An aid for centrifugal purification of an intestinal flora suspension, a purification method and an intestinal flora preparation

Abstract

This invention belongs to the field of biotechnology and discloses an aid for centrifugal purification of intestinal flora suspension, a purification method, and intestinal flora products. The aid for centrifugal purification of intestinal flora suspension provided by this invention includes at least a chitosan oligosaccharide acetic acid solution and sodium bicarbonate. It utilizes the synergistic effect of chitosan oligosaccharide and the buffer system formed by acetic acid and sodium bicarbonate to work together during centrifugation, achieving specific coating of bacteria in the intestinal flora suspension and promoting bacterial aggregation. This protects the intestinal flora while increasing the difference in sedimentation rate between the intestinal flora and other substances, thereby significantly improving the centrifugation recovery rate of the intestinal flora suspension while maintaining the activity of the intestinal sludge obtained by centrifugation. Furthermore, the chitosan oligosaccharide and water-soluble polymer are retained in the intestinal sludge by coating the bacterial surface, effectively improving the stress resistance and intestinal colonization ability of the obtained intestinal sludge, showing excellent application prospects in the preparation of intestinal flora products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to an aid for centrifugal purification of intestinal flora suspension, a purification method, and intestinal flora products. Background Technology

[0002] Intestinal microbiota preparations refer to biological products obtained from the feces of healthy donors through processes such as homogenization, filtration purification, and centrifugation, which retain the diversity of natural intestinal microbiota. During the preparation of intestinal microbiota products, the fecal microbial suspension obtained through homogenization and filtration purification has a complex composition. In addition to a large number of bacteria, it also contains a large amount of food residue, oils, extremely complex bacterial components, and human metabolic products, which will seriously interfere with the centrifugation process, leading to reduced separation efficiency, low yield of intestinal microbiota, and limited impurity removal. This results in poor process stability, unstable quality of the obtained intestinal microbiota products, and less than ideal biosafety and efficacy, thus presenting significant limitations. Summary of the Invention

[0003] The primary objective of this invention is to address the problems of reduced separation efficiency, low yield of intestinal flora, and limited impurity removal in existing centrifugation processes for the preparation of intestinal flora preparations, and to provide an auxiliary agent for centrifugation purification of intestinal flora suspensions.

[0004] The second objective of this invention is to provide a method for purifying intestinal flora suspensions.

[0005] A third objective of this invention is to provide an intestinal flora product.

[0006] Specifically, the adjuvant for centrifugal purification of intestinal flora suspension provided by the present invention includes a chitosan oligosaccharide acetic acid solution with a concentration of 1% (v / v) to 25% (v / v), sodium bicarbonate with a concentration of 0.01% (w / v) to 10% (w / v), and a water-soluble polymer with a concentration of 0 to 20% (w / v).

[0007] Furthermore, in the chitosan oligosaccharide acetic acid solution, the concentration of chitosan oligosaccharide is 0.1% (w / v) to 10% (w / v), and the concentration of acetic acid is 0.3% (v / v) to 0.8% (v / v).

[0008] Furthermore, the weight-average molecular weight of the chitosan oligosaccharide in the chitosan oligosaccharide acetic acid solution is no greater than 3200 Da, and the degree of polymerization is 2~20.

[0009] Furthermore, the water-soluble polymer includes sodium carboxymethyl cellulose and / or sodium alginate.

[0010] Furthermore, the mass ratio of sodium carboxymethyl cellulose to sodium alginate is (0.1~10):(0.1~10).

[0011] Furthermore, the sodium carboxymethyl cellulose has a degree of substitution of 85% to 95% and a viscosity of 100 mPa·s to 500 mPa·s.

[0012] Furthermore, the viscosity of the sodium alginate is 600 mPa·s to 800 mPa·s.

[0013] Furthermore, the concentration of the chitosan oligosaccharide acetic acid solution used is 1% (v / v) to 15% (v / v), and the concentration of the sodium bicarbonate used is 0.05% (w / v) to 1% (w / v).

[0014] Furthermore, the concentration of the chitosan oligosaccharide acetic acid solution is 1% (v / v) to 15% (v / v), the concentration of the sodium bicarbonate is 0.05% (w / v) to 1% (w / v), and the concentration of the water-soluble polymer is 0.1% (w / v) to 20% (w / v).

[0015] The purification method of intestinal flora suspension provided by the present invention includes: mixing sodium bicarbonate with intestinal flora suspension evenly, adding chitosan oligosaccharide acetic acid solution and optional water-soluble polymer, and performing low-temperature static treatment and centrifugation to obtain intestinal sludge.

[0016] Furthermore, in the chitosan oligosaccharide acetic acid solution, the concentration of chitosan oligosaccharide is 0.1% (w / v) to 10% (w / v), and the concentration of acetic acid is 0.3% (v / v) to 0.8% (v / v).

[0017] Furthermore, in the chitosan oligosaccharide acetic acid solution, the weight-average molecular weight of the chitosan oligosaccharide is not greater than 3200 Da, and the degree of polymerization is 2~20.

[0018] Furthermore, the water-soluble polymer comprises sodium carboxymethyl cellulose and sodium alginate in a mass ratio of (0.1~10):(0.1~10).

[0019] Furthermore, the sodium carboxymethyl cellulose has a degree of substitution of 85% to 95% and a viscosity of 100 mPa·s to 500 mPa·s.

[0020] Furthermore, the viscosity of the sodium alginate is 600 mPa·s to 800 mPa·s.

[0021] Furthermore, the concentration of the chitosan oligosaccharide acetic acid solution is 1% (v / v) to 15% (v / v), the concentration of sodium bicarbonate is 0.01% (w / v) to 1% (w / v), and the concentration of the water-soluble polymer is 0.1% (w / v) to 20% (w / v).

[0022] Furthermore, the temperature of the low-temperature static treatment is 1℃~5℃, and the time is 2min~30min.

[0023] Furthermore, the centrifugal force of the centrifugation process is 7500×g to 8500×g, and the time is 1 min to 10 min.

[0024] Furthermore, the preparation of the intestinal flora suspension includes: taking a fecal sample and mixing it with physiological saline, performing stirring and filtration treatment to obtain the intestinal flora suspension.

[0025] Furthermore, the mixing ratio of the fecal sample to physiological saline is 1:(1~15).

[0026] Furthermore, the stirring speed is 800 rpm to 1200 rpm, and the time is 5 min to 15 min.

[0027] Furthermore, in the filtration process, the pore size of the filter membrane is 80μm~120μm.

[0028] The intestinal flora product provided by this invention is prepared by the purification method of the intestinal flora suspension described above.

[0029] Beneficial effects:

[0030] The centrifugal purification aid for intestinal flora suspension provided by this invention specifically includes a chitosan oligosaccharide acetic acid solution and sodium bicarbonate at a specific concentration. The chitosan oligosaccharide works synergistically with the acetic acid-sodium bicarbonate buffer system during centrifugation to specifically coat the bacteria in the intestinal flora suspension and promote bacterial aggregation. This protects the intestinal flora while increasing the difference in sedimentation rate between the intestinal flora and other substances. Therefore, while maintaining the activity of the intestinal sludge obtained by centrifugation, it significantly improves the centrifugation recovery rate of the intestinal flora suspension. Furthermore, the chitosan oligosaccharide is retained in the intestinal sludge by coating the bacterial surface, effectively improving the stress resistance and intestinal colonization ability of the obtained intestinal sludge. It has excellent application prospects in the preparation of intestinal flora products.

[0031] In some specific embodiments, when the auxiliary agent for centrifugal purification of the intestinal flora suspension preferably includes a water-soluble polymer, and the water-soluble polymer specifically includes sodium carboxymethyl cellulose and sodium alginate in a mass ratio of (0.1~10):(0.1~10), the simultaneous introduction of chitosan oligosaccharide, sodium carboxymethyl cellulose and sodium alginate, in synergy with the buffer system formed by acetate-sodium bicarbonate, further enhances the above-mentioned bacterial coating and aggregation process, which can enhance the anti-interference ability of the centrifugation system. The bacterial activity and yield of the bacterial sludge obtained from different donor fecal sources or batches are similar, and it has excellent process stability. Detailed Implementation

[0032] The inventors of this invention, addressing the problems of low intestinal flora yield and poor process stability in existing fecal sample purification processes, selected positively charged natural polymers such as dextran as sedimentation aids during centrifugation. This promotes cell aggregation, enabling rapid sedimentation during centrifugation and thus improving separation efficiency and yield. However, through extensive experiments, the inventors discovered that the addition of these sedimentation aids not only fails to promote intestinal flora sedimentation but may also interact with impurities such as proteins and polysaccharides in the solution, preventing effective sedimentation of the flora or causing it to settle along with impurities. Furthermore, it may damage the activity of the flora, failing to address the problems existing in the aforementioned fecal sample purification process.

[0033] Based on the problems encountered by conventional flocculants applied to complex fecal suspensions, the inventors, after extensive and in-depth consideration and research, and taking into account the interactions between substances introduced during centrifugation and intestinal flora and other impurities, creatively selected chitosan oligosaccharide (commonly considered to have excellent antibacterial properties) as the core substance to enhance the difference in sedimentation rates between intestinal flora and other substances. In the solution prepared using acetic acid as the solute, chitosan oligosaccharide molecules are dispersed in the solution in the form of single chains or low-aggregation molecules. In the presence of alkaline sodium bicarbonate, this chitosan oligosaccharide acetic acid solution comes into contact with intestinal flora and causes them to curl up and / or aggregate, achieving specific coating of bacteria in the intestinal flora suspension and promoting the aggregation of flora, thereby effectively increasing the difference in sedimentation rates between intestinal flora and other substances. While maintaining the activity of intestinal flora, it improves the separation efficiency of intestinal flora from other impurities. Furthermore, the chitosan oligosaccharide coating on the surface of intestinal flora cells and / or cell clusters endows the resulting intestinal sludge with excellent stress resistance and intestinal colonization ability.

[0034] Furthermore, the inventors discovered that introducing sodium carboxymethyl cellulose and sodium alginate in a specific mass ratio during the centrifugation process of the intestinal flora suspension effectively enhances the specific coating of chitosan onto the intestinal flora and promotes bacterial aggregation in an acetate-sodium bicarbonate buffer solution system. This effectively reduces the interference of complex components in the fecal suspension on the coating and aggregation process of the intestinal flora, exhibiting excellent process stability. Based on this, the technical solution of this invention was obtained.

[0035] The intestinal flora suspension centrifugation purification aids provided by this invention specifically include chitosan oligosaccharide acetic acid solution, sodium bicarbonate, and optionally a water-soluble polymer.

[0036] In this invention, the chitosan oligosaccharide acetic acid solution refers to a stable and homogeneous liquid system formed by dissolving chitosan oligosaccharides in acetic acid or an aqueous acetic acid solution. The chitosan oligosaccharide refers to oligosaccharides obtained by enzymatic hydrolysis, acid hydrolysis, or other methods of degradation of chitin and / or chitosan. These oligosaccharides are rich in free amino and hydroxyl groups on their molecular chains and exist as single-chain or low-aggregate molecules in acetic acid or an aqueous acetic acid solution, with a degree of polymerization specifically ranging from 2 to 20.

[0037] In some specific embodiments, the weight-average molecular weight of chitosan oligosaccharides in the chitosan oligosaccharide acetic acid solution is preferably no greater than 3200 Da, more preferably 1000 Da to 2000 Da, specifically 1000 Da, 1350 Da, 1500 Da, 1610 Da, 2000 Da or any value between them.

[0038] In some specific embodiments, the concentration of chitosan oligosaccharide in the chitosan oligosaccharide acetic acid solution is preferably 0.1% (w / v) to 10% (w / v), specifically 0.1% (w / v), 0.3% (w / v), 0.8% (w / v), 1% (w / v), 3% (w / v), 5% (w / v), 7% (w / v), 10% (w / v) or any value between them; the concentration of acetic acid is preferably 0.3% (v / v) to 0.8% (v / v), specifically 0.3% (v / v), 0.35% (v / v), 0.4% (v / v), 0.5% (v / v), 0.6% (v / v), 0.65% (v / v), 0.7% (v / v), 0.8% (v / v) or any value between them. At this point, the concentrations of chitosan oligosaccharide and acetic acid in the solution system are appropriate, allowing them to exist better in the form of extended single-chain molecules, resulting in a better intestinal flora encapsulation effect.

[0039] In some specific embodiments, the concentration of the chitosan oligosaccharide acetic acid solution is preferably 1% (v / v) to 25% (v / v), specifically 1% (v / v), 1.5% (v / v), 2% (v / v), 4% (v / v), 5% (v / v), 8% (v / v), 10% (v / v), 12% (v / v), 15% (v / v), 18% (v / v), 20% (v / v), 22% (v / v), 25% (v / v) or any value between them. The preferred concentration of sodium bicarbonate used is 0.01% (w / v) to 10% (w / v), specifically 0.01% (w / v), 0.05% (w / v), 0.09% (w / v), 0.45% (w / v), 0.95% (w / v), 1% (w / v), 2% (w / v), 2.5% (w / v), 3% (w / v), 4% (w / v), 5.8% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), 10% (w / v), or any value between them.

[0040] In this invention, the water-soluble polymer refers to a class of molecular structures containing a large number of hydrophilic groups and capable of being uniformly dissolved in water to form a stable and homogeneous liquid system, specifically including sodium carboxymethyl cellulose and / or sodium alginate.

[0041] In some specific embodiments, the preferred mass ratio of sodium carboxymethyl cellulose to sodium alginate is (0.1~10):(0.1~10), specifically it can be 0.1:1, 0.1:2, 0.1:5, 0.1:10, 1:1, 1:3, 1:5, 1:10, 2:1, 2:5, 2:9, 3:5, 5:3, 8:1, 9:4, 10:9, 10:1 or any value between them.

[0042] In some specific embodiments, the degree of substitution of the sodium carboxymethyl cellulose is preferably 85% to 95%, specifically 85%, 86%, 87.5%, 89%, 93%, 95%, or any value between them; the viscosity of the sodium carboxymethyl cellulose is preferably 100 mPa·s to 500 mPa·s, specifically 100 mPa·s, 120 mPa·s, 150 mPa·s, 180 mPa·s, 200 mPa·s, 300 mPa·s, 450 mPa·s, 500 mPa·s, or any value between them.

[0043] In some specific embodiments, the viscosity of the sodium alginate is preferably 600 mPa·s to 800 mPa·s, specifically 600 mPa·s, 650 mPa·s, 700 mPa·s, 710 mPa·s, 800 mPa·s or any value between them.

[0044] In some specific embodiments, the concentration of the chitosan oligosaccharide acetic acid solution is preferably 1% (v / v) to 15% (v / v), specifically 1% (v / v), 3% (v / v), 4% (v / v), 8% (v / v), 10% (v / v), 12% (v / v), 14% (v / v), 15% (v / v), or any value between them; the concentration of the sodium bicarbonate is preferably 0.05% (w / v) to 1% (w / v), specifically 0.05% (w / v), 0.09% (w / v), or 0.45% (w / v). The concentration of the water-soluble polymer is preferably 0.1% (w / v) to 20% (w / v), specifically 0.1% (w / v), 0.5% (w / v), 1% (w / v), 1.2% (w / v), 1.5% (w / v), 2% (w / v), 4% (w / v), 5% (w / v), 8% (w / v), 10% (w / v), 15% (w / v), 18% (w / v), 20% (w / v), or any value between them.

[0045] The present invention also provides a method for purifying intestinal flora suspension. The purification method uses the above-mentioned intestinal flora suspension centrifugation purification aid, specifically including: mixing sodium bicarbonate with intestinal flora suspension evenly, adding chitosan oligosaccharide acetic acid solution and optional water-soluble polymer, and performing low-temperature static treatment and centrifugation treatment to obtain intestinal sludge.

[0046] In this invention, the purification method for intestinal flora suspension has excellent treatment effect even on complex intestinal flora suspensions that have only undergone simple filtration.

[0047] In this invention, the preparation of the intestinal flora suspension specifically includes: taking a fecal sample and mixing it with physiological saline, performing stirring and filtration treatment to obtain the intestinal flora suspension.

[0048] In some specific embodiments, in the preparation of the intestinal flora suspension, the preferred mixing volume ratio of the fecal sample to physiological saline is 1:(1~15), specifically it can be 1:1, 1:2.5, 1:4, 1:5, 1:8, 1:10, 1:12, 1:15 or any value between them.

[0049] In some specific embodiments, the stirring conditions in the preparation of the intestinal flora suspension include a rotation speed preferably of 800 rpm to 1200 rpm, specifically 800 rpm, 820 rpm, 850 rpm, 880 rpm, 900 rpm, 950 rpm, 1000 rpm, 1200 rpm or any value between them; and a stirring time preferably of 5 min to 15 min, specifically 5 min, 8 min, 10 min, 12 min, 15 min or any value between them.

[0050] In some specific embodiments, during the preparation of the intestinal flora suspension, the pore size of the filter membrane is preferably 80μm~120μm, specifically 80μm, 85μm, 90μm, 95μm, 100μm, 110μm, 120μm or any value between them.

[0051] In this invention, the sodium bicarbonate is mixed with the intestinal flora suspension to form a weakly alkaline solution environment, which allows the added chitosan oligosaccharide to better contact with the intestinal flora and complete structural transformation during the addition, mixing and low-temperature static treatment, resulting in better intestinal flora encapsulation effect and aggregation promotion effect.

[0052] In some specific embodiments, the conditions for the settling treatment include a temperature preferably of 1°C to 5°C, specifically 1°C, 1.2°C, 1.5°C, 2°C, 3°C, 4°C, 5°C or any value between them; and a time preferably of 2 min to 30 min, specifically 2 min, 2.5 min, 4 min, 5 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min or any value between them.

[0053] In some specific embodiments, the centrifugation conditions include a centrifugal force preferably between 7500×g and 8500×g, specifically 7500×g, 7800×g, 8000×g, 8200×g, 8500×g, or any value between them; and a time preferably between 1 min and 10 min, specifically 1 min, 2 min, 4 min, 5 min, 8 min, 10 min, or any value between them.

[0054] The present invention also provides an intestinal flora product, which is prepared by the purification method of the intestinal flora suspension described above.

[0055] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0056] It should be noted that the intestinal flora suspensions used in the following examples and comparative examples all came from the same fecal sample, and the storage time and processing methods of the fecal samples were kept consistent. The preparation of the intestinal flora suspension included: mixing the fecal sample with 0.9% physiological saline at a volume ratio of 1:8, stirring at 800 rpm for 10 min, filtering through a 100 μm filter membrane, and collecting the filtrate to obtain the intestinal flora suspension.

[0057] Example 1

[0058] This embodiment illustrates a method for purifying an intestinal flora suspension. The purification method specifically includes: adding 1.25g of sodium bicarbonate to 250mL of the intestinal flora suspension and stirring until completely dissolved; then adding 25mL of 5% (w / v) chitosan oligosaccharide acetic acid solution, 0.75g of sodium alginate (Shandong Jiejing Group Co., Ltd., viscosity 700mPa·s, the same below), and 1.25g of sodium carboxymethyl cellulose (Shanghai Shenguang Edible Chemicals Co., Ltd., degree of substitution 91%, viscosity 260.1mPa·s, the same below); stirring at 200rpm and room temperature for 15min; then allowing to stand at 4℃ for 15min; and finally centrifuging at 4℃ and 8000×g for 5min (centrifuge 5910Ri purchased from Eppendorf, speed setting "6, the same below); discarding the supernatant and collecting the intestinal sludge.

[0059] The preparation of the 5% (w / v) chitosan oligosaccharide acetic acid solution includes: adding chitosan oligosaccharide (Jiangsu Caiwei Biotechnology Co., Ltd., MW≤1610Da, the same below) to an acetic acid solution with a final concentration of 5% (w / v) to obtain the 5% (w / v) chitosan oligosaccharide acetic acid solution.

[0060] Example 2

[0061] The purification method of the intestinal flora suspension provided in this embodiment is basically the same as that in Example 1. The difference is that the volume of 5% (w / v) chitosan oligosaccharide acetic acid solution added is 12.5 mL, and the mass of sodium bicarbonate added is 2.5 g. Other conditions remain the same to obtain intestinal sludge.

[0062] Example 3

[0063] The purification method of the intestinal flora suspension provided in this embodiment is basically the same as that in Example 1. The difference is that the volume of 5% (w / v) chitosan oligosaccharide acetic acid solution added is 37.5 mL, and the mass of sodium bicarbonate added is 0.25 g. Other conditions remain the same to obtain intestinal sludge.

[0064] Example 4

[0065] The purification method of the intestinal flora suspension provided in this embodiment is basically the same as that in Example 1. The difference is that sodium alginate is added in place of sodium carboxymethyl cellulose, while other conditions remain the same, to obtain intestinal sludge.

[0066] Example 5

[0067] The purification method of the intestinal flora suspension provided in this embodiment is basically the same as that in Example 1. The difference is that sodium carboxymethyl cellulose is added in place of sodium alginate, while other conditions remain the same, to obtain intestinal sludge.

[0068] Example 6

[0069] The purification method of the intestinal flora suspension provided in this embodiment is basically the same as that in Example 1. The difference is that sodium alginate and sodium carboxymethyl cellulose are not added, while other conditions remain the same, to obtain intestinal sludge.

[0070] Example 7

[0071] The purification method of the intestinal flora suspension provided in this embodiment is basically the same as that in Example 1, except that sodium bicarbonate, 5% (w / v) chitosan oligosaccharide acetic acid solution, sodium alginate, and sodium carboxymethyl cellulose are added simultaneously. Specifically, 1.25 g of sodium bicarbonate, 25 mL of 5% (w / v) chitosan oligosaccharide acetic acid solution, 0.75 g of sodium alginate, and 1.25 g of sodium carboxymethyl cellulose are added to 250 mL of intestinal flora suspension. The mixture is stirred at 200 rpm and room temperature for 15 min, then allowed to stand at 4°C for 15 min, and then centrifuged at 4°C and 8000×g for 5 min (the centrifuge was purchased from Eppendorf, model Centrifuge 5910 Ri, and the acceleration / deceleration setting was set to "6", the same below). The supernatant is discarded, and the intestinal sludge is collected.

[0072] Comparative Example 1

[0073] This comparative example provides a method for purifying intestinal flora suspension, specifically including: adding 25 mL of physiological saline to 250 mL of intestinal flora suspension, stirring at 200 rpm and room temperature for 15 min, then letting it stand at 4°C for 15 min, and then centrifuging at 4°C and 8000×g for 5 min (centrifuge purchased from Eppendorf, model Centrifuge 5910 Ri, with acceleration / deceleration set to "6", the same below), discarding the supernatant, and collecting the intestinal sludge.

[0074] Comparative Example 2

[0075] The purification method of the intestinal flora suspension provided in this comparative example is basically the same as that in Example 1, except that an equal volume of 0.5% (v / v) acetic acid solution was added instead of 5% (w / v) chitosan oligosaccharide acetic acid solution, while keeping other conditions the same, to obtain intestinal sludge.

[0076] Comparative Example 3

[0077] The purification method of the intestinal flora suspension provided in this comparative example is basically the same as that in Example 1, except that an equal volume of 5% (w / v) chitosan oligosaccharide aqueous solution is added instead of 5% (w / v) chitosan oligosaccharide acetic acid solution, while keeping other conditions the same, to obtain intestinal sludge.

[0078] The preparation of the 5% (w / v) chitosan oligosaccharide aqueous solution includes: adding chitosan oligosaccharide to 0.9% physiological saline at a final concentration of 5% (w / v) to obtain a 5% (w / v) chitosan oligosaccharide aqueous solution.

[0079] Comparative Example 4

[0080] The purification method of the intestinal flora suspension provided in this comparative example is basically the same as that in Example 1, except that an equal volume of 5% chitosan acetate solution is added instead of 5% (w / v) chitosan oligosaccharide acetate solution, while keeping other conditions the same, to obtain intestinal sludge.

[0081] The preparation of the 5% (w / v) chitosan acetate solution includes: adding chitosan (Hebei Hongtao Bioengineering Co., Ltd., viscosity 34, DAC 91.8%) to a 0.5% acetic acid solution to obtain the 5% (w / v) chitosan acetate solution.

[0082] Test case

[0083] This test example illustrates the treatment effect of the intestinal flora suspension purification method provided in the above embodiments and the relevant properties of the resulting intestinal sludge. The test specifically includes:

[0084] 1. Recovery rate and activity of intestinal bacterial sludge: The intestinal bacterial suspension and various intestinal bacterial sludge were processed according to the instructions using a bacterial live / dead staining kit (Thermo Fisher Scientific, catalog number L7012). The activity and quantity of the bacterial community were detected by flow cytometry, and the recovery rate was calculated according to the following formula. The results are shown in Table 1.

[0085] Recovery rate = (Encapsulated bacterial count / Total bacterial count) × 100%

[0086] Table 1.

[0087]

[0088] As shown in Table 1, compared with Comparative Examples 1-4, the intestinal flora suspension purification method provided in Examples 1-7 of this invention has an excellent intestinal flora recovery rate and the intestinal flora activity in the resulting intestinal sludge is high, demonstrating excellent purification effect.

[0089] 2. Batch stability of purification methods: The purification methods provided in the above examples and comparative examples were used to process intestinal microbiota suspensions from different donor feces. The microbiota recovery rate was tested using the method provided in "1. Recovery rate of intestinal microbiota and microbiota activity". Each group was tested three times and the average value was taken. The results are shown in Table 2.

[0090] Table 2.

[0091]

[0092] As shown in Table 2, compared with Comparative Examples 1-4, the intestinal flora suspension purification methods provided in Examples 1-7 of this invention have a CV value of no more than 4.87% for the intestinal flora recovery rate of different batches of intestinal sludge, which shows excellent process stability.

[0093] 3. Gastric acid resistance of intestinal bacterial sludge: 1g of each intestinal bacterial sludge was added to 10mL of artificial gastric fluid and treated at 37℃ for 2h. The bacterial activity of the intestinal bacterial sludge before and after treatment with artificial gastric fluid was tested using the method provided in "1. Recovery rate and bacterial activity of intestinal bacterial sludge". The survival rate was calculated according to the following formula. The results are shown in Table 3.

[0094] Survival rate = (Number of viable bacteria after artificial gastric fluid treatment / Number of viable bacteria before artificial gastric fluid treatment) × 100%

[0095] Table 3.

[0096]

[0097] As shown in Table 3, compared with Comparative Examples 1-4, the intestinal microbiota suspension purification method provided in Examples 1-7 of this invention resulted in an intestinal microbiota sludge with a survival rate of over 49.2% after being treated with artificial gastric juice for 2 hours, demonstrating excellent resistance to gastric acid.

[0098] 4. Intestinal colonization effect of intestinal microbial sludge: C57 mice aged 6-8 weeks were used as experimental animals. After one week of acclimatization under the same conditions, they were randomly divided into 10 groups (3 mice in each group, N=3). 0.02g of each intestinal microbial sludge was administered to the mice by gavage. 48h after gavage, colon samples were surgically collected from the mice for 16S rDNA sequencing. The obtained sequencing data were analyzed for biological community diversity (Observed Species). The conditions for α diversity index analysis were kept consistent in each group. The results are shown in Table 4.

[0099] Table 4.

[0100]

[0101] As shown in Table 4, compared with Comparative Examples 1-4, the intestinal microbiota suspension purification method provided in Examples 1-7 of this invention produces intestinal microbiota sludge with similar α-diversity to the original intestinal microbiota suspension. Furthermore, the microbiota diversity in mouse colon samples after gavage treatment with this intestinal microbiota sludge is similar to that of the intestinal microbiota sludge, indicating that the intestinal microbiota sludge has excellent intestinal colonization ability.

[0102] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for purifying an intestinal flora suspension, characterized in that, The purification method includes: mixing sodium bicarbonate with an intestinal flora suspension, adding chitosan oligosaccharide acetic acid solution and water-soluble polymer, and performing low-temperature static treatment and centrifugation to obtain intestinal bacterial sludge; wherein, the temperature of the low-temperature static treatment is 1℃~5℃; In the chitosan oligosaccharide acetic acid solution, the concentration of chitosan oligosaccharide is 0.1% w / v to 10% w / v, and the concentration of acetic acid is 0.3% v / v to 0.8% v / v; In the chitosan oligosaccharide acetic acid solution, the weight-average molecular weight of chitosan oligosaccharide is not greater than 3200 Da, and the degree of polymerization is 2~20. The water-soluble polymer comprises sodium carboxymethyl cellulose and sodium alginate in a mass ratio of (0.1~10):(0.1~10); The sodium carboxymethyl cellulose has a degree of substitution of 85% to 95% and a viscosity of 100 mPa·s to 500 mPa·s. The viscosity of the sodium alginate is 600 mPa·s to 800 mPa·s; The concentration of the chitosan oligosaccharide acetic acid solution added is 1% v / v to 15% v / v, the concentration of sodium bicarbonate added is 0.01% w / v to 1% w / v, and the concentration of the water-soluble polymer added is 0.1% w / v to 20% w / v.

2. The method for purifying intestinal flora suspension according to claim 1, characterized in that, The purification method has at least one of the following (i)-(ii): (i) The time for the low-temperature static treatment is 2 min to 30 min; (ii) The centrifugal force of the centrifugation treatment is 7500×g~8500×g, and the time is 1min~10min.

3. The method for purifying intestinal flora suspension according to claim 1, characterized in that, The preparation of the intestinal flora suspension includes: taking a fecal sample and mixing it with physiological saline, performing stirring and filtration to obtain the intestinal flora suspension; and the preparation of the intestinal flora suspension includes at least one of the following (i)-(iii): (i) The volume ratio of the fecal sample to the saline solution is 1:(1~15); (ii) The stirring speed is 800 rpm to 1200 rpm and the time is 5 min to 15 min; (iii) In the filtration process, the pore size of the filter membrane is 80μm~120μm.

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