Rhizoma gastrodiae fermented by Haemadella coagulatum as well as preparation method and application of rhizoma gastrodiae fermented by Haemadella coagulatum

Abstract

The invention discloses a Haiderriskia coagulum fermented gastrodia elata and a preparation method and application thereof, and relates to the technical field of biological medicines.The preparation method comprises the following steps that 1, gastrodia elata and water are mixed, crushed and homogenized, then sterilized and cooled to 37 DEG C or below, and gastrodia elata slurry is obtained; (2) inoculating the rhizoma gastrodiae serous fluid with the Hahndersella coagulatum liquid, uniformly mixing, sealing, and performing fermentation culture at the temperature of 35-40 DEG C for 5-7 days until the pH value reaches 3.8-4.2, so as to obtain the Hahndersella coagulatum fermented rhizoma gastrodiae. The invention further discloses the gastrodia elata fermented by the Haiderrikia coagulatum and application of the gastrodia elata. The fermented gastrodia elata can effectively relieve physiological symptoms caused by colitis, improve overall health and protect intestinal functions, and has a remarkable effect on prevention of chronic colitis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to Gastrodia elata fermented by coagulans of Bacillus heindrix and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is a type of intestinal disease characterized by inflammation, mainly including ulcerative colitis and Crohn's disease. The causes of these diseases are diverse, involving immune response disorders, genetic factors, intestinal microbial imbalance and various environmental triggers. In a healthy state, the intestinal immune system maintains the mucosal barrier through finely regulated signaling pathways to prevent invasion by pathogens. However, in patients with colitis, the immune system becomes dysregulated, leading to an overreaction to resident bacteria or food proteins, triggering inflammation. Persistent inflammatory response can cause symptoms such as abdominal pain, diarrhea, bloody stools, and weight loss, and may develop into serious complications such as intestinal perforation or colorectal cancer, which has become a common and complex problem in digestive system diseases.

[0003] At present, the treatments for colitis mainly include drug therapy, surgical treatment and adjuvant therapy. The main existing treatments for colitis include corticosteroids, 5-aminosalicylic acid, immunosuppressants (such as azathioprine) and antibodies (such as adalimumab). These drugs control the disease by inhibiting inflammatory responses or regulating the immune system, but long-term use may bring about a series of side effects such as immunosuppression, increased risk of infection, and osteoporosis. Therefore, it is urgent to find new alternatives to fight colitis.

[0004] In recent years, a variety of new therapies have emerged in the field of colitis treatment as its pathogenesis has been deeply understood. Among these new methods, probiotic therapy has attracted widespread attention due to its high safety and low risk of side effects. This therapy not only prevents the occurrence of colitis, but also helps to reduce existing inflammation by adjusting the microbial balance in the intestine, strengthening the intestinal barrier, and inhibiting the growth of harmful microorganisms. In particular, Bacillus coagulans has become a highly respected probiotic due to its unique biological properties and good anti-inflammatory effects. This strain can optimize the composition of the intestinal microbiome, support the growth of beneficial bacteria such as bifidobacteria and lactic acid bacteria, and inhibit the spread of harmful microorganisms such as Escherichia coli and Helicobacter pylori, thereby correcting the imbalance of the flora. Not only that, Bacillus coagulans can also effectively block the invasion of harmful substances and reduce the possibility of inflammation. It can also curb the release of inflammatory factors, directly relieve intestinal inflammation, and help heal damaged mucosa.

[0005] Gastrodia elata Blume belongs to the genus Gastrodia of the orchid family and is a traditional medicinal material with high edible and medicinal value. It is widely distributed in the southwestern plateau areas of my country, especially in Zhaotong and Lijiang, Yunnan. Modern medical research shows that Gastrodia elata is rich in nutrients, has the effects of calming the liver and calming wind, and has a regulatory effect on the central nervous system. It also has multiple effects such as antioxidant, anti-inflammatory, anti-tumor, anti-apoptosis and anti-asthmatic, and is a traditional Chinese herbal medicine widely used in clinical Chinese medicine. Studies have shown that Gastrodia elata can enhance the phagocytic ability of macrophages, improve the body's nonspecific immune effects, and promote antibody production, all of which are of great significance in immune regulation. At the same time, it can increase the expression of intestinal mucosal tight junction proteins and mucins, and enhance the intestinal mucosal barrier function. Therefore, Gastrodia elata can be used as a complementary strategy for the treatment of colitis and effectively alleviate the symptoms of colitis induced by dextran sulfate sodium salt (DSS).

[0006] Given the significant advantages of Bacillus coagulans and Gastrodia elata in the prevention and adjuvant treatment of colitis, the development of a fermentation preparation combining these two ingredients is expected to provide a safe and effective preventive treatment plan for patients with colitis. There are many potential advantages to using probiotics to make fermentation agents. For example, probiotics can improve the taste of Chinese medicinal materials that are both medicinal and edible, and can enhance the biological efficacy of Chinese medicinal materials. Moreover, probiotic fermentation agents can increase the content of active probiotics while retaining the original functional ingredients of Gastrodia elata, and may produce biologically active functional metabolites during the fermentation process, thereby further enhancing the health care function of Gastrodia elata. This therapy is expected to be used for the widespread prevention and treatment of colitis to meet the growing market demand. This innovative preventive treatment plan will bring more treatment options to patients with colitis and provide them with a better quality of life. Summary of the invention

[0007] In order to solve the above technical problems, the purpose of the present invention is to provide a coagulant heindriix Gastrodia elata fermented gastrodia elata and its preparation method and application, which can effectively alleviate the physiological symptoms caused by colitis, improve overall health and protect intestinal function, and has a significant effect in preventing chronic colitis.

[0008] The technical solution of the present invention to solve the above technical problems is as follows: a preparation method of Gastrodia elata fermented by coagulating Bacillus hyendricus is provided, comprising the following steps:

[0009] (1) mixing Gastrodia elata with water, crushing and homogenizing the mixture, sterilizing the mixture, and cooling the mixture to below 37° C. to obtain Gastrodia elata slurry;

[0010] (2) inoculating the coagulated bacteria liquid of Bacillus hyendriculariae into the Gastrodia elata slurry obtained in step (1), mixing them evenly, sealing them, and fermenting and culturing them at a temperature of 35-40° C. for 5-7 days until the pH reaches 3.8-4.2, thereby obtaining the coagulated bacteria liquid of Bacillus hyendriculariae fermented Gastrodia elata.

[0011] Furthermore, in step (1), the mass ratio of Gastrodia elata to water is 1:3-5.

[0012] Furthermore, in step (1), sterilization is performed at 70°C for 30 minutes.

[0013] Further, in step (2), the coagulant Bacillus is coagulant Bacillus AACE9, which was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 15, 2024, with a deposit number of CGMCCNO.31302.

[0014] Furthermore, in step (2), the volume ratio of the coagulated Hyndrickella bacterial liquid to the Gastrodia elata slurry is 1:18-22.

[0015] Furthermore, the number of viable bacteria in the coagulated Hyndrickella hyendriculariae solution was 1.0-5.0×10 9 CFU / mL.

[0016] The present invention also provides coagulated hyendrickii fermented gastrodia elata obtained by the preparation method of coagulated hyendrickii fermented gastrodia elata.

[0017] The present invention also provides the use of the Gastrodia elata fermented by the coagulant Bacillus heindrix in preparing products for preventing or treating chronic colitis.

[0018] Furthermore, the above-mentioned products are food, health products or medicines.

[0019] The present invention has the following beneficial effects:

[0020] 1. The fermented Gastrodia elata of the present invention can alleviate the symptoms of weight loss and loss of appetite caused by colitis, and is helpful to enhance physiological stability.

[0021] 2. The fermented Gastrodia elata of the present invention can reduce the amount of colon intestinal bleeding, relieve symptoms of colon atrophy, reduce colon inflammatory cell infiltration, increase the number of colon goblet cells, reduce the degree of colon crypt destruction, prevent the occurrence of colon inflammatory lesions, reduce intestinal mucosal damage, help protect colon tissue, and maintain stable intestinal function.

[0022] 3. The fermented Gastrodia elata of the present invention can reduce the content of pro-inflammatory factors, increase the level of anti-inflammatory factors, and thus improve the immune regulation ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Figure 2 is the weight change diagram of mice in each group;

[0024] Figure 2 The graphs are the changes in food intake of mice in each group;

[0025] Figure 3 The graphs are the changes in water drinking of mice in each group;

[0026] Figure 4 The disease activity index DAI score results of each group of mice;

[0027] Figure 5 The colon length comparison chart of mice in each group;

[0028] Figure 6 This is a comparison chart of organ indexes of mice in each group;

[0029] Figure 7 The images are of colon tissue sections of mice in each group;

[0030] Figure 8 This is a comparison chart of the anti-inflammatory factor IL-10 levels in the serum of mice in each group;

[0031] Fig. 9 This is a comparison chart of the anti-inflammatory factor TGF-β levels in the serum of mice in each group;

[0032] Fig.10 This is a comparison of the levels of pro-inflammatory factor IL-1β in the serum of mice in each group;

[0033] Fig.11 This is a comparison chart of the pro-inflammatory factor IL-6 levels in the serum of mice in each group;

[0034] Fig.12 This is a comparison chart of the pro-inflammatory factor IL-8 levels in the serum of mice in each group;

[0035] Fig.13 This is a comparison chart of the levels of pro-inflammatory factor TNF-α in the serum of each group of mice. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0037] Example 1 Isolation, identification and preservation of AACE9 of Bacillus heindrix coagulans

[0038] (1) Isolation of AACE9 from Bacillus heindrix

[0039] Weigh 1g of the colon sample of Nandan Yao chicken from a farm in Kunming, suspend it in PBS buffer, spread it on LB solid medium after gradient dilution, pick typical single colonies for expansion culture; take 100μL of the bacterial solution of each single colony and spread it on MRS solid medium containing 5% CaCO3 for culture; pick single colonies with calcium-dissolving rings and expand them in LB liquid medium. The bacterial solution was purified by partitioning and streaking for strain identification, and another 0.5mL of bacterial solution was centrifuged at 3000r / min for 10min, the supernatant was discarded, 30% glycerol was added and stored in a -80℃ refrigerator.

[0040] (2) Identification of AACE9 of Bacillus heindrix

[0041] Using TSINGKE TSP701-50 Bacteria Genomic DNA Kit (Beijing Qingke Biotechnology Co., Ltd.) was used to extract bacterial DNA, and then universal primers 27F and 1492R were used to amplify the 16S rRNA gene. The PCR product was sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with BLAST in NCBI GenBank to determine the classification and name it Heyndrickxia coagulans AACE9.

[0042] (3) Preservation of strains

[0043] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection on July 15, 2024, with the deposit registration number CGMCC NO.31302, and the deposit location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0044] Example 2

[0045] A preparation method of Gastrodia elata fermented with coagulated bacteria Hendrix spp. comprises the following steps:

[0046] (1) Gastrodia elata and water are mixed in a mass ratio of 1:4, crushed and homogenized, sterilized at 70° C. for 30 min, and cooled to below 37° C. to obtain Gastrodia elata slurry;

[0047] (2) The AACE9 bacterial suspension of Hendrix hyandricus (2.0×10 9 CFU / mL) was inoculated into the Gastrodia elata slurry obtained in step (1) at a volume ratio of 1:20, mixed evenly, sealed, and fermented at 37°C for 6 days until the pH reached 4.0 to obtain the Gastrodia elata fermented with coagulated Heindrix spp.

[0048] Example 3 Verification of the preventive effect of Gastrodia elata fermented with Gastrodia elata

[0049] Experimental animals: SPF male C57BL / 6J mice, 6 weeks old, were kept at a temperature of 22-25°C and a humidity of 50% with a 12-h light and 12-h dark cycle to simulate the natural circadian rhythm, and were allowed to drink water and eat freely (the animal experimental protocol was reviewed and approved by the Animal Care and Use Committee of Kunming University of Science and Technology).

[0050] Experimental grouping and treatment: After 7 days of adaptive feeding, the mice were randomly divided into 6 groups (n=6): control group (CK), model group (M), Gastrodia elata group (G), Lactobacillus rhamnosus fermented Gastrodia elata group (LRG), Lactobacillus plantarum fermented Gastrodia elata group (LPG) and Bacillus heindrix coagulans fermented Gastrodia elata group (BCG). The experimental period was 14 days. All groups drank sterile water in the first 7 days, and drank sterile water supplemented with 3% DSS in the last 7 days except for the CK group. The CK group and the M group were gavaged with 200 μL PBS every day; the G group was gavaged with 200 μL Gastrodia elata homogenate every day, the LRG group was gavaged with 200 μL Lactobacillus rhamnosus fermented Gastrodia elata preparation every day, the LPG group was gavaged with 200 μL Lactobacillus plantarum fermented Gastrodia elata preparation every day, and the BCG group was gavaged with 200 μL Bacillus heindrix coagulans fermented Gastrodia elata every day. The mice were fasted but not watered for 12 hours at 9 pm on the 14th day of the experiment, and euthanasia was performed on the morning of the 15th day.

[0051] Sample collection: Blood was collected by sampling from the ophthalmic venous plexus; spleen, kidney and liver were collected; mice were killed by cervical dislocation after blood collection, colons of mice in each group were collected, the distance from the anus to the cecum was measured, and 2 cm from the middle section of the colon without content was sampled and fixed in a neutral universal tissue fixative.

[0052] During the experiment, the mice's food and water intake and body weight changes were monitored every day, and the stool viscosity and occult blood were observed on the 14th day.

[0053] (1) Weight changes

[0054] The weight changes, food intake and water intake of mice in each group during the experiment were as follows: Figure 1-3 shown.

[0055] Depend on Figure 1-3It can be seen that the body weight of mice in each group increased steadily from 1 to 7 days. After the 7th day, all groups except the CK group showed varying degrees of weight loss. Among them, the weight loss trend of mice in the M group was the most obvious. The decrease in food intake and water intake in the M group also indicated that the DSS-induced colitis modeling was successful. The G group and the LPG group also showed a small weight loss phenomenon. In contrast, the weight of mice in the BCG group not only did not decrease, but showed a significant upward trend, close to the CK group. It is worth noting that the weight change of the LRG group tended to be stable throughout the experimental period. In general, each group of fermented preparations can alleviate and reduce the phenomenon of weight loss in mice to varying degrees, and the group treated with Gastrodia elata fermented with Hendrixella coagulans of the present invention (BCG group) is more effective in preventing weight loss than other treatment groups.

[0056] (2) Disease Activity Index (DAI)

[0057] The disease activity index (DAI) score is an important indicator for evaluating colon damage. It is usually combined with weight loss, stool morphology, and fecal occult blood volume for a comprehensive assessment. The specific scoring criteria are shown in Table 1.

[0058] Table 1 DAI scoring criteria

[0059]

[0060] The final DAI score is the sum of the above three scores of each group. The higher the score, the more severe the colitis. Figure 4 shown.

[0061] Depend on Figure 4 It can be seen that in the first 7 days of the experiment, the DAI scores of mice in each group were relatively stable. After the 7th day, the DAI scores of the other groups except the CK group all showed different degrees of increase. Among them, the M group was the most serious, with the highest DAI score of 9.33, indicating that its colitis was the most serious. The highest scores of the G group, LRG group, LPG group and BCG group were 5, 4.5, 4.1 and 3.5, respectively. It can be seen that the DAI score of mice in the BCG group was the lowest. In general, the fermentation preparations of each group can reduce the DAI score to varying degrees, indicating that these fermentation preparations have good prebiotic effects on the prevention and treatment of colon inflammation. In comparison, the preventive and therapeutic effect of the Gastrodia elata fermented by the coagulant Heindrixella of the present invention is more significant.

[0062] (3) Colon length

[0063] After the experiment, the mice were killed, the colons of each group of mice were collected, and their lengths were counted. Figure 5As shown in the figure, different lowercase letters indicate significant differences among the treatment groups (p<0.05). The results showed that compared with the CK group, the colon of the M group was significantly shortened, but the shortening of the colon could be improved after treatment with the fermented Gastrodia preparations of each group. Among them, the colon of the BCG group was the longest compared with the other treatment groups, and the recovery effect was closest to the control group.

[0064] (4) Organ index

[0065] After the experiment, the mice were killed, and the liver, spleen, and kidney of each group were collected and weighed. The organ index (organ index = organ weight / body weight) was used to evaluate the organ health and inflammatory response. Figure 6 As shown in the figure, different lowercase letters indicate significant differences among the treatment groups (p<0.05). The results showed that the indexes of the three organs in the M group were the highest, indicating that the three organs were most affected by inflammation. After being treated with fermented Gastrodia preparations in each group, they all decreased to varying degrees, and the severity of the inflammatory reaction was milder than that in the M group. Among them, the treatment effect of the BCG group was the most obvious, which was close to that of the control group.

[0066] (5) Effects of Gastrodia elata fermented by Bacillus heindrix on colon histopathology in mice

[0067] H&E staining was performed on the cross-section of the mouse colon to observe the structure of each layer of the mouse colon tissue. Figure 7 shown.

[0068] Depend on Figure 7 It can be seen that compared with the CK group, the colon tissue of the M group had large-scale ulcers, connective tissue hyperplasia, unclear mucosal structure, irregular crypt shape, a small amount of pus cells, and a large number of goblet cells missing; the submucosal layer had large-scale edema, loose connective tissue, and a small amount of lymphocyte infiltration; compared with the M group, the G group, LRG group, LPG group, and BCG group all showed different degrees of improvement and prevention, among which the BCG group was better than the other treatment groups, showing that the mucosal epithelium was intact, there were a large number of straight tubular crypts in the lamina propria, which were densely arranged, the number of goblet cells was abundant, and connective tissue hyperplasia was occasionally seen. Compared with the BCG group, the intestinal tissues of the G group, LRG group, and LPG group all had area ulcers, a small amount of residual intestinal gland expansion, and a large number of pus cells were visible in the crypts; the submucosal layer had small-scale edema, loose connective tissue, and a small amount of lymphocytes and granulocytes infiltration. It can be seen that Gastrodia elata fermented with Heindrix coagulans has a more significant probiotic effect on relieving colonic inflammatory response.

[0069] (6) Effects of Gastrodia elata fermented by Bacillus heindrix on serum inflammatory factors in mice

[0070] Cytokine imbalance is closely related to the onset of ulcerative colitis. Cytokines are divided into pro-inflammatory cytokines and anti-inflammatory cytokines. These cytokines have a certain effect on inflammation and immune regulation. The inflammatory factors in the serum of each group of mice were detected. The levels of IL-10, TGF-β, IL-1β, IL-6, IL-8 and TNF-α were as follows: Figure 8-13 Different lowercase letters indicate significant differences among treatment groups (p<0.05).

[0071] Depend on Figure 8-13 It can be seen that the levels of anti-inflammatory cytokines IL-10 and TGF-β in group M were significantly lower than those in the other groups. Group G, LRG, LPG and BCG groups were able to increase the levels of anti-inflammatory factors to varying degrees, among which the BCG group had a higher level of increase than the other treatment groups, which was closest to the control group. The levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8 and TNF-α in group M were significantly higher than those in the other groups, indicating that the inflammatory response was severe. Group G, LRG, LPG and BCG groups were able to decrease the levels of pro-inflammatory factors to varying degrees, especially BCG, which had the most significant decrease. Comprehensive analysis showed that Gastrodia elata and various fermented Gastrodia elata preparations could reduce the inflammation caused by colitis to varying degrees, especially the Gastrodia elata preparation fermented by Heindrix coagulans, which had a more significant anti-inflammatory effect.

[0072] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing Gastrodia elata fermented by coagulating Bacillus hyendriculariae, characterized in that: The following steps are involved: (1) mixing Gastrodia elata with water, crushing and homogenizing the mixture, sterilizing the mixture, and cooling the mixture to below 37° C. to obtain Gastrodia elata slurry; (2) inoculating the coagulated bacteria liquid of Bacillus hyendriculariae into the Gastrodia elata slurry obtained in step (1), mixing them evenly, sealing them, and fermenting and culturing them at a temperature of 35-40° C. for 5-7 days until the pH reaches 3.8-4.2, thereby obtaining the coagulated bacteria liquid of Bacillus hyendriculariae fermented Gastrodia elata.

2. The method for preparing Gastrodia elata fermented by coagulating Bacillus hyendriculariae as claimed in claim 1, characterized in that: In step (1), the mass ratio of Gastrodia elata to water is 1:3-5.

3. The method for preparing Gastrodia elata fermented by coagulating Bacillus hyendriculariae as claimed in claim 1, characterized in that: In step (1), sterilize at 70°C for 30 minutes.

4. The method for preparing Gastrodia elata fermented by coagulating Bacillus hyendriculariae as claimed in claim 1, characterized in that: In step (2), the coagulant Hyendrickella is coagulant Hyendrickella AACE9, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 15, 2024, with a deposit number of CGMCC NO.31302.

5. The method for preparing Gastrodia elata fermented by coagulating Bacillus hyendriculariae as claimed in claim 1, characterized in that: In step (2), the volume ratio of the coagulated Hyndrickella bacterial liquid to the Gastrodia elata slurry is 1:18-22.

6. The method for preparing Gastrodia elata fermented by coagulating Bacillus hyendriculariae as claimed in claim 1, characterized in that: The number of live bacteria in the coagulated Hyndrickella bacterial liquid is 1.0-5.0×10 9 CFU / mL.

7. Coagulated Gastrodia elata fermented by Coagulated Bacillus hyendricus obtained by the method for preparing Gastrodia elata fermented by Coagulated Bacillus hyendricus according to any one of claims 1 to 6.

8. Use of the Gastrodia elata fermented by Bacillus heindrix coagulans as claimed in claim 7 in the preparation of products for preventing or treating colitis.

9. The use according to claim 8, characterized in that The product is food, health product or medicine.

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