Phytobacterium plantarum SP055, afterbiogen and application of phytobacterium plantarum SP055 in maintaining blood sugar and blood fat health

Abstract

The invention provides the plant lactobacillus SP055, the aftergrowth of the plant lactobacillus SP055 and the application of the plant lactobacillus SP055 in maintaining blood sugar and blood fat health. The plant lactobacillus SP055 can be used for treating and / or preventing colitis; treating and / or preventing non-alcoholic fatty liver or atherosclerosis; or treating and / or preventing insulin resistance; the plant saponin or polyphenol raw material is fermented to produce rare saponin and small molecule polyphenol.

Description

Technical Field

[0001] This application relates to the field of microbiology, specifically to Lactobacillus plantarum SP055, its postbiotics, its fermentation products, and their applications in the preparation of food, pharmaceuticals, health foods, cosmetics, and pet foods. Background Technology

[0002] Lactobacillus plantarum ( Lactobacillus plantarum is a type of lactic acid bacteria, a group of Gram-positive, short rod-shaped, microaerophilic, acid-tolerant lactobacilli commonly used in food fermentation. It can ferment a variety of foods, such as yogurt, fermented sausages, bread, and pickles. The application of Lactobacillus plantarum is currently characterized by high academic output but low industrial conversion.

[0003] Genomics and phenomics studies have shown that *Lactobacillus plantarum* possesses one of the world's largest and most open pan-genomes (>3200 core genes, average genome size 3.2-3.4 Mb), adapted to multiple carbon sources and with significant functional module discovery capabilities. Of the more than 3000 traditional fermentation source isolates preserved in China, less than 10% have completed whole-genome sequencing and functional annotation. In the past five years, more than 1200 related papers have been published in China, accounting for 46% of the global total. However, domestic companies hold fewer than 50 patented strains, indicating a serious gap between scientific research and product development.

[0004] Currently, over 80% of the market applications of *Lactobacillus plantarum* are still as a starter culture in traditional fermented foods, especially kimchi; it has not yet been incorporated into mainstream dietary supplement formulations as a probiotic end product. Sampling of shelves in offline pharmacies / baby stores shows that only 2.3% of products are clearly labeled with "*Lactobacillus plantarum* + strain number".

[0005] New strains of Lactobacillus plantarum are still needed in this field. Summary of the Invention

[0006] This invention discovered a strain of Lactobacillus plantarum ( SP055. Lactobacillus plantarum SP055 was deposited on July 2, 2025 at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101), with accession number CGMCC No. 35072.

[0007] Lactobacillus plantarum SP055 has the following functions: (a) prevention and relief of colitis; (b) both live and dead bacteria have lipid-lowering effects; (c) fermented ginseng obtained using Lactobacillus plantarum SP055 has lipid-lowering and insulin resistance-improving effects; (d) fermented ginseng obtained using Lactobacillus plantarum SP055 has flavor-improving effects of "reducing bitterness, increasing flavor, increasing aroma and reducing nitrogen"; (e) as a fermenting agent, it can be converted into rare saponins such as ginseng, Gynostemma pentaphyllum, and Panax notoginseng; or (f) as a fermenting agent, it can convert rutin into quercetin.

[0008] In the first aspect, *Lactobacillus plantarum* is provided, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 35072.

[0009] In the second aspect, the metagener of *Lactobacillus plantarum* from the first aspect is provided.

[0010] In one implementation, the metabiotic is one or more of the following: cell-type metabiotics, cell and metabolite-type metabiotics, and lysate cell-type metabiotics.

[0011] A method for preparing a metabiotic from *Lactobacillus plantarum* according to the first aspect is also provided, comprising the step of inactivating *Lactobacillus plantarum* to prepare the metabiotic. In one embodiment, the metabiotic is inactivated bacterial somatic cells. In one embodiment, inactivation is heat inactivation.

[0012] In a third aspect, a method for fermenting plant material or plant extract is provided, which includes culturing *Lactobacillus plantarum* of the first aspect in the presence of plant material or plant extract to produce ferments.

[0013] In one embodiment, the plant material or plant extract contains one or more of the following active ingredients: saponins and rutin.

[0014] In one embodiment, the plant is one or more of ginseng, astragalus, licorice, gynostemma pentaphyllum, notoginseng, sophora japonica, buckwheat, rue, and dried tangerine peel. In one embodiment, the plant extract is a water-based plant extract.

[0015] In one implementation, the method includes:

[0016] (1) In the presence of extracts of ginseng, astragalus, licorice, and / or gynostemma pentaphyllum or any one thereof, culture *Lactobacillus plantarum* of the first aspect to increase the conversion or content of rare saponins; or

[0017] (2) In the presence of extracts of Sophora japonica buds, buckwheat, rue, and / or dried tangerine peel or any one thereof, *Lactobacillus plantarum* of the first aspect is cultured to convert rutin into quercetin and / or isoquercetin; or

[0018] (3) In the presence of Panax notoginseng or its extracts, culture of Lactobacillus plantarum of the first aspect to increase the content of saponins in the form of protopanaxadiol (PPT) or protopanaxadiol (PPD).

[0019] In the fourth aspect, a fermentation product produced according to the method described in the third aspect is provided.

[0020] In a fifth aspect, a composition is provided comprising one or more of the following: *Lactobacillus plantarum* according to the first aspect, a metabiotic according to the second aspect, and a ferment according to the fourth aspect; and further comprising a pharmaceutically or health-product-acceptable carrier, diluent, or excipient. In one embodiment, the composition is a pharmaceutical, food, health product, or feed.

[0021] In a sixth aspect, the use of *Lactobacillus plantarum* of the first aspect, the metabiotic of the second aspect, and / or the ferment of the fourth aspect in the preparation of a medicament, said medicament being used for:

[0022] (1) Treatment and / or prevention of colitis;

[0023] (2) Treatment and / or prevention of non-alcoholic fatty liver disease or atherosclerosis;

[0024] (3) Treatment and / or prevention of type 2 diabetes or insulin resistance;

[0025] The fermented product mentioned above is ginseng fermented product.

[0026] This application also provides methods for treating and / or preventing colitis in subjects, comprising administering to the subjects *Lactobacillus plantarum* of the first aspect, postbiotics of the second aspect, and / or ferments of the fourth aspect.

[0027] This application also provides methods for treating and / or preventing non-alcoholic fatty liver or atherosclerosis in subjects, comprising administering to the subjects *Lactobacillus plantarum* of the first aspect, postbiotics of the second aspect, and / or ferments of the fourth aspect.

[0028] This application also provides methods for treating and / or preventing type 2 diabetes or insulin resistance in subjects, comprising administering to the subject *Lactobacillus plantarum* of the first aspect, postbiotics of the second aspect, and / or ferments of the fourth aspect.

[0029] In each implementation, the subject is a mammal, such as a human. In each implementation, the colitis is ulcerative colitis.

[0030] In a seventh aspect, the use of *Lactobacillus plantarum* of the first aspect, postbiotics of the second aspect, and / or ferments of the fourth aspect in the preparation of health products is provided, said health products being used for:

[0031] (1) It helps maintain healthy blood sugar levels;

[0032] (2) It helps maintain healthy levels of blood lipids, including cholesterol and / or triglycerides.

[0033] It also provides methods for maintaining healthy blood glucose levels in subjects, which include administering *Lactobacillus plantarum* (a first aspect), postbiotics (a second aspect), and / or ferments (a fourth aspect) to subjects.

[0034] Methods for maintaining healthy blood lipid levels in subjects are also provided, which include administering *Lactobacillus plantarum* (as described in the first aspect), postbiotics (as described in the second aspect), and / or ferments (as described in the fourth aspect) to the subjects. In one embodiment, blood lipids include cholesterol and / or triglycerides.

[0035] In an eighth aspect, a method for converting saponins or rutin is provided, comprising the steps of converting a prototype saponin into a rare saponin using *Lactobacillus plantarum* of the first aspect, or converting rutin into quercetin or isoquercetin. In one embodiment, the prototype saponin comprises one or more of the following: ginsenosides Rb2, Rb3, Rc, Rd, Re, and Rg1. In one embodiment, the rare saponin comprises one or more of the following: ginsenosides Rg5, Rk1, Rg6, Rk3, Rg3, Rh2, Rg2, Rh1, and CK (Compound K, 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol).

[0036] In the ninth aspect, the use of *Lactobacillus plantarum* as a fermenting agent as described in the first aspect is provided.

[0037] In one embodiment, the fermenting agent is used to ferment one or more of ginseng, astragalus, licorice, gynostemma pentaphyllum, sophora japonica, buckwheat, rue, tangerine peel, or extracts thereof.

[0038] In a tenth aspect, a method for improving the sensory pleasantness of saponin-containing plants is provided, comprising a step of fermenting the plant using *Lactobacillus plantarum* of the first aspect, wherein the sensory pleasantness includes taste and / or olfactory pleasantness. In one embodiment, the saponin-containing plant is one or more of ginseng, astragalus, licorice, notoginseng, and gynostemma pentaphyllum.

[0039] The use of *Lactobacillus plantarum*, ferments, and / or postbiotics described herein in the preparation of food, pharmaceuticals, cosmetics, and pet food is also provided.

[0040] The beneficial technical effects of this invention include:

[0041] 1) Compared with other Lactobacillus plantarum strains, Lactobacillus plantarum SP055 can efficiently convert rare ginsenosides.

[0042] 2) Lactobacillus plantarum SP055 can prevent and alleviate colitis. Lactobacillus plantarum SP055 increases the intestinal barrier by regulating immune balance (pro-inflammatory / anti-inflammatory factors), improving short-chain fatty acid composition, and enhancing tight junction protein expression, thereby preventing and alleviating colitis.

[0043] 3) Live or dead Lactobacillus plantarum SP055 can exert lipid-lowering effects, reducing total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels, while increasing high-density lipoprotein cholesterol levels, thereby treating and / or preventing non-alcoholic fatty liver disease or atherosclerosis.

[0044] 4) Ginseng fermented with Lactobacillus plantarum SP055 achieves lipid-lowering function by reducing total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels, while increasing high-density lipoprotein cholesterol content; it also improves insulin resistance by increasing glucose consumption and glycogen synthesis and storage.

[0045] 5) Ginseng fermented with Lactobacillus plantarum SP055 improves its flavor by reducing bitterness and astringency and increasing sourness, saltiness and richness; it enhances aroma and pleasantness by increasing short-chain alkanes, alcohols, aldehydes, ethers and volatile substances and reducing nitrogen oxides.

[0046] 6) Using *Lactobacillus plantarum* SP055 as a fermentation agent increases the variety of rare ginsenosides, including one or more of Rg3, Rh1, Rg5, Rk1, Rg2, Rg6, F4, and Rk3; the proportion of rare saponins in total saponins is ≥80%;

[0047] 7) Lactobacillus plantarum SP055 can convert rutin into quercetin and isoquercetin. Attached Figure Description

[0048] Figure 1 The basic information of *Lactobacillus plantarum* SP055 is shown in Figure A: phylogenetic tree of strain SP055; chromosome map of strain SP055; scanning electron microscope image of strain SP055; and homology comparison table of strain SP055.

[0049] Figure 2The effects of *Lactobacillus plantarum* SP055 on body weight, water intake, disease activity index (DAI), and dietary changes in mice undergoing DSS-induced colitis prevention were shown: Figure A shows the changes in body weight of mice in different treatment groups during the experiment; Figure B shows the changes in disease activity index (DAI) of mice in different treatment groups during the experiment; Figure C shows the changes in dietary intake of mice in different treatment groups during the experiment; and Figure D shows the changes in water intake of mice in different treatment groups during the experiment. CON represents the normal control group, DSS represents the model group, DH and DL represent the high- and low-dose SP055 intervention groups, respectively, and DH+DSS and DSS+DL represent the groups treated with high- and low-dose SP055 via gavage for 7 days followed by DSS induction.

[0050] Figure 3 The study demonstrated the effects of *Lactobacillus plantarum* SP055 on colonic inflammatory factors, anti-inflammatory factors, and oxidative stress-related indicators in preventing DSS-induced colitis in mice: Figures AC represent pro-inflammatory cytokines IL-1β, IL-6, and TNF-α; Figure D represents myeloperoxidase (MPO); Figures EF represent anti-inflammatory cytokines IL-4 and IL-10; and Figures GJ represent aspartate aminotransferase (AST / GOT), alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN). CON represents the normal control group, DSS represents the remission model group, DH and DL represent high-dose and low-dose SP055 intervention groups, respectively, and DH+DSS and DL+DSS represent high-dose and low-dose SP055 gavage for 7 days followed by DSS induction treatment, respectively.

[0051] Figure 4 The study demonstrated the effect of *Lactobacillus plantarum* SP055 on short-chain fatty acid (SCFA) levels in preventing DSS-induced colitis in mice: the A and G plots show the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid, respectively; the H plot shows the total SCFA content. CON represents the normal control group, DSS represents the remission model group, DH and DL represent the high-dose and low-dose SP055 intervention groups, respectively, and DH+DSS and DL+DSS represent the high-dose and low-dose SP055 gavage treatment groups for 7 days followed by DSS induction treatment, respectively.

[0052] Figure 5 The effects of *Lactobacillus plantarum* SP055 on the pathological changes of colon, liver, and thymus tissues in preventing DSS-induced colitis in mice were shown: In colon tissue, black arrows indicate a small number of necrotic cells accompanied by nuclear fragmentation and deep staining due to pyknosis; blue arrows indicate local shedding of mucosal epithelial cells, exposing the lamina propria; yellow arrows indicate a small number of inflammatory cell infiltrations; and red arrows indicate a significant reduction in the number of crypts. CON represents the normal control group, DSS represents the remission model group, and DH+DSS and DL+DSS represent the high- and low-dose P055 gavage treatment groups after 7 days followed by DSS induction, respectively.

[0053] Figure 6 This study demonstrates the effect of *Lactobacillus plantarum* SP055 on the expression of tight junction proteins Claudin-1, Occludin, and ZO-1 in preventing DSS-induced colitis in mice: CON was the normal control group, DSS was the model group, and DH+DSS and DL+DSS were the groups treated with high and low doses of SP055 by gavage for 7 days followed by DSS induction. Brownish-yellow staining indicates positive expression of the target proteins.

[0054] Figure 7 The effects of *Lactobacillus plantarum* SP055 on body weight, water intake, disease activity index (DAI), and dietary changes in mice with DSS-induced colitis were shown: Figure A shows the changes in body weight of mice in different treatment groups during the experiment; Figure B shows the changes in disease activity index (DAI) of mice in different treatment groups during the experiment; Figure C shows the changes in dietary intake of mice in different treatment groups during the experiment; Figure D shows the changes in water intake of mice in different treatment groups during the experiment. CON represents the normal control group, DSS represents the model group, DH and DL represent the high-dose and low-dose SP055 intervention groups, respectively, and DSS+DH and DSS+DL represent the groups treated with SP055 after DSS induction for the first 7 days.

[0055] Figure 8 The effects of *Lactobacillus plantarum* SP055 on inflammatory factors in colonic tissue in alleviating DSS-induced colitis in mice were shown: Figures A and C represent pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, respectively; Figure D represents myeloperoxidase (MPO); Figures E and F represent anti-inflammatory cytokines IL-4 and IL-10, respectively; and Figures G and J represent aspartate aminotransferase (AST / AST), alanine aminotransferase (ALT), creatinine (CRE), and blood urea nitrogen (BUN), respectively.

[0056] Figure 9 The effect of *Lactobacillus plantarum* SP055 on short-chain fatty acid (SCFA) levels in alleviating DSS-induced colitis in mice was shown: the A and G plots show the contents of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and hexanoic acid, respectively; the H plot shows the total short-chain fatty acid content.

[0057] Figure 10 The study demonstrated the effects of *Lactobacillus plantarum* SP055 on the pathological changes of colon, liver, and thymus tissues in alleviating DSS-induced colitis in mice: In the colon tissue, black arrows indicate a small number of necrotic cells accompanied by nuclear fragmentation and deep staining; blue arrows indicate local shedding of mucosal epithelial cells, exposing the lamina propria; yellow arrows indicate a small number of inflammatory cell infiltrations; and red arrows indicate a significant reduction in the number of crypts. CON was the normal control group, DSS was the model group, DH and DL were the high-dose and low-dose SP055 intervention groups, respectively, and DSS+DH and DSS+DL were the groups treated with SP055 after DSS induction for the first 7 days.

[0058] Figure 11 The effects of *Lactobacillus plantarum* SP055 on the expression of tight junction proteins Claudin-1, Occludin, and ZO-1 in alleviating DSS-induced colitis in mice were shown: CON was the normal control group, DSS was the model group, DH and DL were the high- and low-dose SP055 intervention groups, respectively, and DSS+DH and DSS+DL were the DSS combined with SP055 intervention groups. Brownish-yellow staining signal indicates positive expression of the target proteins.

[0059] Figure 12 The study showed the effects of live or inactivated *Lactobacillus plantarum* SP055 on an oleic acid-induced hyperlipidemia model in HepG2 cells: The AD plots represent total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels, respectively; the E plot shows Oil Red O staining of lipid droplets, which appear purplish-red. CON represents the control group, OA represents the oleic acid-induced model group, OA-Or represents the oleic acid-induced model group plus orlistat positive drug treatment group, and OA-LH represents the oleic acid-induced model group plus high-dose live bacteria (1×10⁻⁶). 8 The treatment group (CFU / mL) indicated that OA-LL represented oleic acid modeling with a low dose of live bacteria (1×10⁻⁶ CFU / mL). 7 The treatment group (CFU / mL) indicated that OA-DH represented oleic acid modeling combined with a high dose of inactivated bacteria (1×10⁻⁶ CFU / mL). 8 (cells / mL), OA-DL indicates oleic acid-induced modeling with low-dose inactivated bacteria (1×10⁻⁶ cells / mL). 7 (cells / mL).

[0060] Figure 13 The effects of *Lactobacillus plantarum* SP055 fermented ginseng on an oleic acid-induced hyperlipidemia model in HepG2 cells were shown: AD plots represent total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels, respectively; E plot shows Oil Red O staining of lipid droplets, which appear purplish-red. CON represents the control group, OA represents the oleic acid-induced model group, OA-Or represents the oleic acid-induced model group plus orlistat positive drug treatment group, and OA-Fg represents the oleic acid-induced model group plus fermented ginseng (40 mg / mL) treatment group.

[0061] Figure 14 The study showed the effect of *Lactobacillus plantarum* SP055 fermented ginseng on an insulin-induced HepG2 cell insulin resistance model: Figures A and B show glucose consumption and glycogen content, respectively; Figure C shows glycogen PAS staining, where glycogen appears purple-red; CON represents the control group, INS represents the insulin-induced model group, INS-Met represents the insulin-induced model plus metformin positive drug treatment group, and INS-Fg represents the insulin-induced model plus fermented ginseng (40 mg / mL) treatment group.

[0062] Figure 15 The evaluation of electronic tongue and electronic nose of fermented ginseng by *Lactobacillus plantarum* SP055 is shown: Figure A is the radar chart of electronic tongue score of unfermented ginseng compared with fermented ginseng; Figure B is the radar chart of electronic nose score of unfermented ginseng compared with fermented ginseng; Figure C is the PCA chart of electronic nose.

[0063] Figure 16 The results show the detection of the proportion of rare saponins in ginseng fermented by Lactobacillus plantarum SP055. Detailed Implementation

[0064] definition

[0065] When used in this article, "metogenic postbiotic" refers to inactivated microorganisms and / or cell components with a clearly defined genetic background that are beneficial to the host's health, including or excluding their metabolites; excluding chemically synthesized components and viruses / bacteriophages and their products (see China Biofermentation Industry Association Group Standard T / CBFIA 09001-2023). Metabiotics can include cell-based, cell-and-metabolite-based, and lysed cell-based postbiotics. The strains used must comply with the "List of Microbial Strains that Can Be Used in Food". *Lactobacillus plantarum* used in this article is a strain listed in the "List of Microbial Strains that Can Be Used in Food". Cell-based postbiotics are postbiotics whose main component is inactivated cell-based microorganisms. Cell-and-metabolite postbiotics are postbiotics whose components include inactivated cell-based microorganisms and metabolites. Lysed cell-based postbiotics are postbiotics whose components include cell-wall-broken cell-based microorganisms and metabolites.

[0066] In this article, "saponin" refers to amphoteric glycosides formed by the covalent linkage of a steroidal or triterpenoid sapogenin to one or more glycosyl groups through C-3 sites, acting as a non-glycosylated core. Saponins possess characteristic physicochemical and biological properties such as surface activity and hemolytic activity, and are important defense substances and medicinal active ingredients in plants. Saponins are commonly used as functional components in medicinal and edible raw materials (ginseng, astragalus, licorice, and gynostemma pentaphyllum), where they can regulate intestinal flora and protect the intestinal mucosa.

[0067] When used in this article, "prototype saponin" refers to saponin components that are natively present in plants such as fresh Panax notoginseng and ginseng and are present in high amounts.

[0068] In this article, "rare saponins," or rare ginsenosides, refers to a class of dammarane-type triterpenoid saponin compounds found in extremely low natural concentrations (usually far below 1%, mostly at trace levels) of native medicinal materials such as ginseng, American ginseng, and Panax notoginseng (family Araliaceae). These compounds are difficult to obtain through conventional extraction processes and are mostly transformed from native saponins (the main natural saponins) through structural modification. These saponins are mostly trace secondary metabolites in plant protoplasts, formed primarily through acid hydrolysis, alkaline hydrolysis, microbial transformation, enzymatic hydrolysis, and steaming, resulting from the removal of some sugar groups from native saponins and subsequent configurational transformation. Due to their extremely low natural abundance, high preparation difficulty, and significant and unique physiological activity, they are uniformly defined as rare saponins by the industry and literature.

[0069] When used in this article, "rutin" is a flavonoid glycoside compound widely found in plants such as Sophora japonica, buckwheat, rue, and dried tangerine peel. It is composed of the aglycone quercetin, which forms the flavonoid core structure, and the disaccharide rutin, which are linked by glycosidic bonds. It is one of the most representative natural flavonoid glycoside active ingredients.

[0070] When used in this article, "quercetin" refers to the parent compound of flavonols, which is the aglycone of various flavonoid glycosides (rutin, isoquercetin, etc.). It has a variety of biological activities such as anti-oxidation, anti-inflammation, and cardiovascular protection, and often exists in plants in the form of free state or bound as glycoside.

[0071] When used in this article, "isoquercetin" refers to a flavonol monosaccharide with quercetin as the aglycone and β-D-glucose as the glycosyl group. It has better water solubility and bioavailability than free quercetin and is a common plant-derived natural active ingredient.

[0072] In this article, "colitis" refers to an inflammatory lesion of the colonic mucosa, which can be caused by various factors such as infection and immune disorders. It is characterized by colonic mucosal congestion and edema, erosion, ulceration, crypt abscesses, and inflammatory cell infiltration, accompanied by intestinal barrier damage, intestinal flora imbalance, and intestinal dysfunction. Colitis can be ulcerative colitis. The dextran sulfate sodium (DSS)-induced colitis model is currently the most widely used, simplest to operate, and most reproducible colitis model.

[0073] In this article, "non-alcoholic fatty liver disease" (NAFLD) is defined as a chronic liver disease characterized by diffuse steatosis of hepatocytes, closely associated with insulin resistance and metabolic syndrome, excluding excessive alcohol consumption and other clearly defined causes of liver damage. It encompasses two stages: simple fatty liver and non-alcoholic steatohepatitis (NASH). The most commonly used cell model for NASH is the free fatty acid-induced hepatocyte steatosis model. Hepatocytes can include HepG2 cells, Hep3B cells, etc. Under physiological conditions, free fatty acids are mainly oleic acid and palmitic acid. High concentrations of free fatty acids in vitro can lead to the synthesis and deposition of large amounts of triglycerides (TG) in hepatocytes, while simultaneously inducing lipotoxicity, mitochondrial dysfunction, oxidative stress, and the release of inflammatory factors, simulating the core pathological process from simple steatosis to NASH.

[0074] When used in this article, "atherosclerosis" refers to the deposition of blood components such as lipids in the arterial intima, the proliferation of smooth muscle cells and the increase of collagen fibers, forming atherosclerotic lipid-containing necrotic lesions and hardening of the blood vessel wall.

[0075] In this article, "insulin resistance" is defined as a decreased sensitivity of target tissues to physiological doses of insulin, resulting in a reduced biological effect of insulin in regulating glucose and lipid metabolism, and a pathophysiological state of compensatory hyperinsulinemia. The hyperinsulinemia-induced hepatocyte insulin resistance model is the most commonly used cell model for insulin resistance. This model can also be used to assess the effectiveness of maintaining healthy blood glucose levels.

[0076] In this article, "treatment" refers to a series of professional medical interventions, including medication, surgery, physical therapy, and chemical treatments, for a disease, injury, or pathological condition that has already occurred or exists, aiming to eliminate the cause, alleviate symptoms, correct pathological changes, promote tissue repair, halt disease progression, promote recovery, or improve prognosis. In one implementation plan, treatment is the alleviation of disease, such as colitis.

[0077] When used in this article, "prevention" refers to actions taken to reduce the probability of disease occurrence, delay the onset of disease, block exposure to pathogens, reduce the risk of disease, and prevent the disease from progressing from the incubation period to the clinical stage before the disease has occurred or before the exposure to risk factors has resulted in clinical symptoms and pathological changes.

[0078] As used in this document, “subject” refers to any animal subject, including humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), and domestic pets (e.g., dogs, cats, and rodents). Subjects may have medical conditions such as colitis, non-alcoholic fatty liver disease, etc.

[0079] In this article, fermented ginseng refers to the fermented product obtained by fermenting ginseng or ginseng extract using fermentation strains. For example, fermented ginseng can be prepared as follows: Ginseng less than 5 years old is pulverized through an 80-mesh sieve, or ginseng extract (total saponins 10-80%) is added and diluted with pure water at a material-to-liquid ratio (w / w) of 1:10 to 1:20. Enzymatic hydrolysis is then performed at 0.01% to 1% (w / w) with β-glucosidase and / or cellulase and / or pectinase and / or amylase and / or protease at 40-70°C for 4-8 hours. Finally, inoculation is carried out at a rate of 3-10%, and fermentation is carried out for 1-6 days. After fermentation, the fermented product can also be obtained in powder form by centrifugation and spray drying.

[0080] In this article, fermented Gynostemma pentaphyllum refers to the fermented product obtained by fermenting Gynostemma pentaphyllum or its extract using a fermentation strain. For example, fermented Gynostemma pentaphyllum can be prepared as follows: Gynostemma pentaphyllum is pulverized and passed through an 80-mesh sieve, or the extract (total saponins 10%-80%) is added. Pure water is added at a material-to-liquid ratio (w / w) of 1:10 to 1:20. Enzymatic hydrolysis is then performed at 0.01%-1% (w / w) of β-glucosidase and / or pectinase and / or amylase and / or protease at 40-70°C for 4-8 hours. Finally, inoculation is carried out at a rate of 3-10%, and fermentation is performed for 1-6 days. After fermentation, the fermented product can also be obtained in powder form by centrifugation and spray drying.

[0081] In this article, fermented Sophora japonica buds refer to the fermented product obtained by fermenting Sophora japonica buds or Sophora japonica bud extract with a fermenting strain: Sophora japonica buds are pulverized through an 80-mesh sieve, or Sophora japonica bud extract (rutin 6-95%) is added, and pure water is added at a material-to-liquid ratio (w / w) of 1:10 to 1:20. Enzymatic hydrolysis is then performed at 0.01% to 1% (w / w) with β-glucosidase and / or pectinase and / or amylase and / or protease at 40-70℃ for 4-8 hours. Finally, inoculation is carried out at a 3-10% inoculum for 1-6 days. After fermentation, the fermented product can also be obtained in powder form by centrifugation and spray drying.

[0082] *Lactobacillus plantarum* is a commonly used probiotic in the food industry and is one of the probiotics listed in the "List of Microbial Strains that Can Be Used in Food". This invention discovered a new strain of *Lactobacillus plantarum*, named SP055. Compared to other *Lactobacillus plantarum* strains, SP055 can more effectively convert natural saponins into rare saponins. Specifically, this invention conducted fermentation studies on raw materials such as *Sophora japonica* buds with high rutin content, confirming that fermentation with *Lactobacillus plantarum* SP055 can convert rutin into isoquercitrin and quercetin. After fermentation, the rutin content decreased by 76%, the isoquercitrin content increased by 2 times, and the quercetin content increased by 23%. This invention also used ginseng as a substrate, and fermentation confirmed the production of 9 rare saponins. The conversion effect of rare saponins in fermented ginseng was significant, with the rare saponin / total saponin ratio ≥80%. This invention also discovered that fermentation of *Gynostemma pentaphyllum* increased the content of rare saponins (by up to 2 times).

[0083] This invention also demonstrates that the post-biotics of *Lactobacillus plantarum* SP055 (including dead cells or its plant fermentation products) have good therapeutic effects on intestinal inflammation, non-alcoholic fatty liver disease, and hyperglycemia, with hypoglycemic or lipid-lowering effects even superior to commonly used chemical drugs such as metformin or orlistat. This invention confirms that *Lactobacillus plantarum* SP055 has good efficacy in preventing and alleviating colitis; preventative use can improve tolerance to colitis, and its ability to reduce pro-inflammatory factors during colitis is superior to use after colitis has occurred; higher doses are more effective than lower doses. Although some literature reports that certain *Lactobacillus plantarum* strains may be effective for intestinal inflammation, *Lactobacillus plantarum* encompasses a large number of different strains, of which only a portion actually have therapeutic effects. This invention expands upon the *Lactobacillus plantarum* SP055 strain, making it applicable to the treatment of intestinal inflammation, non-alcoholic fatty liver disease, and hyperglycemia. *Lactobacillus plantarum* SP055 can be prepared as a post-biotic. The post-biotic can also be used to treat the aforementioned intestinal inflammation, non-alcoholic fatty liver disease, and hyperglycemia.

[0084] Lactobacillus plantarum SP055 can be used as a starter culture. For example, Lactobacillus plantarum SP055 can be added to saponin-rich plants (such as ginseng, gynostemma pentaphyllum, or Panax notoginseng) or rutin-rich plants (such as plant powders), or extracts of these plants (such as aqueous extracts) for fermentation culture. Alternatively, Lactobacillus plantarum SP055 can be added to pure saponin compounds or pure rutin compounds for fermentation culture. The fermented product may contain increased levels of rare saponins or increased levels of quercetin or isoquercetin.

[0085] Lactobacillus plantarum SP055 or its metagenes can be formulated into compositions, such as food, health products, feed, or pharmaceuticals. Metagenes of Lactobacillus plantarum SP055 can be prepared using conventional methods. These methods can include inactivation techniques, which are divided into heat inactivation and non-heat inactivation. In industrial production, high-temperature inactivation is generally used to obtain metagenetic formulations.

[0086] Lactobacillus plantarum SP055 or its metagenes can be prepared in liquid, frozen, lyophilized, or freeze-dried forms. For formulations prepared using encapsulation technology, they can be further processed into powder, aerosol, or spray forms. When the formulation is in powder, lyophilized, or freeze-dried form, it can be packaged in conventional pharmaceutical containers such as bottles, tubes, bags, sachets, or pouches. For use, it can be hydrated or reconstituted by adding liquid, including but not limited to water, physiological saline, fruit juice, and dairy products. Specifically, the powder, lyophilized, or freeze-dried formulation can be added to a liquid, or the liquid can be directly injected into the bottle or container containing the formulation, mixed thoroughly, and then administered directly to the individual in need.

[0087] Modifications may be made to the foregoing without departing from the essential aspects of the invention. Although the invention has been described in detail with reference to one or more specific embodiments, those skilled in the art will recognize that modifications can be made to the embodiments specifically disclosed in this application.

[0088] The present invention will be further described below with reference to the embodiments described; however, it should be understood that the present invention is not limited to these embodiments.

[0089] Example

[0090] The following embodiments are provided to further illustrate this application. It should be understood that the embodiments are merely exemplary and not restrictive. The scope of this application is defined by the appended claims. Unless otherwise stated, the materials used in the embodiments are commercially available.

[0091] Animal testing methods

[0092] Reagents, materials and instruments

[0093] DSS (sodium dextran sulfate, molecular weight = 50,000, purchased from Meilun Biotechnology); 4% formaldehyde solution (Aladdin); fecal occult blood test kit (o-toluidine method, purchased from Solarbio); tissue and serum test kits (IL-1β, IL-4, IL-6, IL-10, TNF-α, MPO, AST, ALT CRE, BUN, purchased from Jiangsu Aidisheng Biotechnology Co., Ltd.); anhydrous ethanol, xylene, hydrochloric acid, ammonia, neutral resin (purchased from Sinopharm Group); hematoxylin-eosin staining solution (purchased from Wuhan Baiqiandu Biotechnology); methanol, isopropanol (LC-MS, Thermo Fisher); N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride pyridine, 3-nitrophenylhydrazine, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, 2-ethylbutyric acid (purchased from Shanghai Yuanye Biotechnology). All other analytical grade reagents were purchased from Beijing Solarbio Technology Co., Ltd.

[0094] Dehydrator, embedding machine, freezing stage (Wuhan Junjie Electronics); pathological sectioner (Shanghai Leica); tissue spreader (Zhejiang Jinhua Kedi Instruments); oven (Shanghai Huitai Instruments); glass slides and coverslips (Jiangsu Shitai Experimental Equipment); upright optical microscope and imaging system (Nikon, Japan); freeze dryer 430R (Eppendorf); pure water system Labtower EDI (Thermo Fisher); oscillating thermostatic metal bath (Shanghai Yiheng); liquid chromatograph ExionLC-AE, mass spectrometer TQ6500+ (ABSCIEX).

[0095] 3. HepG2 cell assay method

[0096] Reagents, materials and instruments

[0097] The formula for MRS medium mainly includes the following: 20 g glucose, 5 g yeast extract, 10 g peptone, 10 g beef extract, 5 g anhydrous sodium acetate, 2 g ammonium citrate, 2.62 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.198 g manganese sulfate, 1.0 mL Tween 80, and 1 L pure water.

[0098] Lactobacillus plantarum SP055, human liver cancer cells (HepG2 cells) (purchased from Shanghai Bosheng Biotechnology Co., Ltd.); fetal bovine serum (purchased from Zhejiang Tianhang Biotechnology Co., Ltd.); DEME cell culture medium (purchased from Thermo Fisher Scientific (China) Co., Ltd.); anhydrous ethanol (AR), oleic acid (AR) from Sinopharm Chemical Reagent Co., Ltd.; triglyceride (TG) assay kit, total cholesterol (TC) assay kit, high-density lipoprotein cholesterol (HDL-C) assay kit, low-density lipoprotein cholesterol (LDL-C) assay kit (purchased from Nanjing Jiancheng Biotechnology Co., Ltd.).

[0099] LDZX40AI Vertical Sterilizer (Shenan Medical Devices Co., Ltd.); AIR TECH Workbench (Suzhou Antai Group Co., Ltd.); LRH150 Biological Incubator (Yiheng Instruments Co., Ltd.); Shimadzu AUY 120 Analytical Balance (SHIMADZU Corporation, Japan); ELECTRONIC BALANCE Electronic Balance (Minqiao Precision Instruments Co., Ltd.); HH6 Constant Temperature Water Bath (Guohua Electric Co., Ltd.); HYQ2121A Vortex Mixer (Jiemei Electronics Co., Ltd.); Beckman Avanti JE Refrigerated Centrifuge (BECKEMAN Corporation, USA); STIK Precision Blower Dryer (Steco Instruments (Shanghai) Co., Ltd.); Ultrasonic Cleaner (Kunshan Instruments Co., Ltd.); Carbon Dioxide Constant Temperature Incubator (Thermo Fisher Scientific); XDS-600D Inverted Fluorescence Microscope (Shanghai Caikang Optical Instruments Co., Ltd.); SW-CJ-1FD Ultra-Clean Workbench (Suzhou Antai Air Technology Co., Ltd.); SA402B Taste Analysis System (INSENT Corporation, Japan); MQX 200 ELISA reader (USA).

[0100] Preparation of fermented ginseng, gynostemma pentaphyllum, sophora japonica and other raw materials

[0101] Preparation of Lactobacillus plantarum SP055: Lactobacillus plantarum SP055 glycerol culture was inoculated into MRS medium (20 g glucose, 5 g yeast extract, 10 g peptone, 10 g beef extract, 5 g anhydrous sodium acetate, 2 g ammonium citrate, 2.62 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.198 g manganese sulfate, 1.0 mL Tween 80, and 1 L pure water) for primary activation at 37℃ for 24 h. The culture was then transferred at 4% to 50 mL centrifuge tubes and incubated at 37℃ for approximately 12 h as a secondary activated strain.

[0102] Ginseng, ginseng extract, gynostemma pentaphyllum extract (total saponins 20%), sophora japonica buds and Panax notoginseng extract (total saponins 10%) were purchased from Shaanxi Jiahe Biotechnology Co., Ltd.

[0103] Fermented ginseng: Ginseng is crushed and mixed with pure water at a material-to-liquid ratio of 1:20 (w / w). It is then treated with 0.1% pectinase at 40℃ for 6 hours. Finally, it is inoculated at a 3% inoculum and fermented for 6 days. After fermentation, it is centrifuged and spray-dried.

[0104] Fermentation of Gynostemma pentaphyllum: Take Gynostemma pentaphyllum extract (total saponins 20%), add pure water at a material-to-liquid ratio of 1:20 (w / w), add 0.1% (w / w) pectinase and treat with enzymatic hydrolysis at 40℃ for 6 h. Finally, inoculate with 3% inoculum and ferment for 6 days.

[0105] Fermented Sophora japonica buds: Take Sophora japonica buds, crush them, and add pure water at a material-to-liquid ratio of 1:20 (w / w). Add 0.1% (w / w) of pectinase and treat with enzymatic hydrolysis at 40℃ for 6 hours. Finally, inoculate with 3% inoculum and ferment for 6 days.

[0106] Fermented Panax notoginseng: Take Panax notoginseng extract, add pure water at a material-to-liquid ratio of 1:20 (w / w), add 0.1% (w / w) pectinase and treat with enzymatic hydrolysis at 40℃ for 6 h. Finally, inoculate with 3% inoculum and ferment for 6 days.

[0107] Detection: Accurately weigh an appropriate amount of the well-mixed sample (accurate to 0.0001 g) into a 20 mL volumetric flask, add approximately 15 mL of 40% methanol, extract ultrasonically for 30 min, cool to room temperature, and then dilute to the mark with methanol. Shake well and filter through a 0.45 μm filter membrane into a liquid chromatography injection bottle for analysis. Ginsenoside detection was performed according to the Chinese Pharmacopoeia's method for total ginsenoside detection and the group standard T / CNHFA 001-2021 for the determination of rare ginsenoside content. Rutin, quercetin, and isoquercetin were detected according to the Chinese Pharmacopoeia's method for the detection of Sophora japonica flowers using liquid chromatography.

[0108] Example 1: Basic characteristics of strain SP055

[0109] Screening for the highly efficient conversion strain SP055 of ginseng rare saponins: Ginseng extract containing 10% saponins (actually measured at 12.3%) (purchased from Shaanxi Jiahe Biotechnology Co., Ltd.) was prepared with pure water at a material-to-liquid ratio (w / w) of 1:20. Pectinase (purchased from Xiasheng Biotechnology Development Co., Ltd.) was added at 0.01% (w / w) for enzymatic hydrolysis. Finally, the strain was inoculated at a 3% inoculum and fermented for 6 days. The strain screening process was conducted by Professor Li Wei's team from the College of Food Science and Technology, Nanjing Agricultural University. *Lactobacillus plantarum* SP055 was obtained through screening using an internal strain bank. High conversion strains of rare saponin Rg3 were screened using this rare saponin as the detection index. Total ginseng saponins and rare saponins were detected according to the Chinese Pharmacopoeia's method for total ginseng saponins and the group standard T / CNHFA 001-2021 for the determination of rare ginseng saponin content. Lactobacillus plantarum SP055 was selected, with an Rg3 content of approximately 0.6 mg / g in its fermentation product. Other Lactobacillus plantarum strains showed Rg3 content ranging from 0.1 to 0.25 mg / g in their fermentation products. Lactobacillus plantarum SP055 significantly converted ginseng rare saponin Rg3. Subsequent optimization of the fermentation process was conducted using Lactobacillus plantarum SP055 as the target strain and ginseng as the substrate.

[0110] Sequencing of *Lactobacillus plantarum* SP055: *Lactobacillus plantarum* SP055 was isolated from conventionally fermented dairy products. *Lactobacillus plantarum* SP055 was cultured using MRS at 30°C under static conditions until… After incubating on ice for 10 min, the cells were centrifuged at 4 °C and 5000 × g for 10 min to collect the bacteria. The cells were washed twice with PBS, and the supernatant was discarded. Approximately 200 mg of wet bacterial cells were placed into 2 mL cryovials according to the requirements of Shanghai Meiji Biopharmaceutical Technology Co., Ltd. (hereinafter referred to as Meiji Biopharmaceutical), rapidly frozen in liquid nitrogen, and then shipped on dry ice. Meiji Biopharmaceutical completed DNA extraction, 16S rRNA amplification and sequencing, Illumina and PacBio library construction and sequencing, and subsequent assembly, annotation, and data upload were all performed according to Meiji's standard procedures.

[0111] Scanning electron microscopy: SP055 bacterial sludge was collected, fixed with 2.5% glutaraldehyde at 4 °C for 4 h, washed with PBS, dehydrated with a gradient of ethanol (30→100%) for 15 min each time, air-dried on a silicon wafer, and sputtered with gold at 10 nm. SEM images were taken at 10 kV, and cell morphology was recorded at 10.2 mm × 20 K SE (UL).

[0112] The complete genome of *Lactobacillus plantarum* strain SP055 was sequenced using Illumina, such as... Figure 1 Figure A shows the construction of a phylogenetic tree of the strains, and the homology was compared ( Figure 1Figure D in the diagram shows that the strain was identified as *Lactiplantibacillus plantarum*. Whole-genome sequencing confirmed that the full-length genome of *Lactiplantibacillus plantarum* SP055 is approximately 3.3 Mbp, with a GC content of 44.38%, and contains a circular chromosome approximately 3 Mbp long. Figure 1 (Figure B in the image), the GC content was 44.77%, and 7 complete plasmids were obtained. This experiment was commissioned to Meiji Biotechnology.

[0113] *Lactobacillus plantarum* SP055 is a Gram-positive, non-spore-forming, facultative anaerobic bacterium, acid-resistant (pH 3.0, 2-hour survival rate ≥80%), and bile-resistant (2-hour survival rate ≥80% in a 0.3% bile salt environment). Scanning electron microscopy (SEM) Figure 1 (Figure C) The morphology of the strain is that of regular straight or slightly curved rods with blunt, rounded ends, 1.2–2.4 µm in length and 0.5–0.8 µm in width; it usually appears as a single rod, in pairs, or in short chains, without branches, flagella, or capsules. *Lactobacillus plantarum* strain SP055 was deposited on July 2, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 35072.

[0114] Example 2: The role of *Lactobacillus plantarum* SP055 in the preventive intervention of DSS-induced colonic inflammation in mice and in the alleviation of DSS-induced colonic inflammation in mice.

[0115] Animal experimental design

[0116] Preparation of Lactobacillus plantarum SP055: It was cultured in MRS medium at 37℃ for 24 hours, centrifuged at 4500 rpm for 5 minutes, washed twice with physiological saline, and adjusted to an appropriate concentration according to the growth curve and absorbance at 600 nm.

[0117] In establishing a mouse model of inflammatory bowel disease, female BALB / c mice were administered sodium dextran sulfate (DSS). Upon arrival (day 1), the animals were isolated and adapted to controlled light-cycle conditions (12 h light and 12 h dark) and temperature (25 ± 0.5°C) for 7 days. They were then randomly assigned to groups 1 through 9 (n=10 per group) based on body weight (BW). In establishing a mouse model of *Lactobacillus plantarum*, mice were divided into high-dose and low-dose groups, with the high-dose group (DH) consisting of 10 mice. 9 CFU (0.2 mL / mouse / day), low-dose group (DL) was 10 8CFU (0.2 mL / mouse / day) was administered daily at approximately 9:00 AM. The mice were divided into three groups: a healthy control group (CON), DH, and DL; a SP055 alleviating effect group (DSS, DSS+DH, and DSS+DL); and a SP055 preventative effect group (DSS, DH+DSS, and DL+DSS). During the SP055 administration to the other groups, the CON group received the same volume of sterile PBS via gavage.

[0118] The control group (CON) received only normal drinking water for 16 days. The high-dose group (DH) received only a high dose of SP055 for 14 days, followed by 2 days of normal drinking water. The low-dose group (DL) received only a low dose of SP055 for 14 days, followed by 2 days of normal drinking water.

[0119] Prevention trial (SP055 administered for 14 days from day 8 to day 21, with DSS administered concurrently from day 15 to day 21): DSS group: Normal water was administered from day 8 to day 14, followed by 2.5%-3.5% DSS from day 15 to day 21, then normal water was administered for 2 days; High-dose SP055 + DSS group (DH+DSS): SP055 was administered at a high dose from day 8 to day 21, with DSS administered concurrently from day 15 to day 21, then normal water was administered for 2 days; Low-dose SP055 + DSS group (DL+DSS): SP055 was administered at a low dose from day 8 to day 21, with DSS administered concurrently from day 15 to day 21, then normal water was administered for 2 days.

[0120] Remission Trial (SP055 administered for 14 days from day 8 to day 21, with DSS administered concurrently from day 8 to day 14): DSS group: DSS only administered from day 8 to day 14, followed by normal water only for the remaining 9 days; DSS+DH: SP055 administered at a high dose from day 8 to day 21, with DSS administered concurrently from day 8 to day 14, followed by normal water for 2 days; DSS+DL group: SP055 administered at a low dose from day 8 to day 21, with DSS administered concurrently from day 8 to day 14, followed by normal water for 2 days.

[0121] Measurement of daily diet, drinking water and DAI

[0122] During the experiment, mice were given free access to standard feed and water. The ambient temperature was maintained at 22 ± 2 ℃, the relative humidity at 50–60%, and the light / dark cycle was 12 h. Daily changes in body weight, diet, and water intake were recorded for each group of mice, and their general mental state was observed.

[0123] The Disease Activity Index (DAI) is a comprehensive score based on the degree of weight loss, stool characteristics, and rectal bleeding. Weight change is based on the initial weight at the start of the experiment, scored according to the percentage decrease. Stool characteristics are categorized as normal, soft, or diarrheal. Rectal bleeding is determined through visual observation or occult blood test strips. The DAI score is the sum of these three scores; the specific assessment method is shown in the table below.

[0124]

[0125] Inflammatory factor detection

[0126] After the experiment, the mice were euthanized, and colon tissue samples were collected. The colon tissue was rinsed with pre-cooled physiological saline, weighed, and homogenized with an appropriate amount of lysis buffer. After centrifugation, the supernatant was collected for the detection of inflammatory factors.

[0127] The levels of inflammatory factors (such as IL-1β, IL-6, TNF-α, IL-10, etc.) in tissue homogenates were measured using enzyme-linked immunosorbent assay (ELISA), and the specific procedures were performed according to the kit instructions. The levels of inflammatory factors in the tissues were normalized to bovine serum albumin (BSA) protein concentration. Tissue and serum detection kits (IL-1β, IL-4, IL-6, IL-10, TNF-α, MPO, AST, ALT, CRE, and BUN detection kits) were purchased from Jiangsu Adison Biotechnology Co., Ltd.

[0128] Short-chain fatty acid detection

[0129] Preparation of Standards: Take appropriate amounts of pure standards of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, and 2-ethylbutyric acid (internal standard), and prepare a 100 mg / mL stock solution with methanol and water. Dilute the stock solution with methanol and water to obtain a series of working standard solutions. Prepare a 0.1 mg / mL internal standard with methanol and water. Mix the working standard solutions of the seven acids to prepare sixteen standard curve points: 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 1 ng / mL, 2 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, and 2000 ng / mL. Store the stock solution at -80 ℃, and prepare each working solution fresh before use.

[0130] Metabolite extraction: The entire experiment was performed on ice. 20 mg of fecal solid sample was transferred to a 2 mL centrifuge tube, two steel balls were added, and 800 μL of extraction reagent (containing internal standard) was added. The tube was vortexed for 60 s. The sample was then placed in a tissue homogenizer and homogenized at 55 Hz for 60 s. This process was repeated once. The sample was centrifuged at 4000 g and 10 °C for 10 min. 40 μL of the supernatant was collected and mixed with 20 μL of 200 mM 3-NPH solution. 20 μL of 120 mM (EDC)·HCl-6% pyridine solution was added and mixed. The mixture was reacted at 40 °C and 1200 rpm for 30 min using a thermostat. After the reaction, the sample was cooled on ice for 3 min. The sample was centrifuged at 12000 g and 4 °C for 10 min. 50 μL of the supernatant was collected and diluted to 200 μL with 150 μL of formic acid solution (v / v, containing 0.1% formic acid). The mixture was vortexed for 60 s. The mixture was then centrifuged at 12000 g. Centrifuge at 4℃ for 10 min; filter the supernatant through a 0.22 μm filter membrane, and bottle the filtrate, with a bottle volume of not less than 50 μL / bottle.

[0131] Liquid chromatography detection: A Kinetex C8 column (100 Å, 2.6 µm, 2.1 mm × 100 mm) was used at a flow rate of 0.3 mL / min, a column temperature of 40 ℃, and an autosampler at 4 ℃. The injection volume was 1 μL. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 50% methanol-isopropanol (containing 0.1% formic acid). The mobile phase gradient was as follows: 0–1 min: A / B 80:20 (V / V); 6 min: A / B 60:40 (V / V); 9–10 min: A / B 80:20 (V / V).

[0132] Mass spectrometry: The SCIEX Citrine Triple Quad™ mass spectrometer was used with OS software (version 4.7, SCIEX) in ESI (Electrospray Ionization, ESI) negative mode, employing Multiple Reaction Monitoring (MRM) data acquisition. Ion source parameters: Ionspray (IS) negative mode voltage -4500V, spray gas (GS1) and auxiliary heating gas (GS2) 50psi and 50psi respectively, desolvation gas temperature 550℃; curtain gas (CUR) 30psi, collision gas (CAD) 10.

[0133] All formal samples and QC samples were processed using the chromatographic and mass spectrometric methods described above. Before formal sample injection, 2-4 QC sample tests were performed to confirm the stability of the instrument. During the injection process, one QC sample was injected for every 12 samples for subsequent data evaluation and quality control.

[0134] HE staining and immunohistochemistry of pathological sections

[0135] Paraffin-embedded tissue sections:

[0136] 1. Tissue Collection: Fresh tissue is fixed in 4% paraformaldehyde for at least 24 hours. The tissue is removed from the fixative and trimmed in a fume hood using a scalpel. The trimmed tissue and corresponding labels are then placed in a dehydration box.

[0137] 2. Dehydration: Place the dehydration box into the basket and then into the dehydrator for sequential dehydration with alcohol in a gradient. 75% alcohol 4h - 85% alcohol 2h - 90% alcohol 2h - 95% alcohol 1h - anhydrous ethanol I 30min - anhydrous ethanol II 30min - benzene 5-10min - xylene I 5-10min - xylene II 5-10min - wax I 1h - wax II 1h - wax III 1h.

[0138] 3. Embedding: Embed the paraffin-impregnated tissue in an embedding machine. First, place the molten paraffin into the embedding frame. Before the paraffin solidifies, remove the tissue from the dehydration box, place it into the embedding frame according to the embedding surface requirements, and attach the corresponding label. Cool on a -20°C freezing stage. After the paraffin solidifies, remove the paraffin block from the embedding frame and trim it.

[0139] 4. Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4μm. Float the sections on 40℃ warm water in a slide spreader to flatten the tissue. Use a glass slide to lift the tissue and place it in a 60℃ oven to bake. Once the water has dried and the wax has melted, remove the sections and store them at room temperature for later use.

[0140] HE staining:

[0141] 1. Dewaxing paraffin sections to water: Place the sections in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and then wash with distilled water.

[0142] 2. Hematoxylin staining of cell nuclei: Slices are stained with Harris hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water.

[0143] 3. Eosin staining of cytoplasm: Immerse the sections in eosin staining solution for 1-3 minutes.

[0144] 4. Dehydration and mounting: Place the sections in 95% ethanol I for 5 min, 95% ethanol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min to dehydrate and clear them. Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0145] 5. Microscopic examination, image acquisition and analysis.

[0146] Immunohistochemistry:

[0147] Dewaxing paraffin sections to water: Place the sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and then wash with distilled water.

[0148] Antigen retrieval: Tissue sections were placed in a retrieval container filled with EDTA antigen retrieval buffer (pH 9.0) and microwaved for antigen retrieval. Microwave on medium heat for 8 minutes, then turn off for 8 minutes, and then microwave on medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented; the slides should not be allowed to dry. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each time. (The retrieval solution and conditions are determined based on the specific tissue.)

[0149] Draw circles: After slightly drying the slices, use a histochemical pen to draw circles around the tissue (to prevent antibody migration).

[0150] Serum blocking: Add BSA to the circle and incubate for 30 min.

[0151] Add primary antibody (Claudin 1 antibody (YA3551) catalog number: HY-P81222A; ZO-1 / TJP1 antibody (YA3430) catalog number: HY-P83694; Occludin antibody catalog number: HY-P81231, company MedChemExpress): Gently shake off the blocking solution, add the primary antibody prepared in PBS at a certain ratio to the slide, and incubate the slide flat in a humidified chamber at 4°C overnight.

[0152] Add secondary antibody (AP-conjugated Goat Anti-Rabbit IgG H&L, catalog number: HY-P84940, MedChemExpress): Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker, 5 min each time. After slightly drying the section, add the corresponding species of secondary antibody to the tissue in a circle and incubate at room temperature in the dark for 50 min.

[0153] DAB staining and counterstaining of cell nuclei: Slides were placed in PBS (pH 7.4) and washed three times on a destaining shaker for 5 minutes each time. After drying the sections, DAB staining reagent was added to the inner circle, and staining was controlled under a microscope. After complete staining, the slides were rinsed with distilled or tap water, counterstained with hematoxylin, differentiated with 1% hydrochloric acid alcohol (approximately 1 second), rinsed with tap water, blued with ammonia, and rinsed with running water.

[0154] Dehydration and sealing: Dehydrated with anhydrous ethanol and sealed with xylene transparent neutral resin.

[0155] result

[0156] Prevention test results

[0157] like Figure 2 As shown in Figures A and D, after DSS administration began on day 15 (i.e., day 8 of the intervention period), compared to the control group (labeled CON), the DSS-treated group (labeled DSS) showed a significant decrease in body weight (Figure A), an increase in the disease activity index (DAI) (Figure B), a decrease in food intake (Figure C), and fluctuations in water consumption (Figure D), indicating successful DSS-induced colitis. Compared to the DSS group, early intervention with *Lactobacillus plantarum* SP055 (DSS+DH or DSH and DSS+DL or DSL) significantly improved body weight changes, disease activity index, and feeding and drinking behaviors, demonstrating a good preventive effect, with higher doses being superior to lower doses.

[0158] like Figure 3 As shown in Figures A and J, after DSS treatment, compared with the control group, the levels of pro-inflammatory factors IL-1β (Figure A), IL-6 (Figure B), and TNF-α (Figure C) significantly increased, myeloperoxidase (MPO) level increased (Figure D), and anti-inflammatory factors IL-4 (Figure E) and IL-10 (Figure F) significantly decreased. Liver and kidney function indicators such as aspartate aminotransferase (AST / GLT / ALT) (Figure G), alanine aminotransferase (GPT / ALT) (Figure H), creatinine (CRE) (Figure I), and blood urea nitrogen (BUN) (Figure J) significantly increased. This indicates activated inflammatory response, immune dysregulation, neutrophil infiltration, and impaired liver and kidney function. Compared with the DSS group, prophylactic intervention with *Lactobacillus plantarum* SP055 in DSS treatment (DSH group and DSL group) significantly improved cellular tolerance to DSS, reduced pro-inflammatory factor and MPO levels, increased anti-inflammatory factor levels, balanced immunity, and restored liver and kidney function indicators. The higher dose was more effective than the lower dose. Interestingly, compared to the control group, using Lactobacillus plantarum SP055 alone increased both pro-inflammatory and anti-inflammatory factors to some extent, while also reducing liver and kidney function indicators to some extent.

[0159] like Figure 4As shown in Figure AI, after DSS treatment, compared with the control group, the contents of acetic acid (Figure A), propionic acid (Figure B), butyric acid (Figure C), isobutyric acid (Figure D), valeric acid (Figure E), isovaleric acid (Figure F), hexanoic acid (Figure G), and total short-chain fatty acids (Figure H) all decreased significantly. The preventive intervention of *Lactobacillus plantarum* with DSS treatment restored short-chain fatty acid levels to a certain extent, with significant recovery effects mainly in acetic acid, butyric acid, and total short-chain fatty acid contents, which differed from the changes and response patterns of branched-chain fatty acids isobutyric acid and isovaleric acid, as well as other short-chain fatty acids. There was no significant difference between high and low doses. Interestingly, using *Lactobacillus plantarum* SP055 alone reduced short-chain fatty acid contents to some extent.

[0160] like Figure 5 As shown, in the colon tissue, the colonic mucosa of the control group mice was intact, with regular crypt arrangement and a clear boundary between the mucosa and lamina propria, and no obvious inflammatory cell infiltration or tissue damage was observed. The liver lobule structure of the control group was relatively clear overall. The thymic cortex and medulla of the control group were clearly demarcated and had a dense structure. In contrast, the colon tissue of the DSS model group mice showed significant pathological damage, manifested as local mucosal epithelial cell shedding, exposure of the lamina propria (blue arrow), accompanied by a small amount of cell necrosis, and fragmented and condensed deep-stained nuclei (black arrow). At the same time, local inflammatory cell infiltration (yellow arrow) was observed, the crypt structure was significantly damaged, and the number of crypts was reduced (red arrow), suggesting that DSS can induce significant destruction of the colon tissue structure. The liver lobules of the DSS-treated group showed a mild trend of tissue structure disorder, but no obvious large-scale hepatocyte necrosis or severe inflammatory infiltration. The thymic tissue structure of the DSS-treated group showed a slight degree of disorder. Prophylactic intervention with Lactobacillus plantarum SP055 and DSS treatment resulted in significant restoration of colonic mucosal structure, relatively regular crypt arrangement, marked improvement in epithelial continuity, and a significant reduction in inflammatory cell infiltration and necrosis. The high-dose group was superior to the low-dose group, and it also showed some improvement in hepatocytes and thymus tissue.

[0161] like Figure 6 As shown, in the control group, tight junction proteins Claudin-1, Occludin, and ZO-1 all exhibited continuous and uniform positive staining, indicating an intact colonic epithelial structure and clear intercellular connections, suggesting a healthy tight junction structure. In contrast, the staining intensity of these tight junction proteins in the colonic tissue of the DSS model group was significantly weakened, the positive signal distribution was discontinuous, and some areas showed absence, indicating that DSS treatment significantly disrupted the tight junction structure of the colonic epithelium. Prophylactic intervention with *Lactobacillus plantarum* SP055 after DSS treatment significantly enhanced the positive staining of tight junction proteins in the colonic tissue, with a more continuous staining distribution and significantly improved epithelial structural integrity; higher doses were superior to lower doses.

[0162] According to the results of the prevention trial, preventive intervention with Lactobacillus plantarum SP055 can prevent intestinal inflammation caused by DSS treatment and can be used to prevent colitis.

[0163] mitigation test results

[0164] like Figure 7 As shown in Figures A and D, after DSS treatment began on day 8 (i.e., day 1 of the intervention period), compared to the control group, DSS-treated mice exhibited significant weight loss (Figure A), increased DAI scores (Figure B), decreased food intake (Figure C), and increased water consumption (Figure D) in the mid-to-late stages of the disease. Combined intervention with *Lactobacillus plantarum* SP055 (DSS+DL or DSL, and DSS+DH, or DSH) alleviated the phenotypic symptoms, bringing them closer to those of the control group.

[0165] like Figure 8 As shown in Figures A and J, after DSS treatment, compared with the control group, the levels of pro-inflammatory factors IL-1β (Figure A), IL-6 (Figure B), and TNF-α (Figure C) significantly increased, myeloperoxidase (MPO) (Figure D) level increased, and anti-inflammatory factors IL-4 (Figure E) and IL-10 (Figure F) significantly decreased. Liver and kidney function indicators such as aspartate aminotransferase (AST / GOT / AST) (Figure G), alanine aminotransferase (ALT) (Figure H), creatinine (CRE) (Figure I), and blood urea nitrogen (BUN) (Figure J) significantly increased. The combination of DSS treatment and *Lactobacillus plantarum* SP055 significantly reduced the pro-inflammatory factor IL-1β while increasing MPO, anti-inflammatory factors, and liver and kidney function indicators, but had no significant effect on pro-inflammatory factors IL-6 and TNF-α. This indicates that *Lactobacillus plantarum* SP055 has a certain degree of effect in alleviating the immune response and liver and kidney function associated with DSS.

[0166] like Figure 9 As shown in the AI ​​figure, compared with the control group, short-chain fatty acids decreased significantly after DSS treatment. Both intervention with *Lactobacillus plantarum* SP055 and DSS treatment showed a certain increase in short-chain fatty acids, with no significant dose-response relationship observed in the strain.

[0167] like Figure 10 As shown, compared with the DSS group, intervention with *Lactobacillus plantarum* SP055 significantly alleviated the pathological damage to the colonic tissue after DSS treatment, resulting in relatively intact mucosal structure, significantly reduced epithelial shedding and crypt destruction, and decreased inflammatory cell infiltration and local necrosis. The effect was more pronounced at higher doses. DSS caused mild damage to the liver and thymus, and intervention with *Lactobacillus plantarum* SP055 somewhat mitigated this damage.

[0168] like Figure 11As shown, the immune response of tight junction proteins in the colon tissue of mice treated with DSS was significantly weakened, the staining intensity was reduced and the distribution was discontinuous. After intervention with DSS treatment by Lactobacillus plantarum SP055, the staining signal of tight junction proteins was enhanced and tended to be continuously distributed along the junction region between epithelial cells, suggesting that it alleviated the DSS-induced decrease in tight junction protein expression to some extent.

[0169] According to the results of the mitigation test, Lactobacillus plantarum SP055 can alleviate intestinal inflammation caused by DSS treatment when it is applied to mice simultaneously with DSS, and can be used to relieve or treat colitis.

[0170] Example 3: The role of live and dead Lactobacillus plantarum SP055 in an oleic acid-induced HepG2 high-fat model.

[0171] In this embodiment, the triglyceride (TG) assay kit, total cholesterol (TC) assay kit, high-density lipoprotein cholesterol (HDL-C) assay kit, and low-density lipoprotein cholesterol (LDL-C) assay kit were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0172] Seven groups were set up: a CON blank control group, an OA oleic acid modeling group, an OA-Or orlistat positive group, and a high-dose LH live bacteria group (1×10⁻⁶). 8 CFU / mL), LL low-dose live bacteria group (1×10) 7 CFU / mL), DH high-dose dead bacteria group (1×10) 8 CFU / mL), DL low-dose dead bacteria group (1×10) 7 CFU / mL). Dead bacteria are bacterial samples obtained after heat inactivation of live bacteria (heating at 70°C for 30 min).

[0173] HepG2 cells were cultured in cell culture medium containing 10% (v / v) fetal bovine serum and incubated at 37 ℃ with 5% CO2 for 24 h. Cells were observed under a microscope, and when they reached 90% confluence of the bottom area of ​​the culture flask, they were transferred to well plates, and the cell count was calculated. The experimental group, which did not receive oleic acid or therapeutic agents throughout the experiment, served as the control group.

[0174] A high-lipid HepG2 cell model was established using oleic acid. HepG2 cells (5 × 10⁻⁶) were used. 4 Cells were seeded in 6-well plates at 37 °C and 5% CO2 for 24 h. Cell culture medium containing 1.2 mM (0.34 mg / mL) of oleic acid was added and the cells were incubated for another 24 h. Microscopic observation revealed lipid accumulation in the hepatocytes; if the cells changed from irregular shapes to nearly spherical shapes and had a distinct oily substance at the cell edges.

[0175] Cells from the high-lipid HepG2 cell model were washed, the medium was changed, and a final concentration of 1×10⁻⁶ was added to each treatment group. 8 CFU / mL and 1×10 7 Live and dead bacteria (CFU / mL) were added to the positive control group, and 500 μg / mL of orlistat was added. The mixture was incubated at 37 °C in a 5% CO2 incubator for 24 h.

[0176] For each group of cells, total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured according to the kit instructions. The residual culture medium in each well was washed away with sterile PBS buffer, 1 mL of cell lysis buffer was added to each well, and the cells were lysed for 30 min. The parameters were measured according to the kit instructions.

[0177] Lipid droplet staining: Cell slides were prepared, cells were fixed with 4% paraformaldehyde for 15 min, washed 3 times with ultrapure water, and cell membranes were broken down for 15 min with permeabilization solution. Oil Red O staining was performed for 30 min in the dark. Cell slides were then immersed in 60% isopropanol for 3 s for differentiation, washed 3 times with distilled water, stained with hematoxylin for 10 s and washed with water for 1 s for inversion blue, and mounted with glycerol gelatin. The morphology and accumulation of lipid droplets in each group of cells were observed under a microscope.

[0178] like Figure 12 As shown in Figure AE, compared with the control group (CON), the levels of total cholesterol (TC, Figure A), triglycerides (TG, Figure B), low-density lipoprotein cholesterol (LDL-C, Figure D), and lipid droplet aggregation in HepG2 cells after oleic acid (OA) modeling were significantly increased (p < 0.05), while high-density lipoprotein cholesterol (HDL-C, Figure C) was significantly decreased (p < 0.05). This indicates that the oleic acid modeling of HepG2 hepatocytes successfully established a model of lipid accumulation and cholesterol homeostasis imbalance, which can preliminarily simulate the pathological characteristics of "lipotoxicity" in hepatocytes in the early stages of non-alcoholic fatty liver disease and atherosclerosis. Figure 12 In the AD graph, a, b, c, d, bc, and cd represent significant differences. Different letters between the two groups indicate significant differences (p < 0.05), while the same letter indicates no significant differences (p > 0.05).

[0179] Compared with the model group (OA group), high-dose live bacteria (LH), low-dose live bacteria (LL), high-dose inactivated bacteria (DH), and low-dose inactivated bacteria (DL) all significantly reduced the levels of TC, TG, and LDL-C, alleviated the accumulation load of free cholesterol, improved the lipid deposition phenotype, and reduced the uptake and retention of exogenous cholesterol. There was no significant difference between high and low doses. At the same time, both live and dead bacteria interventions significantly increased HDL-C levels (p < 0.05), and the effect of dead bacteria was better than that of live bacteria.

[0180] All the above results indicate that both live and inactivated *Lactobacillus plantarum* SP055 significantly improved lipid accumulation and restored cholesterol homeostasis, with effects consistent at low and high doses. Based on the effects of live and dead *Lactobacillus plantarum* SP055 in the oleic acid-induced HepG2 hyperlipidemia model, it can be inferred that both live and dead *Lactobacillus plantarum* SP055 can contribute to lipid health, including cholesterol and / or triglycerides. Furthermore, the oleic acid-induced HepG2 hyperlipidemia model is a cell model of non-alcoholic fatty liver disease. Based on these results, live and dead *Lactobacillus plantarum* SP055 can also be used for the treatment and prevention of non-alcoholic fatty liver disease or atherosclerosis-related lipid disorders.

[0181] Example 4: The effect of *Lactobacillus plantarum* SP055 fermented ginseng in an oleic acid-induced HepG2 high-fat model.

[0182] In this embodiment, the triglyceride (TG) assay kit, total cholesterol (TC) assay kit, high-density lipoprotein cholesterol (HDL-C) assay kit, and low-density lipoprotein cholesterol (LDL-C) assay kit were purchased from Nanjing Jiancheng Biotechnology Co., Ltd. Four groups were set up: a CON blank control group, an OA oleic acid model group, an OA-Or orlistat positive group, and an OA-Fg fermented ginseng group. The remaining steps followed the experimental method in Example 3. In short, HepG2 cells were cultured in cell culture medium containing 10% (v / v) fetal bovine serum. After culturing at 37 ℃ and 5% CO2 for 24 h, the cells were observed under a microscope. When the cells covered 90% of the bottom area of ​​the cell culture flask, they were transferred to well plates, and the cell count was counted. A high-lipid HepG2 cell model was established using oleic acid. HepG2 cells (5×10⁶ cells / wells) were used to establish the model. 4 Cells were seeded in 6-well plates (number of cells / mL) and incubated at 37 °C and 5% CO2 for 24 h. Cell culture medium containing 1.2 mM (0.34 mg / mL) oleic acid was added and incubated for another 24 h. Microscopic observation revealed lipid accumulation in hepatocytes if the cells changed from irregular shapes to nearly spherical shapes and had obvious oily substances at the cell edges. Cells were then washed, the medium was changed, and 40 mg / mL fermented ginseng was added to the treatment group, while 500 μg / mL orlistat was added to the positive control group. All cells were incubated at 37 °C and 5% CO2 for 24 h.

[0183] result

[0184] like Figure 13As shown in Figure AE, compared with the control group (CON), the total cholesterol (TC) (Figure A), triglycerides (TG) (Figure B), low-density lipoprotein cholesterol (LDL-C) (Figure C), and lipid droplet aggregation degree (Figure D) of oleic acid (OA) modeled HepG2 cells were significantly increased (p < 0.05), while high-density lipoprotein cholesterol (HDL-C) was significantly decreased (p < 0.05), indicating that oleic acid modeling of HepG2 hepatocytes was successful. Figure 13 In the AD graph, a, b, c, and bc represent significant differences. Different letters between the two groups indicate significant differences (p < 0.05), while the same letter indicates no significant differences (p > 0.05).

[0185] Compared to the OA group, treatment of cells in the OA oleic acid model group with fermented ginseng (Fg) significantly reduced TC, TG, and LDL-C levels, with the reduction in TG and LDL-C being consistent with that of the positive control drug orlistat (Or). Simultaneously, Fg significantly increased HDL-C levels; lipid droplet staining clearly showed that Fg improved lipid droplet aggregation. All results indicate that *Lactobacillus plantarum* SP055 fermented ginseng significantly improves lipid accumulation and restores cholesterol homeostasis, suggesting that fermented ginseng may contribute to lipid health, including cholesterol and / or triglycerides.

[0186] Based on the above results, fermented ginseng can also be used for the treatment and prevention of non-alcoholic fatty liver disease or atherosclerosis-related lipid disorders, and has the potential to be used to intervene in non-alcoholic fatty liver disease or atherosclerosis-related lipid disorders.

[0187] Example 6: The role of *Lactobacillus plantarum* SP055-fermented ginseng in an insulin-induced HepG2 insulin resistance model

[0188] Four groups were set up: CON blank control group, OA oleic acid modeling group, OA-Or orlistat positive group, and OA-Fg fermented ginseng group.

[0189] HepG2 cells were cultured in cell culture medium containing 10% (v / v) fetal bovine serum and incubated at 37 ℃ and 5% CO2 for 24 h. The cells were observed under a microscope. When the cells covered 90% of the bottom area of ​​the cell culture flask, they were transferred to a well plate and the cell count was counted.

[0190] An insulin-resistant HepG2 cell model was established using insulin. HepG2 cells (5 × 10⁻⁶) were used. 4 Cells were seeded at 1 mM (5.81 mg / mL) in 6-well plates and incubated at 37 °C and 5% CO2 for 24 h. Cell culture medium containing 1 mM (5.81 mg / mL) insulin was added and incubated for another 24 h. Glucose consumption was determined using the glucose oxidase method to confirm successful model establishment.

[0191] Cells of the insulin-resistant HepG2 cell model were washed and the medium was changed. 40 mg / mL of fermented ginseng sample and 500 μg / mL of metformin were added to the treatment group and the positive control group, respectively, and incubated at 37 ℃ and 5% CO2 for 24 h.

[0192] Glucose consumption and glycogen content in each group of cells were determined according to the kit instructions. Measurement of intracellular substances first requires cell lysis. Taking a six-well plate as an example, after discarding the culture medium, wash away any remaining culture medium in each well with sterile PBS buffer, add 1 mL of cell lysis buffer to each well, lyse for 30 min, and then measure the parameters according to the kit instructions.

[0193] Glycogen staining: Follow the procedure of the glycogen PAS staining kit. Fix cells with PAS fixative for 15 min, wash with distilled water and air dry; add oxidant and react at room temperature for 20 min, wash twice with distilled water; add Schiff staining solution and stain in the dark for 15 min, rinse with running water for 5 min; add Mayer hematoxylin staining solution, counterstain for 2 min, wash with water and air dry for microscopic examination.

[0194] result

[0195] like Figure 14 As shown in the AC figure, compared with the control (CON), the glucose consumption and glycogen content of HepG2 cells induced by insulin (INS) were significantly reduced (p<0.05), indicating that an insulin resistance model was successfully constructed, which can initially simulate the characteristics of early type 2 diabetes or metabolic syndrome hyperinsulinemia (pathological or drug-induced) caused by insulin receptor desensitization. Figure 14 In the AB diagram, a, b, c, and d represent significance. Different letters between the two groups indicate a significant difference (p < 0.05), while the same letters indicate no significant difference (p > 0.05).

[0196] Treatment of insulin-resistant model cells with fermented ginseng (Fg) significantly improved glucose consumption and cellular glucose uptake; it also significantly increased glycogen content and restored glycogen synthesis reserves; PAS glycogen staining results were consistent with biochemical results. These results indicate that *Lactobacillus plantarum* SP055 fermented ginseng increases glucose consumption and glycogen content. Decreased glucose consumption and glycogen content are direct functional phenotypes of insulin resistance in hepatocytes, leading to elevated blood glucose levels (especially postprandial blood glucose) in type 2 diabetes and insulin resistance. Therefore, *Lactobacillus plantarum* SP055 fermented ginseng helps maintain healthy blood glucose levels. Furthermore, *Lactobacillus plantarum* SP055 fermented ginseng also significantly improves insulin resistance and increases insulin sensitivity, demonstrating potential for delaying or preventing the progression from compensatory hyperinsulinemia to overt hyperglycemia.

[0197] Example 7: Evaluation of ginseng electronic nose and electronic tongue fermented with Lactobacillus plantarum SP055

[0198] Electronic tongue and electronic nose flavor evaluation

[0199] Electronic nose assay: Take 3g of fermented ginseng sample and 3g of unfermented ginseng sample (ginseng powder prepared at a material-to-liquid ratio of 1:20) and place them in 10mL headspace vials. Incubate the sealed headspace vials at 25℃ for 30min. Then, insert the injection needle and the gas injection needle into the sample headspace vials simultaneously for headspace collection and detection. The sensors and their characteristics of the electronic nose (PEN3.5 electronic nose; AIRSENSE GmbH, Germany) are described in the table below.

[0200]

[0201] Electronic tongue (SA402B taste analysis system; INSENT Corporation, Japan) measurements: 60 mL of fermented sample and unfermented ginseng juice (ginseng was prepared with water at a material-to-liquid ratio of 1:20) were taken separately. The electronic tongue, equipped with sensors for CTO (salty taste), AAE (umami taste), CAO (sour taste), COO (bitter taste), and AE1 (astringent taste), was used to measure the electrical signals of the samples. Each sample was measured four times, and the results of the last three measurements were used for data analysis. Radar plots and cluster analyses were performed on the data detected by the electronic nose and electronic tongue.

[0202] result

[0203] like Figure 15As shown in Figures A and C, fermented ginseng from *Lactobacillus plantarum* SP055, compared to unfermented ginseng samples, showed increased sourness, saltiness, and richness, while bitterness and astringency decreased (Figure A). After fermentation, compared to unfermented ginseng juice, the sensitivity to aromatic components, short-chain alkanes such as methane, alcohols, aldehydes, and ethers increased, while nitrogen oxides decreased (Figure B). At the flavor level, the decrease in bitterness and astringency, and the increase in sourness, saltiness, and overall richness, indicate the transformation of bitter precursor saponins (such as Rb1 / Rc) into less bitter rare saponins (such as Rg3), while the release of organic acids and peptides increases, conforming to the classic flavor balance model of "bitterness - reduced saponins, umami - increased organic acid salts." At the aroma level, the enhanced response to short-chain alkanes, alcohols, aldehydes, and ethers indicates that fermentation enhances aroma intensity and complexity. At the safety / pleasure level, the decrease in nitrogen oxides indicates a reduction in potential pungent ammonia / amines and nitrosamines, improving sensory pleasure and product safety (Figure C). In Figure C, PC2 (1.3%) and PC1 (98.6%) represent principal component information, respectively. PC1 is the main dimension distinguishing fermented and unfermented samples, explaining 98.6% of the data variation. The two groups of samples are completely separated on the PC1 axis, indicating that the fermentation process caused significant changes in the odor characteristics of the samples; PC2 is a secondary dimension, explaining only 1.3% of the variation. The two groups of samples overlap along the PC2 direction (both concentrated near 0), indicating that this dimension does not contribute substantially to distinguishing fermentation states. Fermented and unfermented samples show significant separation on PC1 (98.6%), with the unfermented group distributed on the negative PC1 axis (-3 to -2) and the fermented group distributed on the positive PC1 axis (2.5 to 3). The two groups do not overlap and are tightly clustered within each group, indicating that the electronic nose can accurately distinguish different fermentation states. In summary, the evaluation by electronic tongue and electronic nose shows that *Lactobacillus plantarum* SP055 fermented ginseng has a systematic flavor reconstruction effect of "reducing bitterness and increasing flavor, enhancing aroma and reducing nitrogen".

[0204] Example 8: Effects of Lactobacillus plantarum SP055 fermentation of ginseng and Gynostemma pentaphyllum on the conversion of rare saponins.

[0205] The preparation of fermented ginseng, fermented Gynostemma pentaphyllum, fermented Sophora japonica buds, and fermented Panax notoginseng are detailed above. Ginsenoside content in fermented ginseng, fermented Gynostemma pentaphyllum, fermented Sophora japonica buds, and fermented Panax notoginseng were determined according to the Chinese Pharmacopoeia's method for total ginsenoside detection and the group standard T / CNHFA 001-2021 for the determination of rare ginsenoside content.

[0206] result

[0207] As shown in Table 1 and Figure 16 As shown, fermenting ginseng with *Lactobacillus plantarum* SP055 can convert the original saponins into rare saponins. (Table 1 and...) Figure 16The results show the fermentation of ginseng aged less than 5 years. Ginseng aged less than 5 years contains virtually no rare saponins. Fermentation with *Lactobacillus plantarum* SP055 significantly increases the content and types of rare saponins (up to 9 types are shown in Table 1: 20-S-Rg3, 20-R-Rg3, 20-S-Rh1, Rg5, Rk1, 20-S-Rg2, Rg6, F4, and Rk3). Studies have confirmed that after fermentation of ginseng or ginseng extract with *Lactobacillus plantarum* SP055, the ratio of rare saponins to total saponins is ≥80%. Figure 16 ).

[0208] Table 2 shows the effects of fermentation with *Lactobacillus plantarum* SP055 on rare saponins (mainly F2, S-Rg3, R-Rg3, Rg5, CK, S-Rh2, and R-Rh2) of *Gynostemma pentaphyllum* and *Panax notoginseng* extracts. *Gynostemma pentaphyllum* is rich in saponin XVII, which has a structure similar to Rb1. After fermentation with *Lactobacillus plantarum* SP055, the content of rare saponins increased by approximately two times. *Panax notoginseng* contains the characteristic saponin R1 and is also rich in proto-saponins Rb1, Rg1, and Rd. After fermentation with *Lactobacillus plantarum* SP055, the content of rare saponins decreased significantly by 82%, suggesting that *Lactobacillus plantarum* SP055 may further convert rare saponins in *Panax notoginseng* into Rk1, Rk3, Rh4, and even further into protopanaxadiol (PPT) and protopanaxadiol (PPD). This study indicates that *Lactobacillus plantarum* SP055 is also effective for saponin conversion from different sources.

[0209] Table 1: Content of proto-saponins and rare saponins before and after fermentation

[0210]

[0211] Table 2: Rare saponin content before and after fermentation

[0212]

[0213] Example 9: Effects of *Lactobacillus plantarum* SP055 fermentation of Sophora japonica flowers on the conversion of rutin and quercetin.

[0214] Fermented Sophora japonica buds: Sophora japonica buds were crushed and mixed with pure water at a material-to-liquid ratio of 1:20 (w / w). Pectinase was added at 0.1% (w / w) and the mixture was treated with enzymatic hydrolysis at 40℃ for 6 hours. Finally, 3% inoculum was used for fermentation for 6 days. Rutin, quercetin, and isoquercetin were detected according to the liquid chromatography detection method for Sophora japonica flowers in the Chinese Pharmacopoeia.

[0215] As shown in Table 3, *Lactobacillus plantarum* SP055 was used for fermentation using *Sophora japonica* buds, and the contents of rutin, quercetin, and isoquercetin were measured. The results showed that after fermentation, the rutin content decreased significantly by about 76%, the isoquercetin content increased by about 3 times, and the quercetin content increased by about 23%. This study indicates that *Lactobacillus plantarum* SP055 has the ability to convert rutin into isoquercetin and quercetin.

[0216] Table 3: Contents of rutin, quercetin, and isoquercetin before and after fermentation

[0217]

[0218] This application is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure.

[0219] Where a numerical range is provided, it should be understood that, unless the context clearly indicates otherwise, each intermediate value (to one-tenth of the lower limit unit) between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any specified value or intermediate value within the range and any other specified value or intermediate value within the specified range is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in the range or excluded, and any one or both of these limitations are included in each smaller range, which is also included in this disclosure, subject to any specifically excluded limitations within the range. Where the range includes one or two limitations, the range excluding any one or both of those included limitations is also included in this disclosure.

[0220] It should be understood that, as used herein and in the appended claims, unless the context clearly specifies otherwise, the singular forms “a” and “the” include plural references.

[0221] Various modifications and variations of the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications to the described modes of carrying out the invention that will be apparent to those skilled in the art of biochemistry and biotechnology or related fields are intended to fall within the scope of the claims.

Claims

1. Lactiplantibacillus plantarum, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 35072.

2. The method for preparing postbiotics from *Lactobacillus plantarum* according to claim 1, comprising the step of inactivating *Lactobacillus plantarum* to prepare postbiotics.

3. The method according to claim 2, wherein the metagenetic agent is a bacterial metagenetic agent, or the inactivation is heat inactivation.

4. A method for fermenting plant material or plant extract, comprising culturing *Lactobacillus plantarum* as described in claim 1 in the presence of plant material or plant extract to produce a ferment.

5. The method according to claim 4, wherein the plant material or plant extract contains one or more of the following active ingredients: saponins and rutin.

6. The method according to claim 4, wherein the plant is one or more of ginseng, astragalus, licorice, gynostemma pentaphyllum, sophora japonica, buckwheat, rue, tangerine peel, and notoginseng, and the plant extract is a plant water extract.

7. The method of claim 6, further comprising: (1) Culturing *Lactobacillus plantarum* according to claim 1 in the presence of extracts of ginseng, astragalus, licorice, and / or *Gynostemma pentaphyllum* or any one thereof to increase the conversion or content of rare saponins; or (2) Culturing *Lactobacillus plantarum* according to claim 1 in the presence of extracts of *Sophora japonica* buds, buckwheat, rue, and / or dried tangerine peel or any one thereof to convert rutin into quercetin and / or isoquercetin; or (3) Cultivate *Lactobacillus plantarum* according to claim 1 in the presence of *Panax notoginseng* or its extracts to increase the content of saponins in the form of protopanaxadiol (PPT) or protopanaxadiol (PPD).

8. The fermentation product produced by the method according to any one of claims 4-7.

9. The metagene of *Lactobacillus plantarum* according to claim 1.

10. The metabiotic according to claim 9, wherein it is one or more of the following: bacterial cell metabiotic, bacterial cell and metabolite metabiotic, and lysed bacterial cell metabiotic.

11. A composition comprising one or more of the following: *Lactobacillus plantarum* according to claim 1, a ferment according to claim 8, and a metabiotic according to claim 9 or 10; and further comprising a pharmaceutically or health-product-acceptable carrier, diluent, or excipient.

12. The use of *Lactobacillus plantarum* according to claim 1, the fermentation product according to claim 8, and / or the metabiotic according to claim 9 or 10 in the preparation of a medicament, wherein the medicament is used for: (1) Treatment and / or prevention of colitis; or (2) Treatment and / or prevention of non-alcoholic fatty liver disease or atherosclerosis; or (3) Treatment and / or prevention of type 2 diabetes or insulin resistance; The fermented product mentioned above is ginseng fermented product.

13. The use according to claim 12, wherein the colitis is ulcerative colitis.

14. The use of *Lactobacillus plantarum* according to claim 1, the fermentation product according to claim 8, and / or the metabiotic according to claim 9 or 10 in the preparation of health products, wherein the health products are used for: (1) Helps maintain healthy blood sugar levels; or (2) It helps maintain healthy levels of blood lipids, including cholesterol and / or triglycerides.

15. The use of *Lactobacillus plantarum* according to claim 1, the fermentation product according to claim 8, and / or the metabiotic according to claim 9 or 10 in the preparation of food, pharmaceuticals, cosmetics, and pet food.

16. A method for converting saponins or rutin, comprising the steps of converting the prototype saponin into a rare saponin using *Lactobacillus plantarum* as described in claim 1, or converting rutin into quercetin or isoquercetin.

17. The method according to claim 16, wherein the prototype saponins include one or more of the following: ginsenoside Rb2, ginsenoside Rb3, ginsenoside Rc, ginsenoside Rd, ginsenoside Re and ginsenoside Rg1; and the rare saponins include one or more of the following: ginsenoside Rg5, ginsenoside Rk1, ginsenoside Rg6, ginsenoside Rk3, ginsenoside Rg3, ginsenoside Rh2, ginsenoside Rg2, ginsenoside Rh1, compound K, ginsenoside F2 and ginsenoside F4.

18. The use of *Lactobacillus plantarum* as a fermenting agent according to claim 1.

19. The use according to claim 18, wherein the fermenting agent is used to ferment one or more of ginseng, astragalus, licorice, gynostemma pentaphyllum, sophora japonica, buckwheat, rue, tangerine peel, and notoginseng, or one or more of their extracts.

20. A method for improving the sensory pleasure of a saponin-containing plant or its aqueous extract, comprising the step of fermenting the plant or its aqueous extract using *Lactobacillus plantarum* according to claim 1, wherein the sensory pleasure includes taste and / or olfactory pleasure.

21. The method according to claim 20, wherein the plant containing saponins is one or more of ginseng, astragalus, licorice, gynostemma pentaphyllum, and Panax notoginseng.

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