Application of fermentation lactobacillus mucus LFPerfectus001 in preparation of product for improving metabolic syndrome caused by high-fat and high-sugar diet

Abstract

The invention relates to application of fermentation lactobacillus mucus LFPerfectuus001 in preparation of a product for improving metabolic syndrome caused by high-fat and high-sugar diet, and belongs to the technical field of microorganisms. The invention provides application of fermented lactobacillus mucus LFPerfecus001 in preparation of a product for improving metabolic syndrome, the fermented lactobacillus mucus LFPerfecus001 is preserved in Guangdong Microbial Culture Collection Center on April 13, 2022, and the preservation number is GDMCC No: 62335. The invention further provides a preparation method of the fermented lactobacillus mucus LFPerfecus001. Animal experiments report that the fermented lactobacillus mucus LFPerfectus001 has the effects of reducing fat, increasing muscle, improving obesity, hyperglycemia, insulin resistance, dyslipidemia, hyperuricemia and fatty liver for the first time, and can well improve metabolic syndrome caused by high fat and high glucose.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to application of fermented mucus lactobacillus LFPerfectus001 in preparing a product for improving metabolic syndrome caused by a high-fat and high-sugar diet. Background Art

[0002] Excessive intake of nutrients such as carbohydrates or fats can cause disorders of sugar metabolism or lipid metabolism. If sugar metabolism and lipid metabolism disorders occur simultaneously in an individual, it can be called metabolic syndrome (MS). Metabolic syndrome is a complex metabolic disorder syndrome characterized by obesity, insulin resistance, hyperlipidemia, hypertension and hyperglycemia, and is often accompanied by symptoms such as high uric acid, arteriosclerosis, fatty liver, and muscle deficiency. Studies have shown that diet is one of the main environmental factors that induce obesity and related metabolic diseases. In recent years, the economic living standards have gradually improved, the pace of life has accelerated, and fast food diets have become increasingly popular. People are increasingly consuming high-fat, high-sugar and other high-calorie foods. In addition, fructose is widely used in various industrialized foods due to its high cost-effectiveness and pleasant taste, which has led to a significant increase in people's intake of fructose. High fat, high sugar, especially high fructose, are closely related to metabolic abnormalities such as obesity, diabetes, hyperlipidemia, and hyperuric acid. According to epidemiological studies, the current global average incidence of MS is about 30%, and it is expected to increase to 53% by 2035. Each symptom of metabolic syndrome is a risk factor for chronic diseases, greatly increasing the risk of chronic diseases such as cardiovascular and cerebrovascular diseases, type 2 diabetes, and tumors, and can bring significant economic and psychological burdens to patients.

[0003] It is very necessary to find effective and safe means of preventing and treating MS. The occurrence and progression of MS is a chronic and gradual process, which determines that its treatment is also a long-term intervention process. So far, there is no specific drug approved for the treatment of MS in clinical practice. In terms of treatment plans, relevant domestic and foreign guidelines have indicated that lifestyle intervention is the first choice, through the regulation of work and rest, diet and exercise to improve, and then each symptom is treated with targeted drugs. Existing treatment plans have many defects and limitations. It is difficult to maintain long-term regulation of work and rest, diet and exercise, and long-term use of drugs to lower blood sugar, blood lipids and weight can lead to some obvious side effects. In view of these adverse reactions, people are increasingly inclined to look for well-tolerated and safe substances. One of the most promising candidates is probiotics.

[0004] Probiotics are beneficial microorganisms. A few studies have reported that probiotics can be used to improve sugar and lipid metabolism, among which fermented mucus lactobacillus is one of them. Existing studies mainly focus on improving metabolic disorders induced by high-fat diets, and mainly focus on reducing weight gain, visceral fat accumulation, and improving blood lipids, etc. 2-3 symptoms, and the degree of relief is limited or requires a combination of multiple strains. MS is a group of complex metabolic disorder syndromes. The existing fermented mucus lactobacillus has a single effect, fails to improve multiple symptoms of metabolic syndrome at the same time, and has poor effect. In addition, different diets lead to differences in the pathogenesis and physiological state of MS. It is suitable for metabolic abnormalities caused by high-fat diets, but may not be suitable for metabolic abnormalities caused by high fat and high sugar. At the same time, the effect of probiotics has obvious strain specificity (that is, the effect of a certain strain does not mean that other probiotics in this genus or species have this effect), and the same strain of probiotics may not be applicable to all people. Therefore, it is still necessary to develop more other probiotic strains that can improve metabolic syndrome to fill the gaps in existing strains and meet the needs of different populations. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a fermented Lactobacillus mucosa LFPerfectus001 which can inhibit weight gain caused by a high-fat and high-sugar diet, reduce body fat rate, increase lean body mass rate, inhibit the enlargement of epididymal white adipocytes and inguinal adipocytes, improve insulin sensitivity and fatty liver, and reduce serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, total free saturated fatty acids (especially palmitic acid and stearic acid) and blood uric acid content. The fermented Lactobacillus mucosa LFPerfectus001 is used in the preparation of products for improving metabolic syndrome caused by a high-fat and high-sugar diet.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In the first aspect, the present invention provides the use of fermented Lactobacillus mucus LFPerfectus001 in the preparation of a product for improving metabolic syndrome caused by a high-fat and high-fructose diet. The fermented Lactobacillus mucus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with a preservation number of GDMCC No: 62335.

[0008] The present invention reports for the first time through animal experiments that fermented mucus lactobacillus LFPerfectus001 has the effects of reducing fat and increasing muscle, improving obesity, enhancing glucose utilization ability, improving insulin resistance, dyslipidemia, hyperuric acid and fatty liver, and can well improve metabolic syndrome caused by high fat and high fructose, and can make the contents of white fat cells, oral glucose tolerance (OGTT), insulin tolerance (ITT) and serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, total free saturated fatty acids, stearic acid and palmitic acid at or below normal levels.

[0009] As a preferred embodiment of the application of the present invention, the symptoms of metabolic syndrome include at least one of obesity, hyperglycemia, insulin resistance, dyslipidemia, hyperuricemia and fatty liver.

[0010] As a preferred embodiment of the application of the present invention, the product for improving metabolic syndrome can reduce body fat rate, increase lean body mass rate, inhibit white fat cell expansion, improve insulin resistance and fatty liver, and reduce serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, total free saturated free fatty acids and blood uric acid levels.

[0011] In the second aspect, the present invention provides the use of fermented Lactobacillus mucus LFPerfectus001 in the preparation of products for improving obesity, hyperglycemia, insulin resistance, dyslipidemia, hyperuric acid and fatty liver. The fermented Lactobacillus mucus LFPerfectus001 was preserved in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with the preservation number GDMCCNo:62335.

[0012] In the third aspect, the present invention provides the use of fermented mucus Lactobacillus LFPerfectus001 in the preparation of products for improving insulin resistance. The fermented mucus Lactobacillus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with a preservation number of GDMCC No: 62335.

[0013] The present invention found through animal experiments that fermented Lactobacillus mucus LFPerfectus001 can effectively reduce the oral glucose tolerance and insulin tolerance of rats fed with high fat and high fructose, and improve insulin sensitivity, indicating that fermented Lactobacillus mucus LFPerfectus001 has the effect of improving insulin resistance.

[0014] In a fourth aspect, the present invention provides the use of fermented mucus Lactobacillus LFPerfectus001 in the preparation of uric acid-lowering products. The fermented mucus Lactobacillus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with a preservation number of GDMCC No: 62335.

[0015] The present invention found through animal experiments that fermented Lactobacillus mucus LFPerfectus001 can effectively reduce the blood uric acid content of rats fed with high-fat and high-fructose, indicating that fermented Lactobacillus mucus LFPerfectus001 has the effect of lowering uric acid.

[0016] In the fifth aspect, the present invention provides the use of fermented mucus Lactobacillus LFPerfectus001 in the preparation of fat-reducing and muscle-gaining products. The fermented mucus Lactobacillus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with a preservation number of GDMCC No: 62335.

[0017] The present invention discovered through experiments that fermented Lactobacillus mucus LFPerfectus001 can effectively reduce the body fat rate of rats fed a high-fat and high-fructose diet and increase the lean body mass rate, and can also reduce the fat content of obese Caenorhabditis elegans. The improvement effect of fermented Lactobacillus mucus LFPerfectus001 is better than that of the commercialized fermented Lactobacillus mucus CECT5716, indicating that fermented Lactobacillus mucus LFPerfectus001 has the effect of reducing fat and increasing muscle.

[0018] As a preferred embodiment of the application of the present invention, the product includes at least one of biological products, food, medicine, health products and daily chemical products.

[0019] As a preferred embodiment of the application of the present invention, the dosage of the fermented mucus lactobacillus LFPerfectus001 is (1.6-40)×10 9 CFU / d.

[0020] In the sixth aspect, the present invention provides a biological product for improving metabolic syndrome, wherein the biological product includes fermented mucus Lactobacillus LFPerfectus001, and the fermented mucus Lactobacillus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with a collection number of GDMCCNo:62335.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention reports for the first time through nematode experiments and rat experiments that fermented mucus lactobacillus LFPerfectus001 has the effects of reducing fat and increasing muscle, improving glucose utilization ability, improving obesity, insulin resistance, dyslipidemia, hyperuric acid and fatty liver, and can well improve metabolic syndrome caused by high fat and high fructose, and can make the contents of white fat cells, oral glucose tolerance (OGTT), insulin tolerance (ITT) and serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, total free saturated fatty acids, stearic acid, and palmitic acid at or below normal levels, and the effect is better than that of existing reported strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effect of fermented Lactobacillus mucilaginosus LFPerfectus001 on fat in obese Caenorhabditis elegans in Example 2;

[0024] Figure 2 The initial body weight and body weight before intervention of the metabolic syndrome experimental rats in Example 3;

[0025] Figure 3 The effect of the fermented mucus lactobacillus LFPerfectus001 intervention on the body fat rate and lean body mass rate of rats in Example 3, A in the figure is the statistical result of body fat rate, and B is the statistical result of lean body mass rate;

[0026] Figure 4 The effect of fermented Lactobacillus mucilaginosus LFPerfectus001 on the morphology of epididymal and inguinal white adipose tissue in rats in Example 3;

[0027] Figure 5 The effect of the fermented mucus Lactobacillus LFPerfectus001 intervention on the number of fat cells per slice field of rat epididymal and inguinal white fat in Example 3, in which A is the statistical result of the number of fat cells in epididymal white adipose tissue, and B is the statistical result of the number of fat cells in inguinal white adipose tissue;

[0028] Figure 6 The effect of the fermented mucus lactobacillus LFPerfectus001 intervention on oral glucose tolerance in rats in Example 3, in which A is the glucose content at different times of the rat OGTT test, and B is the statistical result of the time-blood glucose area under the curve (AUC) corresponding to the OGTT;

[0029] Figure 7 The effect of fermented mucus lactobacillus LFPerfectus001 intervention on insulin tolerance in rats in Example 3, in which A is the glucose content at different times of the rat ITT test, and B is the statistical result of the area under the time-blood glucose curve (AUC) corresponding to the ITT;

[0030] Figure 8The effect of the fermented mucus Lactobacillus LFPerfectus001 intervention on blood uric acid in rats in Example 3;

[0031] Fig. 9 The effect of the fermented mucus Lactobacillus LFPerfectus001 intervention on blood lipids in rats in Example 3, in which A is the statistical result of triglycerides, B is the statistical result of total cholesterol, C is the statistical result of high-density lipoprotein cholesterol, and D is the statistical result of low-density lipoprotein cholesterol;

[0032] Fig.10 The effect of the fermentation of Lactobacillus mucilaginosus LFPerfectus001 on the free saturated fatty acids in rat serum in Example 3, in which A is the statistical result of total free saturated fatty acids, B is the statistical result of free stearic acid, and C is the statistical result of free palmitic acid;

[0033] Fig.11 The effect of the fermented Lactobacillus mucilaginosus LFPerfectus001 intervention on the liver tissue morphology of rats in Example 3;

[0034] In the above figures, "ns" indicates no significant difference between the two groups, and "*" indicates significant difference between the two groups, where "*" means P < 0.05, "**" means P < 0.01, "***" means P < 0.001, and "****" means P < 0.0001. DETAILED DESCRIPTION

[0035] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0036] In the following embodiments, comparative examples and experimental examples, the experimental methods used are conventional methods unless otherwise specified.

[0037] In the following examples, “fermented Lactobacillus mucilaginosus”, “fermented Lactobacillus mucilaginosus LFPerfectus001”, “LFP001” and “LFP” all refer to fermented Lactobacillus mucilaginosus LFPerfectus001 with a deposit number of GDMCC No.62335.

[0038] Unless otherwise specified, other materials, reagents, etc. used in the embodiments and effect examples can be obtained from commercial sources.

[0039] The formula of SM culture medium is as follows: weigh 5.85 g of sodium chloride, 6 g of potassium dihydrogen phosphate, 1.32 g of potassium dihydrogen phosphate trihydrate, add 960 mL of ultrapure water, shake well, and sterilize with high-pressure steam at 121°C for 30 min. After sterilization, cool to room temperature, add 1 mL of 5 mg / mL cholesterol ethanol solution, 3 mL of 1 mol / L calcium chloride solution, 3 mL of 1 mol / L magnesium sulfate solution, 10 mL of 1 mol / L potassium citrate solution, and 10 mL of trace element solution, shake well, and store in a sealed and cool place for later use.

[0040] The formula of MRS medium is as follows: add 10.0 g of peptone, 5.0 g of beef extract powder, 4.0 g of yeast extract powder, 20.0 g of glucose, 1.0 mL of Tween-80, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 15.0 g of agar, 2.0 g of triammonium citrate, 0.2 g of magnesium sulfate heptahydrate, and 0.05 g of manganese sulfate tetrahydrate to each 1 L of medium, and adjust the pH to 6.2 ± 0.2. If a liquid medium is prepared, agar is not added.

[0041] The normal maintenance feed for rats was purchased from Beijing Keao Xieli Feed Co., Ltd., which contained 23.07% protein, 11.85% fat, and 65.08% carbohydrates.

[0042] The high-fat diet was purchased from Research Diets, Inc., USA, with the product number D12492, which contained 26.02% protein, 34.9% fat, and 26.3% carbohydrates.

[0043] Fructose syrup was purchased from Guangzhou Shuangqiao Co., Ltd., model F55.

[0044] Example 1

[0045] This embodiment provides a biological product for improving metabolic syndrome, and the preparation method of the biological product comprises the following steps:

[0046] 1. Seed activation: Use MRS medium to prepare primary culture medium and secondary culture medium, adjust the pH (6.50±0.02), sterilize at 121℃ for 15min and set aside.

[0047] 1) Primary seed amplification: Inoculate the fermented mucus lactobacillus LFPerfectus001 ampoule tube bacterial powder into MRS medium in the clean bench. Aerobically culture for (24±0.5)h at (37±1℃), observe whether the bacterial morphology is normal, measure its pH to (4.44±0.01), OD600 to (3.81±0.001), and amplify successfully.

[0048] 2) Secondary seed amplification: Take the primary seed solution and continue to inoculate it into MRS medium with an inoculum size of 1-5% (V / V). Culture it aerobically at (37±1)°C for (24±0.5) h. Observe whether the bacterial morphology is normal. When the pH reaches (4.38±0.01) and the OD600 reaches (3.89±0.001), the amplification is successful.

[0049] 2. Fermentation:

[0050] 1) The secondary seed solution was inoculated into the MRS medium. After inoculation, the initial pH was adjusted to (6.50±0.02) with ammonia water, and aerobic fermentation was performed at (37±0.2)°C for 10-12 hours. When the pH value reached (4.50±0.05) and the OD600 reached (8.09±0.01), the tertiary seed solution was obtained. The required tertiary seed solution was transferred into a 5t fermentation tank containing 2t of fermentation solution according to the inoculation amount of 1-5% (V / V) to complete the inoculation.

[0051] 2) After the inoculation, add ammonia water to adjust the initial pH of the fermentation liquid to (6.50±0.02), and stir and ferment. Culture conditions: culture temperature (37±0.2)℃, use ammonia water to control the pH value to be constant (5.50±0.02). During the fermentation process, when the OD600 value of the fermentation liquid reaches (13.00±1.00), and the OD values ​​of two consecutive sampling points (1h interval) are not significantly different (P<0.05), it is judged as the end point of fermentation, that is, the fermentation is over. After the fermentation is over, the culture in the fermentation tank is cooled by cooling water and centrifugation is started.

[0052] 3. Centrifugal drying and crushing:

[0053] The centrifuge was started, and after the centrifuge ran stably for 3 to 5 minutes, the material entered the centrifuge for centrifugal separation, the centrifuged concentrated bacterial sludge was freeze-dried, and the dried bacterial powder was crushed to 40 meshes to obtain the fermented mucus Lactobacillus LFPerfectus001 bacterial powder, which is a biological product for improving metabolic syndrome, and the number of live bacteria in the bacterial powder is 2.5×10 11 CFU / g, moisture content is 6.4%, and water activity is 0.15.

[0054] Example 2

[0055] Oil Red O(C 26 H 24 N 4O) has a strong affinity for neutral lipid components, especially can be combined with triglycerides in tissues and cells to form lipid droplet-shaped red circles. When Oil Red O dye is added to cells or tissues, Oil Red O can combine with triglycerides in cells or tissues to form lipid droplets, which appear red when observed under a microscope. On this basis, in order to verify the fat-reducing effect of fermented mucus lactobacillus LFPerfectus001, this embodiment uses trehalose to establish an obese nematode model, and at the same time compares it with the commercialized star strain fermented mucus lactobacillus CECT5716, and evaluates the fat-reducing ability of the sample by detecting the effect of the sample on the optical density value of fat staining. The specific scheme is as follows:

[0056] 1. Experimental content:

[0057] (1) Collect N2-type Caenorhabditis elegans at the L1 stage and culture them in a 24-well plate (1000 μL / well) until the D1 stage (69 μL of concentrated Escherichia coli NA22 (hereinafter referred to as NA22, the concentration of NA22 is 1×10 10 CFU / mL), incubated at 20°C in a shaker, with approximately 100 cells per well.

[0058] (2) The bacterial powder obtained in Example 1 was mixed with SM medium and centrifuged at 4°C and 10,000 rpm for 5 min. The supernatant was discarded. This was repeated three times and the volume was fixed with SM medium to a bacterial cell concentration of 2 × 10 10 CFU / mL, for future use.

[0059] (3) D1 stage nematodes were collected, trehalose with a final concentration of 10 mg / mL was added, samples were added according to the design in Table 1, and the well plate was placed on a shaker and continued to be cultured until the D4 stage.

[0060] (4) Collect the nematodes into an EP tube, add 40% isopropanol, precipitate and discard the supernatant, add 600 μL of Oil Red O dye, shake at room temperature for 30 min, and wash the nematodes 5 times with 0.5% PBST (absorb 250 μL of Triton, add 50 mL of PBS, and shake well).

[0061] (5) Slides were made and images were taken using a polarizing microscope. ImageJ was used to analyze the images and record the values. The fat staining optical density value reflected the body fat content of Caenorhabditis elegans. The fat staining optical density value = staining area pixel size / nematode pixel size.

[0062] (6) Experimental data are expressed as mean ± standard deviation (Mean ± SD). GraphPad Prism 10.1.1 software was used for statistical analysis and drawing. The differences between the two groups were compared using ordinary one-way analysis of variance. P < 0.05 was considered to be significantly different between the groups. The smaller the P value, the greater the significant difference between the groups. * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001, and ns represents no significant difference. Results are shown in Figure 1 .

[0063] Table 1 Experimental design

[0064]

[0065] 2. Experimental results

[0066] like Figure 1 As shown in the figure, compared with the blank group, the fat staining optical density value of the nematodes in the model group increased significantly (P < 0.0001), indicating that the fat content of the nematodes increased and the model was successfully established. After the intervention of fermented mucus lactobacillus LFPerfectus001, the fat staining optical density value decreased significantly (P < 0.0001), and there was no significant difference with the blank group (P > 0.05), while the fat staining optical density value of the control strain fermented mucus lactobacillus CECT5716 did not show a significant decrease (P > 0.05), indicating that fermented mucus lactobacillus LFPerfectus001 has a fat-reducing effect and can reduce fat to the normal level of the blank group, which is better than the control strain fermented mucus lactobacillus CECT5716.

[0067] Example 3

[0068] This example is to verify the efficacy of the biological product obtained in Example 1 in improving metabolic syndrome. The specific scheme is as follows:

[0069] 1. Experimental method.

[0070] (1) Construction of a rat model of metabolic syndrome induced by high fat and high fructose:

[0071] The animal model of metabolic syndrome (MS) induced by a high-fat, high-fructose diet is more consistent with the pathogenesis of human MS and is often used in MS research. The experimental rats were 58 5-6 week old Sprague-Dawley (SD) healthy SPF male rats bred by the Guangdong Medical Laboratory Animal Center [License No.: SCXK (Yue) 2022-0002]. The experimental animal quality certificate number is No. 44007200107831.

[0072] After 7 days of adaptive feeding, all rats were weighed (week 0) and randomly divided into 2 groups, namely, a blank group (n=10) and a high-fat and high-fructose group (n=48). The blank group was given normal maintenance feed and sterilized water, and the high-fat and high-fructose group was given a high-fat feed and 12.5wt% fructose syrup water. Each group had free access to food and water. After 2 weeks of feeding (week 3), rats with small weight gain and resistance to high-fat and high-fructose diet (20%) were eliminated based on body weight gain. Subsequently, the high-fat and high-fructose group rats were evenly and randomly divided into 4 groups, namely, a model group (n=10), a low-dose group (LFP001-low) (n=10), a medium-dose group (LFP001-medium) (n=10), and a high-dose group (LFP001-high) (n=10).

[0073] (2) Intervention experiment of fermented mucus lactobacillus LFPerfectus001:

[0074] During the intervention, all rats were gavaged once a day. The blank group and the model group were gavaged with 2.5 mL of normal saline each time, and the low-, medium-, and high-dose groups were gavaged with 2.5 mL of bacterial solution each time. The doses of viable bacteria counts were 0.168, 0.833, and 4.17 × 10, respectively. 9 CFU / (kg˙BW) (the converted human dose was 16.3, 81.6, 40.8 billion CFU / person / day, respectively). The blank group was given maintenance feed and normal drinking water, and the other groups were given high-fat feed and 12.5wt% fructose syrup water. All groups were free to eat and drink for 8 weeks.

[0075] The references for rat to human dose conversion are as follows:

[0076] Huang Jihan, Huang Xiaohui, Chen Zhiyang, et al. Conversion of equivalent doses between animals and between animals and humans in pharmacological experiments[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2004, (09): 1069-1072.

[0077] At the end of the experiment (week 10), after weighing and recording data, 6% chloral hydrate was injected intraperitoneally at a dose of 0.5 mL / 100 g for anesthesia, and blood was collected from the abdominal aorta. The time of blood collection was recorded, and the serum was centrifuged and collected after two hours at room temperature, and then packaged and frozen; the liver, epididymal white fat (eWAT) and inguinal white fat (iWAT) were dissected and removed, and quickly rinsed with pre-cooled saline, and the surface moisture was quickly absorbed with absorbent paper, and then weighed and packaged. A part of the tissue was fixed with paraformaldehyde for histopathological analysis, and the rest was quickly frozen in liquid nitrogen and stored at -80°C.

[0078] (3) Detection of indicators:

[0079] 1) Animal status: During the entire experiment, the general survival status of the rats was observed and recorded, mainly including the mental state, movement, defecation and hair of each group of rats, and imaging materials of a single rat were collected before the end of the modeling for direct observation and comparison of the status.

[0080] 2) Body weight and weight gain inhibition rate determination: During the entire experimental period, body weight (g) was measured weekly. Weight gain inhibition rate = (weight gain of model group during intervention period - weight gain of sample group during intervention period) / (weight gain of model group during intervention period - weight gain of blank group during intervention period) * 100%, where weight gain during intervention period = weight at week 10 - weight at week 2. The results are shown in Figure 2 and Table 2.

[0081] 3) Determination of body fat rate and lean body mass rate: One day before killing and sampling, the body composition of rats was determined using Minispec LF90Ⅱ live animal body composition analyzer, including fat weight (g) and muscle weight (g). Body fat rate = fat weight / body weight × 100%, lean body mass rate = muscle weight / body weight × 100%. The results are shown in Figure 3 .

[0082] 4) Oral glucose tolerance test (OGTT): One week before killing and sampling, rats were transferred to clean bedding cages in advance and fasted for 12 hours, but water was not allowed. The rats were weighed and blood was collected by tail-cutting. The first two drops of blood were discarded, and the blood glucose level of the third drop of blood was measured with a blood glucose meter and recorded as the blood glucose level at 0 min. A 50wt% glucose aqueous solution was intragastrically administered at a dose of 2g / (kg˙BW), and the timing was started. The blood glucose level was measured in the same way at 15min, 30min, 60min, 90min, and 120min. A blood glucose curve of 0-120min after intragastric administration of glucose aqueous solution was drawn, and the area under the time-blood glucose curve (AUC) was calculated. The results are shown in the table. Figure 6 .

[0083] 5) Insulin tolerance test (ITT): Three days after the OGTT experiment, the ITT experiment was conducted. The rats were transferred to a clean bedding cage in advance and fasted for 12 hours, but water was not allowed. The rats were weighed and blood was collected by tail-cutting. The first two drops of blood were discarded, and the blood glucose value of the third drop of blood was measured with a blood glucose meter and recorded as the blood glucose value at 0 min. 1U / ml insulin was injected intraperitoneally at a dose of 0.75U / kg, and the timing was started. The blood glucose value was measured in the same way at 15min, 30min, 60min, 90min and 120min. A blood glucose curve of 0-120min for the glucose aqueous solution was drawn, and the area under the time-blood glucose curve (AUC) was calculated. The results are shown in the table. Figure 7 .

[0084] 6) Blood biochemical index determination: Serum uric acid (UA), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were determined using the URIT-8021A fully automatic biochemical analyzer according to the instructions of the corresponding kits. The results are shown in Figure 8-9 .

[0085] 7) Blood free fatty acid determination: Extract and separate serum free fatty acids and determine them by GC-MS. The results are shown in Fig.10 .

[0086] 8) Histopathological morphological observation: Epididymal white fat, inguinal white fat tissue and liver tissue fixed with 4% paraformaldehyde were embedded in paraffin, cut into 5 μm thick sections and stained with H&E; frozen sections of liver tissue were stained with Oil Red O (ORO) to show lipid droplets. The pathological changes of fat and liver tissue were evaluated by optical microscopy using PerkinElmer Vectra 3 (PerkinElmer, USA). The results of H&E staining of epididymal white fat and inguinal white fat tissue sections are shown in Figure 4 The results of fat cell counts in each field of view under an optical microscope are shown in Figure 5 The results of H&E staining and Oil Red O staining of liver tissue sections are shown in Fig.11 .

[0087] 9) Data analysis is the same as in Example 2.

[0088] 2. Experimental results.

[0089] (1) During the experiment, the rats in the blank group maintained a good mental and behavioral state, with smooth fur and a healthy milky white color, and slightly moist ellipsoidal feces. However, after the rats in the model group consumed a high-fat, high-fructose corn syrup diet, their movements gradually became slower. Compared with the blank group, the rats were more likely to enter a quiet or sleepy state in a relatively noisy environment such as changing bedding or feed. The fur began to turn yellow and slightly dirty, and the feces were sticky and easy to deform or break. Compared with the model group, the behavior and mental state of the rats in the low, medium, and high dose groups were greatly improved, and they maintained a relatively good state under the same feeding environment. The fur was slightly smooth and milky white, and the feces were slightly moist and formed.

[0090] (2) Initial body weight of rats in each group Figure 2 A, Body weight of model group, LFP001-low, LFP001-medium and LFP001-high groups Figure 2 B. As Figure 2 There was no statistical difference in the initial body weight of rats in each group (P>0.05), and the body weight was relatively balanced. Figure 2B shows that after 2 weeks of high-fat and high-fructose diet feeding and elimination of some obesity-resistant rats, there was no significant difference in body weight among the model group, LFP001-low, LFP001-medium, and LFP001-high groups, indicating that the obesity status of each mouse was consistent. Figure 2 A. Figure 2 B can eliminate the impact of inconsistent weight on the intervention effect.

[0091] The total weight gain of each rat during the intervention period (week 3-week 10) is shown in Table 2. As shown in Table 2, compared with the blank group, the weight of the rats in the model group increased significantly (P < 0.001); compared with the model group, fermented Lactobacillus mucosa LFPerfectus001 could inhibit the weight gain of rats, and the weight gain inhibition rates of the low, medium and high dose groups were 14.29%, 39.64% and 57.40%, respectively, among which the high dose group had the most obvious reduction effect (P < 0.05), indicating that fermented Lactobacillus mucosa LFPerfectus001 can significantly improve the obesity of rats fed a high-fat and high-fructose diet.

[0092] Table 2 Effect of fermented mucus lactobacillus LFPerfectus001 intervention on body weight of rats

[0093] Group Total weight gain during the intervention period, g / head Weight gain inhibition rate, % Blank group 225.49*** —— Model Group 295.35 —— LFP001-Low 285.23 14.49 LFP001-Medium 267.66 39.64 LFP001-High 255.25* 57.40

[0094] (3) Figure 3 As shown in the results, compared with the blank group, the body fat rate of the rats in the model group was significantly increased (P < 0.0001), and the lean body mass rate was significantly decreased (P < 0.0001); compared with the model group, the body fat rate of the rats in the intervention group was reduced, among which the medium and high dose groups had significant differences (P < 0.05); similarly, compared with the model group, the lean body mass rate of the rats in the intervention group was increased, among which the medium and high dose groups had significant differences (P < 0.05), indicating that fermented Lactobacillus mucosa LFPerfectus001 can significantly reduce the body fat rate of rats fed a high-fat and high-fructose diet, increase the lean body mass rate, and has the effect of reducing fat and increasing muscle.

[0095] (4) Epididymal white fat (eWAT) and inguinal white fat (iWAT) are important visceral and subcutaneous fat in the body, respectively. Both expand by increasing the volume of adipocytes. Typical adipose tissue pathological sections of SD rats are shown in Figure 2. Figure 4 As shown, the number of adipocytes per slice field is Figure 5 As shown. Through pathological sections ( Figure 4 ) It can be seen that compared with the blank group, the fat cells in the white adipose tissue (including epididymal and inguinal white fat) of the model group rats began to swell, the cell volume increased significantly, and the number of fat cells per section decreased significantly ( Figure 5)(P<0.0001); Compared with the model group, the volume of adipocytes in the intervention group became smaller, and the number of adipocytes per section increased. Among them, the improvement effect of the medium and high doses was more significant (P<0.05, P<0.0001), and there was no significant difference in the number of cells in the high dose group compared with the blank group (P>0.05), indicating that fermented Lactobacillus mucinus LFPerfectus001 can significantly inhibit the expansion of adipocytes in rats fed a high-fat and high-fructose diet, and can keep it at the normal level of the blank group.

[0096] (5) Effects of Lactobacillus mucilaginosus LFPerfectus001 intervention on oral glucose tolerance (OGTT) and insulin tolerance (ITT) in rats Figure 6-7 As shown. Figure 6 As shown in B, compared with the blank group, the oral glucose tolerance of the model group was significantly increased (P < 0.0001); compared with the model group, the oral glucose tolerance of the low, medium and high dose groups were significantly reduced (P < 0.05, P < 0.01, P < 0.05), and there was no significant difference in the oral glucose tolerance between the three dose groups and the blank group (P > 0.05). Figure 7 As shown in B, compared with the blank group, the insulin tolerance of the model group was significantly increased (P < 0.0001); compared with the model group, the insulin tolerance of the low, medium and high dose groups was also reduced, among which the medium and high dose groups had better effects (P < 0.01), and there was no significant difference in insulin tolerance between the medium dose group and the blank group (P > 0.05). The above results show that a high-fat and high-sugar diet can reduce the body's ability to utilize glucose and its sensitivity to insulin, leading to insulin resistance, while the intervention of fermented mucus Lactobacillus LFPerfectus001 can significantly improve the ability of rats fed a high-fat and high-fructose diet to utilize glucose and insulin sensitivity, improve insulin resistance, and keep it at a normal level.

[0097] (6) Effects of fermented Lactobacillus mucilaginosus LFPerfectus001 intervention on serum uric acid in rats Figure 8 Compared with the blank group, the blood uric acid content of the rats in the model group was significantly increased (P < 0.0001). Compared with the model group, the blood uric acid in the low, medium and high dose groups was reduced, among which the low, medium and high dose groups had significant differences (P < 0.01), indicating that the intervention of fermented mucus Lactobacillus LFPerfectus001 can significantly reduce the blood uric acid content of rats fed a high-fat and high-fructose diet.

[0098] (7) Effects of fermented Lactobacillus mucilaginosus LFPerfectus001 intervention on blood lipids in rats Fig. 9 As shown. Fig. 9As shown in A, compared with the model group, the serum triglyceride (TG) of rats in the low, medium and high dose groups were significantly reduced (P < 0.01, P < 0.05, P < 0.01), and there was no significant difference between the three dose groups and the blank group (P > 0.05).

[0099] like Fig. 9 As shown in B, compared with the model group, the serum total cholesterol (TC) of rats in the low, medium and high dose groups were significantly reduced (P < 0.001, P < 0.0001, P < 0.0001), among which there was no significant difference between the low and medium dose groups and the blank group (P > 0.05).

[0100] like Fig. 9 As shown in C, compared with the model group, the serum high-density lipoprotein cholesterol (HDL-C) of rats in the low-dose and high-dose groups showed an increasing trend.

[0101] like Fig. 9 As shown in D, compared with the model group, the serum low-density lipoprotein cholesterol (LDL-C) levels of rats in the low, medium and high dose groups were significantly reduced (P < 0.0001, P < 0.001, P < 0.0001), and there was no significant difference between the three dose groups and the blank group (P > 0.05).

[0102] The above shows that fermented Lactobacillus mucilaginosus LFPerfectus001 can significantly reduce the dyslipidemia of rats fed a high-fat and high-fructose diet, and can reduce serum triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) to the normal levels of the blank group.

[0103] (8) Effects of fermented Lactobacillus mucilaginosus LFPerfectus001 intervention on free saturated fatty acids in rat serum Fig.10 Compared with the blank group, the serum total free saturated fatty acids, free stearic acid, and free palmitic acid of the rats in the model group were significantly increased (P < 0.0001); compared with the model group, the serum total free saturated fatty acids of the rats in the high-dose group were significantly decreased (P < 0.0001), especially the contents of palmitic acid and stearic acid (P < 0.0001), and they could be reduced to below the normal level of the blank group.

[0104] (9) Typical liver tissue pathology sections Fig.11As shown. HE staining results showed that the hepatocytes in the liver tissue of the blank group rats were uniform in size, and the hepatocytes were arranged radially around the central vein, with a clear hepatic cord structure; most of the hepatocytes in the model group rats increased in size, large lipid droplet vacuoles appeared in the cytoplasm, the cells were arranged in a disordered manner, and the hepatic cord structure was destroyed; while the liver pathology of the rats in the fermented mucus lactobacillus LFPerfectus001 intervention group improved, among which the low dose improved slightly, and the medium and high dose groups improved more significantly. The hepatic cords of the rats were arranged regularly, and the fat vacuoles were significantly reduced. The results of oil red staining showed that the lipid droplets in the hepatocytes of the model group increased significantly, while the number of lipid droplets in the intervention group decreased significantly. The above results indicate that fermented mucus lactobacillus LFPerfectus001 can reduce the fat content in the liver of rats fed a high-fat and high-fructose diet and improve the symptoms of fatty liver.

[0105] In summary, the fermented mucus lactobacillus LFPerfectus001 of the present invention has the effects of inhibiting weight gain caused by a high-fat and high-fructose diet, reducing body fat rate, increasing lean body mass rate, inhibiting the enlargement of epididymal white adipocytes and inguinal adipocytes, improving glucose utilization ability, improving insulin resistance and fatty liver, reducing serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, total free saturated fatty acids (especially palmitic acid and stearic acid) and uric acid content, and can improve the symptoms of obesity, hyperglycemia, insulin resistance, dyslipidemia, hyperuric acid and fatty liver in metabolic syndrome, and can reduce the content of white adipocytes, oral glucose tolerance (OGTT), insulin tolerance (ITT) and serum triglycerides, total cholesterol, low-density lipoprotein cholesterol, total free saturated fatty acids, stearic acid and palmitic acid to normal levels or below, has a good effect of improving metabolic syndrome, and can be used in products for improving the above symptoms and in products for improving metabolic syndrome.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. The application of fermented Lactobacillus mucus LFPerfectus001 in the preparation of products for improving metabolic syndrome caused by a high-fat and high-sugar diet. The fermented Lactobacillus mucus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with the preservation number GDMCC No: 62335.

2. The use according to claim 1, characterized in that: The symptoms of metabolic syndrome include at least one of obesity, hyperglycemia, insulin resistance, dyslipidemia, hyperuricemia and fatty liver.

3. The use according to claim 1, characterized in that: The metabolic syndrome improving product can inhibit weight gain, reduce body fat percentage, increase lean body mass percentage, inhibit white fat cell expansion, improve insulin resistance and fatty liver, reduce serum triglyceride, total cholesterol, low-density lipoprotein cholesterol, total free saturated fatty acids and uric acid content, and can reduce white fat cells, oral glucose tolerance, insulin tolerance and serum triglyceride, total cholesterol, low-density lipoprotein cholesterol, total free saturated fatty acids, stearic acid, palmitic acid content to normal levels.

4. The application of fermented Lactobacillus mucus LFPerfectus001 in the preparation of products for improving obesity, hyperglycemia, insulin resistance, dyslipidemia, hyperuric acid and fatty liver. The fermented Lactobacillus mucus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with the preservation number GDMCCNo:62335.

5. The application of fermented mucus Lactobacillus LFPerfectus001 in the preparation of products for improving insulin resistance. The fermented mucus Lactobacillus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with the collection number GDMCC No: 62335.

6. The application of fermented mucus Lactobacillus LFPerfectus001 in the preparation of uric acid-lowering products. The fermented mucus Lactobacillus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with the collection number GDMCCNo:62335.

7. The application of fermented mucus Lactobacillus LFPerfectus001 in the preparation of fat-reducing and muscle-gaining products. The fermented mucus Lactobacillus LFPerfectus001 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with the preservation number GDMCCNo:62335.

8. The use according to any one of claims 1 to 7, characterized in that: The product includes at least one of biological products, food, medicine, health products and daily chemical products.

9. The use according to any one of claims 1 to 7, characterized in that: The dosage of the fermented mucus lactobacillus LFPerfectus001 is (1.6-40)×10 9 CFU / d.

10. A biological product for improving metabolic syndrome, characterized in that: The biological product includes fermented mucus Lactobacillus LFPerfectus001, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 13, 2022, with a deposit number of GDMCC No: 62335.

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