A gut microbiota product, capsule, preparation method and apparatus, and its application in precision microbiota transplantation.

Abstract

This invention belongs to the technical field of gut microbiota transplantation, and discloses a gut microbiota product, capsule, preparation method and apparatus, and its application in precision gut microbiota transplantation. The composition provided by this invention includes at least an eluent for purifying donor feces and a protectant for maintaining gut microbiota activity. The eluent comprises plant extracts and Japanese magnolia bark powder in a specific mass ratio, and the protectant comprises small-molecule polyhydroxy compounds, macromolecular polymers, amines, natural emulsifiers, and optionally carbonates in a specific mass ratio. Both constitute an organic whole, working synergistically to achieve efficient odor removal and minimal impact on microbiota activity in the preparation of a gut microbiota product, ultimately obtaining a gut microbiota product with low odor, high microbiota activity, and excellent storage stability.

Description

Technical Field

[0001] This invention belongs to the technical field of gut microbiota transplantation, and particularly relates to a gut microbiota product, capsule, preparation method and device, and its application in precision gut microbiota transplantation. Background Technology

[0002] Microbiota transplantation (CMPT) refers to the transplantation of functional gut microbiota from the feces of healthy individuals into the gastrointestinal tract of patients, reconstructing a normal gut microbiota and achieving the treatment of intestinal and extraintestinal diseases. As a core technology for targeted reconstruction of the gut microbiota, CMPT has become an effective strategy for treating recurrent Clostridium difficile infection, severe Clostridium difficile infection unresponsive to standard treatment, and fulminant Clostridium difficile infection unsuitable for surgery. The cure rate for a single CMPT session is over 90%. Furthermore, CMPT can also be used to relieve intractable constipation, treat symptoms of mild to moderate ulcerative colitis (UC), and treat autism, Parkinson's disease, and various gastrointestinal cancers.

[0003] Clinically, microbial transplantation (MTBG) is mainly performed through surgical infusion of bacterial culture and oral capsule methods. Surgical infusion, which involves inserting bacterial culture into the patient via nasoenteric tube, endoscopy forceps channel, percutaneous endoscopic gastrostomy jejunal tube, colonoscopy, or enema, requires professional personnel and has a high incidence of adverse events, limiting its large-scale practical application. In contrast, oral capsule methods offer advantages such as convenience, high efficiency, low incidence of adverse events, and high patient compliance, making them a promising approach for microbial transplantation.

[0004] Currently, in gut microbiota transplantation, intestinal microbiota products are typically prepared by filling enteric-coated capsule shells in the form of freeze-dried powder or microbial slurry to obtain freeze-dried capsules or microbial slurry capsules. Freeze-dried powder is obtained by freeze-drying purified bacterial solutions. Besides being costly and time-consuming, freeze-drying can also significantly kill bacteria in the intestinal microbiota products, leading to a substantial reduction in the activity and structure of the microbiota within the capsules, lowering the viable bacterial load, affecting clinical treatment efficacy, and in severe cases, even causing side effects. Microbial slurry, on the other hand, is obtained by separating undigested food residues and metabolic products from feces to extract microbial slurry containing functional microbiota. Compared to freeze-drying, it better preserves the microbiota structure and viable bacterial load.

[0005] However, the preparation of bacterial sludge is more complex than that of freeze-dried powder. If the extraction method of bacterial sludge is not appropriate, it will not only have an adverse effect on the activity and viable bacteria of the bacterial community in the extracted bacterial sludge, but may also affect the odor of the bacterial sludge. In addition, the prepared bacterial sludge capsules have a short shelf life at room temperature, and when stored at a low temperature of -80℃, the water in the bacterial sludge will form ice crystals that pierce the bacterial cell walls, causing the bacteria to die, affecting the activity of the bacterial community in the bacterial sludge capsules and changing the bacterial community structure, which has great limitations. Summary of the Invention

[0006] The inventors developed this invention to prepare a microbial mud that efficiently removes odors while minimizing the impact on microbial activity, thereby obtaining a microbial mud capsule that combines low odor, high microbial activity, and excellent storage stability. Through extensive and in-depth research, they creatively arrived at the technical solution of this invention.

[0007] The first objective of this invention is to provide a composition for the extraction of fecal microbiota.

[0008] A second objective of this invention is to provide a method for preparing intestinal flora products.

[0009] A third objective of this invention is to provide an apparatus for preparing intestinal flora products.

[0010] The fourth objective of this invention is to provide an intestinal flora product.

[0011] The fifth objective of this invention is to provide a method for preparing capsules.

[0012] The sixth objective of this invention is to provide a capsule.

[0013] A seventh objective of the present invention is to provide the above-described composition, apparatus for preparing intestinal flora products, and the application of intestinal flora products and / or capsules in precision microbiota transplantation.

[0014] Specifically, the composition for extracting fecal microbiota provided by the present invention comprises: an eluent comprising plant extracts and Japanese magnolia powder in a mass ratio of (0.1–50):(0.001–10); wherein the plant extracts are selected from one or more of Rugosa scutellariae extract, Citrus reticulata peel extract, Citronella extract, Clove extract, Geranium extract, charred Shenqu extract, Licorice extract, charred hawthorn extract, Citrus reticulata peel extract, and charred malt extract; and a protective agent comprising a small molecule polyhydroxy compound, a macromolecule polymer, an amine, a natural emulsifier, and optionally a carbonate in a mass ratio of (5–50):(1–55):(1–35):(1–30):(0–15); wherein the small molecule polyhydroxy compound is selected from mannitol, maltitol, and xylitol. The product comprises one or more of the following: trehalose, stachyose, isomaltulose, mannan oligosaccharide, tea polyphenols, vitamin C, and malic acid; the macromolecular polymer is selected from one or more of fucoidan, konjac mannan, corn starch, ethyl cellulose, starch acetate, and agar powder; the amine is selected from one or more of fish collagen peptide, glutathione, polyacrylamide, N-acetylglucosamine, and taurine; the natural emulsifier is selected from one or more of gum arabic, carrageenan, gellan gum, guar gum, xanthan gum, and medium-chain triglycerides; the carbonate is potassium carbonate and / or sodium carbonate; and, optionally, a separation aid comprising macroporous resin, functional resin, and non-resin adsorbent in a mass ratio of (2–5):(2–6):(2–10).

[0015] Furthermore, the preparation of the plant extract specifically includes: taking plant raw materials and performing water extraction treatment to obtain the plant extract.

[0016] Furthermore, the plant material is selected from one or more of the following: sweet raspberry, orange peel, lemongrass, clove, bay leaf, roasted medicated leaven, licorice, roasted hawthorn, dried tangerine peel, and roasted malt.

[0017] Furthermore, the water extraction treatment method is hot water extraction and / or ultrasonic-assisted extraction.

[0018] Furthermore, maltodextrin is added during the water extraction process, and the mass ratio of the plant raw material to maltodextrin is 1:(1-10).

[0019] Furthermore, the plant extracts include Rugum tsao-ko extract, Citrus peel extract, Citronella extract and Clove extract in a mass ratio of (1-10):(1-10):(0.1-5):(0.1-2).

[0020] Furthermore, the plant extracts include Rugosa stropharia extract, Citrus peel extract, Citronella extract, Clove extract, and roasted malt extract in a mass ratio of (5-10):(0.1-5):(1-3):(0.1-2):(0.1-2).

[0021] Furthermore, the bicarbonate is selected from sodium bicarbonate and / or potassium bicarbonate.

[0022] Furthermore, based on the total mass of the protective agent, the protective agent comprises 5-20 wt% trehalose, 10-30 wt% fucoidan, 15-35 wt% fish collagen peptide, 1-10 wt% vitamin C, 1-5 wt% malic acid, and 5-20 wt% xanthan gum.

[0023] Furthermore, based on the total mass of the protective agent, the protective agent comprises 5-20 wt% mannitol, 0.1-30 wt% gum arabic, 2-10 wt% glutathione, 5-30 wt% maltitol, 1-30 wt% ethyl cellulose and 1-10 wt% carrageenan.

[0024] Furthermore, based on the total mass of the protective agent, the protective agent comprises 20-40 wt% corn starch, 5-15 wt% stachyose, 10-25 wt% xylitol, 1-15 wt% polyacrylamide, 5-15 wt% konjac mannan, and 1-10 wt% gellan gum.

[0025] Further, based on the total mass of the protective agent, the protective agent comprises 5-20 wt% tea polyphenols, 2-10 wt% potassium carbonate, 10-50 wt% starch acetate, 2-25 wt% N-acetylglucosamine, 5-30 wt% medium-chain triglycerides, and 1-5 wt% taurine.

[0026] Furthermore, the macroporous resin is selected from one or more of epoxy resin, polyester resin, polyvinyl chloride resin, polyethylene resin, polypropylene resin, phenolic resin, polyurethane resin, polystyrene resin, acrylic resin, ether-ketone resin, urea-formaldehyde resin, and ketone-formaldehyde resin.

[0027] Furthermore, the functional resin is selected from ion exchange resins and / or chelating resins.

[0028] Furthermore, the non-resin adsorbent is selected from one or more of perlite, diatomaceous earth, activated clay, cellulose, titanium dioxide, activated carbon, coconut shell powder, and maifanite.

[0029] Furthermore, the macroporous resin has an average pore size of 0.4–1.25 mm, the functional resin has an average particle size of 0.5–1 mm, and the non-resin adsorbent has an average particle size of 0.3–1.2 mm.

[0030] The method for preparing intestinal flora products using the above-described composition provided by the present invention includes: S1, taking donor feces and mixing it with the eluent and optional separation aid, and then performing stirring and filtration treatment in sequence to obtain crude bacterial sludge liquid; S2, taking the crude bacterial sludge liquid and performing tangential flow filtration and centrifugation treatment in sequence to obtain bacterial sludge; S3, taking the bacterial sludge and mixing it with a protectant to obtain intestinal flora products.

[0031] Further, in step S1, the mass ratio of the donor feces, eluent, and separation aid is (5-15):(50-75):(1-3).

[0032] Further, in step S1, the stirring speed is 800-1500 r / min and the time is 4-15 min.

[0033] Furthermore, in step S1, the pore size of the filter membrane used for filtration is 20–2000 μm.

[0034] Furthermore, in step S2, the pore size of the filter membrane used in the tangential flow filtration process is 20–2000 μm, and the influent flow rate is 4–6 L / min / m³. 2 The inlet pressure differential is 0.9–1.75 bar, the return pressure differential is 0.05–0.2 bar, the transmembrane pressure is 0.5–0.9 bar, and the average membrane flux is 5.10–24.23 L / m³. 2 h.

[0035] Furthermore, in step S2, the centrifugation speed is 1000-20000g and the time is 2-15min.

[0036] Further, in step S3, the mixing mass ratio of the fungal mud to the protective agent is (5-20):(1-2).

[0037] The apparatus for preparing intestinal flora products provided by the present invention includes a separation tank, a filtration assembly, a collection tank I, a tangential flow filter, a centrifuge, and a collection tank II connected in sequence.

[0038] The intestinal flora product provided by this invention is prepared by the method for preparing bacterial sludge described above.

[0039] The capsule preparation method provided by the present invention includes: coating the capsule shell with a hydrophobic coating material to obtain a hydrophobic capsule shell; and packaging the above-mentioned intestinal flora product with the hydrophobic capsule shell to obtain the capsule.

[0040] The capsule provided by this invention is prepared by the capsule preparation method described above.

[0041] The present invention also provides the above-described compositions, apparatus for preparing intestinal flora products, and the application of intestinal flora products and / or capsules in precision microbiota transplantation.

[0042] Beneficial effects:

[0043] The composition provided by this invention includes at least an eluent for purifying donor feces and a protectant for maintaining the activity of intestinal flora. The eluent comprises plant extracts and magnolia bark powder in a specific mass ratio, and the protectant comprises small-molecule polyhydroxy compounds, macromolecular polymers, amines, natural emulsifiers, and optional carbonates in a specific mass ratio. Together, they form an organic whole and work synergistically to achieve the preparation of a simple-to-operate bacterial sludge with minimal impact on bacterial activity, thereby ultimately obtaining an intestinal flora product with low odor, high bacterial activity, and excellent storage stability.

[0044] The reason why this composition has the above-mentioned effects is speculated to be as follows: the specific plant extracts and Japanese magnolia bark powder included in the eluent can effectively adsorb, decompose or transform odor compounds in feces, thereby achieving a highly efficient odor removal effect. It can also effectively protect cells from mechanical stress damage, thus maintaining the activity of the microbial community. The protective agent, through the synergistic combination of molecular polyhydroxy compounds, macromolecular polymers, amines, natural emulsifiers and optional carbonates, effectively coats the cells in the microbial sludge, effectively protecting the cells from damage caused by ice crystals formed during low-temperature storage. At the same time, it can also provide an anaerobic environment for the storage of the microbial sludge. In synergy with the eluent retained in the intestinal microbial products, it can effectively maintain the abundance of microbial communities in the microbial sludge during low-temperature storage, thereby ultimately improving the storage stability of the intestinal microbial products. Moreover, the microbial abundance in the intestinal microbial products obtained by treating with this composition is close to that of healthy donors. Using this intestinal microbial product is beneficial for the realization of precise microbial transplantation.

[0045] In some specific embodiments, when the composition preferably includes an eluent, a protectant, and a separation aid, the macroporous resin, functional resin, and non-resin adsorbent in the separation aid work synergistically to effectively adsorb large particulate impurities and small molecule impurities in feces. Under the protection of the eluent, the separation aid can generate shear force on the feces to better break down the feces and release more live cells, achieving the transformation from solid to liquid. Furthermore, the separation aid can effectively adsorb non-bacterial micro-impurities such as mucus, bile pigments, and mucins in the feces, thereby improving the quality of the sludge and the number of bacteria per unit while ensuring the activity of the sludge microbiota.

[0046] In some specific embodiments, when the eluent preferably includes extracts of Rugosa scutellariae, Citrus reticulata peel, Citronella extract, Clove extract, and roasted malt extract in a mass ratio of (5-10):(0.1-5):(1-3):(0.1-2):(0.1-2), it can achieve a more ideal synergistic effect with the protectant, thereby giving the final intestinal flora product better storage stability. Attached Figure Description

[0047] Figure 1 This is a schematic diagram of the apparatus for preparing intestinal flora products provided in Example 1 of the present invention;

[0048] Figure 2 This is one of the experimental results of the abundance test of gut microbiota products provided in the test examples of the present invention (Example 2);

[0049] Figure 3 Figure 2 shows the experimental results of the abundance test of gut microbiota products provided in the test examples of this invention (Comparative Example 2);

[0050] Figure 4 Figure 3 shows the experimental results of the abundance test of gut microbiota products provided in the test examples of this invention (Comparative Example 3).

[0051] Reference numerals: 1. Separation tank; 2. Filter assembly; 3. Collection tank I; 4. Tangential flow filter; 5. Centrifuge; 6. Collection tank II. Detailed Implementation

[0052] The composition for extracting fecal microbiota provided by this invention includes an eluent, a protectant, and an optional separation aid. The eluent removes odor substances from the donor feces and protects cells from mechanical stress damage. It also works synergistically with the protectant to improve the storage stability of the microbial sludge. The protectant effectively coats the cells in the microbial sludge, protecting them from ice crystal formation during low-temperature storage. It also provides an anaerobic environment for storage and, in synergy with the eluent retained in the sludge, effectively maintains the abundance of microbial flora during low-temperature storage. The separation aid, used in conjunction with the eluent, effectively breaks down the donor feces and adsorbs impurity molecules, allowing cells to become fully free without affecting microbial activity, thereby improving the quality and bacterial count of the microbial sludge.

[0053] In this invention, the eluent specifically includes plant extracts and Japanese magnolia bark powder, and the mass ratio of the plant extracts to Japanese magnolia bark powder is (0.1-50):(0.001-10), such as 0.1:0.001, 1:5, 10:8, 23:10, 31:5, 40:9, 50:7 or any value between them.

[0054] More specifically, the plant extracts are selected from one or more of the following: Rubus idaeus extract, citrus peel extract, lemongrass extract, clove extract, bay leaf extract, roasted Shenqu extract, licorice extract, roasted hawthorn extract, tangerine peel extract, and roasted malt extract. In this case, through the combined action of one or more of the above-mentioned plant extracts and Magnolia officinalis powder, odor-causing substances are effectively adsorbed and removed. Furthermore, the mixture obtained by mixing the above-mentioned plant extracts in a specific ratio can protect cells from mechanical stress damage, while the active ingredients contained therein promote bacterial growth and reproduction. This allows the mixture to continue to function during the storage stage of the intestinal flora product, thereby improving the activity of the intestinal flora product and maintaining the abundance of intestinal flora, thus giving the prepared intestinal flora product excellent storage stability.

[0055] In this invention, the specific examples of the preparation method of the plant extract may be, but are not limited to, one or more of the following: water extraction, alcohol extraction, carbon dioxide extraction, glycerol extraction, and low-temperature solvent extraction. The preparation methods described above are conventional techniques used in the art, and those skilled in the art can select specific processes and conditions as needed; this invention does not impose any particular limitations on them.

[0056] In this invention, the preferred method for preparing the plant extract is water extraction, specifically including: taking plant raw materials and performing water extraction treatment to obtain the plant extract. In this case, the obtained plant extract is a water extract, and the extracted active ingredients are more conducive to bacterial growth and reproduction, playing a better role in the storage stage of intestinal flora products, thereby giving the prepared intestinal flora products better storage stability.

[0057] In some specific embodiments, the plant raw materials are adaptively selected based on the plant extracts to be obtained. Specific examples include one or more of the following: Rugium affine, orange peel, lemongrass, clove, bay leaf, roasted Shenqu (a type of fermented medicinal herb), licorice, roasted hawthorn, dried tangerine peel, and roasted malt. Each plant extract can be obtained by mixing multiple plant raw materials together or by preparing them independently.

[0058] In some specific embodiments, examples of the water extraction treatment method include, but are not limited to, hot water extraction and / or ultrasonic-assisted extraction. The hot water extraction and ultrasonic-assisted extraction described above are conventional techniques used in the art, and those skilled in the art can select specific processes and conditions as needed; this invention does not impose any particular limitations on them.

[0059] In some specific embodiments, maltodextrin is preferably added to the water extraction process, and the mass ratio of the plant material to maltodextrin is preferably 1:(1-10), such as 1:1, 1:1.5, 1:2.3, 1:4, 1:6, 1:8.7, 1:9, 1:10, or any value between them. In this case, maltodextrin can increase the viscosity and dispersibility of the water extraction system, effectively removing unwanted impurity molecules while improving the extraction rate of active ingredients, thus achieving better extraction results.

[0060] In this invention, the plant extracts in the eluent preferably include extracts of *Rubus idaeus*, *Citrus reticulata* peel, *Citrus medica*, and *Clove* in a mass ratio of (1-10):(1-10):(0.1-5):(0.1-2), such as 1:1:0.1:0.1, 1:3:2:2, 1:10:5:2, 2.5:5.5:0.75:1, 3:1:0.1:2, 3.5:4:1.2:0.55, 4:2:2:1.5, 5.5:2.5:1:0.7, 6:2:1.15:0.5, or any value between them. In this case, the resulting eluent can achieve an ideal synergistic effect with the protectant, thereby endowing the final intestinal flora product with excellent storage stability.

[0061] In this invention, the plant extracts in the eluent preferably include extracts of *Rubus idaeus*, orange peel, lemongrass, clove, and roasted malt in a mass ratio of (5-10):(0.1-5):(1-3):(0.1-2):(0.1-2), such as 5:0.1:1:0.1:2, 8:3:2:2:0.1, 6:2:1.15:0.5:0.5, or any value between them. In this case, the resulting eluent can achieve an ideal synergistic effect with the protectant, thereby endowing the final gut microbiota product with excellent storage stability.

[0062] In this invention, the eluent preferably further includes a bicarbonate, specifically sodium bicarbonate and / or potassium bicarbonate. In this case, the bicarbonate can alter the electrical properties of the solution during donor fecal treatment, thereby achieving a better adsorption and removal effect on odor substances.

[0063] In some specific embodiments, based on the total mass of the eluent, the concentration of bicarbonate is preferably 0.5–10 wt%, such as 0.5 wt%, 0.8 wt%, 1 wt%, 2.4 wt%, 5.5 wt%, 8 wt%, 10 wt%, or any value between them. In this case, the addition of bicarbonate can significantly improve the odor removal effect and is beneficial for enhancing the microbial activity and storage stability of the bacterial sludge.

[0064] In this invention, the protective agent specifically includes small molecule polyhydroxy compounds, macromolecule polymers, amines, natural emulsifiers, and optional carbonates, and the mass ratio of the small molecule polyhydroxy compounds, macromolecule polymers, amines, natural emulsifiers, and optional carbonates is specifically (5-50):(1-55):(1-35):(1-30):(0-15), such as 5:1:1:1, 10:20:15:27:1, 50:55:28:17:13, or any value between them.

[0065] More specifically, the small-molecule polyhydroxy compounds are organic small-molecule compounds containing multiple hydroxyl groups in their molecular structure. They have antioxidant effects and maintain a good state of intestinal flora products. Specific examples include one or more of mannitol, maltitol, xylitol, trehalose, stachyose, isomaltulose, mannan oligosaccharides, tea polyphenols, vitamin C, and malic acid. The macromolecular polymers are a class of high-molecular-weight substances that have a certain viscosity and high biocompatibility after dissolving in water. They can effectively encapsulate the flora and provide a suitable anaerobic environment for their survival. Specific examples include one or more of fucoidan, konjac mannan, corn starch, ethyl cellulose, starch acetate, and agar powder. The amines are products obtained by replacing one or more hydrogen atoms in an ammonia molecule with hydrocarbon groups. Their addition is beneficial to the growth and reproduction of the flora. Specific examples include one or more of fish collagen peptides, glutathione, polyacrylamide, N-acetylglucosamine, and taurine. The natural emulsifier is an emulsifier composed of natural raw materials, which can improve the dispersibility of other substances to achieve a better protective effect on intestinal flora products. Specific examples include one or more of gum arabic, carrageenan, gellan gum, guar gum, xanthan gum, and medium-chain triglycerides. Specific examples of the carbonate include, but are not limited to, potassium carbonate and / or sodium carbonate. In this case, the synergistic effect of small-molecule polyhydroxy compounds, macromolecular polymers, amines, natural emulsifiers, and optional carbonates in the protectant can protect intestinal flora products by reducing physical damage to the flora and improving the condition of the flora. This allows the intestinal flora products to maintain high flora activity and minimal changes in flora abundance during long-term storage at -80°C, exhibiting excellent storage stability.

[0066] In some specific embodiments, based on the total mass of the protective agent, the protective agent comprises: 5-20 wt% trehalose, such as 5 wt%, 8 wt%, 10 wt%, 15 wt%, 20 wt%, or any value between therewith; 10-30 wt% fucoidan, such as 10 wt%, 12.5 wt%, 15 wt%, 18 wt%, 20 wt%, 30 wt%, or any value between therewith; and 15-35 wt% fish collagen peptides, such as 15 wt%, 16 wt%, 18 wt%, 2... 0 wt%, 25 wt%, 30 wt%, 35 wt%, or any value between them; 1 to 10 wt% of vitamin C, such as 1 wt%, 3 wt%, 7 wt%, 10 wt%, or any value between them; 1 to 5 wt% of malic acid, such as 1 wt%, 1.5 wt%, 2 wt%, 3.5 wt%, 4 wt%, 5 wt%, or any value between them; and 5 to 20 wt% of xanthan gum, such as 5 wt%, 8 wt%, 10 wt%, 15 wt%, 20 wt%, or any value between them.

[0067] In some specific embodiments, based on the total mass of the protective agent, the protective agent comprises: 5-20 wt% mannitol, such as 5 wt%, 7.5 wt%, 10 wt%, 15 wt%, 18 wt%, 20 wt%, or any value between therewith; 0.1-30 wt% gum arabic, such as 0.1 wt%, 1 wt%, 5 wt%, 8 wt%, 10 wt%, 13 wt%, 18 wt%, 20 wt%, 25 wt%, 30 wt%, or any value between therewith; 2-10 wt% glutathione, such as 2 wt%, 2.5 wt%, 3 wt%, 4 wt%, ... 8 wt%, 10 wt%, or any value therein; 5 to 30 wt% maltitol, such as 5 wt%, 6.5 wt%, 10 wt%, 12 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, or any value therein; 1 to 30 wt% ethyl cellulose, such as 1 wt%, 3 wt%, 5 wt%, 10 wt%, 20 wt%, 30 wt%, or any value therein; and 1 to 10 wt% carrageenan, such as 1 wt%, 1.5 wt%, 2 wt%, 3.5 wt%, 5 wt%, 8 wt%, 10 wt%, or any value therein.

[0068] In some specific embodiments, based on the total mass of the protective agent, the protective agent comprises: 20–40 wt% corn starch, such as 20 wt%, 21 wt%, 23 wt%, 28 wt%, 30 wt%, 31 wt%, 38 wt%, 40 wt%, or any value between therewith; 5–15 wt% stachyose, such as 5 wt%, 8 wt%, 10 wt%, 12 wt%, 15 wt%, or any value between therewith; and 10–25 wt% xylitol, such as 10 wt%, 15 wt%, 20 wt%, 25 wt%. t% or any value therein; 1 to 15 wt% of polyacrylamide, such as 1 wt%, 3 wt%, 5 wt%, 7 wt%, 9 wt%, 15 wt% or any value therein; 5 to 15 wt% of konjac mannan, such as 5 wt%, 7 wt%, 9 wt%, 10 wt%, 12 wt%, 13 wt%, 15 wt% or any value therein; and 1 to 10 wt% of gellan gum, such as 1 wt%, 2.5 wt%, 5 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt% or any value therein.

[0069] In some specific embodiments, based on the total mass of the protective agent, the protective agent comprises: 5-20 wt% tea polyphenols, such as 5 wt%, 8 wt%, 9 wt%, 10 wt%, 15 wt%, 20 wt%, or any value between therewith; 2-10 wt% potassium carbonate, such as 2 wt%, 2.5 wt%, 5 wt%, 7.5 wt%, 10 wt%, or any value between therewith; and 10-50 wt% starch acetate, such as 10 wt%, 12 wt%, 15 wt%, 20 wt%, 28 wt%, 37 wt%, 40 wt%, 45 wt%, 50 wt%. % or any value therein; 2 to 25 wt% of N-acetylglucosamine, such as 2 wt%, 4.5 wt%, 5 wt%, 8 wt%, 10 wt%, 12 wt%, 18 wt%, 20 wt%, 25 wt% or any value therein; 5 to 30 wt% of medium-chain triglycerides, such as 5 wt%, 8 wt%, 10 wt%, 12 wt%, 18 wt%, 20 wt%, 25 wt%, 30 wt% or any value therein; and 1 to 5 wt% of taurine, such as 1 wt%, 2 wt%, 3.8 wt%, 4 wt%, 5 wt% or any value therein.

[0070] In this invention, the separation aid specifically includes macroporous resin, functional resin and non-resin adsorbent, and the mass ratio of the macroporous resin, functional resin and non-resin adsorbent is (2-5):(2-6):(2-10), such as 1:1:1, 1:3:2, 3:2:2, 3:6:5, 4:5:9, 5:6:10 or any value between them.

[0071] More specifically, the macroporous resin is a type of organic polymer adsorbent with a three-dimensional porous structure, which can effectively adsorb odor substances and other impurity molecules in donor feces. Specific examples include, but are not limited to, one or more of epoxy resin, polyester resin, polyvinyl chloride resin, polyethylene resin, polypropylene resin, phenolic resin, polyurethane resin, polystyrene resin, acrylic resin, ether-ketone resin, urea-formaldehyde resin, and ketone-aldehyde resin. The functional resin is a type of resin in which specific functional groups are incorporated into the macromolecular chain. It can achieve the adsorption of odor substances and other impurity molecules through electrostatic forces and / or complexation. Specific examples include, but are not limited to, one or more of cation exchange resin, anion exchange resin, and chelating resin. The non-resin adsorbent, while having the ability to effectively adsorb odor substances and other impurity molecules in feces, can also provide secondary shear force to break down the donor feces, which is beneficial to the release of bacteria. Specific examples include, but are not limited to, one or more of perlite, diatomaceous earth, activated clay, cellulose, titanium dioxide, activated carbon, coconut shell powder, and maifanite.

[0072] In some specific embodiments, the average pore size of the macroporous resin is preferably 0.4–1.25 mm, such as 0.4 mm, 0.5 mm, 0.63 mm, 0.9 mm, 1 mm, 1.15 mm, 1.2 mm, 1.25 mm, or any value between them; the average particle size of the functional resin is preferably 0.5–1 mm, such as 0.5 mm, 0.55 mm, 0.58 mm, 0.6 mm, 0.67 mm, 0.8 mm, 0.9 mm, 1 mm, or any value between them; the average particle size of the non-resin adsorbent is preferably 0.3–1.2 mm, such as 0.3 mm, 0.38 mm, 0.4 mm, 0.6 mm, 0.9 mm, 1 mm, 1.2 mm, or any value between them. In this case, the macroporous resin, functional resin, and functional adsorbent can better synergize and work together with the eluent to achieve a better deodorization effect.

[0073] The method for preparing intestinal flora products provided by the present invention uses the eluent, protectant and optional separation aid described above to treat donor feces, specifically including: S1, mixing donor feces with the eluent and optional separation aid, and sequentially performing stirring and filtration to obtain crude bacterial sludge liquid; S2, sequentially performing tangential flow filtration and centrifugation on the crude bacterial sludge liquid to obtain bacterial sludge; S3, mixing the bacterial sludge with the protectant to obtain intestinal flora products.

[0074] In this invention, in step S1, the preferred mass ratio of the donor feces, eluent, and separation aid is (5-15):(50-75):(1-3), such as 5:50:1, 10:63:1, 13:75:3, 15:50:3, 15:74:3, or any value between them. This allows for more efficient odor removal with minimal negative impact on bacterial activity, ensuring that the bacteria maintain a high level of activity in subsequent applications.

[0075] In some specific embodiments, the donor feces, eluent, and separation aid can be mixed simultaneously and / or added in batches. Simultaneous addition specifically refers to directly mixing the donor feces, separation aid, and all the eluent, followed by stirring and filtration. Batch addition specifically refers to dividing the eluent into multiple portions, mixing them sequentially with the donor feces and separation aid, stirring after each mixing, and performing multiple filtrations only after all the eluent has been added. In some preferred embodiments, the mixing of the donor feces, eluent, and separation aid is preferably done in batches, which reduces the impact of mixing and stirring processes on microbial activity.

[0076] In this invention, in step S1, the stirring conditions preferably include a rotation speed of 800–1500 r / min, such as 800 r / min, 850 r / min, 900 r / min, 1000 r / min, 1200 r / min, 1500 r / min, or any value between them; and a stirring time preferably of 4–15 min, such as 4 min, 5 min, 8 min, 10 min, 12 min, 15 min, or any value between them. By employing these preferred stirring conditions, the adverse effects on bacterial activity during the stirring process can be reduced, thereby improving the activity of the bacterial community in the sludge.

[0077] In this invention, in step S1, the pore size of the filter membrane used for filtration is preferably 20–2000 μm, such as 20 μm, 25 μm, 50 μm, 100 μm, 237 μm, 500 μm, 750 μm, 1000 μm, 1500 μm, 2000 μm, or any value between them. This effectively separates intestinal flora from unwanted impurities, achieving better purification results.

[0078] In this invention, during step S2, the intestinal flora in the crude liquid of the bacterial sludge is collected through the filter membrane, while impurity particles are effectively retained on the surface of the filter membrane, thereby achieving efficient and gentle separation and collection of intestinal flora, and improving the quality and unit bacterial count of the recovered bacterial sludge.

[0079] In some specific embodiments, the conditions for the tangential flow filtration treatment include a filter membrane pore size preferably between 20 and 2000 μm, such as 20 μm, 30 μm, 50 μm, 150 μm, 250 μm, 1000 μm, 1500 μm, 2000 μm, or any value between them; and an influent flow rate preferably between 4 and 6 L / min / m³. 2 such as 4L / min / m 2 4.5L / min / m 2 5L / min / m 2 5.8L / min / m 2 6L / min / m 2 Or any value between them; the inlet pressure differential is preferably 0.9 to 1.75 bar, such as 0.9 bar, 0.95 bar, 1 bar, 1.23 bar, 1.57 bar, 1.6 bar, 1.75 bar or any value between them; the return pressure differential is preferably 0.05 to 0.2 bar, such as 0.05 bar, 0.08 bar, 0.1 bar, 0.13 bar, 0.15 bar, 0.18 bar, 0.2 bar or any value between them; the transmembrane pressure is preferably 0.5 to 0.9 bar, such as 0.5 bar, 0.55 bar, 0.6 bar, 0.63 bar, 0.68 bar, 0.7 bar, 0.85 bar, 0.9 bar or any value between them; the average membrane flux is preferably 5.10 to 24.23 L / m 2 h, such as 5.1 L / m 2 h, 5.3L / m 2 h, 6L / m 2 h, 7.8L / m 2 h, 10L / m 2 h, 11.5L / m 2 h, 15L / m 2 h, 18.6L / m 2 h, 20L / m 2 h, 23.1 L / m 2 h, 24.23L / m 2 h or any value between them.

[0080] In some specific embodiments, the centrifugation conditions include a rotation speed preferably between 1000 and 20000 g, such as 1000 g, 1001 g, 1500 g, 1800 g, 2000 g, 5000 g, 10000 g, 20000 g or any value between them; and a time of 2 to 15 min, such as 2 min, 2.8 min, 3 min, 5 min, 9 min, 10 min, 12 min, 15 min or any value between them.

[0081] In this invention, in step S3, the preferred mixing mass ratio of the bacterial sludge to the preservative is (5-20):(1-2), such as 5:1, 8:1, 10:1, 20:1, 7:1.5, 17:1.8, 5:2, 19:2, or any value between them. At this time, mixing the bacterial sludge and the preservative according to the above mixing mass ratio can better achieve the protective effect of the preservative on the intestinal flora in the product.

[0082] The apparatus for preparing mycelial sludge provided by this invention is designed based on the method for preparing mycelial sludge described above, and in order to reduce the impact on the microbial community, the apparatus operates in a closed state. Specifically, the apparatus includes: a separation tank, a filter assembly, a collection tank I, a tangential flow filter, a centrifuge, and a collection tank II, connected in sequence.

[0083] In this invention, the filter assembly, tangential flow filter and centrifuge are conventionally used devices in the prior art, limited to those capable of achieving filtration, tangential flow filtration or centrifugation functions. This invention does not impose any particular limitation on their specific structure.

[0084] The present invention also provides an intestinal microbiota product prepared by the method described above for preparing intestinal microbiota products. The intestinal microbiota product has a microbiota activity of more than 70%, and after being stored at -80°C for 360 days, the microbiota activity of the intestinal microbiota product still remains above 53%. The intestinal microbiota product has high microbiota activity and excellent storage stability.

[0085] The capsule preparation method provided by this invention specifically includes: coating a capsule shell with a hydrophobic coating material to obtain a hydrophobic capsule shell; and packaging the above-mentioned intestinal flora product with the hydrophobic capsule shell to obtain the capsule. The hydrophobic coating material includes beeswax, paraffin oil, vegetable oil, and an emulsifier, wherein the mass ratio of beeswax, paraffin oil, vegetable oil, and emulsifier is (5-10):(1-3):(3-5):(1-3), such as 5:1:3:1, 4:3:5:2, 6:1:3:3, 8:2:5:3, 10:3:5:3, or any value between them. At this point, the prepared hydrophobic capsule shell includes a hydrophobic outer shell covering both the inner and outer sides of the capsule shell and a hydrophobic inner coating layer. The hydrophobic outer shell has excellent resistance to gastric acid erosion and can maintain structural integrity in the gastric environment, thereby avoiding premature dissolution. The hydrophobic inner coating layer can provide a better protective environment for the bacterial community. Through the synergistic effect of the outer shell and the inner coating layer, the colonization effect of the bacterial community in the intestine can be significantly enhanced.

[0086] In some specific embodiments, the vegetable oil may be, but is not limited to, one or more of soybean oil, sunflower oil, and olive oil; the emulsifier may include, but is not limited to, glycerol fatty acid esters and / or soybean lecithin.

[0087] In this invention, the coating process can be one or more of immersion, spraying and spin coating, as long as it can achieve the coating of hydrophobic coating material on the inner and outer surfaces of the capsule shell. This invention does not impose any special limitations on the specific process and conditions.

[0088] The capsules provided in this invention are prepared using the capsule preparation methods described above.

[0089] The present invention also provides the above-described compositions, apparatus for preparing intestinal flora products, and the application of intestinal flora products and / or capsules in precision microbiota transplantation.

[0090] 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.

[0091] The raw materials used in the following preparation examples and embodiments, and their sources, are as follows:

[0092] Fucoidan (Qingdao Mingyue Seaweed Bio-Health Technology Group Co., Ltd., product number 9072-19-9);

[0093] Fish collagen peptides (Hebei Kelongduo Biotechnology Co., Ltd., product number 472-61-5);

[0094] Xanthan gum (Guangzhou Yuanchang Trading Co., Ltd., item number 11138-66-2);

[0095] Gum arabic (Jiangsu Caiwei Biotechnology Co., Ltd., product number 9000-01-5);

[0096] Ethyl cellulose (Shanghai Zhongfeng Biotechnology Co., Ltd., product number 618-384-9);

[0097] Carrageenan (Chongqing Tianrun Biological Products Co., Ltd., product number 11114-20-8);

[0098] Polyacrylamide (Henan Shuifangcheng Water Purification Materials Co., Ltd., product number 9003-05-8);

[0099] Konjac glucomannan (Xi'an Lavia Biotechnology Co., Ltd., product number 37220-17-0);

[0100] Gellan gum (Chongqing Tianrun Biological Products Co., Ltd., product number 71010-52-1);

[0101] Acetate starch (Chongqing Tianrun Biological Products Co., Ltd., product number 9045-28-7);

[0102] Medium-chain triglycerides (Shandong Pingju Biotechnology Co., Ltd., product number 538-24-9);

[0103] Macroporous aromatic polymer resin (Shanghai Yuanye Biotechnology Co., Ltd., product number S14162);

[0104] Ion exchange resin (Shanghai Yuanye Biotechnology Co., Ltd., item number 63181-94-2).

[0105] Preparation Example 1

[0106] This preparation example illustrates the preparation of an eluent, specifically including:

[0107] S1. Mix 50g of Rugbysia japonica with 1000mL of distilled water, soak for 10min, heat to boiling, treat at 100℃ for 20min, filter and collect the filtrate, add maltodextrin at 10% (v / v) to the filtrate, and freeze-dry at -40℃ and 0.05MPa to obtain Rugbysia japonica extract.

[0108] S2. Take 50g of lemongrass and mix it with 1000mL of distilled water. Soak for 10min and then heat to boiling. Treat at 100℃ for 20min. Filter and collect the filtrate. Add maltodextrin to the filtrate at a dosage of 10% (v / v). Freeze-dry at -40℃ and 0.05MPa to obtain lemongrass extract.

[0109] S3. Take 50g of cloves and mix them with 1000mL of distilled water. Soak for 10min and then heat to boiling. Treat at 100℃ for 20min. Filter and collect the filtrate. Add maltodextrin to the filtrate at a dosage of 10% (v / v). Freeze-dry at -40℃ and 0.05MPa to obtain clove extract.

[0110] S4. Take 50g of fresh tangerine peel, crush it, add 100mL of distilled water, and perform ultrasonic-assisted extraction at room temperature for 20min. Filter and collect the filtrate, and perform vacuum distillation at -40℃ and 0.05MPa to obtain a paste-like tangerine peel extract.

[0111] S5. Take 10g of Japanese magnolia bark, wash, dry and pulverize it, and then pass it through a 50-mesh sieve to obtain Japanese magnolia bark powder;

[0112] S6. Mix the extracts of Rugbysia japonica, Citrus reticulata peel, Citronella tamariscina, Clove, and Magnolia officinalis powder in a mass ratio of 6:2:1.15:0.5:0.35 to obtain the eluent.

[0113] Preparation Example 2

[0114] This preparation example uses the method provided in Preparation Example 1 to prepare the eluent. The difference is that in step S6, the mass ratio of Rugosa stropharia extract, Citrus peel extract, Citronella extract, Clove extract and Magnolia officinalis powder is 5.5:2.5:1:0.7:0.35, and other conditions are the same, to obtain the eluent.

[0115] Preparation Example 3

[0116] This preparation example uses the method provided in Preparation Example 1 to prepare the eluent. The difference is that in step S6, the mass ratio of Rubus idaeus extract, Citrus reticulata peel extract, Citronella extract, Clove extract and Magnolia officinalis powder is 2.5:5.5:0.75:1:0.25, and other conditions are the same, to obtain the eluent.

[0117] Preparation Example 4

[0118] This preparation example uses the method provided in Preparation Example 1 to prepare the eluent. The difference is that in step S6, the mass ratio of Rutaecarpa extract, Citrus peel extract, Citronella extract, Clove extract and Magnolia officinalis powder is 3.5:4:1.2:0.55:0.75, and other conditions are the same, to obtain the eluent.

[0119] Preparation Example 5

[0120] This preparation example uses the method provided in Preparation Example 1 to prepare the eluent. The difference is that in step S6, the mass ratio of Rugosa stropharia extract, Citrus peel extract, Citronella extract, Clove extract and Magnolia officinalis powder is 4:2:2:1.5:0.5, and other conditions are the same, to obtain the eluent.

[0121] Preparation Example 6

[0122] This preparation example uses the method provided in Preparation Example 1 to prepare the eluent. The difference is that in step S6, roasted malt extract is also added, and the mass ratio of Rutaecarpa extract, Citrus peel extract, Citronella extract, Clove extract, roasted malt extract and Magnolia officinalis powder is 6:2:1.15:0.5:0.5:0.35. Other conditions are the same, and the eluent is obtained.

[0123] In this preparation example, the preparation of the charred malt extract specifically includes: taking 50g of charred malt and mixing it with 1000mL of distilled water, soaking it for 10min, heating it to boiling, treating it at 100℃ for 20min, filtering and collecting the filtrate, adding maltodextrin at a dosage of 10% (v / v) to the filtrate, and freeze-drying it at -40℃ and 0.05MPa to obtain the charred malt extract.

[0124] Preparation Example 7

[0125] This preparation example uses the method provided in Preparation Example 1 to prepare the eluent. The difference is that in step S6, an equal mass of dried tangerine peel extract is used instead of orange peel extract, while other conditions remain the same, to obtain the eluent.

[0126] In this preparation example, the preparation of tangerine peel extract specifically includes: taking 50g of tangerine peel and mixing it with 1000mL of distilled water, soaking it for 10min, heating it to boiling, treating it at 100℃ for 20min, filtering and collecting the filtrate, adding maltodextrin at a dosage of 10% (v / v) to the filtrate, and freeze-drying it at -40℃ and 0.05MPa to obtain tangerine peel extract.

[0127] Preparation Example 8

[0128] This preparation example uses the method provided in Preparation Example 1 to prepare the eluent. The difference is that sodium bicarbonate is added in step S6, and the mass ratio of Rutaecarpa extract, Citrus peel extract, Citronella extract, Clove extract, sodium bicarbonate and Magnolia officinalis powder is 6:2:1.15:0.5:0.5:0.35. Other conditions are the same, and the eluent is obtained.

[0129] Comparative Preparation Example 1

[0130] This comparative preparation example uses the method provided in Preparation Example 1 to prepare the eluent. The difference is that in step S6, equal parts by mass of Artemisia argyi extract are used instead of Rubus idaeus extract, Citrus reticulata peel extract, Citronella extract and Clove extract. That is, the mass ratio of Artemisia argyi extract to Magnolia officinalis powder is 9.65:0.35. Other conditions are the same, and the eluent is obtained.

[0131] In this comparative preparation example, the preparation of Artemisia argyi extract specifically includes: taking 50g of Artemisia argyi and mixing it with 1000mL of distilled water, soaking it for 10min, heating it to boiling, treating it at 100℃ for 20min, filtering and collecting the filtrate, adding maltodextrin at a dosage of 10% (v / v) to the filtrate, and freeze-drying it at -40℃ and 0.05MPa to obtain Artemisia argyi extract.

[0132] Preparation Example 9

[0133] This preparation example provides a method for preparing a protective agent, specifically comprising: taking 20 parts by weight of trehalose, 30 parts by weight of fucoidan, 20 parts by weight of fish collagen peptide, 8 parts by weight of vitamin C, 12 parts by weight of malic acid and 10 parts by weight of xanthan gum and mixing them evenly to obtain the protective agent.

[0134] Preparation Example 10

[0135] This preparation example provides a method for preparing a protective agent, specifically comprising: mixing 15 parts by weight of mannitol, 25 parts by weight of gum arabic, 10 parts by weight of glutathione, 20 parts by weight of maltitol, 25 parts by weight of ethyl cellulose, and 5 parts by weight of carrageenan evenly to obtain the protective agent.

[0136] Preparation Example 11

[0137] This preparation example provides a protective agent preparation, specifically including: taking 35 parts by weight of corn starch, 15 parts by weight of stachyose, 25 parts by weight of xylitol, 5 parts by weight of polyacrylamide, 15 parts by weight of konjac mannan and 5 parts by weight of gellan gum and mixing them evenly to obtain the protective agent.

[0138] Preparation Example 12

[0139] This preparation example provides a method for preparing a protective agent, specifically comprising: mixing 20 parts by weight of tea polyphenols, 10 parts by weight of potassium carbonate, 35 parts by weight of starch acetate, 10 parts by weight of N-acetylglucosamine, 20 parts by weight of medium-chain triglycerides, and 5 parts by weight of taurine evenly to obtain the protective agent.

[0140] Comparative Preparation Example 2

[0141] This comparative preparation example uses the method provided in Preparation Example 9 to prepare the protective agent, except that an equal mass of fucoidan is used instead of xanthan gum, while other conditions remain the same, to obtain the protective agent.

[0142] Comparative preparation example 3

[0143] This comparative preparation example uses the method provided in Preparation Example 9 to prepare the protective agent, except that an equal mass of fucoidan is used instead of fish collagen peptides, while other conditions remain the same, to obtain the protective agent.

[0144] It should be noted that the donor feces used in the following embodiments are the same.

[0145] Example 1

[0146] This embodiment illustrates the apparatus for preparing intestinal flora products and the method for preparing intestinal flora products, referring to... Figure 1The device specifically includes a separation tank 1, a filter assembly 2, a collection tank I3, a tangential flow filter 4, a centrifuge 5, and a collection tank II6. The separation tank 1 is equipped with a stirrer. The filter assembly 2 includes first to fourth stage filter units arranged in series and connected sequentially via sterile conduits. The pore sizes of the filter membranes of the first to fourth stage filter units are 1000 μm, 500 μm, 150 μm, and 100 μm, respectively. The separation tank 1 is connected to the first stage filter unit via a sterile conduit, the fourth stage filter unit is connected to the collection tank I3 via a sterile conduit, the collection tank I3 is connected to the inlet of the tangential flow filter 4 via a sterile conduit, the outlet of the tangential flow filter 4 is connected to the centrifuge 5 via a sterile conduit, and the pore size of the filter membrane of the tangential flow filter 4 is 60 μm. The outlet of the centrifuge 5 is connected to the collection tank II6 via a sterile conduit, and the centrifuge 5 rotates at a speed of 5000 g.

[0147] This embodiment provides a method for preparing intestinal flora products using the above-described apparatus, the method specifically including:

[0148] S1, (1) Qualified donor feces were obtained by screening according to the existing "Chinese Expert Consensus on the Clinical Application and Management of Intestinal Microbiota Transplantation (2022 Edition)";

[0149] (2) Take the eluent provided in Preparation Example 1 and physiological saline and mix them evenly at a mass ratio of 1:9 to obtain the eluent; take 100g of donor feces and 500g of eluent and add them to separation tank 1, stir at 1000r / min for 5min, then add another 500g of eluent and continue stirring at 1000r / min for 5min to obtain the mixture;

[0150] (3) The mixture enters the filter assembly 2 through a sterile conduit and is filtered through the first, second, third and fourth stage filter units. The collection tank I3 is used to collect the filtrate to obtain the coarse liquid of the bacterial sludge.

[0151] S2. The coarse liquid from the bacterial sludge enters the tangential flow filter 4 through a sterile conduit, with a feed flow rate of 5 L / min / m², a feed pressure differential of 1.5 bar, a reflux differential of 0.1 bar, a transmembrane pressure of 0.8 bar, and an average membrane flux of 20 L / m². 2 Under h conditions, tangential flow filtration was performed. Centrifuge 5 was used to collect the bacterial sludge filtrate obtained from the tangential flow filter and centrifuged at 5000g for 10min to obtain bacterial sludge. The bacterial sludge obtained by centrifugation was collected using collection tank II6.

[0152] S3. Take the bacterial sludge and mix it with the protective agent provided in Preparation Example 9 at a mass ratio of 10:1 to obtain the intestinal flora product.

[0153] Example 2

[0154] This embodiment uses the apparatus and method provided in Example 1 to prepare intestinal flora products. The difference is that 50g of separation aid is added in step S1(2). Other conditions are the same, and intestinal flora products are obtained.

[0155] In this embodiment, the separation aids specifically include macroporous aromatic polymer resin, ion exchange resin, and maifanite in a mass ratio of 20:30:50.

[0156] Examples 3-12

[0157] Examples 3-12 use the apparatus and method provided in Example 2 to prepare intestinal flora products. The difference is that the eluent added in step S1(2) and the protective agent added in step S3 are different in amount, as shown in Table 1. Other conditions are the same, and intestinal flora products are obtained.

[0158] Table 1.

[0159]

[0160] Comparative Example 1

[0161] The comparative example uses the method provided in Example 2 to prepare intestinal flora products. The difference is that in step S1(2), an equal mass of physiological saline is used instead of the eluent to mix with the donor feces, and other conditions are the same to obtain intestinal flora products.

[0162] Comparative Example 2

[0163] The comparative example uses the method provided in Example 2 to prepare intestinal flora products. The difference is that in step S1(2), the eluent provided in Comparative Preparation Example 1 of equal mass is used instead of the eluent provided in Preparation Example 1, and the other conditions are the same, to obtain intestinal flora products.

[0164] Comparative Example 3

[0165] The comparative example uses the method provided in Example 2 to prepare intestinal flora products. The difference is that in step S3, the protective agent provided in Comparative Preparation Example 2 of equal mass is used instead of the protective agent provided in Preparation Example 9, and other conditions are the same, to obtain intestinal flora products.

[0166] Comparative Example 4

[0167] The comparative example uses the method provided in Example 2 to prepare intestinal flora products. The difference is that in step S3, the protective agent provided in Comparative Preparation Example 3 of equal mass is used instead of the protective agent provided in Preparation Example 9, and other conditions are the same, to obtain intestinal flora products.

[0168] Example 13

[0169] This embodiment illustrates the preparation of a capsule, specifically including:

[0170] S1. Take 79 parts by weight of anhydrous ethanol and 10 parts by weight of beeswax and heat them in a water bath at 65°C until dissolved. Then add 3 parts by weight of food-grade paraffin oil, 5 parts by weight of soybean oil and 3 parts by weight of soybean lecithin, mix evenly, and sonicate at 65°C for 1 hour to obtain a hydrophobic coating material.

[0171] S2. Immerse the capsule shell completely in the hydrophobic coating material, let it stand for 1 minute, take out the capsule shell and repeatedly turn it over to remove excess hydrophobic coating material, and dry it in an oven at 37°C for 60 minutes to obtain the hydrophobic capsule shell.

[0172] S3. Take 50g of the intestinal flora product provided in Example 1, fill it into a hydrophobic capsule shell, and assemble it into a capsule.

[0173] Example 14

[0174] This embodiment illustrates the preparation of a capsule, specifically including: taking 50g of the intestinal flora product provided in Example 1 and filling it into a capsule shell that has not been impregnated with a hydrophobic coating material, and assembling the capsule. The capsule shell used in this embodiment is the same as that in Example 13.

[0175] Test Case

[0176] This test example is used to illustrate the relevant performance of the intestinal flora products provided in Examples 1-12 and Comparative Examples 1-4, and the capsules provided in Examples 13 and 14, specifically:

[0177] 1. Deodorization effect: The odor of the intestinal flora products provided in Examples 1-12 and Comparative Examples 1-4 was evaluated by ten experimental personnel. Without knowing the control group and the experimental group, they rated the odor of the prepared liquid enteric capsules by sensory evaluation (Grade A: no odor; Grade B: slight odor; Grade C: mild odor; Grade D: moderate odor; Grade E: severe odor). The results are shown in Table 3.

[0178] Table 3.

[0179] Group Sensory rating Group Sensory rating Example 1 Grade A Example 9 Grade B Example 2 Grade A Example 10 Grade A Example 3 Grade A Example 11 Grade B Example 4 Grade A Example 12 Grade A Example 5 Grade A Comparative Example 1 Class E Example 6 Grade A Comparative Example 2 Grade B Example 7 Grade B Comparative Example 3 Grade A Example 8 Grade A Comparative Example 4 Grade A

[0180] As shown in Table 3, the test results indicate that the eluent provided by this invention can effectively remove the odor from intestinal flora products when used to treat donor feces.

[0181] 2. Store the intestinal flora products or capsules in a refrigerator at -80℃, and take samples at 0, 7, 30 and 360 days of storage to test the flora activity and abundance, and calculate the unit bacterial count.

[0182] (1) Microbial activity: After thawing, samples from each group were diluted 100-fold with physiological saline. The microorganisms in the samples were stained with the LIVE / DEAD™ BacLight™ Bacterial Viability Kit dye for 15 min. Microbial activity was detected using a BD Accuri™ C6 flow cytometer, and the changes in activity before and after treatment were analyzed using BD Accuri™ C6 Plus Software. The test results are shown in Table 4.

[0183] Table 4.

[0184]

[0185]

[0186] As shown in Table 4, the test results indicate that, compared to Comparative Examples 1-4, the intestinal flora products prepared using the methods provided in Examples 1-12 of this invention all exhibited a flora activity greater than 69.8%, demonstrating high flora activity. Furthermore, after 360 days of storage at -80℃, the flora activity of the intestinal flora products remained above 53.1%, indicating excellent storage stability. Simultaneously, the introduction of the hydrophobic capsule shell further facilitates air isolation, providing a favorable anaerobic storage environment for the intestinal flora products and further enhancing storage stability.

[0187] (2) Gastric acid resistance: Artificial gastric juice was prepared according to the Chinese Pharmacopoeia and preheated to 37°C. The capsules provided in Examples 13 and 14 were added and subjected to simulated gastric juice digestion for 2 hours at 37°C and 480 r / min. The bacterial activity was tested according to the test method in (1). The results are shown in Table 5.

[0188] Table 5.

[0189]

[0190] As shown in Table 5, the test results indicate that, compared to the ordinary capsule shell used in Example 14, the hydrophobic capsule shell provided in Example 13 of this invention has better resistance to gastric acid digestion, better protection for intestinal flora products, and is conducive to the colonization of flora in the intestine, thus achieving a better effect of precise flora transplantation.

[0191] (3) The method for testing the abundance of gut microbiota is as follows: 0.25g of donor feces and gut microbiota products prepared by the same donors according to the methods provided in Example 2 and Comparative Examples 2 and 3 were taken respectively. The total DNA of the gut microbiota products was extracted using the QIAamp Fast DNA Stool Mini Kit (QIAGEN) according to the instructions. After purifying the extracted total DNA sample, the V4 variable region (515F-806R) in the 16S rRNA of the total DNA sample was amplified by PCR (the reaction system and primer system used in the PCR amplification process are the same and are conventionally used in this field). A qualified sequencing library was constructed, and Paired-End 150bp (PE150) sequencing was performed using the Illumina MiniSeq platform. After sequencing, the sequences obtained by sequencing were spliced ​​using FLAS. Then, the primers were removed from the spliced ​​sequences using cutadapt, and poor-quality sequences were removed. The assembled sequences were used to remove chimeras using usearch, and OTUs were clustered based on 97% similarity. Based on the OTU clustering analysis results, representative sequences of OTUs were classified using the Silva 132 database, R v3.4.1, GraphPad Prism, and SPSS software, with the RDP classifier applied. The richness, diversity, and evenness indices of the microbial community were then calculated. It should be noted that all obtained data are expressed as mean ± standard deviation (SD). The results are as follows... Figures 2-4 As shown.

[0192] Depend on Figures 2-4 The test results show that, compared with Comparative Examples 2 and 3, the intestinal flora product prepared by the method provided in Example 2 of this invention has a high similarity to the donor feces in terms of flora abundance. The use of this intestinal flora product is conducive to the realization of precise flora transplantation. Moreover, after 360 days of low-temperature storage at -80℃, the flora in the intestinal flora product still has a high similarity to the donor feces, indicating that the flora composition of this intestinal flora product changes little after long-term low-temperature storage and has excellent storage stability.

[0193] 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 composition for extracting fecal microbiota, characterized in that, The composition comprises: The eluent comprises plant extracts and Japanese magnolia bark powder in a mass ratio of (9.25–10.15):(0.35–0.75); the plant extracts comprise Rugosa scabra extract, Citrus reticulata peel extract, Citrus medica extract, and Clove extract in a mass ratio of (5–10):(0.1–5):(1–3):(0.1–2); or, the plant extracts comprise Rugosa scabra extract, Citrus reticulata peel extract, Citrus medica extract, Clove extract, and Roasted malt extract in a mass ratio of (5–10):(0.1–5):(1–3):(0.1–2):(0.1–2); the preparation of the plant extracts specifically includes: hot water extraction of plant raw materials to obtain the plant extracts; The protective agent comprises, by mass ratio (5–50):(1–55):(1–35):(1–30):(0–15), a small molecule polyhydroxy compound, a macromolecular polymer, an amine, a natural emulsifier, and optionally a carbonate; wherein the small molecule polyhydroxy compound is selected from one or more of mannitol, maltitol, xylitol, trehalose, stachyose, isomaltulose, mannan oligosaccharide, tea polyphenols, vitamin C, and malic acid; wherein the macromolecular polymer is selected from one or more of fucoidan, konjac mannan, corn starch, ethyl cellulose, starch acetate, and agar powder; wherein the amine is selected from one or more of fish collagen peptides, glutathione, polyacrylamide, N-acetylglucosamine, and taurine; wherein the natural emulsifier is selected from one or more of gum arabic, carrageenan, gellan gum, guar gum, xanthan gum, and medium-chain triglycerides; and wherein the carbonate is potassium carbonate and / or sodium carbonate. And, optionally, separation aids, said separation aids comprising macroporous resins, functional resins, and non-resin adsorbents in a mass ratio of (2-5):(2-6):(2-10); said macroporous resins being selected from one or more of epoxy resins, polyester resins, polyvinyl chloride resins, polyethylene resins, polypropylene resins, phenolic resins, polyurethane resins, polystyrene resins, acrylic resins, ether-ketone resins, urea-formaldehyde resins, and ketone-formaldehyde resins; said functional resins being selected from ion exchange resins and / or chelating resins.

2. The composition for extracting fecal microbiota according to claim 1, characterized in that, Maltodextrin is added during the hot water extraction, and the mass ratio of the plant raw material to maltodextrin is 1:(1-10).

3. The composition for extracting fecal microbiota according to claim 1, characterized in that, The eluent also includes a bicarbonate, which is selected from sodium bicarbonate and / or potassium bicarbonate.

4. The composition for extracting fecal microbiota according to claim 1, characterized in that, Based on the total mass of the protective agent, the protective agent comprises 5-20 wt% trehalose, 10-30 wt% fucoidan, 15-35 wt% fish collagen peptide, 1-10 wt% vitamin C, 1-5 wt% malic acid and 5-20 wt% xanthan gum.

5. The composition for extracting fecal microbiota according to claim 1, characterized in that, Based on the total mass of the protective agent, the protective agent comprises 5-20 wt% mannitol, 0.1-30 wt% gum arabic, 2-10 wt% glutathione, 5-30 wt% maltitol, 1-30 wt% ethyl cellulose and 1-10 wt% carrageenan.

6. The composition for extracting fecal microbiota according to claim 1, characterized in that, Based on the total mass of the protective agent, the protective agent comprises 20-40 wt% corn starch, 5-15 wt% stachyose, 10-25 wt% xylitol, 1-15 wt% polyacrylamide, 5-15 wt% konjac mannan and 1-10 wt% gellan gum.

7. The composition for extracting fecal microbiota according to claim 1, characterized in that, Based on the total mass of the protective agent, the protective agent comprises 5-20 wt% tea polyphenols, 2-10 wt% potassium carbonate, 10-50 wt% starch acetate, 2-25 wt% N-acetylglucosamine, 5-30 wt% medium-chain triglycerides, and 1-5 wt% taurine.

8. The composition for extracting fecal microbiota according to claim 1, characterized in that, The non-resin adsorbent is selected from one or more of perlite, diatomite, activated clay, cellulose, titanium dioxide, activated carbon, coconut shell powder, and maifanite.

9. The composition for extracting fecal microbiota according to claim 1, characterized in that, The macroporous resin has an average pore size of 0.4–1.25 mm, the functional resin has an average particle size of 0.5–1 mm, and the non-resin adsorbent has an average particle size of 0.3–1.2 mm.

10. A method for preparing intestinal flora products using the composition according to any one of claims 1 to 9, characterized in that, The method includes: S1. Take the donor feces and mix them with the eluent and optional separation aid, and then perform stirring and filtration treatment in sequence to obtain crude bacterial mud liquid; S2. Take the crude liquid of the bacterial sludge and sequentially perform tangential flow filtration and centrifugation to obtain bacterial sludge; S3. Mix the bacterial sludge with a preservative to obtain an intestinal flora product.

11. The method for preparing intestinal flora products according to claim 10, characterized in that, In step S1, the mass ratio of the donor feces, eluent, and separation aid is (5-15):(50-75):(1-3).

12. The method for preparing intestinal flora products according to claim 10, characterized in that, In step S1, the stirring speed is 800-1500 r / min and the time is 4-15 min.

13. The method for preparing intestinal flora products according to claim 10, characterized in that, In step S1, the pore size of the filter membrane used for filtration is 20–2000 μm.

14. The method for preparing intestinal flora products according to claim 10, characterized in that, In step S2, the pore size of the filter membrane used in the tangential flow filtration process is 20–2000 μm, and the influent flow rate is 4–6 L / min / m³. 2 The inlet pressure differential is 0.9–1.75 bar, the return pressure differential is 0.05–0.2 bar, the transmembrane pressure is 0.5–0.9 bar, and the average membrane flux is 5.10–24.23 L / m³. 2 h.

15. The method for preparing intestinal flora products according to claim 10, characterized in that, In step S2, the centrifugation speed is 1000-20000g and the time is 2-15min.

16. The method for preparing intestinal flora products according to claim 10, characterized in that, In step S3, the mixing mass ratio of the fungal mud to the protective agent is (5-20):(1-2).

17. An intestinal flora product prepared by the method for preparing intestinal flora products according to any one of claims 10 to 16.

18. A method for preparing a capsule, characterized in that, The preparation method includes: coating a capsule shell with a hydrophobic coating material to obtain a hydrophobic capsule shell; and packaging the intestinal flora product of claim 17 with the hydrophobic capsule shell to obtain the capsule. The hydrophobic coating material comprises beeswax, paraffin oil, vegetable oil, and emulsifier in a mass ratio of (5-10):(1-3):(3-5):(1-3).

19. A capsule prepared by the method of claim 18.

20. The use of the intestinal flora product of claim 17 and / or the capsule of claim 19 in the preparation of a drug for precise flora transplantation.

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