CN111888381A - Intestinal microorganism transplanting process - Google Patents
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- CN111888381A CN111888381A CN202010984720.1A CN202010984720A CN111888381A CN 111888381 A CN111888381 A CN 111888381A CN 202010984720 A CN202010984720 A CN 202010984720A CN 111888381 A CN111888381 A CN 111888381A
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Abstract
The invention discloses an intestinal microorganism transplanting process, which comprises the following steps: 1) collecting a fecal sample; 2) extracting intestinal microorganisms; 3) preparing an intestinal microbial preparation, including an intestinal microbial enema, an intestinal microbial capsule and an intestinal microbial freeze-dried capsule; 4) transplantation of intestinal microorganisms: transplanting the intestinal microbial preparation obtained in the step 3) by means of enema or oral administration. Can be used for treating intestinal diseases caused by intestinal microorganism disorder, and immune system and nervous system diseases caused by intestinal microorganism disorder.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of microbial medicines, in particular to an intestinal microorganism transplanting process.
[ background of the invention ]
At present, the living environment of people is cleaner and more exquisite in food package, and the requirement of sanitation is met, namely, standard bacteria are absolutely not generated. Modern research, however, has shown that there are fewer and fewer beneficial microorganisms in the environment and fewer beneficial microorganisms in the human body, whereas there are more and more "harmful" microorganisms in the environment and in the body. Modern scientific research shows that intestinal flora plays a vital role in human health. The intestinal microorganisms of the human body maintain a dynamic equilibrium state with the internal environment of the intestines, and in some cases, the equilibrium is broken, the intestinal microorganisms are out of order, diseases are caused or the condition is aggravated, and complications and even multiple organ dysfunction syndrome and multiple organ failure occur. If a person has a weight of 60kg and the number of microorganisms in the person is about 2kg, the microorganisms may not be "floating population" that is accompanied by the person, but "permanent population" that is resident, lives and flourishes, labours and functions in the body for a long time.
Scientists have found that human microorganisms are capable of producing vitamins, amino acids, and energy for our use; assisting the immune system to chase away invading pathogens; can also regulate and control the emotion and desire of people. If the microorganism in the human body is eliminated, the human body has short life and poor life. That is, we cannot imagine the existence of a human in the world that is not compatible with the microorganism. From a genetic perspective, we can see how true we are in the same place as microorganisms: we, including other large animals, do not have a so-called "individual", each of which is a "super organism" that is made up of numerous microorganisms.
Intestinal microorganism transplantation, also called Fecal microbiota transplantation (FMT for short), is a technology for separating microorganisms in feces of healthy people, transplanting the microorganisms into intestinal tracts of patients in the forms of enteroscopy, enema, oral administration and the like, reconstructing intestinal microorganisms of the patients and realizing treatment of intestinal and parenteral diseases. Human and human microbiota are a symbiotic relationship. The treatment has been done by human microorganisms internationally, and according to reports of natural journal, scientific journal and the like, many hospitals and scientific research institutions at home and abroad develop flora transplantation FMT treatment research, which is mainly applied to four aspects: 1. intestinal tract aspect: recurrent clostridium difficile infection, slow transit constipation, chronic diarrhea, inflammatory bowel disease, radiation enteritis, irritable bowel syndrome, ulcerative colitis, crohn's disease; 2. child 4A disease: allergies, asthma, hyperactivity and autism; 3. clinical application research of metabolic syndromes such as obesity and type II diabetes; 4. clinical application research of cancer. Furthermore, some unexpected findings when receiving clinical studies of FMT patients: the skin becomes better, and even skin diseases which are difficult to treat by some people are better; the abdominal pain is better and even the sexual function of some people is improved.
Currently, there is research on intestinal microbial transplantation, for example, chinese patent application CN201810612443, a research method of intestinal flora based on stool filtration and transplantation, discloses a research method of interaction between a drug based on stool filtration and transplantation and intestinal flora, and the method includes the following steps: 1. preparation of fecal bacteria transplant recipient experimental animals: the experimental animal of the fecal strain transplant recipient selects a pseudo-aseptic animal or an aseptic animal to form an environment with aseptic or low-bacteria intestinal tract, which is beneficial to the field planting of external microorganisms; 2. preparing a donor experimental animal for coprophilous fungus transplantation, wherein the donor animal is subjected to disease model molding and drug gavage treatment, and positive and negative experimental controls of drug effect are set; 3. the recipient animal is transplanted with filtered and non-filtered fecal bacteria from the donor animal. However, the research is limited to animal experiments at present, and how the effect of human experiments is, the judgment is not easy. For another example, chinese patent application CN201810866487X provides a method for localized transplantation of fecal fungi, which comprises preparing a capsule containing fecal fungi as a main component, and orally administering the capsule, wherein the dissolution pH of the capsule shell is adapted to the pH of the target site for fecal fungi transplantation, so that the capsule is dissolved in the target site for transplantation, and the fecal fungi are implanted in the target site in a localized manner. But each fecal bacteria capsule contains 2.88 multiplied by 1010The number of active bacteria is small.
In summary, the microbial transplantation therapy at home and abroad has some defects:
1) at present, domestic and foreign flora libraries are all prepared into liquid, although the preparation is simple, an enteroscope is required to be used during transplantation, the enteroscope is inserted from the anus under the anesthesia condition, and then goes from the descending colon to the transverse colon to the ascending colon, finally reaches the cecum, and then the bacterium liquid is spread on the whole cecum while being withdrawn, the whole operation is firstly the anesthesia risk, and the possible accidents of the anesthesia can occur. Secondly, enteroscopy can cause physical damage to the intestine.
2) The transplantation of the bacteria liquid is very inconvenient to use and needs hospitalization for transplantation, thus greatly increasing the medical cost.
3) Currently, the intestinal flora donated by a voluntary donor is used at home and abroad, and the flora compatibility among mothers, children, brothers and sisters is the best in families, so that the intestinal flora of the voluntary donor is used intensely and probably not a good transplantation source.
Therefore, it is a technical problem to be solved to develop an intestinal microorganism transplant product with long shelf life and high intestinal microorganism number.
[ summary of the invention ]
Aiming at the defects of short shelf life and low quantity of intestinal microorganisms of an intestinal microorganism transplanting product in the prior art, the invention aims to provide an intestinal microorganism transplanting process, wherein excrement donated by healthy qualified volunteers is filtered, precipitated and concentrated to prepare liquid and capsules (freeze-dried) of high-concentration intestinal flora, and the liquid and capsules are used for treating intestinal diseases caused by intestinal microorganism imbalance and immune system and nervous system diseases caused by intestinal microorganism imbalance.
The intestinal microorganism transplanting process comprises the following steps:
1) collecting a fecal sample:
selecting a feces sample donated by a qualified donor, packaging the feces sample with a disposable food box and a plastic bag, sending the feces sample to a laboratory within one hour, and weighing the feces sample;
secondly, according to the following steps of 1: (3-7) adding normal saline according to the weight ratio, homogenizing, filtering with a filter of 0.2-5.0 μm, and removing residues;
thirdly, detecting the filtrate to obtain qualified filtrate;
2) extraction of intestinal microorganisms:
putting the qualified filtrate into a centrifuge tube, centrifuging at 1000-;
3) the preparation of the intestinal microbial preparation comprises intestinal microbial enema, intestinal microbial capsules and intestinal microbial freeze-dried capsules:
intestinal microorganism enema: mixing the precipitate obtained in the step 2), the freezing storage agent and the normal saline according to the volume ratio of 1:1:50, filling the mixture into a bottle, and storing the bottle in a freezer at the temperature of-80 ℃ for later use;
② intestinal microbial capsules: mixing the precipitate obtained finally in the step 2), the freezing preservative and normal saline according to the volume ratio of 1:1:1, injecting the mixed solution into an enteric capsule by using a liquid transfer device, packaging, filling the intestinal microbial capsule into a medicine bottle, firstly putting 1/4-1/3 volume of dry ice into the medicine bottle, freezing, then putting a deoxidation drying protective agent, and storing in a freezer at-80 ℃ for later use;
or placing the intestinal microbial capsule on a quick-freezing plate, freezing, filling the capsule into a medicine bottle, adding a deoxidation drying protective agent, and storing in a freezer at-80 deg.C for use; the quick-freezing plate is made of heat-conducting high-density aviation alloy with the surface treated by nano silver ions, is placed in a freezer at the temperature of minus 80 ℃ when being flat, is taken out when being used, and is padded with an ice bag below the freezer;
③ enteric microorganism freeze-drying capsules: mixing the precipitate obtained in the step 2), the freezing storage agent and normal saline according to the volume ratio of 1:1:1, placing the mixture into a freeze dryer for freeze drying, filling the intestinal microorganism freeze-dried powder into enteric capsules, packaging, filling the intestinal microorganism freeze-dried capsules into medicine bottles, adding a deoxidation drying protective agent, and placing the medicine bottles in a freezer at the temperature of 80 ℃ below zero for storage;
the cryopreservation agent is obtained by uniformly mixing glycerol, skimmed milk and acetylated distarch phosphate according to the proportion of (9-11) mL, (88-92) mL, (0.01-0.02) g;
4) transplantation of intestinal microorganisms: transplanting the intestinal microbial preparation obtained in the step 3) by means of enema or oral administration.
The qualified donor in the step 1) of the invention is selected to be 18-30 years old, and has no bad taste, no smoking, no alcohol taste, regular diet, regular sleep, good sleep quality and no insomnia; the health volunteers with BMI (body mass index) less than 24 and more than 20, no medical history in half a year, no gastrointestinal symptoms such as diarrhea and constipation in half a year have blood drawing to detect anti-HIV, HBsAg, HBV and syphilis every half a year, or have blood donation to a blood station once for a half year in an uncompensated way, and the blood donation report of the blood station is submitted to a laboratory for archiving; stool was tested monthly for clostridium difficile, salmonella, shigella and parasites and once monthly for calprotectin.
In the laboratory in the step 1), the using rooms are independent units, so that virulent strains or other viruses and pathogenic bacteria cannot be carried or operated, and a production area is disinfected before production; workers must be trained professionally and have good health, and take hepatitis B vaccines, and perform physical examination before production every year, so that patients with infectious hepatitis, dysentery and active tuberculosis cannot enter a laboratory production area, and cannot participate in production activities during diarrhea.
The homogenization in the step 1) refers to medium-speed homogenization for 5-10 minutes.
The qualified filtrate in the step 1) refers to filtrate without obvious precipitation and abnormal color.
The filling amount of the intestinal microorganism enema liquid in the step 1) is 200mL per bottle.
The cryopreservation agent in the step 3) is obtained by uniformly mixing glycerol, skimmed milk and acetylated distarch phosphate according to the proportion of 10mL to 90mL to 0.01 g.
Compared with the prior art, the invention has the following advantages:
1. the acetylated distarch phosphate added into the cryopreservation agent belongs to a variable starch substance, is safe and edible, and can form a compact protective film on the surface of intestinal microorganisms by combining the acetylated distarch phosphate with glycerol and skim milk, so that the intestinal microorganisms are protected; meanwhile, the molecular structure of the acetylated distarch phosphate has hydrophilic groups and hydrophobic groups, so that the acetylated distarch phosphate presents certain properties of a surfactant, micelles can be formed when the acetylated distarch phosphate meets an aqueous solution, the micelles and polymer particles are associated to form a net structure, and one molecule is provided with a plurality of micelles, so that the mobility of water molecules is reduced, the number of viable bacteria of intestinal microbial flora is effectively kept, and the storage life of the intestinal microbial preparation is further prolonged.
2. In the preparation engineering of the intestinal microorganism capsule in the step 3), after the last centrifugation, the intestinal microorganisms are directly exposed in the air, at this time, the wet bacteria after the precipitation, the freezing agent and the normal saline are mixed and are quickly filled into capsules, or rapidly freezing at low temperature to prepare into lyophilized powder, but in the process of producing wet bacteria capsule, particularly, when the capsule is filled into a capsule shell, the time is long, generally about 30 minutes, wet bacteria are fully exposed in the air, in the intestinal microorganisms at this time, aerobic bacteria and facultative aerobic bacteria can survive well and can grow even further, but more than 80% of anaerobic bacteria are very easy to die when exposed to an aerobic environment, for the human body, it is precisely the aerobic bacteria that are mostly harmful bacteria, while the anaerobic bacteria are mostly beneficial bacteria. Therefore, it is desirable to control aerobic bacteria to prevent overgrowth, while anaerobic bacteria should survive as much as possible. Therefore, the wet bacteria are put into the closed container as soon as possible by the method adopted by the invention, and are quickly frozen by the following method: firstly, a wide-mouth heat preservation tank is used for containing food-grade dry ice (solid carbon dioxide) inside, the temperature in the tank can reach about-55 ℃, meanwhile, the solid carbon dioxide in the tank absorbs heat and gasifies, the inner space is filled with the carbon dioxide, the carbon dioxide is heavier than air and can be deposited on the surface of the dry ice, after wet bacteria in a closed container are injected into a capsule, the capsule is rapidly placed into the dry ice tank, under the condition of-80 ℃, the capsule can be frozen within 5-10 minutes, and meanwhile, because the tank is filled with the carbon dioxide, oxygen in the air is isolated, so that anaerobic bacteria are protected; secondly, if dry ice is lacked, a self-made quick-freezing plate is used to achieve the effect of quick freezing, the surface of the quick-freezing plate is made of heat-conducting high-density aviation alloy with nano silver ion surface treatment, the quick-freezing plate is placed in a freezer at the temperature of minus 80 ℃ when being flat, the quick-freezing plate is taken out when being used, an ice bag is padded below the quick-freezing plate, the filled capsule is placed on the quick-freezing plate, and the capsule can be completely frozen after 8-10 minutes of the applicant tests on the quick-freezing plate. If the first or the second is not adopted, the intestinal microbial capsule can be completely frozen within about 20 minutes, and the number of viable bacteria in the capsule can be seriously influenced.
[ detailed description ] embodiments
The following examples are provided to further illustrate the embodiments of the present invention.
Example 1:
an intestinal microorganism transplanting process, which comprises the following steps:
1) collecting a fecal sample:
selecting a feces sample donated by a qualified donor, packaging the feces sample with a disposable food box and a plastic bag, sending the feces sample to a laboratory within one hour, and weighing the feces sample;
secondly, according to the following steps of 1: 3, adding normal saline, homogenizing at medium speed for 5 minutes, filtering at 0.2 mu m, and removing residues;
thirdly, detecting the filtrate to obtain qualified filtrate without obvious precipitation and abnormal color;
2) extraction of intestinal microorganisms:
putting the filtrate into a centrifuge tube, centrifuging at 1000rpm for 20 minutes to remove the precipitate, putting the supernatant into a new centrifuge tube, centrifuging at 3000rpm for 10 minutes, discarding the supernatant, washing the precipitate with normal saline, centrifuging at 3000rpm again for 10 minutes, repeating the steps for three times, filtering the finally obtained liquid with a 60-micron filter membrane to remove possible parasitic ova to obtain a precipitate for later use;
3) the preparation of the intestinal microbial preparation comprises intestinal microbial enema, intestinal microbial capsules and intestinal microbial freeze-dried capsules:
intestinal microorganism enema: mixing the precipitate obtained in the step 2), the freezing storage agent and the normal saline according to the volume ratio of 1:1:50, filling the mixture into bottles, wherein the filling amount of each bottle is 200mL, and storing the bottles in a freezer at the temperature of-80 ℃ for later use;
② intestinal microbial capsules: mixing the precipitate, the freezing preservative and the normal saline obtained in the step 2) according to the volume ratio of 1:1:1, injecting the mixed solution into an enteric-coated capsule (zero-size capsule) by using a liquid transfer machine, packaging, filling the intestinal microbial capsule into a medicine bottle, firstly putting 1/4 volumes of dry ice into the medicine bottle, freezing, then putting a deoxidation drying protective agent, and storing in a freezer at the temperature of-80 ℃ for later use;
or placing the intestinal microbial capsule on a quick-freezing plate, freezing, filling the capsule into a medicine bottle, adding a deoxidation drying protective agent, and storing in a freezer at-80 deg.C for use; the quick-freezing plate is made of heat-conducting high-density aviation alloy with the surface treated by nano silver ions, is placed in a freezer at the temperature of minus 80 ℃ when being flat, is taken out when being used, and is padded with an ice bag below the freezer;
③ enteric microorganism freeze-drying capsules: mixing the precipitate obtained in the step 2), the freezing storage agent and normal saline according to the volume ratio of 1:1:1, placing the mixture into a freeze dryer for freeze drying, filling the intestinal microorganism freeze-dried powder into enteric capsules (zero capsules), packaging, filling the intestinal microorganism freeze-dried capsules into a medicine bottle, adding a deoxidation drying protective agent, and placing the medicine bottle in a freezer at the temperature of-80 ℃ for later use;
the cryopreservation agent is obtained by uniformly mixing glycerol, skimmed milk and acetylated distarch phosphate according to the proportion of 10mL to 90mL to 0.01 g;
4) transplantation of intestinal microorganisms: transplanting the intestinal microbial preparation obtained in the step 3) by means of enema or oral administration;
the qualified donor in the step 1) is selected to be 18-30 years old, and has no bad taste, no smoking, no alcohol taste, regular diet, regular sleep, good sleep quality and no insomnia; the health volunteers with BMI (body mass index) less than 24 and more than 20, no medical history in half a year, no gastrointestinal symptoms such as diarrhea and constipation in half a year have blood drawing to detect anti-HIV, HBsAg, HBV and syphilis every half a year, or have blood donation to a blood station once for a half year in an uncompensated way, and the blood donation report of the blood station is submitted to a laboratory for archiving; detecting clostridium difficile, salmonella, shigella and parasites in the stool once a month, and detecting calprotectin in the stool once a month;
in the laboratory in the step 1), the using rooms are independent units, so that virulent strains or other viruses and pathogenic bacteria cannot be carried or operated, and a production area is disinfected before production; workers must be trained professionally and have good health, and take hepatitis B vaccines, and perform physical examination before production every year, so that patients with infectious hepatitis, dysentery and active tuberculosis cannot enter a laboratory production area, and cannot participate in production activities during diarrhea.
Example 2:
an intestinal microorganism transplanting process, which comprises the following steps:
1) collecting a fecal sample:
selecting a feces sample donated by a qualified donor, packaging the feces sample with a disposable food box and a plastic bag, sending the feces sample to a laboratory within one hour, and weighing the feces sample;
secondly, according to the following steps of 1: 7, adding normal saline, homogenizing at medium speed for 8 minutes, filtering at 5.0 mu m, and removing residues;
thirdly, detecting the filtrate to obtain qualified filtrate without obvious precipitation and abnormal color;
2) extraction of intestinal microorganisms:
putting the filtrate into a centrifuge tube, centrifuging at 2000rpm for 10 minutes to remove the precipitate, putting the supernatant into a new centrifuge tube, centrifuging at 4000rpm for 5 minutes, discarding the supernatant, washing the precipitate with normal saline, centrifuging at 4000rpm for 5 minutes again, repeating the steps for three times, and finally filtering the obtained liquid with a filter membrane of 40 mu m to remove possible parasitic ova to obtain a precipitate for later use;
3) the preparation of the intestinal microbial preparation comprises intestinal microbial enema, intestinal microbial capsules and intestinal microbial freeze-dried capsules:
intestinal microorganism enema: mixing the precipitate obtained in the step 2), the freezing storage agent and the normal saline according to the volume ratio of 1:1:50, filling the mixture into bottles, wherein the filling amount of each bottle is 200mL, and storing the bottles in a freezer at the temperature of-80 ℃ for later use;
② intestinal microbial capsules: mixing the precipitate, the freezing preservative and the normal saline obtained in the step 2) according to the volume ratio of 1:1:1, injecting the mixed solution into an enteric-coated capsule (zero-size capsule) by using a liquid transfer machine, packaging, filling the intestinal microbial capsule into a medicine bottle, firstly putting 1/3 volumes of dry ice into the medicine bottle, freezing, then putting a deoxidation drying protective agent, and storing in a freezer at the temperature of-80 ℃ for later use;
or placing the intestinal microbial capsule on a quick-freezing plate, freezing, filling the capsule into a medicine bottle, adding a deoxidation drying protective agent, and storing in a freezer at-80 deg.C for use; the quick-freezing plate is made of heat-conducting high-density aviation alloy with the surface treated by nano silver ions, is placed in a freezer at the temperature of minus 80 ℃ when being flat, is taken out when being used, and is padded with an ice bag below the freezer;
③ enteric microorganism freeze-drying capsules: mixing the precipitate obtained in the step 2), the freezing storage agent and normal saline according to the volume ratio of 1:1:1, placing the mixture into a freeze dryer for freeze drying, filling the intestinal microorganism freeze-dried powder into enteric capsules (zero capsules), packaging, filling the intestinal microorganism freeze-dried capsules into a medicine bottle, adding a deoxidation drying protective agent, and placing the medicine bottle in a freezer at the temperature of-80 ℃ for later use;
the cryopreservation agent is obtained by uniformly mixing glycerol, skimmed milk and acetylated distarch phosphate according to the proportion of 11mL to 88mL to 0.01 g;
4) transplantation of intestinal microorganisms: transplanting the intestinal microbial preparation obtained in the step 3) by means of enema or oral administration;
the qualified donor in the step 1) is selected to be 18-30 years old, and has no bad taste, no smoking, no alcohol taste, regular diet, regular sleep, good sleep quality and no insomnia; the health volunteers with BMI (body mass index) less than 24 and more than 20, no medical history in half a year, no gastrointestinal symptoms such as diarrhea and constipation in half a year have blood drawing to detect anti-HIV, HBsAg, HBV and syphilis every half a year, or have blood donation to a blood station once for a half year in an uncompensated way, and the blood donation report of the blood station is submitted to a laboratory for archiving; detecting clostridium difficile, salmonella, shigella and parasites in the stool once a month, and detecting calprotectin in the stool once a month;
in the laboratory in the step 1), the using rooms are independent units, so that virulent strains or other viruses and pathogenic bacteria cannot be carried or operated, and a production area is disinfected before production; workers must be trained professionally and have good health, and take hepatitis B vaccines, and perform physical examination before production every year, so that patients with infectious hepatitis, dysentery and active tuberculosis cannot enter a laboratory production area, and cannot participate in production activities during diarrhea.
Example 3:
an intestinal microorganism transplanting process, which comprises the following steps:
1) collecting a fecal sample:
selecting a feces sample donated by a qualified donor, packaging the feces sample with a disposable food box and a plastic bag, sending the feces sample to a laboratory within one hour, and weighing the feces sample;
secondly, according to the following steps of 1:5, adding normal saline, homogenizing at medium speed for 10 minutes, filtering at 3.0 mu m, and removing residues;
thirdly, detecting the filtrate to obtain qualified filtrate without obvious precipitation and abnormal color;
2) extraction of intestinal microorganisms:
putting the qualified filtrate into a centrifuge tube, centrifuging at 1500rpm for 15 minutes to remove the precipitate, putting the supernatant into a new centrifuge tube, centrifuging at 3500rpm for 8 minutes, discarding the supernatant, washing the precipitate with normal saline, centrifuging at 3500rpm for 8 minutes again, repeating the steps for three times, and finally filtering the obtained liquid with a 20-micron filter membrane to remove possible parasitic ova to obtain a precipitate for later use;
3) the preparation of the intestinal microbial preparation comprises intestinal microbial enema, intestinal microbial capsules and intestinal microbial freeze-dried capsules:
intestinal microorganism enema: mixing the precipitate obtained in the step 2), the freezing storage agent and the normal saline according to the volume ratio of 1:1:50, filling the mixture into bottles, wherein the filling amount of each bottle is 200mL, and storing the bottles in a freezer at the temperature of-80 ℃ for later use;
② intestinal microbial capsules: mixing the precipitate, the freezing preservative and the normal saline obtained in the step 2) according to the volume ratio of 1:1:1, injecting the mixed solution into an enteric-coated capsule (zero-size capsule) by using a liquid transfer machine, packaging, filling the intestinal microbial capsule into a medicine bottle, firstly putting 1/4 volumes of dry ice into the medicine bottle, freezing, then putting a deoxidation drying protective agent, and storing in a freezer at the temperature of-80 ℃ for later use;
or placing the intestinal microbial capsule on a quick-freezing plate, freezing, filling the capsule into a medicine bottle, adding a deoxidation drying protective agent, and storing in a freezer at-80 deg.C for use; the quick-freezing plate is made of heat-conducting high-density aviation alloy with the surface treated by nano silver ions, is placed in a freezer at the temperature of minus 80 ℃ when being flat, is taken out when being used, and is padded with an ice bag below the freezer;
③ enteric microorganism freeze-drying capsules: mixing the precipitate obtained in the step 2), the freezing storage agent and normal saline according to the volume ratio of 1:1:1, placing the mixture into a freeze dryer for freeze drying, filling the intestinal microorganism freeze-dried powder into enteric capsules (zero capsules), packaging, filling the intestinal microorganism freeze-dried capsules into a medicine bottle, adding a deoxidation drying protective agent, and placing the medicine bottle in a freezer at the temperature of-80 ℃ for later use;
the cryopreservation agent is obtained by uniformly mixing glycerol, skimmed milk and acetylated distarch phosphate according to the proportion of 9mL to 92mL to 0.02 g;
4) transplantation of intestinal microorganisms: transplanting the intestinal microbial preparation obtained in the step 3) by means of enema or oral administration;
the qualified donor in the step 1) is selected to be 18-30 years old, and has no bad taste, no smoking, no alcohol taste, regular diet, regular sleep, good sleep quality and no insomnia; the health volunteers with BMI (body mass index) less than 24 and more than 20, no medical history in half a year, no gastrointestinal symptoms such as diarrhea and constipation in half a year have blood drawing to detect anti-HIV, HBsAg, HBV and syphilis every half a year, or have blood donation to a blood station once for a half year in an uncompensated way, and the blood donation report of the blood station is submitted to a laboratory for archiving; detecting clostridium difficile, salmonella, shigella and parasites in the stool once a month, and detecting calprotectin in the stool once a month;
in the laboratory in the step 1), the using rooms are independent units, so that virulent strains or other viruses and pathogenic bacteria cannot be carried or operated, and a production area is disinfected before production; workers must be trained professionally and have good health, and take hepatitis B vaccines, and perform physical examination before production every year, so that patients with infectious hepatitis, dysentery and active tuberculosis cannot enter a laboratory production area, and cannot participate in production activities during diarrhea.
Comparative example 1:
in contrast to example 1, no acetylated distarch phosphate was added to the cryopreservation agent, otherwise the same as example 1.
Comparative example 2:
compared with the example 1, the step 3) of preparing the intestinal microbial capsules is the same as the step 1 except that 1/4 volumes of dry ice are not put into the medicine bottles.
Comparative example 3:
compared with the embodiment 1, in the step 3), the intestinal microbial capsules are directly filled into medicine bottles without being placed on a quick-freezing plate in advance in the preparation process of the intestinal microbial capsules, and the rest is the same as the embodiment 1.
Experimental example: detection of the Activity of bacteria after cryopreservation of an intestinal microbial preparation
1. The detection method comprises the following steps: reference to the national Standard for Total bacteria (GB4789.2-2016) for implementation
Table 1: the microbial numbers of the intestinal microbial preparations obtained in examples and comparative examples were measured
2. And (4) analyzing results:
1) the microbial numbers of the intestinal microbial preparations of the examples were measured according to the national bacteria population test standard (GB4789.2-2016), and were not different before and after cryopreservation.
2) Experiments show that the cryopreservation agent of the comparative example is not added with acetylated distarch phosphate, compared with the examples, the reduction of the microbial number in the intestinal microorganism enema liquid is obvious, the reduction of the microbial number in the intestinal microorganism capsule is obvious, and the reduction of the microbial number in the intestinal microorganism freeze-dried capsule is obvious.
3) The cryopreservation agent of the comparative example 1 is not added with acetylated distarch phosphate, and after 12 months of storage, compared with the cryopreservation agent before 12 months, the number of microorganisms in the intestinal microorganism enema liquid is obviously reduced, the number of microorganisms in the intestinal microorganism capsule is obviously reduced, and the number of microorganisms in the intestinal microorganism freeze-dried capsule is obviously reduced.
4) Comparative examples 2 and 3 show that dry ice or a quick-freezing prevention plate is placed in a medicine bottle in advance during the production of the wet bacteria capsule, so that the capsule can be frozen quickly and the viable count in the capsule can be effectively maintained.
Experiments and comparisons show that the acetylated distarch phosphate added into the cryopreservation agent can form a compact protective film on the surface of the intestinal microorganisms by combining the acetylated distarch phosphate with glycerol and skimmed milk, so that the intestinal microorganisms are protected; meanwhile, the molecular structure of the acetylated distarch phosphate has hydrophilic groups and hydrophobic groups, so that the acetylated distarch phosphate presents certain properties of a surfactant, micelles can be formed when the acetylated distarch phosphate meets an aqueous solution, the micelles and polymer particles are associated to form a net structure, and one molecule is provided with a plurality of micelles, so that the mobility of water molecules is reduced, the number of viable bacteria of intestinal microbial flora is effectively kept, and the storage life of the intestinal microbial preparation is further prolonged.
3. The detection method comprises the following steps:
1) at present, the nucleic acid high-throughput sequencing of the intestinal microorganisms detects about 1000 bacteria (the minimum classification of the bacteria) of the intestinal microorganisms, and because of the nucleic acid detection, the bacteria are difficult to judge whether the bacteria are live bacteria or dead bacteria, and because the nucleic acid of the dead bacteria exists in a short time;
2) about 1000 kinds of bacteria can be separated and cultured at present to obtain about 10% of bacteria, such as common escherichia coli, shigella, salmonella, campylobacter jejuni and the like;
3) in about 10% of bacteria capable of isolated culture, aerobic bacteria can be cultured, while anaerobic bacteria can be cultured under anaerobic conditions, and normal culture cannot be performed, such as Campylobacter jejuni.
At present, about 5% of aerobic viable bacteria in the intestinal flora can be detected according to a detection method of the total number of bacteria (GB4789.2-2016), and the overall viable bacteria condition of the intestinal bacteria cannot be reflected, but the activity indexes of the bacteria after the intestinal microbial preparation is frozen can be compared by adopting the same experimental method for the embodiment and the comparative example.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (7)
1. An intestinal microorganism transplanting process, which is characterized in that: the method comprises the following steps:
1) collecting a fecal sample:
selecting a feces sample donated by a qualified donor, packaging the feces sample with a disposable food box and a plastic bag, sending the feces sample to a laboratory within one hour, and weighing the feces sample;
secondly, according to the following steps of 1: (3-7) adding normal saline according to the weight ratio, homogenizing, filtering with a filter of 0.2-5.0 μm, and removing residues;
thirdly, detecting the filtrate to obtain qualified filtrate;
2) extraction of intestinal microorganisms:
putting the qualified filtrate into a centrifuge tube, centrifuging at 1000-;
3) the preparation of the intestinal microbial preparation comprises intestinal microbial enema, intestinal microbial capsules and intestinal microbial freeze-dried capsules:
intestinal microorganism enema: mixing the precipitate obtained in the step 2), the freezing storage agent and the normal saline according to the volume ratio of 1:1:50, filling the mixture into a bottle, and storing the bottle in a freezer at the temperature of-80 ℃ for later use;
② intestinal microbial capsules: mixing the precipitate obtained finally in the step 2), the freezing preservative and normal saline according to the volume ratio of 1:1:1, injecting the mixed solution into an enteric capsule by using a liquid transfer device, packaging, filling the intestinal microbial capsule into a medicine bottle, firstly putting 1/4-1/3 volume of dry ice into the medicine bottle, freezing, then putting a deoxidation drying protective agent, and storing in a freezer at-80 ℃ for later use;
or placing the intestinal microbial capsule on a quick-freezing plate, freezing, filling the capsule into a medicine bottle, adding a deoxidation drying protective agent, and storing in a freezer at-80 deg.C for use; the quick-freezing plate is made of heat-conducting high-density aviation alloy with the surface treated by nano silver ions, is placed in a freezer at the temperature of minus 80 ℃ when being flat, is taken out when being used, and is padded with an ice bag below the freezer;
③ enteric microorganism freeze-drying capsules: mixing the precipitate obtained in the step 2), the freezing storage agent and normal saline according to the volume ratio of 1:1:1, placing the mixture into a freeze dryer for freeze drying, filling the intestinal microorganism freeze-dried powder into enteric capsules, packaging, filling the intestinal microorganism freeze-dried capsules into medicine bottles, adding a deoxidation drying protective agent, and placing the medicine bottles in a freezer at the temperature of 80 ℃ below zero for storage;
the cryopreservation agent is obtained by uniformly mixing glycerol, skimmed milk and acetylated distarch phosphate according to the proportion of (9-11) mL, (88-92) mL, (0.01-0.02) g;
4) transplantation of intestinal microorganisms: transplanting the intestinal microbial preparation obtained in the step 3) by means of enema or oral administration.
2. The intestinal microorganism transplantation process of claim 1, wherein: the qualified donor in the step 1) is selected to be 18-30 years old, and has no bad taste, no smoking, no alcohol taste, regular diet, regular sleep, good sleep quality and no insomnia; the health volunteers are well-balanced, the BMI value is less than 24 and more than 20, no medicine history is used in half a year, no gastrointestinal symptoms such as diarrhea and constipation exist in half a year, blood is drawn every half a year to detect anti-HIV, HBsAg, HBV and syphilis, or blood is donated to a blood station for a long time in a half a year in an uncompensated way, and a blood donation report of the blood station is submitted to a laboratory for archiving; stool was tested monthly for clostridium difficile, salmonella, shigella and parasites and once monthly for calprotectin.
3. The intestinal microorganism transplantation process of claim 1, wherein: in the laboratory in the step 1), the using rooms are independent units, so that virulent strains or other viruses and pathogenic bacteria cannot be carried or operated, and a production area is disinfected before production; workers must be trained professionally and have good health, and take hepatitis B vaccines, and perform physical examination before production every year, so that patients with infectious hepatitis, dysentery and active tuberculosis cannot enter a laboratory production area, and cannot participate in production activities during diarrhea.
4. The intestinal microorganism transplantation process of claim 1, wherein: the homogenization in the step 1) refers to medium-speed homogenization for 5-10 minutes.
5. The intestinal microorganism transplantation process of claim 1, wherein: the qualified filtrate in the step 1) refers to filtrate without obvious precipitation and abnormal color.
6. The intestinal microorganism transplantation process of claim 1, wherein: the filling amount of the intestinal microorganism enema liquid in the step 1) is 200mL per bottle.
7. The intestinal microorganism transplantation process of claim 1, wherein: the cryopreservation agent in the step 3) is obtained by uniformly mixing glycerol, skimmed milk and acetylated distarch phosphate according to the proportion of 10mL to 90mL to 0.01 g.
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| CN112458019A (en) * | 2020-12-02 | 2021-03-09 | 广东南芯医疗科技有限公司 | Fecal bacteria liquid and preparation method and application of freeze-dried powder of fecal bacteria liquid |
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