Pediococcus acidilactici p54 and uses thereof

Abstract

The present application relates to the technical field of microorganism, discloses a pediococcus acidilactici P54 and purposes thereof, the pediococcus acidilactici P54 has the ability of high efficiency, specific degradation group sensing signal molecule 3-oxo-C12-HSL, and can quickly metabolize and produce a large amount of lactic acid, reduce the pH of silage system, in the fermentation of sesbania grandiflora silage, the pediococcus acidilactici P54 provided by the present application is through the dual action of "targeted quenching harmful bacteria + rapid acid production", strongly inhibits the activity of spoilage microorganisms, thereby maximumly reduces the decomposition of crude protein and the generation of ammonia nitrogen in sesbania grandiflora silage, and stabilizes the production of high-quality sesbania grandiflora silage feed.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type of lactic acid cocci P54 and its uses. Background Technology

[0002] Sesbania, a high-quality legume forage, boasts characteristics such as high yield, palatability, and tolerance to salinity, waterlogging, poor soil, and drought, making it a promising new feed resource. Silage is a crucial technology for preserving green fodder in livestock production, playing an irreplaceable role in preserving feed nutrients and ensuring a stable feed supply. Despite its promising feed application prospects, sesbania, as a typical legume, is inherently prone to spoilage due to its low soluble sugar content and high buffering energy. This leads to the proliferation of putrefactive bacteria, consuming large amounts of protein and other nutrients, producing ammonia nitrogen, harmful gases, and potential toxins. This results in a severe decline in the nutritional quality of silage, threatening livestock health. Consequently, the large-scale promotion of sesbania, a high-quality forage with strong resilience and rich nutrients, in livestock feed production is difficult, resulting in a waste of feed resources and failing to fully realize its potential in alleviating feed shortages and optimizing feed structure in saline-alkali areas.

[0003] During the fermentation of sesame silage, Gram-negative putrefactive bacteria such as Enterobacteriaceae easily become dominant, and their growth and metabolism are key factors leading to unstable fermentation and low quality of forage silage. Quorum sensing behavior of Gram-negative putrefactive bacteria is a necessary condition for their adaptive survival in time-varying environments. Studies have found that microbial quorum sensing is closely related to the life activities of Gram-negative putrefactive bacteria, playing a decisive role in their growth and metabolism. Microorganisms possess unique quorum sensing systems, and their quorum sensing signal molecules and receptors are species-specific. Gram-negative putrefactive bacteria mainly maintain high activity during fermentation by secreting AHL quorum sensing signal molecules such as 3-oxo-C12-HSL, increasing their competitiveness as dominant bacteria. During silage fermentation, they deaminate and decarboxylate amino acids, increasing the amount of ammonia and biogenic amines, resulting in low silage quality and toxicity to livestock.

[0004] Targeted degradation of quorum sensing signaling molecules is a way to weaken or eliminate the physiological functions of microorganisms by directly interfering with their quorum sensing system, especially during the active period of the quorum sensing system under unfavorable production and storage conditions (such as high moisture content, hot and humid environments, oxygen, etc.). It is a green and effective new strategy for controlling harmful microorganisms. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a *Pediococcus lactis* P54 and its uses, aiming to provide a method for improving the quality of sesame silage based on a novel regulatory mechanism, thereby increasing the feed application value of sesame and providing a new path for the diversification of livestock feed resources.

[0006] To achieve the above objectives, the present invention provides the following technical solutions.

[0007] In a first aspect, the present invention provides a strain of *Pediococcus acidilactici* P54, which was deposited on February 2, 2026, at the China General Microbiological Culture Collection Center with accession number CGMCC No. 37607.

[0008] Furthermore, the *Pediococcus lactis* P54 was isolated and screened from *Pennisetum affine* silage.

[0009] Furthermore, the screening is conducted based on the mechanism by which quorum sensing signal molecules induce the production of β-galactosidase in strain KYC55.

[0010] In a second aspect, the present invention provides the application of the above-mentioned Pediococcus lactis P54 in the degradation of the quorum sensing signal molecule 3-oxo-C12-HSL.

[0011] In a third aspect, the present invention provides the use of the above-mentioned Pediococcus lactis P54 in the preparation of sesame silage.

[0012] Furthermore, the method for preparing the sesame silage includes: mixing sesame with a microbial agent and fermenting to obtain the sesame silage, wherein the microbial agent contains Pediococcus lactis P54.

[0013] Furthermore, the concentration of Pediococcus lactis P54 in the bacterial agent is ≥10. 6 cfu / mL.

[0014] Furthermore, the sesame seeds and the microbial agent are mixed at a mixing ratio of 1g sesame seeds to 1mL microbial agent and then fermented to prepare the sesame seed silage.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The P54 of Pyrococcus lactis provided by the present invention has the ability to efficiently and specifically degrade the quorum sensing signal molecule 3-oxo-C12-HSL. In response to the problem that harmful microorganisms in sesame silage can synergistically destroy and degrade proteins through the 3-oxo-C12-HSL signal molecule, P54 can effectively block the 3-oxo-C12-HSL quorum sensing signal pathway of putrefactive bacteria during silage fermentation, target and regulate the metabolic activity of putrefactive bacteria, reduce synergistic toxin production (such as ammonia nitrogen generation) and nutrient decomposition behavior, effectively retain crude protein in sesame, and solve the problem of "poor nutrient retention capacity" in sesame silage.

[0016] (2) The P54 lactic acid bacteria provided by the present invention is a homofermentative lactic acid bacteria that can rapidly metabolize and produce a large amount of lactic acid, reduce the pH of the silage system, thereby shortening the fermentation start-up period, timely inhibiting the reproduction of harmful microorganisms (such as putrefactive bacteria and yeast), and solving the problem of slow fermentation start-up and insufficient acidity caused by the silage environment of sesame with "low soluble sugar and high buffer energy".

[0017] In summary, during the fermentation of sesame silage, the P54 lactic acid bacteria provided by this invention strongly inhibits the activity of putrefactive microorganisms through the dual effects of "targeted quenching of harmful bacteria + rapid acid production," thereby minimizing the decomposition of crude protein and the generation of ammonia nitrogen in sesame silage and stabilizing the production of high-quality sesame silage.

[0018] The P54 lactic acid cocci provided by this invention can significantly improve the quality of sesame silage when applied to sesame silage. It has the advantages of high efficiency, strong targeting, stable effect and simple operation. Attached Figure Description

[0019] Figure 1 This is a diagram showing the effect of strain P54, screened in Example 1 of the present invention, on the degradation of the 3-oxo-C12-HSL signaling molecule; Figure 2 This is a colony morphology diagram of strain P54, screened in Example 1 of the present invention, on MRS medium. Figure 3 This is a Gram staining image of strain P54 screened in Example 1 of the present invention; Figure 4 This is a phylogenetic tree diagram of strain P54 screened in Example 1 of the present invention; Figure 5 This is a growth curve diagram measured in Example 2 of the present invention; Figure 6 This is a graph showing the acid production rate measured in Example 2 of the present invention. Detailed Implementation

[0020] To better understand the present invention, the present invention will be further described below with reference to the embodiments. However, the content of the present invention is not limited to the following embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the protection scope of the present invention.

[0021] Example 1: Obtaining the P54 strain of *Pediococcus lactis* Step 1: Isolation of bacterial strains The harvested Napier grass was shredded to 2-3 cm and naturally ensiled for 60 days without any additives. Lactic acid bacteria were then isolated and screened from this silage. Under aseptic conditions, 20 g of the ensiled Napier grass was placed in a 180 mL sterile Erlenmeyer flask containing physiological saline. After thorough shaking for 4 h, the bacterial suspension was picked up with an inoculation loop and streaked onto 1.0% CaCO3 MRS medium. The medium was incubated at 37°C for 24 h. White, moist colonies with a calcium-dissolving zone were picked for isolation and purification. Single colonies were then cultured in liquid form for 24 h, and the strains were preserved using 50% glycerol.

[0022] Step 2: Screening for strains capable of degrading 3-oxo-C12-HSL signaling molecules. The strain "Agrobacterium tumefaciens KYC55" does not produce AHLs itself, but when exogenous AHLs are present, it will induce reporter KYC55 to express the lacZ gene, producing a large amount of β-galactosidase. According to the characteristics of the enzyme, it can hydrolyze X-gal (5-bromo-4-chloro-3-indolegalactoside) to produce blue color. Therefore, this obvious blue color change can be used to screen the strains isolated in step 1 above.

[0023] The KYC55 used in the screening methods described below was provided by Zhili Zhongte (Wuhan) Biotechnology Co., Ltd.

[0024] The screening method used was the double-layer Oxford cup method, with the lower agar layer containing 1.6% and the upper agar layer containing 0.8%. The specific operation is as follows: ① After purifying the lactic acid bacteria strain preserved in step 1 by streaking on an MRS solid plate, pick a single colony with a sterilized pipette tip and inoculate it into 5 mL of MRS liquid medium. Incubate at 37°C for 24 h. Inoculate the cultured lactic acid bacteria suspension at an inoculation rate of 3% into MRS liquid medium containing 2.4 μg / mL 3-oxo-C12-HSL signaling molecules and incubate at 37°C for 24 h.

[0025] ② Pick a single colony of KYC55 and incubate it in liquid LB medium containing 50 µg / mL spectinomycin, 15 µg / mL gentamicin and 4.5 µg / mL tetracycline at 28℃ for 24 h, then set aside for use.

[0026] ③ After heating and melting the freshly prepared and sterilized AT solid medium, cool it to about 45℃. Add 100 µL of 100 mg / mL X-gal and 5 mL of reporter bacterial solution to 100 mL of AT system, mix well and pour onto a pure AT plate. After the plate solidifies, place it in an Oxford cup and add 200 µL of the bacterial solution to be screened into each well. At the same time, use MRS liquid medium as a negative control and a standard solution containing 2.4 μg / mL 3-oxo-C12-HSL as a positive control. Incubate at 28℃ for 24 h and observe the color development of the plate. The quenching effect of the test strain is verified according to the size of the blue halo.

[0027] Based on the above screening method, strain P54 was obtained. Its effect on degrading the signaling molecule 3-oxo-C12-HSL is described in [reference needed]. Figure 1 As shown.

[0028] Step 3: Identification of strain P54 The strain P54 selected in step 2 was streaked on an MRS agar plate and incubated upside down at 37°C for 48 hours. The morphological characteristics of the colonies were then observed. Figure 2 As shown, P54 colonies are white, round, with a smooth surface and regular edges. Further microscopic examination of strain P54 using Gram staining yielded the following results: Figure 3 As shown, Gram staining is positive, and under a microscope, P54 cells appear spherical.

[0029] The target strain was cultured in an anaerobic environment at 37℃ for 24 hours. When the cells reached the late logarithmic growth stage, genomic DNA was extracted for 16S rRNA gene amplification. The successfully amplified product was sequenced at Shanghai Bioengineering Co., Ltd. The sequencing results were compared with known strain sequences from NCBI to identify strains with high homology (similarity >97%). A phylogenetic tree was constructed using MEGA 5.0. The sequences were uploaded to the NCBI website, and after BLAST alignment, the sequences with the highest similarity were downloaded to construct a phylogenetic tree. The results are as follows: Figure 4 As shown in the results, the strain with the highest similarity to strain P54 is... Pediococcus acidilactici strain HBUAS58203 (homology > 99%). Based on the morphological characteristics and molecular biological identification results of strain P54, strain P54 was identified as Pediococcus lactis (…). Pediococcus acidilactici It was named Pediococcus lactis P54.

[0030] Example 2: Determination of the growth rate and acid production rate of Pediococcus lactis P54 The strain P54 obtained in Example 1 and the commercial strain 550 (Lactobacillus plantarum550LPC, purchased from Chengdu Gaofuji Biotechnology Co., Ltd.) were inoculated into sterile MRS broth at an inoculation rate of 3% (v / v) and cultured in a constant temperature shaker (180 rpm) at 37°C. Samples were taken at 48 time points, and the absorbance at 600 nm and the pH at the corresponding time points were measured to determine their growth rate and acid production rate. See [link to results]. Figure 5 and Figure 6 As shown, strain P54 exhibits the same growth rate and acid production rate as commercial strain 550, growing rapidly from 4 to 12 hours and reaching the logarithmic growth phase at 12 hours; the pH decreases rapidly from 4 to 12 hours, and acid production stabilizes after 12 hours.

[0031] Example 3: Application of Pediococcus lactis P54 in sesame silage I. Experimental Materials and Experimental Design (1) Test materials Silage raw material: sesbania.

[0032] Test strain: Pediococcus lactis P54 obtained in Example 1 Preparation of bacterial culture: P54 of *Pediococcus lactis* was transferred to MRS liquid medium and anaerobically cultured overnight at 37°C. The culture was then diluted to 10⁻⁶ with sterile physiological saline. 6 cfu / mL, forming a bacterial suspension containing P54 of Pediococcus lactis.

[0033] (2) Experimental design The experiment included equal volumes of bacterial suspensions containing *Pediococcus lactis* P54 (viable count 1 × 10⁻⁶). 6 CFU / mL treatment, bacterial suspension containing 550 commercial strains (1×10⁻⁶ viable cells) 6 There were three treatments in total: CFU / mL treatment and sterile water treatment. Three parallel controls were set up in each group.

[0034] (3) Silage preparation Sesbania was harvested at the Chongzhou Modern Agricultural Research and Development Base of Sichuan Agricultural University. The harvested sesbania was chopped to 2-3 cm and ensiled. The prepared inoculum solution (1×10⁻⁶) was evenly sprayed onto the surface of the sesbania using a sterilized sprayer. 6 The control group was sprayed with the same volume of sterile water and thoroughly mixed. The material was placed in polyethylene plastic bags (20×30 cm, Shenzhen Sanfeng Plastic Packaging Co., Ltd., China), each bag was accurately weighed to 1000±0.5 g, and three replicates were set up. The bags were vacuum sealed and fermented at room temperature for 45 days. After opening the bags, samples were taken and the quality was determined.

[0035] (4) Quality determination Accurately weigh 20 g of silage sample and mix it with 180 mL of sterile distilled water. Place the mixture in a 4℃ refrigerator for 24 h of extraction. Filter the solution through four layers of gauze and then use a 0.22 μm filter membrane. Use a pH meter (PHS-3C) to quickly determine the pH value of the filtrate. Take a portion of the solution for the determination of ammonia nitrogen and organic acid content.

[0036] Nitrogen content was measured using a fully automated nitrogen analyzer (Rapid Nexceed, Elementar, Germany).

[0037] The content of ammonia nitrogen (AN) was determined using the phenol-sodium hypochlorite colorimetric method.

[0038] The contents of lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were determined using an Agilent 1100 high-performance liquid chromatograph (HPLC): the solution was sputtered at 12000 r·min -1 Centrifuge for 3 minutes and filter the supernatant through a 0.22 µm microporous membrane. The mobile phase is 3 mmol·L⁻¹. -1 Perchloric acid solution, flow rate 1 mL / min -1 The column temperature was 50℃ and the detection wavelength was 210 nm.

[0039] Dry matter (DM) was determined using a conventional drying method: approximately 100g of silage sample was dried in an oven at 65℃ to constant weight.

[0040] The test results are shown in Table 1 below. After 45 days of fermentation, the silage quality indicators of the control group (sterile water sprayed on the surface of sesame), the 550 treatment group (sterile liquid containing commercial strain 550 sprayed on the surface of sesame) and the P54 treatment group (sterile liquid containing P54 lactococcus sprayed on the surface of sesame) showed differences.

[0041] There was no significant difference in dry matter content among the three treatment groups.

[0042] As a key indicator reflecting the nutrient retention capacity of silage, crude protein content was highest in the P54 treatment group (13.63%), and significantly higher than that in the 550 treatment group and the control group (P<0.05), indicating that the P54 treatment group can effectively inhibit the degradation of crude protein during silage.

[0043] pH value is the core indicator for measuring the fermentation effect of silage. The pH values ​​of all groups were below 4.2, which meets the standard of high-quality silage. Among them, the pH value of the P54 treatment group was lower than that of the other two groups, indicating that its lactic acid bacteria fermentation was more active and the degree of acidification was higher, which is conducive to inhibiting the growth of harmful microorganisms such as putrefactive bacteria and Clostridium, and improving the stability of silage.

[0044] The degree of protein degradation was characterized by the ratio of ammonia nitrogen to total nitrogen. Both the control group and the P54 treatment group were significantly lower than the 550 treatment group (P < 0.05), with the P54 treatment group being slightly lower than the control group. The results indicate that the 550 treatment group significantly exacerbated protein degradation, while the P54 treatment group was significantly better than the 550 group in reducing protein breakdown, but not significantly better than the control.

[0045] There was no significant difference in lactic acid and acetic acid content (P > 0.05). The lactic acid content in the control group was 53.57 g·kg⁻¹. - ¹DM, 550 treatment group (51.70 g·kg) - ¹DM) and P54 treatment group (52.31 g·kg) - ¹DM) was slightly lower; regarding acetic acid content, the control group (13.01 g·kg) - ¹DM) was also slightly higher than the 550 treatment group (12.59 g·kg). - ¹DM) and P54 treatment group (12.15 g·kg) - ¹DM). This indicates that bacterial treatment has little effect on the accumulation of lactic acid and acetic acid in sesame silage and does not change the main fermentation type.

[0046] In summary, the P54 treatment group showed the best improvement in the quality of sesame silage. By increasing crude protein content, lowering pH value, and reducing protein degradation, it significantly improved the nutritional quality and fermentation characteristics of sesame silage. Overall, the P54 treatment group exhibited the best quality performance after 45 days of sesame silage fermentation, with high crude protein retention, low pH, low ammonia nitrogen content, and high lactic acid yield, indicating that this treatment effectively improved the fermentation quality of silage.

[0047] Table 1. Quality analysis of sesame silage after 45 days

[0048] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, any improvements and modifications made based on the core ideas of the present invention without departing from the spirit and scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A strain of Pediococcus lactis P54, which was deposited on February 2, 2026, at the China General Microbiological Culture Collection Center with accession number CGMCC No. 37607.

2. The *Pediococcus lactis* P54 according to claim 1, characterized in that: The *Pediococcus lactis* P54 was isolated and screened from *Pennisetum affine* silage.

3. The *Pediococcus lactis* P54 according to claim 2, characterized in that: The screening was conducted based on the mechanism by which quorum sensing signal molecules induce the production of β-galactosidase in strain KYC55.

4. The use of P54 of *Pediococcus lactis* according to any one of claims 1 to 3 in the degradation of the quorum sensing signal molecule 3-oxo-C12-HSL.

5. Use of *Pediococcus lactis* P54 according to any one of claims 1 to 3 in the preparation of sesame silage.

6. The use according to claim 5, characterized in that: The method for preparing the sesame silage includes: mixing sesame with a microbial agent and fermenting to obtain the sesame silage, wherein the microbial agent contains Pediococcus lactis P54 as described in any one of claims 1 to 3.

7. The use according to claim 6, characterized in that: The concentration of *Pediococcus lactis* P54 in the bacterial agent is ≥10. 6 cfu / mL.

8. The use according to claim 7, characterized in that: The sesame seeds and microbial agent were mixed at a ratio of 1g sesame seeds to 1mL microbial agent and then fermented to prepare the sesame seed silage.

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