Lactobacillus plantarum NSLP-1 and complex microbial inoculant and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum NSLP-1 as well as a complex microbial inoculant and application thereof, and discloses the lactobacillus plantarum NSLP-1 with the preservation number of GDMCC NO: 67233. The strain has efficient lipid-lowering capacity and excellent gastrointestinal tract tolerance, and can inhibit intestinal pathogens such as salmonella choleraesuis and staphylococcus aureus. The invention also provides a compound microbial agent containing the strain. The compound microbial agent is prepared by compounding lactobacillus plantarum NSLP-1, bacillus subtilis GL-4 and bacillus velezensis V1 according to the volume ratio of 1: (1-2): (1-2). The strain or the complex microbial inoculant can be used for preparing mulberry twig fermented feed, the crude protein content of the feed can be remarkably increased, the fiber content is reduced, and the fermentation quality is improved. By feeding the fermented feed, the diarrhea rate of the Murrah buffalo can be effectively reduced, and the milk yield and the milk quality can be improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Lactobacillus plantarum NSLP-1 and its compound bacterial agents and applications. Background Technology

[0002] Mulberry branches, a byproduct of mulberry cultivation, are widely available and inexpensive. However, they are high in cellulose, have poor palatability, and low nutritional value, resulting in low utilization as direct feed. Currently, the resource utilization of mulberry branches is mainly focused on the preparation of silage or organic fertilizer. However, commonly used fermentation strains such as lactic acid bacteria and Bacillus have limited effectiveness in degrading cellulose, increasing protein content, and inhibiting intestinal pathogens, especially for high-fiber substrates, where fermentation efficiency and product nutritional value still need improvement. Furthermore, the performance of existing probiotics in tolerating gastrointestinal environments (such as low pH and high bile salts) and inhibiting specific pathogens (such as Salmonella choleraesuis and Staphylococcus aureus) varies, leading to inconsistent practical effects of fermented feed in animal husbandry, higher rates of diarrhea, and low feed conversion efficiency.

[0003] Therefore, it is urgent to screen strains with high cellulose degradation capacity, excellent probiotic properties and high safety, and develop special compound microbial agents suitable for fermentation of fibrous agricultural waste such as mulberry branches, so as to improve the nutritional value and disease resistance of fermented feed and realize the high-value utilization of agricultural waste. Summary of the Invention

[0004] In view of the above, the purpose of this invention is to provide a novel strain of *Lactobacillus plantarum* with highly efficient lipid-lowering ability, excellent probiotic properties, and high safety. Lactiplantibacillus plantarum NSLP-1. Based on this, another object of the present invention is to provide a compound microbial agent containing *Lactobacillus plantarum* NSLP-1. A further object of the present invention is to provide the application of *Lactobacillus plantarum* NSLP-1 and the compound microbial agent in the preparation of fermented mulberry branch feed, so as to significantly improve the nutritional value, digestibility, and disease resistance of the fermented feed, and realize the high-value utilization of mulberry branch resources.

[0005] To achieve the above objectives, this invention screened out a new strain: *Lactobacillus plantarum* strain NSLP-1, which is classified and named as follows: Lactiplantibacillus plantarum NSLP-1, Chinese classification name: Lactobacillus plantarum NSLP-1, accession number GDMCC NO: 67233; this strain is deposited at Guangdong Provincial Center for Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 6, 2025.

[0006] The present invention also provides a compound bacterial agent containing the above-described Lactobacillus plantarum strain NSLP-1.

[0007] To further clarify, the compound microbial agent further includes Bacillus subtilis (B. subtilis). Bacillus subtilis ) strain GL-4 and / or Bacillus belesii ( Bacillus velezensis ) strain V1; the Bacillus subtilis ( Bacillus subtilis The accession number for strain GL-4 is CGMCC NO:21366, and the accession date is December 14, 2020; the *Bacillus belyssii* (… Bacillus velezensis The strain V1 has the accession number CGMCC NO:64843 and the accession date is July 8, 2024.

[0008] To further explain, the compound microbial agent is prepared by mixing Lactobacillus plantarum strain NSLP-1, Bacillus subtilis strain GL-4, and Bacillus belyssus strain V1 in a volume ratio of 1:1-2:1-2.

[0009] To further explain, the compound microbial agent is prepared by mixing Lactobacillus plantarum strain NSLP-1, Bacillus subtilis strain GL-4, and Bacillus belyssus strain V1 in a volume ratio of 1:2:2.

[0010] The present invention also provides the application of the Lactobacillus plantarum strain NSLP-1 as described above or the compound microbial agent as described above in the preparation of fermented mulberry branch feed.

[0011] This invention also provides the application of the *Lactobacillus plantarum* strain NSLP-1 as described above in the preparation of an antibacterial agent against intestinal pathogens, wherein the intestinal pathogen is *Salmonella choleraesuis* (…). Salmonella choleraesuis Staphylococcus aureus Staphylococcus aureus ) and / or Escherichia coli ( Escherichia coli )O157.

[0012] The present invention also provides a fermented feed containing the above-mentioned Lactobacillus plantarum strain NSLP-1 or the compound microbial agent as described above. The fermented feed is characterized in that the preparation method of the fermented feed is as follows: mulberry branches are crushed, treated with distilled water to a moisture content of 40-50%, and then Lactobacillus plantarum strain NSLP-1 or compound microbial agent is inoculated into the mulberry branches at an inoculation rate of 80 ml / kg and fermented for 28 days.

[0013] The present invention also provides the application of the fermented feed described above in feeding Mora buffalo.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1. The Lactobacillus plantarum NSLP-1 provided by the present invention has outstanding triglyceride-lowering function (scavenging rate of 48.13%), and its survival rate is still 73.50% after 4 hours in a pH 2.0 environment and 91.91% after 2 hours in a 0.1% bile salt environment, showing excellent gastrointestinal tolerance.

[0015] 2. This strain has a significant inhibitory effect on pathogens such as Salmonella choleraesuis, Staphylococcus aureus, and Escherichia coli O157, especially on Staphylococcus aureus and Salmonella choleraesuis, which helps maintain the intestinal health of animals.

[0016] 3. Through cytotoxicity experiments and acute oral toxicity tests in mice, strain NSLP-1 was found to be highly safe with no toxic reactions, making it suitable for the development of functional foods and feed additives.

[0017] 4. The selected Lactobacillus plantarum NSLP-1, Bacillus subtilis GL-4, and Bacillus bereaves V1 strains showed no growth inhibition among themselves, achieving stable coexistence and synergistic fermentation. Through optimization of the compounding ratio, the optimal volume ratio (1:1:2) was determined. This compound microbial agent produced high filter paper enzyme (FPA) activity, indicating its potential to synergistically enhance cellulose degradation capabilities.

[0018] 5. Fermenting mulberry branches using the compound microbial agent of this invention can significantly increase the crude protein (CP) content of the fermented feed, effectively reduce the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF), while increasing the accumulation of beneficial lactic acid (LA) and reducing the level of ammonia nitrogen (NH3-N), an indicator of protein putrefaction, thus comprehensively improving the nutritional value and fermentation quality of the feed. Feeding Mora buffalo with mulberry branches fermented using this compound microbial agent significantly reduced the diarrhea rate, even to zero, and significantly increased the average milk yield, feed conversion ratio, and lactose content, demonstrating the comprehensive advantages of this fermented feed in promoting animal growth, improving health, and enhancing economic benefits.

[0019] In summary, this compound microbial agent can effectively solve the technical problem of high fiber and low nutrition in mulberry branches, producing fermented feed with excellent nutritional value and probiotic functions. Ultimately, it can significantly improve animal production performance while reducing the rate of diarrhea, providing an efficient, safe, and feasible new technology path for the resource utilization and high-value utilization of agricultural waste such as mulberry branches. Attached Figure Description

[0020] Figure 1 The lipid-lowering ability of different strains.

[0021] Figure 2 This is a colony morphology diagram of strain NSLP-1.

[0022] Figure 3Phylogenetic tree diagram of the 16S rDNA gene sequence of strain NSLP-1.

[0023] Figure 4 This is a comparison chart of antibacterial effects. In the chart, 546 represents Staphylococcus aureus, 2139 represents Salmonella choleraesuis, and 21530 represents Escherichia coli O157.

[0024] Figure 5 The results of mouse necropsy are shown, with A representing the control group and B representing the NSLP-1 treatment group.

[0025] Figure 6 The results show the antagonistic effects among the three strains. Information on the preservation of biological materials

[0026] The strain information deposited in this application is: Lactobacillus plantarum strain NSLP-1, and its taxonomic name is: Lactiplantibacillus plantarum NSLP-1, Chinese classification name: Lactobacillus plantarum NSLP-1, accession number GDMCC NO: 67233; this strain is deposited at Guangdong Provincial Center for Microbial Culture Collection, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on November 6, 2025.

[0027] Bacillus subtilis ( Bacillus subtilis The accession number of strain GL-4 is CGMCC NO:21366. This strain is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on December 14, 2020.

[0028] Bacillus belesiensis ( Bacillus velezensis The strain is V1, and its classification name is: Bacillus velezensis V1, classified in Chinese as Bacillus belyssus V1, with accession number GDMCC NO:64843, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 8, 2024. Detailed Implementation

[0029] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0030] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features. Example 1

[0031] This example demonstrates the screening and identification of Lactobacillus plantarum NSLP-1.

[0032] Previous laboratory work has isolated multiple probiotic strains from environmental, feed, and dairy cow samples in Guangxi. These strains were then screened for those with strong lipid-lowering abilities, and their cholesterol and triglyceride-lowering capabilities were measured. 16S rDNA sequence analysis identified them as *Lactobacillus plantarum*, and a phylogenetic tree was constructed.

[0033] 1. Activation of the strain The strain preserved at -80℃ was inoculated into MRS liquid medium and nutrient broth medium at an inoculation rate of 1%, and incubated overnight at 37℃. This process was repeated twice to ensure full activation.

[0034] 2. Triglyceride-lowering ability test The activated bacterial strain was inoculated into triglyceride medium at a 3% inoculum and cultured at 37°C for 24 hours. The bacterial culture was then centrifuged at 8000 rpm for 10 minutes to obtain the supernatant. The triglyceride content in the supernatant was determined using a triglyceride kit.

[0035] The experiment was repeated in triplicate, and the data are expressed as mean ± standard deviation. GraphPad Prism software was used for plotting.

[0036] 3. Cholesterol-lowering ability test The activated bacterial strain was inoculated into cholesterol medium at a 3% inoculum and cultured at 37°C for 24 hours. The bacterial culture was centrifuged at 8000 rpm for 10 minutes to obtain the supernatant. The cholesterol content in the supernatant was determined using the phthalaldehyde method: 0.3 mL of the supernatant was thoroughly mixed with 1 mL of phthalaldehyde working solution and allowed to stand for 10 minutes. Then, 0.2 mL of glacial acetic acid and 1 mL of concentrated sulfuric acid were added, and the mixture was allowed to stand at room temperature for 10 minutes. The absorbance of the reaction solution was then measured at a wavelength of 540 nm.

[0037] The method for preparing phthalaldehyde working solution is as follows: Weigh 50 mg of phthalaldehyde, dissolve it in anhydrous ethanol and make up to 50 mL to obtain 1 mg / mL phthalaldehyde working solution, and store it under cold for later use.

[0038] The experiment was repeated in triplicate, and the data are expressed as mean ± standard deviation. GraphPad Prism software was used for plotting.

[0039] 4. Strain identification DNA was extracted according to the bacterial genomic DNA extraction kit instructions and used as a template for PCR amplification using universal primers 16S-F (5'-AGAGTTTGATCATGGCTCAG-3') and 16S-R (5'-GTGTGACGGGCGGTGTGTAC-3'). The amplification volume (20 μL) was 2× Taq PCR Master Mix 10 μL, forward and reverse primers (10 μmol / L) 1 μL each, DNA template 3 μL, and double-distilled water (ddH2O) 5 μL were prepared. The amplification program was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 57℃ annealing for 30 s; 72℃ extension for 2 min; 30 cycles; 72℃ extension for 7 min. The amplified products were analyzed by agarose gel electrophoresis and then sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results were submitted to the GenBank database for sequence alignment. A phylogenetic tree was constructed using neighbor-joining (NJ) in MEGA 7.0 software to determine the strain species.

[0040] The results are as follows: (1) The difference in cholesterol / triglyceride content before and after culturing in a medium containing cholesterol / triglycerides was used to evaluate the lipid-lowering ability of the strain. The experimental results are as follows: Figure 1 As shown, none of the strains had a cholesterol-lowering capacity exceeding 30%, but the probiotic NSLP-1 had a triglyceride clearance rate as high as 48.13% (cholesterol clearance rate of 22.94%). Further experiments were conducted to explore other probiotic properties of strain NSLP-1.

[0041] (2) Identification of strain NSLP-1: ① Morphological identification, such as Figure 2 As shown: the colonies are round or oval, with a smooth and moist surface, neat edges, and a milky white color; ② After extracting and sequencing the strain's DNA, molecular identification was performed. The DNA sequence of the strain was amplified, and the 16S rDNA sequencing results of the NSLP-1 strain were compared for homology in the GenBank gene bank using the BLAST program on NCBI to construct a phylogenetic tree. Figure 3 It is known that strain NSLP-1 is *Lactobacillus plantarum*, and is most closely related to the previously reported *Lactobacillus plantarum* 3762 (GenBank accession number: MT538616.1), with a similarity of 99.78%. Based on morphological identification, this strain is classified and named... Lactobacillus plantarum .

[0042] The applicant has deposited the Lactobacillus plantarum strain NSLP-1 at the Guangdong Provincial Microbial Culture Collection Center, and its classification name is: Lactiplantibacillus plantarumNSLP-1, Chinese classification name: Lactobacillus plantarum NSLP-1, accession number: GDMCC NO: 67233; accession address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, accession date: November 6, 2025. Example 2

[0043] This example verifies the probiotic properties of strain NSLP-1. 1. Tolerance test Acid tolerance test: The pH of MRS liquid culture medium was adjusted to 2.0 and 3.0 respectively using 0.1 mol / L dilute HCl, and sterilized at 121℃ for 15 min. After cooling, strain NSLP-1 was inoculated under aseptic conditions, so that the initial viable count in the culture medium was approximately 1 × 10⁻⁶. 8 CFU / mL, samples were taken after incubation at 37℃ for 0h, 2h, and 4h, and viable bacteria were counted using the plate dilution method.

[0044] Bile salt tolerance test: 0.03%, 0.1%, and 0.3% ox bile salts were added to MRS liquid medium, respectively, and sterilized at 121℃ for 15 min. After cooling, strain NSLP-1 was inoculated under aseptic conditions to maintain the initial viable count in the medium at 1×10⁻⁶. 8 The cfu / mL count was collected after incubation at 37℃ for 0h, 2h, and 4h, and the viable count was determined using the plate dilution method.

[0045] 2. Antibacterial performance test The inhibitory effect of the strains on common pathogens was detected by the agar diffusion method: *Salmonella choleraesuis* (Salmonella cholerae) was used as an example. Salmonella choleraesuis Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli O157, Salmonella enteritidis ( Salmonella enterica subsp. enterica serovar Enteritidis As an indicator bacterium, the concentration was adjusted to 1×10⁻⁶. 6 For CFU / mL, add 1 mL of indicator bacteria to 20 mL of 45℃ nutrient agar medium, shake well, and pour into a sterile plate containing sterile Oxford cups (Φ6mm×8mm×10mm). After the agar solidifies, remove the Oxford cups with sterile forceps, add 200 μL to each well, and incubate at 37℃ and 180 rpm for 24 h with shaking. Centrifuge the bacterial supernatant at 12000 rpm for 5 min. Add nutrient broth to one well as a blank control. After the supernatant has completely permeated, incubate the plate at 37℃ for 18-24 h, and observe and measure the diameter of the inhibition zone. An inhibition zone diameter >7 mm is considered to have antibacterial effect; a diameter of 7-10 mm is considered insensitive; 10-20 mm is considered moderately sensitive; and a diameter greater than 20 mm is considered highly sensitive.

[0046] 3. Comparison of antibacterial properties of Lactobacillus plantarum from different sources Detection of different plant Lactobacillus strains using the agar diffusion method Lactiplantibacillus plantarum CICC6009 and strain NSLP-1 against Salmonella choleraesuis ( Salmonella choleraesuis Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli The antibacterial ability of O157 was assessed. The concentration was adjusted to 1×10⁻⁶. 6 For CFU / mL, add 1 mL of indicator bacteria to 20 mL of 45℃ nutrient agar medium, shake well, and pour into a sterile plate containing sterile Oxford cups (Φ6mm×8mm×10mm). After the agar solidifies, remove the Oxford cups with sterile forceps, add 200 μL to each well, and incubate at 37℃ and 180 rpm for 24 h with shaking. Centrifuge the bacterial supernatant at 12000 rpm for 5 min. Add nutrient broth to one well as a blank control. After the supernatant has completely permeated, incubate the plate at 37℃ for 18-24 h, and observe and measure the diameter of the inhibition zone. An inhibition zone diameter >7 mm is considered to have antibacterial effect; a diameter of 7-10 mm is considered insensitive; 10-20 mm is considered moderately sensitive; and a diameter greater than 20 mm is considered highly sensitive.

[0047] 4. Hydrophobicity test The experimental method was described in the reference. The bacterial culture was centrifuged at 5000 r / min for 10 min to collect the bacterial cells. After washing twice with physiological saline, the bacterial concentration was adjusted to OD. 600nm The value was approximately 1 and recorded as A0. 2 mL of bacterial suspension was taken and mixed with 2 mL of xylene, vortexed for 10 min, and allowed to stand at room temperature for 2 h. Then, 1 mL of bacterial suspension was taken and the OD was measured again. 600nm And record it as A. The formula for calculating the bacterial surface hydrophobicity (CSH, %) is: bacterial surface hydrophobicity (CSH) = (A0 – A) / A0 × 100%.

[0048] The above experiment was repeated 3 times in parallel, and the data are expressed as mean ± standard deviation.

[0049] The results are as follows: (1) Probiotics need to tolerate the adverse environment of the human gastrointestinal tract and survive in order to colonize and exert their effects. Under normal circumstances, the pH of gastric acid is about 1.5-4.5, the concentration of bile salts in the small intestine is about 0.03%-0.3%, and the residence time of food in the intestine is about 1.5-2 hours. Based on this, the acid resistance and bile salt resistance of strain NSLP-1 were tested, and the results are shown in Tables 1 and 2. The survival rate of strain NSLP-1 after culturing in an environment of pH=2.0 for 2 hours and 4 hours was 97.75% and 73.50%, respectively, and the survival rate after 2 hours and 4 hours in an environment with a bile salt concentration of 0.1% was 91.91% and 57.03%, respectively. This indicates that strain NSLP-1 has good acid resistance and can tolerate the bile salt environment for a short period of time.

[0050] Table 1. Acid tolerance of strain NSLP-1

[0051] Table 2. Bile salt tolerance of strain NSLP-1

[0052] (2) Antibacterial properties of Lactobacillus plantarum NSLP-1 Table 3 shows that the culture medium of strain NSLP-1 is effective against Salmonella choleraesuis (Salmonella cholerae). Salmonella choleraesuis Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli O157 has antibacterial effects, particularly against Salmonella enteritidis (Salmonella enteritidis). Salmonella enterica subsp. enterica serovar Enteritidis It has no antibacterial effect, but based on the inhibition zone, it has a strong inhibitory effect on Salmonella choleraesuis and Staphylococcus aureus.

[0053] Table 3. Inhibitory effect of strain NSLP-1 on pathogens

[0054] Note: “—” in the table indicates no inhibitory effect.

[0055] From Table 4 and Figure 4 As can be seen, strain NSLP-1 exhibited moderate to sensitive antibacterial activity against all three tested pathogens, with significantly better antibacterial effect than CICC 6009. CICC 6009 only showed insensitive antibacterial activity against Staphylococcus aureus and Salmonella choleraesuis, and had no antibacterial effect against Escherichia coli O157.

[0056] Table 4. Inhibitory effects of strain NSLP-1 and standard strain CICC 6009 on the pathogen.

[0057] Note: “—” in the table indicates no inhibitory effect.

[0058] (3) Evaluation of the adhesion ability of Lactobacillus plantarum NSLP-1 There is a positive correlation between bacterial hydrophobicity and the adhesion of intestinal epithelial cells. Therefore, three experiments were conducted on strain NSLP-1, with an average hydrophobicity of 30.27%. A hydrophobicity between 20% and 50% is considered moderately hydrophobic, indicating that this strain has the ability to adhere to the surface of the intestinal mucosa. Example 3

[0059] This example is a safety evaluation of strain NSLP-1. The experiment is as follows: Cell experiments: After rupturing the bacterial culture, centrifuge to obtain the supernatant. Dilute the supernatant 2, 4, 8, 16, 32, 64, and 128 times and co-culture it with Vero cells. Calculate the cell viability.

[0060] Acute oral toxicity test in mice: The oral toxicity test in mice was conducted according to the limit method in the National Food Safety Standard for Acute Oral Toxicity Testing (GB15193.3-2014). The bacterial concentration was adjusted to OD0.05. 600 =1.0±0.02 for later use. Purchased mice were randomly divided into two groups (n=6), half male and half female, and weighed before gavage. The gavage dose was 10 mL / kg per mouse. Mice in the experimental group were gavage with the test bacterial solution, while mice in the control group were gavage with the same volume of PBS solution. Mice were observed for 14 days, and their daily condition was recorded. They were given basal feed and free access to water. After the observation period, they were weighed and euthanized to observe for any organ lesions.

[0061] The experiment was repeated three times in parallel, and the data are expressed as mean ± standard deviation.

[0062] The results are as follows: The supernatant from bacterial lysis was diluted 2, 4, 8, 16, 32, 64, and 128 times and co-cultured with Vero cells. Cell viability was calculated to determine the maximum non-toxic dose of the lysis buffer. Results showed that all concentrations of NSLP-1 lysis buffer were non-toxic to Vero cells, with 100% viability. A 14-day acute oral toxicity test was also conducted in mice. No poisoning or death was observed in either the experimental or control groups during the test (Table). Dissection revealed no pathological changes in the internal organs of the mice (see Table 1). Figure 5 .

[0063] Table 5. Results of acute oral toxicity test of strain NSLP-1 Example 4

[0064] This embodiment represents our team's preliminary research on the application effects of some hypotheses regarding the microbial agents obtained in fermented mulberry branch feed. As can be seen from the above examples, the selected strain Lactobacillus plantarum NSLP-1 has good lipid-lowering, acid and bile salt resistance, and antibacterial ability.

[0065] Bacillus subtilis ( Bacillus subtilis The GL-4 strain has high cellulase activity and is heat-resistant. When used in roughage, it can quickly decompose cellulose and improve the animal's ability to utilize cellulose.

[0066] Bacillus belesiensis ( Bacillus velezensis Strain VI has a good ability to produce proteases and cellulases.

[0067] During the fermentation process, it is necessary to rapidly decompose cellulose and improve the antibacterial function of the fermented feed to further enhance the quality of the fermented mulberry branch feed. Therefore, we considered using the above three strains as a compound inoculant to inoculate mulberry branches for fermentation, and then considered the excellent indicators after fermentation of mulberry branches without affecting the decomposition of cellulose.

[0068] 1. We conducted an antagonistic test among the three strains: The three activated bacterial strains were streaked onto LB agar plates and incubated at 37°C for 24 hours. The growth of each pair of strains at the crossover points was then observed. If the growth of both strains at the crossover points was relatively weak or absent, it indicated antagonism between the two strains; if both bacteria at the crossover points grew well, it indicated no antagonism between the two strains, and they could be used for mixed culture. Figure 6 The results showed that no sterile zone was formed among the three strains, and they could grow in contact, indicating that there was no inhibitory effect between them.

[0069] 2. Screening of compound microbial agent combinations Three non-antagonistic bacterial strains were inoculated into LB liquid medium at an inoculum of 2% each. After incubation at 18°C ​​and 160 rpm for 1–3 days with shaking, they were mixed at a certain volume ratio, incubated at 18°C ​​and 160 rpm for 2–3 days to obtain a mixed bacterial suspension. The optimal compound bacterial agent was screened by measuring the activity of various cellulases using the same method.

[0070] Non-antagonistic *Lactobacillus plantarum* strain NSLP-1, *Bacillus subtilis* strain GL-4, and *Bacillus belyssii* strain VI were inoculated into LB liquid medium at a 2% inoculum. After incubation at 37°C and 200 rpm for 24–36 h with shaking, the viable counts of all strains were greater than 10⁻⁶. 7CFU / mL, *Lactobacillus plantarum* strain NSLP-1, *Bacillus subtilis* strain GL-4, and *Bacillus belyssii* strain VI were mixed at volume ratios of 1:1:1 (①), 1:1:2 (②), 1:2:1 (③), 1:2:2 (④), 2:1:1 (⑤), 2:1:2 (⑥), 2:2:1 (⑦), and 2:2:2 (⑧). After mixing, the strains were cultured at 37℃ and 200 rpm for 24 h with shaking to obtain a mixed bacterial suspension with a viable count greater than 10⁻⁶ CFU / mL. 8 CFU / mL. The optimal compound bacterial agent was screened by cellulase activity assay.

[0071] The determination of filter paper enzyme (FPA) was performed according to standard methods, using the inactivated crude enzyme solution as a blank control. Crude enzyme solution, buffer solution, and DNS reagent for each strain were pipetted into separate tubes (three samples per tube number) and mixed thoroughly. The standard tubes were simultaneously placed in a boiling water bath and reacted for 10 min. After removal, the tubes were rapidly cooled to room temperature, and the volume was adjusted to 25 mL with water. The tubes were capped and mixed thoroughly. The absorbance was measured at 540 nm using a 10 mm cuvette on a spectrophotometer. One unit of enzyme activity was defined as the amount of reducing sugar equivalent to 1 μmol of glucose produced by hydrolyzing the cellulose substrate within 1 min at (50 ± 0.1) °C and the corresponding pH, expressed as U / g (or U / mL).

[0072] Table 6. Enzyme Activity Determination of Compound Microbial Agents

[0073] The results showed that the enzyme activity of the compound microbial agent FPA was ranked from high to low as ② > ① > ④ > ③ > ⑥ > ⑤ > ⑧ > ⑦. This indicates that groups ①-④ showed the best results, with group ② being the optimal choice. Under these conditions, the microbial strains may achieve metabolic balance and synergistically promote the secretion of cellulase. Therefore, the applicant applied a mixed microbial agent of *Lactobacillus plantarum* strain NSLP-1, *Bacillus subtilis* strain GL-4, and *Bacillus belyssus* strain VI in a volume ratio of 1:1-2:1-2 in subsequent fermentation experiments on mulberry branches.

[0074] 3. The combination of compound microbial agents obtained in the previous experiment was applied to the fermentation experiment of mulberry branches for verification. Experimental Design: This experiment employed a completely randomized experimental design, with 5 groups and 3 replicates per group: a control group (CK group) without any bacterial agent, Group T1 (Lactobacillus plantarum strain NSLP-1: Bacillus subtilis strain GL-4: Bacillus belysinii strain VI in a volume ratio of 1:1:1, with a viable count greater than 102) 8 cfu / ml), Group T2 (Lactobacillus plantarum strain NSLP-1: Bacillus subtilis strain GL-4: Bacillus belysinus strain VI in a volume ratio of 1:1:2, with a viable count greater than 102) 8 cfu / ml), Group T3 (Lactobacillus plantarum strain NSLP-1: Bacillus subtilis strain GL-4: Bacillus belysinus strain VI in a volume ratio of 1:2:1, with a viable count greater than 102) 8 cfu / ml), Group T4 (Lactobacillus plantarum strain NSLP-1: Bacillus subtilis strain GL-4: Bacillus belyss VI in a volume ratio of 1:2:2, with a viable count greater than 10-10) 8 (cfu / ml).

[0075] Crush mulberry branches, add distilled water to treat to a moisture content of 40-50%, then add 80ml / kg of the corresponding bacterial agent according to the experimental design, mix evenly and pack into polyethylene bags (230 mm × 400 mm) with one-way exhaust valves, 500g per bag, 3 replicates per treatment, seal with a vacuum sealer, ferment at room temperature in the dark for 28 days to obtain the product.

[0076] 2. Measurement indicators and methods.

[0077] (1) Routine nutritional component detection and analysis: The sample was dried at 105℃ for 2 hours, and then dried at 65℃ to constant weight. The dry matter (DM) content was calculated (Zhang Liying, 2003). The sample was pulverized and passed through a 40-mesh sieve for the determination of crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), and water soluble carbohydrates (WSC). CP, NDF, and ADF were determined according to the method of van Soest et al. (1981); WSC was determined by anthrone-colorimetric method; and ammonia nitrogen (NH3-N) was detected by high performance liquid chromatography.

[0078] (2) Determination of fermentation quality of fermented mulberry branches: Take 10g of fresh sample into an Erlenmeyer flask, add 90mL of distilled water, shake well, seal with sealing film, and place in a refrigerator at 4℃ for 24h. After filtering the extract through gauze, centrifuge at 8000r / min for 10min and take the supernatant for the detection of fermentation parameters. Crude protein (CP) was determined by the Kjeldahl method, and neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined by the method of Van Soest et al. (1981). Ammonia nitrogen (NH3-N) and lactic acid (LA) were detected by high performance liquid chromatography.

[0079] The optimal experimental group was selected with the contents of crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), lactic acid (LA) and ammonia nitrogen (NH3-N) as the optimization indexes, as shown in Table 7 and Table 8 specifically. Table 7 Composition Table of Mulberry Branches

[0080] Table 8 Results of Fermenting Mulberry Branches with Different Compound Bacterial Agents

[0081] The CP in all treatment groups increased extremely significantly (T2>T1>T4>T3>CK), with the increase ranging from 42.6% (T3) to 77.96% (T2). This indicates that the compound bacterial agent effectively converts the non-protein nitrogen in mulberry branches into microbial cell protein through the biochemical action of microorganisms, or decomposes fibers to release bound protein, thus greatly enhancing the protein value of the product. The change trends of NDF and ADF were highly consistent, both being T2<T1<T4<T3<CK. The fiber degradation in groups T1 and T2 was the most significant (the NDF degradation rate was about 17%). The degradation of cellulose is one of the important reasons for the increase in CP, and it also improves the digestibility of the fermentation product. Compared with CK, the NH3-N content in all treatment groups decreased (T2<T1<T4<T3<CK). This is usually a positive signal, indicating that less free ammonia is produced by protein degradation, and more nitrogen is assimilated by microorganisms for synthesizing cell protein (echoing the increase in CP), reducing unnecessary nitrogen loss and possible odors. The LA content in all treatment groups increased sharply from almost zero (T2>T1>T4>T3>CK). LA is a marker of excellent fermentation. Its accumulation can rapidly reduce the pH value, inhibit harmful microorganisms, and improve the stability and palatability of fermented feed. Thus, it can be seen that the compound bacterial agent fermentation significantly improves protein, significantly reduces fiber and ammonia nitrogen, and significantly improves the lactic acid fermentation quality. Among them, groups T1 and T2 had the most prominent effects. And group T2 showed balanced and excellent performance in all indexes, which is the optimal fermentation scheme under the current experimental conditions. Example 5

[0082] This example is a feeding experiment of mulberry branch fermented feed on Murrah buffaloes.

[0083] Forty Murrah buffaloes in good body condition, with similar weights and stable production performance during the normal lactation period were randomly divided into 4 groups, with 10 buffaloes in each group.

[0084] Control group (CK): The basal diet was: 69% corn flour, 8% soybean cake, 16% cottonseed cake, 2% baking soda, 5% beef cattle premix; Experimental group 1: Basal diet + adding 10% fermented mulberry branches (fermented without bacterial agent); Experimental Group 2: Basic feed + 10% fermented mulberry branches (compound microbial agent added according to the T2 ratio in Example 4) Experimental Group 3: Basic feed + 15% fermented mulberry branches (compound microbial agent added according to the T2 ratio in Example 4) The mixed feed was fed to each head at a rate of 25 kg per day, ensuring that the feed was consumed daily. The experiment lasted for 40 days, with feeding twice a day, in the morning and at noon. At the beginning and end of the experiment, milk samples were collected from the experimental group and the control group three times a day (morning, noon, and evening). The milk production of the Mora buffaloes was tested at these three times, and the average daily milk production and milk-to-feed ratio per head in each group were calculated. The lactose content of the milk samples was measured using a fully automated milk composition analyzer, and the average value over 40 days was taken. The diarrhea rate was observed and calculated (the diarrhea rate was calculated by counting the number of times diarrhea occurred in the Mora buffaloes during the experimental period: 1 instance of diarrhea was recorded as 1, and 2 instances of diarrhea were recorded as 2). The specific results are shown in Table 9.

[0085] Table 9. Effects of fermented mulberry branches on traits of Mora buffalo.

[0086] As shown in Table 8, the diarrhea rate was 2% in the CK group, increased to 4% in experimental group 1, and 0% in experimental groups 2 and 3. This indicates that the compound microbial agent fermented mulberry branches can effectively improve the intestinal health of Murrah buffalo, while the improperly fermented mulberry branches (experimental group 1) may have led to an increase in diarrhea rate due to bacterial imbalance. In terms of average milk yield, milk-to-feed ratio and lactose content, experimental group 2 (34.33 kg) > experimental group 3 (33.87 kg) > experimental group 1 (30.73 kg) > CK (30.35 kg). Among them, experimental group 2 had the best indicators, indicating that the compound microbial agent fermented mulberry branches can effectively improve the nutritional value and conversion efficiency of feed, and promote milk yield and milk quality. The milk-to-feed ratio and milk yield showed the same trend, with experimental group 2 (4.27) > experimental group 3 (4.14) > experimental group 1 (4.01) > CK (3.94). The results show that fermenting mulberry branches with compound microbial agents can significantly improve the health and production performance of Murrah buffalo. Adding an appropriate amount to the basic diet can reduce costs and increase efficiency, while controlling the fermentation process is the key factor.

[0087] The above series of embodiments demonstrates that this invention completes the entire technical chain from strain screening and identification to practical application verification. First, a novel *Lactobacillus plantarum* strain, NSLP-1, with outstanding triglyceride-lowering ability, was successfully screened and identified from a probiotic library. Subsequently, a systematic review confirmed that this strain possesses excellent acid and bile salt tolerance, inhibitory ability against various intestinal pathogens, and high biocompatibility. Furthermore, through antagonism experiments and enzyme activity assays, NSLP-1 was compounded with complementary *Bacillus subtilis* GL-4 and *Bacillus belyssii* VI, optimizing the ratio of the compound microbial agent (volume ratio 1:1:2) to achieve the best synergistic effect. Experiments using this compound microbial agent to ferment mulberry branches showed that it significantly increased the crude protein content of the feed, reduced the fiber content, and improved fermentation parameters. Finally, a feeding trial with Mora buffalo further confirmed that mulberry branch feed fermented using this compound microbial agent effectively reduced the diarrhea rate in animals and significantly increased milk yield, feed conversion ratio, and milk quality. In summary, the Lactobacillus plantarum NSLP-1 and its compound microbial agent provided by this invention offer an effective microbial solution to the problems of low nutrition, poor function, and unstable effects in the utilization of mulberry branches as feed, and have significant application value and market prospects.

[0088] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Lactobacillus plantarum ( Lactiplantibacillus plantarum ) strain NSLP-1, characterized in that, The Lactobacillus plantarum strain NSLP-1 has the accession number GDMCC NO: 67233 and the accession date is November 6, 2025.

2. A compound bacterial agent comprising the Lactobacillus plantarum strain NSLP-1 as described in claim 1.

3. The compound microbial agent according to claim 2, characterized in that, The compound microbial agent also includes Bacillus subtilis ( Bacillus subtilis ) strain GL-4 and / or Bacillus belesii ( Bacillus velezensis strain V1; the Bacillus subtilis ( Bacillus subtilis The accession number for strain GL-4 is CGMCC NO:21366, and the accession date is December 14, 2020; the *Bacillus belyssii* (… Bacillus velezensis The strain V1 has the accession number GDMCC NO:64843 and the accession date is July 8, 2024.

4. The compound microbial agent according to claim 3, characterized in that, The compound microbial agent was prepared by mixing Lactobacillus plantarum strain NSLP-1, Bacillus subtilis strain GL-4, and Bacillus vesicles strain V1 in a volume ratio of 1:1-2:1-2.

5. The compound microbial agent according to claim 4, characterized in that, The compound microbial agent was prepared by mixing Lactobacillus plantarum strain NSLP-1, Bacillus subtilis strain GL-4, and Bacillus belyssus strain V1 in a volume ratio of 1:1:

2.

6. The application of the Lactobacillus plantarum strain NSLP-1 as described in claim 1 or the compound microbial agent as described in claim 2 in the preparation of fermented mulberry branch feed.

7. The application of the *Lactobacillus plantarum* strain NSLP-1 as described in claim 1 in the preparation of an antibacterial agent against intestinal pathogens, characterized in that... The intestinal pathogen is Salmonella choleraesuis (Swine choleraesuis). Salmonella choleraesuis Staphylococcus aureus Staphylococcus aureus ) and / or Escherichia coli ( Escherichia coli )O157.

8. A fermented feed comprising the *Lactobacillus plantarum* strain NSLP-1 as described in claim 1 or the compound microbial agent as described in claim 2, characterized in that, The fermented feed is prepared by crushing mulberry branches, adding distilled water to treat the moisture content to 40-50%, and then inoculating the mulberry branches with Lactobacillus plantarum strain NSLP-1 or compound bacterial agent at an inoculation rate of 80 ml / kg for fermentation for 28 days.

9. The application of the fermented feed as described in claim 8 in feeding Mora buffalo.

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