Bifidobacterium longum subsp. Longum B53, metagen and preparation method and application of bifidobacterium longum subsp. Longum B53

Abstract

The invention relates to the technical field of microorganisms, and discloses a bifidobacterium longum subsp. Longum B53, a metagen and a preparation method and application of the bifidobacterium longum subsp. Longum B53, the bifidobacterium longum subsp. Longum B53 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35374, and the bifidobacterium longum subsp. Longum B53 is classified and named as bifidobacterium longum subsp. Longum. The invention further discloses application of the bifidobacterium longum subsp. Longum B53 in preparation of products for regulating glucose and lipid metabolism disorder and relieving obesity. The bifidobacterium longum subsp. Longum B53 disclosed by the invention can be used for reducing the generation of SCFAs in a high-fat model; the strain can promote tryptophan metabolism to generate indole and derivatives thereof, has strong anti-inflammatory ability, blood sugar reducing ability and lipid metabolism adjusting ability, is stable in production performance and good in fermentation performance, and has the effects of adjusting glucose and lipid metabolism disorder and relieving obesity.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a strain of Bifidobacterium longum subsp. B53, its postbiotic, its preparation method, and its application. Background Technology

[0002] The global rate of overweight and obesity is currently increasing dramatically. Abnormal weight easily leads to non-communicable diseases such as diabetes, cardiovascular disease, fatty liver, and certain cancers, becoming one of the most serious public health crises. Glucose and lipid metabolism disorders are one of the important mechanisms leading to obesity, and obesity, in turn, exacerbates metabolic disorders, creating a vicious cycle. Commonly used methods for treating obesity include lifestyle interventions, drug therapy, and surgical treatment, which pose a significant challenge to most people. Therefore, it is necessary to develop new weight management methods to improve people's quality of life and physical health.

[0003] Probiotics have the potential to regulate obesity-related metabolic disorders, potentially alleviating obesity by regulating gut microbiota balance, improving the production of metabolites such as short-chain fatty acids, reducing inflammatory responses, improving insulin resistance, and reducing lipid accumulation. However, probiotics require a sufficient number of live bacteria to function effectively, but during storage, a large number of bacteria may be damaged or die, reducing their efficacy. Metabiotics are inactivated microorganisms and / or bacterial components with a clearly defined genetic background beneficial to host health, including or excluding their metabolites. They are usually obtained from inactivated probiotics. Metabiotics have beneficial effects on host health, such as strengthening the epithelial barrier, regulating immune responses, regulating systemic metabolism, and regulating the nervous system. Metabiotics possess the probiotic functions of probiotics while avoiding the management of live bacteria and their potential adverse effects on the human body, making them a safer alternative for people with weakened immune systems. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bifidobacterium longum subsp. B53, its metabolites, preparation methods, and applications. Bifidobacterium longum subsp. B53 can reduce the production of SCFAs in a high-fat model; promote tryptophan metabolism to produce indole and its derivatives, especially ILA, and has strong anti-inflammatory, hypoglycemic, and lipid metabolism regulating abilities. At the same time, this strain has stable production performance and good fermentation performance, and has the effect of regulating glucose and lipid metabolism disorders and alleviating obesity.

[0005] To achieve the above objectives, this invention provides a strain of *Bifidobacterium longum* subsp. longum, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 25, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35374. The classification name is *Bifidobacterium longum* subsp. longum, and the 16S rDNA sequence is shown in SEQ ID NO. 1.

[0006] Furthermore, this invention provides the application of the aforementioned *Bifidobacterium longum* subsp. B53 in the preparation of products for regulating glucose and lipid metabolism disorders and alleviating obesity. These products include dietary supplements, health products, pharmaceuticals, or beverages. Formulations include liquids, tablets, granules, pills, or capsules.

[0007] Furthermore, it is applied to:

[0008] ① Regulate the structure of gut microbiota, reduce the production of SCFAs in a high-fat diet, and maintain the level of SCFAs in the gut;

[0009] ② Reduce the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increase the levels of anti-inflammatory factors IL-10 and IL-22;

[0010] ③ Improve the damage to pancreatic and liver tissues caused by a high-fat diet;

[0011] ④ Promotes tryptophan metabolism to produce indole and its derivatives;

[0012] ⑤ It reduces oral glucose tolerance, insulin levels, and insulin resistance index, reduces the organ index of white adipose tissue, and restores the morphology and structure of adipose tissue.

[0013] Furthermore, the present invention also provides the application of the above-mentioned Bifidobacterium longum subspecies B53 in the preparation of a metabiotic that regulates glucose and lipid metabolism disorders and alleviates obesity.

[0014] Furthermore, it is applied to:

[0015] ① Regulate the structure of gut microbiota, reduce the production of SCFAs in a high-fat diet, and maintain the level of SCFAs in the gut;

[0016] ② Reduce the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increase the levels of anti-inflammatory factors IL-10 and IL-22;

[0017] ③ Improve the damage to pancreatic and liver tissues caused by a high-fat diet;

[0018] ④ Promotes tryptophan metabolism to produce indole and its derivatives;

[0019] ⑤ It reduces oral glucose tolerance, insulin levels, and insulin resistance index, reduces the organ index of white adipose tissue, and restores the morphology and structure of adipose tissue.

[0020] To achieve the above objectives, the present invention also provides a metabiotic, which includes Bifidobacterium longum subsp. B53 and its metabolites.

[0021] Furthermore, the preparation method of the post-genetic agent is as follows:

[0022] Bifidobacterium longum subsp. B53 was inoculated into liquid culture medium and cultured. The bacterial cells were collected by centrifugation. The bacterial cells were resuspended in PBS buffer to obtain a bacterial suspension. The bacterial suspension was sterilized by heating and then spray-dried or freeze-dried to obtain a metabiotic that can regulate glucose and lipid metabolism disorders and alleviate obesity.

[0023] Furthermore, the inoculation amount of Bifidobacterium longum subsp. B53 was 1%–5% v / v;

[0024] The liquid culture medium was MRS liquid medium supplemented with 0.05% L-cysteine ​​hydrochloride;

[0025] The cultivation conditions are: temperature 30℃~37℃, static strict anaerobic culture for 12h~24h;

[0026] Centrifugation conditions were: temperature 4℃, gravitational acceleration 6000g~10000g, and time 5min~10min.

[0027] Furthermore, 1 L of MRS liquid medium supplemented with 0.05% L-cysteine ​​hydrochloride comprises: 5.0 g peptone, 10.0 g trypone, 5.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 0.25 g manganese sulfate, 5.0 g sodium acetate, 2.0 g diammonium citrate, 0.58 g magnesium sulfate, 2.0 g dipotassium hydrogen phosphate, 1.0 g Tween 80, and 0.5 g / L (w / v) L-cysteine ​​hydrochloride.

[0028] Furthermore, the conditions for heat sterilization are: 70℃~90℃, time 10min~30min;

[0029] The spray drying conditions are: inlet air temperature 150℃~180℃, outlet air temperature 80℃~100℃;

[0030] The vacuum freeze-drying conditions are: temperature -50℃ to -10℃, vacuum 2mbar to 5mbar.

[0031] Furthermore, the concentration of *Bifidobacterium longum* subsp. B53 in the metagenin was 1–3 × 10⁻⁶.9 CFU / mL.

[0032] The advantages and positive effects of the Bifidobacterium longum subspecies B53, the metabiotic, the preparation method, and the application described in this invention are as follows:

[0033] 1. The Bifidobacterium longum subspecies B53 provided by this invention has a strong ability to metabolize tryptophan to produce indole and its derivatives, anti-inflammatory ability, hypoglycemic ability and good ability to regulate lipid metabolism. This strain has stable production performance and good fermentation performance, and has the effect of regulating glucose and lipid metabolism disorders and alleviating obesity.

[0034] 2. The Bifidobacterium longum subsp. B53 and its metagener provided by this invention adopts heat inactivation combined with spray drying or vacuum freeze drying technology. The process is simple, convenient, efficient, and easy to preserve, which is conducive to large-scale industrial production. The heat inactivation is carried out at a temperature of 70℃~90℃ for 10min~30min, which can reduce the impact of temperature on the active ingredients in the metagenerer.

[0035] 3. The *Bifidobacterium longum* subsp. B53 and its metabolites provided by this invention effectively alleviate obesity induced by a high-fat diet (HFD) in mice. By regulating the intestinal flora structure, reducing the production of SCFAs, lowering serum levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increasing levels of anti-inflammatory factors IL-10 and IL-22, they improve damage to pancreatic and liver tissues. They also promote tryptophan metabolism to produce indole and its derivatives, especially ILA, thereby activating the AHR / GLP-1 / PDX1 signaling pathway, improving insulin sensitivity, reducing insulin resistance, and lowering blood glucose levels. Furthermore, they activate the AHR / CYP1A1 / LXRα / SREBP1 / FAS / SCD1 signaling pathway, reducing the synthesis of TC, TG, and fatty acids, lowering blood lipid levels, alleviating lipid accumulation in the liver, pancreas, and adipose tissue, regulating glucose and lipid metabolism, and improving host obesity.

[0036] 4. The *Bifidobacterium longum* subsp. B53 and its metabiotic provided by this invention can alleviate the trends of weight gain and energy intake increase in obese mice, significantly reduce oral glucose tolerance test (OGTT), insulin levels, and the homeostasis model of insulin resistance (HOMA-IR), significantly reduce the organ index of white adipose tissue, and restore the morphology and structure of adipose tissue. It significantly reduces serum levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increases levels of anti-inflammatory factors IL-10 and IL-22 (P<0.05), alleviating the host's inflammatory state; it significantly reduces serum LDL-C, TC, and TG levels, increases HDLC levels, and improves blood lipid levels. It significantly reduces malondialdehyde (MDA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels, and increases superoxide dismutase (SOD) and glutathione (GSH) levels, improving host liver oxidative damage. Bifidobacterium longum subspecies B53 and its metabiotics improved the gut microbiota structure in obese mice. At the phylum level, the abundance of Dethiobacteria and Firmicutes decreased, while the abundance of Bacteroidetes increased. At the genus level, the abundance of Lentimicrobium, Anaerorotruncus α, Clostridia_UCG-014, [Eubacterium]_fissicatena_group, Alloprevotella, Roseburia, and Parabacteroides decreased, while the abundance of Prevotella and Bifidobacterium increased.

[0037] Furthermore, *Bifidobacterium longum* subspecies B53 and its metabolites reduced the production of short-chain fatty acids (SCFAs) in the high-fat model; they promoted tryptophan metabolism to produce indole and its derivatives, especially ILA (indolelactic acid). Consequently, *Bifidobacterium longum* subspecies B53 and its metabolites significantly upregulated the expression levels of colonic AHR, liver CYP1A1, pancreatic GLP-1R, and PDX1, and downregulated the expression levels of liver LXRα, SREBP1, FAS, and SCD1, thereby reducing host blood glucose levels, decreasing lipid accumulation, regulating glucose and lipid metabolism, and improving host obesity.

[0038] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0039] Figure 1 The results show the changes in body weight and energy intake of mice; (a) changes in body weight of mice before gavage; (b) changes in body weight of mice during gavage; (c) changes in energy intake of mice before gavage; and (d) changes in energy intake of mice during gavage.

[0040] Figure 2The table shows the OGTT, serum insulin level, and HOMA-IR level of mice; (a) mouse OGTT curve; (b) AUC of mouse OGTT curve; (c) mouse serum INS level; (d) mouse HOMA-IR.

[0041] Figure 3 The adipose organ index of mice; (a) mouse BAT organ index; (b) mouse eWAT organ index; (c) mouse iWAT organ index;

[0042] Figure 4 The image shows the pathological changes in the adipose tissue of the mouse epididymis; the red arrows indicate a significant increase in the volume of intracellular lipid droplets compared to group C; the black arrows indicate visible damage to the lipid droplets.

[0043] Figure 5 The results showed that the levels of lipids and liver damage in mouse serum were as follows: (a) mouse serum HDLC level; (b) mouse serum LDLC level; (c) mouse serum TC level; (d) mouse serum TG level; (e) mouse serum ALT level; and (f) mouse serum AST level.

[0044] Figure 6 The values ​​represent the levels of inflammatory factors in mouse serum; among which, (a) mouse serum TNF-α level; (b) mouse serum IL-1β level; (c) mouse serum IL-6 level; (d) mouse serum IL-10 level; and (e) mouse serum IL-22 level.

[0045] Figure 7 The image shows histopathological changes in mouse pancreas tissue; the red arrows represent circular vacuoles within the cytoplasm.

[0046] Figure 8 The images show histopathological changes in mouse liver tissue; red arrows represent round lipid droplets in the cytoplasm; black arrows indicate significant inflammatory cell infiltration.

[0047] Figure 9 The levels of oxidative stress-related indicators in mouse liver are: (a) GSH level in mouse liver; (b) SOD level in mouse liver; and (c) MDA level in mouse liver.

[0048] Figure 10 The expression levels of genes related to glucose metabolism in mice are shown in the figures; (a) colonic Ahr gene expression level; (b) pancreatic Glp-1r gene expression level; and (c) pancreatic Pdx1 gene expression level.

[0049] Figure 11The expression levels of AHR / GLP-1R / PDX1 proteins in mice are shown below; (a) Western blots of AHR, GLP-1R, and PDX1 proteins in mouse colon and pancreas; (b) GLP expression levels in mouse serum; (c) relative expression levels of AHR protein in mouse colon; (d) relative expression levels of GLP-1R protein in mouse pancreas; and (e) relative expression levels of PDX1 protein in mouse pancreas.

[0050] Figure 12 The expression levels of lipid metabolism-related genes in mice are as follows: (a) colonic Ahr gene expression level; (b) liver Cyp1a1 gene expression level; (c) liver Lxrα gene expression level; (d) liver Srebp1 gene expression level; (e) liver Fas gene expression level; (f) liver Scd1 gene expression level.

[0051] Figure 13 The protein expression levels of CYP1A1 / LXRα / SREBP1 / FAS / SCD1 in mouse liver are shown below. (a) Western blot images of CYP1A1, LXRα, SREBP1, FAS, and SCD1 proteins in mouse liver; (b) relative expression level of CYP1A1 protein in mouse liver; (c) relative expression level of LXRα protein in mouse liver; (d) relative expression level of SREBP1 protein in mouse liver; (e) relative expression level of FAS protein in mouse liver; and (f) relative expression level of SCD1 protein in mouse liver.

[0052] Figure 14 The graph shows the changes in α-diversity of mouse gut microbiota; (a) Chao 1 index of mouse gut microbiota; (b) Shannon index of mouse gut microbiota; (c) Simpson index of mouse gut microbiota.

[0053] Figure 15 The β diversity of mouse gut microbiota; (a) Venn plot; (b) NMDs plot of Bray-Curtis distance;

[0054] Figure 16 Changes in the phylum level of the gut microbiota in mice;

[0055] Figure 17 Changes in the genus level of the intestinal flora in mice;

[0056] Figure 18 LEfSE analysis evolutionary clade plots of mouse gut microbiota C1 and HFD groups;

[0057] Figure 19 Plots showing LDA scores for mouse gut microbiota C1 and HFD groups;

[0058] Figure 20The LDA scores of mouse gut microbiota groups HFD, B, P, D, and I are plotted.

[0059] Figure 21 The levels of SCFAs in mouse intestinal contents include: (a) acetic acid level in mouse intestinal contents; (b) propionic acid level in mouse intestinal contents; (c) butyric acid level in mouse intestinal contents; and (d) total acid level in mouse intestinal contents.

[0060] Figure 22 The results show the targeting effects of indole and its derivatives in mice; including (a) the content of 5-hydroxyindole-3-acetic acid (5-HIAA); (b) the content of β-indole-3-acetic acid (IAA); (c) the content of indole-3-carboxaldehyde (ICAld); (d) the content of L-tryptophan (L-TRP); (e) the content of indole-3-acrylic acid (IDA); (f) the content of indolepropionic acid (IPA); and (g) the content of indolelactic acid (ILA).

[0061] Certificate of Preservation of Biological Materials

[0062] Bifidobacterium longum subsp. longum is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 25, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35374. The classification name is Bifidobacterium longum subsp. longum. The 16S rDNA sequence is shown in SEQ ID NO. 1.

[0063] Detailed Implementation

[0064] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0065] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0066] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0067] This invention provides a strain of *Bifidobacterium longum* subsp. *longum*, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 25, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35374, and classified as *Bifidobacterium longum* subsp. *longum*.

[0068] This invention also provides the application of the above-mentioned Bifidobacterium longum subsp. B53 in the preparation of a metabiotic that regulates glucose and lipid metabolism disorders and alleviates obesity.

[0069] The present invention also provides a metabiotic that regulates glucose and lipid metabolism disorders and alleviates obesity, wherein the metabiotic comprises inactivated Bifidobacterium longum subsp. B53 and its metabolites.

[0070] This invention also provides a method for preparing a metabolite that regulates glucose and lipid metabolism disorders and alleviates obesity, comprising:

[0071] Bifidobacterium longum subsp. B53 was inoculated into liquid culture medium and cultured. The bacterial cells were collected by centrifugation. The preservation number of Bifidobacterium longum subsp. B53 is:

[0072] The bacterial body is resuspended in a buffer solution to obtain a bacterial suspension. The bacterial suspension is then sterilized by heating and combined with spray drying or vacuum freeze drying technology to obtain the metabiotic that regulates glucose and lipid metabolism disorders and alleviates obesity.

[0073] Furthermore, in the preparation method, the inoculation amount of Bifidobacterium longum subsp. B53 is 1% (v / v) to 5% (v / v), which can be 1% (v / v), 2% (v / v), 3% (v / v), 4% (v / v), 5% (v / v), etc., but is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0074] And / or, the liquid culture medium is an MRS liquid culture medium supplemented with 0.05% L-cysteine ​​hydrochloride;

[0075] And / or, the culture conditions are: temperature 30℃~37℃, the temperature can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, etc., but not limited to the listed values, and other unlisted values ​​within this range are also applicable; static strict anaerobic culture for 12h~24h, the time can be 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc., but not limited to the listed values, and other unlisted values ​​within this range are also applicable;

[0076] And / or, the centrifugation conditions are: temperature 4℃, gravitational acceleration 6000g~10000g, which can be 6000g, 6500g, 7000g, 7500g, 8000g, 8500g, 9000g, 9500g, 10000g, etc., but not limited to the listed values, and other unlisted values ​​within this range are also applicable; time 5min~10min, which can be 5min, 6min, 7min, 8min, 9min, 10min, etc., but not limited to the listed values, and other unlisted values ​​within this range are also applicable;

[0077] And / or, the bacterial cells are washed 1 to 3 times with sterile PBS;

[0078] And / or, the buffer solution is PBS buffer;

[0079] Furthermore, the heat sterilization conditions are: 70℃~90℃, which can be 70℃, 75℃, 80℃, 85℃, 90℃, etc., but not limited to the listed values; other unlisted values ​​within this range are also applicable; the time is 10min~30min, which can be 10min, 12min, 14min, 15min, 16min, 18min, 20min, 22min, 24min, 25min, 26min, 28min, 30min, etc., but not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0080] Furthermore, the spray drying conditions are as follows: inlet air temperature 150℃~180℃, which can be 150℃, 160℃, 170℃, 180℃, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable; outlet air temperature 80℃~100℃, which can be 80℃, 90℃, 100℃, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0081] Furthermore, the vacuum freeze-drying conditions are: temperature -50℃ to -10℃, which can be -50℃, -40℃, -30℃, -20℃, -10℃, etc., but is not limited to the listed values, and other unlisted values ​​within this range are also applicable; vacuum 2mbar to 5mbar, which can be 2mbar, 3mbar, 4mbar, 5mbar, but is not limited to the listed values, and other unlisted values ​​within this range are also applicable.

[0082] Further, 1 L of the MRS liquid medium supplemented with 0.05% L-cysteine ​​hydrochloride comprises: 5.0 g peptone, 10.0 g trypone, 5.0 g beef extract, 5.0 g yeast extract, 20.0 g glucose, 0.25 g manganese sulfate, 5.0 g sodium acetate, 2.0 g diammonium hydrogen citrate, 0.58 g magnesium sulfate, 2.0 g dipotassium hydrogen phosphate, 1.0 g Tween 80, and 0.5 g / L (w / v) L-cysteine ​​hydrochloride.

[0083] This invention also provides the above-mentioned metabiotic for regulating glucose and lipid metabolism disorders and alleviating obesity, or the metabiotic prepared by the above-mentioned method for preparing the metabiotic for regulating glucose and lipid metabolism disorders and alleviating obesity, wherein the concentration of Bifidobacterium longum subsp. B53 in the metabiotic is 1-3 × 10⁻⁶. 9 CFU / mL.

[0084] The present invention also provides a formulation comprising the above-mentioned metagenic agent for regulating glucose and lipid metabolism disorders and alleviating obesity, or a metagenic agent prepared by the above-mentioned preparation method for metagenic agent for regulating glucose and lipid metabolism disorders and alleviating obesity, wherein the formulation comprises liquid, tablet, granule, pill or capsule.

[0085] The present invention also provides an application of an epigenetic prepared by the above-mentioned method for regulating glucose and lipid metabolism disorders and alleviating obesity, or an epigenetic prepared by the above-mentioned method for regulating glucose and lipid metabolism disorders and alleviating obesity, the application including preparation of dietary supplements, health products, pharmaceuticals, foods or beverages for alleviating obesity.

[0086] The composition of the MRS culture medium used in this embodiment of the invention is as follows: 5.0g peptone, 2.0g diammonium hydrogen citrate, 10.0g tryptone, 2.0g dipotassium hydrogen phosphate, 5.0g yeast extract, 20.0g glucose, 0.25g manganese sulfate, 1.0g Tween-80, 0.58g magnesium sulfate, 5.0g beef extract, 5.0g sodium acetate, and 0.5g / L (w / v) L-cysteine ​​hydrochloride. Before use, distilled water is added to a final volume of 1L, stirred thoroughly, and autoclaved at 121℃ for 15 minutes. For solid culture medium, 1.5% (w / v) to 1.6% (w / v) agar powder is added.

[0087] Example

[0088] A method for preparing an epigenetic that can alleviate obesity includes the following steps:

[0089] After activation, *Bifidobacterium longum* subsp. B53 was inoculated at a rate of 2% (v / v) into MRS liquid medium supplemented with 0.05% L-cysteine ​​hydrochloride. The culture was then strictly anaerobic and statically incubated at 37°C for 18 h. Cells were collected by centrifugation (4°C, 8000×g, 10 min), washed three times with sterile PBS, and then resuspended in PBS buffer to obtain a concentration of 1×10⁻⁶. 9 A CFU / mL suspension of *Bifidobacterium longum* subsp. B53 in PBS was used to inactivate the bacterial cells by heating at 80°C for 20 min, followed by spray drying or freeze-drying to obtain *B. longum* B53 postbiotic powder. The powder was resuspended in sterile PBS before gavage and prepared fresh each time.

[0090] Test case

[0091] (1) Animal experimental design:

[0092] The experimental animals were 110 five-week-old male C57BL / 6J mice. The environment was maintained with 12 hours of alternating light and dark, a temperature of 23±2℃, and humidity of 55±5%, with bedding changed regularly. All animal experiments in this study were reviewed and approved by the Animal Ethics Committee of Northeast Agricultural University (Review Form No.: NEAUEC20240473) and conducted in accordance with the "Guidelines for Animal Care and Use". Mice had free access to water and a low-fat diet. After one week of acclimatization, they were randomly divided into two groups: a control group (Control, n=20) and a high-fat diet group (HFD) (n=90). The experimental period was 14 weeks, with weeks 1-9 being the modeling period and weeks 10-14 being the experimental period. The control group mice were fed a low-fat diet, while the obese group mice were fed a high-fat diet (60% fat). After 9 weeks of feeding the high-fat diet, an obese mouse model was successfully established (the standard was that the average weight of the HFD group mice exceeded the average weight of the control group by 20%). The comparison of energy and nutrient composition between the low-fat and high-fat diets is shown in Tables 1 and 2.

[0093] Table 1 Comparison of Energy Components between Low-Fat and High-Fat Diets

[0094] Energy components ordinary feed High-fat feed protein 14％ 14％ carbohydrate 76％ 26％ Fat 10％ 60％

[0095] Table 2 Comparison of Nutritional Components of Low-Fat and High-Fat Diets

[0096] Nutritional components Low-fat diet feed (g / kg) High-fat diet feed (g) Protein (casein, L-cysteine) 141 201 Carbohydrates (dextrin, sucrose) 0 320 Carbohydrates (dextrin, sucrose, corn starch) 722 0 Fats (soybean oil, lard) 40 341 Fiber (cellulose) 50 71 Mineral and vitamin complex 47 67 Antioxidant (Tert-butyl hydroquinone, TBHQ) 0.008 0.066 Energy (kcal / g) 3.6 5.1

[0097] Obese mice that successfully developed the model were randomly divided into 6 groups and subjected to a 5-week gavage intervention. The experimental groups were as follows: blank control group 1 (C1, n=10); control group 2 (C2, n=10); high-fat diet group (HFD); spray-dried group (1 mg / mL, P); freeze-dried group (1 mg / mL, D); probiotic group (1×10⁻⁶). 9 CFU / mL, B); Indolelactate group (20 mg / kg, I); Indolelactate (20 mg / kg) + CH223191 group (10 mg / kg, CH) (CH223191 is an aryl hydrocarbon receptor (AhR) antagonist, purchased from AbMole). The specific experimental procedure is shown in Table 3:

[0098] Table 3 Experimental Procedure

[0099] Grouping feed Dosage C1 Adaptive feeding for one week ordinary feed Regular feed + PBS C2 Adaptive feeding for one week ordinary feed Ordinary feed + solubilizer HFD Adaptive feeding for one week HFD feed HFD B Adaptive feeding for one week HFD feed HFD+B.langumB53 P Adaptive feeding for one week HFD feed HFD+ spray-dried metabolites D Adaptive feeding for one week HFD feed HFD + freeze-dried post-biotic I Adaptive feeding for one week HFD feed HFD+ILA CH Adaptive feeding for one week HFD feed HFD+ILA+CH223191 One week 2-10 weeks 11-15 weeks

[0100] Before the end of the experiment, the mice were fasted for 12 hours but allowed to drink water. After anesthetizing them by intraperitoneal injection of ketamine at 100 mg / kg bw, blood was collected from the eyeballs and placed in 1.5 mL centrifuge tubes. The tubes were then allowed to stand at 4°C for at least 30 min. After centrifugation (4°C, 3000×g, 10 min), the supernatant was carefully aspirated to obtain serum, which was then aliquoted and frozen at -80°C for later use.

[0101] After blood collection, mice were euthanized by cervical dislocation. Fresh brown adipose tissue (BAT), white adipose tissue (inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT)), liver, and pancreas were rapidly dissected to obtain these tissues. The adipose tissues were cleaned with physiological saline and weighed. The colon, liver, epididymal adipose tissue, and pancreas of some mice were fixed in 4% paraformaldehyde solution and stored at room temperature. The remaining mouse organs and tissues were flash-frozen in liquid nitrogen and then stored at -80°C. Intestinal contents were collected in 2mL cryovials, rapidly placed in liquid nitrogen, and stored at -80°C for later use. The organ index of the adipose tissue in each group of mice was calculated using the following formula:

[0102]

[0103] (2) Phenomenological changes in mice:

[0104] Record the mice's weight and food intake weekly, and calculate the mice's weight gain rate using the following formula:

[0105] Weight gain (g) = Weight at the end of the experiment - Weight at the beginning of the experiment;

[0106]

[0107] The results are as follows Figure 1 As shown, by Figure 1 As shown in Figure (b), except for groups C1, C2, and HFD, the body weight of the other five groups of mice generally showed a slow upward trend during gavage. In the first week of gavage, compared with the HFD and CH groups, the body weight of the other five intervention groups showed a significant downward trend (P<0.05, not shown in the figure). In weeks 1-2 of gavage, compared with the HFD group, the body weight of mice in groups B, P, D, and I all increased, with significant differences between groups B and I (P<0.05). In weeks 2-3 of gavage, compared with the HFD group, the body weight of the other five groups of mice showed significant differences (P<0.05). The trend was the same as in weeks 1-2 during weeks 3-5 of gavage. Figure 1As shown in (c), during model construction, the energy intake of HFD-fed mice was generally higher than that of control mice, but not significantly (P>0.05). Figure 1 As shown in Figure (d), energy intake in each group of mice was unstable during gavage, but overall, energy intake in the other groups was higher than that in the CON group. These results indicate that B. longum B53, its post-biotic, and ILA can alleviate weight gain in mice induced by a high-fat diet to some extent, but these results are not significantly related to energy intake.

[0108] (3) Oral glucose tolerance test (OGTT):

[0109] In the OGTT, mice were fasted for 12 hours and then administered 2 g / kg of sterile glucose solution by gavage. Blood samples were collected from the tail vein at 0, 15, 30, 60, and 120 minutes, and blood glucose levels were measured using a glucometer. The area under the curve (AUC) was calculated. Serum insulin levels were measured using an ELISA kit, and the HOMA-IR index was calculated using the following formula.

[0110] HOMA-IR = Fasting blood glucose × Fasting insulin ÷ 22.5.

[0111] like Figure 2 As shown in (a), after glucose gavage treatment, the blood glucose levels of obese mice peaked at approximately 30 minutes and then showed a decreasing trend over the next 30-120 minutes. The trends in groups C1 and C2 were largely consistent, while groups B and I showed a faster decreasing trend compared to other treatment groups, indicating that *B. longum* B53 and ILA exhibit better insulin sensitivity. The AUC of the OGTT is shown in... Figure 2 As shown in (b), the AUC of mice in all other groups was significantly lower than that in the HFD group (P<0.05); in the intervention groups, the AUC of groups B and I was significantly lower than that of the other intervention groups (P<0.05), and there was no significant difference between the two groups (P>0.05). Compared with group I, the AUC of group CH was significantly higher (P<0.05). Figure 2 As shown in (c), serum insulin levels in groups C1 and C2 were essentially at the same level (P>0.05). Serum insulin levels in the HFD group and the intervention group were significantly higher than in group C1 (P<0.05). In the intervention group, except for group CH, insulin levels were significantly lower than in group HFD (P<0.05). Compared to group I, insulin levels in group CH were significantly higher (P<0.05). Figure 2As shown in Figure (d), the trends of HOMA-IR and INS levels were similar in all groups of mice, but the difference between the CH group and the HFD group was significant (P<0.05). These results indicate that intervention with B. longum B53, its post-biotic, and ILA can improve glucose tolerance and insulin resistance symptoms in obese mice.

[0112] (4) Changes in fat accumulation in mice:

[0113] Organ indices can reflect the host's organ function to some extent. In this experiment, the adipose organ (BAT, eWAT, iWAT) indices of mice in each group were as follows: Figure 3 As shown.

[0114] Depend on Figure 3 It can be seen that BAT weight is negatively correlated with obesity. Figure 3 As shown in (a), the BAT index of each group was significantly lower than that of group C1 (P<0.05); except for group CH, there was no significant difference in the BAT index between the other intervention groups and the HFD group (P>0.05). Figure 3 As shown in (b), compared with group C1, the eWAT index of all other groups increased significantly (P<0.05); the eWAT index of mice in groups P, D, and CH was not significantly different from that of the HFD group (P>0.05), while the eWAT index of groups B and I was significantly lower than that of the HFD group (P<0.05), but did not return to normal levels, and the eWAT index of group I was significantly lower than that of the CH group (P<0.05); unlike the eWAT index, the iWAT index of mice in groups B, P, D, and I was significantly lower than that of the HFD group (P<0.05). Figure 3 (c)). The above results indicate that the weight of brown adipose tissue in obese mice induced by HFD was significantly reduced, while the weight of white adipose tissue was significantly increased. However, intervention with B. longum B53 and its post-biotics and ILA could alleviate the increase in white adipose tissue weight, but its weight loss effect may not involve the function of brown adipose tissue.

[0115] The epididymal adipose tissue of mice was stained with hematoxylin and eosin (HE) to observe the pathological changes in the epididymal adipose tissue. The results are shown below. Figure 4 .Depend on Figure 4It can be seen that, under the same field of view (200×), the C2 group mice were similar to the C1 group mice, with basically normal tissue structure. The adipocytes in the field of view were clearly structured, abundant, tightly arranged, with flattened nuclei, and no obvious damage or necrosis was observed. The intracellular lipid droplets were large and full; no inflammatory cell infiltration was observed in the tissue. Compared with the C group, the HFD and CH group mice showed moderate abnormalities in adipose tissue structure, with obvious damage to a large number of intracellular lipid droplets (black arrows), a significant decrease in cell number, and a significant increase in intracellular lipid droplet volume (red arrows). The adipose tissue of the B, P, D, and I group mice showed some abnormalities, such as a small number of lipid droplet damages and an increase in the volume of some intracellular lipid droplets.

[0116] (5) Changes in blood lipids and liver function in mice:

[0117] The levels of low-density lipoprotein cholesterol (LDLC), high-density lipoprotein cholesterol (HDLC), total cholesterol (TC), triglycerides (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the serum of mice in each group were determined according to the instructions of the biochemical reagent kit.

[0118] The blood lipid levels and serum AST and ALT levels of mice in each group are as follows: Figure 5 As shown, compared with group C1, the levels of LDLC, TC, and TG in mice in the HFD group were significantly increased (P<0.05), while the level of HDLC was significantly decreased (P<0.05). Compared with the HFD group, mice fed a high-fat diet, after intervention with B. longum B53 and its post-biotics and ILA, had significantly decreased levels of LDLC, TC, and TG (P<0.05), while the level of HDLC was significantly increased (P<0.05). Compared with group I, the levels of LDLC, TC, and TG in the CH group were significantly increased (P<0.05), while the level of HDLC was significantly decreased (P<0.05). ALT and AST are indicators of liver function, and their values ​​reflect the extent of hepatocyte damage. Figure 5 As shown in (e) and (f), compared with the C1 group mice fed a low-fat diet, the ALT and AST levels of the HFD group mice fed a high-fat diet were significantly increased (P<0.05); compared with the HFD group, the ALT and AST levels of the high-fat diet mice were significantly decreased after intervention with B. longum B53, its post-biotic, and ILA (P<0.05); compared with the I group, the ALT and AST levels of the CH group were significantly increased (P<0.05). These results indicate that intervention with B. longum B53, its post-biotic, and ILA can reduce blood lipid levels in mice, decrease lipid accumulation, and alleviate liver damage.

[0119] (6) Changes in serum inflammatory factor levels in mice:

[0120] The serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, and IL-22 were measured according to the instructions in the ELISA kit. The results are as follows: Figure 6 As shown.

[0121] Depend on Figure 6 The results showed that, compared with group C1, the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly increased in the HFD group (P<0.05), while the levels of anti-inflammatory cytokines IL-10 and IL-22 were significantly decreased (P<0.05). Compared with the HFD group, the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 were significantly decreased in groups B, P, D, and I (P<0.05), while the levels of anti-inflammatory cytokines IL-10 and IL-22 were significantly increased (P<0.05). These results indicate that intervention with B. longum B53, its metabolites, and ILA alleviated inflammation in obese mice.

[0122] (7) Changes in the morphology of mouse pancreatic tissue:

[0123] Mouse colon histopathological sections as shown Figure 7 As shown. By Figure 7 As can be seen (200×), the liver tissue structure of groups C1 and C2 was basically normal, with abundant hepatocytes in the field of view, arranged neatly and tightly, with full cytoplasm and clear nuclei; the hepatic cords were neatly arranged, and no obvious dilation of the hepatic sinusoids was observed; no obvious inflammatory cell infiltration was observed in the tissue; the livers of mice in the other experimental groups showed different degrees of abnormality. The liver tissue structure of mice in groups HFD and CH was severely abnormal, with a large number of hepatocytes showing fatty degeneration, and round lipid droplets of varying sizes appearing in the cytoplasm (red arrows), indicating mild inflammation; the liver tissue structure of mice in groups B and I was mildly abnormal, with a small number of hepatocytes showing fatty degeneration and a few round lipid droplets appearing (red arrows); the liver tissue structure of mice in groups P and D was moderately abnormal, with a small number of hepatocytes showing fatty degeneration, a few round lipid droplets appearing (red arrows), and obvious inflammatory cell infiltration (black arrows) observed in the tissue, indicating inflammation.

[0124] (8) Changes in liver damage in mice:

[0125] Mouse liver histopathological sections as shown Figure 8 As shown. By Figure 8As can be seen (200×), the liver tissue structure of groups C1 and C2 was basically normal, with abundant hepatocytes in the field of view, arranged neatly and tightly, with full cytoplasm and clear nuclei; the hepatic cords were neatly arranged, and no obvious dilation of the hepatic sinusoids was observed; no obvious inflammatory cell infiltration was observed in the tissue; the livers of mice in the other experimental groups showed different degrees of abnormality. The liver tissue structure of mice in groups HFD and CH was severely abnormal, with a large number of hepatocytes showing fatty degeneration, and round lipid droplets of varying sizes appearing in the cytoplasm (red arrows), indicating mild inflammation; the liver tissue structure of mice in groups B and I was mildly abnormal, with a small number of hepatocytes showing fatty degeneration and a few round lipid droplets appearing (red arrows); the liver tissue structure of mice in groups P and D was moderately abnormal, with a small number of hepatocytes showing fatty degeneration, a few round lipid droplets appearing (red arrows), and obvious inflammatory cell infiltration (black arrows) observed in the tissue, indicating inflammation.

[0126] The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) in liver tissue were measured according to the instructions of the biochemical reagent kit. Changes in liver oxidative stress-related indicators in each group of mice are shown below. Figure 9 As shown.

[0127] Depend on Figure 9 The results showed that, compared with group C1, the levels of GSH and SOD in the liver of mice in the HFD group were significantly decreased (P<0.05), and the level of MDA was significantly increased (P<0.05). Compared with the HFD group, the levels of GSH and SOD in the liver of mice in groups B, P, D, and I were significantly increased (P<0.05), and the level of MDA was significantly decreased (P<0.05). Compared with group I, the levels of GSH and SOD in the liver of mice in group CH were significantly decreased (P<0.05), and the level of MDA was significantly increased (P<0.05). These results indicate that intervention with B. longum B53, its post-biotic, and ILA can alleviate liver damage induced by HFD in obese mice.

[0128] (9) Changes in the glucose metabolism pathway in mice:

[0129] The expression levels of relevant genes, namely the colonic Ahr gene, the pancreatic Glp-1r gene, and the Pdx1 gene, were detected using RT-qPCR technology. The results are as follows: Figure 10 As shown.

[0130] Depend on Figure 10 It was found that, compared with group C1, the expression levels of Ahr, Glp-1r, and Pdx1 genes in group HFD were significantly decreased (P<0.05); compared with group HFD, the expression levels of Ahr, Glp-1r, and Pdx1 genes in groups B, P, D, and I were significantly increased (P<0.05), and the expression levels of related genes were similar to those in group C1; compared with group I, the expression levels of Ahr, Glp-1r, and Pdx1 genes in group CH were significantly decreased (P<0.05).

[0131] The expression levels of related proteins, namely AHR, GLP-1R, and PDX1, were detected using Western blot. The results are as follows: Figure 11 As shown.

[0132] Depend on Figure 11 As shown in (a) and (c), compared with group C1, the colonic AHR protein expression level of mice in the HFD group was significantly decreased (P<0.05); compared with the HFD group, the colonic AHR protein expression levels of mice in groups P and D were slightly increased, but not significantly (P>0.05), while the colonic AHR protein expression levels of mice in groups B and I were significantly increased (P<0.05); compared with group I, the colonic AHR protein expression level of mice in the CH group was significantly decreased (P<0.05). Figure 11 As shown in (a), (b), (d), and (e), compared with group C1, the expression levels of GLP-1R and PDX1 proteins in the pancreas of mice in the HFD group were significantly decreased (P<0.05). After intervention with B. longum B53, its post-biotic, and ILA, the expression levels of GLP-1R and PDX1 proteins in the pancreas of obese mice were significantly increased (P<0.05). Compared with group I, the expression levels of GLP-1R and PDX1 proteins in the pancreas of mice in the CH group were significantly decreased (P<0.05). The expression level of GLP-1R in the pancreas of mice showed a similar trend to the expression level of GLP-1 in mouse serum. These results indicate that the glucose metabolism pathway in obese mice induced by a long-term high-fat diet is abnormal, and it is restored after intervention with B. longum B53, its post-biotic, and ILA.

[0133] (10) Changes in lipid metabolism pathways in mice:

[0134] The expression levels of relevant genes were detected using RT-qPCR technology, specifically the expression levels of the colonic Ahr gene, and the liver Cyp1a1, Lxrα, Srebp1, Fas, and Scd1 genes. The results are as follows: Figure 12 As shown.

[0135] Depend on Figure 12 It was found that, compared with group C1, the expression level of Cyp1a1 gene was significantly decreased in group HFD (P<0.05), while the expression levels of Lxrα, Srebp1, Fas, and Scd1 genes were significantly increased (P<0.05); compared with group HFD, the expression level of Cyp1a1 gene was significantly increased in groups B, P, D, and I (P<0.05), while the expression levels of Lxrα, Srebp1, Fas, and Scd1 genes were significantly decreased (P<0.05); compared with group I, the expression level of Cyp1a1 gene was significantly decreased in group CH (P<0.05), while the expression levels of Lxrα, Srebp1, Fas, and Scd1 genes were significantly increased (P<0.05).

[0136] Western blot was used to detect the expression levels of related proteins, namely AHR, CYP1A1, LXRα, SREBP1, FAS, and SCD1. The expression level of AHR protein was already... Figure 11 As described in the text, the remaining results are as follows: Figure 13 As shown.

[0137] Depend on Figure 13 Compared with group C1, the HFD group showed significantly decreased CYP1A1 protein expression in the liver (P<0.05) and significantly increased LXRα, SREBP1, FAS, and SCD1 protein expression (P<0.05). Compared with the HFD group, the B, P, D, and I groups showed significantly increased CYP1A1 protein expression in the liver (P<0.05) and significantly decreased LXRα, SREBP1, FAS, and SCD1 protein expression (P<0.05). Compared with group I, the CH group showed significantly decreased CYP1A1 protein expression in the liver (P<0.05) and significantly increased LXRα, SREBP1, FAS, and SCD1 protein expression (P<0.05). These results indicate that the lipid metabolism pathway in obese mice induced by a long-term high-fat diet is abnormal, and this abnormality is somewhat restored after intervention with B. longum B53, its post-biotic, and ILA.

[0138] (11) Changes in the diversity of the gut microbiota in mice:

[0139] Alpha diversity reflects the diversity within the gut microbiota. The alpha diversity of mouse gut microbiota was described using three indices: Chao 1 index, Shannon index, and Simpson index. A higher Chao 1 index indicates a greater total number of species, while higher Shannon and Simpson indices indicate greater species diversity. The results are as follows: Figure 14 As shown.

[0140] Depend on Figure 14 As shown in (a), compared with group C1, the chao 1 index of the HFD group was significantly lower (P<0.05); compared with the HFD group, the chao 1 index of each intervention group increased, but there was no significant difference (P>0.05); there was also no significant difference between group I and group CH (P>0.05). Figure 14As shown in (b) and (c), compared with group C1, the HFD group showed a significant decrease in both Shannon and Simpson indices (P<0.05); compared with the HFD group, groups B, P, D, and I all showed significant increases in both Shannon and Simpson indices (P<0.05); however, there was no significant difference between group I and group CH (P>0.05). These results indicate that the total number and diversity of gut microbiota in obese mice induced by a long-term high-fat diet are reduced. After intervention with B. longum B53 and its postbiotics and ILA, the total number of species did not change significantly, while the diversity increased.

[0141] β-diversity reflects the diversity of bacterial communities among different sample groups, and the results are as follows: Figure 15 As shown.

[0142] Depend on Figure 15 From (a), we know that the number of OUTs in groups C1, C2, HFD, B, P, D, I, and CH are 725, 666, 727, 706, 750, 690, 697, and 635 respectively, and they have a total of 212 identical OUTs; Figure 15 As shown in (b), groups C1 and C2 are closer together, while group C1 and HFD are farther apart; groups I and CH are farther apart from group HFD.

[0143] (12) Changes in the structure of the mouse gut microbiota:

[0144] The effects of different treatment groups on the phylum level of the gut microbiota in mice, such as Figure 16 As shown.

[0145] Depend on Figure 16 It can be seen that the intestinal flora of mice in each group mainly consisted of Firmicutes, Bacteroidetes, Desulfobacterota, and Actinobacteriota. The dominant bacterial flora in C1 group mice were Firmicutes (67.95%), Bacteroidetes (20.74%), and Actinobacteria (6.40%). Compared with C1 group, the abundance of Dethiobacteria (8.68%) and Firmicutes (75.48%) increased, while the abundance of Bacteroidetes (11.48%) and Actinobacteria (2.72%) decreased in the HFD group. Compared with the HFD group, all treatment groups restored this intestinal disorder to some extent, with B. longum B53 and ILA showing the most significant effects. The abundance of Dethiobacteria (8.57%) and Firmicutes (70.74%) and Bacteroidetes (67.90%) decreased in groups B and I, while the abundance of Bacteroidetes (16.64%) increased. Compared with group I, the abundance of Firmicutes (78.82%) increased and the abundance of Bacteroidetes (8.44%) decreased in the CH group.

[0146] The effects of different treatment groups on the level of gut microbiota in mice, such as Figure 17 As shown.

[0147] Depend on Figure 17 It was found that, compared with group C1, the abundance of *Lentimicrobium*, *Anaerotruncus α*, *Clostridia_UCG-014*, *Eubacterium*_fissicatena_group*, *Alloprevotella*, *Roseburia*, and *Parabacteroides* increased in group HFD, while the abundance of *Prevotella* and *Bifidobacterium* decreased. Intervention with *B. longum* B53 and its postbiotics and ILA could restore this disorder to some extent.

[0148] The differences in gut microbiota among the groups of mice were analyzed using the LEfSe method, and the results are as follows: Figures 18-20 As shown.

[0149] Depend on Figure 18 and Figure 19 It was found that 53 significantly different species (LDA>3.5) were detected in both groups C1 and HFD. In the HFD group, Clostridia (c_Clostridia, LDA>4.0), Allobaculum (g_Allobaculum, LDA>4.0), Lachnospiraceae (f_Lachnospiraceae, LDA>4.0), Desulfowibrionaceae (f_Desulfowibrionaceae, LDA>4.0), Desulfovibrio sp (s_Desulfovibrio sp., LDA>4.0), and Mailhella (g_Mailhella, LDA>4.0) were the dominant bacteria. Figure 20The study revealed species differences between the HFD group and each treatment group (B, P, D, I), with a total of 65 significantly different species detected (LDA>2.0). Among them, *Lactobacillus* (g_Lactobacillus, LDA>3.0) and *Verrucomicrobiota* (P_Verrucomicrobiota, LDA>2.5) were the dominant bacteria in the HFD group; *Bifidobacterium longum* (s_Bifidobacterium_longum, LDA>3.0) and *Thermomonas_sp* (s_Thermomonas_sp, LDA>2.5) were the dominant bacteria in group B; and *Pasteurella* (g_Barnesiella, LDA>2.5) were the dominant bacteria in group I. Group P dominant bacteria included *Gordonibacter* (LDA>2.5) and others; Group D dominant bacteria included *Blautia* (LDA>3.0), *Lactobacillales* (LDA>3.0), and *Romboutsia* (LDA>3.0); Group I dominant bacteria included *Porphyromonas* sp (LDA>4.0), *Roseburia intestinalis* (LDA>3.5), and *Lachnospiraceae bacterium* (LDA>3.5).

[0150] (13) Changes in SCFAs in mouse intestinal contents:

[0151] SCFAs are major metabolites of gut microbiota, participating in physiological functions such as digestion and immunity in humans. They mainly include acetic acid, propionic acid, and butyric acid. Therefore, GC-MS was used to detect the levels of SCFAs in the intestinal contents of mice, and the results are as follows: Figure 21 As shown.

[0152] Depend on Figure 21 It was found that compared with group C1, the HFD group had significantly increased acetic acid and butyric acid levels (P<0.05) and significantly decreased propionic acid levels (P<0.05). Compared with the HFD group, the B, P, D, and I groups all had significantly decreased acetic acid levels (P<0.05), significantly increased propionic acid levels except in group P (P<0.05), and significantly decreased butyric acid levels (P<0.05). Compared with group I, the CH group had decreased but not significantly increased propionic acid levels (P>0.05), and significantly increased acetic acid and butyric acid levels (P<0.05). These results indicate that the overall SCFA content in the intestinal contents of obese mice induced by a long-term high-fat diet was increased, and this level recovered somewhat after intervention with B. longum B53, its postbiotics, and ILA.

[0153] (14) Changes in tryptophan metabolism in mouse gut microbiota:

[0154] Thirty-eight tryptophan metabolites in mouse intestinal contents were quantitatively analyzed by LC-MS / MS and divided into eight categories. Metabolites with VIP>1, fold change≥2.0 and fold change≤0.5 were selected as differential metabolites.

[0155] There are 15 differentially expressed tryptophan metabolites between groups C1 and M. Eight of these are upregulated in group M: xanthurenic acid (XA), 5-hydroxyindole-3-acetic acid (5-HIAA), 3-HAA, 5-HT, KYNA, 5-hydroxytryptophol (5-HTOL), indoxylsulfate (3-IS), and N-acetylserotonin (NAS). Seven of these are downregulated: L-TRP, 3-indoleglyoxylic acid (IGA), indole propionic acid (IPA), and 3-indole acrylic acid (IPA). The tryptophol metabolites differentially expressed between groups B and M were 3-HAA, IEt, indole-3-carbaldehyde (ICAld), and DL-indole-3-lactic acid (ILA). Nine metabolites were downregulated in group B: 3-HAA, IEt, 3-hydroxykynurenine (3-HKYN), 5-HTOL, L-kynurenine (L-KYN), KYNA, 5-HIAA, indole-3-acetamide (IAM), and XA. Three metabolites were upregulated: ICA1d and β-indole-3-acetic acid (IAM). There are 11 differentially expressed tryptophan metabolites between groups P and M. Among them, 5 are downregulated in group P: 3-HAA, 5-HIAA, XA, 5-HTOL, and IAM, and 6 are upregulated: IEt, Tryptamine (TRM), IAA, ICA1d, 3-IS, and ILA. There are 12 differentially expressed tryptophan metabolites between groups D and M. Among them, 5 are downregulated in group D: XA, 3-HAA, KYNA, 5-HTOL, and IAM, and 7 are upregulated: ILA, IAA, L-TRP, ICA1d, IEt, N-formylkynurenine (NFK), and 2-Aminophenol (2-AF).There are 10 differentially expressed tryptophan metabolites between groups I and M. Three are downregulated in group I: 3-HAA, 5-HTOL, and IAM; seven are upregulated: IAA, ICA1d, TRM, 2-AF, 5-HT, 2-ketoadipic acid (2-KA), and ILA.

[0156] Statistical analysis was performed on several substances with high content in indole and its derivatives, which are metabolites of tryptophan. The results are as follows: Figure 22 As shown. By Figure 22 It can be seen that, compared with group C1, the HFD group showed no significant change in IAA content (P>0.05), but significantly increased 5-HIAA content (P<0.05), and significantly decreased ICA1d, L-TRP, IDA, IPA, and ILA content. Compared with group HFD, groups B, P, D, and I showed no significant change in IDA and IPA content (P>0.05), but significantly decreased 5-HIAA content (P<0.05), and significantly increased L-TRP (except group B), ICA1d (except group B), ILA, and IAA content (P<0.05). Compared with group I, group CH showed no significant differences except for 5-HIAA and L-TRP (P>0.05).

[0157] In summary, B. longum B53 and its post-biotics described in this invention can alleviate obesity symptoms in mice. They improve the gut microbiota structure of obese mice and reduce the level of total SCFAs in the gut, with significantly reduced levels of acetic acid and butyric acid and significantly increased levels of propionic acid. They alleviate host inflammation by reducing serum levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increasing levels of anti-inflammatory cytokines IL-10 and IL-22. Simultaneously, changes in gut microbiota structure lead to alterations in the levels of indole and its derivatives produced by tryptophan metabolism, particularly a significant increase in ILA levels. This activates the AHR / GLP-1R / PDX1 signaling pathway, improving insulin sensitivity, alleviating insulin resistance in obese mice, and lowering blood glucose levels. Furthermore, activation of the AHR / CYP1A1 / LXRα / SREBP1 / SCD1 signaling pathway significantly reduces levels of LDLC, TC, TG, AST, ALT, and MDA, and significantly increases levels of HDLC, SOD, and GSH, alleviating lipid accumulation and reducing liver oxidative damage in obese mice, thereby alleviating obesity symptoms.

[0158] Therefore, this invention utilizes the aforementioned Bifidobacterium longum subsp. B53, post-biotics, their preparation methods, and applications. Bifidobacterium longum subsp. B53 can reduce the production of SCFAs in a high-fat model; promote tryptophan metabolism to produce indole and its derivatives, especially ILA, exhibiting strong anti-inflammatory, hypoglycemic, and lipid metabolism regulating abilities. Furthermore, this strain demonstrates stable production performance and good fermentation properties, and has the effect of regulating glucose and lipid metabolism disorders and alleviating obesity.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A strain of Bifidobacterium longum subspecies B53, characterized in that: Bifidobacterium longum subsp. longum is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 25, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35374. The classification name is Bifidobacterium longum subsp. longum, and the 16S rDNA sequence is shown in SEQ ID NO.

1.

2. The application of the *Bifidobacterium longum* subsp. B53 strain described in claim 1 in the preparation of products for regulating glucose and lipid metabolism disorders and alleviating obesity, characterized in that: Products include dietary supplements, health products, medicines, or beverages.

3. The application according to claim 2, characterized in that, Applied to: ① Regulate the structure of gut microbiota, reduce the production of SCFAs in a high-fat diet, and maintain the level of SCFAs in the gut; ② Reduce the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increase the levels of anti-inflammatory factors IL-10 and IL-22; ③ Improve the damage to pancreatic and liver tissues caused by a high-fat diet; ④ Promotes tryptophan metabolism to produce indole and its derivatives; ⑤ It reduces oral glucose tolerance, insulin levels, and insulin resistance index, reduces the organ index of white adipose tissue, and restores the morphology and structure of adipose tissue.

4. The application of the Bifidobacterium longum subspecies B53 strain as described in claim 1 in the preparation of a metabiotic that regulates glucose and lipid metabolism disorders and alleviates obesity.

5. The application according to claim 4, characterized in that, Applied to: ① Regulate the structure of gut microbiota, reduce the production of SCFAs in a high-fat diet, and maintain the level of SCFAs in the gut; ② Reduce the levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, and increase the levels of anti-inflammatory factors IL-10 and IL-22; ③ Improve the damage to pancreatic and liver tissues caused by a high-fat diet; ④ Promotes tryptophan metabolism to produce indole and its derivatives; ⑤ It reduces oral glucose tolerance, insulin levels, and insulin resistance index, reduces the organ index of white adipose tissue, and restores the morphology and structure of adipose tissue.

6. An epigenetic agent, characterized by: The metabiotic includes Bifidobacterium longum subsp. B53 and its metabolites as described in claim 1.

7. The epigenetic agent according to claim 6, characterized in that, The preparation method of post-biotics is as follows: Bifidobacterium longum subsp. B53 was inoculated into liquid culture medium and cultured. The bacterial cells were collected by centrifugation. The bacterial cells were resuspended in PBS buffer to obtain a bacterial suspension. The bacterial suspension was sterilized by heating and then spray-dried or freeze-dried to obtain a metabiotic that can regulate glucose and lipid metabolism disorders and alleviate obesity.

8. The epigenetic agent according to claim 7, characterized in that: The inoculation amount of Bifidobacterium longum subsp. B53 is 1%–5% v / v; The liquid culture medium was MRS liquid medium supplemented with 0.05% L-cysteine ​​hydrochloride; The cultivation conditions are: temperature 30℃~37℃, static strict anaerobic culture for 12h~24h; Centrifugation conditions were: temperature 4℃, gravitational acceleration 6000g~10000g, and time 5min~10min.

9. The epigenetic agent according to claim 7, characterized in that: The conditions for heat sterilization are: 70℃~90℃, time 10min~30min; The spray drying conditions are: inlet air temperature 150℃~180℃, outlet air temperature 80℃~100℃; The vacuum freeze-drying conditions are: temperature -50℃ to -10℃, vacuum 2mbar to 5mbar.

10. The epigenetic agent according to claim 6, characterized in that: The concentration of *Bifidobacterium longum* subsp. B53 in the metabiotic was 1–3 × 10⁻⁶. 9 CFU / mL.

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