Plantarum imau40001 and application thereof

Abstract

The application discloses a plant lactobacillus IMAU40001 and application thereof, and belongs to the technical field of probiotics. Lactiplantibacillus plantarum The application provides a new plant lactobacillus with Latin name of IMAU40001, which has a significant antioxidant effect; has good gastric juice, intestinal juice and bile salt tolerance and intestinal adhesion and colonization capacity, can enter the human intestinal tract in a live state, and has a probiotic effect; and through compounding with commercial fermentation agents to prepare fermented milk, the acid production rate, the number of live bacteria and the gel structure stability of the fermented milk can be significantly improved, a large amount of phosphatidylcholine, phosphatidylinositol and other beneficial metabolites can be produced, 20 kinds of significant difference metabolites including amino acids, peptides and organic acids are produced, and the flavor and probiotic potential of the fermented milk are improved.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, and in particular to a strain of Lactobacillus plantarum IMAU40001 and its applications. Background Technology

[0002] Lactobacillus plantarum is an important beneficial microorganism in the human gut, playing a crucial role in maintaining intestinal microecological balance and promoting host health. Numerous studies have confirmed that probiotics not only regulate gut microbiota, enhance immunity, and lower cholesterol, but also possess significant antioxidant activity, showing broad application prospects in the food industry and pharmaceutical and health care fields.

[0003] In recent years, with the in-depth research on the link between oxidative stress and various chronic diseases, *Lactobacillus plantarum* has received widespread attention for its ability to scavenge free radicals and alleviate oxidative damage. Preclinical studies and in vitro experiments have shown that *Lactobacillus plantarum* can effectively reduce lipid peroxidation and plays a positive role in enhancing the body's antioxidant defense system, delaying aging, and preventing atherosclerosis, diabetes, and inflammatory bowel disease.

[0004] Oxidative stress is typically triggered by factors such as environmental pollution, unhealthy diets, and mental stress, leading to an excessive accumulation of reactive oxygen species in the body. This disrupts the balance between the oxidative and antioxidant systems, resulting in cell damage and metabolic disorders. Statistics show that oxidative stress-related diseases have become a significant global health issue. Therefore, developing probiotics and their fermented foods with natural antioxidant functions is of great practical importance for improving population health. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of *Lactobacillus plantarum* IMAU40001 and its applications, so as to provide a new strain with richer functions and better effects.

[0006] To achieve the above objectives, the present invention provides a strain of *Lactobacillus plantarum* IMAU40001, whose Latin name is... Lactiplantibacillus plantarum IMAU40001, taxonomically named Lactiplantibacillus plantarum It was deposited at the China Center for Type Culture Collection on December 10, 2025, with accession number CCTCC NO:M 20252835.

[0007] The cultivation and / or activation method of *Lactobacillus plantarum* IMAU40001 as described above uses MRS solid medium or MRS liquid medium; after streaking on MRS solid medium or inoculating into MRS liquid medium, it is cultured at 37°C under facultative anaerobic conditions.

[0008] The application of *Lactobacillus plantarum* IMAU40001 as described above in the preparation of fermented dairy products.

[0009] The application of *Lactobacillus plantarum* IMAU40001 as described above in the preparation of probiotic agents.

[0010] Preferably, the probiotic preparation includes *Lactobacillus plantarum* IMAU40001; the viable count of *Lactobacillus plantarum* IMAU40001 in the probiotic preparation is not less than 5.0 × 10⁻⁶. 6 CFU / mL.

[0011] The application of *Lactobacillus plantarum* IMAU40001 as described above in the preparation of antioxidant agents.

[0012] Preferably, the antioxidant has the ability to scavenge DPPH free radicals and hydroxyl free radicals.

[0013] The application of the secondary metabolites of *Lactobacillus plantarum* IMAU40001, as described above, in the preparation of antioxidant agents.

[0014] Preferably, the secondary metabolites include lactic acid, pantothenic acid, adenosine, flavin mononucleotide, phosphatidylcholine, and phosphatidylinositol.

[0015] Preferably, the antioxidant has the ability to scavenge DPPH free radicals and hydroxyl free radicals. Therefore, the specific technical effects of the *Lactobacillus plantarum* strain IMAU40001 and its applications provided by this invention are as follows: (1) This invention provides a novel plant lactobacillus strain, whose Latin name is Lactiplantibacillus plantarum IMAU40001, taxonomically named Lactiplantibacillus plantarum It was deposited at the China Center for Type Culture Collection on December 10, 2025, with accession number CCTCC NO: M 20252835; (2) The *Lactobacillus plantarum* IMAU40001 provided by this invention has significant antioxidant effects. Its ability to scavenge DPPH free radicals and hydroxyl free radicals is comparable to that of the LGG strain. It can be used to develop fermented milk or other functional foods / drinks with antioxidant functions. It also has good tolerance to gastric juice, intestinal juice and bile salts. After being taken or consumed, it can enter the human intestine in a live state. Moreover, this strain can effectively adhere to and colonize in the intestine, exert probiotic effects, and can be used to prepare probiotic preparations. (3) The plant lactobacillus IMAU40001 provided by the present invention can be used to prepare fermented dairy products. By compounding with commercial starter cultures, it can significantly improve the physicochemical properties and storage quality of fermented milk, such as acid production rate, viable count, gel structure stability, etc. (4) During the preparation of fermented milk by combining the plant lactobacillus IMAU40001 provided by the present invention with commercial starter, a large number of beneficial metabolites such as phosphatidylcholine and phosphatidylinositol can be produced. Moreover, before and after storage, 20 kinds of significantly different metabolites such as amino acids, peptides and organic acids are produced through pathways such as folic acid-mediated one-carbon unit metabolism, pantothenic acid and coenzyme A biosynthesis metabolism, and riboflavin metabolism, which enhances the flavor and probiotic potential of fermented milk.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a photograph of bacterial colonies from Example 1 of the present invention; Figure 2 The Gram staining results are shown in Example 1 of this invention. Figure 3 The results of the bile salt tolerance test in Example 4 of this invention; Figure 4 This is the genome circular map in Example 5 of the present invention; Figure 5 This is a classification diagram of KEGG function annotations in Embodiment 5 of the present invention; Figure 6 This is a classification diagram of GO function annotations in Embodiment 5 of the present invention; Figure 7 This is a classification diagram of COG function annotations in Embodiment 5 of the present invention; Figure 8 The results of the physicochemical property analysis of fermented milk in Example 7 of the present invention are shown; where (a) is hardness, (b) is consistency, (c) is cohesion, and (d) is viscosity index; A is experimental group A, B is experimental group B; C is experimental group C; and D is experimental group D. Figure 9 The results of pH and titration acidity determination in Example 7 of this invention are shown; where (a) is pH, (b) is titration acidity; A is experimental group A, B is experimental group B; C is experimental group C; D is experimental group D; Figure 10 The results of the fermented milk storage quality analysis in Example 7 of the present invention are shown; where (a) is the number of viable bacteria during storage, (b) is the sensory evaluation during storage; A is experimental group A, B is experimental group B; C is experimental group C; D is experimental group D; Figure 11 The results of metabolite analysis of fermented milk before and after storage in Example 7 of this invention are shown; where (a) is the principal component analysis diagram, (b) is the heatmap cluster analysis diagram, and (c) is the metabolic pathway analysis diagram. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0021] Experimental methods not specified in the embodiments were measured according to national standards. Experimental instruments, equipment and reagents not specified in the embodiments were obtained through commercial means. Unless otherwise defined or stated, all professional and scientific terms used in this invention have the same meaning as those skilled in the art. The method for detecting the number of viable bacteria in the following examples is as follows: the national standard GB 4789.35-2016 Food Safety Standard - Microbiological Testing of Food - Lactic Acid Bacteria Detection.

[0022] Example 1 The basic characteristics of Lactobacillus plantarum IMAU40001 are as follows: Lactobacillus plantarum ( Lactiplantibacillus plantarum IMAU40001, taxonomically named Lactiplantibacillus plantarum It was deposited at the China Center for Type Culture Collection on December 10, 2025, with accession number CCTCC NO: M 20252835; Latin name: Lactobacillus plantarum IMAU40001 is located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0023] Colony images of *Lactobacillus plantarum* IMAU40001 cultured on MRS solid medium for 24 hours are shown below. Figure 1 As shown, the colonies are round and raised with neat edges, smooth and moist surfaces, and are milky white to grayish white in color, with a diameter between 2 and 3 mm.

[0024] Single colonies of bacteria that are dispersed and growing well were Gram-stained and observed under a light microscope. The results are as follows. Figure 2 As shown, the bacteria are Gram-positive, with short rod-shaped cells of relatively uniform thickness. They are arranged in single, chain-like, uniformly dispersed, or clustered forms, without flagella or cilia, and do not move.

[0025] Example 2 The preservation and freeze-dried preparation of *Lactobacillus plantarum* IMAU40001 are detailed below: (1) Preservation.

[0026] Using an inoculation loop, a single *Lactobacillus plantarum* IMAU40001 colony from Example 1 was picked and inoculated into a 5 mL liquid MRS test tube. The culture was then incubated anaerobically (in an anaerobic bag) at 37°C for 24 h. After two passages, the culture was inoculated at a rate of 2% into a 50 mL centrifuge tube (containing 20 mL of MRS liquid medium) for expansion culture. After 24 h of culture, the tube was centrifuged (5000 g, 5 min), the supernatant was discarded, and the bacterial sludge was washed twice with sterile PBS solution. The supernatant was then discarded, and 5 mL of skim milk protectant was added to the bacterial sludge. The bacterial sludge and skim milk protectant were thoroughly mixed and then dispensed into cryovials and stored at -80°C for subsequent experiments.

[0027] (2) Preparation of freeze-dried bacterial powder.

[0028] Using the method in (1), the preserved *Lactobacillus plantarum* was activated and passaged three times in MRS liquid medium at a volume ratio of 2% (v / v). The culture was streaked twice and cultured in liquid medium for 24 h and solid medium for 48 h at 37 °C. The culture was then inoculated into a centrifuge cup containing 700 mL of liquid MRS medium at a volume ratio of 2% and cultured at 37 °C for 24 h. After centrifugation at 5000 × g for 10 min, the culture was washed twice with sterilized physiological saline at 0.85% (w / v) and centrifuged at 5000 × g for 10 min. The culture was then mixed with the bacterial powder protectant at a volume ratio of 1:1 and placed in a sterilized glass dish. The mixture was then freeze-dried in a freeze dryer for 48 h. After being ground into powder, the culture was placed in 50 mL centrifuge tubes and stored at -20 °C for later use.

[0029] Example 3 The free radical scavenging ability of *Lactobacillus plantarum* IMAU40001 was investigated, as follows: (1) DPPH scavenging ability. The strain was activated for two generations (activation conditions: 37℃, anaerobic static culture for 24h, then inoculated into 5mL MRS medium at a 2% inoculum, cultured at 37℃ for 48h to obtain fermentation broth, centrifuged at 5000r / min for 10min, and the supernatant (fermentation supernatant) was collected. 1mL (sample to be tested) was taken and mixed with an equal volume of 0.2mmol / L DPPH-ethanol, shaken for 1min, and allowed to react in the dark at room temperature for 0.5h. After centrifugation at 5000r / min for 10min, the absorbance value A1 of the supernatant was measured at 517nm. An equal volume of anhydrous ethanol was used as a control A0 instead of the sample solution, and an equal volume of anhydrous ethanol was used as a blank A2 instead of DPPH-ethanol. Vitamin C and LGG strains were used as positive controls. The DPPH scavenging rate was calculated using the following formula I, and the results are shown in Table 1.

[0030] DPPH free clearance rate = A0 - (A1 - A2)A0 × 100% (Equation I).

[0031] Table 1 Statistical results of DPPH free radical scavenging rate

[0032] (2) Scavenging ability of hydroxyl radicals. The scavenging ability of fermentation supernatant of Lactobacillus plantarum IMAU40001 (prepared in the same way as in (1)) and bacterial suspension (the part remaining after taking off the fermentation supernatant in (1)) was determined using a hydroxyl radical scavenging ability test kit. The hydroxyl radical scavenging rate was calculated using the following formula II. LGG strain was used as a control. The results are shown in Table 2.

[0033] Hydroxyl radical scavenging rate D% = (A2-A1) ÷ (A0-A1) Formula (Equation II).

[0034] Where A2 represents the absorbance value of the test tube, A1 represents the absorbance value of the control tube, and A0 represents the absorbance value of the blank tube. All of the above are the absorbance values ​​of the sample to be tested at 536 nm.

[0035] Table 2 Statistical results of scavenging rate of hydroxyl radicals

[0036] Lactobacillus plantarum IMAU40001 exhibits a DPPH radical scavenging rate of 70% and a hydroxyl radical scavenging rate of 29%, demonstrating good antioxidant capacity.

[0037] Example 4 The environmental tolerance of *Lactobacillus plantarum* IMAU40001 was investigated, as follows: (1) Tolerance to bile salts.

[0038] After activating the *Lactobacillus plantarum* strain IMAU40001 for two generations (the activation method was the same as in Example 3 (1)), the bacterial culture was inoculated at a 3% inoculum into MRS liquid medium containing 0.3% ox bile salts and cultured at 37°C for 13 hours. Every hour, the bacterial culture was sampled to determine the viable cell count (OD was measured using a UV spectrophotometer). 600 The method of using bile salt tolerance to determine the bile salt tolerance of the tested strains was used, with LGG strain as the control, and three replicates were set for each group.

[0039] The results are as follows Figure 3 As shown, Lactobacillus plantarum IMAU40001 exhibited salt tolerance comparable to the reference strain LGG throughout its entire logarithmic growth phase to stationary phase, providing potential advantages for its application in fermented foods (such as kimchi and pickled products) and in the high-salt intestinal microenvironment.

[0040] (2) Carbohydrate utilization experiment.

[0041] Centrifuge the third-generation strain (5000g, 10min), wash twice with sterile PBS, and resuspend the precipitate in 1mL of API50 CHL medium to prepare a high-concentration bacterial suspension. Open the 5mL ampoule of suspension base and add the above concentrated bacterial suspension dropwise until the turbidity is consistent with the No. 2 McFarland turbidimetric tube provided with the kit. Follow the API50 CHL instructions. Add sterile distilled water to the small groove of the incubator to create a warm temperature. Place the test strip in the incubator tray, add bacterial solution to the test strip, and seal the tube opening with liquid paraffin. Incubate the incubator at 37℃ for 48h and observe the color change of the API50 CHL card. Record the results as shown in Table 3. IMAU40001 can utilize 26 carbohydrates: L-arabinose, D-ribose, D-galactose, D-glucose, D-fructose, D-mannitol, mannitol, sorbitol, methyl-α-D-mannopyranoside, methyl-α-D-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, ferric citrate of aesculin, salicin, D-cellobiose, maltose, D-lactose, D-micobiose, D-sucrose, D-trehalose, D-melatonin, D-raffinose, D-gentiobiose, D-thulene, and potassium gluconate.

[0042] The ability to utilize carbon sources is an important indicator of the metabolic activity of lactic acid bacteria, directly affecting their fermentation process and the generation of metabolites. The wide range of carbon source utilization capabilities endows IMAU40001 with extremely strong environmental adaptability, enabling it to proliferate in a milk matrix where lactose is the primary carbon source. Simultaneously, it can utilize trace amounts of oligosaccharides, polysaccharides, and other carbohydrates in milk, providing ample energy and material basis for the strain's metabolism.

[0043] Table 3. Results of carbohydrate fermentation

[0044] (3) Antibiotic susceptibility test. The susceptibility of the strain to antibiotics was determined using the agar plate disk diffusion method. The activated bacterial culture after two generations was diluted with MRS liquid medium to adjust the bacterial concentration to 10. 8 CFU / mL. Take 100 μL of bacterial suspension and spread it evenly on the surface of MRS solid medium. After letting the plate dry for a short time, use sterile forceps to place the drug sensitivity test strip onto the plate. Once the strip touches the agar, do not move it. Invert the plate and incubate anaerobically at 37℃ for 48 h. Measure the diameter of the inhibition zone. The results are shown in Table 4. IMAU40001 is highly sensitive to ampicillin, piperacillin, cefazolin, cefoperazone, florfenicol, imipenem, tetracycline, doxycycline, minocycline, chloramphenicol, trimethoprim-sulfamethoxazole, and colistin. It is moderately sensitive to ceftriaxone, azithromycin, and erythromycin. It is resistant to cefixime.

[0045] Table 4 Results of Antibiotic Susceptibility Testing

[0046] Note: S indicates sensitive, R indicates resistant, and I indicates moderately sensitive. (4) Gastric juice resistance test.

[0047] Take 2 mL of 1 mol / L hydrochloric acid and adjust the pH to 2.0 with distilled water. Dissolve pepsin in 1 g of pepsin per 100 mL of solution, and sterilize through a 0.22 μm filter to obtain artificial gastric fluid. Inoculate the activated bacterial solution into the artificial gastric fluid at a volume of 10%, mix well, and incubate at 37°C. Measure the viable bacterial count at 0 h and 3 h of incubation (OD value measured using a UV spectrophotometer). 600 The method was used, with *Lactobacillus paracasei* PC-01 as a control. Each treatment was repeated three times, and the results are shown in Table 5. In a simulated human gastric juice environment, there was no significant difference in the survival rate of *Lactobacillus plantarum* IMAU40001 and *Lactobacillus paracasei* PC-01. This indicates that IMAU40001 also possesses excellent gastric acid barrier crossing ability, laying the foundation for its application as a potential probiotic strain in functional foods or oral formulations.

[0048] Table 5 Results of gastric juice resistance test

[0049] (5) Intestinal fluid tolerance test. 0.85 g KH₂PO₄ was dissolved in 62.5 L distilled water, and the pH was adjusted to 7.6 with 1 mol / L NaOH. Trypsin was added at a rate of 1 g / 100 mL in a clean bench, and the solution was sterilized using a 0.22 μm filter. 1.0 mL of activated bacterial solution was mixed with 9.0 mL of artificial intestinal fluid and incubated at 37 °C. Viable bacterial counts were measured at 7 and 11 h using the pour method. *Lactobacillus paracasei* PC-01 was used as a control. Each treatment was repeated three times. The results are shown in Table 6. *Lactobacillus plantarum* IMAU40001 and *Lactobacillus paracasei* PC-01 showed the same tolerance level, with no significant difference in viable bacterial count or survival rate. This indicates that IMAU40001 also possesses excellent intestinal environment tolerance, effectively resisting digestive juices and bile salt stress in the small intestine, providing a key guarantee for its successful colonization of the intestine and the exertion of its potential probiotic functions.

[0050] Table 6 Results of intestinal fluid resistance test

[0051] Example 5 The whole genome of Lactobacillus plantarum 40001 was sequenced, as follows: (1) Whole genome sequencing.

[0052] Lactobacillus plantarum 40001 was inoculated into MRS liquid medium (inoculation amount of 2% (v / v)), cultured in an incubator at 37℃ for 24 h, centrifuged at 4000×g for 10 min, and the bacterial cells were collected and sent to a biotechnology company for whole genome sequencing under dry ice conditions.

[0053] Genomic DNA was collected and purified, processed into 15-18kb fragments, and then expanded using G-Tubes to break them into fragments of varying lengths. These fragments underwent end repair and damage repair, and were then ligated with stem-circular sequencing adapters. Failed ligation fragments were removed using exonucleases, and fragments of suitable length were selected using the BluePippin system. Finally, an SMRTBell library for third-generation sequencing was constructed. The library was loaded onto the PacBioRevio sequencing platform. Based on single-molecule real-time sequencing technology, using the zero-mode waveguide principle, fluorescent labels were used to identify different base signals. Circular consensus sequencing was employed to read multiple subreads of the same DNA molecule to obtain the sequence of the template DNA fragment.

[0054] Third-generation HiFireads obtained from PacBioRevio sequencing were assembled using Hifiasm (version: v0.14.2-r315, https: / / github.com / chhylp123 / hifiasm), an assembly software optimized for third-generation sequencing data. Hifiasm uses a string graph algorithm to correct and splice long reads, resulting in high-quality genome assembly. If there are homologous overlap regions exceeding a certain length at both ends of the assembled sequence, they are joined end-to-end to form a circular structure, and one end of the overlapping sequence is removed, ultimately obtaining complete circular sequences of chromosomes and plasmids. Finally, gene prediction was performed on the assembled sequences to obtain the gene locations and sequence information for each sample. Software such as Glimmer, GeneMarkS, Prodigal, and Barrnap were used to predict the types, locations, and sequence information of all RNAs in the genome of each sample. A total of 3292 genes were predicted, with a total length of 3406895 bp, accounting for 84.13% of the total genes and 44.33% of the total genes. Three plasmids were assembled. The specific genomic composition analysis results are shown in Table 7.

[0055] Table 7 Statistical results of genomic component analysis

[0056] (2) Functional gene annotation.

[0057] The obtained genes were compared with databases through homology alignment to obtain gene annotation descriptions. Functional annotations were performed on the predicted CDS using the GO, COG, KEGG, and CAZy databases. Genome circular maps (e.g., Circos software) were then used to construct genome circular maps. Figure 4 As shown in the figure). Gene function annotation shows that IMAU40001 contains a large number of genes involved in metabolism, information storage and processing, and has outstanding metabolic potential in carbohydrate metabolism, amino acid metabolism and cofactor and vitamin metabolism, indicating that the strain has relatively active metabolic activity and strong biosynthetic capacity. Figures 5-7 ).

[0058] (3) Functional gene characteristics of Lactobacillus plantarum IMAU40001.

[0059] Annotation results from the CAZy database showed that strain IMAU40001 had five classes of carbohydrate-active enzyme-encoding genes annotated. Among them, glycosidase (GH)-related genes were the most numerous, with 143; followed by glycosyltransferases (GTs) with 95; carbohydrate binding module (CBM)-related genes with 41; carbohydrate esterases (CEs) with 6; and helper oxidoreductases (AAs) with 3. No polysaccharide lyase (PLs)-related genes were annotated. The high proportion of GH and GT genes indicates that this strain has strong functional potential in glycosidic bond hydrolysis and glycosyltransferase synthesis, which is closely related to its carbohydrate metabolism capacity. For detailed gene annotation results, see [link to documentation]. Figures 5-7 .

[0060] Example 6 The probiotic properties of *Lactobacillus plantarum* IMAU40001 were investigated, as follows: (1) Determination of bacterial adhesion.

[0061] Hydrophobicity determination: The hydrocarbon adhesion method was used. *Lactobacillus plantarum* IMAU40001 was inoculated into MRS liquid medium and continuously activated up to the third generation. After centrifugation at 5000 g / min for 10 min, the bacterial pellet was collected and a bacterial suspension was prepared with sterile physiological saline. The OD value was adjusted to 0.5 ± 0.02. An equal volume of xylene solution was added, and the mixture was vortexed for 1 min. After standing for 1 h, the absorbance of the lower aqueous phase was measured (600 nm). LGG strain was used as a control. Each treatment was repeated three times. Surface hydrophobicity was calculated using Formula III. The results are shown in Table 8.

[0062] Surface hydrophobicity % = (A0-A1) / A0×100 (Equation III).

[0063] Self-aggregation rate determination: A bacterial suspension was prepared, and the OD value was adjusted to 0.5 ± 0.02. The suspension was then incubated at 37℃ for 20 h, and the absorbance of the supernatant was measured (600 nm). LGG strain and VC were used as controls. Each treatment was repeated three times. The self-aggregation rate was calculated using the following formula IV. The results are shown in Table 9.

[0064] Self-cohesion rate % = (A0-A2) / A0×100 (Equation IV).

[0065] The results showed that *Lactobacillus plantarum* IMAU40001 had excellent adhesion, with low hydrophobicity (3.19%) and a self-aggregation rate of 71.86%, indicating that it could effectively adhere to and colonize in the intestine.

[0066] Table 8 Hydrophobicity Evaluation Results

[0067] Table 9. Evaluation Results of Self-Assembly Ability

[0068] Example 7 Fermented milk was prepared using *Lactobacillus plantarum* IMAU40001, as detailed below: Take fresh skim milk, preheat it, add 6.5% white sugar, homogenize it (20MPa), sterilize it at 95℃ for 30 minutes, and then cool it to 42℃. Inoculate the bacterial strain and starter culture (PYS-010) according to the inoculation amount shown in Table 10, mix well, ferment at 37℃ until pH 4.5-4.6, and store at 4℃.

[0069] Table 10. Grouping of Fermented Milk Samples and Inoculation Amount of Strains

[0070] Samples were taken on storage days 1, 7, 14, and 21 for the following parameters to be measured: (1) Determination of physicochemical properties of fermented milk.

[0071] Determination of changes in microrheological parameters of fermented milk: The elastic index, flow index and solid-liquid equilibrium value were measured using an optical microrheometer, and the measurement was automatically stopped when the measurement time was up.

[0072] pH value: After the fermented milk samples were brought to room temperature, the pH value was measured with a pH meter. Each group of samples was measured in parallel three times and the average value was taken.

[0073] Titration of acidity: Weigh 10.0g of fermented milk, add 20mL of distilled water and mix. Titrate with 0.1mol / L NaOH solution until the color turns slightly red or light pink and does not fade within 30s. Record the amount of NaOH used. Calculate TA according to formula V. Perform three parallel determinations and take the average value.

[0074] Titration acidity (°T) = V / M × 100 (Equation V); In the formula: V represents the volume of NaOH solution consumed (mL); M represents the mass of the sample (g).

[0075] Water retention: Weigh 20g of fermented milk sample, let it stand for 2 hours, weigh the filtrate, and calculate the water retention according to formula VI. Repeat the test 3 times for each sample and take the average value.

[0076] Water holding capacity % = (1 - M1 / M2) × 100% (Equation VI); In the formula: M1 represents the mass of the filtrate (g); M2 represents the mass of the sample (g).

[0077] Viscosity index: After the fermented milk was brought to room temperature, the viscosity was measured for 30 seconds using the #4 rotor of the viscometer at 100 r / min. The data was recorded, and each sample was measured in parallel three times and the average value was taken.

[0078] Determination of textural properties of fermented milk: A TTA XT plus texture analyzer was used. The following settings were used: pre-test moving speed 1.5 mm / s, test speed 1.0 mm / s, retraction speed 1.5 mm / s, initial stress 2.0 g, compression degree 20%, compression time 5 s, and travel distance 20 mm. The hardness, cohesiveness, and viscosity of the fermented milk were measured. Each sample was repeated 3 times and the average value was taken.

[0079] (3) Determination of the storage quality of fermented milk.

[0080] Viable count determination: Weigh 25g of sample and mix with 225mL of physiological saline. After shaking for 15min, dilute the mixture and take 1mL to inoculate into MRS solid medium. After anaerobic culture at 37℃ for 48h, count the viable bacteria.

[0081] Sensory evaluation of fermented milk: Ten students in this field scored the milk based on three aspects: color (20 points), aroma (40 points), and texture (40 points).

[0082] (4) Analysis of metabolites of fermented milk before and after storage.

[0083] Metabolite determination: After thawing, methanol and acetonitrile extract were added at a ratio of 1:4 (v / v), vortexed for 30 s, sonicated for 10 min, and allowed to stand at -40℃ for 1 h before centrifugation. The supernatant was then injected. An AB SCIEX-Triple TOF 6600+ system equipped with an Acquity UPLC BEH Amide column was used. Phase A consisted of an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia, and phase B consisted of acetonitrile. The injection volume was 2 μL, and the sample pan temperature was 4℃. Chromatographic separation and detection were performed.

[0084] The results of the physicochemical property analysis of fermented milk are as follows: Figure 8 and Figure 9 As shown, compared with other groups, the hardness, consistency, cohesiveness, and viscosity index of the fermented milk in experimental group B were significantly improved.

[0085] Results of fermented milk storage quality analysis as follows Figure 10 As shown, on day 21 of storage, the viable bacterial count in experimental group AC was significantly higher than that in control group D. P <0.05), the viable bacterial count of AD in the experimental group was 1.13×10 9 CFU / mL, 1.38×10 9 CFU / mL, 1.28×10 9 CFU / mL and 1.01×10 9 CFU / mL. This indicates that the addition of *Lactobacillus plantarum* IMAU40001 can increase the overall viable count of fermented milk during storage and maintain good stability.

[0086] Results of metabolite analysis of fermented milk before and after storage: Figure 11 As shown in Table 11, 20 significantly different metabolites, including amino acids, peptides, and organic acids, were produced before and after storage through pathways such as one-carbon unit metabolism, pantothenic acid and coenzyme A biosynthesis metabolism, and riboflavin metabolism.

[0087] Table 11 Results of differential metabolite analysis before and after fermented milk storage

[0088] Therefore, this invention provides a novel plant lactobacillus strain, in Latin: Lactiplantibacillus plantarum IMAU40001 exhibits significant antioxidant effects; it possesses good tolerance to gastric juice, intestinal juice, and bile salts, as well as the ability to adhere to and colonize the intestines, enabling it to enter the human gut in a live bacterial state and exert probiotic effects; when combined with commercial starter cultures to prepare fermented milk, it can significantly improve the acid production rate, live bacterial count, and gel structure stability of fermented milk, and can also produce a large number of beneficial metabolites such as phosphatidylcholine and phosphatidylinositol, generating 20 significantly different metabolites including amino acids, peptides, and organic acids, thereby enhancing the flavor and probiotic potential of fermented milk.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications, equivalent substitutions, or combinations thereof can still be made to the technical solutions of the present invention, and such modifications, equivalent substitutions, or combinations thereof cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A strain of *Lactobacillus plantarum* IMAU40001, Latin name: Lactiplantibacillus plantarum IMAU40001, taxonomically named Lactiplantibacillus plantarum It was deposited at the China Center for Type Culture Collection on December 10, 2025, with accession number CCTCC NO: M 20252835.

2. The method for culturing and / or activating *Lactobacillus plantarum* IMAU40001 as described in claim 1, characterized in that: Use MRS solid medium or MRS liquid medium; after streaking on MRS solid medium or inoculating into MRS liquid medium, incubate at 37°C under facultative anaerobic conditions.

3. The application of *Lactobacillus plantarum* IMAU40001 as described in claim 1 in the preparation of fermented dairy products.

4. The application of *Lactobacillus plantarum* IMAU40001 as described in claim 1 in the preparation of probiotic agents.

5. The application according to claim 4, characterized in that: The probiotic preparation includes *Lactobacillus plantarum* IMAU40001; the viable count of *Lactobacillus plantarum* IMAU40001 in the probiotic preparation is not less than 5.0 × 10⁻⁶. 6 CFU / mL.

6. The use of *Lactobacillus plantarum* IMAU40001 as described in claim 1 in the preparation of antioxidant agents.

7. The application according to claim 6, characterized in that: The antioxidant has the ability to scavenge DPPH free radicals and hydroxyl free radicals.

8. The use of the secondary metabolites of *Lactobacillus plantarum* IMAU40001 as described in claim 1 in the preparation of antioxidant agents.

9. The application according to claim 8, characterized in that: The secondary metabolites include lactic acid, pantothenic acid, adenosine, flavin mononucleotide, phosphatidylcholine, and phosphatidylinositol.

10. The application according to claim 8, characterized in that: The antioxidant has the ability to scavenge DPPH free radicals and hydroxyl free radicals.

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