A method for the cryopreservation and thawing of fecal bacteria and a cryoprotective agent

Abstract

The present application relates to a kind of fecal bacteria cryopreservation and thawing method and cryoprotectant.The concentration of permeable protective agent in the fecal bacteria cryoprotectant is 350-400mg / ml, the concentration of impermeable protective agent is 110-130mg / ml, and the concentration of two-dimensional Ti3C2Tx nanosheet is 0.2-0.6mg / ml.The present application also provides a method for cryopreservation and thawing of fecal bacteria using the cryoprotectant, comprising the following steps: (1) preparing fecal bacteria; (2) preparing alginate hydrogel loaded with fecal bacteria; (3) cryopreservation of fecal bacteria; (4) thawing.The cryoprotectant of the present application contains two-dimensional Ti3C2Tx x nanosheet, so it has passive ice inhibition and photothermal active ice inhibition function.During the cryopreservation process, two-dimensional Ti3C2Tx x nanosheet has a significant inhibitory effect on the formation and growth of ice crystals.During the thawing process, two-dimensional Ti3C2Tx x nanosheet fully exerts its own photothermal effect, absorbs the energy of near-infrared laser and converts it into heat, acts as a high-efficiency space heat source, improves the uniformity of internal rewarming of the sample, and reduces the freezing damage caused by recrystallization and devitrification.

Description

Technical Field

[0001] This invention relates to a method for cryopreservation and thawing of fecal microbiota and a cryoprotectant, belonging to the field of fecal microbiota transplantation. Background Technology

[0002] Fecal microbiota transplantation (FMT) is a method that directly alters the recipient's gut microbiota, normalizing its composition and thus providing therapeutic benefits. The history of FMT dates back to the 4th century, when human feces, referred to as "yellow soup," were used for patients with severe diarrhea. As late as the 16th century in Ming Dynasty China, there were descriptions of using fresh or fermented fecal suspensions for patients with gastrointestinal diseases, including diarrhea, constipation, and abdominal pain. Eiseman and his colleagues successfully treated pseudomembranous colitis with FMT in 1958, marking the first report in medical literature. The Italian surgeon Acquapendente coined the term "animal transfer," meaning the transfer of gastrointestinal contents from a healthy animal to a diseased animal, and this term has been widely used in veterinary medicine. In 2013, the U.S. Food and Drug Administration approved FMT for the treatment of recurrent and refractory Clostridium difficile infection, and it has received considerable attention. Since then, the application of FMT has rapidly and extensively expanded beyond gastrointestinal diseases to include extra-gastrointestinal conditions. In conclusion, fecal microbiota transplantation is one of the most effective methods for altering the gut microbiota. However, the problems in the preparation, preservation, and application of fecal microbiota pose challenges to the development of FMT.

[0003] Current research on fecal microbiota preservation methods mainly includes cryopreservation, refrigeration, preservation solution preservation, and vitrification. Cryopreservation is the most common method for long-term preservation of fecal microbiota. However, practical studies have found that ice crystals formed during freezing and thawing can cause irreversible damage to fecal microbiota, leading to mass mortality. Ice damage is the main factor contributing to the failure of long-term cryopreservation of fecal microbiota. Cryopreservation strategies are mainly divided into slow freezing and vitrification. For slow freezing, ice formation and growth during the cooling phase and ice recrystallization during thawing limit the survival quality and efficiency of cryopreserved samples. Although vitrification avoids the devitrification caused by high concentrations of cryoprotectants and ultra-fast cooling rates during freezing, the recrystallization of ice nuclei during thawing still leads to severe freezing damage to the cryopreserved biological samples. In addition, high concentrations of cryoprotectants can cause biotoxicity in frozen samples, especially at the protein and gene levels, which undoubtedly poses a potential risk to the further clinical application of frozen samples.

[0004] Chinese patent document CN111876331A discloses a method for collecting, cryopreserving, and thawing fecal microbiota during fecal microbiota transplantation. This method rapidly cools cells to a "glassy" state, preventing ice crystal formation inside and outside the cells and avoiding excessive damage to the microbiota. Furthermore, antifreeze proteins are added to the cryoprotectant to prevent its cytotoxicity, resulting in a survival rate of over 90% for the microbiota after thawing. However, the patent does not inhibit ice nucleus formation during thawing. Although the patent mentions passing through a dangerous temperature zone instantaneously during thawing and thawing to prevent ice crystal formation, the freezing, thawing, and thawing process is cumbersome and requires highly skilled operators.

[0005] Chinese patent document CN112195103A discloses a freeze-drying protectant, a freeze-dried product of fecal bacteria, and a method for preparing the same. The components include polydextrose, mannitol, glycine, vitamin C, and Tween 80. However, the components used in this patent are still traditional protectant components, and the freeze-drying survival rate remains unsatisfactory.

[0006] In recent years, rapid cooling methods have been proposed due to their advantages such as simple procedures and equipment, and low concentration of penetrating cryoprotectants, which are conducive to their widespread application in clinical medicine and scientific research. Unfortunately, ice nucleation during the cooling process and low-temperature damage caused by recrystallization and devitrification during the heating process remain bottleneck problems for rapid cooling methods. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for cryopreservation and thawing of fecal microorganisms, as well as a cryoprotectant.

[0008] The technical solution of the present invention is as follows:

[0009] A fecal microbiota cryoprotectant, wherein the concentration of the permeable cryoprotectant is 350-400 mg / ml, the concentration of the non-permeable cryoprotectant is 110-130 mg / ml, and the concentration of the two-dimensional Ti3C2Tx nanosheets is 0.2-0.6 mg / ml.

[0010] According to a preferred embodiment of the present invention, the solvent for the fecal microorganism cryoprotectant is sterile purified water.

[0011] According to a preferred embodiment of the present invention, the permeation protectant is one or a mixture of two or more of ethylene glycol, propylene glycol, dimethyl sulfoxide, and glycerol.

[0012] More preferably, the permeability protectant is a mixture of ethylene glycol and propylene glycol, wherein the mass ratio of ethylene glycol to propylene glycol is 1:1.

[0013] According to a preferred embodiment of the present invention, the non-permeable protective agent is one or a mixture of two or more of the following: polyvinylpyrrolidone, monosaccharides, disaccharides, polysaccharides, polyethylene glycol, dextran, trehalose, and ascorbic acid.

[0014] According to a preferred embodiment of the present invention, the two-dimensional Ti3C2T x Nanosheets are prepared according to the following method:

[0015] 2 g of lithium fluoride was added to 40 mL of 9 M hydrochloric acid solution and stirred for 10 min to form a hydrofluoric acid solution. Then, 2 g of MAX-phase ternary carbonitride was slowly added to the hydrofluoric acid solution at 35 °C and stirred for 24 h. The solution was then centrifuged at 4000 r / min for 5 min, and the pH was adjusted to 5.5–6.6. The precipitate was collected. The precipitate was added to sterile ultrapure water and ultrasonically degraded for 20 min. Finally, it was centrifuged at 4000 r / min for 2 h to obtain two-dimensional Ti3C2T. x Nanosheets.

[0016] According to a preferred embodiment of the present invention, the MAX phase ternary carbonitride is Ti3AlC2. The MAX phase is a ternary carbonitride, which can be MAX, M2AX, or M3AX, where M is a transition metal element, A is a group A element, and X is carbon or nitrogen. The stacked structure formed by these three elements exhibits anisotropy. The MAX phase has a hexagonal structure, with the M layer and the group A element layer existing in an alternating layered manner, and X atoms filling the octahedral positions between the structures.

[0017] The preparation method of the above-mentioned fecal microbial cryoprotectant includes the following steps:

[0018] The permeable protective agent, the non-permeable protective agent, and the two-dimensional Ti3C2T x The fecal microorganism cryoprotectant is obtained by mixing the nanosheets evenly according to the specified ratio.

[0019] A method for cryopreserving and thawing fecal microorganisms using the above-mentioned fecal microorganism cryoprotectant includes the following steps:

[0020] (1) Add sterile 0.9% NaCl solution to the donor feces for the first homogenization treatment, filter for the first time to obtain filtrate, add sterile 0.9% NaCl solution to the filtrate for the second homogenization treatment, filter for the second time, and centrifuge the obtained filtrate at 4000-6000 r / min for 10-15 min to obtain fecal bacteria;

[0021] (2) Mix the alginate solution and fecal bacteria evenly, then add CaCl2 solution, centrifuge at 800-1200 r / min for 10-20 min, remove the supernatant, and obtain the alginate hydrogel loaded with fecal bacteria.

[0022] (3) Add fecal bacteria cryoprotectant to the alginate hydrogel loaded with fecal bacteria, mix well and collect it into frozen wheat tubes, place the frozen wheat tubes in liquid nitrogen for rapid cooling, and obtain cryopreserved fecal bacteria.

[0023] (4) The frozen fecal bacteria were heated in a water bath at 35-40°C, and laser-assisted heating was performed at the same time. After thawing, the fecal bacteria cryoprotectant was aspirated, and sugar solution was added to the fecal bacteria cryoprotectant to wash it off. The alginate hydrogel loaded with fecal bacteria was then dissolved with sodium citrate solution. Finally, the fecal bacteria were obtained by centrifugation at 5500-6500 r / min for 10-20 min. The fecal bacteria were placed in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

[0024] According to a preferred embodiment of the present invention, in step (1), the mass-to-volume ratio of donor feces and sterile 0.9% NaCl solution in the first homogenization treatment and the second homogenization treatment is 1:5, unit: g / mL; the first filtration is performed using a 5-10 mm stainless steel filter screen, and the second filtration is performed four times in sequence using stainless steel filter screens of 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm.

[0025] According to a preferred embodiment of the present invention, in step (2), the concentration of the alginate solution is 1.5-2.5%.

[0026] According to a preferred embodiment of the present invention, in step (2), the mass-to-volume ratio of the fecal bacteria and the alginate solution is 2:1, with units of g / mL.

[0027] According to a preferred embodiment of the present invention, in step (2), the concentration of the CaCl2 solution is 1-2%.

[0028] According to a preferred embodiment of the present invention, in step (2), the volume ratio of the alginate solution to the CaCl2 solution is 1:2.

[0029] According to a preferred embodiment of the present invention, in step (3), the mass-to-volume ratio of the alginate hydrogel loaded with fecal bacteria and the fecal bacteria cryoprotectant is 1:2, with units of g / mL.

[0030] According to a preferred embodiment of the present invention, in step (4), during laser-assisted heating, the wavelength of the laser is 800–820 nm and the irradiance is 4500–5500 mW / cm². 2 .

[0031] According to a preferred embodiment of the present invention, in step (4), the sugar solution is added in three separate additions, with concentrations of 0.75M, 0.5M and 0M respectively, and the volumes added in the three additions are the same; the concentration of the sodium citrate solution is 70-80mM, and the volume ratio of the sodium citrate solution to the sugar solution is 1:3.

[0032] According to a preferred embodiment of the present invention, in step (4), the sugar solution is a monosaccharide, disaccharide, polysaccharide or trehalose solution, and the volume ratio of the amount of sugar solution added at one time to the volume of the fecal cryoprotectant is 2:1.

[0033] Beneficial effects:

[0034] 1. The fecal microbial cryoprotectant provided by this invention contains two-dimensional Ti3C2T. x Nanosheets enable cryoprotectants to possess both passive and photothermal active anti-icing functions. During cryopreservation, two-dimensional Ti3C2T... x Nanosheets significantly inhibit the formation and growth of ice crystals. During thawing, two-dimensional Ti3C2T… x Nanosheets also fully utilize their photothermal effect, absorbing the energy of near-infrared lasers and converting it into heat, acting as an efficient space heat source. This greatly improves the uniformity of rewarming inside the sample and also increases the average rewarming rate of the entire sample. It can promote the rapid thawing of frozen samples and reduce freezing damage caused by recrystallization and devitrification.

[0035] 2. The method for cryopreserving fecal microorganisms provided by the present invention does not directly preserve fecal microorganisms. Instead, it first combines fecal microorganisms with a hydrogel, and then uses a cryoprotectant to freeze the alginate hydrogel loaded with fecal microorganisms. This not only avoids direct contact between the cryoprotectant and fecal microorganisms, preventing the cryoprotectant from being toxic to cells, but also eliminates the need to add a thawing protectant during the subsequent thawing process, thus avoiding potential damage to the fecal microorganisms caused by the thawing protectant.

[0036] 3. The fecal microorganism cryoprotectant and cryopreservation and thawing method provided by this invention greatly reduce the risks of cryopreservation and thawing. The survival rate of fecal microorganisms after one month of cryopreservation is over 90%, and the survival rate of fecal microorganisms after six months of cryopreservation is over 80%. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and medicines involved in the embodiments are all commercially available products; unless otherwise specified, the experimental operations involved in the embodiments are all conventional operations in the art.

[0038] Two-dimensional Ti3C2T x The nanosheets were prepared as follows: 2g of lithium fluoride was added to 40mL of 9M hydrochloric acid solution and stirred for 10min to allow for complete reaction and formation of hydrofluoric acid solution. 2g of Ti3AlC2 was then slowly added to the hydrofluoric acid solution at 35℃ and stirred for 24h. During this process, the hydrofluoric acid removed the aluminum layer, and two-dimensional ultrathin Ti3C2 nanosheets were fabricated by etching the MAX phase Ti3AlC2.x Nanosheets were then obtained. The solution was centrifuged at 4000 rpm for 5 min, and the pH was adjusted to 6. The precipitate was collected, added to ultrapure water, and ultrasonically degraded for 20 min. Finally, it was centrifuged at 4000 rpm for 2 h to obtain two-dimensional Ti3C2T nanosheets. x Nanosheets.

[0039] Example 1

[0040] A fecal microbiota cryoprotectant, wherein the fecal microbiota cryoprotectant comprises ethylene glycol 62 mg / ml, 1,2-propanediol 62 mg / ml, trehalose 378.33 mg / ml, and two-dimensional Ti3C2T x Nanosheets 0.2 mg / ml, solvent: sterile water.

[0041] The preparation method of the above-mentioned fecal microbial cryoprotectant includes the following steps:

[0042] The permeable protective agent, the non-permeable protective agent, and the two-dimensional Ti3C2T x The fecal microorganism cryoprotectant is obtained by mixing the nanosheets evenly according to the specified ratio.

[0043] A method for cryopreserving and thawing fecal microorganisms using the above-mentioned fecal microorganism cryoprotectant includes the following steps:

[0044] (1) Add 750ml of sterile 0.9% NaCl solution to 150g of donor feces for the first homogenization treatment. Filter the fecal slurry through a stainless steel filter to remove large particles. Add 750ml of sterile 0.9% NaCl solution to the filtrate for the second homogenization treatment. Then filter through stainless steel filters with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm in succession to remove unabsorbed food residue and small particles. Centrifuge the filtered sample at 6000r / min for 15min to obtain fecal bacteria.

[0045] (2) Mix 16 ml of 2% alginate solution and 8 g of fecal bacteria evenly, then add 32 ml of 1.5% CaCl2 solution, centrifuge at 1000 r / min for 15 min, remove the supernatant, and obtain alginate hydrogel loaded with fecal bacteria.

[0046] (3) Add 16 ml of fecal bacteria cryoprotectant to 8 g of alginate hydrogel loaded with fecal bacteria, mix well and collect it into frozen wheat tubes. Place the frozen wheat tubes in liquid nitrogen for rapid cooling to obtain cryopreserved fecal bacteria.

[0047] (4) Fecal bacteria that have been frozen for one month are heated in a water bath at 37°C, and laser-assisted heating is performed simultaneously with the water bath heating. During the laser-assisted heating, the wavelength of the laser is 808 nm and the irradiance is 5000 mW / cm².2 After thawing, the cryoprotectant for fecal bacteria was aspirated, and then the cryoprotectant was eluted using a gradient of trehalose solutions (0.75M, 0.5M, and 0M), with each concentration of trehalose solution containing 32 ml. The trehalose solution was then aspirated, and 32 ml of a 75 mM sodium citrate solution was added. Finally, the fecal bacteria were obtained by centrifugation at 6000 r / min for 15 min. The fecal bacteria were then placed in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

[0048] Example 2

[0049] A fecal microbiota cryoprotectant, wherein the fecal microbiota cryoprotectant comprises ethylene glycol 62 mg / ml, 1,2-propanediol 62 mg / ml, trehalose 378.33 mg / ml, and two-dimensional Ti3C2T x Nanosheets 0.4 mg / ml, solvent: sterile water.

[0050] A method for cryopreserving and thawing fecal microorganisms using the above-mentioned fecal microorganism cryoprotectant includes the following steps:

[0051] (1) Add 750ml of sterile 0.9% NaCl solution to 150g of donor feces for the first homogenization treatment. Filter the fecal slurry through a stainless steel filter to remove large particles. Add 750ml of sterile 0.9% NaCl solution to the filtrate for the second homogenization treatment. Then filter through stainless steel filters with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm in succession to remove unabsorbed food residue and small particles. Centrifuge the filtered sample at 6000r / min for 15min to obtain fecal bacteria.

[0052] (2) Mix 16 ml of 2% alginate solution and 8 g of fecal bacteria evenly, then add 32 ml of 1.5% CaCl2 solution, centrifuge at 1000 r / min for 15 min, remove the supernatant, and obtain alginate hydrogel loaded with fecal bacteria.

[0053] (3) Add 16 ml of fecal bacteria cryoprotectant to 8 g of alginate hydrogel loaded with fecal bacteria, mix well and collect it into frozen wheat tubes. Place the frozen wheat tubes in liquid nitrogen for rapid cooling to obtain cryopreserved fecal bacteria.

[0054] (4) Fecal bacteria that have been frozen for one month are heated in a water bath at 37°C, and laser-assisted heating is performed simultaneously with the water bath heating. During the laser-assisted heating, the wavelength of the laser is 808 nm and the irradiance is 5000 mW / cm². 2After thawing, the cryoprotectant for fecal bacteria was aspirated, and then the cryoprotectant was eluted using a gradient of trehalose solutions (0.75M, 0.5M, and 0M), with each concentration of trehalose solution containing 32 ml. The trehalose solution was then aspirated, and 32 ml of a 75 mM sodium citrate solution was added. Finally, the fecal bacteria were obtained by centrifugation at 6000 r / min for 15 min. The fecal bacteria were then placed in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

[0055] Example 3

[0056] A fecal microbiota cryoprotectant, wherein the fecal microbiota cryoprotectant comprises ethylene glycol 62 mg / ml, 1,2-propanediol 62 mg / ml, trehalose 378.33 mg / ml, and two-dimensional Ti3C2T x Nanosheets 0.6 mg / ml, solvent: sterile water.

[0057] A method for cryopreserving and thawing fecal microorganisms using the above-mentioned fecal microorganism cryoprotectant includes the following steps:

[0058] (1) Add 750ml of sterile 0.9% NaCl solution to 150g of donor feces for the first homogenization treatment. Filter the fecal slurry through a stainless steel filter to remove large particles. Add 750ml of sterile 0.9% NaCl solution to the filtrate for the second homogenization treatment. Then filter through stainless steel filters with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm in succession to remove unabsorbed food residue and small particles. Centrifuge the filtered sample at 6000r / min for 15min to obtain fecal bacteria.

[0059] (2) Mix 16 ml of 2% alginate solution and 8 g of fecal bacteria evenly, then add 32 ml of 1.5% CaCl2 solution, centrifuge at 1000 r / min for 15 min, remove the supernatant, and obtain alginate hydrogel loaded with fecal bacteria.

[0060] (3) Add 16 ml of fecal bacteria cryoprotectant to 8 g of alginate hydrogel loaded with fecal bacteria, mix well and collect it into frozen wheat tubes. Place the frozen wheat tubes in liquid nitrogen for rapid cooling to obtain cryopreserved fecal bacteria.

[0061] (4) Fecal bacteria that have been frozen for one month are heated in a water bath at 37°C, and laser-assisted heating is performed simultaneously with the water bath heating. During the laser-assisted heating, the wavelength of the laser is 808 nm and the irradiance is 5000 mW / cm². 2After thawing, the cryoprotectant for fecal bacteria was aspirated, and then the cryoprotectant was eluted using a gradient of trehalose solutions (0.75M, 0.5M, and 0M), with each concentration of trehalose solution containing 32 ml. The trehalose solution was then aspirated, and 32 ml of a 75 mM sodium citrate solution was added. Finally, the fecal bacteria were obtained by centrifugation at 6000 r / min for 15 min. The fecal bacteria were then placed in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

[0062] Example 4

[0063] A fecal microbiota cryoprotectant, wherein the fecal microbiota cryoprotectant comprises ethylene glycol 62 mg / ml, 1,2-propanediol 62 mg / ml, trehalose 378.33 mg / ml, and two-dimensional Ti3C2T x Nanosheets 0.4 mg / ml, solvent: sterile water.

[0064] (1) Add 750ml of sterile 0.9% NaCl solution to 150g of donor feces for the first homogenization treatment. Filter the fecal slurry through a stainless steel filter to remove large particles. Add 750ml of sterile 0.9% NaCl solution to the filtrate for the second homogenization treatment. Then filter through stainless steel filters with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm in succession to remove unabsorbed food residue and small particles. Centrifuge the filtered sample at 6000r / min for 15min to obtain fecal bacteria.

[0065] (2) Mix 16 ml of 2.5% alginate solution and 8 g of fecal bacteria evenly, then add 32 ml of 2% CaCl2 solution, centrifuge at 1000 r / min for 15 min, remove the supernatant, and obtain alginate hydrogel loaded with fecal bacteria.

[0066] (3) Add 16 ml of fecal bacteria cryoprotectant to 8 g of alginate hydrogel loaded with fecal bacteria, mix well and collect it into frozen wheat tubes. Place the frozen wheat tubes in liquid nitrogen for rapid cooling to obtain cryopreserved fecal bacteria.

[0067] (4) Fecal bacteria that have been frozen for one month are heated in a water bath at 37°C, and laser-assisted heating is performed simultaneously with the water bath heating. During the laser-assisted heating, the wavelength of the laser is 808 nm and the irradiance is 5000 mW / cm². 2After thawing, the cryoprotectant for fecal bacteria was aspirated, and then the cryoprotectant was eluted using a gradient of trehalose solutions (0.75M, 0.5M, and 0M), with each concentration of trehalose solution containing 32 ml. The trehalose solution was then aspirated, and 32 ml of a 75 mM sodium citrate solution was added. Finally, the fecal bacteria were obtained by centrifugation at 6000 r / min for 15 min. The fecal bacteria were then placed in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

[0068] Example 5

[0069] A fecal microbiota cryoprotectant, with the same specific components as in Example 4.

[0070] A method for cryopreserving and thawing fecal bacteria using the above-mentioned fecal bacteria cryoprotectant is the same as that in Example 4, except that the cryopreservation time in step (4) is 3 months.

[0071] Example 6

[0072] A fecal microbiota cryoprotectant, with the same specific components as in Example 4.

[0073] A method for cryopreserving and thawing fecal bacteria using the above-mentioned fecal bacteria cryoprotectant is the same as that in Example 4, except that the cryopreservation time in step (4) is 4 months.

[0074] Example 7

[0075] A fecal microbiota cryoprotectant, with the same specific components as in Example 4.

[0076] A method for cryopreserving and thawing fecal bacteria using the above-mentioned fecal bacteria cryoprotectant is the same as that in Example 4, except that the cryopreservation time in step (4) is 5 months.

[0077] Example 8

[0078] A fecal microbiota cryoprotectant, with the same specific components as in Example 4.

[0079] A method for cryopreserving and thawing fecal bacteria using the above-mentioned fecal bacteria cryoprotectant is the same as that in Example 4, except that the cryopreservation time in step (4) is 6 months.

[0080] Comparative Example 1

[0081] A method for cryopreserving fecal microbiota, comprising the following steps:

[0082] (1) Place 150g of fresh fecal sample from the donor in a mixer, add 750ml of sterile 0.9% NaCl solution for preliminary homogenization, filter the fecal slurry through a stainless steel mesh to remove large particles; further homogenize within 2 hours; then filter through stainless steel meshes with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm in succession to remove unabsorbed food residue and small particles; centrifuge the filtered sample at 6000r / min for 15min to obtain fecal bacteria;

[0083] (2) Dissolve 8g of fecal bacteria in 16ml of sterile 0.9% NaCl solution, collect directly into frozen wheat tubes, place the wheat tubes directly in liquid nitrogen, and quickly complete the cooling and freezing process to obtain frozen fecal bacteria;

[0084] (3) Fecal bacteria that have been frozen for 1 month were heated in a water bath at 37°C and laser-assisted heating was performed at the same time. After thawing, the NaCl solution was aspirated, and then eluted with a gradient of trehalose solutions (0.75M, 0.5M and 0M). The volume of each trehalose solution was 32 ml. Then, the trehalose solution was aspirated and 32 ml of sodium citrate solution with a concentration of 75 mM was added. Finally, the fecal bacteria were obtained by centrifugation at 6000 r / min for 15 min. The fecal bacteria were placed in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

[0085] Comparative Example 2

[0086] A fecal microbiota cryoprotectant, wherein the fecal microbiota cryoprotectant comprises 62 mg / ml ethylene glycol, 62 mg / ml 1,2-propanediol, and 378.33 mg / ml trehalose, and the solvent is sterile water.

[0087] A method for cryopreserving fecal microorganisms using the above-mentioned cryoprotectant comprises the following steps:

[0088] (1) Place 150g of fresh fecal sample from the donor in a mixer, add 750ml of sterile 0.9% NaCl solution for preliminary homogenization, filter the fecal slurry through a stainless steel mesh to remove large particles; further homogenize within 2 hours; then filter through stainless steel meshes with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm in succession to remove unabsorbed food residue and small particles; centrifuge the filtered sample at 6000r / min for 15min to obtain fecal bacteria;

[0089] (2) Dissolve 8g of fecal bacteria in 16ml of cryoprotectant, collect directly into frozen wheat tubes, place the wheat tubes directly in liquid nitrogen, and quickly complete the cooling and freezing process to obtain cryopreserved fecal bacteria;

[0090] (3) Fecal bacteria that have been frozen for 1 month were heated in a water bath at 37°C and laser-assisted heating was performed at the same time. After thawing, the cryoprotectant of the fecal bacteria was aspirated. Then, the cryoprotectant was eluted by gradient elution with trehalose solutions (0.75M, 0.5M and 0M). The volume of each trehalose solution was 32 ml. Then, the trehalose solution was aspirated and 32 ml of sodium citrate solution with a concentration of 75 mM was added. Finally, the fecal bacteria were obtained by centrifugation at 6000 r / min for 15 min. The fecal bacteria were placed in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

[0091] Comparative Example 3

[0092] A fecal microbiota cryoprotectant, wherein the fecal microbiota cryoprotectant comprises 62 mg / ml ethylene glycol, 62 mg / ml 1,2-propanediol, and 378.33 mg / ml trehalose, and the solvent is sterile water.

[0093] A method for cryopreserving fecal microorganisms using the above-mentioned cryoprotectant comprises the following steps:

[0094] (1) Place 150g of fresh fecal sample from the donor in a mixer, add 750ml of sterile 0.9% NaCl solution for preliminary homogenization, filter the fecal slurry through a stainless steel mesh to remove large particles; further homogenize within 2 hours; then filter through stainless steel meshes with diameters of 2.0mm, 1.0mm, 0.5mm and 0.25mm in succession to remove unabsorbed food residue and small particles; centrifuge the filtered sample at 6000r / min for 15min to obtain fecal bacteria;

[0095] (2) Mix 16 ml of 2% alginate solution and 8 g of fecal bacteria evenly, then add 24 ml of 1.5% CaCl2 solution, centrifuge at 1000 r / min for 15 min, remove the supernatant, and obtain alginate hydrogel loaded with fecal bacteria.

[0096] (3) Add 16 ml of fecal bacteria cryoprotectant to 8 g of alginate hydrogel loaded with fecal bacteria, mix well and collect it into frozen wheat tubes. Place the frozen wheat tubes in liquid nitrogen for rapid cooling to obtain cryopreserved fecal bacteria.

[0097] (4) After freezing and storing fecal bacteria for one month, heat them in a water bath at 37°C and simultaneously perform laser-assisted heating. After thawing, remove the cryoprotectant from the fecal bacteria and then elute the cryoprotectant using a gradient of trehalose solutions (0.75M, 0.5M, and 0M). The volume of each trehalose solution is 32 ml. Then, remove the trehalose solution and add 32 ml of 75 mM sodium citrate solution. Finally, centrifuge at 6000 r / min for 15 min to obtain fecal bacteria. Place the fecal bacteria in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

[0098] Determination of total colony viability in fecal bacteria in experimental cases

[0099] Viable bacteria count before cryopreservation: Fecal bacteria were serially diluted and the viable bacteria count was determined using the pour plate counting method.

[0100] Viable bacteria count after cryopreservation: The thawed fecal bacterial solutions prepared in Examples 1-8 and Comparative Examples 1-3 were serially diluted, and the viable bacteria count was determined by pour plate counting.

[0101] The survival rate was calculated using the following formula: Survival rate = Number of viable bacteria after freezing / Number of viable bacteria before freezing. The results are shown in Table 1.

[0102] Table 1

[0103] Group Fecal bacteria survival rate Example 1 <![CDATA[91.77%±0.36% ## ]]> Example 2 <![CDATA[95.85%±0.33% ##*&& ]]> Example 3 <![CDATA[92.76%±0.40% ## ]]> Example 4 92.18％±0.43％ Example 5 90.42％±0.41％ Example 6 88.92％±0.56％ Example 7 86.74％±0.40％ Example 8 85.47％±0.59％ Comparative Example 1 41.09％±1.80％ Comparative Example 2 <![CDATA[50.81%±0.53% ** <!-- 7 -->]]> Comparative Example 3 <![CDATA[71.54%±1.12% **## ]]>

[0104] Note: * P < 0.05 vs. Comparative Example 1; ** P < 0.01 vs Comparative Example 1; # P < 0.05 vs. Comparative Example 2; ## P < 0.01 vs Comparative Example 2. # P < 0.05 vs. Comparative Example 3; ## P < 0.01 vs Comparative Example 3; * P < 0.05 vs. Example 1; ** P < 0.01 vs. Example 1; & P < 0.05 vs Example 3; && P < 0.01 vs Example 3.

[0105] As shown in Table 1, the fecal microbiota cryoprotectant and the methods for cryopreserving and thawing fecal microbiota provided in Examples 1-3 of this invention all achieved a fecal microbiota survival rate of over 91% after one month of cryopreservation. In contrast, the fecal microbiota survival rate of Comparative Example 1, which was directly frozen, was only 41.09%, and that of Comparative Example 2, which added a commercially available cryoprotectant but did not construct a fecal microbiota-loaded hydrogel structure, was only 50.81%, all significantly lower than that of this invention.

[0106] Furthermore, compared to Comparative Example 3, which uses a commercially available cryoprotectant to construct a fecal microbial-loaded hydrogel structure, the success rate was only 71.54%, significantly lower than Examples 1-3 of this invention. This demonstrates the effectiveness of adding two-dimensional Ti3C2T to the cryoprotectant of this invention. x The nanosheets endow the cryoprotectant with both passive and photothermal active anti-icing functions, promoting rapid thawing of cryopreserved samples, reducing freezing damage caused by recrystallization and devitrification, and further improving the survival rate of fecal microorganisms. A comparison of Examples 2 and 1 & 3 also reveals the presence of two-dimensional Ti3C2T. x The survival rate of fecal bacteria reached its highest when the concentration of nanosheets was 0.4 mg / ml.

[0107] Meanwhile, the fecal microbiota cryoprotectant and the methods for cryopreserving and thawing fecal microbiota provided in Examples 1 to 8 of the present invention can preserve fecal microbiota for a long time. After 6 months of preservation, the survival rate of fecal microbiota is still above 85%.

Claims

1. A fecal microbial cryoprotectant, characterized in that, The fecal microbial cryoprotectant contains a permeable cryoprotectant concentration of 350-400 mg / ml, a non-permeable cryoprotectant concentration of 110-130 mg / ml, and two-dimensional Ti3C2Tx nanosheets concentration of 0.2-0.6 mg / ml.

2. The fecal microbial cryoprotectant as described in claim 1, characterized in that, The permeable protective agent is one or a mixture of two or more of ethylene glycol, propylene glycol, dimethyl sulfoxide, and glycerol; the non-permeable protective agent is one or a mixture of two or more of polyvinylpyrrolidone, monosaccharides, disaccharides, polysaccharides, polyethylene glycol, dextran, trehalose, and ascorbic acid.

3. The fecal microbial cryoprotectant as described in claim 2, characterized in that, The permeability protectant is a mixture of ethylene glycol and propylene glycol, wherein the mass ratio of ethylene glycol to propylene glycol is 1:

1.

4. The fecal microbial cryoprotectant as described in claim 1, characterized in that, The two-dimensional Ti3C2T x Nanosheets are prepared according to the following method: 2 g of lithium fluoride was added to 40 mL of 9 M hydrochloric acid solution and stirred for 10 min to form hydrofluoric acid solution; then 2 g of MAX phase ternary carbonitride was slowly added to hydrofluoric acid solution at 35 °C and stirred for 24 h. The solution was then centrifuged at 4000 r / min for 5 min and the pH was adjusted to 5.5-6.6 before the precipitate was collected. The precipitate was added to sterile ultrapure water and ultrasonically degraded for 20 min, and finally centrifuged at 4000 r / min for 2 h to obtain two-dimensional Ti3C2T. x Nanosheets; The MAX phase ternary carbonitride is Ti3AlC2.

5. A method for cryopreserving and thawing fecal microorganisms using the fecal microorganism cryoprotectant described in claim 1, characterized in that, The steps include the following: (1) Add sterile 0.9% NaCl solution to the donor feces for the first homogenization treatment, filter for the first time to obtain filtrate, add sterile 0.9% NaCl solution to the filtrate for the second homogenization treatment, filter for the second time, and centrifuge the obtained filtrate at 4000~6000 r / min for 10~15min to obtain fecal bacteria; (2) Mix the alginate solution and fecal bacteria evenly, then add CaCl2 solution, centrifuge at 800~1200 r / min for 10~20 min, remove the supernatant, and obtain the alginate hydrogel loaded with fecal bacteria; (3) Add fecal bacteria cryoprotectant to the alginate hydrogel loaded with fecal bacteria, mix well and collect it into frozen wheat tubes, place the frozen wheat tubes in liquid nitrogen for rapid cooling, and obtain cryopreserved fecal bacteria; (4) The frozen fecal bacteria were heated in a water bath at 35~40℃, and laser-assisted heating was performed at the same time. After thawing, the fecal bacteria cryoprotectant was aspirated. Then, the fecal bacteria cryoprotectant on the alginate hydrogel loaded with fecal bacteria was eluted with a sugar solution gradient. The alginate hydrogel loaded with fecal bacteria was then dissolved with sodium citrate solution. Finally, the fecal bacteria were obtained by centrifugation at 5500~6500 r / min for 10~20 min. The fecal bacteria were placed in a sterile 0.9% NaCl solution to obtain the thawed fecal bacteria solution.

6. The method for cryopreserving and thawing fecal microorganisms as described in claim 5, characterized in that, In step (1), the mass-to-volume ratio of donor feces and sterile 0.9% NaCl solution in the first and second homogenization processes is 1:5, unit: g / mL; the first filtration is performed using a 5-10 mm stainless steel filter screen, and the second filtration is performed four times sequentially using stainless steel filter screens of 2.0 mm, 1.0 mm, 0.5 mm and 0.25 mm.

7. The method for cryopreserving and thawing fecal microorganisms as described in claim 5, characterized in that, In step (2), the concentration of the alginate solution is 1.5~2.5%; the mass-to-volume ratio of the fecal bacteria and the alginate solution is 2:1, with units of g / mL.

8. The method for cryopreserving fecal microorganisms and thawing as described in claim 5, characterized in that, In step (2), the concentration of the CaCl2 solution is 1-2%; the volume ratio of the alginate solution to the CaCl2 solution is 1:

2.

9. The method for cryopreserving and thawing fecal microorganisms as described in claim 5, characterized in that, In step (3), the mass-to-volume ratio of the alginate hydrogel loaded with fecal bacteria to the fecal bacteria cryoprotectant is 1:2, with units of g / mL.

10. The method for cryopreserving and thawing fecal microorganisms as described in claim 5, characterized in that, In step (4), during laser-assisted heating, the laser wavelength is 800~820 nm and the irradiance is 4500~5500 mW / cm². 2 The sugar solution was added in three separate additions, with concentrations of 0.75 M, 0.5 M, and 0 M, respectively, and the volumes added in each addition were the same. The concentration of the sodium citrate solution was 70-80 mM, and the volume ratio of the sodium citrate solution to the sugar solution was 1:

3. The volume ratio of the single-use sugar solution addition to the fecal microbial cryoprotectant is 2:1.