Compound probiotic preparation containing natural extracts and used for treating diarrhea and preparation method of compound probiotic preparation

Abstract

The invention discloses a compound probiotic preparation containing natural extracts and used for treating diarrhea and a preparation method of the compound probiotic preparation. The compound probiotic preparation contains a component A, a component B and a component C. The component A is a fermentation extract, the component B is a schisandra chinensis extract, and the component C is probiotic powder. The compound probiotics, the natural extract and the fermentation extract cooperate with one another, play respective advantages and generate a synergistic effect, and compared with a single probiotic preparation or a natural plant extract, the compound microbial agent provided by the invention shows a remarkable curative effect in the aspect of treating diarrhea, can more quickly relieve diarrhea symptoms, and has a good application prospect. The intestinal function recovery is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine preparation, in particular to a compound probiotic preparation containing natural extracts for treating diarrhea and a preparation method thereof. BACKGROUND

[0002] Diarrhea is a common digestive system disease, which brings great trouble to people's health and life. Diarrhea can be caused by various reasons, such as bacterial or viral infection, improper diet, intestinal flora imbalance, etc. At present, the drugs for treating diarrhea mainly include antibiotics, antidiarrheal drugs, etc. Antibiotics are mainly used for diarrhea caused by bacterial infection, however, long-term use of antibiotics can easily lead to drug resistance problem, and can also destroy the balance of normal flora in the intestine, further causing more serious intestinal problems. Traditional antidiarrheal drugs such as montmorillonite powder, etc. can adsorb intestinal pathogenic bacteria and water to achieve the purpose of stopping diarrhea, which can relieve symptoms to a certain extent, but cannot fundamentally relieve intestinal flora imbalance and intestinal function disorder, and the treatment effect is limited.

[0003] It has been shown that probiotics (such as lactobacillus and bifidobacterium) can regulate intestinal microenvironment and inhibit the proliferation of pathogenic bacteria, so probiotics are often used to treat gastrointestinal diseases such as diarrhea. However, the colonization ability and survival time of existing probiotic preparations in the intestine need to be improved, and the treatment effect of diarrhea is still not ideal. Plants contain many active functional components, such as polysaccharides, flavonoids, alkaloids, etc., which have many functions such as antibacterial, anti-inflammatory, regulation of intestinal function, etc., therefore, plant extracts are often used for the treatment of diseases. However, the single target of pure natural plant extracts is relatively single, and the treatment effect on diarrhea is limited. Therefore, exploring and developing new and efficient strategies for treating diarrhea, especially combining probiotics with plant active ingredients, and regulating intestinal health to treat diarrhea through multi-target and multi-pathway synergistic effect, has become an important direction of current research. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a compound probiotic preparation containing natural extracts for treating diarrhea and a preparation method thereof, so as to solve the deficiencies of the existing drugs for treating diarrhea and improve the treatment effect of diarrhea.

[0005] The technical scheme of the present application is as follows:

[0006] A compound probiotic preparation containing natural extracts for treating diarrhea, the compound probiotic preparation contains component A, component B and component C, wherein component A is a fermentation extract, component B is a schisandra chinensis extract, and component C is a probiotic powder;

[0007] The active ingredients in the natural extract and the fermentation extract and the probiotics all have the effect of enhancing intestinal immunity, which can improve the resistance of the body to pathogens and reduce the frequency of diarrhea.

[0008] The preparation method of the fermentation extract comprises the following steps:

[0009] The radix arisaematis is crushed into fine powder, the radix arisaematis fine powder, glucose, beef extract and deionized water are mixed, sterilized to obtain a fermentation medium; Bacillus coagulans and / or Lactobacillus mucosus are inoculated into the fermentation medium for fermentation culture, and after the fermentation is completed, the supernatant is obtained by centrifugation; the organic solvent is added to the supernatant for extraction to obtain the fermentation extract.

[0010] The present scheme utilizes the fermentation of radix arisaematis by probiotics Bacillus coagulans and Lactobacillus mucosus, and extracts the fermentation product to obtain the fermentation secondary metabolite, which can inhibit the growth of harmful bacteria, maintain the balance of intestinal flora, repair the intestinal barrier, and thus achieve the effect of treating diarrhea.

[0011] Preferably, the mass ratio of the component A, the component B and the component C is 2-3:1:7-9.

[0012] More preferably, the mass ratio of the component A, the component B and the component C is 2:1:7.

[0013] Preferably, the preparation method of the Schisandra chinensis extract comprises the following steps:

[0014] After the traditional Chinese medicine Schisandra chinensis is crushed, dimethyl sulfoxide is added, stirring extraction is carried out at 65±2℃ for at least 1h, centrifugation is carried out, the supernatant is collected, and the solvent is removed by vacuum concentration to obtain the Schisandra chinensis extract.

[0015] Preferably, the organic solvent is dimethyl sulfoxide, and the extraction time is at least 1h.

[0016] Preferably, the mass / volume ratio of the radix arisaematis fine powder, glucose, beef extract and deionized water is 12g:20g:5g:1000mL.

[0017] Preferably, the fermentation culture is anaerobic culture at 37±2℃ for 72h.

[0018] Preferably, the volume ratio of the supernatant to the organic solvent is 1:10.

[0019] Preferably, the volume ratio of Schisandra chinensis to dimethyl sulfoxide is 1:10.

[0020] Preferably, the probiotic powder is composed of Lactobacillus acidophilus powder and Bifidobacterium breve powder.

[0021] Preferably, the Lactobacillus acidophilus powder and the Bifidobacterium breve powder are used together, which can effectively regulate the balance of intestinal flora, increase the number of beneficial bacteria, inhibit the growth and reproduction of harmful bacteria, and restore the normal intestinal microecological environment.

[0022] The beneficial results of the present application compared with the prior art are:

[0023] The present application plays the advantages of each other and produces synergistic effect through the cooperation of composite probiotics, natural extracts and fermented extracts. Compared with single probiotic preparation or natural plant extract, the composite microbial agent provided in the present application shows significant efficacy in treating diarrhea, can relieve diarrhea symptoms faster and promote the recovery of intestinal function.

[0024] The composite probiotic agent described in the present application is only composed of probiotics, fermented extracts and plant extracts, has fewer components, and the preparation methods of fermented extracts and plant extracts are relatively simple. Therefore, the preparation process of the present application is simple, the cost is low, and it is more conducive to industrialized production and popularization and application. DETAILED DESCRIPTION

[0025] In order to better understand the technical content of the present application, the present application will be further described below in combination with specific examples.

[0026] The agent source information described in the following examples is as follows: Bacillus coagulans (SHMCC D80587, Shanghai Center for Bio-technology Collection), Limosilactobacillus fermentum (SHMCC D24912, Shanghai Center for Bio-technology Collection), Lactobacillus acidophilus (SHMCC D82496, Shanghai Center for Bio-technology Collection), Bifidobacterium breve (SHMCC D82744, Shanghai Center for Bio-technology Collection).

[0027] Example 1:

[0028] The composite probiotic preparation containing natural extracts is composed of component A, component B and component C with a mass ratio of 2:1:7, wherein component A is a fermented extract, component B is a Schisandra chinensis extract, and component C is a probiotic powder.

[0029] Preparation of fermented extract:

[0030] Step S1: The traditional Chinese medicine Aralia cordata was pulverized into fine powder, 12 g of Aralia cordata fine powder, 20 g of glucose, 5 g of beef extract and 1000 mL of deionized water were mixed, and then the mixture was sterilized at 121℃ for 20 min to obtain a fermentation medium.

[0031] Step S2: 5 w / w% of activated Bacillus coagulans liquid (4.0 x 10 9CFU / mL) and fermented Lactobacillus muiyi liquid (4.0 x 10 9 CFU / mL) was inoculated into the fermentation medium and cultured anaerobically at 37°C for 72 h.

[0032] Step S3: After fermentation, the supernatant was collected by centrifugation at 8000 r / min for 15 min, DMSO (dimethyl sulfoxide) was added at a volume ratio of 1:10, and the mixture was stirred at room temperature for 1 h. Then, the mixture was centrifuged at 10000 r / min for 20 min, and the supernatant was collected and concentrated under reduced pressure at a temperature of 45-55°C to remove the solvent, thereby obtaining the fermentation extract.

[0033] Preparation of Schisandra extract:

[0034] The Schisandra extract was prepared by crushing the Schisandra, adding 10 times the volume of dimethyl sulfoxide, stirring at 65±2°C for 1 h, centrifuging at 8000 r / min for 15 min, collecting the supernatant, and concentrating under reduced pressure at a temperature of 45-55°C to remove the solvent.

[0035] Probiotic powder: Lactobacillus acidophilus powder + Bifidobacterium breve powder, with 1 x 10 9 CFU / g of live Lactobacillus and 1 x 10 9 CFU / g of live Bifidobacterium.

[0036] Example 2:

[0037] The complex probiotic preparation containing natural extracts consists of components A, B and C in a mass ratio of 3:1:9, wherein component A is a fermentation extract, component B is a Schisandra extract, and component C is a probiotic powder. The preparation methods of the components are the same as in Example 1.

[0038] Example 3:

[0039] The complex probiotic preparation containing natural extracts consists of components A, B and C in a mass ratio of 2:1:7, wherein component A is a fermentation extract, component B is a Schisandra extract, and component C is a probiotic powder. Components B and C are the same as in Example 1.

[0040] Preparation of fermentation extract:

[0041] Step S1: The traditional Chinese medicine Aralia rotundata was crushed into fine powder. 12 g of Aralia rotundata fine powder, 20 g of glucose, and 5 g of beef extract were mixed with 1000 mL of deionized water, and the mixture was sterilized at 121°C for 20 min to obtain a fermentation medium.

[0042] Step S2: inoculate the activated Bacillus coagulans bacterial solution (4.0 x 10 9 CFU / mL before inoculation) into the fermentation medium at an inoculation amount of 5 w / w%, and place it in an anaerobic environment at 37°C for 72h.

[0043] Step S3: after the fermentation is completed, centrifuge at 8000r / min for 15min, collect the supernatant, add DMSO (dimethyl sulfoxide) at a volume ratio of 1:10, stir at room temperature for 1h, then centrifuge at 10000r / min for 20min, collect the supernatant, and concentrate and remove the solvent at a temperature of 45°C-55°C under reduced pressure to obtain the fermentation extract.

[0044] Preparation of Schisandra extract:

[0045] After the traditional Chinese medicine Schisandra is crushed, 10 times the volume of dimethyl sulfoxide is added, and stirred at 65±2°C for 1h, centrifuged at 8000r / min for 15min, the supernatant is collected, and the solvent is concentrated and removed at a temperature of 45°C-55°C under reduced pressure to obtain the Schisandra extract.

[0046] Probiotic powder: Lactobacillus acidophilus powder + Bifidobacterium breve powder, the number of viable Lactobacillus in the mixed bacterial powder is 1 x 10 9 CFU / g, and the number of viable Bifidobacterium is 1 x 10 9 CFU / g.

[0047] Example 4:

[0048] The natural extract-containing composite probiotic preparation consists of components A, B and C in a mass ratio of 2:1:7, wherein component A is a fermentation extract, component B is a traditional Chinese medicine Schisandra extract, and component C is a probiotic powder. Components B and C are the same as in Example 1.

[0049] Preparation of fermentation extract:

[0050] Step S1: crush the traditional Chinese medicine Aralia rotundata into fine powder, mix 12g of Aralia rotundata fine powder, 20g of glucose, and 5g of beef extract with 1000mL of deionized water, and sterilize the mixture in a fermentation bottle at 121°C for 20min to obtain a fermentation medium.

[0051] Step S2: inoculate the activated Lactobacillus mucosus bacterial solution (4.0 x 10 9 CFU / mL before inoculation) into the fermentation medium at an inoculation amount of 5 w / w%, and place it in an anaerobic environment at 37°C for 72h.

[0052] Step S3: After fermentation, centrifuge at 8000 r / min for 15 min, collect the supernatant, add DMSO (dimethyl sulfoxide) at a volume ratio of 1:10, stir at room temperature for 1 h, then centrifuge at 10000 r / min for 20 min, collect the supernatant, and concentrate under reduced pressure at a temperature of 45-55°C to remove the solvent to obtain the fermentation extract.

[0053] Preparation of Schisandra extract:

[0054] After the traditional Chinese medicine Schisandra was crushed, 10 times the volume of dimethyl sulfoxide was added, stirred at 65±2°C for 1 h, centrifuged at 8000 r / min for 15 min, the supernatant was collected, and the solvent was removed by concentration under reduced pressure at a temperature of 45-55°C to obtain the Schisandra extract.

[0055] Probiotic powder: Lactobacillus acidophilus powder + Bifidobacterium breve powder, the number of viable Lactobacillus in the mixed bacterial powder is 1×10 9 CFU / g, and the number of viable Bifidobacterium is 1×10 9 CFU / g.

[0056] Compared with Example 1, Example 2 increases the proportion of Component A and Component C, Example 3 uses single-strain Bacillus coagulans for fermentation, and Example 4 uses single-strain Lactobacillus mucosus for fermentation.

[0057] Comparative Example 1:

[0058] The complex probiotic preparation containing natural extracts consists of Component A, Component B and Component C at a mass ratio of 2:1:7, wherein Component A is fine powder of Sarcandra glabra, Component B is Schisandra extract, and Component C is probiotic powder.

[0059] Preparation of Schisandra extract:

[0060] After the traditional Chinese medicine Schisandra was crushed, 10 times the volume of dimethyl sulfoxide was added, stirred at 65±2°C for 1 h, centrifuged at 8000 r / min for 15 min, the supernatant was collected, and the solvent was removed by concentration under reduced pressure at a temperature of 45-55°C to obtain the Schisandra extract.

[0061] Probiotic powder: Lactobacillus acidophilus powder + Bifidobacterium breve powder, the number of viable Lactobacillus in the mixed bacterial powder is 1×10 9 CFU / g, and the number of viable Bifidobacterium is 1×10 9 CFU / g.

[0062] Comparative Example 2:

[0063] The complex probiotic preparation containing natural extracts consists of Component A and Component C at a mass ratio of 2:7, wherein Component A is fermentation extract, and Component C is probiotic powder.

[0064] The preparation method of the fermentation extract is the same as that of Example 1, and the probiotic powder is the same as that of Example 1.

[0065] Comparative Example 3:

[0066] The composite probiotic preparation: Lactobacillus acidophilus powder + Bifidobacterium breve powder, the viable count of Lactobacillus in the mixed powder is 1 x 10 9 CFU / g, and the viable count of Bifidobacterium is 1 x 10 9 CFU / g.

[0067] Comparative Example 1 replaces the fermentation extract with fine powder of Kudzu vine, Comparative Example 2 omits the Schisandra extract, and Comparative Example 3 omits the fermentation extract and the Schisandra extract, compared with Example 1.

[0068] Experimental Example:

[0069] Antibiotics can cause damage to the intestinal mucosa and intestinal microbial flora imbalance, thereby causing diarrhea, which is called antibiotic-associated diarrhea. In this experiment, an antibiotic-associated diarrhea mouse model was established, and the above-described bacterial agents were used for treatment to evaluate the treatment effects of each group.

[0070] Experimental Method:

[0071] Prepare experimental mice (SPF level, 6-week-old, 18-20 g BALB / C mice), and divide the mice into a blank group, a model group, and an experimental group, with 10 mice in each group and half male and half female. The model group and the experimental group are given a mixture of antibiotics (clindamycin 0.8 g / kg + ampicillin sodium 0.9 g / kg + streptomycin 0.5 g / kg) at 0.1 mL / 10 g by gavage once a day for 7 days (success criteria for modeling: compared with the blank group, the mice have rough hair, poor mental state, moist hair around the anus, and obvious dirt). The blank group is given an equal amount of phosphate buffer by gavage.

[0072] After the modeling is completed, the experimental group is given a bacterial agent 1.85 g / kg at 0.1 mL / 10 g, and the blank group and the model group are given phosphate buffer, once a day for 7 days. During the experiment, the mice are normally given food and water for free feeding.

[0073] Index Monitoring:

[0074] The diarrhea condition is evaluated by the water content of feces and the consistency of feces. The consistency of feces is scored as follows: a. shaped, the feces maintain their shape, brown, 1 point; b. semi-shaped or soft, not flowing, yellow, 2 points; c. liquid and unshaped, yellow, 3 points. Fresh feces of the mice in the cage within 2 hours are collected, weighed, and then baked at 100°C for 2 hours, weighed again, and the water content of the feces is calculated.

[0075] Intestinal tight junction proteins occludin and ZO-1 are important proteins on the intestinal tract, which play a role in maintaining the intestinal barrier function. Dysfunction thereof can cause or aggravate diarrhea. The abundance of occludin and ZO-1 can reflect the treatment effect of diarrhea. The relative expression level of mRNA of intestinal barrier tight junction proteins occludin and ZO-1 is detected by RT-qPCR. β-actin is used as an internal reference.

[0076] Table 1

[0077]

[0078]

[0079] Experimental results

[0080] The results in Table 1 show that, compared with the blank group, the relative expression level of mRNA of occludin and ZO-1 in the colon of the model group mice was significantly down-regulated (P<0.01), indicating that the intestinal barrier function was impaired. The stool consistency and fecal water content of the model group were significantly higher than those of the blank group (P<0.01), and the diarrhea was severe. Compared with the model group, the relative expression levels of mRNA of occludin and ZO-1 in the experimental group were significantly up-regulated (P<0.05), and the stool consistency and fecal water content were significantly reduced, indicating that the experimental group could alleviate the intestinal barrier function damage of the diarrhea mice and reduce the intestinal mucosal permeability, thereby playing a role in treating diarrhea.

[0081] Compared with Example 1 and Comparative Example 3, Comparative Example 1 used Artemisia japonica powder instead of the fermentation extract, not only the effect of treating diarrhea was significantly weakened, but also worse than Comparative Example 3 which lacked fermentation and Schisandra extract. It is speculated that the possible reason is that Artemisia japonica is cold in nature, and direct use not only cannot play a role in treating diarrhea, but also has a negative effect, reducing the treatment effect of the complex probiotic. Therefore, the fermentation is the core component of the scheme of the present application.

[0082] Compared with Example 1, the treatment effect of the complex probiotic agent of Comparative Example 2 lacking Schisandra extract was significantly weakened, which showed that the Schisandra extract had a certain effect in treating diarrhea.

[0083] Compared with Example 1 (double-strain fermentation to prepare fermentation), the stool consistency score, fecal water content and the relative expression level of mRNA of intestinal barrier tight junction proteins of Example 3 and Example 4 using single-strain fermentation were significantly higher, and therefore, the preferred scheme of the present application is to use Bacillus coagulans and Lactobacillus gasseri to prepare fermentation by double-strain fermentation.

[0084] The above-mentioned content is only part of the implementation cases of the present application, and the purpose is not to limit the protection scope of the present application; as long as it meets the spirit and basic criteria of the present application, any modification, equivalent replacement, optimization improvement and the like of the technical solution belong to the scope of legal protection of the present application.

Claims

1. A natural extract containing probiotic formulation for the treatment of diarrhea, characterized in that, The composite probiotic preparation contains component A, component B and component C, wherein component A is a fermentation extract, component B is a Schisandra extract, and component C is a probiotic powder; The preparation method of the fermentation extract comprises the following steps: The arisaema cum bengalense is crushed into fine powder, the arisaema cum bengalense fine powder, glucose, beef extract and deionized water are mixed, sterilized to obtain a fermentation medium; Bacillus coagulans liquid and / or Lactobacillus mucosus liquid are inoculated into the fermentation medium for fermentation culture, and after the fermentation is completed, the supernatant is obtained by centrifugation; an organic solvent is added to the supernatant for extraction to obtain the fermentation extract.

2. The composite probiotic formulation according to claim 1, characterized in that, The mass ratio of the component A, the component B and the component C is 2-3:1:7-9.

3. The composite probiotic formulation according to claim 1, characterized in that, The mass ratio of the component A, the component B and the component C is 2:1:

7.

4. The composite probiotic formulation according to claim 1, characterized in that, The preparation method of the Schisandra extract comprises the following steps: After the traditional Chinese medicine Schisandra is crushed, dimethyl sulfoxide is added, stirring extraction is carried out at 65±2℃ for at least 1h, centrifugation is carried out, the supernatant is collected, and the solvent is removed by concentration under reduced pressure to obtain the Schisandra extract.

5. The composite probiotic formulation according to claim 1, wherein, The organic solvent is dimethyl sulfoxide, and the extraction time is at least 1h.

6. The composite probiotic formulation according to claim 1, wherein, The mass / volume ratio of the arisaema cum bengalense fine powder, glucose, beef extract and deionized water is 12g:20g:5g:1000mL.

7. The composite probiotic formulation according to claim 1, wherein, The fermentation culture is anaerobic culture at 37±2℃ for 72h.

8. The composite probiotic formulation according to claim 1, characterized in that, The volume ratio of the supernatant to the organic solvent is 1:

10.

9. The composite probiotic formulation as claimed in claim 4, wherein, The volume ratio of the Schisandra to dimethyl sulfoxide is 1:

10.

10. The composite probiotic formulation according to claim 1, characterized in that, The probiotic powder is composed of lactobacillus acidophilus powder and bifidobacterium breve powder.

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