Phytobacterium plantarum for retarding visceral fat cell expansion, bacterium powder, solid beverage and application

Abstract

The invention discloses plant lactobacillus for slowing down visceral fat cell expansion, bacterial powder and application. The plant lactobacillus PC037 is preserved in the China General Microbiological Culture Collection Center on December 13, 2021, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 24096. The plant lactobacillus PC037 can slow down visceral fat cell expansion, and is specifically embodied in that the size of fat cells can be reduced, the effect in the aspect of reducing the fat absorption speed is also remarkable, obesity and metabolic disorder symptoms induced by obesity can be improved, and the plant lactobacillus PC037 has an obvious promoting effect on reduction of the waistline size and visceral fat area of obese people; and slimming can be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and in particular to a Lactobacillus plantarum for slowing down the expansion of visceral fat cells, bacterial powder, a solid beverage and applications thereof. Background Art

[0002] Visceral adipocytes are fat cells located within the abdominal cavity, surrounding visceral organs such as the liver, stomach, and intestines. These cells expand to store more triglycerides, maintaining homeostasis and preventing metabolic diseases. When energy intake exceeds the fat storage capacity of adipocytes, visceral adipocytes expand to their limit and undergo necrosis, triggering adipose tissue inflammation characterized by the infiltration and activation of numerous inflammatory immune cells, such as giant cells and T cells, and the secretion of large amounts of inflammatory cytokines such as TNF-α, IL-6, IL-1β, and MCP-1. Studies have demonstrated that inflammation in visceral adipocytes contributes to the development of insulin resistance. TNF-α and IL-6 directly inhibit the insulin signaling pathway, leading to insulin resistance in adipose tissue, liver, and muscle. Furthermore, impaired insulin signaling in adipocytes further increases lipid cleavage, adipocyte necrosis, and the release of large amounts of free fatty acids. This leads to the infiltration and activation of more immune cells and the release of inflammatory cytokines, amplifying the inflammatory response and worsening insulin resistance, ultimately contributing to various metabolic syndromes, such as obesity, hypertension, and dyslipidemia. This shows that slowing down the expansion of visceral fat cells is beneficial to improving the symptoms of obesity-induced metabolic disorders.

[0003] Lactobacillus plantarum is a Gram-positive bacterium that is widely found in nature. Its body is a round-ended, straight rod. Its colony characteristics are that the surface colony is about 3 mm wide, convex, round, smooth, dense, and white. It can grow in plants, animals, and natural and artificial environments. It is also one of the important microbial groups in the intestine. Its genome size is between 2.91 and 3.35 Mb, which is larger than the genomes of other lactic acid bacteria. The GC content is about 44.5%, and the number of genes is in the range of 2761 to 3518. It has gene clusters for bacteriocins and exopolysaccharides, as well as functional genes such as proteolytic enzyme systems and sugar metabolism. It has the function of regulating chronic metabolic diseases such as diabetes, hyperlipidemia, obesity, and hypoglycemia. Although the strain of Lactobacillus plantarum has a regulatory effect on the symptoms of metabolic disorders in the human body, the differences between strains also vary in the way and effect of its regulation. Through a large number of experiments, the present application has discovered a strain of Lactobacillus plantarum that can slow down the expansion of visceral fat cells, reduce the rate of fat absorption, and improve obesity and its induced metabolic disorder symptoms in an internal regulation manner. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a Lactobacillus plantarum that slows down the expansion of visceral fat cells, thereby reducing the rate of fat absorption and improving the symptoms of obesity and its induced metabolic disorders.

[0005] A second object of the present invention is to provide a bacterial powder for slowing down the expansion of visceral fat cells.

[0006] A third object of the present invention is to provide a solid beverage that can slow down the expansion of visceral fat cells, thereby slowing down the expansion of visceral fat cells and achieving slimming and weight loss.

[0007] One of the purposes of the present invention is achieved by the following technical solution: A plant lactobacillus for slowing down the expansion of visceral fat cells, wherein the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum ) PC037, the plant lactobacillus PC037 was deposited in the General Microbiology Center of China Culture Collection Administration on December 13, 2021, with the deposit number CGMCC No. 24096.

[0008] As a preferred embodiment of the present invention, the Lactobacillus plantarum PC037 exerts the effect of slowing down the expansion of visceral fat cells through at least one of the following items (I) to (IV): (I) Reduce the size of fat cells; (II) Reduce serum triglyceride levels and slow down fat absorption; (III) reduce the waist size of the subjects; (IV) Reduce visceral fat area The second object of the present invention is achieved by adopting the following technical solution: A bacterial powder for slowing down the expansion of visceral fat cells, wherein the bacterial powder is Lactobacillus plantarum PC037 bacterial powder.

[0009] As a preferred embodiment of the present invention, the bacterial powder is prepared by expanding and culturing Lactobacillus plantarum PC037 in a culture medium, and the culture medium includes the following components: 20-30 g / L of glucose, 12-18 g / L of beef extract, 2-8 g / L of corn flour, 2-8 g / L of potato flour, 1-5 g / L of enoki mushroom powder, 0.1-0.5 g / L of MgSO4, 1-4 g / L of dipotassium hydrogen phosphate, 1-8 g / L of sodium acetate, 1-5 g / L of diammonium hydrogen citrate, and 0.5-2 g / L of Tween 80.

[0010] As a preferred embodiment of the present invention, the culture medium comprises the following components: 25 g / L glucose, 15 g / L beef extract, 5 g / L corn flour, 5 g / L potato flour, 3 g / L enoki mushroom powder, 0.2 g / L MgSO4, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, and 1 g / L Tween 80.

[0011] The present invention also provides a method for preparing bacterial powder for slowing down the expansion of visceral fat cells, comprising the following steps: Prepare a liquid fermentation medium: the components of the liquid fermentation medium include 20-30 g / L glucose, 12-18 g / L beef extract, 2-8 g / L corn flour, 2-8 g / L potato flour, 1-5 g / L enoki mushroom powder, 0.1-0.5 g / L MgSO4, 1-4 g / L dipotassium hydrogen phosphate, 1-8 g / L sodium acetate, 1-5 g / L diammonium hydrogen citrate, and 0.5-2 g / L Tween 80. The solvent is purified water. The above components are mixed uniformly and stirred until dissolved, the pH is adjusted, and sterilized. Preparation of seed solution: Remove the glycerol storage tube of Lactobacillus plantarum PC037, activate it, centrifuge the activated bacterial solution, discard the supernatant, wash the bacterial slurry with sterile saline, and resuspend the washed bacterial slurry with sterile saline. The resulting bacterial suspension is used as the seed solution for subsequent fermentation; Prepare fermentation broth: Fill the fermentation tank to 60-80% liquid volume, sterilize, maintain nitrogen pressure at 0.1-0.2 MPa and reduce to 35-38°C, add seed liquid at a volume ratio of 1-3% inoculum, mix well, and ferment at 35-38°C with constant pH for 10-20 hours, then add liquid fermentation medium at a rate of 0.05-0.2 mL / s for 0.8-1.1 hours, and continue to culture at 35-38°C with constant pH for 1.5-2.3 hours to obtain fermentation broth; Prepare bacterial slurry: centrifuge the fermentation broth and collect the precipitate to obtain the bacterial slurry of Lactobacillus plantarum PC037; Prepare a bacterial sludge mixture: prepare Solution 1, the ingredients of Solution 1 are 3-8% maltodextrin, 4-8% trehalose, 0.1-0.2% ascorbic acid, 1-2% sodium glutamate, and 0.5-1.5% glycerol, and the solvent is purified water. Mix them evenly and stir until dissolved. Then add Lactobacillus plantarum PC037 bacterial sludge. The ratio of Solution 1 to Lactobacillus plantarum PC037 bacterial sludge is 2:(2-4), and stir evenly to obtain Solution 2. Preparation of bacterial cake: pre-freeze solution No. 2 at -85~-75℃ for 1.5-3h, take it out and immediately place it in a vacuum freeze dryer, and continue freeze-drying for 20-28h. The cold trap temperature is -60~-45℃ and the vacuum degree is 18-22Pa to obtain bacterial cake; Preparation of bacterial powder: crush the bacterial cake with a grinder, filter with a sieve, and collect the powder that passes through, which is the Lactobacillus plantarum PC037 bacterial powder.

[0012] The third object of the present invention is achieved by adopting the following technical solution: A solid beverage for slowing down the expansion of visceral fat cells comprises the following components in parts by weight: 30-40 parts of fructooligosaccharides, 20-30 parts of resistant dextrin, 5-15 parts of isomaltooligosaccharides, 5-15 parts of galacto-oligosaccharides, 2-8 parts of xylo-oligosaccharides, 2-8 parts of Lactobacillus plantarum PC037 powder, and 5-15 parts of Lactobacillus plantarum PC037 postbiotics.

[0013] As a preferred embodiment of the present invention, the Lactobacillus plantarum PC037 bacterial powder is the bacterial powder described in any one of the second objectives of the present invention.

[0014] As a preferred embodiment of the present invention, the Lactobacillus plantarum PC037 postbiotic is prepared according to the following method: preparing solution No. 3, the components of solution No. 3 are 15-25% skim milk powder, 1-5% trehalose, and the solvent is purified water, mixing and stirring until dissolved, then adding Lactobacillus plantarum PC037 mud, the ratio of solution No. 3 to Lactobacillus plantarum PC037 mud is 1: (0.8-1.5), stirring evenly, and then heating to 90-98 ° C, maintaining for 2-8 minutes, to obtain solution No. 4; wherein, the Lactobacillus plantarum PC037 mud is the Lactobacillus plantarum PC037 mud described in the second purpose; Solution No. 4 is spray-dried and powder is collected, which is then filtered through a sieve to collect the powder that passes through, which is the Lactobacillus plantarum PC037 postbiotic.

[0015] As a preferred embodiment of the present invention, the solid beverage for slowing down the expansion of visceral fat cells comprises the following components in parts by weight: 35 parts of fructooligosaccharides, 25 parts of resistant dextrin, 10 parts of isomaltooligosaccharides, 10 parts of galacto-oligosaccharides, 5 parts of xylo-oligosaccharides, 5 parts of Lactobacillus plantarum PC037 powder, and 10 parts of Lactobacillus plantarum PC037 postbiotics.

[0016] Fructooligosaccharides, isomaltooligosaccharides, galacto-oligosaccharides, and xylooligosaccharides are all functional polysaccharides. Due to their unique structure, they are not easily digested and degraded in the stomach and small intestine, reaching the colon almost intact. Under the action of bifidobacteria and lactobacilli in the intestine, they produce short-chain fatty acids (SCFAs), creating a slightly acidic environment inside the intestine. This promotes the proliferation of beneficial bacteria such as bifidobacteria and lactobacilli, inhibits the growth of harmful bacteria such as Escherichia coli and Clostridium, and thus promotes the balance of the intestinal microbiome. However, different functional polysaccharides act differently when it comes to weight loss.

[0017] Oligofructose can absorb lipids and bile acids in the intestines, binding to bile salts and promoting their excretion in the feces, indirectly reducing the absorption of cholesterol and fat. Bile acids have hydrophilic hydroxyl groups and hydrophobic hydrocarbon groups, which can reduce the surface tension between water and oil, facilitating the action of pancreatic lipase and cholesterol lipase. Cholesterol esters are emulsified and enzymatically hydrolyzed by bile salts to form free cholesterol. This free cholesterol combines with bile salts, phospholipids, and fat hydrolysis products such as monoglycerides and fatty acids to form mixed micelles, which are absorbed by the small intestinal mucosa. 80% to 90% of the absorbed free cholesterol is further combined with long-chain fatty acids in the intestinal mucosa to form cholesterol esters. Most of the cholesterol is incorporated into chylomicrons, while a small amount is incorporated into very low-density lipoproteins and enters the circulation via the lymphatic system. Unabsorbed cholesterol is reduced to coprosterol by bacteria in the lower small intestine and colon and excreted in the feces.

[0018] Isomaltooligosaccharide can promote the proliferation of bifidobacteria in the human intestine, inhibit the formation of harmful bacteria and corrupt substances in the intestine, reduce intestinal carcinogens, improve serum lipids, and lower cholesterol levels.

[0019] Galacto-oligosaccharides can effectively inhibit the activity of HMG-CoA reductase, which is a key rate-limiting enzyme in the metabolism of lipids in the body. At the same time, it slows down the synthesis of endogenous cholesterol. The short-chain fatty acids after the decomposition of galacto-oligosaccharides can also inhibit fat-producing genes and thus control the synthesis of triglycerides and LDL in the body.

[0020] Xylo-oligosaccharides have strong antioxidant properties and are beneficial for repairing lipid peroxidation damage caused by obesity. Studies have shown that after adding XOS, the levels of oxidized glutathione (GSSH) and malondialdehyde (MDA) in the serum, heart, and liver of mice fed a high-fat diet and mice fed a normal diet were significantly reduced compared to the blank control group, while the addition of reduced GSSH significantly increased the level of antioxidant enzymes. The study also found that the expression levels of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in the hearts of mice fed a high-fat diet with xylo-oligosaccharides were significantly increased compared to the blank control group, while the antioxidant enzyme content was not significantly different from that of normal mice.

[0021] Resistant dextrin is a low-molecular-weight, water-soluble dietary fiber made from natural starch (derived from corn, wheat, potatoes, and cassava flour, among others) through a dextrinization reaction under acidic conditions. It inhibits lipid solubility, slows the diffusion of microparticles formed by dietary fat and bile acids, and reduces the rate at which lipids enter intestinal cells. This inhibits fat digestion, delays the release of fatty acids and glycerol, and promotes lipid excretion in feces, thereby suppressing postprandial triglyceride elevations. Furthermore, resistant dextrin inhibits the digestion and absorption of carbohydrates and fats, reducing the amount of free fatty acids and glycerol entering the liver, leading to a decrease in fat synthesis and, consequently, reduced visceral fat accumulation.

[0022] Postbiotics are preparations of inanimate microorganisms or their components that are beneficial to the host's health. Even after entering the human body and being processed by saliva, gastric acid, etc., they still maintain a high level of physiological activity. The postbiotics of the present invention refer to inactivated bacteria that are rich in peptidoglycan, teichoic acid, polysaccharides, lipids, cell membrane proteins, etc. After being consumed by the human body, they can promote the growth of lactobacilli and bifidobacteria, regulate the intestinal flora structure and bile acid composition and content of obese people, and thus improve the symptoms of lipid metabolism disorders.

[0023] The present invention also provides the use of Lactobacillus plantarum PC037 in the preparation of products for slowing down the expansion of visceral fat cells, wherein the products include foods, health products and medicines; the products include solid products, liquid products and semi-solid products.

[0024] Compared with the prior art, the present invention has the following beneficial effects: (1) The Lactobacillus plantarum provided by the present invention can slow down the expansion of visceral fat cells, thereby reducing the rate of fat absorption and improving the symptoms of obesity and metabolic disorders induced by it.

[0025] (2) The bacterial powder for slowing down the expansion of visceral fat cells provided by the present invention has a significant effect in slowing down the expansion of visceral fat cells.

[0026] (3) The solid beverage for slowing down the expansion of visceral fat cells provided by the present invention can slow down the expansion of visceral fat cells and achieve slimming and weight loss.

[0027] Biomaterial preservation information: Lactobacillus plantarum PC037, preservation number is CGMCC No.24096, classification name: Lactobacillus plantarum Lactobacillus plantarum , was deposited on December 13, 2021 in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101). The abbreviation of the depository is CGMCC No. 24096. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the raw materials, equipment, etc. used in the following embodiments can be purchased through commercial channels.

[0029] The embodiment of the present invention provides a Lactobacillus plantarum PC037 that slows down the expansion of visceral fat cells, which is obtained through animal experiment screening.

[0030] System 1: Screening of Lactobacillus plantarum for slowing down the expansion of visceral fat cells 1. Experimental strains: Lactobacillus plantarum CICC 24936 (Accession number: CICC 24936), Lactobacillus plantarum PC037, Lactobacillus plantarum CICC 25282 (Accession number: CICC 25282), Lactobacillus plantarum CICC 25283 (Accession number: CICC 25283), Lactobacillus plantarum GDMCC 1.4956 (Accession number: GDMCC 1.4956) 2. Sample preparation Remove the glycerol storage tube of Lactobacillus plantarum and inoculate 1 mL into 100 mL of MRS liquid medium for activation. Incubate anaerobically at 37°C for 24 hours. Subculture with a 2% inoculum volume and continue incubating anaerobically at 37°C for 24 hours. Subculture once more and incubate anaerobically at 37°C for 24 hours to complete the activation of the strain. Remove the activated bacterial solution and centrifuge it at 4°C and 6000 r / min for 20 minutes. Discard the supernatant and wash the sludge twice with sterile saline under the same centrifugation conditions. Finally, resuspend the sludge with sterile saline of the same volume of fermentation liquid and adjust the viable count to 1×10 9 CFU / mL, and obtain the bacterial suspension of Lactobacillus plantarum.

[0031] 3. Experimental animals: Male C57BL6 / J mice (6 weeks old) 4. All mice were housed in a constant temperature of 25°C, humidity of 55%, and a 12h / 12h light / dark cycle.

[0032] (1) High-fat model mouse experiment 1) After acclimation for 7 days, the mice were randomly divided into 7 groups of 8 mice each, as shown in Table 1. Mice in Group A were fed a standard diet, while mice in Groups B through G were fed a high-fat diet with a caloric content of 60%. During the experimental period, all mice were gavaged daily for 120 consecutive days. The gavage samples and dosages are shown in Table 1.

[0033] Table 1 Mouse groups and gavage samples Group Oral gavage samples Group A Normal saline (10 mL / kg) Group B Normal saline (10 mL / kg) Group C Lactobacillus plantarum CICC 24936 bacterial suspension (10 mL / kg) Group D Lactobacillus plantarum PC037 bacterial suspension (10 mL / kg) Group E Lactobacillus plantarum CICC 25282 bacterial suspension (10 mL / kg) Group F Lactobacillus plantarum CICC 25283 bacterial suspension (10 mL / kg) Group G Lactobacillus plantarum GDMCC 1.4956 bacterial suspension (10 mL / kg) 2) After oral administration on day 120, the mice were weighed and averaged (Table 3). All mice were then fasted for 12 hours and sacrificed by cervical dislocation. The liver and peri-epididymal adipose tissue were collected. The adipose tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm-thick sections. The sections were then stained with H&E. The size of adipocytes in each field of view was counted using ImageJ software, and the average value was calculated (Table 3).

[0034] H&E staining: Dewaxing: Place the paraffin sections in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol for 10 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 85% alcohol for 5 minutes, 80% alcohol for 5 minutes, and 70% alcohol for 5 minutes.

[0035] Staining: Place the paraffin sections in hematoxylin for 5 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 5 seconds, rinse with tap water, then turn blue with 1% ammonia solution for 1 minute, rinse with running water for 30 seconds, place in eosin staining solution for 5 minutes, and rinse with running water.

[0036] Dehydration: The stained sections were dehydrated with anhydrous ethanol and then with xylene to make the sections transparent.

[0037] Sealing: After sealing with neutral gum, observe and take pictures under a microscope.

[0038] (2) Fat absorption experiment 1) After acclimation for 7 days, the mice were randomly divided into 6 groups of 8 mice each. The grouping is shown in Table 2. During the experiment, all mice were fed a standard diet and administered orally for 15 consecutive days. The gavage samples and dosages are shown in Table 2.

[0039] Table 2 Mouse groups and gavage samples Group Oral gavage samples Group H Normal saline (10 mL / kg) Group I Lactobacillus plantarum CICC 24936 bacterial suspension (10 mL / kg) Group J Lactobacillus plantarum PC037 bacterial suspension (10 mL / kg) Group K Lactobacillus plantarum CICC 25282 bacterial suspension (10 mL / kg) Group L Lactobacillus plantarum CICC 25283 bacterial suspension (10 mL / kg) Group M Lactobacillus plantarum GDMCC 1.4956 bacterial suspension (10 mL / kg) 2) On day 16, all mice were gavaged with 0.8 mL of soybean oil. At 3 h, approximately 100 μL of blood was collected from the tail vein of the mice and centrifuged at 2000 g for 10 min to obtain the supernatant serum. The triglyceride content was measured using a TG kit, and the average value was calculated. The results are shown in Table 4.

[0040] 6 Results and Analysis (1) High-fat model mouse experiment Table 3 Body weight and adipocyte size of mice in each group Group Weight (g) <![CDATA[The size of adipocytes (μm 2 )]]> Group A 27.05±1.14 2469.81±153.27 Group B 43.69±1.53 7581.35±165.63 Group C 40.94±1.01 6905.14±130.76 Group D 29.36±1.37 2850.66±114.19 Group E 33.13±1.40 4937.02±141.35 Group F 36.45±1.65 5624.97±157.98 Group G 39.72±1.28 6178.48±178.01 The results showed that compared with Group A, the weight and adipocyte size of mice in Group B increased significantly, indicating that the high-fat diet model was successfully established. After high-fat feeding, the mice gained weight, developed symptoms of obesity, and had significantly expanded adipocytes. Compared with Group B, the weight and adipocyte size of mice in Groups C to G decreased, indicating that Lactobacillus plantarum alleviated the expansion of visceral adipocytes. However, the degree of improvement varied among different strains. The weight and adipocyte size of mice in Group D were the smallest and closer to those of mice in Group A, indicating that after high-fat feeding, the adipocytes of mice in Group D remained normal. In other words, Lactobacillus plantarum PC037 was most effective in slowing the expansion of visceral adipocytes, which is beneficial for improving obesity symptoms and achieving weight loss.

[0041] (2) Fat absorption experiment Table 4 Triglyceride content in serum of mice Group Triglyceride content (mmol / L) Group H 1.35±0.10 Group I 0.95±0.07 Group J 0.61±0.03 Group K 0.84±0.05 Group L 0.88±0.01 Group M 0.93±0.06 Triglycerides (TG), a type of fat in the blood, are mostly obtained from the diet. Excessive triglycerides accumulate under the skin, leading to obesity and related diseases. For example, accumulation in blood vessels can cause arteriosclerosis, accumulation in the heart can cause cardiomegaly, and accumulation in the liver can cause fatty liver disease. In contrast, cholesterol is mostly synthesized by the body, with a small amount obtained from the diet. Therefore, using TG changes as an evaluation indicator can directly and quickly reflect the body's absorption of fats from food.

[0042] The results showed that compared with group H, the triglyceride content in the serum of mice in groups I to M was lower, indicating that Lactobacillus plantarum can reduce the rate of fat absorption, but the degree of reduction varies with different strains. The triglyceride content in the serum of mice in group J was the lowest, indicating that Lactobacillus plantarum PC037 has the best effect in reducing the rate of fat absorption.

[0043] Combining the experimental results in Table 2 and Table 4, Lactobacillus plantarum PC037 was selected as the best strain to slow down the expansion of visceral fat cells. PC037 is beneficial for reducing the rate of fat absorption and improving the symptoms of obesity and its induced metabolic disorders.

[0044] System 2: Screening of the optimal fermentation time for bacterial powder A method for preparing bacterial powder for slowing down the expansion of visceral fat cells comprises the following steps: 1. Prepare liquid fermentation medium. The components of the medium are glucose 25 g / L, beef extract 15 g / L, corn flour 5 g / L, potato flour 5 g / L, enoki mushroom powder 3 g / L, MgSO4 0.20 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, and Tween 80 1 g / L. The solvent is purified water. Mix well and stir until dissolved. Adjust the pH to 6.5 and sterilize at 121°C for 15 min.

[0045] 2. Remove the glycerol tube of Lactobacillus plantarum PC037 and inoculate 1 mL into 100 mL of MRS liquid medium for activation. Incubate anaerobically at 37°C for 24 hours. Subculture at a 2% inoculum size and continue incubating anaerobically at 37°C for 24 hours. Subculture once more and incubate anaerobically at 37°C for 24 hours to complete activation. Remove the activated culture and centrifuge at 4°C and 6000 rpm for 20 minutes. Discard the supernatant and wash the slurry twice with sterile saline under the same centrifugation conditions. Finally, resuspend the slurry in sterile saline with the same volume of fermentation broth. This suspension serves as the seed medium for subsequent fermentations.

[0046] 3. The fermentation tank was filled with 70% liquid. After sterilization, nitrogen pressure was maintained at 0.1 MPa and dropped to 37°C. The seed liquid was added at an inoculum volume of 2% by volume, mixed evenly, and fermented at 37°C with a constant pH for the time shown in Table 5. Liquid fermentation medium was then added at a rate of 0.1 mL / s for 1 h. The culture was continued at 37°C with a constant pH for 2 h to obtain a fermentation broth.

[0047] 4. Centrifuge the fermentation liquid at a speed of 6000 r / min for 20 min, and collect the precipitate to obtain the Lactobacillus plantarum PC037 sludge.

[0048] 5. Prepare solution No. 1. The ingredients of solution No. 1 are 5% maltodextrin, 6% trehalose, 0.15% ascorbic acid, 1.5% sodium glutamate, and 1% glycerol. The solvent is purified water. Mix them evenly and stir until dissolved. Then add Lactobacillus plantarum PC037 sludge. The ratio of solution No. 1 to Lactobacillus plantarum PC037 sludge is 2:3. Stir evenly to obtain solution No. 2.

[0049] 6. Solution No. 2 was pre-frozen at -80°C for 2 h, and immediately placed in a vacuum freeze dryer for 24 h. The cold trap temperature was -55°C and the vacuum degree was 20 Pa to obtain a bacterial cake.

[0050] 7. Crush the cake with a grinder, filter with an 80-mesh sieve, and collect the powder that passes through, which is the Lactobacillus plantarum PC037 powder.

[0051] Table 5 Fermentation time of bacterial powder in different embodiments Example Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Time (h) 0 4 8 12 16 20 2. High-fat model mouse experiment 1. Experimental animals: Male C57BL6 / J mice (6 weeks old) 2. Preparation of bacterial suspension: Weigh 1 g of bacterial powder and add 200 mL of normal saline to resuspend it.

[0052] 3. All mice were housed in a constant temperature of 25°C, humidity of 55%, and a 12h / 12h light / dark cycle.

[0053] 4. After acclimation for 7 days, the mice were randomly divided into 6 groups of 8 mice each, as shown in Table 6. Mice in groups N through S were fed a high-fat diet with a caloric content of 60%. During the experimental period, all mice were gavage-treated daily for 120 consecutive days. The gavage samples and dosages are shown in Table 6.

[0054] Table 6 Mouse grouping and gavage samples Group Oral gavage samples Group N Bacterial suspension of Example 1 (10 mL / kg) Group O Bacterial suspension of Example 2 (10 mL / kg) Group P Bacterial suspension of Example 3 (10 mL / kg) Group Q Bacterial suspension of Example 4 (10 mL / kg) R Group Bacterial suspension of Example 5 (10 mL / kg) Group S Bacterial suspension of Example 6 (10 mL / kg) 5. After gavage on day 120, the mice were weighed and averaged, as shown in Table 7. All mice were then fasted for 12 hours and sacrificed by cervical dislocation. Liver and peri-epididymal adipose tissue were collected and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm-thick sections. The sections were then stained with H&E. ImageJ software was used to count the size of adipocytes in each field of view, and the average value was calculated. The results are shown in Table 7.

[0055] 6. Results and Analysis Table 7 Body weight and adipocyte size of mice in each group Group Weight (g) Size of fat cells (μm2) Group N 42.65±1.19 7684.70±118.53 Group O 37.37±1.01 5891.68±154.04 Group P 34.06±1.30 5245.43±143.17 Group Q 30.57±1.26 3460.91±115.35 R Group 27.18±1.41 2639.35±130.61 Group S 30.80±1.10 3817.04±171.48 Combining the experimental results in Table 2 and Table 7, compared with Group B, the weight and fat cell size of mice in Groups N to S were reduced, indicating that the Lactobacillus plantarum PC037 powder has a mitigating effect on the expansion of visceral fat cells. However, the degree of improvement varies depending on the powder prepared by different processes. The weight and fat cell size of mice in Group R were the smallest and were closer to those of mice in Group A, indicating that after being fed a high-fat diet, the fat cells of mice in Group R were still in a normal state. That is, the powder of Example 5 had the best effect in slowing the expansion of visceral fat cells. Therefore, 16 hours was selected as the optimal fermentation time.

[0056] System 3: Screening of culture medium formula for bacterial powder A method for preparing bacterial powder for slowing down the expansion of visceral fat cells comprises the following steps: 1. Prepare liquid fermentation medium. The composition of the medium is shown in Table 8. The solvent is purified water. Mix well and stir until dissolved. Adjust the pH to 6.5 and sterilize at 121°C for 15 min.

[0057] 2. Remove the glycerol tube of Lactobacillus plantarum PC037 and inoculate 1 mL into 100 mL of MRS liquid medium for activation. Incubate anaerobically at 37°C for 24 hours. Subculture at a 2% inoculum size and continue incubating anaerobically at 37°C for 24 hours. Subculture once more and incubate anaerobically at 37°C for 24 hours to complete activation. Remove the activated culture and centrifuge at 4°C and 6000 rpm for 20 minutes. Discard the supernatant and wash the slurry twice with sterile saline under the same centrifugation conditions. Finally, resuspend the slurry in sterile saline with the same volume of fermentation broth. This suspension serves as the seed medium for subsequent fermentations.

[0058] 3. Fill the fermentation tank with 70% liquid. After sterilization, maintain the nitrogen pressure at 0.1 MPa and drop it to 37°C. Add the seed liquid at a volume ratio of 2% and mix well. Ferment at 37°C with constant pH for 16 h. Then, add liquid fermentation medium at a rate of 0.1 mL / s for 1 h. Continue to culture at 37°C with constant pH for 2 h to obtain the fermentation liquid.

[0059] 4. Centrifuge the fermentation liquid at a speed of 6000 r / min for 20 min, and collect the precipitate to obtain the Lactobacillus plantarum PC037 sludge.

[0060] 5. Prepare solution No. 1. The ingredients of solution No. 1 are 5% maltodextrin, 6% trehalose, 0.15% ascorbic acid, 1.5% sodium glutamate, and 1% glycerol. The solvent is purified water. Mix them evenly and stir until dissolved. Then add Lactobacillus plantarum PC037 sludge. The ratio of solution No. 1 to Lactobacillus plantarum PC037 sludge is 2:3. Stir evenly to obtain solution No. 2.

[0061] 6. Solution No. 2 was pre-frozen at -80°C for 2 h, and immediately placed in a vacuum freeze dryer for 24 h. The cold trap temperature was -55°C and the vacuum degree was 20 Pa to obtain a bacterial cake.

[0062] The bacterial cake was crushed with a grinder, filtered with an 80-mesh sieve, and the powder that passed through was collected to obtain the Lactobacillus plantarum PC037 bacterial powder.

[0063] Table 8 Content of components in culture medium of different examples (g / L)

[0064] 2. High-fat model mouse experiment 1. Experimental animals: Male C57BL6 / J mice (6 weeks old) 2. Preparation of bacterial suspension: Weigh 1 g of bacterial powder and add 200 mL of normal saline to resuspend it.

[0065] 3. All mice were housed in a constant temperature of 25°C, humidity of 55%, and a 12h / 12h light / dark cycle.

[0066] 4. After acclimation for 7 days, the mice were randomly divided into five groups of eight mice each, as shown in Table 13. Mice in groups N through S were fed a high-fat diet containing 60% of the caloric content. During the experimental period, all mice were gavage-fed daily for 120 consecutive days. The gavage samples and dosages are shown in Table 9.

[0067] Table 9 Mouse grouping and gavage samples Group Oral gavage samples T Group Bacterial suspension of Example 7 (10 mL / kg) Group U Bacterial suspension of Example 8 (10 mL / kg) Group V Bacterial suspension of Example 9 (10 mL / kg) Group W Bacterial suspension of Example 10 (10 mL / kg) Group X Bacterial suspension of Example 11 (10 mL / kg) 5. After gavage on day 120, the mice were weighed and averaged, as shown in Table 10. All mice were then fasted for 12 hours and sacrificed by cervical dislocation. Liver and peri-epididymal adipose tissue were collected and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm-thick sections. The sections were then stained with H&E. ImageJ software was used to count the size of adipocytes in each field of view, and the average value was calculated. The results are shown in Table 10.

[0068] 6. Results and Analysis Table 10 Body weight and fat cell size of mice in each group Group Weight (g) Size of fat cells (μm2) T Group 37.16±1.28 5806.47±137.19 Group U 41.64±1.36 6989.31±130.67 Group V 33.40±1.15 5130.19±138.43 Group W 27.29±1.37 2647.58±141.35 Group X 39.05±1.14 6558.73±145.58 Combining the experimental results in Tables 2 and 10, compared with Group B, the weight and fat cell size of mice in Groups T to X were reduced, but the degree of reduction varied among the groups, indicating that differences in the content of culture medium components significantly affected the effect of the bacterial powder. Among them, the weight and fat cell size of mice in Group W were the smallest, indicating that the degree of fat cell expansion in Group W mice was the smallest, and the weight of the mice was also well controlled. That is, the bacterial powder prepared with the culture medium of Example 11 was most effective in alleviating fat cell expansion. Therefore, Example 11 was selected as the optimal formulation of the culture medium for bacterial powder.

[0069] System 4: Screening for the best formula of a solid beverage A solid beverage containing bacterial powder for slowing down the expansion of visceral fat cells comprises the following steps: 1. Select qualified fructooligosaccharides, resistant dextrin, isomaltooligosaccharides, galacto-oligosaccharides and xylo-oligosaccharides, filter them with an 80-mesh sieve, collect the powder that passes through, and weigh fructooligosaccharides, resistant dextrin, isomaltooligosaccharides, galacto-oligosaccharides and xylo-oligosaccharides according to the formula in Table 11.

[0070] 2. Prepare solution No. 3. The ingredients of solution No. 3 are 20% skim milk powder and 3% trehalose. The solvent is purified water. Mix them evenly and stir until dissolved. Then add Lactobacillus plantarum PC037 sludge. The ratio of solution No. 3 to Lactobacillus plantarum PC037 sludge is 1:1. Stir evenly, then heat to 95°C and maintain for 5 minutes to obtain solution No. 4.

[0071] After spray drying, solutions 3 and 4 were powdered and filtered through an 80-mesh sieve. The powder that passed through was collected to obtain the Lactobacillus plantarum PC037 postbiotic. The spray drying conditions were an inlet temperature of 170°C and an outlet temperature of 60°C.

[0072] 4. Weigh Lactobacillus plantarum PC037 powder and Lactobacillus plantarum PC037 postbiotics according to the formula in Table 11.

[0073] 5. Mix galacto-oligosaccharide and xylo-oligosaccharide evenly, then add isomalto-oligosaccharide, resistant dextrin, fructo-oligosaccharide and Lactobacillus plantarum PC037 postbiotics in sequence and mix evenly. Mix for 5 minutes each time. After cooling to 25°C, add Lactobacillus plantarum PC037 powder and mix evenly. The resulting product is a solid beverage.

[0074] Table 11: Prescriptions of different examples

[0075] 2. Crowd Testing 1. Subjects: 90 obese adults were recruited, with a male-to-female ratio of 1:1, aged 25 to 55 years, and a BMI ≥ 24 kg / m2.

[0076] Exclusion criteria: patients who are pregnant or breastfeeding; patients who have used fibrates, diuretics, weight loss drugs or hypoglycemic drugs in the past 3 months; patients with clear familial hyperlipidemia.

[0077] The subjects were randomly divided into 9 groups, with 10 people in each group, with a male-to-female ratio of 1:1. They consumed solid beverages (mixed with 150 mL of 37°C warm water and drunk) every morning, noon, and half an hour after meals in the evening, 2 g each time, for 60 consecutive days. The grouping and the types of solid beverages consumed by each group are shown in Table 12.

[0078] Table 12 Grouping of subjects Group Edible solid beverages Group A Example 12 Group B Example 13 Group C Example 14 Group D Example 15 Group E Example 16 Group F Example 17 Group G Example 18 Group H Example 19 Group I Example 20 3. On Day 1 and Day 61, measure each person's waist circumference and calculate the average. The results are shown in Table 13. Waist circumference refers to the length (cm) measured by wrapping a soft tape measure around the umbilicus at the end of exhalation.

[0079] 4. On the first and 61st day, the visceral fat area (cm2) of each person was measured using the InBody S10 body composition analyzer, and the average value was calculated. The results are shown in Table 14.

[0080] 5. Results and Analysis (1) Changes in waist circumference Table 13 Changes in waist circumference of subjects (cm)

[0081] Excessive visceral fat in the abdominal cavity can easily lead to central obesity (abdominal obesity). Waist circumference is a commonly used indicator of central obesity. Based on the characteristics of the Chinese adult population and health risk assessment, a normal waist circumference is defined as <85cm (male) and <80cm (female). Central obesity is diagnosed when the waist circumference is ≥90cm (male) and ≥85cm (female).

[0082] The results showed that compared with day 0, after 60 days of consumption of the solid beverage, the waist circumference of both male and female subjects decreased, but the degree of reduction was different, indicating that the difference in the formulation had a significant impact on its effect. Among them, the waist circumference of the subjects in Group I decreased the most, indicating that the solid beverage in Group I (i.e., Example 20) was the most effective in slowing the expansion of visceral fat cells, effectively achieving slimming.

[0083] (2) Changes in visceral fat area Table 14 Changes in visceral fat area of ​​subjects (cm 2 )

[0084] The results showed that compared with day 0, the visceral fat area of ​​the subjects decreased after 60 days of consumption, but the degree of reduction was different, indicating that the difference in the group formula had a significant impact on its effect. Among them, the visceral fat area of ​​the subjects in Group I decreased the most, indicating that the solid beverage in Group I (i.e., Example 20) was the most effective in slowing the expansion of visceral fat cells and effectively reducing the visceral fat area.

[0085] Combining the experimental results in Tables 13 and 14, Example 20 was selected as the optimal formulation for the solid beverage. The resulting solid beverage particularly contains active PC037 strains and postbiotics. After human consumption, the strains can successfully reach the intestines for colonization and proliferation, and the postbiotics can be quickly absorbed and utilized by the intestines. This combination of fast and slow methods effectively slows down the expansion of visceral fat cells, reduces the area of ​​visceral fat, and achieves slimming.

[0086] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A Lactobacillus plantarum for slowing down the expansion of visceral fat cells, characterized in that: The plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum ) PC037, the plant lactobacillus PC037 was deposited in the General Microbiology Center of China Culture Collection Administration on December 13, 2021, with the deposit number CGMCC No. 24096.

2. The Lactobacillus plantarum for slowing down the expansion of visceral fat cells according to claim 1, wherein The Lactobacillus plantarum PC037 exerts the effect of slowing down the expansion of visceral fat cells through at least one of the following items (I) to (IV): (I) Reduce the size of fat cells; (II) Reduce serum triglyceride levels and slow down fat absorption; (III) reduce the waist size of the subjects; (IV) Reduce visceral fat area.

3. A bacterial powder for slowing down the expansion of visceral fat cells, characterized in that: The bacterial powder is Lactobacillus plantarum PC037 bacterial powder; the Lactobacillus plantarum PC037 is the Lactobacillus plantarum described in claim 1.

4. The bacterial powder according to claim 3, wherein The bacterial powder is prepared by expanding and culturing Lactobacillus plantarum PC037 in a culture medium. The culture medium comprises the following components: 20-30 g / L of glucose, 12-18 g / L of beef extract, 2-8 g / L of corn flour, 2-8 g / L of potato flour, 1-5 g / L of enoki mushroom powder, 0.1-0.5 g / L of MgSO4, 1-4 g / L of dipotassium hydrogen phosphate, 1-8 g / L of sodium acetate, 1-5 g / L of diammonium hydrogen citrate, and 0.5-2 g / L of Tween 80.

5. The bacterial powder according to claim 3, wherein The culture medium includes the following components: 25 g / L glucose, 15 g / L beef extract, 5 g / L corn flour, 5 g / L potato flour, 3 g / L enoki mushroom powder, 0.2 g / L MgSO4, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, and 1 g / L Tween 80.

6. A method for preparing the bacterial powder for slowing down the expansion of visceral fat cells according to any one of claims 1 to 5, characterized in that: The following steps are involved: Prepare a liquid fermentation medium: the components of the liquid fermentation medium include 20-30 g / L glucose, 12-18 g / L beef extract, 2-8 g / L corn flour, 2-8 g / L potato flour, 1-5 g / L enoki mushroom powder, 0.1-0.5 g / L MgSO4, 1-4 g / L dipotassium hydrogen phosphate, 1-8 g / L sodium acetate, 1-5 g / L diammonium hydrogen citrate, and 0.5-2 g / L Tween 80, and the solvent is purified water. Mix the above components uniformly and stir until dissolved, adjust the pH, and sterilize. Preparation of seed solution: Remove the glycerol storage tube of Lactobacillus plantarum PC037, activate it, centrifuge the activated bacterial solution, discard the supernatant, wash the bacterial slurry with sterile saline, and resuspend the washed bacterial slurry with sterile saline. The resulting bacterial suspension is used as the seed solution for subsequent fermentation; Prepare fermentation broth: Fill the fermentation tank to 60-80% liquid volume, sterilize, maintain nitrogen pressure at 0.1-0.2 MPa and reduce to 35-38°C, add seed liquid at a volume ratio of 1-3% inoculum, mix well, and ferment at 35-38°C with constant pH for 10-20 hours, then add liquid fermentation medium at a rate of 0.05-0.2 mL / s for 0.8-1.1 hours, and continue to culture at 35-38°C with constant pH for 1.5-2.3 hours to obtain fermentation broth; Prepare bacterial slurry: centrifuge the fermentation broth and collect the precipitate to obtain the bacterial slurry of Lactobacillus plantarum PC037; Prepare a bacterial sludge mixture: prepare Solution 1, the ingredients of Solution 1 are 3-8% maltodextrin, 4-8% trehalose, 0.1-0.2% ascorbic acid, 1-2% sodium glutamate, and 0.5-1.5% glycerol, and the solvent is purified water. Mix them evenly and stir until dissolved. Then add Lactobacillus plantarum PC037 bacterial sludge. The ratio of Solution 1 to Lactobacillus plantarum PC037 bacterial sludge is 2:(2-4), and stir evenly to obtain Solution 2. Preparation of bacterial cake: pre-freeze solution No. 2 at -85~-75℃ for 1.5-3h, take it out and immediately place it in a vacuum freeze dryer, and continue freeze-drying for 20-28h. The cold trap temperature is -60~-45℃ and the vacuum degree is 18-22Pa to obtain bacterial cake; Preparation of bacterial powder: crush the bacterial cake with a grinder, filter with a sieve, and collect the powder that passes through, which is the Lactobacillus plantarum PC037 bacterial powder.

7. A solid beverage for slowing down the expansion of visceral fat cells, characterized in that: The invention comprises the following components in parts by weight: 30-40 parts of oligofructose, 20-30 parts of resistant dextrin, 5-15 parts of isomaltooligosaccharide, 5-15 parts of galacto-oligosaccharide, 2-8 parts of xylo-oligosaccharide, 2-8 parts of Lactobacillus plantarum PC037 powder, and 5-15 parts of Lactobacillus plantarum PC037 postbiotics; the Lactobacillus plantarum PC037 is the Lactobacillus plantarum described in claim 1.

8. The solid beverage according to claim 7, wherein The Lactobacillus plantarum PC037 bacterial powder is the bacterial powder according to any one of claims 3 to 5, or the bacterial powder obtained by the preparation method according to claim 6; The Lactobacillus plantarum PC037 postbiotic is prepared according to the following method: preparing solution No. 3, wherein the components of solution No. 3 are 15-25% skim milk powder, 1-5% trehalose, and purified water as the solvent; the mixture is mixed evenly and stirred until dissolved; then, Lactobacillus plantarum PC037 sludge is added, and the ratio of solution No. 3 to Lactobacillus plantarum PC037 sludge is 1:(0.8-1.5); the mixture is stirred evenly, and the mixture is heated to 90-98° C. and maintained for 2-8 minutes to obtain solution No. 4; wherein the Lactobacillus plantarum PC037 sludge is the Lactobacillus plantarum PC037 sludge according to claim 6; Solution No. 4 is spray-dried and powder is collected, which is then filtered through a sieve to collect the powder that passes through, which is the Lactobacillus plantarum PC037 postbiotic.

9. The solid beverage for slowing down the expansion of visceral fat cells according to claim 7 or 8, characterized in that: The invention comprises the following components in parts by weight: 35 parts of fructooligosaccharides, 25 parts of resistant dextrins, 10 parts of isomaltooligosaccharides, 10 parts of galacto-oligosaccharides, 5 parts of xylo-oligosaccharides, 5 parts of Lactobacillus plantarum PC037 powder and 10 parts of Lactobacillus plantarum PC037 postbiotics.

10. Use of Lactobacillus plantarum PC037 in preparing a product for slowing down the expansion of visceral fat cells, characterized in that: The products include solid products, liquid products, and semi-solid products; the Lactobacillus plantarum PC037 is the Lactobacillus plantarum described in claim 1.

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