A process for removing impurities from fecal suspension using ion exchange resin

Abstract

The present invention relates to a process for removing impurities from fecal suspensions using ion exchange resins. In this process, a bacterial suspension treated by a fecal automatic extractor is taken and treated with ion exchange resins. The present invention utilizes the fact that anion exchange resins carry a large number of negative charges on their outer surfaces. These negative charges enable the resins to effectively adsorb a large amount of positively charged impurities. Therefore, anion exchange resins can selectively adsorb positively charged impurities without affecting the bacterial flora, and then remove the resins and the adsorbed impurities by filtration, thereby achieving the purpose of removing impurities and increasing the unit bacterial quantity.

Description

Technical Field

[0001] The present invention relates to the field of impurity removal from fecal suspensions, and particularly to a process for removing impurities from fecal suspensions using ion exchange resins. Background Art

[0002] Fecal microbiota transplantation is a recognized international treatment method for safely and effectively treating various intestinal diseases, including Clostridium difficile infection (CDI), ulcerative colitis (UC), and Crohn's disease (IBD). In addition to intestinal diseases, more and more research results show that fecal microbiota transplantation technology can also be used to treat some currently incurable human metabolic diseases, including diabetes, and mental and psychological diseases such as autism. The application prospect of this treatment technology is broad, and it is also one of the current popular research fields.

[0003] There are two common dosage forms for fecal microbiota transplantation for treatment. One is a bacterial liquid preparation for invasive treatment by enema, and the other is a capsule preparation for oral delivery. The commonly used preparation process in the current industry is mostly filtration plus centrifugation. This process has the advantages of convenience and speed, but it is difficult to effectively remove tiny impurity particles therein.

[0004] Feces contain a large amount of undigested residues, including proteins, inorganic substances, fats, undigested dietary fibers, dehydrated digestive fluid residues, as well as cells shed from the intestine and dead bacteria. Simple filtration impurity removal processes can only remove some larger impurities that are significantly different from bacteria. Impurities with sizes not much different from bacteria will be carried into the final product together, thereby reducing the purity of the bacteria in the preparation and possibly causing certain adverse effects on the transplant recipient.

[0005] The impurity removal methods described in existing authorized patent technologies such as CN201510304041.4 and CN201911129724.5 are both filtration. The specific impurity removal methods in the two patents are to pass the fecal suspension through sieves with different pore sizes in sequence, and then collect the bacterial sludge by centrifugation.

[0006] However, this impurity removal method inevitably causes smaller impurities to easily pass through multiple sieves and remain in the bacterial sludge together with the bacteria, and the impurity removal effect is not ideal. Summary of the Invention

[0007] To solve the above problems, the primary object of the present invention is to provide a process for removing impurities from fecal suspensions using ion exchange resins. This process combines ion exchange resins and an automatic extractor to quickly process fecal suspensions and achieve the purpose of impurity removal.

[0008] Another object of the present invention is to provide a process for removing impurities from fecal suspensions using ion exchange resins. This process has a fast impurity removal speed, high efficiency, and low cost, and can meet the requirements of quantitative production.

[0009] The inventors have found through research that ion exchange resins are insoluble macromolecular compounds with functional groups (active groups for exchanging ions) and a network structure. According to the different ions they carry, they can be divided into two types: anion exchange resins and cation exchange resins. According to the different functional groups they carry, they can also be divided into strong base anion exchange resins, weak base anion exchange resins, strong acid cation exchange resins, etc. Utilizing the different affinities of ion exchange resins for different ions in a solution and their selective adsorption, they can selectively adsorb impurities with specific charges, thereby achieving the purpose of targeted impurity removal.

[0010] At the same time, 50%-80% of the dry weight of bacteria is protein. Therefore, most bacteria carry a negative charge when the pH is greater than 6.0. And the pH suitable for the survival of bacteria is mostly around 7.0. Therefore, usually, bacteria carry a negative charge. In fecal suspensions, most impurities also carry a certain charge and can be adsorbed by the corresponding ion exchange resins.

[0011] To achieve the above object, the technical solution adopted by the present invention is:

[0012] A process for removing impurities from fecal suspensions using ion exchange resins, taking the bacterial suspension treated by a fecal automatic extractor, and treating the bacterial suspension with ion exchange resins, where the ion exchange resin is an anion exchange resin.

[0013] Further, treating the bacterial suspension with ion exchange resins means filling the exchange resins into a chromatography column after pretreatment respectively, loading the bacterial suspension into the chromatography column at a flow rate of 2.0 ml·min-1, and then eluting the chromatography column with 200 ml of PBS solution.

[0014] Further, the bacterial suspension is previously placed on a magnetic stirrer and stirred evenly for standby.

[0015] Further, the ion exchange resin is any one of Amberlite FPA58Cl strong base anion exchange resin and LEWATIT A365 weak base anion exchange resin.

[0016] Even further, the ion exchange resin is an anion exchange resin, preferably Amberlite FPA58Cl strong base anion exchange resin, to effectively remove impurities and at the same time increase the unit bacterial amount of the bacterial sludge.

[0017] The beneficial effects of the present invention are:

[0018] The anion exchange resin of the present invention has a large number of negative charges on its outer surface. These negative charges enable the resin to effectively adsorb a large amount of positively charged impurities. Therefore, the anion exchange resin can selectively adsorb positively charged impurities without affecting the bacterial flora, and then remove the resin and the adsorbed impurities by filtration, so as to achieve the purpose of impurity removal and increasing the amount of bacteria per unit.

[0019] At the same time, this process is realized by using a feces automatic extractor, which has a fast impurity removal speed, high efficiency and low cost, and can meet the needs of quantitative production.

[0020] Experiments have proved that ion exchange resins all have a certain adsorption effect. Among them, the strong-base anion exchange resin in the ion exchange resin has the best impurity removal effect, effectively increasing the amount of bacteria per unit of bacterial sludge. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the technical circuit diagram of the present invention.

[0022] Figure 2 is the schematic diagram of the weight of the bacterial sludge in the experiment of the present invention.

[0023] Figure 3 is the schematic diagram of the amount of bacteria per unit of the bacterial sludge in the experiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to 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 used to limit the present invention.

[0025] For the process of removing impurities from fecal suspension using ion exchange resin implemented by the present invention, take the bacterial suspension treated by the feces automatic extractor. The bacterial suspension is previously placed on a magnetic stirrer and stirred evenly for standby; then the bacterial suspension is treated with ion exchange resin. The exchange resin is pretreated and then filled into a chromatography column respectively. The bacterial suspension is loaded into the chromatography column at a flow rate of 2.0 ml·min-1, and then the chromatography column is eluted with 200 ml of PBS solution.

[0026] The ion exchange resin is any one of Amberlyst 15WET super-strong acid type cation exchange resin, Amberlite FPC88 strong acid type cation exchange resin, IMAC HP333 weak acid type cation exchange resin, Amberlite FPA58Cl strong base type anion exchange resin, and LEWATIT A365 weak base type anion exchange resin.

[0027] Figure 1 As shown, it is the flow chart of the test method of the present invention. The following experiments are carried out according to this method.

[0028] The implementation of the present invention will be described below in conjunction with experiments.

[0029] I. Reagents and Consumables

[0030] 1. Test Materials

[0031] 1.1 Test Instruments:

[0032] Shaker, centrifuge, flow cytometer, vortex oscillator, pipette, electronic balance, magnetic stirrer, pH meter, constant flow pump.

[0033] 1.2 Test Reagents:

[0034] Normal saline, LIVE / DEAD TM BacLight TM Bacterial Viability Kit live / dead fluorescent dye, dilute hydrochloric acid solution, dilute sodium hydroxide solution.

[0035] 1.3 Test Materials

[0036] Bacterial suspension, ion exchange resins (Amberlyst 15WET super strong acid cation exchange resin, Amberlite FPC88 strong acid cation exchange resin, IMAC HP333 weak acid cation exchange resin, Amberlite FPA58Cl strong base anion exchange resin, LEWATIT A365 weak base anion exchange resin), sterile gauze.

[0037] II. Experimental Methods

[0038] 1. Experimental Preparation

[0039] Take 600 ml of the bacterial suspension processed by the Chengg Ge fecal automatic extractor and place it on a magnetic stirrer for uniform stirring. Use a pipette to dispense 30 ml of the bacterial suspension into 50 ml centrifuge tubes, with a total of 18 portions dispensed. Divide the 18 portions of the bacterial solution into 6 groups, numbered A, B, C, D, E, and F, with three portions in each group.

[0040] 2. Treatment of Bacterial Suspension with Ion Exchange Resin

[0041] After pretreatment, the five ion exchange resins are respectively filled into chromatography columns of the same specification. The bacterial suspension is loaded onto the chromatography column at a flow rate of 2.0 ml·min-1, and then the chromatography column is eluted with 200 ml of PBS solution. Group A passes through the chromatography column without filling resin under the same conditions and also adds 200 ml of PBS solution, as shown in Table 1.

[0042] Table 1. Grouping Table of Ion Exchange Resin Addition

[0043]

[0044] 3. Result Detection

[0045] Take 15 ml of each filtrate and transfer it to a 50-ml centrifuge tube, and centrifuge at 5000×g for 5 min. Discard the supernatant and collect the bacterial sludge. Weigh the bacterial sludge and dilute the bacterial sludge 100 times with normal saline, and stain it with LIVE / DEAD TM BacLight TM Bacterial Viability Kit for 15 min, and count with a flow cytometer.

[0046] V. Result Analysis

[0047] 1. Weight of Bacterial Sludge

[0048] Among the 6 groups of bacterial sludge, the weight of the bacterial sludge of the superacid cation exchange resin is the least (as Figure 2 shown in the figure, in the figure, *. indicates a significant difference between the target group and the control group (P<0.05); **. indicates a very significant difference between the target group and the control group (P<0.01); ***. indicates a very extremely significant difference between the target group and the control group (P<0.001)), and the other four groups all show varying degrees of reduction compared with the control group. It is proved that all 5 kinds of ion exchange resins have a certain adsorption effect, and the superacid cation exchange resin has the strongest adsorption ability.

[0049] 2. Bacterial Quantity per Unit of Bacterial Sludge

[0050] Among the 6 groups of bacterial sludge, the bacterial quantity per unit of the strong-base anion exchange resin is the highest (as Figure 3 shown in the figure, in the figure, *. indicates a significant difference between the target group and the control group (P<0.05); **. indicates a very significant difference between the target group and the control group (P<0.01); ***. indicates a very extremely significant difference between the target group and the control group (P<0.001)), and it is significantly higher than the control group. Among the 5 kinds of ion exchange resins, the strong-base anion exchange resin has the best impurity removal effect and can effectively increase the bacterial quantity per unit of the bacterial sludge. Compared with the control group, the bacterial quantity per unit is increased by 7%-15%.

[0051] VI. Discussion

[0052] The outer surface of the anion exchange resin has a large number of negative charges, which enable the resin to effectively adsorb a large amount of positively charged impurities. Under the condition of pH 7.0, the bacterial population carries negative charges and is difficult to be adsorbed by the anion exchange resin. Therefore, the anion exchange resin can selectively adsorb positively charged impurities without affecting the bacterial population, and then remove the resin and the adsorbed impurities by filtration, so as to achieve the purpose of impurity removal and increasing the bacterial quantity per unit.

[0053] Judging from the results, among the five ion exchange resins, the strong base anion exchange resin has the best impurity removal effect. The strong base anion exchange resin can effectively adsorb and remove impurities in the bacterial suspension, and increase the unit bacterial amount of the bacterial sludge. This result can, to a certain extent, reduce the volume of the bacterial liquid or the number of capsules required for a single transplantation for the transplant recipient during practical application. And this change will be able to well improve the comfort and acceptance of the transplant recipient, reduce discomfort, and bring a better experience. At the same time, this process can also reduce the possibility of negative impacts caused by impurities in the transplant sample and improve the safety of transplantation.

[0054] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A process for removing impurities from fecal suspension using ion exchange resin, characterized in that Take the bacterial suspension treated by the feces automatic extractor, and treat the bacterial suspension with an ion exchange resin, where the ion exchange resin is any one of Amberlite FPA58Cl strong-base anion exchange resin and LEWATIT A365 weak-base anion exchange resin; treating the bacterial suspension with an ion exchange resin means filling the exchange resin into a chromatography column after pretreatment, loading the bacterial suspension into the chromatography column at a flow rate of 2.0 ml·min-1, and then eluting the chromatography column with 200 ml of PBS solution.

2. The process for removing impurities from fecal suspension using ion exchange resin according to claim 1, characterized in that The bacterial suspension is placed on a magnetic stirrer and stirred evenly in advance for standby.

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