Application of lactobacillus mucilaginosus B14.32 in fermentation of traditional Chinese medicine and anti-depression traditional Chinese medicine fermentation composition

Abstract

The invention provides an application of lactobacillus mucus B14.32 in traditional Chinese medicine fermentation and an anti-depression traditional Chinese medicine fermentation composition, a microbial fermentation technology is combined with traditional Chinese medicine extraction, and lactobacillus mucus B14.32 and saccharomycetes are used for carrying out two-step fermentation on traditional Chinese medicine with an anti-depression effect to obtain the anti-depression traditional Chinese medicine fermentation composition. The active ingredients of the traditional Chinese medicines are preserved to the maximum extent, the medicine effect is enhanced, the toxicity is reduced, the obtained anti-depression traditional Chinese medicine fermentation composition has the effects of resisting depression and relieving brain inflammation of depression patients, and a safer and more effective treatment scheme is provided for the depression patients.

Description

Technical Field

[0001] The invention belongs to the technical field of fermented Chinese medicines, and in particular to an application of Lactobacillus mucilaginosus B14.32 in fermented Chinese medicines and an antidepressant Chinese medicine fermentation composition. Background Art

[0002] Depression is a common mental illness, which is mainly manifested by low mood, loss of interest, slow thinking and other symptoms. In severe cases, dangerous behaviors such as self-harm and suicide may occur. Currently, Western medicine is mainly used to treat depression in clinical practice, such as paroxetine, escitalopram, fluoxetine, etc., but these drugs often cause adverse reactions such as nausea, insomnia, headache, vomiting, etc. during treatment.

[0003] Traditional Chinese medicine has become an important means of treating neurological diseases due to its advantages of multiple components, multiple targets, and low side effects. Studies have shown that traditional Chinese medicines such as Bupleurum, Magnolia officinalis, and Ginseng have shown good antidepressant activity in depression animal models and clinically. However, the traditional high-temperature decoction method will destroy the structure of the active ingredients of traditional Chinese medicine to a certain extent, affecting its efficacy.

[0004] In recent years, some researchers have tried to use milder methods such as enzyme method and fermentation method to process traditional Chinese medicine in order to preserve the active ingredients of traditional Chinese medicine to the maximum extent. Among them, the use of probiotics to ferment traditional Chinese medicine can not only promote the transformation of traditional Chinese medicine ingredients and improve the efficacy, but also produce beneficial compounds, thereby enhancing the efficacy and reducing toxicity. Therefore, the combination of probiotics and traditional Chinese medicine fermentation is of great significance in the research and development of antidepressant drugs. However, the existing traditional Chinese medicine antidepressant drugs still have the problem of active ingredients being destroyed and the efficacy being reduced. It is urgent to develop new traditional Chinese medicine antidepressant drug formulas, traditional Chinese medicine fermentation agents and preparation processes to improve the stability and bioavailability of drugs to solve the above problems. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an application of Lactobacillus mucinus B14.32 in fermented traditional Chinese medicine and an antidepressant traditional Chinese medicine fermentation composition. Lactobacillus mucinus B14.32 and yeast are used to perform two-step fermentation on the traditional Chinese medicine with antidepressant effect, which preserves the active ingredients of the traditional Chinese medicine to the maximum extent, enhances the efficacy and reduces the toxicity, and provides a safer and more effective treatment plan for patients with depression.

[0006] To achieve the above object, the present invention provides the following technical solution: application of Lactobacillus mucosa B14.32 in fermented traditional Chinese medicine, wherein Lactobacillus mucosa B14.32 is derived from human vaginal secretions, and the depository of Lactobacillus mucosa B14.32 is: General Microbiological Center of China Microbiological Culture Collection Administration, deposit time: December 25, 2024, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit number is CGMCC NO: 33189, and classification name is: Limosilactobacillus mucosa .

[0007] The present invention also provides a traditional Chinese medicine fermentation agent, specifically Lactobacillus mucosa B14.32, which is derived from human vaginal secretions. The depository of Lactobacillus mucosa B14.32 is: General Microbiology Center of China Microbiological Culture Collection Administration, the deposit time is: December 25, 2024, the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit number is CGMCC NO: 33189, and the classification name is: Limosilactobacillus mucosae .

[0008] Furthermore, yeast is included, and the yeast is used for the primary fermentation, and Lactobacillus mucilaginosus B14.32 is used for the secondary fermentation, and the inoculation amount of the yeast is 10% to 20%, and the inoculation amount of Lactobacillus mucilaginosus B14.32 is 10% to 20%.

[0009] The present invention also provides a method for preparing a fermentation composition, the specific steps of which are as follows: Preparation of yeast inoculum, lactic acid bacteria inoculum and Chinese medicine powder; The Chinese medicine powder is mixed with water to obtain a mixed solution, and yeast inoculum is added to the mixed solution for primary fermentation to obtain a primary fermentation solution; The volume of the primary fermentation liquid is adjusted, and the lactic acid bacteria inoculum is added to carry out secondary fermentation. After the fermentation is completed, a filtrate is obtained; The filtrate is centrifuged and the supernatant is taken as the fermentation composition.

[0010] Furthermore, the Chinese medicine powder is mixed with water in a mass ratio of 1:2 to obtain a mixed solution, and a yeast inoculum with an inoculation amount of 10% to 20% is added to the mixed solution for a fermentation to obtain a primary fermentation liquid; wherein the primary fermentation is fermented at a temperature of 28°C for 48 hours.

[0011] Furthermore, the volume of the primary fermentation liquid was adjusted with sterile water, and a lactic acid bacteria inoculum with an inoculum amount of 10% to 20% was added for secondary fermentation. After the fermentation was completed, a filtrate was obtained; wherein the secondary fermentation was anaerobic fermentation at 37°C for 48 hours.

[0012] Furthermore, the yeast inoculum is commercially available yeast, with a live bacterial count of 2.35×108 CFU / mL; the lactic acid bacteria inoculum adopts mucus Lactobacillus B14.32, and the amount of live bacteria is 7.65×10 10 CFU / mL, Lactobacillus mucosus B14.32 originates from human vaginal secretions. The depository of Lactobacillus mucosus B14.32 is: General Microbiology Center of China Microbiological Culture Collection Administration, deposit date: December 25, 2024, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit number is CGMCC NO: 33189, and classification name is: Limosilactobacillus mucosae .

[0013] The present invention also provides an antidepressant Chinese medicine fermentation composition, the raw materials of which include spiny jujube seeds, notoginseng, peony and liquorice, and the raw materials are crushed and mixed and then fermented using the above preparation method to obtain the composition.

[0014] Furthermore, the raw materials include Chinese jujube seeds, Panax notoginseng, peony root and liquorice in a mass ratio of 1~2:1~2:1:0.25~1.

[0015] Furthermore, the inoculation amount of the yeast inoculum is 20%, and the inoculation amount of the Lactobacillus mucilaginosus B14.32 is 10%.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention adopts mucus lactobacillus B14.32 and yeast to perform two-step fermentation on traditional Chinese medicine, thereby avoiding the problem of traditional high-temperature decoction method destroying the active ingredients of traditional Chinese medicine, preserving the active ingredients of traditional Chinese medicine to the maximum extent, and improving the efficacy of medicine; utilizing probiotics to ferment traditional Chinese medicine can promote the transformation of traditional Chinese medicine ingredients, produce beneficial compounds, enhance the efficacy and reduce toxicity, and after fermentation, the effective ingredients in the traditional Chinese medicine produce a synergistic effect with the probiotics, inhibit the growth of pathogenic bacteria in the intestines of animals, and maintain the balance of intestinal microecology.

[0017] The mucus lactobacillus B14.32 of the present invention originates from human vaginal secretions, has good affinity with the human body, is beneficial to improving the bioavailability of the drug, and expands the scope of applicable population; The antidepressant Chinese medicine fermentation composition of the present invention uses Chinese medicinal materials with antidepressant activity, including Chinese jujube seeds, Panax notoginseng, peony, and liquorice. After fermentation by Lactobacillus mucilaginosus B14.32 and yeast, the active ingredients are released and transformed, and the antidepressant effect is more significant, providing a safer and more effective treatment plan for patients with depression. The preparation method of the fermentation composition provided by the present invention can efficiently extract and transform active ingredients from Chinese medicines such as Semen Ziziphi Spinosae, Radix Notoginseng, Radix Paeoniae Rubrae, and Radix Glycyrrhizae through a two-step fermentation method, i.e., a primary fermentation of yeast and a secondary fermentation of Lactobacillus mucilaginosus B14.32. This fermentation process not only avoids the destruction of the active ingredients of Chinese medicines by traditional high-temperature decoction, but also preserves the active ingredients of Chinese medicines to the maximum extent, thereby improving the efficacy of the medicines. Yeast and Lactobacillus mucilaginosus B14.32 can promote the transformation of Chinese medicine ingredients during the fermentation process and produce new beneficial compounds. These compounds not only enhance the efficacy of the medicines, but also reduce the toxicity of Chinese medicines, making the fermentation composition finally obtained safer and more effective. Compared with the traditional Chinese medicine preparation process, the fermentation process of the present invention not only simplifies the production process, but also reduces the production cost. Animal experimental results show that the antidepressant Chinese medicine fermentation composition of the present invention can effectively improve the survival desire, stimulation sensitivity, and inflammatory response in the hippocampal tissue of CUMS depressed mice. This shows that the composition has a significant antidepressant effect and provides a safer and more effective treatment plan for patients with depression. The antidepressant Chinese medicine fermentation composition of the present invention can be prepared into various forms such as oral liquid and freeze-dried products, which are convenient for patients to take. At the same time, since the composition has significant antidepressant effect and good bioavailability, it can be widely used in clinical treatment, health product development, animal husbandry, aquaculture and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Colony morphology of Lactobacillus mucosa B14.32 (a) and Gram staining microscopic examination (b); Figure 2 : Phylogenetic tree of Lactobacillus mucilaginosus B14.32; Figure 3 :The influence of different ratios of bacterial strains on the soluble solids in the antidepressant traditional Chinese medicine fermentation composition; Figure 4 :The effect of antidepressant Chinese medicine fermentation composition on the forced swimming immobility time of CUMS depression model mice; Figure 5 :Effects of antidepressant Chinese medicine fermentation composition on inflammatory factors in hippocampus tissue of CUMS depression model mice. DETAILED DESCRIPTION The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0019] The present invention provides a method for preparing an antidepressant Chinese medicine fermentation composition, and the specific steps are as follows: 1. Raw material pretreatment: Mix and grind the spiny jujube seeds, Panax notoginseng, peony root and liquorice to obtain a medicine powder; 2. Preparation of antidepressant Chinese medicine fermentation composition The drug powder and water were mixed at a solid-liquid ratio of 1:2 to obtain a mixed solution, 10% yeast inoculum was added to the mixed solution, and the mixture was cultured at 28°C for 48 hours after mixing to obtain a primary fermentation solution; Sterile water and 10% lactic acid bacteria inoculum were added to the primary fermentation liquid, and after being shaken well, the mixture was placed in an anaerobic incubator at 37°C for 48 hours to obtain a secondary fermentation liquid. The secondary fermentation liquid was filtered to obtain a filtrate, the filtrate was centrifuged at 6000 r / min for 5 minutes, and the supernatant was taken to obtain the antidepressant Chinese medicine fermentation composition.

[0020] Preferably, the yeast inoculum is commercially available yeast with a live bacterial count of 2.35×10 8 CFU / mL; the lactic acid bacteria inoculum used was Lactobacillus mucilaginosus B14.32, with a live bacterial count of 7.65×10 10 CFU / mL; The depository of Lactobacillus mucosa B14.32 is: General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is: December 25, 2024, the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101; the deposit number is CGMCCNO: 33189, and the classification name is: Limosilactobacillus mucosae .

[0021] The present invention combines microbial fermentation technology with Chinese medicine extraction to obtain a Chinese medicine fermentation composition, which has the effect of anti-depression and reducing brain inflammation in patients with depression. The soluble solid content of the obtained Chinese medicine fermentation composition is more than 39.5%, which is 19.1% higher than the conventional decoction method. It can be obtained from the results of animal experiments that the anti-depressant Chinese medicine fermentation composition mentioned in the present invention can effectively improve the survival desire, stimulation sensitivity and inflammatory response in the hippocampal tissue of CUMS depressed mice, indicating that the Chinese medicine mentioned in the present invention has a significant anti-depressant effect. Therefore, the anti-depressant Chinese medicine fermentation composition mentioned in the present invention can be prepared into an oral liquid, or used to treat depression after freeze-drying and reasonable proportioning with various excipients.

[0022] Example 1: Screening and identification of lactic acid bacteria capable of fermenting Chinese medicine Physiological saline was added to the human vaginal secretion sample, mixed and diluted 10 times in a gradient and spread on MRS solid medium, cultured in an anaerobic incubator at 37°C for 3 days, and single colonies with different morphologies were selected and named and purified by streaking. Single colonies were picked from the purified plate for liquid culture to the logarithmic phase, and then stored in 50% anaerobic glycerol and placed in a -80°C refrigerator for long-term storage.

[0023] Crush and mix 10 parts by weight of Semen Ziziphi Spinosae, 10 parts by weight of Panax Notoginseng, 10 parts by weight of Paeonia lactiflora and 10 parts by weight of Licorice to make a powder for later use.

[0024] The strain obtained by screening was inoculated into 50 ml of MRS medium and cultured at 37°C anaerobic conditions for 24 hours. After the culture was completed, the supernatant was removed by centrifugation at 6000 rpm / min for 5 minutes, and then 30 ml of sterile water and 10 g of drug powder were added. After culturing at 37°C anaerobic conditions for 48 hours, the drug powder fermentation liquid was obtained by centrifugation. The content of soluble solids in the fermentation liquid was determined. The blank control only contained 30 ml of sterile water and 10 g of drug powder. Finally, a strain of mucus Lactobacillus B14.32 that can ferment traditional Chinese medicine was screened. The colony morphology was as follows: Figure 1 shown.

[0025] The 16s rDNA of strain B14.32 was amplified using bacterial universal primers 27F and 1492R and then sent to Shanghai Bioengineering for sequencing. The sequencing results were uploaded to the EZ Biocloud database for comparison and phylogenetic tree was constructed. Figure 2 is the phylogenetic tree of strain B14.32. The results show that strain B14.32 is related to Limosilactobacillus mucosae S32 is the closest relative, with a similarity of 99.19%, so strain B14.32 can be identified as Limosilactobacillus mucosae The depository of Lactobacillus mucosus B14.32 is: General Microbiology Center of China Microbiological Culture Collection Administration, deposit date: December 25, 2024, deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101; deposit number is CGMCC NO: 33189, classification name: Limosilactobacillus mucosa .

[0026] The sequencing sequence of Lactobacillus mucilaginosus B14.32 that can ferment traditional Chinese medicine is shown in sequence 1: Example 2: Effect of Chinese medicine composition ratio on soluble solids Ratio A: Weigh Chinese jujube seeds, Panax notoginseng, peony root and liquorice in a mass ratio of 1:1:1:1, crush and mix well to make medicinal powder, and set aside.

[0027] Ratio B: Weigh Chinese jujube seeds, Panax notoginseng, peony and liquorice in a mass ratio of 2:2:1:1, crush and mix well to make medicinal powder, and set aside.

[0028] Ratio C: Weigh Chinese jujube seeds, Panax notoginseng, peony and licorice in a mass ratio of 2:2:1:0.3, crush and mix them into powder for later use.

[0029] Ratio D: Weigh Chinese jujube seeds, Panax notoginseng, peony root and licorice in a mass ratio of 2:2:1:0.25, crush and mix them into powder for later use.

[0030] Inoculate Lactobacillus mucosa B14.32 into 50 ml of MRS medium and culture it under anaerobic conditions at 37°C for 24 hours. After the culture is completed, centrifuge at 6000 rpm / min for 5 minutes to remove the supernatant, add 30 ml of sterile water and 10 g of the above-mentioned powder, culture it under anaerobic conditions at 37°C for 48 hours, and then centrifuge to obtain the powder fermentation liquid, and determine the content of soluble solids in the fermentation liquid. The blank control only contains 30 ml of sterile water and 10 g of powder.

[0031] Some Chinese medicines with antidepressant activity have water-soluble active ingredients, including polysaccharides, saponins, polyphenols, etc. Studies have shown that fermenting Chinese medicine with probiotics can effectively increase the content of soluble solids and bioactive ingredients in Chinese medicine liquid, while reducing harmful components in the liquid through microbial metabolism. Therefore, the detection of soluble solid content in Chinese medicine liquid can reflect the content of active substances to a certain extent; as shown in Table 1, after fermentation by Lactobacillus mucosa B14.32, the soluble solid content of each group of powders was higher than that of the control group, among which the soluble solid content of ratio D was the highest, so ratio D was selected for subsequent experiments.

[0032] Table 1 Effect of TCM composition ratio on soluble solids

[0033] Example 3: Effect of fermentation on soluble solids of traditional Chinese medicine According to the mass ratio of 2:2:1:0.25, take 80 g of Ziziphus jujuba seeds, 80 g of Panax notoginseng, 40 g of Paeonia lactiflora, and 10 g of Licorice, grind and mix them into medicinal powder and set aside.

[0034] Yeast culture: The purchased commercial yeast (Angel Yeast) was inoculated into YPD liquid culture medium and cultured at 28°C for 48 hours to obtain inoculum. The live yeast count was measured by plate spreading method and was 2.35×10 8 CFU / mL.

[0035] Expansion of mucus lactobacillus: mucus lactobacillus B14.32 was inoculated into MRS liquid culture medium and cultured in an anaerobic incubator at 37°C for 24 hours to obtain an inoculum. The viable bacterial count of mucus lactobacillus B14.32 was measured by the plate spreading method and was 7.65×10 10 CFU / mL.

[0036] Chinese medicine decoction: add water to the above medicinal materials at a mass ratio of 1:10, boil, then simmer on low heat for 1 hour, filter to obtain the medicinal liquid, add sterile water to the medicinal liquid to make it consistent with the volume of the antidepressant Chinese medicine fermentation composition, and set aside.

[0037] Antidepressant Chinese medicine fermentation composition: Mix the powder and water in a mass ratio of 1:2, add 10% yeast inoculum of the total mass of the powder and water, mix well and culture at 28°C for 48h. Take it out, add 8 parts of sterile water and 10% of the total mass of the powder and water of Lactobacillus mucilaginosus B14.32 inoculum, shake well and put it into a 37°C anaerobic incubator for fermentation for 48h. After the fermentation is completed, take it out and filter it to obtain the filtrate. Then centrifuge the filtrate at 6000r / min for 5min, and take the supernatant to obtain the antidepressant Chinese medicine fermentation composition.

[0038] The soluble solid content of the antidepressant Chinese medicine fermentation composition was detected. It can be seen from Table 2 that the soluble solid content of the decoction prepared by the decoction method was 20.40%, and the soluble solid content of the decoction prepared by the fermentation method was 39.52%. This shows that multiple fermentations using yeast and Lactobacillus mucilaginosus B14.32 are more conducive to the dissolution of soluble solids in Chinese medicine, and are more likely to have better biological activity.

[0039] Table 2 Effects of different treatment methods on soluble solids of traditional Chinese medicine

[0040] Example 4: Effects of different ratios of fermentation strains on soluble solids of traditional Chinese medicine The powder, yeast inoculum and Lactobacillus mucilaginosus B14.32 inoculum were prepared according to the method of Example 3. Yeast and Lactobacillus mucilaginosus B14.32 were inoculated and fermented in the inoculation order of Example 1 at an inoculation rate of 10%:0 (Group 1), 10%:10% (Group 2), 10%:20% (Group 3), 20%:10% (Group 4), and 0:10% (Group 5) to determine the effect of different inoculation ratios on the soluble solid content in the powder. Figure 3As shown: Different inoculation amounts have a significant effect on the dissolution of soluble solids, among which the soluble solid content of the inoculation amount of 20%:10% (Group 4) is the highest, reaching 42.50±1.55%. Therefore, this inoculation amount was selected for subsequent experiments.

[0041] Example 5: Preparation of antidepressant Chinese medicinal fermentation composition Prepare the powder, yeast inoculum and Lactobacillus mucilaginosus B14.32 inoculum according to the method of Example 3. Mix the powder with water in a ratio of 1:2, add 20% of the yeast inoculum, mix well and culture at 28°C for 48h, take out, add 10% of the Lactobacillus mucilaginosus B14.32 inoculum, shake well and place in a 37°C anaerobic incubator for fermentation for 48h. After the fermentation is completed, take out and filter to obtain the filtrate. Centrifuge the filtrate at 6000r / min for 5min, and take the supernatant to obtain the antidepressant Chinese medicine fermentation composition.

[0042] Example 6: Animal grouping and model establishment The model was established according to the internationally recognized CUMS depression modeling method. Adaptive feeding was performed one week before the experiment. In the second week of the experiment, the mice in the CUMS model control group were given more than two stimulations per day, and the stimulation was not repeated for one week, which lasted for 4 weeks. After modeling, the mice showed a decrease in body weight, a decrease in sugar water preference, and a decrease in spontaneous activity and exploratory behavior, indicating that the model was successful.

[0043] After the mice with failed modeling were eliminated, the mice with successful modeling were randomly divided into CUMS model group, normal group, positive group (5 mg / kg fluoxetine), low-dose group (10 g / kg antidepressant Chinese medicine fermentation composition), and high-dose group (20 g / kg antidepressant Chinese medicine fermentation composition). The model group and the normal group were gavaged with normal saline once a day before daily stimulation; the positive group was gavaged with 5 mg / kg fluoxetine once a day before daily stimulation; the high-dose group and the low-dose group were gavaged with 20 g / kg and 10 g / kg respectively before daily stimulation, once a day, for 4 weeks, and the administration volume of each group was 20 mL / kg. After the administration, the mice were tested for behavioral characteristics, and the mice were killed and dissected after the behavioral test.

[0044] Example 7: Effect of the antidepressant Chinese medicinal fermentation composition on the body weight of CUMS depression model mice The same electronic scale was used to weigh the mice in each group at the end of the modeling period (day 35) and the end of the drug treatment period (day 64).

[0045] On day 0 of the experiment, there was no statistically significant difference in body weight between the mice in the normal group and the mice in each group (P>0.05). On day 35 of the experiment, the weight gain of mice in the model group was significantly lower than that of mice in the normal group, indicating that the depression model was successfully established; compared with the model group, the weight gain of mice in the positive group and the high-dose group was significant. After 64 days of the experiment, compared with the model group, the weight of mice in the positive group, low-dose group and high-dose group was significantly higher than that in the model group, and there was no significant difference in the weight of mice in the high-dose and positive drug groups, indicating that the antidepressant Chinese medicine fermentation composition can effectively improve the depressive symptoms of mice and restore the weight of mice.

[0046] Table 3 Effect of antidepressant Chinese medicine fermentation composition on body weight of CUMS depression model mice

[0047] Example 8: Effect of the antidepressant Chinese medicinal fermentation composition on sucrose preference in CUMS depression model mice Sucrose preference experiments were conducted on mice at three points: the adaptation period (7th day), the end of modeling (35th day), and the end of drug treatment (64th day). Adaptation training was performed 3 days before each sucrose preference test, that is, in the first 24 hours, each mouse was given 2 bottles of 1% sucrose solution, and in the second 24 hours, each mouse was given 1 bottle of 1% sucrose solution and 1 bottle of drinking water. After each group of mice adapted to the double-bottle water supply, they were deprived of water for 24 hours, and then given 1 bottle of 1% sucrose solution and 1 bottle of drinking water. After 12 hours, the positions of the sugar water bottle and the drinking water bottle were exchanged. After 24 hours, the remaining amount of sucrose water and drinking water was measured with a measuring cylinder, and the consumption of the two was calculated. Sucrose preference rate = sucrose water consumption / (total liquid consumption)*100%.

[0048] On the 7th day of the experiment, compared with the normal group, the percentage of sucrose consumption of mice in the other groups was significantly reduced. After 35 days of the experiment, compared with the model group, the percentage of sucrose consumption in the positive group, low-dose group, and high-dose group was significantly increased, but still lower than the normal group. On the 64th day of the experiment, compared with the model group, the percentage of sucrose consumption in the positive group, low-dose group, and high-dose group was significantly increased, and there was no significant difference between the positive group and the high-dose group and the normal group. The sucrose preference test is a test to evaluate the degree of depression in rodents after the CUMS modeling process. The main feature of depressive rats is anhedonia (reduced sensitivity to stimulation). The results of this experiment show that the antidepressant Chinese medicine fermentation composition can effectively improve the reward stimulation sensitivity of CUMS depression modeling mice to food (sucrose water).

[0049] Table 4 Effect of the antidepressant Chinese medicine fermentation composition on sucrose preference in CUMS depression model mice

[0050] Example 9: Effect of the antidepressant Chinese medicinal fermentation composition on the forced swimming immobility time of CUMS depression model mice After the drug treatment (day 65), the mice were subjected to a forced swimming test to determine the survival desire of each group of mice. The mice were placed in a glass bar with a height of 24 cm and a diameter of 15 cm, ensuring that the tail of the mouse could not touch the bottom of the bucket. Under the water temperature of 25±1℃, the mice were forced to swim for 6 minutes, and the cumulative immobility time of the mice within 4 minutes (recording stage) was observed.

[0051] like Figure 4 As shown: the time that the model group mice were in an immobile state during the recording phase of the 6-min experiment was significantly longer than that of the normal group mice. The time that the positive group, low-dose group, and high-dose group mice were in an immobile state was longer than that of the normal group, but shorter than that of the model group, and all of them were significantly different (P<0.05). The forced swimming test is often used to evaluate the changes in activity and desire for survival of rodents after CUMS depression modeling. The results of this experiment show that the antidepressant Chinese medicine fermentation composition can effectively improve the negative behavior of CUMS depression modeling mice and enhance their desire for survival.

[0052] Example 10: Effects of the antidepressant Chinese medicinal fermentation composition on inflammatory factors in the hippocampus tissue of CUMS depression model mice After the behavioral experiment was completed, mice in each group were anesthetized with 5% sodium pentobarbital and then dissected. The hippocampal tissue was isolated on ice and washed with physiological saline, placed in 2 ml sterile centrifuge tubes, and stored in a -80°C refrigerator.

[0053] Cut the hippocampal tissue into pieces with sterile scissors and forceps on ice, put it into a 2ml sterile centrifuge tube, add physiological saline at a mass volume ratio of 1:9, and then pour in 0.25g grinding beads. Put the centrifuge tube into a grinder at a frequency of 70Hz / s and grind for 1min to prepare a tissue homogenate. Centrifuge at 4℃ and 3000rpm / min for 15min and collect the supernatant. Take the supernatant and detect the content of inflammatory factors (TNF-α, IL-6, IL-1β, IL-10) in hippocampal tissue according to the instructions of the ELISA kit.

[0054] like Figure 5 As shown: Compared with the normal group, the expression of pro-inflammatory factors TNF-α, IL-6, and IL-1β in the hippocampus tissue of the model group mice increased, while the expression of the anti-inflammatory factor IL-10 was significantly reduced in the model group. Low-dose and high-dose anti-depressant Chinese medicine fermentation compositions reduced the synthesis of pro-inflammatory factors TNF-α, IL-6, and IL-1β in CUMS depressed mice, and increased the expression of anti-inflammatory factor IL-10 in CUMS depressed mice, and showed concentration dependence. It shows that the anti-depressant Chinese medicine fermentation composition prepared by the present invention can reduce the inflammatory response in the hippocampus tissue of depressed patients, thereby alleviating depression.

Claims

1. The application of Lactobacillus mucilaginosus B14.32 in fermented Chinese medicine, characterized in that: Lactobacillus mucus B14.32 originates from human vaginal secretions. The depository of Lactobacillus mucus B14.32 is: General Microbiology Center of China Microbiological Culture Collection Administration Committee. The deposit date is December 25, 2024. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO: 33189, and the classification name is: Limosilactobacillus mucosae .

2. A Chinese medicine fermentation agent, characterized in that: Specifically, it is Lactobacillus mucoides B14.

32. Lactobacillus mucoides B14.32 originates from human vaginal secretions. The depository of Lactobacillus mucoides B14.32 is: General Microbiology Center of China Microbiological Culture Collection Administration. The deposit time is: December 25, 2024. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO: 33189, and the classification name is: Limosilactobacillus mucosae .

3. A Chinese medicine fermentation agent according to claim 2, characterized in that: It also includes yeast, which is used for the primary fermentation, and Lactobacillus mucus B14.32 is used for the secondary fermentation. The inoculation amount of yeast is 10%~20%, and the inoculation amount of Lactobacillus mucus B14.32 is 10%~20%.

4. A method for preparing a fermentation composition, characterized in that: The specific steps are as follows: Preparation of yeast inoculum, lactic acid bacteria inoculum and Chinese medicine powder; The Chinese medicine powder is mixed with water to obtain a mixed solution, and yeast inoculum is added to the mixed solution for primary fermentation to obtain a primary fermentation solution; The volume of the primary fermentation liquid is adjusted, and the lactic acid bacteria inoculum is added to carry out secondary fermentation. After the fermentation is completed, a filtrate is obtained; The filtrate is centrifuged and the supernatant is taken as the fermentation composition.

5. The method for preparing a fermentation composition according to claim 4, characterized in that: The Chinese medicine powder is mixed with water in a mass ratio of 1:2 to obtain a mixed solution, and a yeast inoculum with an inoculation amount of 10% to 20% is added to the mixed solution for a fermentation to obtain a primary fermentation solution; wherein, the primary fermentation is performed at a temperature of 28°C for 48 hours.

6. The method for preparing a fermentation composition according to claim 4, characterized in that: The volume of the primary fermentation liquid was adjusted with sterile water, and a lactic acid bacteria inoculum with an inoculum amount of 10% to 20% was added for secondary fermentation. After the fermentation was completed, a filtrate was obtained; wherein, the secondary fermentation was anaerobic fermentation at 37°C for 48 hours.

7. The method for preparing a fermentation composition according to claim 4, characterized in that: The yeast inoculum was commercially available yeast, with a live bacterial count of 2.35×10 8 CFU / mL; the lactic acid bacteria inoculum adopts mucus Lactobacillus B14.32, and the amount of live bacteria is 7.65×10 10 CFU / mL, Lactobacillus mucosus B14.32 originates from human vaginal secretions. The depository of Lactobacillus mucosus B14.32 is: General Microbiology Center of China Microbiological Culture Collection Administration, deposit date: December 25, 2024, deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit number is CGMCC NO: 33189, and classification name is: Limosilactobacillus mucosae .

8. An antidepressant Chinese medicine fermentation composition, characterized in that: The raw materials include Chinese jujube seeds, Panax notoginseng, peony and liquorice, which are crushed and mixed and then fermented using any one of the preparation methods in claims 4 to 7.

9. The antidepressant Chinese medicinal fermentation composition according to claim 8, characterized in that: The raw materials include Chinese jujube seeds, Panax notoginseng, peony and liquorice in a mass ratio of 1~2:1~2:1:0.25~1.

10. The antidepressant Chinese medicinal fermentation composition according to claim 8, characterized in that: The inoculation amount of the yeast inoculum is 20%, and the inoculation amount of the Lactobacillus mucilaginosus B14.32 is 10%.

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