CN112656766A - Fecal fungus freeze-drying and capsule preparation process for fecal fungus transplantation treatment - Google Patents
Fecal fungus freeze-drying and capsule preparation process for fecal fungus transplantation treatment Download PDFInfo
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Abstract
The invention relates to the technical field of coprophilous fungus transplantation treatment products, and provides coprophilous fungus freeze-drying and capsule preparation processes for coprophilous fungus transplantation treatment. The preparation of the coprinus comatus capsule comprises the following processes: (1) preparing fecal bacteria liquid and reserving a sample; (2) preparing a freeze-drying protective agent; (3) preparing a culture medium; (4) freeze-drying by gradient cooling method; (5) and (5) filling into capsules. The freeze-drying protective agent comprises 10-20% of skimmed milk powder, 1-5% of cane sugar, 1-3% of gelatin and the balance of sterile distilled water. The content of the culture medium is 6.43 percent of MRS powder, and the balance is sterile distilled water. The freeze-drying protective solution can prevent water from generating ice crystals to damage bacteria in the freeze-drying process, and effectively reduces the loss of the total amount of bacteria in the freeze-drying process by matching with gradient cooling; the fecal bacteria capsule enteric capsule shell can ensure that fecal bacteria can reach the intestinal tract to play a role, and the fecal bacteria can be prevented from being killed by gastric acid.
Description
Technical Field
The invention relates to the technical field of coprophilous fungus transplantation treatment products, in particular to coprophilous fungus freeze-drying and capsule preparation processes for coprophilous fungus transplantation treatment.
Background
"Fecal transplantation" (FMT) is defined as the transplantation of functional flora in the feces of healthy people into the intestinal tract of a patient to reconstruct new intestinal flora and realize the treatment of intestinal tract and parenteral diseases.
The fecal strain transplanting technology is to utilize modern medical technology to separate fecal microbes and microbe survival maintaining matters from healthy people, transplant the microorganisms into intestinal tracts of patients and rebuild the intestinal flora of the patients so as to treat clostridium difficile infection, intractable diarrhea, constipation, gastroenteritis, Crohn's disease, ulcerative gastroenteritis and other intestinal diseases and extraintestinal diseases such as obesity, asthma, obesity, autism, depression, diabetes, cancer and other difficult and complicated diseases.
Fecal transplantation (FMT) is of concern for its superior efficacy, but fecal preparation, storage, and logistics limit its widespread use. Meanwhile, in the fecal strain transplanting process, a doctor needs to transplant fecal strains into a patient body through a nasogastric tube, a gastroscope or an enteroscope, and discomfort of the patient is easily caused.
The treatment effect of the fecal bacteria transplantation is definite, but the popularization and application need to reduce the psychological barrier of patients receiving the fecal bacteria transplantation, eliminate discomfort caused in the operation process and reduce the psychological barrier of operators implementing the fecal bacteria transplantation, and the effective technical means can be carried out only when both doctors and patients receive the method. The fecal bacteria capsule can greatly reduce the mental barrier for patients and operators at the same time. On one hand, the fecal bacteria capsule avoids uncomfortable transplanting modes such as a nasogastric tube, a gastroscope and an enteroscope which are needed by a patient, and on the other hand, the fecal bacteria capsule avoids discomfort of an implementer and exposure risk of doctors caused by spreading of diseases caused by regurgitation and cough of food of the patient in the fecal bacteria transplanting process.
A coprinus comatus capsule for coprinus comatus transplantation therapy adopts an enteric gelatin hollow capsule, and the results of experiments of disintegration outside the capsule body are good in 2 hours in an acid environment, the capsule is disintegrated within thirty minutes at pH6.8, the storage condition is simple, and the coprinus comatus capsule can be stored at-20 ℃ for one year.
At present, a plurality of methods for preparing coprinus comatus capsule freeze-drying protective agents are available on the market, such as preparing protective agents from maltodextrin, trehalose, glycerol and the like, but the methods have the following two defects in general: 1. the preservation time is short, and the preservation time of the freeze-dried fungus powder is generally about one month to half a year. 2. The invention adopts a unique freeze-drying protective agent formula, and the freeze-drying protective agent can effectively prevent ice crystals generated by water under low temperature conditions in the freeze-drying process from damaging intestinal bacteria, so that the loss of freeze-dried bacteria is reduced to about 10 percent, the problem of overlarge bacterial loss before and after freeze-drying in the world is solved, and meanwhile, the storage time of bacterial powder is prolonged to about 1 year.
Disclosure of Invention
The invention provides a fecal strain freeze-drying and capsule preparation process for fecal strain transplantation treatment, which is used for solving the problems of short preservation time after freeze-drying and overlarge loss of the total number of the fecal strains after freeze-drying, preventing ice crystals generated by water in the freeze-drying process from damaging intestinal bacteria and effectively ensuring the activity of the intestinal bacteria.
The invention relates to a preparation process of a coprophilous fungi capsule in coprophilous fungi transplantation treatment, which comprises the following steps of:
the method comprises the following steps: taking out the fecal bacteria liquid from a refrigerator at minus 80 ℃, unfreezing the fecal bacteria liquid in a water bath kettle at 37 ℃ for 20-30min, taking out the fecal bacteria liquid, putting the fecal bacteria liquid into a centrifugal machine, and centrifuging the fecal bacteria liquid at 5000r and 4 ℃ for 5 min.
Step two: and (5) preparing a freeze-drying protective agent.
Step three: preparing a culture medium: dissolving 12.9g of MRS culture medium in 200ml of conical flask filled with distilled water, mixing uniformly while stirring, adjusting pH to 7.3 +/-0.2, and sterilizing at 121 ℃ for 20min in an autoclave.
Step four: and taking out the centrifuged bacterial liquid, adding 10ml of freeze-drying protective agent into each tube, and uniformly mixing by using a mixing instrument.
Step five: and (4) sample retention: taking five freezing storage tubes with the serial numbers of 1, 2, 3, 4 and 5, adding 0.5ml of uniformly mixed bacteria liquid into each tube, and respectively performing sample retention experiments on the first day, the first week, the first month, the third month and the first year.
Step six: subpackaging: adding each tube of bacterial liquid into the west forest, wherein the liquid level is about 1cm, simultaneously placing penicillin bottles and five freezing tubes for reserving samples into a freeze dryer, setting the pre-freezing temperature to be-45 ℃, freeze-drying for 48 hours, and controlling the humidity below 40 after freeze-drying.
Step seven: filling: pouring the freeze-dried powder onto a capsule filling plate, and filling a No. 0 capsule. And (4) storing the filled capsules and the preserved freezing tubes in a refrigerator at the temperature of-20 ℃.
The preparation method of the fecal bacteria liquid in the first step comprises the following steps:
s1, uniformly stirring and diluting the excrement:
adding 50g of in vitro fresh excrement into 800ml of 0.9% sterile normal saline with the temperature of 2-6 ℃, manually stirring for 1-2 minutes by using a glass plate, and fully and uniformly stirring for about five minutes by using a magnetic stirrer (the rotating speed of the stirrer is 500 r/min).
S2, filtering:
filtering with stainless steel filter screen on the ultra-clean workbench, sequentially filtering according to 60 mesh and 80 mesh, removing feces residue and large particulate matter, and collecting the final filtrate to obtain the whole feces bacterium solution.
S3, centrifugation:
and (3) subpackaging 45ml of fecal strain liquid into centrifuge tubes, weighing to ensure that the weight deviation of each bottle of fecal strain liquid is not more than 0.5, and symmetrically placing the bottles into a centrifuge after the weighing is finished.
First centrifugation: rotating at a speed of 5000r/min and at a temperature of 10-15 ℃ for 1min, removing supernatant and leaving precipitate;
secondly, resuspending for the first time: adding 0.9% sterile physiological saline at 4 deg.C into the precipitate to 45ml, and shaking with oscillator to obtain resuspension;
third centrifugation: rotating at speed of 5000r/min and temperature of 10 deg.C for 1min, removing supernatant, and collecting precipitate;
fourthly, resuspending for the second time: adding physiological saline with the temperature of 4 ℃, adding 45ML of saline into each tube, and uniformly mixing by using an oscillator;
filtering: filtering the second re-suspended liquid manure with a 80-mesh filter screen;
sixthly, adding 5ML of glycerol with the concentration of 10 percent, and uniformly mixing to obtain a full-fecal bacteria liquid;
and seventhly, preservation: and (3) sticking a freezing label to each tube of the whole manure liquid with the volume of about 45ML, cooling for 1 hour in a refrigerator at the temperature of-4 ℃, and then storing in a refrigerator at the temperature of-70-90 ℃ for 10-15 months.
The freeze-drying protective solution related in the step two mainly comprises skimmed milk powder, cane sugar, glycerol and sterile water, and the freeze-drying protective solution comprises the following components in percentage by weight: 10-20% of skimmed milk powder, 1-5% of sucrose, 1-3% of gelatin and 72-88% of sterile water.
Further, the sucrose is pure sucrose, and the gelatin is 180 freezing strength gelatin.
The preparation and preservation method of the freeze-drying protective solution comprises the following steps:
weighing 18g of skimmed milk powder, 1g of sucrose and 3g of gelatin by using weighing paper, pouring into a conical flask, adding 60 ℃ distilled water, adding water and uniformly mixing until the volume is 100 ml;
② putting the evenly mixed freeze-drying protective solution into a high-temperature high-pressure sterilization pot, and sterilizing for 20min at 121 ℃.
The freeze-drying process of the fecal strain liquid involved in the step six comprises the following steps:
putting the prepared fecal strain liquid into a refrigerator at the temperature of minus 4 ℃ for 30min, taking out, and putting into a refrigerator at the temperature of minus 20 ℃ for 60 min.
Secondly, taking the fecal bacteria liquid out of a refrigerator at the temperature of 20 ℃ below zero, and putting the fecal bacteria liquid into a freeze dryer.
Setting freeze-drying parameters, and freeze-drying through several steps of pre-freezing, vacuumizing and subliming:
s1, pre-freezing: the temperature is-35 ℃ to-45 ℃, and the pre-freezing time is 150-180 min;
s2, vacuumizing: the vacuum degree is 0.2, and the vacuumizing time is 20-30 min;
s3, sublimation: dividing into 9 gradients, increasing the temperature of each gradient by 5 ℃, increasing the temperature from-35 ℃ to 30 ℃, increasing the temperature between each gradient for 10min, and keeping the temperature of each temperature node constant for 30-120 min;
s4, sterilization: sterilizing with ultraviolet lamp for 40-60 min;
and fourthly, taking out the sample, opening an air inlet valve after freeze-drying is finished, filling high-purity nitrogen, sealing the freeze-dried powder, and filling the freeze-dried powder in the next step.
Further, high-purity nitrogen is introduced for protection in the whole freeze-drying process.
The invention has the beneficial effects that:
(1) the freeze-drying protective solution can avoid the damage of bacteria caused by water ice crystals in the freeze-drying process, and the loss of the total amount of bacteria in the freeze-drying process is effectively reduced by matching with gradient cooling;
(2) the coprophilous fungi are freeze-dried and then prepared into an oral capsule, the effect of the oral capsule is not inferior to that of a colonoscope transplantation mode, the psychological barrier of patients receiving coprophilous fungi transplantation is reduced, and the uncomfortable physiological reaction in the treatment process is eliminated;
(3) the fecal bacteria capsule enteric capsule shell can ensure that fecal bacteria can reach the intestinal tract to play a role, and the fecal bacteria can be prevented from being killed by gastric acid.
Drawings
FIG. 1: diluting 1ml of fecal strain liquid by 103 times before freeze-drying, and taking 200 mul of lactobacillus for growth;
FIG. 2: after the lm fecal strain liquid is freeze-dried and diluted to the same times, taking 200 mul of lactobacillus for growth;
FIG. 3: before freeze-drying, 1ml of fecal strain liquid is diluted to 106 times, and 200 mul of whole strain growth condition is taken;
FIG. 4: freeze-drying 1ml of fecal strain liquid, diluting to the same multiple, and taking 200 mul of whole-strain growth condition;
FIG. 5: developing the fecal bacteria capsule under X-ray;
FIG. 6: images of fecal bacteria capsule under capsule gastroscope.
Detailed Description
In order to make the technical scheme and advantages of the embodiment of the invention clearer, the technical scheme is more clearly and thoroughly understood by combining two comparison experiments which are carried out before.
Example 1
And (3) taking a part of excrement of the volunteer according with the requirements of the donor, preparing 6 tubes of excrement liquid, centrifuging, removing supernatant, and adding 10ml of freeze-drying protective agent into each tube. And (3) uniformly mixing by using a mixing instrument, and subpackaging penicillin bottles, wherein the liquid level of each penicillin bottle is about 1 cm. Meanwhile, 0.5ml of sample is kept in a freezing tube and put into a Christ Epsilon 2-6D LSC plus freeze dryer together for freeze drying.
The freeze-drying parameters are as follows:
pre-freezing at-45 deg.C for 180 min;
vacuumizing, wherein the vacuum degree is 0.2, and the vacuumizing time is 20 min;
sublimating at-35 deg.C for 10min for 120 min; heating to-30 deg.C for 10min for 60 min; heating to-25 deg.C for 10min for 60 min; heating at-20 deg.C for 10min for 60 min; heating to-15 deg.C for 10min for 60 min; heating to-10 deg.C for 10min, and maintaining for 180 min; heating at-5 deg.C for 10min for 60 min; heating to-0 deg.C for 10min for 60 min; heating at 5 deg.C for 10min for 60 min; heating at 10 deg.C for 10min for 120 min; heating at 15 deg.C for 10min for 60 min; heating at 20 deg.C for 10min for 30 min; heating at 25 deg.C for 10min for 30 min; heating at 30 deg.C for 10min, and maintaining for 30 min.
Charging high-purity nitrogen to remove vacuum, taking out sample, rapidly encapsulating, and storing in-80 deg.C refrigerator.
Taking out the capsule the next day, adding 0.12g of the capsule into a freezing storage tube, and performing a culture experiment of indicator bacteria lactic acid bacteria and total number of bacteria.
The specific operation steps of the indicator bacterium culture are as follows:
seven test tubes are respectively numbered as 1, 2, 3, 4, 5, 6, 7 and 8, and 9ml of sterile water is added into each test tube;
adding sterile water into the freezing tube, uniformly mixing to 1ml, completely taking out the mixture by using a pipette, adding the mixture into a No. 1 test tube, blowing, beating and uniformly mixing; taking 1ml from the test tube 1, adding into the test tube No. 2, and blowing, beating and uniformly mixing; repeating the steps until the number 8 test tubes are reached, wherein the dilution times of the bacterial liquid in the number 1-8 test tubes are respectively 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8；
③ coating the plates, respectively taking 10-3、10-4、10-5Dripping 200 mul of the three-gradient diluent into an MRS culture medium, and uniformly coating by using a coating rod;
fourthly, putting the coated flat plate into an anaerobic incubator for culture at 37 ℃;
taking out the flat plate after 48h, taking a picture, and counting.
The specific operation steps of total bacteria culture are as follows:
seven test tubes are respectively numbered as 1, 2, 3, 4, 5, 6, 7 and 8, and 9ml of sterile water is added into each test tube;
adding sterile water into the freezing tube, uniformly mixing to 1ml, completely taking out the mixture by using a pipette, adding the mixture into a No. 1 test tube, blowing, beating and uniformly mixing; taking 1ml from the test tube 1, adding into the test tube No. 2, and blowing, beating and uniformly mixing; repeating the steps until the number 8 test tubes are reached, wherein the dilution times of the bacterial liquid in the number 1-8 test tubes are respectively 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8；
③ coating the plates, respectively taking 10-6、10-7、10-8200 μ l of the three-gradient dilutions were added dropwise to the TSA medium and spread evenly with a spreading bar;
fourthly, putting the coated flat plate into an anaerobic incubator for culture at 37 ℃;
taking out the flat plate after 48h, taking a picture, and counting.
The average value of the total number cfu of the lactic acid bacteria after freeze-drying is 3.19 multiplied by 107Mean difference of 9X 105The survival rate of lactobacillus before and after freeze-drying is 95.2 percent. Example 1 the total number of whole bacteria cfu was 8.65X 10 as a result of whole bacteria culture before lyophilization11The average value of the total number cfu of the whole bacteria after freeze-drying is 8.23 multiplied by 1011The average value of viable count N is 11.94, and the survival rate of the whole bacteria before and after freeze-drying is 95.1%.
Example 1 indicators bacteria results of bacterial counts from three parallel experiments, see table below:
the bacterial loss rate before and after freeze-drying is less than 5 percent as can be seen from the index bacterial culture and whole bacterial culture results in example 1.
Clinical trial
The disintegration process of the fecal bacteria capsule in vivo is verified by two methods.
Barium sulfate is filled into a capsule shell for manufacturing the fecal bacteria capsule, two testers take the barium sulfate respectively, one testers takes the barium sulfate and develops the barium sulfate by taking an X-ray film, as shown in figure 5, the barium sulfate enters the small intestine after the capsule stays in the stomach for 24 minutes, and the barium sulfate runs in the small intestine for 4 hours and 05 minutes. Another example was observed under a capsule endoscope, and as shown in FIG. 6, 30ml of drinking water was fed into the barium capsule, left in the stomach for about 20 minutes, and completely dissolved after running in the small intestine for 1 hour and 56 minutes (jejunal segment).
Clinical tests show that the enteric capsule shell of the fecal bacteria capsule can ensure that fecal bacteria can reach the intestinal tract to play a role, and the fecal bacteria can be prevented from being killed by gastric acid.
While the foregoing embodiments are illustrative of the present invention, various modifications and changes may be readily made by those skilled in the art based upon the teachings and principles of this invention, which are intended to be limited not to the details of construction and methods herein shown, but rather to the preferred embodiments described, and therefore all equivalent modifications and changes in light of the above teachings are intended to be included within the scope of the present invention as defined in the appended claims.
Claims (8)
1. A coprophilous fungus freeze-drying and capsule preparation process for coprophilous fungus transplantation treatment is characterized in that the coprophilous fungus freeze-drying comprises the following steps:
the method comprises the following steps: preparing a fecal strain liquid: uniformly stirring and diluting fresh excrement and 0.9% sterile normal saline at the temperature of 2-6 ℃, filtering by a multi-stage filter screen, taking final filtrate to obtain a whole excrement liquid, centrifuging and resuspending the obtained whole excrement liquid for multiple times, centrifuging to remove supernatant, leaving precipitate, shaking the precipitate by a vibrator to obtain a heavy suspension, and finally uniformly mixing the heavy suspension and glycerol and storing in a refrigerator at the temperature of-70-90 ℃ for 10-15 months;
step two: preparing a freeze-drying protective solution: the freeze-drying protective solution comprises the following components in percentage by weight: 10 to 20 percent of skimmed milk powder, 1 to 5 percent of cane sugar, 1 to 3 percent of gelatin and the balance of sterile distilled water;
step three: freeze-drying by using a freeze-dryer, wherein the freeze-drying process and freeze-drying parameters are as follows:
s1, pre-freezing: the temperature is-35 ℃ to-45 ℃, and the pre-freezing time is 150-180 min;
s2, vacuumizing: the vacuum degree is 0.2, and the vacuumizing time is 20-30 min;
s3, sublimation: dividing into 9 gradients, increasing the temperature of each gradient by 5 ℃, increasing the temperature from-35 ℃ to 30 ℃, increasing the temperature between each gradient for 10min, and keeping the temperature of each temperature node constant for 30-120 min;
s4, sterilization: sterilizing with an ultraviolet lamp for 40-60 min.
2. The process of claim 1, wherein the sucrose is pure sucrose and the gelatin is 180 freezing strength gelatin.
3. The process of claim 1, wherein the freeze-drying process of step three is protected by introducing high-purity nitrogen throughout the freeze-drying process.
4. The method for preparing the freeze-drying protective solution for the freeze-drying of coprophila according to claim 1, wherein the method for preparing the freeze-drying protective solution comprises the following steps:
(1) weighing 18g of skimmed milk powder, 1g of sucrose and 3g of gelatin by using weighing paper, pouring into a conical flask, adding 60 ℃ distilled water, adding water and uniformly mixing to 100 ml;
(2) placing the uniformly mixed freeze-dried protective solution into a high-temperature high-pressure sterilization pot, and sterilizing for 20min at 121 ℃.
5. A preparation process of a coprophila fungus capsule for coprophila fungus transplantation treatment is characterized in that the process of the capsule preparation process comprises the following steps:
the method comprises the following steps: taking out the fecal bacteria liquid from a refrigerator at minus 80 ℃, unfreezing the fecal bacteria liquid in a water bath kettle at 37 ℃ for 20-30min, taking out the fecal bacteria liquid, putting the fecal bacteria liquid into a centrifugal machine, and centrifuging the fecal bacteria liquid at 5000r and 4 ℃ for 5 min;
step two: preparing a freeze-drying protective agent;
step three: preparing a culture medium;
step four: taking out the centrifuged bacterial liquid, adding 10ml of freeze-drying protective agent into each tube, uniformly mixing by using an oscillator, respectively reserving 1ml of sample by using two freezing tubes, and performing a bacterial counting experiment before freeze-drying by using one tube;
step five: and (4) sample retention: taking five freezing storage tubes with the serial numbers of 1, 2, 3, 4 and 5, adding 1ml of uniformly mixed bacteria liquid into each tube, and respectively performing a sample-reserving bacteria counting experiment on the first day, the first week, the first month, the third month and the first year;
step six: subpackaging: adding each tube of bacterial liquid into a penicillin bottle, simultaneously placing the penicillin bottle and five freezing tubes for sample preservation into a freeze dryer, setting the pre-freezing temperature to be-45 ℃, freeze-drying for 24 hours, and controlling the humidity below 40 after freeze-drying;
step seven: filling: pouring the freeze-dried powder onto a capsule filling plate, and filling a No. 0 capsule;
and (4) storing the filled capsules and the preserved freezing tubes in a refrigerator at the temperature of-20 ℃.
6. The process for preparing a fecal bacteria capsule for fecal bacteria transplantation therapy according to claim 5, wherein the formula components (g/L) of the culture medium are: 10g of peptone, 5g of beef powder, 4g of yeast powder, 20g of glucose, 801 ml of tween, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate and 15g of agar.
7. The process for preparing a fecal bacteria capsule for fecal bacteria transplantation therapy according to claim 5, wherein the preparation method of the culture medium comprises: dissolving 12.9g of MRS medium in 200ml of conical flask filled with distilled water, mixing uniformly while stirring, adjusting pH to 7.3 + -0.2, and sterilizing at 121 deg.C for 20min in autoclave.
8. The process of claim 5, wherein the fecal bacteria capsule is selected from enteric gelatin hollow capsules.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104922158A (en) * | 2015-06-05 | 2015-09-23 | 中国人民解放军第三军医大学第三附属医院 | Fecal microbiota capsule, as well as preparation method and application thereof |
| CN107574134A (en) * | 2017-10-20 | 2018-01-12 | 南京益恒寿生命科技有限公司 | A kind of caprophyl transplantation treatment Plays caprophyl lyophiled powder preparing technique |
| CN109679877A (en) * | 2019-01-17 | 2019-04-26 | 广州承葛生物科技有限公司 | A kind of preparation method of high activity caprophyl capsule |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104922158A (en) * | 2015-06-05 | 2015-09-23 | 中国人民解放军第三军医大学第三附属医院 | Fecal microbiota capsule, as well as preparation method and application thereof |
| CN107574134A (en) * | 2017-10-20 | 2018-01-12 | 南京益恒寿生命科技有限公司 | A kind of caprophyl transplantation treatment Plays caprophyl lyophiled powder preparing technique |
| CN109679877A (en) * | 2019-01-17 | 2019-04-26 | 广州承葛生物科技有限公司 | A kind of preparation method of high activity caprophyl capsule |
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