Plant lactobacillus and application thereof

Abstract

The invention relates to a plant lactobacillus and application thereof. The preservation number of the plant lactobacillus provided by the invention is GDMCC No.66936, the preservation date is September 8, 2025, and the plant lactobacillus is preserved in Guangdong Microbial Culture Collection Center. Based on whole genome sequencing, it is revealed that the strain contains rich antioxidant related gene clusters (including sodA, katE, gpx, Gor, trxB and the like) and unique metabolic characteristics. Experiments show that the strain has good gastrointestinal tract tolerance and intestinal adhesion capacity, has no hemolytic activity, is sensitive to antibiotics and has high safety. The bacterial cells, the lysate and the fermentation supernatant of the strain all show remarkable scavenging activity on DPPH, ABTS < + > and hydroxyl radicals. The strain can also specifically ferment various matrixes such as grape seed extract and roe extract, and the oxidation resistance of fermentation filtrate is remarkably improved. The strain and metabolites thereof have wide application prospects in the fields of functional food, health care products, cosmetics and the like.

Description

Technical Field

[0001] This invention relates to the fields of microbial engineering and functional food technology, and in particular to a strain of *Lactobacillus plantarum* and its applications. Background Technology

[0002] Oxidative stress is caused by reactive oxygen species (ROS) in the body, such as superoxide anions (O2). - The imbalance between the production and scavenging of hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) leads to the core pathological process of oxidative damage to biological macromolecules (proteins, lipids, nucleic acids), which is closely related to the occurrence and development of aging, neurodegenerative diseases, cardiovascular diseases, diabetes, and various inflammations.

[0003] Currently, the main strategy for dealing with oxidative stress relies on supplementing exogenous antioxidants. Existing antioxidants can be divided into two main categories: (1) Synthetic antioxidants: such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylhydroquinone (TBHQ). Although they have significant antioxidant effects and are inexpensive, more and more studies have shown that they have potential cytotoxicity and carcinogenic risks. Their use in the food and pharmaceutical fields is strictly limited, and consumer acceptance is decreasing. (2) Natural antioxidants: such as vitamin C, vitamin E, polyphenols (such as tea polyphenols), and carotenoids. They are favored because of their high safety. However, they generally have inherent defects such as poor chemical stability, easy photodegradation and oxidation, low bioavailability, and short half-life in vivo, which seriously limit their practical application effects.

[0004] In recent years, microbial antioxidants, especially those derived from recognized as GRAS (Generally Recognized As Safe) lactic acid bacteria (LAB), have become a new research hotspot due to their green, safe, sustainable, and potentially probiotic properties. Lactic acid bacteria exert their antioxidant effects through multiple mechanisms: (i) secreting antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), to directly scavenge reactive oxygen species (ROS); (ii) producing antioxidant metabolites, including extracellular polysaccharides, antioxidant peptides (such as bioactive short peptides produced by fermentation of specific strains), bacteriocins, and organic acids; and (iii) regulating host signaling pathways, such as activating the Nrf2 / Keap1 pathway to upregulate the expression of endogenous antioxidant enzymes, or inhibiting the NF-κB pathway to reduce inflammatory responses.

[0005] However, the development and application of lactic acid bacteria antioxidants still face several serious challenges, which are the technical bottlenecks that this invention aims to solve:

[0006] 1. Limited range of antioxidant activity: Most reported lactic acid bacteria strains and related patents focus on the scavenging of DPPH free radicals, lacking information on ABTS. + Systematic verification of the broad-spectrum and efficient scavenging capabilities of various ROS such as free radicals, hydroxyl radicals (·OH), and superoxide anions (O2-·) is insufficient to address the complex oxidative stress environment in vivo.

[0007] 2. The performance of strains is highly dependent on the substrate and lacks stability: The antioxidant performance of existing strains is often acceptable in simple MRS medium, but once applied to complex natural animal and plant substrates (such as high-protein fish eggs and plant extracts rich in polyphenols), their adaptability and metabolic stability decrease significantly, resulting in unstable antioxidant effects and poor reproducibility of fermentation products, making it difficult to achieve industrial application.

[0008] 3. Vague Mechanism Elucidation and Lack of Genetic Basis: Current research largely focuses on physiological and biochemical phenotypes, with insufficient exploration of the molecular genetic mechanisms underlying the antioxidant capacity of strains. The vast majority of patents do not provide whole-genome sequencing information for the strains, failing to elucidate their potential and unique mechanisms for high-yield antioxidant substances (such as specific EPS and antioxidant peptides) at the gene level. This results in a lack of theoretical guidance and a degree of uncertainty in strain selection and functional optimization.

[0009] 4. Homogeneous application forms and insufficient innovation: Existing technologies are mostly focused on the application development of inactivated bacterial cells (post-biotics) or single fermentation products, and have failed to systematically explore and compare the differentiated advantages of live bacteria, bacterial cells, lysate products, and fermentation supernatant in different application scenarios, thus limiting the scope of application and innovation.

[0010] Therefore, there is an urgent need in this field for a new strain of lactic acid bacteria with a clear genetic background, capable of broadly and efficiently scavenging free radicals at multiple mechanism levels, and stably adaptable to various animal and plant substrates for efficient fermentation and transformation, so as to provide core microbial resources and key technical support for the development of the next generation of efficient, stable and safe natural antioxidants. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention provides a strain of *Lactobacillus plantarum* and its applications. Whole-genome sequencing of this strain reveals that it possesses abundant antioxidant-related gene clusters and unique metabolic characteristics, exhibiting highly efficient and broad-spectrum antioxidant capabilities, excellent gastrointestinal tolerance, and multi-substrate fermentation adaptability. This invention further relates to the application of this strain and its metabolites (cells, lysates, fermentation supernatant, and fermentation filtrate) in the preparation of antioxidant functional foods, health products, cosmetics, and feed additives.

[0012] This invention is achieved through the following technical solution:

[0013] The first objective of this invention is to provide a strain of *Lactiplantibacillus plantarum*, which was deposited on September 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66936.

[0014] A second objective of this invention is to provide a microbial preparation containing the aforementioned *Lactobacillus plantarum*.

[0015] In one embodiment of the present invention, the microbial preparation comprises one or more of the fermentation supernatant of *Lactobacillus plantarum*, lysate, and dead bacteria.

[0016] In one embodiment of the present invention, the microbial preparation is a liquid microbial agent or a solid microbial agent.

[0017] A second objective of this invention is to provide a culture or a processed product thereof containing the aforementioned *Lactobacillus plantarum* or the aforementioned microbial agent.

[0018] A third objective of this invention is to provide an antioxidant preparation comprising the *Lactobacillus plantarum*, the microbial preparation, or a culture or processed form of the microbial agent.

[0019] A fourth objective of this invention is to provide the application of the *Lactobacillus plantarum*, the microbial preparation, the culture of the microbial agent or its processed form, and the antioxidant preparation in the preparation of anti-aging products.

[0020] In one embodiment of the present invention, the product includes food, medicine, health products, feed, or daily chemical products.

[0021] The fifth objective of this invention is to provide a grape seed extract fermentation broth, which is obtained by fermenting grape seed extract using the aforementioned Lactobacillus plantarum.

[0022] The sixth objective of this invention is to provide a fish roe extract fermentation broth, which is obtained by fermenting fish roe using the aforementioned *Lactobacillus plantarum*.

[0023] This invention not only provides the genomic characteristics of *Lactobacillus plantarum* FFLK001 and its genomic composition related to antioxidant activity, but also provides evaluations of the probiotic properties and safety of *Lactobacillus plantarum* FFLK001, including gastrointestinal tolerance, intestinal adhesion, hemolysis, and drug resistance.

[0024] The *Lactobacillus plantarum* FFLK001 described in this invention, after fermentation in various substrates, exhibits filtration activity against DPPH free radicals and ABTS. +Both free radicals and hydroxyl radicals (·OH) exhibited significant and stable scavenging abilities, demonstrating that it has broad-spectrum antioxidant activity and good matrix adaptability.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) High strain safety: Lactobacillus plantarum FFLK001 has no hemolytic activity, is sensitive to common antibiotics, and has good safety.

[0027] (2) Clear genomic characteristics: For the first time, the complete genomic information and genomic composition related to antioxidant activity of this strain were revealed;

[0028] (3) Excellent probiotic properties: This strain has good gastrointestinal tolerance and intestinal adhesion ability;

[0029] (4) Broad-spectrum antioxidant activity: Bacterial cells, lysate and fermentation filtrate have broad-spectrum antioxidant activity against DPPH and ABTS. + Both ·OH radicals have highly efficient scavenging capabilities;

[0030] (5) Broad-spectrum substrate adaptability: This strain can efficiently ferment a variety of animal and plant substrates with minimal loss of functional components and significant enhancement of activity during fermentation;

[0031] (6) Wide range of applications: The resulting products can be used in multiple fields such as functional foods, health products, and cosmetics.

[0032] Preservation of biological materials

[0033] Lactiplantibacillus plantarum FFLK001 was deposited on September 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Its classification name is Lactiplantibacillus plantarum, and its accession number is GDMCC No. 66936. Attached Figure Description

[0034] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0035] Figure 1 This is a colony morphology diagram of Lactiplantibacillus plantarum FFLK001 used in this invention.

[0036] Figure 2This is a cell morphology diagram of Lactiplantibacillus plantarum FFLK001 used in this invention.

[0037] Figure 3 This is a COG classification bar chart of Lactiplantibacillus plantarum FFLK001 in this invention;

[0038] Figure 4 This is a KEGG classification bar chart of Lactiplantibacillus plantarum FFLK001 in this invention;

[0039] Figure 5 This is a bar chart showing the GO classification of Lactiplantibacillus plantarum FFLK001 in this invention;

[0040] Figure 6 The results of the hemolytic evaluation test of Lactiplantibacillus plantarum FFLK001 in this invention;

[0041] Figure 7 The results of the antibacterial test of Lactiplantibacillus plantarum FFLK001 in this invention;

[0042] Figure 8 The hydrophobicity of Lactiplantibacillus plantarum FFLK001 in this invention;

[0043] Figure 9 This relates to the self-aggregation of Lactiplantibacillus plantarum FFLK001 in this invention;

[0044] Figure 10 This is the result of the digestive tract environment resistance test of Lactiplantibacillus plantarum FFLK001 in this invention;

[0045] Figure 11 This invention compares the scavenging rates of DPPH free radicals by *Lactiplantibacillus plantarum* FFLK001 cell cells, lysate products, and fermentation supernatant.

[0046] Figure 12The bacterial cells, lysate products, and fermentation supernatant of *Lactiplantibacillus plantarum* FFLK001 used in this invention are used to evaluate the effects of ABTS. + Comparison of free radical scavenging rates;

[0047] Figure 13 This invention compares the scavenging rates of hydroxyl radicals (·OH) by Lactiplantibacillus plantarum FFLK001 cell cells, lysate products, and fermentation supernatant.

[0048] Figure 14 This invention compares the antioxidant activity of grape seed extract filtrate fermented with Lactiplantibacillus plantarum FFLK001.

[0049] Figure 15 This invention compares the antioxidant activity of the filtrate from the fermented fish roe extract of Lactiplantibacillus plantarum FFLK001. Detailed Implementation

[0050] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0052] The first objective of this invention is to provide a strain of Lactiplantibacillus plantarum FFLK001, which was deposited on September 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with accession number GDMCCNo. 66936.

[0053] Preferably, the culture medium components involved in the activation process of the *Lactiplantibacillus plantarum* FFLK001 strain include: peptone 8-12 g / L, beef meal 6-10 g / L, yeast extract 2-6 g / L, glucose 16-24 g / L, dipotassium hydrogen phosphate 1-3 g / L, diammonium hydrogen citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.02-0.06 g / L, and Tween 80 0.5-2 mL / L, and the pH of the culture medium is 6.0-6.4.

[0054] Preferably, the activation conditions for the bacterial strain are: activation at 37°C for 12–24 hours, until the bacterial suspension density reaches 10. 6 ~10 8 CFU / mL.

[0055] A second objective of this invention is to provide the genomic characteristics of the *Lactobacillus plantarum* FFLK001 and its genomic composition related to antioxidant activity.

[0056] The third objective of this invention is to provide a probiotic characteristic and safety evaluation of the aforementioned *Lactobacillus plantarum* FFLK001, including gastrointestinal tolerance, intestinal adhesion ability, hemolysis, and drug resistance.

[0057] A fourth objective of this invention is to provide the application of *Lactobacillus plantarum* FFLK001 in the preparation of antioxidant agents. The antioxidant agent comprises one or more of the following: fermentation supernatant of the strain, bacterial cells, lysate products, and filtrate products obtained after fermentation of the strain on different substrates.

[0058] The fifth objective of this invention is to provide a method for fermenting *Lactobacillus plantarum* FFLK001 in various substrates, including but not limited to: plant-based and animal-based raw materials such as grape seed extract and fish roe extract.

[0059] The sixth objective of this invention is to provide evaluation results of the antioxidant properties of the fermentation products of *Lactobacillus plantarum* FFLK001. Experiments show that after fermentation on various substrates, the filtrate of this strain exhibits good antioxidant activity against DPPH free radicals and ABTS. + Both free radicals and hydroxyl radicals (·OH) exhibited significant and stable scavenging abilities, demonstrating that it has broad-spectrum antioxidant activity and good matrix adaptability.

[0060] A seventh objective of this invention is to provide a food composition containing one or more of the following: *Lactobacillus plantarum*, antioxidants, and fermentation filtrate.

[0061] An eighth objective of this invention is to provide a functional food composition containing one or more of the following: *Lactobacillus plantarum*, antioxidants, and fermentation filtrate.

[0062] The ninth objective of this invention is to provide a cosmetic containing one or more of the aforementioned *Lactobacillus plantarum*, antioxidants, and fermentation filtrate.

[0063] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0064] The culture medium used in the embodiments of this invention is as follows:

[0065] The components of the MRS liquid culture medium include: peptone 10.0 g / L, beef meal 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, and Tween 80 1.0 mL / L. The pH of the culture medium is 6.2.

[0066] The components of the MRS solid medium include: peptone 10.0 g / L, beef meal 8.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 mL / L, and agar powder 15 g / L. The pH of the medium is 6.2.

[0067] LB liquid medium: Dissolve 10g peptone, 5g yeast extract, and 10g sodium chloride in 950mL deionized water, adjust the pH to 7.0, and bring the volume to 1L.

[0068] Example 1: Isolation and Identification of Microbial Strains

[0069] (1) Strain Isolation: Lactobacillus plantarum FFLK001 (original strain) was isolated from grapes collected in Wuxi City, Jiangsu Province. Specific isolation process: First, 1g of grape sample was taken with sterile tweezers and added to a test tube containing 9mL of sterile physiological saline. The mixture was thoroughly mixed. Then, 100μL of this sample was taken from the test tube using a sterile pipette and added to another test tube containing 900μL of sterile water. The mixture was then thoroughly mixed. This process was repeated to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6Solutions at different dilution ratios.

[0070] Using a sterile pipette, 100 μL of different concentration gradient dilutions were evenly spread onto the surface of an MRS solid medium plate and incubated at 37°C for 24 hours to obtain pure single colonies. Figure 1 The purified lactic acid bacteria were streaked onto MRS solid medium plates and incubated at 37°C for 48 hours. Colony morphology was observed and recorded, and bacterial cell morphology was examined under a light microscope. Figure 2 It was identified as *Lactobacillus plantarum* based on its 16S rRNA gene sequence (as shown in SEQ ID NO. 1).

[0071] The Lactiplantibacillus plantarum FFLK001 strain screened above was deposited on September 8, 2025 at the Guangdong Provincial Center for Microbial Culture Collection (Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou), with accession number GDMCC No. 66936.

[0072] Example 2: Whole Genome Sequencing and Analysis

[0073] (1) Genomic DNA extraction: High-quality genomic DNA of strain FFLK001 was extracted using the CTAB method.

[0074] (2) Sequencing and assembly: Whole genome sequencing was performed using the Illumina NovaSeq and PacBio Sequel platforms, and sequence assembly was performed using Canu software to obtain the complete genome sequence.

[0075] (3) Genome characteristics: The genome size of strain FFLK001 is 3.69Mb, the GC content is 46.2%, and it encodes a total of 3219 genes.

[0076] (4) Functional annotation: Gene function annotation was performed using databases such as COG, KEGG, and GO. Figure 3 , Figure 4 , Figure 5 The strain was found to contain abundant antioxidant-related genes, including:

[0077] Superoxide dismutase (SOD) gene sodA;

[0078] catalase gene katE;

[0079] Glutathione peroxidase gene gpx;

[0080] Glutathione reductase gene gor;

[0081] Thioredoxin reductase gene trxB.

[0082] (5) Secondary metabolic gene clusters: 12 RiPP-like and 40 T3PKS gene clusters were found through antiSMASH analysis.

[0083] (6) Carbohydrate-active enzymes: A total of 74 CAZy enzymes were annotated, including 32 glycoside hydrolases (GH), 15 glycosyltransferases (GT) and 12 carbohydrate esterases (CE), which explains the strain’s adaptability to a variety of substrates.

[0084] Example 3: Evaluation of the strain's drug resistance

[0085] Antibiotic susceptibility of the strains was determined using the disk diffusion method. Logarithmic-phase bacterial suspensions activated to generation 2 were serially diluted (suspension concentration 1×10⁻⁶). 8 (CFU / mL), 0.1 mL was spread onto MRS solid medium. After the plate surface dried, a susceptibility testing disc was placed in the center of each petri dish. The plates were incubated at 37°C for 48 h. The diameter of the inhibition zone around each disc was measured, and the MIC values ​​were compared according to the World Health Organization (WHO) (NCCL S) guidelines. The results are shown in Table 1 below:

[0086] Table 1. Evaluation of drug resistance

[0087]

[0088]

[0089] Note: "S" indicates high sensitivity (inhibition zone diameter > 20.00 mm).

[0090] "I" indicates moderate sensitivity (14 mm < inhibition zone diameter ≤ 20.00 mm)

[0091] “R” indicates drug resistance (inhibition zone diameter ≤ 14.00 mm).

[0092] Analysis showed that this strain exhibited high tolerance to aminoglycoside antibiotics (such as kanamycin and gentamicin), with complete resistance to kanamycin and moderate sensitivity to gentamicin. It also showed high sensitivity to penicillins (such as penicillin), macrolides (such as erythromycin), and chloramphenicol. Furthermore, this strain was also highly sensitive to glycopeptides (vancomycin) and lincosamides (lincomycin).

[0093] Example 4: Evaluation of the hemolytic activity of the strain

[0094] Lactic acid bacteria can be potentially hemolytic bacteria under certain circumstances, which can lead to sepsis in severe cases. Therefore, hemolysis is one of the essential indicators for testing lactic acid bacteria. In this example, the plate test method (GB 4789.11) showed that *Lactobacillus plantarum* FFLK001 did not exhibit hemolysis on any blood agar plates, indicating that this strain has no hemolytic ability. Figure 6 ).

[0095] Example 5: Antibacterial test of the strain

[0096] The activated lactic acid bacteria strain was inoculated into MRS liquid medium at a 1% inoculum and incubated statically at 37°C for 24 hours. The fermentation broth was then centrifuged at 10,000 rpm for 10 minutes at 4°C to collect the supernatant, which was then filtered through a 0.22 μm filter for sterilization. The activated Staphylococcus aureus strain was inoculated into LB liquid medium at a 1% inoculum and incubated statically at 37°C for 24 hours. The concentration was then diluted to 1×10⁻⁶ with physiological saline. 6 CFU / mL, take 100μL 1×10 6 CFU / mL E. coli were spread onto LB agar plates, and 200 μL of the prepared lactic acid bacteria fermentation supernatant was added to Oxford cups. The mixture was diffused at 4°C for 4 h, and then incubated at 37°C for 24 h. The results are as follows: Figure 7 As shown, the fermentation supernatant of *Lactobacillus plantarum* FFLK001 produced a distinct inhibition zone around the Oxford cup, indicating that the metabolites of this strain have a significant inhibitory effect on *Staphylococcus aureus*, and that its secreted bacteriocins or organic acids can effectively inhibit the growth of the pathogen.

[0097] Example 6: Surface characteristics of the strain

[0098] This embodiment systematically evaluates the surface properties of Lactobacillus plantarum FFLK001, including hydrophobicity and self-aggregation, to assess its potential intestinal adhesion ability.

[0099] In the hydrophobicity evaluation, the fermentation broth cultured for 24 hours was collected by centrifugation, resuspended in distilled water, and the OD was adjusted. 600 To 0.8-1.0. Take 3 mL of bacterial suspension and mix it with 1 mL of n-butanol, n-hexane, and ethyl acetate respectively. After shaking for 2 minutes, let it stand at 37℃ for 3 hours. Then measure the absorbance (Ai) of the aqueous phase at 600 nm. Using the initial absorbance (A0) as a reference, calculate the surface hydrophobicity (%) according to the formula "Surface hydrophobicity (%) = (1-Ai / A0) × 100%". The results show that the surface hydrophobicity of this strain in n-butanol, n-hexane, and ethyl acetate are 87.94%, 93.8%, and 92.24%, respectively. Figure 8 ).

[0100] In the self-aggregation evaluation, the bacterial suspension was also adjusted for concentration and incubated at 37°C for 24 hours, and the OD of the supernatant was measured. 600 The self-aggregation rate (%) was calculated using the formula "self-aggregation rate (%) = (1 - Ai / A0) × 100%", and the result was 96.5%. Figure 9 ).

[0101] The two indicators mentioned above—hydrophobicity and self-aggregation rate—are commonly used to indirectly reflect the adhesion potential of lactic acid bacteria to intestinal epithelial cells. Higher values ​​indicate that the strain has a stronger surface adhesion ability. *Lactobacillus plantarum* FFLK001 exhibits high levels in both aspects, indicating that it possesses good intestinal adhesion potential, which may have important physiological significance for this strain to colonize the host gut, maintain microbiota balance, and promote health.

[0102] Example 7: Resistance of the strain to the digestive tract environment

[0103] This embodiment evaluates the digestive tolerance of *Lactobacillus plantarum* FFLK001 by simulating the human gastrointestinal environment. First, artificial gastric and intestinal fluids were prepared with the following compositions: the artificial gastric fluid contained 0.24 g / L KH₂PO₄, 1.44 g / L Na₂HPO₄, 8.00 g / L NaCl, 0.20 g / L KCl, and 3.00 g / L pepsin, with the pH adjusted to 2.5 using 0.1 mol / L HCl, and sterilized through a 0.22 μm filter membrane; the artificial intestinal fluid contained 0.24 g / L KH₂PO₄, 1.44 g / L Na₂HPO₄, 8.00 g / L NaCl, 0.20 g / L KCl, 1.00 g / L trypsin, and 18.00 g / L porcine bile salts, with the pH adjusted to 8.0 using 0.1 mol / L NaOH, and also sterilized through a 0.22 μm filter membrane.

[0104] Activated *Lactobacillus plantarum* FFLK001 was inoculated at a 5% inoculum size into fermentation medium 1 and cultured at pH 6.5 and 37°C for 24 hours to obtain the fermentation broth. This fermentation broth was then inoculated into artificial gastric fluid at a volume ratio of 1:10 and incubated statically at 37°C. Samples were taken at 0, 1, 2, and 3 hours for dilution, spread, and counting. The bacterial suspension treated with gastric fluid for 3 hours was then transferred to artificial intestinal fluid at a 1:10 ratio and incubated statically at 37°C. Samples were taken at 4, 5, 6, 7, and 8 hours to determine the viable cell count (CFU / mL). Finally, the viable cell count was calculated using the common logarithm (lg(CFU / mL)) and analyzed graphically.

[0105] The measurement results are as follows Figure 10As shown, *Lactobacillus plantarum* FFLK001 exhibited good tolerance in a simulated gastrointestinal environment. After 3 hours of treatment in simulated gastric fluid (pH 2.5), the viable bacterial count decreased from an initial approximately 10.92 lg (CFU / mL) to 9.35 lg (CFU / mL); subsequently, after 5 hours of treatment in simulated intestinal fluid (total time 8 hours), the viable bacterial count further decreased slowly to 7.22 lg (CFU / mL). Although the overall bacterial count decreased, the viable bacterial count remained at around 10 throughout the entire process. 7 A concentration of CFU / mL or higher indicates that the strain can effectively tolerate gastric acid and bile salt environments, and has the potential to survive in the digestive tract and exert beneficial functions.

[0106] Example 8: Preparation of *Lactobacillus plantarum* FFLK001 cells, lysate, and fermentation supernatant

[0107] Three samples of strain FFLK001 were prepared: ① Bacterial cells: the bacterial cells were collected by centrifugation, washed with PBS, and resuspended at 10 μL. 9 CFU / mL; ② Lysate: The bacterial cells were treated with lysozyme at 37℃ for 30 min, then sonicated (400W, ice bath, 5s working, 5s rest, total time 30 min), and centrifuged at 10000r / min for 10 min to collect the supernatant; ③ Fermentation supernatant: The fermentation broth was centrifuged at 10000r / min for 10 min, and the supernatant was filtered through a 0.22μm filter membrane.

[0108] Comparative Example 1: Preparation of Lactobacillus rhamnosus LGG cells, lysate and fermentation supernatant

[0109] Preparation of three samples of Lactobacillus rhamnosus LGG: ① Bacterial cells: Collect bacterial cells by centrifugation, wash with PBS and resuspend to 10. 9 CFU / mL; ② Lysate: The bacterial cells were treated with lysozyme at 37℃ for 30 min, then sonicated (400W, ice bath, 5s working, 5s rest, total time 30 min), and centrifuged at 10000r / min for 10 min to collect the supernatant; ③ Fermentation supernatant: The fermentation broth was centrifuged at 10000r / min for 10 min, and the supernatant was filtered through a 0.22μm filter membrane.

[0110] Example 9: Determination of DPPH free radical scavenging rate

[0111] DPPH free radical scavenging rate determination: Mix 2 mL of sample with 2 mL of 0.1 mmol / L DPPH ethanol solution, vortex to mix, and react in the dark for 30 min. Measure the absorbance at 517 nm. Use anhydrous ethanol instead of DPPH solution as the sample background control, and use anhydrous ethanol instead of the sample as the blank control. The DPPH free radical scavenging rate is calculated as follows: Scavenging rate (%) = [1 - (As - Ab) / Ac] × 100%, where As is the sample absorbance, Ab is the sample background absorbance, and Ac is the blank control absorbance.

[0112] The measurement results are as follows Figure 11 As shown, the scavenging rates of DPPH free radicals by *Lactobacillus rhamnosus* LGG fermentation supernatant, bacterial cells, and lysate were 93.23%, 62.34%, and 31.23%, respectively. The scavenging rates of DPPH free radicals by *Lactobacillus plantarum* FFLK001 fermentation supernatant, bacterial cells, and lysate were 97.5%, 87.65%, and 76.09%, respectively. This indicates that the extracellular metabolites secreted by *Lactobacillus plantarum* FFLK001 have significant antioxidant activity, while the lysate exhibits the strongest scavenging ability, suggesting that intracellular antioxidants are fully released after cell disruption and have good application potential in inhibiting free radicals.

[0113] Example 10: ABTS + Free radical scavenging rate determination

[0114] ABTS + Preparation of free radical working solution: Mix 7 mmol / L LABTS solution with an equal volume of 2.45 mmol / L potassium persulfate solution, and react at room temperature in the dark for 12-16 h to generate ABTS. + Stock solution. Dilute with PBS to an absorbance of 0.70 ± 0.02 at 734 nm before use.

[0115] Assay method: Take 0.2 mL of sample and 2.8 mL of ABTS + After mixing the working solutions and vortexing to ensure homogeneity, react in the dark for 6 minutes, then measure the absorbance at 734 nm. Use PBS as a blank control instead of the sample. ABTS + The formula for calculating the free radical scavenging rate is: Scavenging rate (%) = [1-(As-Ab) / Ac]×100%, where As is the absorbance of the sample, Ab is the background absorbance of the sample, and Ac is the absorbance of the blank control.

[0116] The measurement results are as follows Figure 12 As shown: Fermentation supernatant, bacterial cells, and lysate products of *Lactobacillus plantarum* FFLK001 against ABTS +The free radical scavenging rates were 97.61%, 72.42%, and 78.31%, respectively; while the scavenging supernatant, bacterial cells, and lysate of *Lactobacillus rhamnosus* LGG fermentation supernatant and lysate showed positive effects on ABTS. + The free radical scavenging rates were 82.61%, 23.23%, and 42.32%, respectively. This indicates that the extracellular metabolites secreted by *Lactobacillus plantarum* FFLK001 possess significant antioxidant activity in ABTS. + It exhibits the strongest scavenging ability in the free radical scavenging model, further demonstrating that the release of its intracellular antioxidants after lysis can effectively enhance antioxidant performance and has good application potential in inhibiting free radicals.

[0117] Example 11: Determination of scavenging rate of hydroxyl radicals (·OH)

[0118] The Fenton reaction system was used: 1 mL of sample was taken, and 1 mL of 9 mmol / L FeSO4 solution, 1 mL of 9 mmol / L salicylic acid-ethanol solution, and 1 mL of 8.8 mmol / L H2O2 solution were added sequentially. After vortexing and mixing, the mixture was reacted in a water bath at 37℃ for 30 min, and the absorbance was measured at a wavelength of 510 nm. Distilled water was used instead of H2O2 as the sample background control, and distilled water was used instead of the sample as the blank control. The hydroxyl radical scavenging rate was calculated as follows: Scavenging rate (%) = [1 - (As - Ab) / Ac] × 100%, where As is the sample absorbance, Ab is the sample background absorbance, and Ac is the blank control absorbance.

[0119] The measurement results are as follows Figure 13 As shown, the scavenging rates of hydroxyl radicals by *Lactobacillus plantarum* FFLK001 fermentation supernatant, bacterial cells, and lysate were 67.61%, 67.93%, and 77.21%, respectively; while the scavenging rates of hydroxyl radicals by *Lactobacillus rhamnosus* LGG fermentation supernatant, bacterial cells, and lysate were 10.32%, 23.23%, and 25.32%, respectively. These results indicate that all three samples of FFLK001 possess good hydroxyl radical scavenging capabilities.

[0120] Example 14: Preparation of filtrate from grape seed extract fermented by Lactobacillus plantarum FFLK001

[0121] This embodiment is carried out according to the following steps:

[0122] (1) Take 20L of grape seed extract aqueous solution and add 20L of MRS solid culture medium, and mix well.

[0123] (2) Place the above mixture in a fermenter and sterilize at 115°C for 20 minutes. After sterilization, allow it to cool naturally.

[0124] (3) Inoculate Lactiplantibacillusplantarum FFLK001 at a 2% inoculation rate into MRS liquid medium and culture at 37°C.

[0125] (4) Inoculate with fermentation bacteria and ferment at 37℃ and 100r / min for 2 days. The inoculation amount of the bacteria is 2% of the material.

[0126] (5) After fermentation, transfer the fermentation liquid to another container for testing.

[0127] The above steps are strictly required to ensure that all processes are carried out under sterile conditions.

[0128] Comparative Example 2: Preparation of filtrate from grape seed extract fermented with Lactobacillus plantarum FBL002

[0129] This comparison is conducted according to the following steps:

[0130] (1) Take 20L of grape seed extract aqueous solution and add 20L of MRS solid culture medium, and mix well.

[0131] (2) Place the above mixture in a fermenter and sterilize at 115°C for 20 minutes. After sterilization, allow it to cool naturally.

[0132] (3) Inoculate Lactiplantibacillusplantarum FBL002 at a 2% inoculation rate into MRS liquid medium and culture at 37°C.

[0133] (4) Inoculate with fermentation bacteria and ferment at 37℃ and 100r / min for 2 days. The inoculation amount of the bacteria is 2% of the material.

[0134] (5) After fermentation, transfer the fermentation liquid to another container for testing.

[0135] The above steps are strictly required to ensure that all processes are carried out under sterile conditions. Among them, Lactiplantibacillus plantarum FBL002 is disclosed in Chinese patent application number 2025104701564.

[0136] Comparative Example 3: Preparation of filtrate from Lactobacillus rhamnosus LGG fermented grape seed extract

[0137] This comparison is conducted according to the following steps:

[0138] (1) Take 20L of grape seed extract aqueous solution and add 20L of MRS solid culture medium, and mix well.

[0139] (2) Place the above mixture in a fermenter and sterilize at 115°C for 20 minutes. After sterilization, allow it to cool naturally.

[0140] (3) Inoculate Lactobacillus rhamnosus LGG strain into MRS liquid medium and culture at 37°C.

[0141] (4) Inoculate with fermentation bacteria and ferment at 37℃ and 100r / min for 2 days. The inoculation amount of the bacteria is 2% of the material.

[0142] (5) After fermentation, transfer the fermentation liquid to another container for testing.

[0143] The above steps are strictly required to ensure that all processes are carried out under sterile conditions.

[0144] Example 15: Determination of antioxidant activity of grape seed extract filtrate fermented by strain

[0145] Sample processing and determination were performed according to Examples 9, 10, and 11.

[0146] Antioxidant activity assay results are as follows Figure 14 As shown, the filtrate of FFLK001 fermented grape seed extract exhibits superior antioxidant properties compared to other antioxidant strains.

[0147] Example 16: Preparation of filtrate from fish roe extract fermented by Lactobacillus plantarum FFLK001

[0148] Fresh cod eggs were selected as raw material. After pretreatment, low-temperature extraction was performed using a pre-cooled 70% ethanol solution, with antioxidants added during the process to protect the active ingredients. The extract was then defatted and concentrated to obtain a crude active extract of the fish eggs. Subsequently, a fermentation basal culture medium was prepared, containing 40% (v / v) of the fish egg extract stock solution, 3.0% trehalose, 1.5% glucose, 0.8% yeast extract, 0.2% potassium dihydrogen phosphate, and 0.05% magnesium sulfate. The pH was adjusted to 6.5±0.1, and the medium was sterilized at 115℃ for 15 minutes and then cooled to 37℃.

[0149] Aseptic inoculation with 5% (v / v) Lactobacillus plantarum FFLK001 seed culture (viable count ≥1×10⁻⁶) 9 The fish eggs were incubated at 37°C for 48 hours (CFU / mL). After fermentation, the fermentation broth was centrifuged at 10,000 r / min for 15 minutes at 4°C. The supernatant was collected and filtered through a 0.22 μm filter membrane to remove bacteria, yielding the fish egg fermentation filtrate.

[0150] Comparative Example 4: Preparation of filtrate from fish roe extract fermented with Lactobacillus plantarum FBL002

[0151] This comparative example is similar to Example 16, except that: 5% (v / v) *Lactobacillus plantarum* FBL002 seed culture (viable count ≥ 1 × 10⁻⁶) was aseptically inoculated. 9 The fish eggs were incubated at 37°C for 48 hours (CFU / mL). After fermentation, the fermentation broth was centrifuged at 10,000 r / min for 15 minutes at 4°C. The supernatant was collected and filtered through a 0.22 μm filter membrane to remove bacteria, yielding the fish egg fermentation filtrate.

[0152] Comparative Example 5: Preparation of filtrate from Lactobacillus rhamnosus LGG fermented fish roe extract

[0153] This comparative example is similar to Example 16, except that: aseptic inoculation with 5% (v / v) Lactobacillus rhamnosus LGG seed culture (viable count ≥1×10⁻⁶) was performed. 9 The fish eggs were incubated at 37°C for 48 hours (CFU / mL). After fermentation, the fermentation broth was centrifuged at 10,000 r / min for 15 minutes at 4°C. The supernatant was collected and filtered through a 0.22 μm filter membrane to remove bacteria, yielding the fish egg fermentation filtrate.

[0154] Example 17: Determination of antioxidant activity of fermented fish roe extract filtrate

[0155] Sample processing and determination were performed according to Examples 9, 10, and 11.

[0156] The antioxidant activity determination results of Example 16, Comparative Example 4, and Comparative Example 5 are as follows: Figure 15 As shown, compared with other antioxidant strains, the filtrate of FFLK001 fermented fish egg extract exhibits excellent antioxidant properties.

[0157] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A strain of *Lactiplantibacillus plantarum*, characterized in that, The *Lactobacillus plantarum* was deposited on September 8, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNo. 66936.

2. A microbial preparation containing the *Lactobacillus plantarum* as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation includes one or more of the following: fermentation supernatant of *Lactobacillus plantarum*, lysate, and dead bacteria.

4. The microbial preparation according to claim 2, characterized in that, The microbial preparation is a liquid or solid microbial agent.

5. A culture or a processed product thereof containing the *Lactobacillus plantarum* as described in claim 1 or the microbial agent as described in any one of claims 2-4.

6. An antioxidant preparation, characterized in that, A culture or processed product thereof comprising the *Lactobacillus plantarum* of claim 1, the microbial preparation of any one of claims 2-4, or the microbial agent of claim 5.

7. The use of the *Lactobacillus plantarum* of claim 1, the microbial preparation of any one of claims 2-4, the culture of the microbial agent of claim 5 or its processed product, and the antioxidant preparation of claim 6 in the preparation of anti-aging products.

8. The application according to claim 7, characterized in that, The products include food, medicine, health products, feed, or daily chemical products.

9. A grape seed extract fermentation broth, characterized in that, It is obtained by fermentation of grape seed extract using the Lactobacillus plantarum described in claim 1.

10. A fermentation broth of fish roe extract, characterized in that, It is obtained by fermentation of fish eggs using the Lactobacillus plantarum described in claim 1.

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