Codonopsis pilosula and red yeast rice probiotic fermented product as well as preparation method and application of codonopsis pilosula and red yeast rice probiotic fermented product

Abstract

The invention discloses a radix codonopsis and red yeast probiotic fermented product as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The radix codonopsis and red yeast probiotic fermented product is obtained by sequentially inoculating radix codonopsis with monascus purpureus and lactobacillus plantarum, fermenting and concentrating. Experiments prove that the radix codonopsis and red yeast probiotic fermentation product provided by the invention can effectively reduce the contents of TG, TC and LDL-C in serum and improve the content of HDL-C, and has a lipid-lowering effect; the traditional Chinese medicine composition can effectively reduce the levels of IL-6 and TNF-alpha, improve the levels of IL-4 and IL-10, improve immune response and reduce inflammatory response; the intestinal flora can be obviously improved, and the number of harmful bacteria is reduced. Therefore, the radix codonopsis and red yeast rice probiotic fermentation product provided by the invention has the effects of improving immunity, regulating intestinal flora and reducing lipid, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a radix codonopsis and monascus probiotic fermentation product and a preparation method and application thereof. BACKGROUND

[0002] Radix codonopsis is a plant of the family Campanulaceae, and is one of the most common traditional Chinese medicines in clinical use. It has the effects of tonifying the middle energizer, invigorating the spleen and lung, and is mainly used for treating spleen-lung deficiency, shortness of breath, palpitation, poor appetite, loose stool, asthenia panting, cough, and internal heat and thirst. The active ingredients in radix codonopsis mainly include radix codonopsis polysaccharide, radix codonopsis glycosides, alkaloids, flavonoids, ginsenosides, and atractylodes lactones, etc. Among them, radix codonopsis polysaccharide has the effects of anti-gastric ulcer, immune regulation, and intestinal microbial regulation, radix codonopsis glycosides have the effect of protecting gastric mucosa, and atractylodes lactone III has the effect of anti-inflammation, which are important quality evaluation indexes of radix codonopsis. Modern research shows that saponins can prevent the absorption of fat in the intestinal tract, thereby reducing the content of cholesterol and triglyceride, and have the effect of lowering blood lipid. Flavonoids can lower blood lipid level through the pathways of inhibiting the absorption and synthesis of cholesterol, promoting the metabolism and excretion of cholesterol, etc. Most plant polysaccharides have effects on the blood lipid metabolism indexes of animals such as triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and free fatty acid (FFA). Radix codonopsis contains a certain amount of radix codonopsis polysaccharide, saponins, and flavonoids, and has the potential effect of lowering blood lipid on the basis of protecting the gastrointestinal tract and improving immunity.

[0003] Monascus has a history of more than 1,000 years of production and application. Monascus, as the main microorganism for producing red yeast rice, can produce strong enzymes such as amylase, glucoamylase, protease, pectinase, etc. during the growth process. Amylase and glucoamylase can hydrolyze starch into dextrin, oligosaccharide, and glucose, etc. Protease can decompose proteins into polypeptides, amino acids, and other small molecular compounds. Monascus can also produce various useful metabolites such as red yeast rice pigment, Monacolin K, and gamma-aminobutyric acid, etc., which have the health care functions of lowering blood pressure, lowering blood lipid, antioxidant, and anti-cancer, etc. Among them, Monacolin K and its derivatives have been clinically proven to effectively inhibit the synthesis of cholesterol.

[0004] At present, the commonly used drugs for lowering cholesterol and the main lipid-lowering component Monacolin K of red yeast rice are statins. Statins have a certain irritability to the gastrointestinal mucosa, and long-term use can damage the gastrointestinal mucosa, thereby causing symptoms such as indigestion, loss of appetite, etc. It may also be accompanied by discomfort such as nausea, vomiting, etc. There is an urgent need to provide a lipid-lowering medicine with the same origin as food. SUMMARY

[0005] The present application aims to provide a Codonopsis pilosula Monascus yeast probiotic fermentation product, a preparation method and application thereof, so as to solve the problems of the prior art.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0007] The present application provides a Codonopsis pilosula Monascus yeast probiotic fermentation product, which is obtained by sequentially inoculating Codonopsis pilosula with Monascus purpureus and Lactobacillus plantarum and then fermenting and concentrating.

[0008] The present application also provides a preparation method of the Codonopsis pilosula Monascus yeast probiotic fermentation product, comprising the following steps.

[0009] The Codonopsis pilosula is crushed into Codonopsis pilosula powder, and a Codonopsis pilosula aqueous solution is prepared according to a ratio of 30-50 g of Codonopsis pilosula powder to 1 L of distilled water, sterilized, inoculated with Monascus purpureus, and fermented to obtain a Codonopsis pilosula Monascus fermentation product.

[0010] The Codonopsis pilosula Monascus fermentation product is inoculated with Lactobacillus plantarum after sterilization, and continuously fermented to obtain a Codonopsis pilosula fermentation product, which is concentrated to obtain the Codonopsis pilosula Monascus yeast probiotic fermentation product.

[0011] Optionally, the Codonopsis pilosula aqueous solution is prepared according to a ratio of 40 g of Codonopsis pilosula powder to 1 L of distilled water.

[0012] Optionally, the inoculation amount of the Monascus purpureus is 10 wt%.

[0013] The fermentation condition is 30 DEG C for 7 days.

[0014] Optionally, the inoculation amount of the Lactobacillus plantarum is 3 wt%.

[0015] The continuous fermentation condition is 35 DEG C for 1 day.

[0016] Optionally, the concentration step is sterilizing and centrifuging the Codonopsis pilosula fermentation product to obtain a fermentation liquid, and concentrating the fermentation liquid to 10% of the original volume.

[0017] The present application also provides an application of the Codonopsis pilosula Monascus yeast probiotic fermentation product in preparing a lipid-lowering drug.

[0018] The present application also provides an application of the Codonopsis pilosula Monascus yeast probiotic fermentation product in preparing a drug for improving immunity.

[0019] The present application also provides an application of the Codonopsis pilosula Monascus yeast probiotic fermentation product in regulating intestinal flora.

[0020] The present application also provides a lipid-lowering drug comprising the Codonopsis pilosula Monascus yeast probiotic fermentation product.

[0021] The present invention discloses the following technical effects:

[0022] The *Monascus purpureus* used in this invention can use an aqueous solution of *Codonopsis pilosula* powder as the sole culture medium, avoiding the impact of adding other nutrients on the effective components and increasing the concentration of effective components in the fermentation product. Unlike existing bacterial fermentation of medicinal materials using probiotics, yeasts, or *Bacillus subtilis*, *Monascus purpureus* produces a rich enzyme system during its growth process, which can disrupt the cell structure of *Codonopsis pilosula*, more effectively releasing saponins and flavonoids, thus lowering lipids. Simultaneously, through enzymatic action, polysaccharides and proteins in *Codonopsis pilosula* are converted into smaller sugar and peptide molecules, making them more easily absorbed and utilized by the body, thus enhancing immunity. Conventional red yeast rice and statins can damage the gastrointestinal tract. By combining with *Lactobacillus plantarum* and *Codonopsis pilosula*, the active ingredients of probiotics and *Codonopsis pilosula* can protect the gastrointestinal tract and enhance immunity. Furthermore, it synergistically enhances the lipid-lowering effect with *Monascus purpureus*-produced MonacolinK.

[0023] Experimental verification has shown that the Codonopsis pilosula and red yeast rice probiotic fermentation product provided by this invention can effectively reduce the levels of TG, TC, and LDL-C in serum, while increasing the level of HDL-C, thus exhibiting a lipid-lowering effect. It can also effectively reduce the levels of IL-6 and TNF-α, while increasing the levels of IL-4 and IL-10, thereby enhancing the immune response and reducing inflammation. Furthermore, it can significantly improve the intestinal flora and reduce the number of harmful bacteria. Therefore, the Codonopsis pilosula and red yeast rice probiotic fermentation product provided by this invention has the effects of enhancing immunity, regulating intestinal flora, and lowering lipids, and has broad application prospects. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains unless clearly indicated otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. All literature and similar materials cited in this application, including but not limited to, patents, genetic code, scientific and / or technical articles, are expressly incorporated by reference.

[0027] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0028] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or step.

[0029] The probiotic strain used in the present application is Lactobacillus plantarum SXp08, which has been deposited in the China General Microbiological Culture Collection Center on May 19, 2022, with the accession number CGMCC NO. 24928, and has been disclosed in the authorized patent CN115612637B- A strain of Lactobacillus plantarum and its application.

[0030] The Monascus purpureus strain used in the present application is Monascus purpureus MH04, which has been deposited in the China General Microbiological Culture Collection Center on November 19, 2024, with the accession number CGMCC No. 41661.

[0031] Monascus purpureus MH04 was sampled from the soil under shrubs in Xiaodian District, Taiyuan City in August 2013, isolated by PDA medium, has the ability to produce red koji pigment and monacolin K, and does not produce citrinin during fermentation, was identified as Monascus purpureus by ITS sequencing in July 2024, and was deposited in the China General Microbiological Culture Collection Center on November 19, 2024. The ITS sequence is shown as SEQ ID NO. 1.

[0032] SEQ ID NO. 1:

[0033] .

[0034] Example 1

[0035] 1. Preparation of Codonopsis pilosula and red yeast rice probiotic fermentation product

[0036] Three-year-old wild Codonopsis pilosula was cleaned, cut into sections, and dried. The powder was then ground using a traditional Chinese medicine pulverizer. Codonopsis pilosula aqueous solutions were prepared at ratios of 30g, 40g, and 50g of powder to 1L of distilled water, respectively, with a natural pH, and sterilized at 121℃ for 20 minutes. The solutions were inoculated with 10wt% Monascus purpureus and fermented at 30℃ for 7 days. The red pigment titer in the supernatant of the fermentation broth reached over 800u / mL, yielding the Codonopsis pilosula red yeast rice fermented product. After sterilization, the fermented product was inoculated with 3wt% Lactobacillus plantarum and fermented further at 35℃ for 1 day, yielding the Codonopsis pilosula fermented product. The components of the fermented products with different concentrations of Codonopsis pilosula powder were analyzed.

[0037] In this invention, the purple-red Aspergillus mold can be used as the sole culture medium component of Codonopsis pilosula powder aqueous solution, avoiding the influence of adding other nutrients on the effective components and increasing the concentration of effective components in the fermentation product.

[0038] The only inoculation order for preparing the radix ginseng red yeast probiotic fermentation product is to inoculate Monascus purpureus first and then Lactobacillus plantarum. The reason is that the rich enzyme system produced by Monascus purpureus can destroy the cell wall structure of radix ginseng and release more nutrients, and the metabolic products and rich enzyme system produced by Monascus purpureus during growth can decompose sugars and proteins into small molecular polysaccharides and peptides, which is beneficial to the growth of Lactobacillus plantarum which is nutritionally demanding. The above effects do not exist when Lactobacillus plantarum is inoculated first. Lactobacillus plantarum cannot grow in a culture medium with radix ginseng powder water solution as the only nutrient component.

[0039] The ingredient detection results after fermentation of 3%, 4%, and 5% radix ginseng powder are shown in Table 1. As shown in Table 1, the fermentation product has a good state, the extraction rate of radix ginseng effective components is high, and the production of monacolin K is high when 4% radix ginseng powder (radix ginseng powder 40 g, distilled water 1 L to prepare radix ginseng water solution) is used for fermentation. The subsequent experiment is carried out using the fermentation product of 4% radix ginseng powder.

[0040] Table 1. Content of each component after fermentation of radix ginseng powder with different concentrations (mg / mL)

[0041] Concentration End of fermentation state Codonopsis polysaccharide content Flavone content Saponin content Monacolin K content 3％ Codonopsis powder solid all use 8.53 0.60 1.28 0.049 4％ Codonopsis powder solid all use 11.42 0.80 1.70 0.062 5％ Codonopsis powder solid with surplus 12.13 0.89 1.87 0.053

[0042] 2. Preparation of radix ginseng fermentation concentrate

[0043] After fermentation is completed, the radix ginseng fermentation product is sterilized at 120°C for 20 min, centrifuged at 6000 rpm for 15 min, and concentrated to 10% of the original volume at 50-60°C using a reduced pressure concentration device to obtain a radix ginseng fermentation concentrate, which is the radix ginseng red yeast probiotic fermentation product.

[0044] 3. Preparation of radix ginseng dry powder

[0045] The radix ginseng fermentation concentrate is placed in a vacuum drying oven, the vacuum degree is controlled to be -60 to -40 Kpa, the temperature is controlled to be 50-60°C, and the radix ginseng fermentation concentrate is vacuum dried for 3-5 h to form loose dry solid, which is crushed into dry powder using a crusher.

[0046] Example 2. Role of radix ginseng red yeast probiotic fermentation product in improving immunity, regulating intestinal flora, and reducing fat

[0047] 1. Model establishment and grouping for administration

[0048] Select healthy SPF male mice 90 (10 each group), after 1 week of adaptive feeding, randomly divided into control group and experimental group, experimental group into model group, lovastatin group, radix ginseng extract group, monascus purpureus group, lactobacillus plantarum group and radix ginseng fermentation concentrate group. The control group of mice was fed with standard feed (protein 20%, carbohydrate 70%, fat 10%, heat 3.85 kcal / g), and the experimental group of mice was fed with high-fat feed (protein 20%, carbohydrate 20%, fat 60%, heat 5.24 kcal / g). The lovastatin group was orally administered with lovastatin at a dose of 2.0 mg / kg, the radix ginseng extract group was orally administered with radix ginseng extract (radix ginseng: water 1:10 high temperature extraction, concentrated to 10% of the original volume after extraction), the monascus purpureus group was orally administered with monascus purpureus fermentation (monascus purpureus fermentation broth was concentrated to 10% of the original volume), the lactobacillus plantarum group was orally administered with lactobacillus plantarum (10 8 CFU / g), the radix ginseng fermentation concentrate group was orally administered with radix ginseng fermentation concentrate prepared in Example 1 at a dose of 100 mg / kg, 200 mg / kg and 300 mg / kg respectively, and the control group and the model group were orally administered with distilled water. The mice were administered once a day for 6 consecutive weeks. One day before the end of the experiment, the mice were fasted for 12 h, then the mice were sacrificed, the blood was taken from the eyeball and tail, the serum was separated, and the blood lipid content and cytokines in the serum were determined; fresh feces were collected to determine the intestinal flora.

[0049] 2, the influence on blood lipid index

[0050] The blood lipid determination indexes were triglyceride (TG), cholesterol (TC), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C). The triglyceride (TG) detection kit (acetylacetone colorimetric method), total cholesterol (TC) detection kit (o-phenylenediamine colorimetric method), serum high-density lipoprotein (HDL-C) test box (spectrophotometric method) and serum low-density lipoprotein (LDL-C) test box (spectrophotometric method) of Shanghai Enzyme-linked Biological Technology Co., Ltd. were used to determine each index according to the kit instructions. The blood lipid determination results of each group are shown in Table 2.

[0051] Table 2 Serum lipid content (mmol / L) of each group

[0052]

[0053]

[0054] As can be seen from Table 2, the radix codonopsis fermentation concentrate can effectively reduce the content of TG, TC and LDL-C in serum, and increase the content of HDL-C. The mice can basically achieve the lipid-lowering effect of 2.0 mg / kg dose of lovastatin by gavage with radix codonopsis fermentation concentrate at 200 mg / kg, and the effect of reducing TG is better. The radix codonopsis extract and lactobacillus plantarum have little lipid-lowering effect, and the monascus ruber fermentation product has a certain lipid-lowering effect, but the effect is not as good as lovastatin and radix codonopsis monascus probiotic fermentation broth.

[0055] 3. Effect on cytokines

[0056] Cytokines are a class of small molecular proteins with various biological activities, which play a key role in physiological processes such as immune response, inflammatory response and tissue repair. The contents of mouse serum cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor (TNF-α) were determined by conventional ELISA (enzyme-linked immunosorbent assay), and the cytokine determination results of each group are shown in Table 3.

[0057] Table 3 Serum cytokine content of each group (pg / mL)

[0058] Grouping IL-2 IL-6 TNF-α IL-4 IL-10 Control group 10.21 23.58 17.34 14.73 33.56 Model group 7.32 73.24 45.72 8.95 17.64 Lovastatin group 13.41 52.36 38.77 10.94 26.13 Codonopsis extract group 9.77 45.73 32.69 15.62 29.53 Monascus purpureus group 8.73 69.25 50.13 9.28 19.42 Lactobacillus plantarum group 10.64 35.88 27.41 16.00 30.51 100 mg / kg fermentation concentrate 10.37 38.42 26.58 16.22 26.76 200 mg / kg fermentation concentrate 11.55 30.28 29.63 16.94 35.65 300 mg / kg fermentation concentrate 12.06 27.66 25.68 17.05 42.03

[0059] As can be seen from Table 3, compared with the model group, the radix codonopsis fermentation concentrate can effectively reduce the levels of IL-6 and TNF-α, increase the levels of IL-4 and IL-10, improve immune response and reduce inflammatory response. Lovastatin and monascus ruber have little effect on immune and inflammatory response, and radix codonopsis extract and lactobacillus plantarum can have a certain effect, but the effect is not as obvious as radix codonopsis fermentation broth.

[0060] 4. Effect on intestinal flora

[0061] Intestinal flora is closely related to hyperlipidemia, obesity and immunity, involving energy metabolism, inflammation regulation, immune cell function and other aspects, and deeply involved in various regulation processes through metabolic products (SCFAs, LPS), immune cell regulation (Th17 / Treg, M1 / M2) and intestinal barrier function. The changes of intestinal flora of each group were analyzed by 16s RNA high-throughput sequencing, and the results are shown in Tables 4 and 5.

[0062] Table 4 Changes of intestinal flora at the door level of each group

[0063]

[0064] Table 5 Changes of intestinal flora at the genus level of each group

[0065]

[0066]

[0067] From Table 4, at the level of phylum, the Firmicutes of the intestinal flora of the high-fat mice increased, the Bacteroidetes decreased, the intestinal flora adapted to the high-fat diet, further causing the mice to be obese; the Proteobacteria increased, i.e., part of the harmful bacteria increased, which would further strengthen the intestinal inflammation. The lavage of lovastatin and Monascus purpureus could not improve the intestinal flora of the high-fat mice, while the lavage of Codonopsis pilosula extract and Lactobacillus plantarum improved the intestinal flora, and the lavage of 200 mg / kg dose of Codonopsis pilosula fermented concentrate could significantly improve the intestinal flora of the high-fat mice.

[0068] From Table 5, at the level of genus, the Lactobacillus and Lachnospiraceae NK4A136 group of the intestinal flora of the high-fat mice significantly decreased, the Blautia, Erysipelatoclostridium and Enterobacter significantly increased, which increased the risk of intestinal inflammation of the high-fat mice and reduced the intestinal immune capacity. The lavage of lovastatin and Monascus purpureus could not improve the intestinal flora of the high-fat mice, while the lavage of Codonopsis pilosula extract and Lactobacillus plantarum improved the intestinal flora, and the lavage of 200 mg / kg dose of Codonopsis pilosula fermented concentrate could significantly improve the intestinal flora of the high-fat mice.

[0069] The above-described embodiments are only to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A Radix Codonopsis Monascus probiotic ferment, characterized in that, The radix codonopsis is inoculated with Monascus purpureus and Lactobacillus plantarum successively, and then concentrated to obtain the radix codonopsis.

2. The method of claim 1, wherein the preparation of the Radix Codonopsis Monascus probiotic fermentum is characterized by, The method comprises the following steps: The radix codonopsis is crushed into radix codonopsis powder, and then mixed with distilled water to obtain a radix codonopsis water solution, which is sterilized, inoculated with Monascus purpureus, and then fermented to obtain a radix codonopsis Monascus fermentation product. The radix codonopsis Monascus fermentation product is inoculated with Lactobacillus plantarum after sterilization, and then continuously fermented to obtain a radix codonopsis fermentation product, which is concentrated to obtain the radix codonopsis Monascus probiotic fermentation product.

3. The production method according to claim 2, wherein The radix codonopsis powder is mixed with distilled water to obtain a radix codonopsis water solution.

4. The production method according to claim 2, wherein The inoculation amount of the Monascus purpureus is 10 wt%. The fermentation condition is 30℃ for 7 days.

5. The production method according to claim 2, wherein The inoculation amount of the Lactobacillus plantarum is 3 wt%. The continuous fermentation condition is 35℃ for 1 day.

6. The production method according to claim 2, wherein The concentration step is that the radix codonopsis fermentation product is sterilized and centrifuged to obtain a fermentation liquid, and then the fermentation liquid is concentrated to 10% of the original volume.

7. The radix codonopsis Monascus probiotic fermentation product of claim 1 is used for preparing a lipid-lowering drug.

8. The radix codonopsis Monascus probiotic fermentation product of claim 1 is used for preparing a drug for improving immunity.

9. The radix codonopsis Monascus probiotic fermentation product of claim 1 is used for regulating intestinal flora.

10. A lipid lowering agent, characterized by, The radix codonopsis Monascus probiotic fermentation product of claim 1 is contained.

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