A fecal dissolving liquid having a protective effect on intestinal flora

Abstract

The present invention is a feces dissolving liquid having a protective effect on intestinal flora, the dissolving liquid comprising: 0.01%-1% vitamin C, 1%-20% polydextrose, 1%-8% trehalose, 0.3%-10% sorbitol, 0.3%-10% resistant dextrin, and the balance being normal saline. The present invention has a good protective effect on anaerobic bacteria during the homogenization process, and can also improve the activity level of fecal bacteria during the long-term freezing of oral capsules.

Description

Technical Field

[0001] The invention relates to the field of intestinal flora dissolving liquid, and in particular to a feces dissolving liquid having a protective effect on intestinal flora. Background Art

[0002] Fecal microbiota transplantation (FMT) is defined as transplanting the functional flora in the feces of healthy people into the gastrointestinal tract of patients to rebuild new intestinal flora and achieve the treatment of intestinal and extra-intestinal diseases. Fecal microbiota transplantation has considerable advantages in the treatment of intestinal diseases such as Clostridium difficile infection and inflammatory bowel disease. It has a series of advantages such as rapid effect, few side effects, and low recurrence rate.

[0003] The existing fecal microbiota transplantation treatment methods are mainly oral capsules and invasive treatment. Among them, invasive treatment is to infuse bacterial solution through nasogastric tubes, colonoscopy, etc. Oral capsules are prepared by pre-preparing fecal microbiota into oral capsules, and then transplanted by oral delivery during treatment. Compared with invasive treatment, oral capsules have significant advantages such as high patient acceptance, convenient treatment, and high colonization rate.

[0004] The preparation process of oral capsules requires a series of steps such as stirring, dissolving, filtering, and concentration. Each step and the long-term freezing of oral capsules in a -80°C environment may affect the activity of fecal bacteria capsules, thereby reducing the therapeutic effect. Therefore, the optimization of the oral capsule preparation process and the maintenance of a high activity level of the capsules during long-term freezing are both corresponding process difficulties.

[0005] The human intestine is mainly composed of anaerobic bacteria, and oral capsules need to go through a homogenization process for a considerable period of time during preparation, which can easily lead to the death of intestinal anaerobic bacteria. At present, the intestinal flora extraction process uses physiological saline as a dissolving liquid for homogenization production. Physiological saline has no protective effect on intestinal flora, especially anaerobic bacteria, which will greatly affect the therapeutic effect of oral capsules.

[0006] Therefore, it is urgent to upgrade and optimize the existing commonly used dissolving solution (normal saline) in order to achieve a good protective effect during the homogenization process, while also improving the activity level of oral capsule fecal bacteria. Summary of the invention

[0007] To solve the above problems, the primary purpose of the present invention is to provide a fecal dissolving solution that has a protective effect on intestinal flora. The dissolving solution can have a good protective effect on anaerobic bacteria during the homogenization process, and can also improve the activity level of fecal bacteria during long-term frozen storage of oral capsules.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A feces dissolving liquid having a protective effect on intestinal flora, wherein the dissolving liquid comprises, by mass percentage, 0.01%-1% vitamin C, 1%-20% polydextrose, 1%-8% trehalose, 0.3%-10% sorbitol, 0.3%-10% resistant dextrin, and the remainder is physiological saline.

[0010] The dosage of the physiological saline is 51%-97.39%.

[0011] Furthermore, the pH of the dissolving solution is 5.5-9.0.

[0012] The solution needs to be sterilized in a high pressure steam sterilizer at 121°C and then cooled to room temperature before use. High pressure steam sterilization can eliminate the interference of other bacteria contained in the water and further discharge the oxygen molecules contained in the water, thereby constructing an oxygen-free environment.

[0013] After in-depth research, the applicant found that the intestinal flora contained in feces is mainly composed of Ruminococcus, Bacteroides, and Prevotella. Among them, anaerobic bacteria occupy the vast majority, and aerobic bacteria and facultative anaerobic bacteria occupy only a small part; simple physiological saline still contains a certain amount of oxygen, so it cannot protect anaerobic bacteria. The present application uses a dissolving solution composed of vitamin C, polydextrose, trehalose, sorbitol, and resistant dextrin to reduce the dissolved oxygen content in the water and construct a low-oxygen or anaerobic environment, thereby protecting anaerobic bacteria. Vitamin C is a good reducing agent that can react with oxygen in the solution, thereby consuming the oxygen in the solution and reducing the dissolved oxygen content to protect anaerobic bacteria.

[0014] Therefore, the beneficial effects of the present invention are:

[0015] The dissolving liquid of the present invention can better protect anaerobic bacteria in the homogenization stage, and has a good protective effect on anaerobic bacteria, so that the extracted bacterial mud has higher activity; at the same time, after being filled into oral bacterial capsules, the components of the dissolving liquid remaining in the bacterial mud can continue to play the role of a protective agent when frozen at -80°C, so that the survival rate of the oral capsules is maintained at more than 80% within 30 days of storage, and the activity level of fecal bacteria during the long-term freezing of the oral capsules can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the activity detection data of stool sample No. 1 of the present invention.

[0017] Figure 2 This is a schematic diagram of the activity detection data of stool sample No. 2 of the present invention.

[0018] Figure 3 This is a schematic diagram of the activity detection data of stool sample No. 3 of the present invention.

[0019] Figure 4 This is a schematic diagram of the survival rate of the No. 1 stool sample capsule cryopreservation of the present invention.

[0020] Figure 5 It is a schematic diagram of the survival rate of the No. 2 stool sample capsule cryopreservation of the present invention.

[0021] Figure 6 It is a schematic diagram of the survival rate of the No. 3 stool sample capsule cryopreservation of the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] The feces dissolving liquid with protective effect on intestinal flora realized by the present invention comprises, according to mass percentage, 0.01%-1% vitamin C, 1%-20% polydextrose, 1%-8% trehalose, 0.3%-10% sorbitol, 0.3%-10% resistant dextrin, 51%-97.39% physiological saline, and pH 5.5-9.0.

[0024] The implementation of the present invention is described below in conjunction with experiments.

[0025] 1. Test materials

[0026] 1.1 Test equipment

[0027] Water bath, ice maker, -80℃ refrigerator, vortex oscillator, centrifuge, magnetic stirrer, autoclave.

[0028] 1.2 Test reagents:

[0029] Physiological saline, vitamin C, polydextrose, trehalose, sorbitol, resistant dextrin, LIVE / DEAD TM BacLight TM Bacterial Viability Kit dead or alive fluorescent dye.

[0030] 1.3 Test materials

[0031] Donor fresh feces and prepare protective solution.

[0032] 2. Test methods

[0033] 2.1 Dissolving solution composition

[0034] New dissolving solution formula (dissolving solution A): 0.01%-1% vitamin C, 1%-20% polydextrose, 1%-8% trehalose, 0.3%-10% sorbitol, 0.3%-10% resistant dextrin, 51%-97.39% normal saline, pH 5.5-9.0.

[0035] Original dissolving solution formula (dissolving solution B): 0.9% NaCl solution (normal saline).

[0036] Sterilize both solution A and solution B in a high pressure steam sterilizer at 121°C for 20 min, and cool to room temperature for later use.

[0037] 2.2 Fecal bacteria sample preparation

[0038] Weigh the weight of the donor's fresh feces and divide it into two equal parts. One group of feces was added with dissolving solution A at a ratio of 1:5, recorded as group A. The other group of feces was added with dissolving solution B at a ratio of 1:5, recorded as group B. The two groups were homogenized at 150rpm for 30min. Then, they were filtered through 20 mesh, 50 mesh, 100 mesh, 150 mesh, 200 mesh, and 250 mesh sieves to remove large particles. The filtrate obtained by filtration was centrifuged at 5000×g and 4℃ for 5min, and the supernatant was discarded. The bacterial sludge of group A was resuspended with dissolving solution A at a ratio of 1:2, and the bacterial sludge of group B was resuspended with dissolving solution B at a ratio of 1:2, and the bacterial suspension was centrifuged again at 5000×g and 4℃ for 5min, the supernatant was discarded, and the bacterial sludge was collected.

[0039] 2.3 Initial activity detection of bacterial sludge

[0040] Weigh the fecal sample stored in the fecal sample preparation step, dissolve it with saline at a ratio of 1:9, and mix evenly with shaking to prepare a fecal suspension. Pipette 3 portions of 1 ml of the suspension into 3 centrifuge tubes, and dilute it 10 times with saline again to obtain a 100-fold fecal dilution. TM BacLight TM The feces were stained with 100-fold dilutions of Bacterial Viability Kit dead or alive fluorescent dye, and the cells were counted using a BD AccuriC6 PLUS flow cytometer after a 15-min dark reaction.

[0041] After centrifugation and mixing of the bacterial sludge in groups A and B, 3 portions of 1 g of bacterial sludge were respectively placed in centrifuge tubes, diluted 100 times with saline, and then treated with LIVE / DEAD TM BacLight TM The 100-fold dilution of bacterial sludge was stained with the Bacterial Viability Kit live or dead fluorescent dye, and the cells were counted using a BD Accuri C6 PLUS flow cytometer after a dark reaction for 15 minutes.

[0042] 2.4 Oral capsule storage and activity tracking

[0043] The A and B groups of bacterial sludge were filled into capsules and stored in a -80°C ultra-low temperature refrigerator. Three capsules were randomly taken out on the 3rd, 7th, 14th, and 30th days, thawed in a 37°C water bath for 5 minutes, and 0.1g of bacterial sludge was taken from each capsule, diluted 100 times with normal saline, and then frozen with LIVE / DEAD. TM BacLight TM The 100-fold dilution of bacterial sludge was stained with the Bacterial Viability Kit live or dead fluorescent dye, and the cells were counted using a BD Accuri C6 PLUS flow cytometer after a dark reaction for 15 minutes.

[0044] 2.5 Data Analysis

[0045] Graphpad Prism was used to process the data. Two-tailed unpaired Student's t test with Welch's correction was used to evaluate the differences between two independent groups. The significance level of the difference was indicated by *p<0.05, **p<0.01, ***p<0.001.

[0046] 3. Experimental results

[0047] 3.1 Initial activity of bacterial sludge

[0048] The results of the bacterial sludge activity test of groups A and B are as follows Figure 1 , 2 , 3. After being prepared into bacterial sludge, the activity of bacterial sludge in group A (new dissolution solution) was significantly higher than that in group B (original dissolution solution), that is, the new protective solution played a considerable protective role on the bacterial community during the homogenization process.

[0049] 3.2 Oral capsule storage and activity tracking

[0050] The results of the activity test of oral capsules in groups A and B are as follows Figure 1 , 2 , 3. During the preparation of capsules and freezing at -80℃, the activity of oral capsules in group A (new solution) was significantly higher than that in group B (original solution). Figure 4 , 5 , 6), the survival rate of group A within 30 days remained above 80%, while the survival rate of group B was only 40%-60%.

[0051] Results and Discussion

[0052] The dissolving solution in Group A can better protect anaerobic bacteria during the homogenization stage, making the extracted bacterial mud more active. At the same time, after being filled into oral bacterial capsules, the components of the dissolving solution remaining in the bacterial mud can continue to act as a protective agent when frozen at -80°C, so that the survival rate of the oral capsules remains above 80% within 30 days of storage. Therefore, compared with the original dissolving solution, the new dissolving solution can have a good protective effect on fecal bacteria whether in the homogenization process or the -80°C freezing process.

[0053] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and 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 feces dissolving liquid having a protective effect on intestinal flora, characterized in that According to the mass percentage, the dissolving solution is composed of the following substances: 0.01%-1% vitamin C, 1%-20% polydextrose, 1%-8% trehalose, 0.3%-10% sorbitol, 0.3%-10% resistant dextrin, and the amount of normal saline is 51%-97.39%; The pH of the dissolving solution is 5.5-9.

0.

2. The feces dissolving liquid having a protective effect on intestinal flora according to claim 1, characterized in that The dissolving solution needs to be sterilized in a high pressure steam sterilizer and then cooled to room temperature before use.

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