Pediococcus pentosaceus and preparation method and application thereof

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a Pediococcus pentosaceus, which is specifically named as Pediococcus pentosaceus COCPP1-1, the Pediococcus pentosaceus COCPP1-1 is preserved in the Guangdong Microbial Culture Collection Center, and a preservation number is GDMCC No: 65795; a 16S rRNA gene sequence of the Pediococcus pentosaceus COCPP1-1 strain is shown in SEQ ID No: 1. The application further provides a preparation method and application of the Pediococcus pentosaceus. The Pediococcus pentosaceus COCPP1-1 can efficiently degrade tea saponin and tannic acid, and when used for silage of oil tea cake, the effect is better than that of commercial Lactobacillus plantarum and Pediococcus pentosaceus model bacteria, and efficient resource utilization of the oil tea cake is achieved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Pediococcus pentosaceus, its preparation method, and its application. Background Technology

[0002] Camellia oil cake is a byproduct of oil extraction from camellia seeds. It is rich in nutrients such as protein and dietary fiber, making it a potential high-quality feed ingredient and biomass resource. However, camellia oil cake contains anti-nutritional factors such as tea saponins and tannins. Tea saponins have hemolytic and mucosal irritation properties, while tannins easily bind with proteins to form insoluble complexes. These factors not only reduce the nutritional value and palatability of camellia oil cake but also limit its large-scale application in feed and fertilizer fields.

[0003] Currently, the main methods for degrading tea saponins and tannins in camellia oil cake include physical, chemical, and biological methods. Physical methods (such as high temperature and pulverization) and chemical methods (such as acid and alkali treatment) have drawbacks such as high energy consumption, environmental pollution, and damage to nutrients. In contrast, biological methods (using microbial degradation) have advantages such as high efficiency, environmental friendliness, gentleness, and no damage to nutrients, and have become a research hotspot in recent years.

[0004] Lactic acid bacteria are Gram-positive bacteria that ferment sugars to produce lactic acid and are widely used in food fermentation and silage. *Pediococcus pentosaceus*, as a type of lactic acid bacteria, has been shown to have certain metabolic capabilities; however, current reports indicate that *Pediococcus pentosaceus* strains have low degradation efficiency for tea saponins and tannins, and when used for camellia oil cake silage, the silage quality is difficult to meet practical application requirements. While commercially available *Lactobacillus plantarum* is widely used in silage fermentation, its adaptability and silage effect in the specific substrate of camellia oil cake still have room for improvement.

[0005] Therefore, screening a Pediococcus pentosaceus strain that can efficiently degrade tea saponins and tannins and significantly improve the silage quality of camellia oil cake is of great significance for promoting the resource utilization of camellia oil cake, reducing the harm of anti-nutritional factors, and saving feed costs. It can also fill the gap in the existing technology for such efficient strains. Summary of the Invention

[0006] The present invention aims to solve the above-mentioned technical problems and provide a *Pediococcus pentosaceus* strain. This *Pediococcus pentosaceus* COCPP1-1 strain can efficiently degrade tea saponins and tannins, and when used for ensiling camellia oil cake, it is more effective than commercial *Lactobacillus plantarum* and *Pediococcus pentosaceus* model strains, thus realizing the efficient resource utilization of camellia oil cake.

[0007] The technical solution of this invention is as follows:

[0008] A *Pediococcus pentosaceus* strain, named *Pediococcus pentosaceus* COCPP1-1, is deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No: 65795; the 16S rRNA gene sequence of the *Pediococcus pentosaceus* COCPP1-1 strain is shown in SEQ ID No: 1.

[0009] Preferably, the preparation method of Pediococcus pentosaceus according to the present invention comprises the following steps:

[0010] (1) Preparation of Camellia oleifera cake meal sample: Camellia oleifera seeds harvested during the ripening period of Camellia oleifera fruit were dried and physically pressed to extract tea oil to obtain Camellia oleifera cake meal. The Camellia oleifera cake meal was dried at 70℃, crushed, passed through a 60-mesh sieve, and evenly sprayed with 50wt.% purified water. The mixture was then vacuum sealed and anaerobic fermented at 37℃ for 25 days to obtain fermented Camellia oleifera cake meal sample for later use.

[0011] (2) Culture medium preparation:

[0012] 1) Preparation of tea saponin and tannic acid extract: After removing the oil from the crushed camellia cake residue by supercritical carbon dioxide, add 70% ethanol solution at a material-to-liquid ratio of 1g:10mL, and ultrasonically extract at 60℃ and 500w power for 90min. Remove the extraction solvent to obtain camellia cake residue extract.

[0013] 2) Liquid screening medium: Add camellia seed cake extract to MRS broth medium to a final concentration of tea saponin of 5-10 g / L and a final concentration of tannic acid of 2-5 g / L to obtain liquid screening medium;

[0014] (3) Separation and screening:

[0015] 1) Sample dilution: Take the fermented camellia oil cake sample prepared in step (1), add sterile physiological saline at a material-to-liquid ratio of 1g:9mL, shake well for 10min, and prepare 10 -1 The diluent was then serially diluted to 10. -6 10 -7 Diluent;

[0016] 2) Spread culture: Take 10 -6 10 -7 Each dilution of 0.1 mL was spread onto MRS agar plates, with three replicates for each dilution, and anaerobic incubated at 37°C for 48 h.

[0017] 3) Colony selection: Select 10 round, milky white lactic acid bacteria colonies with neat edges and smooth surfaces;

[0018] 4) Screening and purification: Ten selected colonies were inoculated into the liquid screening medium obtained in step (2) 2), anaerobic cultured at 37°C for 72 h, centrifuged at 12000 rpm for 5 min and the supernatant was collected. The content of tea saponin and tannic acid in the supernatant was measured, and the strain with the highest tea saponin and tannic acid degradation efficiency was screened out. The strain was purified by streaking multiple times on MRS medium plates to obtain pure Pediococcus pentosaccharides COCPP1-1.

[0019] Preferably, the parameters of the supercritical carbon dioxide oil removal equipment of the present invention are: extraction pressure 30 MPa, extraction temperature 50℃, CO2 flow rate 2 L / min, extraction 90 min; separation pressure I: 10 MPa / 45℃; separation II: 5 MPa / 30℃.

[0020] This invention also provides the application of *Pediococcus pentosaceus* in the degradation of tea saponins and in the degradation of tannic acid. The *Pediococcus pentosaceus* strain of this invention was isolated from anaerobic fermented camellia oil cake, exhibiting strong adaptability and efficient growth in the environment containing tea saponins and tannic acid derived from camellia oil cake. Its degradation efficiency for tea saponins and tannic acid is significantly higher than that of existing standard *Pediococcus pentosaceus* strains, solving the problem of insufficient degradation capacity of existing strains.

[0021] This invention also provides the application of *Pediococcus pentosaceus* in the preparation of camellia oil cake silage, wherein the concentration of *Pediococcus pentosaceus* is 2 × 10⁻⁶. 6 The inoculum concentration was CFU / mL, and the inoculum amount was 5wt.%-10wt.% of the substrate. When the *Pediococcus pentosaceus* strain of this invention is used for silage fermentation of camellia oil cake, the silage quality is superior to that of commercially available *Lactobacillus plantarum* and *Pediococcus pentosaceus* model strains. It can significantly increase the crude protein and crude fat content of silage, reduce the acid detergent fiber content, and decrease the production of ammonia nitrogen. Simultaneously, it degrades tea saponins and tannin residues in camellia oil cake, improving the palatability and nutritional value of the silage. This provides a highly efficient strain for the resource utilization of camellia oil cake. Due to the adoption of the above technical solution, the beneficial effects of this invention are:

[0022] 1. The *Pediococcus pentosaceus* strain of this invention was isolated from anaerobic fermented camellia seed cake. It has strong adaptability and can grow efficiently in the environment of tea saponins and tannins derived from camellia seed cake. Its degradation efficiency of tea saponins and tannins is significantly higher than that of the existing standard strain of *Pediococcus pentosaceus*, thus solving the problem of insufficient degradation capacity of existing strains.

[0023] 2. When the *Pediococcus pentosaceus* strain of this invention is used for silage fermentation of camellia oil cake, the silage quality is superior to that of commercial *Lactobacillus plantarum* and *Pediococcus pentosaceus* model strains. It can significantly increase the crude protein and crude fat content of silage, reduce the content of acid detergent fiber, reduce the production of ammonia nitrogen, and degrade tea saponin and tannic acid residues in camellia oil cake, thereby improving the palatability and nutritional value of silage. This provides a highly efficient strain for the resource utilization of camellia oil cake.

[0024] 3. The *Pediococcus pentosaceus* strain of this invention is a lactic acid bacterium, which is highly safe, non-pathogenic, and can be directly applied to feed, silage and other fields. Moreover, the cultivation conditions are simple, the cost is low, and it is easy to scale up production and promote application.

[0025] 4. The isolation and identification method of the present invention is simple and easy to implement, and can quickly screen out target strains, providing a reference for the screening of similar highly efficient degradation strains.

[0026] Strain Preservation Notes: The *Pediococcus pentosaceus* COCPP1-1 strain of this invention was deposited on January 15, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology, with accession number GDMCC No: 65795. The 16S rRNA gene sequence of the *Pediococcus pentosaceus* COCPP1-1 strain is shown in SEQ ID No: 1. Attached Figure Description

[0027] Figure 1 This is a morphological diagram of the *Pediococcus pentosaceus* strain from Example 1 of the present invention.

[0028] Figure 2 This is a phylogenetic tree of the 16S rRNA sequence of the strain COCPP1-1 of this invention.

[0029] Figure 3 The growth curves and acid production curves of strain COCPP1-1 of the present invention under different concentrations of tea saponin and tannic acid in Example 2 of the present invention are shown.

[0030] Figure 4 The growth curves and acid production curves of the control strain CICC25165 under different concentrations of tea saponin and tannic acid in Example 2 of this invention are shown.

[0031] Figure 5 The image shows the growth morphology of strain COCPP1-1 of the present invention in Example 2 of this invention on MRS agar medium containing different concentrations of tea saponin and tannic acid; where a: 2.1 wt.% tea saponin, 0.84 wt.% tannic acid; b: 3.5 wt.% tea saponin, 1.4 wt.% tannic acid; c: 7 wt.% tea saponin, 2.8 wt.% tannic acid.

[0032] Figure 6 The images show samples of silage fermented camellia seed cake from different strains in Example 3 of this invention. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] In the following embodiments:

[0035] MRS agar medium (model: 027315) and MRS broth medium (model: 027312) were purchased from Guangdong Huankai Microbial Technology Co., Ltd.

[0036] Example 1: Isolation, screening and identification of Pediococcus pentosaceus COCPP1-1

[0037] 1. Isolation and screening of Pediococcus pentosaceus

[0038] (1) Preparation of Camellia oleifera cake meal samples: Camellia oleifera seeds harvested at the maturity period of Camellia oleifera fruit were collected from the National Camellia oleifera Seed Base of Ganzhou Forestry Research Institute. After drying and physical pressing to extract tea oil, Camellia oleifera cake meal was obtained. The Camellia oleifera cake meal was dried at 70℃, crushed, passed through a 60-mesh sieve, and packed into polyethylene bags. 50wt.% purified water was evenly sprayed into the polyethylene bags. Vacuum sealing was performed using a vacuum sealing machine. Anaerobic fermentation was carried out at 37℃ for 25 days to obtain Camellia oleifera cake meal samples for later use.

[0039] (2) Culture medium preparation:

[0040] 1) Preparation of tea saponin and tannic acid extract: Camellia oil cake was obtained after sun-drying and physical pressing to extract tea oil. The camellia oil cake was dried at 70℃, pulverized, and passed through a 60-mesh sieve. The pulverized camellia oil cake raw material was then subjected to supercritical carbon dioxide degreasing (the parameters of the supercritical carbon dioxide degreasing equipment were: extraction pressure 30MPa, extraction temperature 50℃, CO2 flow rate 2 L / min, extraction 90 min; separation pressure I: 10MPa / 45℃; separation II: 5MPa / 30℃). Then, 70% ethanol solution was added at a material-to-liquid ratio of 1g:10mL, and ultrasonic extraction was performed at 60℃ and 500W for 90 min. The extraction solvent was removed using a rotary evaporator. The tea saponin content was determined using the vanillin-sulfuric acid colorimetric method, and the tannic acid content was determined using the Folin-Ciocalteu colorimetric method. The final camellia oil cake extract was obtained (containing a final volume concentration of 70% tea saponin and a volume concentration of 28% tannic acid).

[0041] 2) Liquid screening medium: Add tea saponin and tannic acid extract from camellia seed cake to MRS broth medium until the final concentration of tea saponin is 7 g / L and the final concentration of tannic acid is 2.8 g / L to obtain liquid screening medium;

[0042] (3) Separation and screening:

[0043] 1) Sample dilution: Take 10g of camellia seed cake sample prepared in step (1), add 90mL of sterile physiological saline, shake well for 10min, and dilute to obtain 10g of sample. -1 The diluent was then serially diluted to 10. -6 10 -7 Diluent;

[0044] 2) Spread culture: Take 10 -6 10 -7 Each dilution of 0.1 mL was spread onto MRS agar plates, with three replicates for each dilution, and anaerobic incubated at 37°C for 48 h.

[0045] 3) Colony selection: Select 10 round, milky white lactic acid bacteria colonies with neat edges and smooth surfaces;

[0046] 4) High-efficiency screening: Ten colonies were inoculated into the liquid screening medium obtained in step (2) 2), anaerobic culture at 37°C for 72 h, centrifuged at 12000 rpm for 5 min and the supernatant was collected. The residual amounts of tea saponin and tannic acid in the liquid screening medium were detected by vanillin-sulfuric acid colorimetric method and Folin-Ciocalteu colorimetric method, respectively. One strain with the highest degradation efficiency of tea saponin and tannic acid was screened out and numbered as strain COCPP1-1.

[0047] 5) Purification culture: The strain COCPP1-1 was purified by streaking multiple times on MRS medium plates to obtain pure strain COCPP1-1, which was stored at 4℃ for later use.

[0048] 2. Identification of Pediococcus pentosaceus

[0049] (1) Morphological identification: The pure strain COCPP1-1 was inoculated onto MRS agar plates and anaerobically cultured at 37℃ for 48 h. The colonies were observed to be round, milky white, with neat edges and smooth surfaces, and about 0.8 mm in diameter. Gram staining was used to observe the bacteria under an optical microscope. The bacteria were spherical, arranged in tetrads, Gram-positive, without spores or flagella, which is consistent with the morphological characteristics of Pediococcus pentosaceus. See details. Figure 1 ;

[0050] (2) Molecular biological identification:

[0051] 1) Genomic DNA extraction: Using a bacterial genomic DNA extraction kit, the genomic DNA of strain COCPP1-1 was extracted according to the instructions. The DNA bands were detected by agarose gel electrophoresis and the purity met the requirements for PCR amplification.

[0052] 2) 16S rRNA gene amplification: PCR amplification was performed using universal primers for lactic acid bacteria 16S rRNA (upstream primer: 5'-AGAGTTTGATCCTGGCTCAG-3', downstream primer: 5'-GGTTACCTTGTTACGACTT-3'); PCR reaction system (25μL): DNA template 1μL, upstream primer 2μL, downstream primer 2μL, PCR Mix 25μL, sterile water 20μL; PCR reaction conditions: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 57℃ annealing for 10 s, 72℃ extension for 30 s, for a total of 35 cycles; final extension at 72℃ for 5 min, stored at 4℃;

[0053] 3) Sequencing and Analysis: The PCR amplification products were sent to a biotechnology company (Shanghai Meiji Biotechnology Co., Ltd.) for sequencing to obtain the 16S rRNA gene sequence, as shown in SEQ ID No:1; this sequence was compared with known sequences in the GenBank database for homology, and a phylogenetic tree was constructed, as shown in... Figure 2 As shown;

[0054] 4) Results Analysis: A phylogenetic tree was constructed based on the 16S rRNA gene sequence (see...). Figure 2 The results showed that strain COCPP1-1 formed a single evolutionary branch with multiple reference strains of Pediococcus pentosaceus (including the type strain DSM 20336 (T)), with a self-development support rate of 94%. The closest related strains were Pediococcus pentosaceus DXNC-3 and HBUAS510123, with sequence homology higher than 99.9%. Based on morphological and physiological and biochemical characteristics, strain COCPP1-1 could be clearly identified as Pediococcus pentosaceus.

[0055] (3) Preservation of strain COCPP1-1:

[0056] The pure strain COCPP1-1 was inoculated into MRS broth medium and anaerobic cultured at 37℃ for 24 h. 1 mL of the bacterial culture was taken, 0.5 mL of 60% glycerol was added, mixed well, and stored at -80℃. Simultaneously, the strain COCPP1-1 was named Pediococcus pentosaceus COCPP1-1 and deposited on January 15, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology, with accession number GDMCC No: 65795 and a deposit period of 30 years.

[0057] Example 2: Application of Pediococcus pentosaceus COCPP1-1 of the present invention in the degradation of tea saponins and tannins

[0058] 1. Experimental Grouping

[0059] To detect the degradation efficiency of Pediococcus pentosacchariformis COCPP1-1 on tea saponins and tannins, the following experimental groups were set up, as shown in Table 1. Among them, the Pediococcus pentosacchariformis COCPP1-1 strain of the present invention, hereinafter referred to as the invention strain; the Pediococcus pentosacchariformis type strain purchased from the China Culture Collection Center for Microbial Cultures, accession number: CICC: 25165, hereinafter referred to as the control strain; blank control group: no addition of tea oil cake extract;

[0060] Each group was treated with different levels of tea saponin and tannic acid by adding camellia seed cake extract to the culture medium (as shown in Table 1), with three replicates for each treatment.

[0061] Table 1. Treatments involving the addition of camellia oil cake extract to the culture medium, resulting in different levels of tea saponin and tannins.

[0062] Unit: wt.%

[0063]

[0064] 2. Test methods

[0065] (1) Cultivation: After each strain is activated, the logarithmic growth phase bacterial solution (the bacterial solution OD600 value is 0.7) is inoculated into the culture medium shown in Table 1. The inoculation amount of the invented strain and the control strain is 1% (v / v) of MRS broth medium. The culture is anaerobic at 37℃ for 60h.

[0066] (2) Detection: The pH value and OD600 absorbance of the bacterial solution were measured at 4, 8, 16, 32, 48 and 60 h of culture, respectively. Acid production curve and growth curve were plotted to analyze the effect of adding camellia seed cake extract on the acid production and growth performance of the strain of the present invention and the control strain. After 60 h of culture, the bacterial solution was centrifuged and the supernatant was collected. The content of tea saponin in the bacterial solution was determined by vanillin-sulfuric acid colorimetric method and the content of tannic acid in the bacterial solution was determined by Folin-Ciocalteu colorimetric method. The degradation of tea saponin and tannic acid was compared and analyzed.

[0067] 3. The results are as follows:

[0068] Figure 3 The results showed that the growth curves and acid production curves of the *Pediococcus pentosaceus* COCPP1-1 strain of this invention, in media supplemented with different concentrations of tea saponin and tannic acid, were consistent with those of the blank group (COPP-0) without the addition of camellia seed cake extract, and the curves highly overlapped. Even as the concentrations of tea saponin and tannic acid in the culture medium gradually increased, the binding... Figure 5 The growth status of the *Pediococcus pentosacchari* COCPP1-1 strain on agar medium of the present invention also shows that the growth, reproduction and acid production capacity of the *Pediococcus pentosacchari* COCPP1-1 strain of the present invention are not significantly affected, indicating that the *Pediococcus pentosacchari* COCPP1-1 strain of the present invention has good tolerance to tea saponins and tannins in camellia oil cake, and these two substances do not have an inhibitory effect on the *Pediococcus pentosacchari* COCPP1-1 strain of the present invention.

[0069] As shown in Figure 4, the performance of the control strain differed significantly from that of the invention strain. In culture systems containing different concentrations of tea saponin and tannic acid, the growth of the control strain was inhibited and its acid-producing capacity decreased. When the concentration of tea saponin increased to 7% and the concentration of tannic acid increased to 2.8%, the pH value of the bacterial culture was 4.58, significantly higher than that of the blank group COPP-0 (3.92). This confirms that as the concentration of tea saponin and tannic acid increases, the growth and acid-producing activity of the control strain are gradually inhibited.

[0070] Table 2 shows that, within the tea saponin concentration range of 2.10%–7.00%, after 60 h of culture, the degradation rate of tea saponin by the *Pediococcus pentosaceus* COCPP1-1 strain of this invention was 91.43%–61.29%; under the same culture conditions, the degradation rate of the control strain was only 25.14%–47.14%. The tea saponin degradation ability of both strains decreased with increasing initial concentration of the substrate tea saponin, but at each concentration gradient, the tea saponin degradation effect of the *Pediococcus pentosaceus* COCPP1-1 strain of this invention was significantly better than that of the control strain (P≤0.01), demonstrating that the *Pediococcus pentosaceus* COCPP1-1 strain of this invention has a significant advantage in the application of tea saponin degradation.

[0071] Table 3 shows that, with an initial tannin concentration in the culture medium ranging from 0.86% to 2.80%, after 60 hours of cultivation, the *Pediococcus pentosaceus* COCPP1-1 strain of this invention exhibited a tannin degradation rate of 63.49%–41.19%, while the control strain's degradation rate was only 49.61%–23.21%. The degradation ability of both strains decreased with increasing tannin concentration, but the *Pediococcus pentosaceus* COCPP1-1 strain of this invention showed superior degradation performance compared to the control strain at all tannin concentration levels, demonstrating significant advantages in application.

[0072] In summary, compared with the control strain, the Pediococcus pentosaceus COCPP1-1 strain of the present invention has stronger substrate tolerance and degradation ability for tea saponins and tannins derived from camellia oil cake.

[0073] Table 2. Degradation of tea saponins by the inventing and control bacteria

[0074]

[0075] Table 3. Degradation of tannins by the inventing and control strains

[0076]

[0077] Example 3: Application of Pediococcus pentosaceus COCPP1-1 of the present invention in the preparation of camellia oil cake silage

[0078] (1) Experimental grouping:

[0079] 1) Experimental group: The *Pediococcus pentosaceus* strain COCPP1-1 of this invention was added;

[0080] 2) Control group 1: Added Pediococcus pentosaceus model strain - CICC25165, with the same inoculum as the experimental group;

[0081] 3) Control group 2: Added with commercial strain of Lactobacillus plantarum—BNCC194165, purchased from Beina Biotechnology Co., Ltd.;

[0082] 4) Blank control group: No bacterial strains were added;

[0083] The experimental and control strains were activated and cultured in MRS broth medium and diluted to 2×10⁻⁶. 6 The CFU / mL used and the inoculum amount were both 7 wt.% of the substrate (camellia oil cake).

[0084] (2) Silage conditions: Camellia oil cake was dried and crushed at 70℃, passed through a 60-mesh sieve, and the moisture content was adjusted to 60wt.%. Each group of bacterial strains was added, mixed evenly, and then packed into 250 mm × 300 mm polyethylene bags, compacted and sealed. Vacuum sealing was performed using a vacuum sealing machine, and silage was carried out at room temperature. After 35 days, the bags were opened and samples were taken for analysis.

[0085] (3) Detection indicators: After silage is completed, 10g of sample is taken from the bag and the dry matter, crude protein, neutral detergent fiber, acid detergent fiber, crude fat, ammonia nitrogen (g / kg), pH, lactic acid, acetic acid, propionic acid, butyric acid, and residual tea saponin and tannic acid are determined. The determination method is in accordance with "DB15T1458—2018 Determination Method of pH, Organic Acids and Ammonia Nitrogen in Silage". The pH is determined immediately after the preparation of the extract using a precision pH meter (Shanghai Leici pHS-2F). Lactic acid, acetic acid, propionic acid and butyric acid are determined using liquid chromatography. The ammonia nitrogen (NH3-N) content is determined by the phenol-sodium hypochlorite colorimetric method. The tea saponin content is determined by the vanillin-sulfuric acid colorimetric method, and the tannic acid content is determined by the Folin-Ciocalteu colorimetric method.

[0086] (5) The results are as follows:

[0087] As shown in Table 4, the crude protein content of the *Pediococcus pentosaceus* strain COCPP1-1 in the experimental group was 44.93% higher than that in the blank control group, and the acid detergent fiber content was 16.6% lower. This was significantly better than the *Pediococcus pentosaceus* model strain in control group 1 and *Lactobacillus plantarum* in control group 2, effectively improving the nutritional value and digestibility of camellia oil cake. As shown in Table 5, the degradation rates of tea saponins and tannins in the experimental group reached 69.0% and 45.59%, respectively, both the highest among all groups, significantly reducing the anti-nutritional level of camellia oil cake and providing crucial support for its safe feeding. From the perspective of fermentation quality, the strain of this invention can rapidly reduce the pH of the fermentation system to 3.99, maintain a high level of lactic acid content, and significantly reduce the content of ammonia nitrogen, acetic acid, and propionic acid, while completely inhibiting butyric acid production. This effectively reduces nitrogen loss and spoilage risk, ensuring the aerobic stability and feeding safety of silage. Figure 6 In terms of appearance quality, both the blank control group and the strain control group showed varying degrees of mold, clumping, and abnormal color. However, the experimental group inoculated with the strain of this invention showed no obvious mold growth, the feed had a uniform texture, no signs of spoilage, and a distinct sour aroma.

[0088] In summary, the strain of this invention has the functions of efficient nutrient conversion, removal of anti-nutritional factors and regulation of silage fermentation. Compared with the commonly used model strains of Lactobacillus plantarum and Pediococcus pentosaceus in silage fermentation, it has significant technical advantages and is a highly efficient fermentation strain for the resource utilization of camellia oil cake.

[0089] Table 4. Effects of different bacterial strains on the nutrient composition of camellia seed cake silage (unit: wt.%)

[0090]

[0091] Note: The significance of the above differences is P ≤ 0.05.

[0092] Table 5. Effects of different bacterial strains on the silage of camellia seed cake.

[0093]

[0094] Note: The significance of the above differences is P ≤ 0.05.

[0095] The above description is a detailed description of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A type of Pediococcus pentosaceus, characterized in that: The Pediococcus pentosaceus is named Pediococcus pentosaceus COCPP1-1, and it is deposited at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCC No: 65795.

2. The *Pediococcus pentosaceus* as described in claim 1, characterized in that: The 16S rRNA gene sequence of the *Pediococcus pentosaceus* COCPP1-1 strain is shown in SEQ ID No:

1.

3. The method for preparing Pediococcus pentosaceus as described in claim 1, characterized in that: The steps are as follows: (1) Preparation of Camellia oleifera cake meal sample: Camellia oleifera seeds harvested during the ripening period of Camellia oleifera fruit were dried and physically pressed to extract tea oil to obtain Camellia oleifera cake meal. The Camellia oleifera cake meal was dried at 70℃, crushed, passed through a 60-mesh sieve, and evenly sprayed with 50wt.% purified water. The mixture was then vacuum sealed and anaerobic fermented at 37℃ for 25 days to obtain fermented Camellia oleifera cake meal sample for later use. (2) Culture medium preparation: 1) Preparation of tea saponin and tannic acid extract: After removing the oil from the crushed camellia cake residue by supercritical carbon dioxide, add 70% ethanol solution at a material-to-liquid ratio of 1g:10mL, and ultrasonically extract at 60℃ and 500w power for 90min. Remove the extraction solvent to obtain camellia cake residue extract. 2) Liquid screening medium: Add camellia seed cake extract to MRS broth medium to a final concentration of tea saponin of 5-10 g / L and a final concentration of tannic acid of 2-5 g / L to obtain liquid screening medium; (3) Separation and screening: 1) Sample dilution: Take the fermented camellia oil cake sample prepared in step (1), add sterile physiological saline at a material-to-liquid ratio of 1g:9mL, shake well for 10min, and prepare 10 -1 The diluent was then serially diluted to 10. -6 10 -7 Diluent; 2) Spread culture: Take 10 -6 10 -7 Each dilution of 0.1 mL was spread onto MRS agar plates, with three replicates for each dilution, and anaerobic incubated at 37°C for 48 h. 3) Colony selection: Select 10 round, milky white lactic acid bacteria colonies with neat edges and smooth surfaces; 4) Screening and purification: Ten selected colonies were inoculated into the liquid screening medium obtained in step (2) 2), anaerobic cultured at 37°C for 72 h, centrifuged at 12000 rpm for 5 min and the supernatant was collected. The content of tea saponin and tannic acid in the supernatant was measured, and the strain with the highest tea saponin and tannic acid degradation efficiency was screened out. The strain was purified by streaking multiple times on MRS medium plates to obtain pure Pediococcus pentosaccharides COCPP1-1.

4. The method for preparing Pediococcus pentosaceus as described in claim 3, characterized in that: The parameters of the supercritical carbon dioxide oil removal equipment are as follows: extraction pressure 30 MPa, extraction temperature 50℃, CO2 flow rate 2 L / min, extraction 90 min; separation pressure I: 10 MPa / 45℃; separation II: 5 MPa / 30℃.

5. The application of Pediococcus pentosaceus as described in claim 1 or 2 in the degradation of tea saponins.

6. The application of Pediococcus pentosaceus as described in claim 1 or 2 in the degradation of tannic acid.

7. The application of Pediococcus pentosaceus as described in claim 1 or 2 in the preparation of camellia oleifera silage.

8. The application of Pediococcus pentosaceus as described in claim 6 in the preparation of camellia oleifera silage, characterized in that: The concentration of *Pediococcus pentosaceus* was 2 × 10⁻⁶. 6 CFU / mL, with inoculum amounts ranging from 5 wt.% to 10 wt.% of the substrate.

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