A probiotic fermentation product and a method for preparing and using the same

Abstract

The present application relates to the field of functional food, and particularly discloses a preparation method of a probiotic fermentation product with functions of helping to maintain a healthy blood sugar level and helping to control body fat, and application thereof. The probiotic fermentation product is obtained by enzymolysis and fermentation of a specific plant material combination of seaweed powder, gamboge fruit powder and flammulina velutipes powder. Animal experiments and human experiments show that the probiotic fermentation product has the functions of helping to control body fat and helping to maintain a healthy blood sugar level, is safe and non-toxic, and is suitable for obese and metabolic syndrome populations, and has great market potential.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, and in particular to a probiotic fermentation product that helps maintain healthy blood sugar levels and helps control body fat, as well as its preparation method and application. Background Technology

[0002] With the global incidence of obesity and type 2 diabetes soaring year by year, these two diseases pose a serious threat to public health. Currently, probiotic preparations and plant-based ingredients and their extracts are the two main solutions.

[0003] However, existing probiotic preparations usually have the following limitations: fermentation conditions conflict between strains with different metabolic characteristics, making it difficult to achieve synergistic effects of multiple strains; some products only focus on a single target that helps maintain healthy blood sugar levels or helps control body fat, lacking a whole-chain regulatory mechanism from intestinal barrier repair, glucose metabolism regulation to lipid breakdown.

[0004] Regarding plant-based raw materials and their extracts, patent KR102800479B1 discloses a composition containing Garcinia Cambogia and natural extracts that has the effects of reducing body fat, inhibiting postprandial blood glucose elevation, and antioxidation. This document teaches the use of hydroxycitric acid (HCA) to inhibit fat synthesis. However, if plant-based raw materials or their extracts and related components are directly used to prepare functional products, there will be problems such as low bioavailability, easy degradation by gastrointestinal enzymes, and long-term use alone may also cause intestinal flora imbalance.

[0005] Based on the above problems, there is an urgent need to provide a probiotic fermentation product that helps maintain healthy blood sugar levels and helps control body fat, in order to meet market demand. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a probiotic fermentation product, its preparation method, and its application, which help maintain healthy blood sugar levels and control body fat. The probiotic fermentation product not only has excellent effects in maintaining healthy blood sugar levels and controlling body fat, but its preparation method is also simple and efficient, and it significantly improves the bioavailability of plant components.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing a probiotic fermentation product that helps maintain healthy blood sugar levels and helps control body fat, comprising the following steps:

[0009] S1: Disperse the mixed raw material powder in deionized water and mix well to obtain a mixed solution. Add a compound enzyme to the mixed solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate.

[0010] S2: Inoculate the mixed bacterial solution into the enzymatic hydrolysate for anaerobic fermentation to obtain the fermentation broth;

[0011] S3: The fermentation broth is sterilized, filtered, concentrated and freeze-dried to obtain probiotic fermentation products;

[0012] The mixed raw material powder includes seaweed powder, Garcinia Cambogia powder, and Enoki mushroom powder;

[0013] The mass ratio of the mixed raw material powder to deionized water is 1:8-10;

[0014] The complex enzyme includes acidic protease, cellulase and pectinase, with a mass ratio of 1:(10-15):(4-6).

[0015] The mixed bacterial solution consists of Rhodotorula glutinis and Akkermansia fasciatus, and the viable count of the mixed bacterial solution is 1*10⁻⁶. 7 -1*10 9 cfu / mL;

[0016] The volume ratio of the mixed bacterial solution to the enzymatic hydrolysate is 1:8-10;

[0017] The ratio of viable cells in the Rhodotorula glutinis culture to the Akkermansia glutinis culture is 1:(3-5).

[0018] The preservation number of the *Rhodotorula glutinis* is CGMCC No. 5081;

[0019] The accession number of the *Ackermania* strain is CGMCC No. 22793.

[0020] Preferably, the mixed raw material powder includes 30-35 parts by weight of seaweed powder, 45-50 parts by weight of enoki mushroom powder, and 18-22 parts by weight of Garcinia Cambogia powder.

[0021] Preferably, the mass ratio of the compound enzyme to the mixture is (1-3):7;

[0022] Preferably, the enzymatic hydrolysis temperature is 40-45℃, the enzymatic hydrolysis time is 2-3h, and the enzymatic hydrolysis pH is 4.0-5.5.

[0023] Preferably, in the fermentation step, the initial pH is 5.0-6.0, the fermentation temperature is 35±2℃, and the fermentation time is 24-48h.

[0024] This invention utilizes a compound enzyme to enzymatically hydrolyze a mixture of seaweed powder, enoki mushroom powder, and Garcinia cambogia powder, followed by anaerobic fermentation with a specific ratio of live bacteria of Rhodotorula glutinis and Akkermansia muciniphila. The fermented broth is then sterilized, filtered, concentrated, and freeze-dried to obtain a probiotic fermented product that helps maintain healthy blood sugar levels and control body fat. The fermentation substrate contains dietary fiber, flavonoids, phenolic compounds, and other components, combined with enzymes and fatty acid metabolites produced by the two bacterial strains. The final fermented product synergistically promotes fat metabolism, inhibits fat absorption and synthesis, and inhibits sugar absorption, thus achieving the dual benefits of maintaining healthy blood sugar levels and controlling body fat. Furthermore, experiments in this application demonstrate that the probiotic fermented product of this invention can safely and effectively control body fat and maintain healthy blood sugar levels, making it suitable for improving energy metabolism in obese individuals and those with high blood sugar.

[0025] Secondly, the present invention provides a probiotic fermentation product prepared by the preparation method described in the first aspect.

[0026] Thirdly, the present invention provides the use of the probiotic fermentation products described in the second aspect in the preparation of foods, pharmaceuticals or health products that help maintain healthy blood sugar levels and help control body fat.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The applicant of this invention conducted extensive experimental screening, selecting a specific mixed raw material combination composed of seaweed powder, Garcinia cambogia powder, and Enoki mushroom powder from hundreds of plant raw materials that help maintain healthy blood sugar levels and control body fat. The mixture was first subjected to enzymatic hydrolysis, in which cellulase and pectinase in the complex enzymes degraded plant cell walls, fully releasing small-molecule sugars and other active substances for use by the microbial community, while acidic protease fully hydrolyzed the protein components into easily bioavailable short peptides. The hydrolysate was then fermented by a specific complex of bacteria composed of Rhodotorula glutinis and Akkermansia muciniphila, resulting in a probiotic fermentation product containing numerous active ingredients such as hydroxycitric acid, polypeptides, dietary fiber, vitamins, and alginate. This product can synergistically achieve the dual effects of maintaining healthy blood sugar levels and controlling body fat through multiple dimensions, including promoting fat metabolism, inhibiting fat absorption and synthesis, and inhibiting sugar absorption.

[0029] Animal experiments have shown that the probiotic fermentation product prepared by this invention is not only safe and non-toxic, but can also significantly reduce the weight of mice and the cholesterol and triglyceride levels in their blood; human experiments have shown that the fermentation product can effectively inhibit the rise in postprandial blood glucose.

[0030] In summary, the probiotic fermentation product of the present invention has the effect of effectively controlling body fat and maintaining healthy blood sugar levels, and is suitable for people with obesity and metabolic syndrome; it has great market potential. Detailed Implementation

[0031] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the described embodiments are only a part of the present invention, and not all of it. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0032] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions comprised of the listed features and open-ended technical solutions that incorporate the listed features. When referring to numerical ranges, unless otherwise specified, the range is considered a continuous range, including its minimum and maximum values, and all values ​​in between. If the range refers to integers, it includes all integers between the minimum and maximum values. Furthermore, when multiple range descriptions exist, these ranges may be merged. Unless otherwise stated, all ranges should be understood to include any of their subranges.

[0033] Unless otherwise specified, all raw materials and instruments used in the embodiments and comparative examples of this invention are commercially available, and the same raw materials are used in parallel experiments. Experimental methods without specific conditions are performed under standard conditions or manufacturer-recommended conditions. Unless otherwise stated, all proportions are calculated in parts by weight or percentages.

[0034] It should also be noted in this invention that:

[0035] Unless otherwise specified, the sterilization, dispersion, filtration, concentration and freeze-drying processes involved in this invention can be implemented using conventional techniques in the field.

[0036] Some of the raw materials and their sources are as follows:

[0037] Seaweed powder: purchased from Shanghai Yuncai Jinke Biotechnology Co., Ltd.;

[0038] Enoki mushroom powder: purchased from Shanghai Weilan Biotechnology Co., Ltd.;

[0039] Garcinia Cambogia powder: purchased from Fufeng Sinote Biotechnology Co., Ltd.;

[0040] Rhodotorula glutinis: Preservation number CGMCC No. 5081;

[0041] Akkermansia myxophilus: accession number CGMCC No. 22793;

[0042] Acidic protease (catalog number: FDG-2237), cellulase (catalog number: FDY-2243), and pectinase (catalog number: FFY-0654) were purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., and are common commercial enzyme preparations. Before the experiment, the purchased enzyme preparations were diluted with deionized water to the enzyme activity required for this experiment. The required enzyme activity for this experiment was 10000 U / mL for acidic protease, 10000 U / mL for cellulase, and 10000 U / mL for pectinase.

[0043] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments, but these embodiments do not constitute a limitation on the scope of protection of the present invention.

[0044] The preparation method of probiotic fermentation products that help maintain healthy blood sugar levels and help control body fat is as follows:

[0045] Fermentation product 1:

[0046] Mixed ingredient powder: By weight, 33 parts seaweed powder, 47 parts enoki mushroom powder, and 20 parts Garcinia cambogia powder, mix well;

[0047] Complex enzyme: acidic protease, cellulase, and pectinase, in a volume ratio of 1:12:5;

[0048] Mixed bacterial culture: Rhodotorula glutinis and Ackermania glutinis, with a live count ratio of Rhodotorula glutinis to Ackermania glutinis of 1:4; the live count in the mixed bacterial culture is 1*10^6 cells / year. 8 cfu / mL;

[0049] Fermentation steps:

[0050] S1: Disperse the mixed raw material powder evenly in deionized water to obtain a mixed solution. Add a compound enzyme to the mixed solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature of 43℃, enzymatic hydrolysis time of 2.5h, and enzymatic hydrolysis pH of 5.0.

[0051] S2: After irradiating and sterilizing the enzymatic hydrolysate, a mixed bacterial solution is inoculated into the sterilized enzymatic hydrolysate for anaerobic fermentation to obtain a fermentation broth; the fermentation conditions are: initial pH 5.5, fermentation temperature 35℃, and fermentation time 36h.

[0052] S3: The fermentation broth is sterilized by irradiation, insoluble matter is filtered out, concentrated by rotary evaporation and freeze-dried to obtain the probiotic fermentation product;

[0053] The mass ratio of the mixed raw material powder to deionized water is 1:9;

[0054] The mass ratio of the compound enzyme to the mixed solution is 2:7;

[0055] The volume ratio of the mixed bacterial culture to the enzyme hydrolysate was 1:9.

[0056] Fermentation product 2:

[0057] Mixed ingredient powder: By weight, 35 parts seaweed powder, 45 parts enoki mushroom powder, and 22 parts Garcinia Cambogia powder, mix well;

[0058] Complex enzymes: acidic protease, cellulase, and pectinase, in a volume ratio of 1:10:6;

[0059] Mixed bacterial culture: Rhodotorula glutinis and Ackermania glutinis, with a live count ratio of Rhodotorula glutinis to Ackermania glutinis of 1:5; the live count in the mixed bacterial culture is 1*10^6 cells / year. 9 cfu / mL;

[0060] Fermentation steps:

[0061] S1: Disperse the mixed raw material powder evenly in deionized water to obtain a mixed solution. Add a compound enzyme to the mixed solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature of 40℃, enzymatic hydrolysis time of 3h, and enzymatic hydrolysis pH of 5.5.

[0062] S2: After irradiating and sterilizing the enzymatic hydrolysate, a mixed bacterial solution is inoculated into the sterilized enzymatic hydrolysate for anaerobic fermentation to obtain the fermentation broth; the fermentation conditions are: initial pH 5.0, fermentation temperature 37℃, and fermentation time 24h.

[0063] S3: The fermentation broth is sterilized by irradiation, insoluble matter is filtered out, concentrated by rotary evaporation and freeze-dried to obtain the probiotic fermentation product;

[0064] The mass ratio of the mixed raw material powder to deionized water is 1:10;

[0065] The mass ratio of the compound enzyme to the mixed solution is 1:7;

[0066] The volume ratio of the mixed bacterial culture to the enzyme hydrolysate was 1:10.

[0067] Fermentation product 3:

[0068] Mixed ingredient powder: By weight, 30 parts seaweed powder, 50 parts enoki mushroom powder, and 18 parts Garcinia Cambogia powder, mix well.

[0069] Complex enzyme: acidic protease, cellulase, and pectinase, in a volume ratio of 1:15:4;

[0070] Mixed bacterial culture: Rhodotorula glutinis and Ackermania glutinis, with a live count ratio of Rhodotorula glutinis to Ackermania glutinis of 1:3; the live count in the mixed bacterial culture is 1*10^6 cells / year. 7 cfu / mL;

[0071] Fermentation steps:

[0072] S1: Disperse the mixed raw material powder evenly in deionized water to obtain a mixed solution. Add a compound enzyme to the mixed solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature of 45℃, enzymatic hydrolysis time of 2h, and enzymatic hydrolysis pH of 4.0.

[0073] S2: After irradiating and sterilizing the enzymatic hydrolysate, a mixed bacterial solution is inoculated into the sterilized enzymatic hydrolysate for anaerobic fermentation to obtain a fermentation broth; the fermentation conditions are: initial pH 6, fermentation temperature 33℃, and fermentation time 48h;

[0074] S3: The fermentation broth is sterilized by irradiation, insoluble matter is filtered out, concentrated by rotary evaporation and freeze-dried to obtain the probiotic fermentation product;

[0075] The mass ratio of the mixed raw material powder to deionized water is 1:8;

[0076] The mass ratio of the compound enzyme to the mixed solution is 3:7;

[0077] The volume ratio of the mixed bacterial culture to the enzyme hydrolysate was 1:8.

[0078] The preparation methods of the probiotic fermentation products obtained in Comparative Examples 1-9 are as follows:

[0079] Comparative Example 1

[0080] Unlike fermentation product 1, the mixed raw material powder used lacks Garcinia Cambogia powder. The missing weight parts are made up by seaweed powder and enoki mushroom powder in a weight ratio of 33:47. The remaining steps and parameters are the same as those of fermentation product 1.

[0081] Comparative Example 2

[0082] Unlike fermentation product 1, the mixed raw material powder used lacks enoki mushroom powder. The missing weight is made up by seaweed powder and Garcinia cambogia powder in a weight ratio of 33:20. The remaining steps and parameters are the same as those of fermentation product 1.

[0083] Comparative Example 3

[0084] Unlike fermentation product 1, the mixed raw material powder used lacks seaweed powder. The missing weight parts are made up by enoki mushroom powder and Garcinia cambogia powder in a weight ratio of 47:20. The remaining steps and parameters are the same as those of fermentation product 1.

[0085] Comparative Example 4

[0086] Unlike fermentation product 1, the compound enzyme used lacks acidic protease. The missing mass is made up by cellulase and pectinase in a mass ratio of 12:5. The remaining steps and parameters are the same as those for fermentation product 1.

[0087] Comparative Example 5

[0088] Unlike fermentation product 1, the mixed bacterial solution used lacked Rhodotorula glutinis, and the missing number of live bacteria was made up with Akkermansia myxophilus. The remaining steps and parameters were the same as those for fermentation product 1.

[0089] Comparative Example 6

[0090] Unlike fermentation product 1, the mixed bacterial culture used lacked Akkermansia myxophilus, and the missing live bacteria were made up with Rhodotorula glutinis. The remaining steps and parameters were the same as those for fermentation product 1.

[0091] Comparative Example 7

[0092] Unlike fermentation product 1, the red yeast used was purchased from Botrytis cinerea, catalog number: HZB124896.

[0093] Comparative Example 8

[0094] Unlike fermentation product 1, the ratio of viable Rhodotorula glutinis to Akkermansia muciniphila in the mixed bacterial culture used was 4:1.

[0095] Comparative Example 9

[0096] Unlike fermentation product 1, bromelain with the same enzyme activity was used to replace the acidic protease, while the remaining steps and parameters were the same as those for fermentation product 1.

[0097] Experiment 1: Weight and blood lipid tests

[0098] Test samples: Fermentation products 1-3 and comparative examples 1-9.

[0099] Experimental animals: SPF-grade KM mice, weighing 23±2g, were observed for 3 days of acclimatization. The animals had free access to water and food. The temperature in the experimental area was 20±2℃, and the humidity was 60±5%. The day and night cycle lasted for 12 hours.

[0100] Establishment of an obesity model: Mice were randomly divided into a normal control group, a model control group, and 12 experimental groups. The normal control group consisted of 10 mice, while the model control group and experimental groups each consisted of 20 mice. The normal control group was fed a basal diet, while the model control group and experimental groups were fed a high-fat diet (45% fat-based energy diet). After two weeks of feeding, 10 heavier mice from each group (excluding obesity-resistant mice) were selected and fed the high-fat diet for another two weeks. The mice in each group were then weighed, and the average weight of each group was calculated and denoted as M0 for subsequent experiments.

[0101] Experiment: Mice in the normal control group, model control group, and experimental group were all fed a basal diet. Mice in the experimental group were administered 6.67 mg / g (body weight) of fermented composition 1-3 and comparative composition 1-9 by gavage daily. Mice in the model group and normal control group were administered the same amount of drinking water by gavage for five consecutive weeks. After the last gavage, the mice were fasted for 6 hours and then weighed. The average body weight of the mice was calculated and denoted as M1. Δbody weight = M1 - M0. The greater the decrease in body weight after the experiment compared to before the experiment, the better the weight loss effect (the smaller the Δ value). The calculation results are expressed as mean ± standard deviation. Blood was collected from the tail vein of the mice, and the serum total cholesterol and triglyceride content were measured. The calculation results were retained to two decimal places.

[0102] Table 1. Effects of fermentation products on mouse body weight and blood total cholesterol and triglycerides.

[0103] Group Δ body weight / g Total cholesterol (mmol / L) Triglycerides (mmol / L) normal control group 13.62±0.94 1.97 0.82 Model control group <![CDATA[22.77±1.03 * ]]> <![CDATA[3.74 * ]]> <![CDATA[1.43 * ]]> Fermentation product 1 <![CDATA[13.93±1.76 # ]]> <![CDATA[2.15 # ]]> <![CDATA[0.85 # ]]> Fermentation product 2 <![CDATA[14.11±1.05 # ]]> <![CDATA[2.27 # ]]> <![CDATA[0.89 # ]]> Fermentation product 3 <![CDATA[14.04±1.27 # ]]> <![CDATA[2.21 # ]]> <![CDATA[0.87 # ]]> Comparative Example 1 <![CDATA[16.57±2.36 # ]]> <![CDATA[2.73 # ]]> <![CDATA[1.16 # ]]> Comparative Example 2 <![CDATA[16.84±1.41 # ]]> <![CDATA[2.78 # ]]> <![CDATA[1.12 # ]]> Comparative Example 3 <![CDATA[16.73±1.03 # ]]> <![CDATA[2.86 # ]]> <![CDATA[1.20 # ]]> Comparative Example 4 <![CDATA[15.71±0.96 # ]]> <![CDATA[2.60 # ]]> <![CDATA[1.07 # ]]> Comparative Example 5 <![CDATA[16.92±1.44 # ]]> <![CDATA[2.92 # ]]> <![CDATA[1.21 # ]]> Comparative Example 6 <![CDATA[19.44±0.72 # ]]> <![CDATA[3.27 # ]]> <![CDATA[1.26 # ]]> Comparative Example 7 <![CDATA[16.43±1.61 # ]]> <![CDATA[2.70 # ]]> <![CDATA[1.15 # ]]> Comparative Example 8 <![CDATA[14.92±1.18 # ]]> <![CDATA[2.45 # ]]> <![CDATA[0.95 # ]]> Comparative Example 9 <![CDATA[15.75±2.41 # ]]> <![CDATA[2.57 # ]]> <![CDATA[0.99 # ]]>

[0104] Note: "#" indicates p < 0.05 compared with the model group; "*" indicates p < 0.05 compared with the normal control group.

[0105] As shown in Table 1, the model control group showed significant differences compared to the normal control group, indicating the successful establishment of the high-fat mouse model. Furthermore, the fermentation product groups 1-3 showed significant differences compared to the model control group, indicating that the fermentation products provided by this invention have significant weight-loss effects and can effectively reduce the levels of total cholesterol and triglycerides in mouse serum.

[0106] Comparing the results of fermentation product 1 with those of comparative examples 1-3, it can be seen that the raw materials defined in this invention have a synergistic effect, which can significantly improve the weight loss effect.

[0107] Comparing the results of fermentation product 1 with those of comparative examples 5-8, it can be seen that the fermentation products obtained using the enzymatic hydrolysis and fermentation methods defined in this invention have significant weight-loss effects; this demonstrates the importance of specific methods and steps for the efficacy of fermentation products.

[0108] Comparing the results of fermentation product 1 with those of comparative examples 4 and 9, it can be seen that the acidic protease, cellulase, and pectinase in the complex enzyme specified in this invention are also irreplaceable.

[0109] Test 2: Toxicity Test

[0110] Test samples: Fermentation products 1-3, Comparative examples 1-9.

[0111] Experimental animals and environment: The experimental animals were SPF-grade ICR mice; the experimental environment was a barrier environment. During the experiment, the ambient temperature was 21-24℃ and the humidity was 54-63%.

[0112] Test method:

[0113] Acute oral toxicity test: The limited dose method was used. Twenty test animals (half male and half female) weighing 18-22g were selected. 20g of test sample was added to 60ml of purified water and administered to mice orally twice a day at 4-hour intervals. The single gavage volume was 0.3mL / 10g·bw, which is equivalent to a dose of 20.00g / kg·bw. The mice were fasted for 6 hours before the first gavage and observed for 2 weeks after gavage. The symptoms of poisoning and mortality were recorded.

[0114] Table 2 Toxicity Test Results

[0115] Group sex ratio Animal number Death count <![CDATA[LD 50 (g / kg·bw)]]> Fermentation product 1 1:1 20 0 ＞20.00 Fermentation product 2 1:1 20 0 ＞20.00 Fermentation product 3 1:1 20 0 ＞20.00 Comparative Example 1 1:1 20 0 ＞20.00 Comparative Example 2 1:1 20 0 ＞20.00 Comparative Example 3 1:1 20 0 ＞20.00 Comparative Example 4 1:1 20 0 ＞20.00 Comparative Example 5 1:1 20 0 ＞20.00 Comparative Example 6 1:1 20 0 ＞20.00 Comparative Example 7 1:1 20 0 ＞20.00 Comparative Example 8 1:1 20 0 ＞20.00 Comparative Example 9 1:1 20 0 ＞20.00

[0116] Results: As shown in Table 2, no obvious poisoning symptoms were observed after gavage administration of the fermentation product at a dose of 10.00 g / kg·bw to both sexes of ICR mice. No animals died during the 14-day observation period. At the end of the observation period, the animals were euthanized and dissected. No obvious abnormalities were found in the major organs, including the liver, spleen, kidneys, stomach, intestines, heart, and lungs. The median lethal dose (LD50) of the sample for both male and female ICR mice was determined. 50 (Greater than 20.00 g / kg·bw. According to the acute toxicity dose grading standard in GB15193.3-2014, it belongs to the practically non-toxic category.)

[0117] The recommended oral dose for humans is approximately 4g per day. For an adult weighing 60kg, this equates to a dose of 0.067g / kg·bw. This dose is significantly lower than that used in the test animals, demonstrating that the fermentation products involved in this invention are safe and non-toxic.

[0118] Experiment 3: Postprandial blood glucose level test

[0119] Sample preparation: Fermentation products 1-3 and comparative examples 1-9 were prepared into a 40g / L mixture using drinking water.

[0120] Each group consisted of 20 healthy adults, half male and half female, aged 25-40 years, with a BMI of 18.8-23.9 kg / m². 2 No history of diabetes (or impaired glucose tolerance), no other metabolic diseases, digestive system diseases, endocrine system diseases, or mental illnesses; no history of allergy or intolerance to the test food, normal oral glucose tolerance test, regular diet, no recent gastrointestinal disorders or medication use, and a satisfactory physical examination. Subjects will be trained and sign an informed consent form before the trial. Food and water intake will be stopped after 10:00 PM the night before the trial, and blood glucose fluctuations after breakfast will be monitored.

[0121] Control group: Breakfast consisted of normal white bread (as a reference food);

[0122] Experimental group: Drink 100mL of the sample within 30 minutes before breakfast, and eat white bread as a reference food for breakfast;

[0123] Plot the blood glucose response curve with time as the x-axis and the blood glucose value minus the fasting blood glucose value at each time point as the y-axis. Calculate the area under the curve (IAUC) above the fasting blood glucose level. Inhibition rate = (IAUC of control group - IAUC of experimental group) / IAUC of control group * 100%. The inhibition rate is retained to two significant figures. The results of the postprandial blood glucose level test are shown in Table 3.

[0124] Blood glucose testing was performed using a Bayer blood glucose meter (Shanghai Jianzhen Medical Technology Co., Ltd.). For specific instructions on measuring fingertip blood glucose, please refer to the blood glucose meter's instruction manual.

[0125] Table 3 Glucose Inhibition Rate

[0126] Group Inhibition rate / % control group - Fermentation product 1 68 Fermentation product 2 62 Fermentation product 3 64 Comparative Example 1 33 Comparative Example 2 31 Comparative Example 3 38 Comparative Example 4 45 Comparative Example 5 42 Comparative Example 6 29 Comparative Example 7 47 Comparative Example 8 52 Comparative Example 9 48

[0127] As shown in Table 3, fermentation products 1-3 all exhibited excellent blood glucose inhibition effects, with fermentation product 1 showing the highest blood glucose inhibition rate. Comparison of fermentation product 1 with comparative examples 1-3 indicates that components Garcinia Cambogia powder, seaweed powder, and Enoki mushroom powder had a synergistic promoting effect on blood glucose inhibition. This may be because the active substances in these components and / or especially the prepared fermentation products have the effect of preventing the hydrolysis and absorption of carbohydrates in the body, inhibiting the absorption of sugar in the small intestine, and promoting the efficiency of glucose utilization by cells, thereby lowering blood glucose levels. Comparison of fermentation product 1 with comparative examples 4 and 9 shows that the enzymatic hydrolysate obtained by the combined enzymatic hydrolysis of acidic protease, cellulase, and pectinase, when controlled by the strains specified in this invention… The fermentation products obtained after fermentation have better blood glucose inhibition effects, indicating that the presence of acidic protease can further decompose the proteins in the raw materials into short peptide chains, thereby enhancing the bioavailability of the raw materials by the strain. Comparing the results of fermentation product 1 with those of Comparative Examples 5-7, it can be seen that the fermentation product obtained by fermentation of Rhodotorula glutinis alone (Comparative Example 6) has limited effect in inhibiting the rise in blood glucose, while the fermentation products obtained by co-fermentation of Rhodotorula glutinis and Akkermansia obliterans (fermentation product 1, Comparative Example 7) have better blood glucose inhibition effects, indicating that Rhodotorula glutinis can enhance the hypoglycemic effect of Akkermansia obliterans fermentation products. Comparing the results of fermentation product 1 with those of Comparative Example 7, it can be seen that Rhodotorula glutinis with preservation number CGMCC No: 5081 as defined in this invention has better blood glucose control effects than other Rhodotorula glutinis, which also shows the irreplaceable role of specific strains when the compound bacteria exert their effects. Comparing the results of fermentation product 1 with those of Comparative Example 8, it can be seen that within the range of live cell ratio of the fermentation strains defined in this invention, the fermentation products obtained have better blood glucose inhibition effects.

[0128] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a probiotic fermentation product that helps maintain healthy blood sugar levels and helps control body fat, characterized in that, Includes the following steps: S1: Disperse the mixed raw material powder in deionized water and mix well to obtain a mixed solution. Add a compound enzyme to the mixed solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate. S2: Inoculate the mixed bacterial solution into the enzymatic hydrolysate for anaerobic fermentation to obtain the fermentation broth; S3: The fermentation broth is sterilized, filtered, concentrated and freeze-dried to obtain probiotic fermentation products; The mixed raw material powder includes seaweed powder, Garcinia Cambogia powder, and Enoki mushroom powder; The mass ratio of the mixed raw material powder to deionized water is 1:8-10; The complex enzyme includes acidic protease, cellulase and pectinase, with a mass ratio of 1:(10-15):(4-6). The mixed bacterial solution consists of Rhodotorula glutinis and Akkermansia fasciatus, and the viable count of the mixed bacterial solution is 1*10⁻⁶. 7 -1*10 9 cfu / mL; The volume ratio of the mixed bacterial solution to the enzymatic hydrolysate is 1:8-10; The ratio of viable cells in the Rhodotorula glutinis culture to the Akkermansia glutinis culture is 1:(3-5). The preservation number of the *Rhodotorula glutinis* is CGMCC No. 5081; The accession number of the *Ackermania* strain is CGMCC No. 22793; The seaweed powder is 30-35 parts, the enoki mushroom powder is 45-50 parts, and the Garcinia Cambogia powder is 18-22 parts.

2. The preparation method according to claim 1, characterized in that, The mass ratio of the compound enzyme to the mixture is (1-3):

7.

3. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis temperature is 40-45℃, the enzymatic hydrolysis time is 2-3 hours, and the enzymatic hydrolysis pH is 4.0-5.

5.

4. The preparation method according to claim 1, characterized in that, During the fermentation, the initial pH was 5.0-6.0, the fermentation temperature was 35±2℃, and the fermentation time was 24-48h.

5. The probiotic fermentation product prepared by the preparation method according to any one of claims 1-4.

6. The use of the probiotic fermentation product according to claim 5 in the preparation of pharmaceuticals or foods that help maintain healthy blood sugar levels and help control body fat.