Preparation method and application of a compound strain of a sugar-controlling probiotic

Abstract

The application discloses a preparation method and application of a composite strain of a sugar-controlling probiotic, and belongs to the technical field of microorganisms. The method comprises the steps of strain stock solution preparation, anaerobic culture, pH value determination and regulation, and composite strain proportioning. Through a dynamic shunting mechanism with pH value 4.3 as a threshold, the culture solution lower than the value is subjected to re-acidification treatment, and the culture solution higher than the value is subjected to differential dilution and coating operation. Finally, Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium breve, Bifidobacterium lactis and Bifidobacterium longum are mixed at a preset ratio, and a sustained-release agent is added for storage. The method can significantly improve the screening efficiency of target probiotics, the stability of the composite preparation and the blood glucose regulation effect, and can be widely applied to the development of sugar-controlling probiotic products.

Description

Technical Field

[0001] This application relates to the field of microbial technology, specifically to a method for preparing a compound strain of sugar-controlled probiotics and its application. Background Technology

[0002] Probiotics have wide applications in the food, pharmaceutical, and health product industries, playing a particularly important role in blood sugar regulation. Current technologies typically employ standardized procedures based on fixed culture parameters for probiotic screening and formulation. This involves mixing a specific carbon source with a nutrient substrate, inoculating the probiotic strain, culturing under constant temperature and anaerobic conditions, monitoring the pH of the culture medium, and performing subsequent processing steps according to preset standards. Finally, the strain combination is determined based on colony identification results. This process involves carbon source preparation, anaerobic culture, pH monitoring, and strain identification, forming a complete system for strain screening and formulation.

[0003] However, in existing technologies, the natural fluctuations in the pH of the culture medium lead to significant differences in the growth status of bacterial strains, and the screening process faces the dilemma of insufficient adaptability of operating parameters. Summary of the Invention

[0004] This application provides a method for preparing a compound strain of sugar-controlled probiotics and its application, which can solve the technical problem of large differences in the growth status of strains and poor screening efficiency caused by different initial pH of the culture medium.

[0005] To achieve the above objectives, this application provides the following technical solution: This application provides a method for preparing a compound strain of sugar-controlling probiotics, including the following steps: S1 Step: Preparation of inoculum stock solution: A mixture containing inulin, resistant dextrin and oligofructose is used as a carbon source and mixed evenly with sterile milk, liquid oil and other excipients. Pre-cooled activated sludge is added, and after stirring evenly, it is allowed to stand at 37°C for 30 minutes to acidify and obtain an acidified mixture. S2 strain anaerobic culture: The acidified mixture was cultured at 37°C for 2 days in an anaerobic environment to obtain the culture medium; S3 pH Measurement and Control: Measure the pH of the culture medium; based on the measured pH, execute different bacterial treatment pathways: if the pH is below 4.3, repeat the acidification treatment in the bacterial stock solution preparation step; if the pH is above 4.3, dilute the culture medium, spread it on anaerobic solid plates, anaerobic incubate at 37°C for 3 days, and count and identify the resulting colonies. S4 Compound Strain Ratio: Based on the identification results, select the bacterial suspensions of Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium breve, Bifidobacterium lactis, and Bifidobacterium longum according to a preset ratio for mixing. After adding a slow-release agent, it is filled into a sealed container for storage.

[0006] In an optional embodiment, among the carbon sources, the purity of inulin is 95%, the purity of resistant dextrin is 98%, and the average degree of polymerization of fructooligosaccharide is 5; the solid content of sterile milk is 88%, and the melting point of liquid oil is 20°C.

[0007] In an optional embodiment, the activated sludge is activated at 37°C for 3 hours before use and pre-cooled to 37°C before adding the mixture.

[0008] In an optional embodiment, if the pH value higher than 4.3 is further divided into different intervals, and different dilution and coating operations are performed according to different intervals: when the pH value satisfies the condition 4.3 < pH ≤ 5.0, the culture solution is diluted 10 times and then coated on an anaerobic plate with a solid content of 1.8%; when the pH value satisfies the condition pH > 5.0, the culture solution is diluted 5 times and then coated on an anaerobic plate with a solid content of 2.5%.

[0009] In an optional embodiment, the preset ratio is: Lactobacillus gasseri 1×10^7 CFU, Streptococcus thermophilus 1×10^9 CFU, Lactobacillus bulgaricus 1×10^9 CFU, Lactobacillus acidophilus 1×10^7 CFU, Lactobacillus plantarum 1×10^7 CFU, Lactobacillus paracasei 1×10^6 CFU, Bifidobacterium breve 3×10^6 CFU, Bifidobacterium lactis 3×10^6 CFU, Bifidobacterium longum 1×10^6 CFU.

[0010] In an optional embodiment, the slow-release agent is polyethylene glycol, and its addition amount is 0.3% of the total mass of the bacterial suspension.

[0011] In an optional embodiment, the mixed bacterial suspension is filled into a rubber stopper bottle, and the filling amount is 85% of the volume of the rubber stopper bottle.

[0012] In an optional embodiment, the sealed container after filling is stored at 30°C.

[0013] On the other hand, the present invention application provides the application of the preparation method of the compound strain of the sugar-control probiotic in any one of the above embodiments in a sugar-control probiotic product.

[0014] This application provides a method for preparing a compound strain of sugar-controlled probiotics and its application. The method involves mixing a mixture of inulin, resistant dextrin, and fructooligosaccharides as a carbon source with sterile milk, liquid oil, and other excipients to provide a stable nutrient substrate for the strain, thus ensuring the uniformity of the initial culture environment. Based on this, a 30-minute acidification treatment at 37°C is used to fully activate the metabolic activity of the activated sludge at a suitable temperature. Furthermore, an anaerobic culture at 37°C for two days forms a culture medium, ensuring that the bacterial community completes key proliferation in an anaerobic environment. The design incorporates a pH of 4.3 as a dynamic control threshold. When the pH of the culture medium falls below 4.3, acidification is repeated to prevent irreversible damage to bacterial activity caused by an overly acidic environment. Conversely, when the pH exceeds 4.3, dilution, coating, and anaerobic culture are initiated, achieving precise adaptation to different acid-base conditions. Finally, based on colony identification results, nine target probiotics are mixed in a specific ratio and a slow-release agent is added for storage. Through the synergistic combination of strains such as *Lactobacillus gasseri* and *Streptococcus thermophilus*, the stability of the compound strain's sugar control function is significantly improved. This method effectively solves the problem of differences in bacterial growth status caused by fluctuations in the culture medium's pH, enabling the screening process to have adaptive adjustment capabilities, thereby improving the enrichment efficiency of target probiotics and the long-term stability of the formulation. Attached Figure Description

[0015] Figure 1 This is a flowchart illustrating a method for preparing a compound strain of sugar-controlling probiotics provided in an embodiment of this application. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to embodiments. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit the invention.

[0017] To achieve the aforementioned objectives, this invention provides a method for preparing and applying a compound strain of sugar-controlled probiotics. The overall technical solution, as described in the embodiments of this application, mainly includes the following core technical elements: a selective culture medium constructed using a composite carbon source system composed of inulin, resistant dextrin, and fructooligosaccharides; a closed-loop process flow involving activated sludge inoculation—acidification start-up—anaerobic enrichment—pH threshold feedback control—gradient dilution separation—quantitative compounding of multiple strains—slow-release encapsulation; and a dynamic diversion decision mechanism based on a pH=4.3 critical value. These technical elements work together to constitute the overall technical solution of this invention.

[0018] refer to Figure 1 As shown, the first aspect of this invention provides a method for preparing a compound strain of sugar-controlling probiotics. The method includes the following S1: preparation of the bacterial stock solution, using a mixture containing inulin, resistant dextrin, and fructooligosaccharides as a carbon source, mixing it evenly with sterile milk, liquid oil, and other excipients, adding pre-cooled activated sludge, stirring evenly, and then... S1: Acidify by standing for 30 minutes to obtain an acidified mixture; S2: Anaerobic culture of the bacterial strain. The acidified mixture is then placed in an anaerobic environment... After 2 days of cultivation, a culture medium was obtained; S3: pH measurement and control, the pH value of the culture medium was measured; based on the measured pH value, different bacterial treatment pathways were implemented: if the pH value was lower than 4.3, the culture medium was acidified again in the bacterial stock solution preparation step; if the pH value was higher than 4.3, the culture medium was diluted, spread on anaerobic solid plates, and... The culture was anaerobic for 3 days, and the resulting colonies were counted and identified. S4: Compound bacterial strain ratio: Based on the identification results, the bacterial suspensions of Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium breve, Bifidobacterium lactis, and Bifidobacterium longum were selected according to the preset ratio, mixed, and then a slow-release agent was added before filling into a sealed container for storage.

[0019] In this invention, a four-stage closed-loop process is established using inulin, resistant dextrin, and fructooligosaccharides as a combined carbon source, coupled with activated sludge inoculation—acidification initiation—anaerobic enrichment—pH threshold feedback regulation. This allows the microbial community to establish a microenvironment favorable for the colonization of target probiotics (especially acid-tolerant Lactobacillus and Bifidobacterium) during the initial acidification stage. When pH < 4.3, a re-acidification mechanism is triggered to prevent strong acid stress from causing inactivation or mutation of functional strains, ensuring that the culture medium entering the screening stage is always in a window period of metabolic activity and stable community structure. When pH > 4.3, the re-acidification mechanism is activated. The dynamic dilution-coating-identification pathway ensures that high-density bacterial communities can be effectively separated and accurately counted. Finally, through the synergistic formulation and sustained-release encapsulation of nine strains, a compound preparation with stable sugar control function is formed, thereby solving the technical defects of the current probiotic screening process, such as unstable bacterial growth, low screening efficiency, and insufficient enrichment of target strains due to the initial pH difference of the culture medium. Its beneficial effects are: in Examples 1-4, the total recovery rate of target strains is increased by about 62% compared with Comparative Example 1, and the batch-to-batch CFU variation coefficient is reduced from 23.7% to 8.1%.

[0020] As a specific implementation method, this invention provides a method for preparing a compound strain of blood sugar-controlling probiotics. In the carbon source, the purity of inulin is [missing information]. The purity of resistant dextrin is The average degree of polymerization of fructooligosaccharides is 5; the solid content of aseptic milk is... The melting point of liquid grease is .

[0021] In this invention, by limiting the purity of inulin to ≥95% and the purity of resistant dextrin to ≥98%, the inhibitory effect of impurity sugars and heavy metal residues on the bacterial metabolic enzyme system can be significantly reduced. The average degree of polymerization of fructooligosaccharides is 5, which gives them sufficient water solubility to support rapid diffusion while retaining an appropriate molecular weight to slow down the degradation rate. This allows them to continuously release short-chain fatty acids such as acetic acid and propionic acid during anaerobic culture, synergistically regulating intestinal pH and enhancing insulin sensitivity. The sterile milk solids content of 88% ensures a stable ratio of casein to whey protein and provides a consistent nitrogen source supply.

[0022] During the preparation process, natural active ingredients such as Schizophyllum commune, snow chrysanthemum polysaccharides from Kunlun Chrysanthemum buds, bitter melon saponins from bitter melon, alkaloids from mulberry leaves, Dendrobium, and oligosaccharides can be introduced. All of these ingredients have been confirmed by modern pharmacological and clinical studies to have a clear blood sugar regulating effect. They can form a synergistic blood sugar control system with nine kinds of compound probiotics, comprehensively improving the body's glucose metabolism level. The active polysaccharides contained in Schizophyllum commune can regulate the body's immune function, enhance the sensitivity of pancreatic islet cells to blood sugar, reduce insulin resistance, and help maintain blood sugar homeostasis. Chrysanthemum polysaccharides extracted from Kunlun Chrysanthemum buds can inhibit the activity of α-glucosidase in the intestine, slow down the rate at which carbohydrates such as starch and sucrose are broken down into glucose, reduce postprandial blood glucose peaks, and achieve stable blood sugar control. Bitter melon saponins, known as "plant insulin," can both mimic the action of insulin to promote glucose uptake by peripheral tissues and protect pancreatic β cells and promote normal insulin secretion. Alkaloids in mulberry leaves (mainly 1-deoxynojirimycin, DNJ) are potent α-glucosidase inhibitors that can block the breakdown and absorption of disaccharides, significantly reducing the rise in postprandial blood glucose. The polysaccharides and phenanthrene components contained in Dendrobium can nourish yin fluids, improve pancreatic microcirculation, enhance glucose tolerance, and delay the progression of abnormal blood glucose levels. Oligosaccharides, as functional prebiotics, can provide targeted nutrition for probiotics, promote bacterial colonization and proliferation in the intestine, regulate the intestinal flora structure, reduce endotoxin entry into the bloodstream, and improve chronic low-grade inflammation-related insulin resistance. When used in combination with the compound probiotics of this application, these components can synergistically exert blood sugar control effects on multiple targets, including inhibiting sugar absorption, promoting insulin secretion, protecting pancreatic function, improving insulin resistance, and regulating the intestinal microecology. This significantly enhances the glycemic regulation efficacy of the formulation, making the product more stable in terms of postprandial blood glucose control, fasting blood glucose optimization, and glycated hemoglobin improvement, thus meeting the long-term conditioning needs of individuals with hyperglycemia, impaired glucose tolerance, and those susceptible to diabetes.

[0023] The melting point of liquid oil is To ensure that it exists as a homogeneous liquid dispersion in the 37℃ culture system, and to avoid solid lipid droplets encapsulating the bacterial cells and causing mass transfer obstacles, a selective culture environment with uniform spatiotemporal distribution of nutrients and controllable metabolic pressure is jointly constructed in steps S1 and S2. The beneficial effect is that the relative abundance of the target strains (Lactobacillus gasseri and Bifidobacterium lactis) in the culture medium obtained using this specification of raw materials is increased by 38.5% and 41.2%, respectively, and the fluctuation range of acid production rate is narrowed to ±5.3%.

[0024] As a specific implementation method, this invention provides a method for preparing a compound strain of sugar-controlling probiotics. Activated sludge is prepared before use... Activate for 3 hours and pre-cool before adding to the mixture. In this invention, by prior to Activation for 3 hours allows dormant functional bacteria to complete energy storage and restore membrane fluidity, followed by isothermal treatment (pre-cooling to...) Integrate into the culture system to avoid abnormal expression of heat shock proteins and cell membrane phase transition damage caused by sudden temperature changes.

[0025] This dual temperature control strategy increased the number of culturable bacterial colonies in the activated sludge to [amount missing]. Compared to the control group that was used directly without activation, the activity was increased by about 4.7 times, and the time to reach pH 4.8 in the S1 acidification stage was shortened from 42 minutes to 26 minutes. This provides a highly active and highly synchronous initial bacterial community basis for subsequent anaerobic enrichment in the S1 step. Its beneficial effects are: in the example, the OD_{600} of the culture medium at the end of S2 reached 1.87, which was significantly higher than that of the control group (OD_{600}=1.13), and the proportion of the target strain in the total bacterial community increased from 51.3% to 76.8%.

[0026] As a specific implementation method, this invention provides a method for preparing a compound strain of sugar-controlled probiotics. If the condition of "pH value higher than 4.3" is further divided into different intervals, and different dilution and coating operations are performed according to different intervals: when the pH value meets the condition... At that time, the culture medium was diluted 10 times and then coated onto a surface with a solid content of [missing information]. On the anaerobic plate; when the pH value meets the condition At that time, the culture medium was diluted 5 times and then coated onto a surface with a solid content of [missing information]. On the anaerobic plate.

[0027] In this invention, a pH gradient-responsive dilution-plate matching mechanism is established to dynamically adapt the bacterial density to the physical support capacity of the culture medium under different acid-base conditions: when the pH is in the range of 4.3–5.0, it indicates that the early acidification and anaerobic enrichment are sufficient and the bacterial density is high. Using a 10-fold dilution with a 1.8% low gel strength plate is beneficial for the full development and morphological differentiation of single colonies; when the pH is >5.0, it indicates that the bacterial proliferation is limited or there is an accumulation of inhibitory metabolites and the bacterial density is low. At this time, a 5-fold dilution with a 2.5% high agar content is used to prevent excessive diffusion of colonies and maintain sufficient mechanical strength to support the stable growth of colonies under 3 days of anaerobic culture.

[0028] This synergistic design resulted in an isolation success rate of over 92.4% for each target strain in Examples 1-4, which is 18.6% higher than that of Comparative Example 4, which uniformly used 10-fold dilution + 2.0% plates. Its beneficial effect is that the culturability of Bifidobacterium breve and Bifidobacterium longum under pH>5.0 conditions increased from 34.1% in Comparative Example 4 to 89.7% in Examples 1-4.

[0029] As a specific implementation method, this invention provides a method for preparing a compound strain of probiotics for blood sugar control. The preset ratio is: *Lactobacillus gasseri*... thermophilic streptococci Lactobacillus bulgaricus Lactobacillus acidophilus Lactobacillus plantarum Lactobacillus paracasei Bifidobacterium breve Bifidobacterium lactis Bifidobacterium longum In this invention, *Streptococcus thermophilus* and *Lactobacillus bulgaricus* are selected as the... High-dose grades constitute the main force in acid production.

[0030] Rapidly lowers the local pH of the intestine and inhibits pathogenic bacteria; *Greaseobacterium*, *Acidophilus*, and *Lactobacillus plantarum*... Medium-dose co-expression of α-glucosidase inhibitory activity and GLP-1 secretion-promoting effect; *Bifidobacterium paracasei*, *Bifidobacterium breve*, *Bifidobacterium lactis*, and *Bifidobacterium longum* as... The combination of nine strains, focusing on butyrate production, upregulation of tight junction protein ZO-1, and inhibition of LPS transport, resulted in a precise ratio of nine strains in terms of both order of magnitude and function. This enabled the compound formulation to achieve a 72-hour survival rate of 83.5% in simulated gastrointestinal fluid in vitro, which is 29.4% higher than that of control group 5, which is a simple mixture of equal amounts of individual strains. Its beneficial effects are as follows: in the db / db mouse model, after continuous gavage for 4 weeks, the fasting blood glucose decreased by 31.2%, which is significantly better than control group 5 (19.7%) and commercially available single-strain products (12.4%).

[0031] As a specific implementation method, this invention provides a method for preparing a compound strain of sugar-controlling probiotics. The sustained-release agent is polyethylene glycol, and its addition amount is [missing information - likely a percentage] of the total mass of the bacterial solution. In this invention, polyethylene glycol with a molecular weight of 6000 is introduced as a sustained-release carrier. Its hydrophilic mesh structure can slowly absorb water and swell in gastric juice (pH 1.5–3.0) without disintegrating and releasing bacteria, while it can dissolve more rapidly in near-neutral intestinal juice (pH 6.8–7.4), achieving a gradient release of probiotics from the stomach to the colon. The optimal addition amount of 0.3% was verified by orthogonal analysis: below 0.2%, the sustained release is insufficient, and the gastric acid inactivation rate reaches 41.3%; above 0.4%, the viscosity is too high, affecting mixing and filling, and some bacteria are embedded too deeply, resulting in delayed release. This parameter works synergistically with the ratio of nine bacterial strains.

[0032] In the embodiment, the number of viable bacteria detected in the colon reached [number missing]. The tissue showed a 3.2-fold increase compared to control group 6 without the added slow-release agent. Its beneficial effect was that the total amount of short-chain fatty acids (acetic acid + propionic acid + butyric acid) in feces reached [a certain level] on day 14 of the intervention. Compared with control group 6, the effect was increased by 57.8%, confirming that the sustained-release structure effectively prolongs the action time of the functional strain at the target site.

[0033] As one specific implementation method, this invention provides a method for preparing a compound strain of probiotics for blood sugar control. The mixed bacterial solution is filled into a rubber-stoppered bottle, the filling amount being [missing information - likely a percentage] of the bottle's volume. In this invention, by controlling the filling amount to 85% and reserving a 15% gas phase space at the top of the rubber-stopped bottle, on the one hand, the liquid volume expansion caused by temperature fluctuations during storage at 30°C (the measured maximum expansion rate is 12.3%) is buffered, preventing the rubber stopper from popping out or the bottle from breaking; on the other hand, after sealing, this limited gas phase space is vacuumed using an anaerobic bag, reducing the residual oxygen concentration to a minimum. This is far below the survival threshold of Bifidobacterium. This parameter, along with the 30°C storage condition, forms a heat-oxygen synergistic protection. In Examples 1-4, the total viable count of the product was 78.4% after 90 days of storage at 30°C, which is significantly higher than Comparative Example 7 (52.1%) with 95% filling and Comparative Example 8 (63.9%) with 70% filling. The beneficial effect is that the survival rates of Bifidobacterium breve and Bifidobacterium lactis reached 76.2% and 79.5%, respectively, which are significantly higher than Comparative Example 7 (41.3% and 44.8%).

[0034] As a specific implementation method, this invention provides a method for preparing a compound strain of probiotics for blood sugar control. The filled sealed container is then... Storage under specific conditions. In this invention, the storage temperature is set to... Between the normal culture temperature ( ) and refrigeration temperature ( Between ) and ), the bacterial metabolic rate is reduced to At 43.7%, it significantly reduced ATP consumption and autolytic protease activation, while avoiding The increased permeability and leakage of contents caused by the lower membrane lipid phase transition; this temperature, combined with 85% filling volume and polyethylene glycol sustained-release structure, achieved a stable viable cell attenuation rate of 0.21–0.24 log_{10}(CFU) / month over a 90-day shelf life in Examples 1-4, which is significantly better than... The storage comparison example 9 (0.58 log_{10} / month) and The storage ratio of 10 (0.41 log_{10} / month) is more ideal, with the beneficial effect that the product can still maintain its quality at the end of the storage period. The total number of live bacteria meets the minimum live bacteria count requirements for probiotic products as stipulated in the "Detailed Rules for the Registration, Review and Approval of Health Foods".

[0035] Unless otherwise specified, all materials, reagents and instruments used in the embodiments of this invention can be obtained through commercial channels.

[0036] Materials and reagents: Inulin (95% purity, Shandong Biotechnology Co., Ltd.); Resistant dextrin (98% purity, Matsutani Chemical Industry Co., Ltd., Japan); Fructooligosaccharides (average degree of polymerization 5, CJ CheilJedang Co., Ltd., South Korea); Aseptic skim milk (88% solids content, Inner Mongolia Yili Industrial Group Co., Ltd.); Liquid fats (melting point...) Coconut oil-based medium-chain triglycerides (AB Sciex, USA); Brain heart infusion agar for anaerobic culture (BHI agar, OXOID Ltd.); Activated sludge was taken from the secondary sedimentation tank return sludge of a sewage treatment plant in Shandong Province, and was centrifuged (4000 rpm, 10 min), washed three times with PBS, and then resuspended in sterile physiological saline; Polyethylene glycol 6000 (Sigma-Aldrich).

[0037] Instruments and equipment: Anaerobic incubator (model: AW100SG, Electrotek Ltd., UK); pH meter (model: FE28, Mettler Toledo, Switzerland); Clean bench (model: SW-CJ-2FD, Suzhou Antai Air Technology Co., Ltd.); Constant temperature shaker (model: ZQTY-70S, Shanghai Zhichu Instrument Co., Ltd.); Inverted microscope (model: CKX53, Olympus, Japan); Fully automated microbial identification system (model: VITEK® 2 COMPACT, bioMérieux, France).

[0038] Characterization and testing methods: Colony counting was performed using the anaerobic pour plate method (GB 4789.35-2016); bacterial species identification was performed using 16S rRNA gene sequencing (primers 27F / 1492R) combined with the VITEK® 2 COMPACT system; viable cell count was determined by anaerobic culture in modified MRS agar (containing 0.1% L-cysteine) for 48 h; in vitro simulated gastrointestinal fluid tolerance was determined according to the "Technical Guidelines for Stability Evaluation of Probiotics" (T / CNFIA 001-2021); fasting blood glucose in mice was measured using a fully automated biochemical analyzer (model: AU5800, Beckman Coulter, USA).

[0039] This embodiment aims to construct a method for formulating a complex of probiotic strains for blood sugar control that conforms to all the technical features of the embodiment, and to verify its feasibility and basic performance.

[0040] Weigh out 12.5 g of inulin, 10.0 g of resistant dextrin, and 7.5 g of fructooligosaccharides, and mix them evenly as a complex carbon source; add 88 g of aseptic skim milk (88% solids content) and 20 g of liquid fat (melting point... Add distilled water to a final volume of 200 g, and stir for 30 minutes until a homogeneous emulsion is formed; take 10 mL of activated sludge (prepared beforehand) Activate for 3 hours and pre-cool to Add the above mixture, stir for 10 minutes, and then... Allow to stand for 30 minutes to acidify, obtaining an acidified mixture; transfer the acidified mixture to an anaerobic incubator (gas composition: 85% N2, 10% H2, 5% CO2), and incubate. After anaerobic culture for 48 h, a culture medium was obtained; a sample was taken and the pH value was measured to be 4.62; according to this application, the culture medium was diluted 10 times, and 100 μL was spread on an anaerobic BHI plate with a solid content of 1.8%. Anaerobic culture for 72 h; 30 typical colonies were picked and identified by 16S rRNA sequencing and VITEK® 2 system, confirming the presence of 9 strains: Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium breve, Bifidobacterium lactis, and Bifidobacterium longum; single bacterial suspensions of each strain were prepared according to the proportions of this application (OD_{600} adjusted to 1.0), mixed, and 0.3% polyethylene glycol 6000 (w / w) was added and mixed well; the mixed bacterial suspensions were filled into 10mL rubber-stoppered bottles at 85% fill rate and sealed; and placed in a container. Store in a constant temperature chamber.

[0041] The results showed that the total viable count of the final product was: After treatment with simulated gastrointestinal fluid in vitro for 2 hours, the survival rate was 83.5%; After 90 days of storage, the number of live bacteria was The survival rate was 46.5%; the strain composition was verified by 16S sequencing and the deviation from the feeding ratio was ≤8.2%.

[0042] This embodiment successfully achieved all the technical features defined in the embodiment, and the product has a high initial viable count, good gastrointestinal tolerance and long-term storage stability, which proves that the compound strain ratio method is completely feasible.

[0043] The purpose of this embodiment is to verify the feasibility of the carbon source purity and auxiliary material parameter boundary conditions in this application.

[0044] Under the same preparation conditions as in the example, only the purity of inulin was adjusted from 95% to 92%, the purity of resistant dextrin from 98% to 95%, and the average degree of polymerization of fructooligosaccharides from 5 to 4, while the specifications of other raw materials and operating parameters remained unchanged, to obtain the product. The results showed that the relative abundance of the target strains (Lactobacillus gasseri and Bifidobacterium lactis) in the culture medium of this product was 32.1% and 28.7%, respectively, a decrease of 38.5% and 41.2% compared to the example; the total viable count of the final product was... The decrease was 39.1% compared to the previous embodiment; however, it still remained within the range of... The above satisfies the basic functional requirements, proving that the technical solution of this invention has good feasibility and stability within the parameter range defined in this application.

[0045] The purpose of this embodiment is to verify the effectiveness of the pH range division and corresponding dilution / plate parameters in this application under boundary values.

[0046] With all other preparation conditions the same as in the examples, only the pH of the culture medium was adjusted to 5.01 (i.e., falling into the range of 5.01). The culture medium was diluted 5-fold and spread onto anaerobic plates with a solid content of 2.5%. Results showed that under these conditions, the single colony count was 217 CFU / plate, with clear colony morphology and good separation. All nine target strains were successfully isolated, with culturable rates of 89.7% and 86.3% for *Bifidobacterium breve* and *Bifidobacterium longum*, respectively, showing no significant difference compared to the example (pH=4.62) (P>0.05). The final product had a total viable count of [missing information]. Even under pH range boundary conditions, the gradient dilution-plate matching strategy of this invention can still ensure efficient isolation and accurate quantification of the target strain, thus proving that this technical feature has sufficient support for the range.

[0047] The purpose of this embodiment is to verify the functional robustness of the strain ratio in this application under non-preferred values.

[0048] With all other preparation conditions the same as in the examples, only the amount of Streptococcus thermophilus and Lactobacillus bulgaricus added was changed from... Adjusted to (i.e., in this application) The lower limit of allowable deviation was set, and the dosage of other strains and process parameters remained unchanged to obtain the product. The results showed that the product had a 2-hour survival rate of 76.2% in simulated gastrointestinal fluid, a decrease of 8.7% compared to the previous example; after continuous gavage for 4 weeks in the db / db mouse model, the fasting blood glucose level decreased by 26.8%, a decrease of 4.4 percentage points compared to the previous example (31.2%), but still significantly better than Comparative Example 5 (19.7%, P<0.01); the fecal butyrate content was... Compared to the embodiment ( The difference was slightly lower but not statistically significant (P>0.05). Within the range specified in this application, even when the dosage of the key acid-producing strain was reduced to the lower limit, the compound preparation could still maintain its core sugar-controlling efficacy, demonstrating that the ratio range is reasonably broad and technically feasible.

[0049] The purpose of this embodiment is to verify the performance of the sustained-release agent dosage under boundary values ​​in this application.

[0050] Under the same preparation conditions as in the example, only the amount of polyethylene glycol added was adjusted from 0.3% to 0.2% to obtain the product. The results showed that the product had a 2-hour survival rate of 71.4% in simulated gastrointestinal fluid, a decrease of 14.5% compared to the example; the number of viable bacteria detectable in colon tissue was [missing information]. Compared to the embodiment ( The total short-chain fatty acid content in feces decreased by 38.1%; The decrease was 21.1% compared to the previous example. Although the addition of 0.2% still had a certain sustained-release effect, it was significantly weaker than the optimal value of 0.3%. This result confirms that 0.3% is the key threshold for ensuring the functional effect in this application, and provides experimental support for limiting its range.

[0051] The purpose of this embodiment is to verify the storage stability of the 85% filling amount in this application at the boundary value. With other preparation conditions the same as in the embodiment, only the filling amount of the rubber stopper bottle was adjusted from 85% to 80% to obtain the product. The results show that the product exhibits good storage stability at the boundary value. The total number of viable bacteria after 90 days of storage was The survival rate was 40.0%, a decrease of 6.5 percentage points compared to the previous example (46.5%); among which, the survival rate of Bifidobacterium breve was 68.4%, a decrease of 7.8 percentage points compared to the previous example (76.2%). Although an 80% filling volume could still maintain basic sealing, the excessive top space led to an increase in residual oxygen concentration (measured at 0.28 v / v%), exacerbating oxidative damage from anaerobic bacteria; this data confirms that 85% is an optimized filling ratio that balances safety and stability, supporting the limitations of the previous example.

[0052] The purpose of this embodiment is to verify the applicability of the 30°C storage temperature in this application under boundary values. With other preparation conditions the same as in the embodiment, only the storage temperature is changed from... Adjusted to The product was obtained. Results showed that the total viable bacterial count of the product was [value missing] after 90 days of storage. The survival rate was 50.0%, which was slightly higher than that of the previous example (46.5%) but there was no statistical difference (P>0.05); however, its production energy consumption and storage costs increased significantly, and it did not bring substantial performance gains. It is the optimal storage temperature that balances stability, economy and practicality, and the implementation examples are based on sufficient technical evidence.

[0053] This embodiment aims to systematically evaluate the blood sugar control efficacy and related biological properties of the product obtained by the compound microbial strain formulation method of the present invention, and to compare and analyze it with the control sample.

[0054] The following comparative examples were set up: Comparative Example 1 (blank control): No activated sludge was added, and the remaining steps were the same as in the example; Comparative Example 2 (raw material specifications not conforming): Carbon source with inulin purity of 90%, resistant dextrin purity of 95%, and fructooligosaccharide polymerization degree of 3 was used, and the remaining steps were the same as in the example; Comparative Example 3 (unactivated sludge): Activated sludge was used directly without activation, and the remaining steps were the same as in the example; Comparative Example 4 (uniform dilution): When pH>4.3, a uniform 10-fold dilution + 2.0% plate was used, and the remaining steps were the same as in the example; Comparative Example 5 (equal mixing): Nine strains were mixed according to... Equal volumes were mixed, but the S1–S3 screening steps were not performed; otherwise, the process was the same as in the examples. Comparative Example 6 (no sustained release): No polyethylene glycol was added; otherwise, the process was the same as in the examples. Comparative Example 7 (overfilled): 95% fill level; otherwise, the process was the same as in the examples. Comparative Example 8 (low-temperature storage): Storage, otherwise the same as in the embodiments; Comparative Example 9 (high temperature storage): Storage, otherwise the same as in the examples; commercially available product A (single Lactobacillus gasseri preparation, labeled with viable count) ); Commercially available product B (compound probiotic powder, containing 6 strains, labeled with total live bacteria count) All samples were treated with the same in vitro simulated gastrointestinal fluid for 2 h, and the survival rate was measured. They were then used in db / db mouse models (n=12 / group) for gavage intervention for 4 weeks, with fasting blood glucose measured weekly. Fecal samples were collected 24 h after the last administration to measure short-chain fatty acids. The mice were then sacrificed, and colon tissue was collected to determine the viable bacterial colonization. The test results are shown in Table 1.

[0055] The results in Table 1 show that the embodiment significantly outperformed all comparative examples and commercially available products in all key performance indicators (P<0.01); especially in in vitro gastrointestinal fluid survival rate (83.5%), blood glucose intervention effect (reduction of 31.2%), and colonic colonization ability (P<0.01). In terms of the three core indicators, the improvements were 29.4%, 11.5 percentage points, and 2.2 times compared to the closest comparative example 5, respectively. Moreover, the effect was not a simple summation of individual improvements, but rather the result of the synergistic effect of the entire process from S1 to S4. For example, comparative examples 2 and 3, when improved individually, only improved survival rates by 12.3% and 8.7%, respectively, while the overall improvement of this example reached 29.4%, indicating a significant synergistic effect among the technical features of this invention. This unexpected technical effect stems from the precise shaping of the bacterial community structure by the pH threshold feedback regulation mechanism, and the multi-target intervention of the host's sugar metabolism pathway by the complementary functional ratio of the nine strains.

[0056] In this application example, the test samples included the products prepared according to each implementation method in Examples 1-7, and the blood glucose control efficacy was uniformly evaluated in the db / db mouse model. The results showed that after 4 weeks of intervention, the fasting blood glucose reduction of all sample examples was no less than 26.8% (Example), significantly higher than that of Comparative Example 5 (19.7%) and commercially available products (12.4%–15.8%); among them, the reduction in Examples 3, 4, 6, and 7 was all above 30.0%, with Example 2 showing 26.8%; the fecal butyrate content of all samples was higher than... The number of viable bacteria detected in colon tissue was higher than that of other tissues. Experimental results show that the probiotic compound preparation for blood sugar control prepared in this invention exhibits good blood sugar regulation effects in the db / db diabetic mouse model, and therefore can be used to prepare drugs for the prevention and / or treatment of type 2 diabetes.

[0057] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a compound strain of probiotics for blood sugar control, characterized in that, Includes the following steps: Preparation of S1 inoculum stock solution: A mixture containing inulin, resistant dextrin, and fructooligosaccharides was used as a carbon source and mixed evenly with aseptic milk, liquid oil, and other excipients. Pre-cooled activated sludge was then added and stirred thoroughly. The mixture was left to stand and acidified for 30 minutes to obtain an acidified mixture. S2 strain anaerobic culture: The acidified mixture is cultured in an anaerobic environment... Culture medium was obtained after 2 days of incubation; S3 pH Measurement and Control: The pH value of the culture medium is measured; characterized in that, different bacterial treatment paths are executed according to the measured pH value: if the pH value is lower than 4.3, the culture medium is re-acidified in the bacterial stock solution preparation step; if the pH value is higher than 4.3, the culture medium is diluted, spread on anaerobic solid plates, and... The cells were cultured anaerobically for 3 days, and the resulting colonies were counted and identified. S4 compound bacterial strain ratio: Based on the identification results, select Lactobacillus gasseri, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium breve, Bifidobacterium lactis and Bifidobacterium longum bacterial suspensions according to the preset ratio, mix them, add a slow-release agent and fill them into a sealed container for storage.

2. The method for preparing the compound strain of blood sugar-controlling probiotics according to claim 1, characterized in that, The purity of inulin in the carbon source is: The purity of resistant dextrin is The average degree of polymerization of fructooligosaccharides is 5; the solid content of the aseptic milk is... The melting point of the liquid grease is .

3. The method for preparing the compound strain of blood sugar-controlling probiotics according to claim 1, characterized in that, The activated sludge is prepared before use. Activate for 3 hours and pre-cool before adding to the mixture. .

4. The method for preparing the compound strain of blood sugar-controlling probiotics according to claim 1, characterized in that, The condition of "pH value higher than 4.3" is further divided into different intervals, and different dilution and coating operations are performed according to different intervals: when the pH value meets the condition... At that time, the culture medium was diluted 10 times and then coated onto a surface with a solid content of [missing information]. On the anaerobic plate; when the pH value meets the condition At that time, the culture medium was diluted 5 times and then coated onto a surface with a solid content of [missing information]. On the anaerobic plate.

5. The method for preparing the compound strain of blood sugar-controlling probiotics according to claim 1 or 4, characterized in that, The preset ratio is: Lactobacillus gasseri CFU, Streptococcus thermophilus CFU, Lactobacillus bulgaricus CFU, Lactobacillus acidophilus CFU, Lactobacillus plantarum CFU, Lactobacillus paracasei CFU, Bifidobacterium breve CFU, Bifidobacterium lactis CFU, Bifidobacterium longum CFU.

6. The method for preparing the compound strain of blood sugar-controlling probiotics according to claim 1, characterized in that, The sustained-release agent is polyethylene glycol, and its addition amount is equal to the total mass of the bacterial solution. .

7. The method for preparing the compound strain of blood sugar-controlling probiotics according to claim 1, characterized in that, Fill the rubber-stopped bottle with the mixed bacterial solution, the filling amount being [percentage]% of the bottle's volume. .

8. The method for preparing the compound strain of blood sugar-controlling probiotics according to claim 1, characterized in that, The filled, sealed container should be stored at 30°C.

9. The application of the method for preparing the compound strain of blood sugar-controlling probiotics as described in any one of claims 1-8 in blood sugar-controlling probiotic products.

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