Acid-producing plant lactobacillus and application thereof

Abstract

The invention relates to the field of microorganisms and fermentation engineering, in particular to acid-producing plant lactobacillus and application thereof. Specifically, the bacterial strain is named as the lactobacillus plantarum BLSCX-2, and the bacterial strain is named as the lactobacillus plantarum BLSCX-2. Tests prove that the yield of lactic acid and phenyllactic acid is obviously higher than that of reported strains, so that the strain is suitable for efficient fermentation food production. Meanwhile, the plant lactobacillus BLSCX-2 disclosed by the invention has a broad-spectrum bacteriostatic effect on various food-borne pathogenic bacteria and putrefying bacteria, and can be used for biological preservation or development of medical bacteriostatic agents. Besides, the strain can still keep activity under the conditions of low pH, high bile salt, low temperature and high salt, and the application range of the strain in a complex environment is widened, so that the strain has good practical application value.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and fermentation engineering, specifically to a strain of acid-producing plant lactobacillus and its applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Lactic acid bacteria, as an important group of probiotics, have wide applications in food fermentation, pharmaceuticals, health products, and industrial production. *Lactobacillus plantarum* (…) Lactiplantibacillus plantarum As a typical representative of lactic acid bacteria, it has become a research hotspot in recent years due to its good acid and bile salt resistance, rich metabolites and probiotic properties.

[0004] Acid production is one of the core functions of *Lactobacillus plantarum*. The organic acids it produces are crucial not only for food flavor formation and preservation performance but also for emerging fields such as bio-preservation, pharmaceutical antibacterial agents, and environmental remediation. However, the inventors have discovered that although various *Lactobacillus plantarum* strains have been isolated and studied, these strains exhibit significant differences in acid production capacity, antibacterial effects, and environmental tolerance. For example, existing *Lactobacillus plantarum* strains have limited performance in lactic acid production, phenyllactic acid synthesis, and antibacterial properties, making it difficult to meet the needs of efficient production and practical applications. Furthermore, some strains show low survival rates and metabolic activity in extreme environments (such as low pH, high bile salt concentrations, or low temperatures), limiting their application in complex environments.

[0005] In the food industry, *Lactobacillus plantarum* is commonly used in the production of fermented foods. However, its fermentation efficiency is often insufficient in low-temperature or high-salt environments, leading to prolonged production cycles or unstable product quality. In the pharmaceutical field, the antibacterial spectrum and efficacy of *Lactobacillus plantarum* still need further improvement to address the challenges posed by various pathogens. Therefore, developing a *Lactobacillus plantarum* strain with high acid production capacity, efficient antibacterial properties, and excellent environmental tolerance is of great significance for promoting technological progress in related fields. Summary of the Invention

[0006] The purpose of this invention is to provide an acid-producing *Lactobacillus plantarum* strain and its applications. This strain is safe to obtain and easy to cultivate, exhibiting not only significant acid-producing capacity but also efficient synthesis of phenyllactic acid. Furthermore, it demonstrates excellent antibacterial effects against various pathogenic and putrefactive bacteria. In addition, this *Lactobacillus plantarum* strain maintains high survival rates and metabolic activity under low pH, high (bile) salt concentrations, and low temperature conditions, showing broad application prospects. Based on the above research results, this invention has been completed.

[0007] Specifically, the technical solutions of the present application are as follows: In the first aspect of the present application, the present application provides a plant Lactobacillus plantarum strain, which is named Lactobacillus plantarum (Lactobacillus plantarum) BLSCX-2, which has been preserved in the China Center for Type Culture Collection on July 11, 2025, the address is Wuhan University in Wuhan, and the preservation number is CCTCC NO: M 20251576. Lactiplantibacillus plantarum ) BLSCX-2, which has been preserved in the China Center for Type Culture Collection on July 11, 2025, the address is Wuhan University in Wuhan, and the preservation number is CCTCC NO: M 20251576.

[0008] The above-mentioned Lactobacillus plantarum is a strain of Lactobacillus plantarum with high acid-producing capacity, broad-spectrum antibacterial capacity, excellent environmental tolerance, and easy cultivation, high industrialization potential, which is isolated by the applicant from sour pickles through screening, purification.

[0009] Specifically, the Lactobacillus plantarum has the following characteristics: (1) High acid-producing capacity: lactic acid and phenyllactic acid synthesis efficiency is outstanding The core advantage of strain BLSCX-2 is high-efficiency acid-producing performance. The organic acid produced by it is not only the key to food flavor and preservation performance, but also provides a material basis for antibacterial and fresh-keeping. The lactic acid yield of strain BLSCX-2 can reach 25.622 g / L in MRS liquid medium at 37℃ for 24h, which is significantly higher than that of other strains screened at the same period. This yield can quickly reduce the pH of the fermentation system, effectively shorten the fermentation period of food, and also inhibit the contamination of miscellaneous bacteria through the acidic environment. At the same time, as a natural antibacterial substance, the synthesis capacity of strain BLSCX-2 is much higher than that of existing strains, which provides an advantage for its antibacterial performance, and also effectively expands its application field.

[0010] (2) Broad-spectrum antibacterial activity: effectively covering 11 common pathogenic bacteria and spoilage bacteria, with significant targeted advantage The fermentation supernatant of Lactobacillus plantarum BLSCX-2 of the present application has inhibitory effect on a variety of pathogenic bacteria, with wide antibacterial spectrum and excellent effect. Specifically, it has inhibitory effect on 11 kinds of pathogenic bacteria and spoilage bacteria such as Candida albicans, Shigella, Escherichia coli, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa. Especially worth noting is that it has particularly outstanding inhibitory effect on common pathogenic bacteria in aquaculture (such as Vibrio alginolyticus, Vibrio parahaemolyticus and Aeromonas hydrophila), so it can be used for inhibiting bacteria in aquaculture water or preserving aquatic products.

[0011] (3) Excellent environmental tolerance: adapt to complex scenes and broaden the application boundary The plant lactobacillus BLSCX-2 of the present application can still maintain activity in an extreme environment (such as a low pH, high choline salt, low temperature and high salt environment), thereby effectively solving the problem of the "limited application scene" of the existing strain: in terms of specific analysis, the survival rate thereof in an acidic environment of pH 2.5 is as high as 100%, which is much higher than that of ordinary lactobacillus; meanwhile, the survival rate thereof under a high choline salt concentration of 0.3% is still 8.93%, and therefore, it can tolerate the digestive tract environment and has the potential to be used as a probiotic preparation. Meanwhile, it has been verified through research that it can grow normally at a low temperature of 10 DEG C, the number of live bacteria after 5 days of fermentation is 240.00 x 10 7 CFU / mL, and the lactic acid yield is 16.722 g / L, which is close to the fermentation effect under the condition of 15 DEG C; In addition, it still maintains metabolic activity after being refrigerated at 4 DEG C for 21 days, is suitable for low-temperature fermented food (such as fermented fruit juice and low-temperature yogurt) and application in cold regions, and avoids the loss of nutrients or the deterioration of taste caused by high-temperature fermentation. Meanwhile, it can still survive (the number of live bacteria is 4.45 x 10 8 CFU / mL) in an 8% sodium chloride environment and still has the ability to produce lactic acid, and therefore, it can be used for fermentation of high-salt food (such as pickles and preserved vegetables), without the need for additional adjustment of the salt concentration, thereby effectively simplifying the production process.

[0012] (4) Culture and morphological characteristics: easy to culture, high industrialization potential The plant lactobacillus BLSCX-2 of the present application has good growth performance in a conventional MRS culture medium, and has good fermentation stability, and through test verification, the number of live bacteria and the lactic acid content do not decrease significantly after 10 generations of continuous passage, and it has the basis for large-scale industrial culture.

[0013] For convenience of statement, the plant lactobacillus BLSCX-2 described in the present application will also be abbreviated as strain BLSCX-2 or BLSCX-2 in the application file. Lactiplantibacillus plantarum

[0014] The strain BLSCX-2 of the present application can be cultured in an MRS culture medium. In an MRS solid culture medium, it is cultured at 37 DEG C for 48 h to form a milky white, convex, smooth, moist and easy-to-pick round-shaped colony. It is found through observation under a microscope that the cell morphology thereof is rod-shaped, arranged alone, in pairs or in short chains.

[0015] Preferably, the MRS liquid culture medium comprises the following components: glucose 20 g / L, proteose peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 5 g / L, manganese sulfate 0.2 g / L, magnesium sulfate 0.5 g / L, Tween-80 1 g / L, pH 6.0, and agar 15 g / L needs to be added to the solid culture medium, and sterilized at 121 DEG C for 30 min.​

[0016] In a second aspect of the present application, the present application provides a bacterial agent comprising the Lactobacillus plantarum or the fermentation product or the metabolite thereof of the first aspect described above.

[0017] The metabolite of the present application includes intracellular metabolite and / or extracellular metabolite.

[0018] The term "fermentation product" refers to the total product of the fermentation process of Lactobacillus plantarum BLSCX-2 under suitable conditions. The fermentation product can include, but is not limited to, the following components: viable bacteria, dead bacteria, extracellular metabolite, culture medium components. In the present application, the fermentation product can be in liquid, semi-solid or processed solid form. That is, the corresponding fermentation product can be a liquid obtained from the process of fermenting Lactobacillus plantarum BLSCX-2 bacteria, and thus can also be referred to as fermentation broth. The liquid can contain bacteria (bacterial cells), but it is not necessarily required to contain bacteria. The liquid preferably contains metabolites produced by BLSCX-2 bacteria of the present application. Lactiplantibacillus plantarum )BLSCX-2 under suitable conditions. The fermentation product can include, but is not limited to, the following components: viable bacteria, dead bacteria, extracellular metabolite, culture medium components. In the present application, the fermentation product can be in liquid, semi-solid or processed solid form. That is, the corresponding fermentation product can be a liquid obtained from the process of fermenting Lactobacillus plantarum BLSCX-2 bacteria, and thus can also be referred to as fermentation broth. The liquid can contain bacteria (bacterial cells), but it is not necessarily required to contain bacteria. The liquid preferably contains metabolites produced by BLSCX-2 bacteria of the present application.

[0019] In an embodiment of the present application, the fermentation broth or culture broth containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation or other means known in the art to obtain bacterial cells grown in the fermentation broth or culture broth, and the liquid remaining after removing the bacterial cells is "supernatant", and in the present application, the supernatant contains extracellular metabolites of BLSCX-2. In an embodiment of the present application, the bacterial agent can also contain the supernatant.

[0020] In an embodiment of the present application, the fermentation broth or culture broth containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation or other means known in the art to obtain bacterial cells grown in the fermentation broth or culture broth, and the liquid remaining after removing the bacterial cells is "supernatant", and in the present application, the supernatant contains extracellular metabolites of BLSCX-2. In an embodiment of the present application, the bacterial agent can also contain the supernatant.

[0021] In a third aspect of the present application, the present application provides a product comprising the Lactobacillus plantarum of the first aspect described above or the bacterial agent of the second aspect described above.

[0022] In an embodiment of the present application, the product can be a bacteriostatic agent, a preservative, a fermentation agent, a feed additive, a food, a food additive or a pharmaceutical product.

[0023] In a fourth aspect of the present application, there is provided use of the Lactobacillus plantarum as described in the first aspect above or the bacterial agent as described in the second aspect above and / or the product as described in the third aspect above in the preparation of a product having bacteriostatic ability.

[0024] The bacteriostatic ability includes bacteriostatic ability against Candida albicans, Shigella, Aeromonas hydrophila, Salmonella enteritidis, Escherichia coli, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Vibrio parahaemolyticus, Vibrio alginolyticus and Pseudomonas aeruginosa.

[0025] The product can be a drug or a feed additive.

[0026] The beneficial technical effects of the above technical solutions are as follows: The Lactobacillus plantarum BLSCX-2 in the above technical solution has good fermentation performance and high acid production capacity, and the lactic acid and phenyllactic acid production of the Lactobacillus plantarum BLSCX-2 are significantly higher than those of the existing reported strains, so the Lactobacillus plantarum BLSCX-2 is suitable for high-efficiency fermentation food production. Meanwhile, the Lactobacillus plantarum BLSCX-2 has broad-spectrum bacteriostatic effect on a plurality of foodborne pathogenic bacteria and spoilage bacteria, and can be used for biological preservation or development of a medical bacteriostatic agent. In addition, the strain can still maintain activity under low pH, high bile salt, low temperature and high salt conditions, thereby widening the application range of the strain in complex environments, and therefore the Lactobacillus plantarum BLSCX-2 has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0027] The drawings constituting a part of the specification of the present application are used to provide further understanding of the present application, and the schematic embodiments of the present application and the description thereof are used to explain the present application, and do not constitute improper limitation on the present application. In the following, the embodiments of the present application are described in detail in combination with the drawings, in which: Figure 1 The figure is a calcium-dissolving ring for strain screening in Example 1 of the present application.

[0028] Figure 2 The figure is a colony morphology of the strain BLSCX-2 in Example 1 of the present application.

[0029] Figure 3 The figure is a cell morphology of the strain BLSCX-2 in Example 1 of the present application (oil lens observation). DETAILED DESCRIPTION

[0030] The present application is further described below in combination with specific examples. It should be understood that the examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods not specified in the following examples are usually carried out according to conventional conditions or according to the conditions suggested by the manufacturers.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The materials used to practice the present application, unless otherwise indicated, are commercially available from standard sources and are prepared according to conventional methodologies, using conventional techniques, unless otherwise indicated. In addition, any method and material similar or equivalent to those described herein can be used in the practice of the present application. The methods and materials described herein are illustrative only and not intended to be limiting.

[0032] In each embodiment, the determination of the content of organic acid is carried out according to the method described in the national standard GB5009.157-2016 "Determination of Organic Acids in Food Safety National Standard", and the determination of the content of phenyllactic acid is carried out according to the method described in the literature (Zhang Zhonghua, Li Xiaoran, Li Hongmei, et al. Rapid detection method of DL-3-phenyllactic acid in lactic acid bacteria fermentation broth by high performance liquid chromatography, 2013, 38 (2): 80-83, 87.).

[0033] Example 1: Isolation and screening of strains and identification 1 Test materials 1.1 MRS medium The composition of MRS liquid medium is: glucose 20 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, potassium phosphate dibasic 5 g / L, manganese sulfate 0.2 g / L, magnesium sulfate 0.5 g / L, proteose peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, Tween-80 1 g / L, pH 6.0, 121 ℃ sterilization for 30 min.

[0034] The composition of MRS solid medium is: add agar 15 g / L in MRS liquid medium, 121 ℃ sterilization for 30 min.

[0035] 1.2 MRS agar medium containing calcium carbonate Add 0.5-1.0% calcium carbonate in 1.1 MRS liquid agar medium.

[0036] 1.3 Test method 1.3.1 Isolation sample: commercially available pickled cabbage in Shandong, Sichuan, Xinjiang and other places.

[0037] 1.3.2 Determination of lactic acid content: the yield of lactic acid in fermentation broth is determined by high performance liquid chromatography (HPLC) method.

[0038] 1.3.3 Lactic acid producing bacteria screening method In a sterile operating table, the commercially available pickled vegetables were crushed separately. 10 g of each was added to a 90 mL Erlenmeyer flask containing sterile physiological saline and glass beads. The flasks were shaken thoroughly at 25 °C for 60 min. Then, they were serially diluted with sterile physiological saline to a 10⁻⁶ concentration. -4 -10 -6 0.1 mL of different dilution gradients were injected onto MRS agar medium containing calcium carbonate, spread evenly with a spreader, and incubated at 37 ℃ for 24-48 h. Single colonies were then picked and streaked onto MRS solid medium for purification. After incubation at 37 ℃ for 24 h, single colonies were picked for Gram staining. Gram-positive strains were selected and preserved on MRS slant agar.

[0039] The strains preserved on MRS slant were inoculated into MRS liquid medium and cultured at 37 ℃ for 24 h. After centrifugation at 10000 rpm for 5 min, the supernatant was filtered through a 0.22 µm filter membrane to obtain the test sample. The content of organic acids in the fermentation supernatant was detected by high performance liquid chromatography (HPLC).

[0040] 1.3.4 Strain Identification (1) Morphological identification Pure cultures of strains with good lactic acid production were inoculated into MRS solid medium plates and incubated at 37 ℃ for 48 h. Colony morphology was then observed.

[0041] (2) Molecular biological identification The target strain was inoculated into fresh MRS liquid medium and cultured at 37 °C for 24 h. Bacterial DNA was extracted using a kit from Tiangen Biotech, and its 16S rDNA sequence was amplified. Universal primers were used. 1492r: 5'-ggttaccttgttacgactt-3' (SEQ ID NO: 1); 27f: 5'-agagttgatcctggctcag-3' (SEQ ID NO: 2).

[0042] The PCR reaction system (50 μL) consisted of: Mixture 25 μL (containing Taq DNA polymerase and dNTPs, etc., Tiangen Biotech). The following reagents were prepared: 1 μL each of forward and reverse primers, 2 μL of template DNA, and 21 μL of ultrapure water. The PCR amplification program was: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 1 min, 52℃ annealing for 1 min, 72℃ extension for 2 min, 25 cycles, followed by a 72℃ extension for 10 min. The PCR products were sent to Beijing Boshan Biotechnology Co., Ltd. for sequencing.

[0043] 2. Experimental Results 2.1 Isolation and screening of bacterial strains: Pickled vegetable samples were plated on MRS agar plates containing calcium carbonate, and colonies with large calcium dissolution zones were picked and streaked for purification. Figure 1 The purified strains were inoculated into MRS liquid medium and cultured at 37°C for 24 h. After centrifugation at 10,000 rpm for 5 min, the supernatant was filtered through a 0.22 µm filter membrane to obtain the test sample. The organic acid content in the fermentation supernatant was detected by high performance liquid chromatography (HPLC) to obtain a high lactic acid-producing strain, BLSCX-2.

[0044] Table 1. Results of lactic acid content determination (g / L) of isolated bacterial strains in pickled vegetable samples.

[0045] Table 1 shows that BLSCX-2 had the highest lactic acid content (25.602 g / L in fermentation supernatant) after 24 h of culture in MRS liquid medium, followed by BLSC-19 with a lactic acid content of 20.301 g / L in fermentation supernatant. Therefore, BLSCX-2 will be used as the research subject for further studies.

[0046] 2.2 Identification of BLSCX-2 strain 2.2.1 Morphological identification When strain BLSCX-2 was cultured at 37 ℃ for 48 h, the colony morphology was milky white, raised, smooth, moist, and round (e.g., Figure 2 As shown in the image, under an optical microscope, the bacterial cells appear as rod-shaped, arranged singly, in pairs, or in short chains, such as... Figure 3 As shown, based on the colony morphology and cell morphology, the bacterium was preliminarily identified as Lactobacillus.

[0047] 2.2.2 Molecular biological identification Electrophoresis of the 16S rDNA PCR product of strain BLSCX-2 showed a highly specific band at approximately 1500 bp, consistent with expectations. Sequencing was performed, and the sequence is shown in SEQ ID NO:3. Comparison of the sequenced sequence with the 16S rDNA gene sequences of some strains already registered on the website http: / / www.ncbi.nlm.nih.gov showed that strain BLSCX-2 is consistent with previously reported... Lactobacillus plantarum The sequence homology of strains (CP039121.1, NR_115605.1, and NR_113338.1, etc.) is greater than 99%. Based on the latest nomenclature rules for *Lactobacillus plantarum*, strain BLSCX-2 was identified as belonging to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum Its 16S rDNA sequence is as follows: SEQ ID NO: 3 2.3 Passage stability of strain BLSCX-2 Continuous liquid subculturing was used. BLSCX-2 bacterial culture in the logarithmic growth phase was inoculated into fresh MRS liquid medium at an inoculum of 2.0% and cultured statically at 37 ℃ until the logarithmic growth phase (usually 12-16 hours / generation). The viable cell count and lactic acid content in the fermentation supernatant were measured by taking the fermentation broth of the original strain in generation 0, generation 5 and generation 10 respectively.

[0048] Table 2. Effects of passage number on viable count and lactic acid content of *Lactobacillus plantarum* BLSCX-2

[0049] As shown in Table 2, compared with the original strain in generation 0, there was no significant difference in the viable cell count and lactic acid content of the fermentation broth after continuous subculturing to the 5th and 10th generations, indicating that the strain has good subculturing stability.

[0050] Example 2: Quantitative comparison of the ability of different Lactobacillus plantarum fermentation broths to produce organic acids and phenyllactic acid 1. Materials and Methods 1.1 Materials 1.1.1 DL-3 phenyllactic acid standard (analytical grade, purity ≥98%) was purchased from Sigma-Aldrich, USA.

[0051] 1.1.2 Test strain: *Lactobacillus plantarum* obtained from screening in Example 1 of this invention (… Lactiplantibacillus plantarum )BLSCX-2. Lactobacillus plantarum BLCC2-0881, BLCC2-1014 and BLCC2-1015 were all preserved and provided by the strain resource bank of the Science and Technology Innovation Center of Shandong Baolai Lailai Biotechnology Co., Ltd.

[0052] 1.2 Test Methods 1.2.1 Obtaining the sample to be tested The test strains were inoculated into MRS liquid medium and incubated at 37°C overnight. Then, they were transferred to MRS liquid medium at a 2.0% inoculation rate and incubated at 37°C for 24 hours. The pH, viable cell count, and OD of the fermentation broth were then measured. 600nm Value, organic acid and phenyl lactic acid content.

[0053] 1.2.2 Pretreatment of Fermentation Broth Samples Take 5.0 mL of the 24 h fermentation broth of the above-mentioned test strains, centrifuge at 10000 rpm for 5 min, and filter the supernatant through a 0.22 µm filter membrane to obtain the test sample. The content of organic acids and phenyl lactic acid in the fermentation supernatant is detected by high performance liquid chromatography (HPLC).

[0054] 2 Results 2.1 The influence of 4 strains of Lactobacillus plantarum on pH and viable bacteria count Table 3 Comparison of pH and viable bacteria count of 4 strains of Lactobacillus plantarum

[0055] Note: The same column with different lowercase letters indicates significant difference (p < 0.05), and the same or no letter indicates no significant difference (p > 0.05). P P

[0056] From Table 3, it can be seen that the viable bacteria count of the 4 strains of Lactobacillus plantarum is at the 9th power level, and the fermentation performance is good. The pH of the fermentation broth of BLSCX-2 is the lowest, which is 3.503, which is significantly lower than that of the other three strains of Lactobacillus plantarum, indicating that the strain has strong acid-producing ability and excellent pH-lowering performance.

[0057] 2.2 The influence of 4 strains of Lactobacillus plantarum on the content of organic acids Table 4 Influence of 4 strains of Lactobacillus plantarum on the content of organic acids (g / L)

[0058] From Table 4, it can be seen that the main organic acids in the fermentation supernatant of the 4 strains of Lactobacillus plantarum are lactic acid, acetic acid, citric acid and succinic acid. The content of lactic acid in BLSCX-2 is the highest, which is 25.622 g / L, which is 57.18%, 37.67% and 45.74% higher than that of BLCC2-0881, BLCC2-1014 and BLCC2-1015, respectively.

[0059] 2.3 The influence of 4 strains of Lactobacillus plantarum on the content of phenyl lactic acid Table 5 Influence of 4 strains of Lactobacillus plantarum on the content of phenyl lactic acid (g / L)

[0060] Note: The same column with different lowercase letters indicates significant difference (p < 0.05), and the same or no letter indicates no significant difference (p > 0.05). P P

[0061] From Table 5, it can be seen that the content of phenyl lactic acid produced by BLSCX-2 among the 4 strains of Lactobacillus plantarum is the highest, which is 1.452 g / L, which is significantly higher than that of the other three strains of Lactobacillus plantarum.

[0062] Example 3: Determination of the antibacterial performance of Lactobacillus plantarum BLSCX-2 in vitro 1 Materials and methods 1.1 Test strain ​​​​The plant lactobacillus obtained by screening in Example 1 of this invention ( Lactiplantibacillus plantarum )BLSCX-2. Lactobacillus plantarum BLCC2-0881, BLCC2-1014 and BLCC2-1015 were all preserved and provided by the strain resource bank of the Science and Technology Innovation Center of Shandong Baolai Lailai Biotechnology Co., Ltd.

[0063] 1.2 Determination of in vitro antibacterial properties 1.2.1 Preparation of fermentation supernatant The four tested strains of Lactobacillus plantarum were inoculated into MRS liquid medium and incubated at 37 ℃ for 24 h. The fermentation broth was centrifuged at 4000 rpm for 10 min and the supernatant was collected for later use.

[0064] 1.2.2 Indicator bacteria The following bacteria, including Candida albicans BLCC8-0018, Bacillus cereus BLCC8-0140, Pseudomonas aeruginosa BLCC8-0199, Shigella BLCC8-0071, Escherichia coli BLCC8-0135, Staphylococcus aureus BLCC8-0138, Salmonella enteritidis BLCC8-0129, Aeromonas hydrophila BLCC8-0121, Vibrio parahaemolyticus BLCC8-0145, Vibrio alginolyticus BLCC8-0146, and Clostridium perfringens BLCC8-0136, are all preserved and provided by the bacterial strain resource bank of the Science and Technology Innovation Center of Shandong Baolai Lailai Biotechnology Co., Ltd.

[0065] 1.2.3 Determination of the antibacterial properties of Lactobacillus plantarum BLSCX-2 The antibacterial activity of *Lactobacillus plantarum* BLSCX-2 against indicator bacteria was determined using the perforation method.

[0066] Candida albicans BLCC8-0018, Bacillus cereus BLCC8-0140, Pseudomonas aeruginosa BLCC8-0199, Shigella BLCC8-0071, Escherichia coli BLCC8-0135, Staphylococcus aureus BLCC8-0138, Salmonella enteritidis BLCC8-0129, and Aeromonas hydrophila BLCC8-0121 were inoculated into NB broth medium and incubated at 37 ℃ and 180 rpm for 12–18 h. The viable cell count was then adjusted to 1.0 × 10⁻⁶ cells / mL with sterile physiological saline. 7 Prepare CFU / mL for later use. Simultaneously, take 1 mL of diluted indicator bacteria and 17 mL of NA medium into a petri dish, mix well, and let it stand until solidified. Then, punch holes with a punch and add 90 µL of the fermentation supernatant of the test strain to each dish. Incubate at 37 ℃ for 12 h. Perform 3 parallel plates for each sample.

[0067] V. fischeri BLCC8-0136 was inoculated into liquid sulphite medium and incubated at 45 °C for 12-18 h. The viable cell count was adjusted to 1.0 x 10 7 CFU / mL. Meanwhile, 1 mL of the diluted indicator bacteria and 17 mL of the iron sulphite agar were taken into a Petri dish, mixed evenly, and left to solidify. Then, the holes were punched with a puncher, and 90 μL of the fermentation supernatant of the tested strain was added. The Petri dish was placed in a 37 °C incubator for anaerobic culture for 12 h, and 3 parallel plates were prepared for each sample.

[0068] V. alginolyticus BLCC8-0146 and V. parahaemolyticus BLCC8-0145 were inoculated into 2216E liquid medium, respectively, and incubated at 37 °C and 180 rpm for 12-18 h. Then, the viable cell count was adjusted to 1.0 x 10 7 CFU / mL. Meanwhile, 1 mL of the diluted indicator bacteria and 17 mL of the 2216E agar medium were taken into a Petri dish, mixed evenly, and left to solidify. Then, the holes were punched with a puncher, and 90 μL of the fermentation supernatant of the tested strain was added. The Petri dish was placed in a 37 °C incubator for culture for 12 h, and 3 parallel plates were prepared for each sample.

[0069] The size of the inhibition zone was measured with a vernier caliper, and the antibacterial activity was evaluated.

[0070] 2 Test results Table 6 In vitro antibacterial results of L. plantarum BLSCX-2

[0071] As shown in Table 6, L. plantarum BLSCX-2 had antibacterial effects on the 11 tested pathogenic bacteria, and the diameter of the inhibition zone was 15 mm or more. Among them, the antibacterial effect on V. alginolyticus, V. parahaemolyticus and Aeromonas hydrophila was better, and the diameter of the inhibition zone was more than 20 mm.

[0072] Example 4: Determination of the acid, alkali and bile salt resistance of L. plantarum BLSCX-2 1 Materials and methods Lactobacillus plantarum BLSCX-2 and Lactobacillus plantarum BLCC2-0881, BLCC2-1014 and BLCC2-1015 were respectively inoculated into 100 mL of MRS liquid medium from the slant, and cultured at 37 ℃ for 24 h. 10 mL of the fermentation liquid was centrifuged at 4000 rpm for 10 min, and the supernatant was discarded. 10 mL of normal saline was added to prepare a bacterial suspension as the original bacterial liquid. Lactobacillus plantarum BLCC2-0881, BLCC2-1014 and BLCC2-1015 were preserved and provided by the Microbial Resource Bank of the Science and Technology Innovation Center of Shandong Baolailailai Biological Engineering Co., Ltd.

[0073] 1.1 Acid tolerance test The above-mentioned original bacterial liquid was inoculated into normal saline (pH about 6.5), normal saline with pH values of 2.0, 2.5, 3.0 and 4.0, respectively, at an inoculation amount of 2.0%, and cultured at 37 ℃ for 2 h. The viable cell count was determined by ten-fold gradient dilution method, and the survival rate was calculated.

[0074] 1.2 Cholate salt tolerance test The above-mentioned original bacterial liquid was inoculated into normal saline with cholate salt concentrations of 0, 0.1%, 0.2%, 0.3% and 0.5%, respectively, at an inoculation amount of 2.0%, and cultured at 37 ℃ for 4 h. The viable cell count was determined by ten-fold gradient dilution method, and the survival rate was calculated.

[0075] 1.3 Alkaline tolerance test The above-mentioned original bacterial liquid was inoculated into MRS medium with pH values of 7.0, 8.0 and 10.0, respectively, at an inoculation amount of 2.0%, and cultured at 37 ℃ for 24 h. The pH value and viable cell count were detected.

[0076] 2. Results Table 7. Results of acid tolerance test

[0077] As shown in Table 7, Lactobacillus plantarum BLSCX-2 could tolerate the environment with the lowest pH value of 2.5, and the survival rate was 100% under the condition of pH 2.5. Lactobacillus plantarum BLCC2-1015 had a survival rate of 95% under the condition of pH 2.5 for 2 h. The survival rates of the other two Lactobacillus plantarum strains were less than 90%.

[0078] Table 8. Results of cholate salt tolerance test

[0079] As shown in Table 8, the plant Lactobacillus BLSCX-2 has the best bile salt tolerance performance, and the survival rates are 93.25%, 50.00%, 8.93% and 0.60% respectively after being cultured in 0.1%, 0.2%, 0.3% and 0.5% bile salt concentrations for 4 hours.

[0080] Table 9: Results of alkali tolerance performance determination

[0081] As shown in Table 9, the plant Lactobacillus BLSCX-2 has the best alkali tolerance performance, and the lowest pH value and the highest viable cell count are obtained when the pH value is 7.0, 8.0 and 10.0. When the pH value is 10.0, the pH value of the fermentation broth can reach 4.73, and the viable cell count is 6.87×10 8 CFU / mL, which has strong alkali tolerance performance.

[0082] Example 5: Effect of temperature on the fermentation performance of plant Lactobacillus BLSCX-2 1. Materials and methods 1.1 Test strain: plant Lactobacillus BLSCX-2 screened in Example 1 of the present application. Lactiplantibacillus plantarum BLCC2-0881, BLCC2-1014 and BLCC2-1015 are preserved and provided by the Strain Resource Library of the Innovation Center of Shandong Baolailailai Biological Engineering Co., Ltd.

[0083] 1.2 Test method The test strain was inoculated into MRS liquid medium at 37℃ and incubated overnight, then inoculated into MRS liquid medium at 5.0% inoculation amount, and cultured at 10℃ and 15℃ respectively. The fermentation broth pH value, viable cell count and lactic acid content in the fermentation supernatant were detected at 0 d, 1 d, 3 d, 5 d and 7 d respectively. 2. Results 2.1 Effect of temperature on the pH value of the fermentation broth of plant Lactobacillus BLSCX-2 Table 10: Effect of temperature on the pH value of the fermentation broth of plant Lactobacillus BLSCX-2

[0084] From Table 10, compared with 10℃ fermentation, 15℃ is more conducive to Lactobacillus plantarum fermentation. In the 15℃ incubator, the pH values of the other three strains except BLCC2-1015 can be reduced to about 4.5 after 1 d of fermentation, and then the pH value decreases slowly with the extension of fermentation time. The pH values of BLSCX-2 are 3.89 and 3.63 after 5 d and 7 d of fermentation, respectively, indicating that the strain can also better reduce the pH value of the fermentation broth under low temperature conditions by extending the fermentation time.

[0085] 2.2 Effect of temperature on the viable count of Lactobacillus plantarum BLSCX-2 fermentation broth Table 11 Effect of temperature on the viable count of Lactobacillus plantarum BLSCX-2 fermentation broth (×10 7 CFU / mL)

[0086] As shown in Table 11, the viable count of Lactobacillus plantarum BLSCX-2 is the highest under the same temperature and time. Compared with the initial viable count, the viable count changes little after 1 d of fermentation at 10℃, and increases by one order of magnitude after 3 d, reaching 1.7767 billion CFU / mL, which is comparable to the viable count after 1 d of fermentation at 15℃. Then, the viable count increases slowly with the extension of fermentation time. After 5 d of fermentation, the viable counts at 10℃ and 15℃ are 2.4 billion CFU / mL and 2.58 billion CFU / mL, respectively, with little difference. This indicates that the strain can achieve good fermentation effect by extending the fermentation time at 10℃.

[0087] 2.3 Effect of temperature on the lactic acid content of Lactobacillus plantarum BLSCX-2 fermentation broth Table 12 Effect of temperature on the lactic acid content of Lactobacillus plantarum BLSCX-2 fermentation broth (g / L)

[0088] As shown in Table 12, the lactic acid content of the fermentation broth of the four strains of Lactobacillus plantarum increases with the increase of fermentation temperature and the extension of fermentation time. The lactic acid content of Lactobacillus plantarum BLSCX-2 increases with the extension of fermentation time when fermented at 10℃, and reaches 16.722 g / L and 19.144 g / L after 5 d and 7 d of fermentation, respectively. The lactic acid content is 19.194 g / L after 3 d of fermentation at 15℃, which is comparable to the lactic acid content after 7 d of fermentation at 10℃. This indicates that the strain of Lactobacillus plantarum can achieve better fermentation effect by extending the fermentation time under low temperature conditions.

[0089] Example 6: Effect of low temperature on the fermentation performance of Lactobacillus plantarum BLSCX-2 1. Materials and methods 1.1 Test strains: Lactobacillus plantarum BLSCX-2 screened in Example 1 of the present application. Lactiplantibacillus plantarum Lactobacillus plantarum BLCC2-0881, BLCC2-1014 and BLCC2-1015 are preserved and provided by the Strain Resource Library of the Innovation Center of Shandong Baolailailai Biological Engineering Co., Ltd.

[0090] 1.2 Test method The test strains were inoculated into MRS liquid medium at 37°C and incubated overnight, then transferred into MRS liquid medium at an inoculation amount of 5.0%, and incubated in a refrigerator at 4°C. Samples were taken at 0 d, 7 d, 14 d and 21 d, and the pH value and viable cell count of the fermentation broth were detected.

[0091] 2 Results 2.1 Effect of 4°C on the pH value of Lactobacillus plantarum BLSCX-2 fermentation broth Table 1 Effect of 4°C on the pH value of Lactobacillus plantarum BLSCX-2 fermentation broth

[0092] The results showed that the pH value of the fermentation broth of the four strains cultured at 4°C for 21 d was lower than that cultured for 14 d, indicating that although the viable cells did not significantly increase at 4°C, the strains still had metabolic activity.

[0093] 2.2 Effect of 4°C on the viable cell count of Lactobacillus plantarum BLSCX-2 fermentation broth Table 2 Effect of 4°C on the viable cell count of Lactobacillus plantarum BLSCX-2 fermentation broth (×10 7 CFU / mL)

[0094] The results showed that the viable cell count of the four Lactobacillus plantarum strains cultured at 4°C for 21 d was in the same order of magnitude as the initial viable cell count.

[0095] Example 7: Effect of sodium chloride on the fermentation performance of Lactobacillus plantarum BLSCX-2 1 Materials and methods 1.1 Test strains: Lactobacillus plantarum BLSCX-2 screened in Example 1 of the present application. Lactiplantibacillus plantarum Lactobacillus plantarum BLCC2-0881, BLCC2-1014 and BLCC2-1015 are preserved and provided by the Strain Resource Library of the Innovation Center of Shandong Baolailailai Biological Engineering Co., Ltd.

[0096] 1.2 Test method The test strains were inoculated into MRS liquid medium and incubated at 37℃ overnight, then inoculated into MRS liquid medium containing different concentrations of sodium chloride (the concentrations of sodium chloride were 0%, 5% and 8% respectively) at 5.0%, and incubated at 37℃ for 24 hours. The pH value and viable count of the fermentation broth were detected, and the lactic acid content in the fermentation supernatant was detected.

[0097] 2Results Table 15 Effect of sodium chloride on the fermentation performance of Lactobacillus plantarum BLSCX-2

[0098] As shown in Table 15, with the increase of the concentration of sodium chloride, the viable count and lactic acid content of Lactobacillus plantarum BLSCX-2 showed a downward trend, and the viable count and lactic acid content of Lactobacillus plantarum BLSCX-2 were higher than those of the other three Lactobacillus plantarum under the same concentration of sodium chloride.

[0099] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of *Lactobacillus plantarum* is named *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum BLSCX-2, this strain was deposited at the China Center for Type Culture Collection on July 11, 2025, at Wuhan University, Wuhan, with accession number CCTCC NO: M 20251576.

2. A microbial agent comprising *Lactobacillus plantarum* as described in claim 1.

3. A microbial agent comprising the fermentation product of *Lactobacillus plantarum* as described in claim 1.

4. A microbial agent comprising the metabolites of *Lactobacillus plantarum* as described in claim 1.

5. The microbial agent as described in claim 4, characterized in that, The metabolites are intracellular and / or extracellular metabolic products of the bacteria.

6. A product comprising *Lactobacillus plantarum* as described in claim 1 or any one of claims 2-5.

7. The product according to claim 6, characterized in that, The product is any one or more of the following: antibacterial agent, preservative, fermentation agent, feed additive, food, food additive, or medicine.

8. The use of *Lactobacillus plantarum* as described in claim 1, or the bacterial agent as described in any one of claims 2-5, and / or the product as described in any one of claims 6-7, in the preparation of a product with antibacterial ability.

9. The application as described in claim 8, characterized in that, The antibacterial ability includes the ability to inhibit Candida albicans, Shigella, Aeromonas hydrophila, Salmonella enteritidis, Escherichia coli, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, Vibrio parahaemolyticus, Vibrio alginolyticus, and Pseudomonas aeruginosa.

10. The application as described in claim 9, characterized in that, The product is a drug or feed additive.

Images