Method for purifying and enriching intestinal flora based on freeze-thaw pore-forming technology

Abstract

The invention discloses a method for purifying and enriching intestinal flora based on a freeze-thaw pore-forming technology, and belongs to the technical field of porous material modification and flora transplantation. The process comprises the following steps: firstly, putting a porous material into liquid nitrogen for quick freezing, and then after the liquid nitrogen volatilizes, performing freeze-drying treatment to obtain an internal and external pore-forming modified material; and mixing the internal and external pore-forming modified material with the bacterial suspension, incubating, filtering, and centrifuging to obtain purified and enriched bacterial sludge. A liquid nitrogen circulation quick-freezing technology and a vacuum freeze-drying technology are combined, a freeze-thawing pore-forming technology is developed, the porosity of an original substance structure is further improved, a porous structure with developed inside and outside is constructed, and the method is used for separating and removing various impurities in the intestinal flora suspension. Finally, the extraction and enrichment of the pure intestinal flora are completed.

Description

Technical Field

[0001] The present invention belongs to the technical field of porous material modification and flora transplantation, and specifically relates to a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology. Background Art

[0002] Microbiota transplantation is a new technology that transplants intestinal flora from the feces of healthy donors to patients, thereby adjusting or reconstructing the balance of the patient's intestinal flora. Currently reported technologies for intestinal flora extraction and purification and impurity separation include centrifugation, resuspension and washing, filtration and impurity removal, and adsorption resin. Although the above methods separate and remove impurities in feces in different ways and achieve extraction and purification of intestinal flora to a certain extent, they all have corresponding disadvantages. First, centrifugation may not be able to effectively remove all non-bacterial impurities, especially those with small particles or substances similar to the flora; the resuspension and washing method requires repeated washing and resuspension steps, which is cumbersome, time-consuming, and inefficient; when facing a bacterial suspension containing a large amount of impurities, the filter screen of the filtration and impurity removal method can easily reach a saturated state, causing blockage during the treatment process, which greatly limits the sample loading and increases production costs; and the adsorption resin method is easily limited by saturation capacity. The adsorption capacity of macroporous resins is limited. Once saturated, it needs 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 links.

[0003] Based on the above background, developing a method for purifying and enriching intestinal flora can reduce production and operating costs while being simple, time-saving and efficient, so that it can be scaled up and applied industrially, and ultimately complete the extraction and purification of intestinal flora and the separation and removal of impurities, thereby ensuring the efficacy and stability of the transplantation technology and the patient's experience, and reducing related risks and safety hazards. This is one of the key points to break through the current technical bottlenecks and barriers in the field of flora transplantation. Summary of the invention

[0004] In view of the content of the background technology, the purpose of the present invention is to provide a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, which is specifically achieved through the following technical solutions: The present invention provides a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, comprising the following steps: Step 1: Take the porous material and put it into liquid nitrogen for quick freezing, then wait for the liquid nitrogen to volatilize, and then freeze-dry it to obtain the internal and external pore-forming modified material; Step 2: Mix the internal and external pore-forming modified material obtained in step 1 with the bacterial suspension, incubate, and then filter and centrifuge to obtain purified and enriched bacterial mud.

[0005] Furthermore, the porous material in step 1 is at least one of sawdust, corn cobs, straw, loofah or rice husk, and has a particle size of 3-30 mm.

[0006] Furthermore, the porous material is wood chips with a particle size of 3-10 mm, and the wood chips are at least one of oak chips, pine chips or hardwood chips.

[0007] Furthermore, in the quick freezing process in step 1, the liquid nitrogen must not cover the porous material; quick freezing and volatilization must be repeated multiple times before freeze drying. In a specific implementation case, the freeze drying process parameters are shown in Table 1: Table 1 Freeze-drying process parameters

[0008] Furthermore, in step 2, the ratio of the internal and external pore-forming modification materials to the bacterial suspension is 0.1%-20%, g:mL; preferably 2%-15%, more preferably 5%-10%.

[0009] Furthermore, the incubation speed in step 2 is 200 rpm and the incubation time is 15 min.

[0010] Furthermore, the centrifugal speed in step 2 is 6000 g / min and the time is 10 min.

[0011] Liquid nitrogen quick freezing technology is a method that uses liquid nitrogen at an extremely low temperature (-196 ℃) to quickly freeze food and biological samples. It is often used for rapid freezing and preservation of food, and it is also often used for cryopreservation of cells. Due to the extremely low temperature of liquid nitrogen, when a substance with a certain water content is placed in liquid nitrogen, the water inside it will quickly cool and solidify into ice, causing volume expansion. This expansion will cause physical pressure on the internal structure of the substance, resulting in the formation of tiny cracks and pores inside it. When the liquid nitrogen evaporates, the ice crystal structure inside the substance will partially melt due to the rise in temperature. If the liquid nitrogen treatment is circulated multiple times (i.e., liquid nitrogen is naturally evaporated after quick freezing, and then liquid nitrogen is quick frozen again), new ice crystals formed after each quick freezing will form in the original cracks and pores, thereby increasing the complexity and connectivity of the pores, making the internal pore structure of the substance more developed.

[0012] Vacuum freeze drying technology is a processing technology that removes moisture from materials through sublimation under low temperature and low pressure. It is often used to produce foods that are easy to store for a long time, light in weight, small in size, and can be quickly rehydrated. During the vacuum freeze drying process, the moisture in the material is usually directly sublimated in the solid form of ice crystals to form water vapor and thus removed, so gaps will be left on the surface and inside of the material, forming a porous sponge structure.

[0013] Based on this, the inventors used a material with a porous structure as the base, adopted liquid nitrogen cycle quick freezing technology to enhance the complexity and connectivity of its original pores, and used vacuum freeze-drying technology to further create pores on the outside while retaining the internal porous structure of the material, thereby achieving the preparation of an adsorption material with high porosity, which was ultimately applied to the purification and enrichment of intestinal flora.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention combines liquid nitrogen cycle quick freezing technology with vacuum freeze-drying technology to develop a freeze-thaw pore-making technology; the porosity of the original material is further improved on the structure of the original material, and a porous structure material with developed internal and external structures is constructed, which is used to separate and remove various impurities in the intestinal flora suspension, and finally completes the extraction and enrichment of pure intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the microscopic examination results of the sawdust material of test group A in Example 1.

[0016] Figure 2 The microscopic examination results of the sawdust material of test group B in Example 1 are shown.

[0017] Figure 3 The microscopic examination results of the sawdust material of test group C in Example 1 are shown.

[0018] Figure 4 The microscopic examination results of the sawdust material of test group D in Example 1 are shown.

[0019] Figure 5 The following are the results of microscopic examination of the bacterial mud of the control group in Example 1.

[0020] Figure 6 The results of microscopic examination of bacterial mud of test group D in Example 1 are shown.

[0021] Figure 7 The microscopic examination results of the loofah material of test group A in Example 2 are shown.

[0022] Figure 8 The microscopic examination results of the loofah material of test group B of Example 2 are shown.

[0023] Fig. 9 The microscopic examination results of the loofah material of test group C in Example 2 are shown.

[0024] Fig.10 The microscopic examination results of the loofah material of test group D in Example 2 are shown.

[0025] Fig.11 This is the bacterial sludge precipitation situation of the control group in comparative example 1.

[0026] Fig.12 This is the bacterial sludge precipitation situation of experimental group A in comparative example 1.

[0027] Fig.13 This is the bacterial sludge precipitation situation of experimental group B in comparative example 1. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] Example 1 Collect fresh stool samples from healthy donors, add physiological saline and stir to mix, then use a sieve to remove large particles of food residues and other substances to obtain the original bacterial suspension.

[0031] The original bacterial suspension was divided into 5 equal portions by volume, each with 50 mL. One portion of the untreated bacterial suspension was taken as the control group, and the remaining four portions were used as experimental group A, experimental group B, experimental group C, and experimental group D, respectively.

[0032] The experimental setup was divided into the following groups: Control group: The original bacterial suspension was placed on a shaker and incubated for 15 min at a speed of 200 rpm. After incubation, the bacterial suspension was processed once with a sieve, the filtrate was collected and centrifuged at 6000 g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group A: Untreated oak sawdust (particle size: 3-5 mm) was added to the bacterial suspension at a ratio of 10% (g:ml, sawdust weight: bacterial suspension volume), and incubated on a shaker for 15 min at a speed of 200 rpm. After incubation, the sawdust was removed with a sieve, the filtrate was collected and centrifuged at 6000 g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group B: Oak sawdust (particle size: 3-5 mm) was put into liquid nitrogen (just enough to cover the material), and then the liquid nitrogen was naturally volatilized, and then it was put into liquid nitrogen again, and the cycle was repeated 3 times. Then it was taken out and put into the bacterial suspension at a ratio of 10% (g:ml, sawdust weight: bacterial suspension volume), and incubated on a shaker for 15 min at a speed of 200 rpm. After incubation, the sawdust was removed with a sieve, and the filtrate was collected and centrifuged at 6000 g / min for 10 min. The supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group C: Oak sawdust (particle size: 3-5 mm) was placed in a vacuum freeze dryer with a set program for freeze drying, then taken out and added to the bacterial suspension at a ratio of 10% (g:ml, sawdust weight: bacterial suspension volume), and incubated on a shaker for 15 min at a speed of 200 rpm. After incubation, the sawdust was removed with a sieve, the filtrate was collected and centrifuged at 6000 g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group D: Oak sawdust (particle size: 3-5 mm) was put into liquid nitrogen (just enough to cover the material), then the liquid nitrogen was allowed to evaporate naturally, and then it was put into liquid nitrogen again, and the cycle was repeated three times. Then it was placed in a vacuum freeze dryer with a set program for freeze drying, taken out, and put into the bacterial suspension at a ratio of 10% (g:ml, sawdust weight: bacterial suspension volume), and incubated on a shaker for 15 min at a speed of 200 rpm. After incubation, the sawdust was removed with a sieve, the filtrate was collected and centrifuged at 6000g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; The experimental results show that the average unit bacterial count of the control group of this example is 1.74×10 11 cells / g, and the average bacterial count of test group A was 2.03×10 11 cells / g, an average increase of 16.67% compared with the control group; the average unit bacterial count of the experimental group B was 2.19×10 11 cells / g, an average increase of 25.86% compared with the control group; the average unit bacterial count of the experimental group C was 2.49×10 11 cells / g, an average increase of 43.10% compared with the control group; the average unit bacterial count of the experimental group D was 2.77×10 11 cells / g, an average increase of 59.20% compared with the control group.

[0033] The results show that sawdust treated with liquid nitrogen and freeze-drying has the best effect in extracting and enriching intestinal flora, and can effectively remove related impurities in the original bacterial suspension. The purification effect of sawdust treated with freeze-drying alone is better than that of sawdust treated with liquid nitrogen alone. It is worth noting that sawdust without any treatment also has a certain adsorption and impurity removal ability.

[0034] Based on this, it is speculated that liquid nitrogen cycle quick freezing technology can enhance the complexity and connectivity of the internal pores of the material on the basis of the original material, completing the internal pore modification, while vacuum freeze drying technology can further form a porous structure on the surface of the material to complete the external pore modification. When the two technologies are combined, an adsorption material with a well-developed internal and external pore structure will be formed, thereby separating and removing impurities in the bacterial suspension and completing the extraction and enrichment of the intestinal flora. From the existing results, the effect of sawdust treated with liquid nitrogen combined with freeze drying on the extraction and enrichment of intestinal flora is better than that of liquid nitrogen treatment or freeze drying alone, and the extraction and enrichment effect of sawdust treated with liquid nitrogen or freeze drying alone is better than that of untreated sawdust. Therefore, on the one hand, this result has been able to prove the effect of liquid nitrogen cycle quick freezing technology and vacuum freeze drying technology on the internal and external pores of the material, and on the other hand, it needs to be further verified by microscopic examination (see for details). Figure 1-Figure 4 ).

[0035] from Figure 1 It can be seen that although there are arranged gaps formed by the fiber structure on the surface of the untreated sawdust, there is no obvious pore structure. Figure 2 It can be clearly seen that after the sawdust was treated with liquid nitrogen, a large number of pores appeared inside the fiber, proving that the internal pores have been formed. Figure 3 It can be observed that the surface of the freeze-dried sawdust presents a neatly arranged mesh structure, proving that the external pores have been completed. Figure 4 The interior of the sawdust (i.e., sawdust treated with liquid nitrogen combined with freeze-drying) maintains a vacuum fiber structure, while the outside forms a relatively uniform porous network morphology, forming a modified sawdust with high porosity, proving the feasibility of using liquid nitrogen cycle quick freezing technology and vacuum freeze-drying technology to create pores inside and outside the material. Figure 5 , Figure 6 The results of microscopic examination of bacterial mud obtained from the control group and the experimental group D are as follows: Figure 6 (liquid nitrogen combined with freeze-drying) compared to Figure 5 (Without any treatment) The impurity particles are greatly reduced.

[0036] Example 2 The experimental setup was divided into the following groups: Control group: The original bacterial suspension was placed on a shaker and incubated for 15 min at a speed of 200 rpm. After incubation, the bacterial suspension was processed once with a sieve, the filtrate was collected and centrifuged at 6000 g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group A: Untreated loofah (particle size: 10-15 mm) was added to the bacterial suspension at a ratio of 10% (g: ml, loofah weight: bacterial suspension volume), and incubated on a shaker for 15 min at a speed of 200 rpm. After incubation, the loofah was removed with a sieve, the filtrate was collected and centrifuged at 6000 g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group B: The loofah (particle size: 10-15 mm) was put into liquid nitrogen (just enough to cover the material), and then the liquid nitrogen was naturally volatilized, and then it was put into liquid nitrogen again, and the cycle was repeated 3 times. Then it was taken out and put into the bacterial suspension at a ratio of 10% (g:ml, loofah weight: bacterial suspension volume), and placed on a shaker for incubation for 15 min at a speed of 200 rpm. After incubation, the loofah was removed with a sieve, and the filtrate was collected and centrifuged at 6000 g / min for 10 min. The supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group C: The loofah (particle size: 10-15 mm) was placed in a vacuum freeze dryer with a set program for freeze drying, then taken out and added to the bacterial suspension at a ratio of 10% (g:ml, loofah weight: bacterial suspension volume), and incubated on a shaker for 15 min at a speed of 200 rpm. After incubation, the loofah was removed with a sieve, the filtrate was collected and centrifuged at 6000g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group D: The loofah (particle size: 10-15 mm) was put into liquid nitrogen (just enough to cover the material), and then the liquid nitrogen was naturally volatilized, and then it was put into liquid nitrogen again, and the cycle was repeated 3 times. Then it was placed in a vacuum freeze dryer with a set program for freeze drying, taken out, and put into the bacterial suspension at a ratio of 10% (g:ml, loofah weight: bacterial suspension volume), and placed on a shaker for incubation for 15 min at a speed of 200 rpm. After incubation, the loofah was removed with a sieve, and the filtrate was collected and centrifuged at 6000 g / min for 10 min. The supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; The experimental results show that the average unit bacterial count of the control group in this example is 1.27×10 11 cells / g, and the average bacterial count of test group A was 1.44×1011 cells / g, an average increase of 13.39% compared with the control group; the average unit bacterial count of the experimental group B was 1.57×10 11 cells / g, an average increase of 23.62% compared with the control group; the average unit bacterial count of the experimental group C was 1.67×10 11 cells / g, an average increase of 31.50% compared with the control group; the average unit bacterial count of the experimental group D was 1.72×10 11 cells / g, an average increase of 35.43% compared with the control group.

[0037] The results show that the extraction and enrichment of intestinal flora by loofah after liquid nitrogen combined with freeze-drying is better than that of the two technologies alone. Microscopic examination can also prove the feasibility of liquid nitrogen cycle quick freezing technology and vacuum freeze drying technology in the internal and external pore formation of materials (see Figure 7-10 ).

[0038] Comparative Example 1 Collect fresh stool samples from healthy donors, add physiological saline and stir to mix, then use a sieve to remove large particles of food residues and other substances to obtain the original bacterial suspension.

[0039] The original bacterial suspension was divided into three equal parts by volume, each with 80 mL. One untreated bacterial suspension was taken as the control group, and the other two were used as experimental group A and experimental group B, respectively.

[0040] The experimental setup was divided into the following groups: Control group: The original bacterial suspension was placed on a shaker and incubated for 15 min at a speed of 200 rpm. After incubation, the bacterial suspension was processed once with a sieve, the filtrate was collected and centrifuged at 6000 g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Test group A: Untreated hardwood chips (particle size: 4-8 mm) were put into liquid nitrogen (just cover the material), and then the liquid nitrogen was naturally volatilized, and then put into liquid nitrogen again, and the cycle was repeated 3 times, and then placed in a vacuum freeze dryer with a set program for freeze drying, taken out, and put into the bacterial suspension at a ratio of 10% (g:ml, sawdust weight: bacterial suspension volume), and placed on a shaker for incubation for 15 min at a speed of 200 rpm. After incubation, the sawdust was removed with a sieve, and the filtrate was collected and centrifuged at 6000 g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; Experimental group B: Porous resin (type: non-polar; particle size: 0.25-1 mm; pore size: 2-10 μm) was added to the bacterial suspension at a ratio of 10% (g: ml, porous resin weight: bacterial suspension volume), and incubated on a shaker for 15 min at a speed of 200 rpm. After incubation, the resin was removed using a sieve, the filtrate was collected and centrifuged at 6000 g / min for 10 min, the supernatant was removed, the bacterial sludge was collected, and the unit bacterial count of the bacterial sludge was detected by flow cytometry; The experimental results show that the average unit bacterial count of the control group is 1.38×10 11 cells / g, and the average bacterial count of test group A was 2.48×10 11 cells / g, an average increase of 79.71% compared with the control group; the average unit bacterial count of the experimental group B was 4.94×10 8 cells / g, an average decrease of 99.64% compared with the control group.

[0041] The results show that the sawdust treated with liquid nitrogen combined with freeze-drying is still effective in adsorbing impurities and improving the purity of intestinal flora. The samples treated with porous resin showed a significant decrease in the amount of bacteria per unit compared with the control group, which indicates that most of the bacteria were not collected with the filtrate, but adsorbed on the surface or inside of the resin. Further analysis showed that the weight of the precipitated bacterial mud after centrifugation of the resin-treated samples was significantly lower than that of the sawdust treatment group and the control group (control group bacterial mud weight: 1.7329 g; experimental group A bacterial mud weight: 1.1953 g; experimental group B bacterial mud weight: 0.0257 g, the details of bacterial mud precipitation are shown in Figure 11-13 ), which further shows that after resin treatment, in addition to the flora, other non-bacterial impurities will also be enriched on the resin. Therefore, porous resin cannot achieve the purification effect of separating intestinal flora from impurities and specifically enriching intestinal flora.

[0042] The reason for this phenomenon is that porous resins are artificially pre-designed synthetic materials. Their single pore size, porosity and pore size distribution make them lack selectivity and specificity in adsorption capacity. They are easy to adsorb bacteria and impurities at the same time, and cannot achieve the effect of separation and purification. The sawdust treated with liquid nitrogen combined with freeze-drying forms a "dynamic filter" in pore distribution. On the one hand, the large pore area can intercept large particles of impurities such as undigested food residues and fibers. On the other hand, the small pore area allows bacteria to pass through and be collected together with the filtrate, achieving the effect of graded filtration. In addition, due to the natural material characteristics of sawdust, lignin, cellulose and other components may preferentially adsorb hydrophobic impurities (such as lipids) through hydrophobic-hydrophilic effects, electrostatic effects, etc., while the adsorption of bacteria (negatively charged surface or with specific hydrophilicity) is weaker, and freeze-thaw pore formation may expose more active groups, enhancing the binding ability to specific impurities.

[0043] 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 method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, characterized in that: The steps include: Step 1: Take the porous material and put it into liquid nitrogen for quick freezing, then wait for the liquid nitrogen to volatilize, and then freeze-dry it to obtain the internal and external pore-forming modified material; Step 2: Mix the internal and external pore-forming modified material obtained in step 1 with the bacterial suspension, incubate, and then filter and centrifuge to obtain purified and enriched bacterial mud.

2. According to claim 1, a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, characterized in that: The porous material in step 1 is at least one of sawdust, corn cobs, straw, loofah or rice husk, and has a particle size of 3-30 mm.

3. According to claim 2, a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, characterized in that: The porous material is wood chips with a particle size of 3-10 mm, and the wood chips are at least one of oak chips, pine chips or hardwood chips.

4. According to claim 1, a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, characterized in that: In step 1, the liquid nitrogen in the quick freezing process must not cover the porous material; quick freezing and volatilization must be repeated several times before freeze drying.

5. According to claim 1, a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, characterized in that: Step 2: The ratio of the internal and external pore-forming modification materials to the bacterial suspension is 0.1%-20%, g:mL.

6. According to claim 1, a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, characterized in that: The incubation speed in step 2 is 200 rpm and the incubation time is 15 min.

7. According to claim 1, a method for purifying and enriching intestinal flora based on freeze-thaw pore-making technology, characterized in that: The centrifugation speed in step 2 is 6000 g / min and the time is 10 min.

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