The above-mentioned unexpected technical effects are detailed in Tables 3, 4, 5, and 6 and related data in the examples.

[0017] The preservation information is as follows:

[0018] The strain Siccibacter turicensis CCRCGPMR002 was deposited on September 27, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 65222), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Attached Figure Description

[0019] Figure 1 Photographs of the colony morphology of Siccibacter turicensis CCRCGPMR002 on tryptic soybean agar (TSA medium);

[0020] Figure 2Microscopic morphology image of Siccibacter turicensis CCRCGPMR002 under a scanning electron microscope;

[0021] Figure 3 Phylogenetic tree of Siccibacter turicensis CCRCGPMR002 constructed based on 16S rRNA sequence results;

[0022] Figure 4 The growth and acid production curves of Siccibacter turicensis CCRCGPMR002 from Zurich;

[0023] Figure 5 Hydrolysis zones produced by *Siccibacter turicensis* CCRCGPMR002 on Congo Red plates;

[0024] Figure 6 Hydrolysis zones produced by *Siccibacter turicensis* CCRCGPMR002 on aniline blue plates. Detailed Implementation

[0025] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0026] In this application:

[0027] Tryptic soybean agar medium (TSA medium) (g / L): 15 g tryptic peptone, 5 g soybean peptone, 5 g sodium chloride, 20 g agar, 1000 mL distilled water, pH 7.3;

[0028] Tryptic soybean peptone liquid medium (TSB medium) (g / L): 17 g tryptic peptone, 3 g soybean peptone, 5 g sodium chloride, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, 20 g agar, 1000 mL distilled water, pH 7.3;

[0029] Congo red medium: sodium nitrate 1.0 g, disodium hydrogen phosphate 1.2 g, potassium dihydrogen phosphate 0.9 g, magnesium sulfate 0.5 g, potassium chloride 0.5 g, yeast extract 0.5 g, acid-hydrolyzed casein 0.5 g, Congo red 0.2 g, cellulose powder 5.0 g, agar 15.0 g, distilled water 1000 mL, pH 7.0;

[0030] Aniline blue medium (g / L): 200.0 g peeled potato, 15.0 g yeast extract, 5.0 g peptone, 10.0 g glucose, 5.0 g sodium chloride, 0.1 g aniline blue, 15.0 g agar, 1000 mL distilled water, pH 7.0;

[0031] Lactose-peptone medium (g / L): Peptone: 10 g; Lactose: 5 g; Beef extract: 3 g; Sodium chloride: 5 g, pH 7.3;

[0032] Artificial intestinal fluid: Dissolve 6.8 g of dipotassium hydrogen phosphate in 500 mL of distilled water, adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution, add 10 g of trypsin and dissolve it completely, bring the volume to 1000 mL with distilled water, and finally filter it through a sterile 0.22 μm microporous membrane for sterilization before use;

[0033] Artificial gastric fluid: Take 16.4 mL of 10% hydrochloric acid, dilute with distilled water, adjust the pH to 2.0, add 10g of pepsin and dissolve it completely, make up to 1000 mL with distilled water, and finally filter it through a sterile 0.22 μm microporous membrane for sterilization before use.

[0034] Columbia blood agar: purchased from Hunan Changde Bickman Biotechnology Co., Ltd.

[0035] The following specific embodiments further illustrate the present invention in detail. These descriptions are intended to explain the invention and not to limit it.

[0036] Example 1: Isolation and identification of Siccibacter turicensis CCRCGPMR002 (Zurich-derived strain)

[0037] 1.1 Isolation of Siccibacter turicensis CCRCGPMR002 from Zurich

[0038] Fresh bamboo leaf samples (including *Phyllostachys nigra*, *Phyllostachys pubescens*, and *Phyllostachys aurea*) were collected aseptically. The bamboo leaves were chopped and placed in sterile PBS buffer, shaken at 180 rpm for 30 min to fully elute the epiphytic bacteria on the leaf surface. The eluent was then serially diluted 10 times. -1 Up to 10 -5 From 10 -3 10 -4 10 -5Take 100 μL of each liquid and spread it evenly on tryptic soy agar (TSA) medium. Incubate the plates at 37 ℃ for 48 h. Select rapidly growing strains with large colonies and purify them using the streak plate method until purified strains are obtained. Transfer the purified strains to tryptic soy agar (TSA) medium and incubate at 37 ℃ for 48 h. Then preserve the culture with 30% glycerol and freeze at -80 ℃.

[0039] 1.2 Identification of *Siccibacter turicensis* (Zurich strain)

[0040] 1.2.1 Strain morphology

[0041] Single colonies of strain CCRCGPMR002 were picked and inoculated onto tryptic soybean agar (TSA medium) and incubated in an anaerobic jar at 37 °C for 24 h. The colony morphology of the strain was observed, and the cell morphology of the strain was observed by Gram staining and scanning electron microscopy.

[0042] 1.2.2 DNA extraction and PCR amplification of the strain

[0043] (1) Reagents: sterile double-distilled water, Mix (Tiangen Biotech (Beijing) Co., Ltd.).

[0044] (2) Primers

[0045] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3 and 1492R: 5'-TACGACTTAACCCCAATCGC-3'.

[0046] (3) Bacterial DNA extraction

[0047] Using a disposable inoculation loop, pick a small amount of bacterial cells into a sterile 1.5 mL centrifuge tube, then add 500 µL of sterile double-distilled water. Quickly freeze the centrifuge tube and place it in a 99 ℃ metal bath for 5 min. After removing it, vortex for 30 s. Repeat the above operation 1-2 times. Then, centrifuge the resulting bacterial cells and sterile double-distilled water mixture at 12000 rpm for 3 min. Use the supernatant as a template and store it at -20 ℃. Detect the bacterial cells by 1% agarose gel electrophoresis.

[0048] (4) Amplification of bacterial 16S rRNA gene

[0049] 16S rRNA gene amplification conditions: 95 ℃ pre-denaturation for 3 min, 94 ℃ denaturation for 30 s, 56 ℃ annealing for 30 s, 72 ℃ extension for 1.5 min, 30 cycles, total extension at 72 ℃ for 20 min. PCR products were purified using the Shanghai Sangon EZ Spin Column PCR Product Purification Kit UNlQ-1 (SK1142-N) according to the operating instructions. The purified products were sent to Sangon Biotech Co., Ltd. for sequencing.

[0050] 1.2.3 16S rRNA gene sequence analysis and phylogenetic tree construction

[0051] The sequence obtained after sequencing (SEQ ID No. 1) was searched for similarity in NCBI using BLAST software. 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 standard strain of Bacillus subtilis (AB042061) was used as the outgroup. Multiple sequence alignment analysis was performed using Clustal X software. A phylogenetic tree was constructed using the nearest neighbor method (NJ method) in MEGA 7.0 software to determine the taxonomic position of the strain.

[0052] 1.3 Experimental Results

[0053] The bacterial strain CCRCGPMR002 was isolated from bamboo leaves, the staple food of giant pandas. After inoculation on tryptic soybean agar (TSA medium) and incubation at 37°C for 24 h, the colonies were 1–2 mm in size, round and convex, with neat edges, smooth and moist surfaces, and a light yellow color. They were easily picked up. Figure 1 The bacteria are rod-shaped, measuring 1.0–1.1 × 1.5–2.5 μm in size, and are Gram-negative. Figure 2 ).

[0054] The 16S rRNA sequence of strain CCRCGPMR002 was determined, and BLAST homology comparison was performed in the NCBI database. Phylogenetic analysis was then conducted using MEGA 7.0 software. The 16S rRNA nucleotide sequence of this strain showed 99.44% homology with that of *Siccibacter turicensis*. Based on the combined results of morphological characteristics, physiological and biochemical characteristics, and 16S rRNA sequence homology analysis, strain CCRCGPMR002 was identified as *Siccibacter turicensis*. Figure 3 ).

[0055] Example 2: Growth and safety determination of Siccibacter turicensis CCRCGPMR002 (Zurich strain).

[0056] 2.1 Growth curves and acid production capacity determination of the strain

[0057] The strain CCRCGPMR002 was inoculated into a test tube containing 10 mL of lactose peptone medium and placed in an anaerobic jar at 37 °C for 48 h. During the culture period, the OD600 absorbance and pH value of the culture medium were measured every 2 h, and the growth curve and acid production curve of the strain were plotted.

[0058] 2.2 Safety Measurement

[0059] 2.2.1 Antibiotic susceptibility test

[0060] The bacterial suspension of strain CCRCGPMR002 was evenly spread on tryptic soy agar (TSA). Antibiotic susceptibility testing discs were placed on TSA according to the Clinical and Laboratory Standards Institute (CLSI) 2012 recommended method. The plates were then incubated in an anaerobic jar at 37 °C for 48 h. The diameter of the inhibition zone of each disc was recorded and compared with the CLSI standard to assess the susceptibility of the strain to antibiotics.

[0061] 2.2.2 Hemolysis test

[0062] The prepared test bacteria were inoculated into blood agar basal medium containing 5% defibrinated sheep blood using an MIT-60P multi-point bacterial inoculation instrument, with 3 replicates per group. The plates were placed in an anaerobic jar and incubated at 37 °C for 48 h. The presence or absence of hemolytic zones was observed.

[0063] 2.3 Experimental Results

[0064] 2.3.1 Growth curve of Siccibacter turicensis CCRCGPMR002

[0065] After 8 h of anaerobic culture, strain CCRCGPMR002 entered the logarithmic growth phase. With increasing growth rate, the pH of the culture medium decreased, reaching the stationary growth phase after 26 h of culture. The final pH of the fermentation broth was 4.23, indicating that this strain exhibits good growth and acid production performance under anaerobic conditions. Figure 4 ).

[0066] 2.3.2 Safety Measurement

[0067] (1) Antibiotic susceptibility test

[0068] According to the Clinical and Laboratory Standards Institute (CLSI) 2012 criteria for determining susceptibility or resistance, the strain was sensitive to polymyxin, chloramphenicol, erythromycin, cephalexin, rifampin, and ampicillin, and moderately sensitive to tetracycline and vancomycin (Table 1).

[0069] Table 1. Antibiotic susceptibility of strains

[0070]

[0071] (2) Hemolysis test

[0072] The strain CCRCGPMR002 does not produce a hemolytic zone on blood agar plates, indicating that this strain is non-hemolytic.

[0073] Example 3: Determination of the probiotic properties of *Siccibacter turicensis* CCRCGPMR002 (Zurich strain).

[0074] 3.1 Tolerance of the strain

[0075] (1) Preparation of bacterial culture

[0076] The strain CCRCGPMR002 was inoculated into lactic acid bacteria liquid medium (MRS medium) and cultured in an anaerobic tank for 48 h. After centrifugation at 10000 r / min and 4 °C for 5 min, the culture was washed twice with 2 mL PBS solution and resuspended to prepare a bacterial suspension for later use.

[0077] (2) Acid resistance: 5% of the bacterial suspension was inoculated into MRS medium with pH values ​​of 2.0 and 3.0. After incubation at 37℃ for 0 h and 4 h respectively, viable bacteria were counted and the survival rate was calculated according to the formula.

[0078] (3) Bile salt tolerance: 5% of the bacterial suspension was inoculated into MRS medium containing bovine bile salt at a mass concentration of 3 g / L. After incubation at 37 °C for 0 h and 4 h, viable bacteria were counted and the survival rate was calculated according to the formula.

[0079] (4) Test for resistance to artificial gastrointestinal fluid: 5% of the bacterial suspension was inoculated into artificial intestinal fluid and artificial gastric fluid respectively. After incubation at 37 ℃ for 0 h and 4 h respectively, viable bacteria were counted and the survival rate was calculated according to the formula.

[0080] The strain's resistance to acid, bile salts, and artificial gastrointestinal fluids was calculated using the following formula:

[0081]

[0082] Where A0 is the number of viable bacteria at 0 h and A1 is the number of viable bacteria at 4 h.

[0083] 3.2 Antibacterial activity test

[0084] 2.5 mL of bacterial culture was inoculated into 50 mL of tryptic soybean agar (TSA medium) and incubated in an anaerobic jar at 37 °C for 48 h. After centrifugation at 4 °C and 8000 r / min for 10 min, the supernatant was collected in an Oxford cup to determine the antibacterial activity of the strain against Staphylococcus aureus (ATCC 27217), enteropathogenic Escherichia coli (Escherichiacoli ATCC 25922, EPEC), and Salmonella enterica (ATCC 13076).

[0085] 3.3 Experimental Results

[0086] 3.2.1 Tolerance of the strain

[0087] The tolerance test results of strain CCRCGPMR002 showed that the strain had different degrees of tolerance to various test items, and its survival rate was >70%. The strain could grow under acidic conditions of pH 2.0 and pH 3.0, showing good acid resistance. The survival rate of the strain after treatment with 0.3% bile salts for 4 h was 71.21%, which showed good bile salt tolerance. The survival rates of the strain after treatment with artificial simulated intestinal fluid and gastric fluid for 4 h were 84.30% and 72.59%, respectively (Table 2).

[0088] Table 2. Tolerance of strains

[0089]

[0090] 3.2.2 Antibacterial ability of the strain

[0091] The antibacterial results showed that strain CCRCGPMR002 had antibacterial effects against three indicator bacteria: Staphylococcus aureus, enteropathogenic Escherichia coli, and Salmonella enteritidis (Table 3).

[0092] Table 3. Antibacterial ability of strains

[0093]

[0094] Example 4: Determination of enzyme activity of *Siccibacter turicensis* CCRCGPMR002 (Zurich strain)

[0095] 4.1 Determination of Lignocellulose Degradation Capacity

[0096] 4.1.1 Qualitative determination

[0097] The strain CCRCGPMR002 was evenly spread on tryptic soybean agar (TSA medium) and placed in a 37 ℃ biochemical incubator until the strain grew evenly on the entire surface of the medium. Mycelial cakes were made using a 5 mm diameter punch and placed upside down on Congo red medium and aniline blue medium, respectively. After incubation in an anaerobic jar at 37 ℃ for 3-5 days, the diameter of the hydrolysis zone (D) and the diameter of the colony (d) were measured, and the ratio (D / d) was calculated. Three replicates were set up.

[0098] 4.1.2 Quantitative determination

[0099] The strain CCRCGPMR002 was inoculated into liquid culture medium and cultured at 37°C for 24 h. The bacterial cells were collected by centrifugation at 6000 g for 10 min, the supernatant was discarded, and the bacterial suspension was resuspended in physiological saline and adjusted to OD200. 600 The value was 0.6. 1 mL of bacterial suspension was inoculated into 9 mL of bamboo powder degradation medium with bamboo powder as the sole carbon source and cultured at 37°C for 24 h. Subsequently, the fermentation broth was centrifuged at 4°C (8000 g, 10 min), and the supernatant was collected for enzyme activity analysis.

[0100] Hemicellulose degradation capacity was determined using a neutral xylanase activity assay kit; cellulose degradation capacity was determined using a β-glucosidase activity assay kit; and lignin degradation capacity was determined using a manganese peroxidase assay kit. All kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0101] 4.2 Experimental Results

[0102] Tests showed that strain CCRCGPMR002 possessed the ability to produce hemicellulose, cellulose, and lignin degradation products, as well as the ability to produce lipase. Figure 5 , Figure 6 (Table 4) The enzyme activities were 27.18 U / mL, 39.57 U / mL and 19.32 U / mL, respectively (Table 4).

[0103] Table 4 Enzyme production capacity of strains

[0104]

[0105] 4.3 Technical Effect Analysis

[0106] The results of this embodiment demonstrate that the strain CCRCGPMR002 of this invention is the first to quantitatively confirm, using an enzyme activity assay kit, that it simultaneously possesses three enzyme activities: hemicellulase, cellulase, and lignin-degrading enzyme. In contrast, while the *Siccibacter turicensis* strain isolated from kangaroo feces by Dhakal et al. in 2020 showed signs of cellulose degradation via hydrolysis zones on CMC plates, its enzyme production capacity was too weak to be detected in culture supernatant and cell lysate, and this study completely neglected the detection of hemicellulase and lignin-degrading enzyme activities. The three enzyme activities of the strain of this invention were accurately quantified using commercially available kits, reaching 27.18 U / mL, 39.57 U / mL, and 19.32 U / mL, respectively. Furthermore, hydrolysis zones on Congo red and aniline blue plates further corroborate the lignocellulose degradation ability of this strain. The results showed that not all strains of *Siccibacter erturicensis* from Zurich possessed practically applicable lignocellulose degradation capabilities, and there were significant differences in enzyme activity intensity among strains from different isolation sources. The unique ecological niche of bamboo leaves, the staple food of giant pandas, provides a unique microbial resource for screening lignocellulose-degrading bacteria with practical application value.

[0107] Example 5 Preparation of microbial inoculants

[0108] This embodiment provides a microbial inoculant, the preparation method of which is as follows:

[0109] The strain CCRCGPMR002 was streaked onto tryptic soy agar (TSA) medium, and single colonies were picked and cultured on TSA medium. The culture was then incubated in an anaerobic chamber at 37 °C for 48 h to obtain a seed culture. This seed culture was then inoculated onto TSA medium at a 5% (v / v) inoculum for scale-up culture, and incubated in an anaerobic chamber at 37 °C for 48 h to obtain the microbial inoculum, with an effective viable count of 5.42 × 10⁻⁶. 9 CFU / g, pH value 6.8~7.0.

[0110] Example 6: Application of *Siccibacter turicensis* CCRCGPMR002 in the preparation of silage.

[0111] 6.1 Preparation of Silage

[0112] Fresh, fully ripe corn plants were naturally air-dried until their moisture content reached 60%–65%. The stalks were then chopped to 2–3 cm. One kilogram of the chopped corn stalks was weighed and evenly sprayed onto the wilted corn using the microbial inoculant from Example 5. The inoculant solution and silage material were thoroughly mixed to achieve an inoculum size of 1 × 10⁻⁶. 6 CFU / g was packed into vacuum-sealed polyethylene bags, compacted, and then vacuum-sealed to produce bagged silage. The treatment group was supplemented with *Siccibacter turicensis* CCRCGPMR002, while the control group underwent natural fermentation (with an equal amount of sterile water added). Each treatment was replicated three times. Silage was stored for 30–90 days at a fermentation temperature of 25–35°C.

[0113] 6.2 Determination of Fermentation Quality of Silage

[0114] After fermentation at each time point, the silage was opened, and 10 g of fresh sample was immediately weighed and added to 90 ml of distilled water. The mixture was then extracted overnight at 4 °C. The extract was transferred to a centrifuge tube and centrifuged at 10,000 r / min for 5 min at 4 °C to obtain the supernatant. A portion of the supernatant was used for pH and ammonia nitrogen determination, while another portion was filtered through a 0.22 μm organic filter membrane for organic acid content determination. The pH of the silage extract was measured using a pH meter, the ammonia nitrogen (NH3-N) content was determined using the phenol-hypochlorous acid colorimetric method, and the lactic acid (LA) and acetic acid (AA) contents were determined using high-performance liquid chromatography (HPLC).

[0115] 6.3 Determination of nutrient composition of silage

[0116] The silage was dried to constant weight, and the dry matter (DM) content was determined. The dried sample was then ground through a 40-mesh sieve, and the relevant nutrient components of the silage were determined. The Kjeldahl method was used to determine the crude protein (CP) content, the anthrone-sulfuric acid colorimetric method was used to determine the soluble carbohydrate (WSC) content, and the Panthen fiber assay was used to determine the neutral detergent fiber (NDF) and acid detergent fiber (ADF) content.

[0117] 6.4 Experimental Results

[0118] 6.4.1 Determination of Fermentation Quality of Silage

[0119] After 60 days of ensiling, samples were taken from the bags to test the effect of added microbial agents on the fermentation quality of corn silage. As shown in Table 5, compared with the group without added microbial agents, the addition of Zürich Siccibacter turicensis CCRCGPMR002 significantly increased the content of lactic acid and acetic acid in the silage, reduced the pH value, decreased the production of ammonia nitrogen, and improved the quality of corn silage.

[0120] Table 5 Fermentation quality of silage

[0121]

[0122] Note: Different lowercase letters in the superscript of the same data indicate significant differences (P<0.05), and no letter indicates no significant differences (P>0.05).

[0123] 6.4.2 Determination of nutrient composition of silage

[0124] Table 6 shows that the addition of *Siccibacter turicensis* CCRCGPMR002 significantly affected the contents of dry matter, crude protein, soluble carbohydrates, neutral detergent fiber, and acid detergent fiber in corn silage. This indicates that the addition of *Siccibacter turicensis* CCRCGPMR002 not only retains more dry matter and crude protein, reducing the loss of dry matter and nutrients, but also promotes the degradation of cellulose in corn, thereby improving the utilization rate of corn silage.

[0125] Table 6 Nutritional quality of silage

[0126]

[0127] Note: Different lowercase letters in the superscript of data from the same row indicate significant differences (P<0.05).

[0128] 6.5 Technical Effect Analysis

[0129] The results of this embodiment demonstrate that the strain of the present invention produced technical effects in silage applications that exceeded reasonable expectations:

[0130] (1) Significantly increased lactic acid content: The lactic acid content increased from 35.23 g / kg to 47.53 g / kg, an increase of 34.9%. Existing studies have shown that Pediococcus pentosaceus PL12 can increase the lactic acid content of corn silage by 68.41%; although the lactic acid increase of the strain of this invention is lower than that level, the core contribution of this invention is to provide a novel microbial resource for silage additives, rather than replacing the effects of existing lactic acid bacteria additives.

[0131] (2) Significant increase in soluble carbohydrates (WSC) against the trend: WSC content increased from 22.74 g / kg to 57.83 g / kg, an increase of 154.3%. Conventional lactic acid bacteria additives usually consume WSC during silage, resulting in a decrease in its content; however, the strain of this invention degrades cellulose in corn stalks into soluble sugars by secreting three enzyme activities: hemicellulase, cellulase, and lignin-degrading enzyme, achieving a significant increase in WSC against the trend, which has not been reported in conventional silage lactic acid bacteria additives.

[0132] (3) Significant decrease in pH and ammonia nitrogen: The pH value decreased from 4.89 to 4.54, and the ammonia nitrogen content decreased from 2.58 g / kg to 2.29 g / kg. The low pH value effectively inhibited the growth of putrefactive bacteria; the decrease in ammonia nitrogen reflected the reduction in protein degradation, which is conducive to the retention of more nutrients.

[0133] (4) Uniqueness of the overall effect: In 2020, Dhakal et al. isolated the same strain from kangaroo feces, which had cellulase activity, but it could not be detected in the culture supernatant and cell lysate. The strain isolated from bamboo leaves, the staple food of giant pandas, achieved accurate quantification of the activities of three lignocellulose-degrading enzymes for the first time, and successfully applied the strain to silage for the first time. Through the integrated mode of one strain and multiple enzymes, it synergistically acts on the plant cell wall of corn silage raw material, effectively destroying the structure of cellulose and lignin, releasing more soluble carbohydrates, and ultimately achieving a comprehensive improvement in silage quality. It has significant advantages in reducing additive costs and operational complexity.

Claims

1. Siccibacter turicensis CCRCGPMR002, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO: 65222.

2. A microbial agent containing *Siccibacter turicensis* CCRCGPMR002 as described in claim 1.

3. The application of the Zurich Siccibacter turicensis CCRCGPMR002 as described in claim 1 or the microbial agent as described in claim 2 in the preparation of silage.

4. The application according to claim 3, characterized in that, The silage is corn silage.

5. The application according to claim 3, characterized in that, The preparation refers to inoculating whole-plant corn at the waxy maturity stage with *Siccibacter turicensis* as described in claim 1 or the microbial agent as described in claim 2, and then carrying out silage fermentation.

6. The application according to claim 5, characterized in that, The preparation includes the following steps: (1) Naturally sun-dry fresh whole corn plants at the waxy ripening stage until they wilt to a moisture content of 60%~65%, then chop them into pieces of 2~3 cm; (2) Spray the wilted corn treated in step (1) evenly with the bacterial solution of *Siccibacter turicensis* CCRCGPMR002 as described in claim 1, with an inoculation amount of not less than 1×10⁻⁶. 6 CFU / g, vacuum packaged and ensiled for 30-90 days, fermentation temperature 25-35℃.

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