Application of Lactobacillus rhamnosus NKU FL1-8 in resisting obesity, serum inflammation and cognitive impairment caused by high-fat diet

Abstract

The present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )The application of NKU FL1‑8 in resisting obesity, serum inflammation and cognitive impairment caused by a high-fat diet belongs to the field of microbial technology. The present invention finds that Lactobacillus rhamnosus NKU FL1‑8 can significantly reduce the increase of adipose tissue caused by a high-fat diet, alleviate liver damage caused by a high-fat diet, inhibit serum inflammation caused by a high-fat diet, improve cognitive impairment caused by a high-fat diet, strengthen the intestinal barrier, increase the content of short-chain fatty acids in the cecum and feces, regulate the intestinal flora, and increase the diversity of the intestinal flora, thereby effectively resisting related diseases caused by a high-fat diet.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of Lactobacillus rhamnosus NKU FL1-8 in resisting obesity, serum inflammation and cognitive impairment caused by a high-fat diet. Background Art

[0002] Unreasonable eating habits are one of the main factors causing obesity. It is characterized by unhealthy fat accumulation due to energy imbalance in the body's adipose tissue. The increase in fat accumulation gradually stimulates the imbalanced use and storage of energy, leading to glucose, protein and lipid metabolic dysfunction. Obesity is an important risk factor for metabolic disorders, including insulin resistance, type 2 diabetes, dyslipidemia, non-alcoholic fatty liver disease and cardiovascular disease. In addition to bariatric surgery, some drugs can also be used to treat obesity, among which appetite suppressants are commonly prescribed, but they usually have side effects related to the central nervous system.

[0003] As a symbiotic microorganism in the human intestine, probiotics are increasingly attracting widespread attention from researchers in this field due to their relative safety and diverse efficacy. The intestinal flora can convert indigestible carbohydrates into digestible energy, and the regulatory effect of microorganisms on energy metabolism may be a convenient and effective way to lose weight. Different probiotics have different therapeutic mechanisms in the treatment of obesity. Some strains improve the composition of the intestinal flora, while others can stimulate leptin production through the central nervous system and related mechanisms, and the gut-brain axis inhibits appetite and energy intake. Other probiotics have been shown to exert anti-obesity effects by inhibiting oxidative stress, endoplasmic reticulum stress, regulating lipid, cholesterol and glucose metabolism, and reducing chronic low-grade inflammatory markers and cytokines. The anti-obesity effect of probiotics is strain, genus and species specific. Therefore, screening probiotics that can effectively resist obesity caused by a high-fat diet has become the key to solving metabolic diseases caused by obesity in modern people. Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )NKU FL1-8, deposited in Guangdong Microbial Culture Collection Center, with the deposit number GDMCC No: 64053 and the deposit date November 23, 2023. Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus NKU FL1-8 has been shown to alleviate alcoholic liver damage, but its effects on obesity caused by a high-fat diet and the metabolic diseases further caused have not been reported. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the object of the present invention is to provide the use of Lactobacillus rhamnosus NKUFL1-8 in resisting obesity, serum inflammation and cognitive impairment caused by a high-fat diet.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) Use of NKU FL1-8 or rhamnosus Lactobacillus NKU FL1-8 bacterial agent in the preparation of products for preventing and / or treating related diseases caused by a high-fat diet, the preservation number of the rhamnosus Lactobacillus NKU FL1-8 is GDMCC No: 64053.

[0007] Preferably, the related diseases are any one or more of obesity, serum inflammation and cognitive impairment.

[0008] Preferably, the Lactobacillus rhamnosus NKU FL1-8 bacterial agent includes a Lactobacillus rhamnosus NKU FL1-8 bacterial suspension and / or a Lactobacillus rhamnosus NKU FL1-8 postbiotic.

[0009] Preferably, the method for preparing the bacterial suspension of Lactobacillus rhamnosus NKU FL1-8 comprises the following steps: culturing Lactobacillus rhamnosus NKU FL1-8 in a liquid culture medium, centrifuging the culture solution, and resuspending the precipitate to obtain a bacterial suspension of Lactobacillus rhamnosus NKUFL1-8; the method for preparing the postbiotics of Lactobacillus rhamnosus NKU FL1-8 comprises the following steps: heating the bacterial suspension of Lactobacillus rhamnosus NKU FL1-8 in a water bath to inactivate the postbiotics of Lactobacillus rhamnosus NKU FL1-8 to obtain postbiotics of Lactobacillus rhamnosus NKU FL1-8.

[0010] Preferably, the Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent reduces the increase in adipose tissue and weight gain caused by a high-fat diet.

[0011] Preferably, the Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent reduces the MDA level of liver tissue.

[0012] Preferably, the Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent reduces the levels of GOT, GPT, TNF-α, IL-1β, IL-6, TC, TG, LDL, leptin and endotoxin in serum.

[0013] Preferably, the Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent increases the content of short-chain fatty acids in the cecum and feces.

[0014] Preferably, the Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent increases the abundance and diversity of beneficial intestinal flora.

[0015] Preferably, the Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent improves cognitive impairment caused by a high-fat diet.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0017] The present invention finds that the Lactobacillus rhamnosus NKU FL1-8 and its bacterial agent can alleviate oxidative stress caused by obesity; reduce the weight gain of adipose tissue and reduce body weight; increase the length of ileal villi and crypt depth; regulate the level of liver and serum lipid metabolism; reduce serum and liver inflammation levels; alleviate liver oxidative stress levels; strengthen the intestinal barrier, increase the content of short-chain fatty acids in the cecum and feces; regulate intestinal flora and improve the diversity of intestinal flora. The Lactobacillus rhamnosus NKU FL1-8 and its bacterial agent of the present invention can effectively resist obesity, serum inflammation and cognitive impairment caused by a high-fat diet. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : The colony count of Lactobacillus rhamnosus NKU FL1-8 under different prebiotic addition conditions;

[0019] Figure 2 : Relative cumulative oil droplet volume of Caco-2 cells in each group after co-culture with different bacteria;

[0020] Figure 3 : HSA synergy index of each strain combination;

[0021] Figure 4 : Bar graph of relative abundance of species at the phylum level in the intestinal flora of each group of mice;

[0022] Figure 5 : Bar graph of relative abundance of species at genus level in the intestinal flora of mice in each group;

[0023] Figure 6 : Bar graph of relative abundance of Lactobacillus in the intestinal flora of mice in each group;

[0024] Figure 7 : Alpha diversity index of intestinal flora of mice in each group;

[0025] Figure 8 : Alpha diversity dilution curves of intestinal flora of mice in each group;

[0026] Fig. 9 : Unweighted Unifrac analysis results of intestinal microorganisms of each group of mice;

[0027] Fig.10 : POCA analysis results of beta-diversity of intestinal microorganisms in each group of mice;

[0028] Fig.11 : Body weight change curves of mice in each group;

[0029] Fig.12 : Feed utilization rate of mice in each group;

[0030] Fig.13 : Lee's index of mice in each group;

[0031] Fig.14 :Adipose tissue near the epididymis of mice in each group, the background scale in the figure is 1 cm;

[0032] Fig.15 : Epididymal adipose tissue weight of mice in each group;

[0033] Fig.16 : HE staining of epididymal tissue sections of mice in each group, scale bar is 200 μm;

[0034] Fig.17 : Liver weight and total cholesterol content in liver of mice in each group;

[0035] Fig.18 : Levels of GSH, MDA and SOD in the liver of mice in each group;

[0036] Fig.19 : GOT and GPT levels in the liver of mice in each group;

[0037] Fig. 20 : The levels of inflammatory factors IL-6, IL-1β, TNF-α, TC and TG, LDL-C and HDL-C in the serum of mice in each group;

[0038] Fig.21 : The levels of leptin and endotoxin in the serum of mice in each group;

[0039] Fig. 22 : Statistics of the cecum length of some mice and each group of mice. The background scale in the figure is 1 cm;

[0040] Fig.23 :HE staining of ileum sections of mice in each group, scale bar is 100 μm;

[0041] Fig.24 : ileum villus length, crypt depth, and villus length / crypt depth of mice in each group;

[0042] Fig.25 : Contents of short-chain fatty acids and five major acids in the cecal contents of mice in each group;

[0043] Fig.26 : Changes in fecal short-chain fatty acid content in each group of mice;

[0044] Fig. 27 : Nesting scoring results of mice in each group;

[0045] Fig.28 : Results of open field test of mice in each group;

[0046] Fig.29 : The levels of IGF-1 and BDNF in serum of mice in each group.

[0047] Biological Deposit Description

[0048] Lactobacillus rhamnosus NKU FL1-8 ( Lacticaseibacillus rhamnosus NKU FL1-8), deposited in: Guangdong Microbiological Culture Collection Center, deposited in: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit number: GDMCC No: 64053, deposit date November 23, 2023. DETAILED DESCRIPTION

[0049] The present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) Use of NKU FL1-8 or rhamnosus lactobacillus NKU FL1-8 bacterial agent in the preparation of products for preventing and / or treating related diseases caused by a high-fat diet. The related diseases described in the present invention are any one or more of obesity, serum inflammation and cognitive impairment. The rhamnosus lactobacillus NKU FL1-8 described in the present invention has a deposit number of GDMCC No: 64053, is isolated and purified from infant fecal samples, and is proved to belong to Lacticaseibacillus rhamnosus , and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 23, 2023. The address of the deposit unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences.

[0050] The Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention comprises a Lactobacillus rhamnosus NKU FL1-8 bacterial suspension and / or a Lactobacillus rhamnosus NKU FL1-8 postbiotic. Preferably, the concentration of Lactobacillus rhamnosus NKU FL1-8 in the Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention is 1×10 4 ~9×10 10 cfu / mL, more preferably 1×10 6 ~9×10 9 cfu / mL.

[0051] The preparation method of the bacterial suspension of Lactobacillus rhamnosus NKU FL1-8 of the present invention comprises the following steps: culturing Lactobacillus rhamnosus NKU FL1-8 in a liquid culture medium, centrifuging the culture medium, and resuspending the precipitate to obtain a bacterial suspension of Lactobacillus rhamnosus NKU FL1-8; the preparation method of the postbiotic of Lactobacillus rhamnosus NKU FL1-8 comprises the following steps: heating and inactivating the bacterial suspension of Lactobacillus rhamnosus NKU FL1-8 in a water bath to obtain the postbiotic of Lactobacillus rhamnosus NKU FL1-8. In the present invention, the liquid culture medium can be selected as an MRS liquid culture medium or a prebiotic culture medium. The prebiotic culture medium of the present invention is obtained by removing the carbon source (glucose) in the MRS liquid culture medium and adding prebiotics with a final concentration of 15-25 g / L, wherein the final concentration of the prebiotics is preferably 20 g / L, and the prebiotics are composed of oligosaccharides and polyglucose, and the mass ratio of the oligosaccharides to the polyglucose is preferably 1-3:1-3, and more preferably 1:3; the inoculation amount of the rhamnosus lactobacillus NKUFL1-8 is 1-3%, and preferably 2%; the culture temperature is 36-38°C, and preferably 37°C; the culture time is 15-20h, and preferably 16-18h; the culture method is preferably anaerobic culture in a constant temperature incubator; the centrifugation conditions are: 4000-6000rpm, centrifugation for 10-15min, the centrifugation speed is preferably 5000-5500rpm, and the centrifugation time is preferably 12-14min. The bacterial precipitate is preferably washed 2 to 3 times with a sterile saline buffer solution, and the bacterial sludge is resuspended with a sterile saline buffer solution to obtain a bacterial suspension. The sterile saline buffer solution is preferably physiological saline. The water bath heating temperature is 78 to 90° C., preferably 80 to 85° C.; the water bath heating time is 15 to 25 minutes, preferably 20 minutes.

[0052] The Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention can reduce the increase of adipose tissue caused by a high-fat diet, reduce the Lee's index (reduce the degree of obesity), reduce the weight gain caused by excessive energy intake, reduce the area of ​​a single fat cell, improve the compactness and orderliness of the arrangement of fat cells, and resist obesity caused by a high-fat diet.

[0053] The Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention can alleviate liver damage caused by a high-fat diet. The intake of a high-fat diet can lead to fatty liver. The strain and the bacterial agent of the present invention can reduce liver weight, reduce liver tissue cholesterol, and alleviate the formation of liver fat droplets; downregulate the MDA level in serum, upregulate the GSH level and maintain a normal SOD level, and alleviate the liver oxidative stress level; reduce the GOT and GPT levels in serum, and resist obesity and metabolic disorders caused by a high-fat diet.

[0054] The Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention can inhibit serum inflammation caused by a high-fat diet. The intake of a high-fat diet can cause inflammation. The strain and the bacterial agent of the present invention can downregulate the level of inflammatory factor TNF-α in serum and significantly reduce the levels of IL-1β and IL-6, prevent the increase of serum total triglyceride and total cholesterol levels, reduce serum low-density lipoprotein (LDL) content, and reduce the LDL / HDL ratio, thereby resisting obesity and serum inflammation caused by a high-fat diet.

[0055] The Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention can strengthen the intestinal barrier, reduce the serum endotoxin level, reduce the penetration of bacterial endotoxin in the intestine, increase the length of ileal villi and the depth of crypts, and reduce the length of villi / crypt depth.

[0056] The Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention can increase the content of short-chain fatty acids in the cecum and feces, and the short-chain fatty acids are preferably acetic acid, propionic acid, isobutyric acid, n-butyric acid, and valeric acid, which improves the integrity of the intestinal barrier function, maintains the homeostasis of the intestinal flora, and resists metabolic disorders caused by a high-fat diet.

[0057] The Lactobacillus rhamnosus NKU FL1-8 or Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention can improve the abundance and diversity of beneficial intestinal flora. At the phylum level, the Proteobacteria ( Proteobacteria ) and Bacteroidetes ( Bacteroidota ) and Iron-reducing bacteria ( Deferribacterota ) and reduced the abundance of Actinobacteria ( Actinobacteriota ) abundance; at the genus level, the genus Lactobacillus ( Lacticaseibacillus ) and Bacteroides ( Bacteroides ) and reduced the abundance of Dubcea nycticola ( Dubosiella ) and Blautia ( Faecalibaculum ) abundance; Lactobacillus rhamnosus NKU FL1-8 can be successfully colonized in the intestine, significantly increasing the abundance of the genus Lactobacillus. The strain and its bacterial agent of the present invention increase the α-diversity and β-diversity of the intestinal flora and resist metabolic disorders caused by a high-fat diet.

[0058] A high-fat diet has a certain impact on the cognitive development ability, environmental adaptability and problem-solving ability of mice. The Lactobacillus rhamnosus NKU FL1-8 or the Lactobacillus rhamnosus NKU FL1-8 bacterial agent of the present invention can improve the score of nesting behavior, adjust the levels of IGF-1 and BDNF in serum, improve the cognitive ability of mice, reduce the anxiety level of mice to the new environment, and have a healthier mental state.

[0059] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] In an embodiment of the present invention, MRS solid culture medium: peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, ammonium citrate 2.0 g / L, manganese sulfate 0.05 g / L, magnesium sulfate 0.2 g / L, agar 15.0 g / L, Tween 80 1.0 g / L; MRS liquid culture medium: peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, sodium acetate 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, ammonium citrate 2.0 g / L, manganese sulfate 0.05 g / L, magnesium sulfate 0.2 g / L, Tween 80 1.0 g / L.

[0061] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0062] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0063] Example 1

[0064] Effect of prebiotic composition on proliferation of Lactobacillus rhamnosus NKU FL1-8 in vitro:

[0065] Based on MRS medium, carbon-deficient medium, prebiotic medium and control medium were set up respectively.

[0066] Carbon-deficient culture medium: Removing the carbon source (glucose) from the MRS culture medium results in carbon-deficient culture.

[0067] Prebiotic culture medium: Add prebiotics with a final concentration of 20 g / L to the carbon-deficient culture medium to obtain the prebiotic culture medium; there are 5 groups of prebiotic culture medium, and the prebiotics added are: galacto-oligosaccharide (GOS), polydextrose (PDX), GOS:PDX=2:2, GOS:PDX=1:3, GOS:PDX=3:1;

[0068] Control culture medium: MRS medium (Qingdao Haibo).

[0069] Activation of strains: streak the Lactobacillus rhamnosus NKU FL1-8 (GDMCC No: 64053) frozen at -80℃ on the MRS medium, pick a single colony after culturing for 24 hours and inoculate it into 10mL MRS liquid medium, and culture it at 37℃ for 12 hours to obtain the primary fermentation liquid. According to the inoculation amount of 3% (v / v), the cultured primary fermentation liquid was inoculated into the carbon-deficient medium, prebiotic medium and control medium respectively. The liquid volume of the culture medium was 90% of the volume of the centrifuge tube, and the culture was kept at a constant temperature of 37℃ incubator for 24 hours and 48 hours. The absorbance of the bacterial liquid was measured at a wavelength of 600nm by a spectrophotometer, and the plate was diluted and coated at the same time to calculate the number of colonies. The results are shown in Table 1 and Figure 1 .

[0070] Table 1 Colony counts of Lactobacillus rhamnosus NKU FL1-8 under different prebiotic addition conditions

[0071]

[0072] It can be seen that when the mass ratio of GOS to PDX is 3:1 and 1:3, the prebiotic composition can significantly improve the proliferation ability of Lactobacillus rhamnosus NKU FL1-8, and the number of colonies of Lactobacillus rhamnosus NKU FL1-8 is more than that of the colonies grown in the medium containing GOS alone, and significantly higher than that of the colonies grown in the medium containing PDX alone; at the same time, its OD 600 The values ​​were significantly higher than the OD values ​​of the cells grown in the medium containing GOS alone and the medium containing PDX alone. 600 This indicates that the probiotic effect is better when the mass ratio of GOS to PDX is 3:1 and 1:3.

[0073] Example 2

[0074] Effects of Lactobacillus rhamnosus NKU FL1-8 on fatty acid absorption in intestinal epithelial cells:

[0075] Lactobacillus rhamnosus NKU FL1-8 (GDMCC No: 64053), Lactobacillus plantarum LZ015 (CGMCC No: 29395), Lactobacillus paracasei GF009 (CGMCC No. 24259), Lactobacillus reuteri LL029 (CGMCC No. 23648), and Bifidobacterium lactis BR008 (CGMCC No. 26481) were inoculated separately. Lactobacillus rhamnosus NKU FL1-8, Lactobacillus reuteri LL029, Lactobacillus plantarum LZ015, and Lactobacillus paracasei GF009 were inoculated into MRS broth culture medium, and Bifidobacterium lactis BR008 was inoculated into BS broth culture medium, and cultured at 37°C for 18 hours. The cultures were centrifuged at 4°C and 3000 rpm for 5 min, the supernatant was removed and the precipitate was washed with an appropriate amount of PBS, and then suspended with an appropriate amount of DMEM medium [supplemented with 2% BSA and 500 μM oleic acid (purchased from Sigma-Aldrich, catalog number SI-T8158-100MG)] to obtain Lactobacillus rhamnosus NKUFL1-8, Lactobacillus reuteri LL029, Bifidobacterium lactis BR008, Lactobacillus plantarum LZ015, and Lactobacillus paracasei GF009 (all at a concentration of 4 × 10 8 CFU / mL (obtained by counting the number of bacteria using plate counting medium)].

[0076] The Caco-2 cells were divided into 19 groups, including 1 blank control group, 1 blank plus oil acid control group, and 17 intervention experimental groups. 6 The number of cells / well was added to a 6-well culture plate containing 2 mL of DMEM medium (supplemented with 10% FBS and 1% penicillin-streptomycin), and 0.5 mL of bacterial solution or mixed bacterial solution was added to each experimental group according to the formula shown in Table 2, and an appropriate amount of oleic acid was added to a final concentration of 500 μM (4 μL of oleic acid was added to 2.5 mL of the culture system). The blank control group was not treated. The cells in each group were placed in an incubator set at 37°C and 5% CO2 for 6 hours.

[0077] Table 2 Bacterial solution containing oleic acid added to each experimental group

[0078]

[0079] The culture medium of each group of cultures was removed, and 1 mL of 10% (v / v) formaldehyde solution was added to fix the cells at 37°C for 10 min, and the cells were washed with an appropriate amount of PBS. 1 mL of Oil-Red-O staining reagent (purchased from Beijing Solebow, item number G1206) was added at 37°C to stain the oil droplets accumulated in the cells. The cells were rinsed with PBS, 1 mL of isopropanol was added to each group of cells, and the cells were incubated at 25°C for 10 min. The absorbance value (OD) of the reaction mixture formed by each group was read with a spectrophotometer at a wavelength of 520 nm. 520 ).

[0080] The relative cumulative oil droplet volume (%) of each group of Caco-2 cells was obtained by standardizing the absorbance values ​​measured in each group with the absorbance values ​​measured in the oleic acid control group. Each group of experiments was repeated 3 times, and the experimental data obtained were expressed as "mean ± standard deviation (SD)" and analyzed by Student's t-test to evaluate the differences between the groups. If the statistical analysis results are P <0.05, indicating statistical significance.

[0081] The results are as follows Figure 2 As shown. It can be seen that the cumulative oil droplet amount measured by the oleic acid control group is significantly higher than that of the blank control group, showing that Caco-2 cells absorb extracellular oleic acid and accumulate in the cell. Compared with the oleic acid control group, the cumulative oil droplet amount of the intervention group is significantly reduced, and the intervention group of Lactobacillus rhamnosus NKU FL1-8 alone and the rhamnose compound group have a relatively obvious reduction in lipid droplet accumulation. In comparison, there is no significant difference between the Lactobacillus reuteri LL029, the Bifidobacterium lactis BR008 alone intervention group and its low-dose, high-dose compound group and the oleic acid control group, and there is a significant difference between the Lactobacillus plantarum LZ015, the Lactobacillus paracasei GF009 alone intervention group and its low-dose, high-dose compound group and the oleic acid control group. It shows that the Lactobacillus rhamnosus NKU FL1-8 of the present invention can effectively inhibit the intestinal epithelial cells from absorbing fatty acids, and can show more excellent efficacy when used in combination with Lactobacillus plantarum LZ015 and Lactobacillus paracasei GF009.

[0082] The HSA (Highest Single Agent) synergy index is an indicator used to evaluate the effect of two drugs when used in combination relative to when used alone. It is based on the assumption that the effect of the drug combination should not exceed the maximum effect of the single drug of the two drugs. The calculation formula is E HSA =E AB −max(E A ,E B ), where EAB is the effect of the combination of two drugs, E A and E B are the effects of the two drugs when used alone; the effect value is obtained by: (measured absorbance value - blank absorbance value) / measured absorbance value. HSA If it is positive, the drug combination is considered to be synergistic; if it is negative or close to zero, it may indicate antagonism or additive effects.

[0083] The HSA synergy index of each group of strain combinations is as follows Figure 3 As shown. Combining the absorbance data and the HSA synergy index, it can be seen that rhamnosus casei NKU FL1-8 itself has a good effect on alleviating the accumulation of fatty acids in cells, while Lactobacillus reuteri LL029 and Bifidobacterium lactis BR008 have no significant effect, Lactobacillus plantarum LZ015 and Lactobacillus paracasei GF009 have certain effects, and after compounding the bacterial liquid in different proportions, it can be seen that the overall synergistic effect is not as good as the intervention of rhamnosus casei NKU FL1-8 alone, but the compounding of rhamnosus casei NKU FL1-8 has made a significant improvement in the intervention effect, reflecting the superior effect of rhamnosus casei NKU FL1-8: whether it is used for intervention alone or compound intervention, it can alleviate the accumulation of fatty acids in cells to a certain extent, and rhamnosus casei NKU FL1-8 has the effect of fatty acid metabolism.

[0084] Example 3

[0085] 1. Establishment of C57BL / 6J mouse obesity model:

[0086] Four-week-old, SPF-grade male C57BL / 6J mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and housed in a constant temperature of 25°C, relative humidity of 55%, and an artificial 12-h light-dark cycle. The mice had free access to food and water. After a one-week adaptation period, 60 mice were randomly divided into 5 groups: blank group (SD), model group (HFD), Lactobacillus rhamnosus NKU FL1-8 intervention group (FL1-8), Lactobacillus rhamnosus NKU FL1-8 postbiotic preparation intervention group (POT), and GDMCC1.325 purchased from Guangdong Institute of Microbiology (ATCC 7469 in other collection centers) as the strain control group (BSC) for baseline data. After one week of adaptive feeding, the mice entered the intervention period. The intervention period lasted for 8 weeks. The blank group continued to be fed with standard maintenance diet after the adaptation period, and was gavaged with 0.2 mL of normal saline every morning. The model group and other groups fasted for 12 hours after the adaptation period, and began to be fed with high-fat diet (60% fat), and were gavaged with 0.2 mL of normal saline every morning. The FL1-8 group fasted for 12 hours after the adaptation period, and began to be fed with high-fat diet (60% fat), and was gavaged with 0.2 mL of rhamnosus casei NKU FL1-8 bacterial suspension (1×10 10 CFU / mL), Lactobacillus rhamnosus NKU FL1-8 postbiotic preparation (1×10 10 CFU / mL), strain GDMCC1.325 bacterial suspension (1×10 10 CFU / mL), modeling and intervention at the same time.

[0087] Preparation of Lactobacillus rhamnosus NKU FL1-8 bacterial suspension: Take out the strain NKU FL1-8 (GDMCC No: 64053) at -80℃, inoculate the strain into prebiotic medium at a 2% inoculation rate (remove glucose from MRS medium, and then add prebiotics with a final concentration of 20g / L, and the prebiotics are composed of GOS and PDX at a mass ratio of 3:1), culture at 37℃ for 18h, take 1mL of culture solution and measure the absorbance at OD=600nm. When OD=2, the number of viable bacteria is about 10 9 , centrifuge 30 mL of the bacterial suspension at 4000×g for 15 min, wash the precipitate three times with a sterile salt buffer solution, and resuspend the bacterial slurry with 2 mL of physiological saline to obtain a rhamnosus Lactobacillus casei NKU FL1-8 bacterial suspension.

[0088] Preparation of the postbiotic preparation of Lactobacillus rhamnosus NKU FL1-8: The bacterial suspension of Lactobacillus rhamnosus NKU FL1-8 was heated in a water bath at 80° C. for 20 min to obtain the postbiotic preparation of Lactobacillus rhamnosus NKU FL1-8.

[0089] Preparation of bacterial suspension of strain GDMCC1.325: strain GDMCC1.325 was taken out at -80℃, and the strain was inoculated into prebiotic medium at a 2% inoculation rate (MRS medium was depleted of glucose, and then prebiotics with a final concentration of 20 g / L were added, and the prebiotics were composed of GOS and PDX at a mass ratio of 3:1). After culturing at 37℃ for 18 hours, 1 mL of culture medium was taken to measure the absorbance at OD=600nm. When OD=2, the number of viable bacteria was about 10 9 , centrifuge 30 mL of the bacterial suspension at 4000×g for 15 min, wash the precipitate three times with sterile salt buffer solution, and resuspend the bacterial mud with 2 mL of physiological saline to obtain the bacterial suspension of strain GDMCC1.325.

[0090] On the evening of the eighth week of the animal experiment, the mice were fasted but not watered. After 12 hours, the mice were killed, and the abdominal cavity was opened to collect blood and tissue (liver, ileum, cecum) samples. Some of the collected samples were fixed in paraformaldehyde, and some were immediately frozen in liquid nitrogen and stored at -80°C.

[0091] 2. Effect of mouse intestinal flora

[0092] Cecal contents were collected and total DNA was extracted using the QIAamp DNA stool minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total DNA content was normalized to 1 ng / μL. The V3-V4 region of 16S rRNA was amplified using the upstream primer 341F and the downstream primer 802R. The PCR reaction program was as follows: 10 ng of pure DNA, 15 μL of high-fidelity DNA polymerase and PCR master mix, 200 nmol / L of upstream and downstream primers, and nuclease-free water in a final volume of 30 μL. PCR cycling conditions included an initial denaturation at 98°C for 1 min, 30 cycles of 98°C for 10 s, 50°C for 30 s, and 72°C for 5 min. PCR products were quantified, mixed in equimolar ratios, and then purified for sequencing using the Illumina MeSeq platform with a sequencing depth of at least 20,000 reads per sample.

[0093] The QIIME analysis software used the FLASH process to merge sequences. The paired-end joined sequences were merged into operational taxonomic units (OTUs) at 97% sequence similarity using the GreenGenes dataset and the UPARSE algorithm. The representative sequences of each OTU were aligned, and then the RDP classifier was used to annotate the taxonomic information of each representative sequence. The analysis included each taxonomic level (such as phylum, class, order, family, genus, etc.). In-house Perl scripts were used to analyze alpha diversity within samples and beta diversity between samples. Principal coordinate analysis (PCoA) was used to evaluate the differences between experimental samples using the Unweighted unifrac method. All samples were used to calculate alpha diversity including richness and diversity. The taxonomic information of 16S rRNA gene sequence information was analyzed using UCLUST version 1.2.22 for the Silva119 16S rRNA dataset using 90% confidence intervals. PICRUSt analysis was used to predict microbial functions, and the abundance of annotated OTUs was used using the LEfSe online tool to clarify the highly representative bacterial communities in different treatment groups. The results are shown in Figure 4~Figure 10 shown.

[0094] Figure 4 The species richness bar graph and hotspot map at the phylum level show that the microbiota of mice in the model group decreased Verrucomicrobiota ) and Proteobacteria ( Proteobacteria ) and Bacteroidetes ( Bacteroidota ) abundance; increased Firmicutes ( Firmicutes ) and Iron-reducing bacteria ( Desulfovibrio ) abundance. Compared with the model group, the FL1-8 group significantly increased the abundance of Proteobacteria ( Proteobacteria ) and Bacteroidetes ( Bacteroidota ) and Iron-reducing bacteria ( Deferribacterota ) and reduced the abundance of Actinobacteria ( Actinobacteriota ) abundance, and the abundance of iron-reducing bacteria was increased in the BSC group.

[0095] Figure 5 The species richness bar chart and heat map at the genus level show that the FL1-8 group significantly increased the Lactobacillus genus ( Lacticaseibacillus ) and Bacteroides ( Bacteroides ); reduced New York Dubcea ( Dubosiella ) and Blautia ( Faecalibaculum ), POT and BSC groups had no significant regulatory effect.

[0096] Figure 6This is a comparison chart of the relative abundance of Lactobacillus. The FL1-8 group significantly enriched the abundance of Lactobacillus, which may be due to the successful colonization of FL1-8 in the mouse intestine. The POT group also promoted the proliferation of lactic acid bacteria to a certain extent and had a small prebiotic function. In contrast, the BSC group only slightly increased the colonization of Lactobacillus, indicating that the strain GDMCC1.325 itself has poor customization ability and its prebiotic ability is not as good as FL1-8.

[0097] Figure 7 The results of the Alpha diversity analysis of intestinal microorganisms in mice are shown in Figure 2. Chao1 and Shannon index are used for microbial abundance and diversity analysis. Chao1 is used to estimate the total number of species contained in the community sample; the higher the community diversity, the more even the species distribution, and the larger the Shannon index. Compared with the blank group, Chao1 and Shannon index of mice in the model group were significantly reduced, while the abundance in the intestine was adjusted after intervention with FL1-8 and its postbiotics, with the POT group adjusting slightly more, and the FL1-8 group and the BSC group adjusting to about the same degree.

[0098] Figure 8 The Rarefaction Curve is a common curve that describes the diversity of samples within a group. It randomly extracts a certain amount of sequencing data from the sample, and counts their alpha diversity index values. The curve is constructed based on the amount of sequencing data extracted and the corresponding index value (cutoff=49342). When the curve tends to be flat, it means that the amount of sequencing data is asymptotically reasonable, and more data will not have a significant impact on the alpha diversity index. Figure 8 It can be seen that the intervention of FL1-8 and its postbiotics increased the diversity of species within the group, which was roughly the same as that of the BSC group.

[0099] Fig. 9This is the result of unweighted unifrac analysis. In Beta diversity studies, the Unifrac distance measures the dissimilarity coefficient between two samples. The smaller the value, the smaller the difference in species diversity between the two samples. Beta Diversity is a comparative analysis of the microbial community composition of different samples. First, based on the species annotation results of all samples and the abundance information of the characteristic sequences, the characteristic sequence information of the same classification is merged to obtain the species abundance information table (Profiling Table). At the same time, the phylogenetic relationship between the characteristic sequences is used to further calculate the Unifrac distance. The Unifrac distance is a method of calculating the distance between samples using the evolutionary information between the microbial sequences in each sample. For more than two samples, a distance matrix is ​​obtained. Then, the Weighted unifrac distance is further constructed using the abundance information of the characteristic sequences for the Unifrac distance. It can be seen that there is a smaller difference between the intervention group and the blank group.

[0100] Fig.10 Table 3 shows the results of β-diversity analysis of inter-group differences in mouse intestinal microorganisms. Principal coordinate analysis (PCoA) was used to perform phylogenetic analysis (β diversity) of intestinal microorganisms between different samples, and the algorithm used was Unweightedunifrac.

[0101] Table 3 POCA analysis of mouse intestinal microorganisms

[0102]

[0103] It can be seen that the microbial community of the model group is clustered separately from the blank group, and the model group is also included in the FL1-8 intervention group to form a smaller cluster. The clustering of the FL1-8 group is different from the blank group and covers the clustering of the model group, POT group, and BSC group, suggesting that the strain FL1-8 has a certain effect on enriching the intestinal flora while regulating the intestinal flora and resisting obesity, serum inflammation, and cognitive impairment caused by a high-fat diet.

[0104] 3. Effects of mouse weight gain and feed utilization

[0105] During the modeling process, the body weight of mice was measured every week. Fig.11 .Depend on Fig.11It can be seen that the body weight of the model group was significantly higher than that of the blank group, indicating that the obesity model induced by a high-fat diet was successfully established, and the FL1-8 group could significantly resist the weight gain caused by a high-fat diet, indicating that the strain FL1-8 has a mitigating effect on the weight gain of mice on a high-fat diet. In terms of final body weight, the FL1-8 group adjusted back to the same level as the blank group, and both the FL1-8 group and the blank group were significantly different from the model group. In terms of the difference from the model group, FL1-8 was the most significant, followed by the POT group, and finally the BSC group. The BSC group had almost no mitigation of weight gain and was not significantly different from the model group.

[0106] During the modeling process, the feed consumption of the mice was weighed. After the experiment, the feed utilization rate of the mice was calculated according to the weight gain (g) / feed consumption (g) × 100%. The results are shown in Fig.12 It can be observed that the feed utilization rate of the intervention group was significantly decreased compared with the high-fat obesity group, indicating that the intervention of FL1-8 and its postbiotics reduced the weight gain of mice caused by excessive energy intake. The effects were as follows: FL1-8 group was better than POT group, and POT group was better than BSC group.

[0107] 4. Effect of Lee's Index on Mice

[0108] Lee's index: (weight g) 1 / 3 / body length cm×1000, the obesity degree of mice was evaluated by Lee's index, the results are shown in Fig.13 .Depend on Fig.13 It can be seen that the obesity level of the model group was higher than that of the blank group, while that of the FL1-8 group was significantly lower than that of the model group, indicating that the strain FL1-8 alleviated the obesity caused by a high-fat diet. The BSC group was only slightly lower, and the difference from the model group was the most significant in FL1-8, slightly significant in the POT group, and no significant difference in the BSC group.

[0109] 5. Effect of epididymal fat weight on mice

[0110] After the mouse was dissected, the fat tissue near the epididymis was taken and photographed. Fig.14 The fat tissue in the model group was larger, the fat tissue in the FL1-8 group was roughly the same as that in the blank group, and both were significantly smaller than the model group. The fat tissue in the POT group was smaller, and the fat tissue in the BSC group was larger. Fig.15 .Depend on Fig.15 It can be seen that the body weight of the model group was significantly higher than that of the blank group, while the body weight of the FL1-8 group and the POT group returned to normal levels, indicating that the strain FL1-8 and its postbiotics reduced the weight gain of adipose tissue caused by a high-fat diet, and the BSC group slightly improved the volume and weight of adipose tissue.

[0111] 6. Pathological analysis of mouse epididymal adipose tissue HE staining

[0112] After the mouse was dissected, the fat tissue near the epididymis was taken and placed in a paraformaldehyde fixative, soaked, and rinsed overnight. The samples were dehydrated in 70%, 80%, and 90% ethanol solutions for 30 minutes each, and then placed in 95% and 100% ethanol solutions twice, each for 20 minutes for dehydration. Placed in a mixture of 1 / 2 pure alcohol and 1 / 2 xylene for 15 minutes, and washed in xylene before and after washing for 15 minutes until transparent. Placed in a mixture of half xylene and half paraffin for 15 minutes, and then placed in paraffin I and paraffin II for 55 minutes each, and the transparent agent was removed. The processed samples were embedded, sliced, spread, baked, H&E stained, and sealed.

[0113] The results of HE staining of mouse epididymal fat are shown in Fig.16 .Depend on Fig.16 It can be seen that compared with the blank group, the area of ​​single fat cells in the model group was significantly larger than that in the blank group, while the FL1-8 group and the POT group returned to the same state as the blank group, with cells arranged tightly and orderly and a small area of ​​single fat cells, indicating that strain FL1-8 can alleviate obesity caused by a high-fat diet. The BSC group slightly reduced the size of fat cells, which was consistent with the appearance of adipose tissue.

[0114] 7. Effect of mouse liver tissue weight and cholesterol content

[0115] After the mice were dissected, the whole liver tissue was weighed and statistically calculated. The liver tissue was ground and the supernatant was taken for physical and chemical analysis of cholesterol content. The cholesterol in the supernatant was determined using a commercial kit (Nanjing Jiancheng Bioengineering Institute). Fig.17 .Depend on Fig.17 It can be seen that the liver weight of the model group was higher than that of the blank group and the intervention group, indicating that the intervention of strain FL1-8 and POT group reduced the liver weight. The cholesterol content in the model group was higher than that in the blank group, while the cholesterol content in the FL1-8 group and POT group was even lower than that in the blank group, indicating that the intervention of strain FL1-8 played a role in reducing cholesterol in liver tissue. The BSC group was effective but not as good as the FL1-8 group.

[0116] 8. Mouse liver oxidative stress assay

[0117] The determination of liver tissue lipid metabolism related indicators SOD, MDA and GSH was detected using commercial kits (Nanjing Jiancheng Bioengineering Institute). The results are shown in Fig.18 .Depend on Fig.18 It can be seen that the MDA level in the model group was significantly higher than that in the control group, and the GSH level was significantly lower than that in the control group, indicating that a high-fat diet affects the level of liver oxidative stress. The FL1-8 group can downregulate the MDA level, upregulate the GSH level and maintain a normal SOD level, indicating that the strain FL1-8 can alleviate the liver oxidative stress level. The POT group and the BSC group are effective, but not as good as the FL1-8 group.

[0118] 9. Levels of aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) in mouse serum

[0119] GOT and GPT in serum were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute), and the results are shown in Fig.19 .Depend on Fig.19 It can be seen that the GOT and GPT levels in the model group were significantly higher than those in the blank group, indicating that liver function was impaired; the GOT and GPT levels in both intervention groups were significantly reduced, indicating that strain FL1-8 and POT groups could alleviate liver function damage.

[0120] 10. Determination of serum inflammatory factors and lipid metabolism-related indicators in mice

[0121] Serum inflammatory factors TNF-α, IL-1β, IL-6, TC and TG, LDL and HDL were measured using commercial kits (Jiangsu Enzyme Biotechnology Co., Ltd.), and the results are shown in Fig. 20 .Depend on Fig. 20 It can be seen that the levels of TNF-α, IL-1β, and IL-6 in the model group were significantly higher than those in the blank group, indicating that the intake of a high-fat diet can cause inflammation. The FL1-8 group can downregulate the level of TNF-α and significantly reduce the levels of IL-1β and IL-6, indicating that the strain FL1-8 can inhibit inflammation. The BSC group is similar to the FL1-8 group, and the POT group is not as effective as the FL1-8 group. The TG and TC levels in the model group were significantly higher than those in the blank group, indicating that a high-fat diet affects liver lipid metabolism. The FL1-8 group and the POT group can downregulate the levels of TG and TC, indicating that the strain FL1-8 and its postbiotics can prevent the increase in TG and TC levels, and the BSC group has no obvious effect. Compared with the blank group mice, the model group mice showed hypercholesterolemia, and the levels of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were increased, resulting in an increase in the LDL / HDL ratio, while the mice given the strain FL1-8 and its postbiotics intervention significantly reduced the LDL level and LDL / HDL ratio, and the BSC group also showed a similar effect, but the effect was slightly worse.

[0122] 11. Determination of serum leptin and endotoxin concentrations in mice

[0123] Serum leptin and endotoxin concentrations were determined using commercial kits (Jiangsu ELISA Biotechnology Co., Ltd.), and the results are shown in Fig.21 .Depend on Fig.21It can be seen that the leptin level in the FL1-8 group was significantly lower than that in the model group, indicating that the intervention of FL1-8 reduced energy intake by suppressing appetite, thereby resisting the weight gain caused by a high-fat diet; there was no significant difference in serum endotoxin levels among the groups, and the serum endotoxin concentrations in the FL1-8 group and the POT group were lower than those in the blank group and the model group, indicating that the high-fat diet did not cause obvious intestinal damage. Compared with the blank group, the FL1-8 group and the POT group had the effect of strengthening the intestinal barrier and reducing the penetration of bacterial endotoxins, and the effect of the BSC group was not obvious.

[0124] 12. Effect of the length of the cecum and colon on mice

[0125] After the mice were dissected, the cecum part was taken and photographed. Typical photos of the cecum part of mice and the statistical results of the average length of the cecum are shown in the figure. Fig. 22 The length of the colon reflects possible colon inflammation. The high-fat diet caused the colon length of the model group and the BSC group to shorten, while the FL1-8 and its postbiotic intervention group corrected the shortening of the colon length caused by the high-fat diet and resisted ileal inflammation, which corresponded to the serum inflammation level. The effect of the BSC group was not obvious.

[0126] 13. Pathological analysis of mouse ileum HE staining

[0127] After the mouse was dissected, fresh ileum tissue was taken and placed in paraformaldehyde fixative, soaked, and rinsed overnight. The samples were dehydrated in 70%, 80%, and 90% ethanol solutions for 30 minutes each, and then placed in 95% and 100% ethanol solutions twice, each for 20 minutes for dehydration. Placed in a mixture of 1 / 2 pure alcohol and 1 / 2 xylene for 15 minutes, and washed in xylene before and after washing for 15 minutes until transparent. Placed in a mixture of half xylene and half paraffin for 15 minutes, and then placed in paraffin I and paraffin II for 50-60 minutes each to remove the transparent agent. The processed samples were embedded, sliced, spread, baked, H&E stained, and sealed.

[0128] The results of HE staining of mouse ileum are shown in Fig.23 The statistical results of mouse villus length and crypt depth are shown in Fig.24 .Depend on Fig.23 , Fig.24It can be seen that there is no significant difference in the villus length between the model group and the blank group, while the villus length of the FL1-8 group and the POT group became longer. Generally speaking, the villus length is related to the ability of the intestine to absorb nutrients, indicating that the villus growth in the model group promotes fat absorption and weight gain, while the intervention group has better absorption of nutrients and does not absorb excessive fat substances. The crypt depth reflects that the crypts in the model group are shallower, which is the reason for the increased absorption of fat substances. In the villus-crypt ratio, the model group was significantly higher than the blank group and the FL1-8 group, indicating that its enhanced absorption capacity may lead to more energy intake and weight gain, while the strain FL1-8 significantly reduced the villus-crypt ratio.

[0129] 14. Effects of short-chain fatty acids in cecal contents

[0130] After the mouse was dissected, the cecum was taken, 100 mg was weighed and dissolved in 0.9 mL ultrapure water, magnetic beads were added for grinding, and the homogenate supernatant was centrifuged and added with 100 μL 10% sulfuric acid for digestion. 0.5 mL ether was vortexed for extraction, centrifuged, and the supernatant was passed through a 0.22 μL organic filter membrane and collected in a gas phase vial for the detection of short-chain fatty acids. On-machine detection: Agilent 7890 gas chromatograph was used for gas phase analysis. Gas chromatography conditions: DB-FFAB capillary column (inner diameter 30 mm × 250 μm, membrane thickness 0.25 μm) was used for separation, and the injection volume was 1 μL. Column box heating program: initial temperature 80 ° C, maintained for 2 min, then increased to 180 ° C at a rate of 10 ° C / min, and maintained for 5 min. Aliengt's MSD software was used for data processing.

[0131] As metabolic markers of intestinal flora, short-chain fatty acids are absorbed and utilized by intestinal epithelial cells as energy substances, and can also enter the blood to affect systemic metabolism; short-chain fatty acids can enhance the proliferation and differentiation of intestinal epithelial cells and promote the maintenance of intestinal barrier function; short-chain fatty acids also play an immunomodulatory role by binding to G protein-coupled receptors such as GPR41 and GPR43, affecting intestinal inflammatory response. Fig.25 ,Depend on Fig.25 It can be seen that the intake of high-fat diet affected the intestinal short-chain fatty acid metabolism of the model group, FL1-8 group and POT group. The content of short-chain fatty acids in the cecum of the model group was significantly lower than that of the blank group. FL1-8 and postbiotics adjusted the content of short-chain fatty acids, and increased the levels of the five major short-chain fatty acids (acetic acid, propionic acid, isobutyric acid, n-butyric acid, and valeric acid), thereby improving the integrity of the intestinal barrier function of mice. The effect of the BSC group was similar.

[0132] 15. Effects of fecal short-chain fatty acids

[0133] Mouse feces were collected weekly and frozen in a -80°C refrigerator. Feces were collected at the beginning of the intervention and at the last week of the intervention. 200 mg was weighed and dissolved in 0.9 mL ultrapure water, and magnetic beads were added for grinding. The homogenate supernatant was centrifuged and added with 100 μL of 10% sulfuric acid for digestion. 0.5 mL of ether was vortexed and extracted. The supernatant was centrifuged and passed through a 0.22 μL organic filter membrane and collected in a gas phase vial for the detection of short-chain fatty acids. On-machine detection: Agilent 7890 gas chromatograph was used for gas phase analysis. Gas chromatography conditions: DB-FFAB capillary column (inner diameter 30 mm × 250 μm, membrane thickness 0.25 μm) was used for separation, and the injection volume was 1 μL. Column box heating program: initial temperature 80°C, maintained for 2 min, then increased to 180°C at a rate of 10°C / min and maintained for 5 min. Aliengt's MSD software was used for data processing.

[0134] Results Fig.26 ,Depend on Fig.26 It can be seen that the intake of high-fat diet has an impact on the metabolism of intestinal short-chain fatty acids in the model group and the intervention group, especially in the first two weeks of high-fat diet intake. The metabolism of short-chain fatty acids is low, and the model group is at a relatively low level throughout the experiment. With the intervention of strain FL1-8 and its postbiotics, the short-chain fatty acids in the cecum of the intervention group gradually recovered, indicating the establishment of intestinal flora homeostasis.

[0135] 16. The impact of mouse nesting behavior

[0136] The evaluation criteria for mouse nesting behavior usually involve the evaluation of the mouse's ability to build nests under specific conditions. Nesting behavior is not only related to the mouse's physiological health, but also to their psychological state. Use absorbent cotton as nesting material to score the quality of the resulting nest. The specific scoring system uses a 5-point system: 1 point: flat nest, the material is slightly torn but no nest structure is formed. 2 points: The nest covers the mouse, but the structure is relatively loose. 3 points: The nest structure is relatively complete, more materials are used, and the shape is relatively regular. 4 points: The nest structure is complete, the shape is regular, and the materials are fully used. 5 points: A perfect nest with a high degree of structural integrity and regular shape, and the materials are very fully used.

[0137] The results of mouse nesting scoring are as follows Fig. 27 It can be seen that the high-fat diet has a certain impact on the cognitive development ability, environmental adaptability and problem-solving ability of mice. Compared with the model group, the FL1-8 and postbiotic intervention groups have improved the scores of nesting behavior, improved the cognitive ability of mice, and had a healthier mental state. The situation of the BSC group is worse.

[0138] 17. Effects of the open field test on mice

[0139] The mouse open field test (OFT) is a behavioral test method used to evaluate the autonomous behavior, exploratory behavior, and tension of mice in a novel environment. The preference of mice for the center and edge areas of the open field can reflect their adaptability to the new environment and their anxiety level. Activities in the center area can be regarded as an indicator of anxiety. The design of the open field reaction box allows mice to move freely in it. The inner wall is painted black and divided into multiple small squares to facilitate the observation and recording of the behavior of mice. The mouse is placed in the center of the open field, and video and timing are performed at the same time. The video is stopped after a certain period of observation. The observation time is 3 minutes. The behavior of the mouse, including its position preference, activity pattern, and behavioral characteristics in the open field, is analyzed by computer software.

[0140] The results are as follows Fig.28 It can be seen that the total distance of movement in each group is roughly the same, and compared with the blank group, the model group has a reduced movement time and distance in the central area, while the FL1-8 intervention group significantly increased the time and distance of mice in the central area, indicating that they have a lower level of anxiety about the new environment or they are better adapted to the new environment. There was no significant effect in the POT group and the BSC group.

[0141] 18. Effects of IGF-1 and BDNF in mouse serum

[0142] The levels of IGF-1 and BDNF in mouse serum were detected using a commercial kit (Jiangsu Enzyme Biotechnology Co., Ltd.). Fig.29 As shown. It can be seen that compared with the blank group, the intervention of FL1-8 and its postbiotics adjusted the level of IGF-1 in serum, while the effect of the BSC group was not good; the BDNF in the model group was lower than that in the blank group, indicating that the high-fat diet had a certain impact on cognitive ability, while the intervention of FL1-8 and its postbiotics adjusted the level of BDNF in serum, and the BSC group also had similar performance, indicating that FL1-8 and its postbiotics can improve cognitive function by increasing the levels of IGF-1 and BDNF, which echoes the previous behavioral experiments.

[0143] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) Use of NKU FL1-8 or Lactobacillus rhamnosus NKU FL1-8 bacterial agent in the preparation of a product for preventing and / or treating cognitive impairment caused by a high-fat diet, characterized in that: The deposit number of the Lactobacillus rhamnosus NKU FL1-8 is GDMCC No: 64053; the cognitive disorder is cognitive ability disorder.

2. The use according to claim 1, characterized in that: The Lactobacillus rhamnosus NKU FL1-8 bacterial agent includes a Lactobacillus rhamnosus NKU FL1-8 bacterial suspension and / or a Lactobacillus rhamnosus NKU FL1-8 postbiotic.

3. The use according to claim 2, characterized in that: The preparation method of the rhamnosus casei bacteria suspension of NKU FL1-8 comprises the following steps: culturing the rhamnosus casei bacteria NKU FL1-8 in a liquid culture medium, centrifuging the culture medium, and resuspending the precipitate to obtain a rhamnosus casei bacteria suspension of NKU FL1-8; and the preparation method of the rhamnosus casei bacteria NKU FL1-8 postbiotic comprises the following steps: heating and inactivating the rhamnosus casei bacteria suspension of NKU FL1-8 in a water bath to obtain the rhamnosus casei bacteria NKU FL1-8 postbiotic.

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