Lactobacillus plantarum capable of degrading cellulose and DON and application of lactobacillus plantarum

Abstract

The invention relates to lactobacillus plantarum capable of degrading cellulose and DON and application of the lactobacillus plantarum, belongs to the technical field of microorganisms, and is used for solving at least one of the problems that the utilization rate of silage is low due to the fact that the content of cellulose and the content of DON in existing silage are high, and no special lactic acid bacterium agent capable of degrading fiber and DON at the same time exists in the silage fermentation process. The lactobacillus plantarum LAB-GS-CSR1 disclosed by the invention has the characteristics of high acid production speed and high growth speed, effectively solves the problems of high fiber and DON (deoxynivalenol) in silage raw materials, and overcomes the problem of adaptability of lactic acid bacteria to the raw materials, so that the activity of the lactic acid bacteria can be improved, and the silage fermentation quality can be improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more particularly to a plant lactobacillus with cellulose and DON degradation capabilities and its applications. Background Technology

[0002] Silage is a common feed preservation method in modern animal husbandry. It extends the shelf life of roughage through anaerobic fermentation while improving palatability and nutrient conversion efficiency. However, in actual production, silage raw materials often suffer from high fiber content and severe mycotoxin contamination. In particular, frequent extreme weather events worldwide have exacerbated DON (vomiting toxin) contamination, making it the most frequently detected of various mycotoxins. Furthermore, during the silage process, DON content increases with fermentation stimulation. This not only severely inhibits the growth of beneficial fermenting bacteria but also affects the final silage quality, thereby harming animal health and production performance, becoming a significant bottleneck restricting the improvement of silage quality.

[0003] Currently, numerous studies have focused on screening and utilizing silage functional microorganisms capable of degrading fiber and mycotoxins. Among the many microbial resources, *Trichoderma* spp. fungi are widely used in the screening of silage functional microorganisms due to their natural cellulase and multiple degradation enzyme systems. Traditional random isolation (such as soil and ordinary silage) is like "finding a needle in a haystack," with extremely low efficiency. In particular, no publicly available information has been published on screening lactic acid bacteria that possess both high efficiency in fiber degradation and strong degradation of DON toxins. Furthermore, traditional antibacterial screening often targets bacteria (such as *Escherichia coli*) or non-toxin-producing fungi, which is misaligned with the control needs of Fusarium oxysporum, the most harmful fungus in silage. Additionally, the functions of strains isolated using traditional methods are often verified under idealized in vitro conditions, and their activity may significantly decrease in the actual anaerobic environment of silage. Summary of the Invention

[0004] In view of the above analysis, the present invention aims to provide a *Lactobacillus plantarum* and its application to solve at least one of the following problems: high cellulose and DON content in existing silage, resulting in low silage utilization rate; and the lack of a dedicated lactic acid bacteria agent that can simultaneously degrade cellulose and DON during silage fermentation.

[0005] In a first aspect, the present invention provides a Lactobacillus plantarum LAB-GS-CSR1 with accession number CGMCC No.34170.

[0006] Furthermore, the Lactobacillus plantarum LAB-GS-CSR1 contains the 16S rDNA shown in SEQ ID NO.1.

[0007] Furthermore, the Lactobacillus plantarum LAB-GS-CSR1 was extracted, isolated, and cultured from corn silage containing Trichoderma.

[0008] Secondly, the present invention provides a method for extracting, isolating, and culturing the above-mentioned *Lactobacillus plantarum* LAB-GS-CSR1, the method comprising:

[0009] The corn silage containing Trichoderma was mixed with physiological saline, homogenized, and the supernatant was transferred to solid MRS medium for culture.

[0010] Furthermore, the cultivation temperature is 30–37°C.

[0011] Thirdly, the present invention provides a silage additive comprising the aforementioned Lactobacillus plantarum LAB-GS-CSR1.

[0012] Fourthly, the present invention provides a silage comprising the aforementioned Lactobacillus plantarum LAB-GS-CSR1.

[0013] Fifthly, the present invention provides a method for preparing the above-mentioned silage, comprising the following steps:

[0014] (1) Chop and mix the silage raw materials to be fermented evenly;

[0015] (2) Add the Lactobacillus plantarum LAB-GS-CSR1 to the mixed raw materials, and vacuum seal and store to obtain the silage.

[0016] Furthermore, in step (2), the effective viable count of *Lactobacillus plantarum* LAB-GS-CSR1 added per gram of raw material is at least 1.0 × 10⁻⁶. 6 indivual.

[0017] Sixthly, the present invention provides an application of the above-mentioned Lactobacillus plantarum LAB-GS-CSR1 in the preparation of silage.

[0018] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0019] (1) The Lactobacillus plantarum LAB-GS-CSR1 described in this invention has the characteristics of rapid acid production and rapid growth, and effectively solves the problems of high fiber and DON (vomiting toxin) in silage raw materials, and overcomes the problem of lactic acid bacteria adaptability to raw materials, thereby improving the activity of lactic acid bacteria and improving the quality of silage fermentation.

[0020] (2) The Lactobacillus plantarum LAB-GS-CSR1 of the present invention reduced NDF by ≥11.03%, ADF by ≥7.60%, HCEL by ≥12.00%, cellulose by ≥9.03%, lignin by ≥42.65%, and DON by ≥32.26% after silage storage for 30 days.

[0021] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0022] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0023] Figure 1 The fiber-degrading ability of strains 1 to 9 screened in this invention;

[0024] Figure 2 The growth curves of bacteria 1-7 and the CK group of this invention are shown.

[0025] Figure 3 The results of the cellulase activity test of the present invention;

[0026] Figure 4 Antibacterial test of the strains screened for this invention;

[0027] Figure 5 This is a graph showing the acid production and growth rate of the degrading bacteria screened in this invention. Detailed Implementation

[0028] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which constitute a part of the present invention and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0029] A specific embodiment of the present invention discloses a strain of *Lactobacillus plantarum* LAB-GS-CSR1 (hereinafter referred to as LAB1) with accession number CGMCC No. 34170. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No. 34170, and the deposit date is April 11, 2025.

[0030] Specifically, the Lactobacillus plantarum LAB-GS-CSR1 contains the 16S rDNA shown in SEQ ID NO.1.

[0031] Specifically, the Lactobacillus plantarum LAB-GS-CSR1 was extracted, isolated, and cultured from corn silage containing Trichoderma.

[0032] It should be noted that the silage containing Trichoderma mentioned above is corn silage stored for 148 days.

[0033] Another specific embodiment of the present invention discloses a method for extracting, isolating, and culturing *Lactobacillus plantarum* LAB-GS-CSR1, the specific culturing process including:

[0034] The corn silage containing Trichoderma was mixed with 0.85% physiological saline, homogenized, and the supernatant was transferred to solid MRS medium for culture.

[0035] For example, 20g of corn silage containing Trichoderma was mixed with 0.85% physiological saline and homogenized at 4°C for more than 4 hours. 30μL of the supernatant was then transferred to solid MRS medium for culture.

[0036] Trichoderma has the ability to transfer genes horizontally and also possesses the ability to degrade fiber and antagonize all pathogens. Through extensive experimentation, this invention has discovered that functional microorganisms can be isolated directly from silage containing Trichoderma.

[0037] Specifically, the cultivation temperature is 30-37℃.

[0038] Specifically, after culturing the isolated Lactobacillus plantarum for 24 hours, the pH of the MRS liquid culture medium can be reduced to 3.64–3.67.

[0039] It should be noted that the biological characteristics of *Lactobacillus plantarum* LAB1 described in this invention are Gram-positive bacteria, undergoing glucose homofermentation, and can grow normally at pH 3.65, indicating its strong acid resistance; after culturing in MRS liquid medium at 37°C for 12 hours, the OD... 600 The nm value is 1.6383, indicating that its growth rate is fast.

[0040] Another specific embodiment of the present invention discloses a silage additive, comprising the aforementioned Lactobacillus plantarum LAB-GS-CSR1.

[0041] Preferably, the silage additive is a corn silage additive.

[0042] Another specific embodiment of the present invention discloses a silage feed comprising the aforementioned Lactobacillus plantarum LAB-GS-CSR1.

[0043] Another specific embodiment of the present invention discloses a method for preparing silage, comprising: mixing and fermenting the silage raw material to be fermented with the Lactobacillus plantarum LAB-GS-CSR1 to obtain the silage.

[0044] Specifically, the method for preparing silage includes the following steps:

[0045] (1) Chop and mix the silage raw materials to be fermented evenly;

[0046] (2) Add the Lactobacillus plantarum LAB-GS-CSR1 to the mixed raw materials, and vacuum seal and store to obtain the silage.

[0047] Specifically, in step (1), the silage material to be fermented is whole corn. Preferably, it is chopped to a length of 1-2 cm.

[0048] Specifically, in step (2), the effective viable count of Lactobacillus plantarum LAB-GS-CSR1 added per gram of raw material is at least 1.0 × 10⁻⁶. 6 indivual.

[0049] Specifically, in step (2), the storage temperature is 20-25℃, and the pH value of the system is reduced to 3.49-3.50 after 30 days of storage.

[0050] In another specific embodiment of the present invention, the application of the aforementioned Lactobacillus plantarum LAB-GS-CSR1 in the preparation of silage is disclosed.

[0051] It should be noted that the Lactobacillus plantarum LAB1 described in this invention has the characteristics of rapid acid production and fast growth rate, and effectively solves the problems of high fiber and DON (vomiting toxin) in silage raw materials, and overcomes the problem of lactic acid bacteria adaptability to raw materials, thereby improving the activity of lactic acid bacteria and improving the quality of silage fermentation.

[0052] The silage of the present invention overcomes the problem of the adaptability of lactic acid bacteria to raw materials during silage by using plant epiphytic lactic acid bacteria (Lactobacillus plantarum LAB1), thereby reducing the fiber and DON content.

[0053] The silage of the present invention utilizes lactic acid bacteria to improve the fermentation quality and nutritional quality of the silage, and is low in cost, safe, reliable and easy to use.

[0054] The technical solution of the present invention will be further explained below with reference to specific embodiments.

[0055] The whole-column corn stalk rot material containing Trichoderma described in this invention was taken from Shuangshu Village, Baiyin City, Gansu Province (37°N, 103°E, altitude 1600m, average annual temperature 6.6℃, average annual precipitation 212.8mm).

[0056] The whole-plant maize material used in this invention to verify the fiber degradation ability was taken from the experimental field in Dongchengfang Town, Zhuozhou City, Hebei Province (39.50°N, 115.97°E, altitude 40m, average annual temperature 13-14℃, average annual precipitation 600-700mm).

[0057] The MRS liquid culture medium used in the isolation, culture, and determination of physiological and biochemical indicators such as growth rate and acid production rate of Lactobacillus plantarum LAB1 is shown in Table 1 below.

[0058] Table 1. Composition of MRS liquid culture medium

[0059]

[0060] In this invention, 15g of agar is added to the MRS liquid culture medium to obtain the MRS solid culture medium. All the above culture media are sterilized in an autoclave at 121°C for 15 minutes.

[0061] Example 1

[0062] Isolation and screening of Lactobacillus plantarum LAB1

[0063] 1. Isolation and purification of bacterial strains

[0064] Take 20g of whole-plant corn silage containing Trichoderma (stored for 148 days) and add it to a clean, sealed bag containing 180mL of sterile physiological saline (0.85% NaCl solution). After thorough shaking, place it in a 4℃ refrigerator for 4 hours for extraction. Take the supernatant (recorded as 10). -1 (Concentration gradient) 1 mL and then diluted sequentially to 10-fold dilutions. -2 Concentration gradient, 10 -3 Concentration gradient and 10 -4 For each concentration gradient, 100 μL of the diluted solution was plated on MRS solid medium and incubated at 37°C for 48 h. Based on colony morphology (morphology, color, and size), typical colonies were selected and isolated and purified at least twice using the streak plate method until single-colony lactic acid bacteria were obtained. The lactic acid bacteria were then transferred to 20% glycerol using a colony enrichment method and stored at -80°C. The composition of the MRS solid medium was as described above.

[0065] 2. Initial screening of biodegradable fiber-rich lactic acid bacteria

[0066] Six lactic acid bacteria were selected from the screened lactic acid bacteria based on their acid production rate and growth curve (e.g., ...). Figure 1 Strains 1-6 (shown in the text) are fast-producing and high-growth bacteria, and are used with commercially available inoculants (such as...) Figure 1 strain 7 and strain 8 (such as strain 7) Figure 1 strain 8 in the sample (laboratory inoculum) and water (such as...) Figure 1 Strain No. 9 was used as a control in subsequent experiments. The commercially available bacterial agent was Yiqing No. 3, provided by Beijing Zhongqing Yicao Technology Co., Ltd.

[0067] The screening method for strain No. 8 was similar to that for strains No. 1 to No. 6. The difference was that the raw material was replaced with whole-plant silage containing Trichoderma and corn silage infected with head smut.

[0068] Strains 7 and 8 were inoculated at a rate of 1% (v / v) into fresh 3 mL LMR Slurry medium and cultured at 30°C and 250 rpm on a shaker. After 24 hours, their pH values ​​were 3.7 and 5.76, respectively. This indicates that strain 8, compared to the laboratory inoculum 7, did not produce acid. 190 μL of the supernatant of the screened lactic acid bacteria culture was added to a sterile 96-well plate, followed by 10 μL of fungal spores at concentrations of 2.83 × 10⁵ CFU / ml - 7.87 × 10⁵ CFU / ml. The supernatant without fungal spores served as a control. The plates were incubated at 30°C for 24 hours, and OD₆₀₀ was measured. The inhibition rate was calculated using the formula: OD₆₀₀₁(LAB + fungal spores) / OD₆₀₀₁(control) × 100. The 24-hour antagonism rates against Fusarium graminearum 120 in 96-well plates were 86.06% and 27.13%, respectively, and the antagonism rates against Fusarium graminearum 209 were 91.02% and 37.21%, respectively. This indicates that No. 8 does not inhibit bacterial growth compared to laboratory inoculum No. 7.

[0069] Streaking 1% CMC MRS medium into eight equal parts, then activating the selected lactic acid bacteria. 50 μL of the activated lactic acid bacteria solution was inoculated onto the corresponding locations on the surface. After the surface bacterial solution dried and no longer dispersed, the inoculated medium was slowly placed in a 30°C incubator for 12 hours. The inoculated bacteria were then rinsed thoroughly with running water. The surface of the medium was then completely covered with 1% Congo red for 30 minutes. Decolorization was performed 3-4 times with 1% sodium chloride solution, 5-6 minutes each time. The size of the discoloration zone around the colonies was observed. Colonies showing a clear zone were preliminarily considered to have the ability to degrade fibers. This strain was then selected. Figure 1 As shown, subsequent screening of cellulose-degrading bacteria was conducted by measuring their cellulase activity. The 1% CMC MRS medium was prepared by adding 1% sodium carboxymethyl cellulose (CMC) to the MRS solid medium. The composition of the MRS solid medium is as shown above.

[0070] from Figure 1 As can be seen, strains 1 through 3 all have distinct transparent zones. Figure 2 The growth curves for strains 1 through 7 are shown, with CK representing the MRS control group (blank control group). The graph shows that strains 1 and 3 had the fastest growth rates at 12 hours. Therefore, strains 1 and 3 were selected for screening.

[0071] 3. Secondary screening of biodegradable fiber-rich lactic acid bacteria

[0072] Determination of cellobiase (CBH exonuclease) and β-1,3-glucan endonuclease: The activated bacterial suspensions of the above-screened strains that degrade cellulase were inoculated into 100 mL of MRS liquid medium containing 0.5 g of corn stalk powder. The medium was incubated at 37 °C and 250 rpm for 12 h on a shaker. The supernatant was collected by centrifugation at 8000 rpm and used to test the cellulase activity.

[0073] Enzyme activity was measured using a kit from BYabscience.

[0074] The specific steps are as follows: place the kit at room temperature for 20 minutes, mix the reagents in the sample tube and the test tube according to the kit operation steps, and after mixing, incubate in a 95°C water bath for 5 minutes for color development. After removing and cooling to room temperature, take 200 μL and measure the absorbance value A at 540 nm.

[0075] The formula for calculating enzyme activity is: Enzyme activity (μg / h / mL) = ((△A + 0.0747) / 0.0099) / V1 / T, where △A is the difference between the test tube and the control tube, V1 is the volume of sample added, and T is the reaction time. Cellulase activity results are as follows... Figure 3 As shown, ABC and abc represent significant differences, where A, B, and C represent significant differences at different number of days (differences compared to 12h, 24h, and 36h), and a, b, and c represent significant differences between different strains (7 different strains, i.e., strains 1-7). The experiment found that strain 1 had the highest enzyme activity, which continued to increase. Therefore, it was initially determined that strain 1 (i.e., LAB1) had the strongest ability to degrade fibers.

[0076] Further screening will be conducted to determine if it also possesses the ability to inhibit toxin-producing strains (from *Fusarium graminearum* and *Fusarium verticillatum*, which are pathogens of corn stalk rot). The composition of the MRS liquid culture medium is as described above. 0.5g of corn stalk powder is placed in a fiber bag and then placed in an Erlenmeyer flask. After sterilization, liquid culture medium is added for shaker culture.

[0077] 4. Screening for lactic acid bacteria that can inhibit DON.

[0078] Pour an appropriate amount of MRS medium into the bottom layer of the culture medium. After the medium solidifies, place sterile Oxford cups on a plate (divided into eight equal parts, one Oxford cup per region). Pour the liquid containing fungal spores (Fusarium graminearum and Fusarium verticillatum) into PDA medium (composition shown in Table 2) to achieve a concentration of 2.83 × 10⁻⁶. 5 CFU / mL ~ 7.87 × 10 5 CFU / mL, then pipette 25mL of the mixed PDA medium into an Oxford cup plate. After the PDA medium solidifies, remove the Oxford cup and add 50μL of lactic acid bacteria solution (strains 1-6 are 6 strains of initially screened degradable fiber-degrading lactic acid bacteria, strain 7 is commercially available inoculant Yiqing No. 3, strain 8 is laboratory inoculant, and strain 9 is water as control CK). Incubate at 30℃ for 48h and observe the size of the inhibition zone, which indicates the antibacterial ability. Figure 4 As shown. Figure 4 In the test, 7 and 218 were for antagonistic effects against Fusarium verticillatum. Figure 4 In the assays, samples 209 and 120 were used to test the antagonism of Fusarium graminearum. It can be seen that the screened lactic acid bacteria showed a stronger ability to inhibit Fusarium graminearum than Fusarium verticillatum, which means they may have the ability to degrade DON (Fusarium graminearum mainly produces DON). Based on the calculation of the inhibition zone size, degrading bacteria were ultimately screened out as well.

[0079] Table 2. Components of PDA liquid culture medium

[0080]

[0081] 5. Physiological and chemical characteristics of the screened degrading bacteria

[0082] (1) The testing procedures for Gram staining, cell morphology observation, and catalase experiment are as follows:

[0083] Gram staining method: Place a loopful of water in the center of a glass slide, then use an inoculation loop to pick up a small amount of bacteria and mix it evenly with the water droplets on the slide to form a thin bacterial film. Allow it to air dry. Fix the slide upwards over a low flame. After initial staining with crystal violet for 1 minute, rinse thoroughly with water (be gentle and avoid direct water flow onto the bacterial block). Mordant with iodine solution for 1-2 minutes, decolorize with 95% ethanol and rinse with water, counterstain with safranin for 1-2 minutes and rinse with water. Allow to air dry. Observe under a 1000x oil immersion microscope: blue-purple indicates Gram-positive bacteria, and red indicates Gram-negative bacteria.

[0084] Catalase test: Use a pipette tip to draw 3% (volume fraction) hydrogen peroxide solution onto a plate. Use an inoculation loop to pick up a small amount of bacteria and mix it thoroughly with the hydrogen peroxide solution. After 2-3 minutes, observe whether bubbles are produced. If bubbles are produced, the result is positive; otherwise, the result is negative.

[0085] (2) Determination and screening of acid production and growth rate of degrading bacteria: The isolated and purified bacteria were inoculated into 3 mL of MRS liquid medium and cultured overnight at 30℃ and 250 rpm for about 14-16 h. The inoculum was then transferred to a fresh 3 mL MRS liquid medium at a rate of 1% (V / V) and cultured at 30℃ and 250 rpm. The pH of the MRS liquid medium was measured at 0 h, 12 h, 24 h, and 48 h after inoculation, and the absorbance was measured at a wavelength of 600 nm. Three replicates were required for each strain at each time point, and the test tubes used for culturing the bacteria should be of the same size.

[0086] Based on the above experiments, the morphology and physiological and biochemical characteristics of the degrading bacteria are shown in Table 3. The acid production and growth rate of the degrading bacteria and the commercially available bacterial agent (Yiqing No. 3 provided by Beijing Zhongqing Yicao Technology Co., Ltd.) are shown in Table 3. Figure 5 As shown.

[0087] Table 3 Morphological and physiological-biochemical characteristics of the degrading strains

[0088]

[0089] Note: +: positive; -: negative.

[0090] According to Table 3 Figure 2 and 5 Yes, the degrading bacteria are Gram-positive, homofermentative bacilli with fast growth rates and strong overall acid production capacity. Compared to commercially available bacterial agents, these bacteria have a stronger ability to lower pH.

[0091] 6. Perform 16S rDNA gene homology analysis on the bacteria.

[0092] The bacterial strain was cultured overnight at 35°C in 5 mL of MRS medium. The bacterial culture was then transferred to a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 3–5 min to collect the bacteria. The collected bacteria were sent to BGI Genomics Co., Ltd. (China) for colony PCR sequencing. The 16S rDNA is shown in SEQ ID NO.1. The results were compared with the NCBI gene database. The comparison results showed that the degrading bacteria had a similarity of over 99% with *Lactobacillus plantarum*. Based on physiological and biochemical indicators, the degrading bacteria and *Lactobacillus plantarum* were determined to be the same species. The degrading bacteria in this invention is *Lactobacillus plantarum* LAB1 as described in this invention.

[0093] Experimental Example 1

[0094] 1. Preparation of Silage

[0095] Two types of whole-plant corn harvested in Baiyin City, Gansu Province, were manually chopped to 1-2 cm using a guillotine cutter and mixed thoroughly. The two types of whole-plant corn were selected, one containing Trichoderma and the other free of it. GCJF consisted of normal plants (free of Trichoderma), while GJF consisted of plants containing Trichoderma. The uniformly mixed raw materials were packed into 28cm × 35cm polyethylene silage bags, with 3 bags per treatment, each bag containing approximately 500g. The bags were vacuum-sealed and stored at 20–25℃ for 30 days of fermentation before being opened.

[0096] 2. Testing of fiber and toxin content in whole corn raw materials and silage

[0097] The fiber and toxin content of whole-plant corn raw material and 6-day silage were sampled and analyzed. Specific results are shown in Tables 4 and 5. The testing procedure was as follows: 100-200g of silage sample (raw material) was dried in an oven at 65℃ to constant weight, and the dry matter (DM) content was determined. The dried sample was then pulverized and passed through a 1mm sieve, and the neutral detergent fiber (NDF) and acid detergent fiber (ADF) content were determined using the fenestration method. HCEL was calculated by subtracting NDF from ADF. Another 100-200g of silage sample (raw material) was freeze-dried to constant weight, then pulverized and passed through a 1mm sieve. Six mycotoxins (AFB1, ZEN, DON, OTA, T-2, and FUM) were quantitatively analyzed using an ELISA kit (Jiangsu Meibiao Biotechnology Co., Ltd.).

[0098] The fiber content results (Table 4) show that, compared with their respective raw materials, the NDF, ADF, and HCEL of GCJF (silage without Trichoderma) after 60 days of ensiling decreased by 4.13%, 7.77%, and -0.36%, respectively, while the NDF, ADF, and HCEL of GJF (silage with Trichoderma) after 60 days of ensiling decreased by 24.88%, 25.30%, and 24.31%, respectively. This indicates that silage containing Trichoderma contains microorganisms that degrade fiber. No fiber-degrading lactic acid bacteria were subsequently screened in GCJF.

[0099] The toxin content results (Table 5) show that, regardless of whether it is silage raw material or silage silage, the content of various toxins in GJF is consistently lower than that in GCJF, especially DON, OTA, and T-2. This indicates that silage containing Trichoderma possesses microorganisms capable of degrading toxins.

[0100] Table 4 Fiber Content

[0101]

[0102] Note: SEM represents the standard error, and p-value represents the significance of the difference.

[0103] Table 5 Toxin Content

[0104]

[0105] Note: SEM represents the standard error, and p-value represents the significance of the difference.

[0106] Application Example 1

[0107] 1. Preparation of Silage

[0108] Whole-plant corn harvested from Dongchengfang Town, Zhuozhou, Hebei Province, was chopped to 1-2 cm using a shredder and pulverizer, mixed evenly, and inoculated with *Lactobacillus plantarum* LAB1 strain from Example 1. Approximately 1 × 10⁻⁶ LAB1 strain was added to each gram of fresh material. 6 Each treatment was prepared by filling 3 polyethylene silage bags (approximately 500g each) with the uniformly mixed raw materials into 28cm×35cm bags. The bags were vacuum-sealed and stored at 20-25℃. The bags were opened after 14 and 30 days of fermentation.

[0109] 2. Testing of fiber and toxin content in whole corn raw materials and silage

[0110] The fiber and toxin content of whole-plant corn raw materials and silage treated with Lactobacillus plantarum LAB1 were analyzed after 30 days of silage (18 toxins were tested, only DON was detected). Specific results are shown in Table 6. The testing procedure is as follows:

[0111] Take 100-200g of silage sample (raw material), dry it in an oven at 65℃ to constant weight, and determine the dry matter (DM) content. After pulverizing the dried sample and passing it through a 1mm sieve, it is used to determine neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose (HCEL), cellulose (ADL), and lignin: NDF and ADF are determined using the Van der Waals fiber method with an ANKOMA2000 instrument; lignin is obtained by soaking the residue remaining after acid washing in 72% sulfuric acid for 3 hours with continuous stirring, washing it to neutral after soaking, drying and weighing, and carbonizing the remaining residue and then calcining it in a muffle furnace at 550℃ for 3 hours (determination method reference: 1991, Yang Sheng, Feed Analysis and Feed Quality Testing Technology). ADL is calculated by the difference between ADF and lignin, and HCEL is calculated by the difference between NDF and ADF.

[0112] Take another 100-200g of silage sample (raw material) and freeze-dry it to constant weight in a freeze dryer. Then, pulverize it and pass it through a 1mm sieve. Detect the content of 18 toxins using ultra-high performance liquid chromatography-tandem mass spectrometry (Reference: Zhang Dawei, 2018, Simultaneous Detection of 18 Mycotoxins in Feed Raw Materials and Feed Products by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry). Specifically, weigh (25±0.1)g of the sample to be used into a 250mL Erlenmeyer flask, add 100mL of 50% acetonitrile aqueous solution, shake on a shaker (250r / min) for 1h, and filter at least 2mL of filtrate. Transfer 2mL of filtrate to a test tube and simultaneously add 100μL of acetic acid. Vortex to achieve an acetic acid concentration of 5%. After mixing, transfer 750μL of the sample solution from the test tube to the bottom of a MycoSpin™ 400 multifunctional purification column, place it on a 2mL centrifuge tube, and centrifuge at 10000r / min for 1min. After centrifugation, the filtrate is retained in the centrifuge tube. 75 μL of the sample solution is transferred from the centrifuge tube, and 75 μL of positive ion internal standard and 75 μL of negative ion internal standard are added respectively. The mixture is vortexed for 20 s. After centrifugation at 10000 r / min for 3 min, 120 μL of the positive and negative ion mixture is transferred to a sample vial with an inner liner and injected into the centrifuge.

[0113] As shown in Tables 6 and 7, after 30 days of whole-plant corn silage with added LAB1, compared with the control group (Yiqing No. 3) and the unadded (ck) silage, the levels of NDF, ADF, and HCEL were the lowest, with reduction rates of 11.03%, 7.60%, and 12.00%, respectively (compared to silage without added microbial agents). The contents of Pro (proline in silage is positively correlated with toxins), pH, NH3-N%TN, and AA were the lowest, while the contents of WSC and LA were the highest. This indicates that LAB1 significantly improved the nutritional quality and fermentation quality of silage and was superior to the commercial microbial agent Yiqing No. 3.

[0114] After 30 days of silage, whole-plant maize with added LAB1 showed significantly lower cellulose and lignin content compared to maize without LAB1, with reduction rates of 9.03% and 42.65%, respectively. DON content also decreased significantly at 30 days, with a reduction rate of 32.26%.

[0115] Table 6 Results of silage quality and fermentation quality

[0116]

[0117] Note: CK is silage without added microbial agents, LAB-1 is silage with added LAB, the comparative example (Yiqing No. 3) is silage with added commercial microbial agents, SEM is the standard error, and p-value is the significance of the difference.

[0118] Table 7 Degradation of other cellulose and DON

[0119]

[0120] Application Example 2

[0121] 1. Preparation of Silage

[0122] Whole corn plants harvested from Dongchengfang Town, Zhuozhou, Hebei Province were manually chopped to 1-2 cm using a guillotine cutter, mixed thoroughly, and inoculated with *Lactobacillus plantarum* LAB1 from Example 1. Approximately 1 × 10⁻⁶ LAB1 strains were added to each gram of fresh material. 6 Each treatment was prepared by filling 3 polyethylene silage bags (approximately 500g each) with the uniformly mixed raw materials into 28cm×35cm bags. The bags were vacuum-sealed and stored at 20-25℃ for 60 days before being opened.

[0123] 2. Testing of fiber in whole-plant corn raw materials and silage.

[0124] The fiber content of whole-plant corn raw materials and silage treated with Lactobacillus plantarum LAB1 was analyzed after 60 days of silage. The specific results are shown in Table 8. The testing procedure is the same as in Application Example 1.

[0125] The fiber content of whole-plant maize silage after LAB1 supplementation is shown in Table 5. Compared with the control group, specifically, the NDF and ADF were significantly reduced at 60 days of silage, with reduction rates of 5.25% and 16.19%, respectively.

[0126] Table 8 Fiber Degradation Status

[0127]

[0128] In Application Example 1, the material was shredded by machine, while in Application Example 2, the material was shredded by hand using a guillotine. The machine shredded the material more finely, resulting in more thorough fermentation, more complete application of LAB1, and higher fiber degradation efficiency.

[0129] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A Lactobacillus plantarum LAB-GS-CSR1 with accession number CGMCC No.34170.

2. The *Lactobacillus plantarum* according to claim 1, characterized in that, The Lactobacillus plantarum LAB-GS-CSR1 contains the 16S rDNA shown in SEQ ID NO.

1.

3. The *Lactobacillus plantarum* according to claim 1 or 2, characterized in that, The Lactobacillus plantarum LAB-GS-CSR1 was extracted, isolated, and cultured from corn silage containing Trichoderma.

4. A method for extracting, isolating, and culturing *Lactobacillus plantarum* LAB-GS-CSR1 according to any one of claims 1-3, characterized in that, The method includes: The corn silage containing Trichoderma was mixed with physiological saline, homogenized, and the supernatant was transferred to solid MRS medium for culture.

5. The method for extracting, isolating, and culturing *Lactobacillus plantarum* according to claim 4, characterized in that, The cultivation temperature is 30–37℃.

6. A silage additive, characterized in that, Includes Lactobacillus plantarum LAB-GS-CSR1 as described in any one of claims 1-3.

7. A type of silage, characterized in that, Includes Lactobacillus plantarum LAB-GS-CSR1 as described in any one of claims 1-3.

8. A method for preparing silage according to claim 7, characterized in that, Includes the following steps: (1) Chop and mix the silage raw materials to be fermented evenly; (2) Add the Lactobacillus plantarum LAB-GS-CSR1 to the mixed raw materials, and vacuum seal and store to obtain the silage.

9. The method for preparing silage according to claim 8, characterized in that, In step (2), the effective viable count of Lactobacillus plantarum LAB-GS-CSR1 added per gram of raw material is at least 1.0 × 10⁻⁶. 6 indivual.

10. The use of Lactobacillus plantarum LAB-GS-CSR1 as described in any one of claims 1-3 in the preparation of silage.

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