Lactobacillus plantarum-1 and application thereof

Abstract

This invention relates to the field of microbial fermentation technology, and specifically discloses a strain of Lactobacillus plantarum. L. plantarum -1 and its bacterial suspension and applications, the Lactobacillus plantarum L. plantarum -1 was deposited on May 9, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No. 66300, and classified as follows: Lactiplantibacillus plantarum. The Lactobacillus plantarum provided by this invention L. plantarum -1 High safety profile; fermentation of apple juice reduces the ratio of Firmicutes to Bacteroidetes in the gut and lowers... Lysinibacillus and Bilophila Increased abundance of [something] can help reduce obesity.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a strain of Lactobacillus plantarum. L.plantarum -1 and its bacterial solution and applications. Background Technology

[0002] Obesity is considered a persistent metabolic disorder resulting from the combined effects of genetic, biological, environmental, behavioral, sociocultural, and economic factors. It is well-known that obesity is a significant risk factor for cardiovascular disease, type 2 diabetes, hypertension, cancer, chronic kidney disease, a range of musculoskeletal disorders, and mental illnesses, primarily because obesity is a systemic chronic inflammatory state. The obesity epidemic is the result of both fixed factors (genes, age, sex) and variable factors (intake of high-calorie foods and reduced physical activity), with dietary factors playing a crucial 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, making it necessary to obtain a microorganism with obesity-alleviating effects. Summary of the Invention

[0003] To develop a microorganism that alleviates obesity, this invention provides a strain of *Lactobacillus plantarum*. L.plantarum -1 and its bacterial suspension and applications. The *Lactobacillus plantarum* provided by this invention. L.plantarum -1 has the function of alleviating obesity, the plant lactobacillus L.plantarum -1 Fermented apple juice can improve gut microbiota structure and alleviate obesity.

[0004] This invention provides a strain of *Lactobacillus plantarum*, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 9, 2025, with accession number GDMCC No. 66300, and classified as... Lactiplantibacillus plants .

[0005] The Lactobacillus plantarum provided by this invention L.plantarum -1 has the function of alleviating obesity, the plant lactobacillus L.plantarum -1 Fermented apple juice can improve gut microbiota structure and alleviate obesity.

[0006] The Lactobacillus plantarum provided by this invention L.plantarum -1 High safety profile; reduces the ratio of Firmicutes to Bacteroidetes in the gut through apple juice fermentation and reduces... Lysinibacillus and Bilophila Increased abundance of [something] can help reduce obesity.

[0007] The present invention also provides a bacterial solution containing the aforementioned *Lactobacillus plantarum*. L.plantarum -1.

[0008] The present invention also provides a fermented apple juice, made from the aforementioned *Lactobacillus plantarum*. L.plantarum -1. Obtained through fermentation. Utilizing *Lactobacillus plantarum* of the present invention. L.plantarum -1 Fermented apple juice produces a richer variety of flavor compounds (sulfides, alcohols, methyl groups, organosulfur compounds, and nitrogen oxides).

[0009] Furthermore, the preparation process of the fermented apple juice is as follows: Lactobacillus plantarum... L.plantarum -1 The bacterial culture was activated by inoculation into LB liquid medium and cultured to obtain a bacterial suspension. Then, the bacterial suspension was inoculated into MRS broth medium for fermentation to obtain a fermentation broth. The fermentation broth was then inoculated into apple juice and cultured until the viable count reached 10^6. 8 A seed solution was obtained by adding CFU / mL or higher. The seed solution was then inoculated into apple juice and fermented to obtain fermented apple juice.

[0010] The present invention also provides the aforementioned *Lactobacillus plantarum*. L.plantarum -1 or the application of the bacterial solution described above in the preparation of products that alleviate obesity.

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

[0012] Furthermore, improving the gut microbiota manifests as a reduction in the ratio of Firmicutes to Bacteroidetes.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: The aforementioned Lactobacillus plantarum L.plantarum Compared to other *Lactobacillus plantarum* species, this *Lactobacillus* species exhibits lower amine production capacity, does not show β-hemolytic activity, and is safer, making it suitable for intestinal interventions. Compared to other *Lactobacillus plantarum* species, the *Lactobacillus plantarum* species utilized in this invention... L.plantarum -1 fermentation of apple juice produces a richer variety of flavor compounds.

[0014] The Lactobacillus plantarum provided by this invention L.plantarum -1 has the function of alleviating obesity, the plant lactobacillus L.plantarum -1 Fermented apple juice can improve gut microbiota structure and alleviate obesity.

[0015] Information on the Preservation of Biological Materials L.plantarum -1, referred to in this application as L.plantarum- 1. Deposited on May 9, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 66300). The address of the depository is 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510075, China. The classification name is... Lactiplantibacillus plants . Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0017] Figure 1 In this invention L.plantarum -1 strain hemolytic activity; In the figure, A represents the invention. L.plantarum -1 strain hemolytic activity; B is the control strain. Virgibacillus halodenitricanicans Hemolytic activity.

[0018] Figure 2 The growth characteristic curves of different Lactobacillus plantarum and their growth characteristic curves during the fermentation of apple juice are shown. In the figure, A represents the growth characteristic curves of four strains of Lactobacillus plantarum in MRS broth medium; B represents the growth characteristic curves of four strains of Lactobacillus plantarum during the fermentation of apple juice.

[0019] Figure 3 Results of electronic nose response values ​​for apple juice fermented with different Lactobacillus plantarum strains; In the figure, A is a radar chart showing the response values ​​of the electronic nose for fermenting apple juice with different Lactobacillus plantarum strains; B is a bar chart showing the mean response values ​​of the electronic nose for fermenting apple juice with different Lactobacillus plantarum strains.

[0020] Figure 4 The effect of fermented apple juice on the gut microbiota level was investigated, where O represents the obese group and O+FAJ represents the fermented apple juice group; * indicates a significant difference. In the figure, A is a heatmap showing the effect of fermented apple juice on the gut microbiota phylum level; B represents the effect of fermented apple juice on the relative abundance of Firmicutes; C represents the effect of fermented apple juice on the relative abundance of Proteobacteria; D represents the effect of fermented apple juice on the relative abundance of Bacteroidetes; E represents the effect of fermented apple juice on the Firmicutes / Bacteroidetes ratio.

[0021] Figure 5The effect of fermented apple juice on gut microbiota levels was investigated, where O represents the obese group, O+FAJ represents the fermented apple juice group, and * indicates a significant difference. In the figure, A is a genus-level heatmap for the fermented apple juice group and the obese group; B represents the fermented apple juice group and the obese group. Escherichia-Shigell a. Relative abundance; C represents the fermented apple juice group and the obese group. Lactobacillus Relative abundance; D represents the fermented apple juice group and the obese group. Bacteroides Relative abundance; E represents the fermented apple juice group and the obese group. Fusobacterium Relative abundance; F represents the fermented apple juice group and the obese group. Paeniclostridium Relative abundance; G represents the fermented apple juice group and the obese group. Lysinibacillus Relative abundance; H represents the fermented apple juice group and the obese group. Phascolarctobacterium Relative abundance; I represents the fermented apple juice group and the obese group. Bilophila Relative abundance; J represents the fermented apple juice group and the obese group. Parabacteroids Relative abundance; K represents the fermented apple juice group and the obese group. Bifidobacterium Relative abundance; L represents the fermented apple juice group and the obese group. Enterococcus Relative abundance.

[0022] Figure 6 For functional analysis and species network correlation diagrams; The image shows the default display. P Species with a correlation coefficient <0.05; the size of the nodes in the graph represents the species abundance, and different colors represent different species; the color of the connecting lines represents positive and negative correlation, with red indicating a positive correlation and green indicating a negative correlation; the thickness of the lines represents the magnitude of the correlation coefficient, with thicker lines indicating a higher correlation between species; the more lines there are, the closer the connection between that species and other species. In the figure, A is the MetaCyc pathway heatmap; B is the Network diagram. Detailed Implementation

[0023] 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 to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0024] Example 1: A strain of Lactobacillus plantarum, its bacterial culture, and its application.

[0025] I. Isolation and Identification of Strains A strain was isolated from naturally fermented yogurt and identified as *Lactobacillus plantarum* by 16S rDNA analysis. Lactobacillus plantarum. Recorded as L.plantarum- 1.

[0026] II. Analysis of the bioamine production capacity, hemolytic activity, and biofilm formation capacity of *Lactobacillus plantarum* Lactobacillus plantarum CGMCC 1.568, purchased from the China General Microbiological Culture Collection Center, is designated as... L.plantarum 1.568; Lactobacillus plantarum CGMCC 1.124, denoted as L.plantarum 1.124; Lactobacillus plantarum CGMCC 1.572, denoted as L.plantarum 1.572. The results obtained by separating the samples according to this invention were analyzed separately. L.plantarum- 1. The bio-amine production capacity, hemolytic activity, and bio-membrane production capacity of three commercially available strains of Lactobacillus plantarum were compared and analyzed.

[0027] 1. Detection of bioamine production capacity of Lactobacillus plantarum Separate this embodiment L.plantarum- 1 strain and commercially available strain L.plantarum 1.568 L.plantarum 1.124 and L.plantarum1.572 μL was inoculated into 5 mL of LB liquid medium and incubated at 37°C for 24 h to obtain the first bacterial culture. 100 µL of the first bacterial culture was then inoculated into 5 mL of fresh LB liquid medium and incubated at 37°C for 24 h to obtain the second bacterial culture. To eliminate the influence of inoculum size on the production of amines by the strain, this step was repeated three times to obtain the third bacterial culture. 100 µL of the third bacterial culture 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 precursor, and 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℃ 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 water bath at 45℃ for 40 min; add 50 µL of ammonia water and let stand at room temperature for 30 min; add 550 µL of acetonitrile and incubate at 3000× g Centrifuge at 4℃ for 5 min, filter twice through a 0.22 µm filter, and then perform high performance liquid chromatography analysis.

[0028] The results of the bioamine production capacity tests of different Lactobacillus plantarum are shown in Table 1.

[0029] Table 1. Results of bioamine production capacity of different Lactobacillus plantarum strains. Note: Different lowercase letters indicate significant differences between groups. P <0.05).

[0030] As shown in Table 1, none of the four *Lactobacillus plantarum* strains produced histamine or tyramine, and the levels of putrescine and cadaverine were low. Among them, the strain of the present invention… L.plantarum- Strain 1 produced the lowest levels of biogenic amines, meaning L.plantarum- Strain 1 is safer.

[0031] 2. Qualitative analysis of hemolysis For the β-hemolytic activity test, 5% (v / v) sheep blood was added to MRS agar. Colonies were picked up with an inoculation loop and streaked onto blood-containing MRS agar plates. The plates were incubated at 37°C for 48 h, followed by a 24 h cold shock at 4°C. The hemolytic activity of the strain was determined by observing the clear lysis zone around each colony on the blood-containing MRS agar plate. A moderately halophilic bacterium (Hib) purchased from the China General Microbiological Culture Collection Center was used. Virgibacillus halodenitrifyingCGMCC 1.8915 was used as a positive control.

[0032] Qualitative analysis results of hemolysis as follows Figure 1 As shown, known hemolytic strains Virgibacillus halodenitricanicans When grown on MRS agar, a very clear area forms around the colony, while the isolated... L.plantarum strain -1 did not exhibit β-hemolytic activity. Therefore L.plantarum The -1 strain has high safety and can be used as a food fermentation bacteria.

[0033] 3. Analysis of the ability to generate biofilm Biofilm formation was measured using the 96-well microplate method: the components separated in this invention... L.plantarum- 1 strain and commercially available strain L.plantarum 1.568 L.plantarum 1.124 and L.plantarum 1.572 was inoculated into 5 mL of LB liquid medium and incubated at 37°C until the bacterial culture reached OD. 600nm The value was 0.4, and 200 μL of OD was taken respectively. 600nm A bacterial culture solution of 0.4 g was added to the well plates, with sterile LB liquid medium used as a negative control. Staphylococcus aureus (S. aureus) Staphylococcus aureus ATCC6538 was used as a positive control, and the culture was carried out at 37°C for 48 h. Eight replicates were performed for each strain. The OD of the bacterial culture was measured using a microplate reader. 600nm Absorbance; discard the bacterial suspension, add 250 μL of 1×PBS to each well, wash 3 times to completely remove liquid; add 200 μL of anhydrous methanol to each well, fix at room temperature for 15 min, then discard the liquid; place in a 60℃ oven with the lid open for 15 min; stain with 200 μL of 0.1% crystal violet, place at room temperature for 15 min, then discard the liquid; wash with ultrapure water to remove excess staining solution, repeat this step 3-5 times, and place in a 60℃ oven with the lid open for 30 min; add 200 μL of 33% glacial acetic acid to each well, and shake evenly for 20 min; measure the OD of the liquid using a microplate reader. 590nm Absorbance.

[0034] Criteria for evaluating biofilm formation: ODc: using the negative control group OD 590nm Mean + 3 × negative control group OD 590nm Standard deviation; OD 590 nm ≤ODc, no biofilm formation; ODc < OD 590nm ≤2×ODc, weak biofilm; 2×ODc<OD 590nm ≤4×ODc, medium-strength biofilm; 4×ODc<OD 590nm Strong biofilm. Five independent experiments were conducted. The results are shown in Table 2.

[0035] Table 2. Results of biofilm assay (n=5) This invention evaluated the biofilm formation ability of four strains of Lactobacillus plantarum, and the results showed that all four strains of Lactobacillus plantarum had the ability to produce biofilms.

[0036] II. Utilization L.plantarum- Fermented apple juice with 1 strain 1. Preparation of apple juice Fuji apples purchased from Dalian, China, were used as the fermentation substrate. The apples were washed and cut into 4cm x 4cm pieces, then pressed 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 carried out at 55°C for 2 hours, followed by enzyme inactivation at 90°C for 15 minutes to obtain apple juice. The apple juice was stored at −20°C.

[0037] 2. Fermentation of apple juice (1) The present invention is separated L.plantarum -1 strain prepared the third bacterial culture according to the above steps, and then inoculated the third bacterial culture into 8 mL of MRS broth medium at an inoculation rate of 1%, and subcultured at 37 ℃ for 18 h to obtain the first generation fermentation broth. The first generation fermentation broth was inoculated into new MRS broth medium at an inoculation rate of 1%, and subcultured at 37 ℃ for 18 h to obtain the second generation fermentation broth.

[0038] 1L of MRS broth medium contains: 10 g peptone, 8 g beef meal, 4 g yeast powder, 20 g glucose, 2 g dimethyl hydrogen 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 remainder distilled water.

[0039] (2) The second-generation fermentation broth was inoculated at an inoculation rate of 1% (v / v) into 10 mL of sterile apple juice that had been pasteurized at 80°C for 15 min and cooled to 25°C. The mixture was then incubated at 37°C until the viable count measured by the plate pour method exceeded 10. 8 CFU / mL was used to obtain a seed solution.

[0040] Then count the viable bacteria to 10. 8 A seed solution of CFU / mL was inoculated into 300 mL of sterile apple juice at an inoculum rate of 3% (v / v, 9 mL) and incubated at 37°C for 24 h to obtain fermented apple juice. Finally, the fermented apple juice was stored at -20°C.

[0041] III. Detection of flavor compounds in fermented apple juice and in vitro colonic fermentation 1. Analysis of the growth characteristics of different Lactobacillus plantarum strains (1) Growth characteristics of different Lactobacillus plantarum The growth characteristic curves of four strains of Lactobacillus plantarum cultured in MRS broth are as follows: Figure 2 As shown in Figure A, samples were taken every 2 hours during the culture process to measure OD. 600nm Changes in OD values. During the first 2 hours of culture, probiotics need to adapt to the new environment; OD... 600nm The value did not change significantly; it rapidly entered the logarithmic growth phase 2 hours after inoculation; the growth rate slowed down after 8 hours, and reached the maximum growth at 14 hours.

[0042] (2) Growth characteristics of different Lactobacillus plantarum in fermented apple juice Samples were taken every 2 hours during the fermentation process to measure OD. 600nm Changes in value ( Figure 2 (B). As fermentation time increases, the OD of apple juice... 600nm The values ​​showed an upward trend. During the initial fermentation stage (0h–8h), the OD values ​​of each experimental group were... 600nm The relatively stable value indicates that the probiotics are in the environmental adaptation phase. During this phase, the bacteria synthesize essential enzyme systems and metabolites through metabolic reprogramming, but have not yet entered the proliferation phase. After 8 hours of fermentation, the bacterial community enters the logarithmic growth phase (8-16 hours), as shown by the OD value. 600nm The value increased significantly, the number of viable bacteria increased exponentially, and metabolic activity was significantly enhanced. At 16 hours of fermentation, the count showed that the total number of viable bacteria reached its highest level, indicating that the culture system had reached its maximum cell density. Afterward, the number of viable bacteria remained relatively stable, and the OD value of the apple juice... 600nm The value changes slowly. Furthermore, the experimental results also show that... L.plantarum The growth rate of strain -1 was higher than that of the other three *Lactobacillus plantarum* strains, indicating that the *Lactobacillus plantarum* isolated in this invention... L.plantarum -1 strain is better adapted to the growth environment of apple juice.

[0043] 2. Response of the electronic nose to flavor compounds in apple juice fermented by different Lactobacillus plantarum strains Volatile flavors were determined 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. Triple 20 mL portions of the fermented apple juice sample were precisely measured for electronic nose analysis. The measuring vials were sealed with three layers of plastic film and allowed to stand at room temperature for 30 min before analysis. The sensors were cleaned with clean air for 120 s, then sample gas was drawn into the electronic nose using a vacuum pump at an inlet rate of 0.3 L / min. The detection time was 150 s. The substances corresponding to each sensor of the electronic nose are shown in Table 3.

[0044] Table 3 Performance of each sensor The electronic nose system integrates an array of 10 metal-oxide-semiconductor (MOS) sensors with specific responses, mimicking the human olfactory perception mechanism. Each sensor exhibits differentiated sensitivity to specific volatile organic compounds, and their response patterns can be visualized and analyzed using characteristic radar maps for pattern recognition. The odor characteristics of cloudy apple juice were analyzed using a portable PEN-3 electronic nose, and the response characteristics of each sensor are shown in Table 3.

[0045] An electronic nose was used to detect the apple juice fermented by four different strains of Lactobacillus plantarum. For ease of comparison, the relative resistivity (G / G0) measured from 58 s to 60 s was expressed using a radar graph. Figure 3 (A) In the radar chart, the 10 axes represent 10 different sensors, and the magnitude on 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, organosulfur compounds, and nitrogen oxides contribute the most to the flavor of fermented apple juice. While the four different *Lactobacillus plantarum* strains had similar overall flavors, only... L.plantarum-1 The response value of [specific strain name] is located on the outermost edge of the radar chart, indicating that its expression of the above five flavor compounds is superior to that of the other three *Lactobacillus plantarum* strains. The other five sensors—W1C, W3C, W6S, W5C, and W3S—detected benzene, ammonia, hydrides, short-chain alkanes, and long-chain alkanes, which, while contributing to the flavor of fermented apple juice, belong to the category of secondary flavor compounds. Among these compounds, [further details needed]. L.plantarum-1 The strain showed no significant differentiation in its apple juice fermentation responses compared to other strains, indirectly reflecting its superior ability to regulate core flavor compounds (sulfides, alcohols, methyl groups, organosulfur compounds, and nitrogen oxides), while the common characteristics of secondary flavor compounds did not mask the differentiated performance of its key flavor components. Furthermore, due to... Figure 3 As can be seen from B, the present invention separates L.plantarum- The apple juice fermented with strain 1 has the most intense flavor.

[0046] In summary, L.plantarum- Strain 1 has low biogenic amine production, can produce biofilms, is non-hemolytic, and contributes the most to the flavor of fermented apple juice.

[0047] Four, L.plantarum- The effect of fermented apple juice obtained by fermenting apple juice with strain 1 on gut microbiota 1. Test Methods (1) In vitro colonic fermentation Fresh fecal samples were obtained from four obese volunteers (n=4, aged 24–32 years, BMI > 28%) who had no intestinal diseases and had not used antibiotics in the previous three months. 1.0 g of collected fresh fecal material was soaked in 10 mL of sterile PBS (pH 7.2), shaken thoroughly, and then centrifuged at 600×g, 4°C for 5 min to remove undigested food and small particles. Four parallel fecal samples were treated in the same manner to obtain four supernatants, which were then 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.

[0048] Will L.plantarum- Fermented apple juice prepared by strain 1 was dissolved in PBS and filtered through a 0.22 μm sterile filter. It was then added to autoclaved basal nutrient growth medium (2.0 g / L peptone, 2.0 g / L yeast extract, 0.02 g / L heme chloride, 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 dipotassium 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) resazu solution, and 10 μL / L vitamin K) as a single carbon source, with a final concentration of 5.0 mg / mL (w / v). Before fermentation, 1.0 mL of fecal microbiota solution was added to 5.0 mL of prepared basal nutrient growth medium in a sterile tube, and in vitro fermentation was carried out in an anaerobic chamber at 37°C for 96 h. After fermentation, the supernatant was collected after centrifugation at 8000 g for 15 min and used for further analysis. The control group consisted of the prepared basal nutrient growth medium, which did not contain fermented apple juice.

[0049] (2) Determination of intestinal flora DNA from gut bacteria was extracted from fecal microbiota solutions containing fermented apple juice using the EZNA® Soil DNA Kit. DNA quality was assessed by 1% agarose gel electrophoresis, and the concentration was determined using a NanoDrop spectrophotometer after purification. Genomic DNA was amplified using standard barcoding primer pairs 338F (5'-ACTCCTACGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGTWTCTAAT-3') based on 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).

[0050] (3) Statistical analysis All experiments were performed in at least three biological replicates. Data are presented as mean ± standard deviation (SD) to ensure reproducibility and statistical reliability. Data were processed using OriginPro 2024 (OriginLab software company, USA). Results were analyzed using ANOVA, followed by Tukey's multiple comparison test. p <0.05 indicates a statistically significant difference.

[0051] 2. Test Results (1) Effects of fermented apple juice on the level of gut microbiota The obesity group and the fermented apple juice intervention group were dominated by Proteobacteria, Firmicutes, and Bacteroides, with a total relative abundance exceeding 80%. Figure 4 The percentage of A in the fermented apple juice reached 90%. Figure 4 As can be seen from B to D in groups B to 4, compared with the obese group, the changes in Firmicutes, Proteobacteria, and Bacteroidetes were not significant after the fermented apple juice intervention, but the ratio of Firmicutes to Bacteroidetes (F / B) was significantly reduced. P <0.05)( Figure 4 (E). It is known that an increase in the F / B ratio is associated with obesity, Firmicutes are more abundant in obese individuals, while Bacteroidetes are the opposite; a decrease in the F / B ratio to some extent indicates weight loss. Therefore, it is explained that the present invention... L.plantarum- Intervention with fermented apple juice prepared by strain 1 can significantly reduce the Firmicutes to Bacteroidetes ratio (F / B), indicating that... L.plantarum-Fermented apple juice prepared with strain 1 was beneficial for weight loss after intervention. It has a certain weight loss effect.

[0052] (2) Effects of fermented apple juice on gut microbiota genera like Figure 5 As shown, intervention in fermented apple juice can... Lysinibacillus and Bilophila Significantly reduced ( P <0.001), Parabacteroides and Bifidobacterium Abundance increased significantly ( P <0.001), Lactobacillus , Bacteroides , Phascolarctobacterium and Enterococcus The abundance increased to some extent, but not significantly. Therefore, L.plantarum- Fermented apple juice prepared from strain 1 has the effect of improving gut microbiota and alleviating obesity.

[0053] five, L.plantarum- Study on the mechanism of fermented apple juice prepared by strain 1 in alleviating obesity 1. MetaCyc Function Predictive Analysis The pathway analysis results from the MetaCyc database show the distribution of functional abundance across different samples. Figure 6 Figure A) visually illustrates the distribution of key functionalities across different samples. Color gradients are used to represent changes in the abundance of different functionalities 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 intervention with fermented apple juice, the abundance of each function significantly increased, with the NONOXIPENT-PWY pathway being particularly prominent. This is a common glycolytic pathway used by most organisms for the breakdown and metabolism of glucose and related sugars. Increased glycolysis levels can promote the browning of white adipose tissue, not only by promoting lipolysis to combat obesity induced by a high-fat diet but also by increasing the body's glucose uptake and utilization, and improving insulin resistance. Fermented apple juice promoted glycolysis in obese samples, rapidly converting intestinal sugars into energy, thereby alleviating obesity. Therefore, this result further suggests that fermented apple juice has the potential to prevent and alleviate obesity.

[0054] 2. Network Correlation Analysis This invention further understands the interactions among dominant species through species correlation networks. The 20 genera with the highest abundance were selected, and Spearman correlation coefficients were calculated to reflect the correlations between species. The interactions among gut microbiota species after processing fermented apple juice are shown below. Figure 6 As shown in Figure B, in the species-related network graph, "degree" represents the number of nodes connected to a given node, and "clustering" represents the connections between a node and its neighbors. If a node is fully connected to all its neighbors, the clustering coefficient is 1. Conversely, if a node has almost no connections to its neighbors, the clustering coefficient is close to 0. A higher clustering coefficient indicates a more important 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 It shows a significant positive correlation; while Acidaminococcus and Bifidobacterium Then there is a significant negative correlation. Bifidobacterium and Enterococcus It is positively correlated with, and with Parabacteroide、 Acidaminococcus and Megamonas Negative correlation; Megamonas and Acidaminococcus and Parabacteroides Positively correlated with Enterococcus and Streptococcus A negative correlation was observed. These genera all play important roles in the fermentation system network, among which... Bifidobacterium and Acidaminococcus , Megamonas and Parabacteroides Negative correlation Bifidobacterium It is a key species in the gut microbiome, serving as a major degrader and promoting the balance of the gut environment in various ways; Acidaminococcus and Megamonas The abundance of both is higher in obese individuals, and their presence has an additive effect on obesity. Meanwhile, Enterococcus As symbiotic bacteria, they colonize the digestive systems of humans and animals and participate in immune regulation. Streptococcus It can prevent obesity caused by excessive sucrose intake by producing extracellular polysaccharides (EPS). Therefore, combining the experimental results and the above analysis, it is shown that when obese groups are treated with fermented apple juice, the beneficial bacteria in the gut increase, while the obese bacteria that are negatively correlated with it decrease significantly, thereby achieving the effect of alleviating obesity by improving the gut microbiota.

[0055] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0056] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A strain of Lactobacillus plantarum L. plantarum -1, characterized in that, The Lactobacillus plantarum L. plantarum -1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 9, 2025, with accession number GDMCC No. 66300, and classified as... Lactiplantibacillus plantarum .

2. A bacterial solution, characterized in that, The bacterial solution contains *Lactobacillus plantarum* as described in claim 1. L. plantarum -1.

3. A fermented apple juice, characterized in that, Lactobacillus plantarum as described in claim 1 L. plantarum -1 is 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 The bacterial culture was activated by inoculation into LB liquid medium and cultured to obtain a bacterial suspension. Then, the bacterial suspension was inoculated into MRS broth medium for fermentation to obtain a fermentation broth. The fermentation broth was then inoculated into apple juice and cultured until the viable count reached 10^6. 8 A seed solution was obtained by adding CFU / mL or higher. The seed solution was then inoculated into apple juice and fermented to obtain fermented apple juice.

5. A *Lactobacillus plantarum* as described in claim 1 L. plantarum -1 or the use of the bacterial solution according to claim 2 in the preparation of products that improve intestinal flora.

6. The application according to claim 5, characterized in that, Improving the gut microbiota is manifested by reducing the ratio of Firmicutes to Bacteroidetes.

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