CN112094735B - Simulated digestion system device and method for detecting microorganisms in medicine - Google Patents
Simulated digestion system device and method for detecting microorganisms in medicine Download PDFInfo
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/58—Reaction vessels connected in series or in parallel
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/16—Vibrating; Shaking; Tilting
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
- C12M37/04—Seals
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
- C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
Abstract
The invention discloses a simulated digestion system device and a simulated digestion system method for detecting microorganisms in medicines, wherein the simulated digestion system device comprises a first reaction tank and a second reaction tank which are mutually communicated, the two reaction tanks are communicated through a connecting pipe, a one-way valve is arranged between the first reaction tank and the connecting pipe, a liquid feeding sampling port is arranged on the side surface of each reaction tank, a filtering valve is arranged on the side surface of the bottom of the first reaction tank, and a heating oscillation box is arranged outside each reaction tank. Every structure of the device all has a inspection hole that can extract the sample, can detect the microorganism situation in the different device positions at any time, realizes the real-time supervision of the sample that awaits measuring, need not plus known bacterium to verify whether the sample has the antibacterial nature, and the accessible in-process discharges unnecessary reaction liquid and gets rid of the operation of plus microorganism to can simplify the experimentation, reduce experimental operation step.
Description
Technical Field
The invention relates to the field of drug and food inspection and detection, in particular to a simulated digestive system device and a method for inspecting microorganisms in drugs.
Background
The current examination of microorganisms in drugs in the "chinese pharmacopoeia" is based on examination of the limits of microorganisms in drugs, i.e. to examine whether the total number of colonies of microorganisms in a certain amount of drug exceeds the limit standards stipulated by the pharmacopoeia. The examination method comprises dissolving or diluting a certain amount of drug to be measured with a certain amount of buffer solution, placing in a culture dish, pouring corresponding culture medium, culturing for a period of time, and counting the number of microorganisms on the dish. This method only allows to check the number of microorganisms that may be contaminated in the drug, and cannot detect microorganisms that remain alive and harmful to the human body after the environmental effects of the digestive system in vivo. In the actual medical treatment process, most of the medicines are disintegrated and dispersed in the stomach of a human body and digested and absorbed in the small intestine, so that the growth state of microorganisms and the pathogenic effect of the microorganisms can be changed under the in-vivo environmental conditions, particularly under the influence of gastric acid and intestinal juice. On the other hand, if the total number of microorganisms in the drug does not exceed the standard range specified in the pharmacopeia, the existing testing method considers that the microorganism testing result of the drug is in accordance with the specification even if the microorganisms contained in the drug can cause diseases of human bodies, because the existing method cannot examine the in vivo process of the microorganisms in the drug.
The main digestive system of the human body, which affects absorption and utilization of drugs, is mainly composed of the stomach and small intestine, and a very small part of the drugs are absorbed by the large intestine. Although the process of human body's digestion and absorption of drugs is complex, the pathogenic process of microorganisms in the body in the drugs is relatively simple and clear. After the medicine enters a digestive system through the mouth and is dispersed in the stomach, polluted microorganisms in the medicine can be separated out, microorganisms which are not inactivated by gastric acid or microorganisms capable of resisting gastric acid enter the small intestine through the pylorus of the stomach, and then enter blood and lymph through the mucous membrane of the digestive tract under proper conditions in the small intestine to cause bacteremia, and part of microorganisms can further enter the large intestine through the small intestine to destroy the colony environment in the large intestine, so that diarrhea or other intestinal diseases are caused. Throughout the environment of the digestive system in the body, the stomach, small intestine and large intestine are the major distribution sites for microorganisms in the body. In this sense, if a set of simulative devices of stomach, small intestine and large intestine are established, the process of microorganisms in human body can be effectively simulated by adding artificially synthesized gastric juice and intestinal juice, and the improvement from the current single inspection of the total number of bacterial colonies in the medicine to the comprehensive inspection of pathogenicity of microorganisms in the in vivo environment in the medicine can be realized, thereby realizing the multi-aspect inspection of the microorganisms in the medicine including the in vivo process.
. In order to verify whether a medicine has antibacterial activity, the existing microbial inspection method in Chinese pharmacopoeia is to add a certain amount of known microbes into a sample, examine whether the sample has antibacterial activity by counting the number of microbial colonies in a sample plate and the number of microbial colonies in a non-sample plate, and if the ratio of the number of the microbial colonies grown in the sample plate to the number of the microbial colonies in the non-sample plate is less than 0.5 or more than 2, the verification method is considered to be false and a microbial inspection method needs to be established again. The initial aim of the test method is to eliminate the possible bacteriostatic activity of the drug itself, and the methods generally adopted are to increase the dilution ratio of the sample and to use membrane filtration, and besides the time and labor for the operation and the need to prepare a suitable amount of added microorganisms in advance, the test method has the following problems: the additional amount of microorganisms is not necessarily contained in the drug itself but cannot be detected due to the bacteriostatic activity of the drug itself. If the drug itself contains microorganisms other than those to be additionally verified, current methods of microorganism verification are somewhat meaningless, since the methods of excluding the bacteriostatic activity of the drug are not exactly the same for different microorganisms. In addition, the existing inspection method only inspects the number of microorganisms because the change of the microorganisms generated by the influence of gastric acid and intestinal juice after the medicine enters the human body is not considered, and actually, the bacterial colony which can survive and proliferate under the action of gastric juice or intestinal juice in the medicine enters the human body is the foundation for inspecting and ensuring the safety of the medicine, and obviously, the existing pharmacopeia microorganism inspection method does not consider more important factors of the medicine in the human body.
In terms of safety and practical operability, the microbiological examination of the drug should be conducted by a set of devices simulating the digestion environment in human body in vitro to examine whether there is microbiological contamination in the drug. The device can not only check the total number of microorganisms in the medicine, but also check the condition of the microorganisms in the medicine after simulating gastric acid environment and intestinal fluid environment treatment change. The device needs to keep aseptic, can be a disposable aseptic plastic product, and also can be other material products which can be repeatedly sterilized by high pressure, and the intermediate link can extract a sample for detection so as to comprehensively investigate the change process of microorganisms in the medicine. According to the retrieval, no method for testing the microorganisms in the medicines exists at present, and no medicine microorganism simulation digestive system device which meets the testing requirements exists.
Disclosure of Invention
Aiming at the problems, the invention discloses a simulated digestion system device and a method for detecting microorganisms in medicines, wherein each structure of the device is provided with a detection hole capable of extracting a sample, so that the conditions of the microorganisms in different device parts can be detected at any time, the real-time monitoring of the sample to be detected is realized, known bacteria are not required to be added to verify whether the sample has antibacterial activity, and the operation of removing the added microorganisms by discharging redundant reaction liquid in the process can be realized, so that the test process can be simplified, and the test operation steps can be reduced.
The utility model provides a simulation digestive system device for examining microorganism in medicine, includes first retort 1 and second retort 3 that communicate each other, through connecting pipe 8 intercommunication between two retort, is provided with check valve 7 between first retort and the connecting pipe, and every retort side all is provided with liquid feeding sample connection 4, first retort bottom side is provided with filter valve 6, and every retort outside all is provided with the oscillation case 2 of heating.
Preferably, the connecting pipe is a flexible pipe.
The invention also discloses a method for detecting microorganisms in a medicine by using the digestive system device, which comprises the following steps:
step 1, adding a set amount of medicine to be measured into a first reaction tank, adding artificial gastric juice, and then carrying out heat preservation oscillation to uniformly disperse the medicine;
step 4, adding the artificial intestinal juice into a second reaction tank 3 through a liquid adding sampling port 4, and carrying out heat preservation oscillation;
and 5, sampling from the liquid adding sampling port 4 of the second reaction tank 3 for inspection and determination.
And 6, filtering the reaction liquid in the second reaction tank 3, and counting the number and the types of the microorganisms contained in the sample by using a filter membrane at the bottom of the test.
Preferably, the adding amount of the medicine in the step 1 is 1g, the adding amount of the artificial gastric juice is 200ml, the heat preservation temperature is 37 ℃, and the oscillation time is 30 min.
Preferably, the step 2 is to extract a sample in the first reaction tank for inspection and measurement according to experimental requirements during the reaction process.
The invention has the beneficial effects that:
1. the device is a split structure with the reaction tanks connected, and can be used for combining one-connection, two-connection and three-connection tank bodies according to the test requirements. Each structure is provided with a liquid adding sampling port capable of extracting samples, so that the microorganism conditions in different device parts can be detected at any time, and the samples to be detected can be monitored in real time.
2. The side wall of the tank body close to the bottom is provided with a filtering valve, and when the valve is opened, redundant reaction liquid in the reaction tank can be filtered and discharged. The bottom of the reaction tank is designed by adopting a one-way valve which is opened downwards, and when the one-way valve is opened, the liquid to be detected of the medicine dispersed in the reaction tank can be transmitted into the next-stage reaction tank through a connecting pipe.
3. Compared with the existing test method, the inventive device and the test method disclosed by the invention do not need to add known bacteria to verify whether the sample is bacteriostatic or not. As described above, the conventional test method is a test for establishing a test result in the sense that a microorganism possibly contaminated in a pharmaceutical product is not necessarily an added microorganism. If the washing amount must be increased to eliminate the bacteriostatic activity of the medicine, the first group of reaction tanks of the device can be completed. The device and the inspection method can remove the additional microorganism by discharging redundant reaction liquid (artificial gastric juice or artificial intestinal juice) in the process, thereby simplifying the test process and reducing the test operation steps. 4. Compared with the existing inspection method, the device and the inspection method can simulate the in-vivo process of the medicine, really inspect the limit of microorganisms in the medicine from the perspective of safety, and particularly analyze the types and the quantity of microorganisms still growing after the treatment of the gastrointestinal environment of a human body, so that the device and the inspection method have good practical significance for controlling the quality of the medicine and preventing the microbial contamination of the medicine which is easy to occur clinically, but the existing inspection method of pharmacopoeia cannot realize the method.
5. Compared with the existing inspection method, the device of the invention can realize the compatibility of the existing method and can also solve the problem which cannot be solved by the existing method.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic view of the overall structure of the present invention;
in the figure: 1. the device comprises a first reaction tank, 2, a heating oscillation box, 3, a second reaction tank, 4, a sampling hole, 5, a liquid feeding sampling port, 6, a filtering valve, 7, a one-way valve, 8, a connecting pipe, 9 and a third reaction tank.
Detailed Description
The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present application without making any creative effort, shall fall within the protection scope of the present application.
The artificial gastric juice related to the invention can adopt a commercially available sterile artificial gastric juice, and can also adopt the following preparation method: diluted hydrochloric acid with the concentration of 1mol/ml is taken, water is added for dilution, and the pH is adjusted to 1.5. Adding 1g of pepsin into every 100ml of liquid, mixing uniformly, filtering and sterilizing for later use.
The artificial intestinal juice related to the invention can adopt a commercially available sterile artificial intestinal juice, and can also adopt the following preparation method: take KH2PO46.8g were dissolved in 500ml of water and the pH was adjusted back to 6.8 with 0.4% (w/w) NaOH. Adding 1g of trypsin into every 100ml of liquid, mixing uniformly, and filtering and sterilizing for later use.
As shown in figure 1, the invention discloses a simulated digestion system device and a method for detecting microorganisms in medicines, the device comprises a first reaction tank 1 and a second reaction tank 2 which are communicated with each other, and a third reaction tank 9 can be added at the tail end of the second reaction tank according to actual needs to simulate the large intestine digestion system. The heating oscillation box 2 is arranged outside each reaction tank and used for heating and oscillating liquid in the tank to simulate a real in-vivo digestive system environment. The heating oscillation box is an air sleeve type closed heating system, the temperature control range is 25-42 ℃, and the temperature can be adjusted to meet the requirements of different tests. The reaction tank is fixed in the heating oscillation box through a spring net bag, and can perform transverse, longitudinal and rotary oscillation. Set up sample addition port 5 at first retort top for add the medicine, set up liquid feeding sample connection 4 to the side of first retort, be used for adding gastric juice and sample test, be provided with filter valve 6 to the side of first retort bottom, be used for filtering unnecessary reaction liquid, the valve of 1 bottom of retort has certain distance apart from the retort bottom, and this distance can be guaranteed to persist certain volume from valve department to the retort bottom, this volume can mark out when making the retort, this volume is fixed volume. The end of first retort is through connecting pipe 8 and second retort intercommunication, and this connecting pipe 8 is the flexible pipe, produces the linkage to another retort when preventing that one of them retort from shaking. A one-way valve 7 is arranged between the second reaction tank and the connecting pipe, and a liquid feeding sampling port 4 is also arranged on the side surface of the second reaction tank.
The specific detection method comprises the steps of adding 1g of a medicine to be detected into a first reaction tank, adding about 200ml of artificial gastric juice, then carrying out heat preservation and oscillation at 37 ℃ for 30 minutes, so that the medicine is uniformly dispersed, and microorganisms possibly existing in the medicine can be fully dispersed by oscillation. According to the experimental requirements, the sample in the first reaction tank is extracted from the liquid adding sampling port for inspection and measurement. After the oscillation finishes, open the filter valve 6 of first retort lateral wall, filter most reaction solution (artificial gastric juice) and discharge the back, open the check valve 7 of 1 bottom of first retort, the one-way flashboard of medicine after the dispersion and the small part artificial gastric juice accessible bottom passes through the connecting pipe with the sample and gets into the second retort, add the second retort with reaction solution (artificial intestinal juice) through liquid feeding sample connection 4 on the second retort, 2 lateral walls of second retort also can set up the filter valve as required by the experiment, can keep warm the oscillation to second retort mechanical energy. After the test is finished, the sample can be sucked from the liquid adding sampling port 4 of the second reaction tank 3 for inspection and determination, if no subsequent test is available, the reaction liquid in the second reaction tank 3 can be filtered, and the filter membrane at the bottom is taken to count the number and the type of the microorganisms (if any) contained in the sample.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A simulated digestion system device for detecting microorganisms in medicines is characterized by comprising a first reaction tank (1) and a second reaction tank (3) which are communicated with each other, wherein the two reaction tanks are communicated with each other through a connecting pipe (8), a one-way valve (7) is arranged between the first reaction tank and the connecting pipe, the side surface of each reaction tank is provided with a liquid adding sampling port (4), the side surface of the bottom of the first reaction tank is provided with a filtering valve (6), the valve at the bottom of the first reaction tank (1) is at a certain distance from the bottom end of the reaction tank, and a heating oscillation box (2) is arranged outside each reaction tank; the reaction tank is fixed in the heating oscillation box through a spring net bag, and can perform transverse, longitudinal and rotary oscillation.
2. The simulated digestion system apparatus for testing pharmaceutical products for microorganisms according to claim 1, wherein the connecting tube is a flexible tube.
3. A method for testing a pharmaceutical product for microorganisms using the simulated digestive system device of claim 1 or 2, comprising the steps of:
step 1, adding a set amount of medicine to be measured into a first reaction tank, adding artificial gastric juice, and then carrying out heat preservation oscillation to uniformly disperse the medicine;
step 2, after the oscillation is finished, opening a filtering valve (6) of the first reaction tank (1), and filtering and discharging a part of the reacted artificial gastric juice;
step 3, opening a one-way valve (7) at the tail end of the first reaction tank (1), and enabling the dispersed medicine and the other part of the reacted artificial gastric juice to enter a second reaction tank (3);
step 4, adding the artificial intestinal juice into a second reaction tank (3) through a liquid adding sampling port (4), and carrying out heat preservation oscillation;
step 5, sampling from a liquid adding sampling port 4 of the second reaction tank (3) for inspection and determination;
and 6, filtering the reaction liquid in the second reaction tank (3), and counting the number and the types of the microorganisms contained in the sample by using a filter membrane at the bottom of the test.
4. The method for detecting microorganisms in a drug according to claim 3, wherein the drug is added in an amount of 1g, the artificial gastric juice is added in an amount of 200ml, the incubation temperature is 37 ℃, and the shaking time is 30min in step 1.
5. The method for detecting microorganisms in a pharmaceutical product according to claim 3, wherein in the step 2, the sample in the first reaction tank is extracted for detection and determination according to the experimental requirements during the reaction.
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| JP4429069B2 (en) * | 2004-05-06 | 2010-03-10 | 三洋電機株式会社 | Microorganism detection apparatus and method |
| JP5620257B2 (en) * | 2010-12-24 | 2014-11-05 | 積水化学工業株式会社 | Microbial contaminant detection container, microbial contaminant detection system, and microbial contaminant detection method |
| CN202315914U (en) * | 2011-11-14 | 2012-07-11 | 四川汇宇制药有限公司 | Inspection isolator |
| CN106834416A (en) * | 2015-12-03 | 2017-06-13 | 康普药业股份有限公司 | A kind of microorganism detection method of antibiotics |
| CN107043803A (en) * | 2017-05-24 | 2017-08-15 | 中检科（北京）实验室能力评价有限公司 | Yeast and mold sum numerical ability verification sample and preparation method thereof in medicine |
| CN109652334A (en) * | 2019-01-11 | 2019-04-19 | 谭瑛 | A kind of complex microbial inoculum and its preparation method and application |
| CN110231437A (en) * | 2019-04-26 | 2019-09-13 | 合肥工业大学 | A kind of device that test-type automatic imitation stomach and intestine continuously digest and application method |
| CN110093397A (en) * | 2019-06-10 | 2019-08-06 | 甘肃省药品检验研究院 | A kind of microbial limit method applicability inspection method, system and equipment |
| CN110305781B (en) * | 2019-07-05 | 2022-03-18 | 山东大学 | Microbial community co-culture device and evaluation method for evaluating in-vitro activity of microecological preparation |
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