Postbiotic preparation with weight-reducing and fat-reducing effects as well as preparation method and application thereof

Abstract

The invention discloses a postbiotic preparation with weight and fat reducing effects as well as a preparation method and application of the postbiotic preparation, and belongs to the technical field of microbial fermentation preparations and food. Comprising inactivated probiotics, a compound fermentation substrate and metabolites generated by fermenting the compound fermentation substrate by the probiotics, the probiotics are casei paracasei; the composite fermentation substrate comprises apples, potatoes, corn stigma and oranges. According to the invention, the lactobacillus paracasei is adopted as the probiotics to ferment the composite fermentation substrate containing a plurality of natural dietary components, so that the postbiotic preparation with dual effects of reducing weight and fat is successfully developed. The metagen preparation can help people with weight management requirements to reduce fat and weight, and has good practical application value.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation preparations and food technology, specifically to a postbiotic preparation with weight loss and fat reduction effects, its preparation method, and its application. Background Technology

[0002] With socio-economic development and improved living standards, changes in dietary structure have led to a rapid increase in overweight and obese individuals. Obesity is not only closely related to chronic metabolic diseases such as hypertension, diabetes, and cardiovascular and cerebrovascular diseases, but it also exacerbates the medical burden and affects working capacity and life expectancy.

[0003] Currently, common weight management methods mainly include diet control, beauty treatments, and medical interventions, but all have certain limitations. For example, many people lose weight by controlling their diet or even intermittent fasting, but the effect is limited, rebound is common, and it can easily lead to malnutrition. Beauty treatments guide clients to lose weight through massage, acupuncture, and meal replacement powders, but the effect is unstable, and some institutions use products with hidden ingredients that can cause side effects. Medical interventions, such as weight loss drugs and liposuction, are expensive, only achieve localized fat reduction, and consumers must bear the side effects of drugs and the risks of surgery. In addition, in recent years, more and more health products and functional foods claiming to have weight loss effects have emerged on the market. However, these products generally achieve weight loss by promoting defecation and emptying the intestines. However, this method of weight loss through defecation is only a temporary solution, and the anthraquinones in the defecation-promoting ingredients can irritate the colon, and in severe cases, even induce colon cancer. The side effects cannot be ignored. Therefore, how to achieve scientific, side-effect-free, healthy, and sustainable weight loss without rebound has become an urgent problem to be solved.

[0004] From the perspective of obesity mechanisms, its essence is that energy intake consistently exceeds expenditure, leading to fat accumulation. Therefore, an ideal weight loss strategy needs to balance reducing calorie intake with promoting energy expenditure. Recent studies have found that gut peptides such as glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) play key roles in regulating appetite, delaying gastric emptying, and enhancing satiety. For example, GLP-1 receptor agonists (such as smegglutinin) can suppress appetite through the central nervous system, but as prescription drugs, their applicability is limited and they are accompanied by adverse reactions. CCK, as a classic gut peptide, can regulate eating behavior through the gut-brain axis feedback, synergistically enhancing weight loss with GLP-1. That is, CCK can help regulate GLP-1 to some extent, and the two work synergistically to suppress appetite, thereby reducing calorie intake. Although existing technologies, such as Chinese patent CN112608378A, have developed a dual agonist of GLP-1 / CCK-1 receptors, they belong to the pharmaceutical field, which has a high application threshold and thus limits their application scope. Another example is Chinese patent CN113288997A, which proposes a probiotic composition that regulates intestinal flora and promotes weight loss, but the effect is greatly affected by individual differences, which may lead to unstable effects of the composition.

[0005] Studies have shown that CCK secretion can be regulated by dietary components, providing a new approach to effectively regulate metabolism through the consumption of functional foods. Therefore, developing a weight loss program based on natural dietary components that can synergistically activate the GLP-1 and CCK pathways has significant application value. Summary of the Invention

[0006] The purpose of this invention is to provide a post-biotic preparation with weight loss and fat reduction effects to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An epibiotic preparation with weight loss and fat reduction effects is made by fermentation of probiotics; the epibiotic preparation includes inactivated probiotics, a complex fermentation substrate, and metabolites produced by the fermentation of the complex fermentation substrate by the probiotics; the probiotics are Lactobacillus paracasei; the complex fermentation substrate includes a variety of natural dietary components.

[0009] Preferably, the probiotic is Lactaseibacillus paracasei HBC003, deposited at the China Center for Type Culture Collection (CCTCC) on June 23, 2025, with accession number CCTCCNO: M 20251447, and deposited at Wuhan University, Wuhan, China.

[0010] Preferably, the composite fermentation substrate includes apples, potatoes, corn silk, and citrus fruits.

[0011] Preferably, the composite fermentation base comprises the following components by weight: 25-35 parts apple, 25-35 parts potato, 1-5 parts corn silk, and 1-5 parts citrus.

[0012] Another object of the present invention is to provide a method for preparing the above-mentioned postbiotic preparation with weight loss and fat reduction effects, comprising the following steps:

[0013] The probiotics were streaked onto an activation medium for activation to obtain activated strains;

[0014] Select activated bacterial strains and incubate them statically in activation culture medium to obtain seed culture;

[0015] The seed culture was inoculated into a seed tank containing an activated culture medium for cultivation to obtain a culture medium. The culture medium was then centrifuged and resuspended in sterile physiological saline to obtain a bacterial suspension.

[0016] The bacterial suspension was inoculated into a composite fermentation substrate for fermentation, and then inactivated to obtain the postbiotic preparation.

[0017] Preferably, the activation culture medium comprises the following components in concentration: peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 3-5 g / L, glucose 18-22 g / L, dipotassium hydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.04-0.06 g / L, Tween-80 0.5-1.5 g / L, and agar 12-18 g / L.

[0018] Preferably, the activated culture medium comprises the following components in concentration: peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 3-5 g / L, glucose 18-22 g / L, dipotassium hydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.04-0.06 g / L, Tween-80 0.5-1.5 g / L, and L-cysteine ​​hydrochloride 0.4-0.6 g / L.

[0019] Preferably, the preparation method of the composite fermentation substrate includes the following steps: 25-35 parts of apple, 25-35 parts of potato, 1-5 parts of corn silk and 1-5 parts of citrus are washed with sterile water to remove impurities, crushed into a homogenate, diluted in 1000-1500 parts of pure water, and then sterilized to obtain the composite fermentation substrate.

[0020] Preferably, the mass ratio of the bacterial suspension to the composite fermentation substrate is 1:(10-20).

[0021] Preferably, after inoculating the bacterial suspension into the composite fermentation substrate for fermentation and then performing an inactivation treatment, the process further includes a vacuum low-temperature drying treatment step.

[0022] Another object of the present invention is to provide the application of the above-mentioned postbiotic preparation in the preparation of functional foods for weight loss.

[0023] This invention screened and obtained a strain of *Lactobacillus paracasei* HBC003. Based on this strain and a compound fermentation substrate made from various natural dietary ingredients, a postbiotic preparation was successfully developed, which can help people with weight management needs to lose fat and weight. The postbiotic preparation of this invention can be made into functional foods with fat-reducing and weight-loss effects in different dosage forms such as liquids, powders, and compressed candies, and has good practical application value. This invention, based on the inherent efficacy characteristics of natural plants, screened and scientifically compounded various raw materials to prepare a compound fermentation substrate. Simultaneously, utilizing the positive effect of probiotic fermentation, the compound fermentation substrate was fermented under suitable conditions to release the active substances and produce the postbiotic preparation. After being taken into the human body, this postbiotic preparation can scientifically amplify the satiety signal of food and prolong gastric emptying time by simultaneously stimulating GLP-1 receptors and CCK receptors, thereby reducing calorie intake by enhancing the feeling of food satisfaction. At the same time, it promotes the browning of white fat and enhances calorie consumption. It can also alleviate insulin resistance, protect pancreatic function, inhibit α-amylase activity, and prolong physical endurance, among other things, to safely achieve the dual effects of fat reduction and weight loss through multiple dimensions and physiological mechanisms. Attached Figure Description

[0024] Figure 1 This is a colony morphology diagram of the HBC003 strain isolated in an embodiment of the present invention on an MRS plate.

[0025] Figure 2 This is a morphological image of the HBC003 strain isolated in an embodiment of the present invention under an optical microscope.

[0026] Figure 3 This is a comparison chart of blood glucose concentrations in zebrafish from different groups during the efficacy verification of this invention embodiment; compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0027] Figure 4 This is a comparison chart of cholesterol levels in zebrafish from different groups during the efficacy verification of this invention embodiment; compared with the model control group, *p<0.05, **p<0.01, ***p<0.001.

[0028] Figure 5 This is a comparison of the expression of appetite-suppressing genes in zebrafish during the efficacy verification of this invention embodiment; compared with the model control group, *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] In one embodiment of the present invention, a postbiotic preparation with weight loss and fat reduction effects is provided, which is made by fermentation of probiotics; specifically, the postbiotic preparation includes inactivated probiotics, a complex fermentation substrate, and metabolites produced by the fermentation of the complex fermentation substrate by the probiotics. The probiotics are *Lactobacillus paracasei*; the complex fermentation substrate includes various natural dietary components; and the metabolites include, but are not limited to, extracellular polysaccharides, polypeptides, and amino acids.

[0031] In practical applications, the preferred probiotic is *Lactaseibacillus paracasei* HBC003. *Lactaseibacillus paracasei* HBC003 is deposited at the China Center for Type Culture Collection (CCTCC) on June 23, 2025, with accession number CCTCC NO: M 20251447, located at Wuhan University, Wuhan, China. *Lactaseibacillus paracasei* HBC003 can be purchased directly from the collection.

[0032] In a preferred embodiment of the present invention, the compound fermentation substrate includes apples, potatoes, corn silk, and citrus fruits. The combination of various natural components in the compound fermentation substrate can achieve functional synergy, simultaneously affecting two important metabolic hormone receptors, CCK and GLP-1. Apple polyphenols, a collective term for the polyphenolic substances contained in apples, can effectively promote GLP-1 release and upregulate the expression of hypothalamic appetite-inhibiting neuropeptides, thereby suppressing appetite. Apple polyphenols can also reduce food intake by remodeling the gut microbiota and increase the body's thermogenesis by promoting fat browning. Potato protein components include potato protease inhibitors, which have a regulatory effect on diet; they can stimulate the release of CCK receptors, thereby increasing satiety. Corn silk contains various chemical components, such as volatile oils, saponins, alkaloids, flavonoids, pentosans, allantoin, organic acids, and vitamin E. Modern pharmacological studies have proven that corn silk has significant effects in lowering blood sugar, inhibiting bacteria, lowering blood pressure, and enhancing immunity. In addition, corn silk polysaccharides can effectively prolong gastric emptying time and inhibit appetite and weight gain in mice. Citrus polyphenols in citrus fruits can safely and effectively promote fat burning. The selection and rational combination of these natural raw materials with glycemic metabolism regulating functions lays the foundation for the preparation of safe and effective post-biotic formulations.

[0033] Of course, the compound fermented base may also include other natural dietary ingredients that regulate blood sugar metabolism and increase satiety, such as white kidney beans and bitter melon. Preferably, the compound fermented base is made from the following components by weight: 25-35 parts apple, 25-35 parts potato, 1-5 parts corn silk, and 1-5 parts citrus.

[0034] However, since the human body's absorption efficiency of polyphenols is not high, it is necessary to improve the bioactivity and bioavailability of polyphenols in raw materials in order to maximize their efficacy. Microbial fermentation technology can alter the composition and content of polyphenols in raw materials. Through fermentation, not only can bound polyphenols be released, but they can also be converted into more active substances, thereby improving their bioavailability and influencing two important metabolic hormone receptors, CCK and GLP-1.

[0035] Specifically, in another embodiment of the present invention, a method for preparing the above-mentioned postbiotic preparation with weight loss and fat reduction effects is also provided, comprising the following steps:

[0036] The probiotics were streaked onto an activation medium for activation to obtain activated strains;

[0037] Select activated bacterial strains and incubate them statically in an activated culture medium to obtain seed culture. The static incubation temperature is 35-37℃, pH = 6.2±0.2, and the time is 18-22h. It should be noted that the seed culture can be further expanded according to the size of the culture system to prepare secondary seed culture.

[0038] The seed culture was inoculated into a seed tank containing activated culture medium for cultivation to obtain a culture medium. After centrifugation, the culture medium was resuspended in sterile physiological saline to obtain a bacterial suspension. The inoculation amount of the seed culture was 1%-5%, v / v. The cultivation temperature for this step was 35-37℃, pH=6.2±0.2, pressure was 0.03-0.05MPa, and the cultivation time was 18-22h.

[0039] The bacterial suspension was inoculated into a composite fermentation substrate for fermentation, and then inactivated to obtain the post-biotic preparation. The fermentation temperature was 35-37℃, pH = 6.2±0.2, pressure was 0.03-0.05MPa, and time was 24-30h. The specific method for inactivation treatment was to maintain the temperature at 85℃ for 50min.

[0040] Preferably, the activated culture medium comprises the following components in concentration: peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 3-5 g / L, glucose 18-22 g / L, dipotassium hydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.04-0.06 g / L, Tween-80 0.5-1.5 g / L, and agar 12-18 g / L. The activated culture medium must be sterilized at 121°C for 20 minutes before use.

[0041] Preferably, the activated culture medium comprises the following components in concentration: peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 3-5 g / L, glucose 18-22 g / L, dipotassium hydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.04-0.06 g / L, Tween-80 0.5-1.5 g / L, and L-cysteine ​​hydrochloride 0.4-0.6 g / L. The activated culture medium must be sterilized at 121°C for 20 min before use.

[0042] In a preferred embodiment of the present invention, the preparation method of the composite fermentation substrate includes the following steps: 25-35 parts of apple, 25-35 parts of potato, 1-5 parts of corn silk, and 1-5 parts of citrus are washed with sterile water to remove impurities, crushed into a homogenate, diluted in 1000-1500 parts of pure water, sterilized by high-pressure steam, cooled to 25-35°C and held for 20-40 minutes to obtain the composite fermentation substrate.

[0043] Preferably, the mass ratio of the bacterial suspension to the composite fermentation substrate is 1:(10-20).

[0044] Preferably, after inoculating the bacterial suspension into a composite fermentation substrate for fermentation and then performing an inactivation treatment, a vacuum low-temperature drying step is also included. During the vacuum low-temperature drying process, the temperature of the drying chamber can be controlled to remain below 60°C, and the product can be made into powder through vacuum low-temperature drying.

[0045] In another embodiment of the present invention, the application of the above-mentioned postbiotic preparation in the preparation of functional foods for weight loss is also provided. The food includes, but is not limited to, ordinary foods and special dietary foods, and the dosage form can be liquid or solid dosage forms; the food ingredients may also include at least one other component; other components may be carriers, excipients, and diluents commonly used in the food industry, or additives such as sweet and sour flavorings. According to conventional methods, it can be made into solid dosage forms such as solid beverages and compressed candies; the carrier, sweet and sour flavorings, and other components are well known to those skilled in the art and will not be described in detail here.

[0046] In an embodiment of the present invention, by using *Lactobacillus paracasei* as a probiotic to ferment a complex fermentation substrate containing various natural dietary components, a postbiotic preparation with dual effects of weight loss and fat reduction was successfully developed. The *Lactobacillus paracasei* HBC003 provided in this embodiment of the invention, after functional evaluation, showed an increase in polyphenol content in the fermented complex fermentation substrate, indicating that this strain can activate the release of effective components in each part of the raw materials. This helps to amplify the efficacy of natural raw materials while truly enabling users to reduce calorie intake and increase calorie expenditure, achieving safe and effective fat reduction and weight loss without digestive system side effects. The postbiotic preparation obtained in this embodiment of the invention can be used as a compound raw material combined with other flavor components, or it can be added to solid dosage forms such as solid beverages and compressed candies through vacuum low-temperature drying powdering, facilitating the customization of different types of weight loss products for use by various groups, expanding the product's application range, and thus possessing good application value.

[0047] Example 1: This example provides a method for isolating probiotics, specifically including the following steps:

[0048] Homemade kimchi broth was used as the separation sample, specifically the broth from the bottom of a kimchi jar that had been stored for a while but had not spoiled. Lactic acid bacteria are concentrated during the long kimchi-making process.

[0049] Prepare sterile physiological saline and dilute the samples using a serial dilution method. Prepare MRS solid agar plates, spread 0.1 mL of each dilution gradient sample onto the plates, and incubate anaerobically at 37°C for 48 h. Select colonies with different calcium dissolution zones, colony morphology, size, and color, streak them on MRS solid agar plates for purification, and incubate for 48 h. Select single colonies, repeat the purification and isolation process 3-5 times, and store the purified single colonies in 20% glycerol tubes at -80°C for later use.

[0050] A purified single colony preserved in glycerol tubes was activated and centrifuged. The acid production in the fermentation supernatant was measured to infer the basic fermentation properties of the colony. The target strain was used to ferment mixed fruit and vegetable juices of apple, potato, corn silk, and citrus. The changes in flavor and total phenolic content of the fruit and vegetable juices before and after fermentation were evaluated. Strain HBC003, which showed significant acid production, marked flavor improvement, and a significant increase in the total phenolic content of the fruit and vegetable juices, was selected. After 16S rRNA gene extraction and identification, this strain was identified as *Lactaseibacillus paracasei* and deposited at the China Center for Type Culture Collection (CCTCC) on June 23, 2025, with accession number CCTCC NO: M 20251447, at Wuhan University, Wuhan, China.

[0051] Example 2: This example provides a post-natal vitamin preparation with weight loss and fat reduction effects. The preparation method specifically includes the following steps:

[0052] S1. In a sterile operating table, streak *Lactobacillus paracasei* HBC003, stored in an ultra-low temperature freezer at -80℃, onto an activation medium plate. Pick a single colony and repeat the streaking activation process three times to obtain the activated strain. The colony morphology and cell morphology of *Lactobacillus paracasei* HBC003 under an optical microscope are shown below. Figure 1 and Figure 2 As shown. The components of the activation culture medium are as follows: peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, agar 15.0 g / L, Tween-80 1.0 g / L, and the pH of the culture medium is 6.5 ± 0.2 (25℃).

[0053] S2. Single colonies of the activated strain were picked and inoculated into 100 mL of activation culture medium in a blue-capped bottle. The culture was incubated at 37°C for 18 h to obtain a primary seed culture. This primary seed culture was then transferred again to 2 L of activation culture medium for further expansion to obtain a secondary seed culture. The activation culture medium consisted of the following components: peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L, and the pH of the culture medium was 6.5 ± 0.2 (25°C).

[0054] S3. Under aseptic conditions, inoculate the prepared secondary seed culture at a rate of 3% into a seed tank containing sterilized activated culture medium. Incubate at 37℃, pH 6.2, and a pressure of 0.03-0.05 MPa for 18 hours to obtain a culture medium. Centrifuge the culture medium and resuspend it in pure water to obtain a bacterial suspension. Inoculate the bacterial suspension into a steam-sterilized composite fermentation substrate at a mass ratio of 1:20. Ferment at 37℃, pH 6.2, and a tank pressure maintained at 0.03-0.05 MPa for 26 hours. Stop fermentation and maintain the temperature at 85℃ for 50 minutes. After cooling, the post-fermentation agent is obtained. The preparation method of the composite fermentation substrate includes the following steps: Wash 25 kg of apples, 25 kg of potatoes, 3 kg of corn silk, and 1 kg of citrus with sterile water to remove impurities, then crush them into a homogenate. Dilute the homogenate in 1000 kg of pure water, then sterilize by high-pressure steam and cool to room temperature for 30 minutes to obtain the composite fermentation substrate.

[0055] The post-biotic preparation obtained in Example 2 can have its taste adjusted by combining it with other ingredients such as crystalline trehalose. The post-biotic preparation was tested and found to contain 70 kJ of energy, 1.2g of protein, and 0g of fat per 100mL.

[0056] Example 3: This example provides a post-natal vitamin preparation with weight loss and fat reduction effects. The preparation method specifically includes the following steps:

[0057] S1. In a sterile operating table, streak *Lactobacillus paracasei* HBC003, stored in an ultra-low temperature freezer at -80℃, onto an activation medium plate. Pick a single colony and repeat the streak to obtain the activated strain. The activation medium consists of the following components: peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, agar 15.0 g / L, Tween-80 1.0 g / L, and the pH of the medium is 6.5 ± 0.2 (25℃).

[0058] S2. Select a single colony of the activated strain and inoculate it into 100 mL of activation culture medium in a blue-capped bottle. Incubate at 35°C for 22 h to obtain the primary seed culture. Transfer the primary seed culture to 2 L of activation culture medium again for further expansion to obtain the secondary seed culture. The composition of the activation culture medium is as follows: peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L, and the pH of the culture medium is 6.5 ± 0.2 (25°C).

[0059] S3. Under aseptic conditions, inoculate the prepared secondary seed culture at a 3% inoculation rate (v / v) into a seed tank containing sterilized activated culture medium. Incubate at 35℃, pH=6.2, and tank pressure maintained at 0.03-0.05MPa for 22 hours to obtain a culture medium. Then, centrifuge the culture medium and resuspend it in pure water to obtain a bacterial suspension. Inoculate the bacterial suspension into a steam-sterilized composite fermentation substrate at a mass ratio of 1:15. Incubate at 35℃, stirring speed of 30rpm, pH=6.2, and tank pressure maintained at 0.03-0.05MPa for 3 hours. After stirring, maintain the tank pressure and continue fermentation. After 30 hours, stop fermentation and maintain 85℃ for 50 minutes. After cooling to room temperature, add resistant dextrin to the fermentation system in a mixing tank and mix well. Then, use a vacuum low-temperature drying oven for low-temperature drying, keeping the drying temperature below 60℃. Finally, pulverize and prepare a powder for the preparation of a bioactive agent. The preparation method of the compound fermentation substrate includes the following steps: 30 kg of apples, 30 kg of potatoes, 5 kg of corn silk and 5 kg of citrus are washed with sterile water to remove impurities, crushed into a homogenate, diluted in 1500 kg of pure water, sterilized by high pressure steam, cooled to 35°C and kept for 30 min to obtain the compound fermentation substrate.

[0060] Example 4: This example provides a post-natal vitamin preparation with weight loss and fat reduction effects. The preparation method specifically includes the following steps:

[0061] S1. In a sterile operating table, streak *Lactobacillus paracasei* HBC003, stored in an ultra-low temperature freezer at -80℃, onto an activation medium plate. Pick a single colony and repeat the streak to obtain the activated strain. The components of the activation medium are as follows: peptone 8 g / L, beef extract 4 g / L, yeast extract 3 g / L, glucose 18 g / L, dipotassium hydrogen phosphate 1 g / L, triammonium citrate 1 g / L, sodium acetate 4 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.04 g / L, agar 12 g / L, Tween-80 0.5 g / L, and the pH of the medium is 6.5 ± 0.2 (25℃).

[0062] S2. Select a single colony of the activated strain and inoculate it into 100 mL of activation culture medium in a blue-capped bottle. Incubate at 37°C for 18 h to obtain the primary seed culture. Transfer the primary seed culture to 2 L of activation culture medium for further expansion to obtain the secondary seed culture. The components of the activation culture medium are as follows: peptone 8 g / L, beef extract 4 g / L, yeast extract 3 g / L, glucose 18 g / L, dipotassium hydrogen phosphate 1 g / L, triammonium citrate 1 g / L, sodium acetate 4 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.04 g / L, Tween-80 0.5 g / L, L-cysteine ​​hydrochloride 0.4 g / L, and the pH of the culture medium is 6.5 ± 0.2 (25°C).

[0063] S3. The prepared secondary seed culture was inoculated into a seed tank containing sterilized activated culture medium at an inoculation rate of 1% (v / v) under aseptic conditions. The culture was then incubated at 37°C, pH=6.2, and tank pressure maintained at 0.03-0.05 MPa for 20 h to obtain a culture medium. The culture medium was then centrifuged and resuspended in sterile physiological saline to obtain a bacterial suspension. The bacterial suspension was inoculated into a steam-sterilized composite fermentation substrate at a mass ratio of 1:10. Fermentation was carried out at 37°C, pH=6.2, stirring speed of 30 rpm, and tank pressure maintained at 0.03-0.05 MPa for 28 h. After fermentation was stopped, the mixture was kept at 85°C for 50 min and then cooled to room temperature to obtain the post-biotic preparation. The preparation method of the compound fermentation substrate includes the following steps: 25kg of apples, 25kg of potatoes, 10kg of white kidney beans, 4kg of chia seeds, 3kg of corn silk and 3kg of citrus are washed with sterile water to remove impurities, crushed into a homogenate, diluted in 1200kg of pure water, sterilized by high pressure steam and cooled to room temperature to obtain the compound fermentation substrate.

[0064] Efficacy Verification: I. Zebrafish Experiment: Wild-type AB strain zebrafish aged 5 days post-fertilization (dpf) were selected and bred through natural pair mating. The zebrafish were housed in aquarium water at 28℃ (water quality: conductivity 500-800 μS / cm; pH 7.0-8.0). Zebrafish aged 5 days post-fertilization were used for the determination of the maximum detectable dose (MTC) and efficacy evaluation of the weight loss effect.

[0065] Wild-type AB zebrafish with normal development were placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). The liquid post-biotic preparation prepared in Example 2 was administered in water. A blank control group and five sample exposure groups with varying concentrations were set up, with a volume of 3 mL per well. The microplates were placed in a constant temperature and light incubator and treated at 28°C for 5 days post-exposure (dpf). The MTC of the post-biotic preparation on zebrafish was measured, and the experimental concentrations for efficacy evaluation were determined to be 0.625 μL / mL (high concentration) and 0.3125 μL / mL (low concentration). Wild-type AB zebrafish at 5 dpf were randomly divided into four groups (normal control group, model control group, metformin group, and post-biotic preparation group). Except for the normal control group, all other groups were fed a high-sugar, high-fat diet. The positive control group (metformin) and the post-biotic preparation group were simultaneously treated with the drug for 5 days. Zebrafish samples were collected from each experimental group, and their blood glucose levels were measured.

[0066] Wild-type AB zebrafish at 5dpf were randomly divided into four groups (normal control group, model control group, atorvastatin calcium tablet group, and post-biotic preparation group). Except for the normal control group, all other groups were fed a high-sugar, high-fat diet. In addition, the positive control group (atorvastatin calcium tablet) and the post-biotic preparation group were simultaneously treated with the drug for 5 days. Zebrafish samples were collected from each experimental group. After the treatment, the total cholesterol (TG) level of the samples was measured to evaluate the weight loss effect of the post-biotic preparation.

[0067] Wild-type AB zebrafish at 5 dpf were randomly divided into three groups (normal control group, model control group, and metagenic agent group). Except for the normal control group, all other groups were fed a high-sugar, high-fat diet. The metagenic agent group was treated with different concentrations of samples. After 5 days of treatment, zebrafish samples were collected from each experimental group, and the expression level of the zebrafish leptin gene (which encodes a protein secreted by white adipocytes into the circulation and plays a major role in energy homeostasis regulation. Circulating leptin binds to leptin receptors in the brain, activating downstream signaling pathways, inhibiting feeding, and promoting energy expenditure) was detected by qPCR to evaluate the effect of samples on the regulation of this gene expression. The relevant primer sequences for the leptin gene and the internal reference β-actin are shown in Table 1.

[0068] Table 1

[0069]

[0070] The above-mentioned zebrafish blood glucose concentration, cholesterol concentration, and leptin expression levels are as follows: Figure 3 , Figure 4 , Figure 5 As shown in the figure, the post-biotic preparation prepared by fermenting a compound fermentation substrate with Lactobacillus paracasei HBC003 in this embodiment of the invention has the effects of lowering blood sugar, lowering cholesterol, and significantly increasing the expression of the leptin gene, and has the effect of reducing fat and weight.

[0071] II. Population Experiment: Thirty adults aged 22-60 years were selected to participate in the experiment voluntarily and complete the questionnaire and record the data. 25 mL of the post-biotic preparation prepared in Example 2 was taken orally on an empty stomach one hour before lunch or dinner daily for 10 days to 16 weeks. Questionnaires were conducted before and after the course of treatment. Participants were allowed to eat freely during the treatment period and were required to monitor their appetite. The results are shown in Table 2.

[0072] Table 2

[0073]

[0074]

[0075] The statistical results of the small-scale population experiment showed that 25 out of 30 subjects experienced varying degrees of weight and body fat percentage reduction after taking the postbiotic preparation prepared in Example 2. Furthermore, the subjects reported reduced appetite and increased satiety after taking the postbiotic preparation, without experiencing nausea, acid reflux, or other gastrointestinal discomfort. These efficacy verification experiments demonstrate that the postbiotic preparation prepared in this invention can achieve fat reduction and weight loss effects.

[0076] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. A post-natal vitamin preparation with weight loss and fat reduction effects, characterized in that, It is made by fermentation of probiotics; the postbiotic preparation includes inactivated probiotics, a complex fermentation substrate, and metabolites produced by the fermentation of the complex fermentation substrate by the probiotics; the probiotics are Lactobacillus paracasei; the complex fermentation substrate includes a variety of natural dietary components.

2. The post-natal vitamin preparation with weight loss and fat reduction effects according to claim 1, characterized in that, The probiotic is Lacticaseibacillus paracasei HBC003, deposited at the China Center for Type Culture Collection (CCTCC) on June 23, 2025, with accession number CCTCC NO: M 20251447.

3. The post-natal vitamin preparation with weight loss and fat reduction effects according to claim 1, characterized in that, The compound fermentation substrate includes apples, potatoes, corn silk, and citrus fruits.

4. The post-natal vitamin preparation with weight loss and fat reduction effects according to claim 3, characterized in that, The compound fermentation base comprises the following components by weight: 25-35 parts apple, 25-35 parts potato, 1-5 parts corn silk, and 1-5 parts citrus.

5. A method for preparing a post-biotic preparation with weight loss and fat reduction effects as described in any one of claims 1-4, characterized in that, Includes the following steps: The probiotics were streaked onto an activation medium for activation to obtain activated strains; Select activated bacterial strains and incubate them statically in activation culture medium to obtain seed culture; The seed culture was inoculated into a seed tank containing an activated culture medium for cultivation to obtain a culture medium. The culture medium was then centrifuged and resuspended in sterile physiological saline to obtain a bacterial suspension. The bacterial suspension was inoculated into a composite fermentation substrate for fermentation, and then inactivated to obtain the postbiotic preparation.

6. The method for preparing the post-natal vitamin preparation with weight loss and fat reduction effects according to claim 5, characterized in that, The activation culture medium comprises the following components in concentration: peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 3-5 g / L, glucose 18-22 g / L, dipotassium hydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.04-0.06 g / L, and Tween-80. 0.5-1.5 g / L, agar 12-18 g / L; the activated culture medium comprises the following components in concentration: peptone 8-12 g / L, beef extract 4-6 g / L, yeast extract 3-5 g / L, glucose 18-22 g / L, dipotassium hydrogen phosphate 1-3 g / L, triammonium citrate 1-3 g / L, sodium acetate 4-6 g / L, magnesium sulfate 0.1-0.3 g / L, manganese sulfate 0.04-0.06 g / L, Tween-80 0.5-1.5 g / L, L-cysteine ​​hydrochloride 0.4-0.6 g / L.

7. The method for preparing the post-natal vitamin preparation with weight loss and fat reduction effects according to claim 5, characterized in that, The preparation method of the composite fermentation substrate includes the following steps: 25-35 parts apple, 25-35 parts potato, 1-5 parts corn silk and 1-5 parts citrus are washed with sterile water to remove impurities, crushed into a homogenate, diluted in 1000-1500 parts pure water, and then sterilized to obtain the composite fermentation substrate.

8. The method for preparing the post-natal vitamin preparation with weight loss and fat reduction effects according to claim 5 or 7, characterized in that, The mass ratio of the bacterial suspension to the composite fermentation substrate is 1:(10-20).

9. The method for preparing the post-natal vitamin preparation with weight loss and fat reduction effects according to claim 5, characterized in that, The bacterial suspension is inoculated into a composite fermentation substrate for fermentation, and after an inactivation treatment step, a vacuum low-temperature drying treatment step is also included.

10. The use of an epigenetic preparation as described in any one of claims 1-4 in the preparation of functional foods for weight loss and fat reduction.

Images