CN110452887A - A kind of bacteriophage protective agent and its application - Google Patents
A kind of bacteriophage protective agent and its application Download PDFInfo
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- CN110452887A CN110452887A CN201910647890.8A CN201910647890A CN110452887A CN 110452887 A CN110452887 A CN 110452887A CN 201910647890 A CN201910647890 A CN 201910647890A CN 110452887 A CN110452887 A CN 110452887A
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- bacteriophage
- protective agent
- inulin
- preparation
- sodium
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
Abstract
The present invention relates to a kind of bacteriophage protective agent and its application, the bacteriophage protective agent includes following components: inulin, trehalose, polyvinylpyrrolidone and skimmed milk power.The bacteriophage protective agent can also include sodium alginate.Bacteriophage protective agent is applied in the protection of bacteriophage HS15, Vibrio Parahaemolyticus Bacteriophages or vibrio alginolyticus bacteriophage etc.; may make bacteriophage at normal temperature has good survival rate; by then passing through after addition sodium alginate is prepared into micro-capsule, survival benefit of the bacteriophage in room temperature even at 37 DEG C can be obviously improved.It efficiently solves the problems, such as that bacteriophage can only save at low temperature, and the preservation of a variety of bacteriophages can be suitable for.
Description
Technical field
The present invention relates to microorganisms technical fields, and in particular to a kind of bacteriophage protective agent and its application.
Background technique
The problem of superbacteria caused by the abuse of antibiotic and drug residue of food, causes the extensive concern in the world,
Multinational government has also promulgated that corresponding laws and regulations or measure promote " nonreactive process ".Bacteriophage is bacterial infection, fungi, algae
The viral general name of the microorganisms such as class, actinomyces or conveyor screw, because part can cause the cracking of host strain, therefore referred to as bacteriophage.By
Have many advantages, such as that safe and efficient, high specificity, self duplication are fast, the screening period is short in it, therefore, in recent years about by phagocytosis
Body receives common concern as the research of antibiotic preparation, and the especially application in fields such as medicine, cultivation and food receives
The extensive concern of domestic and foreign scholars becomes current one of research hotspot.There is phage preparation product in food in succession
It is applied in processing and fresh-keeping, animal productiong, purification of water quality and Human clinical, it can be seen that, the development prospect of bacteriophage
It is boundless.However, in the application process of bacteriophage, since bacteriophage is easy to be contaminated, and must be under cryogenic
(2~8 DEG C) preservations, so that bacteriophage development, largely by being limited, therefore, the Techniques of preserving of bacteriophage is to restrict to bite
The widely applied important technology problem of thallus.
Trehalose is a kind of nonreducing sugar being made of 2 glucose molecules with 1,1- glycosidic bond, has 3 kinds of isomers i.e. extra large
Algae sugar (α, α), isotrehalose (β, β) and neotrehalose (α, β), and there is non-specific protection to make various bioactivators
With.Since trehalose can be formed solely under the severe environmental conditions such as high temperature, high and cold, hyperosmosis and dry dehydration in cell surface
Special protective film is effectively protected protein molecule invariance inactivation, thus the life process and biological characteristic of the body that sustains life.
Therefore, compared with other function oligosaccharide, trehalose glass transition temperature is higher, hygroscopicity is lower and without reproducibility, has more
Broad application prospect.Chinese invention patent application file CN201610415395.0 discloses a kind of bacteriophage preservation protective agent
And the preparation method and application thereof, wherein contain the Multiple components such as trehalose in the preservation protective agent and preparation process is complicated, because
This, finds out a kind of ingredient and the simple bacteriophage protective agent of preparation process is of great significance.
Summary of the invention
The first technical problem to be solved by the present invention is: providing a kind of ingredient and the simple bacteriophage of preparation process protects
Protect agent.
Second technical problem to be solved by this invention is: providing a kind of protectant application of above-mentioned bacteriophage.
In order to solve above-mentioned first technical problem, the technical solution adopted by the present invention are as follows: a kind of bacteriophage protective agent, institute
Stating bacteriophage protective agent includes following components: inulin, trehalose, polyvinylpyrrolidone and skimmed milk power.
Further, the bacteriophage protective agent further includes sodium alginate and sodium carboxymethylcellulose.
Further, the mass ratio of the sodium alginate and sodium carboxymethylcellulose is 1:(0.5~1.5)；Preferably 1:
1。
Preferably, the protective agent is dissolved in buffer, and the mass concentration of the inulin is (50~150) g/L；It is preferred that
Ground, the mass concentration of the inulin are (100~150) g/L.
Further, the buffer is SM buffer；Preferably, the SM buffer is conventionally prepared, tool
Body composition is as follows: NaCl 5.89g, MgSO4·7H2O 29g, Tris-HCl (1mol/L, pH7.5) 50ml, 2% gelatin 5ml steams
Distilled water 1000ml.
The beneficial effects of the present invention are: the present invention program is made by compounding the functional oligoses such as inulin and trehalose
The resilience that bacteriophage damages variation of ambient temperature bring can be enhanced for protective agent, improve its survival rate, it can
Suitable for a variety of bacteriophages；The preservative agent component of the present invention program is simple, reduces production cost and technical complexity, and this hair
The protective agent of bright scheme makes phagocytosis physical efficiency still keep higher survival rate at room temperature, overcomes the bacteriophage in traditional technology
It is only capable of the limitation saved at low temperature, reduces the cost in bacteriophage transport and storage process.
In order to solve above-mentioned second technical problem, the technical solution adopted by the present invention are as follows: a kind of phage preparation, it is described
Preparation includes above-mentioned protective agent and bacteriophage.
Preferably, the volume ratio of the protective agent and bacteriophage is 1:(1.5~2.5)；Preferably 1:2.
Further, the bacteriophage includes bacteriophage HS15, Vibrio Parahaemolyticus Bacteriophages or vibrio alginolyticus bacteriophage
At least one of.
A kind of preparation method of phage preparation, the raw material of the phage preparation include bacteriophage and protective agent, described
Protective agent includes inulin, trehalose, polyvinylpyrrolidone, skimmed milk power, sodium alginate and sodium carboxymethylcellulose, including with
Lower step: taking the bacteriophage of inulin, trehalose, polyvinylpyrrolidone, skimmed milk power, sodium alginate, after being dissolved in buffer,
It is mixed in instillation calcium chloride solution fixed-type.
Further, the ratio between mass concentration of the calcium chloride and sodium alginate is 1:(1~2)；Preferably 1:1.5.
Further, the weight concentration of calcium chloride is (10~20) g/L, preferably 15g/L in the calcium chloride solution.
The beneficial effects of the present invention are: the present invention program passes through single step reaction method for bacteriophage, sodium carboxymethylcellulose
And sodium alginate, it is fixed-type to instill calcium chloride mixing.The method preparation process is simple, and encystation speed is fast, the thickness of film, hole
The mechanical strength of diameter, surface charge and packing can be controlled by adjusting the concentration of sodium alginate and calcium chloride, this packing
Active material can be fixed to improve its stability, the rate of release of bacteriophage, Neng Gouti can also be regulated and controled by technique for packing
For the more growing spaces of bacteriophage and better mass exchange, phage preparation in application process, can preferably discharge.
Specific embodiment
To explain the technical content, the achieved purpose and the effect of the present invention in detail, it is explained below in conjunction with embodiment.
The embodiment of the present invention one are as follows: a kind of bacteriophage protective agent, including following prepare raw material inulin, trehalose, polyethylene
Pyrrolones and skimmed milk power, preparation method are that the above-mentioned raw material for preparing is dissolved in SM buffer, and the formula of SM buffer is as follows:
NaCl 5.89g, MgSO4·7H2O 29g, 1M Tris-HCl (pH 7.5) 50ml, 2% gelatin 5ml, distilled water 1000ml, system
The addition of standby raw material is such as follows: inulin (5%, 10% or 15%) w/v, trehalose 5%w/v, polyvinylpyrrolidone 1%w/
V, skimmed milk power 5%w/v.
The protective agent is applied to the preservation of bacteriophage HS15:
One, the preparation of phage suspensions:
Host strain ATCC17802 is inoculated into 2216E fluid nutrient medium, shaken cultivation 10h (bacterial population about 108/ ml),
Bacteriophage HS15 is inoculated, continues culture and thoroughly clarifies 5 000 × g centrifugation 5min to bacterium solution, collect the supernatant containing bacteriophage and make
For phage suspensions.
Two, it saves:
Bacteriophage protective agent is added in phage suspensions plus by volume for 1:2,0.15ml volume dispenses multitube, considers
Preservation problem in actual application is stored in 4 DEG C, room temperature (25 DEG C), 37 DEG C respectively.
Three, it detects:
It uses double-layer agar technique to detect preservation effect: saving pipe with the suspension of original volume (100 μ l) SM buffer solution, Qi Tabao
It deposits group and also respectively takes 100 μ l of phage suspensions, make 10 times of serial dilutions with SM buffer solution respectively, take each 100 μ l of pipe dilution, point
Each 900 μ l host strain solution (bacterial concentration 10 is not added7/ ml), it mixes, 37 DEG C are incubated overnight.Then appropriate volume is taken
The phagocytosis body fluid and host's bacterium solution of (200 μ l) mix, and 2216E semisolid culturemedium is added and topples over double-layer plate, 37 DEG C of trainings overnight
It supports, counts plaque number.Each sample is in triplicate.
Testing result is as follows:
Influence of the different inulin concentration to bacteriophage preservation effect in 1 embodiment of the present invention 1 of table
Bacteriophage preservation effect difference in 10% and 15% inulin is not significant, but is significantly better than in 5% inulin,
In view of the use cost of reagent, 10% inulin is selected to use concentration as preservation.
Select preservation of 10% inulin for vibrio parahaemolyticus phage, preservation effect such as the following table 2 institute under different temperatures
Show:
Table 2 uses the preservation effect of protectant vibrio parahaemolyticus phage of the embodiment of the present invention 1
From Table 2, it can be seen that added with protectant bacteriophage survival rate be substantially better than plus, especially in room temperature
Under, effect is more prominent, can promote 2~3 orders of magnitude.
The embodiment of the present invention two are as follows: a kind of bacteriophage protective agent, including following prepare raw material inulin, trehalose, polyethylene
Pyrrolones, skimmed milk power, sodium alginate and sodium carboxymethylcellulose, preparation method are to prepare raw material and bacteriophage is molten for above-mentioned
In SM buffer (NaCl 5.89g, MgSO4·7H2O 29g, 1M Tris-HCl (pH 7.5) 50ml, 2% gelatin 5ml, distillation
Water 1000ml) in be then added dropwise to the micro-capsule that spherical particle is fixed into 1.5%w/v calcium chloride.Wherein, inulin 10%w/v, sea
Algae sugar 5%w/v, polyvinylpyrrolidone 1%w/v, skimmed milk power 5%w/v, sodium alginate 1%w/v, sodium carboxymethylcellulose
1%w/v.
The protective agent is applied to the preservation of vibrio parahaemolyticus phage and vibrio alginolyticus bacteriophage, wherein phage suspensions
Preparation process referring to embodiment 1, weigh 0.1g spherical particle and be placed in 1.5mlEP pipe, add 1ml SM buffer solution,
It is stored in 4 DEG C, room temperature (25 DEG C), 37 DEG C respectively.
Similarly, preservation effect is measured using double-layer plate detection method based on use of bacteriophages for detection hiological, operating procedure is similar to embodiment
One, the measurement result of vibrio parahaemolyticus phage is as shown in table 3 below:
The protectant protecting effect of bacteriophage of 3 embodiment of the present invention 2 of table
As can be seen from the above table, protective agent is prepared into micro-capsule with bacteriophage may make the survival rate of bacteriophage further
It is promoted.
The measurement result of vibrio alginolyticus bacteriophage is as shown in table 4 below:
The protectant protecting effect of bacteriophage of 4 embodiment of the present invention 2 of table
It is demonstrated experimentally that the protective agent of the component can reduce the rate of bacteriophage inactivation, the survival rate of bacteriophage is improved, especially
It is the survival rate that can be improved bacteriophage under room temperature state.This experiment used in bacteriophage, can high temperature resistant, therefore,
After two months with 37 DEG C of processing, bacteriophage still being capable of large number of viable.The technology of bacteriophage is wrapped up in particular by sodium alginate,
Survival rate of the bacteriophage at 37 DEG C can be significantly improved.
Inulin is a kind of biological polyoses for being widely present in nature, and various plants and vegetables are present in the form of energy
In, especially largely it is present in compositae plant such as jerusalem artichoke, in the root tuber of witloof etc., is mainly made of fructose and glucose, wherein
Fructose molecule passes through β-(2,1) glucosides key connection.For extent of polymerization from 2~60 (usually 10), terminal is glucose unit, point
Minor can use GFnIt indicates, wherein G is terminal glucose unit, and F represents fructose molecule, and n then represents fructose molecular number.Work as polymerization
(Degree of Polymerization, DP=2~9) are properly termed as oligofructose (Fructo Oligo when spending lower
Saccharide).Bacterium can be enhanced to the resilience of Freeze-drying Damage as freeze drying protectant in functional oligose, improves it
Survival rate.In addition, inulin, which due to its special structure, can selectively promote Bifidobacterium and Bacillus acidi lactici etc. in enteron aisle, to be had
The growth of beneficial bacteria inhibits the growth and breeding of the pathogenic microorganisms such as Escherichia coli, creates ideal animal intestinal micro-ecology environment, mention
High breeding performonce fo animals and immune function.Inulin is not digested utilization in enteron aisle, it is a kind of prebiotics, is a kind of feed addition
Agent promotes beneficial bacterium growth, and internal noresidue do not develop drug resistance, and inulin is a kind of worth development and application in aquaculture
Resource.
Sodium alginate (Sodium alginate, abbreviation SA) be by beta-D-mannuronic acid and α-L- guluronic acid by
(1 → 4) polysaccharide made of being keyed.Method using sodium alginate as microcapsule wall material is that it at room temperature can shape
At thermal stability gel, the gelation that SA has had due to its aqueous solution, stability and high viscosity, therefore, in food, cosmetics
With additive and adjuvant are commonly used in pharmaceuticals industry.In addition, SA is wide due to its hypotoxicity and excellent biocompatibility
It is general to be applied to prepare biomedical material.Alginate can be used as filmogen in preparation microbial capsules, which is
By being cross-linked to form between the carboxylate anion and calcium ion of guluronic acid, when the sodium ion in sodium alginate is chlorinated calcium
In calcium ion replace after can form gel.
Bacteriophage HS15 in above-described embodiment is isolated from the sewage of Guangzhou fish market acquisition, host strain pair haemolysis arc
Bacterium ATCC17802 is purchased from Institute of Micro-biology, Guangdong Province Culture Collection Center.2216E fluid nutrient medium and 2216E Semi-solid cell culture
Base is commercially available.The representative " g/100ml " of " %w/v " in embodiment.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalents made by bright description are applied directly or indirectly in relevant technical field, are similarly included in this hair
In bright scope of patent protection.
Claims (10)
1. a kind of bacteriophage protective agent, it is characterised in that: the bacteriophage protective agent includes following components: inulin, gathers trehalose
Vinylpyrrolidone and skimmed milk power.
2. bacteriophage protective agent according to claim 1, it is characterised in that: the bacteriophage protective agent further includes alginic acid
Sodium and sodium carboxymethylcellulose.
3. bacteriophage protective agent according to claim 1, it is characterised in that: the sodium alginate and sodium carboxymethylcellulose
Mass ratio be 1:(0.5~1.5)；Preferably 1:1.
4. bacteriophage protective agent according to claim 1, it is characterised in that: the protective agent is dissolved in buffer, institute
The mass concentration for stating inulin is (50~150) g/L；Preferably, the mass concentration of the inulin is (100~150) g/L.
5. bacteriophage protective agent according to claim 1, it is characterised in that: the buffer is SM buffer.
6. a kind of phage preparation, it is characterised in that: the preparation includes the protective agent and phagocytosis as described in Claims 1 to 5
Body.
7. phage preparation according to claim 6, it is characterised in that: the volume ratio of the protective agent and bacteriophage is 1:
(1.5~2.5)；Preferably 1:2.
8. phage preparation according to claim 6, it is characterised in that: the bacteriophage includes bacteriophage HS15, secondary molten
At least one of hemorrhagic Vibriophage or vibrio alginolyticus bacteriophage.
9. a kind of preparation method of phage preparation, the raw material of the phage preparation includes bacteriophage and protective agent, feature
Be: the protective agent includes inulin, trehalose, polyvinylpyrrolidone, skimmed milk power, sodium alginate and carboxymethyl cellulose
Sodium, comprising the following steps: the bacteriophage for taking inulin, trehalose, polyvinylpyrrolidone, skimmed milk power, sodium alginate is dissolved in
After buffer, mixed in instillation calcium chloride solution fixed-type.
10. the preparation method of phage preparation according to claim 9, it is characterised in that: the calcium chloride and alginic acid
The ratio between mass concentration of sodium is 1:(1~2)；Preferably 1:1.5.
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