A method for improving the purity of intestinal flora using macroporous resin

Abstract

The present invention is a method for improving the purity of intestinal flora by using macroporous resin, wherein the method obtains an intestinal flora suspension sample, and then purifies the sample by wet column loading with food-grade macroporous resin. The present invention utilizes the adsorption of macroporous resin to remove other components other than intestinal flora, thereby achieving the purpose of increasing the bacterial content in fecal sludge.

Description

Technical Field

[0001] The present invention relates to the field of purifying intestinal flora in FMT, and in particular to a method for improving the purity of intestinal flora by using macroporous resin. Background Art

[0002] Fecal microbiota transplantation (FMT) is an effective method to reconstruct the patient's overall intestinal microbiota with a wide range of therapeutic applications. It has been recommended in clinical guidelines, consensus, and joint experts for the treatment of recurrent Clostridium difficile infection (CDI). More and more experimental studies and literature have shown that FMT also has important therapeutic value for diseases other than CDI, such as ulcerative colitis, Crohn's disease (CDI), hepatic encephalopathy, and autism.

[0003] However, this method still has many problems. So far, most studies and consensus recommend that doctors determine the therapeutic dose of microbiota based on the weight of the donor's feces. Many studies recommend a single dose of 30-50 grams of feces to treat patients. How to increase the unit bacteria in feces so that patients can take less feces has become an urgent problem to be solved.

[0004] Macroporous adsorption resin, also known as macroporous resin, is a new type of polymer adsorption material produced in the 1960s. It is spherical particles with a macroporous network structure. The particle size is concentrated between 0.25-0.84mm. It has a certain adsorption capacity for organic matter and substances with smaller molecular weight.

[0005] At present, the functional bacteria used in FMT are generally collected from the feces of healthy people after washing and filtration. The bacterial sludge contains more impurities, and the bacteria account for about half of the sludge. The rest is undigested cellulose in food, upper gastrointestinal secretions, connective tissue, mucus, bile pigments, mucin, digestive juices, fragments of gastrointestinal mucosa shed, and epithelial cells.

[0006] However, there are no reports on the application of macroporous resins in FMT to purify intestinal flora. Summary of the invention

[0007] To solve the above problems, the purpose of the present invention is to provide a method for improving the purity of intestinal flora by using macroporous resin. This method is applied to FMT, and the adsorption of macroporous resin is used to remove other components other than intestinal flora, thereby achieving the purpose of increasing the bacterial content in fecal sludge.

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

[0009] A method for improving the purity of intestinal flora by using macroporous resin, wherein the method obtains an intestinal flora suspension sample, and then purifies the sample by wet column loading with food-grade macroporous resin.

[0010] The above-mentioned macroporous resin is any one of the three resins AMBERLITE XAD4, AMBERLITE XAD7HP and AMBERLITEXAD16; after purification, the filtered bacterial suspension is collected, 1 ml of the bacterial suspension is centrifuged to collect the bacterial mud, and then the weight, unit bacterial amount, activity, bacterial amount per gram and total bacterial amount of the bacterial mud after centrifugation are determined, so as to evaluate the effects of different resins on the bacterial mud.

[0011] Furthermore, the macroporous resin needs to be pre-treated, specifically including: first rinsing the macroporous resin with deionized water three times, pouring out the remaining water, then adding 10% NaCl twice the volume of the resin, sealing with plastic wrap, and soaking for more than 20 hours; pouring out the 10% NaCl solution, then rinsing with deionized water three times, pouring out the remaining water, adding 75% alcohol twice the volume of the resin, soaking for more than 20 hours, pouring out the remaining alcohol solution, then rinsing with deionized water three times, pouring out the remaining water, putting into an opaque plastic bottle, sealing and storing in a 4°C refrigerator.

[0012] Furthermore, the macroporous resin uses its surface area for adsorption, and the high surface area provides a high adsorption capacity. The resin AMBERLITEXAD16 is preferably used, which can effectively and stably increase the unit bacterial amount in the bacterial mud to achieve the effect of removing impurities.

[0013] The beneficial effects of the present invention are:

[0014] The macroporous resin of the present invention has basically no effect on the activity of the bacterial flora, and the method will not cause damage to the intestinal flora; after resin treatment, the bacterial mass per gram of all donors is increased, and the bacterial mass per gram of the bacterial mud can be significantly increased. The macroporous resin treatment can effectively remove impurities, leaving a purer bacterial flora, thereby achieving the effect of improving the purity of the bacterial flora.

[0015] Moreover, as a material for separating and purifying organic matter, macroporous resin has the characteristics of low price, high selectivity, convenient operation and reusability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The technical roadmap implemented by the present invention.

[0017] Figure 2 Schematic diagram of the bacterial sludge activity in the test of the present invention.

[0018] Figure 3 Schematic diagram of the weight of bacterial mud in the experiment of the present invention.

[0019] Figure 4 Schematic diagram of the total bacterial count of the bacterial sludge in the experiment of the present invention.

[0020] Figure 5Schematic diagram of the amount of bacteria per gram of bacterial mud in the test of the present invention. DETAILED DESCRIPTION

[0021] 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.

[0022] like Figure 1 As shown, the method implemented by the present invention obtains intestinal flora suspension samples from volunteers, and then purifies the samples through wet column loading with food-grade macroporous resins, wherein the above-mentioned macroporous resins are resin AMBERLITE XAD4, resin AMBERLITE XAD7HP, and resin AMBERLITEXAD16, which are purified separately; after purification, the filtered bacterial suspension is collected, and then 1 ml of the bacterial suspension is centrifuged to collect bacterial mud, and then the weight, unit bacterial count, activity, bacterial count per gram, and total bacterial count of the bacterial mud after centrifugation are determined, and the effects of different resins on the bacterial mud are evaluated accordingly.

[0023] The implementation of the present invention is described below in conjunction with experiments.

[0024] Experiment 1: Effects of different resins on fecal sludge

[0025] 1. Test materials

[0026] 1.1 Test instruments: 4℃ refrigerator, vortex oscillator, centrifuge, magnetic stirrer, pipette, biosafety cabinet, centrifuge tube, glass chromatography column

[0027] 1.2 Test reagents: LIVE / DEAD TM BacLight TM Bacterial Viability Kit dead or alive fluorescent dye, physiological saline, NaCl, 75% alcohol.

[0028] 1.3 Experimental materials: fresh stool samples from three healthy volunteers, donors 125, 124, and 123, AMBERLITE XAD4 resin, AMBERLITE XAD7HP resin, and AMBERLITE XAD16 resin.

[0029] Table 1 Resin Model

[0030]

[0031] 2. Test methods:

[0032] 2.1 Resin pretreatment

[0033] First, place the macroporous resin in a beaker, rinse it three times with deionized water, pour out the remaining water, then add 10% NaCl twice the volume of the resin, seal it with plastic wrap, soak it for more than 20 hours, pour out the 10% NaCl solution, rinse it three times with deionized water, pour out the remaining water, add 75% alcohol twice the volume of the resin, soak it for more than 20 hours, pour out the remaining alcohol solution, rinse it three times with deionized water, pour out the remaining water, put it into a light-proof plastic bottle, seal it and store it in a 4°C refrigerator.

[0034] 2.2 Collection of donor cells

[0035] 100-200 g of fresh feces were collected from three healthy volunteers, respectively, dissolved in 750-1000 mL of physiological saline, and processed by an automatic feces extractor to remove the residue to obtain 600-850 mL of bacterial suspension.

[0036] 2.3 Experimental Grouping

[0037] The macroporous resin treated in step 2.1 was loaded into the column, and No. 4 (AMBERLITE XAD4), No. 7 (AMBERLITE XAD7HP) and No. 16 (AMBERLITE XAD16) resins were added, as well as a control group without added resin. Then, the bacterial suspension collected in step 2.2 was loaded into the chromatography column at a flow rate of 2.0 ml min-1.

[0038] 2.4 Centrifugal collection of bacterial sludge

[0039] Collect the bacterial suspension after filtration in step 2.3 into a 50 ml centrifuge tube, vortex to mix, and take 1 ml of the bacterial suspension for centrifugation. Repeat three times in each group, with a centrifugal force of 5000 g and a time of 5 minutes. After centrifugation, pour out the supernatant, collect the precipitated bacterial sludge, and weigh and record it.

[0040] 2.5. Live bacteria detection

[0041] The bacterial sludge collected in step 2.4 was gradiently diluted 100 times. The specific steps were as follows: the bacterial sludge collected in step 2.4 was first diluted 10 times with physiological saline, and vortexed to obtain fecal bacteria dilution solution A. 100 μL of fecal bacteria dilution solution A was added to a new 2 ml sterile centrifuge tube and mixed with 900 μL of physiological saline to obtain fecal bacteria dilution solution B.

[0042] Follow LIVE / DEAD TM BacLight TMThe diluted bacterial sludge was stained with the Bacterial Viability Kit. The specific steps were as follows: 987 μL of 0.9 NaCl, 1.5 μL of SYT09 and 1.5 μL of PI dye were added to a 2 ml sterilized tube, and finally 10 μL of fecal bacteria dilution solution B was added, mixed and incubated for 15 min under dark conditions. After the dark reaction, the BD Accuri C6 flow cytometer was used to set the parameters to analyze the viability rate and unit bacterial count of the bacterial sludge.

[0043] 2.6 Data Analysis

[0044] The experimental data were analyzed by SPSS 22.0 Univariate method, and the factors that reached the significant level in the F test were subjected to variance analysis and Duncan's multiple comparisons between groups. The experimental data of each group were expressed as (mean ± standard deviation). The statistical significance level was P < 0.05, and the extremely significant level was P < 0.01.

[0045] Results Analysis

[0046] 3.1 Effects of different protective solutions on activity and unit bacterial count.

[0047] The activity, weight, total bacterial count and bacterial count per gram of the bacterial sludge collected after the bacterial suspension was soaked in resin were tested.

[0048] Depend on Figure 2 It can be seen that in donor 125, after soaking in macroporous resin, the activity of the control group was 63.53%, the activity of resin group No. 4 was 63.13%, and the activity of resin group No. 7 was 63.23%. There was no significant difference among the three groups (P>0.05), while the activity of resin group No. 16 was 60.77%, which was significantly lower than that of the control group and resin groups No. 4 and No. 7 (P<0.05); in donors 124 and 123, there was no significant difference in activity (P>0.05).

[0049] Depend on Figure 3 It can be seen that after resin treatment, the weight of the collected bacterial sludge was lower than that of the control group (P < 0.05). In 125 donors, the weight of the bacterial sludge in the control group was 0.16 g, which was 35% lower than that of the control group in the No. 4 resin group, 36% lower than that of the No. 7 resin group, and 37% lower than that of the No. 16 resin group; in 124 donors, the weight of the bacterial sludge in the control group was 0.31 g, which was 45% lower than that of the control group in the No. 4 resin group, 57% lower than that of the No. 7 resin group, and 58% lower than that of the No. 16 resin group; in 123 donors, the weight of the bacterial sludge in the control group was 0.19 g, which was 30% lower than that of the control group in the No. 4 resin group, 30% lower than that of the No. 7 resin group, and 17% lower than that of the No. 16 resin group.

[0050] Depend on Figure 4It can be seen that from the perspective of total bacterial count, there was no significant difference in the total bacterial count between the donor 125 and 124 groups (P>0.05), but in donor 123, the total bacterial count of the control group was 1.86×1010cells, and the total bacterial count of the No. 16 resin group was 1.76×1010cells, and there was no significant difference between the two groups (P>0.05), but both were significantly higher than the No. 4 resin and No. 7 resin groups (P<0.05), the total bacterial count of the No. 4 resin group was 1.56×1010cells, and the total bacterial count of the No. 7 resin group was 1.46×1010cells, and there was no significant difference in the total bacterial count between the No. 4 resin and No. 7 resin groups (P>0.05).

[0051] Depend on Figure 5 It can be seen that after resin treatment, the bacterial count per gram of all donors increased, which was significantly higher than that of the control group (P < 0.05). Among the 125 donors, the bacterial count per gram of the control group was 4.81×1010 (cells / g), the bacterial count per gram of the No. 4 resin group increased by 42.80%, and the bacterial count per gram of the No. 7 resin group increased by 48.28%. There was no significant difference in the bacterial count per gram between the No. 4 resin group and the No. 7 resin group (P > 0.05). The bacterial count per gram of the No. 16 resin group increased by 60.89%, which was significantly higher than the other groups (P < 0.05); among the 124 donors, the bacterial count per gram of the control group was 5.55×1010 (cells / g), the bacterial count per gram of the No. 7 resin group increased by 98.49%, and the bacterial count per gram of the No. 16 resin group increased by 101.16%. There was no significant difference in the bacterial count per gram between the No. 7 resin group and the No. 16 resin group (P > 0.05). The bacterial count per gram of resin group No. 4 increased by 98.49%, which was significantly lower than that of resin groups No. 7 and No. 16 (P < 0.05). Among 123 donors, the bacterial count per gram of the control group was 9.92×1010 (cells / g), the bacterial count per gram of resin group No. 4 increased by 21.19%, the bacterial count per gram of resin group No. 7 increased by 22.64%, and the bacterial count per gram of resin group No. 16 increased by 22.64%. There was no significant difference in the bacterial count per gram among the three resin groups (P > 0.05).

[0052] 4. Results and Discussion

[0053] The value of FMT has gradually been highly recognized by the market and medicine. However, the current dosage of FMT is still based on the weight of the bacterial sludge. How to increase the unit bacterial count in the bacterial sludge has always been a difficult problem to be solved. Fecal bacteria contain a large number of microorganisms, which play an important role in improving host immunity, food digestion, intestinal endocrine function and neural signal regulation, drug function and metabolism, endotoxin clearance and affecting the production of host metabolism-related substances. The number of live bacteria in the bacterial sludge is a key factor in judging the quality of the product and the size of its effectiveness. While improving the efficacy, it can also reduce the number of times patients take it. Therefore, a high bacterial count is often the key to the efficacy of FMT. As a material for separating and purifying organic matter, macroporous resin has the characteristics of low price, high selectivity, convenient operation, and reusability. It is widely used in various fields, especially in extraction and purification. This experiment also provides new thinking for the application of macroporous resin in the field of FMT.

[0054] The effects of three types of food-grade macroporous resins on the removal of impurities from bacterial sludge were tested, and it was found that macroporous resins had basically no effect on the activity of the bacterial flora, indicating that this method would not cause damage to the intestinal flora. In this experiment, the weight of the bacterial sludge collected by centrifugation decreased after adsorption by macroporous resins. This is because the macroporous resin can adsorb more impurities in the bacterial sludge, such as bile pigments, mucins, inorganic salts, fats, cholesterol, purine groups and a small amount of vitamins, and use the intermolecular force to adsorb impurities into the pores, resulting in a decrease in the weight of the bacterial sludge. The unit bacterial amount was converted, and it was found that after treatment with macroporous resins, although the weight of the bacterial sludge was reduced, the unit bacterial amount of the bacterial sludge could be significantly increased, indicating that the treatment with macroporous resins can effectively remove impurities, leaving a purer bacterial flora, thereby achieving the goal of improving the purity of the bacterial flora. Testing of different types of resins also found that different resins have different enhancement effects on different donors. This may be because the human intestinal microecology is in a dynamic balance, which is not only affected by internal factors such as physical health and psychological factors, but also changes with external factors such as time, diet, and environment. Therefore, the content of impurities in the intestinal flora donated by volunteers is different each time, resulting in different impurity removal effects of different resins.

[0055] According to the above indicators, high surface area provides high adsorption capacity. The surface area of ​​No. 16 resin is higher than that of the other two resins, and the improvement effect is the highest in 125 and 124 donors, but the improvement trend is not as good as No. 4 resin in 123 donor, but there is no significant difference (P>0.05). The improvement effect is stable in different donors, and the comprehensive effect is better than that of No. 4 and No. 7 resins. In summary, No. 16 resin can effectively and stably increase the unit bacterial amount in the bacterial sludge and achieve the effect of removing impurities.

[0056] 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 method for improving the purity of intestinal flora using macroporous resin, characterized in that Obtain an intestinal flora suspension sample, and then purify the sample by wet column loading with food-grade macroporous resin; the macroporous resin is any one of the three resins AMBERLITE XAD4, AMBERLITE XAD7HP, and AMBERLITEXAD16.

2. The method for improving the purity of intestinal flora using macroporous resin according to claim 1, characterized in that The macroporous resin needs to be pre-treated, specifically including: first rinse the macroporous resin with deionized water three times, pour out the remaining water, then add 10% NaCl twice the volume of the macroporous resin, seal with plastic wrap, and soak for more than 20 hours; pour out the 10% NaCl solution, rinse with deionized water three times, pour out the remaining water, add 75% alcohol twice the volume of the macroporous resin, soak for more than 20 hours, pour out the remaining alcohol solution, rinse with deionized water three times, pour out the remaining water, put into an opaque plastic bottle, seal and store in a 4°C refrigerator.

3. The method for improving the purity of intestinal flora using macroporous resin according to claim 1, characterized in that The macroporous resin is resin AMBERLITEXAD16.

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