Probiotic preparation with fat reducing function and application thereof

Abstract

The invention discloses a preparation method and application of a composite probiotic preparation which has a fat reducing function and comprises Lactobacillus rhamnosum D8A, and belongs to the technical field of probiotics. The probiotics can inhibit the growth of obesity-related pathogenic bacteria lactobacillus cloacae, and the content of chlorogenic acid and polysaccharide is remarkably increased through specific fermentation of honeysuckle. The extracted polysaccharide shows fat-reducing activity through verification of a 3T3-L1 cell model, and animal experiments show that the effect of the probiotic preparation prepared based on the polysaccharide embedding technology developed on the basis is obviously better than that of other preparations. The invention provides an innovative solution for developing a fat-reducing probiotic preparation, and has important application value in the fields of functional food and weight management.

Description

Technical Field

[0001] The present invention belongs to the field of probiotics, and in particular relates to a probiotic with fat-reducing function and application of a preparation thereof. Background Art

[0002] In recent years, obesity rates have continued to rise globally, making it a major threat to human health. Obesity not only affects appearance but is also closely linked to a variety of metabolic diseases, such as type 2 diabetes, cardiovascular disease, and hypertension. Therefore, the development and promotion of effective fat-reducing products is of great social and economic significance.

[0003] Currently, fat reduction methods mainly include diet control, exercise, drug intervention and surgical treatment. However, these methods often have the following limitations:

[0004] 1. Dietary control and exercise require long-term persistence, and the effects vary from person to person;

[0005] 2. Drug intervention may be accompanied by side effects, and long-term use may be risky;

[0006] 3. Surgical treatment is costly and carries certain risks.

[0007] Therefore, developing a safe, effective and easy-to-promote fat reduction method has become the focus of current research.

[0008] In recent years, probiotics—active microorganisms that benefit host health—have been widely used in food, health supplements, and medicine. Research has shown breakthroughs in understanding the relationship between the gut microbiome and obesity. Probiotics and their metabolites (such as polysaccharides and short-chain fatty acids) have multiple physiological functions, including regulating the gut microbiome, improving metabolic function, and enhancing immunity.

[0009] Further research revealed that changes in the abundance of specific bacterial species are closely associated with obesity. The proliferation of opportunistic pathogens is highly correlated with obesity. For example, Staphylococcus aureus is enriched in the intestines of overweight people, while Enterobacter cloacae B29 strain accounts for up to 35% of the intestines of obese patients, and its colonization can directly induce obesity in germ-free mice. These findings suggest that obesity-related intestinal dysbiosis is not only manifested by a decrease in beneficial bacteria, but may also be accompanied by the abnormal proliferation of obesity-promoting pathogens.

[0010] Currently commercialized specific probiotic strains (such as Morinaga Milk's Bifidobacterium breve B3, DuPont Danisco's Bifidobacterium animalis B420, Snow Brand Milk's Lactobacillus gasseri SP strain, and Calpis' Lactobacillus amylolique CP1563) have been shown through clinical studies to have the potential to regulate body fat or weight. Their mechanisms involve lowering cholesterol, improving lipid metabolism, or promoting the colonization of beneficial bacteria such as Akkermansia muciniphila. However, the effects of existing strains strictly depend on strain specificity, and individual intestinal microecology varies significantly, making it difficult for a single strain to cover the needs of all populations. For example, the B3 strain is effective in regulating body fat in sub-obese people, while B420 works by regulating specific metabolic pathways. Therefore, although existing strains have partially met market demand, the continued development of new probiotics is still necessary.

[0011] In the prior art, plant polyphenolic compounds (including brass, phenolic acids, stilbenes, lignans, and tannins) have attracted the attention of scientific researchers due to their various biological activities, including anti-lipidemia. Numerous studies have found that polyphenolic compounds have a significant inhibitory effect on the activity of enzymes associated with human diseases, such as PL (pancreatic lipase, obesity), cholinesterase (Alzheimer's disease), xanthine oxidase (gout), tyrosinase (pigmentation), α-amylase and α-glucosidase (diabetes), angiotensin-converting enzyme (hypertension), and α-synuclein (Parkinson's disease). Therefore, by studying the biological functions and mechanisms of action of various polyphenolic compounds, corresponding references can be provided for the comprehensive development of polyphenolic products. Studies have shown that chlorogenic acid can significantly reduce body weight, visceral fat mass, and plasma leptin and insulin levels in obese mice induced by a high-fat diet. It also reduces the concentrations of triglycerides (in plasma, liver, and heart) and cholesterol (in plasma, liver, and heart), which can improve fat metabolism and thus alleviate obesity. Chlorogenic acid comes from dicotyledonous plants and ferns such as honeysuckle and eucommia leaves, and is widely used in medicine, food and other fields.

[0012] Research on probiotics and their metabolites has primarily focused on regulating intestinal health and enhancing immune function, while relatively little research has focused on the application of probiotic fermentation as both a medicine and a food for fat loss. Polysaccharides are one of the key metabolites of probiotics and possess a variety of biological activities, including anti-inflammatory, antioxidant, and immunomodulatory properties.

[0013] The market demand for safe, natural products with fat-reducing effects is growing. Therefore, the development of probiotic-based preparations in fat-reducing products has broad prospects.

[0014] Related existing technologies are as follows:

[0015] Publication No. CN113322216A discloses a Lactobacillus paracasei B111 H strain and its use in treating metabolic syndrome. The strain, isolated and screened from human feces, significantly inhibits lipid accumulation in hepatocytes, reduces body weight and blood lipid levels, and improves hepatic steatosis. This strain has important application implications for alleviating hyperlipidemia, obesity, and fatty liver. The discovery of this strain provides a new microbial resource for lipid-lowering foods or pharmaceuticals.

[0016] The invention patent application with publication number CN109251877A discloses a probiotic effect of Bacillus natto with functions such as fat reduction and its application. The application discloses Bacillus natto SJLH001 (Bn-SJLH001 for short) and its application in fat reduction and probiotic foods. The strain was obtained through targeted screening and has significant in vitro probiotic activities, including fibrin decomposition, anti-coagulation and antioxidant functions (such as scavenging DPPH and ABTS free radicals). In a high-fat diet mouse model, Bn-SJLH001 exhibited multiple fat-reducing effects: reduced body weight gain and serum triglyceride levels, reduced the weight of epididymal, perirenal and mesenteric fat and the diameter of fat cells, and downregulated the expression of lipogenesis genes (GSK-3α / β), inhibiting the accumulation of uric acid in perirenal adipose tissue. Its mechanism of action includes: (1) regulating the structure of intestinal flora, increasing the abundance of Bacteroidetes, reducing the ratio of Firmicutes / Bacteroidetes (F / B), and improving obesity-related flora imbalance; (2) regulating the expression of genes involved in intestinal nutrient transport (such as SGLT1), metal ion channels (such as ZIP4), and metabolite transporters (such as MCT1), inhibiting lipid absorption and promoting metabolism; (3) synergistically reducing the level of inflammatory factors and blocking the adipogenesis pathway. The invention patent application with publication number CN111254089A discloses a strain of Lactobacillus plantarum with weight loss function and its application. The application discloses that the strain Lactobacillus plantarum 1701 was isolated from yogurt powder and has good tolerance and adhesion, can reduce serum leptin levels (promote lipolysis and inhibit fat synthesis), reduce body weight, fat weight, body fat ratio, and reduce total cholesterol and triglyceride levels. Inactivated bacteria are also effective, reducing body weight, fat weight, and body fat ratio. In addition, live bacteria and inactivated bacteria compositions can reduce liver weight. Studies have shown that the strains achieve fat reduction, weight control and metabolic regulation functions through multi-pathway synergistic effects.

[0017] Patent application CN119120247A discloses a strain of Bifidobacterium lactis BLYR55 with weight loss and fat reduction properties, and its applications. The application discloses that a strain of Bifidobacterium lactis BLYR55 was isolated from yogurt and is highly effective in reducing weight and fat, both in live and sterilized forms. Furthermore, it can alleviate lactose intolerance, suggesting potential applications in a wide range of medicinal and healthcare products.

[0018] The invention patent application with publication number CN118685300A discloses a strain of Bifidobacterium longum subspecies longum and its use in preventing, alleviating, regulating or treating diseases related to lipid metabolism. The application discloses that Bifidobacterium longum subspecies longum dipro-0 produces a dual mechanism of indole-3-lactic acid (ILA) through high production of bile salt hydrolase (BSH) and metabolism of tryptophan, showing multi-dimensional metabolic regulation function: it can significantly reduce weight gain induced by a high-fat diet, improve insulin resistance, inhibit the expression of the pro-inflammatory factor TNF-α, and regulate blood lipid profile (reduce total cholesterol and low-density lipoprotein, and increase high-density lipoprotein levels); at the same time, it reshapes the balance of intestinal flora, alleviates liver lipid deposition and intestinal barrier damage. Its pasteurized form still retains the activity of regulating lipid metabolism, can effectively reduce body weight and dyslipidemia, and provides a new strategy for the development of inactive probiotic preparations, which is suitable for the intervention of metabolic diseases such as hyperlipidemia. Summary of the Invention

[0019] Based on the above background, the present invention can effectively inhibit pancreatic lipase, obesity-causing bacteria such as Lactobacillus cloacae and Staphylococcus aureus through specific screening, use probiotics to ferment honeysuckle to produce chlorogenic acid exopolysaccharides, and use the exopolysaccharides produced by the bacteria to specifically encapsulate and protect the strain, thereby obtaining a probiotic preparation and verifying its significant fat-reducing effect. The technical solution provided by the present invention has the following advantages:

[0020] High safety: Derived from natural probiotics, the fermentation of honeysuckle with probiotics has no toxic side effects;

[0021] Significant effect: Experimental verification shows that it has a significant effect on the decomposition of lipid droplets and the inhibition of fat accumulation;

[0022] Wide application: It can be widely used in food, health care products and medicine fields.

[0023] The present invention is achieved through the following technical solutions:

[0024] The results were obtained by in vitro screening by measuring the antibacterial properties of probiotics against pathogenic bacteria such as Lactobacillus cloacae, which are common pathogens that cause obesity, and the content of extracellular polysaccharides produced.

[0025] A fermentation broth with fat-reducing properties, obtained by fermenting honeysuckle powder with Lactobacillus rhamnosus D8A. Lactobacillus rhamnosus D8A was deposited with the General Microbiology Center of the China National Culture Collection Administration on March 7, 2024, with the deposit number CGMCC No. 29961. This was disclosed in a Chinese invention patent application with publication number CN118389363A, entitled "Lactobacillus rhamnosus, a probiotic preparation that aids sleep, and its preparation method."

[0026] The method for preparing the fermentation broth comprises the following steps:

[0027] 3 vol% of the bacterial liquid of the adaptively evolved Lactobacillus rhamnosus D8A was inoculated into a liquid fermentation medium and fermented at 37° C. for 24 hours to obtain a fermentation liquid.

[0028] The preparation method of the liquid fermentation medium comprises the steps of mixing 10.0 g of casein enzymatic digest, 2.0 g of triammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 15 g of glucose, 10 g of honeysuckle dry powder, and 1.08 g of Tween-80, and then diluting the mixture to 1 L with water.

[0029] The pH value of the liquid fermentation medium is 5.8.

[0030] The adaptively evolved Lactobacillus rhamnosus D8A is obtained by sequentially fermenting honeysuckle dry powder soaking solutions with increasing weight ratios of honeysuckle dry powder to water by Lactobacillus rhamnosus D8A.

[0031] The fermentation broth is used to prepare medicines or health products for inhibiting lipid accumulation in 3T3-L1 preadipocytes.

[0032] A probiotic preparation comprises the freeze-dried powder of the fermentation broth.

[0033] A method for preparing a probiotic preparation comprises the following steps:

[0034] After sterilizing and cooling the culture medium containing honeysuckle powder to room temperature, the culture medium is inoculated with adaptively evolved Lactobacillus rhamnosus D8A, fermented and cultured, centrifuged, and a freeze-drying protective agent is added, followed by freeze-drying; or

[0035] The culture medium containing honeysuckle powder was sterilized and cooled to room temperature, and then inoculated with adaptively evolved Lactobacillus rhamnosus D8A, fermented and cultured, inactivated, and then concentrated and freeze-dried.

[0036] The inoculation concentration of the adaptively evolved Lactobacillus rhamnosus D8A is not less than 1×10 7 CFU / mL;

[0037] The fermentation temperature is 37°C and the fermentation time is 24 hours;

[0038] The lyoprotectant comprises the following components by weight: 10-15% skim milk powder, 0.6-2.5% calcium alginate, 2-4% pectin, 1-2% inulin, and the balance water. Alternatively, the lyoprotectant comprises the following components by weight: 10-15% skim milk powder, 0.6-2.5% calcium alginate, 2-4% pectin, 0.5-1.5% inulin, 0.5-1% exopolysaccharide from Lactobacillus rhamnosus D8A, and the balance water. The probiotic preparation is used to prepare a medicine or health product that inhibits lipid synthesis or promotes lipid metabolism.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention obtains a probiotic preparation by fermenting honeysuckle with rhamnosus lactobacillus D8A, and the content of chlorogenic acid and polysaccharide in the preparation is significantly improved. Since honeysuckle dry powder can significantly increase the expression of phenylpropanoid metabolism genes (PAL, C4H, 4CL, HCT) in rhamnosus lactobacillus D8A, honeysuckle dry powder can produce a synergistic effect with rhamnosus lactobacillus D8A, improve the ability of rhamnosus lactobacillus D8A to produce polysaccharides, and then enhance the antioxidant and lipid metabolism regulating function of the probiotic preparation. The in vitro experiments provided by the present invention confirm that the probiotic preparation provided by the present invention has the function of inhibiting 3T3-L1 preadipocyte differentiation and lipid droplet accumulation; in the animal model, the high-activity probiotic preparation and the polysaccharide-embedded probiotic preparation significantly improved the body weight of high-fat mice, reduced serum TG, TCHO, LDL-C and liver lipid deposition, while restoring HDL-C levels and alleviating liver damage (ALT / AST decreased). The present invention shows that the probiotic preparation can reduce the body weight of obese mice induced by a high-fat diet, improve abnormal blood lipid levels, and alleviate liver fat accumulation.

[0041] The present invention utilizes the extracellular polysaccharide produced by D8A fermentation as an embedding protective agent for the probiotic preparation. Research on the fat-reducing effect of the low-activity preparation (1 billion CFU) is higher than that of the high-activity preparation (5 billion CFU) using a conventional embedding method. DETAILED DESCRIPTION

[0042] The present invention is further described below with reference to the following examples. The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0043] In the present embodiment, the strain involved includes: Lactobacillus rhamnosus D8A, information about which is detailed in the Chinese invention patent application with publication number CN118389363B, entitled "Lactobacillus rhamnosus, a probiotic preparation that helps sleep, and a method for preparing the same." The strain was obtained by the inventors using the screening method disclosed in the invention patent application with publication number CN118389363B.

[0044] Example 1

[0045] In vitro screening of strains

[0046] 1.1 Evaluation of antibacterial properties and polysaccharide content

[0047] Four laboratory bacterial strains were activated using solid MRS medium and then enriched using MRS liquid medium. Their antibacterial activity against Staphylococcus aureus and Lactobacillus cloacae was determined. Specific results are shown in Table 1.

[0048] The reagent configuration method:

[0049] MRS solid medium components: casein digest 10.0g, ammonium citrate tribasic 2.0g, beef extract powder 10.0g, magnesium sulfate 0.2g, yeast extract powder 4.0g, manganese sulfate 0.05g, sodium acetate 5.0g, potassium dihydrogen phosphate 2.0g, glucose 20.0g, Tween-80 1.08g, agar 18g, dilute to 1L, final pH: 5.7±0.2, sterilize at 121℃ for 15min.

[0050] MRS liquid medium components: casein digest 10.0 g, ammonium citrate tribasic 2.0 g, beef extract powder 10.0 g, magnesium sulfate 0.2 g, yeast extract powder 4.0 g, manganese sulfate 0.05 g, sodium acetate 5.0 g, potassium dihydrogen phosphate 2.0 g, glucose 20.0 g, Tween-80 1.08 g, dilute to 1 L, final pH: 5.7, sterilize at 121°C for 15 min.

[0051] Extraction of exopolysaccharide (crude polysaccharide) after culture in MRS liquid medium: Extraction was carried out according to the method of "DB15 / T 2033-2020 Technical Specification for Isolation, Purification and Identification of Exopolysaccharides of Lactic Acid Bacteria". The glucose standard curve was used for calculation during detection, y = 0.024x + 0.0479, R 2 =0.9961.

[0052] Table 1 Evaluation of antibacterial performance and polysaccharide content

[0053]

[0054] According to the data in Table 1, strains 15M9 (D8A) and 225M4 showed the best antibacterial activity, followed by 19M5. These strains showed significant inhibitory effects on conditional pathogens (such as Staphylococcus aureus and Enterobacter cloacae) that are highly abundant in the intestines of obese people. It is worth noting that these three strains also have high exopolysaccharide (EPS) production. Based on existing studies confirming the probiotic functional properties of EPS (including pathogen inhibition, intestinal barrier enhancement, etc.), the results of the study showed that 15M9 (D8A), 225M4 and 19M5 showed good prebiotic potential in in vitro experiments and can be used as potential probiotic candidate strains for subsequent functional evaluation.

[0055] 1.2 Determination of intracellular and cell membrane beta-glucosidase Experimental method: seed liquid is inoculated into MRS liquid culture medium with 2% (V / V) inoculum size, after taking culture fluid and centrifuging 5min under 4 DEG C, 6000r / min conditions, bacterial sediment is washed 2 times with normal saline, is suspended in 2mL, pH=5 acetic acid-sodium acetate buffer, then carries out ultrasonic cell crushing under ice bath conditions, after all crushing, centrifuges 10min at 4 DEG C, 12000r / min speed. Take supernatant, be used for measuring intracellular key enzyme beta-glucosidase activity; Take the centrifugal precipitation of cell pulverization liquid, after washing 2 times with pH=5.0 acetic acid-sodium acetate buffer, be suspended in 1mL, pH=5.0 buffer and then draw 100 μ L suspension in test tube, be used for measuring cell membrane beta-glucosidase activity. Use beta-glucosidase activity detection kit to measure.

[0056] Table 2 β-glucosidase activity determination

[0057] strain Enzyme activity of cell disruption supernatant (U / mL) Enzyme activity of cell disruption supernatant (U / mL) 15M9(D8A) 0.05 50.21 225M4 0.31 46.21 19M5 0.01 39.21 Comparison of commercially available strains 0.30 40.31

[0058] The cell membrane-bound β-glucosidase activity of strains 15M9 (D8A) and 225M4 was significantly higher than that of the commercially available control strain, followed by 19M5. Combined with the key role of β-glucosidase in glycosidic bond hydrolysis and the conversion of bioactive substances, this characteristic further enhances its potential as a probiotic candidate. β-glucosidase can catalyze the degradation of dietary fiber (such as cellulose and glucan), promote the production of short-chain fatty acids, and activate plant-derived flavonoid aglycones, enhancing their bioavailability, thereby playing an important role in regulating the intestinal microecology and host health.

[0059] 1.3 Evaluation of pancreatic lipase inhibitory activity

[0060] Prepare 5 U / mL lipase solution and 0.005 mol / L 4-nitrophenyl octanoate (PNP-C) solution separately. Adjust the pH of the sterile MRS liquid medium and the test bacterial suspension to 7.2-7.4 with 2 mol / L NaOH solution, then dilute 5-fold with PBS buffer (pH 7.4). Perform the experimental procedures according to the reaction system shown in the table.

[0061] Table 3 Pancreatic lipase inhibition performance evaluation reaction system

[0062] Samples to be tested PBS buffer (mL) Lipase solution (mL) PNP-C (mL) A 0.5(MRS) 0.1 0.2 0.2 a 0.5(MRS) 0.3 - 0.2 B 0.5 (bacterial suspension) 0.1 0.2 0.2 b 0.5 (bacterial suspension) 0.3 - 0.2

[0063] In a dark environment, sequentially add all reaction solutions to a centrifuge tube and mix thoroughly. Place the centrifuge tube in a 37°C water bath for 60 minutes. Mix 600 μL of the reaction solution with 1.2 mL of acetonitrile and 100 μL of formic acid and centrifuge at 10,000 rpm for 3 minutes. Measure the optical density of the supernatant at OD316. Perform the assay in triplicate for each sample, and calculate the pancreatic lipase inhibition rate as indicated. Chlorogenic acid and orlistat were used as positive controls.

[0064] Wherein A: enzyme activity without adding inhibitors; a: blank control (no enzyme); B: enzyme activity with adding inhibitors; b: negative control (no enzyme).

[0065]

[0066] Table 4 Pancreatic lipase inhibition rate

[0067] Serial number strain number Pancreatic lipase inhibition rate 1 15M9(D8A) 64.83 2 225M4 53.21 3 19M5 49.71 4 Chlorogenic acid 69.78 5 Rosiglitazone 75.43

[0068] This experiment screened out strains with the potential to inhibit pancreatic lipase, such as 15M9 (D8A), whose effect is close to that of the natural inhibitor chlorogenic acid. Combined with the efficacy of pancreatic lipase, inhibiting this enzyme can reduce the breakdown and absorption of fat, thereby reducing calorie intake. Therefore, substances with high inhibition rates may be used as potential weight loss drugs or functional food ingredients. These strains may help weight management by reducing fat absorption. Subsequent animal experiments (such as high-fat diet models) are needed to verify the in vivo effects and evaluate safety (such as intestinal tolerance) to provide a scientific basis for product development.

[0069] 1.4 Key gene analysis

[0070] In order to further explore the potential functions of Lactobacillus rhamnosus D8A, the present invention enriched and extracted the mRNA of D8A. The expression levels of PAL / C4H / 4CL / HCT genes, which activate the phenylpropanoid metabolic pathway, were detected by qRT-PCR. The results are shown in Table 6. Compared with DBA, 225M4 and the commercially available strain Lactobacillus rhamnosus LGG, the expression of these four key genes increased after fermentation. However, PAL, as the rate-limiting enzyme in phenylpropanoid metabolism, was significantly upregulated in the D8A strain, indicating that the phenylpropanoid metabolic pathway is active in this strain. C4H is responsible for cinnamic acid hydroxylation, and its expression level is the highest in the D8A strain, which may be related to the specific metabolic characteristics of this strain. 4CL activates hydroxycinnamic acid and participates in the synthesis of various secondary metabolites. Its expression pattern is similar to that of C4H, and it may act synergistically in the D8A strain. HCT, as a key enzyme in chlorogenic acid synthesis, is also highly expressed in the D8A strain, suggesting that this strain may have a higher chlorogenic acid synthesis capacity. By detecting the expression levels of these genes, we can not only provide metabolic regulation targets for probiotic fermentation of medicinal and edible substances (such as honeysuckle), but also increase the production of chlorogenic acid through targeted optimization of the synthesis system, serving the development of functional products such as anti-obesity and antioxidant, which has important scientific value and application prospects.

[0071] Table 5 Primer design

[0072]

[0073] Table 6 mRNA expression levels of four genes

[0074]

[0075] Example 2

[0076] Determination of the concentration of honeysuckle dry powder fermented by rhamnosus Lactobacillus D8A and preparation method of its probiotic preparation Honeysuckle is rich in chlorogenic acid (up to 3-8%). Based on this, this patent uses probiotics to ferment honeysuckle to prepare a probiotic preparation with weight loss effect.

[0077] 2.1 Adaptive evolution of lactic acid bacteria and confirmation of honeysuckle concentration

[0078] Honeysuckle dry powder was added with water in a ratio of 1:1, 1:10, 1:20, 1:50, and 1:100 and soaked for 2 h. The mixture was heated in a water bath at about 80°C and soaked for 16 h to obtain honeysuckle pollen soaking solutions of different concentrations.

[0079] Adaptive evolution fermentation: Lactobacillus rhamnosus D8A was inoculated into the cooled honeysuckle powder soaking liquid at a strain concentration of (2-8) × 10 11CFU / mL, an inoculation ratio of 2%-5%, a constant fermentation pH of about 5.9, a temperature of 37°C, a nutrient medium of (2.5% glucose, 2% soy peptone, 0.03% magnesium sulfate, 0.2% dipotassium hydrogen phosphate), and normal pressure fermentation for 24-48 hours. First, the bacterial liquid is inoculated into a 1:100 ratio of soaking liquid for fermentation. After the fermentation is completed, the fermentation liquid is enriched in MRS medium. After one day of enrichment, it is inoculated into a 1:50 ratio of soaking liquid for fermentation. According to the above method, the concentration of the fermentation liquid is gradually increased, and finally inoculated into a 1:1 fermentation liquid to enhance the adaptability of the strain.

[0080] According to the culture medium formula, 10.0 g of casein digest, 2.0 g of triammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 10.0 g of glucose, 1.08 g of Tween-80, 5-20 g of honeysuckle dry powder, dilute to 1 L with water, and adjust the pH to 5.8.

[0081] The addition amount of honeysuckle dry powder was 5g, 10g, 15g and 20g respectively. After the bacterial solution was cultured at 37℃ for 24h, the absorbance value of OD600 of the bacterial solution was measured and the OD values ​​were compared. The results are shown in Table 7. When the addition amount was 10g, the OD value was the highest and the number of viable bacteria in the bacterial solution was the highest.

[0082] Table 7 OD values ​​of different amounts of honeysuckle dry powder added

[0083]

[0084] 2.2 Preparation of probiotic preparations and detection of effective ingredients

[0085] After streaking, single probiotics were picked and placed in liquid MRS, cultured at 37°C, and activated twice with an inoculum volume of 3% (volume ratio) to a concentration of no less than 1×10 7 CFU / mL, inoculate with culture medium (10.0 g of casein digest, 2.0 g of triammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 15 g of glucose, 10 g of honeysuckle powder, 1.08 g of Tween-80, pH 5.8, dilute to 1 L with water, and sterilize at 121°C for 21 min), and ferment at 37°C for 24 h to obtain the fermentation broth.

[0086] Comparative Example 1

[0087] The difference from Example 2 is that 3% of commercially available Lactobacillus rhamnosus GG was used and fermented at 37° C. for 24 h.

[0088] Comparative Example 2

[0089] The difference from Example 2 is that 3% of commercially available Lactobacillus rhamnosus UP180 was used and fermented at 37° C. for 24 h.

[0090] Comparative Example 3

[0091] The difference from Example 2 is that 3% of commercially available Lactobacillus acidophilus CICC6089 was used and fermented at 37° C. for 24 h.

[0092] Comparative Example 4

[0093] The difference from Example 2 is that 3% of commercially available Bifidobacterium longum ATCC 1570 was used and fermented at 37° C. for 24 h.

[0094] Comparative Example 5

[0095] The difference from Example 2 is that 10 g of honeysuckle dry powder was added during the preparation of the probiotic preparation and the detection of the effective ingredients.

[0096] Comparative Example 6

[0097] The difference from Example 2 is that the honeysuckle dry powder added during the preparation of the probiotic preparation and the detection of the effective ingredients is 10g of Eucommia leaf powder.

[0098] Take the fermentation broth before and after fermentation, add 5mL of 50% methanol, ultrasonicate at 400W for 20min, centrifuge for 10min (8000r), take 0.1mL of supernatant and dilute it with 50% methanol in a 5ml volumetric flask, and use HPLC for quantitative analysis and detection. When performing chromatographic analysis on it, use a C18 column with a specification of 4.6mL×250mL and 5 microns as the stationary phase. At the same time, acetonitrile is used as mobile phase A, 0.1% phosphoric acid water is used as mobile phase B, the detection wavelength is set at 327nm, and 15% acetonitrile is used for isocratic liquid phase detection. Purchase 99% chlorogenic acid standard to make a standard curve. Its linear range is 0.2-100mg / mL, and the detection standard curve is y=0.5163x+0.0129, R 2 =0.9944.

[0099] The polysaccharide content detection method was as described in Example 1, step 1.1. The fermentation broth was treated with trichloroacetic acid and centrifuged to obtain the supernatant, which was then precipitated with anhydrous ethanol. The supernatant was placed in a dialysis bag with a molecular weight cutoff of 8 kDa (the dialysate volume was 100 times the sample volume; one end was clamped with a clamp, and the other end was added with the sample solution, leaving 20% ​​of the space open and clamped). After 2 to 4 hours of dialysis, the dialysate was replaced, and then the dialysate was replaced every 6 to 8 hours for 2 consecutive days. The polysaccharide solution in the dialysis bag was collected and lyophilized to obtain the extracellular polysaccharide.

[0100] Table 8 Changes in chlorogenic acid and polysaccharide content before and after fermentation

[0101]

[0102] After fermentation with rhamnosus Lactobacillus D8A, the chlorogenic acid content increased from 0.31 mg / g to 0.71 mg / g (an increase of 129%), and the extracellular polysaccharide production increased from 41.21 ug / mL to 59.32 ug / mL (an increase of 43.95%), indicating that the D8A strain can efficiently convert the honeysuckle substrate to synthesize chlorogenic acid. At the same time, the extracellular polysaccharide production increased, confirming that the strain has the ability to efficiently synthesize polysaccharides, further supporting its potential application value as a probiotic.

[0103] However, after fermentation with different rhamnosus Lactobacillus or other genera, it was found that the chlorogenic acid and polysaccharide contents were not significantly increased.

[0104] Fermentation of other plant species, such as honeysuckle or Eucommia ulmoides leaves, using Lactobacillus rhamnosus D8A also resulted in lower increases in chlorogenic acid and polysaccharide production. 2.3 Gene Expression Detection Before and After Fermentation: qPCR was used to detect and quantify the expression of the PAL / C4H / 4CL / HCT genes before and after fermentation with Lactobacillus rhamnosus D8A. The primers are shown in Table 5. The results showed that, compared to pre-fermentation, the expression levels of the PAL / C4H / 4CL / HCT genes in Lactobacillus rhamnosus D8A after fermentation were significantly increased by 1.9-fold, 2.1-fold, 2.6-fold, and 1.7-fold, respectively. This demonstrates that during fermentation of honeysuckle by D8A, PAL acts as the rate-limiting enzyme to initiate the metabolic pathway, while 4CL and HCT accelerate precursor activation and final product synthesis, directly driving increased chlorogenic acid production. In contrast, no significant increases were observed in fermentations of other plant species or by D8A. These results reveal the mechanism by which the D8A strain enhances product synthesis through gene expression regulation during specific fermentation of honeysuckle, providing promising insights into the development of functional fermented products that combat obesity and promote intestinal health.

[0105]

[0106] Example 3

[0107] Study on the Differentiation Effect of 3T3-L1 Preadipocytes

[0108] Based on the results of Example 2, it can be seen that after fermentation of honeysuckle by Lactobacillus rhamnosus D8A, the content of chlorogenic acid and polysaccharides in the fermentation broth can be increased. The effect of the fermentation broth on adipogenesis and lipid accumulation was evaluated using a cell model.

[0109] 1. 3T3-L1 cell culture and differentiation

[0110] 3T3-L1 cells were cultured at 2×10 5The cells were seeded into 6-well plates at a density of 2 μm per well and cultured with complete medium [DMEM (high glucose) + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin] until they grew to contact inhibition (2 days).

[0111] The cells were cultured for 2 days using differentiation medium [high-glucose DMEM medium containing 1 M DEX, 0.5 mM IBMX, 10 μg / mL INS, 10% FBS, and 1% P / S] to induce differentiation for 1-2 days.

[0112] Then, the cells were switched to differentiation maintenance medium (high-glucose DMEM medium containing 10 μg / mL INS, 10% FBS, and 1% P / S) to maintain differentiation for 2-4 days.

[0113] Referring to Table 9, after adding samples from different groups and differentiation induction medium to induce differentiation, obvious oil droplets were observed in most cells. Culture was continued for 48 hours, and lipid droplet accumulation was detected.

[0114] Table 93T3-L1 cell differentiation effect evaluation group

[0115] Serial number Group Treatment 1 Blank control group Contains only complete culture medium (DMEM + 10% FBS + 1% P / S) 2 Negative control group 1 Unfermented honeysuckle dry powder water extract 3 Negative control group 2 Probiotic group without honeysuckle in fermentation medium 4 Experimental Group 1 / 2 / 3 D8A fermentation broth of honeysuckle (gradient concentration: 0.1 / 0.25 / 0.5 mg / mL, calculated as chlorogenic acid content) 5 Experimental Group 4 Exopolysaccharide group (50uM) 6 Positive control group 1 Chlorogenic acid standard 0.25mg / mL 7 Positive control group 2 Rosiglitazone 10 μM

[0116] 2. Fat reduction effect index detection

[0117] ① Quantification of lipid accumulation--Oil Red O staining

[0118] When the cells differentiated to maturity, they were stained with Oil Red O. The specific operation steps were strictly followed according to the instructions of the Oil Red O staining kit. The OD value was measured at a wavelength of 520 nm for quantitative analysis.

[0119] Table 10 Effects on lipid deposition

[0120] Serial number Group OD 1 Blank control group 2.917 2 Negative control group 1 2.312 3 Negative control group 2 1.996 4 Experimental Group 1 1.832 5 Experimental Group 2 1.661 6 Experimental Group 3 1.534 7 Experimental Group 4 1.783 8 Positive control group 1 1.688 9 Positive control group 2 1.203

[0121] ②Triglyceride (TG) detection

[0122] Triglyceride (TG) content was determined using a triglyceride assay kit. The prepared cell suspension was removed and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the cell pellet was washed twice with PBS. The cells were centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The cell pellet was then washed with 100 μL of RIPA lysis buffer. After thorough lysis, the cells were assayed according to the kit protocol. TG content was corrected based on the protein concentration per gram.

[0123] Table 11 Effect on triglyceride content

[0124]

[0125]

[0126] The experimental results showed that the honeysuckle fermentation broth obtained by D8A probiotic fermentation can significantly inhibit lipid accumulation in 3T3-L1 preadipocytes. Oil red O staining showed that as the concentration of the fermentation broth increased (0.1-0.5 mg / mL), the intracellular lipid droplets decreased significantly, even lower than the pure chlorogenic acid group (OD=1.688). The OD value dropped from 2.917 in the blank group to 1.534 in experimental group 3 (a decrease of 47.4%). It is worth noting that experimental group 4 (OD=1.783, TG=0.203 mmol / gprot), which was added with exopolysaccharides alone, also showed a certain fat-reducing effect. Although the effect was slightly lower than that of the D8A fermented honeysuckle group, it was still significantly better than the negative control group, indicating that exopolysaccharides themselves may participate in lipid regulation by regulating cellular metabolism. TG content detection further confirmed that the triglyceride level of the fermentation broth-treated group (0.157-0.218mmol / g prot) was significantly reduced by 44.5%-56.0% compared with the blank group (0.357mmol / g prot), and was dose-dependent. The effect of the simple probiotic group without the addition of honeysuckle (OD=1.996, TG=0.298) was significantly weaker than that of the D8A fermented honeysuckle group, indicating that there is a synergistic effect between honeysuckle and the D8A strain. These data confirm that the fermentation broth effectively reduces intracellular lipid deposition through a dual mechanism - inhibiting adipocyte differentiation and promoting lipid decomposition. Its effect is close to that of the positive control drug rosiglitazone (OD=1.203, TG=0.101), providing an important basis for the development of natural fat-reducing preparations based on probiotic fermentation.

[0127] Example 4

[0128] Preparation method of probiotic preparation

[0129] Probiotic fermentation culture: After streaking the probiotics, pick out a single bacterium and put it into liquid MRS, culture it at 37℃, and after two rounds of activation, use a 3% inoculation volume and a concentration of not less than 1×10 7 CFU / mL, the culture medium was inoculated and fermented at 37°C for 24 hours. The fermentation culture was divided into two parts. The first part was centrifuged to obtain probiotic sludge, which was divided into two parts. The probiotic sludge was evenly mixed with different embedding protective agents and freeze-dried to obtain the first probiotic preparation and the second probiotic preparation; in the second part, the fermentation broth was directly inactivated and then concentrated and freeze-dried to obtain postbiotics.

[0130] The culture medium includes the following components by weight percentage: 10.0g of casein digest, 2.0g of triammonium citrate, 10.0g of beef extract powder, 0.2g of magnesium sulfate, 4.0g of yeast extract powder, 0.05g of manganese sulfate, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 15g of glucose, 10g of honeysuckle powder, 1.08g of Tween-80, pH 5.8, and water is used to adjust the volume to 1L. The probiotic slurry and the embedding protective agent and the freeze-drying protective agent in the first embodiment include the following components by weight percentage: 10-15% of skim milk powder, 0.6-2.5% of calcium alginate, 2-4% of pectin, 1-2% of inulin, and the balance of water to obtain a first probiotic preparation.

[0131] The second solution is based on the following components: 10-15% skim milk powder, 0.6-2.5% calcium alginate, 2-4% pectin, 0.5-1.5% inulin, 0.5-1% exopolysaccharide (extraction method 2.2 in Example 2), and the balance water to obtain a second probiotic preparation.

[0132] Due to their high viscosity and film-forming ability, extracellular polysaccharides can synergize with ingredients such as calcium alginate and pectin to form a dense microcapsule network, effectively encapsulating probiotics and shielding them from adverse external factors like gastric acid and bile salts, thereby enhancing bacterial survival in the gastrointestinal tract. Their high molecular weight and branched structure can slow the disintegration of microcapsules, enabling targeted release of probiotics in the intestine and enhancing colonization. Furthermore, cell-based experiments have demonstrated that these polysaccharides can significantly promote fat metabolism, acting as a protective agent while also directly exerting their functional activity, enhancing the overall health benefits of the preparation.

[0133] Example 5

[0134] Animal performance testing

[0135] 1. Animal experiment design

[0136] 115 SPF male C57BL / 6J mice (4-6 weeks) were adapted for feeding for 1 week. During the entire experimental period, the mice were housed in a specific pathogen-free environment with a temperature of 23±2°C and a humidity of 55±10%, a 12-h light / dark cycle, and free access to water.

[0137] Fifteen mice were randomly selected from the blank control group (C) and maintained on a normal diet daily. The remaining 85 mice were fed a 60% kilocalorie high-fat diet according to the standard. Their body weight was recorded weekly and their food intake was recorded every two days. After four weeks of feeding, the model group was judged to have formed an obesity model based on the body weight of the mice being 120% of the blank group. 90 mice with qualified body weight were selected and randomly divided into six groups: model group (M), positive drug group (PCG, positive control group), probiotic preparation group 1 low concentration group (P-D1), probiotic preparation group 1 high concentration group (P-H1), probiotic preparation group 2 (P-D2), probiotic preparation group 2 high concentration group (P-H2) and probiotic inactivation group (IP, inactivation group). The model group was gavaged with the same volume of sterile distilled water every day, the positive drug group was gavaged with 5 mg / kg / d, and the probiotic preparation group 1 / 2 low concentration group was gavaged with 1×10 bacterial powder every day after modeling. 9 CFU, probiotic preparation group 1 / 2 high concentration group were gavaged with bacterial powder 5×10 every day after modeling. 9 CFU. The postbiotic group was gavaged daily with 100 mg / kg / day for 6 weeks after model establishment. All groups except the blank group continued to be fed a high-fat diet with unlimited water.

[0138] 2. Effects on the body weight of obese mice

[0139] To investigate the effects of the untreated group on obese mice, monitor their daily diet and behavior, and keep records. Weigh the mice weekly.

[0140] Table 12 Mouse weight changes

[0141] Serial number Group Initial body weight (g) Final body weight (g) 1 C 19.31±1.02 22.35±2.11 2 M 24.31±1.44 39.81±2.91 3 PCG 24.61±2.09 25.33±1.06 4 P-D1 25.11±1.58 29.28±2.01 5 P-H1 25.35±2.09 28.71±2.10 6 P-D2 25.37±2.01 28.91±2.35 7 P-H2 25.01±1.99 25.17±2.17 8 IP 24.22±2.71 30.56±1.94

[0142] According to the mouse weight change table, there were significant differences in weight gain among the groups, reflecting the effects of different intervention measures. The initial weight of the model group (M) was 24.31g, and eventually increased to 39.81g (an increase of 63.8%), indicating that the obesity model was successfully established. The positive control group (PCG) only gained 2.9% in weight (finally 25.33g) through standard intervention, confirming its strong inhibitory effect. Among the experimental groups, the P-H2 group performed outstandingly, with a final weight of 25.17g (an increase of 0.6%), and the effect was close to that of the PCG group; while the weight gains of other experimental groups (such as P-D1 and IP) were between 15% and 26%, and the effect was second best. It is worth noting that the excellence of the P-H2 group may be related to the specific component of the extracellular polysaccharide combined with the probiotic preparation.

[0143] 3. Mouse serum collection and biochemical index detection

[0144] At the end of the experimental intervention, the mice were eyeballed and blood was collected. The supernatant was centrifuged and placed in a 4°C refrigerator for later use. After blood collection, the mice were killed by cervical dislocation and dissected. After the samples were collected, they were placed in an ultra-low temperature refrigerator for later use. Six serum samples were randomly selected from each group and the serum triglyceride (TG), total cholesterol (TCHO), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured according to the instructions of the Nanjing Jiancheng kit.

[0145] Table 13 Test results of various indicators in serum

[0146]

[0147]

[0148] Experimental results showed that a high-fat diet successfully induced lipid disorders and liver damage in mice, manifested by significant increases in triglycerides (PG), total cholesterol (TCHO), and low-density lipoprotein cholesterol (LDL-C), decreases in high-density lipoprotein cholesterol (HDL-C), and concomitant increases in liver enzymes (ALT / AST). Following probiotic intervention, the live bacteria group (particularly the polysaccharide-encapsulated probiotic preparation) demonstrated comprehensive metabolic improvements: lipid profiles approached normal levels, LDL-C decreased while HDL-C increased, and liver enzyme levels decreased significantly, suggesting that probiotics work by regulating lipid metabolism and alleviating liver inflammation. The inactivated bacteria group showed weaker effects than the live bacteria group, suggesting that active metabolites of probiotics (such as short-chain fatty acids) are key to improving metabolism. While the positive drug group demonstrated significant lipid-lowering effects, its cholesterol homeostasis regulation and safety profile were inferior to those of probiotics. The overall trend suggests that live probiotic intervention can synergistically alleviate metabolic abnormalities induced by a high-fat diet through multiple pathways, with a superior safety profile.

[0149] 4. Liver lipid content detection

[0150] Mouse liver tissue was frozen and ground in physiological saline buffer, and the supernatant was taken. The TG and T-CHO contents in the liver were determined by diluting the supernatant according to a certain ratio and strictly following the operating procedures of the kit.

[0151] Table 14 Effects on lipids in mouse liver

[0152] Serial number Group TG (mmol / gprot) TCHO (mmol / gprot) 1 C 0.912±0.098 0.521±0.062 2 M 2.357±0.209 1.135±0.106 3 PCG 1.109±0.130 0.679±0.101 4 P-D1 1.775±0.137 0.821±0.069 5 P-H1 1.543±0.163 0.701±0.006 6 P-D2 1.573±0.129 0.775±0.401 7 P-H2 1.209±0.116 0.690±0.205 8 IP 1.734±0.216 0.801±0.201

[0153] Experimental data showed that a high-fat diet significantly increased the triglyceride (TG) and total cholesterol (TCHO) content in the liver of mice, and the model group had the most serious lipid accumulation in the liver. After intervention, the liver lipid level of the positive drug group (PCG) was close to that of the normal control group, indicating its potent lipid-lowering effect. Among the probiotic live bacteria intervention groups, the live bacteria group, especially the probiotic preparation group encapsulated in polysaccharides, showed excellent P-H2 performance, and the TG and TCHO content was close to that of the positive drug group, indicating that its intervention measures (such as high-dose extracellular polysaccharide preparations) may have a significant effect by inhibiting lipid synthesis or promoting metabolism.

[0154] The TG and TCHO contents of the probiotic preparation groups without extracellular polysaccharide encapsulation (P-D1 and P-H1) were significantly lower than those in the model group, but still slightly higher than those in the positive drug group, indicating that probiotics can effectively alleviate liver lipid deposition, but the effect is weaker than that of chemical drugs. It is worth noting that the improvement effect of the inactivated bacteria group (IP) was significantly inferior to that of the live bacteria group, further confirming the importance of the metabolic activity of live bacteria (such as regulating lipid synthesis or decomposition pathways). In addition, the effect of the low concentration of probiotic preparation group 2 (P-D2) was inferior to that of the high concentration group. Taken together, the intervention of live probiotics and the protection of live bacteria by metabolites-polysaccharides has a fat-reducing effect on high-fat model mice, and its effect trend is consistent with the improvement of serum indicators.

Claims

1. A fermentation liquid with fat-reducing function, characterized in that: Obtained by fermenting honeysuckle dry powder with Lactobacillus rhamnosus D8A.

2. The method for preparing a fermentation liquid according to claim 1, wherein: The steps include: 3 vol% of the bacterial liquid of the adaptively evolved Lactobacillus rhamnosus D8A was inoculated into a liquid fermentation medium and fermented at 37° C. for 24 hours to obtain a fermentation liquid.

3. The method for preparing a fermentation liquid according to claim 2, wherein: The preparation method of the liquid fermentation medium comprises the steps of mixing 10.0 g of casein enzyme digest, 2.0 g of triammonium citrate, 10.0 g of beef extract powder, 0.2 g of magnesium sulfate, 4.0 g of yeast extract powder, 0.05 g of manganese sulfate, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 15 g of glucose, 10 g of honeysuckle dry powder, and 1.08 g of Tween-80, and then diluting the mixture to 1 L with water; The pH value of the liquid fermentation medium is 5.

8.

4. The method for preparing a fermentation liquid according to claim 2, wherein: The adaptively evolved Lactobacillus rhamnosus D8A is obtained by sequentially fermenting honeysuckle dry powder soaking solutions with increasing weight ratios of honeysuckle dry powder to water by Lactobacillus rhamnosus D8A.

5. The use of the fermentation liquid according to claim 1, wherein: The method is used for preparing medicines, health products or foods for inhibiting lipid accumulation in 3T3-L1 preadipocytes.

6. A probiotic preparation, characterized in that: The freeze-dried powder of the fermentation broth according to claim 1.

7. A method for preparing a probiotic preparation, characterized in that: The following steps are involved: After the culture medium containing honeysuckle powder is sterilized and cooled to room temperature, the adaptively evolved Lactobacillus rhamnosus D8A is inoculated, fermented, centrifuged, a freeze-protectant is added, and then freeze-dried; or The culture medium containing honeysuckle powder was sterilized and cooled to room temperature, and then inoculated with adaptively evolved Lactobacillus rhamnosus D8A, fermented and cultured, inactivated, and then concentrated and freeze-dried.

8. The method for preparing the probiotic preparation according to claim 7, wherein: The inoculation concentration of the adaptively evolved Lactobacillus rhamnosus D8A is not less than 1×10 7 CFU / mL; The fermentation temperature is 37° C. and the fermentation time is 24 hours.

9. The method for preparing the probiotic preparation according to claim 7, wherein: The freeze-drying protective agent comprises the following components in percentage by weight: 10-15% skim milk powder, 0.6-2.5% calcium alginate, 2-4% pectin, 1-2% inulin, and the balance water, or The freeze-drying protective agent comprises the following components in percentage by weight: 10-15% skim milk powder, 0.6-2.5% calcium alginate, 2-4% pectin, 0.5-1.5% inulin, 0.5-1% exopolysaccharide of Lactobacillus rhamnosus D8A, and the balance water.

10. The use of the probiotic preparation according to claim 6, characterized in that: The invention can be used to prepare medicines, foods or health products that inhibit lipid synthesis or promote lipid metabolism.

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