Phytobacterium plantarum capable of inhibiting growth of monascus and application of phytobacterium plantarum

Abstract

The invention relates to the technical field of microorganisms, and discloses a plant lactobacillus capable of inhibiting growth of monascus and application of the plant lactobacillus. The preservation number of the plant lactobacillus is CGMCC (China General Microbiological Culture Collection Center) No.34851. According to the present invention, the plant lactobacillus CCNH295 has strong bacterial inhibition ability on common soybean meal contaminating molds, such as aspergillus flavus, aspergillus ochraceus, fusarium moniliforme and penicillium; the plant lactobacillus CCNH295 has excellent bacteriostatic ability to monascus, and when the plant lactobacillus CCNH295 is used for fermentation production of soybean meal, growth of monascus can be remarkably inhibited, so that mildewing of the fermented soybean meal is effectively inhibited, and the prepared fermented soybean meal does not mildew after being placed at 37 DEG C for 6 months; besides, the fermented soybean meal produced by solid state fermentation of the plant lactobacillus CCNH295 is used as a feed raw material, so that the stability of the feed can be improved, and the palatability of the feed can be improved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of *Lactobacillus plantarum* that inhibits the growth of *Monascus purpureus* and its applications. Background Technology

[0002] Fermented soybean meal, through a microbial fermentation process, effectively degrades anti-nutritional factors (such as antigenic proteins and oligosaccharides) in soybean meal, improves protein digestibility, and produces beneficial metabolites. Lactic acid bacteria, due to their strong acid-producing capacity, high safety, and ability to improve the intestinal microecology, are widely used in solid-state fermentation of soybean meal. Ideally, fermented soybean meal products will ultimately have lactic acid bacteria as the dominant microbial community, achieving effective storage. However, due to the weak growth performance of fermentation strains and improper fermentation process control, the original microorganisms in the soybean meal raw material may become the dominant microbial community during fermentation, leading to problems such as mold growth and crusting, and easily producing mycotoxins, posing a potential threat to animal health. Current technologies often use methods such as adding chemical preservatives, drying, and sterilization to inhibit unwanted microorganisms, which are costly, complex to operate, affect fermentation efficiency, and result in chemical residues. Therefore, developing a fermentation technology that can effectively inhibit the growth of mold and in-situ bacteria in soybean meal raw materials, while allowing rapid growth and efficient acid production during soybean meal fermentation, and establishing a fermentation microecological system dominated by lactic acid bacteria after fermentation, is of great significance for ensuring the stability, safety, and quality improvement of fermented soybean meal products. Summary of the Invention

[0003] The purpose of this invention is to overcome the technical problem that fermented soybean meal is prone to mold growth, resulting in a short storage time. This invention provides a strain of *Lactobacillus plantarum* that inhibits the growth of *Monascus purpureus* and its application. This strain can inhibit the growth of mold, especially significantly inhibiting the growth of *Monascus purpureus*, thus extending the storage period of fermented soybean meal.

[0004] To achieve the above objectives, the first aspect of the present invention provides a strain of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The preservation number of the plant lactobacillus is CGMCC No. 34851.

[0005] A second aspect of the present invention provides a fermentation agent, wherein the fermentation agent comprises *Lactobacillus plantarum* as described in the present invention.

[0006] A third aspect of the present invention provides the application of the *Lactobacillus plantarum* or the fermentation agent described in the present invention in fermented soybean meal.

[0007] A fourth aspect of the present invention provides a method for preparing fermented soybean meal, the method comprising inoculating the *Lactobacillus plantarum* or the fermentation agent described in the present invention into a fermentation material containing soybean meal for fermentation.

[0008] The fifth aspect of the present invention provides fermented soybean meal prepared by the method described herein.

[0009] Through the above technical solution, the present invention achieves at least the following beneficial effects: (1) The plant lactobacillus CCNH295 of the present invention has a strong antibacterial ability against molds, such as Aspergillus flavus, Aspergillus ochraceus, Fusarium moniliforme and Penicillium. In addition, the plant lactobacillus CCNH295 of the present invention has excellent antibacterial ability against molds that cause soybean meal to mold (Monascus purpureus). When it is used in the fermentation production of soybean meal, it can significantly inhibit the growth of Monascus purpureus, thereby effectively inhibiting the mold growth of fermented soybean meal. In the preferred embodiment, the plant lactobacillus CCNH295 of the present invention is used in the production of fermented soybean meal, and the prepared fermented soybean meal can be placed at 37°C for 3 months without mold growth. (2) The plant lactobacillus CCNH295 of the present invention has excellent broad-spectrum antibacterial properties and has a very strong antibacterial ability against Staphylococcus aureus, Staphylococcus kohlii, Staphylococcus carinatum, Staphylococcus saprophyticus, Escherichia coli, Salmonella and Staphylococcus aureus.

[0010] Biological Preservation The strain provided by this invention is classified and named *Lactobacillus plantarum*. Lactiplantibacillus plantarum It was isolated from samples from the brewing process of Shaoxing rice wine in Zhejiang Province and deposited on June 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34851. Attached Figure Description

[0011] Figure 1 This is a colony morphology diagram of *Lactobacillus plantarum* CCNH295, which is the present invention.

[0012] Figure 2 The images show the Gram staining results and cell morphology diagram of *Lactobacillus plantarum* CCNH295 of this invention.

[0013] Figure 3 This is the lactic acid standard curve for detecting lactic acid content using bromophenol blue.

[0014] Figure 4 This is a test diagram of the antibacterial performance of *Lactobacillus plantarum* CCNH295 against *Monascus purpureus* according to the present invention.

[0015] Figure 5 These are images showing the effects of soaking and mold growth tests on different fermented soybean meal samples; Figure 5From top to bottom and from left to right, the control group (formulation raw materials), Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (commercially available bacterial enzyme pack), and Comparative Example 3 (CGMCC 1.12934) are respectively.

[0016] Figure 6 These are product status images of different fermented soybean meal samples after being placed at 37℃ for 3 months. Figure 6 From left to right, the figures are Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (market bacterial enzyme package), and Comparative Example 3 (CGMCC1.12934).

[0017] Figure 7 This is a bar chart showing the microbial (bacterial) composition analysis of different fermented soybean meal samples after being stored at 37°C for 3 months; among them, Figure 7 From left to right: Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (commercial bacterial enzyme pack), Comparative Example 2 - mold spots (mold spots on commercially available bacterial enzyme pack), and Comparative Example 3 (CGMCC 1.12934). Figure 8 This is a bar chart showing the microbial (fungal) composition analysis of different fermented soybean meal samples after being stored at 37°C for 3 months; among them, Figure 8 From left to right: Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (commercial bacterial enzyme pack), Comparative Example 2 - mold spots (mold spots on commercially available bacterial enzyme pack), and Comparative Example 3 (CGMCC 1.12934).

[0018] Figure 9 This is a graph showing the bacterial growth of different fermented soybean meal samples after soaking. Figure 9 From top to bottom and from left to right, the ingredients are: formulation raw materials (control group), Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (market bacterial enzyme package), and Comparative Example 3 (CGMCC 1.12934). Detailed Implementation

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] In this invention, "CCNH295" is the number of *Lactobacillus plantarum* provided by the inventors during the research process, and "CGMCC No." is the preservation number of the strain. The two represent the same strain, and their numbers can be used interchangeably in the following text.

[0021] The first aspect of this invention provides a strain of *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The preservation number of the plant lactobacillus is CGMCC No. 34851.

[0022] A second aspect of the present invention provides a fermentation agent, wherein the fermentation agent comprises *Lactobacillus plantarum* as described in the present invention.

[0023] According to some embodiments of the present invention, the content of *Lactobacillus plantarum* is ≥10 based on the total mass of the fermentation agent. 5 CFU / g.

[0024] Preferably, the fermentation agent further includes Bacillus subtilis and Saccharomyces cerevisiae.

[0025] Preferably, the Bacillus subtilis has the accession number CGMCC No. 21218 (published in CN116478891A); and the Saccharomyces cerevisiae has the accession number CGMCC No. 22616 (published in CN113717870A).

[0026] Preferably, in the fermentation agent, the ratio of viable counts of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* is (4-10):(3-10):1.

[0027] A third aspect of the present invention provides the application of the *Lactobacillus plantarum* or the fermentation agent described in the present invention in fermented soybean meal.

[0028] A fourth aspect of the present invention provides a method for preparing fermented soybean meal, the method comprising inoculating the *Lactobacillus plantarum* or the fermentation agent described in the present invention into a fermentation material containing soybean meal for fermentation.

[0029] In this invention, preferably, the fermentation material further includes wheat bran and glucose.

[0030] Preferably, the mass ratio of soybean meal, wheat bran and glucose in the fermentation material is (10-20):1:(0.01-0.05).

[0031] More preferably, the mass ratio of soybean meal, wheat bran and glucose in the fermentation material is (15-20):1:(0.02-0.03).

[0032] Preferably, the fermentation material also contains water, and the amount of water used is such that the water content of the fermentation material is 30-35 wt%.

[0033] According to some embodiments of the present invention, the amount of *Lactobacillus plantarum* or the fermentation agent is such that the viable count of *Lactobacillus plantarum* in the inoculated fermentation material is ≥10⁻⁶. 5 CFU / g.

[0034] In this invention, the method further includes seed culture of the *Lactobacillus plantarum* CCNH295 before inoculation to obtain seed solution.

[0035] Preferably, the seed culture method includes: inoculating glycerol tubes of *Lactobacillus plantarum* CCNH295 into fresh, sterile MRS liquid medium and culturing overnight at 33-37°C for 24-30 hours; then inoculating the tubes into fresh MRS liquid medium at an inoculation rate of 2-5% (v / v) and culturing at 33-37°C for 24-30 hours to obtain the seed culture of *Lactobacillus plantarum* CCNH295.

[0036] In this invention, the MRS liquid culture medium is obtained commercially available.

[0037] Preferably, the inoculation amount of the seed liquid is 1-5 (v / w) based on the mass of the fermentation material.

[0038] In this invention, "(v / v)%" refers to volume percentage; "(v / w)%" refers to volume mass percentage.

[0039] According to some embodiments of the present invention, the fermentation conditions include: a temperature of 30-37°C and a time of 15-72 hours.

[0040] The fifth aspect of the present invention provides fermented soybean meal prepared by the method described herein.

[0041] According to a particularly preferred embodiment of the present invention, a method for preparing fermented soybean meal is provided, the method comprising the following steps: (1) Glycerol tubes containing Bacillus subtilis CGMCC No. 21218, Saccharomyces cerevisiae CGMCC No. 22616, and Lactobacillus plantarum CCNH295 were inoculated into fresh, sterile LB, YPD, and MRS liquid media, respectively, and cultured overnight at 35-37℃ for 24-30 h. Then, at an inoculation rate of 2-3 (v / v)%, the culture was inoculated into fresh LB, YPD, and MRS liquid media and cultured statically at 35-37℃ for 24-30 h to obtain seed culture of Bacillus subtilis CGMCC No. 21218 (with a viable count of ≥5×10⁻⁶). 7CFU / mL), seed culture of Saccharomyces cerevisiae CGMCC No. 22616 (viable count of Saccharomyces cerevisiae CGMCC No. 22616 ≥ 5 × 10⁻⁶ CFU / mL), 7 CFU / mL), seed culture of *Lactobacillus plantarum* CCNH295 (live count of *Lactobacillus plantarum* CCNH295 ≥ 5 × 10⁻⁶ CFU / mL), 8 (CFU / mL) (2) Inoculate the seed culture of Bacillus subtilis CGMCC No.21218, the seed culture of Saccharomyces cerevisiae CGMCC No.22616 and the seed culture of Lactobacillus plantarum CCNH295 into the sterilized fermentation material containing soybean meal at 3-5 (v / w)%, place it in a breathing bag, and ferment at 35-37℃ for 48-72h to obtain fermented soybean meal; The ratio of viable counts of Bacillus subtilis CGMCC No.21218, Saccharomyces cerevisiae CGMCC No.22616 and Lactobacillus plantarum CCNH295 in the initial fermentation material is (4-5):(3-4):1; In the fermentation material, the mass ratio of soybean meal, wheat bran and glucose is (18-20):1:(0.025-0.03). The moisture content of the fermentation material is 33-35 wt%.

[0042] The present invention will be described in detail below through embodiments.

[0043] In the following embodiments, "(v / v)%" refers to volume percentage; "(v / w)%" refers to volume mass percentage.

[0044] In the following examples, the composition of the MRS liquid culture medium includes: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL Tween-80, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, pH 6.2, and the balance being deionized water.

[0045] In the following examples, the composition of the MRS solid culture medium includes: 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L diammonium citrate, 5 g / L sodium acetate, 20 g / L glucose, 1 mL Tween-80, 0.5 g / L magnesium sulfate, 0.25 g / L manganese sulfate, 20 g / L agar, pH 6.2, and the balance being deionized water.

[0046] In the following examples, the composition of LB liquid culture medium includes: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, and the balance being deionized water, pH 6.8.

[0047] In the following examples, the composition of the Bengal Red culture medium includes: 5 g / L peptone, 10 g / L glucose, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, 0.033 g / L Bengal Red, 20 g / L agar, and 50 mg / L chloramphenicol.

[0048] Unless otherwise specified, all reagents and materials used in the following examples and comparative examples are commercially available products purchased from reputable chemical or biological reagent and material suppliers, and all reagents are of analytical grade.

[0049] Preparation Example 1 Isolation, purification, identification, performance testing, and biological preservation of Lactobacillus plantarum CCNH295.

[0050] (I) Isolation and purification of strains A sample of lees from the brewing process of Shaoxing rice wine was collected from a rice wine factory in Zhejiang Province. 1g of sample was weighed and placed in 9mL of sterile physiological saline. The sample was shaken thoroughly and centrifuged at 900rpm for 1min to remove particulate matter. The supernatant was collected and counted using a cell counter. Based on the viable cell count, the sample was spread on MRS solid medium plates and incubated at 37℃ for 48h under anaerobic conditions. After colonies grew on the plates, the sample was purified.

[0051] The isolated single colonies were streaked three times on MRS solid medium plates. The obtained single colonies were then placed on MRS solid medium plates containing 1 g / L calcium carbonate. Strains with calcium dissolution zones were inoculated into MRS liquid medium. The bacterial suspensions were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for sequencing. A total of 64 strains were identified as feed additives, including 43 strains of *Lactobacillus plantarum*, 4 strains of *Lactobacillus acidophilus*, 5 strains of *Lactobacillus casei*, and 12 strains of *Lactobacillus paracasei*.

[0052] (1) Evaluation of acid production performance Sixty-four cryopreserved tubes of lactic acid bacteria were transferred to MRS liquid medium at an inoculation rate of 0.2% and cultured overnight. After OD normalization, they were transferred to fresh MRS liquid medium and cultured for 18 hours. The lactic acid content in the supernatant of the fermentation broth was rapidly detected using bromophenol blue. A strain with high lactic acid production was obtained and numbered CCNH295.

[0053] Detection method: Bromophenol blue was used as an indicator, with a system of 180 μL + 20 μL bromophenol blue solution and an absorption wavelength of 422 nm. The fermentation broth after 18 hours of fermentation was used as the detection point, and MRS liquid medium was mixed with the fermentation broth at a 1:1 ratio (i.e., 90 μL + 90 μL). Bromophenol blue preparation method: 0.1 g of bromophenol blue was dissolved in 3 mL of 0.05 mol / L sodium hydroxide solution, and then the volume was adjusted to 200 mL. A bromophenol blue standard curve was prepared (e.g., ...). Figure 3 (As shown).

[0054] The lactic acid production of *Lactobacillus plantarum* CCNH295 was quantitatively determined by liquid chromatography. The method for quantitative determination of lactic acid production was in accordance with the "Chen Ying, Yang Xin, Sun Haoxuan, et al. Characteristics of *Lactobacillus plantarum* and its application in feed [J]. Modern Chemical Industry, 2024, 53(11):2673-2677". The results showed that *Lactobacillus plantarum* CCNH295 could produce 20.1 g / L of lactic acid in MRS liquid medium after fermentation at 37℃ for 15 h.

[0055] (ii) Strain identification (1) Morphological identification: Observing the colony morphology of strain CCNH295, it was found that its colonies on MRS plates were round with steam at the edges, about 2 mm in diameter, raised, moist, smooth, glossy, and milky white (the colony morphology of Lactobacillus plantarum CCNH295 is as follows). Figure 1 (As shown); then, Gram staining was performed on strain CCNH295, and the staining results and cell morphology were observed under a microscope. The cells were short rod-shaped and arranged singly, in pairs, or in chains (Gram staining results and cell morphology of *Lactobacillus plantarum* CCNH295 are shown in the figure). Figure 2 (As shown).

[0056] (2) Molecular identification Strain CCNH295 was sequenced, and the sequencing results were compared with those in the NCBI database. It was found that the 16S rDNA (SEQ ID NO.1) of strain CCNH295 was similar to that of *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The homology between the strain CCNH295 and *Lactobacillus plantarum* reached 99.58%, thus identifying CCNH295 as *Lactobacillus plantarum*. The identification result was also confirmed as *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The selected strains were inoculated into MRS medium and cultured for 24 hours to preserve the bacteria, resulting in a total of 64 strains.

[0057] 16S rDNA (SEQ ID NO.1) sequence information of strain CCNH295:

[0058] (III) Basic performance evaluation (1) Evaluation of antibacterial properties 1. Evaluation of antibacterial properties against Staphylococcus and common opportunistic pathogens Indicator strains of *Staphylococcus aureus*, *Staphylococcus coli*, *Staphylococcus fleshyus*, *Staphylococcus saprophyticus*, *Escherichia coli* CICC25922 (purchased from the China Industrial Microbial Culture Collection Center), *Salmonella* CICC14028 (purchased from the China Industrial Microbial Culture Collection Center), and *Staphylococcus aureus* ATCC26001 (purchased from the American Center for Type Culture Collection) were inoculated into LB broth, respectively. After incubating the indicator cultures overnight, they were transferred to fresh LB broth and cultured until OD500. 600 =1 or the number of viable bacteria is approximately 10. 8 CFU / mL, dilute the viable bacteria count in LB medium to 10. 5 CFU / mL was used as the indicator bacterial solution; among them, Staphylococcus aureus, Staphylococcus kohlii, Staphylococcus carinatum, and Staphylococcus saprophyticus were isolated from soybean meal raw materials.

[0059] Glyceryl ester tubes of *Lactobacillus plantarum* CCNH295 and *Lactobacillus plantarum* CGMCC 1.12934 (purchased from the China General Microbiological Culture Collection Center) were inoculated into fresh, sterile MRS liquid medium and cultured overnight at 37°C for 24 h. Then, at a 2 (v / v)% inoculation rate, the inoculum was added to fresh MRS liquid medium and incubated statically at 37°C for 24 h. The supernatant was then centrifuged, sterilized by membrane filtration, and used as the test sample. MRS liquid medium served as a negative control. The reaction system consisted of: 50% (S1): 100 μL fermentation broth supernatant + 100 μL indicator bacteria dilution; 25% (S2): 100 μL fermentation broth supernatant diluted 2 times + 100 μL indicator bacteria dilution; and 12.5% ​​(S3): 100 μL fermentation broth supernatant diluted 4 times + 100 μL indicator bacteria dilution.

[0060] Inhibition rate (%) = (A0 - A) / A0 × 100%, where: A0 is the OD of the negative control sample. 600 Value; A is the sample OD. 600 Values ​​(where A0 and A are the OD values ​​after culture) 600 and OD before culture 600 The results are shown in Table 1.

[0061] Table 1

[0062] As shown in Table 1, compared with *Lactobacillus plantarum* CGMCC 1.12934, the *Lactobacillus plantarum* CCNH295 provided by this invention has a strong antibacterial ability against the native strains of soybean meal raw materials, including *Staphylococcus aureus*, *Staphylococcus kohlii*, *Staphylococcus carinatum*, and *Staphylococcus saprophyticus*. Even at an addition amount of 12.5%, it still has a strong antibacterial ability.

[0063] Furthermore, the *Lactobacillus plantarum* CCNH295 provided by this invention also exhibits very strong antibacterial activity against common pathogenic bacteria, including *Escherichia coli*, *Salmonella*, and *Staphylococcus aureus*. Therefore, using *Lactobacillus plantarum* CCNH295 in soybean meal fermentation will effectively extend the shelf life of soybean meal, inhibit the proliferation of spoilage and pathogenic bacteria, maintain the stability of raw material quality, and improve the disease resistance of fed animals.

[0064] 2. Evaluation of antibacterial properties against Monascus purpureus Fermented soybean meal products often exhibit "white mold," a phenomenon characterized by the growth of white, flocculent, fluffy, or powdery mold on or inside the raw materials. This is a typical manifestation of microbial contamination during fermentation or storage. The isolated white mold strain was identified as Monascus purpureus.

[0065] The bacterial culture of *Lactobacillus plantarum* CCNH295, after static incubation, was filtered through a membrane, centrifuged, and the supernatant was used as the test sample. The Oxford cup method was used to evaluate its inhibitory ability against *Monascus purpureus*: a substrate was prepared with 1.2% agar solution, Oxford cups were evenly placed on the substrate, and LB solid medium containing 6.4 × 10⁶ oz. was added. 5 CFU / mL of Monascus purpureus spores were mixed and poured onto a substrate. The culture medium was allowed to solidify in a clean bench. After solidification, the Oxford cups were removed using sterile forceps, and the test sample, 10 g / L lactic acid, and 20 g / L lactic acid (as controls) were added to the wells respectively. Two replicates were performed for each sample. Results are shown below. Figure 4 As shown.

[0066] The inhibitory activity against *Monascus purpureus* was further evaluated using the minimum inhibitory concentration (MIC) method. *Monascus purpureus* spores were diluted to 6.4 × 10⁻⁶ using PDB liquid medium. 5 CFU / mL, 100 μL of the dilution was placed in a 96-well cell culture plate, with MRS liquid medium as a blank control. The test sample and MRS medium were diluted 1.14, 1.33, 2, 4, and 8 times with sterile water, and 100 μL of each was placed in a 96-well cell culture plate. The plates were incubated for 24 h and 48 h, respectively. Because mycelia are present during mold growth, the culture conditions of the 96-well plates after 24 h and 48 h were photographed to evaluate the inhibitory effect on mold. The results are as follows: Figure 4 As shown.

[0067] Depend on Figure 4It can be seen that the loading concentration of Monascus purpureus spore suspension in LB solid medium is 6.4 × 10⁻⁶. 5 At a concentration of 10 g / mL, *Lactobacillus plantarum* CCNH295 produced a significant inhibition zone against *Monascus purpureus*, indicating that *Lactobacillus plantarum* CCNH295 has a strong antibacterial ability against *Monascus purpureus*. However, no significant antibacterial activity was detected in *Monascus purpureus* at concentrations of 10 g / L and 20 g / L of lactic acid.

[0068] Because of the presence of mycelia on the surface of the liquid culture of mold, turbidity is used to determine the antibacterial ability of the strain. Figure 4 It can be seen that when the supernatant of CCNH295 centrifugation was added at a concentration of 25%, it had 100% inhibition of the strain. The addition of 12.5% ​​had a certain inhibitory effect, but not complete inhibition. Therefore, under the experimental conditions, the minimum inhibitory concentration was 25%. After 48 hours of cultivation, at the addition of 25%, Monascus purpureus grew some mycelia, and the inhibitory effect weakened. At the addition of 50% or more, it still had 100% inhibitory effect.

[0069] 3. Evaluation of antibacterial properties against common mold contaminants in soybean meal The inhibitory activity of *Lactobacillus plantarum* CCNH295 against common mold contaminants in soybean meal was evaluated using the Oxford cup method described in "CN11394361A - A strain of *Bacillus laterosporus*, its composition and its uses". The specific method included: inoculating glycerol tubes of *Lactobacillus plantarum* CCNH295 and *Lactobacillus plantarum* CGMCC 1.12934 into fresh, sterile MRS liquid medium and incubating overnight at 37°C for 24 hours; then inoculating at a 2 (v / v)% inoculum into fresh MRS liquid medium and incubating statically at 37°C for 24 hours; centrifuging and collecting the supernatant; sterilizing by membrane filtration; and using this as the test sample. 100 μL of the supernatant was then added to an inoculum containing 1×10⁻⁶... 5 PDA solid culture media containing CFU / mL of indicator bacteria Aspergillus flavus CICC40375 (purchased from China Industrial Microbial Culture Collection Center), Aspergillus ochraceus CICC2471 (purchased from China Industrial Microbial Culture Collection Center), Fusarium moniliforme CICC2490 (purchased from China Industrial Microbial Culture Collection Center), and Penicillium ATCC16025 (purchased from American Center for Type Culture Collection) were poured onto an agar plate with Oxford cups evenly distributed. After standing for 10 min, the Oxford cups were removed using sterile forceps, and 100 μL of the test sample was added to the well. After being placed in a 4°C refrigerator for 4 h, the plates were incubated at 37°C for 24 h, and the diameter of the inhibition zone was measured. The results are shown in Table 2.

[0070] As shown in Table 2, the *Lactobacillus plantarum* CCNH295 provided by this invention has a strong inhibitory effect on common contaminating molds in soybean meal, including *Aspergillus flavus* CICC40375, *Aspergillus ochraceus* CICC2471, *Fusarium moniliforme* CICC2490, and *Penicillium* ATCC16025, and its inhibitory performance is better than that of *Lactobacillus plantarum* CGMCC 1.12934.

[0071] Table 2

[0072] (iv) Biological preservation The strain CCNH295 provided by this invention is classified as *Lactobacillus plantarum*. Lactiplantibacillus plantarum It was isolated from samples from the brewing process of Shaoxing rice wine in Zhejiang Province and deposited on June 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34851.

[0073] Example 1-1 (1) Glycerol tubes of *Lactobacillus plantarum* CCNH295 were inoculated into fresh, sterile MRS liquid medium and cultured overnight at 37°C for 24 h. Then, the inoculum was inoculated into fresh MRS liquid medium at a rate of 2 (v / v)% and cultured statically at 37°C for 24 h to obtain seed culture of *Lactobacillus plantarum* CCNH295 (the viable count of *Lactobacillus plantarum* was 10⁻⁶). 9 (CFU / mL) (2) The seed culture of *Lactobacillus plantarum* CCNH295 was inoculated into sterilized fermentation material containing soybean meal at 4 (v / w)% and fermented at 37℃ for 48 h to obtain fermented soybean meal. The viable count in the fermented soybean meal reached 2.04 × 10⁻⁶. 10 CFU / g, lactic acid production reached 30.42g / kg, pH was 4.07; The amount of seed liquid used is such that the viable count of *Lactobacillus plantarum* CCNH295 in the fermented material after inoculation is ≥10⁻⁶. 5 CFU / g; The fermentation material comprises 72 wt% soybean meal, 4 wt% wheat bran, 0.1 wt% glucose, and 23.9 wt% water, with a moisture content of approximately 33 wt%.

[0074] Examples 1-2 (1) Glycerol tubes of *Lactobacillus plantarum* CCNH295 were inoculated into fresh, sterile MRS liquid medium and cultured overnight at 37°C for 24 h. Then, the inoculum was inoculated into fresh MRS liquid medium at a rate of 2 (v / v)% and cultured statically at 37°C for 24 h to obtain seed culture of *Lactobacillus plantarum* CCNH295 (the viable count of *Lactobacillus plantarum* CCNH295 was 10⁻⁶). 9 (CFU / mL) (2) The seed culture of *Lactobacillus plantarum* CCNH295 was inoculated into sterilized fermentation material containing wheat bran at 4 (v / w)% and fermented at 37°C for 48 h to obtain fermented wheat bran. The viable count in the fermented wheat bran reached 2.72 × 10⁻⁶. 9 CFU / g, lactic acid production reached 23.97g / kg, pH was 3.98; The fermentation material comprises 61.5 wt% wheat bran, 35 wt% corn flour, 2 wt% glucose, and the remainder is water.

[0075] Example 2 (1) Glycerol tubes containing Bacillus subtilis CGMCC No. 21218, Saccharomyces cerevisiae CGMCC No. 22616 (cultured at 30℃), and Lactobacillus plantarum CCNH295 were inoculated into fresh, sterile LB, YPD, and MRS liquid media, respectively, and cultured overnight at 37℃ for 24 h. After inoculation at 2 (v / v)% into fresh LB, YPD, and MRS liquid media, and incubated statically at 37℃ for 24 h, seed culture of Bacillus subtilis CGMCC No. 21218 was obtained (the viable count of Bacillus subtilis CGMCC No. 21218 was 10⁻⁶). 8 CFU / mL), seed culture of Saccharomyces cerevisiae CGMCC No. 22616 (the viable count of Saccharomyces cerevisiae CGMCC No. 22616 is 10). 8 CFU / mL), seed culture of *Lactobacillus plantarum* CCNH295 (live count of *Lactobacillus plantarum* CCNH295 is 10-1). 9 (CFU / mL) (2) At 4 (v / w)% of the seed culture of Bacillus subtilis CGMCC No.21218, the seed culture of Saccharomyces cerevisiae CGMCC No.22616 and the seed culture of Lactobacillus plantarum CCNH295, the sterilized fermentation material containing soybean meal was inoculated into the fermentation material, placed in a breathing bag, and fermented at 37°C for 72 h to obtain fermented soybean meal. The viable counts of Bacillus subtilis CGMCC No. 21218, Saccharomyces cerevisiae CGMCC No. 22616, and Lactobacillus plantarum CCNH295 in the initial fermentation material were 1.5 × 10⁻⁶. 5 CFU / g, 5×104 CFU / g and 2×10 5 CFU / g; The fermentation material comprises 72 wt% soybean meal, 4 wt% wheat bran, 0.1 wt% glucose, and 23.9 wt% water, with a moisture content of approximately 33 wt%.

[0076] Comparative Example 1 The method of Example 1 is the same as in Example 1, except that the seed liquid of Lactobacillus plantarum CCNH295 is replaced with commercially available fermented soybean meal protease (purchased from Henan Xinyangshao Biotechnology Co., Ltd., enzyme activity 200000U / g), and the amount of protease used is 3g / kg of material (i.e. 600 U / g of material). The other steps are the same as in Example 1.

[0077] Comparative Example 2 The method is the same as in Example 1, except that the seed culture of *Lactobacillus plantarum* CCNH295 is replaced with a commercially available fermented soybean meal inoculant, added at a rate of 100 g / t of fermentation material. The fermented soybean meal inoculant contains lactic acid bacteria, yeast, and *Bacillus subtilis*, with viable counts of 2 × 10⁻⁶ for each of the lactic acid bacteria, yeast, and *Bacillus subtilis*. 9 CFU / g, 5×10 8 CFU / g and 1.5×10 9 CFU / g, and other steps are consistent with Example 1.

[0078] Comparative Example 3 The method of Example 2 is the same as that of Example 2, except that Bacillus plantarum CCNH295 is replaced with Bacillus plantarum CGMCC 1.12934.

[0079] Test Example 1 The fermented soybean meal from the above examples and comparative examples was cultured at 37°C for 3 days. Samples were taken and the pH, lactic acid production, viable lactic acid bacteria count, and viable staphylococcus count of the fermented soybean meal were measured. The results are shown in Table 3.

[0080] The pH, lactic acid yield, viable count of lactic acid bacteria, and viable count of staphylococci in fermented soybean meal were determined according to the methods described in (Chen Ying, Lu Zongmei, Zhang Lin, et al. Analysis and detection technology of fermented feed [J]. Modern Chemical Industry, 2019, 48(9):2060-2063. Chen Ying, Lu Zongmei, Yu Jianliang, et al. Counting method of probiotics in microbial fermented feed [J]. Modern Chemical Industry, 2018, 47(5):991-994.). Lactic acid yield was detected by HPLC. Viable counts of lactic acid bacteria and staphylococci were determined by diluting and plating MRS solid medium and distinguishing lactic acid bacteria and staphylococci by microscopic examination.

[0081] Table 3 Performance indicators of fermented soybean meal after 3 days of storage

[0082] As shown in Table 3, the pH of fermented soybean meal prepared using *Lactobacillus plantarum* CCNH295 (Example 2) of this invention can be reduced to 4.79, while that prepared using commercially available inoculant packets can only be reduced to 5.21. In terms of acid production, *Lactobacillus plantarum* CCNH295 (Example 2) of this invention produced the highest acid content in the fermented soybean meal, reaching 32.14 g / kg, and had a high number of viable lactic acid bacteria, with no viable staphylococci detected. In contrast, commercially available inoculant packets showed a large number of staphylococci, and *Lactobacillus plantarum* CGMCC 1.12934 produced less acid and grew poorly under this fermentation system, exhibiting relatively poor inhibition of staphylococci. Compared to the enzyme preparation group, fermentation of soybean meal without the addition of inoculant did not occur because the viable counts of both lactic acid bacteria and staphylococci were low. The material itself contained a certain amount of staphylococci; if the added lactic acid bacteria had no inhibitory effect on staphylococci, the staphylococci would multiply rapidly.

[0083] Test Example 2 (a) Immersion mold test Take 50g of the fermented soybean meal from the above examples and comparative examples, place it in a clean paper cup, add sterile water, and soak until the material is moist to obtain the soaking material; using the formula raw material (soybean meal raw material) as the control group, place it at 37℃ for 3 months and observe the mold growth of the soaking material. The results are as follows. Figure 5 .

[0084] Figure 5 These are images showing the effects of soaking and mold growth tests on different fermented soybean meal samples; Figure 5 From top to bottom and left to right, the groups are: control group (formulation ingredients), Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (commercially available bacterial enzyme pack), and Comparative Example 3 (CGMCC 1.12934). Figure 5 It can be seen that after soaking and placing the fermented soybean meal samples at room temperature for 3 months, mold quickly grew on the surface of the control group (formula raw materials) (the arrows in the figure indicate the mold spots of the soaked material); Comparative Example 1 (enzyme preparation group) also had a certain amount of mold; Comparative Example 2 (commercially available enzyme pack) also had a certain amount of mold, but it was slightly better than the control group (formula raw materials), with fewer types of mold; while the fermented soybean meal of Example 2 (CCNH295) of this application, after soaking and placing for 3 months, still did not show mold growth, and the taste was only that of grain, while other groups could be clearly smelled of mold and decay.

[0085] (II) Performance indicators and microbial composition analysis The performance indicators (including pH and lactic acid content) and microbial composition (abundance of bacteria and fungi) of fermented soybean meal stored at 37℃ for 3 months were analyzed. The results are shown in Table 4 and 5. Figure 6-8 .

[0086] Performance parameters (including pH and lactic acid content) were determined according to the method in Test Example 1.

[0087] Microbial composition analysis (bacterial and fungal abundance): High-throughput sequencing of fermentation samples was commissioned to Shanghai Paisennong Biotechnology Co., Ltd. Bacterial community composition was determined by amplifying the V3-V4 hypervariable region of the 16S rRNA gene, and fungal community composition by amplifying the ITS1 region. Sequencing was performed using the Illumina MiSeq platform. After quality control, noise reduction, clustering (or ASV analysis), and species annotation, the relative abundance of each taxonomic unit was calculated.

[0088] Table 4 Performance indicators of fermented soybean meal after 3 months of storage

[0089] As shown in Table 4, the pH of the fermented soybean meal in different examples and comparative examples was around 4.5 after being placed at 37°C for 3 months, and the lactic acid yield was greater than 25 g / kg.

[0090] Figure 6 These are product status images of different fermented soybean meal samples after being placed at 37℃ for 3 months. Figure 6 From left to right, the images are: Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (commercial bacterial enzyme package), and Comparative Example 3 (CGMCC1.12934). Figure 6 It can be seen that the fermented soybean meal samples in Comparative Examples 1-3 all had mold spots after being placed at 37°C for 3 months (the red circles in the figure indicate the moldy areas), while Example 2 was in good condition and no mold was found.

[0091] Figure 7 This is a bar chart showing the microbial (bacterial) composition analysis of different fermented soybean meal samples after being stored at 37°C for 3 months; among them, Figure 7 From left to right: Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (commercial bacterial enzyme pack), Comparative Example 2 - mold spots (mold spots on commercially available bacterial enzyme pack), and Comparative Example 3 (CGMCC 1.12934). Figure 8 This is a bar chart showing the microbial (fungal) composition analysis of different fermented soybean meal samples after being stored at 37°C for 3 months; among them, Figure 8 From left to right: Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (commercially available enzyme pack), Comparative Example 2 - mold spots (mold spots on commercially available enzyme pack), and Comparative Example 3 (CGMCC 1.12934). Figure 7 It can be seen that, from the perspective of bacterial composition, after fermented soybean meal in different examples and comparative examples was placed at 37°C for 3 months, the abundance of harmful bacteria was effectively controlled in Example 2 (CCNH295), while the abundance of beneficial probiotics (specifically including Lactobacillus plantarum, Lactobacillus, Pediococcus lactis, etc.) was increased. Figure 8 It can be seen that, in terms of fungal composition, after fermented soybean meal in different examples and comparative examples was placed at 37°C for 3 months, Example 2 (CCNH295) had Saccharomyces cerevisiae as the main fungal component, with other fungi having a very small abundance; while Comparative Example 1 (enzyme preparation group) had... Aspergillaceae The primary fungus was Aspergillus; in contrast, the commercially available enzyme package showed signs of mold growth. The moldy material was mainly composed of Aspergillus purpureus, while the commercially available enzyme package contained other types of molds and other miscellaneous bacteria.

[0092] In summary, the microbial composition of fermented soybean meal varied significantly among the different examples and comparative examples after being stored at 37°C for 3 months. This is not simply due to low pH and high lactic acid content regulating the microbial composition of the fermented soybean meal, but rather the regulation of the entire fermentation process by the metabolic products of the bacterial strains. Example 2 (CCNH295) showed detectable abundances of *Lactobacillus plantarum*, *Saccharomyces cerevisiae*, and *Bacillus subtilis* in the fermented soybean meal. All added fermentation strains grew and functioned in the material. Example 2 (CCNH295) showed a relatively low abundance of *Staphylococcus* compared to other groups, while the enzyme preparation group and the commercially available enzyme package both showed a certain abundance of *Staphylococcus*, with the enzyme preparation group showing a higher abundance. Therefore, the addition of *Lactobacillus plantarum* CCNH295 can effectively inhibit the growth of harmful bacteria in fermented soybean meal, regulate the entire fermentation process, and ensure that the added beneficial bacteria dominate the microbial community.

[0093] Test Example 3 Three samples were taken from each soaking medium in Test Example 2, and evenly placed on Bengal Red Agar. The samples were incubated at 25°C for 3 days, and the growth of the bacterial strains was observed. The results are as follows: Figure 9 .

[0094] Figure 9 This is a graph showing the bacterial growth of different fermented soybean meal samples after soaking. Figure 9 From top to bottom and left to right, the table shows the formulation ingredients (control group), Example 2 (CCNH295), Comparative Example 1 (enzyme preparation group), Comparative Example 2 (market-grown bacterial enzyme package), and Comparative Example 3 (CGMCC 1.12934). Figure 9It can be seen that molds grew in Comparative Example 1 (enzyme preparation group), Comparative Example 2 (commercially available bacterial enzyme package), Comparative Example 3 (Bacillus plantarum CGMCC 1.12934), and soybean meal raw material (control group), while the morphology of the strain growing in Example 2 (CCNH295) was observed to be yeast.

[0095] Test Example 4 The fermented materials in the above embodiments and comparative examples were subjected to feed palatability tests. The specific test methods included: 120 weaned piglets of the same breed were selected and divided into 3 treatments. They were fed fermented soybean meal as in Example 2 and fermented soybean meal as in Comparative Example 2, respectively. Soybean meal was used as a blank control. Each treatment had 8 replicates, with 5 pigs per pen. Each replicate was randomly separated. The experimental period was 42 days. The daily amount of fermented soybean meal added was 10% in the early nursery stage and 5% in the later nursery stage.

[0096] The body weights of piglets at 0, 14, 28, and 42 days of age in the three treatments were recorded. The average daily weight gain, average daily feed intake, feed conversion ratio, and diarrhea rate were calculated. The results are shown in Table 5.

[0097] Table 5 Growth performance indicators of piglets

[0098] As shown in Table 5, feeding piglets with fermented soybean meal prepared using the method of Example 2 of this invention can further reduce the diarrhea rate of piglets and improve their growth indicators (such as body weight, average daily weight gain, average daily feed intake, and feed conversion ratio). Clearly, using fermented soybean meal produced by solid-state fermentation with *Lactobacillus plantarum* CCNH295 of this invention as a feed ingredient can improve feed stability and palatability.

[0099] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum ), characterized in that, The preservation number of the *Lactobacillus plantarum* is CGMCC No. 34851.

2. A fermentation agent, characterized in that, The fermentation agent includes *Lactobacillus plantarum* as described in claim 1.

3. The fermentation agent according to claim 2, wherein, Based on the total mass of the fermentation agent, the content of *Lactobacillus plantarum* is ≥10. 5 CFU / g.

4. The fermentation agent according to claim 2, wherein, The fermentation agent also includes Bacillus subtilis and Saccharomyces cerevisiae.

5. The fermentation agent according to claim 4, wherein, The Bacillus subtilis has the accession number CGMCC No. 21218; the Saccharomyces cerevisiae has the accession number CGMCC No. 22616; Preferably, in the fermentation agent, the ratio of viable counts of *Lactobacillus plantarum*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* is (4-10):(3-10):

1.

6. The application of *Lactobacillus plantarum* as described in claim 1 or the fermentation agent as described in any one of claims 2-5 in fermented soybean meal.

7. A method for preparing fermented soybean meal, characterized in that, The method includes inoculating the *Lactobacillus plantarum* of claim 1 or the fermentation agent of any one of claims 2-5 into a fermentation material containing soybean meal for fermentation.

8. The method according to claim 7, wherein, The fermentation materials also include wheat bran and glucose; Preferably, the mass ratio of soybean meal, wheat bran, and glucose in the fermentation material is (10-20):1:(0.01-0.05). Preferably, the fermentation material also contains water, and the amount of water used is such that the water content of the fermentation material is 30-35 wt%.

9. The method according to claim 7, wherein, The amount of *Lactobacillus plantarum* or the fermentation agent used is such that the viable count of *Lactobacillus plantarum* in the inoculated fermentation material is ≥10⁻⁶. 5 CFU / g; Preferably, the fermentation conditions include: a temperature of 30-37°C and a time of 15-72 hours.

10. Fermented soybean meal prepared by the method according to any one of claims 7-9.

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