Pediococcus acidilactici NCUF214.4-A33-12 and application thereof in relieving ETEC infectious diarrhea

Abstract

The application provides a pediococcus acidilactici NCUF214.4-A33-12 and application thereof in relieving ETEC infectious diarrhea, and relates to the technical field of microorganisms.The pediococcus acidilactici NCUF214.4-A33-12 provided by the application is preserved in the China Center for Type Culture Collection on January 20, 2026, and the preservation number is CCTCC NO: M 2026164.The pediococcus acidilactici with bacteriostatic activity to escherichia coli and capable of growing in a pH=3.7 environment is screened by an ARTP mutagenesis combined with an acid stress directional adaptive evolution strategy.The pediococcus acidilactici has strong bacteriostatic performance, high acid tolerance, and can relieve the diarrhea symptoms of mice caused by ETEC K88.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of lactic acid cocci NCUF214.4-A33-12 and its application in alleviating ETEC infectious diarrhea. Background Technology

[0002] Diarrhea is a significant public health problem worldwide, with enterotoxigenic Escherichia coli (ETEC) being one of the leading pathogens causing diarrhea in infants, travelers, and foodborne illnesses, resulting in hundreds of thousands of deaths annually. For a long time, antibiotics have been the primary treatment for ETEC infections. However, the widespread use of antibiotics has led to the emergence of drug-resistant strains, and while antibiotics kill pathogens, they also disrupt the balance of normal gut flora, further weakening the host's defenses. Therefore, developing safe and effective non-antibiotic alternatives, particularly probiotic-based microecological preparations, has become a current research hotspot and an industry demand.

[0003] Probiotics are live bacteria that can have beneficial effects on humans or animals by regulating the gut microbiota. They have been widely reported to treat diarrhea caused by pathogens by maintaining or improving gut microbiota balance. Lactic acid bacteria, due to their ability to produce various antibacterial substances, have a good ability to antagonize diarrhea-causing pathogens. Pediococcus acidilactici is a Gram-positive lactic acid bacterium that lowers the intestinal pH by producing lactic acid through fermentation, inhibiting pathogen colonization. Its bacteriocins and other antibacterial substances can target and kill harmful bacteria while promoting the proliferation of beneficial bacteria and optimizing the gut microbiota structure. Studies have shown that it has probiotic functions such as relieving diarrhea, enhancing immunity, and improving intestinal inflammation, showing good application potential in relieving diarrhea.

[0004] However, the probiotic functions of *Pediococcus lactis* isolated and screened directly from nature or feces in existing technologies are generally limited by the following two key defects, which seriously restrict their practical application in oral probiotic preparations: (1) Insufficient resistance to gastric acid. Oral probiotics must first tolerate the extremely acidic environment in the stomach (the pH of gastric juice before meals can be as low as 1.5) in order to reach the intestines with a sufficient number of live bacteria to colonize and exert probiotic functions. The survival rate of most *Pediococcus lactis* reported in the present study is less than 50% in the pH=3.0 environment, and the survival rate is generally less than 40% in the pH=1.5 environment. The low survival rate leads to a large number of probiotics being inactivated in the stomach and unable to be effectively delivered to the intestines. Even if they have excellent antibacterial activity in vitro, it is difficult to convert them into actual diarrhea relief effects in vivo; (2) Single probiotic function, it is difficult to obtain both high acid resistance and strong antibacterial properties. Traditional strain improvement methods, such as natural screening or single mutagenesis breeding, can usually only optimize a certain trait. In lactic acid bacteria, acid resistance and antibacterial properties often exhibit a trade-off—excessive emphasis on acid resistance may affect the strain's metabolic activity and bacteriocin synthesis capacity, leading to a decrease in targeted antibacterial efficacy against ETEC; conversely, pursuing high antibacterial activity may weaken the strain's adaptability to environmental stress. Current technologies lack effective strategies for synergistic screening and systematic evaluation of dual functional traits of acid resistance and strong antibacterial activity. Simultaneously, most strains face the dual challenge of insufficient acid resistance and limited efficacy against pathogens (such as ETEC). For example, the survival rate of some strains in the extreme acidic environment of pH 1.5 is generally below 40%, and their targeted antibacterial efficacy against ETEC is poor, making it difficult to simultaneously meet the dual requirements of acid resistance and strong antibacterial activity, thus limiting their practical application in the treatment of diarrhea.

[0005] Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0006] The purpose of this invention is to provide a *Pediococcus lactis* NCUF214.4-A33-12 strain and its application in alleviating ETEC infectious diarrhea.

[0007] In a first aspect, the present invention provides a *Pediococcus acidilactici* NCUF214.4-A33-12 strain, which is classified as *Pediococcus acidilactici* NCUF214.4-A33-12. This strain was deposited on January 20, 2026, at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 2026164, at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Secondly, the present invention provides a method for screening and obtaining the above-mentioned *Pediococcus lactis* NCUF214.4-A33-12, comprising the following steps:

[0009] (1) The starting strain, Pediococcus lactis PA-214.4, was subjected to atmospheric pressure and room temperature plasma mutagenesis for 120s, and the mutagenesis strain with enhanced antibacterial activity was obtained by screening.

[0010] (2) The mutant strain obtained in step (1) was subjected to acid stress-directed adaptive evolution culture in a medium with continuously decreasing pH, and the target strain, Pediococcus lactis NCUF214.4-A33-12, was screened to improve acid resistance and maintain antibacterial activity.

[0011] Optionally, the pH range of the culture medium in step (2) that continuously decreases pH is gradually reduced from 6.0 to 3.7.

[0012] Optionally, the number of generations of acid stress-directed adaptive evolution culture described in step (2) is 40-50.

[0013] Optionally, the survival rate of the *Pediococcus lactis* NCUF214.4-A33-12 is 85.95% at pH 3.0 and 57.88% at pH 1.5; the inhibition zone of *Pediococcus lactis* NCUF214.4-A33-12 against enterotoxigenic *Escherichia coli* (ETEC) is 14.79 mm, and it has a relieving effect on diarrhea symptoms in mice infected with enterotoxigenic *Escherichia coli*.

[0014] Optionally, the mutant genes in the whole genome of *Pediococcus lactis* NCUF214.4-A33-12 include genes aguD, gtfA, rpoC, and rpoB; the nucleotide sequence of gene aguD is shown in SEQ ID NO.2; the mutation of gene aguD specifically involves a T-to-G mutation at position 160883 relative to the whole genome sequence of *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of gene gtfA is shown in SEQ ID NO.3; the mutation of gene gtfA specifically involves a T-to-G mutation at position 194609 relative to the whole genome sequence of *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of gene rpoC is shown in SEQ ID NO.2. As shown in NO.4; the specific mutation of the rpoC gene is a mutation from T to C at position 1473663 relative to the whole genome sequence of the *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of the rpoB gene is shown in SEQ ID NO.5; the specific mutation of the rpoB gene is a mutation from T to G at position 1475092 relative to the whole genome sequence of the *Pediococcus lactis* NCUF214.4-A33-12.

[0015] Thirdly, the present invention provides an application of the above-mentioned *Pediococcus lactis* NCUF214.4-A33-12 in relieving ETEC infectious diarrhea, the application comprising using *Pediococcus lactis* NCUF214.4-A33-12 to prepare a microecological preparation for relieving ETEC infectious diarrhea; the microecological preparation is one of the following forms: solution, powder, suspension, granules or tablets.

[0016] Optionally, the *Pediococcus lactis* NCUF214.4-A33-12 can be used alone or in combination with a compound freeze-drying protectant containing 8.2% inulin at a volume ratio of 1:1 to prepare a microecological preparation.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention uses an ARTP mutagenesis and acid stress adaptive evolution coupling strategy to selectively screen and obtain the lactic acid cocci strain NCUF214.4-A33-12 with synergistic enhancement of acid resistance and antibacterial function. Its inhibition diameter against enterotoxigenic Escherichia coli is 14.79 mm, and its survival rate is 85.95% at pH=3 and 57.88% at pH=1.5.

[0019] (2) This invention identifies the mutation sites, mutated bases and mutation types of mutant strains through whole-genome resequencing analysis, and associates the superior functional traits of the strains with specific gene mutations (aguD, gtfA, rpoB, rpoC), revealing the molecular basis of the synergistic enhancement of acid resistance and antibacterial activity, providing a theoretical basis and technical support for the rapid screening of subsequent strains, molecular marker-assisted breeding and further rational modification.

[0020] (3) The Porphyromonas lactis NCUF214.4-A33-12 provided by the present invention has been confirmed by safety evaluation to have no hemolytic activity (γ-hemolysis) and is sensitive to a variety of commonly used clinical antibiotics such as erythromycin, penicillin, and cefotaxime. It has no potential pathogenic risk and can be used as a safe and usable strain for the production of microecological preparations.

[0021] (4) The microecological preparation prepared in this invention can be used alone or combined with a compound freeze-drying protectant containing 8.2% inulin at a volume ratio of 1:1 to form a synbiotic preparation. Animal experiments have verified that Porphyromonas lactis NCUF214.4-A33-12 and its synbiotic preparation can significantly alleviate the weight loss and diarrhea symptoms in mice caused by ETEC K88 (reducing fecal water content), inhibit organ (liver and spleen) enlargement caused by ETEC, effectively alleviate colonic atrophy and inflammation, repair intestinal barrier function, regulate flora balance, and the intervention effect of mutant strain NCUF214.4-A33-12 is significantly better than that of the original strain. The effect is even better after being combined with inulin. It has good potential for the development of microecological preparations and industrial application value. Attached Figure Description

[0022] Figure 1 This is a diagram showing the adaptive evolution of the strain under different pH conditions during acid tolerance-oriented selection in Example 1.

[0023] Figure 2 The graph shows the antibacterial activity results of the starting strain and the stressed strain in Example 1;

[0024] Figure 3 The figures show the acid tolerance and bile salt tolerance results of the starting strain and the stressed strain in Example 1; among them, Figure 3 In Figure A, the results of the acid resistance of the starting strain and the stress strain in Example 1 are shown. Figure 3 B in the figure represents the bile salt tolerance results of the starting strain and the stress strain in Example 1;

[0025] Figure 4 The image shows the colony morphology of *Pediococcus lactis* NCUF214.4-A33-12 on agar plates.

[0026] Figure 5 Growth curve of Pediococcus lactis NCUF214.4-A33-12;

[0027] Figure 6 The genetic stability results of Porphyromonas lactis NCUF214.4-A33-12 are shown in the figure.

[0028] Figure 7 The image shows the results of hemolytic activity assay for the stressed strain NCUF214.4-A33-12.

[0029] Figure 8 The figure shows the effect of pH 3.7 on the growth curves of the starting strain and the stressed strain NCUF214.4-A33-12.

[0030] Figure 9 This is a graph showing the effects of different experimental groups on mouse body weight in Example 5;

[0031] Figure 10 This is a graph showing the effect of different experimental groups on the water content of mouse feces in Example 5;

[0032] Figure 11 This is a graph showing the effects of different experimental groups on the morphology of the mouse colon in Example 5;

[0033] Figure 12 This is a graph showing the effects of different experimental groups on mouse organ indices in Example 5; where, Figure 12 Figure A in the graph shows the effects of different experimental groups on the liver index in mice. Figure 12 In the figure, B represents the effect of different experimental groups on the spleen index of mice.

[0034] Figure 13 This diagram illustrates the mechanisms of action of the mutant genes aguD, gtfA, rpoC, and rpoB. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0036] This invention provides a *Pediococcus acidilactici* NCUF214.4-A33-12, which is classified as *Pediococcus acidilactici* NCUF214.4-A33-12. It was deposited at the China Center for Type Culture Collection on January 20, 2026, with accession number CCTCC NO: M 2026164, and the deposit address is: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0037] In fact, this invention utilizes an ambient-pressure room-temperature plasma (ARTP) mutagenesis combined with a directed adaptive evolution strategy under acid stress to screen and obtain the target strain *Pediococcus lactis* NCUF214.4-A33-12, which possesses both strong antibacterial properties and high acid resistance. Whole-genome resequencing of this strain identified mutant genes such as aguD, gtfA, rpoB, and rpoC. Animal experiments showed that this strain and its synbiotic formulation with inulin significantly alleviated weight loss, diarrhea, colonic atrophy, and inflammatory responses induced by ETEC K88 in mice. The *Pediococcus lactis* NCUF214.4-A33-12 strain provided by this invention is safe and reliable, offering an ideal candidate strain for the development of probiotics to alleviate infectious diarrhea caused by ETEC.

[0038] Specifically, *Pediococcus lactis* NCUF214.4-A33-12 has the following biological characteristics:

[0039] (1) Bacterial characteristics: Gram-positive, spherical cells, non-motile;

[0040] (2) Colony characteristics: On MRS medium, the colonies are small, milky white, opaque, smooth and uniform in texture;

[0041] (3) Growth characteristics: Under constant temperature of 37℃, the growth reaches the end of the logarithmic phase after 15h of culture in MRS medium;

[0042] (4) Antibacterial activity: It has an antibacterial activity of 14.79 mm against enterotoxigenic Escherichia coli (ETEC);

[0043] (5) Acid resistance test: The survival rate was 85.95% at pH=3.0 and 57.88% at pH=1.5.

[0044] The present invention also provides a method for screening to obtain the above-mentioned *Pediococcus lactis* NCUF214.4-A33-12, comprising the following steps:

[0045] (1) The starting strain, Pediococcus lactis PA-214.4, was subjected to atmospheric pressure and room temperature plasma mutagenesis for 120s, and the mutagenesis strain with enhanced antibacterial activity was obtained by screening.

[0046] (2) The mutant strain obtained in step (1) was subjected to acid stress-directed adaptive evolution culture in a medium with continuously decreasing pH, and the strain of Pediococcus lactis NCUF214.4-A33-12 with improved acid resistance and maintained antibacterial activity was screened.

[0047] In some embodiments, the pH range of the continuously decreasing culture medium used in step (2) is gradually reduced from 6.0 to 3.7.

[0048] In some embodiments, the acid stress-directed adaptive evolution culture in step (2) is passaged 40-50 times.

[0049] In fact, step (2) is to conduct targeted selection for acid tolerance of the mutant strain. The initial pH value for acid tolerance acclimatization is 6.0, which is gradually decreased until the final pH value is 3.7. The mutant strain is acclimatized for a long time to improve its acid tolerance.

[0050] Specifically, in step (2), it is preferable to use MRS liquid culture medium with continuously decreasing pH gradients (pH=6.0, 5.6, 5.2, 4.9, 4.6, 4.3, 4.0, 3.7) to continuously culture for 46 generations, and measure the growth at each stage, and combine the results of antibacterial activity, acid resistance and bile salt resistance.

[0051] In some embodiments, the survival rate of *Pediococcus lactis* NCUF214.4-A33-12 was 85.95% at pH 3.0 and 57.88% at pH 1.5; the inhibition zone of *Pediococcus lactis* NCUF214.4-A33-12 against enterotoxigenic *Escherichia coli* was 14.79 mm, and it had a relieving effect on diarrhea symptoms in mice infected with enterotoxigenic *Escherichia coli*.

[0052] In some embodiments, the mutant genes in the whole genome of *Pediococcus lactis* NCUF214.4-A33-12 include genes aguD, gtfA, rpoC, and rpoB; the nucleotide sequence of gene aguD is shown in SEQ ID NO.2; the mutation of gene aguD specifically involves a T-to-G mutation at position 160883 relative to the whole genome sequence of *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of gene gtfA is shown in SEQ ID NO.3; the mutation of gene gtfA specifically involves a T-to-G mutation at position 194609 relative to the whole genome sequence of *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of gene rpoC is shown in SEQ ID NO.2. As shown in NO.4; the specific mutation of the rpoC gene is a mutation from T to C at position 1473663 relative to the whole genome sequence of the *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of the rpoB gene is shown in SEQ ID NO.5; the specific mutation of the rpoB gene is a mutation from T to G at position 1475092 relative to the whole genome sequence of the *Pediococcus lactis* NCUF214.4-A33-12.

[0053] The present invention also provides an application of the above-mentioned *Pediococcus lactis* NCUF214.4-A33-12 in relieving ETEC infectious diarrhea, the application comprising using *Pediococcus lactis* NCUF214.4-A33-12 to prepare a microecological preparation for relieving ETEC infectious diarrhea; the microecological preparation is one of the following forms: solution, powder, suspension, granules or tablets.

[0054] In some embodiments, the *Pediococcus lactis* NCUF214.4-A33-12 can be used alone or in combination with a compound freeze-drying protectant containing 8.2% inulin at a volume ratio of 1:1 to prepare a microecological preparation.

[0055] Example 1: Screening and identification of Pyorrhea-resistant Lactococcus NCUF214.4-A33-12

[0056] (1) Activation and culture of microbial strains

[0057] The starting strain of *Pediococcus lactis* PA-214.4, which has strong antibacterial activity, used in this invention was isolated from live bacterial slides and stored at 4°C. The stored strain was transferred to MRS solid medium and cultured for 48 hours. Subsequently, single colonies on the medium were inoculated into MRS liquid medium and cultured (37°C, 24 hours), activating the strain twice. Then, the activated bacterial solution was transferred to fresh MRS liquid medium at an inoculation rate of 2% for later use.

[0058] The indicator bacterium used in this invention is enterotoxigenic Escherichia coli (ETEC), purchased from BNCC. The indicator bacterium was revived and activated. Single colonies from the second activation were picked and inoculated into LB liquid medium. After incubation at 37°C on a shaker for 24 hours, the culture was centrifuged at 8000 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in PBS to achieve a viable count of approximately 10⁻⁶. 8 CFU / mL, ready for use.

[0059] (2) Selection of ARTP strains of Pietrococcus lactis with high antibacterial activity

[0060] Collect bacterial culture in the logarithmic growth phase by centrifugation (8000 r / min, 4℃), wash with physiological saline and dilute to OD200. 600 The bacterial suspension was prepared with a concentration between 0.6 and 0.8 μg. The strain was then mutagenized using the ARTP technique with a mutagenesis time of 120 seconds. The bacterial suspension was serially diluted and evenly spread onto MRS solid medium, incubated at 37°C for 48 hours, and single colonies with plump morphology and large particles were randomly selected for activation culture. After activation, the antibacterial activity against *Escherichia coli* was determined using the perforation method. Mutagenic strains of *Pediococcus lactis* with enhanced antibacterial activity were selected for subsequent acid stress tolerance evolution.

[0061] (3) Acid stress-oriented adaptive evolution of highly antibacterial Pyrococcus lactis

[0062] The mutant strains selected above were subjected to acid tolerance-oriented selection to obtain stress-tolerant strains. The initial pH for acid tolerance acclimatization was 6.0, which was then decreased sequentially (pH=6.0, 5.6, 5.2, 4.9, 4.6, 4.3, 4.0, 3.7), with a final pH of 3.7. The growth of the strains under different acid concentrations was measured through 46 consecutive subcultures. The results of the targeted selection are as follows: Figure 1 As shown, the four strains with the best growth performance in the mutant library samples were named NCUF214.4-A33-7, NCUF214.4-A33-10, NCUF214.4-A33-12, and NCUF214.4-A33-39, respectively. These four strains were plated and streaked 2-3 times. Single colonies were selected and inoculated into slant agar, incubated at 30°C for 48 hours, and then stored at 4°C.

[0063] The antibacterial activity, acid tolerance, and bile salt tolerance of the starting strain and the stressed strain were determined separately. The antibacterial activity was determined using the perforation method, taking 100 μL of indicator bacterial suspension (10... 8The cfu / mL solution was spread onto the surface of LB solid medium. Four 5mm holes were punched in each petri dish using a sterile puncher. 100μL of the experimental bacterial culture was added to each hole, with MRS medium used as a negative control. After diffusion at 4℃ for 2-4 hours, the culture was incubated at 37℃ for 24-48 hours. The diameter of the inhibition zone was measured and recorded using calipers. The determination of acid resistance and bile salt resistance was the same as in (2) the selection of ARTP strains of highly antibacterial Pediococcus lactis.

[0064] The results of the antibacterial activity, acid resistance, and bile salt resistance of the starting strain and the stressed strain are as follows: Figure 2 , Figure 3 As shown in the figure, strain NCUF214.4-A33-12 exhibited the strongest antibacterial activity, with an inhibition diameter of 14.79 mm. Its survival rate at pH 3 was 85.95%, an increase of 36.45% compared to before stress. Furthermore, its survival rate after 3 hours of incubation at pH 1.5 was 57.88%, both the strongest among all stressed strains. This strain also showed strong survival rates at 0.3% and 0.5% bile salts, at 75.75% and 58.25%, respectively. Therefore, this strain was ultimately selected as the target strain.

[0065] (4) Strain identification

[0066] The selected target strain was sent to Shanghai Paisennong Biotechnology Co., Ltd. for 16S rRNA gene sequencing. The 16S rRNA gene sequence of *Pediococcus lactis* NCUF214.4-A33-12 is shown in SEQ ID NO.1. The identification results showed that the target strain was *Pediococcus lactis*, and it was named *Pediococcus lactis* NCUF214.4-A33-12. It was subsequently deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2026164.

[0067] The colony morphology of *Pediococcus lactis* NCUF214.4-A33-12 plate is as follows: Figure 4 As shown, its growth performance is as follows Figure 5 As shown, the genetic stability results are as follows: Figure 6 As shown, its antibacterial and acid-resistant properties remained largely unchanged after continuous subculturing, indicating excellent genetic stability.

[0068] Example 2: Whole-genome resequencing analysis of gene loci that may promote antibacterial and acid-resistant properties

[0069] Whole-genome sequencing was performed on the dominant mutant strain *Pediococcus lactis* NCUF214.4-A33-12 obtained in Example 1, and whole-genome resequencing analysis was performed on the originating strain *Pediococcus lactis* PA-214.4. Specifically, genomic DNA was extracted from *Pediococcus lactis* NCUF214.4-A33-12, and after library construction, sequencing, sequence assembly, and gene function annotation, the whole-genome sequence of *Pediococcus lactis* NCUF214.4-A33-12 was obtained and used as the reference genome. Subsequently, resequencing analysis was performed on the originating strain *Pediococcus lactis* PA-214.4. Integrational alignment analysis was performed on differentially expressed genes and genes related to acid tolerance and antimicrobial activity in the starting strain *Pediococcus lactis* PA-214.4 and the stressed strain *Pediococcus lactis* NCUF214.4-A33-12. Preliminary identification of variant genes related to acid tolerance and antimicrobial activity in *Pediococcus lactis* NCUF214.4-A33-12 was conducted, as shown in Table 1 below. The mechanisms of action are as follows: Figure 13 As shown. The nucleotide sequences of the mutant genes aguD, gtfA, rpoC, and rpoB are shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively.

[0070] Table 1: Mutant genes associated with the gastric acid resistance and diarrhea-relieving effect of P. lactis NCUF214.4-A33-12

[0071]

[0072] Example 3: Safety evaluation of Pyotrophic lactis NCUF214.4-A33-12

[0073] (1) Hemolytic activity assay: Activated *Pediococcus lactis* strains were streaked onto Columbia blood agar medium and incubated at 37°C for 24 hours. The hemolytic activity of *Staphylococcus aureus* was used as a positive control to observe whether hemolysis occurred. Figure 7 The figures show the hemolytic activity of Staphylococcus aureus (positive control) and the stressed strain NCUF214.4-A33-12 on Columbia blood agar, respectively. In the positive control strain, the red blood cells surrounding the Staphylococcus aureus colonies completely ruptured, forming a well-defined, completely transparent hemolytic zone, indicating β-hemolysis. In contrast, no hemolytic zone appeared around the stressed strain NCUF214.4-A33-12, indicating γ-hemolysis. This demonstrates that the strain does not possess hemolytic activity and has good in vitro safety.

[0074] (2) Antibiotic susceptibility testing: The susceptibility of the strains to antibiotics was determined using the paper disc agar diffusion method. The strains, activated twice (10... 8CFU / mL of antibiotics were evenly spread on MRS plates. Then, susceptibility testing discs for 12 antibiotics—erythromycin, cefotaxime, penicillin, norfloxacin, tetracycline, ciprofloxacin, chloramphenicol, vancomycin, clindamycin, streptomycin, ofloxacin, and gentamicin—were evenly affixed to the surface of the MRS plates. The plates were then incubated at 37°C for 24 hours. The diameter of the inhibition zone on each disc was measured, and three measurements were taken, with the average value taken. Based on the diameter of the inhibition zone, the resistance of each isolate was classified as resistant (R), sensitive (S), or intermediate (I). The results of the resistance testing of the stressed strain NCUF214.4-A33-12 to 12 commonly used antibiotics are shown in Table 2 below.

[0075] The test results showed that the stressed strain NCUF214.4-A33-12 was resistant to norfloxacin, ciprofloxacin, streptomycin and ofloxacin, moderately sensitive to tetracycline and vancomycin, and sensitive to erythromycin, cefotaxime, penicillin, chloramphenicol and clindamycin and gentamicin.

[0076] Based on the above results, the stressed strain NCUF214.4-A33-12 was inhibited by different types of antibiotics and did not pose a potential pathogenic risk, thus it can be determined to be a safe and usable probiotic strain.

[0077] Table 2: Susceptibility of Pediococcus lactis NCUF214.4-A33-12 to 12 antibiotics

[0078]

[0079] Note: S indicates sensitive, I indicates intermediate, and R indicates drug resistance.

[0080] Example 4: Evaluation of acid stress tolerance, colonization ability and in vitro digestion survival rate of *Pediococcus lactis* NCUF214.4-A33-12

[0081] 4.1 Growth characteristics under acid stress

[0082] The growth characteristics of the starting strain PA-214.4 and the stressed strain NCUF214.4-A33-12 under acid stress were evaluated by measuring their growth curves in acid stress medium (pH 3.7). The results are as follows: Figure 8 As shown.

[0083] Under pH 3.7 acid stress, the lag phase of all strains was prolonged from 3 hours under normal conditions to 9 hours. The starting strain reached a stationary phase at approximately 24 hours, while the target strain NCUF214.4-A33-12 reached a stationary phase at 36 hours. The growth of the starting strain was significantly inhibited at pH 3.7, with its biomass decreasing by 57.71% after 24 hours compared to the normal environment, indicating its sensitivity to acidic conditions. In contrast, the target strain NCUF214.4-A33-12 showed significantly reduced growth inhibition under pH 3.7 acidic conditions, with its OD value increasing by 61.75% after 36 hours compared to the starting strain under acid stress. These results indicate that the target strain NCUF214.4-A33-12, obtained through acid tolerance-oriented selection, possesses significantly enhanced acid tolerance.

[0084] 4.2 Self-aggregation and surface hydrophobicity

[0085] The aggregation ability and hydrophobic properties of cell surfaces are key indicators for evaluating whether probiotics can adhere to intestinal epithelial cells. The self-aggregation and surface hydrophobicity results of the starting strain and the target strain are shown in Table 3 below.

[0086] The self-aggregation rate of both strains increased with prolonged incubation time. After 18 hours of incubation, the self-aggregation rate of both strains reached over 60%, demonstrating strong self-aggregation ability. After 24 hours of incubation, the self-aggregation rate of the target strain NCUF214.4-A33-12 was 76.96%, an increase of 8.39% compared to the 71% of the starting strain, indicating that mutagenesis and acid stress treatment did not weaken the surface adhesion characteristics of the strains. There was no significant difference in the xylene hydrophobicity of the two strains at 1 hour, at 56.84% and 59.48%, respectively. Considering both the self-aggregation rate (≥50%) and surface hydrophobicity (≥50%), the two strains significantly exceeded the reported threshold for probiotics, suggesting that they can rapidly form microcolonies in the intestine and establish stable adhesion to epithelial cells, with the target strain NCUF214.4-A33-12 showing the best performance.

[0087] Table 3: Self-aggregation and surface hydrophobicity of strains

[0088]

[0089] Note: * and ** indicate that P<0.05 and P<0.01 are compared within the same column.

[0090] 4.3 Survival rate of in vitro simulated digestion

[0091] Probiotics can only exert their biological functions such as antibacterial activity, colonization, and regulation of the gut microbiota if they maintain a high level of viable bacteria after passing through the gastrointestinal tract. The survival ability of strains under simulated gastric juice, intestinal juice, and bile salt stress was determined by simulating the human gastrointestinal digestive environment in vitro. The results are shown in Table 4 below.

[0092] The initial viable cell counts of the two strains before digestion were similar, with no significant difference (P>0.05). After treatment with simulated gastric juice, the viable cell counts of both strains decreased significantly, with the target strain NCUF214.4-A33-12 maintaining a higher viable cell count than the starting strain. This difference further increased after continuous simulated intestinal digestion, with the final viable cell counts of the target and starting strains reaching 4.82 × 10⁻⁶. 7 CFU / mL and 2.09×10 7 The CFU / mL concentration of the target strain NCUF214.4-A33-12 was significantly different from that of the starting strain (P<0.05). The final survival rates of the two strains after continuous simulated gastrointestinal digestion were 69.34% and 44.83%, respectively, showing a trend of NCUF214.4-A33-12 > starting strain PA-214.4. These results indicate that after mutagenesis and subsequent acid stress-directed evolution treatment, the gastrointestinal adaptability of the target strain NCUF214.4-A33-12 was enhanced, exhibiting strong survival ability in in vitro simulated digestion.

[0093] Table 4: Survival rate of strains after in vitro simulated digestion

[0094]

[0095] Note: * and ** indicate that P<0.05 and P<0.01 are compared within the same column.

[0096] Example 5: In vivo experiment on the antidiarrheal effect of Pyotrophic cephalosporin NCUF214.4-A33-12

[0097] (1) High-density culture of strains: The starting strain and the stress strain NCUF214.4-A33-12 were cultured at high density in a 5L small fermenter. Each group of strains was further divided into a probiotic group and a synbiotic group, with inulin added as a prebiotic to the synbiotic group. After the culture was completed, the obtained lactic acid bacteria suspension was mixed with a compound freeze-drying protectant at a volume ratio of 1:1. The concentrations of each component of the compound freeze-drying protectant were: inulin (8.2%), trehalose (4.47%), skim milk powder (9.24%), and D-sorbitol (13.42%), and then subjected to vacuum freeze-drying. After the freeze-drying was completed, the bacterial powder in the vial was taken out and stored at 4℃ for later use.

[0098] (2) Effects of probiotics and their synbiotic preparations on mice with diarrhea

[0099] The probiotic powder and synbiotic powder prepared above were reconstituted with sterile 0.9% physiological saline to adjust the live bacteria concentration to 1×10⁻⁶. 9 CFU / mL bacterial suspensions were prepared for use. Four-week-old female ICR mice weighing 19±2g were selected and acclimatized for 7 days under standard laboratory conditions with a 12-hour light / dark cycle and free access to food and water. Seventy mice were then randomly divided into seven groups of ten mice each: a blank control group (CON), a model group (MOD), a positive drug control group (CIP), a starting strain bacterial powder group (214.4-P), a starting strain synbiotic bacterial powder group (214.4-S), an NCUF214.4-A33-12 bacterial powder group (214.4-A33-12-P), and an NCUF214.4-A33-12 synbiotic bacterial powder group (214.4-A33-12-S). All groups were fed the same diet. During the 12-day experimental period, from day 1 to day 7, the blank control group, model group, and positive control group were administered 0.1 mL / 10 g body weight of sterile 0.9% saline via gavage daily, while the four experimental groups were administered 0.1 mL / 10 g body weight of the corresponding bacterial suspension (1 × 10⁻⁶). 9 (CFU / mL); From day 8 to 10, all mice were switched to drinking water containing 6.7% fructose and 5 g / L streptomycin, and were fasted for 12 hours starting from day 10, after which they were switched to sterile water without streptomycin; From day 11 to 12, except for the blank control group, all other groups were administered 0.1 mL / 10 g body weight of 1×10 9 Infection model was established using CFU / mL ETEC bacterial suspension. One hour after modeling, the positive control group was administered 0.1 mL / 10 g body weight of ciprofloxacin solution (5 g / L) by gavage, while each experimental group was administered the corresponding probiotic or synbiotic suspension by gavage. The blank control group and the model group were administered an equal volume of physiological saline by gavage. Mouse weight was recorded daily, and feces were collected from each mouse before and after modeling. The water content of the feces was determined using the direct drying method. At the end of the experiment, the length of the mouse colon was measured and photographed. The spleen and liver were weighed, and the organ index was calculated using the following formula:

[0100]

[0101]

[0102] The effects of different experimental groups on mouse body weight and fecal water content, as follows: Figure 9 , Figure 10As shown, during the infection modeling period on days 11-12, all groups except the CON group experienced varying degrees of weight loss. The MOD group showed the most significant weight loss, and its fecal water content increased by 27.58% compared to pre-infection levels, indicating successful diarrhea modeling. The 214.4-S and A33-12-S groups showed significant effects in alleviating ETEC K88-induced weight loss and diarrhea symptoms in mice. The effects of different experimental groups on mouse colon morphology are shown below. Figure 11 As shown, colonic atrophy and inflammation in mice in the CIP group and each probiotic / synbiotic intervention group were alleviated to varying degrees. Among them, the colon length recovery was most significant in the synbiotic group and the CIP group, with an appearance similar to the control group. The effects of different experimental groups on mouse organ indices are shown in the figure below. Figure 12 As shown, the liver and spleen indices of the synbiotic intervention groups 214.4-S and A33-12-S were significantly lower than those of the MOD group (P<0.05), which can inhibit ETEC-induced organ enlargement in mice and maintain their normal physiological state. These results indicate that all probiotic intervention groups can alleviate diarrhea symptoms in mice to varying degrees, with the synbiotic intervention group showing the strongest effect. Furthermore, the allergic reaction in the NCUF214.4-A33-12 mutant strain intervention group was superior to that in the original strain intervention group.

[0103] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A type of *Pediococcus lactis* NCUF214.4-A33-12, characterized in that, The *Pediococcus lactis* NCUF214.4-A33-12 strain was deposited at the China Center for Type Culture Collection (CCTCC) on January 20, 2026, with accession number CCTCC NO: M2026164, at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province. The 16S rRNA gene sequence of the *Pediococcus lactis* NCUF214.4-A33-12 strain is shown in SEQ ID NO.

1.

2. The *Pediococcus lactis* NCUF214.4-A33-12 according to claim 1, characterized in that, The mutant genes in the whole genome of *Pediococcus lactis* NCUF214.4-A33-12 include genes aguD, gtfA, rpoC, and rpoB; the nucleotide sequence of gene aguD is shown in SEQ ID NO.2; the mutation of gene aguD is specifically a T-to-G mutation at position 160883 relative to the whole genome sequence of *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of gene gtfA is shown in SEQ ID NO.3; the mutation of gene gtfA is specifically a T-to-G mutation at position 194609 relative to the whole genome sequence of *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of gene rpoC is shown in SEQ ID NO.

2. As shown in SEQ ID NO. 4; the specific mutation of the rpoC gene is a mutation from T to C at position 1473663 relative to the whole genome sequence of *Pediococcus lactis* NCUF214.4-A33-12; the nucleotide sequence of the rpoB gene is shown in SEQ ID NO. 5; the specific mutation of the rpoB gene is a mutation from T to G at position 1475092 relative to the whole genome sequence of *Pediococcus lactis* NCUF214.4-A33-12.

3. The *Pediococcus lactis* NCUF214.4-A33-12 according to claim 1, characterized in that, The survival rate of the *Pediococcus lactis* NCUF214.4-A33-12 was 85.95% at pH 3.0 and 57.88% at pH 1.

5. The inhibition zone of *Pediococcus lactis* NCUF214.4-A33-12 against enterotoxigenic *Escherichia coli* was 14.79 mm, and it had a relieving effect on diarrhea symptoms in mice infected with enterotoxigenic *Escherichia coli*.

4. A method for screening to obtain the *Pediococcus lactis* NCUF214.4-A33-12 as described in claim 1, characterized in that, Includes the following steps: (1) The starting strain, Pediococcus lactis PA-214.4, was subjected to atmospheric pressure and room temperature plasma mutagenesis for 120s, and the mutagenesis strain with enhanced antibacterial activity was obtained by screening. (2) The mutant strain obtained in step (1) was subjected to acid stress-directed adaptive evolution culture in a medium with continuously decreasing pH, and the target strain, Pediococcus lactis NCUF214.4-A33-12, was screened to improve acid resistance and maintain antibacterial activity.

5. The method according to claim 4, characterized in that, The pH range of the culture medium that is continuously reduced in step (2) is gradually reduced from 6.0 to 3.

7.

6. The method according to claim 4, characterized in that, The number of generations of acid stress-directed adaptive evolution culture described in step (2) is 40-50.

7. The application of *Pediococcus lactis* NCUF214.4-A33-12 as described in claim 1 in alleviating enterotoxigenic *Escherichia coli* infectious diarrhea, characterized in that... The application includes using Pediococcus lactis NCUF214.4-A33-12 to prepare a microecological preparation for relieving enterotoxigenic Escherichia coli-induced infectious diarrhea; the microecological preparation is in one of the following forms: solution, powder, suspension, granules, or tablets.

8. The application according to claim 7, characterized in that, The lactic acid cocci NCUF214.4-A33-12 can be used alone or in combination with a compound freeze-drying protectant containing 8.2% inulin at a volume ratio of 1:1 to prepare microecological preparations.

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