Lactobacillus animalis strain ccrcgpyq001 and applications thereof

Abstract

The application discloses an animal lactobacillus CCRCGPYQ001 and application thereof, and belongs to the field of microorganisms, wherein the strain is preserved in the Guangdong Microbial Culture Collection Center, and has a preservation number of GDMCC NO: 65224; the strain of the application is separated from bamboo culms of bamboo eaten by giant pandas, has the abilities of decomposing lignocellulose, producing extracellular polysaccharide and gamma-aminobutyric acid, has good inhibiting effects and coaggregation abilities on pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella and pseudomonas aeruginosa, has moderate to high sensitivity to commonly used antibiotics in clinic, and does not produce hemolysis phenomenon. The microbial agent prepared from the strain can retain more dry matter and crude protein content in corn silage feed, and reduce the loss of dry matter and nutrients through the application in corn silage feed fermentation.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of animal lactobacillus (Ligilactobacillus sanimalis) and its applications. Background Technology

[0002] The preparation of silage relies on lactic acid bacteria converting soluble sugars into organic acids under anaerobic conditions, resulting in a rapid decrease in pH, thereby inhibiting the proliferation of putrefactive bacteria and ensuring long-term preservation of the feed. In the cell walls of silage materials such as corn, cellulose, hemicellulose, and lignin are tightly cross-linked, forming a natural barrier that hinders microbial enzymatic hydrolysis and animal digestion. Therefore, obtaining lactic acid bacteria strains with both high acid-producing capacity and lignocellulose-degrading activity is an important direction for developing novel silage additives.

[0003] Bamboo culms, the staple food of giant pandas (common species include arrow bamboo, walking stick bamboo, and white-striped bamboo), are rich in cellulose, hemicellulose, and lignin, and their surfaces and interiors are colonized by a large number of epiphytic and endophytic microorganisms. Studies have already isolated endophytic bacteria with cellulose-degrading capabilities from these bamboo culms. *Ligilactobacillus animalis*, an anaerobic or facultative anaerobic bacterium, can metabolize normally under anaerobic conditions, making it well-suited to the anaerobic environment of silage fermentation. The low-oxygen microenvironment within the bamboo culm tissue also provides natural conditions for the colonization of facultative anaerobic bacteria. Therefore, isolating lactic acid bacteria from bamboo culms and applying them to corn silage has both ecological niche and functional rationale and scientific basis.

[0004] The probiotic functions of *Ligilactobacillus animalis* (formerly known as *Lactobacillus animalis*) have been partially revealed, including acid and bile salt tolerance, antibacterial properties, and antioxidant effects. However, no reports have been found in the existing technology regarding the lignocellulose degradation ability of this strain—although it can ferment cellobiose, the mechanism of cellobiose fermentation is completely different from the active secretion of cellulase, hemicellulase, or lignin-degrading enzymes to degrade lignocellulose macromolecules. Furthermore, no quantitative data on EPS and GABA production by this strain have been found in the existing technology. It is noteworthy that although there have been preliminary studies on the use of *Lactobacillus animalis* for alfalfa silage, alfalfa (legume) and maize (grass) differ significantly in cell wall composition and soluble sugar content, and the previous studies did not involve an evaluation of lignocellulose degradation ability; therefore, it is not a simple raw material substitution.

[0005] There are currently no reports on the isolation source of *Ligilactobacillus sanimalis* from bamboo stalks, the staple food of giant pandas, and its application in corn silage. There are also no reports on the quantitative data of the activities of three enzymes—hemicellulase, cellulase, and lignin-degrading enzyme—as well as the yield data of EPS and GABA. Summary of the Invention

[0006] The technical problem to be solved by this invention is: in order to isolate and screen probiotic lactic acid bacteria with cellulose degradation function and apply them to the preparation of corn silage, one of the objectives of this invention is to provide a probiotic lactic acid bacteria with cellulose degradation function, and another objective is to provide the application of this strain.

[0007] The technical solution of the present invention is: Ligilactobacillus animalis CCRCGPYQ001, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 27, 2024, with accession number GDMCC NO: 65224. The address of the depository is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0008] Microbial agents containing the aforementioned *Ligilactobacillus animalis* CCRCGPYQ001.

[0009] The application of the aforementioned *Ligilactobacillus animalis* CCRCGPYQ001 or microbial inoculants in the preparation of silage.

[0010] Furthermore, the silage is corn silage.

[0011] Further, the preparation involves inoculating whole corn plants at the waxy maturity stage with the aforementioned *Ligilactobacillus animalis* CCRCGPYQ001 or a microbial agent for silage fermentation.

[0012] Further, the preparation includes the following steps:

[0013] (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;

[0014] (2) Spray the wilted corn treated in step (1) evenly with the bacterial solution of *Siccibacter turicensi* CCRCGPYQ001 described above, with an inoculation amount of not less than 1×10⁻⁶. 8CFU / g, vacuum packaged and ensiled for 30-90 days, fermentation temperature 25-35℃.

[0015] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0016] This invention uses bamboo stalks, the staple food of giant pandas, as the isolation source to screen and obtain a strain of *Ligilactobacillus animalis*, CCRCGPYQ001. This strain is the first to quantitatively demonstrate the simultaneous presence of three enzymes: hemicellulase, cellulase, and lignin-degrading enzyme, filling a gap in existing research on the lignocellulose degradation capabilities of this species. Simultaneously, this strain exhibits excellent acid production capacity, acid resistance, bile salt resistance, resistance to artificial intestinal fluid, and resistance to artificial gastric fluid, among other beneficial properties. It shows significant antibacterial effects against pathogens such as *Staphylococcus aureus*, enteropathogenic *Escherichia coli*, *Salmonella enteritidis*, and *Pseudomonas aeruginosa*, with a self-aggregation rate of 39.16% and a co-aggregation rate against pathogens reaching a maximum of 25.85%. Its DPPH free radical scavenging rate is 45.43%, and its hydroxyl free radical scavenging rate is 62.41%. It does not produce hemolysis and is sensitive to commonly used clinical antibiotics. This strain also possesses the ability to produce extracellular polysaccharides and γ-aminobutyric acid (GABA). When the microbial agent prepared using this strain was applied to the fermentation of corn silage, the lactic acid content increased by 111.8%, the soluble carbohydrate (WSC) content increased by 247.1%, the pH value decreased from 4.89 to 4.12, the ammonia nitrogen content decreased from 2.58 g / kg to 2.02 g / kg, the dry matter content increased by 21.6%, and the crude protein content increased by 31.1%, resulting in a comprehensive improvement in silage quality.

[0017] The concentrated presence of the aforementioned multiple properties in a single strain of *Ligilactobacillus animalis* constitutes an unexpected technical effect of this invention compared to existing technologies. Specifically: (1) Although existing technologies have reported various probiotic properties of this strain, they have never addressed its lignocellulose degradation ability. This invention is the first to quantitatively confirm the activity of three lignocellulose-degrading enzymes in this strain; (2) Existing technologies have not provided data on the yield of EPS and GABA in this strain. This invention is the first to provide quantitative data; (3) Although existing technologies involve alfalfa silage, alfalfa belongs to the legume family, while corn belongs to the grass family. The two differ significantly in cell wall composition, soluble sugar content, and buffer capacity, and their performance in alfalfa silage cannot predict their effect in corn silage. This invention is the first to successfully apply this strain to corn silage and achieve a fermentation improvement effect significantly superior to conventional lactic acid bacteria additives.

[0018] The preservation information is as follows:

[0019] The strain of *Ligilactobacillus animalis* CCRCGPYQ001 was deposited on September 27, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 65224), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Attached Figure Description

[0020] Figure 1 Photographs showing the colony morphology of *Ligilactobacillus animalis* CCRCGPYQ001 on lactic acid bacteria medium (MRS medium);

[0021] Figure 2 Microscopic morphological image of Ligilactobacillus animalis CCRCGPYQ001 under a scanning electron microscope;

[0022] Figure 3 Phylogenetic tree of Ligilactobacillus animalis CCRCGPYQ001 constructed based on 16S rRNA sequence results;

[0023] Figure 4 The growth and acid production curves of Ligilactobacillus animalis CCRCGPYQ001 are shown.

[0024] Figure 5 Hydrolysis zones produced by *Ligilactobacillus animalis* CCRCGPYQ001 on Congo red plates;

[0025] Figure 6 Hydrolysis zones produced by *Ligilactobacillus animalis* CCRCGPYQ001 on aniline blue plates. Detailed Implementation

[0026] 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 products.

[0027] In this application:

[0028] Lactic acid bacteria culture medium (MRS medium): 10 g peptone, 5 g beef extract powder, 4 g yeast extract powder, 20 g glucose, 1 g Tween-80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar, pH 7.0;

[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 powder 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] 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.

[0032] 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, 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] Columbia blood agar: purchased from Hunan Changde Bickman Biotechnology Co., Ltd.

[0034] The following specific embodiments further illustrate the present invention in detail. These descriptions are intended to explain the invention and not to limit it.

[0035] Example 1: Isolation and identification of *Ligilactobacillus animalis* CCRCGPYQ001

[0036] 1.1 Isolation of *Ligilactobacillus animalis* CCRCGPYQ001

[0037] Fresh bamboo culms (including *Phyllostachys nigra*, *Phyllostachys pubescens*, and *Phyllostachys aurea*) were aseptically collected from the bamboo culms. The epidermis and phloem tissue were scraped off with a sterile scalpel, chopped, and placed in sterile PBS buffer. The culms were then thoroughly shaken to release surface epiphytes and superficial endophytes. The eluent was serially diluted 10^-10 times. -1 Up to 10 -5 From 10 -3 10 -4 10 -5 Take 100 μL of each liquid and spread it evenly on lactic acid bacteria medium (MRS 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 lactic acid bacteria medium (MRS medium), incubate at 37 ℃ for 48 h, then preserve them with 30% glycerol and freeze at -80 ℃.

[0038] 1.2 Identification of *Ligilactobacillus animalis* CCRCGPYQ001

[0039] 1.2.1 Strain morphology

[0040] Single colonies of strain CCRCGPYQ001 were picked and inoculated onto lactic acid bacteria medium (MRS medium), and placed in an anaerobic jar at 37 ℃ for 24 h. The colony morphology of the strain was observed, Gram staining was performed, and the cell morphology of the strain was observed under a scanning electron microscope.

[0041] 1.2.2 DNA extraction and PCR amplification of the strain

[0042] (1) Reagents: sterile double-distilled water, Mix (Tiangen Biotech (Beijing) Co., Ltd.).

[0043] (2) Primers

[0044] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3 and

[0045] 1492R: 5'-TACGACTTAACCCCAATCGC-3'.

[0046] (3) Bacterial DNA extraction

[0047] A small amount of bacterial cells was picked up using a disposable inoculation loop and centrifuged in a sterile 1.5 mL container. Then, 500 µL of sterile double-distilled water was added, and the centrifuged mixture was quickly frozen. The mixture was then placed in a 99 °C metal bath for 5 min, removed, and vortexed for 30 s. This process was repeated 1-2 times. The resulting bacterial cells and sterile double-distilled water mixture was then centrifuged at 12000 rpm for 3 min. The supernatant was used as a template and stored at -20 °C. The mixture was then analyzed by 1% agarose gel electrophoresis.

[0048] (4) Bacterial 16S rRNA gene amplification

[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 strain CCRCGPYQ001 of *Ligilactobacillus animalis* was isolated from the bamboo stalks, the staple food of giant pandas. After inoculation onto lactic acid bacteria medium (MRS medium) and incubation at 37 °C for 24 h in an anaerobic jar, the colonies were 1–1.5 mm in size, round and convex in shape, with neat edges, a smooth and moist surface, and a milky white color. They were easily picked up. Figure 1 The bacteria are short rod-shaped, measuring 0.9–1.2 × 3.1–4.3 μm in size, and are Gram-positive. Figure 2 ).

[0054] The 16S rRNA sequence of strain CCRCGPYQ001 was determined, and BLAST homology comparison was performed in the NCBI database. Phylogenetic analysis was then conducted using MEGA 7.0 software. The strain showed 100% homology with the 16S rRNA nucleotide sequence of *Ligilactobacillus animalis*. Based on the combined results of morphological characteristics, physiological and biochemical characteristics, and 16S rRNA sequence homology analysis, strain CCRCGPYQ001 was identified as *Ligilactobacillus animalis*. Figure 3 ).

[0055] Example 2: Growth and safety determination of Ligilactobacillus animalis CCRCGPYQ001

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

[0057] The strain CCRCGPYQ001 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 CCRCGPYQ001 was evenly spread on lactic acid bacteria medium (MRS medium). According to the Clinical and Laboratory Standards Institute (CLSI) recommended method in 2012, antibiotic susceptibility test strips were placed on lactic acid bacteria medium (MRS medium). The plates were placed in an anaerobic jar and incubated at 37 °C for 48 h. The diameter of the inhibition zone of each susceptibility test strip was recorded and compared with the CLSI standard to evaluate the susceptibility of the strain to antibiotics.

[0061] 2.2.2 Hemolysis test

[0062] The prepared test bacteria were inoculated into Columbia blood agar medium 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 and acid production curves of *Ligilactobacillus animalis* CCRCGPYQ001

[0065] After 6 h of anaerobic culture, strain CCRCGPYQ001 entered the logarithmic growth phase. With increasing growth rate, the pH of the culture medium decreased, reaching a stable growth phase after 14 h of culture. The final pH of the fermentation broth was 3.87, 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, ampicillin, and vancomycin, and moderately sensitive to tetracycline (Table 1).

[0069] Table 1. Antibiotic susceptibility of strains

[0070]

[0071] (2) Hemolysis test

[0072] The strain CCRCGPYQ001 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 Ligilactobacillus animalis CCRCGPYQ001

[0074] 3.1 Tolerance of the strain

[0075] (1) Preparation of bacterial culture

[0076] The strain CCRCGPYQ001 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 bacterial suspension was prepared by washing twice with 2 mL PBS solution and resuspending.

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

[0078] (3) Bile salt tolerance: 5% of the lactic acid bacteria 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 tolerance to artificial gastrointestinal fluid: 5% of the lactic acid bacteria 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 Determination of the antibacterial ability of the strain

[0084] 2.5 mL of bacterial culture was inoculated into 50 mL of lactic acid bacteria medium (MRS medium), and cultured 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 (ATCC25922, EPEC), Salmonella enterica (ATCC 13076), and Pseudomonas aeruginosa (ATCC 27853).

[0085] 3.3 Detection of the self-aggregation and co-aggregation abilities of the strain

[0086] 3.3.1 Detection of the self-aggregation ability of the strain

[0087] The bacterial culture to be tested, after being cultured for 12 h, was prepared into OD using PBS solution. 600 For a dilution of 0.6 ± 0.05 (denoted as A0), take 1 mL of the dilution into a centrifuge tube, shake for 10 s, incubate at 37℃ for 5 h, and then take the supernatant to measure the OD. 600 (denoted as A), the self-aggregation rate is (1-A / A0)×100%.

[0088] 3.3.2 Detection of bacterial coagulation ability

[0089] The test bacteria and the pathogen marker strains from section 3.2, cultured for 12 h, were adjusted to OD values ​​using PBS solution. 600 For a dilution of 0.6 ± 0.05, take 0.5 mL of each solution and transfer it to the same centrifuge tube. Mix and vortex for 10 s, then record the initial OD. 600 (Recorded as A0), let the mixture stand at 37℃ for 5 hours, then take the upper layer of liquid to measure the OD. 600 (denoted as A), the co-aggregation rate is (1-A / A0)×100%.

[0090] 3.5 Antioxidant capacity determination

[0091] Pick a single colony and place it in MRS medium. After incubating for 24 h, centrifuge at 8000 r / min for 10 min at 4℃ and collect the supernatant.

[0092] (1) Determination of DPPH free radical scavenging ability

[0093] Take 0.5 mL of bacterial suspension and 0.5 mL of DPPH (0.2 mmol / L, prepared with anhydrous ethanol), mix thoroughly, and react at room temperature in the dark for 30 min, then centrifuge at 6000 r / min for 10 min. Take the supernatant and measure the OD value at 517 nm. Use an equal volume of PBS solution instead of the sample solution as a control group, and use an equal volume of PBS solution and anhydrous ethanol mixture as a blank to zero the sample. The experiment was repeated 3 times. The scavenging rate of DPPH free radicals was calculated as follows:

[0094] DPPH radical scavenging rate = (1-A) 517（样品） / A 517（对照） ) × 100%

[0095] (2) Determination of hydroxyl radical scavenging ability

[0096] Take 0.5 mL of *Lactobacillus plantarum* suspension, 0.5 mL of 2.5 mmol / L 1,10-phenanthroline, 0.5 mL of PBS (pH 7.4), and 0.5 mL of 2.5 mmol / L FeSO4, respectively. After mixing, add 0.5 mL of 20 mmol / L H2O2 and incubate at 37 ℃ for 90 min. Measure the OD value at 536 nm. Replace 0.5 mL of *Lactobacillus plantarum* suspension with 0.5 mL of PBS as the blank group; replace 0.5 mL of H2O2 with 0.5 mL of distilled water as the control group. The experiment was repeated 3 times. The ability to scavenge hydroxyl radicals was calculated as follows:

[0097] Hydroxyl radical scavenging rate = (A 536（样品） - A 536（空白） ) / (A 536（对照） -A 536（空白）) × 100%

[0098] In the formula: A 536（样品） A 536（对照） and A 536（空白） The absorbance values ​​measured at 536 nm are for the sample group, control group, and blank group, respectively.

[0099] 3.3 Experimental Results

[0100] 3.2.1 Tolerance of the strain

[0101] The tolerance test results of strain CCRCGPYQ001 showed that strain Ligilactobacillus sanimalis CCRCGPYQ001 exhibited varying degrees of tolerance to all test items, with a survival rate >80%. Strain CCRCGPYQ001 could grow under acidic conditions at pH 2.0 and pH 3.0, demonstrating good acid tolerance. After treatment with 0.3% bile salts for 4 h, the survival rate was 93.27%, indicating excellent bile salt tolerance. The survival rates of strain CCRCGPYQ001 after treatment with simulated intestinal and gastric fluids for 4 h were 81.46% and 90.12%, respectively (Table 2).

[0102] Table 2. Tolerance of strains

[0103]

[0104] 3.2.2 Antibacterial ability of the strain

[0105] The antibacterial results showed that strain Ligilactobacillus animalis CCRCGPYQ001 had antibacterial effects against four indicator bacteria: Staphylococcus aureus, enteropathogenic Escherichia coli, Salmonella enteritidis, and Pseudomonas aeruginosa (Table 3).

[0106] Table 3. Antibacterial ability of strains

[0107]

[0108] 3.3 Detection of the self-aggregation and co-aggregation abilities of the strain

[0109] The strain CCRCGPYQ001 exhibited good self-aggregation and co-aggregation abilities, with a self-aggregation rate of 39.16%. The co-aggregation rates against four indicator bacteria—Staphylococcus aureus (ATCC 27217), enteropathogenic Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076), and Pseudomonas aeruginosa (ATCC 278534)—were 25.85%, 9.73%, 2.90%, and 17.53%, respectively.

[0110] 3.4 Detection of the antioxidant capacity of the strain

[0111] The antioxidant capacity of lactic acid bacteria was evaluated by detecting their scavenging rates of DPPH and hydroxyl radicals. The results showed that strain CCRCGPYQ001 had scavenging rates of 45.43% and 62.41% for DPPH and hydroxyl radicals, respectively.

[0112] Example 4: Determination of major metabolites of *Ligilactobacillus animalis* CCRCGPYQ001

[0113] 4.1 Determination of major metabolites of the strain

[0114] 4.1.1 Detection of Extracellular Polysaccharide (EPS) Production Capacity

[0115] Pick a single colony of the test bacteria and place it in MRS medium, then incubate for 12 h (adjusting the OD of the bacterial culture). 600 (1) Detection of water-soluble EPS: Mix the collected supernatant with 800 mg / mL trichloroacetic acid (TCA) solution to make the TCA concentration 40 mg / mL after mixing. Let stand overnight at 4℃, then centrifuge at 8000 r / min for 10 min at 4℃. Transfer the supernatant into a dialysis bag and dialyze at 4℃ for 24 h. Take 250 µL of the dialyzed supernatant and mix it with 250 µL of 6% (w / v) phenol solution and 1 mL of concentrated sulfuric acid. Let stand at room temperature. After cooling, measure the OD of the reaction solution. 490 Value. (2) Detection of alkali-soluble EPS: Collect the centrifuged bacterial cells, wash three times with PBS solution, add 1 mL of 1 mol / L sodium hydroxide solution, mix well at room temperature, let stand at 37℃ for 2 h, take the supernatant after centrifugation, take 250 µL of the supernatant and mix it with 250 µL of 6% (w / v) phenol solution and 1 mL of concentrated sulfuric acid, let stand at room temperature. After cooling, measure the OD of the reaction solution. 490 Values. Standard curve construction: A standard curve was constructed using glucose concentrations of 3.125 mg / L, 6.25 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, and 100 mg / L, and the sugar content was determined.

[0116] 4.1.2 Determination of the strain's ability to produce γ-aminobutyric acid (GABA)

[0117] Pick a single colony of the test bacteria and incubate it in GYP medium for 12 h. Centrifuge at 8000 rpm for 10 min at 4 °C. Transfer 500 µL of the supernatant to a centrifuge tube and add 200 µL of borate buffer (0.2 M pH 9.0), 1 mL of 6% (w / v) phenol solution, and 400 µL of 5.5% (w / v) sodium hypochlorite solution. After mixing the liquids in the tube, heat in a boiling water bath for 10 min, then immediately incubate on ice for 20 min. Once a blue color appears, add 2 mL of 95% ethanol to the tube, shake well, and measure the OD. 645 Values ​​were determined by preparing solutions of 0 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, and 1.0 g / L using GABA standards, plotting standard curves, and calculating the corresponding GABA content based on the standard curve equation.

[0118] 4.2 Determination of Lignocellulose Degradation Capacity

[0119] 4.2.1 Qualitative determination

[0120] The strain CCRCGPYQ001 was evenly spread on lactic acid bacteria medium (MRS 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 inverted on Congo red medium and aniline blue medium, respectively. After being placed in an anaerobic jar and incubated 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 of the two (D / d) was calculated. Three replicates were set up.

[0121] 4.2.2 Quantitative determination

[0122] The bacterial strain 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.

[0123] 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.

[0124] 4.3 Experimental Results

[0125] 4.3.1 Determination of major metabolites of the strain

[0126] The strain CCRCGPYQ001 produced 25.14 mg / L of water-soluble EPS and 15.75 mg / L of non-water-soluble EPS, and 0.52 g / L of GABA.

[0127] 4.3.2 Determination of the strain's ability to degrade lignocellulose

[0128] Tests showed that strain CCRCGPYQ001 possessed the ability to produce hemicellulose, cellulose, and lignin degradation products, as well as the ability to produce lipase. Figures 5-6 (Table 4) The enzyme activities were 29.61 U / mL, 53.26 U / mL and 25.59 U / mL, respectively (Table 4).

[0129] Table 4 Enzyme production capacity of strains

[0130] 4.3.3 Technical Effect Analysis

[0131] The results of this embodiment demonstrate for the first time that the strain CCRCGPYQ001 of this invention quantitatively confirms that *Ligilactobacillus animalis* CCRCGPYQ001 simultaneously possesses three enzyme activities: hemicellulase (29.61±1.17 U / mL), cellulase (53.26±1.84 U / mL), and lignin-degrading enzyme (25.59±0.65 U / mL), and forms distinct hydrolysis zones on Congo red and aniline blue plates (D / d 5.33 and 2.56, respectively). Furthermore, this invention provides for the first time quantitative data on EPS (water-soluble 25.14 mg / L, insoluble 15.75 mg / L) and GABA (0.52 g / L) of this strain.

[0132] Compared to existing technologies, although the probiotic properties of *Ligilactobacillus animalis*, such as acid and bile salt tolerance, antibacterial activity, antioxidant properties, and self-aggregation, have been reported, and the earliest reported isolate of this strain can ferment cellobiose, none of the published studies have addressed its ability to degrade lignocellulose. Furthermore, while there are existing studies on recombinant expression of glucan sucrase from this strain, this enzyme is used for glucan synthesis and is unrelated to lignocellulose degradation. This invention represents a substantial leap from known probiotics to multifunctional biocatalysts, demonstrating that the unique ecological niche of bamboo stalks, the staple food of giant pandas, provides a unique resource for discovering new microbial functions.

[0133] Example 5 Preparation of microbial inoculants

[0134] This embodiment provides a microbial inoculant, the preparation method of which is as follows:

[0135] Ligilactobacillus animalis CCRCGPYQ001 was streaked onto a lactic acid bacteria medium (MRS medium), and single colonies were picked and cultured in an anaerobic tank at 37 °C for 48 h to obtain a seed culture. This seed culture was then inoculated into MRS medium at a 5% (v / v) inoculum for expansion culture, and cultured in an anaerobic tank at 37 °C for 48 h to obtain a microbial inoculum with an effective viable count of 7.85 × 10⁻⁶. 10 CFU / g, pH value 6.8~7.0.

[0136] Example 6: Application of strain Lactobacillus animalis CCRCGPYQ001 in the preparation of silage.

[0137] 6.1 Preparation of Silage

[0138] Fresh, waxy-ripe whole-plant corn was naturally air-dried until its moisture content reached 60-65%, then chopped into 2-3 cm pieces. 1 kg 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. 8 CFU / g was packed into vacuum-sealed polyethylene bags, compacted, and then vacuum-sealed to produce bagged silage. The group treated with *Ligilactobacillus animalis* CCRCGPYQ001 was the inoculum group, while the control group was the group that underwent natural fermentation (with an equal amount of sterile water added). Each treatment was replicated three times. Silage was kept for 30–90 days at a fermentation temperature of 25–35°C.

[0139] 6.2 Determination of Fermentation Quality of Silage

[0140] 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).

[0141] 6.3 Determination of nutrient composition of silage

[0142] 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.

[0143] 6.4 Experimental Results

[0144] 6.4.1 Determination of Fermentation Quality of Silage

[0145] 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 Ligilactobacillus animalis CCRCGPYQ001 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.

[0146] Table 5 Fermentation quality of silage

[0147]

[0148] 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).

[0149] 6.4.2 Determination of nutrient composition of silage

[0150] Table 6 shows that the addition of *Ligilactobacillus animalis* CCRCGPYQ001 inoculant 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 *Ligilactobacillus animalis* inoculant not only retains more dry matter and crude protein content and reduces the loss of dry matter and nutrients, but also promotes the degradation of cellulose in corn and improves the utilization rate of corn silage.

[0151] Table 6 Nutritional quality of silage

[0152]

[0153] Note: Different lowercase letters in the superscript of data from the same row indicate significant differences (P<0.05).

[0154] 6.4.3 Technical Effect Analysis

[0155] The results of this embodiment demonstrate that the strain CCRCGPYQ001 of the present invention produced significant technical effects in the application of corn silage, exceeding reasonable expectations:

[0156] (1) Significantly increased lactic acid content: The lactic acid content increased from 35.23 g / kg to 74.62 g / kg, an increase of 111.8%. Existing studies have shown that Pediococcus pentosaceus PL12 can increase the lactic acid content of corn silage by 68.41%, and compound lactic acid bacteria additives can increase the lactic acid content by 52.1%; the lactic acid increase of the strain of this invention is significantly higher than the levels reported above.

[0157] (2) A significant increase in soluble carbohydrates (WSC) against the trend: The WSC content increased from 22.74 g / kg to 78.92 g / kg, an increase of 247.1%. Conventional lactic acid bacteria additives usually consume WSC during silage, resulting in a decrease in its content; however, the strain of this invention degrades the 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.

[0158] (3) Significantly reduced pH, decreased ammonia nitrogen, and retained dry matter: The pH decreased from 4.89 to 4.12, and the ammonia nitrogen content decreased from 2.58 g / kg to 2.02 g / kg; the dry matter (DM) content increased from 29.16 g / kg to 35.47 g / kg, an increase of 21.6%, which is significantly better than the effect of conventional lactic acid bacteria inoculants, which usually have no significant effect on DM content. The low pH effectively inhibits the growth of putrefactive bacteria, and the reduction of ammonia nitrogen and the increase of DM are both conducive to the retention of more nutrients.

[0159] (4) Uniqueness and unpredictability of the overall effect: In the prior art, although the probiotic function of *Ligilactobacillus animalis* has been reported, its ability to degrade lignocellulose has never been involved. Although this strain can ferment cellobiose, the mechanism of cellobiose fermentation is completely different from that of secreting cellulase to actively degrade cellulose macromolecules. This invention is the first to concentrate multiple probiotic properties and the newly discovered lignocellulose degradation ability, EPS production ability, and GABA production ability into a single strain, and has been successfully applied to corn silage for the first time. Although there have been preliminary studies on the use of this strain in alfalfa silage, these studies only used it as a conventional lactic acid bacteria and did not involve corn silage, let alone the activity of the three enzymes and the synergistic function of EPS and GABA. The application of this invention in corn silage and the synergistic effect of multifunctional integration have achieved significantly better results than those reported in the prior art in key indicators such as lactic acid enhancement, WSC growth against the trend, and DM retention.

Claims

1. *Ligilactobacillus animalis* CCRCGPYQ001, deposited at Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC NO: 65224.

2. A microbial agent containing *Ligilactobacillus animalis* CCRCGPYQ001 as described in claim 1.

3. The application of the *Ligilactobacillus animalis* CCRCGPYQ001 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 *Ligilactobacillus animalis* CCRCGPYQ001 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 turicensi* CCRCGPYQ001 as described in claim 1, with an inoculation amount of not less than 1×10⁻⁶. 8 CFU / g, vacuum packaged and ensiled for 30-90 days, fermentation temperature 25-35℃.

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