CN110305781A - Microbiologic population's co-culture device and evaluation method for the evaluation of probiotics external activity - Google Patents
Microbiologic population's co-culture device and evaluation method for the evaluation of probiotics external activity Download PDFInfo
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Abstract
The present invention relates to the activity rating fields of probiotics drug, in particular to a kind of microbiologic population's co-culture device and activity rating method for the evaluation of probiotics external activity.Microbiologic population's co-culture device includes pure culture pipe and co-cultures disk, and the pure culture pipe realizes Anaerobic culturel by connection oxygen-eliminating device；The co-cultivation disk is connected by connecting tube with pure culture pipe；The upper wall for co-culturing disk has a culture medium thief hatch and culture medium re-injection mouth, and small ventilative rubber plug is arranged at the top of the culture medium thief hatch and culture medium re-injection mouth；There is communicating valve in the connecting tube.The co-culture system that the present invention designs realizes aerobic bacteria, anaerobic bacteria and amphimicrobe and cultivates in same cultivating system, that is, keeps relatively independent, and is interconnected.Under the conditions of the shaken cultivation of mitigation, each culture solution cultivated between chamber is slowly exchanged, and is interacted, is simulated survival condition of the microorganism in enteron aisle to the full extent.
Description
Technical field
The present invention relates to the activity rating fields of probiotics drug, are used for Tiny ecosystem system in particular to one kind
The microbiologic population's co-culture device and activity rating method of agent external activity evaluation.
Background technique
Tiny ecosystem viable bacteria product is processed by human normal or harmless foreign nationality microorganism, for treating flora imbalance class disease
Disease.Its action pathway, which mainly passes through, inhibits harmful bacteria growth, promotes growth of probiotics, Lai Shixian vivo environment rebalancing.It is clinical
It is widely used.The probiotics of China's approved listing have 22 kinds, are related to 13 kinds of microorganisms, annual sales amount is billions of.Micro- life
The possible mechanism of action of state preparation includes: bacteriocin, polypeptide, albumen, habitat change, Reverse transcriptase etc., and the mechanism of action is multiple
It is miscellaneous, and dynamic change is presented with the growth of microbiologic population in active constituent.Therefore, it is explicitly chemical to be different from effective component
Medicine, the pharmaceutical activity for evaluating Tiny ecosystem active bacteria formulation are extremely difficult.
The method for thering is document report to use animal experiment, by detecting the intracorporal micro- life of animal subject after chronic oral administration
The variation of object group, and compared with the control group, evaluate the activity of drug.This method is by individual difference, test duration, sample
The factors such as amount, criterion, economy influence, and the quality standard of drug can not be applied to as a kind of detection method of standard
In.
In comparison, external activity evaluation method is easy to operate, result is clear.It is with specific certain or several microorganisms
For research object, experimental animal individual difference, sample size size bring uncertain factor are eliminated.Pass through dynamic monitoring culture
The situation of change of microbiologic population in system, compared with blank control group, in conjunction with statistical analysis, overall merit pharmaceutical activity.
This method result is objective, clear.Due to lacking the cultivating system and culture apparatus of profession, currently, the Tiny ecosystem system that document records
The external activity evaluation method of agent, mostly using single microorganism as research object, such as to the inhibiting effect of staphylococcus aureus or
To the growth promoting function etc. of Bacillus acidi lactici, do not fully consider the interaction between enteric microorganism, evaluation result have compared with
Big limitation.
With the continuous development of probiotics pharmaceutical industries, quality control standard will also be correspondinglyd increase, and establish Tiny ecosystem
The external activity evaluation method of preparation develops in a healthy way to probiotics industry significant.
Summary of the invention
The invention discloses a set of microbiologic population's co-culture devices for the evaluation of probiotics external activity.It will be more
Kind test microbial inoculant is cultivated into co-culture device, is detected under drug effect, the situation of change of microbiologic population, is evaluated medicine
Effect of the object to microorganism is co-cultured.
It is a kind of for probiotics external activity evaluation microbiologic population's co-culture device, including pure culture pipe and altogether
Culture plate, the pure culture pipe are connected by connecting tube with disk is co-cultured, and main material is glass, a small amount of metal, rubber, pvc
Deng；The upper wall for co-culturing disk has culture medium thief hatch and culture medium re-injection mouth, and the culture medium thief hatch and culture medium return
Small ventilative rubber plug is arranged at the top of geat；Have communicating valve in the connecting tube, communicating valve can close pure culture pipe and co-culture disk it
Between access, pure culture pipe can be removed from connecting tube after closing.Culture medium thief hatch and culture medium return gate diameter
5mm is sealed using small ventilative rubber plug, the pressure co-cultured in disk is effectively adjusted, simultaneously as culture medium thief hatch and culture medium
Re-injection mouth pipeline is more elongated, effectively prevents outside air and is excessively entering into the oxygen content influenced in co-cultivation disk.
Connecting tube around the co-cultivation disk is uniformly distributed；The quantity of connecting tube is 4-14；The pure culture pipe
Diameter is 4cm, is highly 11cm, is that graduation mark is arranged at 7-9cm in height；The diameter for co-culturing disk is 10cm, is highly
2.5cm；Connect pipe diameter 5mm, length 3cm.The co-culture system, pure culture pipe and co-cultivation disk are not limited to above-mentioned volume,
It can be scaled up or reduce.Connecting tube is elongated design, can both guarantee culture medium or microorganism in two culture vessels
Effectively exchange, and exchange capacity can be limited, keep aerobic or anaerobic condition stable in pure culture pipe.
Rubber plug is arranged at the top of the pure culture pipe；The rubber plug is ventilative or half is ventilative.
The pure culture pipe connects oxygen-eliminating device, and the oxygen-eliminating device passes through filter membrane and pure culture pipe Gai Zucheng by oxygen scavenger room,
Pure culture is covered there are also decompression capsule, and the gas that microbial metabolism generates during the cultivation process can drain into decompression capsule, guarantee pure training
Overpressure is supported to stablize.
The oxygen scavenger room and pure culture pipe lid material are pvc, and there are pp material in oxygen scavenger room and the junction pure culture Guan Gai
Filter membrane, prevent oxygen scavenger particle from entering pure culture lid while keeping deoxygenation room and the good permeability of pure culture pipe.Specifically
, only need to be by pure culture pipe cover buckle on the pure culture pipe for needing to carry out Anaerobic culturel when operation, sealing rubber ring can be tight by the two
Anaerobic culture environment is realized in closely connected conjunction.
The pure culture pipe lid is connected with pure culture pipe using sealing rubber ring.
A kind of evaluation method that microbe colony co-cultures, using following steps:
A kind of evaluation method that microbe colony co-cultures, which is characterized in that use following steps:
(1) above-mentioned co-culture system is placed in Biohazard Safety Equipment, adds fluid nutrient medium from any pure culture pipe, sets sample
Product group and control group, evenly distributed in culture plate and pure culture pipe to culture medium, culture medium reaches graduation mark in pure culture pipe, closes
Close communicating valve；Wherein sample sets are the culture medium containing drug, and control group is the culture medium that drug is not added；
(2) it is inoculated with corresponding test microorganism in pure culture pipe, co-culture device is moved into incubator, first preculture
0.5-1 hours, then open communicating valve, 10~20r/min shaken cultivation；
(3) it counts to respectively testing microorganism in culture, calculate the lg value added of each microorganism in sample sets or subtracts
Few value；
(4) χ is used2The method of inspection examines control group and sample sets probiotics and harmful bacteria composition ratio, evaluates drug pair
The function and effect of microbial flora.
Further, drug described in step (1) includes: lactein, active bacteria formulation, Chinese patent drug and chemicals etc.,
With the drug for improving intestinal flora effect；It is also possible to microbial metabolic products extract, with its activity of present invention verifying.
Further, the specific steps of the method for inspection described in step (4) are as follows:
(1) after cultivating, the content of each microorganism in analysis of accounts control group and sample sets culture；
(2) compared with the control group, it calculates in the every 1ml culture of sample sets, the logarithm value added or reduced value of each microorganism；
(3) in every 10ml, harmful bacteria in sample sets and control group, probiotics composition ratio carry out χ2It examines.
(4) result judgement is carried out according to result judgement standard；Wherein result judgement standard are as follows:
For being sampled during the cultivation process to the culture co-cultured in disk, sampling amount is the culture medium thief hatch
1~5ml, sampling simultaneously, the aseptic culture medium of sampling amount same volume are added from culture medium re-injection mouth.
After the completion of inoculation described in step (2), the microorganism of anaerobic condition is needed, seals pure culture pipe with ventilative rubber plug
Mouthful, oxygen-eliminating device is then connected into pure culture pipe, forms anaerobic condition；The microorganism for needing micro- aerobic condition, with half ventilative rubber plug
Pure culture nozzle is sealed, micro- aerobic condition is formed；The microorganism for needing aerobic conditions seals pure culture nozzle with ventilative rubber plug.
The microorganism of anaerobic condition is needed to have Anaerobic indicator in oxygen scavenger room when culture.Such as Anaerobic indicator in incubation
Discoloration, then need to change oxygen-eliminating device.After the completion of test, the deoxygenation of failure can be replaced by the oxygen scavenger room bottom cover of oxygen-eliminating device bottom end
Agent, oxygen-eliminating device are reusable.
Further, the formula of the fluid nutrient medium are as follows: tryptone 15g, soybean Papain hydrolysate 3g, ox
Meat extract powder 3g, yeast extract powder 10g, glucose 10g, lactose 5g, maltose 1.5g, cellobiose 1.5g, soluble starch 1g are sweet
Reveal alcohol 0.5g, cysteine-hydrochloric acid 0.25g, dipotassium hydrogen phosphate 0.5g, potassium dihydrogen 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 sulphate 0.01g, vitamin K1500ml is added in 0.5ml, sodium chloride 2g, folic acid 1mg, niacin 1mg, biotin 2mg
Roll leaching liquor, distilled water 500ml.
The Roll leaching liquor the preparation method comprises the following steps: take calf intestinal 250g, ox colon 250g, aseptic water washing for several times, powder
Broken machine crushes, and 1000ml distilled water is added, stirs evenly, and sets 4 DEG C and stores 48 hours, six layers of hospital gauze filtering take filtrate i.e.
?.
Step (2) evaluation method according to claim 7, which is characterized in that test microorganism described in step (2)
It must simultaneously include probiotics, neutral bacterium and harmful bacteria；Wherein the probiotics is bifidobacterium longum, bifidobacterium infantis, youth
One or more of Bifidobacterium, lactobacillus buchneri, Lactobacillus rhamnosus and lactobacillus plantarum；The neutrality bacterium is butyric acid shuttle
Bacterium, enterococcus faecalis, streptococcus fecalis, bacteroides vulgatus, bacillus coagulans, bacillus licheniformis, clostridium difficile and the true bar of mucus
One or more of bacterium；The harmful bacteria be moscow' paratyphi B, vibrio parahemolyticus, staphylococcus aureus,
One or more of escherichia coli and shigella flexneri；It can also be used in the probiotics, neutral bacterium and harmful bacteria
His type strain or the non-mode bacterial strain for being isolated from human or animal's enteron aisle, but have to comply with probiotics, neutral bacterium and harmful bacteria
Test microorganism compositional model.
Beneficial effect
(1) co-culture device that designs of the present invention fully considers diversity (including the harmful bacteria, probiotics of enteric microorganism
And neutral bacterial), microorganism the intestinal wall field planting dominance in region, microorganism with the migration of intestinal juice, microorganism to oxygen not
Interaction, drug between same demand, microorganism is to the effect of microorganism, the wriggling of enteron aisle and mobility of intestinal juice etc..
Evaluation result changes closer to true intestinal microflora.
(2) co-culture system that the present invention designs, realizes aerobic bacteria, anaerobic bacteria and amphimicrobe in same cultivating system
Culture, that is, keep relatively independent, and is interconnected.Under the conditions of the shaken cultivation of mitigation, each culture solution cultivated between chamber
Slowly exchange, interaction, simulates Natural Survival state of the microorganism in enteron aisle to the full extent.
Detailed description of the invention
Fig. 1 is the overall diagram of co-culture device of the invention；
Fig. 2 is the aerial view of co-culture device of the invention；
Fig. 3 is the sectional side elevation of co-culture device of the invention；
Fig. 4 is the oxygen-eliminating device decomposition view of co-culture device of the invention；
Fig. 5 is growth curve of 8 kinds of test organisms under the conditions of pure culture in embodiment 1；
In figure: 1, pure culture pipe, 2, co-cultivation disk, 3, connecting tube, 4, communicating valve, 5, culture medium thief hatch, 6, culture medium
Re-injection mouth, 7, small ventilative rubber plug, 8, ventilative rubber plug (or half ventilative rubber plug), 9, oxygen-eliminating device, 10, oxygen scavenger room, 11, filter membrane,
12, pure culture pipe lid, 13, decompression capsule, 14, oxygen indicator, 15, deoxygenation room bottom cover, 16, sealing rubber ring, 17, graduation mark.
Specific embodiment
The present invention is further illustrated with comparative example in conjunction with the embodiments, it should explanation, following the description be only for
It explains the present invention, its content is not defined.
Embodiment 1
Effect of the present embodiment using present invention evaluation lactein to enteric microorganism.
Lactein is the metabolite of Bacillus acidi lactici, and drug lactein particle is using lactein as primary raw material
It is made, description of the pharmacology effect partial for this product in package insert --- " selective kill pathogenic entero becteria, protection
Promote the growth of beneficial bacterium ".
Shown in Fig. 1, the structural schematic diagram of the co-culture device of the embodiment of the present invention 1, including pure culture pipe 1 and co-cultivation disk
2, the pure culture pipe 1 is by connecting tube 3 and co-cultures the connection of disk 2；The upper wall for co-culturing disk 2 has culture medium thief hatch 5
With culture medium re-injection mouth 6, small ventilative rubber plug 7 is arranged at the top of the culture medium thief hatch 5 and culture medium re-injection mouth 6；The connection
There is communicating valve 4 on pipe 3.
The connecting tube 3 co-cultured around disk 2 is uniformly distributed, and the quantity of connecting tube is 8；The pure culture pipe 1
Diameter is 4cm, is highly 11cm, is that graduation mark 17 is arranged at 7-9cm in height；The diameter for co-culturing disk 2 is 10cm, is highly
2.5cm；Connecting tube 3 diameter 5mm, length 3cm；The culture medium thief hatch 5 and 6 diameter of culture medium re-injection mouth are 5mm.
The pure culture pipe 1 connects oxygen-eliminating device 9, and the oxygen-eliminating device 9 passes through filter membrane 11 and pure culture pipe by oxygen scavenger room 10
Lid 12 forms, and there are also decompression capsules 13 on pure culture lid 12.
The oxygen scavenger room 10 and 12 material of pure culture pipe lid are pvc, 12 junction of oxygen scavenger room 10 and pure culture pipe lid
There is the filter membrane 11 of pp material.
The pure culture pipe lid 12 and pure culture pipe 1 are connected using sealing rubber ring 16.
(1) the present embodiment lactein solution the preparation method comprises the following steps: take lactein raw material appropriate, diluted with aqua sterilisa,
20% lactein material solution is made, 4000r/min is centrifuged 5min, removes cell, fiber etc. in solution, takes and be rich in
The supernatant of active constituent is spare.
(2) the present embodiment test strain is bifidobacterium longum (BNCC3337084), clostridium butyricum (BNCC337239), excrement
Enterococcus (BNCC186300), Lactobacillus rhamnosus (BNCC134266), bacteroides vulgatus (BNCC186191), lichens gemma bar
Bacterium (BNCC1326654), staphylococcus aureus (CMCC26003), moscow' paratyphi B (CMCC50094).
Above-mentioned strain is seeded in culture medium respectively, 37 DEG C of culture 18h are clear by centrifugal enrichment, sterile saline
It washes, dilute again, turbidimetric analysis turbidimetry, each test organisms is made about 107The bacteria suspension of cfu/ml, it is spare.
(3) the enrichment medium formula of the present embodiment are as follows: tryptone 15g, soybean Papain hydrolysate 3g, beef extract
Powder 3g, yeast extract powder 10g, glucose 10g, lactose 5g, maltose 1.5g, cellobiose 1.5g, soluble starch 1g, mannitol
0.5g, cysteine-hydrochloric acid 0.25g, dipotassium hydrogen phosphate 0.5g, potassium dihydrogen 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, sulphur
Sour copper 0.01g, vitamin K1The extraction of 500ml Roll is added in 0.5ml, sodium chloride 2g, folic acid 1mg, niacin 1mg, biotin 2mg
Liquid, 115 DEG C, sterilize 30min, spare.
Culture medium is that 2 times of enrichment mediums are used after dilution with sterile purified water or sample solution at this time.
(4) the lactein solution of 500ml sterilizing enrichment medium and 500ml20% is uniformly mixed, is slowly added into altogether
Culture apparatus, it is evenly distributed to culture medium, close communicating valve.In 8 pure culture pipes, it is inoculated with 1ml above-mentioned 10 respectively7cfu/ml
Bacteria suspension.It is inoculated with the pure culture of bifidobacterium longum, clostridium butyricum, Lactobacillus rhamnosus, bacteroides vulgatus, bacillus licheniformis
Pipe connects oxygen-eliminating device after the sealing of ventilative rubber plug, forms anaerobic environment；Be inoculated with enterococcus faecalis, Salmonella paratyphi B it is pure
Culture tube simulates micro- aerobic environment with half ventilative rubber plug sealing；It is inoculated with the pure culture pipe of staphylococcus aureus, with ventilative glue
Plug sealing, forms aerobic condition.
(5) co-culture device is transferred in shaken cultivation case, 50~60r/min, after shaken cultivation 1 hour, is opened each
Communicating valve adjusts hunting speed to 10~20r/min, continues to cultivate.
Microorganism growth, breeding and metabolism in logarithmic phase are more vigorous, more sensitive to extraneous environmental change, right
Periods after number is interim, microorganisms gradually generate secondary metabolite, at this point, effect of the drug to microbiologic population, microorganism
Between interaction have typical performance.Therefore, choose as far as possible each test organisms be in logarithmic phase, the culture of rear period
Object sample detection.
According to 8 kinds of test organisms (initial concentrations 105Cfu/ml) growth curve under the conditions of pure culture (See Figure 5),
It was sampled at 10 hours, counts the content of each microorganism in culture medium.
Control group
Sterilizing enrichment medium 500ml in (4) the step of embodiment 1 500ml aqua sterilisa is diluted, is mixed, remaining
Step is the same as embodiment 1.
The counting of microorganism is respectively tested in culture
Selective medium counting method: after sampling, selective medium can be used to count the microorganism in culture
Number.Bifidobacterium longum is counted using mupirocin lithium salts and cysteine hydrochloride modified MRS agar medium；Using containing for many years
Rhzomorph B and the clostridium enriched medium of ampicillin count clostridium butyricum；Using the beef agar containing sodium azide and bilein
Culture medium counts enterococcus faecalis；Lactobacillus rhamnosus is counted using Lincomycin Hydrochloride modified MRS agar medium；Using addition
The xylan carbon source agar medium of kanamycins and vancomycin counts bacteroides vulgatus；Using 55 DEG C of height of propionic acid salt culture medium
Warm cultivation counts bacillus licheniformis；Staphylococcus aureus is counted using BP agar medium；It is selected using SXT4 sramana
Property culture medium count paratyphoid B sramana when bacterium.
Selective medium can be prepared voluntarily, and commercially available finished product culture medium also can be selected.
Nonsele ctive culture media counting method: the culture in the agar medium of non-selectivity, such as above-mentioned " (3) " is used
Base adds 1.5% agar powder, or suitable commercially available culture medium, counts the microorganism in culture.Select colony density suitable
Suitable plate, according to colony characteristics, dyeing microscopic examination or biochemical test of the test microorganism on plate, to therein 50~100
A microorganism is identified, parallel 2 plates, calculates ratio shared by each test microorganism.
Also alternative plate is used in combination with non-selective plates.
Result judgement standard
Probiotics are extremely complex for the adjustment mechanism of enteric microorganism, are reflected in vitro test, can simply manage
Xie Weisan kind mode: the first, probiotics promote growth of probiotics to breed first, probiotics absolute quantity in the unit time
Increase, harmful bacteria accounting relative reduction, probiotics inhibits harmful bacteria by habitat competition, antibiosis metabolic product etc., continuous and harmful
Bacterium absolute quantity reduces；Second, probiotics directly inhibit or kill harmful bacteria, and harmful bacteria absolute quantity reduces, prebiotic
Bacterium is reduced by the Reverse transcriptase pressure from harmful bacteria, mass propagation, and absolute quantity increases；The third, probiotics
Promote growth of probiotics breeding, kills or inhibit harmful bacteria, while probiotics absolute quantity increases, harmful bacteria absolute quantity drop
It is low.
Probiotics are continuous, dynamic, the drug effect possibility from be administered orally to playing for the adjustment process of flora
The time of a couple of days is wanted, and experiment in vitro just provides result within a few hours.Certainly, in vitro test can not simulated gastrointestinal tract digestion
Absorption, the adjusting intervention of immune system and successive administration, drug metabolism etc., only drug directly acts on flora, to bacterium
The rapid contribution that group generates represents flora variation in vivo, developing direction.The probiotics of different probiotics effect or
Harmful bacteria is also not quite similar.Therefore, judgement as a result should not simply with a certain or several specific varied numbers of microorganism come
It indicates, flora should be able to be embodied and totally constituted, increase the trend development of harmful bacteria reduction towards probiotics.
It to sum up analyzes, χ can be used2The method of inspection is examined in control group and sample sets, probiotics and harmful bacteria composition ratio
Between have an indifference, analyze the variation tendency that flora is constituted in sample sets, and combine the actual bacterium amount changing value of every kind of microorganism,
Drug is evaluated to the function and effect of microbial flora.Specific step is as follows: 1. under the action of various factors, testing the numerous of microorganism
Speed difference is grown, causes different microorganisms quantity variance excessive, causes the loss of information in analytic process to amplify, by taking logarithm
Value eliminates this species diversity；2. compared with the control group, calculate the every ml culture of sample sets in, the logarithm value added of each microorganism or
Reduced value；3. carrying out χ with the amount of microorganism in every 1ml culture2Check analysis, sample size are on the low side, in fact it could happen that and theoretical value 1≤
The case where grid number of T≤5 is more than 1/5 can be handled by way of increasing sample size, with microorganism in every 10ml culture
Amount carries out statistical analysis, i.e. 10lg；4. carrying out χ to the composition of probiotics and harmful bacteria in two groups2It examines.5. being sentenced according to result
Calibration is quasi- to carry out result judgement.
1 probiotics activity test in vitro result judgement standard of table
Lactein particle activity test in vitro result:
Content of microorganisms in 2 two groups of cultures of table
The composition of harmful bacteria and probiotics compares in 3 two groups of table
| Group | Bifidobacterium longum | Lactobacillus rhamnosus | Staphylococcus aureus | Salmonella paratyphi B | It is total |
| Sample | 78 | 80 | 45 | 73 | 276 |
| Control | 66 | 75 | 76 | 81 | 298 |
| It is total | 144 | 155 | 121 | 154 | 574 |
4 test result of table
Claims (13)
1. microbiologic population's co-culture device for the evaluation of probiotics external activity, which is characterized in that including pure culture
It manages (1) and co-cultures disk (2), the pure culture pipe (1) is by connecting tube (3) and co-cultures disk (2) connection；The co-cultivation disk
(2) upper wall has culture medium thief hatch (5) and culture medium re-injection mouth (6), the culture medium thief hatch (5) and culture medium re-injection mouth
(6) small ventilative rubber plug (7) is arranged at top；There are communicating valve (4) on the connecting tube (3).
2. co-culture device according to claim 1, which is characterized in that the connecting tube (3) co-cultured around disk (2)
It is uniformly distributed, the quantity of connecting tube is 4-14；The diameter of the pure culture pipe (1) is 4cm, is highly 11cm, is highly being
Graduation mark (17) are set at 7-9cm；The diameter for co-culturing disk (2) is 10cm, is highly 2.5cm；Connecting tube (3) diameter 5mm, it is long
Spend 3cm；The culture medium thief hatch (5) and culture medium re-injection mouth (6) diameter are 5mm.
3. co-culture device according to claim 1 or 2, which is characterized in that rubber plug is arranged at the top of the pure culture pipe (1)
(8)；The rubber plug (8) is ventilative or half is ventilative.
4. co-culture device according to claim 1 or 2, which is characterized in that the pure culture pipe (1) connects oxygen-eliminating device
(9), the oxygen-eliminating device (9) is made up of oxygen scavenger room (10) filter membrane (11) and pure culture pipe lid (12), pure culture lid (12)
It is upper that there are also decompressions capsule (13).
5. co-culture device according to claim 4, which is characterized in that the oxygen scavenger room (10) and pure culture pipe lid
(12) material is pvc, and there is the filter membrane (11) of pp material in oxygen scavenger room (10) and pure culture pipe lid (12) junction.
6. co-culture device according to claim 4, which is characterized in that the pure culture pipe lid (12) and pure culture pipe
(1) it is connected using sealing rubber ring (16).
7. the evaluation method that a kind of microbe colony co-cultures, which is characterized in that use following steps:
(1) the described in any item co-culture systems of claim 2-6 are placed in Biohazard Safety Equipment, from any pure culture pipe (1)
Add fluid nutrient medium, parallel to set sample sets and control group, wherein sample sets are the culture medium containing drug, and are uniformly mixed；
Control group is the culture medium that drug is not added；To culture medium in culture plate (2) and pure culture pipe (1) evenly distributed, pure culture pipe (1)
Middle culture medium reaches graduation mark (17), closes communicating valve (4)；
(2) the corresponding test microorganism of inoculation in pure culture pipe (1), co-culture device is moved into incubator, first preculture
After 0.5-1 hours, communicating valve is opened, continues to mitigate shaken cultivation；
(3) it counts to respectively testing microorganism in culture, calculates the lg value added or reduced value of each microorganism in sample sets；
(4) control group and sample sets probiotics and harmful bacteria composition ratio are examined using the method for chi-square criterion, evaluates drug to micro- life
The function and effect of object flora.
8. evaluation method according to claim 7, which is characterized in that the specific steps of the method for inspection described in step (4)
Are as follows:
(1) after cultivating, the content of each microorganism in analysis of accounts control group and sample sets culture；
(2) compared with the control group, it calculates in the every 1ml culture of sample sets, the logarithm value added or reduced value of each microorganism；
(3) in every 10ml, harmful bacteria in sample sets and control group, probiotics composition ratio carry out chi-square criterion；
(4) result judgement is carried out according to result judgement standard；Wherein result judgement standard are as follows:
。
9. evaluation method according to claim 7, which is characterized in that from culture medium thief hatch (5) in the incubation
Sampling, sampling amount are 1 ~ 5ml, and sampling simultaneously, the aseptic culture medium of sampling amount same volume is added from culture medium re-injection mouth (6).
10. according to evaluation method described in right 7, which is characterized in that after the completion of inoculation described in step (2), need anaerobism item
The microorganism of part seals pure culture pipe (1) mouth with ventilative rubber plug (8), oxygen-eliminating device (9) is then connected pure culture pipe (1), formed
Anaerobic condition has Anaerobic indicator (14) in the oxygen scavenger room (10) of oxygen-eliminating device (9)；The microorganism of micro- aerobic condition is needed, is used
Pure culture pipe (1) mouth is sealed in half ventilative rubber plug (8), forms micro- aerobic condition；The microorganism for needing aerobic conditions, with ventilative rubber plug
(8) pure culture pipe (1) mouth is sealed.
11. according to the described in any item evaluation methods of claim 7-10, which is characterized in that the formula of the fluid nutrient medium
Are as follows: tryptone 15g, soybean Papain hydrolysate 3g, beef extract powder 3g, yeast extract powder 10g, glucose 10g, lactose 5g,
Maltose 1.5g, cellobiose 1.5g, soluble starch 1g, mannitol 0.5g, cysteine-hydrochloric acid 0.25g, dipotassium hydrogen phosphate
0.5g, potassium dihydrogen 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 sulphate 0.01g, vitamin K10.5ml, sodium chloride
500ml Roll leaching liquor, distilled water 500ml is added in 2g, folic acid 1mg, niacin 1mg, biotin 2mg.
12. evaluation method according to claim 11, which is characterized in that the Roll leaching liquor the preparation method comprises the following steps: taking
Calf intestinal 250g, ox colon 250g, aseptic water washing crush, and 1000ml distilled water is added, stirs evenly, and it is small to set 4 DEG C of storages 48
When, filtering takes filtrate to obtain the final product.
13. evaluation method according to claim 7, which is characterized in that test microorganism described in step (2) must wrap simultaneously
Containing probiotics, neutral bacterium and harmful bacteria；Wherein the probiotics be bifidobacterium longum, bifidobacterium infantis, bifidobacterium adolescentis,
One or more of lactobacillus buchneri, Lactobacillus rhamnosus and lactobacillus plantarum；The neutrality bacterium is clostridium butyricum, excrement intestines ball
One of bacterium, streptococcus fecalis, bacteroides vulgatus, bacillus coagulans, bacillus licheniformis, clostridium difficile and mucus Eubacterium
Or it is several；The harmful bacteria is moscow' paratyphi B, vibrio parahemolyticus, staphylococcus aureus, escherichia coli
One or more of bacterium and shigella flexneri；Other type strains can also be used in the probiotics, neutral bacterium and harmful bacteria
Or it is isolated from the non-mode bacterial strain of human or animal's enteron aisle.
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| CN109207406A (en) * | 2018-10-24 | 2019-01-15 | 广东绿百多生物开发有限公司 | A kind of method and culture medium for improving bacillus coagulans cell and forming gemma |
| CN112094735A (en) * | 2020-09-02 | 2020-12-18 | 山东省食品药品检验研究院 | Simulated digestion system device and method for detecting microorganisms in medicine |
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