Lactobacillus plantarum L.plantarum-1 as well as bacterial liquid and application of lactobacillus plantarum L.plantarum-1

Abstract

The invention relates to the technical field of microbial fermentation, and particularly discloses lactobacillus plantarum L.plantarum 1 and a bacterial liquid and application thereof, the lactobacillus plantarum L.plantarum 1 is preserved in Guangdong Microbial Culture Collection Center on May 9, 2025, the preservation number is GDMCC No.66300, and the lactobacillus plantarum L.plantarum 1 is classified and named as Lactobacillus plantarum. The lactobacillus plantarum L.plantarum-1 provided by the invention is high in safety, the ratio of the intestinal tract thick-wall mycophylum to bacteroides is reduced by fermenting the apple juice, the abundance of Lysinibacillus and Bilophila is reduced, and the obesity is retarded.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a strain of Lactobacillus plantarum. L.plantarum -1 and its bacterial solution and application. Background Art

[0002] Obesity is considered a persistent metabolic disorder resulting from a combination of genetic, biological, environmental, behavioral, sociocultural, and economic factors. It is widely recognized that obesity is a significant risk factor for cardiovascular disease, type 2 diabetes, hypertension, cancer, chronic kidney disease, a range of musculoskeletal disorders, and psychiatric illnesses. This is primarily due to obesity being a systemic, chronic inflammatory state. The obesity epidemic is driven by a combination of fixed factors (genetics, age, and gender) and modifiable factors (high-calorie food intake and reduced physical activity), with diet playing a key role. Therefore, obesity can be prevented by adjusting dietary habits. The search for functional products that can reduce weight and increase the abundance of beneficial bacteria to alleviate obesity is urgent. Microorganisms play a significant role in the fermentation of functional products, and it is essential to identify strains of microorganisms with obesity-alleviating properties. Summary of the Invention

[0003] In order to develop a microorganism to alleviate obesity, the present invention provides a strain of Lactobacillus plantarum L.plantarum -1 and its bacterial liquid and application. The present invention provides Lactobacillus plantarum L.plantarum -1 has the function of alleviating obesity, the plant lactobacillus L.plantarum -1 Fermenting apple juice can improve the structure of intestinal flora and relieve obesity.

[0004] The present invention provides a strain of Lactobacillus plantarum, which was deposited in Guangdong Provincial Microbial Culture Collection Center on May 9, 2025, with a deposit number of GDMCC No.66300 and a classification name of Lactiplantibacillus plantarum .

[0005] Lactobacillus plantarum provided by the present invention L.plantarum -1 has the function of alleviating obesity, the plant lactobacillus L.plantarum -1 Fermenting apple juice can improve intestinal microbial structure and alleviate obesity.

[0006] Lactobacillus plantarum provided by the present invention L.plantarum -1 High safety, by fermenting apple juice to reduce the ratio of intestinal Firmicutes to Bacteroidetes and reduce Lysinibacillus and Bilophila abundance, slowing down obesity.

[0007] The present invention also provides a bacterial solution, which contains the plant lactobacillus L.plantarum -1.

[0008] The present invention also provides a fermented apple juice, which is prepared by the plant lactobacillus L.plantarum -1 fermented. Utilize the plant lactobacillus of the present invention L.plantarum -1 fermented apple juice produces richer flavor substances (sulfides, alcohols, methyls, organic sulfides and nitrogen oxides).

[0009] Furthermore, the preparation process of the fermented apple juice is as follows: Lactobacillus plantarum L.plantarum -1 was inoculated into LB liquid medium for activation culture to obtain bacterial liquid, and then the bacterial liquid was inoculated into MRS broth medium for fermentation to obtain fermentation liquid, and the fermentation liquid was inoculated into apple juice and cultured until the number of viable bacteria was 10 8 CFU / mL or above, a seed solution is obtained, and then the seed solution is inoculated into apple juice, and fermentation is continued to obtain fermented apple juice.

[0010] The present invention also provides a plant lactobacillus L.plantarum -1. Use of the bacterial liquid or the fermented apple juice in the preparation of a product that reduces fertilizer consumption.

[0011] Furthermore, the product has the effect of improving intestinal flora.

[0012] Furthermore, the improvement of intestinal flora is manifested by reducing the ratio of Firmicutes to Bacteroidetes and reducing Lysinibacillus and Bilophila abundance.

[0013] Furthermore, the product is obtained by dissolving the fermented apple juice in PBS and filtering and collecting the filtrate.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Described Lactobacillus plantarum L.plantarum Compared with other plant lactobacilli, Lactobacillus plantarum has low bioamine production ability, no β-hemolytic activity, higher safety, and is suitable for intestinal intervention. L.plantarum -1 Fermented apple juice produces richer flavor substances.

[0015] Lactobacillus plantarum provided by the present invention L.plantarum -1 has the function of alleviating obesity, the plant lactobacillus L.plantarum -1 Fermenting apple juice can improve intestinal microbial structure and alleviate obesity.

[0016] Information on the deposit of biological materials L.plantarum -1, referred to in this application as L.plantarum-1. It has been deposited in Guangdong Provincial Microbiological Culture Collection Center on May 9, 2025, with the deposit number GDMCC No.66300. The deposit address is 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code: 510075, and is classified as Lactiplantibacillus plantarum . BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 For the present invention L.plantarum -1 hemolytic activity of the strain; In the figure, A is the L.plantarum -1 hemolytic activity of the strain; B is the control strain Virgibacillus halodenitricanicans hemolytic activity.

[0019] Figure 2 The growth characteristic curves of different Lactobacillus plantarum and their growth characteristic curves during the fermentation of apple juice; In the figure, A is the growth characteristic curve of four strains of Lactobacillus plantarum in MRS broth medium; B is the growth characteristic curve of four strains of Lactobacillus plantarum during the fermentation of apple juice.

[0020] Figure 3 The electronic nose response results of apple juice fermented by different Lactobacillus plantarum; In the figure, A is a radar chart of the response values ​​of the electronic nose to apple juice fermented by different Lactobacillus plantarum; B is the bar graph of the mean response values ​​of the electronic nose for apple juice fermented by different Lactobacillus plantarum.

[0021] Figure 4 Figure 2 shows the effect of fermented apple juice on the level of intestinal flora, where O represents the obese group and O+FAJ represents the fermented apple juice group; * indicates significant difference; In the figure, A is a heat map of the effect of fermented apple juice on the phylum level of intestinal flora; B is the effect of fermented apple juice on the relative abundance of Firmicutes; C is the effect of fermented apple juice on the relative abundance of Proteobacteria; D is the effect of fermented apple juice on the relative abundance of Bacteroidetes; E is the effect of fermented apple juice on the ratio of Firmicutes / Bacteroidetes.

[0022] Figure 5 The effect of fermented apple juice on the genus level of intestinal flora, where O represents the obesity group, O+FAJ represents the fermented apple juice group, and * indicates significant difference; In the figure, A is the genus-level heat map of the fermented apple juice group and the obesity group; B is the fermented apple juice group and the obese group Escherichia-Shigell aRelative abundance; C is the fermented apple juice group and the obese group Lactobacillus relative abundance; D is the difference between the fermented apple juice group and the obese group Bacteroides relative abundance; E is the difference between the fermented apple juice group and the obese group Fusobacterium relative abundance; F is the fermented apple juice group and the obese group Paeniclostridium relative abundance; G is the fermented apple juice group and the obese group Lysinibacillus relative abundance; H is the difference between the fermented apple juice group and the obese group Phascolarctobacterium relative abundance; I is the fermented apple juice group and the obese group Bilophila relative abundance; J is the fermented apple juice group and the obese group Parabacteroids relative abundance; K is the difference between the fermented apple juice group and the obese group Bifidobacterium relative abundance; L is the fermented apple juice group and the obese group Enterococcus Relative abundance.

[0023] Figure 6 for functional analysis and species network correlation diagrams; The default display in the figure P <0.05; the size of the node in the figure represents the species abundance, and different colors represent different species; the color of the connecting line represents positive and negative correlation, red represents positive correlation, and green represents negative correlation; the thickness of the line represents the size of the correlation coefficient, the thicker the line, the higher the correlation between species; the more lines, the closer the connection between the species and other species; In the figure, A is the MetaCyc pathway heat map; B is the Network analysis diagram. DETAILED DESCRIPTION

[0024] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0025] Example 1: A strain of Lactobacillus plantarum, its bacterial liquid and application.

[0026] 1. Isolation and identification of strains A strain was isolated from naturally fermented yogurt and identified as Lactobacillus plantarum by 16S rDNA. Lactobacillus plantarum. Recorded as L.plantarum- 1.

[0027] 2. Analysis of the ability of Lactobacillus plantarum to produce bioamines, hemolysis, and biofilm Lactobacillus plantarum CGMCC 1.568 was purchased from China General Microbiological Culture Collection Center and was recorded as L.plantarum 1.568; Lactobacillus plantarum CGMCC 1.124, recorded as L.plantarum 1.124; Lactobacillus plantarum CGMCC1.572, recorded as L.plantarum 1.572. The isolated samples were detected L.plantarum- 1 and three commercially available Lactobacillus plantarum strains were compared and analyzed for their ability to produce bioamines, hemolysis and biofilm.

[0028] 1. Detection of bioamine production ability of Lactobacillus plantarum The separation of this embodiment L.plantarum- 1 strain and commercially available strains L.plantarum 1.568, L.plantarum 1.124 and L.plantarum1.572 was inoculated into 5 mL of LB liquid medium and cultured at 37°C for 24 h to obtain the first bacterial suspension. 100 µL of the first bacterial suspension was inoculated into 5 mL of fresh LB liquid medium and cultured at 37°C for 24 h to obtain the second bacterial suspension. To eliminate the influence of inoculum size on bioamine production, this step was repeated three times to obtain the third bacterial suspension. 100 µL of the third bacterial suspension was inoculated into 5 mL of LB liquid medium containing 0.5% (w / v) L-histidine monohydrochloride monohydrate, 0.5% (w / v) L-tyrosine disodium salt monohydrate, 0.5% (w / v) L-ornithine monohydrochloride, and 0.5% (w / v) L-lysine hydrochloride precursors. The culture was incubated at 37°C for 48 h to obtain the culture medium. 1 mL of the culture medium was added to 9 mL of 10% TCA, vortexed, and incubated at 4°C for 2 h. 3000× g Centrifuge at 4°C for 10 min, take 400 µL of the liquid, add 80 µL of 2M sodium hydroxide, 120 µL of saturated sodium bicarbonate and 800 µL of dansyl chloride, and incubate in a 45°C water bath for 40 min; add 50 µL of ammonia water and let stand at room temperature for 30 min; add 550 µL of acetonitrile and 3000× g The solution was centrifuged at 4°C for 5 min, filtered twice through a 0.22 µm filter, and then analyzed by high performance liquid chromatography.

[0029] The results of the bioamine production ability test of different Lactobacillus plantarum strains are shown in Table 1.

[0030] Table 1 Test results of bioamine production ability of different Lactobacillus plantarum Note: Those marked with different lowercase letters indicate significant differences between groups ( P <0.05).

[0031] As shown in Table 1, the four strains of Lactobacillus plantarum did not produce histamine and tyramine, but produced low levels of putrescine and cadaverine. L.plantarum- Strain 1 produced the lowest content of biogenic amines, which means L.plantarum- Strain 1 is safer.

[0032] 2. Qualitative analysis of hemolysis For the β-hemolytic assay, 5% (v / v) sheep blood was added to MRS agar. Colonies were streaked onto the MRS agar plates containing blood using an inoculation loop. The plates were incubated at 37°C for 48 hours and then placed in a 4°C cold shock for 24 hours. The hemolytic activity of the strains was determined by observing the formation of a clear lysis zone around each colony on the MRS agar plates containing blood. Moderately halophilic bacteria ( Virgibacillus hal denitrifyingCGMCC 1.8915) was used as a positive control.

[0033] The results of hemolytic qualitative analysis are as follows Figure 1 As shown, known hemolytic strains Virgibacillus halodenitri canary When grown on MRS agar, a very clear zone forms around the colonies, while the isolated L.plantarum -1 strain did not show β-hemolytic activity. L.plantarum -1 strain is highly safe and can be used as a food fermentation bacterium.

[0034] 3. Analysis of the ability to produce biofilm The formation of biofilm was measured by 96-well plate method: the isolated L.plantarum- 1 strain and commercially available strains L.plantarum 1.568, L.plantarum 1.124 and L.plantarum 1.572 were inoculated into 5 mL LB liquid medium and cultured at 37°C until the bacterial solution OD 600nm was 0.4, and 200 μL of OD 600nm The bacterial solution was added to the well plate at a concentration of 0.4, and sterile LB liquid medium was used as a negative control. Staphylococcus aureus ( Staphylococcus aureus ATCC6538) was used as a positive control and cultured at 37°C for 48 h. Each strain was replicated eight times. The OD value of the bacterial solution was measured using a microplate reader. 600nm Absorbance; discard the bacterial suspension, add 250 μL 1× PBS to each well, wash three times, and completely remove the liquid; add 200 μL anhydrous methanol to each well, fix at room temperature for 15 minutes, and then discard the liquid; place in a 60°C oven with the lid open for 15 minutes; stain with 200 μL 0.1% crystal violet, place at room temperature for 15 minutes, and then discard the liquid; wash away the excess staining solution with ultrapure water, repeat this step 3-5 times, and place in a 60°C oven with the lid open for 30 minutes; add 200 μL 33% glacial acetic acid to each well, shake at a constant speed for 20 minutes; measure the liquid OD using a microplate reader 590nm absorbance.

[0035] The evaluation criteria of biofilm formation amount: ODc: negative control group OD 590nm Average value + 3× OD of negative control group 590nm Standard deviation; OD 590nm ≤ODc, no biofilm produced; ODc<OD 590nm ≤2×ODc, weak biofilm; 2×ODc<OD 590nm ≤4×ODc, moderate biofilm; 4×ODc<OD 590nm , strong biofilm. Five independent experiments were performed. The results are shown in Table 2.

[0036] Table 2 Biofilm assay results (n=5) The present invention evaluated the biofilm forming ability of 4 strains of Lactobacillus plantarum, and the results showed that all 4 strains of Lactobacillus plantarum had the ability to produce biofilm.

[0037] Utilization L.plantarum- 1 strain fermented apple juice 1. Preparation of apple juice Fuji apples purchased from Dalian, China, were used as the fermentation substrate. The apples were washed and cut into 4 cm × 4 cm pieces and squeezed in a juicer (HX-PB 956, Blenders, China) for 5 minutes. The crude juice was passed through an 80-mesh filter, and the filtrate was collected. 0.08% (w / v) pectinase and 0.8% (w / v) cellulase were added to the filtrate. Enzymatic hydrolysis was performed at 55°C for 2 hours, followed by enzyme inactivation at 90°C for 15 minutes to obtain apple juice. The juice was stored at −20°C.

[0038] 2. Fermentation of apple juice (1) The present invention is separated L.plantarum -1 strain according to the above steps to prepare the third bacterial liquid, then inoculate the third bacterial liquid into 8 mL of MRS broth medium at a 1% inoculum size and subculture at 37°C for 18 h to obtain the first-generation fermentation liquid. The first-generation fermentation liquid was inoculated into new MRS broth medium at a 1% inoculum size and subcultured at 37°C for 18 h to obtain the second-generation fermentation liquid.

[0039] 1L of MRS broth medium contains: 10 g peptone, 8 g beef powder, 4 g yeast powder, 20 g glucose, 2 g dimethyl phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1 g Tween, and the balance distilled water.

[0040] (2) The second generation fermentation broth was inoculated into 10 mL of sterile apple juice pasteurized at 80°C for 15 min and cooled to 25°C at a 1% (v / v) inoculum volume, and cultured at 37°C until the viable count measured by the plate pour method exceeded 10 8 CFU / mL to obtain the seed solution.

[0041] Then count the viable bacteria to 10 8 The seed solution with a CFU / mL concentration of 3% (v / v, 9 mL) was inoculated into 300 mL of sterile apple juice and incubated at 37°C for 24 h to obtain fermented apple juice. Finally, the fermented apple juice was stored at -20°C.

[0042] 3. Detection of flavor compounds in fermented apple juice and in vitro colonic fermentation 1. Analysis of growth characteristics of different Lactobacillus plantarum (1) Growth characteristics of different Lactobacillus plantarum strains The growth characteristic curves of four strains of Lactobacillus plantarum cultured in MRS broth are shown in Figure 2. Figure 2 As shown in A, samples were taken every 2 h during the culture process to measure OD 600nm 2h before culture, probiotics need to adapt to the new environment, OD 600nm There was no significant change in the value; 2 hours after inoculation, it quickly entered the logarithmic growth phase; 8 hours later, the growth rate slowed down and reached the maximum growth at 14 hours.

[0043] (2) Growth characteristics of different Lactobacillus plantarum strains in fermented apple juice During the fermentation process, samples were taken every 2 h to measure OD 600nm Change in value ( Figure 2 B). As the fermentation time increases, the OD 600nm The value showed an upward trend. At the initial stage of fermentation (0h~8h), the OD 600nm The value remains relatively stable, indicating that the probiotics are in the stage of environmental adaptation. In this stage, the bacteria synthesize the necessary enzymes and metabolites through metabolic reprogramming, but have not yet entered the proliferation stage. After 8 hours of fermentation, the bacterial colony enters the logarithmic growth phase (8h-16h), which is manifested by OD 600nm The value increased significantly, the number of viable bacteria increased exponentially, and the metabolic activity was significantly enhanced. At 16 h of fermentation, the counting results showed that the total number of viable bacteria reached the highest level, indicating that the culture system had reached the maximum bacterial density. After that, the number of viable bacteria remained relatively stable. The OD value of apple juice was 0.05, which was 0.01. 600nm The value changes slowly. In addition, according to the experimental results, L.plantarum -1 had a higher growth rate than the other three plant lactobacillus strains, indicating that the isolated L.plantarum -1 strain is more adaptable to the growing environment of apple juice.

[0044] 2. Response of the electronic nose to flavor signals of different Lactobacillus plantarum fermented apple juices Volatile flavors were measured using a PEN-3 electronic nose system (E-nose) and pattern recognition software (Win-Muster 1.6.2) on fermented apple juice samples stored at 4°C. Precision-measured 20 mL of fermented apple juice sample was used for electronic nose analysis in triplicate. The vial was sealed with three layers of plastic film and allowed to stand at room temperature for 30 minutes before analysis. The sensor was purged with clean air for 120 seconds, and then the sample gas was drawn into the electronic nose using a vacuum pump at an intake rate of 0.3 L / min for 150 seconds. The substances corresponding to each sensor in the electronic nose are shown in Table 3.

[0045] Table 3 Performance of each sensor The electronic nose system mimics the human olfactory perception mechanism and integrates an array of 10 metal oxide semiconductor (MOS) sensors with specific responses. Each sensor exhibits differentiated sensitivity to specific volatile organic compounds, and its response pattern can be visualized and recognized using characteristic radar maps. The odor characteristics of cloudy apple juice were analyzed using a portable PEN-3 electronic nose. The response characteristics of each sensor are shown in Table 3.

[0046] The electronic nose was used to detect apple juice fermented by four different strains of Lactobacillus plantarum. For the convenience of comparison, the relative resistivity (G / G0) measured at 58s to 60s was expressed as a radar chart ( Figure 3 In the radar chart, the 10 axes represent 10 different sensors, and the size of the axes indicates the sensitivity of the sensor to each sample. Five sensors: W1W, W2W, W1S, W2S, and W5S showed relatively high responses. This indicates that sulfides, alcohols, methyl groups, organic sulfides, and nitrogen oxides contribute most to the flavor of fermented apple juice. Although the four different strains of Lactobacillus plantarum have similar overall flavors, only L.plantarum-1 The response value of is at the outermost edge of the radar chart, indicating that it is better than the other three strains of Lactobacillus plantarum in expressing the above five flavor substances. The other five sensors: W1C, W3C, W6S, W5C and W3S sensors detected five types of substances, such as benzene, ammonia, hydride, short-chain alkanes and long-chain alkanes. Although they have a certain contribution to the flavor of fermented apple juice, they belong to the category of minor flavor substances. Among these substances, L.plantarum-1 There was no significant difference in the response of the strain to the apple juice fermented by other strains, which indirectly reflects that the strain has a more prominent advantage in regulating the core flavor substances (sulfide, alcohol, methyl, organic sulfide, nitrogen oxides), and the common characteristics of the secondary flavor substances did not mask the differentiated performance of its key flavor components. Figure 3 It can be seen from B that the present invention separates L.plantarum- The flavor of apple juice fermented by strain 1 is the strongest.

[0047] In summary, L.plantarum- Strain 1 has low biogenic amine production, can produce biofilm, is non-hemolytic and contributes the most to the flavor of fermented apple juice.

[0048] Four, L.plantarum- Effects of fermented apple juice obtained by fermenting apple juice with 1 strain on intestinal flora 1. Test method (1) In vitro colonic fermentation Fresh fecal material was collected from obese volunteers (n = 4, aged 24 to 32 years, BMI > 28%) who had no intestinal disease and no antibiotic use within the previous three months. 1.0 g of fresh fecal material was immersed in 10 mL of sterile PBS (pH 7.2), shaken thoroughly, and then centrifuged at 600 × g at 4°C for 5 minutes to remove undigested food and small particles. Four replicate fecal samples were processed identically, and the resulting supernatants were mixed in equal volumes, resuspended in sterile PBS, and immediately mixed with 40% (v / v) sterile glycerol to a final concentration of 20% (v / v) to obtain the fecal microbiota solution.

[0049] Will L.plantarum- Fermented apple juice prepared from strain 1 was dissolved in PBS and filtered through a 0.22 μm sterile filter. It was then added to autoclaved minimal nutrient growth medium (2.0 g / L peptone, 2.0 g / L yeast extract, 0.02 g / L hemin, 0.5 g / L cysteine ​​hydrochloride, 0.5 g / L bile salts, 0.1 g / L sodium chloride, 0.04 g / L potassium dihydrogen phosphate, 0.04 g / L potassium hydrogen phosphate, 0.01 g / L magnesium sulfate, 0.01 g / L calcium chloride, 2 g / L sodium bicarbonate, 2.0 mL / L Tween 80, 1.0 mL / L 1% (w / v) resazurin solution, and 10 μL / L vitamin K) as the sole carbon source to a final concentration of 5.0 mg / mL (w / v). Before fermentation, 1.0 mL of the fecal microbiota solution was added to 5.0 mL of the prepared basal nutrient growth medium in a sterile tube. In vitro fermentation was carried out in an anaerobic chamber at 37°C for 96 h. After fermentation, the supernatant was collected by centrifugation at 8000 g for 15 minutes and used for further analysis. A control group consisted of the prepared basal nutrient growth medium without fermented apple juice.

[0050] (2) Determination of intestinal flora Enteric bacterial DNA was extracted from fecal microbiota solution samples containing fermented apple juice using the EZNA® Soil DNA Kit. DNA quality was assessed by 1% agarose gel electrophoresis, purified, and concentration was determined using a NanoDrop spectrophotometer. Extracted genomic DNA was amplified using the conventional barcoded primer pair 338F (5'-ACTCCTACGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGTWTCTAAT-3'), targeting the V3-V4 hypervariable region of the bacterial 16S rRNA gene. Sequencing libraries were generated using the NEXTFLEX Rapid DNA-Seq Kit. Sequencing was performed on an Illumina Miseq PE300 platform at Shanghai Majorbio Bio-pharm Technology Co. Ltd (Shanghai, China).

[0051] (3) Statistical analysis All experiments were performed with at least three biological replicates, and the measured data were expressed as mean ± standard deviation (SD) to ensure the repeatability and statistical reliability of the experimental results. The data were processed using OriginPro 2024 (OriginLab Software, USA). The results were analyzed by variance analysis (ANOVA) followed by Tukey's multiple comparison test. p The difference was statistically significant when the value was <0.05.

[0052] 2. Test results (1) Effects of fermented apple juice on the level of intestinal flora The obesity group and the fermented apple juice intervention group were mainly composed of Proteobacteria, Firmicutes, and Bacteroidota, with a total relative abundance of more than 80% ( Figure 4 A), after the intervention of fermented apple juice, it reached 90%. Figure 4 As can be seen from B to D of 4, compared with the obese group, the changes in Firmicutes, Proteobacteria and Bacteroidetes were not significant after the intervention of fermented apple juice, but the ratio of Firmicutes to Bacteroidetes (F / B) was significantly reduced ( P <0.05) ( Figure 4 E). It is known that the increase of F / B is related to obesity. Firmicutes is more abundant in obese people, while Bacteroidetes is the opposite. The decrease of F / B means weight loss to a certain extent. Therefore, the present invention can L.plantarum- 1 strain can significantly reduce the ratio of Firmicutes to Bacteroidetes (F / B). L.plantarum-The fermented apple juice prepared by strain 1 is beneficial to weight loss after intervention and has a certain weight loss effect.

[0053] (2) Effects of fermented apple juice on the genus level of intestinal flora like Figure 5 As shown, intervention with fermented apple juice can Lysinibacillus and Bilophila Significantly reduced ( P <0.001), Parabacteroides and Bifidobacterium The abundance increased significantly ( P <0.001), Lactobacillus 、 Bacteroides 、 Phascolarctobacterium and Enterococcus The abundance has increased to a certain extent, but not significantly. L.plantarum- 1. The fermented apple juice prepared by the strain has the effect of improving intestinal flora and alleviating obesity.

[0054] five, L.plantarum- Study on the mechanism of alleviating obesity by fermented apple juice prepared by 1 strain 1. MetaCyc functional prediction analysis The pathway analysis results of the MetaCyc database show the distribution of functional abundance in different samples ( Figure 6 Figure A) intuitively shows the distribution of the main dominant functions in different samples. The color gradient of the color blocks shows the changes in the abundance of different functions in the samples / groups. The functions on the right side of the figure are mainly related to glycolysis, including NONOXIPENT-PWY, PWY-7111, CALVIN-PWY, PWY-7663, PWY-5973, P42-PWY, ILEUSYN-PWY, VALSYN-PWY, PWY-6126, and DTDPRHAMSYN-PWY. After fermented apple juice intervention, the abundance of each function increased significantly, among which the NONOXIPENT-PWY pathway was particularly prominent. It is a common glycolysis pathway used by most organisms for the breakdown and metabolism of glucose and related sugars. Increased glycolysis levels can promote the browning of white fat, which not only resists high-fat diet-induced obesity by promoting lipolysis, but also increases the body's uptake and utilization of glucose, improving insulin resistance. Fermented apple juice promoted glycolysis in obese subjects, rapidly converting sugar in the intestines into energy, thereby alleviating obesity. Therefore, this result further suggests that fermented apple juice has the potential to prevent and alleviate obesity.

[0055] 2. Network Correlation Analysis The present invention further understands the interaction between dominant species through the species correlation network, selects the 20 genera with the highest abundance, and calculates the Spearman correlation coefficient to reflect the correlation between species. Figure 6 As shown in Figure B, in the species correlation network diagram, "degree" represents the number of nodes connected to a node, and "clustering" represents the connection between a node and its adjacent nodes. If a node is completely connected to its adjacent nodes, the clustering coefficient is 1. On the contrary, if a node has almost no connection with its adjacent nodes, the clustering coefficient is close to 0. The larger the clustering coefficient, the more important the node. Streptococcus、 Parabacteroides、Enterococcus、Acidaminococcus、Megamonas、Bifidobacterium The degrees are 7, 6, 5, 5, 6 and 6 respectively ( Figure 6 B), as shown in the figure, Streptococcus and Bifidobacterium、 Acidaminococcus and Megamonas There is a significant positive correlation; Acidaminococcus and Bifidobacterium There is a significant negative correlation. Bifidobacterium and Enterococcus is positively correlated with Parabacteroide、 Acidaminococcus and Megamonas negatively correlated; Megamonas and Acidaminococcus and Parabacteroides Positively correlated with Enterococcus and [[ID= These bacterial genera all play an important role in the fermentation system network, among which, ​ and ​ 、 ​ and ​ negatively correlated, ​ It is a key species in the intestinal microbiome, acting as a major degrader and promoting the balance of the intestinal environment in various ways; ​ and ​ The abundance of these two proteins is higher in obese people, and their presence has an additive effect on obesity. ​ As commensal bacteria, they colonize the digestive systems of humans and animals and participate in immune regulation. ​ It can prevent obesity caused by excessive sucrose intake by producing extracellular polysaccharides (EPS). Therefore, combined with the experimental results and the above analysis, when fermented apple juice is given to obese people, the beneficial bacteria in the intestines increase, while the obesogenic bacteria that are negatively correlated with it decrease significantly, thereby achieving the effect of alleviating obesity by improving the intestinal flora.

[0056] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0057] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A strain of Lactobacillus plantarum L. plantarum -1, characterized in that Lactobacillus plantarum L. plantarum -1 was deposited in Guangdong Provincial Microbial Culture Collection Center on May 9, 2025, with the deposit number GDMCC No.66300 and the classification name Lactiplantibacillusplantarum .

2. A bacterial liquid, characterized in that: The bacterial liquid contains the Lactobacillus plantarum according to claim 1 L. plantarum -1.

3. A fermented apple juice, characterized in that: The plant lactobacillus according to claim 1 L. plantarum -1 obtained from fermentation.

4. The fermented apple juice according to claim 3, characterized in that The preparation process of the fermented apple juice is as follows: Lactobacillus plantarum L. plantarum -1 was inoculated into LB liquid medium for activation culture to obtain bacterial liquid, and then the bacterial liquid was inoculated into MRS broth medium for fermentation to obtain fermentation liquid, and the fermentation liquid was inoculated into apple juice and cultured until the number of viable bacteria was 10 8 CFU / mL or above, a seed solution is obtained, and then the seed solution is inoculated into apple juice, and fermentation is continued to obtain fermented apple juice.

5. The plant lactobacillus of claim 1 L. plantarum -1. Use of the bacterial solution according to claim 2 or the fermented apple juice according to any one of claims 3 to 4 in the preparation of a product for alleviating obesity.

6. The use according to claim 5, characterized in that The product has the effect of improving intestinal flora.

7. The use according to claim 6, characterized in that Improvement of intestinal flora is manifested by: reducing the ratio of Firmicutes to Bacteroidetes and reducing Lysinibacillus and Bilophila abundance.

8. The use according to claim 7, characterized in that The product is obtained by dissolving the fermented apple juice in PBS and collecting the filtrate by filtration.

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