CN109943486A - Microbial co culture method, micro-fluidic chip and purposes - Google Patents
Microbial co culture method, micro-fluidic chip and purposes Download PDFInfo
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Abstract
The invention discloses a kind of microbial co culture method, micro-fluidic chip and purposes, are related to technical field of biological culture.Microbial co culture method method the following steps are included: prepare the hydrogel microsphere of embedding different microorganisms using micro-fluidic chip respectively, and the hydrogel microsphere for the embedding different microorganisms prepared is delivered in described micro-fluidic chip the same area, it is co-cultured；Wherein, microorganism is at least two kinds.Microbial co culture method of the invention realizes the co-cultivation of multiple-microorganism by microfluidic chip technology and hydrogel microsphere wrapper technology, it being capable of interaction process complicated between two kinds or more of microorganism in simulation true environment very well, being separately separated after being easy to later period microculture simultaneously, it realizes the co-cultivation of multiple-microorganism and is separately separated, and microfluidic chip technology parameter is controllable, convenient for co-culturing the research of process.
Description
Technical field
The present invention relates to technical field of biological culture, in particular to a kind of microbial co culture method, micro-fluidic core
Piece and purposes.
Background technique
The discovery and use of antibiotics be 20th century medical and health field it is greatest contribution one of, it reduce the mankind because
Bacterium or virus infection and have the function of brilliance in terms of bring morbidity and mortality.However, abuse of antibiotics is also people
Class brings new crisis, and according to statistics, the amount by the end of the antibiotic that 2002 use every year reaches 100,000-20 ten thousand tons, from two
Since tenth century the forties, the total output of antibiotic has been more than 1,000,000 tons, this has largely promoted a large amount of multidrug resistances
" superbacteria " appearance, declare that the mankind formally enter " rear antibiotic " epoch.
The sciences problems that multidrug resistance pathogenic bacterial infection is clinically urgent need to resolve are treated, and probiotics is not only adjustable
The intracorporal microecological balance of host, and various active factors secreted by probiotics, can upset, slow down bacterial drug resistance
Generate and diffusion, be a kind of great prospect can partially or completely substitute antibiotics treatment multidrug resistance pathogenic bacterial infection
" green remedy ".Therefore, it studies probiotics and the interaction of pathogen is very necessary.
However, the existing research for probiotics and pathogenic fungi interaction, it is single that the overwhelming majority is built upon change
In the condition of the culture environment of one microorganism, lead to not simulate well in true environment complicated mutual between two kinds of microorganisms
Mechanism.It is the reality in the way of being directly blended that minority studies probiotics with the interaction of pathogen in the way of co-cultivation
Existing, but this mode incubation is uncontrollable, and can not be separately separated to two kinds of microorganisms after cultivating, no
Conducive to the development of follow-up study.There are also only a fews to utilize special device, such as the intermediate U-tube added with semi-permeable membrane, although can be
Two kinds of microorganisms are separated by way of physical barrier, but incubation not easy-regulating, processing difficulties.
In view of this, the present invention is specifically proposed.
Summary of the invention
One of the objects of the present invention is to provide a kind of microbial co culture methods, pass through microfluidic chip technology and water-setting
Glue microsphere encapsulation technology realizes the co-cultivation of multiple-microorganism, being capable of two kinds or more of micro- life in simulation true environment very well
Complicated interaction process between object, while being easy to being separately separated after later period microculture, realize being total to for multiple-microorganism
It cultivates and is separately separated.
The second object of the present invention is to provide a kind of micro-fluidic chip for realizing mentioned microorganism co-culture method, pass through
Microbial co culture process can be realized using the micro-fluidic chip of the structure well.
The third object of the present invention is to provide a kind of mentioned microorganism co-culture method in monitoring microbial co culture mistake
Application in journey in biomass, gene or protein expression variation.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
In a first aspect, the present invention provides a kind of microbial co culture methods, comprising the following steps:
Prepare the hydrogel microsphere of embedding different microorganisms respectively using micro-fluidic chip, and not by the embedding prepared
Hydrogel microsphere with microorganism is delivered in described micro-fluidic chip the same area, is co-cultured；Wherein, microorganism is at least
It is two kinds.
Preferably, on the basis of technical solution provided by the invention, the microorganism be two kinds, preferably probiotics and
Pathogen.
Preferably, on the basis of technical solution provided by the invention, the hydrogel microsphere for embedding different microorganisms is only
On the spot it is prepared by emulsifying/internal gelation reaction.
Preferably, on the basis of technical solution provided by the invention, the system of the hydrogel microsphere of different microorganisms is embedded
Preparation Method independently the following steps are included:
Microorganism is suspended in water phase in micro-fluidic chip, water phase and oil mix to form Water-In-Oil microlayer model, micro-
Drop causes internal gelation reaction under the action of gel triggering agent, forms the hydrogel microsphere for embedding the microorganism；
Preferably, the water phase is the Polymer Solution for being dispersed with insoluble calcium salts；
Preferably, the concentration of the Polymer Solution is 0.1-5g/L；Concentration of the insoluble calcium salts in Polymer Solution is
1-50g/L；
Preferably, the macromolecule is one or more of sodium alginate, pectin, gelatin, agar or hyaluronic acid；
Preferably, concentration of the microorganism in water phase is 104-109CFU/mL；
Preferably, the oil mutually includes one or more of atoleine, olive oil or grape seed oil；
Preferably, contain surfactant in the oily phase；
Preferably, the additional amount of the surfactant is 1-5% (v/v)；
Preferably, the surfactant is one or more of Tween 80, polysorbas20 or sorbester p17；
Preferably, the gel triggering agent of 0.01-0.5% (v/v) is added in oily phase；
Preferably, gel triggering agent is glacial acetic acid；
Preferably, water phase and the oily mutually flow velocity in micro-fluidic chip are 0.01-1mL/min.
Preferably, on the basis of technical solution provided by the invention, the method also includes: embed different microorganisms
After hydrogel microsphere is formed, at least by one of imbedded microbe hydrogel microsphere and said polycation solution hybrid reaction, obtain
To imbedded microbe polyanion-polycation microcapsules, then carry out conveying co-cultivation；
Preferably, the mass percentage concentration of said polycation solution is 0.1-10%；
Preferably, polycation is one or more of chitosan, polylysine or polysulfonate acidic group styrene；
Preferably, the volume ratio of hydrogel microsphere and said polycation solution is 1:1~1:40；
Preferably, mixed reaction time 1-60min.
Preferably, on the basis of technical solution provided by the invention, described micro-fluidic chip the same area is micro-fluidic
Co-cultivation room on chip co-cultures interior and has been passed through culture medium；
Preferably, the volume for co-culturing room is 100-10000mm3；
Preferably, the co-cultivation hydrogel microsphere for the embedding different microorganisms prepared being delivered on micro-fluidic chip
Indoor co-cultivation 2-12h.
Preferably, on the basis of technical solution provided by the invention, the microorganism be probiotics and pathogen, including
Following steps:
(a) probiotics and insoluble calcium salts are suspended in Polymer Solution in micro-fluidic chip, water phase are formed, to oily phase
Middle addition surfactant, water phase and oil mix to form Water-In-Oil microlayer model, and gel triggering agent is added in oily phase, are formed
Embed the hydrogel microsphere of probiotics；
(b) pathogen and insoluble calcium salts are suspended in Polymer Solution in micro-fluidic chip, water phase are formed, to oily phase
Middle addition surfactant, water phase and oil mix to form Water-In-Oil microlayer model, and gel triggering agent is added in oily phase, are formed
Embed the hydrogel microsphere of pathogen；
(c) hydrogel microsphere of the hydrogel microsphere of obtained embedding probiotics and embedding pathogen is delivered to described micro-
It is co-cultured in co-cultivation room on fluidic chip containing culture medium；
In step (c), the hydrogel microsphere and said polycation solution hybrid reaction of probiotics will be embedded, obtain embedding prebiotic
The polyanion-polycation microcapsules of bacterium will embed polyanion-polycation microcapsules and the embedding pathogen of probiotics
Hydrogel microsphere be delivered in the co-cultivation room on the micro-fluidic chip containing culture medium and co-cultured；Or, step (c)
In, the hydrogel microsphere and said polycation solution hybrid reaction of pathogen will be embedded, the polyanion-for obtaining embedding pathogen is poly-
Cationic microgel capsule, the polyanion-polycation microcapsules and the hydrogel microsphere for embedding probiotics that embed pathogen are defeated
It send into the co-cultivation room containing culture medium on the micro-fluidic chip and is co-cultured；Or, will be embedded prebiotic in step (c)
The hydrogel microsphere and said polycation solution hybrid reaction of bacterium, obtain the polyanion-polycation microcapsules of embedding probiotics,
The hydrogel microsphere and said polycation solution hybrid reaction of pathogen will be embedded, obtains the poly- sun of polyanion-of embedding pathogen
Ion microcapsules, will embed the poly- sun of polyanion-of the polyanion-polycation microcapsules and embedding pathogen of probiotics from
Sub- microcapsules are delivered in the co-cultivation room on the micro-fluidic chip containing culture medium and are co-cultured.
Second aspect, it is described micro- the present invention provides a kind of micro-fluidic chip for realizing mentioned microorganism co-culture method
Fluidic chip includes the sealing-in chip that upper layer microfluidic control chip and lower layer are used for sealing-in, and upper layer and lower layer pass through irreversible envelope shape
At micro-fluidic chip；
Microfluidic control chip includes at least two kinds of microbe microsphere preparation modules and co-cultures room；Interior is co-cultured to be connected with
Culture medium；
Culturing room is connected to every kind of microbe microsphere preparation module together, and every kind of microbe microsphere preparation module is independently wrapped
Water phase sample holes and oily phase sample holes are included, water phase sample holes are connected to oily phase sample holes by microfluidic channel.
Preferably, on the basis of technical solution provided by the invention, the microfluidic control chip includes that probiotics is micro-
Ball prepares module, pathogen microballoon preparation module and co-cultures room；
Probiotics microballoon prepares module, and culturing room is connected to together with pathogen microballoon preparation module, the preparation of probiotics microballoon
Module and pathogen microballoon preparation module independently include water phase sample holes and oily phase sample holes, water phase sample holes and oil mutually into
Sample hole is connected to by microfluidic channel；
Polymer Solution, insoluble calcium salts and probiotics enter microfluidic channel, oil phase and gel from water phase sample holes respectively
Initiator enters microfluidic channel from oily phase sample holes respectively, and Water-In-Oil microlayer model is formed after mixing, and microlayer model causes in gel
Cause internal gelation reaction under the action of agent, forms the hydrogel microsphere of embedding probiotics；
Polymer Solution, insoluble calcium salts and pathogen enter microfluidic channel, oil phase and gel from water phase sample holes respectively
Initiator enters microfluidic channel from oily phase sample holes respectively, and Water-In-Oil microlayer model is formed after mixing, and microlayer model causes in gel
Cause internal gelation reaction under the action of agent, forms the hydrogel microsphere of embedding pathogen；
The hydrogel microsphere of the hydrogel microsphere and embedding pathogen that embed probiotics is delivered to altogether by microfluidic channel
Culturing room co-cultures room and is communicated with delivery outlet, for separating the hydrogel microsphere of embedding probiotics and the water-setting of embedding pathogen
Glue microballoon；
Preferably, probiotics microballoon preparation module further includes polycation sample holes；Polycation sample holes, water phase sample introduction
Hole and oily phase sample holes are interconnected by microfluidic channel；
Said polycation solution enters microfluidic channel from polycation sample holes, mixed with the hydrogel microsphere of embedding probiotics
It closes, forms the polyanion-polycation microcapsules of embedding probiotics；Embed the micro- glue of polyanion-polycation of probiotics
Capsule and the hydrogel microsphere of embedding pathogen are delivered to by microfluidic channel and co-culture room；Or,
It further includes polycation sample holes that opportunistic pathogen microballoon, which prepares module,；Polycation sample holes, water phase sample holes and oily phase
Sample holes are interconnected by microfluidic channel；
Said polycation solution enters microfluidic channel from polycation sample holes, mixed with the hydrogel microsphere of embedding pathogen
It closes, forms the polyanion-polycation microcapsules of embedding pathogen；Embed the micro- glue of polyanion-polycation of pathogen
Capsule and the hydrogel microsphere of embedding probiotics are delivered to by microfluidic channel and co-culture room.
The third aspect, the present invention provides a kind of mentioned microorganism co-culture methods during monitoring microbial co culture
Application in biomass, gene or protein expression variation.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method for the present invention is a kind of co-culture method of two or more microorganism, passes through micro-fluidic chip skill
Art and hydrogel microsphere wrapper technology realize the co-cultivation of multiple-microorganism, and the water of corresponding microorganism is prepared for different microorganisms
Then the microballoon for embedding different microorganisms is mixed on micro-fluidic chip and is co-cultured, by this way can by gel micro-ball
Interaction process complicated between two or more microorganisms in enough fine simulation true environments.
(2) co-culture method of the invention, which passes through, is prepared into microballoon for every kind of microorganism embedding, has micropore on microballoon, both
The bioactivity and interaction of microorganism are not influenced, and can effectively be separated microorganism, after being easy to later period microculture
It is separately separated, realizes the co-cultivation of multiple-microorganism and is separately separated, and micro-fluidic includes flow velocity, condition of culture, cell ratio
Example etc. parameters be it is controllable, be conducive to further investigate co-cultivation process, can be used for investigating multiple-microorganism in total training
The variation of biomass, gene and protein expression during supporting.
Detailed description of the invention
Fig. 1 is two kinds of microbial co culture method schematics of one embodiment of the present invention；
Fig. 2 is that the probiotics-pathogen of one embodiment of the present invention co-cultures micro-fluidic chip schematic diagram；
Fig. 3 is the calcium alginate microsphere laser confocal scanning microscope figure (A that dyeing is embedded with pseudomonas aeruginosa anyway
For the calcium alginate microsphere laser confocal scanning microscope figure for being embedded with pseudomonas aeruginosa of life or death dyeing survival, B is dead
The calcium alginate microsphere laser confocal scanning microscope figure for being embedded with pseudomonas aeruginosa of vital staining death)；
Fig. 4 is the present invention using the embedding Lactobacillus rhamnosus microballoon of emulsifying/internal gelation preparation and its point of partial size
(A is embedding Lactobacillus rhamnosus microballoon schematic diagram of the present invention using emulsifying/internal gelation preparation to divergence schematic diagram, and B is this
The dispersion degree schematic diagram for the embedding Lactobacillus rhamnosus microspherulite diameter that invention is prepared using emulsifying/internal gelation)；
Fig. 5 is pseudomonas aeruginosa green fluorescent protein after Lactobacillus rhamnosus of the present invention and pseudomonas aeruginosa co-cultivation
The variation of expression quantity.
Appended drawing reference: 100- probiotics microballoon prepares module；200- pathogen microballoon prepares module；300- co-cultures room；
110- water phase sample holes；120- oil phase sample holes；130- polycation sample holes；140- delivery outlet.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
According to the first aspect of the invention, a kind of microbial co culture method is provided, comprising the following steps:
Prepare the hydrogel microsphere of embedding different microorganisms respectively using micro-fluidic chip, and not by the embedding prepared
Hydrogel microsphere with microorganism is delivered in described micro-fluidic chip the same area, is co-cultured；Wherein, microorganism is at least
It is two kinds.
Microbial co culture method of the invention is the co-culture method of the microorganism with two or more, to micro-
The type of biology is not construed as limiting, and typical but non-limiting microorganism is, for example, bacterium, fungi, virus or some small-sized originals
Raw biology, microalga etc..
Different microorganisms described in the present invention can be to be belonged to not from inhomogeneous microorganism, such as bacterium and fungi
Same microorganism is also possible to belong to the different microorganisms in same category, such as belongs to different microorganisms between different bacterium, no
Belong to different microorganisms etc. with fungi.
Microfluidic chip technology is bases such as biology, chemistry, the sample preparation of medical analysis process, reaction, separation, detections
This operating unit is integrated on the chip of one piece of micro-meter scale, is automatically performed analysis overall process.Micro-fluidic chip is to realize miniflow
The carrier of chip technology is controlled, micro-fluidic chip is using micron order fluid channel come the device for the treatment of fluid.
The present invention embeds the hydrogel microsphere of different microorganisms using microfluidic chip technology preparation, on micro-fluidic chip
It realizes the preparation process of the hydrogel microsphere of embedding different microorganisms and co-cultures process, the hydrogel for embedding different microorganisms is micro-
The preparation process and co-cultivation process of ball can be realized on same micro-fluidic chip.It is may be implemented on the micro-fluidic chip for not
With the preparation of the hydrogel microsphere of microorganism embedding, while can will be prepared by the flowing realization of liquid in micro-fluidic chip
The hydrogel microspheres of different microorganisms be delivered on micro-fluidic chip in the same area, the same area can be in micro-fluidic core
On piece setting co-cultures room, realizes the co-cultivation of multiple-microorganism.
The structure of the micro-fluidic chip is not limited, if can be realized different microorganisms hydrogel microsphere preparation and
Co-cultivation process.
Typical but non-limiting microfluidic chip structure is micro- including the first microbe microsphere preparation module, the second microorganism
Ball prepare module ..., N microbe microsphere preparation module and co-culture module, the first microbe microsphere prepare module, second
Microbe microsphere preparation module ..., N microbe microsphere preparation module independently with co-culture module be connected to, every kind of micro- life
The structure that object microballoon prepares module can use identical structure, and preparation method can use same procedure, the micro- life only embedded
Object is different.The structure of every kind of microbe microsphere preparation module is designed according to the preparation method of the microballoon of typical imbedded microbe
It arrives, structure corresponding to the preparation method of the conventional microballoon that can prepare imbedded microbe can be used to carry out, typical case's embedding
The preparation method of the microballoon of microorganism is, for example, the method for sodium alginate embedding bioactive substance.
For studying the interaction of multiple-microorganism, currently used system is built upon mostly changes single micro- life
In the culture environment of object, and can not interaction complicated between two or more microorganisms in simulation true environment very well
Journey.The mode incubation being directly blended is uncontrollable, and can not be separately separated to two kinds of microorganisms after cultivating, unfavorable
In the development of follow-up study.By the way of although the special device using the U-tube for example with semi-permeable membrane can be by physical barrier
Two kinds of microorganisms are separated, but incubation not easy-regulating, processing difficulties.
The co-culture method of multiple-microorganism of the invention is using microfluidic chip technology for different microorganisms preparation pair
The hydrogel microsphere of microorganism is answered, the microballoon for embedding different microorganisms is mixed on micro-fluidic chip then and is co-cultured,
By this way can interaction process complicated between two or more microorganisms in simulation true environment very well, while this
The co-culture method of invention, which passes through, is prepared into microballoon for every kind of microorganism embedding, has micropore, neither influence microorganism on microballoon
Bioactivity and interaction, and effectively microorganism can be separated, being separately separated after being easy to later period microculture, be realized
It the co-cultivation of multiple-microorganism and is separately separated, and micro-fluidic include the parameters such as flow velocity, condition of culture, cell proportion is controllable
, be conducive to further investigate co-cultivation process.
In a preferred embodiment, microorganism is two kinds, preferably probiotics and pathogen.
As a kind of typical case, which is suitable for two kinds of microorganisms, such as probiotics and pathogen, convenient for research
Interaction between two kinds of microorganisms.
When microorganism is two kinds, typical but non-limiting microfluidic chip structure is prepared including the first microbe microsphere
Module, the second microbe microsphere preparation module and co-cultivation module, the first microbe microsphere prepare module and the second microorganism
Microballoon preparation module is connected to co-cultivation module (co-culturing room) respectively, and the first microbe microsphere preparation module is used to prepare embedding
The hydrogel microsphere of first microorganism, the hydrogel that the second microbe microsphere preparation module is used to prepare the second microorganism of embedding are micro-
Ball, the hydrogel microsphere of the second microorganism of hydrogel microsphere and embedding for the first microorganism of embedding being prepared pass through micro-fluidic
Liquid flowing on chip is delivered to co-culture to be co-cultured in module.First microbe microsphere prepares module and second micro- life
The counter structure of the hydrogel microsphere wrapper technology of existing achievable imbedded microbe can be used in object microballoon preparation module.
In a preferred embodiment, the hydrogel microspheres of different microorganisms is embedded independently by emulsification/interior
Portion's gelation reaction is prepared.
The hydrogel microsphere of imbedded microbe is prepared on micro-fluidic chip using emulsifying/internal gelation technique.
"/" in emulsifying/internal gelation indicates "-", that is, the meaning of sum.Pass through emulsion process and gelation
Journey prepares hydrogel microsphere, and wherein gelation uses internal gelation.
It is sodium alginate packet that sodium alginate extruding, which is added dropwise in ionic calcium soln, with syringe at present and forms gel micro-ball
Bury the main method of bioactive substance.Since calcium ion gradually penetrates into inside from droplet surface, this method is referred to as
Exterior gel.
Internal gelation method, which refers to, mixes insoluble calcium salt (such as calcium carbonate) with sodium alginate, is then formed in oily phase steady
Determine lotion, adds the molten glacial acetic acid of oil and cause calcium carbonate dissociation, discharge calcium ion, form gel micro-ball from drop internal.The party
Method can prepare the lesser gel micro-ball of partial size, it is easy to accomplish.
Preferably, the partial size for the hydrogel microsphere prepared is 50-500 μm.
In a preferred embodiment, the preparation method for embedding the hydrogel microsphere of different microorganisms independently includes
Following steps:
Microorganism is suspended in water phase in micro-fluidic chip, water phase and oil mix to form Water-In-Oil microlayer model, micro-
Drop causes internal gelation reaction under the action of gel triggering agent, forms the hydrogel microsphere for embedding the microorganism.
Emulsifying/internal gelation technique is carried out in micro-fluidic chip, microorganism is suspended in water phase, with microorganism
Water phase forms Water-In-Oil microlayer model in oily phase, then the gel triggering agent by being added causes internal gelation reaction, forms packet
Bury the hydrogel microsphere of microorganism.
Preferably, water phase is the Polymer Solution for being dispersed with insoluble calcium salts.
Typical insoluble calcium salts are calcium carbonate.
Preferably, the concentration of Polymer Solution is 0.1-5g/L；Concentration of the insoluble calcium salts in Polymer Solution is 1-
50g/L。
Polymer Solution is typical but non-limiting concentration be, for example, 0.1g/L, 0.2g/L, 0.5g/L, 1g/L, 2g/L,
3g/L、4g/L、5g/L。
Concentration of the insoluble calcium salts in Polymer Solution be, for example, 1g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L,
30g/L, 35g/L, 40g/L, 45g/L or 50g/L.
Preferably, macromolecule is natural polymer.
Preferably, macromolecule is one or more of sodium alginate, pectin, gelatin, agar or hyaluronic acid.
Preferably, concentration of the microorganism in water phase is 104-109CFU/mL。
Typical but non-limiting concentration of the microorganism in water phase is, for example, 104CFU/mL、105CFU/mL、106CFU/
mL、107CFU/mL、108CFU/mL or 109CFU/mL。
Preferably, oil mutually includes one or more of atoleine, olive oil or grape seed oil.
Preferably, contain surfactant in oily phase.
Preferably, the additional amount of surfactant is 1-5% (v/v).
V/v refers to the ratio between addition volume and oil phase volume of surfactant herein, typical but non-limiting to be, for example,
1%, 2%, 3%, 4% or 5%.
Preferably, surfactant is one or more of Tween 80, polysorbas20 or sorbester p17.
Preferably, the gel triggering agent of 0.01-0.5% (v/v) is added in oily phase.
V/v refers to the ratio between addition volume and oil phase volume of gel triggering agent herein, typical but non-limiting to be, for example,
0.01%, 0.02%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%.
Preferably, gel triggering agent is glacial acetic acid.
Cause insoluble calcium salts dissociation by the molten glacial acetic acid of oil, discharge calcium ion, forms gel micro-ball from drop internal.
Preferably, water phase and the oily mutually flow velocity in micro-fluidic chip are 0.01-1mL/min, such as 0.01mL/min,
0.02mL/min, 0.05mL/min, 0.1mL/min, 0.2mL/min, 0.5mL/min, 0.8mL/min or 1mL/min.
Flow rate of liquid can realize controllable adjustment by additional liquid pump in micro-fluidic chip, pass through the parameters such as coutroi velocity
It can be realized the controllable of incubation.
In a preferred embodiment, in method further include: embed different microorganisms hydrogel microsphere formed after,
At least by one of imbedded microbe hydrogel microsphere and said polycation solution hybrid reaction, obtain the poly- yin of imbedded microbe from
Son-polycation microcapsules, then carry out conveying co-cultivation.
Since hydrogel microsphere has tridimensional network, there is porous structure, water-setting is typically formed by sodium alginate
Carboxylate radical is had in glue microballoon monomer residue, is a kind of polyanionic structure.Hydrogel microsphere hole is larger, microorganism volume one
As it is smaller, in order to avoid microorganism is flowed out out of microballoon hole, after the hydrogel microsphere of imbedded microbe is formed, allow microballoon
Reacted with said polycation solution, formed polyanion-polycation be complexed film, ultimately form be embedded with microorganism it is poly- yin from
Son-polycation microcapsules, typical microcapsules are sodium alginate-chitosan microcapsules.
The differentiation standard of polycation (polycation) or polyanion (polyanion) is consolidating on macromolecular chain
Determine charge, it is exactly polycation that fixed charge, which is positive charge, and fixed charge is that negative electrical charge is exactly polyanion.It is typical but
Unrestricted polycation is chitosan, polylysine or polysulfonate acidic group styrene.
The step can be the hydrogel microsphere progress polycation processing for one of microorganism, and forming embedding should
The polyanion-polycation microcapsules of kind microorganism, then carry out conveying co-cultivation with other microballoons again, can also be for every
A kind of hydrogel microsphere of microorganism carries out polycation processing, and the polyanion-for being respectively formed embedding different microorganisms is poly-
Cationic microgel capsule, then the polyanion-polycation microcapsules for embedding different microorganisms are subjected to the step of conveying co-cultures.
Preferably, the mass percentage concentration of said polycation solution be 0.1-10%, such as 0.1%, 0.5%, 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.
Preferably, the volume ratio of microballoon and said polycation solution is 1:1~1:40, such as 1:1,1:2,1:5,1:10,1:
15,1:20,1:25,1:30,1:35 or 1:40.
Preferably, mixed reaction time 1-60min, for example, 1min, 5min, 10min, 15min, 20min, 25min,
30min, 40min, 50min or 60min.
In a preferred embodiment, micro-fluidic chip the same area is the co-cultivation room on micro-fluidic chip, altogether
Culture medium has been passed through in culturing room.
Room is co-cultured by setting on micro-fluidic chip, the microballoon for embedding different microorganisms is carried out in co-culturing room
It co-cultures, to facilitate simulation and study the interaction between different microorganisms.
Preferably, the volume for co-culturing room is 100-10000mm3, for example, 100mm3、1000mm3Or 10000mm3。
Preferably, the co-cultivation hydrogel microsphere for the embedding different microorganisms prepared being delivered on micro-fluidic chip
Indoor co-cultivation 2-12h, such as 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h or 12h.
By culture a period of time, the interaction between microorganism can be investigated, it is micro- during being co-cultured convenient for research
The variation of biotinylated biomolecule amount, gene and protein expression.
As a kind of typical microbial co culture method, i.e. probiotics-pathogen co-culture method, as shown in Figure 1, packet
Include following steps:
(a) probiotics and insoluble calcium salts are suspended in Polymer Solution in micro-fluidic chip, water phase are formed, to oily phase
Middle addition surfactant, water phase and oil mix to form Water-In-Oil microlayer model, and gel triggering agent is added in oily phase, are formed
Embed the hydrogel microsphere of probiotics；
(b) pathogen and insoluble calcium salts are suspended in Polymer Solution in micro-fluidic chip, water phase are formed, to oily phase
Middle addition surfactant, water phase and oil mix to form Water-In-Oil microlayer model, and gel triggering agent is added in oily phase, are formed
Embed the hydrogel microsphere of pathogen；
(c) hydrogel microsphere of the hydrogel microsphere of obtained embedding probiotics and embedding pathogen is delivered to described micro-
It is co-cultured in co-cultivation room on fluidic chip containing culture medium.
Typical but non-limiting probiotics is, for example, bifidobacterium lactis, lactobacillus acidophilus, sandlwood saccharobacillus, long bifid bar
Bacterium or Lactobacillus casei etc..
Typical but non-limiting pathogen is, for example, pseudomonas aeruginosa, staphylococcus glucose coccus, enterococcus or lung
Scorching streptococcus etc..
The probiotics-pathogen co-culture method is realized in micro-fluidic chip, first prepares embedding probiotics respectively
Obtain two kinds of microbe microspheres, are then delivered to and co-culture in room by the hydrogel microsphere of hydrogel microsphere and embedding pathogen
It is co-cultured.
The structure of micro-fluidic chip is not construed as limiting, as long as can be realized the preparation of probiotics and pathogen hydrogel microsphere
It, can be according to the hydrogel microsphere of the hydrogel microsphere and embedding pathogen of above-mentioned embedding probiotics with co-cultivation process
Preparation method is voluntarily related to microfluidic chip structure and pipeline, or uses the existing miniflow that above-mentioned preparation process may be implemented
Control chip.
Preferably, micro-fluidic chip includes probiotics microballoon preparation module, pathogen microballoon preparation module and co-cultures mould
Block (co-cultures room).
By preparing the hydrogel microsphere of embedding probiotics and pathogen respectively using microfluidic chip technology, pass through miniflow
Two kinds of microballoons are transported to by the flow rate of liquid in control chip to be co-cultured room and co-cultures, this mode realize to probiotics and
The controllable encapsulating of pathogen, microballoon inner cell activity is good, can probiotics and pathogen are multiple in simulation true environment very well
Miscellaneous interaction process, while two kinds of microorganisms after co-cultivation can be separately separated, it is conducive to follow-up study.
Preferably, in step (c), the hydrogel microsphere and said polycation solution hybrid reaction of probiotics will be embedded, obtained
The polyanion-polycation microcapsules for embedding probiotics will embed the polyanion-polycation microcapsules and packet of probiotics
The hydrogel microsphere for burying pathogen, which is delivered in the co-cultivation room on the micro-fluidic chip containing culture medium, to be co-cultured；
Or, the hydrogel microsphere and said polycation solution hybrid reaction of pathogen being embedded in step (c), obtaining embedding pathogen
Polyanion-polycation microcapsules will embed the water of the polyanion-polycation microcapsules and embedding probiotics of pathogen
Gel micro-ball is delivered in the co-cultivation room on the micro-fluidic chip containing culture medium and is co-cultured；Or, in step (c),
The hydrogel microsphere and said polycation solution hybrid reaction of probiotics will be embedded, obtains the poly- sun of polyanion-of embedding probiotics
Ion microcapsules will embed the hydrogel microsphere and said polycation solution hybrid reaction of pathogen, obtain the poly- of embedding pathogen
Anion-polycation microcapsules will embed the poly- yin of the polyanion-polycation microcapsules and embedding pathogen of probiotics
Ion-polycation microcapsules are delivered in the co-cultivation room on the micro-fluidic chip containing culture medium and are co-cultured.
In step (c), the hydrogel microsphere that step (a) embeds probiotics can further be reacted with polycation, be obtained
It is defeated to the polyanion-polycation microcapsules of embedding probiotics, then with the hydrogel microsphere progress of step (b) embedding pathogen
It send and co-cultures；Alternatively, the hydrogel microsphere that step (b) embeds pathogen can further be reacted with polycation, obtain
The polyanion-polycation microcapsules of pathogen are embedded, then are conveyed with the hydrogel microsphere of step (a) embedding probiotics
And co-cultivation；Alternatively, can be simultaneously by the water of the hydrogel microsphere of step (a) embedding probiotics and step (b) embedding pathogen
Gel micro-ball is further reacted with polycation, obtains the polyanion-polycation microcapsules and embedding disease of embedding probiotics
The polyanion-polycation microcapsules of opportunistic pathogen, then the two is conveyed and co-cultured.
By at least one of hydrogel microsphere for will embedding the hydrogel microsphere of probiotics and pathogen will be embedded into
The processing of row polycation, further makes probiotics and pathogen individualism after processing, avoids microorganism out of, microballoon hole
Outflow.
According to the second aspect of the invention, a kind of micro-fluidic chip for realizing microbial co culture method is provided, is wrapped
It includes upper layer microfluidic control chip and lower layer is used for the sealing-in chip of sealing-in, upper layer and lower layer form micro-fluidic core by irreversible envelope
Piece；
Microfluidic control chip includes at least two kinds of microbe microsphere preparation modules and co-cultures room；Interior is co-cultured to be connected with
Culture medium；
Culturing room is connected to every kind of microbe microsphere preparation module together, and every kind of microbe microsphere preparation module is independently wrapped
Water phase sample holes and oily phase sample holes are included, water phase sample holes are connected to oily phase sample holes by microfluidic channel.
Culture medium is co-culture medium, can select the conventional medium for being suitable for cultivating microorganism.
In use, Polymer Solution, insoluble calcium salts and every kind of microorganism enter microfluidic channel from water phase sample holes respectively,
Oily phase, surfactant and gel triggering agent enter microfluidic channel from oily phase sample holes respectively, and it is micro- that Water-In-Oil is formed after mixing
Drop, microlayer model cause internal gelation reaction under the action of gel triggering agent, and the hydrogel that formation embeds the microorganism is micro-
Ball；The hydrogel microsphere of every kind of microorganism is delivered to co-cultivation room by microfluidic channel and is co-cultured, and co-cultures room connection
There is delivery outlet, for separating every kind of microorganism hydrogel microsphere.
Preferably, the flow rate of liquid in channel is regulated and controled by additional liquid pump.
Preferably as a kind of typical realization probiotics-pathogen co-culture method micro-fluidic chip, such as Fig. 2 institute
Show, microfluidic control chip includes probiotics microballoon preparation module 100, pathogen microballoon preparation module 200 and co-cultures room
300；
Probiotics microballoon prepares module 100, and culturing room 300 is connected to together with pathogen microballoon preparation module 200, prebiotic
It independently includes water phase sample holes 110 and oily phase sample holes that bacterium microballoon, which prepares module 100 and pathogen microballoon preparation module 200,
120, water phase sample holes 110 are connected to oily phase sample holes 120 by microfluidic channel；
Polymer Solution, insoluble calcium salts and probiotics enter microfluidic channel from water phase sample holes 110 respectively, and oil is mutually and solidifying
Glue initiator enters microfluidic channel from oily phase sample holes 120 respectively, Water-In-Oil microlayer model is formed after mixing, microlayer model is in gel
Cause internal gelation reaction under the action of initiator, forms the hydrogel microsphere of embedding probiotics；
Polymer Solution, insoluble calcium salts and pathogen enter microfluidic channel from water phase sample holes 110 respectively, and oil is mutually and solidifying
Glue initiator enters microfluidic channel from oily phase sample holes 120 respectively, Water-In-Oil microlayer model is formed after mixing, microlayer model is in gel
Cause internal gelation reaction under the action of initiator, forms the hydrogel microsphere of embedding pathogen；
The hydrogel microsphere of the hydrogel microsphere and embedding pathogen that embed probiotics is delivered to altogether by microfluidic channel
Culturing room 300 co-cultures room 300 and is communicated with delivery outlet 140, for separating the hydrogel microsphere and embedding cause of disease of embedding probiotics
The hydrogel microsphere of bacterium.
The hydrogel microsphere of embedding probiotics and the hydrogel microsphere of embedding pathogen, can be real by the difference of particle size
Now it is separately separated.
It should be noted that description and the mentioned microorganism of Polymer Solution, insoluble calcium salts, oil phase and gel triggering agent are total
Description in cultural method is identical, and details are not described herein.
In a preferred embodiment, probiotics microballoon preparation module further includes polycation sample holes 130；Poly- sun
Ion sample holes, water phase sample holes and oily phase sample holes are interconnected by microfluidic channel；
Said polycation solution enters microfluidic channel from polycation sample holes 130, micro- with the hydrogel of embedding probiotics
Ball mixing, forms the polyanion-polycation microcapsules of embedding probiotics；Embed the polyanion-polycation of probiotics
Microcapsules and the hydrogel microsphere of embedding pathogen are delivered to by microfluidic channel and co-culture room；Or,
It further includes polycation sample holes 130 that opportunistic pathogen microballoon, which prepares module,；Polycation sample holes, water phase sample holes and oil
Phase sample holes are interconnected by microfluidic channel；
Said polycation solution enters microfluidic channel from polycation sample holes 130, micro- with the hydrogel of embedding pathogen
Ball mixing, forms the polyanion-polycation microcapsules of embedding pathogen；Embed the polyanion-polycation of pathogen
Microcapsules and the hydrogel microsphere of embedding probiotics are delivered to by microfluidic channel and co-culture room.
It should be noted that the description of said polycation solution is identical as the description in mentioned microorganism co-culture method,
This is repeated no more.
According to the third aspect of the present invention, a kind of mentioned microorganism co-culture method is provided to train altogether in monitoring microorganism
Application during supporting in biomass, gene or protein expression variation.
It, being capable of phase complicated between two kinds or more of microorganism in simulation true environment very well by using the above method
Interaction process, convenient for investigating two kinds and two or more microorganisms biomass, gene or protein expression during co-cultivation
Variation, be conducive to provide more scientific foundation.
In order to further appreciate that the present invention, the method for the present invention and effect are done further in detail combined with specific embodiments below
Explanation.
Embodiment 1-5 is to use to realize with the micro-fluidic chip of flowering structure: micro-fluidic chip includes probiotics microballoon system
Standby module, pathogen microballoon preparation module and co-cultivation room；Probiotics microballoon prepares module and pathogen microballoon preparation module is equal
Culturing room is connected to together, and probiotics microballoon preparation module includes water phase sample holes, oily phase sample holes and polycation sample holes, water
Phase sample holes, oily phase sample holes are connected to polycation sample holes by microfluidic channel；Pathogen microballoon prepares module
Water phase sample holes and oily phase sample holes, water phase sample holes and oily phase sample holes are connected to by microfluidic channel.
Embodiment 1
A kind of co-culture method of probiotics-pathogen, probiotics are sandlwood saccharobacillus；Pathogen is P. aeruginosa
Bacterium；The following steps are included:
(a) it the hydrogel microsphere of preparation embedding probiotics: is added into the water phase sample holes of probiotics microballoon preparation module
Sodium alginate soln, calcium carbonate and probiotics；To probiotics microballoon preparation module oily phase sample holes in be added atoleine and
Tween 80, and glacial acetic acid is added, water phase and oil form Water-In-Oil microlayer model, effect of the microlayer model in gel triggering agent after mixing
Lower initiation internal gelation reaction, forms the hydrogel microsphere of embedding probiotics；
Wherein, the concentration of sodium alginate soln is 0.1g/L；Concentration of the calcium carbonate in Polymer Solution is 1g/L；It is prebiotic
The inoculum density of bacterium is 106CFU/mL；
The additional amount of Tween 80 is 1% (v/v)；The additional amount of glacial acetic acid is 0.01% (v/v)；
(b) the polyanion-polycation microcapsules of preparation embedding probiotics: to the poly- of probiotics microballoon preparation module
Chitosan solution is added in cationic sample holes, embeds the hydrogel microsphere and chitosan solution hybrid reaction of probiotics, is formed
Embed the polyanion-polycation microcapsules of probiotics；
Wherein, the mass percentage concentration of chitosan solution is 0.1%；The volume ratio of hydrogel microsphere and chitosan solution is
1:1；Mixed reaction time is 1min；
(c) it the hydrogel microsphere of preparation embedding pathogen: is added into the water phase sample holes of pathogen microballoon preparation module
Sodium alginate soln, calcium carbonate and probiotics；To pathogen microballoon preparation module oily phase sample holes in be added atoleine and
Tween 80, and glacial acetic acid is added, water phase and oil form Water-In-Oil microlayer model, effect of the microlayer model in gel triggering agent after mixing
Lower initiation internal gelation reaction, forms the hydrogel microsphere of embedding pathogen；
Wherein, the concentration of sodium alginate soln is 0.1g/L；Concentration of the calcium carbonate in Polymer Solution is 1g/L；It is prebiotic
The inoculum density of bacterium is 106CFU/mL；
The additional amount of Tween 80 is 1% (v/v)；The additional amount of glacial acetic acid is 0.01% (v/v)；
(d) the polyanion-polycation microcapsules and the hydrogel microsphere for embedding pathogen that embed probiotics are defeated
It send to interior is co-cultured, is passed through 10mL culture medium, cultivate 2h.
Embodiment 2
A kind of co-culture method of probiotics-pathogen, probiotics is bifidobacterium lactis；Pathogen is staphylococcus glucose
Coccus；The following steps are included:
(a) it the hydrogel microsphere of preparation embedding probiotics: is added into the water phase sample holes of probiotics microballoon preparation module
Pectin solution, calcium carbonate and probiotics；Atoleine and tween is added into the oily phase sample holes of probiotics microballoon preparation module
20, and glacial acetic acid is added, water phase and oil form Water-In-Oil microlayer model after mixing, and microlayer model draws under the action of gel triggering agent
Internal gelation reaction is sent out, the hydrogel microsphere of embedding probiotics is formed；
Wherein, the concentration of pectin solution is 5g/L；Concentration of the calcium carbonate in Polymer Solution is 50g/L；Probiotics
Inoculum density is 108CFU/mL；
The additional amount of polysorbas20 is 5% (v/v)；The additional amount of glacial acetic acid is 0.5% (v/v)；
(b) the polyanion-polycation microcapsules of preparation embedding probiotics: to the poly- of probiotics microballoon preparation module
Polyacrylic acid solution is added in cationic sample holes, embeds the hydrogel microsphere and polyacrylic acid solution hybrid reaction of probiotics,
Form the polyanion-polycation microcapsules of embedding probiotics；
Wherein, the mass percentage concentration of polyacrylic acid solution is 10%；The volume of hydrogel microsphere and polyacrylic acid solution
Than for 1:40；Mixed reaction time is 60min；
(c) it the hydrogel microsphere of preparation embedding pathogen: is added into the water phase sample holes of pathogen microballoon preparation module
Pectin solution, calcium carbonate and probiotics；Atoleine and tween is added into the oily phase sample holes of pathogen microballoon preparation module
20, and glacial acetic acid is added, water phase and oil form Water-In-Oil microlayer model after mixing, and microlayer model draws under the action of gel triggering agent
Internal gelation reaction is sent out, the hydrogel microsphere of embedding pathogen is formed；
Wherein, the concentration of pectin solution is 5g/L；Concentration of the calcium carbonate in Polymer Solution is 50g/L；Probiotics
Inoculum density is 108CFU/mL；
The additional amount of polysorbas20 is 1-5% (v/v)；The additional amount of glacial acetic acid is 0.01-0.5% (v/v)；
(d) the polyanion-polycation microcapsules and the hydrogel microsphere for embedding pathogen that embed probiotics are defeated
It send to interior is co-cultured, is passed through 0.1mL culture medium, cultivate 12h.
Embodiment 3
A kind of co-culture method of probiotics-pathogen, probiotics is lactobacillus acidophilus；Pathogen is enterococcus；Including
Following steps:
(a) it the hydrogel microsphere of preparation embedding probiotics: is added into the water phase sample holes of probiotics microballoon preparation module
Gelatin solution, calcium carbonate and probiotics；Atoleine and sapn is added into the oily phase sample holes of probiotics microballoon preparation module
80, and glacial acetic acid is added, water phase and oil form Water-In-Oil microlayer model after mixing, and microlayer model draws under the action of gel triggering agent
Internal gelation reaction is sent out, the hydrogel microsphere of embedding probiotics is formed；
Wherein, the concentration of gelatin solution is 1g/L；Concentration of the calcium carbonate in Polymer Solution is 5g/L；Probiotics connects
Kind density is 107CFU/mL；
The additional amount of sorbester p17 is 2% (v/v)；The additional amount of glacial acetic acid is 0.1% (v/v)；
(b) the polyanion-polycation microcapsules of preparation embedding probiotics: to the poly- of probiotics microballoon preparation module
Polysulfonate acidic group styrene solution is added in cationic sample holes, hydrogel microsphere and the polysulfonate acidic group styrene for embedding probiotics are molten
Liquid hybrid reaction forms the polyanion-polycation microcapsules of embedding probiotics；
Wherein, the mass percentage concentration of polysulfonate acidic group styrene solution is 0.5%；Hydrogel microsphere and poly- sulfonic benzo second
The volume ratio of alkene solution is 1:10；Mixed reaction time is 10min；
(c) it the hydrogel microsphere of preparation embedding pathogen: is added into the water phase sample holes of pathogen microballoon preparation module
Gelatin solution, calcium carbonate and probiotics；Atoleine and sapn is added into the oily phase sample holes of pathogen microballoon preparation module
80, and glacial acetic acid is added, water phase and oil form Water-In-Oil microlayer model after mixing, and microlayer model draws under the action of gel triggering agent
Internal gelation reaction is sent out, the hydrogel microsphere of embedding pathogen is formed；
Wherein, the concentration of gelatin solution is 1g/L；Concentration of the calcium carbonate in Polymer Solution is 5g/L；Probiotics connects
Kind density is 107CFU/mL；
The additional amount of sorbester p17 is 2% (v/v)；The additional amount of glacial acetic acid is 0.1% (v/v)；
(d) the polyanion-polycation microcapsules and the hydrogel microsphere for embedding pathogen that embed probiotics are defeated
It send to interior is co-cultured, is passed through 1mL culture medium, cultivate 5h.
Embodiment 4
A kind of co-culture method of probiotics-pathogen, probiotics is Lactobacillus casei；Pathogen is streptococcus pneumonia；
The following steps are included:
(a) it the hydrogel microsphere of preparation embedding probiotics: is added into the water phase sample holes of probiotics microballoon preparation module
Agar solution, calcium carbonate and probiotics；Grape seed oil and sapn is added into the oily phase sample holes of probiotics microballoon preparation module
80, and glacial acetic acid is added, water phase and oil form Water-In-Oil microlayer model after mixing, and microlayer model draws under the action of gel triggering agent
Internal gelation reaction is sent out, the hydrogel microsphere of embedding probiotics is formed；
Wherein, the concentration of agar solution is 2g/L；Concentration of the calcium carbonate in Polymer Solution is 10g/L；Probiotics
Inoculum density is 107CFU/mL；
The additional amount of sorbester p17 is 3% (v/v)；The additional amount of glacial acetic acid is 0.2% (v/v)；
(b) the polyanion-polycation microcapsules of preparation embedding probiotics: to the poly- of probiotics microballoon preparation module
Chitosan solution is added in cationic sample holes, embeds the hydrogel microsphere and chitosan solution hybrid reaction of probiotics, is formed
Embed the polyanion-polycation microcapsules of probiotics；
Wherein, the mass percentage concentration of chitosan solution is 1%；The volume ratio of hydrogel microsphere and chitosan solution is 1:
20；Mixed reaction time is 20min；
(c) it the hydrogel microsphere of preparation embedding pathogen: is added into the water phase sample holes of pathogen microballoon preparation module
Agar solution, insoluble calcium salts and probiotics；Grape seed oil and department is added into the oily phase sample holes of pathogen microballoon preparation module
Disk 80, and glacial acetic acid is added, water phase and oil form Water-In-Oil microlayer model after mixing, and microlayer model is under the action of gel triggering agent
Cause internal gelation reaction, forms the hydrogel microsphere of embedding pathogen；
Wherein, the concentration of agar solution is 2g/L；Concentration of the calcium carbonate in Polymer Solution is 10g/L；Probiotics
Inoculum density is 107CFU/mL；
The additional amount of sorbester p17 is 3% (v/v)；The additional amount of glacial acetic acid is 0.2% (v/v)；
(d) the polyanion-polycation microcapsules and the hydrogel microsphere for embedding pathogen that embed probiotics are defeated
It send to interior is co-cultured, is passed through 2mL culture medium, cultivate 6h.
Embodiment 5
A kind of co-culture method of probiotics-pathogen, probiotics are sandlwood saccharobacillus；Pathogen is P. aeruginosa
Bacterium；The following steps are included:
(a) it the hydrogel microsphere of preparation embedding probiotics: is added into the water phase sample holes of probiotics microballoon preparation module
Hyaluronic acid solution, calcium carbonate and probiotics；Olive oil is added into the oily phase sample holes of probiotics microballoon preparation module and spits
Temperature 80, and is added glacial acetic acid, and water phase and oil form Water-In-Oil microlayer model after mixing, and microlayer model is under the action of gel triggering agent
Cause internal gelation reaction, forms the hydrogel microsphere of embedding probiotics；
Wherein, the concentration of hyaluronic acid solution is 4g/L；Concentration of the calcium carbonate in Polymer Solution is 20g/L；It is prebiotic
The inoculum density of bacterium is 108CFU/mL；
The additional amount of Tween 80 is 4% (v/v)；The additional amount of glacial acetic acid is 0.5% (v/v)；
(b) the polyanion-polycation microcapsules of preparation embedding probiotics: to the poly- of probiotics microballoon preparation module
Chitosan solution is added in cationic sample holes, embeds the hydrogel microsphere and chitosan solution hybrid reaction of probiotics, is formed
Embed the polyanion-polycation microcapsules of probiotics；
Wherein, the mass percentage concentration of chitosan solution is 5%；The volume ratio of hydrogel microsphere and chitosan solution is 1:
30；Mixed reaction time is 30min；
(c) it the hydrogel microsphere of preparation embedding pathogen: is added into the water phase sample holes of pathogen microballoon preparation module
Hyaluronic acid solution, calcium carbonate and probiotics；Olive oil is added into the oily phase sample holes of pathogen microballoon preparation module and spits
Temperature 80, and is added glacial acetic acid, and water phase and oil form Water-In-Oil microlayer model after mixing, and microlayer model is under the action of gel triggering agent
Cause internal gelation reaction, forms the hydrogel microsphere of embedding pathogen；
Wherein, the concentration of hyaluronic acid solution is 4g/L；Concentration of the calcium carbonate in Polymer Solution is 20g/L；It is prebiotic
The inoculum density of bacterium is 108CFU/mL；
The additional amount of Tween 80 is 4% (v/v)；The additional amount of glacial acetic acid is 0.5% (v/v)；
(d) the polyanion-polycation microcapsules and the hydrogel microsphere for embedding pathogen that embed probiotics are defeated
It send to interior is co-cultured, is passed through 5mL culture medium, cultivate 10h.
Embodiment 6
Sandlwood saccharobacillus and pseudomonas aeruginosa are co-cultured according to the co-culture method of embodiment 1, monitors co-cultivation process
The variation of middle biomass, gene or protein expression, as a result as shown in Figure 5.
As a result, it has been found that: the pseudomonas aeruginosa of expressing green fluorescent protein plasmid is carried in co-culture system, it is green
The expression quantity of color fluorescin is significantly lowered, and great variety has occurred in its biomass during co-cultivation of indirect reaction.
Probiotics and pathogen are co-cultured in co-cultivation interior by the method for embodiment 1-5, study it mutually
Interactively, can interaction process complicated between two kinds or more of microorganism in simulation true environment very well, simultaneously
Being separately separated after being easy to later period microculture is realized the co-cultivation of multiple-microorganism and is separately separated, and micro-fluidic chip
Technical parameter is controllable, by adjustment parameter, convenient for co-culturing the research of process.
Fig. 3 is the calcium alginate microsphere laser confocal scanning microscope figure that dyeing is embedded with pseudomonas aeruginosa anyway,
A is embedding Lactobacillus rhamnosus microballoon schematic diagram of the present invention using emulsifying/internal gelation preparation in Fig. 3, and B is this in Fig. 3
The dispersion degree schematic diagram for the embedding Lactobacillus rhamnosus microspherulite diameter that invention is prepared using emulsifying/internal gelation；Fig. 4 is this hair
It is bright using the embedding Lactobacillus rhamnosus microballoon of emulsifying/internal gelation preparation and its dispersion degree schematic diagram of partial size, A in Fig. 4
The embedding Lactobacillus rhamnosus microballoon schematic diagram of emulsifying/internal gelation preparation is used for the present invention, B adopts in Fig. 4 for the present invention
With the dispersion degree schematic diagram of embedding Lactobacillus rhamnosus microspherulite diameter prepared by emulsifying/internal gelation.
The method of the present invention can be realized using micro-fluidic chip to probiotics and pathogen it can be seen from Fig. 3 and Fig. 4
Effectively embedding, microballoon inner cell activity is good, be embedded with pseudomonas aeruginosa calcium alginate microsphere dye anyway after 90% with
Upper activity is very well.
The above results can be seen that the probiotics that the method for the present invention is realized and pathogen co-culture system, can be fine
Be applied to co-cultivation during, the inspection of the variation for the one or two kinds of and above microbes biomass or gene expression amount
It surveys.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from of the invention
Many other change and modification can be made in the case where spirit and scope.It is, therefore, intended that wrapping in the following claims
Include all such changes and modifications belonged in the scope of the invention.
Claims (10)
1. a kind of microbial co culture method, which comprises the following steps:
Prepare the hydrogel microsphere of embedding different microorganisms respectively using micro-fluidic chip, and the embedding difference prepared is micro-
The hydrogel microsphere of biology is delivered in described micro-fluidic chip the same area, is co-cultured；Wherein, microorganism is at least two
Kind.
2. microbial co culture method described in accordance with the claim 1, which is characterized in that the microorganism is two kinds, preferably
Probiotics and pathogen.
3. microbial co culture method according to claim 1 or 2, which is characterized in that embed the water-setting of different microorganisms
Glue microballoon is prepared independently by emulsifying/internal gelation reaction.
4. microbial co culture method described in accordance with the claim 3, which is characterized in that the hydrogel for embedding different microorganisms is micro-
The preparation method of ball independently the following steps are included:
Microorganism is suspended in water phase in micro-fluidic chip, water phase and oil mix to form Water-In-Oil microlayer model, microlayer model
Cause internal gelation reaction under the action of gel triggering agent, forms the hydrogel microsphere for embedding the microorganism；
Preferably, the water phase is the Polymer Solution for being dispersed with insoluble calcium salts；
Preferably, the concentration of the Polymer Solution is 0.1-5g/L；Concentration of the insoluble calcium salts in Polymer Solution is 1-
50g/L；
Preferably, the macromolecule is one or more of sodium alginate, pectin, gelatin, agar or hyaluronic acid；
Preferably, concentration of the microorganism in water phase is 104-109CFU/mL；
Preferably, the oil mutually includes one or more of atoleine, olive oil or grape seed oil；
Preferably, contain surfactant in the oily phase；
Preferably, the additional amount of the surfactant is 1-5% (v/v)；
Preferably, the surfactant is one or more of Tween 80, polysorbas20 or sorbester p17；
Preferably, the gel triggering agent of 0.01-0.5% (v/v) is added in oily phase；
Preferably, gel triggering agent is glacial acetic acid；
Preferably, water phase and the oily mutually flow velocity in micro-fluidic chip are 0.01-1mL/min.
5. microbial co culture method according to claim 4, which is characterized in that the method also includes: embedding is different
After the hydrogel microsphere of microorganism is formed, at least one of imbedded microbe hydrogel microsphere is mixed with said polycation solution
Reaction, obtains imbedded microbe polyanion-polycation microcapsules, then carry out conveying co-cultivation；
Preferably, the mass percentage concentration of said polycation solution is 0.1-10%；
Preferably, polycation is one or more of chitosan, polylysine or polysulfonate acidic group styrene；
Preferably, the volume ratio of hydrogel microsphere and said polycation solution is 1:1~1:40；
Preferably, mixed reaction time 1-60min.
6. microbial co culture method according to claim 1 or 2, which is characterized in that the same area of micro-fluidic chip
Domain is the co-cultivation room on micro-fluidic chip, co-cultures interior and has been passed through co-culture medium；
Preferably, the volume for co-culturing room is 100-10000mm3；
Preferably, the hydrogel microsphere for the embedding different microorganisms prepared is delivered to the interior of the co-cultivation on micro-fluidic chip
Co-culture 2-12h.
7. microbial co culture method according to claim 1 or 2, which is characterized in that the microorganism be probiotics and
Pathogen, comprising the following steps:
(a) probiotics and insoluble calcium salts are suspended in Polymer Solution in micro-fluidic chip, water phase are formed, to oily Xiang Zhongjia
Enter surfactant, water phase and oil mix to form Water-In-Oil microlayer model, and gel triggering agent is added in oily phase, form embedding
The hydrogel microsphere of probiotics；
(b) pathogen and insoluble calcium salts are suspended in Polymer Solution in micro-fluidic chip, water phase are formed, to oily Xiang Zhongjia
Enter surfactant, water phase and oil mix to form Water-In-Oil microlayer model, and gel triggering agent is added in oily phase, form embedding
The hydrogel microsphere of pathogen；
(c) hydrogel microsphere of the hydrogel microsphere of obtained embedding probiotics and embedding pathogen is delivered to described micro-fluidic
It is co-cultured in co-cultivation room on chip containing culture medium；
In step (c), the hydrogel microsphere and said polycation solution hybrid reaction of probiotics will be embedded, obtain embedding probiotics
Polyanion-polycation microcapsules will embed the water of the polyanion-polycation microcapsules and embedding pathogen of probiotics
Gel micro-ball is delivered in the co-cultivation room on the micro-fluidic chip containing culture medium and is co-cultured；Or, in step (c),
The hydrogel microsphere and said polycation solution hybrid reaction of pathogen will be embedded, obtains the poly- sun of polyanion-of embedding pathogen
Ion microcapsules convey the hydrogel microsphere of the polyanion-polycation microcapsules for embedding pathogen and embedding probiotics
It is co-cultured in the co-cultivation room containing culture medium on to the micro-fluidic chip；Or, probiotics will be embedded in step (c)
Hydrogel microsphere and said polycation solution hybrid reaction, obtain embedding probiotics polyanion-polycation microcapsules, will
The hydrogel microsphere and said polycation solution hybrid reaction for embedding pathogen, obtain the poly- sun of polyanion-of embedding pathogen from
Sub- microcapsules will embed the polyanion-polycation of the polyanion-polycation microcapsules and embedding pathogen of probiotics
Microcapsules are delivered in the co-cultivation room on the micro-fluidic chip containing culture medium and are co-cultured.
8. a kind of micro-fluidic chip for realizing the described in any item microbial co culture methods of claim 1-7, which is characterized in that
The micro-fluidic chip includes the sealing-in chip that upper layer microfluidic control chip and lower layer are used for sealing-in, and upper layer and lower layer pass through can not
Inverse envelope forms micro-fluidic chip；
Microfluidic control chip includes at least two kinds of microbe microsphere preparation modules and co-cultures room；It co-cultures interior and is connected with culture
Base；
Culturing room is connected to every kind of microbe microsphere preparation module together, and every kind of microbe microsphere preparation module independently includes water
Phase sample holes and oily phase sample holes, water phase sample holes and oily phase sample holes are connected to by microfluidic channel.
9. micro-fluidic chip according to claim 8, which is characterized in that the microfluidic control chip includes that probiotics is micro-
Ball prepares module, pathogen microballoon preparation module and co-cultures room；
Probiotics microballoon prepares module, and culturing room is connected to together with pathogen microballoon preparation module, and probiotics microballoon prepares module
It independently include water phase sample holes and oily phase sample holes with pathogen microballoon preparation module, water phase sample holes and oily phase sample holes
It is connected to by microfluidic channel；
Polymer Solution, insoluble calcium salts and probiotics enter microfluidic channel from water phase sample holes respectively, and oil mutually causes with gel
Agent enters microfluidic channel from oily phase sample holes respectively, Water-In-Oil microlayer model is formed after mixing, microlayer model is in gel triggering agent
Effect is lower to cause internal gelation reaction, forms the hydrogel microsphere of embedding probiotics；
Polymer Solution, insoluble calcium salts and pathogen enter microfluidic channel from water phase sample holes respectively, and oil mutually causes with gel
Agent enters microfluidic channel from oily phase sample holes respectively, Water-In-Oil microlayer model is formed after mixing, microlayer model is in gel triggering agent
Effect is lower to cause internal gelation reaction, forms the hydrogel microsphere of embedding pathogen；
The hydrogel microsphere of the hydrogel microsphere and embedding pathogen that embed probiotics is delivered to co-cultivation by microfluidic channel
Room co-cultures room and is communicated with delivery outlet, and the hydrogel microsphere and the hydrogel for embedding pathogen for separating embedding probiotics are micro-
Ball；
Preferably, probiotics microballoon preparation module further includes polycation sample holes；Polycation sample holes, water phase sample holes and
Oily phase sample holes are interconnected by microfluidic channel；
Said polycation solution enters microfluidic channel from polycation sample holes, mixes with the hydrogel microsphere of embedding probiotics,
Form the polyanion-polycation microcapsules of embedding probiotics；Embed probiotics polyanion-polycation microcapsules and
The hydrogel microsphere for embedding pathogen is delivered to by microfluidic channel co-cultures room；Or,
It further includes polycation sample holes that opportunistic pathogen microballoon, which prepares module,；Polycation sample holes, water phase sample holes and oily phase sample introduction
Hole is interconnected by microfluidic channel；
Said polycation solution enters microfluidic channel from polycation sample holes, mixes with the hydrogel microsphere of embedding pathogen,
Form the polyanion-polycation microcapsules of embedding pathogen；Embed pathogen polyanion-polycation microcapsules and
The hydrogel microsphere for embedding probiotics is delivered to by microfluidic channel co-cultures room.
10. the described in any item microbial co culture methods of claim 1-7 during monitoring microbial co culture biomass,
Application in gene or protein expression variation.
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