Strain of bifidobacterium longum subsp. longum and use thereof in preventing, alleviating, regulating, or treating lipid metabolism-related diseases

Abstract

Disclosed is a strain of Bifidobacterium longum subsp. longum named dipro-O. The Bifidobacterium longum subsp. longum dipro-O used in the present invention has the ability to overproduce BSH, the ability to reduce cholesterol, and the ability to metabolize tryptophan into indole-3-lactic acid, can effectively reduce body weight, significantly lower insulin resistance levels resulting from high-fat diets, reduce the expression of the inflammatory cytokine TNF-α, significantly lower the levels of total cholesterol (TC) and low-density lipoprotein (LDL) in the blood, and increase the level of high-density lipoprotein (HDL), and can ameliorate disorders of the gut microbiome structure. In addition, the strain can also have an ameliorating effect on other lipid metabolism-related diseases such as hyperlipidemia, thereby providing health interventions for the host's body weight, liver, intestinal health, and blood lipid levels. Moreover, pasteurized dipro-O can also have the effect of regulating lipid metabolism, further extending the range of applications of the strain.

Description

A strain of Bifidobacterium longum and its application in the prevention, alleviation, regulation or treatment of lipid metabolism-related diseases. Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bifidobacterium longum and its application in the prevention, relief, regulation or treatment of lipid metabolism-related diseases. Background Technology

[0002] With economic development and improved living standards, dietary structures have also changed. Data from the 2015-2017 China National Nutrition and Health Survey shows that the average daily energy intake of Chinese residents was 2007.4 kcal, with the proportion of fat increasing year by year, exceeding the upper limit of 20%-30% recommended by dietary guidelines since 2012. Excessive lipid intake leads to a disproportionate energy intake and expenditure, causing fat to accumulate in the body, resulting in lipid metabolism disorders and ultimately obesity. Besides obesity, excessive lipid intake can also lead to abnormal blood sugar / lipid levels. Long-term obesity increases the risk of cardiovascular diseases (such as atherosclerosis), non-alcoholic fatty liver disease, and hyperuricemia. Follow-up studies have shown that in people aged 40-59 (BMI ≥ 22.5), for every 5-point increase in BMI, the future risk of CVD mortality increases by 37%. Therefore, excessive lipid intake and secondary diseases not only increase the cost of residents' health care services, but also increase the burden on the medical and health system. More importantly, the amplification of this series of problems will have a negative impact on social and psychological issues, which is not conducive to social stability and development.

[0003] To address these issues, current market treatments often have limited indications and significant side effects. For example, oseltira, used only for obesity, works by inhibiting lipase activity in the gastrointestinal tract, reducing lipid absorption and thus controlling weight. However, it frequently causes gastrointestinal side effects such as diarrhea, fecal incontinence, and abdominal pain. Smegglutide, which has gained popularity in recent years, offers advantages in both blood sugar control and weight loss, but its effects are addictive. While it provides good control of blood sugar and weight during administration, weight rebound is common after discontinuation. This drug also has side effects, with common gastrointestinal side effects including nausea and vomiting, and less frequently, tachycardia and gallstones. Therefore, although drugs exist for different indications, their limitations and side effects hinder widespread adoption, leaving a significant unmet market demand. While major pharmaceutical companies continue to conduct clinical trials on new drugs, their targeted mechanisms of action remain within the realm of pharmaceuticals, making it difficult to expect these unapproved drugs to overcome the side effects and limitations of continued use currently existing medications.

[0004] Throughout history, the relationship between microorganisms and health has been a topic of great interest to humankind. The gut is the organ with the highest concentration of human microorganisms, containing over a thousand species and approximately 10... 14Countless microorganisms inhabit this environment, their composition influenced by delivery method, feeding method, lifestyle, drug use, and genetic factors, but they play important roles in host immunity, metabolism, endocrine function, and neural signal regulation. Recent studies have gradually revealed the correlation between changes in the content of specific gut microbiota and the occurrence of metabolic diseases such as obesity, type II diabetes, hyperlipidemia, and non-alcoholic fatty liver disease. Probiotics are live microorganisms that are beneficial to the human body when present in sufficient numbers. Supplementing with probiotics to rebuild gut microbiota homeostasis is the most direct and effective method. Clinical trials have demonstrated that probiotics can participate in human metabolism and immune responses. For example, supplementing with probiotics (a mixture of Lactobacillus and Bifidobacterium) can significantly reduce fasting blood glucose and insulin resistance index in patients with type II diabetes; supplementing children with severe acute malnutrition with probiotics (Lactobacillus rhamnosus GG and Bifidobacterium animalis Bifidobacterium lactis subsp. Bb-12) can improve gut microbiota imbalance and significantly reduce the incidence of diarrhea. In recent years, as the relationship between specific microorganisms and diseases has become increasingly clear, probiotics (or in combination with prebiotics and postbiotics) have demonstrated their potential as a substitute for drugs in restoring the balance of gut microbiota and maintaining healthy homeostasis. Due to their inherent characteristics, they possess natural advantages over medications. Research on probiotics has grown rapidly since the beginning of the 21st century. Based on current research findings and future development trends in the field, some scholars have proposed the concept of "probacine," using live bacteria as a drug to "add" to the host's health and "subtract" from disease, maintaining gut homeostasis and overall health. This academic concept suggests that probiotics may gradually increase their role in disease treatment in the future, evolving from health supplements to complementary therapies, to rivaling drugs, and ultimately becoming drug alternatives, achieving worry-free treatment of diseases. However, current research on probiotic intervention in lipid metabolism is relatively limited. For example, patent CN116814501B mentions that *Bifidobacterium longum* subsp. *longum* BL36 can reduce the weight of mice on a high-fat diet for 8 weeks (25% lower percentage of weight gain compared to the control group), but this strain did not analyze weight changes under obesity conditions, nor was it validated in other lipid metabolism-related disease models. Another example is patent CN116286551B, which mentions *Bifidobacterium longum* subsp. *infant* NKUFB3-14, which can reduce mouse weight after 8 weeks of bacterial treatment. However, similar to BL36, the experiment was conducted simultaneously with bacterial treatment and high-fat diet modeling; therefore, the mice's weight did not exceed 32g by the end of the experiment, indicating a low degree of obesity, thus limiting its significance. Furthermore, the hypoglycemic effect of this strain is limited to fasting blood glucose, so its ability to control postprandial blood glucose is unclear. In summary, the existing intervention effects of probiotics or their preparations on lipid metabolism-related diseases caused by high-fat diets are still very limited, and their effectiveness needs further improvement.Therefore, how to provide a probiotic preparation that is effective and does not produce adverse reactions during treatment has become an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings of the existing technology, the purpose of this invention is to provide a strain of Bifidobacterium longum subsp. longum and its application in lowering lipids and losing weight or improving lipid metabolism-related diseases.

[0006] Technical solution: To solve the above technical problems, the present invention provides a strain of Bifidobacterium longum subsp. longum dipro-O, which was deposited at the China General Microbiological Culture Collection Center on December 25, 2023, with the accession number CGMCC No. 29383.

[0007] The *Bifidobacterium longum* subsp. *longum* dipro-O strain described in this invention can produce bile salt hydrolase with a bile salt hydrolysis ring diameter of 15–35 mm; the strain can utilize tryptophan metabolism to produce indole-3-lactic acid with a content of 5000–25000 ng / ml; the strain can directly reduce the cholesterol level in the culture medium by 10%–100%.

[0008] The present invention also includes products containing the aforementioned Bifidobacterium longum subsp. longum dipro-O, said products including fermented products, food, health products, pharmaceuticals or feed.

[0009] The fermented products include fermentation supernatant, fermentation precipitate, or bacterial suspension of Bifidobacterium longum subsp. longum dipro-O.

[0010] The dosage forms of the products include powders, capsules, tablets, pills, film-coated tablets, aerosols, granules, liquids, liposomes, transdermal preparations, suppositories, or lyophilized powder injections.

[0011] The product also includes protective agents, functional auxiliaries / or auxiliary additives, drug carriers and / or pharmaceutical excipients.

[0012] The present invention also includes the aforementioned Bifidobacterium longum subsp. longum dipro-O and the application of the aforementioned product in the preparation of products for the prevention, relief, regulation or treatment of lipid metabolism-related diseases.

[0013] The lipid metabolism-related diseases mentioned include obesity, diabetes, non-alcoholic fatty liver disease, hyperlipidemia, or hyperuricemia.

[0014] This invention also includes products for regulating lipid metabolism prepared from *Bifidobacterium longum subsp. longum* dipro-O, which are products having at least one of the following effects:

[0015] 1) Products that help reduce weight;

[0016] 2) Products that improve blood sugar;

[0017] 3) Products that improve intestinal immunity;

[0018] 4) Products that improve lipid metabolism disorders;

[0019] 5) Products that improve liver tissue damage induced by a high-fat diet;

[0020] 6) Products that improve the intestinal barrier;

[0021] 7) Products that can improve the disordered structure of the intestinal flora.

[0022] Specifically, the product that reduces weight is a product that reduces weight gain caused by a high-fat diet.

[0023] Specifically, the product that improves blood sugar is a product that regulates the rise in blood sugar caused by a high-fat diet.

[0024] Specifically, the product that improves intestinal immunity is a product that regulates the level of TNF-α in the intestine.

[0025] Specifically, the product that improves lipid metabolism disorders is one that reduces the levels of total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the blood, while increasing the levels of high-density lipoprotein cholesterol (HDL-C) in the blood.

[0026] Specifically, the product that improves liver tissue damage caused by a high-fat diet is a product that improves aspartate aminotransferase and hepatocellular steatosis.

[0027] Specifically, the product that improves the intestinal barrier enhances the expression of intestinal barrier genes mTff3, Muc2, and ZO-1 in the intestine.

[0028] Specifically, the product that improves gut microbiota structure disorder is a product that regulates β-diversity and genus-level changes in gut microbiota. The application includes treatment with *Bifidobacterium longum* subsp. *longum* dipro-O to reduce the expression of the inflammatory cytokine TNF-α in the colon of obese mice induced by a high-fat diet, while simultaneously promoting intestinal barrier repair and maintaining the expression of related genes mTff3, Muc2, and ZO-1.

[0029] The application includes treatment with *Bifidobacterium longum* subsp. *dipro-O* to reduce the level of aspartate aminotransferase in the blood of mice with hyperlipidemia induced by a high-fat diet, while also reducing fat vacuoles and the degree of inflammatory cell aggregation in the mice.

[0030] The high-fat diet modeled obese mice include HFD diet-induced high-fat diet obese mouse models or ASHF diet-induced mouse hyperlipidemia models.

[0031] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: The *Bifidobacterium longum* subsp. *longum* dipro-O used in this invention has the ability to produce high levels of BSH, lower cholesterol, and metabolize tryptophan to produce indole-3-lactic acid. It can effectively reduce weight, significantly reduce insulin resistance caused by a high-fat diet, reduce the expression of the inflammatory cytokine TNF-α, significantly reduce total cholesterol (TC) and low-density lipoprotein (LDL) levels in the blood, and increase high-density lipoprotein (HDL) levels; it also improves intestinal flora imbalance; and it can also play a beneficial role in other lipid metabolism-related diseases such as hyperlipidemia, thereby providing health intervention for the host's weight, liver, intestinal health, and blood lipid levels. Furthermore, pasteurized dipro-O can also regulate lipid metabolism, further broadening the application scope of this strain. Attached Figure Description

[0032] Figure 1 shows the ability to produce bile salt hydrolase (BSH); Bifidobacterium longum subsp. longum has a stronger BSH production capacity with dipro-O (dipro-O: d = 24.5 mm, BPL1: d = 21.5 mm);

[0033] Figure 2 shows the results of cholesterol-lowering ability determination; Bifidobacterium longum subsp. longum has a stronger cholesterol-lowering ability (mean dipro-O: 19.93%, BPL1: 9.1%).

[0034] Figure 3 shows the ability of dipro-O to produce indole-3-lactic acid (ILA) through tryptophan metabolism; Bifidobacterium longum subsp. longum dipro-O has a stronger ability to produce BSH (the average ILA production capacity of dipro-O is 11110.3 ng / mL, while the average ILA production capacity of BPL1 is 2782.29 ng / mL);

[0035] Figure 4 shows the weight gain rate of mice on a high-fat diet. The weight gain rate of obese mice on a high-fat diet model treated with Bifidobacterium longum subsp. longum dipro-O was lower and slower (dipro-O group: 54.898%, reference BPL1 group: 60.154%, PBS group: 100.01%).

[0036] Figure 5 shows the OGTT experiment in a high-fat diet mouse model. Bifidobacterium longum dipro-O can significantly reduce the insulin resistance level in high-fat diet model mice (significant differences were observed at 30 min, 60 min, and 90 min compared with the PBS group, and significant differences were observed between the dipro-O group and the reference bacteria BPL1 group at 30 min).

[0037] Figure 6 shows liver sections of mice induced by a high-fat diet. Both live Bifidobacterium longum subsp. longum dipro-O and pasteurized inactivated Bifidobacterium longum subsp. longum dipro-O can improve lipid accumulation and inflammatory cell aggregation in the liver, and significantly reduce the liver NAS score used to analyze hepatocellular steatosis, hepatocellular ballooning degeneration, and intralobular inflammation.

[0038] Figure 7 shows the serum AST levels in mice induced by a high-fat diet.

[0039] Figure 8 shows the changes in total cholesterol (TC), triglycerides (TG), and high-density lipoprotein (HDL) in the blood of mice induced by a high-fat diet. Compared with the BPL1 group, treatment with *Bifidobacterium longum subsp. longum* dipro-O significantly reduced the level of total cholesterol in the blood of obese mice induced by a high-fat diet. Compared with the PBS group, it significantly increased the level of high-density lipoprotein (HDL) in the blood. At the same time, compared with the PBS group, the level of triglycerides in the blood of mice in the dipro-O and Inactive dipro-O groups was significantly reduced.

[0040] Figure 9 shows the expression of intestinal barrier-related genes in mice induced by a high-fat diet. Treatment with *Bifidobacterium longum* subsp. *longum* dipro-O can reduce the expression of the inflammatory cytokine TNF-α in the colon of obese mice induced by a high-fat diet, while promoting the expression of genes related to intestinal barrier repair and maintenance (mTff3, Muc2, ZO-1).

[0041] Figure 10 shows the results of 16S rRNA sequencing of feces from mice modeled on a high-fat diet and analysis of gut microbiota. The results show that the content of Clostridium species in mice increased after dipro-O treatment.

[0042] Figure 11 shows that Bifidobacterium longum subsp. dipro-O can effectively reduce lipid accumulation in the liver in an ASHF-induced hyperlipidemic mouse model. According to the slice data, the dipro-O group mice have reduced fat vacuoles, low inflammatory cell aggregation, and significantly lower NAS scores than the PBS group.

[0043] Figure 12 shows that intervention with Bifidobacterium longum subsp. dipro-O in an ASHF diet-induced hyperlipidemia mouse model can significantly reduce the level of aspartate aminotransferase (AST) in the blood.

[0044] Figure 13 shows that intervention with Bifidobacterium longum subsp. dipro-O in an ASHF-induced hyperlipidemic mouse model can significantly reduce the levels of total cholesterol (TC) and low-density lipoprotein (LDL) in the blood. Detailed Implementation

[0045] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0046] Example 1: Screening and microbiological identification of strain dipro-O

[0047] Bifidobacterium longum subsp. longum dipro-O was isolated from the feces of healthy children. After isolation, it was identified by MALDI-TOF, 16S rRNA sequencing and whole genome sequencing. The species identification results were consistent with the MALDI-TOF results, and all strains were identified as Bifidobacterium longum subsp. longum.

[0048] Bifidobacterium longum subsp. longum dipro-O was deposited on December 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 29383.

[0049] Whole-genome sequencing results showed that the genome of the dipro-O strain is a circular chromosome without plasmids, containing a total of 8 rRNAs, namely 4 23S and 4 16S; as well as 55 tRNAs and one tmRNA.

[0050] dipro-O 16S rRNA sequence:

[0051] 5'→3'

[0052] The antibiotic resistance of strain dipro-O was analyzed according to the "National Food Safety Standard - Safety Evaluation Procedure for Food Microbial Strains (Draft for Comments)". The results are as follows: dipro-O is sensitive to antibiotics such as kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, and vancomycin. The conclusion is that it meets the national requirements.

[0053] In functional experiments, glycerol-preserved *Bifidobacterium longum* subsp. *longum* dipro-O was removed from a -80°C freezer, thawed in a 37°C water bath, and 20 μL was spread onto MRS (MRS broth + 0.5 g / L cysteine) plates and anaerobically cultured at 37°C for 48 h. Subculturing was then performed using an inoculation loop. After 48 h of culture, single colonies were picked and cultured in MRS broth liquid medium supplemented with 0.5 g / L cysteine ​​for expansion, after which subsequent experiments could be conducted.

[0054] Example 2: Experiment on the ability of dipro-O bile salt hydrolase (BSH)

[0055] The experiment used BSH screening plates: MRS solid medium (MRS solid medium + 0.5 g / L cysteine) + 0.37 g / L CaCl2 (calcium chloride) + 5 g / L sodium taurine deoxycholate (TDCA); 6 mm antimicrobial susceptibility testing paper was placed in the center of the BSH screening plate, and the OD600 of *Bifidobacterium longum* subsp. *dipro-O* in the logarithmic growth phase was adjusted to 0.5. The plate was washed twice with PBS buffer (Procell, 10 mM, pH 7.2-7.4, catalog number: PB180327), then resuspended in 500 μL PBS. 10 μL of the resuspended solution was added dropwise to the antimicrobial susceptibility testing paper, and the plate was anaerobically incubated at 37 °C for 48 h. The diameter of the hydrolysis loop was then measured.

[0056] Comparative Example 1: BPL1's ability to produce bile salt hydrolase (BSH)

[0057] Using the same BSH screening plate as in Example 1, a 6 mm antimicrobial susceptibility test disc was placed in the center of the BSH screening plate. BPL1 (ADM, Bifidobacterium CECT8145) in the logarithmic growth phase was then adjusted to OD600 = 0.5 and washed twice with PBS. The disc was then resuspended in 500 μL of PBS buffer. 10 μL of the resuspended solution was added dropwise to the antimicrobial susceptibility test disc and anaerobically incubated at 37°C for 48 h. The diameter of the hydrolysis loop was then measured.

[0058] As shown in Figure 1, in Example 2, the hydrolysis ring diameter of dipro-O is d = 24.5 mm, and in Comparative Example 1, the hydrolysis ring diameter of BPL1 is d = 21.5 mm. By comparing the hydrolysis ring size to determine the BSH production capacity of each strain, it can be found that the BSH production capacity of dipro-O is 13.95% higher than that of BPL1.

[0059] Example 3: Experiment on the cholesterol-lowering ability of dipro-O

[0060] Log-phase Bifidobacterium longum dipro-O was adjusted to OD600 = 2.5 and inoculated at a 2% inoculation ratio into freshly prepared MRS broth containing 0.3% (w / v) porcine bile salts and 0.1 g / L cholesterol (soluble in anhydrous ethanol, Sigma-Aldrich) supplemented with 0.5 g / L cysteine, and incubated at 37°C for 24 hours. Then, it was centrifuged at 4°C, 4000g for 15 minutes, and the cholesterol level in the supernatant was measured using a cholesterol level detection kit (Elaret, E-BC-K109-M). A blank culture medium without bacterial culture but in parallel was used as a control.

[0061] Comparative Example 2: BPL1 Cholesterol-Lowering Ability Experiment

[0062] The BPL1 strain in logarithmic growth phase was adjusted to OD600 = 2.5 and inoculated at a 2% inoculation ratio into freshly prepared MRS broth containing 0.3% (w / v) bile salts and 0.1 g / L cholesterol (soluble in anhydrous ethanol, Sigma-Aldrich) supplemented with 0.5 g / L cysteine. The culture was incubated at 37°C for 24 hours. Then, the culture was centrifuged at 4°C, 4000 g for 15 minutes, and the cholesterol level in the supernatant was measured using a cholesterol level detection kit. A blank culture medium without bacterial culture but in parallel was used as a control.

[0063] The results are shown in Figure 2. In Example 3, the cholesterol content in the culture supernatant of dipro-O decreased by 20.07%. Compared with the cholesterol-lowering ability of BPL1 (13.67%) in Comparative Example 2, the cholesterol-lowering ability of dipro-O was 46.82% higher than that of BPL1, which is stronger than BPL1. This Example 3 proves that dipro-O has the ability to lower cholesterol, and from another perspective, it proves that dipro-O has the potential to become a probiotic for weight loss.

[0064] Example 4: Detection of dipro-O's tryptophan metabolism capacity

[0065] Dipro-O in the exponential growth phase was enriched in MRS broth liquid medium supplemented with 0.5 g / L cysteine, washed once with PBS, and adjusted to OD600 = 2.5. Then, it was inoculated at a 2% inoculum ratio in M9 medium containing 0.12 g / L tryptophan and cultured at 37°C for 48 h. Subsequently, the production of indole derivatives was detected using a high performance liquid chromatography system (Waters 2498, USA).

[0066] Comparative Example 3: Detection of tryptophan metabolism capacity of BPL1

[0067] BPL1 cells in the exponential growth phase were enriched in MRS liquid medium supplemented with 0.5 g / L cysteine, washed once with PBS, and adjusted to OD600 = 2.5. They were then inoculated at a 2% inoculum ratio in M9 medium containing 0.12 g / L tryptophan and cultured at 37°C for 48 h. The production of indole derivatives was then detected using a high performance liquid chromatography system (Waters 2498, USA).

[0068] The results of the determination in Example 4 and Comparative Example 3 are shown in Figure 3. dipro-O mainly produces indole-3-lactic acid (ILA) through metabolism, with an average yield of 11110.3 ng / mL. BPL1's tryptophan metabolites are mainly ILA, with an average yield of 2782.29 ng / mL. dipro-O's ILA yield is 400% of that of BPL1 (2782.29 ng / mL), indicating that dipro-O has a strong ability to produce ILA.

[0069] Example 5: dipro-O can effectively reduce the body weight of a high-fat diet-induced obesity (DIO) animal model.

[0070] Eight-week-old male C57BL / 6J mice were used in the experiment, divided into a negative control (NC) group, a high-fat control (PBS) group, and an experimental (dipro-O) group. Obesity was induced for 10 weeks using a high-fat mouse diet (Dyets, catalog number: HF60) with 60% kcal fat. Subsequently, Bifidobacterium longum dipro-O was administered via gavage. The gavage method was as follows: Live Bifidobacterium longum dipro-O in its exponential growth phase was anaerobic fermented at 37°C, and the fermentation broth was centrifuged and resuspended in PBS buffer. The OD600 was adjusted to 2.5, and 200 μL (approximately 1*10^6 ml) was administered to each mouse via gavage. 9 CFU levels were maintained for 9 weeks. The NC group and the high-fat control group were administered the same dose of PBS buffer by gavage. Mice were weighed weekly. After 9 weeks, relevant indicators of the probiotic group with biological effects were analyzed and measured: the levels of total cholesterol (TC), triglycerides (TG), and high-density lipoprotein (HDL) in the blood were measured using a Cardick dry biochemical analyzer, the level of aspartate aminotransferase (AST) in serum was detected using the Elite Aspartate Aminotransferase (AST / GOT) colorimetric test kit (#E-BC-K236-S), and liver tissue sections were analyzed to determine the amount of fat accumulation in the liver.

[0071] Analysis of the weight loss ability of reference strain BPL1 in the high-fat diet-induced obesity (DIO) animal model, Comparative Example 4

[0072] Eight-week-old male C57BL / 6J mice were fed a high-fat diet (Dyets, catalog number: HF60) with 60% kcal fat to induce obesity for eight weeks. Following this, BPL1 was administered via gavage. The gavage procedure was as follows: live BPL1 in its exponential growth phase was anaerobic fermented at 37°C. The fermentation broth was centrifuged, resuspended in PBS buffer, and adjusted to OD600 = 2.5. 200 μL (approximately 1*10^6 ml) was administered to each mouse via gavage. 9CFU bacterial count was maintained for 9 weeks. Mice were weighed weekly, and relevant indicators were analyzed after 9 weeks: total cholesterol (TC), triglycerides (TG), and high-density lipoprotein (HDL) levels in the blood were measured using a Cardick dry biochemical analyzer; serum aspartate aminotransferase (AST) levels were detected using the Elite Aspartate Aminotransferase (AST / GOT) colorimetric test kit (#E-BC-K236-S); and liver tissue sections were analyzed to determine the extent of liver fat accumulation.

[0073] The results of Example 5 and Comparative Example 4 are shown in Figure 4: The weight of mice in the PBS group increased by 100.01% compared with the first day of gavage, the weight of mice in the dipro-O group increased by 54.90% compared with the first day of gavage, and the weight of mice in the reference strain BPL1 group increased by 60.15% compared with the first day of gavage. It can be concluded that dipro-O has a better ability to control the weight of mice under high-fat diet conditions than the reference strain BPL1, and can effectively reduce the weight gain.

[0074] Example 6: Reduced insulin resistance in DIO animal model after dipro-O intervention

[0075] In the 19th week after the mice were modeled for high-fat diets according to the method in Example 5 and administered Bifidobacterium longum dipro-O by gavage, oral glucose tolerance test (OGTT) was performed: blood glucose levels were measured by collecting blood from the tail tip of the mice after fasting for 12 hours using a Roche Smart Glucose Meter, and then the mice were administered 20% glucose solution by gavage at a dose of 1 g / kg. Blood glucose levels were measured at 30, 60, 90 and 120 minutes after gavage.

[0076] Comparative Example 5: Analysis of BPL1 oral glucose tolerance in DIO animal models

[0077] In the 19th week after the mice were modeled for high-fat diets according to the method in Example 5 and administered BPL1 strain by gavage, an oral glucose tolerance test (OGTT) was performed: blood glucose levels were measured by collecting blood from the tail tip of mice after fasting for 12 hours using a Roche Smart Glucose Meter. The mice were then administered 20% glucose solution by gavage at a dose of 1 g / kg. Blood glucose levels were measured at 30, 60, 90, and 120 minutes after gavage.

[0078] Example 7: Reduced insulin resistance in DIO animal model after Inactive dipro-O intervention

[0079] Following the method described in Example 5, mice were induced to develop a high-fat diet. After 10 weeks, they were treated with pasteurized inactivated dipro-O (Inactive dipro-O) by gavage for 9 weeks. Inactive dipro-O was obtained by incubating dipro-O in a 70°C water bath for 30 minutes. At the end of the 19th week, an oral glucose tolerance test (OGTT) was performed: blood glucose levels were measured using a Roche Smart Glucose Meter after fasting for 12 hours. The mice were then gavaged with 20% glucose solution at a dose of 1 g / kg. Blood glucose levels were measured at 30, 60, 90, and 120 minutes after gavage.

[0080] The results of Examples 6 and 7 and Comparative Example 5 are shown in Figure 5. The blood glucose levels of the dipro-O group mice were significantly lower than those of the PBS group at three time points of 30 min, 60 min and 90 min, indicating that the insulin resistance level of the mice after dipro-O intervention was lower than that of the PBS group. Combined with the OGTT experiment of the BPL1 group mice, the blood glucose of the dipro-O group mice was significantly lower than that of the BPL1 group at 30 min, indicating that dipro-O has better sensitivity to blood glucose changes in a short period of time than BPL1.

[0081] The mice in the Inactive dipro-O group had the lowest blood glucose level at 30 minutes, indicating that the mice's insulin sensitivity increased after Inactive dipro-O intervention, making them more sensitive to changes in blood glucose levels in the short term. Combined with the results of Examples 6 and 7, it can be shown that both live and pasteurized bacteria of dipro-O can effectively regulate blood glucose homeostasis.

[0082] Example 8: Reduced liver lipid accumulation levels in a DIO animal model after dipro-O and Inactive dipro-O intervention.

[0083] Following the method described in Example 5, a high-fat diet model was established in mice. At the end of week 19 after gavage administration of Bifidobacterium longum (dipro-O), mouse livers were harvested, fixed in 4% paraformaldehyde, and H&E-stained sections were prepared. Lipid accumulation was analyzed, and the results were analyzed based on the sections and liver NAS scores.

[0084] Comparative Example 6: Liver slice analysis after BPL1 treatment in the DIO animal model

[0085] At the end of week 19 after mouse high-fat modeling was performed according to the method in Example 5 and BPL1 strain was administered by gavage, mouse livers were collected, fixed in 4% paraformaldehyde, and H&E stained sections of the liver were prepared and lipid accumulation was analyzed. The results were obtained from the sections and liver NAS scores.

[0086] The results of Example 8 and Comparative Example 6 are shown in Figure 6. As can be seen from Figure 6, the livers of mice in the PBS-treated group showed a large number of vacuoles caused by lipid droplet accumulation. The NAS scores of the livers of mice in the dipro-O group and the Inactive dipro-O group were significantly lower than those in the PBS group, indicating that lipid accumulation in the livers of mice was reduced after administration of probiotics. Combined with the section results of mice in the BPL1 group, it can be found that the number of vacuoles in the livers of mice in the BPL1 group was reduced compared with the PBS group, but no significant difference was found in the NAS scores. In summary, compared with BPL1, dipro-O and Inactive dipro-O have excellent ability to remove lipid accumulation.

[0087] Example 9: Serum AST levels decreased in a DIO animal model after dipro-O intervention.

[0088] At the end of the 19th week after the mice were induced into a high-fat diet model and administered Bifidobacterium longum dipro-O via gavage according to the method in Example 5, the mice were sampled, and their blood was collected in 1.5 mL sterile centrifuge tubes. The serum was collected by centrifugation at 7000 rpm for 10 minutes at 4°C, and the AST level in the mouse serum was detected.

[0089] Comparative Example 7: Serum AST Level Analysis After BPL1 Treatment in DIO Animal Model

[0090] At the end of the 19th week after the mouse high-fat model was established by gavage with BPL1 strain according to the method in Example 5, the mice were sampled, and the mouse blood was collected in a 1.5 mL sterile centrifuge tube. The serum was collected by centrifugation at 7000 rpm for 10 minutes at 4°C, and the AST level in the mouse serum was detected.

[0091] The results of Example 9 and Comparative Example 7 are shown in Figure 7. As can be seen from Figure 7, compared with the PBS group, the serum AST level of mice in the dipro-O group was reduced by 45.85%, while the AST level of the BPL1 group was found to be not statistically different from that of the PBS group. This indicates that compared with BPL1, the intervention of dipro-O can reduce the level of liver damage in DIO model mice, while the BPL1 group does not have a protective effect on the liver.

[0092] Example 10: After dipro-O intervention, serum cholesterol, triglyceride, and high-density lipoprotein levels decreased in the DIO animal model.

[0093] At the end of the 19th week after the mouse high-fat model was established by gavage with Bifidobacterium longum dipro-O according to the method of Example 5, serum of each group of DIO model mice was collected (see Example 9 for specific steps) and the changes in total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL) in the blood were detected.

[0094] Analysis of serum cholesterol, triglyceride and high-density lipoprotein levels after BPL1 treatment in the comparative 8DIO animal model

[0095] At the end of the 19th week after the mouse high-fat model was established by gavage with BPL1 strain according to the method of Example 5, serum of DIO model mice in each group was collected (see Example 9 for specific steps) and the changes in total cholesterol (TC), triglycerides (TG) and high-density lipoprotein (HDL) in the blood were detected.

[0096] The results of Example 10 and Comparative Example 8 are shown in Figure 8. As can be seen from Figure 8, compared with the data of the BPL1 group, the cholesterol level in the blood of mice in the dipro-O group was significantly reduced (by 16.28%). At the same time, the analysis of the triglyceride level in the blood of mice showed that compared with the PBS group, the serum triglyceride level in the dipro-O group was reduced by 25.00%, and the serum triglyceride level in the inactive dipro-O group was reduced by 25.03%. In the high-density lipoprotein analysis, it was found that dipro-O treatment increased the HDL level in the blood of mice by 27.87%. There was no significant difference between the other groups and the PBS group. These results indicate that compared with BPL1, the intervention of dipro-O resulted in lower cholesterol and triglyceride levels in the serum of mice, while also increasing the level of high-density lipoprotein, which is beneficial to humans.

[0097] Example 11: Dipro-O intervention improved intestinal barrier health and reduced inflammatory cytokine levels in DIO mice.

[0098] At the end of week 19 after mouse high-fat diet modeling and gavage administration of Bifidobacterium longum dipro-O, colon tissue was collected from mice according to the method in Example 5. The expression of colon-related genes was analyzed. The specific steps are as follows: A 5 mm piece of mouse colon tissue was placed in a 1.5 mL EP tube. 1 mL of Trizol reagent (Invitrogen, catalog number: 15596026) and 4 2.5 mm stainless steel beads were added to each tube. The mixture was homogenized at 60 MHz for 45 seconds, with a 15-second interval, and repeated 5 times. RNA was then extracted and reverse transcribed. The gene expression level was quantitatively analyzed using the QuantStudio™ 5 Real-Time PCR System. The genes detected included ZO-1, mTff3, TNF-α, Muc2, and the internal reference gene beta-Actin.

[0099] Comparative Example 9: Analysis of changes in intestinal gene expression levels after BPL1 treatment in the DIO animal model

[0100] Following the method in Example 5, a high-fat diet model was established in mice, and BPL1 strain was administered via gavage at the end of week 19. Colon tissue was collected from the mice, and the expression of colon-related genes was analyzed. The specific steps are described in Example 11.

[0101] The experimental results of Example 11 and Comparative Example 9 are shown in Figure 9. Figure 9 shows the results of intestinal barrier and inflammation-related gene analysis in high-fat model obese mice: the expression level of mouse trefoil peptide factor (mTff3), which plays a role in mucosal repair, was increased by 55.59% in the dipro-O group, indicating that dipro-O can promote intestinal mucosal repair, while the level of mTff3 was significantly decreased in the BPL1 group; analysis of the expression of the intestinal barrier tight junction protein gene ZO-1 showed that compared with the NC group, ZO-1 was significantly decreased in the PBS group and the BPL1 group (45.77% decrease in the PBS group and 48.85% decrease in the BPL1 group), while the expression of ZO-1 in the dipro-O group was not different from that in the NC group, indicating inactive... The expression level of ZO-1 in the dipro-O group was significantly increased (by 76.66%), indicating that dipro-O treatment can promote the expression of tight junction proteins. Analysis of the expression level of mucin Muc2 revealed that Muc2 expression was reduced by 75.96% in the BPL1 group compared to the NC group, while the expression levels of Muc2 in the dipro-O and Inactive dipro-O groups were not different from those in the NC group. Analysis of pro-inflammatory cytokine levels showed that the levels of the pro-inflammatory cytokine TNF-α in the colon tissue of mice in the PBS and BPL1 groups were significantly increased compared to the blank treatment (NC) group (by 372.74% in the PBS group and 564.79% in the BPL1 group), while there was no statistically significant difference between the dipro-O and Inactive dipro-O groups and the NC group. This suggests that the use of dipro-O or Inactive dipro-O treatment... dipro-O can reduce the level of inflammation in the colon of mice caused by a high-fat diet. Based on the above data, we believe that dipro-O can maintain the health of the intestinal barrier by promoting the expression of genes related to the maintenance and repair of the intestinal barrier, while inhibiting the expression of inflammatory cytokines, which is more advantageous than BPL1.

[0102] Example 12 Analysis of gut microbiota changes in a dipro-O-treated hyperlipidemic mouse model

[0103] At week 19 after mouse hyperlipidemia modeling and gavage administration of Bifidobacterium longum dipro-O according to the method in Example 5, mouse feces were collected, and microbial DNA was extracted using a fecal genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP328). The DNA was amplified using universal 16S rRNA primers (sequencing primers are shown in Table 1) and sent to Qingke Biotechnology Co., Ltd. for sequencing. Bioinformatics analysis software (such as QIIME2) was then used to analyze the samples from different dimensions, comparing the differences between groups. As shown in Figure 10, changes were observed in mouse β-diversity (Figure 10-A) and genus-level (Figure 10-B) gut microbiota structure. Further LEFSE analysis of the differentially expressed bacteria (Figure 10-C) revealed that dipro-O treatment increased the abundance of Clostridium bacteria in the mouse gut. This indicates that dipro-O, while exerting its weight-loss and lipid-lowering effects, can also regulate the dysbiosis in obese mice to a certain extent.

[0104] Table 1. 16S amplification primers

[0105] Example 13: Reduced liver lipid accumulation in a hyperlipidemic animal model after dipro-O intervention

[0106] Eight-week-old male C57BL / 6J mice were used to establish a hyperlipidemia model for 8 weeks using a high-fat (40kcal%), high-cholesterol (1.25%), and high-cholate sodium (0.5%) diet (Dyets, catalog number: ASHF4). The gavage conditions were the same as those for the DIO model mice in Example 5. After 8 weeks of gavage, the livers of the mice were sampled, and liver tissue sections were stained with H&E. The results are shown in Figure 11. It can be seen that after the hyperlipidemia model was established, a small number of cavities caused by lipid droplets and some inflammatory cell aggregation appeared in the liver (PBS group). However, after intervention with dipro-O, the vacuolar structure and the degree of inflammatory cell aggregation were significantly reduced (dipro-O group), indicating that dipro-O intervention effectively reduced the degree of lipid accumulation in the liver.

[0107] Example 14: Serum AST levels decreased in a hyperlipidemic animal model after dipro-O intervention.

[0108] Eight-week-old male C57BL / 6J mice were used to establish a hyperlipidemia model by feeding them a high-fat (40kcal%), high-cholesterol (1.25%), and high-cholate sodium (0.5%) diet (Dyets, catalog number: ASHF4). After eight weeks of modeling, the mice were administered the diet via gavage under the same conditions as the DIO model in Example 5. Blood was collected after eight weeks of gavage, and serum was separated for analysis of serum AST levels. As shown in Figure 12, the serum AST level in the PBS group increased by 108.30%, indicating an increased degree of liver damage. After intervention with Bifidobacterium longum dipro-O, the AST level in the PBS group decreased by 35.45% compared to the PBS group, indicating that the intervention of dipro-O alleviated the liver damage caused by the high-fat diet in mice.

[0109] Example 15: Serum cholesterol (TC) and low-density lipoprotein (LDL) levels decreased in a hyperlipidemic animal model after dipro-O intervention.

[0110] Eight-week-old male C57BL / 6J mice were fed a high-fat (40kcal%), high-cholesterol (1.25%), and high-cholate sodium (0.5%) diet (Dyets, catalog number: ASHF4) to induce hyperlipidemia for eight weeks. The gavage conditions were the same as those for the DIO model mice in Example 5. After eight weeks of gavage, blood was collected from the mice, and serum cholesterol and low-density lipoprotein were analyzed. The results are shown in Figure 13. In the ASHF-induced hyperlipidemia mouse model, intervention with Bifidobacterium longum dipro-O reduced blood cholesterol levels by 40.50% and low-density lipoprotein levels by 60.25%, indicating that dipro-O intervention can effectively improve the blood health of mice.

Claims

1. A strain of Bifidobacterium longum subsp. longum dipro-O, characterized in that, The Bifidobacterium longum subsp.longum dipro-O was preserved in China General Microbiological Culture Collection Center on December 25, 2023, and the preservation number is CGMCC No.29383.

2. Bifidobacterium longum subsp. longum dipro-O according to claim 1, characterized in that, The strain can produce a bile salt hydrolase with a bile salt hydrolysis ring diameter of 15-35 mm; the strain can produce indole-3-lactic acid by metabolizing tryptophan, and the content of the produced indole-3-lactic acid is 5000-25000 ng / ml; and the strain can directly reduce the level of cholesterol in the culture medium, and the proportion of the reduced cholesterol is 10%-100%.

3. Product containing Bifidobacterium longum subsp. longum dipro-O of claim 1, characterized in that, The product is a fermentation product.

4. The product according to claim 3, wherein the fermentation product comprises a fermentation precipitate or a bacterial suspension of Bifidobacterium longum subsp.longum dipro-O.

5. Use of the Bifidobacterium longum subsp.longum dipro-O according to claim 1 or the product according to any one of claims 3-4 in the preparation of a fermentation product for regulating lipid metabolism, wherein the fermentation product for regulating lipid metabolism is a fermentation product having at least one of the following effects: 1) a fermentation product for reducing body weight; 2) a fermentation product for improving blood glucose; 3) a fermentation product for improving intestinal immunity; 4) a fermentation product for improving blood lipid metabolism disorder; 5) a fermentation product for improving liver tissue damage induced by a high-fat diet; 6) a fermentation product for improving intestinal barrier; 7) a fermentation product for improving intestinal flora structure disorder.

6. Use according to claim 5, characterized in that, The fermentation product for reducing body weight is specifically a fermentation product for reducing body weight increase caused by a high-fat diet.

7. Use according to claim 5, characterized in that, The fermentation product for improving blood glucose is specifically a fermentation product for reducing blood glucose increase caused by a high-fat diet.

8. Use according to claim 5, characterized in that, The fermentation product for improving intestinal immunity is specifically a fermentation product for reducing TNF-α level in the intestine.

9. Use according to claim 5, characterized in that, The fermentation product for improving blood lipid metabolism disorder is specifically a fermentation product for reducing the content of total cholesterol TC, total triglyceride TG, and low-density lipoprotein LDL-C in blood and increasing the content of high-density lipoprotein cholesterol HDL-C in blood.

10. Use according to claim 5, characterized in that, The fermentation product for improving liver tissue damage caused by a high-fat diet is specifically a fermentation product for improving glutamic-oxalacetic transaminase and hepatocyte steatosis.

11. Use according to claim 5, characterized in that, The fermentation product for improving intestinal barrier is specifically a fermentation product for increasing the expression of intestinal barrier genes mTff3, Muc2, and ZO-1 in the intestine.

12. The use according to claim 5, characterized in that, The fermentation product for improving intestinal flora structure disorder is specifically a fermentation product for regulating the beta diversity and genus level changes of intestinal flora.

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