A probiotic fermented roselle juice with auxiliary hypoglycemic function and a preparation method thereof

Abstract

The present application relates to a kind of functional food preparation and the development technology field of homology resource of medicine and food, specifically a kind of probiotic fermented roxburgh rose juice with auxiliary hypoglycemic function and its preparation method.Roxburgh rose original juice high natural acidity, initial pH value is low, strong acid matrix environment can seriously inhibit the physiological activity and metabolic capacity of fermenting microorganism, lead to conventional probiotic fermentation process difficult to smoothly carry out.The roxburgh rose original juice probiotic fermentation process of the present application is different from the fermentation slurry formed by mixing roxburgh rose original juice and water or multi-component compound roxburgh rose juice as fermentation matrix in prior art, the present application uses roxburgh rose original juice as fermentation substrate, and resource comprehensive utilization rate is greatly improved, the solid concentration and nutrient component density of roxburgh rose original have.The single-stage fermentation is used, process flow is simplified, operation is convenient, and it is unnecessary to inoculate twice and intermediate treatment, significantly reduce the equipment investment, manpower cost and contamination risk of industrial production.

Description

Technical Field

[0001] This invention relates to the field of functional food preparation and the development of medicinal and edible resources, specifically a probiotic fermented prickly pear juice with auxiliary hypoglycemic function and its preparation method. Background Technology

[0002] Diabetes mellitus (DM) is a major chronic metabolic disease that seriously threatens human health worldwide. According to the International Diabetes Federation (IDF), there were 537 million adults with diabetes globally in 2021, with type 2 diabetes (T2DM) accounting for over 90%, and this number is projected to reach 783 million by 2045. my country has the largest number of diabetes patients in the world, with an adult prevalence rate as high as 12.8%. Currently, clinical treatment for T2DM mainly relies on chemically synthesized drugs (such as metformin, sulfonylureas, and DPP-4 inhibitors) and insulin injections. Long-term medication often results in side effects such as gastrointestinal adverse reactions, hypoglycemia risk, and weight gain. Therefore, developing functional foods derived from natural ingredients that are safe and suitable for long-term consumption to help lower blood sugar has significant social demand and economic value.

[0003] Rosa roxburghii Tratt, a plant belonging to the genus Rosa in the family Rosaceae, is a unique medicinal and edible resource in my country, rich in vitamin C, superoxide dismutase (SOD), polyphenols, flavonoids, and triterpenoids. Modern pharmacological studies have shown that Rosa roxburghii extract possesses antioxidant, anti-inflammatory, immunomodulatory, and glucose and lipid metabolism-improving bioactivities. Probiotic fermentation technology can effectively improve the flavor and quality of fruit and vegetable products, while simultaneously enriching active ingredients and generating new functional metabolites through microbial metabolic transformation, making it an important technical means for the development of functional foods. However, current evaluations of the efficacy of fermented Rosa roxburghii products in the prevention and treatment of type 2 diabetes are mostly limited to in vitro antioxidant effects or single animal indicators, lacking comprehensive in vivo evaluations covering multiple dimensions such as glycemic homeostasis regulation, insulin sensitivity, systemic inflammation, and target organ pathological protection.

[0004] There has been considerable research on the fermentation preparation of prickly pear fermented products. For example, patent application 1: Prickly pear fermented nutrient solution and its application (CN110419696A): Hu Ping et al. of Guizhou University (application date: June 28, 2019, publication number: CN110419696A) disclosed a prickly pear fermented nutrient solution and its preparation method. The patented technical solution includes the following steps: (1) Raw material processing: Select mature fresh prickly pear fruits, wash and juice them, and keep the fruit residue. After pulping the fruit residue, mix the prickly pear pulp with water to obtain prickly pear pulp for later use; (2) Inoculation fermentation: Inoculate with lactic acid bacteria and yeast for compound inoculation. The inoculation amount is 1-5%. After fermentation, the fermentation product is obtained; (3) Sterilization and filling: Centrifuge the fermentation product to extract the supernatant, filter it with an ultrafiltration membrane, and sterilely bottle it to obtain the finished prickly pear fermented nutrient solution.

[0005] Patent Application 2: A Prickly Pear Probiotic Enzyme and Its Preparation Method (CN109043511A): Yangsheng (Nanzhao) Biotechnology Co., Ltd., Li Heng et al. (Application Date: July 20, 2018, Publication No.: CN109043511A) disclosed a prickly pear probiotic enzyme and its preparation method. The main raw materials used in this patent are prickly pear, hawthorn, and dried plum, which are fermented in three stages—the first stage is yeast fermentation, the second stage is lactic acid bacteria fermentation, and the third stage is acetic acid bacteria fermentation.

[0006] Patent Application 3: An Enzymatic Prickly Pear Health Drink and Its Preparation Process (CN106261361A): Yang Xiaosheng et al. from the Key Laboratory of Natural Product Chemistry, Chinese Academy of Sciences, Guizhou Province (Application Date: August 5, 2016, Publication No.: CN106261361A) disclosed an enzymatic prickly pear health drink and its preparation process. The fermentation raw materials of this patent include: 20-30 parts prickly pear, 5-10 parts white sugar, 5-10 parts medicinal and edible plant extracts, and 60-80 parts purified water, wherein the weight ratio of prickly pear fruit to water is 1:2-3. This patent adopts a three-stage enzymatic process—fermentation with yeast, fermentation with acetic acid bacteria, and fermentation with alcohol koji—under three different temperature conditions.

[0007] Current fermentation technologies have the following shortcomings: (1) The fermentation substrate is all made of water-diluted prickly pear juice or compound fermentation system, not pure prickly pear juice single mechanism.

[0008] In existing technologies, the preparation of the substrate for prickly pear fermentation generally adopts the process of "prickly pear fresh fruit being pulped / juiced with water". For example, patent application 1 (CN110419696A) describes pulping prickly pear pulp and then mixing it with water to form a fermentation slurry; patent application 3 (CN106261361A) describes a weight ratio of prickly pear fruit to water of 1:2-3. Although this water dilution operation reduces the difficulty of pulping and the viscosity of the material to some extent, it substantially changes the original solids concentration and nutrient density of the prickly pear.

[0009] (2) Fermentation substrates are mostly multi-component compound systems.

[0010] The aforementioned patented technologies all belong to multi-component compound fermentation systems, such as compounding hawthorn, dried plum, etc. on the basis of prickly pear juice, rather than pure substrate fermentation systems with prickly pear as a single raw material. Although compounding helps to enrich the flavor and function, it changes the intrinsic fermentation characteristics of prickly pear raw material, and the established process parameters cannot be directly applied to the prickly pear juice system.

[0011] (3) Lack of scientific quantitative indicators for process control Existing patents lack a combined pH-viable cell count process control system for monitoring the fermentation process. pH is a real-time indicator reflecting acid production intensity, while viable cell count is a core parameter for evaluating probiotic function; both must be used together to accurately determine the fermentation endpoint. Existing patents do not provide systematic single-factor process optimization schemes (inoculum size, temperature, time), and the process parameters lack scientific basis.

[0012] (4) Lack of in vivo studies on the hypoglycemic activity of fermented prickly pear juice Currently, there are no publicly available documents regarding the efficacy evaluation of fermented prickly pear products in animal models of type 2 diabetes, and there is a lack of an integrated R&D chain that fully covers "process parameter optimization → characterization of active ingredients before and after fermentation → comprehensive evaluation of efficacy in diabetic models".

[0013] Therefore, it is necessary to find a method that can utilize the natural high acidity and low initial pH of prickly pear juice. A strongly acidic substrate environment severely inhibits the physiological activity and metabolic capacity of fermenting microorganisms, making conventional probiotic fermentation processes difficult to carry out smoothly. Summary of the Invention

[0014] To address the aforementioned technical problems in the existing technology, this invention provides a probiotic-fermented prickly pear juice with auxiliary blood sugar-lowering function and its preparation method. Specifically, this is achieved through the following technical solution: A method for preparing probiotic fermented prickly pear juice with auxiliary hypoglycemic function includes the following steps: (1) Preparation of prickly pear juice: Prickly pear juice is obtained by directly pressing prickly pear; (2) Preparation of prickly pear juice: Adjust the initial pH of the system to 4.5-5.5, then add exogenous carbon source and nitrogen source, and stir thoroughly. (3) Sterilization; (4) Cooling inoculation and fermentation: After the juice temperature has cooled to room temperature, inoculate the bacterial strain into the fruit and vegetable juice; (5) Fruit juice preparation: Add auxiliary ingredients to flavor the juice; (6) Sterilization: After the fermentation liquid is inactivated by enzyme inactivation, it is quickly cooled to 4°C to obtain the probiotic fermented prickly pear product.

[0015] Furthermore, the prickly pear juice is made by selecting fresh prickly pear fruits that are moderately ripe and free from mold and spoilage. After washing and removing the stems, the fruits are crushed using a high-speed blender or a pulverizer. Physical pressing is then used until no obvious juice seeps out of the pulp. The juice is then filtered or centrifuged to obtain the prickly pear juice.

[0016] Furthermore, step (2) involves adjusting the initial pH of the system using sodium bicarbonate.

[0017] Furthermore, step (2) involves adjusting the initial pH of the system to 5.0.

[0018] Furthermore, the exogenous carbon source is anhydrous glucose, specifically 3% anhydrous glucose.

[0019] Furthermore, the nitrogen source is MRS broth.

[0020] Furthermore, in step (3), pasteurization is used to bring the juice to a core temperature of 105°C for 1 minute.

[0021] Furthermore, the inoculation in (4) is carried out at a ratio of 1‰ of the freeze-dried powder.

[0022] Furthermore, the fermentation in step (4) is carried out at a temperature of 30-35°C for a time of 45-50 hours. Furthermore, in step (5), the excipients are erythritol and mogroside. The mixing ratio is 6.5% erythritol and 0.02% mogroside.

[0023] Furthermore, the sterilization step (6) involves pasteurizing and inactivating the fermentation broth by pasteurizing and inactivating the enzymes at 60°C for 30 minutes, followed by rapid cooling to 4°C.

[0024] A probiotic-fermented prickly pear juice with blood sugar-lowering function was prepared by the above method.

[0025] Compared with the prior art, the technical effects of this invention are reflected in: (1) Realize the high-value utilization of the whole fruit resources of prickly pear The natural high acidity and low initial pH of prickly pear juice create a highly acidic substrate environment that severely inhibits the physiological activity and metabolic capacity of fermenting microorganisms, making conventional probiotic fermentation processes difficult to implement. This is a critical technical challenge that urgently needs to be overcome in the industrial processing of prickly pear fermentation. Existing fermentation substrates are limited to multi-component compound systems, such as prickly pear juice mixed with other plant components like hawthorn and dried plum, or diluted prickly pear juice, resulting in low utilization of the original juice. This invention provides a probiotic fermentation process using prickly pear juice as the fermentation substrate, achieving full utilization of prickly pear and avoiding nutrient loss and resource waste. Unlike existing technologies that use a fermentation slurry formed by mixing prickly pear juice with water or multi-component compound prickly pear juice as the fermentation substrate, this invention uses prickly pear juice as the fermentation substrate, significantly improving the comprehensive utilization rate of resources and preserving the original solids concentration and nutrient density of the prickly pear.

[0026] (2) Simplified process and improved production efficiency Compared to the existing two-stage mixed fermentation of yeast and lactic acid bacteria, which is complex and time-consuming, this invention employs a single-stage fermentation process, simplifying the workflow and making it more convenient to operate. Under optimal conditions (inoculation amount 1‰, temperature 32℃, time 48h), the fermentation cycle for a single batch is 48h, and no double inoculation or intermediate treatment is required, significantly reducing equipment investment, labor costs, and the risk of contamination in industrial production.

[0027] (3) Establish a fermentation process control system To address the shortcomings of existing process control indicators, this invention establishes a fermentation process monitoring system using pH value and viable cell count as joint evaluation indicators. Through single-factor experimental system optimization of three core parameters—inoculum size, fermentation temperature, and fermentation time—the optimal process conditions for prickly pear juice probiotic fermentation are determined. This invention is the first to combine pH value and viable cell count as core monitoring indicators for the prickly pear juice fermentation process. pH value measurement is simple and inexpensive, enabling online real-time monitoring; viable cell count reflects the core parameters of probiotic function. The combined use of these two indicators achieves precise control of the fermentation process, ensuring the stability and consistency of product quality between batches, and providing a reliable control basis for industrial-scale production.

[0028] (4) Achieve targeted regulation of active ingredients The optimal process of this invention achieves selective regulation of the active ingredients of prickly pear, significantly reducing the neutral sugar content, thereby lowering the digestible sugar content and calorie value of the product, which aligns with the trend of low-sugar healthy consumption and is especially suitable for people controlling their blood sugar. The total flavonoid content is significantly increased, the content of free flavonoids is increased, the antioxidant capacity is enhanced, and the bioavailability is improved. The total phenol and SOD enzyme activities remain stable: the original levels of the core active ingredients of prickly pear are preserved, and the nutritional loss caused by excessive fermentation is avoided.

[0029] (5) Fermented prickly pear juice has the effects of regulating blood sugar, reducing insulin resistance and protecting pancreatic β cells. A type II diabetic mouse model was established by inducing a high-fat, high-sugar diet combined with STZ. Basic indicators such as body weight, food and water intake, fasting blood glucose, insulin tolerance, and oral glucose tolerance were measured. Organ indices, glycated serum protein, serum insulin, and insulin resistance index were analyzed. The changes in the volume and number of liver and pancreatic cells were observed by hematoxylin-eosin staining. The effects of fermented prickly pear juice on regulating blood sugar, reducing insulin resistance, and protecting pancreatic β cells were studied.

[0030] (6) To address the lack of in vivo functional activity evaluation of fermented prickly pear in existing technologies, a complete technical solution was established, encompassing fermentation process optimization, precise characterization of active ingredients, and multi-level efficacy verification in diabetic animal models. It was fully confirmed that the fermented prickly pear juice of this application can effectively improve the hyperglycemic state of type II diabetic mice induced by a high-fat, high-sugar diet combined with STZ; effectively improve glucose tolerance in diabetic mice and enhance the body's blood glucose regulation capacity; effectively improve insulin resistance in diabetic mice and enhance insulin sensitivity; improve kidney and liver indices, demonstrating potential application value in improving liver and kidney health in mice; and possesses the ability to improve insulin resistance and insulin sensitivity. Attached Figure Description

[0031] Figure 1 pH changes in fermented prickly pear juice under different inoculation amounts.

[0032] Figure 2 viable cell counts in fermented prickly pear juice at different inoculation amounts.

[0033] Figure 3 pH changes in fermented prickly pear juice at different fermentation times.

[0034] Figure 4 Changes in viable bacterial count in fermented prickly pear juice at different fermentation times.

[0035] Figure 5 pH changes in fermented prickly pear juice at different fermentation temperatures.

[0036] Figure 6 The number of viable bacteria in fermented prickly pear juice at different fermentation temperatures.

[0037] Figure 7 Comparison of total phenol content before and after fermentation of prickly pear juice.

[0038] Figure 8 Comparison of SOD content before and after fermentation of prickly pear juice.

[0039] Figure 9 Comparison of sugar content before and after fermentation of prickly pear juice.

[0040] Figure 10 Comparison of sugar content before and after fermentation of prickly pear juice.

[0041] Figure 11 Weight changes in the normal diet group and the high-fat diet group after six weeks of high-fat, high-sugar diet.

[0042] Figure 12 Mouse body weight change curve.

[0043] Figure 13 Blood glucose change curve in mice.

[0044] Figure 14 Blood glucose levels in mice at week 5 of intervention.

[0045] Figure 15 Changes in blood glucose concentration during OGTT.

[0046] Figure 16 Area under the blood glucose concentration curve in OGTT.

[0047] Figure 17 Changes in blood glucose concentration during ITT.

[0048] Figure 18 Area under the glucose concentration line in ITT.

[0049] Figure 19 Mouse liver index.

[0050] Figure 20 Mouse kidney index.

[0051] Figure 21 Serum glycosylated serum proteins.

[0052] Figure 22 Serum insulin levels.

[0053] Figure 23 Insulin resistance index. Detailed Implementation

[0054] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0055] 1. Main Materials and Reagents Table 1 Main Materials and Reagents

[0056] 2. Main Instruments and Equipment Table 2 Main Instruments and Equipment

[0057] 3. Preparation of fermentation bacteria Based on project requirements and preliminary research, suitable microbial strains for prickly pear fermentation were selected. These strains must possess excellent fermentation performance, be able to grow and reproduce rapidly in prickly pear juice, and produce beneficial metabolites.

[0058] 4. Preparation of Fermented Prickly Pear Juice Step 1: Preparation of Prickly Pear Juice Select fresh prickly pear fruits that are moderately ripe and free from mold and spoilage. After washing and removing the stems, crush the whole fruit using a high-speed blender or a pulverizer. Use physical pressing until no obvious juice seeps out of the pulp. Filter or centrifuge to obtain prickly pear juice.

[0059] Step Two: Preparation of Prickly Pear Juice The initial pH of the system was adjusted to 5.0 using sodium bicarbonate, followed by the addition of 3% anhydrous glucose as an exogenous carbon source and 0.1% MRS broth as a nitrogen source, and the mixture was stirred thoroughly.

[0060] Step 3: Sterilization Pasteurization brings the juice to a core temperature of 105°C for 1 minute. Step 4: Cooling, inoculation, and fermentation After the juice has cooled to room temperature, 1‰ of the freeze-dried bacterial powder is inoculated into the fruit and vegetable juice. The fermentation temperature is 32℃ and the time is 48 hours.

[0061] Step 5: Juice Preparation The blending ratio is 6.5% erythritol and 0.02% mogroside.

[0062] Step Six: Sterilization The fermentation broth was pasteurized at 60℃ for 30 minutes to inactivate enzymes, and then rapidly cooled to 4℃ to obtain the probiotic fermented prickly pear product. Step 7: Filling While still hot, perform aseptic filling in an ultra-clean workbench.

[0063] 5 Single-factor experiments Using the preparation of 200 mL of prickly pear fermented juice as the standard, and with viable cell count and pH as evaluation indicators, the effects of inoculum amount (0.03%, 0.1%, 0.5% and 1%), fermentation time (12, 24, 48, 72 h), and fermentation temperature (30, 32, 35, 37 ℃) on the quality of prickly pear fermented juice were investigated.

[0064] 5.1 Effects of different inoculation amounts Under aerobic conditions at 37℃, bacterial cultures were inoculated into prickly pear juice at volume ratios of 0.03%, 0.1%, 0.5%, and 1%, with other conditions remaining constant. After 48 hours, bacterial colony growth was observed by plate plating. The viable cell count of the fermented prickly pear juice at different inoculum amounts was determined, and the corresponding pH values ​​were also measured. Each sample was diluted three times, with three replicates for each dilution. The results are as follows: Figure 1-2 As shown, at an inoculum size of 0.10%, the pH of the fermented prickly pear juice drops to its lowest level, while the viable cell count reaches its maximum.

[0065] 5.2 Effect of different fermentation times Under aerobic conditions at 37℃, fermentation time experiments were conducted on the proportion of samples with excellent fermentation and superior product indicators from the single-factor inoculum size experiment. Fermentation times were controlled at 12, 24, 48, and 72 hours, with other conditions remaining constant. Viable cell counts were determined by plate plating at each time point. Each sample was diluted three times, with three replicates for each dilution. pH values ​​were also measured at each fermentation stage. Results are as follows: Figure 3-4 As shown, the pH of the system dropped to 3.88 and tended to stabilize after 48 hours of fermentation, indicating that the microbial metabolic activities (such as acid production) entered a plateau phase and the substrate conversion efficiency reached a high level. Further extending the fermentation time did not result in significant changes in pH or significant progress in the metabolic process. Therefore, 48 hours was chosen as the fermentation endpoint to balance fermentation efficiency and cost.

[0066] 5.3 Effect of different fermentation temperatures Under aerobic conditions, the proportions of samples from the above single-factor experiments that exhibited excellent fermentation and superior product indicators were selected. Fermentation temperatures were controlled at 30, 32, 35, and 37℃, with other conditions remaining constant. Viable cell counts were determined by plate plating, with each sample diluted three times, and each dilution performed in triplicate. pH values ​​were also measured. Results are as follows: Figure 5-6 As shown, 32℃ is the optimal temperature for this fermentation process. Under this condition, the microbial proliferation activity is the strongest, and the pH of the system is stable within a suitable range.

[0067] 6. Functional component analysis of fermentation products 6.1 Determination of total phenol content Depend on Figure 7 The total phenolic content of the original prickly pear juice and the fermented prickly pear juice were approximately 13.57±0.12 mg / mL and 14.12±0.42 mg / mL, respectively. After probiotic fermentation, the total phenolic content of the fermented group increased only slightly compared to the unfermented juice. Statistical analysis showed no significant difference between the two groups (ns, p>0.05), indicating that probiotic fermentation under the experimental conditions did not cause a statistically significant change in the total phenolic content of the prickly pear system.

[0068] 6.2 SOD content determination The results are as follows Figure 8As shown, the SOD content of the original prickly pear juice and the fermented prickly pear juice were approximately 9746.34±147.16 and 9998.36±67.46 U / mL, respectively. After probiotic fermentation, the SOD content of the fermented group only increased slightly compared with the unfermented juice. Statistical analysis showed no significant difference between the two groups (ns, p>0.05), indicating that probiotic fermentation under the experimental conditions did not cause a statistically significant change in the SOD content of the prickly pear system.

[0069] 6.3 Determination of sugar content like Figure 9 As shown, the neutral sugar content of the original prickly pear juice was approximately 64 mg / mL, which significantly decreased to approximately 18 mg / mL after fermentation. Compared with the unfermented juice, the neutral sugar content in the fermented group decreased significantly (***, P < 0.001), with a degradation rate exceeding 70%. This indicates that the probiotic fermentation process can efficiently metabolize small-molecule free neutral sugars in the original prickly pear juice, significantly reducing the glycemic load of the system.

[0070] 6.4 Determination of total flavonoid content From columnar Figure 10 The results showed that the total flavonoid content of the unfermented prickly pear juice was approximately 9.1 mg / mL. After probiotic fermentation, the total flavonoid content in the fermented group increased to approximately 9.6 mg / mL. Statistical difference analysis showed a significant difference between the two groups (P < 0.05), indicating that this fermentation process can increase the total flavonoid level of the prickly pear juice system.

[0071] 7. Study on the hypoglycemic activity of fermented prickly pear juice in vivo 7.1 Construction of a type 2 diabetic mouse model Male C57BL / 6 mice (16–18 g, 6–8 weeks old) were housed in an environment with a temperature of 24±1℃, relative humidity of 40–80%, and adequate water, food, and light. Four groups were established: a normal control group (NC), a diabetic model group (DC), a metformin group (Met), a fermented prickly pear intervention group (FN), and a prickly pear juice group (CL), with 12 mice in each group. A type 2 diabetic mouse model was induced by injection of streptozotocin (STZ). Mice in the normal control group received intraperitoneal injections of citrate buffer.

[0072] Mice were acclimatized for 7 days and then randomly divided into a normal control group (n=12) and an experimental group (n=68). The normal control group was fed a standard diet, while the experimental group was fed a high-fat diet. Mice were weighed regularly. After 6 weeks of feeding, 20 mice (5 groups of 4 each) were fasted for 12 hours and then injected intraperitoneally with STZ. Mice in the experimental group were further divided into 5 groups of 4 each to establish a type 2 diabetes mellitus (T2DM) model. After 12 hours of fasting, STZ was injected intraperitoneally at doses of 0, 75, 80, 85, and 90 mg / kg body weight (BW). One week later, blood glucose levels were measured. A T2DM model was considered successful if the blood glucose level was ≥11.1 mmol / L, and the most suitable dose was selected to establish the T2DM model. The experimental group received STZ-citrate buffer (pH 4.5, 0.1 M) at a dose of 75 mg / kg, while the normal control group received an equal volume of citrate buffer. Fasting blood glucose was measured via tail vein sampling on day 7 post-injection. Mice with FBG > 11.1 mmol / L were designated as diabetic mice. Individuals that did not meet the diagnostic criteria for diabetes were given STZ supplementation (dose ≤ 15 mg / mL) and observed until the model was successfully established.

[0073] Weight changes after 7 weeks of a high-fat, high-sugar diet: Figure 11 As shown, the body weight of the NC group increased slightly from 20.9g to 24.1g, while the body weight of the mice on the high-fat diet increased from 20.9g to 30.1g. A high-fat, high-sugar diet causes a rapid rise in blood sugar, stimulating insulin secretion. Insulin then promotes the uptake of glucose by cells and may also affect the appetite regulation center, causing the mice to consume more food, resulting in a significant increase in body weight compared to the normal diet control group.

[0074] 7.2 Trial Period Seven days later, blood glucose was measured after a 12-hour fast. Mice with a fasting blood glucose (FBG) level exceeding 11.1 mmol / L were considered type II diabetic mice. The diabetic mice were randomly divided into five groups: a normal control group, a diabetic model group, a metformin group (70 mg / kg), a fermented prickly pear intervention group, and an unfermented prickly pear group (10 mL / kg), with 12 mice in each group. During the experiment, mice in each group were administered the corresponding solvent by gavage for 5 weeks, with free access to food and water, administered by gavage at 9:00 AM daily. On the last day, after a 12-hour fast, the mice were sacrificed, and serum, liver, kidney, and pancreas samples were collected and stored at -80℃ for analysis.

[0075] 7.3 Weight, diet, water intake, and fasting blood glucose measurement In a type 2 diabetic mouse model, the mice exhibit typical characteristics in their diet, water intake, weight, and blood glucose levels. A high-fat, high-sugar diet leads to a sharp rise in blood glucose, triggering a surge in insulin secretion and promoting glucose uptake by cells. It may also affect the appetite regulation center, causing the mice to consume more food and water, thus resulting in fluctuations in the weight of the diabetic mice.

[0076] like Figure 13 As shown, in the first 1-2 weeks after the intervention, the blood glucose levels of mice in each group fluctuated significantly. In the model group, blood glucose levels continued to rise with a high-fat, high-sugar diet, while no significant changes in blood glucose levels were observed in any group at the beginning of the intervention. Starting from week 1, except for the NC group, the blood glucose levels in the DC group remained high and showed a slow upward trend, while the fasting blood glucose levels in the Met, FN, and CL intervention groups all began to show a continuous downward trend, which may be related to the cumulative regulatory effect of the active ingredients in vivo. Figure 14 As can be seen, by week 5, the fasting blood glucose levels in the Met, FN, and CL groups were significantly lower than those in the DC group (p<0.05), demonstrating a good hypoglycemic effect. Among them, the Met group showed the most significant hypoglycemic effect, followed by the FN group, while the CL group showed a relatively weaker hypoglycemic effect. In summary, the fasting blood glucose results indicate that both Met and FN interventions can effectively improve the hyperglycemic state in type 2 diabetic mice induced by a high-fat, high-sugar diet combined with STZ.

[0077] Persistent hyperglycemia can lead to intestinal dysfunction and metabolic imbalance, thereby affecting food intake and weight changes in mice. Figure 12 As shown, at week 0 of gavage, there were significant differences in body weight between the normal control group (NC), the diabetic model group (DC), and each intervention group, with the model group mice having a significantly higher body weight than the normal group. The body weight of the DC group mice showed a continuous decreasing trend during gavage, reaching the lowest level among all groups by week 5. The Met, FN, and CL intervention groups all experienced varying degrees of body weight loss in the early stages of gavage (weeks 1-3). From week 3 onwards, the body weight of the Met and FN groups gradually stabilized and showed a slow recovery trend, with the FN group showing the most significant recovery. While the CL group experienced fluctuations, its body weight continued to decrease overall. The decrease in body weight in the Met and FN groups was significantly smaller than that in the DC group (p<0.05), suggesting that these two interventions can effectively alleviate the continuous weight loss in diabetic mice. This result is consistent with the findings of Li et al.: in a high-fat diet combined with STZ-induced diabetic model, insulin deficiency and metabolic disorders lead to progressive weight loss in mice, while effective intervention can improve their metabolic state and slow down weight loss.

[0078] 7.4 Oral Glucose Tolerance Test (OGTT) Oral glucose tolerance test (OGTT) is an important clinical method for determining whether there are abnormalities in glucose metabolism. It is mainly used to assess pancreatic islet cell function and the body's ability to regulate blood glucose. The area under the curve (AUC) reflects the degree of glucose metabolism impairment; the more severe the impairment, the higher the AUC value. OGTT results are as follows: Figure 15As shown, blood glucose levels significantly increased in mice after gavage administration of 1.5 g / kg BW glucose solution: the NC group reached its peak at approximately 15 min, followed by a rapid decline, with blood glucose levels returning to near initial levels by 90 min; the DC group maintained high blood glucose levels for 15–30 min and remained at a high level even at 90 min, indicating significantly impaired glucose metabolism regulation. Blood glucose levels in the Met and FN groups peaked at 15–30 min and then showed a significant downward trend, with blood glucose levels significantly lower than those in the DC group at 90 min; while the CL group had an even higher peak blood glucose level, which remained high throughout the observation period, with no significant downward trend. Figure 16 As can be seen, compared with the DC group, the AUC of OGTT blood glucose concentration in the Met and FN groups was significantly lower (p<0.05), suggesting that the two can effectively improve glucose tolerance in diabetic mice and enhance the body's ability to regulate blood glucose; the AUC of the CL group was not significantly different from that of the DC group (p>0.05), indicating that the intervention had no significant effect on improving glucose tolerance.

[0079] 7.5 Insulin Resistance (ITT) Measurement Intermittent insulin therapy (ITT) is used to assess the body's sensitivity to insulin and the peripheral tissue's response to insulin. In diabetic models, blood glucose levels decreased significantly and slowly after insulin injection. Results were as follows... Figure 17 As shown, the DC group did not show a significant decrease in blood glucose after insulin injection, indicating severe insulin resistance, which may be related to the long-term hyperglycemic state of the DC group mice and a significant decrease in the sensitivity of peripheral tissues to insulin. Except for the DC group, blood glucose levels decreased to varying degrees after insulin injection in all other groups: the NC group showed a rapid decrease in blood glucose after injection, followed by a slow recovery after about 60 minutes; the Met group reached its lowest value at 30 minutes; the FN group showed a continuous decrease in blood glucose after injection, reaching its lowest value at 60 minutes and then slowly recovering, demonstrating a good improvement in insulin sensitivity; the CL group showed some decrease in blood glucose, but the overall decrease was small, and the recovery was also slow. Figure 18 The AUC of blood glucose concentration showed that the AUC of blood glucose concentration in the DC group was significantly higher than that in the NC group, Met group and FN group (p<0.05), while there was no significant difference in AUC between the CL group and the DC group (p>0.05). This indicates that Met and FN intervention can effectively improve insulin resistance and enhance insulin sensitivity in diabetic mice.

[0080] 7.6 Evaluation of related organ indices High-fat diets combined with STZ-induced diabetes models typically lead to increased organ indices, fat accumulation, inflammatory infiltration, and potential organ damage risks.

[0081] The results are as follows Figure 19This indicates that the liver index in the NC group was significantly lower than that in the DC group (p<0.05). Compared with the DC group, the organ indices in the Met, FN, and CL groups all decreased to varying degrees, with the FN group showing the most significant decrease (p<0.05), followed by the Met group (p<0.05), and the CL group also showing a certain trend of improvement (p<0.05). Kidney indices, such as... Figure 20 As shown, the NC, Met, FN, and CL groups all had significantly lower levels of renal indices compared to the DC group (p), indicating that metformin, fermented prickly pear, and prickly pear juice can all improve renal indices. In conclusion, metformin and fermented prickly pear juice intervention demonstrate potential application value in improving liver and kidney health in mice.

[0082] 7.7 Measurement of glycated serum protein and serum insulin and evaluation of insulin resistance Glycated serum protein (GSP) is an important indicator for evaluating the effectiveness of blood glucose control. Long-term hyperglycemia promotes non-enzymatic glycosylation of serum proteins and hemoglobin, forming glycosylated proteins. These proteins then undergo a series of reactions to become advanced glycation end products (AGEs), a significant cause of diabetic complications. The levels of glycosylated proteins in the blood are relatively stable and not easily affected by transient changes in blood glucose; therefore, GSP has become a clinical indicator for assessing the glycemic status of diabetic patients. This study measured GSP levels in mice. Figure 21 It was found that the GSP in the FN group was significantly lower than that in the Met, CL, and DC groups (p). GSP in mice was significantly reduced after intervention with fermented prickly pear (p). This indicates that fermented prickly pear can alleviate persistent hyperglycemia. Therefore, GSP, as an indicator for evaluating hypoglycemic efficacy, is more stable than fasting blood glucose and is less susceptible to external factors.

[0083] The homeostasis model of insulin resistance (HOMA-IR) is an important indicator reflecting the functional status of pancreatic β-cells. From... Figure 22 It can be seen that in the diabetes model group (DC group), serum insulin levels were significantly higher than those in healthy controls (p). After metformin intervention, serum insulin levels were significantly lower than those in the DC group (P<0.05). Compared with the DC group, insulin levels in the FN group showed a decreasing trend, but the difference was not statistically significant (P>0.05). Figure 23As can be seen, the HOMA-IR level of diabetic model group (DC) mice was significantly increased. Compared with the DC group, the HOMA-IR level of fermented prickly pear group (FN) mice was significantly decreased, while the prickly pear juice group (CL) showed no significant improvement. This indicates that fermentation is the key to the effect of prickly pear in improving insulin resistance. This may be because the fermentation process converts the bound polyphenols, polysaccharides, and other macromolecules in prickly pear into easily absorbed small molecule active ingredients, while producing metabolites such as short-chain fatty acids, which enhances its ability to regulate glucose metabolism and improve insulin sensitivity. In contrast, the bioavailability of active ingredients in prickly pear juice is low, and its natural sugars may increase the burden of glucose metabolism, thus failing to effectively improve insulin resistance.

[0084] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for preparing probiotic-fermented prickly pear juice with auxiliary hypoglycemic function, characterized in that, Includes the following steps: (1) Preparation of prickly pear juice: Prickly pear juice is obtained by directly pressing prickly pear; (2) Preparation of prickly pear juice: Adjust the initial pH of the system to 4.5-5.5, then add exogenous carbon source and nitrogen source, and stir thoroughly. (3) Sterilization; (4) Cooling inoculation and fermentation: After the juice temperature has cooled to room temperature, inoculate the bacterial strain into the fruit and vegetable juice; (5) Fruit juice preparation: Add auxiliary ingredients to flavor the juice; (6) Sterilization: After the fermentation liquid is inactivated by enzyme inactivation, it is quickly cooled to 4°C to obtain the probiotic fermented prickly pear product.

2. The preparation method according to claim 1, characterized in that, The prickly pear juice is made by selecting fresh prickly pear fruits that are moderately ripe and free from mold and spoilage. After washing and removing the stems, the whole fruit is crushed using a high-speed blender or a pulverizer. Physical pressing is then used until no obvious juice seeps out of the pulp. The juice is then filtered or centrifuged to obtain the prickly pear juice.

3. The preparation method according to claim 1, characterized in that, Step (2) involves adjusting the initial pH of the system using sodium bicarbonate.

4. The preparation method according to claim 1, characterized in that, Step (2) involves adjusting the initial pH of the system to 5.

0.

5. The preparation method according to claim 1, characterized in that, The exogenous carbon source is 3% anhydrous glucose.

6. The preparation method according to claim 1, characterized in that, The nitrogen source is MRS broth.

7. The preparation method according to claim 1, characterized in that, Step (3) involves pasteurizing the juice to a core temperature of 105°C for 1 minute.

8. The preparation method according to claim 1, characterized in that, The inoculation in (4) is carried out at a ratio of 1‰ of freeze-dried powder.

9. The preparation method according to claim 1, characterized in that, The fermentation in step (4) is carried out at a temperature of 30-35℃ for 45-50 hours.

10. The preparation method according to claim 1, characterized in that, The excipients in step (5) are erythritol and mogroside. Its blending ratio is 6.5% erythritol and 0.02% mogroside.

11. The preparation method according to claim 1, characterized in that, The sterilization step (6) involves pasteurizing and inactivating the enzymes in the fermentation broth, specifically by pasteurizing and inactivating the enzymes at 60°C for 30 minutes, followed by rapid cooling to 4°C.

12. A probiotic-fermented prickly pear juice with auxiliary hypoglycemic function, characterized in that, It is prepared by the method described in any one of claims 1-11.

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