Amino acid coccus and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an amino acid coccus strain and application thereof. The specific technical scheme is as follows: an amino acid coccus sp. LBK-2, wherein the amino acid coccus is preserved in the China General Microbiological Culture Collection Center on March 7, 2025, and the amino acid coccus is preserved in the China General Microbiological Culture Collection Center on March 7, 2025; the preservation number of the strain is CGMCC NO: 46388. The amino acid coccus LBK-2 has good salt resistance, acid resistance and high temperature resistance, and has the potential of adjusting the fat level.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to an Acidaminococcus and its application. Background Art

[0002] Research shows that the gut microbiota plays an important role in the occurrence and development of obesity. There are significant differences in the gut microbiota structure between obese and non-obese individuals, and this difference may affect processes such as energy metabolism, sugar decomposition, and fat storage. Imbalance of specific microbiota, such as an overly high proportion of "fat bacteria" (Firmicutes) and an overly low proportion of "thin bacteria" (Bacteroidetes), may lead to obesity.

[0003] Fecal microbiota transplantation for weight loss is a method of transplanting the gut microbiota of healthy donors into the gut of patients, aiming to reshape the normal functional gut microbiota of patients and achieve the treatment of obesity. There have been many clinical cases using fecal microbiota transplantation to treat obesity and certain effects have been achieved.

[0004] However, the current research on "thin bacteria" is still relatively preliminary and very limited. The known types of "thin bacteria" are few, and there are certain safety hazards, and the effects are also uncertain.

[0005] Therefore, if new, safe, and effective gut microorganisms with the potential to regulate obesity can be developed, it will have important research and application value. Summary of the Invention

[0006] The object of the present invention is to provide an Acidaminococcus and its application.

[0007] To achieve the above object of the invention, the technical solution adopted by the present invention is: an Acidaminococcus sp. LBK-2, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 7, 2025; the deposit number is: CGMCC NO: 46388.

[0008] Correspondingly, an Acidaminococcus sp. LBK-2, the 16S rDNA sequence of which is shown in SEQ ID NO: 1.

[0009] Correspondingly, a bacterial preparation prepared using the Acidaminococcus.

[0010] Correspondingly, a bacterial preparation containing the Acidaminococcus.

[0011] Correspondingly, a food, health product, or medicine prepared using the Acidaminococcus.

[0012] Correspondingly, a food, health product, or medicine containing the Acidaminococcus.

[0013] Correspondingly, foods, health products or drugs prepared using the said bacterial preparation.

[0014] Correspondingly, foods, health products or drugs containing the said bacterial preparation.

[0015] The present invention has the following beneficial effects: The present invention provides a new strain of Acidaminococcus LBK-2, which has been identified as a new species under the genus Acidaminococcus sp. The LBK-2 strain has no obvious biological and cytotoxicity, and has the effect of slowing down the weight gain induced by a high-fat diet. Description of the Drawings

[0016] Figure 1 It is a colony morphology diagram of LBK-2;

[0017] Figure 2 It is a scanning electron microscope diagram of LBK-2;

[0018] Figure 3 It is a schematic diagram of the phylogenetic tree of LBK-2;

[0019] Figure 4 It is a schematic diagram of the normal growth of HT 29 cells;

[0020] Figure 5 It is a schematic diagram of the adhesion of DH5α to HT 29 cells;

[0021] Figure 6 It is a schematic diagram of the adhesion of LBK-2 to HT 29 cells;

[0022] Figure 7 It is a schematic diagram of the hemolysis of LBK-2; on the left side in the figure is the strain LBK-2, and on the right side is Staphylococcus aureus;

[0023] Figure 8 It is a jejunum section diagram of the normal mouse CON group;

[0024] Figure 9 It is a jejunum section diagram of the normal mouse LBK-2 group;

[0025] Figure 10 It is a schematic diagram of the effect of LBK-2 on the body weight change of high-fat diet mice;

[0026] Figure 11 It is a schematic diagram of the effect of LBK-2 on the body weight gain of high-fat diet mice;

[0027] Figure 12 It is a schematic diagram of the effect of LBK-2 on the blood lipid-related indexes in the serum of high-fat diet mice;

[0028] Figure 13Schematic diagram of the effect of LBK-2 on the changes of inflammatory factors in the serum of high-fat diet mice;

[0029] Figure 14 Liver tissue section diagrams of mice under different treatments;

[0030] Figure 15 Adipose section diagrams of mice under different treatments;

[0031] Figure 16 Schematic diagram of the effect of LBK-2 on the expression levels of LPL and CYP7A1 in high-fat diet mice. Specific implementation manners

[0032] The present invention provides a new strain of Acidaminococcus sp. LBK-2, which was deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 7, 2025; the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101; the deposit number is: CGMCC NO: 46388. The 16S rRNA sequence of the Acidaminococcus sp. LBK-2 is shown as SEQ ID NO:1. The Acidaminococcus sp. LBK-2 has good salt tolerance, acid tolerance and high temperature tolerance, and has the potential to regulate fat levels.

[0033] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The obtained data are all the averages obtained after at least 3 repetitions, and all the repetitions obtained are valid data.

[0034] The culture media and reagents used in each embodiment are as follows:

[0035] Anaerobic basal medium: tryptone (5.68 g), yeast extract (5.0 g), Tris (tris(hydroxymethyl)aminomethane, 3.0 g), peptone (5.0 g), NaCl (1.67 g), soy peptone (1.0 g), K2HPO4 (0.83 g), resazurin (1 mL), water (1000 mL); after boiling, add L-cysteine hydrochloride (0.5 g), fill with nitrogen and dispense, sterilize at 121 °C for 20 min, and then add hemin (4 mL) and vitamin K (2 mL).

[0036] Coccus fermentans amino acid medium: Tryptone (5.0 g), acid-hydrolyzed casein (10.0 g), yeast extract (5.0 g), glucose (5.0 g), sodium glutamate (4.0 g), arginine (1.0 g), glycine (1.0 g), tryptophan (0.1 g), Tween-80 (0.5 mL), KH2PO4 (2.0 g), resazurin (1 mL), water (1000 mL); after boiling, add L-cysteine hydrochloride (0.5 g), sub-pack under nitrogen, and sterilize at 121 °C for 15 min.

[0037] Blood agar plate: Columbia blood agar plate.

[0038] Example 1: Screening and identification of microorganisms

[0039] 1. Screening: Collect the intestinal contents of raccoon dogs, transfer them to the anaerobic basal medium, and anaerobically culture them at 37 °C for 2 - 4 weeks to allow sufficient fermentation and enrichment. Take the mixed culture solution and use PBS to perform serial dilutions at gradients of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 . Take 100 μL of the culture solutions at two gradients of 10 -6 、10 -7 and spread them on the Coccus fermentans amino acid medium (plate). Place the spread plates in an anaerobic environment and culture them at 37 °C. After colonies grow on the plates, pick single colonies and purify them three times continuously to obtain a pure culture of strain LBK-2.

[0040] 2. Physiological and biochemical characteristics: The colony morphology diagram of strain LBK-2 after culturing on the Coccus fermentans amino acid medium at 37 °C in an anaerobic environment for 48 h is as shown in Figure 1 and the electron microscopy scanning diagram is as shown in Figure 2 . Strain LBK-2 is a Gram-negative bacterium, strictly anaerobic, with spherical cells, non-motile, and non-spore-forming; the colonies are milky white, semi-transparent at the edges, round, and raised. Strain LBK-2 can grow normally under the conditions of 20 °C - 50 °C, pH = 3.5 - 8, and sodium chloride concentration of 0.5% - 2%, and the optimal growth conditions are: 37 °C, pH = 7.0.

[0041] 3. DNA Identification: The genomic DNA of strain LBK-2 was extracted using a bacterial genomic DNA extraction kit. The complete 16S rRNA gene was amplified using universal primers 27F and 1492R, and its 16S rRNA is shown as SEQ ID NO:1. The 16S rRNA gene was sequenced by Tianjin Qingke Biotechnology Co., Ltd. The 16S rRNA gene sequence of LBK-2 was analyzed on the EzBioCloud platform, trimmed and aligned using the software EZeditor, and then a phylogenetic tree was constructed using the MEGA 11 software. As Figure 3 shown, Propionispora hippei KS T (AJ508928) was used as the outgroup in the figure.

[0042] Tianjin Qingke Biotechnology Co., Ltd. performed whole-genome sequencing and assembly on strain LBK-2. The assembled genes were uploaded to the website https: / / tygs.dsmz.de / to calculate its dDDH value and the website https: / / www.ezbiocloud.net / tools / ani to calculate its ANI value. The calculation results are shown in Table 1.

[0043] Table 1 Comparison table of the genetic relationship between LBK-2 and publicly available strains

[0044]

[0045] It can be seen from Table 1 that both the dDDH and ANI values of strain LBK-2 and the reference strains are lower than the recommended and generally accepted species boundaries (dDDH is 70%, ANI is 97%). Therefore, it can be proved that strain LBK-2 is a new species under the genus Acidaminococcus.

[0046] Example 2: Demonstration of the tolerance of strain LBK-2 to liquids in organisms

[0047] 1. Bile salt tolerance: Bile salts were added additionally to the fermentative amino acid coccus medium, and the bile salt concentrations were set at 0%, 0.1%, 0.3%, and 0.5% respectively. 2% of LBK-2 was inoculated into each medium, and its OD was measured at 0 h, 4 h, and 8 h respectively under anaerobic conditions. 600 value, with each group repeated 3 times and the average value taken. According to the formula: survival rate % = 100% × (OD of the experimental group 600 / OD of the control group 600 ), calculate the survival rate of bacteria LBK-2 at 4 h and 8 h under different bile salt concentrations.

[0048] The results showed that: at 4 h and 8 h, the survival rate of the bacterium under different bile salt concentrations could remain above 85%, indicating that the bacterium had good bile salt tolerance.

[0049] 2. Gastrointestinal fluid tolerance: The preparation method of simulated gastrointestinal fluid was as follows. Simulated gastric fluid: Take 16.4 mL of dilute hydrochloric acid, add about 800 mL of sterile water, adjust the pH value to 3.0, add 10 g of pepsin, shake well and then add sterile water to make up to 1 L, filter and sterilize for later use. Simulated intestinal fluid: Dissolve 6.8 g of potassium dihydrogen phosphate in 500 mL of distilled water, then adjust the pH value of the solution to 6.8, take it out after autoclaving at 121 °C for 20 min, add trypsin at a concentration of 10 g / L, and finally add sterile water to make up to 1 L, filter and sterilize for later use. Centrifuge the bacterial liquid (obtained by anaerobic overnight culture of the fermented amino acid coccus medium at 37 °C), resuspend it with an equal volume of gastric fluid or intestinal fluid, place it in an anaerobic incubator at 37 °C for 3 h, and perform serial dilution and plating for plate counting at 0 h and 3 h respectively. Each treatment was repeated 3 times, and the results were averaged.

[0050] The results were as follows: When treated with gastric fluid for 0 h, the viable count was 5.68×10 9 cfu / mL. After treating with gastric fluid for 3 h, the viable count was 1.8×10 8 cfu / mL. When treated with intestinal fluid for 0 h, the viable count was 6.72×10 9 cfu / mL. After treating with intestinal fluid for 3 h, the viable count was 1.48×10 9 cfu / mL. It indicated that strain LBK-2 had good tolerance to gastrointestinal fluid and could be orally delivered into the organism to play a role.

[0051] Example 3: Demonstration of the cell adhesion effect of strain LBK-2

[0052] Resuscitation of HT-29 cells: Place the cryopreservation tube containing 500 μL of cell suspension in a 37 °C water bath and shake it evenly to thaw. Immediately take it out after melting, then add it to a 15 mL centrifuge tube containing 3 mL of complete medium, mix it well with the cell suspension by pipetting, place it in a low-speed centrifuge at 1000 rpm for 3 - 5 min, discard the supernatant, and resuspend it with complete culture medium again. Transfer it into a T25 cell culture flask, make up to 5 mL with complete medium, and place it in a 37 °C carbon dioxide incubator for overnight culture. Observe the cell growth status, cell attachment and cell density under an inverted microscope the next day.

[0053] Among them, the complete medium refers to: high-glucose DMEM liquid medium (brand Biosharp, catalog number BL301A) + 10% premium fetal bovine serum (brand Analysis Quiz / Aoqing, catalog number AQmv09900) + 1% penicillin-streptomycin mixture (brand Analysis Quiz / Aoqing, catalog number AQ512).

[0054] Subculture of HT-29 cells: Pour out the medium in the cell culture flask, add 1 mL of trypsin digestion solution to slowly rinse the cells, then add another 1 mL of trypsin digestion solution, place it in a 37°C carbon dioxide incubator for digestion, observe the digestion situation of the cells. After digestion is completed, add 3 - 5 mL of complete medium to terminate digestion. Subculture according to a ratio of 1:2.

[0055] Preparation of bacterial suspension: Take the LBK-2 bacterial solution cultured overnight in a fermented amino acid coccus medium under anaerobic conditions at 37°C, centrifuge and resuspend it with PBS. After washing twice, adjust the bacterial solution concentration to 1 - 3×10 8 CFU / mL using a turbidimeter. At the same time, activate DH5α and place it in an LB solution for overnight culture at 37°C. After centrifugation, resuspend it with PBS. After washing twice, adjust the bacterial solution concentration to 1 - 3×10 8 CFU / mL (as a negative control).

[0056] Place sterile cover slips of 22×22 mm in a six-well cell culture plate in advance. Transfer the cells into the six-well cell culture plate and culture them in an incubator at 37°C, 5% CO2, and 95% humidity to allow the bacteria to adhere to the cover slips and grow. When the cells grow into a confluent monolayer, rinse the cells twice with sterile PBS buffer. Then add 1 mL of DMEM-H medium and 1 mL of the above-prepared bacterial suspension with adjusted concentration to each well. Repeat three wells for each strain. At the same time, set up a blank control group with 1 mL of DMEM-H medium and 1 mL of PBS, gently shake evenly, and continue to culture in a carbon dioxide incubator. After 2 hours of culture, take out the six-well cell culture plate, wash it 5 times with sterile PBS buffer to remove unadhered bacteria, and add absolute methanol for fixation for 20 minutes. Then take out the cell cover slips fixed with absolute methanol, perform Gram staining and observe under a microscope.

[0057] The results are as Figure 4 、 5 shown in Figures 6. The adhesion and colonization of bacteria in the intestinal epithelium are prerequisites for their normal physiological functions. Bacteria with high adhesion ability can, to a certain extent, extend the interaction time with the host, and thus more fully exert their functions on the host organism. The results show that strain LBK-2 has a certain adhesion ability to HT-29 cells.

[0058] Example 5: Safety Test of Strain LBK-2

[0059] 1. Hemolysis Test

[0060] Inoculate the strain LBK-2 by streaking on a blood agar plate. At the same time, use Staphylococcus aureus as a positive control, and culture it in an anaerobic environment at 37°C for 48 h, then observe the hemolysis situation.

[0061] The results are as Figure 7 shown. On the left in the figure is the strain LBK-2, and on the right is Staphylococcus aureus. A well-defined and completely transparent hemolysis ring appears around the colonies of Staphylococcus aureus; while there is no hemolysis phenomenon around the colonies of the strain LBK-2; indicating that the strain LBK-2 does not cause cell hemolysis.

[0062] 2. Safety Test in Mice

[0063] Preparation of the bacterial suspension: Take the bacterial liquid cultured overnight in the fermented Acidaminococcus liquid medium in an anaerobic environment at 37°C, centrifuge it, resuspend it with 0.9% normal saline, wash it twice, and then use a turbidimeter to adjust the bacterial liquid concentration to 1×10 8 CFU / mL.

[0064] Select 10 male Balb / c mice at 75 days old, with a body weight of 24.37 g ± 2.27 g. The breeding environment uses 12 h / 12 h intermittent lighting day and night, with a constant humidity and the temperature controlled at about 25°C. The mice are housed separately in cages and start to adapt to the environment 1 week before the experiment. During this period, the mice can eat and drink freely, and the room temperature and humidity are strictly controlled. After the 1-week adaptation period, randomly divide the 10 mice into 2 groups (experimental group and control group) evenly, with 5 mice in each group. The initial body weights of the mice in each group have no significant difference (P > 0.05). The control group (CON group) is given 0.2 mL of 0.9% normal saline by gavage per mouse per day, and the experimental group (LBK-2 group) is given 0.2 mL of the 1.0×10 8 CFU / mL bacterial suspension by gavage per mouse per day for 14 d. Throughout this stage, the mice can drink and eat freely, and the bedding is cleaned regularly. Before the start and end of the experiment, the mice are fasted overnight (12 h, fasting but not water deprivation).

[0065] (1) Effect on body weight. Weigh the initial body weight and final body weight of the mice, and calculate the body weight gain (Body weight, BW) and average daily gain (Average daily Gain, ADG). The results are shown in Table 2.

[0066] Table 2 Comparison Table of the Effect of Strain LBK-2 on the Body Weight of Mice

[0067]

[0068]

[0069] The results showed that there was no significant difference in the initial body weights between the control group and the experimental group (P > 0.05). After 14 days of continuous gavage, the body weights of mice in each group increased to varying degrees. There were no significant differences in the initial weight, final weight, body weight gain, and average daily weight gain between the LBK-2 group and the CON group (P > 0.05).

[0070] (2) Effects on blood biochemical indices. After the experiment ended and the mice were weighed, blood was collected by puncturing the eye balls of the mice, and anticoagulated blood was collected in EDTA tubes. Blood leukocytes (WBC), lymphocytes (LYM), red blood cells (RBC), hemoglobin (HGB), and platelets (PLT) were immediately measured. After blood was collected in ordinary centrifuge tubes, the blood was centrifuged at 3500 rpm for 10 min to separate the serum, which was then stored at -80 °C. The serum was sent to Jiangsu Addison Biotechnology Co., Ltd. using dry ice for the detection of total protein (TP), albumin (ALB), blood glucose (GLU), triglyceride (TG), low-density lipoprotein (LDL), and total cholesterol (TC) in blood biochemical indices, as well as the detection of cytokines such as IL-2, IL-6, and IL-12 in the serum. The results are shown in Tables 3 - 5.

[0071] Table 3 Comparison Table 1 of the Effects of LBK-2 on Blood Biochemical Indices in Mice

[0072] Test Items CON LBK-2 <![CDATA[White blood cell count WBC (10 9 / L)]]> 3.03±0.30 5.32±2.13 <![CDATA[Red blood cell count RBC (10 12 / L)]]> 11.46±0.31 11.31±0.55 Hemoglobin HGB (g / L) 195.5±5.45 196.0±8.92 <![CDATA[Platelet count PLT (10 9 / L)]]> 511.0±68.36 504.2±70.69 <![CDATA[Lymphocyte count Lym (10 9 / L)]]> 1.80±0.22 3.26±1.65

[0073] Table 4 Comparison Table 2 of the Effects of LBK-2 on Blood Biochemical Indices in Mice

[0074] Test Items CON LBK-2 Total Protein TP (g / L) 70.42±5.16 69.55±3.54 Albumin ALB (g / L) 40.70±0.51 38.28±2.55 Glucose GLU-YA (mmol / L) 5.49±1.35 4.75±1.18 Total Cholesterol TC (mmol / L) 3.58±0.68 3.49±0.43 Low Density Lipoprotein LDL-C (mmol / L) 0.39±0.03 0.30±0.05 Triglyceride TG (mmol / L) 1.06±0.13 1.47±0.35

[0075] Table 5 Comparison Table of the Effects of LBK-2 on Serum Cytokines in Mice

[0076]

[0077]

[0078] The results showed that there were no significant differences in the levels of white blood cells (WBC), lymphocytes (LYM), red blood cells (RBC), hemoglobin (HGB), platelets (PLT), total protein (TP), albumin (ALB), glucose (GLU-YA), total cholesterol (TC), and triglyceride (TG) between the LBK-2 group and the CON group (P > 0.05). However, the level of low-density lipoprotein (LDL-C) in the LBK-2 group was significantly lower than that in the CON group (P < 0.05). There were no significant differences in the levels of IL-6 and IL-12 in the serum compared with the control group (P > 0.05); while the level of IL-2 in the serum of the LBK-2 group was significantly lower than that in the CON group (P < 0.05).

[0079] (3) Effect on organ indices. After blood collection, the mice were sacrificed, and their thymus, spleen, liver, and kidneys were removed and weighed using an electronic balance. The organ index was calculated according to the following formula:

[0080] Organ index (mg / g) = Organ weight (mg) / Fasting body weight (g)

[0081] The results are shown in Table 6.

[0082] Table 6 Effect of LBK-2 on organ indices of mice

[0083] Group Thymus Index Liver Index Spleen Index Kidney Index CON 1.27±0.16 34.85±2.91 3.54±0.19 11.73±1.00 LBK-2 1.36±0.18 36.06±2.51 3.33±0.49 11.52±1.08

[0084] The results showed that there were no significant differences in thymus index, liver index, spleen index, and kidney index between the CON group and the LBK-2 group (P > 0.05).

[0085] (4) Effect on jejunum histology of mice.

[0086] Approximately 1.5 cm of jejunum was intercepted, gently washed with normal saline to remove the contents, and then fixed in 10% formaldehyde solution. Paraffin sections stained with hematoxylin-eosin (H&E) were prepared according to the conventional method. The results are as Figure 8 , 9 and Table 7 show. Figure 8 Figure is the jejunum section of the CON group, Figure 9 Figure is the jejunum section of the LBK-2 group.

[0087] Table 7 Effect of LBK-2 on jejunum of mice

[0088] Group Villus Length (μm) Crypt Depth (μm) Villus Length / Crypt Depth CON 605.57±57.48 242.83±21.82 2.49±1.12 LBK-2 688.08±95.16 242.23±49.27 2.84±1.37

[0089] Figure 8 , 9 No obvious pathological changes were observed in and , and there were no significant differences in villus length, crypt depth, and their ratio between the CON group and the LBK-2 group (P > 0.05).

[0090] In summary, the results showed that continuous intragastric administration of LBK-2 bacterial solution for 14 days had no significant effect on the body weight change of mice; had no obvious effect on the measured blood routine of mice; had no significant effect on thymus index, spleen index, liver index, and kidney index of mice; had no obvious effect on the morphological structure and villus growth of mouse jejunum. However, administration of LBK-2 bacterial solution could reduce the contents of interleukin-2 and low-density lipoprotein in the serum of mice, indicating that strain LBK-2 may be related to animal obesity.

[0091] Example 6: Effect of strain LBK-2 on high-fat diet mice

[0092] Experimental animals and feed: Twenty-three 4-week-old male C57BL / 6J mice were selected and adaptively fed for 2 weeks. The feed was purchased from Kuibu Qianli Biotechnology (Xuancheng) Co., Ltd. During the adaptive feeding stage and in the blank control group, standard feed was fed, while in the high-fat control group and the experimental group during the experimental stage, a high-fat feed with 60% fat energy supply was used.

[0093] Preparation of bacterial suspension: The LBK-2 bacterial solution cultured overnight in the fermented Acidaminococcus liquid medium was centrifuged. After the precipitate was washed twice, it was resuspended in 0.9% sterile physiological saline, and the turbidity of the bacterial solution was adjusted to 1×10 9 CFU / mL.

[0094] Grouping of experimental animals: After 2 weeks of adaptive feeding, the mice were randomly selected and divided into 3 groups, and the initial body weights of the mice were recorded. Gavage was performed at a fixed time every morning, and all gavage reagents were freshly prepared and used immediately. The intervention continued for 8 weeks. The specific grouping and experimental methods are shown in Table 8.

[0095] Table 8 Comparison table of mouse grouping and treatment

[0096] Group Quantity Gavage Dose Feed CON 7 rats Normal Saline / 0.2 mL / rat / day Standard Feed HFD 9 rats Normal Saline / 0.2 mL / rat / day High-Fat Feed LBK 7 rats <![CDATA[1×10 9 CFU / mL LBK-2 bacterial suspension / 0.2 mL / per mouse / per day]]> High-Fat Feed

[0097] Sample collection and treatment: At the end of the experiment, the mice were fasted but allowed to drink water for 12 h. After blood collection from the eyeballs, tissues such as the heart, liver, spleen, and abdominal fat were dissected and separated. They were washed clean with sterile physiological saline, weighed after blotting with filter paper. Mouse liver tissue, adipose tissue, and colon feces were frozen in liquid nitrogen and stored in a -80°C refrigerator. Another part of the liver tissue and adipose tissue was immersed in 10% formaldehyde solution for fixation and placed at room temperature for standby.

[0098] (1) Effect of strain LBK-2 on body weight

[0099] During the experiment, the growth status of the mice in each group was observed. It was found that the hair of the mice in each group was bright and shiny, the mental state was good, the diet and water intake were normal. The fur of the mice in the HFD group and the LBK group was bright and greasy, and their body size was slightly larger than that of the CON group. No adverse physiological reactions were observed. During the experimental period, the growth status of the mice was observed every day, and the body weights of the mice were weighed at a fixed time every week. The changes in body weights of each group are as Figure 10 shown, and after the experiment, the weight increments are as Figure 11 shown.

[0100] Results showed that there were no significant differences in the initial body weights among the CON group, HFD group, and LBK group. During the experiment, the body weights of all groups showed an upward trend. Among them, the body weight of the HFD group increased significantly faster than that of the CON group, and the body weight growth trend of the LBK group was between that of the CON group and the HFD group. At the end of the experiment, the body weight of the HFD group was significantly higher than that of the CON group (P < 0.05); the weight gain of the HFD group was also significantly higher than that of the other two groups (P < 0.05). This indicates that after a high-fat diet, the body weight of mice will increase significantly, and the intervention of LBK-2 will slow down the weight gain induced by a high-fat diet.

[0101] (2) Effects of strain LBK-2 on the weights of organs and fat

[0102] After the experiment, the mice in each group were sacrificed, and the weights of the organs and fat of each group were weighed. The results are shown in Table 9.

[0103] Table 9 Effects of LBK-2 on the weights of organs and fat

[0104] Group CON HFD LBK Heart Weight (g) 0.11±0.005 0.16±0.027 0.12±0.017 Liver Weight (g) 0.89±0.019 1.04±0.197 0.84±0.112 Spleen Weight (g) 0.048±0.0080 0.077±0.0170 0.065±0.0101 Kidney Weight (g) 0.28±0.022 0.35±0.054 0.31±0.023 Fat Weight (g) 0.32±0.090 1.28±0.339 1.04±0.364

[0105] Results showed that compared with the CON group, the heart weight of the HFD group was significantly increased (P < 0.05), while the LBK group significantly reduced the increase in heart weight (P < 0.05). Compared with the CON group, the spleen weights of the HFD group and the LBK group were significantly increased (P < 0.05), and the spleen weight of the LBK group was lower than that of the HFD group. Compared with the CON group, the kidney weights of the HFD group and the LBK group were significantly increased (P < 0.05), and the kidney weight of the LBK group was lower than that of the HFD group. Compared with the CON group, the fat weights of the HFD group and the LBK group were significantly increased (P < 0.01), and the fat weight of the LBK group was lower than that of the HFD group. This indicates that the intervention of LBK-2 can significantly reduce the effects of a high-fat diet on the weights of organs and fat.

[0106] (3) Effects of strain LBK-2 on blood lipid-related indicators in serum

[0107] A high-fat diet can lead to abnormal blood lipid levels, usually manifested as increased levels of total cholesterol (TC), low-density lipoprotein (LDL-C), and decreased levels of high-density lipoprotein (HDL-C). The blood collected by eyeball puncture from the mice was allowed to stand at room temperature for 1 h, centrifuged at 3000 rpm for 10 min, and then centrifuged at 12000 rpm at 4°C for 10 min. The supernatant was aliquoted and stored at -80°C. The serum was sent to Jiangsu Addison Biotechnology Co., Ltd. to measure the contents of blood lipid-related indicators TC, LDL-C, HDL-C, and cellular immune factors IL-1β, IL-10, TNF-α in the serum of mice. The results of the blood lipid-related indicators are as Figure 12 shown.

[0108] Results showed that the TC level in the serum of mice in the HFD group was significantly higher than that in the CON group (P < 0.05), the LDL-C level was extremely significantly higher than that in the CON group (P < 0.01), and the HDL-C level was significantly lower than that in the CON group (P < 0.05). Compared with the HFD group, the TC and LDL-C levels in the serum of mice in the LBK group decreased to varying degrees; the HDL-C level in the serum of mice in the LBK group was significantly higher than that in the HFD group (P < 0.05). This indicates that the intervention of LBK-2 can significantly reduce the impact of a high-fat diet on the serum index HDL-C in mice.

[0109] (4) Effects of strain LBK-2 on inflammatory factors in serum

[0110] The results were as Figure 13 shown. Results showed that the content of the pro-inflammatory factor TNF-α in the serum of mice in the HFD group was higher than that in mice in the CON group, and the content of IL-1β was extremely significantly higher than that in mice in the CON group (P < 0.01), suggesting that the mice were in an inflammatory state, while the content of the anti-inflammatory factor IL-10 in the HFD group was extremely significantly lower than that in the CON group (P < 0.01). The contents of TNF-α and IL-1β in the serum of mice in the LBK group decreased compared with those in the HFD group, while the content of IL-10 in the serum of mice in the LBK group was extremely significantly higher than that in the HFD group (P < 0.01). This indicates that the intervention of strain LBK-2 may be able to inhibit the inflammatory response of high-fat diet-fed mice by increasing the content of IL-10 in the serum.

[0111] (5) Effects of strain LBK-2 on the pathological status of the liver and adipose tissue

[0112] A high-fat diet usually causes abnormal lipid metabolism in the liver, increased lipid accumulation, and thus leads to fatty degeneration of hepatocytes (fatty liver). Take the liver tissue and adipose tissue immersed in paraformaldehyde solution, cut them into tissue blocks of appropriate size, dehydrate them with gradient alcohol until the tissue is moderately transparent, then embed them in paraffin, make 5-μm-thick sections, and finally stain them with hematoxylin-eosin (H&E), dehydrate, make transparent, and seal to complete the production of tissue sections. Observe the sections with a slice microscope and conduct pathological analysis. The liver sections of each group were as Figure 14 shown; the adipose tissue sections of each group were as Figure 15 shown.

[0113] Figure 14 It was shown that the liver texture of the CON group was orderly, the boundaries between hepatocyte cords were obvious, and there were no lipid droplets. Many obvious white vacuole structures of various sizes appeared in the HFD group, the boundaries between hepatocyte cords were unclear, and the arrangement was irregular. In the LBK group, the size and number of lipid droplets decreased accordingly. This indicates that a high-fat diet can lead to lipid accumulation, resulting in a large number of lipid vacuoles in the liver tissue sections, but the intervention of strain LBK-2 can reduce the accumulation of lipid droplets, helping the boundaries between hepatocyte cords to become clearer and the arrangement to become more regular.

[0114] Figure 15 Results: The abdominal adipose tissue of mice was subjected to staining analysis. After H&E staining, the adipose tissue showed a vacuolated morphology. The abdominal adipose cells of the CON group mice were intact in morphology, arranged neatly, with a smaller cell area and a larger number of adipose cells under the field of view. The number of abdominal adipose cells in the HFD group mice was significantly reduced, the area was increased, the arrangement was irregular, and the cell structure was incomplete. This indicates that long-term intake of a high-fat diet induced lipid accumulation in the adipose tissue of mice. After intervention with strain LBK-2, the morphology of the abdominal adipose cells of mice was restored to some extent. The size of the adipose cells observed under the same field of view was significantly reduced, the number was significantly increased, and the cell structure also tended to be complete. This indicates that the intervention of strain LBK-2 can improve the fat accumulation caused by a high-fat diet and maintain the normal morphology of adipose tissue.

[0115] (6) Effect of strain LBK-2 on the expression levels of lipid metabolism-related genes

[0116] Low expression of CYP7A1 mRNA can lead to cholesterol metabolism disorders, and further lead to liver fat accumulation and obesity. LPL is a proteolytic enzyme and a key enzyme in the lipid metabolism pathway. If the activity of this enzyme is reduced, it will lead to an increase in the content of TG in serum and a decrease in the content of HDL-C, thereby increasing the blood lipid level and inducing obesity.

[0117] The expression levels of LPL and CYP7A1 in each group of mice were determined by RT-qPCR method. The results are as Figure 16 shown. The results showed that the expression levels of LPL and CYP7A1 in the liver tissues of the CON group mice were significantly higher than those of the HFD group, and strain LBK-2 could up-regulate the expression levels of LPL and CYP7A1 in the liver tissues of mice.

[0118] A study on the improvement of glucose metabolism in high-fat diet mice by liver-specific overexpression of lipoprotein lipase pointed out that in high-fat diet mice, overexpression of LPL can reduce liver fat accumulation and improve glucose metabolism. After the intervention of strain LBK-2, the expression level of LPL in the liver tissues of the LBK group was much higher than that of the CON group and the HFD group. This may be because the intervention of strain LBK-2 triggered certain mechanisms, resulting in overexpression of LPL in liver tissues, thereby reducing the accumulation of lipids in the liver and alleviating the negative effects brought by a high-fat diet.

[0119] The embodiments described above are only descriptions of the preferred modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Acidaminococcus sp. LBK-2, characterized in that: The amino acid coccus was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 7, 2025; the deposit number is: CGMCCNO: 46388.

2. A strain of Acidaminococcus sp. LBK-2, characterized in that: The 16SrDNA sequence of the aminoacidococcus is shown in SEQ ID NO:

1.

3. A bacterial preparation prepared using the amino acid coccus according to claim 1 or 2.

4. A bacterial preparation comprising the aminoacidococcus according to claim 1 or 2.

5. Food, health product or medicine prepared using the amino acid coccus according to claim 1 or 2.

6. Food, health product or medicine comprising the aminoacidococcus according to claim 1 or 2.

7. Food, health product or medicine prepared using the bacterial preparation according to claim 3 or 4.

8. Food, health product or medicine comprising the bacterial preparation according to claim 3 or 4.

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