Negative pressure hierarchical filtering method for concentration and enrichment of intestinal active flora

Abstract

The invention discloses a negative pressure hierarchical filtering method for concentration and enrichment of intestinal active flora, and belongs to the technical field of biology. The surface activation technology, the particle size hierarchical separation system, the membrane separation system and the charge separation technology are adopted to form a composite technology, the unit bacterial quantity and activity can be remarkably improved, operation is easy, consumed time is short, only 0.5-1 min is needed for treating 100 mL of bacterial suspension on average, the situation that blockage is caused by too high membrane loading capacity is avoided, the filtering efficiency is greatly improved, and the method is suitable for large-scale popularization and application. The sample loading amount of the bacterial suspension is ensured, so that the process is closer to actual production, and amplification and industrial application are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a negative pressure laminar filtration method for concentrating and enriching active intestinal flora. Background Art

[0002] Microbiota transplantation is a new technology that transplants the intestinal flora from the feces of healthy donors to patients, thereby adjusting or reconstructing the balance of the patient's intestinal flora. However, since this field is in its infancy both at home and abroad, there are many technical bottlenecks and barriers.

[0003] First, although donors undergo rigorous screening before transplantation to ensure the safety and diversity of their microbial communities, potential risks and safety hazards are still inevitable. The reason is that in addition to intestinal flora, donor feces also contain various intestinal contents, undigested food residues, and bacterial metabolites. Intestinal contents and undigested food residues may carry pathogens, such as viruses or parasites, which may be transmitted to patients during the transplantation process, increasing the risk of infection. In addition, these substances may also contain anti-nutritional factors, which can inhibit the absorption of nutrients, affect the nutritional value intake of patients, and may cause malnutrition or aggravate existing nutritional problems. For microbiota metabolites, such as short-chain fatty acids, amino acids and their derivatives, bile acids, hydrogen sulfide, etc., although they are beneficial to the host under normal physiological conditions, during the microbiota transplantation process, if the amount and proportion of these metabolites are unbalanced, it may have an adverse effect on the patient's physiological and pathological processes. For example, the production of short-chain fatty acids is related to diet, microbiota composition and the patient's own health system. If the metabolite circulation level is inappropriate, it may affect the patient's health, leading to metabolic disorders or impaired intestinal barrier function. For example, hydrogen sulfide has been found to inhibit the production of glucagon-like peptide-1 (GLP-1) in the intestine, thereby damaging the body's metabolism and promoting the occurrence of metabolic diseases such as obesity.

[0004] Secondly, how to ensure that the donor's intestinal flora remains highly active before being transplanted into the patient's body is also an important technical barrier. On the one hand, the donor may maintain a low intestinal flora activity for a long time due to lifestyle, eating habits or health conditions. On the other hand, the donor's fecal samples will be affected by the corresponding environment in all links from collection, processing to storage and transportation, such as temperature changes and oxygen content, which may cause the activity of different types of bacteria to decay at different rates, ultimately leading to a decrease in the overall flora activity. The high activity state of the flora or the proportion of live bacteria is crucial in the transplantation process. The reason is that live bacteria are the key factor in achieving the therapeutic effect. They can colonize in the patient's intestine and play their physiological functions, such as helping digest food, synthesizing vitamins, stimulating the patient's immune system, promoting the patient's immune response, inhibiting the growth of pathogens, removing harmful microorganisms and toxins, affecting the absorption and metabolism of nutrients, affecting hormone levels, affecting appetite and energy consumption, affecting the body's endocrine level, and affecting the growth and differentiation of fat cells. These functions have significant therapeutic effects on the treatment of various diseases related to intestinal flora disorders, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), metabolic syndrome (MS), etc. In addition, live bacteria have shown great potential in the treatment of cardiovascular diseases such as hypertension, neurological diseases such as depression and autism, and even in the auxiliary treatment of tumors. They can affect the central nervous system through the gut-brain axis, thereby improving the symptoms of patients with depression and autism. In cancer treatment, active intestinal flora can reverse the condition of patients who do not respond to PD-1 therapy and help cancer treatment. However, excessive proportion of dead bacteria during transplantation will reduce the treatment effect and trigger immune response. Since dead bacteria cannot colonize and reproduce in the patient's intestines, they cannot effectively restore the balance of intestinal flora or improve the symptoms of related diseases. Repeated transplantation is required, which increases the complexity and cost of treatment and leads to various adverse reactions, such as abdominal pain and diarrhea.

[0005] Based on this, in various cases of using microbiota transplantation technology to treat related diseases, in order to ensure the efficacy, each research team has put forward strict requirements on the total amount of live bacteria transplanted. If the microbiota amount and activity decrease due to a large proportion of sample impurities and dead bacteria, which ultimately affects the total amount of live bacteria in a single transplant, it is necessary to increase the dosage or increase the number of transplants. Taking the oral intestinal flora liquid enteric-coated capsules, which are currently one of the transplantation routes, as an example, due to the large particle size of the capsules, increasing the dosage will cause the patient to have difficulty swallowing each time, especially for the elderly and children, which can easily cause the patient to be unable to swallow or even get stuck in the throat.

[0006] The technologies reported so far for extracting and purifying intestinal flora and separating impurities include centrifugation, resuspension and washing, filtration and impurity removal, and adsorption resin. The first is centrifugation, which uses the force generated by high-speed centrifugation to separate solid particles and liquids in fecal samples. Solid particles such as cells and undigested food residues in feces will settle at the bottom due to their large mass, while microorganisms such as flora and viruses will remain in the supernatant. This method can effectively remove large particles of impurities in feces; the second is the resuspension and washing method, which mixes fecal samples with physiological saline or other buffers, and removes impurities by repeated resuspension and washing. This method can remove some large particles of physical impurities; the third is the filtration and impurity removal method, which uses sieves of different apertures to filter fecal samples, intercept impurities larger than the sieve aperture, such as undigested food residues, and then remove large particles in feces; and the adsorption resin method uses the macroporous structure and high specific surface area of ​​the adsorption resin to remove impurities in feces, such as metabolites, through physical adsorption, so as to achieve the extraction and purification of intestinal flora.

[0007] Although the above methods separate and remove impurities in feces in different ways and achieve the extraction and purification of intestinal flora to a certain extent, they all have corresponding disadvantages. First, the centrifugation method may not be able to effectively remove all non-bacterial impurities, especially those with smaller particles or substances similar to the flora. During actual separation, due to the convection and diffusion generated by centrifugation, some components with similar sedimentation coefficients cannot be completely separated and purified. In addition, due to the repeated washing, dissolution, and reprecipitation of the precipitate, intermediate losses are easily caused, resulting in poor centrifugal resolution, and multiple centrifugation may affect the activity of the flora; the resuspension and washing method requires repeated washing and resuspension steps, which is cumbersome and time-consuming. Especially when dealing with a large number of samples, its efficiency is low, and this method may dilute the concentration of the flora, and cannot guarantee the complete removal of all impurities. For impurities with strong adhesion, simple resuspension and washing may not be able to effectively remove them; when facing a bacterial suspension containing a large amount of impurities, the filtration and impurity removal method has a filter screen. It is easy to reach a saturated state, which may cause blockage during the treatment process, resulting in a decrease in filtration efficiency, which greatly limits the sample loading amount and increases production and operating costs. It is only suitable for laboratory treatment and is difficult to scale up, making it difficult to apply in industry. The adsorption resin method is easily limited by saturation capacity. The adsorption capacity of macroporous resins is limited. Once saturated, they need to be regenerated or replaced, which means that a large amount of resin and additional operating costs may be required, and the process is complicated and cumbersome, which is not suitable for actual production. In addition, the adsorption resin may not be able to completely remove all types of impurities, especially those that do not match the adsorption capacity of the resin, and may even easily adsorb the bacterial colony, resulting in a decrease in the bacterial collection rate, which is contrary to the expected effect. Some resins, because they carry specific functional groups, will kill the bacterial colony and affect the activity of the bacterial colony.

[0008] In addition, the above-mentioned purification and impurity removal methods also have a common problem, that is, it is impossible to extract and enrich the active bacteria from the dead bacteria and impurities, resulting in the inability of the donor's intestinal flora to maintain a high activity state before transplantation into the patient's body, which will directly affect the efficacy, stability, risk and patient experience of the transplantation technology. The reason for this problem is that there are differences in the integrity of the cell wall and membrane between live bacteria and dead bacteria. Live bacteria have complete cell membranes and metabolic activity, which can maintain their physiological functions and use specific substrates for energy metabolism, while the cell membrane of dead bacteria may have been broken or damaged, so they do not have relevant metabolic capabilities. The above-mentioned methods such as centrifugation, resuspension washing, and filtration and impurity removal are mainly based on physical properties for purification and impurity removal, not on the physiological and biochemical properties of dead and live bacteria, so it is impossible to complete the extraction of active bacteria. The adsorption resin method is mainly based on the size of inactive substances, functional group structure and other related properties to complete extraction and separation. However, these properties may not be significantly different between live bacteria and dead bacteria, so it is also impossible to extract and enrich live bacteria from dead bacteria and impurities. Therefore, developing a method for concentrating and enriching active intestinal flora is simple to operate, time-saving and efficient. On the one hand, it does not affect the activity and collection rate of the flora. On the other hand, it improves the filtration efficiency, ensures the sample loading amount, and reduces the production cost and operation cost, so that it can be scaled up and applied industrially. Ultimately, the extraction and purification of active intestinal flora, the separation and removal of dead bacteria and impurities, and the guarantee of the efficacy and stability of the transplantation technology and the patient's experience of taking the transplantation technology are completed, and the related risks and safety hazards are reduced. This is one of the key points to solve the current technical bottlenecks and barriers in the field of flora transplantation. Summary of the invention

[0009] In view of the problems raised in the background technology, the purpose of the present invention is to provide a negative pressure laminar filtration method for concentrating and enriching active intestinal flora. The present invention specifically adopts the following technical solutions: The present invention provides a negative pressure laminar filtration method for concentrating and enriching active intestinal flora, comprising the following steps: Step 1: Take the donor's feces and add physiological saline to mix, filter, and collect the filtrate as the bacterial suspension; Step 2: Add the surfactant to the bacterial suspension obtained in step 1 and mix well to obtain an activated bacterial suspension for later use; Step 3: Use the filter membrane and the Buchner funnel to construct a vacuum negative pressure membrane separation system, then add separation materials of different particle sizes into the Buchner funnel in sequence, and complete the construction of the particle size hierarchical separation system on the vacuum negative pressure membrane separation system; Step 4: Pass the activated bacterial suspension of step 2 through the particle size grading separation system of step 3. After filtration is completed, add cationic solution to the particle size grading separation system and collect all the filtrate.

[0010] Furthermore, the filtration in step 1 uses 4 layers of sieves, and the mesh sizes from top to bottom are 50, 80, 100, and 120 meshes respectively.

[0011] Furthermore, the surfactant in step 2 is at least one of Tween 20, Tween 40, Tween 60, Tween 80, and Span 80; preferably Tween 40, Tween 60, Tween 80, and more preferably Tween 80.

[0012] Furthermore, in step 2, the volume ratio of the surfactant solution to the bacterial suspension is 1:(60-300); preferably 1:(80-200), and more preferably 1:(100-150).

[0013] Furthermore, the filter membrane in step three is any one of a nylon filter membrane, a polypropylene filter membrane, and a polyethersulfone filter membrane, preferably a nylon filter membrane; the pore size of the filter membrane is 6-18 μm, preferably 8-15 μm, and more preferably 10-12 μm.

[0014] Furthermore, the separation material in step three is at least two of perlite, diatomaceous earth, quartz sand, cellulose, bentonite, kaolin, silicon dioxide, magnesium silicate, and polyacrylamide; preferably at least two of perlite, diatomaceous earth, quartz sand, cellulose, kaolin, and magnesium silicate, and more preferably perlite, diatomaceous earth, and quartz sand.

[0015] Furthermore, the particle size of the separation material in step three is 20-3000 μm, preferably 20-2000 μm, and more preferably 20-1000 μm; according to the different particle sizes of the separation materials, they are arranged in order from small to large in the Buchner funnel from bottom to top.

[0016] Furthermore, the addition ratio of the single material to the bacterial suspension in the separation material in step three is 5%-25%, preferably 8%-20%, more preferably 10%-15%, g:mL.

[0017] Furthermore, the cationic solution in step 4 is at least one of a chitosan solution, a carboxymethyl chitosan solution, a cationized soybean polysaccharide solution, and a polylysine solution, preferably a chitosan solution and a polylysine solution, and more preferably a chitosan solution; the concentration is 0.1%-10%, preferably 0.5%-8%, and more preferably 1%-5%.

[0018] Furthermore, in step 4, the volume ratio of the cationic solution to the bacterial suspension is (1-10):1, preferably (1-5):1, and more preferably (1-2):1.

[0019] Furthermore, the treatment processes from step 1 to step 4 are all carried out at 4° C. under anaerobic conditions.

[0020] The present invention first uses the pressure generated by the positive and negative pressure difference as a power source, and utilizes surface activation technology, a particle size hierarchical separation system, a membrane separation system, and a charge separation technology to complete the concentration and enrichment of intestinal active flora. First, the intestinal flora suspension is pretreated with a surfactant, which can reduce the surface tension of the bacterial suspension, improve wettability and dispersibility, and reduce aggregation. On the one hand, it enables it to better penetrate into the subsequent particle size hierarchical separation system, thereby more effectively removing impurities. On the other hand, it can increase the solubility of some difficult-to-dissolve substances in the bacterial suspension, and help these substances disperse in the particle size hierarchical separation system by forming micelles or increasing the hydrophilicity of the dissolved substances, preventing excessive compaction, maintaining filtration efficiency, and facilitating the separation and removal of impurities. Then, the particle size difference of different substances is used to construct a hierarchical separation system. Due to the gap effect between the same substance and different substances, impurities with a particle size greater than or equal to a certain substance will be intercepted by the substance, thereby completing the effect of step-by-step separation of impurities in the bacterial suspension. At the same time, since live bacteria have a certain degree of motility, and dead bacteria cannot carry out behavioral activities due to the loss of vital signs, they are more likely to be intercepted together with impurities, and the irregular arrangement between substances can also play a role in drainage and filtration, making the bacterial suspension pass more smoothly. After the bacterial suspension is processed by the particle size hierarchical separation system, it is further passed through a filter membrane with a smaller pore size to achieve membrane separation. Since a part of the large particle impurities and dead bacteria have been pre-intercepted, the impurity capacity pressure carried by the membrane is smaller at this time, and the membrane separation efficiency is higher. At this point, the effect of separating and removing most of the impurities and dead bacteria in the intestinal flora suspension has been achieved. In order to further improve the collection rate of active flora, cationic solution is added to the particle size hierarchical separation and membrane separation system. Since the cell walls and membranes of active intestinal flora contain more acidic phospholipids and other acidic polysaccharides, these molecules easily lose protons and thus carry a negative charge, while the cell membranes of dead bacteria have been broken or damaged, and metabolic activities have stopped, and normal charge balance cannot be maintained, resulting in changes in surface charge, which usually appear to be uncharged or positively charged. Based on this, the present invention utilizes the charge separation effect generated by the cationic solution to further collect the active flora remaining in the particle size hierarchical separation system and on the membrane, and ultimately completes the concentration and enrichment of the intestinal active flora.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The technical method of the present invention is simple, easy and time-saving. Under the action of surface activation technology and particle size grade separation system, it takes only 0.5-1 min on average to process 100 mL of bacterial suspension, and there will be no blockage caused by excessive membrane load, which greatly improves the filtration efficiency and ensures the amount of bacterial suspension sample, making the process closer to actual production and convenient for scale-up and industrial application. In addition, the main instruments and equipment used in the treatment process, such as Buchner funnel, suction filtration bottle, etc., are low-priced and simple to operate, which reduces the production cost and operating cost to a certain extent, and does not require expensive instruments and labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a flow chart of the technical solution of the present invention.

[0023] Figure 2 This is the microscopic examination picture of donor C control group.

[0024] Figure 3 This is the microscopic examination picture of donor C test group H. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0027] Example 1) Preparation of bacterial suspension 250 g of fresh feces from a healthy donor was placed in a sampling box, and 1000 mL of normal saline was added at a ratio of 1:4 (g:mL, feces weight: normal saline volume). The feces were broken up and homogenized under the action of a propeller at a speed of 3000 g / min. The fecal homogenate was then passed through a vibrating screen to remove large particles of food residues, such as undigested food fiber, to avoid subsequent blockage. The vibration frequency was 600 g / min, and the screen consisted of 4 layers, with mesh sizes of 50, 80, 100, and 120 from top to bottom, respectively. The vibration time was between 10-15 min, and the filtrate collected was the bacterial suspension, totaling 930 mL. The temperature of the preparation process was controlled at 4°C, and the anaerobic state was maintained throughout the process.

[0028] 2) Surface activation pretreatment of bacterial suspension After the bacterial suspension was vortexed at 5000 g / min for 1 min, it was divided into 9 equal parts with a volume of 100 mL each, and named as control group, experimental group A, experimental group B, experimental group C, experimental group D, experimental group E, experimental group F, experimental group G, and experimental group H, respectively. The bacterial suspensions of experimental group B, experimental group E, experimental group F, and experimental group H were taken at a ratio of 1:100 (mL:mL, Tween 80 volume: bacterial suspension volume), and 1 mL of Tween 80 solution was added to each of the four groups, and vortexed at a speed of 4000 g / min and a vortex time of 2 min. The surface activation technology pretreatment of the bacterial suspension was completed. The temperature was controlled at 4°C throughout the process and maintained in an anaerobic state.

[0029] 3) Construction of membrane separation system Prepare 8 250 mL suction flasks and 8 60 mm Büchner funnels. Place the Büchner funnel on top of the suction flask, take 8 circular nylon filter membranes with a pore size of 10 μm and a diameter of 40 mm and put them into the Büchner funnel. Use a vacuum rubber tube to connect the suction flask to a circulating water vacuum pump. Then add 10 mL of physiological saline to moisten each filter membrane, turn on the vacuum pump, and use the pressure generated by the positive and negative pressure difference to suck the filter membrane tightly onto the Büchner funnel. The construction of the membrane separation system is completed. The 8 membrane separation systems are named A, B, C, D, E, F, G, and H respectively.

[0030] 4) Construction of particle size separation system 10% (g:mL, weight of perlite / diatomaceous earth / quartz sand: subsequent bacterial suspension loading volume) of perlite, diatomaceous earth and quartz sand were added to membrane separation systems C, E, G and H, i.e. 10 g of each component, arranged in order from top to bottom as quartz sand, diatomaceous earth and perlite, among which the particle size of quartz sand was between 0.6-1.0 mm, the particle size of diatomaceous earth was between 40-80 μm, and the particle size of perlite was between 20-30 μm. After leveling, the particle size hierarchical separation system was completed.

[0031] 5) Preparation of cationic solution Take 5 g of chitosan powder, add it to 500 mL of normal saline, and stir to dissolve at room temperature. The stirring speed is 300 g / min and the stirring time is 10 min. After the dissolution is complete, the preparation of the cationic solution is completed.

[0032] 6) Negative pressure layer filtration technology treatment The corresponding treatment methods of the control group and each experimental group are as follows: Control group: bacterial suspension without any treatment; Experimental group A: membrane separation system treatment group; Experimental group B: membrane separation system combined with surface activation technology treatment group; Experimental group C: membrane separation system combined with particle size fractionation system treatment group; Experimental group D: membrane separation system combined with charge separation technology treatment group; Experimental group E: membrane separation system combined with surface activation technology and particle size fractionation system treatment group; Experimental group F: membrane separation system combined with surface activation technology and charge separation technology treatment group; Experimental group G: membrane separation system combined with particle size separation system and charge separation technology treatment group; Experimental group H: membrane separation system combined with surface activation technology, particle size classification system, and charge separation technology treatment group; Note: Surface activation technology, particle size separation system and charge separation technology require membrane separation system to complete and cannot be processed alone; Specifically, in test group A, 100 mL of the corresponding bacterial suspension was directly added to membrane separation system A, and then the filtrate was collected.

[0033] In test group B, 100 mL of bacterial suspension treated with surface activation technology was added to membrane separation system B, and the filtrate was collected.

[0034] Experimental group C added 100 mL of the corresponding bacterial suspension into the particle size graded separation system built on membrane separation system C, and collected the filtrate.

[0035] In test group D, 100 mL of the corresponding bacterial suspension was directly added to membrane separation system D. After filtration was completed, 100 mL of cationic solution was added to membrane separation system D at a ratio of 1:1 (mL:mL, cationic solution volume: bacterial suspension volume), and all the filtrate was collected.

[0036] Experimental group E added 100 mL of bacterial suspension treated with surface activation technology into the particle size graded separation system built on membrane separation system E, and collected the filtrate.

[0037] In test group F, 100 mL of bacterial suspension treated with surface activation technology was added to membrane separation system F. After filtration was completed, 100 mL of cationic solution was added to membrane separation system F at a ratio of 1:1 (mL:mL, cationic solution volume: bacterial suspension volume), and all the filtrate was collected.

[0038] Experimental group G added 100 mL of the corresponding bacterial suspension to the particle size graded separation system built on membrane separation system G. After the filtration was completed, 100 mL of cationic solution was added to the particle size graded separation system at a ratio of 1:1 (mL:mL, cationic solution volume: bacterial suspension volume), and all the filtrate was collected.

[0039] Experimental group H added 100 mL of bacterial suspension treated with surface activation technology to the particle size graded separation system built on membrane separation system H. After filtration was completed, 100 mL of cationic solution was added to the particle size graded separation system at a ratio of 1:1 (mL:mL, cationic solution volume: bacterial suspension volume), and all the filtrate was collected.

[0040] The above treatment processes were all completed at 4°C and maintained in anaerobic conditions.

[0041] 7) Detection of sample activity and related indicators of unit bacterial count After the filtrates of the control group and each experimental group were collected, they were centrifuged at 8000 g / min for 10 min at 4°C, and then the supernatant was removed and the bacterial sludge was collected. The bacterial sludge was diluted 1000 times with physiological saline and then stained with Syto-9 and PI (obtained from LIVE / DEAD ™ BacLight ™ Bacterial Viability Kit was used to detect the activity and unit bacterial count of each group of samples after dark reaction for 15 min.

[0042] Test example A total of three fresh feces from different healthy donors were selected for relevant tests. The donors were numbered and named as donor A, donor B, and donor C. The test results are shown in Tables 1 to 3, respectively.

[0043] Table 1 Test results of donor A

[0044] Table 2 Test results of donor B

[0045] Table 3 Donor C related test results

[0046] Table 4 Comprehensive test results of three donors

[0047] Based on the combined results of the three donors: The activity of test group A was 2.52% higher than that of the control group, the unit bacterial count was 26.28% higher than that of the control group, and the unit viable bacterial count was 29.45% higher than that of the control group. The activity of test group B was increased by an average of 0.62% compared with the control group, the unit bacterial count was increased by an average of 30.87% compared with the control group, and the unit viable bacterial count was increased by an average of 31.74% compared with the control group; The activity of test group C was 43.97% higher than that of the control group, the unit bacterial count was 174.35% higher than that of the control group, and the unit viable bacterial count was 293.99% higher than that of the control group. The activity of the experimental group D was increased by an average of 27.57% compared with the control group, the unit bacterial count was increased by an average of 61.91% compared with the control group, and the unit viable bacterial count was increased by an average of 107.20% compared with the control group; The activity of the experimental group E was 44.78% higher than that of the control group, the unit bacterial count was 196.90% higher than that of the control group, and the unit viable bacterial count was 326.98% higher than that of the control group. The activity of the experimental group F was increased by an average of 27.63% compared with the control group, the unit bacterial count was increased by an average of 112.18% compared with the control group, and the unit viable bacterial count was increased by an average of 171.72% compared with the control group; The activity of the experimental group G was increased by an average of 52.26% compared with the control group, the unit bacterial count was increased by an average of 429.51% compared with the control group, and the unit viable bacterial count was increased by an average of 709.25% compared with the control group; The activity of the experimental group H was increased by an average of 56.88% compared with the control group, the unit bacterial count was increased by an average of 456.80% compared with the control group, and the unit viable bacterial count was increased by an average of 775.22% compared with the control group.

[0048] Through further analysis of the results, it was found that in terms of activity improvement rate, from high to low, they were test group H, test group G, test group E, test group C, test group F, test group D, test group A, and test group B. After further comparison of test group D with test group A, it was found that charge separation technology for treating bacterial suspension can separate and remove some dead bacteria, and further extract and enrich more active bacteria. Similarly, comparison of test group C with test group A can also draw the same conclusion, that is, the role of particle size separation system in enriching live bacteria, and its effect is better than that of charge separation technology treatment.

[0049] In terms of the rate of increase in unit bacterial count, from high to low, they are respectively test group H, test group G, test group E, test group C, test group F, test group D, test group A, and test group B. After comparing test group D with test group A, and test group C with test group A, it was found that both the particle size separation system and the charge separation technology can intercept and remove impurities in the bacterial suspension to a certain extent, greatly increasing the unit bacterial count, and the purification effect of the particle size separation system is better than that of the charge separation technology.

[0050] After comparing the unit bacterial count of test group A with that of the control group, it was found that the membrane separation system can effectively remove some impurities and increase the unit bacterial count when treating the bacterial suspension, further proving the effect of the membrane separation system. In addition, by observing the experimental process of each group, it was found that the filtration speed of the test groups (test group B, test group E, test group F, test group H) treated with surface activation technology was faster than that of other groups, with an average treatment time of 0.53-1.07 min, while the average treatment time of other groups was between 2.93-3.70 min. This result proves the role of surface activation technology in improving filtration effect.

[0051] In general, although the surface activation technology, particle size separation system, and charge separation technology (based on the membrane separation system) have good effects in terms of activity enhancement rate / unit bacterial quantity enhancement rate / filtration effect when used alone, the combined use of the technologies (i.e., test group H) will further improve the effect and achieve more efficient concentration and enrichment of intestinal active bacteria. See the relevant microscopic examination pictures. Figure 2 , 3 .

[0052] The composite technology formed by the surface activation technology, particle size grading separation system, membrane separation system and charge separation technology of the present invention, on the one hand, has a more significant improvement rate per unit bacterial amount, indicating that it has a better effect on removing non-bacterial impurities in the bacterial suspension, and will not adsorb the bacterial flora and thus cause a decrease in the bacterial collection rate, which is contrary to the expected result. On the other hand, the bacterial suspension treated with this technical method has a much higher activity improvement rate than other groups, and is significantly improved compared with the untreated control group. This effect cannot be achieved by the existing technology. It is mainly due to the dual effects of the particle size grading separation system and the charge separation technology, which not only avoids the impact on the activity of the bacterial flora during the treatment process, but also achieves further concentration and enrichment of the active intestinal flora.

[0053] The embodiments described above only express several preferred embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but are not intended to limit the present invention. It should be noted that for those skilled in the art, the present invention may also have various changes and modifications, and any modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A negative pressure laminar filtration method for concentrating and enriching active intestinal flora, characterized in that: The steps include: Step 1: Take the donor's feces and add physiological saline to mix, filter, and collect the filtrate as the bacterial suspension; Step 2: Add the surfactant to the bacterial suspension obtained in step 1 and mix well to obtain an activated bacterial suspension for later use; Step 3: Use the filter membrane and the Buchner funnel to construct a vacuum negative pressure membrane separation system, then add separation materials of different particle sizes into the Buchner funnel in sequence, and complete the construction of the particle size hierarchical separation system on the vacuum negative pressure membrane separation system; Step 4: Pass the activated bacterial suspension of step 2 through the particle size grading separation system of step 3. After filtration is completed, add cationic solution to the particle size grading separation system and collect all the filtrate.

2. The negative pressure laminar filtration method for concentrating and enriching intestinal active flora according to claim 1, characterized in that: In step 2, the surfactant is at least one of Tween 20, Tween 40, Tween 60, Tween 80, and Span 80.

3. The negative pressure laminar filtration method for concentrating and enriching active intestinal flora according to claim 1, characterized in that: The volume ratio of the surfactant solution to the bacterial suspension in step 2 is 1:(60-300).

4. The negative pressure laminar filtration method for concentrating and enriching active intestinal flora according to claim 1, characterized in that: The filter membrane in step 3 is any one of a nylon filter membrane, a polypropylene filter membrane, and a polyethersulfone filter membrane; the pore size of the filter membrane is 6-18 μm.

5. The negative pressure laminar filtration method for concentrating and enriching active intestinal flora according to claim 1, characterized in that: In step 3, the separation material is at least two of perlite, diatomaceous earth, quartz sand, cellulose, bentonite, kaolin, silicon dioxide, magnesium silicate, and polyacrylamide.

6. The negative pressure laminar filtration method for concentrating and enriching active intestinal flora according to claim 1, characterized in that: The particle size of the separation material in step 3 is 20-3000 μm. According to the different particle sizes of the separation materials, they are arranged from small to large in order from bottom to top in the Buchner funnel.

7. The negative pressure laminar filtration method for concentrating and enriching active intestinal flora according to claim 1, characterized in that: In step 3, the addition ratio of the single material to the bacterial suspension in the separation material is 5%-25%, g:mL.

8. The negative pressure laminar filtration method for concentrating and enriching active intestinal flora according to claim 1, characterized in that: In step 4, the cationic solution is at least one of a chitosan solution, a carboxymethyl chitosan solution, a cationic soybean polysaccharide solution, and a polylysine solution, and the concentration is 0.1%-10%.

9. The negative pressure laminar filtration method for concentrating and enriching active intestinal flora according to claim 1, characterized in that: The volume ratio of the cationic solution to the bacterial suspension in step 4 is (1-10):

1.

10. The negative pressure laminar filtration method for concentrating and enriching active intestinal flora according to claim 1, characterized in that: The treatment processes from step 1 to step 4 are all carried out at 4°C under anaerobic conditions.

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