CN110305781B - Microbial community co-culture device and evaluation method for evaluating in-vitro activity of microecological preparation - Google Patents

Microbial community co-culture device and evaluation method for evaluating in-vitro activity of microecological preparation Download PDF

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CN110305781B
CN110305781B CN201910602847.XA CN201910602847A CN110305781B CN 110305781 B CN110305781 B CN 110305781B CN 201910602847 A CN201910602847 A CN 201910602847A CN 110305781 B CN110305781 B CN 110305781B
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丁勃
刘新泳
李军
胡德福
国明
沈振
徐晓洁
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Shandong University
Shandong Institute for Food and Drug Control
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract

The invention relates to the field of activity evaluation of a microecological preparation medicament, in particular to a microbial community co-culture device and an activity evaluation method for evaluating the in-vitro activity of a microecological preparation. The microbial community co-culture device comprises a pure culture pipe and a co-culture tray, wherein the pure culture pipe is connected with a deaerator to realize anaerobic culture; the co-culture tray is connected with the pure culture tube through a connecting tube; the upper wall of the co-culture tray is provided with a culture medium sampling port and a culture medium reinjection port, and the upper parts of the culture medium sampling port and the culture medium reinjection port are provided with small air-permeable rubber plugs; and the connecting pipe is provided with a communicating valve. The co-culture system designed by the invention realizes that aerobic bacteria, anaerobic bacteria and facultative bacteria are cultured in the same culture system, and are relatively independent and mutually communicated. Under the mild shaking culture condition, the culture solution between each culture chamber is slowly communicated and interacted, and the survival state of the microorganisms in the intestinal tract is simulated to the maximum extent.

Description

Microbial community co-culture device and evaluation method for evaluating in-vitro activity of microecological preparation
Technical Field
The invention relates to the field of activity evaluation of a microecological preparation medicament, in particular to a microbial community co-culture device and an activity evaluation method for evaluating the in-vitro activity of a microecological preparation.
Background
The microbial product is prepared from normal or harmless exogenous microorganisms, and can be used for treating dysbacteriosis. The action way of the probiotic bacteria preparation is mainly to realize the rebalance of the internal environment by inhibiting the growth of harmful bacteria and promoting the growth of probiotics. Has wide clinical application. The micro-ecological preparation approved to be on the market in China has 22 types, relates to 13 types of microorganisms, and has billions of annual sales. Possible mechanisms of action of the probiotics include: bacteriocins, polypeptides, proteins, habitat changes, competitive inhibition, etc., have a complex mechanism of action and the active ingredients exhibit dynamic changes with the growth of the microbial community. Therefore, it is very difficult to evaluate the medicinal activity of the live microbial preparation, unlike chemical agents having a definite active ingredient.
There is a report in the literature that the activity of a drug is evaluated by measuring the change in the microbial community in a test animal after long-term oral administration by a method of animal test, and comparing with a control group. The method is influenced by factors such as individual difference, test time, sample size, judgment standard, economy and the like, and cannot be used as a standard detection method to be applied to the quality standard of the medicine.
In contrast, the in vitro activity evaluation method is simple and convenient to operate and has clear results. It uses a specific microorganism or several microorganisms as research objects, and eliminates uncertain factors caused by individual difference and sample size of experimental animals. The change condition of the microbial community in the culture system is dynamically monitored, and the activity of the medicine is comprehensively evaluated by combining with statistical analysis and comparing with a blank control group. The method has objective and definite result. Due to the lack of professional culture systems and culture devices, currently, the in vitro activity evaluation method of the microbial ecological agents collected in the literature mostly takes a single microorganism as a research object, such as the inhibition effect on staphylococcus aureus or the growth promotion effect on lactobacillus, and the interaction among intestinal microorganisms is not fully considered, so that the evaluation result has great limitation.
With the continuous development of the microecological preparation pharmaceutical industry, the quality control standard is also improved correspondingly, and the establishment of the in vitro activity evaluation method of the microecological preparation has great significance for the healthy development of the microecological preparation industry.
Disclosure of Invention
The invention discloses a microbial community co-culture device for evaluating the in-vitro activity of a microecological preparation. Inoculating multiple test microorganisms into a co-culture device for culture, detecting the change of microbial community under the action of the drug, and evaluating the effect of the drug on the co-culture microorganisms.
A microbial community co-culture device for evaluating the in-vitro activity of a microecological preparation comprises a pure culture tube and a co-culture disc, wherein the pure culture tube is connected with the co-culture disc through a connecting tube, and the main material of the pure culture tube is glass, a small amount of metal, rubber, pvc and the like; the upper wall of the co-culture tray is provided with a culture medium sampling port and a culture medium reinjection port, and the upper parts of the culture medium sampling port and the culture medium reinjection port are provided with small air-permeable rubber plugs; the connecting pipe is provided with a communicating valve which can close a passage between the pure culture pipe and the co-culture tray, and the pure culture pipe can be taken down from the connecting pipe after being closed. Culture medium sampling opening and culture medium reinjection mouth diameter are 5mm, adopt little ventilative plug to seal, effectively adjust the pressure in the coculture dish, simultaneously, because culture medium sampling opening and culture medium reinjection mouth pipeline are slender, have effectively avoided the oxygen content that the external air excessively got into the influence coculture dish.
The connecting pipes around the co-culture disc are uniformly distributed; the number of the connecting pipes is 4-14; the diameter of the pure culture tube is 4cm, the height of the pure culture tube is 11cm, and a scale mark is arranged at the position with the height of 7-9 cm; the diameter of the co-culture disc is 10cm, and the height of the co-culture disc is 2.5 cm; the diameter of the connecting pipe is 5mm, and the length is 3 cm. The co-culture system, the pure culture tubes and the co-culture trays are not limited to the above volumes and may be scaled up or down. The connecting pipe is designed to be slender, which can ensure the effective exchange of culture medium or microbe in the two culture containers, and can limit the exchange amount and maintain the stable aerobic or anaerobic condition in the pure culture pipe.
The upper part of the pure culture tube is provided with a rubber plug; the rubber plug is air-permeable or semi-air-permeable.
The pure culture tube is connected with a deaerator, the deaerator is composed of a deaerator chamber, a filter membrane and a pure culture tube cover, the pure culture tube cover is also provided with a decompression bag, gas generated by microorganism metabolism in the culture process can be discharged into the decompression bag, and the pressure stability in the pure culture tube is ensured.
The deoxidant room is managed lid material for pvc with pure cultivation, and deoxidant room and pure cultivation pipe lid junction have the filtration membrane of pp material, prevent that the deoxidant granule from getting into pure cultivation lid and keep the good permeability of deoxidant room and pure cultivation pipe simultaneously. During the concrete operation, only need to carry out the pure culture tube lid with pure culture tube cover knot on the pure culture tube that the anaerobism was cultivateed, the sealed rubber circle can closely laminate both, realizes the anaerobism and cultivates the environment.
The pure culture tube cover and the pure culture tube are connected by a sealing rubber ring.
A method for evaluating the co-culture of microbial colonies comprises the following steps:
a method for evaluating the co-culture of microbial colonies is characterized by comprising the following steps:
(1) placing the co-culture system in a biological safety cabinet, adding a liquid culture medium from any pure culture tube, setting a sample group and a control group, and closing a communication valve when the culture medium is uniformly distributed in a culture tray and the pure culture tube and reaches a scale mark in the pure culture tube; wherein the sample group is culture medium containing medicine, and the control group is culture medium without medicine;
(2) inoculating corresponding test microorganisms into a pure culture tube, transferring a co-culture device into an incubator, pre-culturing for 0.5-1 hour, then opening a communication valve, and performing shaking culture at 10-20 r/min;
(3) counting each test microorganism in the culture, and calculating the lg increase value or lg decrease value of each microorganism in the sample group;
(4) by means of x2The test method tests the ratio of probiotics to harmful bacteria in the control group and the sample group, and evaluates the effect of the medicament on microbial flora.
Further, the medicament of step (1) comprises: lactein, viable bacteria preparation, Chinese patent medicine, chemical medicine and the like, and has the function of improving intestinal flora; or a microorganism metabolite extract, the activity of which is verified by the present invention.
Further, the specific steps of the inspection method in the step (4) are as follows:
(1) after the culture is finished, counting and analyzing the content of each microorganism in the culture of the control group and the sample group;
(2) calculating the log increase or decrease of each microorganism per 1ml of culture in the sample group compared to the control group;
(3) per 10ml, the composition ratio of harmful bacteria and probiotic bacteria in the sample group and the control group is subjected to Chi2And (6) checking.
(4) Judging the result according to the result judgment standard; wherein the result judgment standard is as follows:
Figure BDA0002119609280000041
the culture medium sampling port is used for sampling the culture in the co-culture disc in the culture process, the sampling amount is 1-5 ml, and sterile culture medium with the same sampling amount and volume is added from the culture medium reinjection port during sampling.
After the inoculation in the step (2) is completed, sealing the pure culture pipe orifice by using a breathable rubber plug for microorganisms needing anaerobic conditions, and then connecting a deaerator with the pure culture pipe to form anaerobic conditions; sealing the pure culture tube mouth with a semi-permeable rubber plug to form micro-aerobic conditions; the pure culture tube mouth is sealed by a breathable rubber plug, and microorganisms in aerobic conditions are needed. Microorganisms requiring anaerobic conditions have an anaerobic indicator in the oxygen scavenger chamber at the time of culturing. If the anaerobic indicator changes color in the culture process, the deaerator needs to be replaced. After the test is finished, the deoxidant chamber bottom cover at the bottom end of the deoxidant device can be used for replacing the deoxidant which is out of work, and the deoxidant device can be repeatedly used.
Further, the formula of the liquid culture medium is as follows: 15g of tryptone, 3g of soybean and pawpaw protein hydrolysate, 3g of beef extract powder, 10g of yeast extract powder, 10g of glucose, 5g of lactose, 1.5g of maltose, 1.5g of cellobiose, 1g of soluble starch, 0.5g of mannitol,cysteine-hydrochloric acid 0.25g, dipotassium hydrogen phosphate 0.5g, monopotassium phosphate 0.5g, sodium bicarbonate 6g, calcium chloride 0.15g, magnesium sulfate 0.1g, sodium acetate 0.15g, ammonium citrate 0.1g, ferrous sulfate 0.02g, manganese sulfate 0.02g, zinc sulfate 0.02g, copper sulfate 0.01g, vitamin K10.5ml, 2g of sodium chloride, 1mg of folic acid, 1mg of nicotinic acid and 2mg of biotin are added into 500ml of cattle intestine leaching liquor and 500ml of distilled water.
The preparation method of the cattle intestine leaching liquor comprises the following steps: taking 250g of small intestine and 250g of colon of cattle, washing with sterile water for several times, crushing by a crusher, adding 1000ml of distilled water, stirring uniformly, storing at 4 ℃ for 48 hours, filtering by six layers of medical gauze, and taking filtrate to obtain the traditional Chinese medicine composition.
The evaluation method according to claim 7, wherein the test microorganism of step (2) comprises probiotic bacteria, neutrophilic bacteria and harmful bacteria; wherein the probiotic is one or more of Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus buchneri, Lactobacillus rhamnosus and Lactobacillus plantarum; the neutral bacteria are one or more of clostridium butyricum, enterococcus faecalis, streptococcus faecalis, bacteroides vulgatus, bacillus coagulans, bacillus licheniformis, clostridium difficile and eubacterium mucilaginosus; the harmful bacteria are one or more of salmonella paratyphi B, vibrio parahaemolyticus, staphylococcus aureus, escherichia coli and shigella flexneri; other model strains or non-model strains isolated from the human or animal gut may also be used, but must conform to the test microbial composition patterns of probiotics, neutrophiles and harmful bacteria.
Advantageous effects
(1) The co-culture device designed by the invention fully considers the diversity of intestinal microorganisms (including harmful bacteria, probiotics and neutral bacteria), the dominance of the microorganisms in the colonization area of the intestinal wall, the migration of the microorganisms along with intestinal fluid, different requirements of the microorganisms on oxygen, interaction among the microorganisms, the action of drugs on the microorganisms, the peristalsis of the intestinal tract, the fluidity of the intestinal fluid and the like. The results of the evaluation are closer to the real changes in the intestinal microflora.
(2) The co-culture system designed by the invention realizes that aerobic bacteria, anaerobic bacteria and facultative bacteria are cultured in the same culture system, and are relatively independent and mutually communicated. Under the mild shaking culture condition, the culture solution between each culture chamber is slowly communicated and interacted, and the natural survival state of the microorganisms in the intestinal tract is simulated to the maximum extent.
Drawings
FIG. 1 is an overall view of a co-culture apparatus of the present invention;
FIG. 2 is a perspective view of a co-cultivation device according to the present invention;
FIG. 3 is a longitudinal sectional view of the co-culture apparatus of the present invention;
FIG. 4 is an exploded plan view of the deaerator of the co-cultivation apparatus of the present invention;
FIG. 5 is a graph showing the growth curves of 8 test bacteria in example 1 under pure culture conditions;
in the figure: 1. pure culture tube, 2, co-culture dish, 3, connecting tube, 4, communicating valve, 5, culture medium sampling port, 6, culture medium reinjection port, 7, small permeable rubber plug, 8, permeable rubber plug (or semi-permeable rubber plug), 9, deaerator, 10, deaerator chamber, 11, filter membrane, 12, pure culture tube cover, 13, decompression bag, 14, oxygen indicator, 15, deaerator chamber bottom cover, 16, sealing rubber ring, 17 and scale mark.
Detailed Description
The present invention will be further described with reference to examples and comparative examples, but it should be noted that the following description is only for the purpose of explaining the present invention and does not limit the contents thereof.
Example 1
This example uses the present invention to evaluate the effect of lactobacilli on intestinal microorganisms.
The lactobacillin is a metabolite of lactobacillus, the lactobacillin granules are prepared from the lactobacillin serving as a main raw material, and the physical and functional parts in the medicament specification describe the product, namely the product can be used for selectively killing intestinal pathogenic bacteria and protecting and promoting the growth of beneficial bacteria.
FIG. 1 is a schematic view showing the structure of a co-culture apparatus according to example 1 of the present invention, which comprises a pure culture tube 1 and a co-culture tray 2, wherein the pure culture tube 1 is connected to the co-culture tray 2 via a connecting tube 3; the upper wall of the co-culture tray 2 is provided with a culture medium sampling port 5 and a culture medium re-injection port 6, and the upper parts of the culture medium sampling port 5 and the culture medium re-injection port 6 are provided with small air-permeable rubber plugs 7; the connecting pipe 3 is provided with a communicating valve 4.
The connecting pipes 3 around the co-culture disc 2 are uniformly distributed, and the number of the connecting pipes is 8; the diameter of the pure culture tube 1 is 4cm, the height is 11cm, and a scale mark 17 is arranged at the position with the height of 7-9 cm; the diameter of the co-culture disc 2 is 10cm, and the height is 2.5 cm; the diameter of the connecting pipe 3 is 5mm, and the length is 3 cm; the diameter of the culture medium sampling port 5 and the diameter of the culture medium reinjection port 6 are 5 mm.
The pure culture tube 1 is connected with a deaerator 9, the deaerator 9 consists of a deaerator chamber 10, a filter membrane 11 and a pure culture tube cover 12, and a pressure reducing bag 13 is arranged on the pure culture cover 12.
The material of deoxidant room 10 and pure culture tube lid 12 is pvc, and there is the filtration membrane 11 of pp material at deoxidant room 10 and pure culture tube lid 12 junction.
The pure culture tube cover 12 and the pure culture tube 1 are connected by a sealing rubber ring 16.
(1) The preparation method of the lactein solution of this example is as follows: taking a proper amount of a lactein raw material, diluting with sterilized water to prepare a 20% lactein raw material solution, centrifuging at 4000r/min for 5min, removing cells, fibers and the like in the solution, and taking a supernatant rich in active ingredients for later use.
(2) The test strains in this example were Bifidobacterium longum (BNCC3337084), Clostridium butyricum (BNCC337239), enterococcus faecalis (BNCC186300), Lactobacillus rhamnosus (BNCC134266), Bacteroides vulgatus (BNCC186191), Bacillus licheniformis (BNCC1326654), Staphylococcus aureus (CMCC26003) and Salmonella paratyphi B (CMCC 50094).
Respectively inoculating the above strains into culture medium, culturing at 37 deg.C for 18 hr, centrifuging for enrichment, washing with sterile physiological saline, diluting, and measuring turbidity to obtain about 10 of test bacteria7cfu/ml bacterial suspension for use.
(3) The concentrated medium formulation of this example was: tryptone 15g, soybean papaya protein hydrolysis3g of the compound, 3g of beef extract powder, 10g of yeast extract powder, 10g of glucose, 5g of lactose, 1.5g of maltose, 1.5g of cellobiose, 1g of soluble starch, 0.5g of mannitol, 0.25g of cysteine-hydrochloric acid, 0.5g of dipotassium hydrogen phosphate, 0.5g of monopotassium phosphate, 6g of sodium bicarbonate, 0.15g of calcium chloride, 0.1g of magnesium sulfate, 0.15g of sodium acetate, 0.1g of ammonium citrate, 0.02g of ferrous sulfate, 0.02g of manganese sulfate, 0.02g of zinc sulfate, 0.01g of copper sulfate, 0.01g of vitamin K10.5ml, 2g of sodium chloride, 1mg of folic acid, 1mg of nicotinic acid and 2mg of biotin are added into 500ml of cattle intestine leaching liquor, and the mixture is sterilized for 30min at 115 ℃ for later use.
At this time, the medium was 2-fold concentrated medium, diluted with sterilized purified water or sample solution, and used.
(4) 500ml of the sterilized concentrated culture medium and 500ml of 20% lactein solution are mixed uniformly, slowly added into a co-culture device, and a communication valve is closed when the culture medium is distributed uniformly. In 8 pure culture tubes, 1ml of the above 10 cells were inoculated7cfu/ml bacterial suspension. Inoculating pure culture tubes of bifidobacterium longum, clostridium butyricum, lactobacillus rhamnosus, common bacteroides and bacillus licheniformis, sealing the culture tubes by using a breathable rubber plug, and connecting a deaerator to form an anaerobic environment; inoculating a pure culture tube of enterococcus faecalis and salmonella paratyphi B, sealing the pure culture tube by using a semi-permeable rubber plug, and simulating a microaerophilic environment; the pure culture tube inoculated with staphylococcus aureus is sealed by a breathable rubber plug to form aerobic conditions.
(5) And transferring the co-culture device into a shaking culture box, carrying out shaking culture for 1 hour at 50-60 r/min, opening each communication valve, adjusting the shaking speed to 10-20 r/min, and continuing to culture.
The microorganisms in the logarithmic phase are vigorous in growth, propagation and metabolism and sensitive to external environment changes, and the microorganisms gradually generate secondary metabolites in a later period of the logarithmic phase, so that the action of the drugs on microbial communities and the interaction among the microorganisms have typical performances. Therefore, the culture samples of all the test bacteria in the logarithmic phase and the later period are selected as much as possible for detection.
According to 8 test bacteria (initial concentration of 10)5cfu/ml) growth curves under pure culture conditions (see FIG. 5 below) inSamples were taken for 10 hours and the content of each microorganism in the medium was counted.
Control group
500ml of the sterilized concentrated medium in the step (4) of example 1 was diluted with 500ml of sterilized water and mixed well, and the rest of the procedure was the same as in example 1.
Enumeration of individual test microorganisms in culture
Selective medium counting method: after sampling, the microorganisms in the culture can be counted using selective media. Adopting mupirocin lithium salt and cysteine hydrochloride modified MRS agar culture medium to count Bifidobacterium longum; counting clostridium butyricum by adopting a clostridium intensified culture medium containing perennial rhzomorph B and ampicillin; counting enterococcus faecalis by adopting a beef agar culture medium containing sodium azide and cow bile powder; adopting a lincomycin hydrochloride improved MRS agar culture medium to count the lactobacillus rhamnosus; counting bacteroides vulgatus by adopting a xylan carbon source agar culture medium added with kanamycin and vancomycin; counting the bacillus licheniformis by adopting a propionate culture medium high-temperature culture method at 55 ℃; counting staphylococcus aureus by adopting a BP agar culture medium; and (4) counting salmonella paratyphi B time bacteria by adopting an SXT4 salmonella selective medium.
The selective culture medium can be prepared by itself, or a commercial finished culture medium can be selected.
Non-selective medium counting method: the microorganisms in the culture were counted using a non-selective agar medium, such as the medium described in "(3)" above, supplemented with 1.5% agar powder, or a suitable commercially available medium. Selecting a flat plate with proper colony density, identifying 50-100 microorganisms according to colony characteristics, staining microscopy or biochemical tests of the test microorganisms on the flat plate, paralleling 2 plates, and calculating the proportion of each test microorganism.
Selective plates may also be used in combination with non-selective plates.
Criteria for determination of results
The microecologics firstly promote the growth and the propagation of probiotics, the absolute quantity of the probiotics in unit time is increased, the proportion of harmful bacteria is relatively reduced, the probiotics inhibits the harmful bacteria through ecological competition, antibacterial metabolites and the like, and the absolute quantity of the harmful bacteria is reduced; secondly, the microecological preparation directly inhibits or kills harmful bacteria, the absolute quantity of the harmful bacteria is reduced, the competitive inhibition pressure of probiotics from the harmful bacteria is reduced, the probiotics are propagated in large quantities, and the absolute quantity is increased; thirdly, the microecological preparation promotes the growth and the reproduction of the probiotics and kills or inhibits harmful bacteria, and the absolute quantity of the probiotics is increased while the absolute quantity of the harmful bacteria is reduced.
The microbial population regulation process is continuous and dynamic, and it may take several days from oral administration to exerting the drug effect, while in vitro experiments give results within hours. Certainly, in vitro tests cannot simulate the digestion and absorption of the gastrointestinal tract, the adjustment and intervention of the immune system, continuous administration, drug metabolism and the like, but only the drugs directly act on the flora to quickly influence the flora, which represents the change and development direction of the flora in vivo. Different probiotics also have different effects on probiotics or harmful bacteria. Therefore, the determination of the result should not be expressed simply by the specific change of the number of one or more microorganisms, and should show the overall flora composition and the trend toward increasing the number of the probiotics and decreasing the number of the harmful bacteria.
In summary, χ can be used2The detection method comprises the steps of detecting whether the composition ratio of the probiotics to the harmful bacteria in a control group and a sample group is different, analyzing the change trend of the composition of the microbial community in the sample group, and evaluating the action effect of the medicament on the microbial community by combining the actual bacterial quantity change value of each microorganism. The method comprises the following specific steps: under the action of various factors, the propagation speeds of test microorganisms are different, so that the number difference of different microorganisms is overlarge, the loss of information in the analysis process is amplified, and the difference is eliminated by taking a logarithm value; comparing with the control group, calculating the logarithmic increase value or decrease value of each microorganism in each ml of culture of the sample group; ③ Per 1ml of culture medium of microorganisms2The inspection and analysis show that the sample amount is less, the situation that the number of lattices with the theoretical value of 1 ≦ T ≦ 5 exceeds 1/5 can occur, and the sample amount can be increasedTreatment in such a way that a statistical analysis is carried out on the amount of microorganisms per 10ml of culture, i.e. 10 lg; fourthly, the components of the probiotics and the harmful bacteria in the two groups are subjected to Chi2And (6) checking. Judging the result according to the result judgment standard.
TABLE 1 determination criteria for results of in vitro activity tests of microecological preparations
Figure BDA0002119609280000111
Results of the in vitro activity test of Lactobacilli particles:
TABLE 2 microbial content in the two groups of cultures
Figure BDA0002119609280000112
TABLE 3 comparison of the composition of harmful bacteria and probiotic bacteria in the two groups
Group of Bifidobacterium longum Lactobacillus rhamnosus Staphylococcus aureus Salmonella paratyphi B Total up to
Sample (I) 78 80 45 73 276
Control 66 75 76 81 298
Total up to 144 155 121 154 574
TABLE 4 test results
Figure BDA0002119609280000121

Claims (9)

1. A microbial community co-culture device for evaluating the in-vitro activity of a microecological preparation is characterized by comprising a pure culture tube (1) and a co-culture disc (2), wherein the pure culture tube (1) is connected with the co-culture disc (2) through a connecting tube (3); the upper wall of the co-culture tray (2) is provided with a culture medium sampling port (5) and a culture medium reinjection port (6), and the upper parts of the culture medium sampling port (5) and the culture medium reinjection port (6) are provided with small air-permeable rubber plugs (7); a communicating valve (4) is arranged on the connecting pipe (3); the diameter of the pure culture tube (1) is 4cm, the height of the pure culture tube is 11cm, and a scale mark (17) is arranged at the position with the height of 7-9 cm; the diameter of the co-culture disc (2) is 10cm, and the height is 2.5 cm; the diameter of the connecting pipe (3) is 5mm, and the length is 3 cm;
the pure culture tube (1) is connected with a deaerator (9), the deaerator (9) consists of a deaerator chamber (10) and a pure culture tube cover (12) through a filter membrane (11), and a decompression bag (13) is arranged on the pure culture cover (12); the deoxidant room (10) and the pure culture tube cover (12) are made of pvc, and the joint of the deoxidant room (10) and the pure culture tube cover (12) is provided with a filter membrane (11) made of pp.
2. Co-cultivation device according to claim 1, characterised in that the connecting tubes (3) around the co-cultivation tray (2) are evenly distributed, the number of connecting tubes being 4-14; the diameters of the culture medium sampling port (5) and the culture medium reinjection port (6) are 5 mm.
3. Co-cultivation device according to claim 1 or 2, characterized in that the upper part of the pure cultivation tube (1) is provided with a plug (8); the rubber plug (8) is breathable or semi-breathable.
4. A co-cultivation device as claimed in claim 1, wherein the pure cultivation tube lid (12) and the pure cultivation tube (1) are connected with a sealing rubber ring (16).
5. A method for evaluating the co-culture of microbial colonies is characterized by comprising the following steps:
(1) placing the co-culture system of any one of claims 1-4 in a biosafety cabinet, adding liquid culture medium from any pure culture tube (1), setting a sample group and a control group in parallel, wherein the sample group is culture medium containing drugs, and mixing uniformly; the control group is culture medium without drug; when the culture medium is uniformly distributed in the culture tray (2) and the pure culture tube (1), the culture medium in the pure culture tube (1) reaches the scale mark (17), and the communicating valve (4) is closed;
(2) inoculating corresponding test microorganisms into the pure culture tube (1), transferring the co-culture device into an incubator, pre-culturing for 0.5-1 hour, then opening the communication valve, and performing shaking culture at 10-20 r/min;
after the inoculation in the step (2) is completed, sealing the opening of the pure culture tube (1) by using a breathable rubber plug (8) for microorganisms needing anaerobic conditions, then connecting a deaerator (9) with the pure culture tube (1) to form anaerobic conditions, wherein an anaerobic indicator (14) is arranged in a deaerator chamber (10) of the deaerator (9); the micro-aerobic condition is formed by sealing the mouth of the pure culture tube (1) by using a semi-permeable rubber plug (8) for the microorganisms needing the micro-aerobic condition; the mouth of the pure culture tube (1) is sealed by a breathable rubber plug (8) when the microorganism needs aerobic conditions;
(3) counting each test microorganism in the culture, and calculating the lg increase value or lg decrease value of each microorganism in the sample group;
(4) adopting a Chi 2 inspection method to inspect the composition ratio of the probiotics to the harmful bacteria of the control group and the sample group, and evaluating the effect of the medicament on the microbial flora; the detection method in the step (4) comprises the following specific steps:
counting and analyzing the content of each microorganism in the culture of a control group and a sample group after the culture is finished;
comparing with the control group, calculating the logarithmic increase value or decrease value of each microorganism in each 1ml of culture of the sample group;
carrying out chi 2 inspection on the composition ratio of harmful bacteria and probiotics in each 10ml sample group and control group;
fourthly, judging the result according to the result judgment standard; wherein the result judgment standard is as follows:
Figure 160427DEST_PATH_IMAGE002
。
6. the method according to claim 5, wherein the culture medium is sampled from the medium sampling port (5) in an amount of 1 to 5ml and the sterile medium is added from the medium refilling port (6) in the same volume as the sampled amount during the culture.
7. The evaluation method according to claim 5 or 6, wherein the liquid medium has a formulation of: 15g of tryptone, namely 15g of tryptone,3g of soybean and papaya protein hydrolysate, 3g of beef extract powder, 10g of yeast extract powder, 10g of glucose, 5g of lactose, 1.5g of maltose, 1.5g of cellobiose, 1g of soluble starch, 0.5g of mannitol, 0.25g of cysteine-hydrochloric acid, 0.5g of dipotassium hydrogen phosphate, 0.5g of potassium dihydrogen phosphate, 6g of sodium bicarbonate, 0.15g of calcium chloride, 0.1g of magnesium sulfate, 0.15g of sodium acetate, 0.1g of ammonium citrate, 0.02g of ferrous sulfate, 0.02g of manganese sulfate, 0.02g of zinc sulfate, 0.01g of copper sulfate, 0.01g of vitamin K10.5ml, 2g of sodium chloride, 1mg of folic acid, 1mg of nicotinic acid and 2mg of biotin are added into 500ml of cattle intestine leaching liquor and 500ml of distilled water.
8. The evaluation method according to claim 7, wherein the preparation method of the bovine intestine leachate comprises: taking 250g of small intestine and 250g of colon of cattle, washing with sterile water, crushing, adding 1000ml of distilled water, stirring uniformly, storing at 4 ℃ for 48 hours, and filtering to obtain filtrate.
9. The evaluation method according to claim 5, wherein the test microorganism of step (2) comprises probiotic bacteria, neutrophilic bacteria and harmful bacteria; wherein the probiotic is one or more of Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus buchneri, Lactobacillus rhamnosus and Lactobacillus plantarum; the neutral bacteria are one or more of clostridium butyricum, enterococcus faecalis, streptococcus faecalis, bacteroides vulgatus, bacillus coagulans, bacillus licheniformis, clostridium difficile and eubacterium mucilaginosus; the harmful bacteria are one or more of salmonella paratyphi B, vibrio parahaemolyticus, staphylococcus aureus, escherichia coli and shigella flexneri; other model strains or non-model strains isolated from the human or animal intestine may also be used for the probiotic, neutrophilic and harmful bacteria.
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