A probiotic compound for metabolic intervention and its preparation method and application

Abstract

This invention discloses a probiotic complex for metabolic intervention, its preparation method, and its application, belonging to the field of microbial technology. The probiotic complex includes *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum ), fermenting lactobacillus ( Limosilactobacillus fermentum ), Lactobacillus reuteri ( Limosilactobacillus reuteri Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) and Bifidobacterium breve ( Bifidobacterium breve The viable count ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* is (2–3):(2–2.5):(2–3.5):(1–2):(0.8–1). These probiotics work synergistically to effectively regulate the balance of the intestinal flora, improve glucose and lipid metabolism, regulate energy metabolism, and inhibit inflammatory factors, achieving precise intervention in metabolic disorders. Furthermore, the strains exhibit good compatibility and high stability, allowing for the preparation of various probiotic formulations suitable for the treatment of metabolic syndrome, obesity, hyperlipidemia, hyperglycemia, and other metabolic abnormalities, with broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a probiotic complex for metabolic intervention, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Obesity and related metabolic diseases, such as type 2 diabetes, hyperlipidemia, and non-alcoholic fatty liver disease, have become a global public health problem. Among existing interventions, diet control and exercise are basic measures, but patient compliance is poor and the treatment period is long. Drug treatment, such as orlistat and metformin, can quickly improve metabolic indicators, but they are often accompanied by side effects such as gastrointestinal adverse reactions and liver damage, and rebound is likely after discontinuation of the drug.

[0004] In recent years, significant progress has been made in research on the relationship between gut microbiota and host metabolism. Evidence suggests that obese individuals and those with metabolic disorders commonly exhibit gut microbiota dysbiosis, characterized by a reduction in beneficial bacteria and an increase in opportunistic pathogens, leading to impaired intestinal barrier function, chronic low-grade inflammation, and abnormal energy absorption. Therefore, supplementing with exogenous probiotics to reshape the gut microbiota is considered a safe and sustainable metabolic intervention strategy.

[0005] However, most current probiotic products use single strains, which can only act on a specific metabolic pathway and are difficult to comprehensively correct multi-target imbalances in metabolic disorders. Although some studies have attempted to combine several probiotics, whether there are antagonistic effects between different strains, whether they can synergistically colonize in the gut, and how to optimize the live bacteria ratio of each strain are all technical challenges faced by those skilled in the art. Therefore, how to screen compound probiotic formulations that can synergistically act in the gut environment and precisely intervene in different metabolic targets is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a probiotic complex for metabolic intervention, its preparation method, and its application. The complex utilizes multiple strains to synergistically intervene in metabolism, precisely regulating the balance of intestinal flora and achieving efficient metabolic intervention.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a probiotic complex for metabolic intervention, the probiotic complex comprising *Lactobacillus plantarum* (… Lactiplantibacillus plantarum ), fermenting lactobacillus ( Limosilactobacillus fermentum), Lactobacillus reuteri ( Limosilactobacillus reuteri Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) and Bifidobacterium breve ( Bifidobacterium breve ); The viable count ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* is (2–3):(2–2.5):(2–3.5):(1–2):(0.8–1).

[0008] Preferably, the viable count mass ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* is 2:2.5:3:1.5:1.

[0009] Preferably, the total viable count of the probiotic complex is not less than 1×10⁻⁶. 9 ~1×10 11 CFU / mL.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned probiotic complex, comprising the following steps: S1. The freeze-dried powder cultures of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* were respectively inoculated into liquid MRS medium and cultured. The operation was repeated twice to obtain five single-strain seed cultures. S2. The seed cultures of the five single bacteria were inoculated into liquid MRS medium for fermentation culture. After fermentation, the bacterial sludge was collected by centrifugation and washed with sterile physiological saline to obtain wet bacterial sludge of the five single bacteria. S3. Mix the five types of single-strain wet sludge according to the mass ratio of live bacteria, and stir evenly to obtain the final product.

[0011] Preferably, in S1, the inoculation amount is 1-3%; The culture conditions for *Lactobacillus plantarum* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the fermented Lactobacillus mucinus were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus reuteri* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus rhamnosus* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the *Bifidobacterium breve* were: 37±2℃, 180~220r / min, anaerobic culture for 18~24h.

[0012] Preferably, in S2, the inoculum size is 1-3%, and the fermentation pH is controlled at 6.0-6.5; The culture conditions for *Lactobacillus plantarum* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the fermented Lactobacillus mucinus were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus reuteri* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus rhamnosus* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the *Bifidobacterium breve* were: 37±2℃, 180~220r / min, anaerobic culture for 18~24h.

[0013] Preferably, in S2, the centrifugation speed is 8000 r / min and the centrifugation time is 8 to 12 min.

[0014] Thirdly, the present invention provides a probiotic preparation, the active ingredient of which is the above-mentioned probiotic complex and the probiotic complex prepared by the above-mentioned preparation method.

[0015] Preferably, the dosage form of the probiotic preparation is any one of powder, granules, capsules, tablets, or oral liquid.

[0016] Fourthly, the present invention provides the application of the above-mentioned probiotic complex, the probiotic complex prepared by the above-mentioned preparation method, or the above-mentioned probiotic preparation in the preparation of metabolic intervention-related products, wherein the products are food, health food, or dietary supplements, and the metabolic intervention includes regulating the balance of intestinal flora, improving glucose and lipid metabolism, regulating energy metabolism, alleviating obesity, reducing inflammatory factors, reducing blood lipids, or reducing blood sugar.

[0017] The beneficial effects of this invention are as follows: This invention achieves a breakthrough in multi-target synergistic intervention through a combination of five probiotic strains in a specific ratio. The five probiotic strains exhibit significant synergistic effects, with complementary functions, colonization compatibility, and no antagonistic effects. They can form a stable symbiotic system in the gut, jointly exerting metabolic intervention effects. Specifically, *Lactobacillus plantarum* inhibits the proliferation of harmful intestinal bacteria, creating a suitable environment for the colonization of other beneficial bacteria, while also initially regulating lipid metabolism; *Lactobacillus fermentum* removes excess cholesterol from the intestine, reduces oxidative stress damage to pancreatic β-cells, and helps improve glucose and lipid metabolism disorders; *Lactobacillus reuteri* produces reuterin, enhancing intestinal barrier integrity, reducing intestinal fat absorption, and regulating bile acid metabolism, further optimizing lipid metabolism; *Lactobacillus rhamnosus* inhibits chronic intestinal inflammation, alleviates inflammation-mediated metabolic imbalance, and helps regulate glucose metabolism; and *Bifidobacterium breve* produces short-chain fatty acids such as acetic acid through fermentation, protecting the intestinal mucosal barrier, regulating lipopolysaccharide translocation, and reducing metabolic endotoxemia, laying a good foundation for metabolic intervention. Crucially, when combined according to a specific ratio of live bacteria, they can mutually promote colonization in the intestinal environment, synergistically metabolize substrates, and jointly regulate the intestinal microecological structure, achieving efficient metabolic intervention. Attached Figure Description

[0018] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0019] Figure 1 The effect on mouse body weight gain; Figure 2 This is a comparison chart of mouse body sizes; Figure 3 The effect on blood biochemical parameters in mice; Figure 4 The effect of IL-6, an inflammatory factor in mouse serum; Figure 5 Representative images of mouse adipocytes stained with H&E (scale bar = 100 μm). Figure 6 This represents the fasting blood glucose level in mice. Figure 7 Oral glucose tolerance test; Figure 8 This is an insulin tolerance test. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] This invention provides a probiotic complex for metabolic intervention, the probiotic complex comprising *Lactobacillus plantarum* (… Lactiplantibacillus plantarum ), fermenting lactobacillus ( Limosilactobacillus fermentum ), Lactobacillus reuteri ( Limosilactobacillus reuteri Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) and Bifidobacterium breve ( Bifidobacterium breve ); The viable count ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* is (2–3):(2–2.5):(2–3.5):(1–2):(0.8–1).

[0023] The five strains mentioned above are all beneficial bacteria native to the human gut, possessing strong colonization capabilities and complementary functions, with no inter-strain antagonism, thus providing a prerequisite for synergistic intervention. Specifically, *Lactobacillus plantarum* can inhibit the proliferation of harmful intestinal bacteria, creating a favorable environment for the colonization of other beneficial bacteria, while also regulating lipid metabolism; *Lactobacillus fermentum* can remove cholesterol, reduce oxidative stress damage to pancreatic β-cells, and help improve glucose and lipid metabolism; *Lactobacillus reuteri* can produce reuterin, enhancing intestinal barrier function and reducing fat absorption; *Lactobacillus rhamnosus* can inhibit intestinal inflammatory responses and alleviate inflammation-mediated metabolic disorders; and *Bifidobacterium breve* can produce acetic acid through fermentation, protecting the intestinal mucosa, regulating lipopolysaccharide translocation, and reducing metabolic endotoxemia. The functional complementarity of these strains lays the foundation for multi-target synergistic intervention.

[0024] Specifically, the live bacteria ratio in this invention is scientifically optimized based on the growth characteristics, colonization ability, and functional strength of each strain, maximizing the synergistic effect of each strain. Among them, *Lactobacillus reuteri* and *Lactobacillus plantarum* constitute a relatively high proportion, fully leveraging their core roles in intestinal barrier repair and harmful bacteria inhibition, laying the foundation for metabolic intervention; *Lactobacillus fermentum* assists in regulating lipid metabolism, synergizing with the first two strains to enhance the lipid metabolism improvement effect; *Lactobacillus rhamnosus* and *Bifidobacterium breve* constitute a moderate proportion, precisely regulating intestinal inflammation and endotoxin levels, avoiding interference from inflammatory factors on metabolism, and not competing with other strains for intestinal colonization sites. When the proportion of a particular strain is too low, it cannot effectively colonize due to its inability to resist the gastrointestinal environment, its function is diluted, leading to a decrease in overall efficacy; at the same time, an excessive amount of a single strain may also be utilized by other microorganisms as an exogenous carbon source, increasing the metabolic burden on the host. An excessively high proportion of a particular strain can lead to niche monopoly, inhibiting the activity of other strains and weakening synergistic effects; abnormal accumulation of its metabolites can disrupt intestinal homeostasis, inducing bloating and diarrhea; at the same time, continuous antigen stimulation may overactivate the immune system, especially in people with allergies or immune disorders, which can easily trigger unexpected or excessive immune responses.

[0025] This specific ratio enables the five strains to form a synergistic chain of barrier repair, inflammation suppression, and glycolipid regulation. Each strain promotes and complements the others, achieving multi-target, systemic intervention for metabolic disorders. Its overall metabolic intervention effect is significantly better than that of single strains and arbitrary ratios of compound schemes, effectively solving the technical problems of poor synergy and unsatisfactory intervention effects of existing compound complexes.

[0026] In some embodiments, the viable count mass ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* is 2:2.5:3:1.5:1.

[0027] In some embodiments, the total live bacteria count of the probiotic complex is not less than 1 × 10⁻⁶. 9 ~1×10 11 CFU / mL.

[0028] This invention provides a method for preparing the above-mentioned probiotic complex, comprising the following steps: S1. The freeze-dried powder cultures of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* were respectively inoculated into liquid MRS medium and cultured. The operation was repeated twice to obtain five single-strain seed cultures. S2. The seed cultures of the five single bacteria were inoculated into liquid MRS medium for fermentation culture. After fermentation, the bacterial sludge was collected by centrifugation and washed with sterile physiological saline to obtain wet bacterial sludge of the five single bacteria. S3. Mix the five types of single-strain wet sludge according to the mass ratio of live bacteria, and stir evenly to obtain the final product.

[0029] In some embodiments, in S1, the inoculation amount is 1-3%; The culture conditions for *Lactobacillus plantarum* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the fermented Lactobacillus mucinus were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus reuteri* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus rhamnosus* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the *Bifidobacterium breve* were: 37±2℃, 180~220r / min, anaerobic culture for 18~24h.

[0030] In some embodiments, in S2, the inoculum size is 1-3%, and the fermentation pH is controlled at 6.0-6.5; The culture conditions for *Lactobacillus plantarum* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the fermented Lactobacillus mucinus were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus reuteri* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus rhamnosus* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the *Bifidobacterium breve* were: 37±2℃, 180~220r / min, anaerobic culture for 18~24h.

[0031] In some embodiments, in S2, the centrifugation speed is 8000 r / min and the centrifugation time is 8 to 12 min.

[0032] This invention provides a probiotic preparation, the active ingredients of which are the above-mentioned probiotic complex and the probiotic complex prepared by the above-mentioned preparation method.

[0033] In some embodiments, the dosage form of the probiotic preparation is any one of powder, granules, capsules, tablets, or oral liquid.

[0034] In some embodiments, the probiotic preparation comprises the above-described probiotic complex and a pharmaceutically acceptable carrier; The carrier is selected from one or more of maltodextrin, inulin, fructooligosaccharides, galactooligosaccharides, dietary fiber, and skim milk powder; In the probiotic preparation, the probiotic complex accounts for 10-50% of the mass, and the carrier accounts for 50-90% of the mass.

[0035] In some embodiments, the probiotic preparation further includes resistant dextrin and fructooligosaccharides; The total amount of the polyfructose, resistant dextrin, and phospholipids used is 10%-20% of the probiotic complex.

[0036] Specifically, resistant dextrin and fructooligosaccharides, as prebiotics, can promote the proliferation of probiotics and increase their colonization rate in the intestines.

[0037] This invention provides the application of the above-mentioned probiotic complex, the probiotic complex prepared by the above-mentioned method, or the above-mentioned probiotic preparation in the preparation of metabolic intervention-related products. The products are food, health food, or dietary supplements. The metabolic intervention includes regulating the balance of intestinal flora, improving glucose and lipid metabolism, regulating energy metabolism, alleviating obesity, reducing inflammatory factors, reducing blood lipids, or reducing blood sugar.

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0039] All strains used in the following examples and comparative examples are publicly available commercial strains or preserved strains. *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The following bacteria are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 1.2469; *Lactobacillus fermentum* ( Limosilactobacillus fermentum ) is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 1.15608; Lactobacillus reuteri ( Limosilactobacillus reuteri ) is deposited at the China Agricultural Microbial Culture Collection Center, with accession number ACCC 0349; Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus Bifidobacterium breve ( ) is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 1.2467; Bifidobacterium breve It is deposited in the China Industrial Microbial Culture Collection Center, with accession number CICC 6182.

[0040] Example 1: S1. Preparation of Single-Bacterial Seed Cultures: Lyophilized powder cultures of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* were inoculated at 2% (w / v) into liquid MRS medium. *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, and *Lactobacillus rhamnosus* were cultured aerobically at 37℃ and 200 rpm for 20 h; *Bifidobacterium breve* was cultured anaerobically at 37℃ and 200 rpm for 20 h. The above activation process was repeated twice to obtain five single-bacterial seed cultures.

[0041] S2. Single-strain fermentation culture: Five single-strain seed cultures were inoculated into liquid MRS medium at an inoculum volume of 2%. The fermentation pH was controlled at 6.2, and fermentation was carried out at 37℃ and 200 rpm for 24 h (Bifidobacterium breve is anaerobic). After fermentation, the bacterial sludge was collected by centrifugation at 4℃ and 8000 rpm for 10 min, and washed three times with sterile physiological saline to obtain wet bacterial sludge of the five single strains.

[0042] S3. Compound Mixing: The viable cell concentration of each single-strain wet sludge was determined using the plate count method. Then, the viable cell masses were mixed according to the following ratio: *Lactobacillus plantarum*: *Lactobacillus fermentum*: *Lactobacillus reuteri*: *Lactobacillus rhamnosus*: *Bifidobacterium breve* = 2:2.5:3:1.5:1, and stirred thoroughly to obtain a liquid probiotic complex. The total viable cell count was 1 × 10⁻⁶. 9 CFU / mL.

[0043] Example 2: The preparation method is the same as in Example 1, except that in step S3, the viable bacteria are mixed in a mass ratio of *Lactobacillus plantarum*: *Lactobacillus fermentum*: *Lactobacillus reuteri*: *Lactobacillus rhamnosus*: *Bifidobacterium breve* = 2:2:2:1:0.8. The total viable bacteria count is 1 × 10⁻⁶. 9 CFU / mL.

[0044] Example 3: The preparation method is the same as in Example 1, except that in step S3, the viable bacteria are mixed in a mass ratio of *Lactobacillus plantarum*: *Lactobacillus fermentum*: *Lactobacillus reuteri*: *Lactobacillus rhamnosus*: *Bifidobacterium breve* = 3:2.5:3.5:2:1. The total viable bacteria count is 1 × 10⁻⁶. 9 CFU / mL.

[0045] Example 4: Preparation of solid probiotic formulation Take 100g of the probiotic complex prepared in Example 1, add 700g of maltodextrin, 90g of resistant dextrin, and 30g of fructooligosaccharide (the ratio of resistant dextrin to fructooligosaccharide is 3:1), mix evenly, process by freeze drying, pulverize and pass through an 80-mesh sieve, and package to obtain solid probiotic powder.

[0046] Comparative Example 1 Following steps S1 and S2 of Example 1, only *Lactobacillus plantarum* was cultured to obtain *Lactobacillus plantarum* wet bacterial mud, without further processing.

[0047] The same carriers (maltodextrin, resistant dextrin, and fructooligosaccharides) as in Example 4 were directly added to prepare a solid powder. This powder was then dissolved in sterile saline to prepare a gavage solution, to be prepared fresh for each use. The total viable count was 1 × 10⁻⁶. 9 The CFU / mL count is consistent with the total viable count of the probiotic complex in Example 1.

[0048] Comparative Example 2 Following steps S1 and S2 of Example 1, only *Lactobacillus mucinus* was cultured to obtain fermented *Lactobacillus mucinus* wet sludge, without further compounding.

[0049] The same carrier as in Example 4 was directly added to prepare a solid powder, which was then dissolved in sterile physiological saline to prepare a gavage solution. The solution was prepared fresh for each use, and the total viable count was 1 × 10⁻⁶. 9 The CFU / mL count is consistent with the total viable count of the probiotic complex in Example 1.

[0050] Comparative Example 3 Following steps S1 and S2 of Example 1, only *Lactobacillus reuteri* was cultured to obtain *Lactobacillus reuteri* wet bacterial mud, without further processing.

[0051] The same carrier as in Example 4 was directly added to prepare a solid powder, which was then dissolved in sterile physiological saline to prepare a gavage solution. The solution was prepared fresh for each use, and the total viable count was 1 × 10⁻⁶. 9 The CFU / mL count is consistent with the total viable count of the probiotic complex in Example 1.

[0052] Comparative Example 4 Following steps S1 and S2 of Example 1, only Lactobacillus rhamnosus is cultured to obtain wet Lactobacillus rhamnosus sludge, without further processing.

[0053] The same carrier as in Example 4 was directly added to prepare a solid powder, which was then dissolved in sterile physiological saline to prepare a gavage solution. The solution was prepared fresh for each use, and the total viable count was 1 × 10⁻⁶. 9 The CFU / mL count is consistent with the total viable count of the probiotic complex in Example 1.

[0054] Comparative Example 5 Following steps S1 and S2 of Example 1, only Bifidobacterium breve is cultured to obtain Bifidobacterium breve wet sludge, without further processing.

[0055] The same carrier as in Example 4 was directly added to prepare a solid powder, which was then dissolved in sterile physiological saline to prepare a gavage solution. The solution was prepared fresh for each use, and the total viable count was 1 × 10⁻⁶. 9The CFU / mL count is consistent with the total viable count of the probiotic complex in Example 1.

[0056] Comparative Example 6: The preparation method is the same as in Example 1, except that in step S3, the viable bacteria are mixed in the following mass ratio: *Lactobacillus plantarum*: *Lactobacillus fermentum*: *Lactobacillus reuteri*: *Lactobacillus rhamnosus*: *Bifidobacterium breve* = 1.7:1.7:1.7:0.5:0.5.

[0057] The same carriers (maltodextrin, resistant dextrin, and fructooligosaccharides) as in Example 4 were added to prepare a solid powder. This powder was then dissolved in sterile physiological saline to prepare a gavage solution, to be prepared fresh for each use. The total viable count was 1 × 10⁻⁶. 9 The CFU / mL count is consistent with the total viable count of the probiotic complex in Example 1.

[0058] Comparative Example 7: The preparation method is the same as in Example 1, except that in step S3, the viable bacteria are mixed in a mass ratio of 5:3:5:3:1: *Lactobacillus plantarum*: *Lactobacillus fermentum*: *Lactobacillus reuteri*: *Lactobacillus rhamnosus*: *Bifidobacterium breve*.

[0059] The same carriers (maltodextrin, resistant dextrin, and fructooligosaccharides) as in Example 4 were added to prepare a solid powder. This powder was then dissolved in sterile physiological saline to prepare a gavage solution, to be prepared fresh for each use. The total viable count was 1 × 10⁻⁶. 9 The CFU / mL count is consistent with the total viable count of the probiotic complex in Example 1.

[0060] III. Animal Experiment Verification Experimental Example 1: Laboratory animals and grouping Sixty 6-week-old SPF-grade male C57BL / 6J mice were selected and, after one week of acclimatization feeding, were randomly divided into 10 groups (n=6): Normal control group (NC group): fed with ordinary feed throughout the treatment period, and administered 200μL of sterile saline by gavage at regular intervals every day during the treatment period; Obesity model group (HFD group): fed with 60% high-fat diet throughout the treatment period, and administered 200μL of sterile saline by gavage at regular intervals every day during the treatment period; Example 1 Group (Group E): Feeded with a 60% high-fat diet throughout the treatment period, and administered 200 μL of the solid probiotic powder prepared in Example 4 (dissolved in physiological saline) (live bacteria count 1 × 10⁻⁶) daily by gavage at regular intervals during the treatment period. 9 (CFU / mL) Comparative Examples 1-7 (D1-D7): 200 μL of the solid powder prepared in Comparative Examples 1-7 (dissolved in physiological saline) was administered by gavage (concentration of 1×10⁻⁶ for each group). 9 (CFU / mL) The gavage treatment lasted for 8 weeks, with mouse body weight recorded weekly. After the treatment period, mice were fasted for 12 hours, anesthetized with pentobarbital, and blood was collected from the orbital rim to measure serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and the inflammatory factor IL-6. Epididymal adipose tissue was harvested for H&E staining to observe adipocyte morphology.

[0061] Experimental results The representative effect test results of the E group and D1-D7 groups in obese mice are shown in Table 1.

[0062] Table 1 Test Results

[0063] (1) Weight gain like Figure 1 and Figure 2 As shown, after 8 weeks of treatment, the body weight of mice in the HFD group was significantly increased compared to that in the NC group. Compared to the HFD group, the body weight gain of mice in the E group was significantly inhibited.

[0064] As shown in Table 1, the weight reduction effect of the five-strain complex of the present invention is significantly better than that of any single strain, and the weight reduction effect of Example 1 is significantly better than that of combinations that deviate from the ratio, proving that a specific ratio is the key to achieving synergistic weight reduction.

[0065] (2) Serum biochemical indicators like Figure 3 As shown in Table 1, compared with the HFD group, the serum TC, TG, and LDL-C levels of mice in group E decreased by 16.22%, 42.35%, and 17.86%, respectively. The average reductions in TC, TG, and LDL-C in the single-strain groups from D1 to D5 were only 6.5%, 12.9%, and 10%, respectively. The lipid-lowering effects of groups D6 and D7 were also significantly inferior to those of group E.

[0066] (3) Levels of inflammatory factors like Figure 4 As shown in Table 1, the serum IL-6 level in group E mice was 40.80% lower than that in the HFD group, which was significantly better than that in the single strain group and the deviated ratio group.

[0067] (4) Morphology of adipose tissue like Figure 5 As shown, the adipocytes in the NC group were regular in shape and small in size; the adipocytes in the HFD group were significantly enlarged and irregular in shape; and the adipocytes in the E group were significantly smaller in size, close to that of the NC group.

[0068] The experimental data above show that the five-strain compound scheme of this invention, at the optimal ratio (Example 1), significantly improves the body weight, blood lipids, inflammatory factors, and fat accumulation in obese mice, and this effect is significantly better than that of single strains and combinations deviating from the ratio. This verifies that the ratio range is the key technical boundary for achieving synergistic effects of the five strains. Among them, the single strains in Comparative Examples 1-7 cannot cover multiple target metabolic pathways, and the effect is limited. Even if all five strains are present, if the ratio is unbalanced, the intervention effect will be significantly reduced due to the antagonism of the five compound strains or insufficient effective dose.

[0069] Experimental Example 2: The effect of probiotic complex on blood glucose regulation in type 2 diabetic mice Laboratory animals and grouping Sixty 6-week-old SPF-grade male C57BL / 6J mice were selected. After one week of acclimatization, all groups except the NC group were fed a 60% high-fat diet for 10.5 weeks to induce a type 2 diabetes model. After successful model establishment, they were randomly divided into 10 groups (n=6): Normal control group (NC group): fed with ordinary feed throughout the entire process, and gavaged with 200μL of physiological saline; Diabetes model group (T2DM group): 200 μL of normal saline was administered by gavage; Example 1 Group (Group E): 200 μL of the solid probiotic powder prepared in Example 4 (dissolved in physiological saline) was administered by gavage. Comparative Examples 1-7 (D1-D7): Each group was administered 200 μL of the solid powder prepared in Comparative Examples 1-7 (dissolved in physiological saline) by gavage.

[0070] The gavage treatment lasted for 8 weeks. After the treatment was completed, fasting blood glucose (FBG) was measured, and oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed.

[0071] Experimental results Table 2 shows the representative effect test results of the E group and the D1-D7 group in diabetic mice.

[0072] Table 2 Test Results

[0073] (1) Fasting blood glucose like Figure 6 As shown in Table 2, after 8 weeks of treatment, the fasting blood glucose level in the T2DM group was as high as 8.1 mmol / L. The fasting blood glucose level in the E group decreased to 6.5 mmol / L, which was close to that of the NC group (4.9 mmol / L). As shown in Table 2, the fasting blood glucose levels in the single-strain groups from D1 to D5 were 7.9-10.2 mmol / L, while those in the D6 and D7 groups were 8.9 mmol / L and 9.1 mmol / L, respectively, all of which were significantly higher than those in the E group.

[0074] (2) Oral glucose tolerance test like Figure 7 As shown in Table 2, the OGTT results showed that the peak blood glucose levels and area under the curve (AUC) of mice in group E at 60 min and 120 min after gavage were significantly lower than those in the T2DM group, each single strain group, and the deviation ratio group.

[0075] (3) Insulin tolerance like Figure 8 As shown in Table 2, the ITT results showed that the E group mice experienced the greatest decrease in blood glucose and a significant improvement in insulin sensitivity after insulin injection. The improvement in insulin sensitivity in the single strain group and the deviated ratio group was inferior to that in the E group.

[0076] The experimental data above show that the probiotic complex prepared in this invention (especially the optimal ratio) can significantly reduce fasting blood glucose, improve glucose tolerance and insulin sensitivity in type 2 diabetic mice, and its effect is significantly better than that of single strains and combinations deviating from the optimal ratio. This further verifies the synergistic effect of the five-strain compound and specific ratio range in glucose metabolism intervention.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A probiotic complex for metabolic intervention, characterized in that, The probiotic complex includes Lactobacillus plantarum ( Lactiplantibacillus plantarum ), fermenting lactobacillus ( Limosilactobacillus fermentum ), Lactobacillus reuteri ( Limosilactobacillus reuteri Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) and Bifidobacterium breve ( Bifidobacterium breve ); The viable count ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* is (2–3):(2–2.5):(2–3.5):(1–2):(0.8–1).

2. The probiotic complex according to claim 1, characterized in that, The viable count mass ratio of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* is 2:2.5:3:1.5:

1.

3. The probiotic complex according to claim 1, characterized in that, The total live bacteria count of the probiotic complex is not less than 1×10⁻⁶. 9 ~1×10 11 CFU / mL.

4. A method for preparing a probiotic complex as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. The freeze-dried powder cultures of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Lactobacillus reuteri*, *Lactobacillus rhamnosus*, and *Bifidobacterium breve* were respectively inoculated into liquid MRS medium and cultured. The operation was repeated twice to obtain five single-strain seed cultures. S2. The seed cultures of the five single bacteria were inoculated into liquid MRS medium for fermentation culture. After fermentation, the bacterial sludge was collected by centrifugation and washed with sterile physiological saline to obtain wet bacterial sludge of the five single bacteria. S3. Mix the five types of single-strain wet sludge according to the mass ratio of live bacteria, and stir evenly to obtain the final product.

5. The preparation method according to claim 4, characterized in that, In S1, the inoculation amount is 1-3%; The culture conditions for *Lactobacillus plantarum* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the fermented Lactobacillus mucinus were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus reuteri* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus rhamnosus* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the *Bifidobacterium breve* were: 37±2℃, 180~220r / min, anaerobic culture for 18~24h.

6. The preparation method according to claim 4, characterized in that, In S2, the inoculum size is 1-3%, and the fermentation pH is controlled at 6.0-6.5; The culture conditions for *Lactobacillus plantarum* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the fermented Lactobacillus mucinus were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus reuteri* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for *Lactobacillus rhamnosus* were: 37±2℃, 180~220r / min, aerobic culture for 18~24h; The culture conditions for the *Bifidobacterium breve* were: 37±2℃, 180~220r / min, anaerobic culture for 18~24h.

7. The preparation method according to claim 4, characterized in that, In S2, the centrifugation speed is 8000 r / min and the centrifugation time is 8 to 12 min.

8. A probiotic preparation, wherein the active ingredient is the probiotic complex according to any one of claims 1 to 3 and the probiotic complex prepared by the preparation method according to any one of claims 4 to 7.

9. The probiotic preparation according to claim 8, wherein the dosage form of the probiotic preparation is any one of powder, granules, capsules, tablets, or oral liquid.

10. The use of the probiotic complex according to any one of claims 1 to 3, the probiotic complex prepared by the preparation method according to any one of claims 4 to 7, or the probiotic preparation according to any one of claims 8 to 9 in the preparation of metabolic intervention-related products, characterized in that, The product is a food, health food, or dietary supplement, and the metabolic intervention includes regulating the balance of intestinal flora, improving glucose and lipid metabolism, regulating energy metabolism, alleviating obesity, reducing inflammatory factors, lowering blood lipids, or lowering blood sugar.

Images