Probiotics with weight-losing effect and application thereof

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to a probiotic with a weight reducing effect and application of the probiotic, the probiotic is Limosibacillus fermentum ZKAW03 and is preserved in Guangdong Microbiological Culture Collection Center, the preservation number is GDMCC NO: 65680, the strain is separated from healthy human intestinal tracts, the gastrointestinal tract tolerance and bile salt hydrolase activity are high, and the weight reducing effect is good. Fat metabolism can be regulated through multi-target chemical reaction; the application of the probiotics comprises preparation of probiotic fat-reducing tablets, compound beverages and capsules, functional components (such as tea polyphenol, whey protein and chitosan) and strains are subjected to a synergistic chemical reaction to form a hydrogen bond network, a protein-polysaccharide compound and a polyelectrolyte complex, so that the effects of strain colonization, protection and weight loss are enhanced; and a novel solution is provided for obesity management.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to a probiotic with weight loss effect and its application. Background Technology

[0002] Obesity is a major global public health problem. Its core mechanism is the excessive accumulation of fat due to an imbalance between energy intake and expenditure, accompanied by gut microbiota dysbiosis, increased inflammatory response, and abnormal metabolic pathways. Traditional weight loss methods, such as diet control, exercise, and drug intervention, often have limitations such as poor adherence, significant side effects, or easy rebound.

[0003] Probiotics, as a microecological regulator, are considered a potential obesity management strategy by improving intestinal barrier function, regulating immunity, and influencing host energy metabolism. However, current probiotic strains still have significant shortcomings in weight loss applications: First, most strains lack specific regulatory mechanisms targeting fat synthesis and breakdown pathways, resulting in limited functionality; second, strains have low survival rates in the gastrointestinal tract, with gastric acid and bile salts easily leading to a sharp decline in the number of viable bacteria, making it difficult to colonize the intestine and maintain efficacy; third, strain culture processes are not optimized for weight loss characteristics, failing to efficiently induce the production of enzymes related to fat metabolism regulation.

[0004] Therefore, there is an urgent need in this field for a new probiotic strain with high gastrointestinal tolerance and the ability to regulate lipid metabolism through synergistic effects of multi-target chemical reactions, so as to provide an innovative solution for obesity management. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a probiotic with weight-loss effects and its application. It involves isolating and screening probiotic strains with weight-loss functions from the gut of healthy individuals, then culturing these strains. Utilizing the chemical effects between these strains and functional components, the duration of the probiotics' action is increased, regulating human energy metabolism and reducing fat absorption. This solves the problems of limited functional mechanisms and low gastrointestinal survival rates of probiotics in existing technologies, providing a more efficient solution for obesity management.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a probiotic with weight loss effect and its application. The probiotic with weight loss effect is Limosilactobacillus fermentum ZKAW03 (hereinafter abbreviated as ZKAW03), which was isolated from the intestine of a healthy human and deposited on December 26, 2024 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 65680 and taxonomic name Limosilactobacillus fermentum. The viability test result showed that it was viable.

[0007] Furthermore, the method for isolating and identifying the probiotics with weight-loss effects is as follows: S1: Take an intestinal contents sample with a mass-to-volume ratio of 1:10 and mix it with sterile phosphate-buffered saline to obtain a sample suspension. Dilute the bacterial suspension using a 10-fold serial dilution method and spread it on MRS solid medium containing 0.1% (w / v) L-cysteine. Incubate anaerobicly at 37°C for 48 h. Pick single colonies with typical morphological characteristics of Lactobacillus and purify them by streaking on MRS solid medium. Repeat the operation until the colony growth characteristics on MRS solid medium are consistent to obtain purified colonies with different characteristics. S2: After obtaining purified single colonies with different phenotypic characteristics, Gram staining was performed and microscopic observation was conducted to screen for Gram-positive strains. The Gram-positive strains were further subjected to catalase experiment to screen for Gram-positive and catalase-negative strains, namely Lactobacillus characteristic strains. S3: The characteristic strains of Lactobacillus were inoculated into MRS liquid medium and cultured anaerobically at 37°C for 24 hours. After three generations of continuous activation, the third generation bacterial solution was centrifuged at 4°C and 8000 rpm for 10 minutes. The supernatant was discarded to obtain bacterial sludge. Sterile physiological saline was added to the bacterial sludge for washing to obtain the test strain. The test strain was identified by genus identification to obtain Lactobacillus strains. S4: 16S rDNA sequencing, sequence alignment, and homology analysis were performed on Lactobacillus strains to obtain a novel Lactobacillus strain, Limosilactobacillus fermentum ZKAW03, which is a probiotic with weight loss effects.

[0008] The present invention also provides an application of probiotics with weight loss effect, wherein the application is a probiotic weight loss tablet prepared using probiotics with weight loss effect.

[0009] Furthermore, the probiotic weight-loss tablets with weight-loss effects comprise the following ingredients in parts by weight: 50-70 parts of freeze-dried probiotic powder, 10-20 parts of tea polyphenol extract, 20-30 parts of fructooligosaccharides, 15-20 parts of microcrystalline cellulose, and 0.5-2 parts of magnesium stearate. The preparation method of the probiotic freeze-dried powder is as follows: (1) The activated ZKAW03 strain is inoculated into MRS liquid medium and cultured anaerobically at 37℃ for 18 hours until the late logarithmic growth stage. Then, the bacterial sludge is collected by centrifugation at 4℃ and 8000 rpm for 10 minutes and washed twice with sterile physiological saline. (2) The washed bacterial sludge is resuspended in a freeze-drying protectant containing 10% (w / v) trehalose and 5% (w / v) skim milk to make the bacterial concentration reach 1×10¹¹ CFU / mL, and then dispensed into freeze-drying bottles. (3) The dispensed samples are pre-frozen in an ultra-low temperature freezer at -80℃ for 4 hours, and finally freeze-dried in a freeze dryer at -50℃ and 0.1 mBar for 24 hours to obtain highly active ZKAW03 freeze-dried powder, which is the probiotic freeze-dried powder.

[0010] The tea polyphenol extract is a standardized extract extracted from green tea with a tea polyphenol content of ≥98%.

[0011] Furthermore, the preparation process of the probiotic weight-loss tablets with weight-loss effects is as follows: A1 Mixing: Weigh the probiotic freeze-dried powder, tea polyphenol extract, fructooligosaccharides and microcrystalline cellulose accurately according to the above ratio and place them in a V-type mixer. Mix at 30 rpm for 30 minutes until the mixture is uniform to obtain the main drug mixture. A2 Granulation: Slowly spray a 70% ethanol solution as a wetting agent into the active pharmaceutical ingredient mixture while stirring until a soft material that can be formed by squeezing into a ball but crumbles easily when pressed is formed. Granulate the soft material through a 20-mesh sieve to obtain wet granules. A3 Drying: Spread the wet granules evenly on a tray and dry them in a 45°C hot air circulating oven for 40 minutes. A4 Granulation and Mixing: The dried granules are granulated again through a 20-mesh sieve, and then magnesium stearate is added according to the formula. The mixture is then mixed in a mixer at 20 rpm for 5 minutes to obtain the mixed granules. A5 tableting: The total granules are compressed using a rotary tableting machine, with the tablet weight adjusted to 0.5 g / tablet and the pressure set to 5 kN, to obtain unprocessed tablets; A6 Coating: Using a high-efficiency coating pan, the pressed tablets are placed in the pan and enteric coating is performed using an enteric coating solution prepared by dissolving polyacrylic acid resin II in 95% ethanol by volume. The concentration of polyacrylic acid resin II is 6% (w / v), and the weight gain is controlled at 3%. After coating, the tablets are cured at 40°C for 2 hours to obtain the probiotic weight loss tablets.

[0012] This invention also provides an application of probiotics with weight-loss effects, specifically a probiotic compound beverage prepared using probiotics with weight-loss effects. The probiotic compound beverage comprises the following ingredients in parts by weight: 30-50 parts probiotic fermentation broth, 10-20 parts whey protein, 5-10 parts inulin, 0.1-0.3 parts citric acid, and 0.05-0.1 parts steviol glycosides. The preparation method of the probiotic fermentation broth is as follows: ZKAW03 strain is inoculated into sterilized MRS liquid culture medium at an inoculum rate of 2% (v / v), and then subjected to anaerobic fermentation at 37°C for 24 hours until the viable cell count reaches 1×10⁻⁶. 9 A probiotic fermentation broth with a concentration of CFU / mL or higher is obtained.

[0013] Furthermore, the preparation process of the probiotic compound beverage with weight loss effect is as follows: B1: Dissolve whey protein powder and inulin in purified water at 50°C to prepare a 35% (w / v) solution, and stir until completely dissolved to obtain an aqueous matrix; B2: Dissolve citric acid and steviol glycosides completely in warm water to obtain the flavoring liquid; B3: Cool the aqueous phase matrix to room temperature, then mix it with the specified proportions of probiotic fermentation liquid and flavoring liquid in the mixing tank. Use a shear emulsifier to stir at 2000 rpm for 15 minutes to ensure uniform mixing and obtain a mixture. B4: Use pure water to bring the mixture to the required total volume, then transfer it to a high-pressure homogenizer and homogenize it three times under a pressure of 20 MPa to obtain a uniform and stable liquid. B5: The homogenized liquid is aseptically filtered and cold-filled to obtain the probiotic compound beverage.

[0014] This invention also provides an application of probiotics with weight-loss effects, specifically a probiotic capsule prepared using these probiotics. The probiotic capsule comprises the following ingredients in parts by weight: 20-40 parts of freeze-dried probiotic powder, 2-5 parts of chitosan, 1-3 parts of sodium alginate, 10-20 parts of inulin, and 0.5-2 parts of silica. The preparation method of the freeze-dried probiotic powder is the same as that used in probiotic weight-loss tablets. The preparation method of the probiotic capsule with weight-loss effects is as follows: (a) The probiotic freeze-dried powder, inulin and silica were mixed in a mixer for 25 minutes according to the specified ratio to obtain a uniform core material mixture; (b) Dissolve chitosan in a 1% (v / v) acetic acid solution and stir to prepare a 3% (w / v) chitosan solution. At the same time, dissolve sodium alginate in purified water and stir to prepare a 2% (w / v) sodium alginate solution. (c) The core material mixture is dispersed in a sodium alginate solution of 2 times its weight, and then added dropwise to a calcium chloride solution by a peristaltic pump. After standing and solidifying for 30 minutes, gel microspheres are formed. After washing with sterile water, they are immersed in chitosan solution for 15 minutes for composite coating, and then washed with sterile water to obtain encapsulated probiotic capsules. (d) The washed probiotic capsules are freeze-dried and then filled into hydroxypropyl methylcellulose plant hollow capsule shells to obtain the probiotic capsules.

[0015] Compared with the prior art, the beneficial effects of the present invention are: The *Lactobacillus fermentum* ZKAW03 provided in this invention originates from the intestines of healthy humans. It possesses excellent resistance to gastric acid and bile salts, enabling it to reach and colonize the intestines with a high survival rate. This provides a fundamental guarantee for its probiotic functions of regulating intestinal flora and promoting metabolic health. In different application products, this strain exhibits a multi-level synergistic mechanism with functional components: in probiotic weight-loss tablets, the phenolic hydroxyl groups of tea polyphenols form a hydrogen bond network with the surface proteins of probiotic ZKAW03, which not only enhances the adhesion and colonization of the strain in the intestines but also activates the bile salt hydrolytic enzymes produced by the strain, significantly enhancing fat decomposition efficiency; in probiotic compound beverages, whey protein and the extracellular polysaccharides secreted during the fermentation of ZKAW03 form a stable protein-polysaccharide complex through hydrophobic interactions and disulfide bond exchange. This structure not only protects the activity of the bacterial strain, but more importantly, it forms a physical barrier in the stomach after entering the human body, slowing down the erosion of probiotics by gastric acid and prolonging the duration of action. In addition, in the probiotic capsule, chitosan and sodium alginate form a polyelectrolyte complex through iontophoresis, which builds a protective layer on the surface of the probiotics, effectively shielding the bacteria from the erosion of gastric acid. Inulin, as a prebiotic, has its fructose chains undergo Maillard reaction with the glycoproteins on the surface of bacterial cells, and the resulting novel glycoprotein complex can significantly enhance the survival rate of the strain in the gastrointestinal tract. Attached Figure Description

[0016] Figure 1 This is a scanning electron microscope image of the ZKAW03 probiotic strain isolated and identified in Example 1 of this invention. Figure 2 These are scanning electron microscope images of the microstructure of Lactobacillus fermentum ZKAW03 used in Examples 2-4 of the present invention and the common Lactobacillus fermentum of Comparative Example 1 after treatment with gastric juice. Figure 3 This is a graph showing the experimental results of the weight loss effect evaluation of this invention. Detailed Implementation

[0017] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0019] Example 1: This example provides a method for isolating and identifying probiotics with weight loss effects as follows: S1: Take 10 intestinal contents samples and mix them with 100 sterile phosphate-buffered saline to obtain a sample suspension. Dilute the bacterial suspension using a 10-fold serial dilution method. Spread the suspension on MRS solid medium containing 0.1% (w / v) L-cysteine ​​and incubate anaerobicly at 37°C for 48 h. Select typical Lactobacillus colonies (milky white, round, smooth surface, and regular edges) for streak purification. Repeat the streak 3 times until the colony morphology is consistent to obtain purified single colonies. S2: After Gram staining of purified single colonies, microscopic observation was performed to screen for Gram-positive strains. Further catalase experiments were conducted by adding 3% hydrogen peroxide solution to the surface of the colonies to screen for 12 Gram-positive, catalase-negative Lactobacillus strains. S3: The above-mentioned Lactobacillus characteristic strains were inoculated into MRS liquid medium and cultured anaerobically at 37°C for 24 hours. After three generations of continuous activation, the third generation bacterial solution was centrifuged at 4°C and 8000 rpm for 10 minutes, and the supernatant was discarded to obtain bacterial sludge. Sterile physiological saline was added to the bacterial sludge for washing to obtain the test strains. The genus was identified using the API 50 CHL identification system, and 8 strains were confirmed to be Lactobacillus. S4: 16S rDNA sequencing, sequence alignment, and homology analysis were performed on eight Lactobacillus strains. One strain showed the highest homology with Limosilactobacillus fermentum, resulting in a novel Lactobacillus strain, Limosilactobacillus fermentum ZKAW03, which is a probiotic with weight-loss effects. Its morphology and structure are as follows: Figure 1 As shown.

[0020] Example 2: This invention provides an application of probiotics with weight loss effects, wherein the application is a probiotic fat-reducing tablet prepared using probiotics with weight loss effects.

[0021] The probiotic weight-loss tablets with weight-loss effects include the following ingredients in parts by weight: 50 parts of freeze-dried probiotic powder, 10 parts of tea polyphenol extract, 20 parts of fructooligosaccharides, 15 parts of microcrystalline cellulose, and 0.5 parts of magnesium stearate. The preparation method of the probiotic freeze-dried powder is as follows: (1) The activated ZKAW03 strain is inoculated into MRS liquid medium and cultured anaerobically at 37℃ for 18 hours until the late logarithmic growth stage. Then, the bacterial sludge is collected by centrifugation at 4℃ and 8000 rpm for 10 minutes and washed twice with sterile physiological saline. (2) The washed bacterial sludge is resuspended in a freeze-drying protectant containing 10% (w / v) trehalose and 5% (w / v) skim milk to make the bacterial concentration reach 1×10¹¹ CFU / mL, and then dispensed into freeze-drying bottles. (3) The dispensed samples are pre-frozen in an ultra-low temperature freezer at -80℃ for 4 hours, and finally freeze-dried in a freeze dryer at -50℃ and 0.1 mBar for 24 hours to obtain highly active ZKAW03 freeze-dried powder, which is the probiotic freeze-dried powder.

[0022] The tea polyphenol extract is a standardized extract extracted from green tea with a tea polyphenol content of ≥98%.

[0023] The preparation process of the probiotic weight-loss tablets with weight-loss effect is as follows: A1 Mixing: Weigh the probiotic freeze-dried powder, tea polyphenol extract, fructooligosaccharides and microcrystalline cellulose accurately according to the above ratio and place them in a V-type mixer. Mix at 30 rpm for 30 minutes until the mixture is uniform to obtain the main drug mixture. A2 Granulation: Slowly spray a 70% ethanol solution as a wetting agent into the active pharmaceutical ingredient mixture while stirring until a soft material that can be formed by squeezing into a ball but crumbles easily when pressed is formed. Granulate the soft material through a 20-mesh sieve to obtain wet granules. A3 Drying: Spread the wet granules evenly on a tray and dry them in a 45°C hot air circulating oven for 40 minutes. A4 Granulation and Mixing: The dried granules are granulated again through a 20-mesh sieve, and then magnesium stearate is added according to the formula. The mixture is then mixed in a mixer at 20 rpm for 5 minutes to obtain the mixed granules. A5 tableting: The total granules are compressed using a rotary tableting machine, with the tablet weight adjusted to 0.5 g / tablet and the pressure set to 5 kN, to obtain unprocessed tablets; A6 Coating: Using a high-efficiency coating pan, the pressed tablets are placed in the pan and coated with an enteric coating solution prepared by dissolving polyacrylic acid resin II in 95% ethanol by volume. The concentration of polyacrylic acid resin II is 6% (w / v), and the weight gain is controlled at 3%. After coating, the tablets are cured at 40°C for 2 hours to obtain the probiotic weight loss tablets.

[0024] This embodiment also provides an application of probiotics with weight-loss effects. The application is a probiotic compound beverage prepared using probiotics with weight-loss effects. The probiotic compound beverage contains the following ingredients in parts by weight: 30 parts probiotic fermentation broth, 10 parts whey protein, 5 parts inulin, 0.1 parts citric acid, and 0.05 parts steviol glycosides. The preparation method of the probiotic fermentation broth is as follows: ZKAW03 strain is inoculated into sterilized MRS liquid culture medium at an inoculation rate of 2% (v / v), and then statically fermented at 37°C for 24 hours until the viable cell count reaches 1×10⁻⁶. 9 A probiotic fermentation broth with a concentration of CFU / mL or higher is obtained.

[0025] The preparation process of the probiotic compound beverage with weight loss effect is as follows: B1: Dissolve whey protein powder and inulin in purified water at 50°C to prepare a 35% (w / v) solution, and stir until completely dissolved to obtain an aqueous matrix; B2: Dissolve citric acid and steviol glycosides completely in warm water to obtain the flavoring liquid; B3: Cool the aqueous phase matrix to room temperature, then mix it with the specified proportions of probiotic fermentation liquid and flavoring liquid in the mixing tank. Use a shear emulsifier to stir at 2000 rpm for 15 minutes to ensure uniform mixing and obtain a mixture. B4: Use pure water to bring the mixture to the required total volume, then transfer it to a high-pressure homogenizer and homogenize it three times under a pressure of 20 MPa to obtain a uniform and stable liquid. B5: The homogenized liquid is aseptically filtered and cold-filled to obtain the probiotic compound beverage.

[0026] This embodiment also provides an application of probiotics with weight-loss effects. The application involves preparing probiotic capsules with weight-loss effects using these probiotics. The probiotic capsules contain the following ingredients in parts by weight: 20 parts lyophilized probiotic powder, 2 parts chitosan, 1 part sodium alginate, 10 parts inulin, and 0.5 parts silica. The preparation method of the lyophilized probiotic powder is the same as that used in probiotic weight-loss tablets. The preparation method of the probiotic capsules with weight-loss effects is as follows: (a) The probiotic freeze-dried powder, inulin and silica were mixed in a mixer for 25 minutes according to the specified ratio to obtain a uniform core material mixture; (b) Dissolve chitosan in a 1% (v / v) acetic acid solution and stir to prepare a 3% (w / v) chitosan solution; dissolve sodium alginate in purified water and stir to prepare a 2% (w / v) sodium alginate solution; (c) The core material mixture is dispersed in a sodium alginate solution of 2 times its weight, and then added dropwise to a calcium chloride solution by a peristaltic pump. After standing and solidifying for 30 minutes, gel microspheres are formed. After washing with sterile water, they are immersed in chitosan solution for 15 minutes for composite coating, and then washed with sterile water to obtain encapsulated probiotic capsules. (d) The washed probiotic capsules are freeze-dried and then filled into hydroxypropyl methylcellulose plant hollow capsule shells to obtain the probiotic capsules.

[0027] Example 3: This example provides an application of probiotics with weight loss effects, wherein the application is a probiotic weight loss tablet prepared using probiotics with weight loss effects.

[0028] The probiotic weight-loss tablets with weight-loss effects include the following ingredients in parts by weight: 60 parts of freeze-dried probiotic powder, 15 parts of tea polyphenol extract, 25 parts of fructooligosaccharides, 17.5 parts of microcrystalline cellulose, and 0.25 parts of magnesium stearate. The preparation method of the probiotic freeze-dried powder is as follows: (1) The activated ZKAW03 strain is inoculated into MRS liquid medium and cultured anaerobically at 37℃ for 18 hours until the late logarithmic growth stage. Then, the bacterial sludge is collected by centrifugation at 4℃ and 8000 rpm for 10 minutes and washed twice with sterile physiological saline. (2) The washed bacterial sludge is resuspended in a freeze-drying protectant containing 10% (w / v) trehalose and 5% (w / v) skim milk to make the bacterial concentration reach 1×10¹¹ CFU / mL, and then dispensed into freeze-drying bottles. (3) The dispensed samples are pre-frozen in an ultra-low temperature freezer at -80℃ for 4 hours, and finally freeze-dried in a freeze dryer at -50℃ and 0.1 mBar for 24 hours to obtain highly active ZKAW03 freeze-dried powder, which is the probiotic freeze-dried powder.

[0029] The tea polyphenol extract is a standardized extract extracted from green tea with a tea polyphenol content of ≥98%.

[0030] The preparation process of the probiotic weight-loss tablets with weight-loss effect is as follows: A1 Mixing: Weigh the probiotic freeze-dried powder, tea polyphenol extract, fructooligosaccharides and microcrystalline cellulose accurately according to the above ratio and place them in a V-type mixer. Mix at 30 rpm for 30 minutes until the mixture is uniform to obtain the main drug mixture. A2 Granulation: Slowly spray a 70% ethanol solution as a wetting agent into the active pharmaceutical ingredient mixture while stirring until a soft material that can be formed by squeezing into a ball but crumbles easily when pressed is formed. Granulate the soft material through a 20-mesh sieve to obtain wet granules. A3 Drying: Spread the wet granules evenly on a tray and dry them in a 45°C hot air circulating oven for 40 minutes. A4 Granulation and Mixing: The dried granules are granulated again through a 20-mesh sieve, and then magnesium stearate is added according to the formula. The mixture is then mixed in a mixer at 20 rpm for 5 minutes to obtain the mixed granules. A5 tableting: The total granules are compressed using a rotary tableting machine, with the tablet weight adjusted to 0.5 g / tablet and the pressure set to 5 kN, to obtain unprocessed tablets; A6 Coating: Using a high-efficiency coating pan, the pressed tablets are placed in the pan and coated with an enteric coating solution prepared by dissolving polyacrylic acid resin II in 95% ethanol by volume. The concentration of polyacrylic acid resin II is 6% (w / v), and the weight gain is controlled at 3%. After coating, the tablets are cured at 40°C for 2 hours to obtain the probiotic weight loss tablets.

[0031] This embodiment also provides an application of probiotics with weight-loss effects. The application is a probiotic compound beverage prepared using probiotics with weight-loss effects. The probiotic compound beverage contains the following ingredients by weight: 40 parts probiotic fermentation broth, 15 parts whey protein, 7.5 parts inulin, 0.2 parts citric acid, and 0.075 parts steviol glycosides. The preparation method of the probiotic fermentation broth is as follows: ZKAW03 strain is inoculated into sterilized MRS liquid culture medium at an inoculation rate of 2% (v / v), and then statically fermented at 37°C for 24 hours until the viable cell count reaches 1×10⁻⁶. 9 A probiotic fermentation broth with a concentration of CFU / mL or higher is obtained.

[0032] The preparation process of the probiotic compound beverage with weight loss effect is as follows: B1: Dissolve whey protein powder and inulin in purified water at 50°C to prepare a 35% (w / v) solution, and stir until completely dissolved to obtain an aqueous matrix; B2: Dissolve citric acid and steviol glycosides completely in warm water to obtain the flavoring liquid; B3: Cool the aqueous phase matrix to room temperature, then mix it with the specified proportions of probiotic fermentation liquid and flavoring liquid in the mixing tank. Use a shear emulsifier to stir at 2000 rpm for 15 minutes to ensure uniform mixing and obtain a mixture. B4: Use pure water to bring the mixture to the required total volume, then transfer it to a high-pressure homogenizer and homogenize it three times under a pressure of 20 MPa to obtain a uniform and stable liquid. B5: The homogenized liquid is aseptically filtered and cold-filled to obtain the probiotic compound beverage.

[0033] This embodiment also provides an application of probiotics with weight-loss effects. The application involves preparing probiotic capsules with weight-loss effects using these probiotics. The probiotic capsules contain the following ingredients in parts by weight: 30 parts lyophilized probiotic powder, 3.5 parts chitosan, 2 parts sodium alginate, 15 parts inulin, and 1.25 parts silica. The preparation method of the lyophilized probiotic powder is the same as that of the probiotic weight-loss tablets. The preparation method of the probiotic capsules with weight-loss effects is as follows: (a) The probiotic freeze-dried powder, inulin and silica were mixed in a mixer for 25 minutes according to the specified ratio to obtain a uniform core material mixture; (b) Dissolve chitosan in a 1% (v / v) acetic acid solution and stir to prepare a 3% (w / v) chitosan solution; dissolve sodium alginate in purified water and stir to prepare a 2% (w / v) sodium alginate solution; (c) The core material mixture is dispersed in a sodium alginate solution of 2 times its weight, and then added dropwise to a calcium chloride solution by a peristaltic pump. After standing and solidifying for 30 minutes, gel microspheres are formed. After washing with sterile water, they are immersed in chitosan solution for 15 minutes for composite coating, and then washed with sterile water to obtain encapsulated probiotic capsules. (d) The washed probiotic capsules are freeze-dried and then filled into hydroxypropyl methylcellulose plant hollow capsule shells to obtain the probiotic capsules.

[0034] Example 4: This example provides an application of probiotics with weight loss effect, wherein the application is a probiotic weight loss tablet prepared using probiotics with weight loss effect.

[0035] The probiotic weight-loss tablets with weight-loss effects include the following ingredients in parts by weight: 70 parts of freeze-dried probiotic powder, 20 parts of tea polyphenol extract, 30 parts of fructooligosaccharides, 20 parts of microcrystalline cellulose, and 2 parts of magnesium stearate. The preparation method of the probiotic freeze-dried powder is as follows: (1) The activated ZKAW03 strain is inoculated into MRS liquid medium and cultured anaerobically at 37℃ for 18 hours until the late logarithmic growth stage. Then, the bacterial sludge is collected by centrifugation at 4℃ and 8000 rpm for 10 minutes and washed twice with sterile physiological saline. (2) The washed bacterial sludge is resuspended in a freeze-drying protectant containing 10% (w / v) trehalose and 5% (w / v) skim milk to make the bacterial concentration reach 1×10¹¹ CFU / mL, and then dispensed into freeze-drying bottles. (3) The dispensed samples are pre-frozen in an ultra-low temperature freezer at -80℃ for 4 hours, and finally freeze-dried in a freeze dryer at -50℃ and 0.1 mBar for 24 hours to obtain highly active ZKAW03 freeze-dried powder, which is the probiotic freeze-dried powder.

[0036] The tea polyphenol extract is a standardized extract extracted from green tea with a tea polyphenol content of ≥98%.

[0037] The preparation process of the probiotic weight-loss tablets with weight-loss effect is as follows: A1 Mixing: Weigh the probiotic freeze-dried powder, tea polyphenol extract, fructooligosaccharides and microcrystalline cellulose accurately according to the above ratio and place them in a V-type mixer. Mix at 30 rpm for 30 minutes until the mixture is uniform to obtain the main drug mixture. A2 Granulation: Slowly spray a 70% ethanol solution as a wetting agent into the active pharmaceutical ingredient mixture while stirring until a soft material that can be formed by squeezing into a ball but crumbles easily when pressed is formed. Granulate the soft material through a 20-mesh sieve to obtain wet granules. A3 Drying: Spread the wet granules evenly on a tray and dry them in a 45°C hot air circulating oven for 40 minutes. A4 Granulation and Mixing: The dried granules are granulated again through a 20-mesh sieve, and then magnesium stearate is added according to the formula. The mixture is then mixed in a mixer at 20 rpm for 5 minutes to obtain the mixed granules. A5 tableting: The total granules are compressed using a rotary tableting machine, with the tablet weight adjusted to 0.5 g / tablet and the pressure set to 5 kN, to obtain unprocessed tablets; A6 Coating: Using a high-efficiency coating pan, the pressed tablets are placed in the pan and coated with an enteric coating solution prepared by dissolving polyacrylic acid resin II in 95% ethanol by volume. The concentration of polyacrylic acid resin II is 6% (w / v), and the weight gain is controlled at 3%. After coating, the tablets are cured at 40°C for 2 hours to obtain the probiotic weight loss tablets.

[0038] This embodiment also provides an application of probiotics with weight-loss effects. The application is a probiotic compound beverage prepared using probiotics with weight-loss effects. The probiotic compound beverage contains the following ingredients in parts by weight: 50 parts probiotic fermentation broth, 20 parts whey protein, 10 parts inulin, 0.3 parts citric acid, and 0.1 parts steviol glycosides. The preparation method of the probiotic fermentation broth is as follows: ZKAW03 strain is inoculated into sterilized MRS liquid culture medium at an inoculation rate of 2% (v / v), and then statically fermented at 37°C for 24 hours until the viable cell count reaches 1×10⁻⁶. 9 A probiotic fermentation broth with a concentration of CFU / mL or higher is obtained.

[0039] The preparation process of the probiotic compound beverage with weight loss effect is as follows: B1: Dissolve whey protein powder and inulin in purified water at 50°C to prepare a 35% (w / v) solution, and stir until completely dissolved to obtain an aqueous matrix; B2: Dissolve citric acid and steviol glycosides completely in warm water to obtain the flavoring liquid; B3: Cool the aqueous phase matrix to room temperature, then mix it with the specified proportions of probiotic fermentation liquid and flavoring liquid in the mixing tank. Use a shear emulsifier to stir at 2000 rpm for 15 minutes to ensure uniform mixing and obtain a mixture. B4: Use pure water to bring the mixture to the required total volume, then transfer it to a high-pressure homogenizer and homogenize it three times under a pressure of 20 MPa to obtain a uniform and stable liquid. B5: The homogenized liquid is aseptically filtered and cold-filled to obtain the probiotic compound beverage.

[0040] This embodiment also provides an application of probiotics with weight-loss effects. The application involves preparing probiotic capsules with weight-loss effects using probiotics with weight-loss effects. The probiotic capsules contain the following ingredients in parts by weight: 40 parts of freeze-dried probiotic powder, 5 parts of chitosan, 3 parts of sodium alginate, 20 parts of inulin, and 2 parts of silica. The preparation method of the freeze-dried probiotic powder is the same as that of the probiotic weight-loss tablets. The preparation method of the probiotic capsules with weight-loss effects is as follows: (a) The probiotic freeze-dried powder, inulin and silica were mixed in a mixer for 25 minutes according to the specified ratio to obtain a uniform core material mixture; (b) Dissolve chitosan in a 1% (v / v) acetic acid solution and stir to prepare a 3% (w / v) chitosan solution; dissolve sodium alginate in purified water and stir to prepare a 2% (w / v) sodium alginate solution. (c) The core material mixture is dispersed in a sodium alginate solution of 2 times its weight, and then added dropwise to a calcium chloride solution by a peristaltic pump. After standing and solidifying for 30 minutes, gel microspheres are formed. After washing with sterile water, they are immersed in chitosan solution for 15 minutes for composite coating, and then washed with sterile water to obtain encapsulated probiotic capsules. (d) The washed probiotic capsules are freeze-dried and then filled into hydroxypropyl methylcellulose plant hollow capsule shells to obtain the probiotic capsules.

[0041] Comparative Example 1: The difference between Comparative Example 1 and Example 2 is that the probiotic ZKAW03 is replaced with ordinary Lactobacillus fermentum, and the rest is exactly the same as Example 2.

[0042] Experimental methods: Gastrointestinal tolerance test: The seed culture of Lactobacillus fermentum ZKAW03 in Examples 2-4 and the common Lactobacillus fermentum used in Comparative Example 1 were inoculated into simulated gastric juice at pH 2.5 (containing 0.3% pepsin), and cultured at 37°C. Samples were taken at 1 hour and 2 hours for plate colony counting, and the gastric acid tolerance survival rate was calculated. The results are shown in Table 1.

[0043] Observation of probiotic surface morphology: The morphology of the ZKAW03 probiotics isolated and identified in Example 1 was observed using scanning electron microscopy. The results are as follows: Figure 1 As shown; in addition, the *Lactobacillus fermentum* ZKAW03 used in Examples 2-4 and the common *Lactobacillus fermentum* of Comparative Example 1 were treated with simulated gastric juice (pH 2.5) for 2 hours, and the cells were collected by centrifugation. After fixation with glutaraldehyde, gradient dehydration with ethanol, and critical point drying, the integrity and changes of the ultrastructure on the cell surface were observed using scanning electron microscopy. The results are shown in the figure. Figure 2 As shown.

[0044] Bile salt hydrolase activity assay: ZKAW03 from Examples 2-4 and Lactobacillus fermentum from Comparative Example 1 were taken, resuspended in phosphate buffer at pH 6.5, ultrasonically disrupted in an ice bath, and centrifuged to obtain crude enzyme solution. A 10 mL reaction system (containing 5 mL phosphate buffer, 1 mL substrate, and 2 mL crude enzyme solution) was constructed using sodium taurocholate as substrate. The reaction was carried out in a water bath at 37℃ for 1 h and then terminated with 30% trichloroacetic acid. The filtrate was titrated to pH 7.0 with 0.05 mol / L NaOH. The bile salt hydrolase activity was calculated based on the volume of NaOH consumed, and the results are shown in Table 2.

[0045] Evaluation of weight loss effect: A high-fat diet-induced obese C57BL / 6J mouse model was used. Successfully modeled mice were randomly divided into a model group (a), an intervention group (Example 2), and a control group (c). A control group fed a normal diet (d) was also included. The Example 2 interference group received daily gavage containing 1×10⁻⁶ fat.9 Mice in the control group were given the same amount of CFU ZKAW03 bacterial suspension daily. The model group and control group were given the same volume of physiological saline by gavage. The intervention period was 8 weeks. Body weight was measured weekly during the experiment. After the intervention, blood samples were collected to detect lipid levels (total cholesterol TC, triglycerides TG), and epididymal and retroperitoneal adipose tissues were separated and weighed. The results are as follows: Figure 3 As shown.

[0046] Table 1 Gastrointestinal Tolerance Test ; Table 2 Results of bile salt hydrolytic enzyme activity assay .

[0047] As can be seen from the data in Table 1, ZKAW03 in all examples exhibited significantly higher gastric acid tolerance than Comparative Example 1, and there was no significant difference in gastric acid tolerance among the different examples, demonstrating the acid stability of the strain itself. Figure 2 As can be seen, compared to Comparative Example 1, the bacterial cells in Examples 2-4 maintained their basic short rod-shaped morphology, with only slight surface wrinkling and good structural integrity, further demonstrating their good tolerance. Bile salt hydrolase is a key enzyme regulating lipid metabolism, and its activity directly affects the efficiency of bile salt decomposition. Table 2 shows that the ZKAW03 strains used in Examples 2-4 all exhibited high bile salt hydrolase activity, enabling them to more efficiently catalyze bile salt decomposition and promote cholesterol excretion, providing core enzymatic support for their weight-loss function. Figure 3 As can be seen, the ZKAW03 provided in Example 2 can significantly improve obesity, body fat accumulation and abnormal blood lipids caused by a high-fat diet, with excellent results.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0049] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A probiotic with weight-loss effects, characterized in that, The probiotic with weight loss effect is Limosilactobacillus fermentum ZKAW03, with accession number GDMCC NO: 65680; the isolation and identification method of the probiotic with weight loss effect is as follows: S1: Prepare a suspension of healthy human intestinal contents, dilute, spread, and culture anaerobically, then purify to obtain purified colonies; S2: The purified colonies were subjected to Gram staining and catalase experiments to obtain characteristic strains of Lactobacillus. S3: The characteristic strains of Lactobacillus were identified by genus to obtain Lactobacillus strains; S4: Sequencing, sequence comparison, and homology analysis of Lactobacillus strains were performed to obtain probiotics with weight loss effects.

2. An application of a probiotic with weight-loss effect according to claim 1, characterized in that, The application refers to probiotic weight-loss tablets, probiotic compound beverages, and probiotic capsules prepared using probiotics with weight-loss effects; the probiotics with weight-loss effects are in the form of freeze-dried probiotic powder in the preparation of probiotic weight-loss tablets and probiotic capsules; the probiotics with weight-loss effects are in the form of probiotic fermentation liquid in the preparation of probiotic compound beverages.

3. The application of a probiotic with weight-loss effect according to claim 2, characterized in that, The preparation method of the probiotic freeze-dried powder is as follows: (1) The activated Limosilactobacillus fermentum ZKAW03 strain is anaerobically cultured to the late logarithmic growth stage, and then centrifuged and washed with sterile physiological saline; (2) The washed bacterial sludge is resuspended in a freeze-drying protectant and dispensed into freeze-drying bottles; (3) The dispensed samples are pre-freeze-dried to obtain highly active Limosilactobacillus fermentum ZKAW03 freeze-dried powder, which is the probiotic freeze-dried powder.

4. The application of a probiotic with weight-loss effect according to claim 3, characterized in that, The probiotic weight loss tablets comprise the following ingredients in parts by weight: 50-70 parts of freeze-dried probiotic powder, 10-20 parts of tea polyphenol extract, 20-30 parts of fructooligosaccharides, 15-20 parts of microcrystalline cellulose, and 0.5-2 parts of magnesium stearate; the tea polyphenol extract is a standardized extract extracted from green tea with a tea polyphenol content ≥98%.

5. The application of a probiotic with weight-loss effect according to claim 4, characterized in that, The preparation process of the probiotic weight loss tablets is as follows: A1 Mixing: Accurately weigh the probiotic freeze-dried powder, tea polyphenol extract, fructooligosaccharides and microcrystalline cellulose according to the weight ratio, mix them evenly to obtain the main drug mixture; A2 Granulation: Slowly spray an ethanol solution into the active pharmaceutical ingredient mixture while stirring to form a soft mass. Granulate the soft mass to obtain wet granules. A3 Drying: Drying wet granules; A4 Granulation and Mixing: The dried granules are screened, and then magnesium stearate is added according to the formula to obtain the mixed granules; A5 tableting: The total mixture of granules is compressed into tablets to obtain plain tablets; A6 Coating: The compressed tablets are coated with enteric coating to obtain the probiotic weight loss tablets.

6. The application of a probiotic with weight-loss effect according to claim 2, characterized in that, The probiotic compound beverage comprises the following ingredients in parts by weight: 30-50 parts probiotic fermentation broth, 10-20 parts whey protein, 5-10 parts inulin, 0.1-0.3 parts citric acid, and 0.05-0.1 parts steviol glycosides. The probiotic fermentation broth is prepared by inoculating strain ZKAW03 into MRS liquid culture medium and anaerobic fermenting at 37°C for 24 hours to obtain the probiotic fermentation broth.

7. The application of a probiotic with weight-loss effect according to claim 6, characterized in that, The preparation process of the probiotic compound beverage is as follows: B1: Dissolve whey protein powder and inulin in purified water and stir until completely dissolved to obtain an aqueous matrix; B2: Dissolve citric acid and steviol glycosides in warm water to obtain a flavoring liquid; B3: Cool the aqueous phase matrix to room temperature, and then mix it with the specified amount of probiotic fermentation broth and flavoring liquid to obtain a mixture; B4: Use purified water to bring the mixture to the required total volume, and then homogenize it to obtain a uniform and stable liquid. B5: The homogenized liquid is aseptically filtered and cold-filled to obtain the probiotic compound beverage.

8. The application of a probiotic with weight-loss effect according to claim 3, characterized in that, The probiotic capsules contain the following ingredients in parts by weight: 20-40 parts of freeze-dried probiotic powder, 2-5 parts of chitosan, 1-3 parts of sodium alginate, 10-20 parts of inulin, and 0.5-2 parts of silicon dioxide.

9. The application of a probiotic with weight-loss effect according to claim 8, characterized in that, The preparation method of the probiotic capsules is as follows: (a) The probiotic freeze-dried powder, inulin and silica are mixed in the specified proportions to obtain a uniform core material mixture; (b) Dissolve chitosan in acetic acid solution and stir to prepare chitosan solution; at the same time, dissolve sodium alginate in pure water to prepare sodium alginate solution. (c) The core material mixture is dispersed in a sodium alginate solution of 2 times its weight, then added dropwise to a calcium chloride solution, allowed to stand and solidify to form gel microspheres, washed with sterile water, immersed in chitosan solution for reaction and composite coating, and then washed with sterile water to obtain encapsulated probiotic capsules. (d) The washed probiotic capsules are freeze-dried and then filled into hydroxypropyl methylcellulose plant hollow capsule shells to obtain the probiotic capsules.

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