Application of secondary metabolite of plant lactobacillus YN-06 in lowering cholesterol

Abstract

The invention relates to the technical field of microorganisms, and discloses an application of a secondary metabolite of phytobacterium plantarum YN-06 in lowering cholesterol, the phytobacterium plantarum YN-06 is preserved in China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, the preservation time is March 13, 2025, the preservation number is CCTCC NO: M 2025471, and the classification name is Lactobacillus plantarum YN-06. According to the application of the secondary metabolite of the phytobacterium plantarum YN-06 in lowering cholesterol, the YN-06 has a remarkable cholesterol degradation effect, can produce various beneficial metabolites such as L-beta-homoserine and 5-amino valeraldehyde, can be used for developing fermented dairy products and other functional foods with a cholesterol lowering function, and has a wide application prospect. And new strategies and products are provided for preventing and improving hypercholesteremia and related cardiovascular diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to application of secondary metabolites of Lactobacillus plantarum YN-06 in lowering cholesterol. Background Art

[0002] Probiotics have numerous physiological benefits for the body, such as regulating intestinal flora, lowering cholesterol, providing antioxidants, and enhancing immunity. They hold significant application value in the food industry and healthcare. Lactobacillus plantarum, a beneficial bacterium in the human intestine, plays a key role in maintaining intestinal microbiome balance and promoting health. In recent years, numerous preclinical and clinical studies, as well as in vitro experiments, have demonstrated that Lactobacillus plantarum has significant therapeutic effects on a variety of conditions, such as inflammatory bowel disease, irritable bowel syndrome, and cardiovascular disease. It can modulate intestinal flora structure, enhance intestinal barrier function, and maintain homeostasis. Hypercholesterolemia is a major threat to human health and is closely associated with cardiovascular and cerebrovascular diseases, such as atherosclerosis and coronary heart disease. According to statistics, the prevalence of hypercholesterolemia in adults in my country is as high as 18.6%, making it a leading risk factor for cardiovascular and cerebrovascular diseases. Changes in modern lifestyles, unhealthy diets (such as excessive intake of high-fat foods), lack of exercise, and other environmental factors, as well as genetic susceptibility, contribute to the high incidence of hypercholesterolemia. Hypercholesterolemia can also lead to numerous complications, severely impacting quality of life and healthy life expectancy. This study found that Lactobacillus plantarum YN-06 has significant cholesterol-degrading capabilities, with a cholesterol degradation rate of (71.04±0.81)%. It also exhibits good antioxidant properties and gastrointestinal tolerance. Furthermore, Lactobacillus plantarum YN-06 produces a variety of beneficial metabolites, such as L-β-homoserine and 5-aminovaleraldehyde. These metabolites not only help maintain intestinal microbial balance but also lower cholesterol levels through various mechanisms, including inhibiting cholesterol absorption and promoting cholesterol breakdown and excretion. The application of Lactobacillus plantarum YN-06 in fermented milk can significantly improve the quality and functionality of fermented milk, providing new ideas and methods for the development of novel cholesterol-lowering foods. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of secondary metabolites of Lactobacillus plantarum YN-06 in lowering cholesterol. YN-06 has a significant cholesterol-degrading effect and can produce a variety of beneficial metabolites such as L-β-homoserine and 5-aminovaleraldehyde. It can be used to develop fermented dairy products and other functional foods with cholesterol-lowering functions, providing new strategies and products for preventing and improving hypercholesterolemia and related cardiovascular diseases.

[0004] To achieve the above object, the present invention provides the use of secondary metabolites of Lactobacillus plantarum YN-06 in lowering cholesterol. Lactobacillus plantarum YN-06 is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 13, 2025, with a deposit number of CCTCC NO: M 2025471 and a classification name of Lactobacillus plantarum YN-06.

[0005] Furthermore, the secondary metabolites of Lactobacillus plantarum YN-06 include L-β-homoserine, 3-keto-inositolamine and 5-aminovaleraldehyde.

[0006] Furthermore, when used, the viable count of Lactobacillus plantarum YN-06 is not less than 5.0×10 6 CFU / mL.

[0007] Further, it is applied to: ① Reduce serum total cholesterol levels; ② Reduce the level of low-density lipoprotein cholesterol.

[0008] The present invention also provides a probiotic agent for lowering cholesterol, the active ingredient of which includes Lactobacillus plantarum YN-06, which is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 13, 2025, with a deposit number of CCTCC NO: M 2025471 and a classification name of Lactobacillus plantarum YN-06.

[0009] Furthermore, the viable count of Lactobacillus plantarum YN-06 is not less than 5.0×10 6 CFU / mL.

[0010] The present invention also provides the use of a secondary metabolite of Lactobacillus plantarum YN-06 in the preparation of an anti-Staphylococcus aureus preparation. The secondary metabolite of Lactobacillus plantarum YN-06 has an inhibition rate of 68.46±0.53% on Staphylococcus aureus. Lactobacillus plantarum YN-06 is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 13, 2025, with a deposit number of CCTCC NO: M 2025471 and a classification name of Lactobacillus plantarum YN-06.

[0011] The advantages and positive effects of the use of the secondary metabolites of Lactobacillus plantarum YN-06 in lowering cholesterol are: 1. The Lactobacillus plantarum YN-06 in the present invention has a significant cholesterol degradation effect, with a cholesterol degradation rate of (71.04±0.81)%, and has good gastrointestinal tolerance, with gastric acid resistance and bile salt survival rates both exceeding 80%, and can produce a variety of beneficial metabolites such as L-β-homoserine and 5-aminovaleraldehyde.

[0012] 2. When the Lactobacillus plantarum YN-06 of the present invention is used in fermented milk, the combination of YN-06 and commercial starter culture can significantly improve the acid production rate, viable cell count and gel structure stability of the fermented milk, shorten the fermentation time, enhance the flavor characteristics of the fermented milk, and exhibit excellent cholesterol-lowering ability. On the 14th day of storage, the cholesterol degradation rate of the fermented milk is as high as 67.92±0.48%.

[0013] 3. The fermented milk formulated with Lactobacillus plantarum YN-06 in the present invention contains beneficial metabolites such as glutamate, acetyleucine and malic acid, and produces 20 significantly different metabolites such as amino acids, peptides, and organic acids through pathways such as the tricarboxylic acid cycle and carbohydrate metabolism before and after storage, which helps to enhance the flavor and probiotic potential of the fermented milk.

[0014] 4. The Lactobacillus plantarum YN-06 of the present invention can be used to develop fermented dairy products and other functional foods with cholesterol-lowering function, providing new strategies and products for preventing and improving hypercholesterolemia and related cardiovascular diseases.

[0015] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments.

[0016] Biomaterial Deposit Lactobacillus plantarum ( Lactobacillus plantarum ) YN-06, taxonomically named Lactobacillus plantarum , was deposited in China Center for Type Culture Collection on March 13, 2025, with the deposit number CCTCC NO:M 2025471; the Latin name is Lactobacillus plantarum YN-06, deposited at Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A is the cholesterol removal rate of Lactobacillus plantarum YN-06, B is the triglyceride removal rate, C is the survival rate of Lactobacillus plantarum YN-06 in intestinal fluid and pH 2.0, D is the growth curve of Lactobacillus plantarum YN-06, E is the survival rate of Lactobacillus plantarum YN-06 in gastric fluid and 0.3% bile salt, and F is the inhibition rate of Staphylococcus aureus by Lactobacillus plantarum YN-06 metabolites; Figure 2 This is a circle map of the genome of Lactobacillus plantarum YN-06 in the embodiment of the present invention; Figure 3 This is the KEGG functional annotation classification diagram of Lactobacillus plantarum YN-06 in the embodiment of the present invention; Figure 4 GO functional annotation classification diagram of Lactobacillus plantarum YN-06 in the embodiment of the present invention; Figure 5 A is a map showing the drug resistance genes of Lactobacillus plantarum YN-06 in the embodiment of the present invention, and B is a map showing the virulence genes; Figure 6 This is the phylogenetic tree of Lactobacillus plantarum YN-06 in the embodiment of the present invention; Figure 7 The probiotic properties of Lactobacillus plantarum YN-06 in the embodiment of the present invention are shown in Figure 1, where A represents hydrophobicity, B represents self-aggregation rate, C represents 2-amino-4-methoxyphenol content, and D represents 1-deoxymannosyl inositol content. Figure 8 Figure A is a pie chart of the classification and proportion of metabolites of Lactobacillus plantarum YN-06 in the embodiment of the present invention, and Figure B is a Venn diagram of the metabolites of each group of strains; Figure 9 The physical and chemical properties of the Lactobacillus plantarum YN-06 fermented milk in the embodiment of the present invention, wherein A is hardness, B is consistency, C is cohesion, D is viscosity index, E is cholesterol degradation rate, and F is triglyceride degradation rate; Figure 10 This is the storage quality analysis of the Lactobacillus plantarum YN-06 fermented milk in the embodiment of the present invention, where A is the number of viable bacteria during storage, B is the sensory evaluation during storage, C is the cholesterol degradation rate during storage, and D is the triglyceride degradation rate during storage; Figure 11 This is the metabolite analysis of Lactobacillus plantarum YN-06 fermented milk before and after storage in the embodiment of the present invention, where A is a volcano plot, B is a heat map cluster analysis diagram, and C is a metabolic pathway analysis diagram; Figure 12 The following are the secondary metabolite analysis results of Lactobacillus plantarum YN-06 in the examples of the present invention, where A is the heat map of the difference in secondary metabolites between the YN-06 strain and the control strain, and B is the cholesterol degradation rate of the YN-06 secondary metabolite; Figure 13 A is the TIC diagram in the negative ion mode, and B is the TIC diagram in the positive ion mode; Figure 14 A is the standard mass charge-to-charge ratio diagram of 3-keto-inositolamine, B is the standard mass charge-to-charge ratio diagram of 5-aminovaleraldehyde, and C is the standard mass charge-to-charge ratio diagram of L-β-homoserine. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0019] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0020] All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally determined in accordance with national standards. Experimental instruments, equipment, and reagents in the following examples where the sources are not specified are all commercially available raw materials.

[0021] Unless otherwise defined or indicated, all technical and scientific terms used in this invention have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of the present invention. It should be noted that, unless there is a conflict, the embodiments and features of the embodiments of the present invention may be combined with each other.

[0022] The method for detecting the number of viable bacteria involved in the following embodiments is: using the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection".

[0023] The Lactobacillus plantarum YN-06 of the present invention is isolated by the following method: The collected yogurt samples were smeared on site to isolate the strains. The samples were first diluted in multiples and 10 -5 and 10 -6 Evenly spread 200 μL of the dilution gradient sample onto a plate of MRS solid medium and incubate aerobically at 37°C for 48–72 hours. Select individual colonies of varying morphology, size, and color and inoculate them into MRS liquid medium. Incubate aerobically at 37°C for 24–36 hours. Once the strains have grown well, perform Gram staining and microscopic examination. Preserve the isolates and extract genomic DNA for subsequent analysis.

[0024] The Lactobacillus plantarum YN-06 of the present invention forms colonies on an MRS solid culture medium with a diameter of 2 to 3 mm, round protrusions, neat edges, a smooth and moist surface, and a milky white to grayish white color.

[0025] The Lactobacillus plantarum YN-06 of the present invention has the following biological characteristics: the bacteria appear in the shape of short rods under a microscope, with relatively uniform thickness, and are arranged in single, chain-like, evenly dispersed or clustered states. It has no flagella or cilia and does not move.

[0026] The Lactobacillus plantarum YN-06 of the present invention is a strain with significant cholesterol degradation ability that is preliminarily screened out from 5 strains of Lactobacillus plantarum in a lactic acid bacteria resource library.

[0027] The present invention's Lactobacillus plantarum YN-06 exhibits a survival rate exceeding 80% after 3 hours of treatment in simulated gastric fluid at pH 2.5 and over 85% after 8 hours of treatment in simulated intestinal fluid at pH 8.0. This demonstrates good gastrointestinal tolerance and allows it to enter the human intestine as a viable bacterium, exerting its probiotic effects. These properties give it the potential to serve as a probiotic.

[0028] Example 1 Lactobacillus plantarum ( Lactobacillus plantarum ) Isolation and identification of YN-06: a. Lactobacillus plantarum ( Lactobacillus plantarum ) Isolation of YN-06: The strain was activated for two generations and then inoculated at a 3% inoculum into 5 mL of MRS-CHOL medium and 5 mL of triglyceride medium, respectively. Each experiment was performed in triplicate. A control group was supplemented with MRS liquid medium containing equal amounts of cholesterol and triglycerides. After incubation at 37°C for 48 hours, the fermentation broth was centrifuged at 5000 rpm for 10 minutes. The supernatant was collected and assayed for total cholesterol and triglyceride levels using assay kits.

[0029] The formula for calculating the cholesterol / triglyceride removal rate is: ; Wherein: A: cholesterol / triglyceride concentration in fermentation broth supernatant (mmol / L); B: cholesterol / triglyceride concentration in culture medium without inoculation of strain (mmol / L); D: cholesterol / triglyceride removal rate.

[0030] Acid resistance test: Adjust the pH of MRS medium to 2.0 with 1 mol / L hydrochloric acid, inoculate with 3% activated two-generation high-lipid-lowering lactic acid bacteria fermentation broth, and incubate at 37°C for 3 hours. Take the culture broth at 0 and 3 hours to measure the number of viable bacteria and determine the acid resistance. Repeat 3 times for each group ( Figure 1 (as shown in C in the figure).

[0031] Bile salt tolerance test: After activating a lactic acid bacteria strain with a high lipid-lowering rate for two generations, take the second generation of bacterial culture and inoculate it at a 3% inoculum into MRS liquid medium containing 0.3% ox bile salts. Incubate at 37°C for 9 hours. Take the bacterial culture at 0, 3, 6, and 9 hours to measure the number of viable bacteria to determine the bile salt tolerance of the test strain. Set up 3 replicates for each group ( Figure 1 (as shown in E in the figure).

[0032] Gastric fluid resistance test: Take 2 mL of 1 mol / L hydrochloric acid and adjust the pH to 2.0 with distilled water. Dissolve with pepsin at 1 g / 100 mL and sterilize with a 0.22 μm filter. Add 10% of the activated bacterial solution to artificial gastric fluid, mix thoroughly, and incubate at 37°C and 100 rpm. Measure the number of viable cells at 0 and 3 hours of incubation. Repeat three times. Figure 1 (as shown in E in the figure).

[0033] Intestinal fluid resistance test: Dissolve 0.85g KH2PO4 in 62.5L distilled water, adjust the pH to 7.6 with 1mol / L NaOH, and dissolve with trypsin at a rate of 1g / 100mL on a clean bench. Sterilize with a 0.22μm filter. Mix 1.0mL of activated bacterial solution with 9.0mL of artificial intestinal fluid, incubate at 37°C and 100r / min, and measure the viable bacterial count at 0, 3, and 6h using the pouring method. Repeat three times ( Figure 1 (as shown in C in the figure).

[0034] Growth characteristics determination: The strain was inoculated into MRS liquid medium and cultured for 28 h. The number of viable bacteria and OD were measured every 2 h. 600 Absorbance value, repeated three times, and the growth curve was drawn ( Figure 1 (as shown in D in the figure).

[0035] Morphological observation: After the selected strains are separated by streaking on the plate, the colony morphology, size, and color are observed and recorded. Single colonies with dispersed colonies and good growth are selected for Gram staining. The bacterial morphology is observed under an optical microscope and photographed for record.

[0036] Lipid-lowering ability assay: The initial viable bacterial count ratios of YN-06 (A) and SMN3-2 (B) were set at (0:1, 1:0, 1:1, 1:2, and 2:1) and inoculated into MRS-CHOL medium and triglyceride medium, respectively. Uninoculated medium was used as the control group. The cells were incubated at 37°C for 48 h and centrifuged at 5000 rpm for 10 min. The supernatant was collected and the cholesterol removal rate and triglyceride removal rate were measured using a total cholesterol test kit and a triglyceride test kit, respectively.

[0037] From 80 test strains, the lactic acid bacteria strain YN-06 with significant cholesterol removal rate, triglyceride degradation rate and gastrointestinal tolerance was screened and obtained. The cholesterol removal rate was (71.04±0.81)%, and the triglyceride removal rate was (35.21±0.68)%. Figure 1 (as shown in A and B).

[0038] Figure 1YN-06 is Lactobacillus plantarum, derived from yogurt in Xilin Gol League, Inner Mongolia; QS6-1 is Lactobacillus delbrueckii, derived from yogurt in Zhenglan Banner, Xilin Gol League, Inner Mongolia; 2018-1 is Lactobacillus helveticus, derived from yogurt in Zhenglan Banner, Xilin Gol League, Inner Mongolia; 9-8 is Lactococcus lactis, derived from milk tofu in Zhenglan Banner, Xilin Gol League, Inner Mongolia; 10-5 is Lactococcus lactis, derived from milk tofu in Zhenglan Banner, Xilin Gol League, Inner Mongolia; 9-4 is Lactobacillus kumiss, derived from yogurt in Xilin Gol League, Inner Mongolia; QS1-3 is Lactobacillus plantarum, derived from yogurt in Zhenglan Banner, Xilin Gol League, Inner Mongolia; 3-1 is Pediococcus acidilactici, derived from pickled cabbage in Xilin Gol League, Inner Mongolia; NDF11-5 is lactic acid S6-7 is Lactobacillus acidophilus, sourced from sauerkraut in Xilin Gol League, Inner Mongolia; S12-2 is Lactococcus lactis, sourced from mare's milk in Mongolia; S10-7 is Lactobacillus plantarum, sourced from sauerkraut in Xilin Gol League, Inner Mongolia; MN8-3 is Lactobacillus helveticus, sourced from mare's milk in Mongolia; STM1 is Lactobacillus acidophilus, sourced from sauerkraut in Xilin Gol League, Inner Mongolia; S12-6 is Lactobacillus helveticus, sourced from mare's milk in Xilin Gol League, Inner Mongolia; S10-7 is Lactobacillus plantarum, sourced from mare's milk in ...

[0039] b. Preservation and freeze-dried powder preparation of Lactobacillus plantarum: Lactobacillus plantarum YN-06 was removed from the -80°C freezer and inoculated into 4.5 mL of MRS liquid medium. The culture was then incubated in a 37°C incubator under facultative anaerobic conditions for 24 hours to complete the initial activation. The activated culture was then transferred to fresh MRS liquid medium at a 3% (v / v) volume ratio and cultured and subcultured. The activation and subculture steps were repeated three times before the culture was expanded in 50 mL centrifuge tubes. After incubation, the culture was centrifuged at 4200 × g for 5 minutes. The supernatant was discarded, and the precipitated sludge was washed with PBS twice. The supernatant from the second centrifugation was discarded, and skim milk preservative was added to the remaining sludge. The sludge and preservative were thoroughly mixed by pipetting and shaking. Finally, the mixed culture was aliquoted into cryovials and ampoules and stored in a -80°C freezer for subsequent experiments.

[0040] The preserved plant lactobacillus was activated and passaged three times with MRS liquid culture medium at a volume ratio of 2% (v / v), streaked twice, cultured in liquid at 37°C for 24 hours and in solid culture for 48 hours, inoculated into a centrifuge cup of 700 ml liquid MRS culture medium according to the original proportion, cultured at 37°C for 24 hours, centrifuged at a speed of 4200×g, washed twice with 0.85% (w / v) sterilized saline, centrifuged, mixed evenly with bacterial powder protective agent in a 1:1 ratio, placed in a sterilized glass dish, placed in a freeze dryer for freeze drying for 48 hours, and the sample was ground into powder, placed in a 50 ml centrifuge tube, and placed at -20°C for use.

[0041] c. Whole genome sequencing: The purified strain was cultured in an incubator at 37°C for 24 h, centrifuged at 4200×g for 10 min, and the cells were collected and stored on dry ice before being sent to Meiji Biotech for whole genome sequencing.

[0042] Genomic DNA is collected and purified, then processed into 8-10k fragments. Using Covaris software, the DNA is broken into fragments of varying lengths. The ends of these fragments are then linked, and fragments of appropriate length are selected based on specific criteria. This is then used to construct a library for genome sequencing. Fluorescent labeling is used to read and polymerize nucleotides to determine the sequence of the template DNA fragments.

[0043] Based on the raw data obtained from sequencing on the Illumina sequencing platform, genome sequences were assembled de novo. Third-generation sequence assembly was performed using the assembly software Unicycler v0.4.8. Sequence correction was performed using Pilon v1.22 software during the assembly process. If any overlap at either end of the assembled sequence exceeded a specific length, the sequence was joined end to end to form a circular structure. The overlapping sequence at one end was then removed to obtain complete chromosome and plasmid sequences. Finally, gene prediction was performed on the assembled sequence to determine the gene location and sequence information for each sample. Software such as Glimmer, GeneMarkS, Prodigal, and Barrnap were used to predict the type, location, and sequence of all RNA in the genome of each sample. 3474 genes were predicted, totaling 2.83 Mb in length. The shortest gene fragment was 860 bp, with coding genes accounting for 82.99% and a GC content of 45.43%. A 0.96 Mb plasmid was assembled. Most of its coding genes were distributed between 200 and 1000 bp, with only 178 genes under 200 bp. Detailed genome component analysis results are shown in Table 1.

[0044] Table 1 Statistics of genome analysis results of YN-06 ;

[0045] The obtained genes were compared with the database by homology alignment to obtain the annotation description information of the genes. The predicted CDS were functionally annotated using the GO, COG, KEGG, and CAZy databases, and the Circos software was used to draw the genome circular map ( Figure 2 ). Gene function annotation showed that YN-06 has outstanding potential in carbohydrate and amino acid metabolism. Resistance gene prediction found that it contains 196 resistance genes related to 11 types of antibiotics, but there are unverified potential resistance genes. Virulence gene prediction found that it contains 277 virulence genes, but whether these genes are expressed needs further verification. The specific gene functions and gene predictions are as follows. Figure 3 、 Figure 4 and Figure 5 shown.

[0046] By constructing a phylogenetic tree ( Figure 6

[0047] d. Functional gene characteristics of Lactobacillus plantarum YN-06: Tolerance-related functional genes of Lactobacillus plantarum YN-06 were annotated, and 10 acid tolerance-related genes (lysC, dapA, argC, argH, metE, argR, argJ, argS, murE, lysS); 8 heat tolerance-related genes (dnaK, hrcA, dnaJ, dnaA, dnaG, dnaB, dnaE, dnaN); 2 cold tolerance-related genes (cspA, clpC); and 3 bile salt tolerance-related genes (cbh, purD, gcvH) were annotated.

[0048] Lactobacillus plantarum YN-06 was compared with the VFDB (Virulence Factors of Pathogenic Bacteria) database and the VirulenceFinder database (https: / / cge.cbs.dtu.dk / services / VirulenceFinder), using sequence similarity >90% and sequence coverage >60% as screening criteria. The results showed that no potential virulence-related genes were detected in the YN-06 genome.

[0049] Genes related to probiotic functions were detected in the genome of Lactobacillus plantarum YN-06 and annotated, and functional genes related to riboflavin, lactic acid, bioactive peptide synthesis, and acid and bile resistance were obtained. The specific genes are shown in Table 2.

[0050] Table 2 Probiotic-related genes ;

[0051] Example 2 Study on the probiotic properties of Lactobacillus plantarum YN-06 1. Experimental methods: Determination of strain adhesion: Hydrophobicity was determined using the hydrocarbon adhesion method. YN-06 was inoculated into MRS medium and activated continuously until the third generation. The culture was centrifuged at 4200 rpm for 10 minutes. The precipitated cells were collected and resuspended in sterile saline, adjusting the OD to 0.5 ± 0.02. A xylene solution was added and vortexed for 1 minute. The mixture was allowed to stand for 1 hour, and the lower aqueous phase was aspirated and the absorbance measured at 600 nm. Surface hydrophobicity (%) = (A0 - A1) / A0 × 100.

[0052] Determination of autoaggregation rate: prepare bacterial suspension, adjust the OD value to 0.5±0.02, and let it stand at 37℃ for 20 hours. Measure the absorbance of the supernatant (600nm). The autoaggregation rate (%) = (A0-A2) / A0×100.

[0053] Determination of the antioxidant activity of strains: Determination of DPPH free radical scavenging rate: Pipette 2 mL of the strain suspension into a centrifuge tube containing 1 mL of DPPH solution. Mix thoroughly and incubate in the dark for 30 minutes. After centrifugation, collect the supernatant and measure absorbance. Calculate the DPPH free radical scavenging rate (%) using the formula: [1 - (A1 - A0) / A2] × 100.

[0054] Determination of hydroxyl radical (-OH) scavenging rate: Prepare a salicylic acid ethanol solution, ferric sulfate solution, hydrogen peroxide solution, and strain suspension. Mix and incubate at 37°C in a water bath for 15 minutes. Centrifuge and measure the absorbance of the supernatant at 510 nm. Calculate the hydroxyl radical (-OH) scavenging rate (%) = (1-A1 / A0) × 100.

[0055] 2. Experimental results: like Figure 7 As shown in the study of probiotic properties, Lactobacillus plantarum YN-06 showed excellent adhesion, with a hydrophobicity of 59.56% ( Figure 7 A), the self-aggregation rate is 82.80% ( Figure 7 B), indicating that it can effectively adhere to and colonize in the intestine. In the antioxidant experiment, the DPPH free radical scavenging rate was 59.92%, and the hydroxyl free radical scavenging rate was 48.31%, indicating good antioxidant capacity. Metabolite analysis showed that the strain can produce unique metabolites such as L-β-homoserine and 5-aminovaleraldehyde, as well as beneficial metabolites such as aspartyl-arginine, threonyl-alanine, and 2-amino-4-methoxyphenol ( Figure 7 C) and 1-deoxymannosyl inositol ( Figure 7 D), etc., which helps to improve the flavor and nutritional value of fermented milk.

[0056] Figure 7 IMAU12337 is a strain of Lactobacillus plantarum derived from mare's milk in Xilin Gol League, Inner Mongolia; IMAU12339 is a strain of Lactobacillus plantarum derived from milk tofu in Zhenglan Banner, Xilin Gol League, Inner Mongolia; IMAU12340 is a strain of Lactobacillus plantarum derived from milk tofu in Zhenglan Banner, Xilin Gol League, Inner Mongolia; and P-8 is a strain of Lactobacillus plantarum derived from yogurt in Urad Middle Banner, Bayannur City, Inner Mongolia. These strains are publicly available and can be obtained by contacting the applicant if required.

[0057] Example 3 Study on the metabolic characteristics of Lactobacillus plantarum YN-06 1. Experimental methods: Obtain fermentation broth containing secondary metabolites: activate the strain for two generations, inoculate 5 mL of MRS-CHOL medium and 5 mL of triglyceride medium at a 3% inoculum, and culture at 37°C for 48 hours to obtain fermentation broth containing secondary metabolites.

[0058] Isolation and identification of secondary metabolites: The fermentation broth containing secondary metabolites was added with an extract of methanol and acetonitrile (1:1) in a ratio of 1:4, vortexed for 30 seconds, ultrasonicated for 10 minutes, and allowed to stand at -40°C for 1 hour before centrifugation to obtain the supernatant and bacterial cells. The supernatant was transferred to a sample injection bottle for detection on the instrument.

[0059] Standard solutions of 3-keto-inositolamine, 5-aminovaleraldehyde, and L-β-homoserine were prepared, and secondary metabolites were separated using ultra-high performance liquid chromatography (UPLC) equipped with a Waters ACQUITY UPLC BEH Amide column. 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 sample tray temperature was maintained at 4°C, and the injection volume was precisely controlled to 2 μL.

[0060] 2. Experimental results: like Figure 13 As shown in Figure A, in the negative ion mode, 3-keto-inositolamine was detected at a retention time of 0.96 minutes. Its mass-to-charge ratio (Feature M177T58) matched that of the standard library (L1131). The characteristic peaks appeared in the m / z range of 40–160, with few unmatched fragments and high qualitative confidence ( Figure 14 Middle A); 5-aminopentanal was detected at a retention time of 3.22 minutes. Its mass-to-charge ratio (Feature M152T193) matched the standard library (B9A9, D1V9), and its m / z range was centered at 60–140, corresponding to the typical fragment ions of the substance ( Figure 14 At the retention time of 3.66 minutes, L-β-homoserine was detected, and its mass-to-charge ratio graph showed a high-intensity ion peak at m / z 70–100, which was consistent with the molecular structure characteristics of the substance ( Figure 14 Middle C).

[0061] like Figure 13 As shown in Figure B, in the positive ion mode, 3-keto-inositolamine was detected at a retention time of 0.96 minutes. Its mass-to-charge ratio diagram (Feature M177T58) showed a main peak at m / z 100–160, which was consistent with the fragment information of the standard library (L1131). The ionization efficiency was high ( Figure 14A); 5-aminovaleraldehyde was detected at a retention time of 3.22 minutes. Its mass-to-charge ratio diagram showed that it had characteristic ion peaks in the range of m / z 80–120, which matched the standard library, indicating that the substance can also be effectively ionized in the positive ion mode ( Figure 14 At a retention time of 3.66 minutes, L-β-homoserine was detected, and its mass-to-charge ratio showed a high relative intensity peak in the range of m / z 70–90, which was consistent with the protonated ion characteristics of this substance in the positive ion mode ( Figure 14 Middle C).

[0062] like Figure 8 and Figure 12 As shown in Figure A, in metabolic profile analysis, the metabolite composition of YN-06 is similar to that of other strains, but there are significant differences. PCA and OPLS-DA analyses revealed significant differences in the metabolites of YN-06 and other strains. YN-06-specific metabolites include L-β-homoserine and 5-aminovaleraldehyde, which confer unique functions or characteristics to the strain. Differential metabolites, such as aspartyl-arginine and threonyl-alanine, promote the growth and fermentation of lactic acid bacteria, enhancing the quality and nutritional value of fermented milk. Figure 12 As shown in Figure B, secondary metabolites are mainly present in the supernatant, and the supernatant has a higher cholesterol degradation rate.

[0063] L-β-homoserine is an intermediate metabolite of the aspartate family of amino acids and participates in multiple biosynthetic pathways. Through specific enzymatic reactions, it participates in the synthesis or conversion of other amino acids, thereby optimizing their metabolic pathways, improving the growth efficiency and metabolite production of lactic acid bacteria. This is crucial for maintaining amino acid balance and normal energy metabolism during lactic acid fermentation. L-β-homoserine also contributes to the synthesis of lactic acid bacterial cell walls, specific active substances, and secondary metabolites with antimicrobial activity (such as bacteriocins and antimicrobial peptides).

[0064] 5-Aminovaleraldehyde is an aldehyde compound that is typically volatile and can produce a distinctive odor. It participates in the Maillard reaction, thereby contributing to the flavor of food or beverages. For example, aldehydes are important flavor components in certain fermented foods (such as cheese and wine) or heat-treated foods (such as grilled meats). Due to the volatile flavor it produces, 5-Aminovaleraldehyde can be used to improve the flavor and mouthfeel of foods. For example, in the flavor and fragrance industry, it can be used as a flavor enhancer or flavor precursor.

[0065] As a signaling molecule or metabolic regulator, 3-keto-inositolamine participates in the metabolic regulation process within lactic acid bacteria cells. It can interact with specific receptors or enzymes within the cells and is associated with the activity of related metabolic pathways. By regulating the growth, reproduction, and synthesis of metabolites of lactic acid bacteria, it influences their utilization of carbon and nitrogen sources, as well as their energy metabolism. Furthermore, 3-keto-inositolamine affects fermentation characteristics, influencing the pH value, viscosity, and other physicochemical properties of the fermentation broth, thereby influencing the growth environment and fermentation process of lactic acid bacteria. It also interacts with other fermentation products, indirectly affecting the flavor, texture, and other quality characteristics of the fermented product.

[0066] It can be seen that L-β-homoserine and 3-keto-inositolamine in the metabolites of Lactobacillus plantarum YN-06 give the strain a certain antibacterial potential, and 5-aminovaleraldehyde gives the strain a certain aroma-producing characteristic.

[0067] The inhibition rate of Lactobacillus plantarum YN-06 metabolites on Staphylococcus aureus is as follows Figure 1 As shown in F.

[0068] Cholesterol degradation rate of Lactobacillus plantarum YN-06 metabolites Figure 12 As shown in Figure B, the cholesterol degradation rate of the fermentation broth was 71.04±0.81%, the cholesterol degradation rate of the metabolites (supernatant) was 68.58±0.62%, and the cholesterol degradation rate of the bacteria was 23.68±0.24%, indicating that the cholesterol degradation effect of the fermentation broth of the strain mainly comes from the metabolites.

[0069] Example 4 Application of Lactobacillus plantarum YN-06 in fermented milk Experimental method: Preparation of fermented milk: Skim milk was preheated, added with 6.5% white sugar, homogenized (20 MPa), sterilized at 95°C for 30 min, and then cooled to 42°C. The strains and starter cultures were inoculated according to the inoculum amount in Table 3 and fermented at 37°C to a pH of 4.5-4.6. The milk was stored at 4°C and samples were collected at 1, 7, 14, 21, and 28 days.

[0070] Table 3 Fermented milk sample groups and strain inoculation amounts ;

[0071] Determination of changes in microrheological parameters of fermented milk: The elasticity index, flow index and solid-liquid balance value were measured with an optical micro-rheometer, and the fermentation process was automatically stopped at the end point.

[0072] Determination of physical and chemical indicators of fermented milk: pH value: The fermented milk samples were restored to room temperature and measured with a pH meter. Each group of samples was measured in parallel 3 times and the average value was taken.

[0073] Titrate acidity: Weigh 10.0 g of fermented milk, add 20 mL of distilled water, and titrate with 0.1 mol / L NaOH solution until the solution turns slightly red or light pink and does not fade within 30 s. Record the amount of NaOH used and calculate the acidity according to the formula. Repeat the determination three times and take the average value.

[0074] Water holding capacity: Weigh 20 g of fermented milk sample, let it stand for 2 h, weigh the mass of the filtrate, and calculate the water holding capacity according to the formula. Repeat each group of samples 3 times and take the average value.

[0075] Viscosity: The fermented milk was returned to room temperature and measured at 100 r / min for 30 s using a #4 rotor of the viscometer. The data were recorded. Each group of samples was measured three times in parallel and the average value was obtained.

[0076] Determination of textural properties of fermented milk: The hardness, cohesion, and viscosity of fermented milk were measured using a TTA XT plus texture analyzer with the following settings: pre-test speed of 1.5 mm / s, test speed of 1.0 mm / s, retraction speed of 1.5 mm / s, initial stress of 2.0 g, compression degree of 20%, compression time of 5 s, and travel distance of 20 mm. Each group of samples was repeated three times and the average value was taken.

[0077] Determination of viable bacterial count: Weigh 25 g of sample and mix with 225 mL of normal saline, shake for 15 min, dilute, take 1 mL and inoculate MRS solid medium, incubate anaerobically at 37°C for 48 h, and then count.

[0078] Sensory index determination of fermented milk: 10 postgraduate students scored based on color (20 points), flavor (40 points) and texture (40 points).

[0079] Lipid-lowering ability assay: Cholesterol degradation rate: A cholesterol standard curve was prepared according to the national standard "GB 5009.128-2016". Fermented milk was inoculated into MRS-CHOL medium and cultured at 37°C for 24 h. The culture was then centrifuged (4200 rpm, 10 min) and the supernatant was collected to calculate the cholesterol degradation rate according to the formula.

[0080] Triglyceride degradation rate: Use a triglyceride test kit, inoculate fermented milk into triglyceride culture medium, and culture at 37℃ for 48 h. Calculate the triglyceride degradation rate according to the formula.

[0081] Metabolite determination: After thawing, samples were extracted with methanol and acetonitrile in a 1:4 ratio. The samples were vortexed for 30 seconds, sonicated for 10 minutes, and allowed to stand at -40°C for 1 hour before centrifugation. The supernatant was sampled for injection. Chromatographic separation and detection were performed using an AB SCIEX-Triple TOF 6600+ system equipped with an Acquity UPLC BEH Amide column. 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 tray temperature was 4°C.

[0082] Experimental results: Analysis of the physical and chemical properties of fermented milk Figure 9 As shown, compared with other groups, the hardness, consistency, cohesion and viscosity index of the fermented milk in the compound group were significantly improved, and the cholesterol degradation rate and triglyceride degradation rate of the fermented milk in the compound group were significantly improved.

[0083] Fermented milk storage quality analysis Figure 10 As shown in the figure, after 28 days of storage, the number of viable bacteria in the compound group remained at 10 10 The CFU / mL was higher than that of the single bacteria group and the commercial group. After storage, the sensory score, cholesterol degradation rate, and triglyceride degradation rate of the compound group were significantly higher than those of the single bacteria group and the commercial group.

[0084] Metabolite analysis before and after milk storage Figure 11 As shown in the figure, 20 significantly different metabolites such as amino acids, peptides, and organic acids were produced through pathways such as the tricarboxylic acid cycle and carbohydrate metabolism before and after storage.

[0085] Lactobacillus plantarum YN-06 and commercial starter culture PYS-010 were compounded in a specific ratio (YN-06 order of magnitude was 5.0×10 6 CFU / mL, PYS-010 (containing thermophilic Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) was purchased from Beijing Ketuo Hengtong Biotechnology Co., Ltd. and added at a rate of 0.03% to prepare fermented milk. The time for the pH to reach the fermentation endpoint (4.6) was 3.5 hours, which significantly improved the production efficiency of fermented milk. After electronic nose detection, the response value of W5S (nitrogen oxides) in the compound group was higher than that of the A0 single bacteria and B0 commercial groups. Nitrogen oxides participate in the formation of the flavor of fermented milk, giving the fermented milk a richer and unique flavor characteristic. A certain concentration of nitrogen oxides has an antibacterial effect, which helps maintain the quality and safety of fermented milk. After storage for 1d-28d, the number of viable bacteria in the compound group remained at 10 10 CFU / mL and above, which was significantly higher than that of the single bacteria group and the commercial group.

[0086] In summary, the combination of Lactobacillus plantarum YN-06 and a commercial starter culture significantly improved the fermented milk's acid production rate, viable cell count, gel structure, flavor, and cholesterol degradation (67.92 ± 0.48%), while maintaining stable storage quality. Fermented milk containing Lactobacillus plantarum YN-06 exhibited beneficial metabolites such as glutamate, acetyleucine, and malic acid. Furthermore, 20 significantly different metabolites, including amino acids, peptides, and organic acids, were produced before and after storage through pathways such as the tricarboxylic acid cycle and carbohydrate metabolism, contributing to the enhanced flavor and beneficial potential of the fermented milk.

[0087] Therefore, the present invention uses the secondary metabolites of the above-mentioned Lactobacillus plantarum YN-06 for use in lowering cholesterol. YN-06 has a significant cholesterol degradation effect and can produce a variety of beneficial metabolites such as L-β-homoserine and 5-aminovaleraldehyde, which can be used to develop fermented dairy products and other functional foods with cholesterol-lowering functions; during fermentation, YN-06 is compounded with commercial starter cultures to significantly improve the acid production rate, viable cell count and gel structure stability of fermented milk, shorten the fermentation time, and enhance the flavor characteristics of the fermented milk, providing new strategies and products for preventing and improving hypercholesterolemia and related cardiovascular diseases.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of a secondary metabolite of Lactobacillus plantarum YN-06 in lowering cholesterol, characterized in that: Lactobacillus plantarum YN-06 was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 13, 2025, with the deposit number CCTCC NO: M 2025471. Lactobacillus plantarum YN-06.

2. The use of the secondary metabolite of Lactobacillus plantarum YN-06 according to claim 1 in lowering cholesterol, characterized in that: The secondary metabolites of Lactobacillus plantarum YN-06 include L-β-homoserine, 3-keto-inositolamine and 5-aminovaleraldehyde.

3. The use of the secondary metabolites of the Lactobacillus plantarum YN-06 strain according to claim 1 in lowering cholesterol, characterized in that: When used, the viable count of Lactobacillus plantarum YN-06 should not be less than 5.0×10 6 CFU / mL.

4. The use of the secondary metabolites of the Lactobacillus plantarum YN-06 according to claim 1 in lowering cholesterol, characterized in that: Applies to: ① Reduce serum total cholesterol levels; ② Reduce the level of low-density lipoprotein cholesterol.

5. A cholesterol-lowering probiotic agent, characterized in that: Its active ingredients include Lactobacillus plantarum YN-06, which is deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 13, 2025, with a deposit number of CCTCC NO: M 2025471, and a classification name of Lactobacillus plantarum YN-06.

6. The cholesterol-lowering probiotic agent according to claim 5, characterized in that: The viable count of Lactobacillus plantarum YN-06 is not less than 5.0×10 6 CFU / mL.

7. Use of a secondary metabolite of Lactobacillus plantarum YN-06 in the preparation of an anti-Staphylococcus aureus preparation, characterized in that: The secondary metabolites of Lactobacillus plantarum YN-06 have an inhibition rate of 68.46±0.53% against Staphylococcus aureus. Lactobacillus plantarum YN-06 was deposited in China Center for Type Culture Collection, Wuhan University, Wuhan, China, on March 13, 2025, with a deposit number of CCTCC NO: M 2025471 and a classification name of Lactobacillus plantarum YN-06.

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