Plant lactobacillus producing acid and application thereof

Abstract

This invention relates to the fields of microbiology and fermentation engineering, specifically to an acid-producing *Lactobacillus plantarum* strain and its applications. Specifically, this strain is named *Lactobacillus plantarum* (…). Lactiplantibacillus plants BLSCX-2. Experiments have verified that its lactic acid and phenyllactic acid yields are significantly higher than those of existing reported strains, making it suitable for high-efficiency fermented food production. Simultaneously, *Lactobacillus plantarum* BLSCX-2 in this invention exhibits broad-spectrum antibacterial activity against various foodborne pathogens and spoilage bacteria, making it suitable for biological preservation or pharmaceutical antibacterial agent development. Furthermore, this strain maintains its activity under low pH, high bile salt, low temperature, and high salt conditions, broadening its application range in complex environments and thus possessing significant 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 solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a strain of *Lactobacillus plantarum*, 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.

[0009] The aforementioned *Lactobacillus plantarum* is a strain isolated by the applicant from pickled vegetables through screening and purification. It possesses high acid production capacity, broad-spectrum antibacterial ability, excellent environmental tolerance, ease of cultivation, and high industrialization potential.

[0010] Specifically, the *Lactobacillus plantarum* has the following characteristics:

[0011] (1) High acid production capacity: The synthesis efficiency of lactic acid and phenyllactic acid is outstanding.

[0012] The core advantage of strain BLSCX-2 lies in its highly efficient acid production. The organic acids it produces are not only crucial for food flavor and preservation but also provide the material basis for antibacterial and freshness-preserving effects. When cultured in MRS liquid medium at 37°C for 24 hours, its lactic acid yield reaches 25.622 g / L, significantly higher than other strains screened at the same time. This yield can rapidly lower the pH of the fermentation system, effectively shortening the food fermentation cycle, and also inhibiting contamination by other microorganisms through an acidic environment. Furthermore, as a natural antibacterial substance, phenyllactic acid is synthesized by strain BLSCX-2 at a much higher rate than existing strains, providing an advantage in its antibacterial performance and effectively expanding its application areas.

[0013] (2) Broad-spectrum antibacterial activity: Effectively covers 11 common pathogenic and putrefactive bacteria, with significant targeted advantages.

[0014] The fermentation supernatant of *Lactobacillus plantarum* BLSCX-2 of this invention exhibits inhibitory effects against a variety of pathogenic bacteria, demonstrating a broad antibacterial spectrum and excellent efficacy. Specifically, it shows inhibitory effects against 11 pathogenic and putrefactive bacteria, including *Candida albicans*, *Shigella*, *Escherichia coli*, *Staphylococcus aureus*, *Bacillus cereus*, and *Pseudomonas aeruginosa*. Of particular note is its outstanding inhibitory effect on common pathogenic bacteria in aquaculture (such as *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, and *Aeromonas hydrophila*), thus making it suitable for antibacterial purposes in aquaculture water or for preserving aquatic products.

[0015] (3) Excellent environmental tolerance: adaptable to complex scenarios and broadens application boundaries

[0016] The *Lactobacillus plantarum* BLSCX-2 strain of this invention maintains its activity even in extreme environments (such as low pH, high bile salt, low temperature, and high salinity), effectively solving the problem of "limited application scenarios" for existing strains. Specifically, its survival rate is as high as 100% in an acidic environment of pH 2.5, a survival rate far exceeding that of ordinary *Lactobacillus plantarum*. Simultaneously, its survival rate remains at 8.93% even at a high bile salt concentration of 0.3%, thus demonstrating its tolerance to the digestive tract environment and its potential as a probiotic preparation. Furthermore, studies have confirmed that it can grow normally at a low temperature of 10℃, with a viable cell count reaching 240.00 × 10⁻⁶ after 5 days of fermentation. 7 The CFU / mL yielded a lactic acid production of 16.722 g / L, which is close to the fermentation effect at 15℃.

[0017] Furthermore, it retains its metabolic activity after 21 days of refrigeration at 4℃, making it suitable for low-temperature fermented foods (such as fermented fruit juices and low-temperature yogurt) and applications in cold regions, avoiding nutrient loss or taste deterioration caused by high-temperature fermentation. It can also survive in an 8% sodium chloride environment (4.45 × 10⁻⁶ viable bacteria). 8 It contains CFU / mL and still has the ability to produce lactic acid, so it can be used for the fermentation of high-salt foods (such as kimchi and pickles) without the need for additional salt concentration adjustment, thus effectively simplifying the production process.

[0018] (4) Cultivation and morphological characteristics: Easy to cultivate and has high industrialization potential.

[0019] The *Lactobacillus plantarum* BLSCX-2 of this invention exhibits good growth performance in conventional MRS medium and good fermentation stability. Experiments have verified that after 10 consecutive generations, the number of viable bacteria and lactic acid content did not decrease significantly, thus providing a basis for large-scale industrial cultivation.

[0020] For ease of description, the *Lactobacillus plantarum* (Lactobacillus plantarum) described in this invention... Lactiplantibacillus plantarum BLSCX-2 is also abbreviated as strain BLSCX-2 or BLSCX-2 in the application documents.

[0021] The strain BLSCX-2 of this invention can be cultured in MRS medium. After culturing in MRS solid medium at 37°C for 48 h, it forms milky-white, raised, smooth, moist, and easily pickable round colonies. Microscopic observation reveals that the bacterial cells are rod-shaped, arranged singly, in pairs, or in short chains.

[0022] Preferably, the MRS liquid culture medium consists of: 20 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L ammonium citrate, 5 g / L sodium acetate, 5 g / L dipotassium hydrogen phosphate, 0.2 g / L manganese sulfate, 0.5 g / L magnesium sulfate, and 1 g / L Tween-80, with a pH of 6.0. The solid culture medium requires the addition of 15 g / L agar and is sterilized at 121°C for 30 min.

[0023] In a second aspect, the present invention provides a microbial agent comprising *Lactobacillus plantarum* or its ferments or metabolites as described in the first aspect above.

[0024] The metabolites described in this invention include intracellular and / or extracellular metabolic products of bacteria.

[0025] The term "fermentation product" refers to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The fermentation product is the collective term for all products generated during the fermentation process of BLSCX-2 under suitable conditions. The fermentation product may include, but is not limited to, the following components: live bacterial cells, dead bacterial cells, extracellular metabolites, and culture medium components. In this invention, 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 fermentation culture of *Lactobacillus plantarum* BLSCX-2 bacteria, and therefore can also be called fermentation broth; the liquid may contain bacteria (bacterial cells), but does not necessarily need to contain bacteria. Preferably, the liquid contains metabolites produced by the BLSCX-2 bacteria of this invention.

[0026] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is separated from the liquid by centrifugation, filtration, sedimentation, or other means known in the art. The liquid remaining after removing the bacterial cells is called the "supernatant," and in the present invention, the supernatant contains extracellular metabolites of BLSCX-2. In embodiments of the present invention, the bacterial agent may also contain this supernatant.

[0027] Furthermore, in embodiments of the present invention, the fermentation broth or culture medium containing bacterial cells is centrifuged, filtered, settled, or otherwise known in the art to separate the bacterial cells grown in the fermentation broth or culture medium from the liquid to obtain bacterial cells. The bacterial cells can be broken up to obtain bacterial cell fragments. The breaking up method can be ultrasound (e.g., ice bath ultrasound to break up cells) or other methods known in the art. Alternatively, the bacterial cell fragments can be centrifuged to collect the supernatant, which is designated as the cell-free extract. In the present invention, the bacterial cell fragments or cell-free extract contain intracellular metabolites of BLSCX-2. In embodiments of the present invention, the bacterial agent may also contain the bacterial cell fragments or cell-free extract.

[0028] In a third aspect, the present invention provides a product comprising *Lactobacillus plantarum* as described in the first aspect or the microbial agent as described in the second aspect.

[0029] In embodiments of the present invention, the product may be an antibacterial agent, a preservative, a fermentation agent, a feed additive, a food, a food additive, or a pharmaceutical.

[0030] In a fourth aspect of the invention, the use of *Lactobacillus plantarum* as described in the first aspect, or the microbial agent as described in the second aspect, and / or the product as described in the third aspect, in the preparation of a product with antibacterial capabilities is provided.

[0031] 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.

[0032] The product may be a drug or a feed additive.

[0033] The beneficial technical effects of the above technical solution are as follows:

[0034] This invention first screened and obtained a strain of *Lactobacillus plantarum* BLSCX-2 with excellent fermentation performance and high acid production capacity. Its lactic acid and phenyllactic acid yields are significantly higher than those of existing reported strains, making it suitable for high-efficiency fermented food production. Simultaneously, the *Lactobacillus plantarum* BLSCX-2 in the above technical solution exhibits broad-spectrum antibacterial activity against various foodborne pathogens and spoilage bacteria, and can be used for biological preservation or pharmaceutical antibacterial agent development. Furthermore, this strain maintains its activity under low pH, high bile salt, low temperature, and high salt conditions, broadening its application range in complex environments, thus possessing good practical application value. Attached Figure Description

[0035] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

[0036] Figure 1 This refers to the strain screening-calcification zone in Example 1 of the present invention.

[0037] Figure 2 This is a colony morphology diagram of strain BLSCX-2 in Example 1 of the present invention.

[0038] Figure 3 This is a cell morphology diagram (oil immersion) of strain BLSCX-2 in Example 1 of the present invention. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. The reagents and raw materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods or product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0041] In each embodiment, the determination of organic acid content was carried out in accordance with the method described in the national standard GB5009.157-2016 "National Food Safety Standard - Determination of Organic Acids in Food", and the determination of phenyllactic acid content was carried out in accordance with the method described in the literature (Zhang Zhonghua, Li Xiaoran, Li Hongmei, et al. Establishment of a rapid detection method for DL-3-phenyllactic acid by high performance liquid chromatography in lactic acid bacteria fermentation broth, 2013, 38(2): 80-83, 87.).

[0042] Example 1: Isolation, Screening and Identification of Strains

[0043] 1. Experimental Materials

[0044] 1.1 MRS medium

[0045] The MRS liquid culture medium consisted of: 20 g / L glucose, 2 g / L ammonium citrate, 5 g / L sodium acetate, 5 g / L dipotassium hydrogen phosphate, 0.2 g / L manganese sulfate, 0.5 g / L magnesium sulfate, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 1 g / L Tween-80, pH 6.0, and was sterilized at 121 °C for 30 min.

[0046] The composition of MRS solid medium is as follows: add 15 g / L agar to MRS liquid medium and sterilize at 121 °C for 30 min.

[0047] 1.2 MRS agar medium containing calcium carbonate

[0048] Add 0.5-1.0% calcium carbonate to 1.1 MRS liquid agar medium.

[0049] 1.3 Test Methods

[0050] 1.3.1 Separation of samples: commercially available pickled vegetables from Shandong, Sichuan, Xinjiang and other places.

[0051] 1.3.2 Determination of lactic acid content: The yield of lactic acid in the fermentation broth was determined by high performance liquid chromatography (HPLC).

[0052] 1.3.3 Screening methods for lactic acid-producing bacteria

[0053] 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.

[0054] 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).

[0055] 1.3.4 Strain Identification

[0056] (1) Morphological identification

[0057] 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.

[0058] (2) Molecular biological identification

[0059] 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.

[0060] 1492r: 5'-ggttaccttgttacgactt-3' (SEQ ID NO: 1);

[0061] 27f: 5'-agagttgatcctggctcag-3' (SEQ ID NO: 2).

[0062] The PCR reaction system (50 μL) consisted of: Mixture 25 μL (containing Taq DNA polymerase and dNTPs, etc., Tiangen Biotech).

[0063] 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.

[0064] 2. Experimental Results

[0065] 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.

[0066] Table 1. Results of lactic acid content determination (g / L) of isolated strains in pickled vegetable samples.

[0067]

[0068] 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.

[0069] 2.2 Identification of BLSCX-2 strain

[0070] 2.2.1 Morphological identification

[0071] 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.

[0072] 2.2.2 Molecular biological identification

[0073] 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:

[0074] SEQ ID NO:3

[0075]

[0076] 2.3 Passage stability of strain BLSCX-2

[0077] 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.

[0078] Table 2. Effects of passage number on viable count and lactic acid content of *Lactobacillus plantarum* BLSCX-2

[0079]

[0080] 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.

[0081] Example 2: Quantitative comparison of the ability of different Lactobacillus plantarum fermentation broths to produce organic acids and phenyllactic acid

[0082] 1. Materials and Methods

[0083] 1.1 Materials

[0084] 1.1.1 DL-3 phenyllactic acid standard (analytical grade, purity ≥98%) was purchased from Sigma-Aldrich, USA.

[0085] 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.

[0086] 1.2 Test Methods

[0087] 1.2.1 Obtaining the sample to be tested

[0088] 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.

[0089] 1.2.2 Pretreatment of Fermentation Broth Samples

[0090] 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).

[0091] 2 Results

[0092] 2.1 Effects of four tested Lactobacillus plantarum strains on pH and viable cell count

[0093] Table 3 Comparison of pH value and viable cell count of the four Lactobacillus plantarum strains tested.

[0094]

[0095] Note: Different lowercase letters in the same column's subscript indicate significant differences. P <0.05), the same or no letter subscript indicates no significant difference ( P >0.05).

[0096] Table 3 shows that the viable cell counts of all four tested *Lactobacillus plantarum* strains were at the 9th power level, indicating good fermentation performance. The lowest pH value (3.503) was found in the fermentation broth of BLSCX-2, significantly lower than the other three strains, suggesting that this strain has strong acid-producing ability and excellent pH-lowering properties.

[0097] 2.2 Effects of fermentation broth from four tested Lactobacillus plantarum strains on organic acid content

[0098] Table 4. Effects of four strains of Lactobacillus plantarum on organic acid content (g / L)

[0099]

[0100] As shown in Table 4, the main organic acids in the fermentation supernatant of the four tested Lactobacillus plantarum strains were lactic acid, acetic acid, citric acid and succinic acid. Among them, the lactic acid content was highest in BLSCX-2 at 25.622 g / L, which was 57.18%, 37.67% and 45.74% higher than that in BLCC2-0881, BLCC2-1014 and BLCC2-1015, respectively.

[0101] 2.3 Effect of fermentation broth from four tested Lactobacillus plantarum strains on phenyllactic acid content

[0102] Table 5. Effects of four strains of Lactobacillus plantarum on phenyllactic acid content (g / L)

[0103]

[0104] Note: Different lowercase letters in the same column's subscript indicate significant differences. P<0.05), the same or no letter subscript indicates no significant difference ( P >0.05).

[0105] As shown in Table 5, among the four tested Lactobacillus plantarum strains, BLSCX-2 produced the highest content of phenyl lactic acid after 24 h of fermentation, which was 1.452 g / L, significantly higher than the other three Lactobacillus plantarum strains.

[0106] Example 3: Determination of the in vitro antibacterial properties of Lactobacillus plantarum BLSCX-2

[0107] 1. Materials and Methods

[0108] 1.1 Test strains

[0109] 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.

[0110] 1.2 Determination of in vitro antibacterial properties

[0111] 1.2.1 Preparation of fermentation supernatant

[0112] 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.

[0113] 1.2.2 Indicator bacteria

[0114] 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.

[0115] 1.2.3 Determination of the antibacterial properties of Lactobacillus plantarum BLSCX-2

[0116] The antibacterial activity of *Lactobacillus plantarum* BLSCX-2 against indicator bacteria was determined using the perforation method.

[0117] 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.

[0118] Clostridium perfringens BLCC8-0136 was inoculated into liquid thioglycolate medium and incubated at 45 °C for 12–18 h. The viable count was then adjusted to 1.0 × 10⁻⁶ with sterile physiological saline. 7 Prepare CFU / mL for later use. Simultaneously, take 1 mL of diluted indicator bacteria and 17 mL of ferrous sulfite agar 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 anaerobically at 37 ℃ for 12 h. Perform 3 parallel plates for each sample.

[0119] Vibrio alginolyticus BLCC8-0146 and Vibrio parahaemolyticus BLCC8-0145 were inoculated into 2216E liquid medium and cultured at 37 ℃ and 180 rpm for 12–18 h. The viable cell counts were then adjusted to 1.0 × 10⁻⁶ cells / year with sterile physiological saline. 7 CFU / mL was prepared for use. Simultaneously, 1 mL of diluted indicator bacteria and 17 mL of 2216E agar medium were added to a petri dish, mixed thoroughly, and allowed to solidify. Then, holes were punched, and 90 µL of the fermentation supernatant of the test strain was added to each hole. The dishes were incubated at 37 °C for 12 h. Three replicates were prepared for each sample.

[0120] The size of the inhibition zone was measured using vernier calipers, and the antibacterial activity was evaluated.

[0121] 2. Experimental Results

[0122] Table 6. In vitro antibacterial results of Lactobacillus plantarum BLSCX-2

[0123]

[0124] As shown in Table 6, BLSCX-2 of *Lactobacillus plantarum* had antibacterial effects against all 11 tested pathogens, with inhibition zone diameters of 15 mm and above. Among them, the antibacterial effects against *Vibrio alginolyticus*, *Vibrio parahaemolyticus* and *Aeromonas hydrophila* were better, with inhibition zone diameters of 20 mm and above.

[0125] Example 4: Determination of the acid, alkali and bile salt resistance of Lactobacillus plantarum BLSCX-2

[0126] 1. Materials and Methods

[0127] Lactobacillus plantarum BLSCX-2 and Lactobacillus plantarum BLCC2-0881, BLCC2-1014, and BLCC2-1015 were revived from slant culture and transferred to 100 mL of MRS liquid medium. The cultures were incubated at 37 ℃ for 24 h. 10 mL of each fermentation broth was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and 10 mL of physiological saline was added and mixed thoroughly to prepare a bacterial suspension, which served as the initial bacterial suspension. 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.

[0128] 1.1 Determination of acid resistance

[0129] The original bacterial culture was inoculated at a rate of 2.0% into physiological saline solutions with pH of natural (approximately 6.5), 2.0, 2.5, 3.0, and 4.0, respectively. The solutions were incubated at 37 °C for 2 h, and viable cell counts were performed using a tenfold serial dilution method to calculate the survival rate. Here, "natural pH" refers to the pH value of the solution itself, formed naturally without human intervention.

[0130] 1.2 Test of bile salt tolerance

[0131] The original bacterial culture was added to physiological saline solutions with bile salt concentrations of 0%, 0.1%, 0.2%, 0.3%, and 0.5% at an inoculation rate of 2.0%, respectively, and cultured at 37 °C for 4 h. Viable bacteria were counted using a tenfold serial dilution method, and the survival rate was calculated.

[0132] 1.3 Alkali resistance test

[0133] The original bacterial culture was added to MRS medium with pH values ​​of 7.0, 8.0 and 10.0 at an inoculation rate of 2.0%, respectively, and cultured at 37°C for 24 h. The pH value and viable cell count were then measured.

[0134] 2 Results

[0135] Table 7 Results of acid resistance test

[0136]

[0137] As shown in Table 7, Lactobacillus plantarum BLSCX-2 can tolerate a minimum pH of 2.5, with a survival rate of 100% at pH 2.5. BLCC2-1015 is the next best, with a survival rate of 95% after 2 hours at pH 2.5. The survival rates of the other two Lactobacillus plantarum strains are both below 90%.

[0138] Table 8 Results of bile salt tolerance test

[0139]

[0140] As shown in Table 8, Lactobacillus plantarum BLSCX-2 exhibited the best bile salt tolerance, with survival rates of 93.25%, 50.00%, 8.93%, and 0.60% after 4 h of culture in bile salt concentrations of 0.1%, 0.2%, 0.3%, and 0.5%, respectively.

[0141] Table 9 Results of Alkali Resistance Test

[0142]

[0143] Table 9 shows that *Lactobacillus plantarum* BLSCX-2 exhibits the best alkali tolerance, with the lowest pH and highest viable cell count at pH 7.0, 8.0, and 10.0. At pH 10.0, the fermentation broth achieved a pH of 4.73 and a viable cell count of 6.87 × 10⁻⁶. 8 It has a concentration of CFU / mL and exhibits strong alkali resistance.

[0144] Example 5: Effect of temperature on the fermentation performance of Lactobacillus plantarum BLSCX-2

[0145] 1. Materials and Methods

[0146] 1.1 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.

[0147] 1.2 Test Methods

[0148] The test strains were inoculated into MRS liquid medium and incubated at 37°C overnight. Then, they were transferred to MRS liquid medium at an inoculation rate of 5.0% and incubated at 10°C and 15°C, respectively. Samples were taken at 0, 1, 3, 5 and 7 days to detect the pH value of the fermentation broth, the number of viable cells, and the lactic acid content in the fermentation supernatant.

[0149] 2 Results

[0150] 2.1 Effect of temperature on pH value of Lactobacillus plantarum BLSCX-2 fermentation broth

[0151] Table 10 Effect of temperature on pH value of Lactobacillus plantarum BLSCX-2 fermentation broth

[0152]

[0153] As shown in Table 10, 15℃ is more conducive to the fermentation of Lactobacillus plantarum than 10℃. In a 15℃ incubator, the pH value of the three strains except BLCC2-1015 can be reduced to about 4.5 after 1 day of fermentation. Subsequently, the pH value decreased slowly as the fermentation time was extended. The pH values ​​of BLSCX-2 were 3.89 and 3.63 after 5 days and 7 days of fermentation, respectively, indicating that this strain can also effectively reduce the pH value of the fermentation broth by extending the fermentation time under low temperature conditions.

[0154] 2.2 Effect of temperature on viable cell count of Lactobacillus plantarum BLSCX-2 fermentation broth

[0155] Table 11 Effect of temperature on viable cell count of *Lactobacillus plantarum* BLSCX-2 fermentation broth (×10⁻⁶) 7 CFU / mL

[0156]

[0157] Table 11 shows that *Lactobacillus plantarum* BLSCX-2 had the highest viable cell count when sampled at the same time and temperature. Compared to the initial viable cell count, the viable cell count did not change significantly after 1 day of cultivation at 10℃. At 3 days, the viable cell count increased by an order of magnitude, reaching 1.7767 billion CFU / mL, which is comparable to the viable cell count after 1 day of cultivation at 15℃. Afterward, the viable cell count showed a slow increasing trend with prolonged fermentation time. At 5 days of fermentation, the viable cell counts at 10℃ and 15℃ were 2.4 billion CFU / mL and 2.58 billion CFU / mL, respectively, with little difference. This indicates that extending the fermentation time at 10℃ can achieve better fermentation results for this strain.

[0158] 2.3 Effect of temperature on lactic acid content of Lactobacillus plantarum BLSCX-2 fermentation broth

[0159] Table 12 Effect of temperature on lactic acid content (g / L) of Lactobacillus plantarum BLSCX-2 fermentation broth

[0160]

[0161] Table 12 shows that the lactic acid content in the fermentation broth of the four *Lactobacillus plantarum* strains increased with increasing fermentation temperature and fermentation time. For *Lactobacillus plantarum* BLSCX-2, at 10℃, the lactic acid content increased with fermentation time, reaching 16.722 g / L and 19.144 g / L after 5 and 7 days of fermentation, respectively. At 15℃ for 3 days, the lactic acid content was 19.194 g / L, which is comparable to the lactic acid content at 10℃ for 7 days. This indicates that extending the fermentation time under low-temperature conditions can achieve better fermentation results for this *Lactobacillus plantarum* strain.

[0162] Example 6: Effect of low temperature on the fermentation performance of Lactobacillus plantarum BLSCX-2

[0163] 1. Materials and Methods

[0164] 1.1 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.

[0165] 1.2 Test Methods

[0166] The test strains were inoculated into MRS liquid medium and incubated at 37°C overnight. They were then transferred to MRS liquid medium at a 5.0% inoculation rate and incubated at 4°C. Samples were taken at 0, 7, 14, and 21 days to detect the pH value and viable cell count of the fermentation broth.

[0167] 2 Results

[0168] 2.14℃ Effect of pH on Fermentation Broth of Lactobacillus plantarum BLSCX-2

[0169] Table 1. Effect of 34℃ on pH value of Lactobacillus plantarum BLSCX-2 fermentation broth

[0170]

[0171] The results showed that the pH of the fermentation broth decreased after 21 days of culture at 4℃ compared to 14 days, indicating that although the number of viable bacteria did not increase significantly at 4℃, the strains still had metabolic activity.

[0172] 2.24℃ Effect on viable cell count of Lactobacillus plantarum BLSCX-2 fermentation broth

[0173] Table 1. Effect of 44℃ on viable cell count of *Lactobacillus plantarum* BLSCX-2 fermentation broth (×10⁻⁶) 7 CFU / mL

[0174]

[0175] The results showed that the viable count of the four Lactobacillus plantarum strains after culturing at 4°C for 21 days was on the same order of magnitude as the initial viable count.

[0176] Example 7: Effect of sodium chloride on the fermentation performance of Lactobacillus plantarum BLSCX-2

[0177] 1. Materials and Methods

[0178] 1.1 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.

[0179] 1.2 Test Methods

[0180] The test strains were inoculated into MRS liquid medium and incubated at 37°C overnight. Then, they were transferred at a 5.0% inoculum to MRS liquid medium containing different sodium chloride concentrations (0%, 5%, and 8%) and incubated at 37°C for 24 h. The pH value and viable cell count of the fermentation broth were measured, and the lactic acid content in the fermentation supernatant was also measured.

[0181] 2 Results

[0182] Table 15 Effect of sodium chloride on the fermentation performance of Lactobacillus plantarum BLSCX-2

[0183]

[0184] As shown in Table 15, the viable count and lactic acid content of *Lactobacillus plantarum* BLSCX-2 decreased with increasing sodium chloride concentration. At the same sodium chloride concentration, the viable count and lactic acid content of *Lactobacillus plantarum* BLSCX-2 were higher than those of the other three *Lactobacillus plantarum* strains.

[0185] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

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. The use of *Lactobacillus plantarum* as described in claim 1 or the bacterial agent as described in any one of claims 2-3 in the preparation of products with antibacterial capabilities; 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.

Images