阅读说明：本技术 降血糖、降血脂组合物、制备方法及其应用 (Composition for reducing blood sugar and blood fat, preparation method and application thereof ) 是由 范代娣 宋养斌 段志广 马晓轩 于 2020-07-01 设计创作，主要内容包括：本发明公开了一种降血糖、降血脂组合物、制备方法及其应用。所述降血糖、降血脂组合物包含稀有人参皂苷和益生菌粉,所述益生菌粉含有冠突散囊菌、枯草芽孢杆菌和保加利亚乳杆菌。本发明所述的组合物能够充分发挥稀有人参皂苷及冠突散囊菌的降血糖、降血脂功效,与单纯冠突散囊菌或经转化后的稀有人参皂苷作用相比,组合物具有相互促进、协同增效的效果。并且所述的组合物能够实现一药多治,能够减少同时患有糖尿病、高血脂症患者的服药种类,增强患者的医从性,方便患者服药；同时可以兼顾调节肠道菌群中益生菌比例,补充益生菌,促进吸收等多重功效。(The invention discloses a composition for reducing blood sugar and blood fat, a preparation method and application thereof. The blood sugar and blood fat reducing composition comprises rare ginsenoside and probiotic powder, wherein the probiotic powder contains eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus. The composition can fully exert the blood sugar and blood fat reducing effects of the rare ginsenoside and eurotium cristatum, and has the effects of mutual promotion and synergistic effect compared with the effect of the pure eurotium cristatum or the transformed rare ginsenoside. The composition can realize multi-treatment by one medicine, can reduce the medicine taking types of patients suffering from diabetes and hyperlipidemia at the same time, enhances the medical compliance of the patients and facilitates the patients to take medicines; meanwhile, the probiotic proportion in intestinal flora can be adjusted, the probiotics can be supplemented, and the absorption can be promoted.)

1. A method for preparing rare ginsenoside, characterized in that the method comprises fermenting active raw materials by probiotics.

2. The method of claim 1, wherein the probiotic bacteria comprise Eurotium cristatum, Bacillus subtilis, and Lactobacillus bulgaricus.

3. The method according to any one of claims 1 to 2, wherein the active ingredient is selected from one or more of ginseng, notoginseng flower, american ginseng, gynostemma pentaphylla or an extract thereof, preferably ginseng or an extract thereof.

4. The production method according to any one of claims 1 to 3, characterized by comprising:

respectively carrying out activation and amplification culture on eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus to obtain seed solutions;

fermentation: respectively inoculating eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus seed liquid into a fermentation tank, adding active raw materials into the fermentation tank, and fermenting to obtain fermentation liquid;

and carrying out solid-liquid separation on the fermentation liquor, collecting supernatant, and concentrating to obtain rare ginsenoside.

5. The method according to claim 4, wherein the fermentation step comprises inoculating the seed solution of Eurotium cristatum into the culture medium in a fermenter; then adding active raw materials for fermentation; respectively inoculating the seed liquid of the lactobacillus bulgaricus and the bacillus subtilis to a culture medium in a fermentation tank for further fermentation to obtain fermentation liquid.

6. The process according to claim 4 or 5, wherein the fermentation temperature is 28 to 30 ℃ and the fermentation pH is 4 to 8.

7. The method according to claim 4 or 5, wherein the fermentation pH is 5 to 7.

8. The method according to claim 4 or 5, wherein the dissolved oxygen amount is 20% or more, preferably 50 to 70% or less, in the fermentation step.

9. A method for preparing probiotic powder, characterized in that the method comprises fermenting active raw materials by probiotics.

10. The method of claim 9, wherein the probiotic bacteria comprise eurotium cristatum, bacillus subtilis, and lactobacillus bulgaricus.

Technical Field

The invention relates to the technical field of bioengineering, in particular to a composition for reducing blood sugar and blood fat, a preparation method and application thereof.

Background

Diabetes is a metabolic disorder syndrome which is related to abnormal production and action of insulin and is mainly characterized by hyperglycemia, is a chronic disease seriously harming health, and is one of the main health problems faced by human beings at present; hyperlipidemia is a clinical symptom in which lipids in blood are higher than normal levels due to abnormal fat metabolism or abnormal operation, and is usually manifested as hyperlipoproteinemia, i.e., hypercholesterolemia, hypertriglyceridemia or both, and in addition, hyperlipidemia can also induce the onset of diabetes and impaired glucose tolerance, and is also an important inducing factor for fatty liver, liver cirrhosis, cholelithiasis and pancreatitis, so that prevention, alleviation and treatment of diabetes and hyperlipidemia are inevitable.

The Fuzhuan tea is a traditional tea drink in northwest regions of China, is a special product in dark tea in six major tea, and researches show that the Fuzhuan tea has good effects on reducing blood fat and blood sugar. Eurotium cristatum belongs to probiotics, is natural probiotic dominant thallus grown by a 'flower growing' process of Fuzhuan tea under specific temperature and humidity conditions, is commonly called 'golden flower', is an unusual place of Fuzhuan tea, and various effects of the Fuzhuan tea need to be realized by depending on the Eurotium cristatum. The blood fat and blood sugar reducing activity of the golden flower is also researched and confirmed.

Ginseng is a traditional rare medicinal material in China, and has the effects of greatly invigorating primordial qi, tonifying spleen and lung, promoting the production of body fluid, soothing nerves, and improving intelligence. Ginsenoside is the main active ingredient of ginseng, and scientists all over the world find that ginseng contains 128 monomeric saponins, and can be extracted by modern high-tech technology and has forty clinical efficacies. The rare ginsenoside only has the difference of glycosidic bond at the end of sugar chain, and high-activity ginsenoside can be carried out by hydrolyzing the glycosidic side group, and a large amount of pharmacological studies show that the rare ginsenoside can show unique curative effect in the aspects of certain intractable diseases such as tumor treatment, diabetes and hyperlipidemia, so the rare ginsenoside has more excellent clinical application value as a candidate drug.

At present, the preparation method for obtaining rare ginsenoside mainly comprises a heating method, an acidolysis method, an alkaline hydrolysis method, a Smith degradation method, a microbial transformation method and an enzyme method. The saponin can be hydrolyzed by chemical method. But the hydrolysis conditions of the raw materials are severe, and aglycone is easy to dehydrate or cyclize, so that the structure of the aglycone is changed, byproducts are increased, the product is difficult to purify, and the environment is polluted. The method for preparing rare ginsenoside by a microbial transformation method has the problems that the safety of microorganisms is difficult to ensure, the process is complicated, the obtained product needs to be separated and purified and cannot be directly eaten, the production period is long, the transformation rate needs to be improved and the like, and the requirement of developing novel medicines cannot be met by taking the traditional pure culture microorganisms as the sources of the obtained compounds.

Disclosure of Invention

In order to solve the problems, the inventor provides a method for transforming rare ginsenoside by using food-safe probiotics composite fermentation, specifically adopting the coencystic coronary disease, bacillus subtilis and lactobacillus bulgaricus for co-culture, so that three strains form competitive ecological pressure and activate silent gene clusters and other non-traditional methods, thereby forming pressure induction by co-culture, activating a complicated regulation mechanism of fungi and silent gene clusters, and synthesizing secondary metabolites with rich structural types and diverse activities, the process is simple, the period is shorter, the environmental pollution is less, the production cost is low, and the obtained fermentation composition has excellent effects of reducing blood fat and reducing blood sugar, can reduce blood sugar and control blood fat while regulating the proportion of probiotics in intestinal flora, the probiotics and the traditional Chinese medicine active substance are used as main components, so that the health-care food has high safety and good application prospect.

The composition provided by the invention can fully exert the blood sugar and blood fat reducing effects of the rare ginsenoside RH4 and the eurotium cristatum, mutually promote and mutually enhance, realize the treatment effect superior to the single action, simultaneously realize multiple effects of regulating the probiotic proportion in intestinal flora, supplementing probiotics, promoting absorption and the like, and provide the proportion of two substances in the composition and the application of a product containing the composition.

The specific technical scheme of the invention is as follows:

1. a method for preparing rare ginsenoside, characterized in that the method comprises fermenting active raw materials by probiotics.

2. The method according to item 1, wherein the probiotic bacteria include Eurotium cristatum, Bacillus subtilis, and Lactobacillus bulgaricus.

3. The method according to any one of claims 1 to 2, wherein the active ingredient is selected from one or more of ginseng, notoginseng flower, american ginseng, gynostemma pentaphylla or an extract thereof, preferably ginseng or an extract thereof.

4. The production method according to any one of claims 1 to 3, characterized by comprising:

respectively carrying out activation and amplification culture on eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus to obtain seed solutions;

fermentation: respectively inoculating eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus seed liquid into a fermentation tank, adding active raw materials into the fermentation tank, and fermenting to obtain fermentation liquid;

and carrying out solid-liquid separation on the fermentation liquor, collecting supernatant, and concentrating to obtain rare ginsenoside.

5. The process according to item 4, wherein in the fermentation step, the Eurotium cristatum seed solution is inoculated into the medium in the fermenter; then adding active raw materials for fermentation; respectively inoculating the seed liquid of the lactobacillus bulgaricus and the bacillus subtilis to a culture medium in a fermentation tank for further fermentation to obtain fermentation liquid.

6. The production method according to item 4 or 5, wherein the fermentation temperature is 28 to 30 ℃ and the fermentation pH is 4 to 8.

7. The production method according to item 4 or 5, wherein the fermentation pH is 5 to 7.

8. The production method according to item 4 or 5, wherein the dissolved oxygen amount is 20% or more, preferably 50 to 70% or less in the fermentation step.

9. A method for preparing probiotic powder, characterized in that the method comprises fermenting active raw materials by probiotics.

10. The method according to item 9, wherein the probiotic bacteria include eurotium cristatum, bacillus subtilis, and lactobacillus bulgaricus.

11. The method according to any one of claims 9 to 10, wherein the active ingredient is selected from one or more of ginseng, notoginseng flower, american ginseng, gynostemma pentaphylla or an extract thereof, and preferably ginseng or an extract thereof.

12. The production method according to any one of claims 9 to 11, characterized by comprising:

respectively carrying out activation and amplification culture on eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus to obtain seed solutions;

fermentation: respectively inoculating eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus seed liquid into a fermentation tank, adding active raw materials into the fermentation tank, and fermenting to obtain fermentation liquid;

and (4) carrying out solid-liquid separation on the fermentation liquor, collecting the solid matter and freeze-drying to obtain probiotic powder.

13. The process according to item 12, wherein in the fermentation step, the Eurotium cristatum seed solution is inoculated into the medium in the fermenter; then adding active raw materials for fermentation; respectively inoculating the seed liquid of the lactobacillus bulgaricus and the bacillus subtilis to a culture medium in a fermentation tank for further fermentation to obtain fermentation liquid.

14. The production method according to item 12 or 13, wherein the fermentation temperature is 28 to 30 ℃ and the fermentation pH is 4 to 8.

15. The production method according to item 12 or 13, wherein the fermentation pH is 5 to 7.

16. The production method according to item 12 or 13, wherein the dissolved oxygen amount is 20% or more, preferably 50 to 70% or less in the fermentation step.

17. The rare ginsenoside prepared by the preparation method according to any one of items 1 to 8.

18. The probiotic powder prepared by the preparation method according to any one of the items 9 to 16.

19. A probiotic powder, characterized in that it comprises Eurotium cristatum, Bacillus subtilis and Lactobacillus bulgaricus; preferably, the ratio of the total number of viable bacteria of the eurotium cristatum to the total number of viable bacteria of the bacillus subtilis and the lactobacillus bulgaricus in unit volume is 1:1-10, preferably 1:1-5, and more preferably 1: 1-2.

20. The probiotic powder according to item 19, characterized in that the ratio of the total number of viable bacteria of the bacillus subtilis to the total number of viable bacteria of the lactobacillus bulgaricus is 1-5:1, preferably 1-2: 1.

21. A composition comprising rare ginsenosides and the probiotic powder according to item 19 or 20.

22. The composition of item 21, wherein the rare ginsenoside is Rh 4.

23 the composition of item 21 or 22, wherein the mass ratio of the rare ginsenoside to the probiotic powder is 1:1-10, preferably 1:1-5, more preferably 1: 2-3.

24. Use of the composition according to any one of claims 21 to 23 in food, health products or pharmaceuticals, preferably in pharmaceuticals, further preferably in the manufacture of a medicament for the prevention or treatment of diabetes, hyperlipidemia or complications caused by diabetes and hyperlipidemia.

25. A nutraceutical composition comprising an effective amount of the composition of any one of items 21 to 24 and an additive.

26. A food composition comprising an effective amount of the composition according to any one of items 21 to 24 and an additive.

27. A pharmaceutical composition comprising an effective amount of the composition according to any one of items 21 to 24 and a pharmaceutically acceptable excipient.

28. The pharmaceutical composition of claim 27, wherein the pharmaceutically acceptable excipients comprise pharmaceutically acceptable carriers, excipients and diluents.

29. A preparation for preventing or treating diabetes, hyperlipidemia, or complications caused by diabetes and hyperlipidemia, which is prepared from the raw materials comprising the pharmaceutical composition according to claim 27 or 28.

30. The formulation of claim 29, wherein the formulation is in the form of a solid formulation or a liquid formulation.

31. A formulation according to claim 30, wherein the solid formulation is a tablet, pill, capsule, powder or granule.

32. The formulation of claim 30, wherein the liquid formulation is a suspension, tincture, syrup, emulsion, or suspension.

33. Use of the composition according to any one of claims 25 to 28 or the formulation according to any one of claims 29 to 32 for reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load in the blood.

ADVANTAGEOUS EFFECTS OF INVENTION

(1) The rare ginsenoside is converted by the action of exogenous enzymes generated by the composite fermentation of three bacteria, namely probiotics bacillus subtilis, lactobacillus bulgaricus and eurotium cristatum, and the safety is high.

(2) The blood sugar and blood fat reducing composition prepared by the invention can fully exert the blood sugar and blood fat reducing effects of rare ginsenoside and eurotium cristatum, and has the effects of mutual promotion and synergistic effect compared with the effect of pure eurotium cristatum or the transformed rare ginsenoside.

(3) The composition provided by the invention can realize multi-treatment by one medicine, can reduce the medicine taking types of patients suffering from diabetes and hyperlipidemia at the same time, enhances the medical compliance of the patients and facilitates the patients to take medicines; meanwhile, the probiotic proportion in intestinal flora can be adjusted, the probiotics can be supplemented, and the absorption can be promoted.

Detailed Description

The present invention will be described in detail below. While specific embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It should be noted that certain terms are used throughout the description and claims to refer to particular components. As one skilled in the art will appreciate, various names may be used to refer to a component. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, however, the description is given for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.

The invention provides a preparation method of rare ginsenoside, wherein the preparation method comprises fermenting active raw materials by probiotics.

Wherein, the probiotics are active microorganisms which are beneficial to a host and change the composition of flora at a certain part of the host by colonizing in a human body. The health of the intestinal tract is kept by promoting the absorption of nutrients by regulating the immune function of the host mucous membrane and the system or by regulating the balance of flora in the intestinal tract, so that single microorganisms or mixed microorganisms with definite compositions which are beneficial to the health are generated.

In a preferred embodiment of the invention, the probiotic bacteria comprise eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus.

The eurotium cristatum is commonly called as 'golden flower', belongs to a fungus of eurotium of trichothecaceae of eurotiales, consists of ascocarp and hypha, has low nutritional requirement, can grow on a potato glucose agar culture medium and a Bengal culture medium, has strong adaptability, can utilize various nitrogen sources and carbon sources, is a dominant strain in the fermentation process of the Fuzhuan tea, obtains nutrient substances from the tea, generates various enzymes through self metabolism to catalyze various substances in the tea to carry out conversion, forms the unique color, fragrance and taste quality of the Fuzhuan tea, and is an important index for evaluating the quality of the Fuzhuan tea.

The Eurotium cristatum is selected from mycelium, spore or combination of the mycelium and the spore which are prepared by large-scale fermentation and culture of Eurotium cristatum, and the mycelium and the spore can be in a wall-broken state of breaking cell wall forms and releasing intracellular contents through physical, biological or chemical methods, or in an unprocessed non-wall-broken state, preferably in a wall-broken state. The physical, biological and chemical treatment for breaking cell walls refers to the wall breaking method commonly adopted by the technical personnel in the field, such as: high-pressure homogenizing, ultrasonic wall breaking, biological enzyme wall breaking, acid-base wall breaking, etc.;

the bacillus subtilis powder and the lactobacillus bulgaricus powder are obtained by fermenting and culturing the bacillus subtilis powder and the lactobacillus bulgaricus powder respectively.

In a preferred embodiment of the present invention, the active ingredient is selected from one or more of ginseng, notoginseng flower, american ginseng, gynostemma pentaphylla or an extract thereof, preferably ginseng or an extract thereof.

In a more preferred embodiment of the present invention, the preparation method comprises:

respectively carrying out activation and amplification culture on eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus to obtain seed solutions;

fermentation: respectively inoculating eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus seed liquid into a fermentation tank, adding active raw materials into the fermentation tank, and fermenting to obtain fermentation liquid;

and carrying out solid-liquid separation on the fermentation liquor, collecting supernatant, and concentrating to obtain rare ginsenoside.

In a more preferred embodiment of the present invention, in the fermentation step, the eurotium cristatum seed solution is inoculated into the culture medium in the fermentation tank; then adding active raw materials for fermentation; respectively inoculating the seed liquid of the lactobacillus bulgaricus and the bacillus subtilis into a culture medium in a fermentation tank for further fermentation to obtain a fermentation liquid, wherein the fermentation temperature is 28-30 ℃, the fermentation pH is 4-8, preferably 5-7, and the dissolved oxygen is more than or equal to 20%, preferably 50-70% in the fermentation step.

In a more preferred embodiment of the present invention, the step of strain activation and expanded culture of Eurotium cristatum comprises: inoculating eurotium cristatum strains to a slant of a PDA culture medium, culturing at 28 ℃ for activation, washing the hyphae with 5mL of sterile normal saline after the hyphae grow over the slant, inoculating 0.2mL of the hyphae into a 500-mL triangular flask containing 200mL of PDA liquid culture medium, and culturing on a shaking table at 28 ℃ and 160rpm for 120 hours; then inoculating the second-level shake flask seeds according to the inoculation amount of 10 percent, and culturing the second-level shake flask seeds on a shaking table at the temperature of 28 ℃ and the rpm of 160 for 72 hours for later use;

the strain activation and amplification culture steps of the lactobacillus bulgaricus comprise: inoculating the strain of lactobacillus bulgaricus to a modified MRS agar culture medium plate, placing the plate on an activation culture for 48h at 37 ℃, scraping 1 ring by using an inoculating ring, inoculating the ring into a 250mL triangular flask filled with 80mL of MRS liquid culture medium, and placing the bottle on a shaking table at 30 ℃ and 160r/min for culture for 48h for later use;

the activation and amplification culture steps of the bacillus subtilis strain are as follows: the Bacillus subtilis strain is inoculated on an agar culture medium (the components of the culture medium are 1L of distilled water, 20g of glucose, 15g of peptone, 0.5g of beef extract and 20g of agar) inclined plane, the inclined plane is placed at 30 ℃ for activation culture for 48h, 1 ring is scraped by an inoculating ring and inoculated into a 250mL triangular flask filled with 80mL of liquid culture medium (the components of the culture medium are 1L of distilled water, 20g of glucose, 15g of peptone and 0.5g of beef extract), and the triangular flask is placed on a shaking table at 30 ℃ and 160r/min for culture for 36h for later use.

In a preferred embodiment of the present invention, wherein, in the fermentation step, the inoculum size of the eurotium cristatum is 5-15%, preferably, the fermentation temperature is 28-30 ℃, further preferably, the fermentation pH is 4-7, further preferably, the fermentation time is 66-78 h.

In a preferred embodiment of the present invention, wherein, in the fermentation step, the lactobacillus bulgaricus is inoculated in an amount of 2-5%, preferably, the fermentation temperature is 28-30 ℃, and more preferably, the fermentation pH is 5-8.

In a preferred embodiment of the present invention, wherein, in the fermentation step, the amount of the bacillus subtilis is 2 to 5%, preferably, the fermentation temperature is 28 to 30 ℃, and more preferably, the fermentation pH is 5 to 8.

In a more preferable embodiment of the invention, in the fermentation step, a fermentation medium (1% of sugar and milk powder) is filled into a fermentation tank according to about 60% of liquid filling amount, the fermentation tank is subjected to high-pressure steam sterilization, 10% of expanded eurotium cristatum strains are inoculated into the cooled fermentation medium, active raw materials are fed in the whole fermentation process, the DO is controlled to be not less than 20%, the temperature is 28-30 ℃, the pH is 5-6, the activated raw materials are cultured for 66-78h, the expanded lactobacillus bulgaricus and bacillus subtilis strains are inoculated according to 3% of inoculum sizes respectively, the temperature of the fermentation culture is controlled to be 28-30 ℃, the pH is 6-7, the rotating speed is controlled to be 160r/min, the dissolved oxygen is controlled to be not less than 20%, and the composite fermentation liquid is obtained after continuous culture for 60-80 h.

Wherein, the DO is the dissolved amount (unit: mg/L) of oxygen in water. The dissolved oxygen concentration is usually expressed in terms of percent air saturation in the fermentation (100% air saturation of oxygen in the fermentation broth at 28 ℃ is only about 0.25 mmol/L).

In a preferred embodiment of the present invention, in the solid-liquid separation step, the supernatant contains rare ginsenosides. In the solid-liquid separation step, solid-liquid separation can be realized through equipment such as a plate frame, ultrafiltration and ceramic membrane, solid matters are collected to obtain probiotics, and the supernatant is concentrated, separated and purified to obtain rare saponin.

In a preferred embodiment of the present invention, the active ingredient is fed in a feeding manner, and the active ingredient and ultrapure water are prepared into a suitable mother liquor, wherein the feeding amount of the active ingredient is determined by the mass of the solid active substance and the volume after feeding, and the proportion of the final active ingredient is 5-50% (w/v), preferably 10-30%, more preferably 25%, and the final active ingredient is fed into the mixed culture solution in a feeding manner in the whole course of fermentation.

The invention provides a preparation method of probiotic powder, wherein the preparation method comprises the step of fermenting active raw materials by probiotics.

In a preferred embodiment of the invention, the probiotic bacteria comprise eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus.

The eurotium cristatum is commonly called as 'golden flower', belongs to a fungus of eurotium of trichothecaceae of eurotiales, consists of ascocarp and hypha, has low nutritional requirement, can grow on a potato glucose agar culture medium and a Bengal culture medium, has strong adaptability, can utilize various nitrogen sources and carbon sources, is a dominant strain in the fermentation process of the Fuzhuan tea, obtains nutrient substances from the tea, generates various enzymes through self metabolism to catalyze various substances in the tea to carry out conversion, forms the unique color, fragrance and taste quality of the Fuzhuan tea, and is an important index for evaluating the quality of the Fuzhuan tea.

The Eurotium cristatum is selected from mycelium, spore or combination of the mycelium and the spore which are prepared by large-scale fermentation and culture of Eurotium cristatum, and the mycelium and the spore can be in a wall-broken state of breaking cell wall forms and releasing intracellular contents through physical, biological or chemical methods, or in an unprocessed non-wall-broken state, preferably in a wall-broken state. The physical, biological and chemical treatment for breaking cell walls refers to the wall breaking method commonly adopted by the technical personnel in the field, such as: high-pressure homogenizing, ultrasonic wall breaking, biological enzyme wall breaking, acid-base wall breaking, etc.;

the bacillus subtilis powder and the lactobacillus bulgaricus powder are obtained by fermenting and culturing the bacillus subtilis powder and the lactobacillus bulgaricus powder respectively.

In a preferred embodiment of the present invention, the active ingredient is selected from one or more of ginseng, notoginseng flower, american ginseng, gynostemma pentaphylla or an extract thereof, preferably ginseng or an extract thereof.

In a preferred embodiment of the present invention, the preparation method comprises:

respectively carrying out activation and amplification culture on eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus to obtain seed solutions;

fermentation: respectively inoculating eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus seed liquid into a fermentation tank, adding active raw materials into the fermentation tank, and fermenting to obtain fermentation liquid;

and (4) carrying out solid-liquid separation on the fermentation liquor, collecting the solid matter and freeze-drying to obtain probiotic powder.

In a more preferred embodiment of the present invention, in the fermentation step, the eurotium cristatum seed solution is inoculated into the culture medium in the fermentation tank; then adding active raw materials for fermentation; respectively inoculating the seed liquid of the lactobacillus bulgaricus and the bacillus subtilis to a culture medium in a fermentation tank for further fermentation to obtain fermentation liquid. The fermentation temperature is, for example, 28 to 30 ℃, the fermentation pH is, for example, 4 to 8, preferably 5 to 7, and the dissolved oxygen amount is 20% or more, for example, 50 to 70%.

In a more preferred embodiment of the present invention, the step of strain activation and expanded culture of Eurotium cristatum comprises: inoculating eurotium cristatum strains to a slant of a PDA culture medium, culturing at 28 ℃ for activation, washing the hyphae with 5mL of sterile normal saline after the hyphae grow over the slant, inoculating 0.2mL of the hyphae into a 500-mL triangular flask containing 200mL of PDA liquid culture medium, and culturing on a shaking table at 28 ℃ and 160rpm for 120 hours; then inoculating the second-level shake flask seeds according to the inoculation amount of 10 percent, and culturing the second-level shake flask seeds on a shaking table at the temperature of 28 ℃ and the rpm of 160 for 72 hours for later use;

the strain activation and amplification culture steps of the lactobacillus bulgaricus comprise: inoculating the strain of lactobacillus bulgaricus to a modified MRS agar culture medium plate, placing the plate on an activation culture for 48h at 37 ℃, scraping 1 ring by using an inoculating ring, inoculating the ring into a 250mL triangular flask filled with 80mL of MRS liquid culture medium, and placing the bottle on a shaking table at 30 ℃ and 160r/min for culture for 48h for later use;

the activation and amplification culture steps of the bacillus subtilis strain are as follows: the Bacillus subtilis strain is inoculated on an agar culture medium (the components of the culture medium are 1L of distilled water, 20g of glucose, 15g of peptone, 0.5g of beef extract and 20g of agar) inclined plane, the inclined plane is placed at 30 ℃ for activation culture for 48h, 1 ring is scraped by an inoculating ring and inoculated into a 250mL triangular flask filled with 80mL of liquid culture medium (the components of the culture medium are 1L of distilled water, 20g of glucose, 15g of peptone and 0.5g of beef extract), and the triangular flask is placed on a shaking table at 30 ℃ and 160r/min for culture for 36h for later use.

In a preferred embodiment of the present invention, wherein, in the fermentation step, the inoculum size of the eurotium cristatum is 5-15%, preferably, the fermentation temperature is 28-30 ℃, further preferably, the fermentation pH is 4-7, further preferably, the fermentation time is 66-78 h.

In a preferred embodiment of the present invention, wherein, in the fermentation step, the lactobacillus bulgaricus is inoculated in an amount of 2-5%, preferably, the fermentation temperature is 28-30 ℃, and more preferably, the fermentation pH is 5-8.

In a preferred embodiment of the present invention, wherein, in the fermentation step, the amount of the bacillus subtilis is 2 to 5%, preferably, the fermentation temperature is 28 to 30 ℃, and more preferably, the fermentation pH is 5 to 8.

In a more preferable embodiment of the invention, in the fermentation step, a fermentation medium (1% of sugar and milk powder) is filled into a fermentation tank according to about 60% of liquid filling amount, the fermentation tank is subjected to high-pressure steam sterilization, 10% of expanded eurotium cristatum strains are inoculated into the cooled fermentation medium, active raw materials are fed in the whole fermentation process, the DO is controlled to be not less than 20%, the temperature is 28-30 ℃, the pH is 5-6, the activated raw materials are cultured for 66-78h, the expanded lactobacillus bulgaricus and bacillus subtilis strains are inoculated according to 3% of inoculum sizes respectively, the temperature of the fermentation culture is controlled to be 28-30 ℃, the pH is 6-7, the rotating speed is controlled to be 160r/min, the dissolved oxygen is controlled to be not less than 20%, and the composite fermentation liquid is obtained after continuous culture for 60-80 h.

Wherein, the DO is the dissolved amount (unit: mg/L) of oxygen in water. The dissolved oxygen concentration is usually expressed in terms of percent air saturation in the fermentation (100% air saturation of oxygen in the fermentation broth at 28 ℃ is only about 0.25 mmol/L).

In a preferred embodiment of the present invention, in the solid-liquid separation step, the supernatant contains rare ginsenosides. In the solid-liquid separation step, solid-liquid separation can be realized through equipment such as a plate frame, ultrafiltration and ceramic membrane, solid matters are collected to obtain probiotics, and the supernatant is concentrated, separated and purified to obtain rare saponin.

In a preferred embodiment of the present invention, the active ingredient is fed in a feeding manner, and the active ingredient and ultrapure water are prepared into a suitable mother liquor, wherein the feeding amount of the active ingredient is determined by the mass of the solid active substance and the volume after feeding, and the proportion of the final active ingredient is 5-50% (w/v), preferably 10-30%, more preferably 25%, and the final active ingredient is fed into the mixed culture solution in a feeding manner in the whole course of fermentation.

The invention provides rare ginsenoside prepared by the method.

The invention provides the probiotic powder prepared by the method.

The invention provides probiotic powder, wherein the probiotic powder contains eurotium cristatum, bacillus subtilis and lactobacillus bulgaricus; preferably, the ratio of the total number of viable bacteria of the eurotium cristatum to the total number of viable bacteria of the bacillus subtilis and the lactobacillus bulgaricus in unit volume is 1:1-10, preferably 1:1-5, and more preferably 1: 1-2.

The ratio of the total viable count of eurotium cristatum to the total viable count of the sum of bacillus subtilis and lactobacillus bulgaricus in a unit volume may be, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any range therebetween.

The Eurotium Cristatum is a selenium-rich strain (Eurotium Cristatum XD-01) of Eurotium Cristatum, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 4 and 2 days in 2018, the preservation number is CGMCC No.15395, the preservation address is No. 3 of Xilu No.1 of Beijing area on the sunny region, and the strain appears in a publication document with the Chinese patent application number of CN201911381242.9 for the first time.

The bacillus subtilis is commercially purchased from China Industrial microbial culture Collection center (CICC), and the strain with the preservation number of CICC10071 is deposited at No. 6 Hospital No. 24 of the Zhonghao Luxiaoqiao of the Korean-Yang district, Beijing.

The lactobacillus bulgaricus is commercially available from the China agricultural microbial culture Collection center (CICC) with the preservation number of ACCC03598, and the preservation address is resource building 206 of the institute of agricultural and agricultural regionalism, No. 12 China agricultural academy of sciences, south China southern China, in Haidian, Beijing.

In a preferred embodiment of the present invention, the ratio of the total viable count of bacillus subtilis to the total viable count of lactobacillus bulgaricus is 1-5:1, preferably 1-2: 1.

For example, the ratio of the total viable count of bacillus subtilis to the total viable count of lactobacillus bulgaricus may be, for example, 1:1, 2:1, 3:1, 4:1, 5:1, or any range therebetween.

The invention provides a composition, which comprises rare ginsenoside and the probiotics, wherein the mass ratio of the rare ginsenoside to the probiotics is 1:1-10, preferably 1:1-5, and more preferably 1: 2-3.

The mass ratio of the rare ginsenoside to the probiotic bacteria may be, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, or any range therebetween.

In a preferred embodiment of the present invention, the rare ginsenoside is Rh 4.

The invention provides application of the composition in food, health products or medicines, preferably application in medicines, and further preferably application in preparing medicines for preventing or treating diabetes, hyperlipidemia or complications caused by diabetes and hyperlipidemia.

The invention provides the use of the composition described above for reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load in the blood.

The invention provides a health-care product composition, which comprises an effective dose of the composition and an additive.

The term "effective dose" refers to a dose corresponding to a desired function, and the effective dose can be obtained by a person skilled in the art in limited experiments according to actual needs, and the additive is well known to the person skilled in the art, and can be, for example, magnesium stearate, sodium carboxymethyl cellulose, microcrystalline cellulose or sodium carboxymethyl starch.

The invention provides a food composition, which comprises an effective dose of the composition and additives.

The term "effective dose" refers to a dose corresponding to a desired function, and the effective dose can be obtained by a person skilled in the art in limited experiments according to actual needs, and the additive is well known to the person skilled in the art, and can be, for example, magnesium stearate, sodium carboxymethyl cellulose, microcrystalline cellulose or sodium carboxymethyl starch.

The invention provides a pharmaceutical composition, which comprises an effective dose of the composition and pharmaceutically acceptable auxiliary materials.

The "effective dose" refers to a dose that exerts a pharmaceutical function when used as a drug.

The pharmaceutically acceptable excipients include pharmaceutically acceptable carriers, excipients, diluents and the like, which are compatible with the active ingredient. The pharmaceutically acceptable excipients are well known to those skilled in the art and may be, for example, magnesium stearate, sodium carboxymethylcellulose, microcrystalline cellulose or sodium starch glycolate.

The invention provides a preparation for preventing or treating diabetes, hyperlipidemia or complications caused by diabetes and hyperlipidemia, which is prepared from the raw materials comprising the pharmaceutical composition.

In a preferred embodiment of the invention, the dosage form is a solid preparation, such as a tablet, a pill, a capsule (including sustained release or delayed release forms), a powder or a granule, or a liquid preparation, such as a suspension, a tincture, a syrup, an emulsion or a suspension.

In a more preferred embodiment of the present invention, the dosage form is a solid preparation.

The invention provides application of the health care product composition in reducing blood sugar concentration, total cholesterol, triglyceride or lipoprotein loading in blood.

The invention provides the use of the food composition as described above for reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load in the blood.

The invention provides the application of the pharmaceutical composition in reducing blood sugar concentration, total cholesterol, triglyceride or lipoprotein load in blood.

The invention provides the use of the above-described formulation for reducing blood glucose concentration, total cholesterol, triglyceride or lipoprotein load in the blood.

The invention is described generally and/or specifically for the materials used in the tests and the test methods, in the following examples,% means wt%, i.e. percent by weight, unless otherwise specified. The reagents or instruments used are not indicated by the manufacturer, and are all conventional reagent products or conventional laboratory instruments which are commercially available.