Probiotic composition for regulating blood sugar and blood fat and preparation method thereof

Abstract

The invention provides a probiotic composition for regulating blood sugar and blood fat and a preparation method thereof, and belongs to the technical field of probiotics. The preparation method comprises the following steps: extracting and degrading low-molecular-weight fucoidin sulfate from undaria pinnatifida, performing water extraction and alcohol precipitation on the low-molecular-weight fucoidin sulfate and traditional Chinese medicines to obtain traditional Chinese medicine polysaccharide mixed chelating metal ions, and preparing a polysaccharide-Zn / Cr chelate; the preparation method comprises the following steps: fermenting residual solid residues, washing solids to obtain concentrated bacterial liquid, mixing fermentation liquid and traditional Chinese medicine liquid, purifying through ionic liquid to obtain an active component, embedding the concentrated bacterial liquid in a polysaccharide-Zn / Cr chelate and sodium alginate to obtain probiotic / polypeptide / polysaccharide nanospheres, and preparing the probiotic / polypeptide / polysaccharide nanospheres from the probiotic / polypeptide / polysaccharide nanospheres. The probiotic composition for regulating blood sugar and blood fat is prepared by mixing the probiotic powder with the active components, the fermented protein peptide, the panax notoginseng saponins and the ginsenoside Rg4, can play a good role in resisting inflammation, resisting oxidation, reducing blood sugar and blood fat and regulating immunity, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically to a probiotic composition for regulating blood sugar and blood lipids and its preparation method. Background Technology

[0002] With rapid economic development and an increasingly fast-paced lifestyle, people's dietary structure has undergone significant changes, leading to overnutrition and nutritional imbalances. This has resulted in a marked increase in the incidence of metabolic diseases, with diabetes being one of the fastest-growing categories. It is well known that diabetes is a serious threat to human health and quality of life. 60% of patients die from diabetic heart disease, 36% from diabetic viral complications, and the blindness rate within five years exceeds 80%. Almost 100% of patients experience lifelong numbness and pain in their hands and feet due to peripheral mental illness. Many patients with diabetic foot suffer from untreatable conditions leading to amputation, causing severe health and economic losses to individuals, families, and society. Therefore, serious attention and treatment of diabetes are urgently needed. Currently, there is no ideal treatment for this disease; therefore, continuously searching for new prevention and treatment methods is the dream of researchers, and they will continue to explore this goal.

[0003] Meanwhile, dyslipidemia / hyperlipidemia is a fundamental vascular disease underlying many cardiovascular-related illnesses, with complex mechanisms that make treatment cumbersome and difficult. Scientific research indicates that gut microbiota is related to blood lipids, and its mechanisms of influencing or exacerbating cardiovascular disease may include, but are not limited to, promoting the production of gut microbiota metabolites and activating immune inflammatory responses.

[0004] Alterations in glucose and lipid metabolism and the occurrence of oxidative stress, along with gut microbiota-related metabolites and their producing bacteria, can respectively promote / inhibit hyperglycemia, hyperlipidemia, and related diseases. Previously, the most direct methods for treating hyperglycemia and hyperlipidemia through gut microbiota were drug or dietary interventions.

[0005] In recent years, probiotics have been widely used in beverages. Data shows that approximately 400 to 500 million people worldwide consume probiotic drinks. The market is flooded with various probiotic beverages, and major brands are launching their own to compete for market share. While there are many types of these beverages on the market, they suffer from severe homogenization in function. In particular, there is a lack of probiotic products that effectively lower blood sugar and cholesterol. Although most products contain probiotics, the effects are poor due to long-term storage or the inactivation of most probiotics after entering the body, thus limiting the expansion of this market segment.

[0006] Chinese patent application CN105707896A discloses a probiotic composition with hypoglycemic function, its preparation method, and its application. The composition, by weight percentage, comprises the following components: 60-70% probiotic dry powder, 10-15% Coptis chinensis extract powder, 10-15% Astragalus membranaceus extract powder, and 5-10% Lonicera japonica extract powder.

[0007] Chinese patent application CN114343097A discloses a probiotic solid beverage that can help lower blood sugar and its preparation method. The raw materials include, by weight ratio: 10-20 parts of traditional Chinese medicine complex, 30-40 parts of compound probiotic powder, 5-10 parts of oligomannose, 1-3 parts of freeze-dried powder, 3-5 parts of prebiotics, 1-3 parts of stachyose, 1-3 parts of lactitol, 1-3 parts of L-arabinose, 3-5 parts of Jerusalem artichoke powder, 1-3 parts of maltodextrin, 1-3 parts of green tea powder, and 1-3 parts of citric acid.

[0008] However, these compositions are simply a mixture of probiotics and other ingredients. Not only are they ineffective, but they also suffer from probiotic inactivation. Typically, after entering the body, many probiotics die and become inactive in the stomach due to the acid, preventing them from successfully reaching the intestines and colonizing, thus failing to achieve their intended effect of regulating blood sugar and blood lipids. Summary of the Invention

[0009] The purpose of this invention is to propose a probiotic composition for regulating blood sugar and blood lipids and its preparation method. It can play a good role in anti-inflammatory, antioxidant, blood sugar and blood lipid reduction, and has a certain immune regulation effect. It can also improve the preservation time of probiotics and increase the number of live bacteria after entering the human intestine, which greatly improves the metabolic regulation effect of the composition and has broad application prospects.

[0010] The technical solution of this invention is implemented as follows:

[0011] This invention provides a method for preparing a probiotic composition for regulating blood sugar and blood lipids. Low molecular weight fucoidan sulfate is extracted and degraded from wakame seaweed. Traditional Chinese medicine is extracted with water and precipitated with alcohol to obtain medicinal polysaccharides, which are then mixed with low molecular weight fucoidan sulfate to chelate metal ions, forming a polysaccharide-Zn / Cr chelate. The remaining solid residue is fermented, filtered, and washed to obtain a concentrated bacterial solution. The fermentation broth and the medicinal solution are mixed and purified using an ionic liquid to obtain the active component. The concentrated bacterial solution is then encapsulated in the polysaccharide-Zn / Cr chelate and sodium alginate to obtain probiotic / peptide / polysaccharide nanospheres. These nanospheres are then mixed with the active component, fermented protein peptides, total saponins of Panax notoginseng, and ginsenoside Rg4 to obtain the probiotic composition for regulating blood sugar and blood lipids.

[0012] As a further improvement to the present invention, the following steps are included:

[0013] S1. Preparation of low molecular weight fucoidan sulfate: Wakame seaweed was washed, dried, and pulverized to obtain wakame powder. The powder was added to water and transferred to a closed hydrothermal reactor. The mixture was heated and pressurized for extraction, filtered, and the residue was retained. Calcium chloride was added to the filtrate, and the mixture was stirred and mixed evenly. The mixture was then filtered again. Vitamin C and hydrogen peroxide were added to the filtrate, and the mixture was heated and stirred for reaction. The mixture was dialyzed, the dialysate was concentrated, and the solution was freeze-dried to obtain low molecular weight fucoidan sulfate.

[0014] S2. Water extraction of traditional Chinese medicine: Astragalus membranaceus, Gynostemma pentaphyllum, hawthorn and cornus officinalis were washed, dried and pulverized to obtain traditional Chinese medicine powder. The powder was added to water, heated to boiling and extracted. The powder was filtered, and the residue was kept. Ethanol was added to the filtrate to precipitate. The solid was washed and dried to obtain traditional Chinese medicine polysaccharide. The liquid was kept.

[0015] S3. Preparation of polysaccharide-Zn / Cr chelate: The low molecular weight fucoidan sulfate ester obtained in step S1 and the traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water, chromium salt and zinc salt were added, the pH value of the solution was adjusted, the reaction was stirred, dialyzed, the dialysate was concentrated, and freeze-dried to obtain polysaccharide-Zn / Cr chelate.

[0016] S4. Fermentation: Add the filter residue from step S1, the filter residue from step S2, and whey protein to water, sterilize, inoculate with Lactobacillus casei and Lactobacillus acidophilus seed culture, ferment, filter, wash with solids, collect the bacterial solution, concentrate, and obtain concentrated bacterial solution; perform liquid dialysis, concentrate the dialysis solution, freeze dry, and obtain fermented protein peptides, retaining the permeate;

[0017] S5. Extraction of active components: Mix the liquid in step S2 and the permeate in step S4 evenly, add ionic liquid, introduce CO2 gas to increase the polarity of ionic liquid, extract with ultrasound, separate the ionic liquid layer, heat to allow CO2 to escape, separate the ionic liquid to remove the liquid, and collect the active components.

[0018] S6. Preparation of probiotic / peptide / polysaccharide nanospheres: Add the polysaccharide-Zn / Cr chelate, sodium alginate and lecithin obtained in step S3 to the concentrated bacterial solution obtained in step S4, stir and mix evenly, add to fish oil, emulsify, add calcium chloride solution dropwise, solidify at room temperature, centrifuge, wash, dry, and obtain probiotic / peptide / polysaccharide nanospheres.

[0019] S7. Preparation of probiotic composition for regulating blood sugar and blood lipids: The probiotic / peptide / polysaccharide nanospheres obtained in step S6, the active components obtained in step S5, the fermented protein peptides obtained in step S4, the total saponins of Panax notoginseng, and the ginsenoside Rg4 are mixed evenly to obtain the probiotic composition for regulating blood sugar and blood lipids.

[0020] As a further improvement of the present invention, in step S1, the solid-liquid ratio of wakame powder to water is 1:5-7 g / mL; the temperature of the heating and pressurizing extraction is 110-130℃, the pressure is 1.2-1.5 atmospheres, and the extraction time is 2-4 hours; the amount of calcium chloride added is such that the calcium chloride concentration after addition is 0.1-0.12 mol / L; the amount of vitamin C added is such that the vitamin C concentration after addition is 25-35 mmol / L; the amount of hydrogen peroxide added is such that the hydrogen peroxide concentration after addition is 25-35 mmol / L; the temperature of the heating and stirring reaction is 65-75℃, and the time is 1-3 hours; the dialysis bag used for dialysis has a pore size of 2-3 kDa, and the dialysis time is 3-5 hours.

[0021] As a further improvement of the present invention, the mass ratio of Astragalus membranaceus, Gynostemma pentaphyllum, Crataegus pinnatifida, and Cornus officinalis in step S2 is 3-5:7-10:5-7:1-2, the solid-liquid ratio of the herbal powder and water is 1:5-10 g / mL, the heating and boiling extraction time is 2-4 h, the addition of ethanol to the system to an ethanol content of 80-90 wt% and the precipitation time is 5-7 h.

[0022] As a further improvement of the present invention, the mass ratio of low molecular weight fucoidan sulfate, traditional Chinese medicine polysaccharide, chromium salt, and zinc salt in step S3 is 10-12:7-10:0.5-1:1-2, wherein the chromium salt is chromium chloride, and the zinc salt is selected from at least one of zinc chloride, zinc sulfate, zinc nitrate, and zinc acetate. The pH value of the solution is adjusted to 7.5-8, the stirring reaction time is 20-40 min, the dialysis bag pore size is 2-3 kDa, and the dialysis time is 2-4 h.

[0023] As a further improvement of the present invention, in step S4, the mass ratio of the filter residue in step S1, the filter residue in step S2, and the whey protein is 10-20:12-15:3-5, and the bacterial count of the Lactobacillus casei and Lactobacillus acidophilus seed solution is 10. 8 -10 9 The inoculum concentration of *Lactobacillus casei* and *Lactobacillus acidophilus* was 2-3 v / v% and 1-3 v / v%, respectively. The fermentation conditions were anoxic: 36-38°C, 100-200 rpm, for 48-56 hours. The concentrated bacterial solution contained 10... 10 -10 11 cfu / mL.

[0024] As a further improvement of the present invention, in step S5, the mass ratio of the liquid in step S2, the permeate in step S4, and the deep eutectic solvent is 20-30:15-20:30-50, the CO2 gas flow rate is 3-5 L / min, the ultrasonic power is 1000-1200 W, the extraction time is 1-2 h, the heating temperature is 50-60 °C, and the ionic liquid is selected from at least one of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, 1-butyl-3-methylimidazolium hexafluoroantimonate, 1-hexyl-3-methylimidazolium tetrafluoroborate, 1-hexyl-3-methylimidazolium hexafluorophosphate, and 1-hexyl-3-methylimidazolium hexafluoroantimonate.

[0025] As a further improvement of the present invention, the mass ratio of the concentrated bacterial solution, polysaccharide-Zn / Cr chelate, sodium alginate and lecithin in step S6 is 100:5-7:12-15:0.5-1, and the solidification time at room temperature is 20-30 min; the mass ratio of the probiotic / peptide / polysaccharide nanospheres, active components, fermented protein peptides, total saponins of Panax notoginseng and ginsenoside Rg4 in step S7 is 100:12-15:2-4:1-2:0.5-1.

[0026] As a further improvement to the present invention, the specific steps include:

[0027] S1. Preparation of low molecular weight fucoidan sulfate: Wakame seaweed was washed, dried, and pulverized to obtain wakame powder. The powder was added to water at a solid-liquid ratio of 1:5-7 g / mL. The mixture was transferred to a closed hydrothermal reactor, heated to 110-130℃, and pressurized to 1.2-1.5 atm for 2-4 hours. After extraction, the mixture was filtered, and the residue was retained. Calcium chloride was added to the filtrate to a final concentration of 0.1-0.12 mol / L, and the mixture was stirred until homogeneous. After filtration, vitamin C was added to the filtrate to a final concentration of 25-35 mmol / L, and hydrogen peroxide was added to a final concentration of 25-35 mmol / L. The mixture was heated to 65-75℃ and stirred for 1-3 hours. Dialysis was performed using a dialysis bag with a pore size of 2-3 kDa for 3-5 hours. The dialysate was concentrated and freeze-dried to obtain low molecular weight fucoidan sulfate.

[0028] S2. Water extraction of traditional Chinese medicine: Wash 3-5 parts by weight of Astragalus membranaceus, 7-10 parts by weight of Gynostemma pentaphyllum, 5-7 parts by weight of Crataegus pinnatifida, and 1-2 parts by weight of Cornus officinalis, dry them, pulverize them to obtain traditional Chinese medicine powder, add them to water, the solid-liquid ratio of the traditional Chinese medicine powder to water is 1:5-10 g / mL, heat to boiling and extract for 2-4 hours, filter, keep the filter residue, add ethanol to the filtrate until the ethanol content of the system is 80-90 wt%, precipitate for 5-7 hours, wash the solid, dry it to obtain traditional Chinese medicine polysaccharide, and keep the liquid for use;

[0029] S3. Preparation of polysaccharide-Zn / Cr chelate: 10-12 parts by weight of low molecular weight fucoidan sulfate obtained in step S1 and 7-10 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water. 0.5-1 parts by weight of chromium chloride and 1-2 parts by weight of zinc salt were added. The pH of the solution was adjusted to 7.5-8. The mixture was stirred for 20-40 min and dialyzed for 2-4 h using a dialysis bag with a pore size of 2-3 kDa. The dialysate was concentrated and freeze-dried to obtain polysaccharide-Zn / Cr chelate.

[0030] S4. Fermentation: Add 10-20 parts by weight of the filter residue from step S1, 12-15 parts by weight of the filter residue from step S2, and 3-5 parts by weight of whey protein to 500 parts by weight of water, sterilize, and inoculate with Lactobacillus casei and Lactobacillus acidophilus seed cultures at inoculation rates of 2-3 v / v% and 1-3 v / v%, respectively. Under anaerobic conditions, ferment at 36-38℃ and 100-200 r / min for 48-56 h. Filter, wash the solids, collect the bacterial culture, concentrate, and obtain a concentrated bacterial culture with a bacterial count of 10. 10 -10 11 cfu / mL; liquid to be retained;

[0031] The bacterial count of the Lactobacillus casei and Lactobacillus acidophilus seed solution was 10. 8 -10 9 cfu / mL;

[0032] S5. Extraction of active components: Mix 20-30 parts by weight of the liquid from step S2 and 15-20 parts by weight of the permeate from step S4 evenly, add 30-50 parts by weight of ionic liquid, and pass CO2 gas through at a flow rate of 3-5 L / min to increase the polarity of the ionic liquid. Extract with ultrasonic waves at 1000-1200W for 1-2 hours, separate the ionic liquid layer, heat to 50-60℃ to allow CO2 to escape, separate the liquid from the ionic liquid, and collect the active components.

[0033] S6. Preparation of probiotic / peptide / polysaccharide nanospheres: Add 5-7 parts by weight of the polysaccharide-Zn / Cr chelate obtained in step S3, 12-15 parts by weight of sodium alginate and 0.5-1 parts by weight of lecithin to 100 parts by weight of the concentrated bacterial solution obtained in step S4, stir and mix evenly, add to 200 parts by weight of fish oil, emulsify, add 10-15 parts by weight of 3-5 wt% calcium chloride solution dropwise, solidify at room temperature for 20-30 min, centrifuge, wash, dry, and obtain probiotic / peptide / polysaccharide nanospheres;

[0034] S7. Preparation of a probiotic composition for regulating blood sugar and blood lipids: 100 parts by weight of the probiotic / peptide / polysaccharide nanospheres obtained in step S6, 12-15 parts by weight of the active component obtained in step S5, 2-4 parts by weight of the fermented protein peptide obtained in step S4, 1-2 parts by weight of total Panax notoginseng saponins, and 0.5-1 parts by weight of ginsenoside Rg4 are mixed evenly to obtain a probiotic composition for regulating blood sugar and blood lipids.

[0035] The present invention further protects a probiotic composition for regulating blood sugar and blood lipids prepared by the above-described preparation method.

[0036] The present invention has the following beneficial effects:

[0037] This invention prepares a low molecular weight fucoidan sulfate. Fucoidan sulfate typically possesses various biological activities, but these activities are limited by its structural characteristics and a limited number of functional groups, resulting in low water solubility and limited biological activity. This invention utilizes vitamin C and hydrogen peroxide for oxidative degradation, reducing the molecular weight of fucoidan sulfate and improving its water solubility. Simultaneously, it enhances its antioxidant, anti-inflammatory, blood glucose and lipid-regulating, and immunomodulatory effects. It can effectively improve glucose homeostasis by inhibiting α-amylase activity and improving glucose tolerance, regulating liver glucose metabolism, and alleviating hyperglycemia and hyperlipidemia. Furthermore, it is a good prebiotic, helping to promote the utilization of probiotics and exert its blood glucose and lipid-regulating effects.

[0038] This invention selects Astragalus membranaceus, Gynostemma pentaphyllum, Crataegus pinnatifida, and Cornus officinalis as raw materials. Water extraction was used to obtain water extracts. The Astragalus membranaceus extract lowers lipids by inhibiting the biosynthesis of triglycerides and cholesterol in the body. The Crataegus pinnatifida extract inhibits cholesterol synthesis by regulating the NF-κB signaling pathway. The Gynostemma pentaphyllum extract inhibits the upregulation of HMGCR, sterol regulatory element-binding protein-1c, FAS, and ACC-1 expression in liver tissue, and inhibits the downregulation of PPARα and CPT-1 expression, significantly reducing the formation of lipid droplets in the liver and regulating blood lipid homeostasis. The Cornus officinalis extract inhibits the activity of α-amylase and α-glucosidase, has an insulin-sensitizing effect, increases insulin secretion, and promotes the proliferation of pancreatic β-cells, effectively combating insulin resistance and regulating blood lipid disorders.

[0039] First, this invention involves adding ethanol to the aqueous extract of traditional Chinese medicine to precipitate it, obtaining polysaccharides. These polysaccharides exhibit good blood glucose and lipid regulation effects, promote the growth of beneficial bacteria in the gut, regulate gut microbiota homeostasis, and may help regulate abnormal lipid metabolism. Simultaneously, this invention mixes the obtained polysaccharides with low molecular weight fucoidan sulfate, and chelates them with chromium and zinc ions. The chelates form by complexing with free groups such as -OH and -NH2 on the polysaccharide backbone that can provide lone pairs of electrons. Chromium, in its trivalent state, participates in the composition of glucose tolerance factor, assisting insulin in regulating glucose and lipid metabolism. Inorganic zinc ions, upon entering the gastrointestinal tract, easily precipitate upon encountering substances such as oxalic acid and phytic acid, making them difficult for the body to absorb and utilize. Selecting a safe and stable carrier to bind with zinc ions helps improve the bioavailability of zinc supplements. Polysaccharide zinc complexes are highly water-soluble and have a high absorption and utilization rate. They possess good biological activity, promote the secretion of insulin by pancreatic cells, reduce blood glucose levels, regulate the sensitivity of hepatocytes to insulin, combine with lipids in the blood, participate in the cholesterol metabolism process, promote lipid transport and excretion, and improve hyperglycemia and hyperlipidemia.

[0040] Furthermore, this invention mixes the solid residue of traditional Chinese medicine and wakame seaweed with whey protein, and then inoculates it with *Lactobacillus casei* and *Lactobacillus acidophilus* for fermentation. On the one hand, the probiotics can promote the rupture of the cell walls of the solid residue, dissolving more active components into the fermentation product. On the other hand, it can also promote the proliferation of probiotics, obtaining a high-concentration bacterial solution. *Lactobacillus casei* can regulate glucose metabolism and lower blood sugar through mechanisms such as protecting pancreatic islet structure, increasing the sensitivity of target tissues to insulin, improving insulin sensitivity, accelerating glucose transport, reducing endotoxins, and anti-inflammation. *Lactobacillus acidophilus* can inhibit the body's fat synthesis and enhance fat breakdown, hydrolyzing conjugated bile acids into free bile acids. The solubility of free bile acids decreases, making them less likely to be reabsorbed and reused by the liver through enterohepatic circulation. This promotes the body's use of cholesterol to synthesize new bile acids, reduces cholesterol absorption and clearance, promotes the conversion of cholesterol into bile acids, and promotes the excretion of bile acids, thereby lowering blood and cholesterol levels. The addition of both has a synergistic effect.

[0041] Whey protein can provide a nitrogen source for the fermentation of probiotics. At the same time, under the fermentation action of probiotics, whey protein peptides are generated, which can inhibit the activity of dipeptidyl peptidase IV, thereby increasing the secretion of insulin, reducing atherosclerotic lesions and hepatic steatosis damage, inhibiting lipid accumulation in the liver, reducing the level of liver inflammation and oxidative stress damage, and playing a role in assisting in the regulation of blood sugar and blood lipids.

[0042] The aqueous extracts and fermentation permeate of traditional Chinese medicine contain abundant active components such as polyphenols, flavonoids, and triterpenes, which have excellent activity in regulating blood sugar and blood lipids. However, there are also some other impurities that reduce the regulatory activity. Therefore, this invention uses ionic liquids for separation and purification. After CO2 is introduced, the polarity of the ionic liquid increases, which helps to extract these active substances from the aqueous solution into the ionic liquid layer. After heating, the ionic liquid changes from high polarity to normal polarity, and these active components are separated from the ionic liquid. After removing the ionic liquid, the active components are well separated and purified, and the regulatory activity is greatly enhanced.

[0043] This invention utilizes the synergistic encapsulation of concentrated bacterial culture with polysaccharide-Zn / Cr chelates and sodium alginate. On one hand, the polysaccharide-Zn / Cr chelate forms a relatively stable shell with sodium alginate under the cross-linking effect of calcium chloride, encapsulating probiotics within microspheres. The dense cross-linked structure effectively protects the probiotics against the effects of gastric acid and choline, increasing the viable count of probiotics and significantly enhancing their regulatory effect. On the other hand, both the polysaccharide-Zn / Cr chelate and sodium alginate are excellent prebiotics. After rupturing in the intestines, they can be directly utilized by probiotics, promoting their proliferation. Simultaneously, while being utilized, the polysaccharide-Zn / Cr chelate exerts excellent antioxidant, anti-inflammatory, and blood glucose and lipid-regulating effects, while the released Zn and Cr ions exert excellent metabolic regulatory effects.

[0044] The composition of this invention also includes total saponins of Panax notoginseng and ginsenoside Rg4. Total saponins of Panax notoginseng possess various biological activities and may exert anti-inflammatory, antioxidant, hypoglycemic, and hypolipidemic effects in vivo. Ginsenoside Rg4 has health benefits by enhancing the body's immunity, improving physical fitness, promoting normal metabolism, lowering blood sugar (helping alleviate symptoms in diabetic patients), and lowering blood lipids (helping improve blood circulation, thus helping to alleviate cardiovascular diseases). The addition of both has a synergistic effect.

[0045] The probiotic composition for regulating blood sugar and blood lipids prepared by this invention can play a good role in anti-inflammatory, antioxidant, blood sugar and blood lipid reduction, and has a certain immunomodulatory effect. It can also improve the storage time of probiotics and increase the number of live bacteria after entering the human intestine, which greatly improves the metabolic regulation effect of the composition and has broad application prospects. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Preparation of Lactobacillus casei and Lactobacillus acidophilus (purchased from Weikaiheis (Shandong) Biotechnology Co., Ltd.) inoculum: The inoculum was inoculated separately into Gao's medium and activated under anaerobic conditions at 37℃ and 100 r / min for 24 h to obtain a bacterial count of 10. 8 -10 9 CFU / mL bacterial seed solution.

[0048] Whey protein, purchased from Chongqing Tianrun Biological Products Co., Ltd.

[0049] Total saponins of Panax notoginseng, purity >70%, purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0050] Ginsenoside Rg4 was purchased from Chengdu Gepute Biotechnology Co., Ltd.

[0051] Example 1

[0052] This embodiment provides a method for preparing a probiotic composition for regulating blood sugar and blood lipids, specifically including the following steps:

[0053] S1. Preparation of low molecular weight fucoidan sulfate: Wakame seaweed was washed, dried, and pulverized to obtain wakame powder. The powder was added to water at a solid-liquid ratio of 1:5 g / mL and transferred to a closed hydrothermal reactor. The mixture was heated to 110°C and pressurized to 1.2 atm for 2 hours. After extraction, the mixture was filtered, and the residue was retained. Calcium chloride was added to the filtrate to a final concentration of 0.1 mol / L. The mixture was stirred and mixed evenly and then filtered. Vitamin C and hydrogen peroxide were added to the filtrate to a final concentration of 25 mmol / L. The mixture was heated to 65°C and stirred for 1 hour. The mixture was dialyzed for 3 hours using a dialysis bag with a pore size of 2 kDa. The dialysate was concentrated and freeze-dried to obtain low molecular weight fucoidan sulfate.

[0054] S2. Water extraction of traditional Chinese medicine: 3 parts by weight of Astragalus membranaceus, 7 parts by weight of Gynostemma pentaphyllum, 5 parts by weight of Crataegus pinnatifida, and 1 part by weight of Cornus officinalis were washed, dried, and pulverized to obtain traditional Chinese medicine powder. The powder was added to water at a solid-liquid ratio of 1:5 g / mL and heated to boiling for 2 hours. The powder was filtered, and the residue was kept. Ethanol was added to the filtrate until the ethanol content of the system was 80 wt%. The mixture was allowed to precipitate for 5 hours. The solid was washed and dried to obtain traditional Chinese medicine polysaccharide. The liquid was kept.

[0055] S3. Preparation of polysaccharide-Zn / Cr chelate: 10 parts by weight of low molecular weight fucoidan sulfate obtained in step S1 and 7 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water. 0.5 parts by weight of chromium chloride and 1 part by weight of zinc chloride were added, the pH of the solution was adjusted to 7.5, the reaction was stirred for 20 min, dialyzed for 2 h using a dialysis bag with a pore size of 2 kDa, the dialysate was concentrated, and freeze-dried to obtain polysaccharide-Zn / Cr chelate;

[0056] S4. Fermentation: Add 10 parts by weight of the filter residue from step S1, 12 parts by weight of the filter residue from step S2, and 3 parts by weight of whey protein to 500 parts by weight of water, sterilize, and inoculate with Lactobacillus casei and Lactobacillus acidophilus seed culture at inoculation amounts of 2 v / v% and 1 v / v%, respectively. Fermentation is carried out under anaerobic conditions at 36°C and 100 r / min for 48 h. After filtration and solid washing, the bacterial culture is collected, concentrated, and a concentrated bacterial culture with a bacterial count of 10 10 -10 11 cfu / mL; liquid to be retained;

[0057] S5. Extraction of active components: Mix 20 parts by weight of the liquid from step S2 and 15 parts by weight of the permeate from step S4 evenly, add 30 parts by weight of ionic liquid, introduce CO2 gas at a flow rate of 3L / min to increase the polarity of the ionic liquid, extract with ultrasonic at 1000W for 1h, separate the ionic liquid layer, heat to 50℃ to allow CO2 to escape, separate the ionic liquid to remove the liquid, and collect the active components.

[0058] S6. Preparation of probiotic / peptide / polysaccharide nanospheres: Add 5 parts by weight of the polysaccharide-Zn / Cr chelate obtained in step S3, 12 parts by weight of sodium alginate and 0.5 parts by weight of lecithin to 100 parts by weight of the concentrated bacterial solution obtained in step S4, stir and mix for 20 min, add to 200 parts by weight of fish oil, emulsify at 10000 r / min for 15 min, add 10 parts by weight of 3wt% calcium chloride solution, solidify at room temperature for 20 min, centrifuge, wash and dry to obtain probiotic / peptide / polysaccharide nanospheres;

[0059] S7. Preparation of probiotic composition for regulating blood sugar and blood lipids: 100 parts by weight of probiotic / peptide / polysaccharide nanospheres obtained in step S6, 12 parts by weight of active component obtained in step S5, 2 parts by weight of fermented protein peptide obtained in step S4, 1 part by weight of total Panax notoginseng saponins, and 0.5 parts by weight of ginsenoside Rg4 are stirred and mixed for 30 minutes to obtain probiotic composition for regulating blood sugar and blood lipids.

[0060] Example 2

[0061] This embodiment provides a method for preparing a probiotic composition for regulating blood sugar and blood lipids, specifically including the following steps:

[0062] S1. Preparation of low molecular weight fucoidan sulfate: Wakame seaweed was washed, dried, and pulverized to obtain wakame powder. The powder was added to water at a solid-liquid ratio of 1:7 g / mL and transferred to a closed hydrothermal reactor. The mixture was heated to 130°C and pressurized to 1.5 atm for 4 hours. After filtration, the residue was retained. Calcium chloride was added to the filtrate to a final concentration of 0.12 mol / L and stirred until homogeneous. The mixture was then filtered again. Vitamin C and hydrogen peroxide were added to the filtrate to a final concentration of 35 mmol / L and heated to 75°C. The mixture was stirred for 3 hours and dialyzed through a 3 kDa dialysis bag for 5 hours. The dialysate was concentrated and freeze-dried to obtain low molecular weight fucoidan sulfate.

[0063] S2. Water extraction of traditional Chinese medicine: 5 parts by weight of Astragalus membranaceus, 10 parts by weight of Gynostemma pentaphyllum, 7 parts by weight of Crataegus pinnatifida, and 2 parts by weight of Cornus officinalis were washed, dried, and pulverized to obtain traditional Chinese medicine powder. The powder was added to water at a solid-liquid ratio of 1:10 g / mL and heated to boiling for 4 hours. The powder was filtered, and the residue was kept. Ethanol was added to the filtrate until the ethanol content of the system was 90 wt%. The mixture was allowed to precipitate for 7 hours. The solid was washed and dried to obtain traditional Chinese medicine polysaccharide. The liquid was kept.

[0064] S3. Preparation of polysaccharide-Zn / Cr chelate: 12 parts by weight of low molecular weight fucoidan sulfate obtained in step S1 and 10 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water. 1 part by weight of chromium chloride and 2 parts by weight of zinc sulfate were added, the pH of the solution was adjusted to 8, and the reaction was stirred for 40 min. The solution was dialyzed for 4 h using a dialysis bag with a pore size of 3 kDa. The dialysate was concentrated and freeze-dried to obtain polysaccharide-Zn / Cr chelate.

[0065] S4. Fermentation: Add 20 parts by weight of the filter residue from step S1, 15 parts by weight of the filter residue from step S2, and 5 parts by weight of whey protein to 500 parts by weight of water, sterilize, and inoculate with Lactobacillus casei and Lactobacillus acidophilus seed cultures at inoculation rates of 3 v / v% and 3 v / v%, respectively. Fermentation is carried out under anaerobic conditions at 38°C and 200 r / min for 56 h. After filtration and solid washing, the bacterial culture is collected, concentrated, and a concentrated bacterial culture with a bacterial count of 10 is obtained. 10 -10 11 cfu / mL; liquid to be retained;

[0066] S5. Extraction of active components: Mix 30 parts by weight of the liquid from step S2 and 20 parts by weight of the permeate from step S4 evenly, add 50 parts by weight of ionic liquid, introduce CO2 gas at a flow rate of 5 L / min to increase the polarity of the ionic liquid, extract with ultrasonic at 1200W for 2 hours, separate the ionic liquid layer, heat to 60℃ to allow CO2 to escape, separate the ionic liquid to remove the liquid, and collect the active components.

[0067] S6. Preparation of probiotic / peptide / polysaccharide nanospheres: Add 7 parts by weight of the polysaccharide-Zn / Cr chelate prepared in step S3, 15 parts by weight of sodium alginate and 1 part by weight of lecithin to 100 parts by weight of the concentrated bacterial solution obtained in step S4, stir and mix for 20 min, add to 200 parts by weight of fish oil, emulsify at 10000 r / min for 15 min, add 15 parts by weight of 5 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash and dry to obtain probiotic / peptide / polysaccharide nanospheres;

[0068] S7. Preparation of probiotic composition for regulating blood sugar and blood lipids: 100 parts by weight of probiotic / peptide / polysaccharide nanospheres obtained in step S6, 15 parts by weight of active component obtained in step S5, 4 parts by weight of fermented protein peptide obtained in step S4, 2 parts by weight of total Panax notoginseng saponins, and 1 part by weight of ginsenoside Rg4 are stirred and mixed for 30 minutes to obtain probiotic composition for regulating blood sugar and blood lipids.

[0069] Example 3

[0070] This embodiment provides a method for preparing a probiotic composition for regulating blood sugar and blood lipids, specifically including the following steps:

[0071] S1. Preparation of low molecular weight fucoidan sulfate: Wakame seaweed was washed, dried, and pulverized to obtain wakame powder. The powder was added to water at a solid-liquid ratio of 1:7 g / mL and transferred to a closed hydrothermal reactor. The mixture was heated to 120°C and pressurized to 1.35 atm for 3 hours. After extraction, the mixture was filtered, and the residue was retained. Calcium chloride was added to the filtrate to a final concentration of 0.11 mol / L. The mixture was stirred and mixed evenly, and then filtered. Vitamin C and hydrogen peroxide were added to the filtrate to a final concentration of 30 mmol / L. The mixture was heated to 70°C and stirred for 2 hours. The mixture was dialyzed for 4 hours using a dialysis bag with a pore size of 2.5 kDa. The dialysate was concentrated and freeze-dried to obtain low molecular weight fucoidan sulfate.

[0072] S2. Water extraction of traditional Chinese medicine: 4 parts by weight of Astragalus membranaceus, 8.5 parts by weight of Gynostemma pentaphyllum, 6 parts by weight of Crataegus pinnatifida, and 1.5 parts by weight of Cornus officinalis were washed, dried, and pulverized to obtain traditional Chinese medicine powder. The powder was added to water at a solid-liquid ratio of 1:7 g / mL and heated to boiling for 3 hours. The mixture was filtered, and the residue was kept. Ethanol was added to the filtrate until the ethanol content of the system was 85 wt%. The mixture was allowed to precipitate for 6 hours. The solid was washed and dried to obtain traditional Chinese medicine polysaccharide. The liquid was kept.

[0073] S3. Preparation of polysaccharide-Zn / Cr chelate: 11 parts by weight of low molecular weight fucoidan sulfate obtained in step S1 and 8.5 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water. 0.7 parts by weight of chromium chloride and 1.5 parts by weight of zinc nitrate were added, the pH of the solution was adjusted to 7.7, the reaction was stirred for 30 min, dialyzed for 3 h using a dialysis bag with a pore size of 2.5 kDa, the dialysate was concentrated, and freeze-dried to obtain polysaccharide-Zn / Cr chelate;

[0074] S4. Fermentation: Add 15 parts by weight of the filter residue from step S1, 13.5 parts by weight of the filter residue from step S2, and 4 parts by weight of whey protein to 500 parts by weight of water, sterilize, and inoculate with Lactobacillus casei and Lactobacillus acidophilus seed culture at inoculation amounts of 2.5 v / v% and 2 v / v%, respectively. Fermentation is carried out under anaerobic conditions at 37°C and 150 r / min for 52 h. After filtration and solid washing, the bacterial culture is collected, concentrated, and a concentrated bacterial culture with a bacterial count of 10 is obtained. 10 -10 11 cfu / mL; liquid to be retained;

[0075] S5. Extraction of active components: Mix 25 parts by weight of the liquid from step S2 and 17 parts by weight of the permeate from step S4 evenly, add 40 parts by weight of ionic liquid, and introduce CO2 gas at a flow rate of 4 L / min to increase the polarity of the ionic liquid. Extract with ultrasonic waves at 1100 W for 1.5 h, separate the ionic liquid layer, heat to 55 °C to allow CO2 to escape, separate the liquid to remove the ionic liquid, and collect the active components.

[0076] S6. Preparation of probiotic / peptide / polysaccharide nanospheres: Add 6 parts by weight of the polysaccharide-Zn / Cr chelate obtained in step S3, 13.5 parts by weight of sodium alginate and 0.7 parts by weight of lecithin to 100 parts by weight of the concentrated bacterial solution obtained in step S4, stir and mix for 20 min, add to 200 parts by weight of fish oil, emulsify at 10000 r / min for 15 min, add 12 parts by weight of 4wt% calcium chloride solution, solidify at room temperature for 25 min, centrifuge, wash and dry to obtain probiotic / peptide / polysaccharide nanospheres;

[0077] S7. Preparation of probiotic composition for regulating blood sugar and blood lipids: 100 parts by weight of probiotic / peptide / polysaccharide nanospheres obtained in step S6, 13.5 parts by weight of active component obtained in step S5, 3 parts by weight of fermented protein peptide obtained in step S4, 1.5 parts by weight of total Panax notoginseng saponins, and 0.7 parts by weight of ginsenoside Rg4 are stirred and mixed for 30 minutes to obtain probiotic composition for regulating blood sugar and blood lipids.

[0078] Comparative Example 1

[0079] The difference from Example 3 is that no oxidative degradation was performed in step S1.

[0080] Specifically as follows:

[0081] S1. Preparation of fucoidan sulfate: Wakame seaweed was washed, dried, and pulverized to obtain wakame powder. The powder was added to water at a solid-liquid ratio of 1:7 g / mL and transferred to a closed hydrothermal reactor. The mixture was heated to 120°C and pressurized to 1.35 atm for 3 hours. After extraction, the mixture was filtered, and the residue was retained. Calcium chloride was added to the filtrate to a final concentration of 0.11 mol / L. The mixture was stirred and mixed evenly, filtered, and dialyzed using a dialysis bag with a pore size of 2.5 kDa for 4 hours. The dialysate was concentrated and freeze-dried to obtain fucoidan sulfate.

[0082] Comparative Example 2

[0083] The difference from Example 3 is that low molecular weight fucoidan sulfate was not added in step S3.

[0084] Specifically as follows:

[0085] S3. Preparation of polysaccharide-Zn / Cr chelate: 19.5 parts by weight of the traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water, 0.7 parts by weight of chromium chloride and 1.5 parts by weight of zinc nitrate were added, the pH of the solution was adjusted to 7.7, the reaction was stirred for 30 min, dialyzed for 3 h using a dialysis bag with a pore size of 2.5 kDa, the dialysate was concentrated and freeze-dried to obtain polysaccharide-Zn / Cr chelate.

[0086] Comparative Example 3

[0087] The difference from Example 3 is that no traditional Chinese medicine polysaccharides were added in step S3.

[0088] Specifically as follows:

[0089] S3. Preparation of polysaccharide-Zn / Cr chelate: 19.5 parts by weight of low molecular weight fucoidan sulfate obtained in step S1 was dissolved in water, 0.7 parts by weight of chromium chloride and 1.5 parts by weight of zinc nitrate were added, the pH of the solution was adjusted to 7.7, the reaction was stirred for 30 min, dialyzed for 3 h using a dialysis bag with a pore size of 2.5 kDa, the dialysate was concentrated and freeze-dried to obtain polysaccharide-Zn / Cr chelate.

[0090] Comparative Example 4

[0091] The difference from Example 3 is that chromium chloride was not added in step S3.

[0092] Specifically as follows:

[0093] S3. Preparation of polysaccharide-Zn chelate: 11 parts by weight of low molecular weight fucoidan sulfate obtained in step S1 and 8.5 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water. 2.2 parts by weight of zinc nitrate were added, the pH of the solution was adjusted to 7.7, and the mixture was stirred for 30 min. Dialysis was performed for 3 h using a dialysis bag with a pore size of 2.5 kDa. The dialysate was concentrated and freeze-dried to obtain polysaccharide-Zn chelate.

[0094] Comparative Example 5

[0095] The difference from Example 3 is that zinc nitrate was not added in step S3.

[0096] Specifically as follows:

[0097] S3. Preparation of polysaccharide-Cr chelate: 11 parts by weight of low molecular weight fucoidan sulfate obtained in step S1 and 8.5 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water. 2.2 parts by weight of chromium chloride were added, the pH of the solution was adjusted to 7.7, and the mixture was stirred for 30 min. The mixture was dialyzed for 3 h using a dialysis bag with a pore size of 2.5 kDa. The dialysate was concentrated and freeze-dried to obtain polysaccharide-Cr chelate.

[0098] Comparative Example 6

[0099] The difference from Example 3 is that chromium chloride and zinc nitrate were not added in step S3.

[0100] Specifically as follows:

[0101] S3. Preparation of polysaccharides: 11 parts by weight of the low molecular weight fucoidan sulfate obtained in step S1 and 8.5 parts by weight of the traditional Chinese medicine polysaccharide obtained in step S2 are mixed to prepare polysaccharides.

[0102] Comparative Example 7

[0103] The difference from Example 3 is that no Lactobacillus casei seed solution was inoculated in step S4.

[0104] Specifically as follows:

[0105] S4. Fermentation: Add 15 parts by weight of the filter residue from step S1, 13.5 parts by weight of the filter residue from step S2, and 4 parts by weight of whey protein to 500 parts by weight of water, sterilize, and inoculate with Lactobacillus acidophilus seed culture at an inoculation rate of 4.5 v / v%. Fermentation is carried out under anaerobic conditions at 37°C and 150 r / min for 52 h. After filtration and solid washing, the bacterial culture is collected, concentrated, and a concentrated bacterial culture with a bacterial count of 10 is obtained. 10 -10 11 cfu / mL; liquid to be kept for future use.

[0106] Comparative Example 8

[0107] The difference from Example 3 is that Lactobacillus acidophilus seed solution was not inoculated in step S4.

[0108] Specifically as follows:

[0109] S4. Fermentation: Add 15 parts by weight of the filter residue from step S1, 13.5 parts by weight of the filter residue from step S2, and 4 parts by weight of whey protein to 500 parts by weight of water, sterilize, and inoculate with Lactobacillus casei seed culture at an inoculation rate of 4.5 v / v%. Fermentation is carried out under anaerobic conditions at 37°C and 150 r / min for 52 h. After filtration and solid washing, the bacterial culture is collected, concentrated, and a concentrated bacterial culture with a bacterial count of 10 is obtained. 10 -10 11 cfu / mL; liquid to be kept for future use.

[0110] Comparative Example 9

[0111] The difference from Example 3 is that ionic liquid purification was not performed in step S5.

[0112] Specifically as follows:

[0113] S5. Extraction of active components: Mix 25 parts by weight of the liquid from step S2 and 17 parts by weight of the permeate from step S4 evenly, freeze-dry, and obtain the active components.

[0114] Comparative Example 10

[0115] The difference from Example 3 is that total saponins of Panax notoginseng were not added in step S7.

[0116] Specifically as follows:

[0117] S7. Preparation of probiotic composition for regulating blood sugar and blood lipids: 100 parts by weight of probiotic / peptide / polysaccharide nanospheres obtained in step S6, 13.5 parts by weight of active component obtained in step S5, 3 parts by weight of fermented protein peptide obtained in step S4, and 2.2 parts by weight of ginsenoside Rg4 are stirred and mixed for 30 minutes to obtain probiotic composition for regulating blood sugar and blood lipids.

[0118] Comparative Example 11

[0119] The difference compared to Example 3 is that ginsenoside Rg4 was not added in step S7.

[0120] Specifically as follows:

[0121] S7. Preparation of probiotic composition for regulating blood sugar and blood lipids: 100 parts by weight of probiotic / peptide / polysaccharide nanospheres obtained in step S6, 13.5 parts by weight of active component obtained in step S5, 3 parts by weight of fermented protein peptide obtained in step S4, and 2.2 parts by weight of Panax notoginseng total saponins are stirred and mixed for 30 minutes to obtain probiotic composition for regulating blood sugar and blood lipids.

[0122] Comparative Example 12

[0123] The difference from Example 3 is that ginsenoside Rg4 and total Panax notoginseng saponins were not added in step S7.

[0124] Specifically as follows:

[0125] S7. Preparation of probiotic composition for regulating blood sugar and blood lipids: 100 parts by weight of probiotic / peptide / polysaccharide nanospheres obtained in step S6, 13.5 parts by weight of active component obtained in step S5, and 3 parts by weight of fermented protein peptide obtained in step S4 are mixed for 30 minutes to obtain probiotic composition for regulating blood sugar and blood lipids.

[0126] Test Example 1

[0127] The probiotic / peptide / polysaccharide nanospheres prepared in step S6 of Examples 1-3, and commercially available probiotic agents (Lactobacillus casei and Lactobacillus acidophilus, mass ratio 2.5:2, without encapsulation) were subjected to performance tests for resistance to gastric acid, choline, and intestinal fluid. The results are shown in Table 1.

[0128] Choline tolerance test:

[0129] Add 0.3 wt% ox bile salt to LB liquid medium, sterilize, add 1 g of probiotic / peptide / polysaccharide nanospheres and commercially available probiotic agent to the above medium, and incubate at 37℃ and 180 r / min for 2 h. Take samples at 0 and 2 h for plate colony counting and calculate the survival rate.

[0130] Gastric acid resistance test:

[0131] Preparation of artificial gastric juice: Take 16.4 mL of 10% hydrochloric acid, dilute with water to make the pH value 2, sterilize, add 1 g of pepsin to every 100 mL, mix well, and the artificial gastric juice is prepared.

[0132] 1g of probiotic / peptide / polysaccharide nanospheres and commercially available probiotic agent were added to artificial gastric fluid and cultured in a shaker at 37℃ and 180r / min for 2h. Samples were taken at 0 and 2h for plate colony counting and survival rate was calculated.

[0133] Intestinal fluid resistance test:

[0134] Preparation of artificial intestinal fluid: Dissolve 6.8g of Na2HPO4 in 500mL of water, adjust the pH to 6.8 with sodium hydroxide solution, dilute with water to 1000mL, sterilize, add 1g of trypsin to each 100mL of liquid, mix well, and the artificial intestinal fluid is obtained.

[0135] Add 1g of probiotic / peptide / polysaccharide nanospheres and commercially available probiotic agent to artificial intestinal fluid, and incubate at 37℃ and 180r / min for 2h in a shaker. Take samples at 0 and 2h for plate colony counting and calculate the survival rate.

[0136] Strain survival rate (%) = N t / N0×100%.

[0137] N t : Viable bacteria count after 2 hours; N0: Viable bacteria count after 0 hours.

[0138] Table 1

[0139]

[0140] As shown in the table above, the probiotic / peptide / polysaccharide nanospheres prepared in Examples 1-3 of this invention, under the synergistic encapsulation effect of polysaccharide-Zn / Cr chelate and sodium alginate, can form a relatively stable shell with sodium alginate under the cross-linking effect of calcium chloride, thus encapsulating the probiotics in the microspheres. The cross-linked structure is relatively dense, which can effectively protect the probiotics from the effects of gastric acid, choline, and intestinal fluid, and the viable bacteria count is high.

[0141] Test Example 2: Determination of ABTS Free Radical Scavenging Rate

[0142] The probiotic compositions for regulating blood glucose and lipids prepared in Examples 1-3 and Comparative Examples 1-12 were formulated into 50 mg / L aqueous solutions and tested using an ABTS kit. 200 μL of the reaction solution was transferred to a 96-well plate, and the absorbance at 734 nm was measured. Ultrapure water was used as a blank control, and vitamin C as a positive control. The scavenging rate (%) of ABTS free radicals was calculated. The results are shown in Table 2.

[0143] ABTS radical scavenging rate (%) = (A0 - A1) / A0 × 100%

[0144] Where A0 is the absorbance of the blank group and A1 is the absorbance of the experimental group.

[0145] Table 2

[0146] Group ABTS radical scavenging rate (%) Vitamin C 61.7 Example 1 85.9 Example 2 85.1 Example 3 86.4 Comparative Example 1 82.2 Comparative Example 2 77.8 Comparative Example 3 79.6 Comparative Example 4 84.1 Comparative Example 5 83.9 Comparative Example 6 82.6 Comparative Example 7 82.9 Comparative Example 8 81.5 Comparative Example 9 78.2 Comparative Example 10 84.2 Comparative Example 11 84.5 Comparative Example 12 83.1

[0147] As can be seen from the table above, the probiotic compositions for regulating blood sugar and blood lipids prepared in Examples 1-3 of the present invention have good antioxidant activity.

[0148] Test Example 3

[0149] 1. α-Glucosidase activity inhibition experiment

[0150] Using 0.05 mol / L PBS (pH = 6.8) as a solvent, the probiotic compositions for regulating blood glucose and lipids prepared in Examples 1-3 and Comparative Examples 1-12 were prepared into 50 mg / L aqueous solutions. 0.5 mL of each sample solution was taken, and 0.5 mL of 0.2 mol / L α-glucosidase solution was added. The mixture was incubated at 37°C for 20 min, followed by the addition of 0.5 mL of 0.02 mol / L p-nitrophenyl α-D-glucopyranoside. The mixture was thoroughly mixed and incubated at 37°C for 20 min. Finally, 2 mL of 1 mol / L Na₂CO₃ was added to terminate the reaction. The absorbance was measured at 700 nm. Acarbose was used as a positive control.

[0151] Inhibition rate of α-glucosidase activity (%) = 1 - (AB) / (CD) × 100%

[0152] Where A is the absorbance of the sample solution; B is the absorbance of the α-glucosidase solution; C is the absorbance of PBS instead of the polysaccharide solution; and D is the absorbance of PBS only.

[0153] 2. α-Amylase activity inhibition experiment

[0154] Using 0.05 mol / L PBS (pH = 6.8) as solvent, the probiotic compositions for regulating blood glucose and lipids prepared in Examples 1-3 and Comparative Examples 1-12 were prepared into 50 mg / L aqueous solutions. 0.25 mL of each sample solution was taken, and 0.25 mL of α-amylase (1 U / mL) was added and mixed. The mixture was reacted at 37°C for 15 min. 500 μL of 1% starch solution was added to the mixture, and the reaction continued for 10 min. 600 μL of DNS reagent was added, and the mixture was treated in a boiling water bath for 15 min. After cooling to room temperature, the absorbance was measured at 540 nm. Acarbose was used as a positive control.

[0155] Inhibition rate of α-amylase activity (%) = 1 - (AB) / (CD) × 100%

[0156] Where A is the absorbance of the sample solution; B is the absorbance of the α-amylase solution; C is the absorbance of the PBS solution instead of the polysaccharide solution; and D is the absorbance of the PBS solution alone.

[0157] The results are shown in Table 3.

[0158] Table 3

[0159] Group Inhibition rate of α-glucosidase activity (%) Inhibition rate of α-amylase activity (%) Acarbose 72.1 57.6 Example 1 92.5 88.9 Example 2 92.1 88.5 Example 3 93.2 89.6 Comparative Example 1 90.3 85.1 Comparative Example 2 86.7 80.2 Comparative Example 3 88.9 83.4 Comparative Example 4 86.1 82.2 Comparative Example 5 85.8 83.1 Comparative Example 6 83.2 78.9 Comparative Example 7 80.1 74.8 Comparative Example 8 82.8 76.5 Comparative Example 9 82.0 75.9 Comparative Example 10 86.0 83.6 Comparative Example 11 85.5 83.2 Comparative Example 12 84.4 82.1

[0160] As shown in the table above, the probiotic compositions for regulating blood sugar and blood lipids prepared in Examples 1-3 of this invention have good inhibitory activity against α-glucosidase and α-amylase.

[0161] Test Example 3

[0162] C57BL / 6 mice were randomly divided into a normal group, a model group, Examples 1-3, and Comparative Examples 1-12. During the experiment, the normal group was fed a basal diet, while the other groups were fed a high-fat diet. Examples 1-3 and Comparative Examples 1-12 were administered 1 g / kg of the prepared probiotic composition for regulating blood glucose and lipids via gavage daily, while the other groups were given an equal volume of water. The experiment lasted for 8 weeks. One day after the end of the experiment, mice were fasted for 10 hours, then anesthetized, and blood was collected from the abdominal aorta. Serum was collected by centrifugation.

[0163] Lipid-related markers were measured: Serum levels of TC, TG, LDL-C, and HDL-C were determined according to the instructions of the commercial kits. The results are shown in Table 4.

[0164] Table 4

[0165] Group TC content (mg / L) TG content (mg / L) LDL-C content (mg / L) HDL-C content (mg / L) normal group 128.82±5.82 39.04±2.01 29.14±4.22 95.52±4.97 Model group 160.21±3.91* 56.28±1.02* 50.17±6.28* 68.10±8.25* Example 1 135.56±4.82# 42.15±1.87# 33.28±3.72# 90.01±4.82# Example 2 134.99±4.18# 42.27±1.27# 33.41±2.87# 91.07±4.17# Example 3 132.85±3.72# 41.78±1.07# 31.05±4.58# 89.78±4.37# Comparative Example 1 135.21±2.18 44.17±1.12 36.63±3.82 85.62±3.82 Comparative Example 2 142.10±1.98 48.29±1.09 40.25±4.28 78.29±3.48 Comparative Example 3 140.47±2.25 46.79±1.14 38.57±3.76 80.16±2.75 Comparative Example 4 139.51±1.75 45.09±1.02 39.15±4.21 79.19±2.17 Comparative Example 5 138.57±2.14 45.25±1.24 38.05±3.27 78.54±2.25 Comparative Example 6 143.28±2.25 47.98±2.25 41.12±3.28 75.25±3.28 Comparative Example 7 141.12±2.91 46.32±1.28 40.28±2.84 76.18±3.41 Comparative Example 8 145.21±1.58 49.21±2.04 42.58±3.19 73.09±2.85 Comparative Example 9 144.91±4.29 48.62±3.29 42.08±2.75 74.45±2.14 Comparative Example 10 137.28±3.87 45.78±4.25 37.82±1.82 78.11±2.81 Comparative Example 11 138.04±2.19 46.09±3.28 38.57±2.27 78.01±1.75 Comparative Example 12 140.19±3.85 47.11±3.19 40.11±3.16 76.95±3.27

[0166] Note: * indicates P < 0.05 compared to the normal group; # indicates P < 0.05 compared to the model group.

[0167] A decrease in TC levels indicates a reduction in cholesterol accumulation and a lessening of lipid accumulation in the body. TG participates in lipid synthesis and accumulation, and a decrease in its levels can also inhibit lipid synthesis. HDL-C can promote the breakdown and metabolism of TC and TG. As shown in the table above, the probiotic compositions for regulating blood sugar and lipids prepared in Examples 1-3 of this invention have a good lipid-lowering effect.

[0168] Oral glucose tolerance test: The oral glucose tolerance test was conducted after a 10-hour fast on the day of the experiment. Blood glucose levels were measured by tail vein sampling at 30 and 60 minutes after glucose ingestion (2 g / kg). The results are shown in Table 5.

[0169] Table 5

[0170]

[0171]

[0172] Note: * indicates P < 0.05 compared to the normal group; # indicates P < 0.05 compared to the model group.

[0173] As can be seen from the table above, the probiotic compositions for regulating blood sugar and blood lipids prepared in Examples 1-3 of the present invention have a good blood sugar lowering effect.

[0174] Compared to Example 3, Comparative Example 1 did not involve oxidative degradation in step S1. This resulted in decreased antioxidant, lipid-lowering, and blood glucose-lowering properties. This invention prepares a low molecular weight fucoidan sulfate. While fucoidan sulfate typically possesses various biological activities, these are limited by its structural characteristics and limited functional groups, leading to low water solubility and bioactivity. This invention utilizes vitamin C and hydrogen peroxide for oxidative degradation, reducing the molecular weight of fucoidan sulfate and increasing its water solubility. Simultaneously, it enhances its antioxidant, anti-inflammatory, blood glucose and lipid-regulating, and immunomodulatory effects. It can effectively improve glucose homeostasis by inhibiting α-amylase activity and improving glucose tolerance, regulating liver glucose metabolism, and alleviating hyperglycemia and hyperlipidemia. Furthermore, it is a good prebiotic, promoting the utilization of probiotics and exerting its blood glucose and lipid-regulating effects.

[0175] Compared with Example 3, Comparative Examples 2 and 3 did not include the addition of low molecular weight fucoidan sulfate or traditional Chinese medicine polysaccharides in step S3. The antioxidant, lipid-lowering, and hypoglycemic properties were significantly reduced. The traditional Chinese medicine polysaccharides of this invention lower lipids by inhibiting the biosynthetic pathways of triglycerides and cholesterol in vivo, regulate the NF-κB signaling pathway, inhibit cholesterol synthesis, inhibit the upregulation of HMGCR, sterol regulatory element-binding protein-1c, FAS, and ACC-1 expression in liver tissue, inhibit the downregulation of PPARα and CPT-1 expression, significantly reduce the formation of liver lipid droplets, regulate blood lipid homeostasis, inhibit the activity of α-amylase and α-glucosidase, and have an insulin-sensitizing effect, increasing insulin secretion and promoting pancreatic β-cell proliferation, effectively combating insulin resistance and regulating blood lipid disorders. Low molecular weight fucoidan sulfate effectively improves glucose homeostasis by inhibiting α-amylase activity and improving glucose tolerance, regulating liver glucose metabolism, and improving hyperglycemia and hyperlipidemia.

[0176] Compared with Example 3, Comparative Examples 4 and 5 did not add chromium chloride or zinc nitrate in step S3. Comparative Example 6, compared with Example 3, did not add chromium chloride or zinc nitrate in step S3. The lipid-lowering and blood glucose-lowering effects were decreased. This invention mixes the prepared traditional Chinese medicine polysaccharide with low molecular weight fucoidan sulfate, and through chelation with chromium and zinc ions, it forms chelates with free groups such as -OH and -NH2 on the polysaccharide backbone that can provide lone pair electrons. The trace element chromium participates in the composition of glucose tolerance factor in a trivalent state, assisting insulin in regulating the metabolism of glucose and lipids. Inorganic zinc ions easily precipitate upon encountering substances such as oxalic acid and phytic acid after entering the gastrointestinal tract, making them difficult for the body to absorb and utilize. Choosing a safe and stable carrier to bind with zinc ions helps improve the bioavailability of zinc supplements. Polysaccharide zinc complexes are highly water-soluble and have a high absorption and utilization rate. They possess good biological activity, promote the secretion of insulin by pancreatic cells, reduce blood glucose levels, regulate the sensitivity of hepatocytes to insulin, combine with lipids in the blood, participate in the cholesterol metabolism process, promote lipid transport and excretion, and improve hyperglycemia and hyperlipidemia.

[0177] Compared with Example 3, Comparative Examples 7 and 8 did not include Lactobacillus casei or Lactobacillus acidophilus seed culture in step S4. This resulted in decreased antioxidant, lipid-lowering, and blood glucose-lowering properties. In this invention, traditional Chinese medicine residue and wakame seaweed residue are mixed with whey protein and then inoculated with Lactobacillus casei and Lactobacillus acidophilus for fermentation. On one hand, the probiotics promote the rupture of the cell walls of the residue, releasing more active components into the fermentation product. On the other hand, they also promote the proliferation of probiotics, resulting in a high-concentration bacterial culture. Lactobacillus casei can regulate glucose metabolism and lower blood glucose through mechanisms such as protecting pancreatic islet structure, increasing the sensitivity of target tissues to insulin, improving insulin sensitivity, accelerating glucose transport, reducing endotoxins, and anti-inflammation. Lactobacillus acidophilus can inhibit fat synthesis and enhance fat breakdown, hydrolyzing conjugated bile acids into free bile acids. The reduced solubility of free bile acids makes them less susceptible to reabsorption and reuse by the liver through enterohepatic circulation. This promotes the body's synthesis of new bile acids from cholesterol, reduces cholesterol absorption and clearance, promotes the conversion of cholesterol into bile acids, and facilitates bile acid excretion, thereby lowering blood and cholesterol levels. The addition of both has a synergistic effect.

[0178] Compared to Example 3, Comparative Example 9 did not undergo ionic liquid purification in step S5. This resulted in decreased antioxidant, lipid-lowering, and blood glucose-lowering properties. The aqueous extract and fermentation permeate of the traditional Chinese medicine also contain abundant polyphenols, flavonoids, triterpenes, and other active components, exhibiting excellent activity in regulating blood glucose and lipids. However, some other impurities reduce this regulatory activity. Therefore, this invention uses ionic liquids for separation and purification. After introducing CO2, the polarity of the ionic liquid increases, facilitating the extraction of these active substances from the aqueous solution into the ionic liquid layer. Upon heating, the ionic liquid returns from high polarity to normal polarity, allowing these active components to separate from the ionic liquid. Removing the ionic liquid results in better separation and purification of the active components, significantly enhancing the regulatory activity.

[0179] Compared with Example 3, Comparative Examples 10 and 11 did not add Panax notoginseng total saponins or ginsenoside Rg4 in step S7. Compared with Example 3, Comparative Example 12 did not add ginsenoside Rg4 or Panax notoginseng total saponins in step S7. Antioxidant properties, lipid-lowering properties, and blood sugar-lowering properties decreased. The composition of this invention also contains Panax notoginseng total saponins and ginsenoside Rg4. Panax notoginseng total saponins have various biological activities and may exert anti-inflammatory, antioxidant, blood sugar-lowering, and lipid-lowering effects in vivo. Ginsenoside Rg4 has health-promoting functions such as enhancing immunity, improving physical fitness, promoting normal metabolism, lowering blood sugar (helping to alleviate symptoms in diabetic patients), and lowering blood lipids (helping to improve blood circulation, thereby helping to alleviate cardiovascular diseases). The addition of both has a synergistic effect.

[0180] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 probiotic composition for regulating blood sugar and blood lipids, characterized in that, Low molecular weight fucoidan sulfate was extracted and degraded from wakame seaweed. Traditional Chinese medicine was extracted with water and precipitated with alcohol to obtain medicinal polysaccharides, which were then mixed with low molecular weight fucoidan sulfate to chelate metal ions, thus preparing a polysaccharide-Zn / Cr chelate. The remaining solid residue was fermented, filtered, and washed to obtain a concentrated bacterial solution. The fermentation broth and the medicinal solution were mixed and purified using ionic liquids to obtain the active component. The concentrated bacterial solution was encapsulated in the polysaccharide-Zn / Cr chelate and sodium alginate to prepare probiotic / peptide / polysaccharide nanospheres. These nanospheres were then mixed with the active component, fermented protein peptides, total saponins of Panax notoginseng, and ginsenoside Rg4 to prepare a probiotic composition for regulating blood sugar and blood lipids.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of low molecular weight fucoidan sulfate: Wakame seaweed was washed, dried, and pulverized to obtain wakame powder. The powder was added to water and transferred to a closed hydrothermal reactor. The mixture was heated and pressurized for extraction, filtered, and the residue was retained. Calcium chloride was added to the filtrate, and the mixture was stirred and mixed evenly. The mixture was then filtered again. Vitamin C and hydrogen peroxide were added to the filtrate, and the mixture was heated and stirred for reaction. The mixture was dialyzed, the dialysate was concentrated, and the solution was freeze-dried to obtain low molecular weight fucoidan sulfate. S2. Water extraction of traditional Chinese medicine: Astragalus membranaceus, Gynostemma pentaphyllum, hawthorn and cornus officinalis were washed, dried and pulverized to obtain traditional Chinese medicine powder. The powder was added to water, heated to boiling and extracted. The powder was filtered, and the residue was kept. Ethanol was added to the filtrate to precipitate. The solid was washed and dried to obtain traditional Chinese medicine polysaccharide. The liquid was kept. S3. Preparation of polysaccharide-Zn / Cr chelate: The low molecular weight fucoidan sulfate ester obtained in step S1 and the traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water, chromium salt and zinc salt were added, the pH value of the solution was adjusted, the reaction was stirred, dialyzed, the dialysate was concentrated, and freeze-dried to obtain polysaccharide-Zn / Cr chelate. S4. Fermentation: Add the filter residue from step S1, the filter residue from step S2, and whey protein to water, sterilize, inoculate with Lactobacillus casei and Lactobacillus acidophilus seed culture, ferment, filter, wash with solids, collect the bacterial solution, concentrate, and obtain concentrated bacterial solution; perform liquid dialysis, concentrate the dialysis solution, freeze dry, and obtain fermented protein peptides, retaining the permeate; S5. Extraction of active components: Mix the liquid in step S2 and the permeate in step S4 evenly, add ionic liquid, introduce CO2 gas to increase the polarity of ionic liquid, perform ultrasonic extraction, separate the ionic liquid layer, heat to allow CO2 to escape, separate the ionic liquid to remove the liquid, and collect the active components. S6. Preparation of probiotic / peptide / polysaccharide nanospheres: Add the polysaccharide-Zn / Cr chelate, sodium alginate and lecithin obtained in step S3 to the concentrated bacterial solution obtained in step S4, stir and mix evenly, add to fish oil, emulsify, add calcium chloride solution dropwise, solidify at room temperature, centrifuge, wash, dry, and obtain probiotic / peptide / polysaccharide nanospheres. S7. Preparation of probiotic composition for regulating blood sugar and blood lipids: The probiotic / peptide / polysaccharide nanospheres obtained in step S6, the active components obtained in step S5, the fermented protein peptides obtained in step S4, the total saponins of Panax notoginseng, and the ginsenoside Rg4 are mixed evenly to obtain the probiotic composition for regulating blood sugar and blood lipids.

3. The preparation method according to claim 2, characterized in that, In step S1, the solid-liquid ratio of wakame powder to water is 1:5-7 g / mL. The temperature for the heating and pressurizing extraction is 110-130℃, the pressure is 1.2-1.5 atmospheres, and the extraction time is 2-4 hours. The amount of calcium chloride added is such that the concentration of calcium chloride after addition is 0.1-0.12 mol / L. The amount of vitamin C added is such that the concentration of vitamin C after addition is 25-35 mmol / L. The amount of hydrogen peroxide added is such that the concentration of hydrogen peroxide after addition is 25-35 mmol / L. The temperature for the heating and stirring reaction is 65-75℃, and the time is 1-3 hours. The dialysis bag used for dialysis has a pore size of 2-3 kDa, and the dialysis time is 3-5 hours.

4. The preparation method according to claim 2, characterized in that, In step S2, the mass ratio of Astragalus membranaceus, Gynostemma pentaphyllum, Crataegus pinnatifida, and Cornus officinalis is 3-5:7-10:5-7:1-2, the solid-liquid ratio of the herbal powder to water is 1:5-10 g / mL, the heating and boiling extraction time is 2-4 h, the addition of ethanol until the ethanol content of the system is 80-90 wt%, and the precipitation time is 5-7 h.

5. The preparation method according to claim 2, characterized in that, In step S3, the mass ratio of low molecular weight fucoidan sulfate, traditional Chinese medicine polysaccharide, chromium salt, and zinc salt is 10-12:7-10:0.5-1:1-2. The chromium salt is chromium chloride, and the zinc salt is selected from at least one of zinc chloride, zinc sulfate, zinc nitrate, and zinc acetate. The pH of the solution is adjusted to 7.5-8, the stirring reaction time is 20-40 min, the dialysis bag pore size is 2-3 kDa, and the dialysis time is 2-4 h.

6. The preparation method according to claim 2, characterized in that, In step S4, the mass ratio of the filter residue from step S1, the filter residue from step S2, and the whey protein is 10-20:12-15:3-5, and the bacterial count of the *Lactobacillus casei* and *Lactobacillus acidophilus* seed culture is 10. 8 -10 9 The inoculum concentration of *Lactobacillus casei* and *Lactobacillus acidophilus* was 2-3 v / v% and 1-3 v / v%, respectively. The fermentation conditions were anoxic: 36-38°C, 100-200 rpm, for 48-56 hours. The concentrated bacterial solution contained 10... 10 -10 11 cfu / mL.

7. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of the liquid in step S2, the permeate in step S4, and the deep eutectic solvent is 20-30:15-20:30-50; the CO2 gas flow rate is 3-5 L / min; the ultrasonic power is 1000-1200 W; the extraction time is 1-2 h; the heating temperature is 50-60 °C; and the ionic liquid is selected from at least one of 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide, 1-butyl-3-methylimidazolium hexafluoroantimonate, 1-hexyl-3-methylimidazolium tetrafluoroborate, 1-hexyl-3-methylimidazolium hexafluorophosphate, and 1-hexyl-3-methylimidazolium hexafluoroantimonate.

8. The preparation method according to claim 2, characterized in that, In step S6, the mass ratio of concentrated bacterial solution, polysaccharide-Zn / Cr chelate, sodium alginate, and lecithin is 100:5-7:12-15:0.5-1, and the solidification time at room temperature is 20-30 min; in step S7, the mass ratio of probiotics / peptide / polysaccharide nanospheres, active components, fermented protein peptides, total saponins of Panax notoginseng, and ginsenoside Rg4 is 100:12-15:2-4:1-2:0.5-1.

9. The preparation method according to claim 2, characterized in that, Specifically, the following steps are included: S1. Preparation of low molecular weight fucoidan sulfate: Wakame seaweed was washed, dried, and pulverized to obtain wakame powder. The powder was added to water at a solid-liquid ratio of 1:5-7 g / mL. The mixture was transferred to a closed hydrothermal reactor, heated to 110-130℃, and pressurized to 1.2-1.5 atm for 2-4 hours. After extraction, the mixture was filtered, and the residue was retained. Calcium chloride was added to the filtrate to a final concentration of 0.1-0.12 mol / L, and the mixture was stirred until homogeneous. After filtration, vitamin C was added to the filtrate to a final concentration of 25-35 mmol / L, and hydrogen peroxide was added to a final concentration of 25-35 mmol / L. The mixture was heated to 65-75℃ and stirred for 1-3 hours. Dialysis was performed using a dialysis bag with a pore size of 2-3 kDa for 3-5 hours. The dialysate was concentrated and freeze-dried to obtain low molecular weight fucoidan sulfate. S2. Water extraction of traditional Chinese medicine: Wash 3-5 parts by weight of Astragalus membranaceus, 7-10 parts by weight of Gynostemma pentaphyllum, 5-7 parts by weight of Crataegus pinnatifida, and 1-2 parts by weight of Cornus officinalis, dry them, pulverize them to obtain traditional Chinese medicine powder, add them to water, the solid-liquid ratio of the traditional Chinese medicine powder to water is 1:5-10 g / mL, heat to boiling and extract for 2-4 hours, filter, keep the filter residue, add ethanol to the filtrate until the ethanol content of the system is 80-90 wt%, precipitate for 5-7 hours, wash the solid, dry it to obtain traditional Chinese medicine polysaccharide, and keep the liquid for use; S3. Preparation of polysaccharide-Zn / Cr chelate: 10-12 parts by weight of low molecular weight fucoidan sulfate obtained in step S1 and 7-10 parts by weight of traditional Chinese medicine polysaccharide obtained in step S2 were mixed and dissolved in water. 0.5-1 parts by weight of chromium chloride and 1-2 parts by weight of zinc salt were added. The pH of the solution was adjusted to 7.5-8. The mixture was stirred for 20-40 min and dialyzed for 2-4 h using a dialysis bag with a pore size of 2-3 kDa. The dialysate was concentrated and freeze-dried to obtain polysaccharide-Zn / Cr chelate. S4. Fermentation: Add 10-20 parts by weight of the filter residue from step S1, 12-15 parts by weight of the filter residue from step S2, and 3-5 parts by weight of whey protein to 500 parts by weight of water, sterilize, and inoculate with Lactobacillus casei and Lactobacillus acidophilus seed cultures at inoculation rates of 2-3 v / v% and 1-3 v / v%, respectively. Under anaerobic conditions, ferment at 36-38℃ and 100-200 r / min for 48-56 h. Filter, wash the solids, collect the bacterial culture, concentrate, and obtain a concentrated bacterial culture with a bacterial count of 10. 10 -10 11 cfu / mL; liquid to be retained; The bacterial count of the Lactobacillus casei and Lactobacillus acidophilus seed solution was 10. 8 -10 9 cfu / mL; S5. Extraction of active components: Mix 20-30 parts by weight of the liquid from step S2 and 15-20 parts by weight of the permeate from step S4 evenly, add 30-50 parts by weight of ionic liquid, and introduce CO2 gas at a flow rate of 3-5 L / min to increase the polarity of the ionic liquid. Extract with ultrasonic waves at 1000-1200W for 1-2 hours, separate the ionic liquid layer, heat to 50-60℃ to allow CO2 to escape, separate the liquid from the ionic liquid, and collect the active components. S6. Preparation of probiotic / peptide / polysaccharide nanospheres: Add 5-7 parts by weight of the polysaccharide-Zn / Cr chelate obtained in step S3, 12-15 parts by weight of sodium alginate and 0.5-1 parts by weight of lecithin to 100 parts by weight of the concentrated bacterial solution obtained in step S4, stir and mix evenly, add to 200 parts by weight of fish oil, emulsify, add 10-15 parts by weight of 3-5 wt% calcium chloride solution dropwise, solidify at room temperature for 20-30 min, centrifuge, wash, dry, and obtain probiotic / peptide / polysaccharide nanospheres; S7. Preparation of a probiotic composition for regulating blood sugar and blood lipids: 100 parts by weight of the probiotic / peptide / polysaccharide nanospheres obtained in step S6, 12-15 parts by weight of the active component obtained in step S5, 2-4 parts by weight of the fermented protein peptide obtained in step S4, 1-2 parts by weight of Panax notoginseng total saponins, and 0.5-1 parts by weight of ginsenoside Rg4 are mixed evenly to obtain a probiotic composition for regulating blood sugar and blood lipids.

10. A probiotic composition for regulating blood sugar and blood lipids prepared by the preparation method according to any one of claims 1-9.

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