Freeze-drying protective agent and freeze-drying process of fecal bacteria

Abstract

The application provides a freeze-drying protective agent and a freeze-drying process of fecal bacteria, and the preparation raw materials of the freeze-drying protective agent include sugar substances and high molecular protective agents, and the sugar substances include a combination of xylo-oligosaccharides, trehalose and mannitol. The freeze-drying protective agent and the freeze-drying process of fecal bacteria provided by the application can protect the activity of the extracted bacteria liquid in the feces, and effectively improve the freeze-drying survival rate of the fecal bacteria flora.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a freeze-drying protectant and a freeze-drying process for fecal bacteria. Background Technology

[0002] Fecal microbiota transplantation (FMT) involves transplanting functional bacteria from the feces of healthy individuals into the gastrointestinal tract of a patient, thereby rebuilding the patient's gut microbiota and enabling the diagnosis and treatment of intestinal and extraintestinal diseases. Currently, FMT is primarily performed via instrument-assisted intervention, requiring multiple transplants and demanding a high level of technical skill. Furthermore, the prepared fecal samples have a short shelf life at room temperature and can cause some discomfort to patients, limiting its application.

[0003] The preparation of freeze-dried fecal microbiota powder can avoid the need for surgical intervention in transplantation, reduce the volume and weight of the drug, and facilitate transportation and storage.

[0004] Because the object of drying is live microorganisms, they are more fragile and sensitive to drying conditions compared to other non-living active substances (such as proteins and enzymes). Therefore, the freeze-drying process for microorganisms is more challenging. For example, the freeze-drying of the most common lactic acid bacteria and probiotics usually requires the addition of protective agents in addition to common fillers and excipients.

[0005] CN105602875A discloses a preservative for storing lyophilized lactic acid bacteria powder at room temperature, comprising 2-4 wt% skim milk powder, 2-4 wt% monosodium glutamate, 5-8 wt% trehalose, 2-5 wt% maltitol, and 12-18 wt% polydextrose.

[0006] CN110964640A discloses a freeze-drying protectant for protecting the cells of a fermentation strain, the components of which include skim milk powder, tamarind polysaccharide, polydextrose and water.

[0007] Unlike traditional lactic acid bacteria and probiotics, the microbiota derived from feces is not a single, clearly defined strain or species, but rather a complex and abundant group of bacteria. Compared to lactic acid bacteria and probiotics, the microorganisms in fecal microbiota are more anaerobic, and many species are sensitive to environmental conditions, making it difficult for them to survive long-term in a laboratory environment. Therefore, freeze-drying is an important method for preserving and producing fecal microbiota, and exploring suitable freeze-drying protectants to safeguard the stability of the microbiota in feces is a significant technological barrier to the industrialization of human gut microbiota. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a freeze-drying protectant and a freeze-drying process for fecal microorganisms. The freeze-drying protectant provided by this invention can protect the activity of bacterial solutions extracted from feces, effectively improving the freeze-drying survival rate of fecal microorganisms.

[0009] To achieve this objective, the present invention employs the following technical solution:

[0010] In a first aspect, the present invention provides a freeze-drying protectant, wherein the raw materials for preparing the freeze-drying protectant include sugars and polymeric protectants, and the sugars include a combination of xylooligosaccharides, trehalose and mannitol.

[0011] In this invention, the raw materials for preparing the freeze-drying protectant include, by weight, 10-20 parts of carbohydrates (e.g., 10, 12, 14, 16, 18, 20, etc.) and 5-10 parts of polymer protectant (e.g., 5, 6, 7, 8, 9, 10, etc.).

[0012] Preferably, the mass ratio of xylooligosaccharide, trehalose, and mannitol is (5-10):(3-8):(1-5);

[0013] Among them, "5-10" can be 5, 6, 7, 8, 9, 10, etc.;

[0014] "3-8" can be 3, 4, 5, 6, 7, 8, etc.;

[0015] "1-5" can be 1, 2, 3, 4, 5, etc.

[0016] Preferably, the polymeric protective agent includes skim milk powder.

[0017] In this invention, the raw materials for preparing the freeze-drying protectant also include any one or a combination of at least two of unsaturated fatty acids, amino acids, or salts.

[0018] Preferably, the raw materials for preparing the freeze-drying protectant also include unsaturated fatty acids, amino acids, and salts.

[0019] Preferably, the raw materials for preparing the freeze-drying protectant further include, by weight, 0.005-0.015 parts of unsaturated fatty acids (e.g., 0.005 parts, 0.007 parts, 0.009 parts, 0.011 parts, 0.013 parts, 0.015 parts, etc.), 1-3 parts of amino acids (e.g., 1 part, 1.5 parts, 2 parts, 2.5 parts, 3 parts, etc.) and 0.1-1 parts of salt (e.g., 0.1 parts, 0.3 parts, 0.5 parts, 0.7 parts, 0.9 parts, 1 part, etc.).

[0020] Preferably, the freeze-drying protectant further includes a solvent, which comprises 90-110 parts by weight of water (e.g., 90, 93, 96, 99, 103, 106, 109, 110, etc.).

[0021] In this invention, the unsaturated fatty acid includes oleic acid.

[0022] Preferably, the amino acid includes polyγ-glutamic acid.

[0023] Preferably, the salt comprises sodium alginate.

[0024] In a second aspect, the present invention provides the application of the freeze-drying protectant according to the first aspect in the preparation of freeze-dried fecal bacteria products.

[0025] In this invention, fecal bacteria refers to bacteria obtained by extracting human feces through a fecal bacteria separation system.

[0026] Thirdly, the present invention provides a freeze-drying process for fecal microorganisms, wherein the freeze-drying protectant described in the first aspect is used to freeze-dry the fecal microorganisms.

[0027] In this invention, the freeze-drying process specifically includes the following steps: mixing fecal microbial liquid with a freeze-drying protectant and then freeze-drying to obtain freeze-dried fecal microbial.

[0028] In this invention, the fecal microbial solution is obtained by extracting human feces through a fecal microbial separation system;

[0029] Preferably, the solid-liquid ratio of the fecal microbial solution and the freeze-drying protectant is 1:(1-1.5);

[0030] Among them, "1-1.5" can be 1, 1.1, 1.2, 1.3, 1.4, 1.5, etc.

[0031] The optimal mixing ratio of bacterial culture to freeze-drying protectant is w:v = 1:1.2.

[0032] In this invention, after freeze-drying, post-processing is also included, which includes the following steps: pulverizing the freeze-dried fecal bacteria obtained after freeze-drying and sieving it to obtain freeze-dried fecal bacteria powder.

[0033] Preferably, the pulverization is carried out in an environment with a humidity of less than 30% (e.g., 28%, 25%, 21%, 15%, 13%, 10%, 8%, 5%, 3%, etc.).

[0034] Preferably, the sieving uses a sieve with a mesh size of 100-300 (e.g., 100 mesh, 130 mesh, 160 mesh, 190 mesh, 230 mesh, 260 mesh, 290 mesh, 300 mesh, etc.).

[0035] In this invention, the freeze-dried fecal microbiota powder can be packaged into capsules to make oral capsules. The oral capsules are no less effective than colonoscopy transplantation, reducing the psychological barriers of patients undergoing fecal microbiota transplantation and eliminating the uncomfortable physiological reactions that may occur during the treatment process.

[0036] As a preferred technical solution of the present invention, the preparation method of the fecal microorganism freeze-dried powder capsules includes the following steps:

[0037] (1) Separation: Obtain human feces and extract bacterial solution through fecal microbial separation system.

[0038] (2) Freeze-drying: The obtained bacterial solution and freeze-drying agent are mixed at a solid-liquid ratio of 1:(1-1.5) and then freeze-dried to obtain freeze-dried bacterial solution;

[0039] (3) Pulverization: Pulverize the freeze-dried bacterial solution in an environment with a humidity of less than 30%, and pass it through a 100-300 mesh sieve to obtain freeze-dried bacterial solution powder;

[0040] (4) Filling: The lyophilized bacterial solution is dispensed into capsules to prepare lyophilized fecal bacteria capsules.

[0041] In this invention, the freeze-drying specifically includes the following steps:

[0042]

[0043]

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The freeze-drying protectant and freeze-drying process for fecal bacteria provided by this invention can protect the activity of the bacterial solution extracted from feces and effectively improve the freeze-drying survival rate of fecal bacteria. Attached Figure Description

[0046] Figure 1 Images showing the appearance of live and dead bacteria. Detailed Implementation

[0047] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0048] The xylooligosaccharides used in the following examples were purchased from the Yobojia Food Flagship Store.

[0049] Example 1

[0050] This embodiment provides a freeze-drying protectant, the preparation method of which includes the following steps: mixing 8% (w / v) xylooligosaccharide, 5% (w / v) trehalose, 3% (w / v) mannitol, 7% (w / v) skim milk powder, 2% (w / v) polyγ-glutamic acid, 0.5% (w / v) sodium alginate and 0.01% (w / v) oleic acid to obtain the freeze-drying protectant, wherein the solvent of the freeze-drying protectant is water.

[0051] Example 2

[0052] This embodiment provides a freeze-drying protectant. The preparation method of the freeze-drying protectant includes the following steps: mixing 5% (w / v) xylooligosaccharide, 7% (w / v) trehalose, 2% (w / v) mannitol, 5% (w / v) skim milk powder, 3% (w / v) polyγ-glutamic acid, 0.3% (w / v) sodium alginate and 0.008% (w / v) oleic acid to obtain the freeze-drying protectant. The solvent of the freeze-drying protectant is water.

[0053] Example 3

[0054] This embodiment provides a freeze-drying protectant. The preparation method of the freeze-drying protectant includes the following steps: mixing 10% (w / v) xylooligosaccharide, 3% (w / v) trehalose, 5% (w / v) mannitol, 10% (w / v) skim milk powder, 3% (w / v) polyγ-glutamic acid, 0.8% (w / v) sodium alginate and 0.012% (w / v) oleic acid to obtain the freeze-drying protectant. The solvent of the freeze-drying protectant is water.

[0055] Example 4

[0056] This embodiment provides a freeze-drying protectant. The preparation method of the freeze-drying protectant includes the following steps: mixing 8% (w / v) sucrose, 5% (w / v) trehalose, 3% (w / v) mannitol and 7% (w / v) skim milk powder to obtain the freeze-drying protectant. The solvent of the freeze-drying protectant is water.

[0057] Example 5

[0058] This embodiment provides a freeze-drying protectant. The preparation method of the freeze-drying protectant includes the following steps: mixing 8% (w / v) xylooligosaccharide, 5% (w / v) trehalose, 3% (w / v) mannitol, 0.5% (w / v) sodium bicarbonate and 7% (w / v) skim milk powder to obtain the freeze-drying protectant. The solvent of the freeze-drying protectant is water.

[0059] Example 6

[0060] This embodiment provides a freeze-drying protectant. The preparation method of the freeze-drying protectant includes the following steps: mixing 8% (w / v) xylooligosaccharide, 5% (w / v) trehalose, 3% (w / v) mannitol and 7% (w / v) skim milk powder to obtain the freeze-drying protectant. The solvent of the freeze-drying protectant is water.

[0061] Example 7

[0062] This embodiment provides a freeze-drying protectant. The preparation method of the freeze-drying protectant includes the following steps: mixing 8% (w / v) soybean polysaccharide, 5% (w / v) trehalose, 3% (w / v) mannitol, 7% (w / v) skim milk powder, 2% (w / v) lysine, 0.5% (w / v) sodium alginate and 0.01% (w / v) oleic acid to obtain the freeze-drying protectant. The solvent of the freeze-drying protectant is water.

[0063] Example 8

[0064] This embodiment provides a freeze-drying protectant, the preparation method of which includes the following steps: mixing 8% (w / v) xylooligosaccharide, 5% (w / v) trehalose, 3% (w / v) mannitol, 7% (w / v) skim milk powder, 2% (w / v) lysine, 0.5% (w / v) sodium alginate and 0.01% (w / v) oleic acid to obtain the freeze-drying protectant, wherein the solvent of the freeze-drying protectant is water.

[0065] Example 9

[0066] This embodiment provides a freeze-drying protectant, which differs from Example 1 only in that polyγ-glutamic acid is replaced with an equal amount of glycine, while the other preparation methods are the same as in Example 1.

[0067] Comparative Example 1

[0068] This embodiment provides a freeze-drying protectant, which differs from Example 1 only in that it does not contain xylooligosaccharides, the trehalose content is increased to 10% (w / v), the mannitol content is increased to 6% (w / v), and the other preparation methods are the same as in Example 1.

[0069] Comparative Example 2

[0070] This embodiment provides a freeze-drying protectant, which differs from Example 1 only in that it does not contain trehalose, the content of xylooligosaccharides is increased to 11.6% (W / V), and the content of mannitol is increased to 4.4% (W / V). The other preparation methods are the same as in Example 1.

[0071] Comparative Example 3

[0072] This embodiment provides a freeze-drying protectant, which differs from Example 1 only in that it does not contain mannitol, the content of xylooligosaccharides is increased to 9.8% (W / V), the content of trehalose is increased to 6.2% (W / V), and the other preparation methods are the same as in Example 1.

[0073] Application Example 1

[0074] This application example provides a freeze-dried fecal microorganism powder, the preparation method of which includes the following steps:

[0075] (1) Separation: Obtain human feces and extract bacterial solution through fecal microbial separation system.

[0076] (2) Freeze-drying: The obtained bacterial solution was mixed with the freeze-drying protectant provided in Example 1 at a ratio of w:v = 1:1.2 and then freeze-dried to obtain the freeze-dried bacterial solution;

[0077] (3) Pulverization: Pulverize the freeze-dried bacterial solution in an environment with a humidity of 25% and pass it through a 100-mesh sieve to obtain freeze-dried bacterial solution powder;

[0078] In step (2), the freeze-drying specifically includes the following steps:

[0079] 1 4 -40.0 2 12 -30.0 3 4 -20.0 4 4 0.0 5 5 10.0 6 3 15.0 7 2 17.0 8 8 25.0 9 (Concluding paragraph) 1 30.0 .

[0080] Application Example 2

[0081] This application example provides a freeze-dried fecal microorganism powder, the preparation method of which includes the following steps:

[0082] (1) Separation: Obtain human feces and extract bacterial solution through fecal microbial separation system.

[0083] (2) Freeze-drying: The obtained bacterial solution was mixed with the freeze-drying protectant provided in Example 2 at a ratio of w:v = 1:1.1 and then freeze-dried to obtain the freeze-dried bacterial solution;

[0084] (3) Pulverization: The freeze-dried bacterial solution is pulverized in an environment with a humidity of 22% and passed through a 300-mesh sieve to obtain freeze-dried bacterial solution powder;

[0085] In step (2), the freeze-drying specifically includes the following steps:

[0086]

[0087]

[0088] Application Example 3

[0089] This application example provides a freeze-dried fecal microorganism powder, the preparation method of which includes the following steps:

[0090] (1) Separation: Obtain human feces and extract bacterial solution through fecal microbial separation system.

[0091] (2) Freeze-drying: The obtained bacterial solution was mixed with the freeze-drying protectant provided in Example 3 at a ratio of w:v = 1:1.3 and then freeze-dried to obtain the freeze-dried bacterial solution;

[0092] (3) Pulverization: Pulverize the freeze-dried bacterial solution in an environment with a humidity of 20% and pass it through a 200-mesh sieve to obtain freeze-dried bacterial solution powder;

[0093] In step (2), the freeze-drying specifically includes the following steps:

[0094]

[0095]

[0096] Application Example 4-9

[0097] Application Examples 4-9 each provide a fecal microbial freeze-dried powder, the only difference from Application Example 1 being that the freeze-drying protectant provided in Example 1 is replaced with the freeze-drying protectant provided in Examples 4-9.

[0098] Application Example 10

[0099] This application example provides a freeze-dried powder of fecal bacteria, which differs from application example 1 only in that, in step (2), the freeze-drying specifically includes the following steps:

[0100] 1 4 -40.0 2 16 -30.0 3 4 0.0 4 5 10.0 5 3 15.0 6 2 17.0 7 8 25.0 8 (Concluding paragraph) 1 30.0 .

[0101] Application Example 11

[0102] This application example provides a freeze-dried powder of fecal bacteria, which differs from application example 1 only in that, in step (2), the freeze-drying specifically includes the following steps:

[0103]

[0104]

[0105] Application Example 12

[0106] This application example provides a freeze-dried powder of fecal bacteria, which differs from application example 1 only in that, in step (2), the freeze-drying specifically includes the following steps:

[0107] 1 4 -32 2 12 -20 3 4 -10 4 4 6 5 5 15 6 3 18 7 2 18 8 8 28 9 (Concluding paragraph) 1 37 .

[0108] Comparative Application Examples 1-3

[0109] Comparative Application Examples 1-3 each provide a fecal microbial freeze-dried powder. The only difference from Application Example 1 is that the freeze-drying protectant provided in Example 1 is replaced with the freeze-drying protectant provided in Comparative Examples 1-3.

[0110] Test case

[0111] Survival test

[0112] Test samples: freeze-dried fecal bacteria powder provided in Application Examples 1-12 and Comparative Application Examples 1-3.

[0113] Test method:

[0114] (1) Freeze-dried survival rate test

[0115] Test method: Dilute the bacterial suspension 10 times or weigh 50mg of bacterial powder into a 1.5mL EP tube, add 1mL of sterile physiological saline to dissolve thoroughly, and mix the diluted bacterial suspension with 0.4% trypan blue at a 9:1 ratio. Count the bacteria using a hemocytometer. Using a 16*25 type counting chamber, count the bacteria in four diagonally opposite squares: top left, bottom left, top right, and bottom right. When counting, if bacteria are found on a square, follow the principle of "count the top, not the bottom; count the left, not the right." Count the number of live bacteria (clear) and dead bacteria (black) separately to calculate the bacterial survival rate (e.g., ...). Figure 1 (See the images of live and dead bacteria). After obtaining the raw data, take the average value and calculate as follows: average value * 16 * 10000 to obtain the corresponding total bacterial count. The count result N of dissolved bacterial powder, (N / weight) gives the final count result.

[0116] Freeze-dried survival rate (%) = (number of viable bacteria before freeze-drying / number of viable bacteria after freeze-drying) × 100%.

[0117] (2) Survival rate test after storage at -20℃ for 30 days

[0118] Test method: The freeze-dried fecal bacteria powder provided in Application Examples 1-12 and Comparative Application Examples 1-3 were dispensed into centrifuge tubes and stored at -20°C for 30 days. The survival rate was then calculated.

[0119] Survival rate (%) after storage at -20℃ for 30 days = (Number of viable bacteria after storage at -20℃ for 30 days / Number of viable bacteria before storage) × 100%.

[0120] The test results are shown in Table 1 below:

[0121] Table 1

[0122]

[0123] As shown in Table 1, the freeze-drying protectant and drying process provided by this invention can improve the survival rate of fecal microorganisms. Specifically, the fecal microorganism freeze-dried powder provided in Application Example 1 can achieve a viable bacterial count of 7.97 × 10⁻⁶. 11 CFU / g. The various components in the freeze-drying protectant work synergistically to enhance the activity of fecal bacteria.

[0124] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. The application of a freeze-drying protectant in the preparation of freeze-dried fecal microorganisms products, characterized in that, The raw materials for preparing the freeze-drying protectant include, by weight, 10-20 parts of carbohydrates, 5-10 parts of polymer protectant, 0.005-0.015 parts of unsaturated fatty acids, 1-3 parts of amino acids, and 0.1-1 parts of salt. The sugar substance is a combination of xylooligosaccharides, trehalose, and mannitol; The polymeric protective agent includes skim milk powder; The unsaturated fatty acids include oleic acid; The amino acid includes polyγ-glutamic acid; The salt includes sodium alginate.

2. The application according to claim 1, characterized in that, The mass ratio of xylooligosaccharide, trehalose and mannitol is (5-10):(3-8):(1-5).

3. The application according to claim 1, characterized in that, The freeze-drying protectant also includes a solvent, which comprises 90-110 parts by weight of water.

4. A freeze-drying process for fecal bacteria, characterized in that, The freeze-drying agent described in any one of claims 1-3 is mixed with fecal microbial solution at a mass-volume ratio of (1-1.5):1 and then freeze-dried to obtain freeze-dried fecal microbial solution. The freeze-drying process specifically includes the following steps:

5. The freeze-drying process according to claim 4, characterized in that, The fecal microbial solution is obtained by extracting human feces through a fecal microbial separation system.

6. The freeze-drying process according to claim 4, characterized in that, The freeze-drying process also includes post-processing, which includes the following steps: pulverizing the freeze-dried fecal bacteria obtained after freeze-drying and sieving it to obtain freeze-dried fecal bacteria powder.

7. The freeze-drying process according to claim 6, characterized in that, The pulverization is carried out in an environment with a humidity of less than 30%.

8. The freeze-drying process according to claim 6, characterized in that, The sieving process uses a 100-300 mesh sieve.

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