CN110452887B - Phage protective agent and application thereof - Google Patents
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- CN110452887B CN110452887B CN201910647890.8A CN201910647890A CN110452887B CN 110452887 B CN110452887 B CN 110452887B CN 201910647890 A CN201910647890 A CN 201910647890A CN 110452887 B CN110452887 B CN 110452887B
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Abstract
The invention relates to a phage protective agent and application thereof, wherein the phage protective agent comprises the following components: inulin, trehalose, polyvinylpyrrolidone and skimmed milk powder. The phage protective agent may also include sodium alginate. The phage protective agent is applied to the protection of phage HS15, vibrio parahaemolyticus phage or vibrio alginolyticus phage and the like, so that the phage has good survival rate at normal temperature, and the survival effect of the phage at room temperature even 37 ℃ can be remarkably improved after the phage is prepared into the microcapsule by adding sodium alginate. Effectively solves the problem that the bacteriophage can only be preserved at low temperature, and can be suitable for the preservation of various bacteriophages.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a phage protective agent and application thereof.
Background
The problem of drug residues in superbacteria and food products caused by abuse of antibiotics has attracted much attention in the world, and corresponding laws and regulations or measures have been promulgated by various governments to promote "nonreactive processes". The phage is a generic term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, and spirochetes, and is called phage because it partially causes lysis of host bacteria. Because of the advantages of safety, high efficiency, strong specificity, fast self-proliferation, short screening period and the like, the method has attracted general attention in recent years on the research of using the phage as an antibacterial agent, especially has attracted wide attention of scholars at home and abroad in the fields of medicine, cultivation, food and the like, and becomes one of the current research hotspots. The application of phage preparation products in food processing and preservation, animal production, water purification and human clinical treatment is sequentially appeared, so that the development prospect of the phage is very wide. However, in the application process of the phage, the phage is easily polluted and must be stored at low temperature (2-8 ℃), so that the development of the phage is greatly limited, and therefore, the phage storage technology is an important technical problem which restricts the wide application of the phage.
Trehalose is a non-reducing sugar consisting of 2 glucose molecules with 1, 1-glycosidic bonds, has 3 isomers, namely trehalose (α, α), isophyiosaccharide (β, β) and neotrehalose (α, β), and has a non-specific protective effect on a variety of bioactive substances. The trehalose can form a unique protective film on the cell surface under severe environmental conditions of high temperature, high cold, high osmotic pressure, drying and dehydration and the like, so that the protein molecules are effectively protected from being inactivated, and the life process and biological characteristics of a living body are maintained. Therefore, compared with other functional oligosaccharides, the trehalose has higher glass transition temperature, lower hygroscopicity, no reducibility and wider application prospect. The Chinese patent application document CN201610415395.0 discloses a phage preservation protective agent and a preparation method and application thereof, wherein the preservation protective agent contains various components such as trehalose and the like and has a complex preparation process, so that finding out a phage protective agent with simple components and a simple preparation process is of great significance.
Disclosure of Invention
The first technical problem to be solved by the invention is as follows: provides a phage protective agent with simple components and preparation process.
The second technical problem to be solved by the invention is: provides an application of the phage protectant.
In order to solve the first technical problem, the invention adopts the technical scheme that: a phage protective agent comprising the following components: inulin, trehalose, polyvinylpyrrolidone and skimmed milk powder.
Further, the phage protective agent also comprises sodium alginate and sodium carboxymethyl cellulose.
Further, the mass ratio of the sodium alginate to the sodium carboxymethyl cellulose is 1 (0.5-1.5); preferably 1:1.
Preferably, the protective agent is dissolved in a buffer solution, and the mass concentration of the inulin is (50-150) g/L; preferably, the mass concentration of the inulin is (100-150) g/L.
Further, the buffer solution is SM buffer solution; preferably, the SM buffer is in accordance with the conventional methodThe preparation method comprises the following steps: NaCl 5.89g, MgSO4·7H2O29 g, Tris-HCl (1mol/L, pH7.5)50ml, 2% gelatin 5ml, distilled water 1000 ml.
The invention has the beneficial effects that: according to the scheme, inulin, trehalose and other functional oligosaccharides are compounded to serve as a protective agent, so that the resistance of the phage to damage caused by environmental temperature change can be enhanced, the survival rate of the phage is improved, and the protective agent can be suitable for various phages; the preservative provided by the scheme of the invention has simple components, reduces the production cost and the technical complexity, and the protective agent provided by the scheme of the invention enables the bacteriophage to maintain a higher survival rate at room temperature, overcomes the limitation that the bacteriophage in the traditional technology can be preserved only at low temperature, and reduces the cost in the transportation and storage processes of the bacteriophage.
In order to solve the second technical problem, the invention adopts the technical scheme that: a phage preparation comprising the protective agent and phage described above.
Preferably, the volume ratio of the protective agent to the phage is 1 (1.5-2.5); preferably 1: 2.
Further, the bacteriophage comprises at least one of bacteriophage HS15, Vibrio parahaemolyticus bacteriophage, or Vibrio alginolyticus bacteriophage.
A preparation method of a phage preparation, wherein the raw materials of the phage preparation comprise phage and a protective agent, the protective agent comprises inulin, trehalose, polyvinylpyrrolidone, skim milk powder, sodium alginate and sodium carboxymethylcellulose, and the preparation method comprises the following steps: dissolving phage of inulin, trehalose, polyvinylpyrrolidone, skimmed milk powder and sodium alginate in buffer solution, dropping into calcium chloride solution, mixing, fixing and molding.
Further, the mass concentration ratio of the calcium chloride to the sodium alginate is 1 (1-2); preferably 1: 1.5.
Furthermore, the weight concentration of calcium chloride in the calcium chloride solution is (10-20) g/L, and preferably 15 g/L.
The invention has the beneficial effects that: the invention adopts the scheme that phage, sodium carboxymethylcellulose and sodium alginate are dripped into calcium chloride to be mixed, fixed and molded by a one-step reaction method. The method has simple preparation process and high capsulizing speed, the thickness, the pore diameter, the surface charge and the mechanical strength of the membrane can be controlled by adjusting the concentrations of sodium alginate and calcium chloride, the capsulizing can fix active substances to improve the stability of the active substances, the release rate of the phage can be regulated and controlled by a wrapping technology, more growth spaces and better substance exchange of the phage can be provided, and the phage preparation can be better released in the application process.
Detailed Description
In order to explain the technical content, the objects and the effects of the present invention in detail, the following description will be given with reference to the embodiments.
The first embodiment of the invention is as follows: a phage protectant comprises the following preparation raw materials of inulin, trehalose, polyvinylpyrrolidone and skim milk powder, and the preparation method is to dissolve the preparation raw materials in SM buffer solution, wherein the formula of the SM buffer solution is as follows: NaCl 5.89g, MgSO4·7H2O29 g, 1M Tris-HCl (pH 7.5)50ml, 2% gelatin 5ml, distilled water 1000ml, the addition of preparation raw materials was as follows: inulin (5%, 10% or 15%) w/v, trehalose 5% w/v, polyvinylpyrrolidone 1% w/v, skimmed milk powder 5% w/v.
The protective agent is applied to preservation of phage HS 15:
firstly, preparing a phage suspension:
inoculating host bacteria ATCC17802 into 2216E liquid culture medium, and shake culturing for 10h (bacterial count is about 10)8And/ml), inoculating bacteriophage HS15, continuously culturing until bacterial liquid is thoroughly clarified to 5000 × g, centrifuging for 5min, and collecting the supernatant containing the bacteriophage as bacteriophage suspension.
II, storage:
the phage protective agent is added into the phage suspension according to the volume ratio of 1:2, and the phage suspension is divided into multiple tubes with the volume of 0.15ml, and the multiple tubes are respectively stored at 4 ℃, room temperature (25 ℃) and 37 ℃ in consideration of the storage problem in the practical application process.
Thirdly, detection:
the preservation effect is detected by adopting a double-layer flat plate method: the storage tubes were buffered with SM at the original volume (100. mu.l)Suspending with dissolving solution, respectively taking 100 μ l phage suspension for other preservation groups, respectively diluting with SM buffer solution by 10 times, respectively, taking 100 μ l dilution, respectively adding 900 μ l host bacteria solution (bacteria concentration is 10)7Ml), mixed well and incubated overnight at 37 ℃. Then, a moderate volume (200 μ l) of phage liquid is mixed with the host bacterium liquid, 2216E semisolid culture medium is added, the mixture is poured onto a double-layer plate, overnight culture is carried out at 37 ℃, and the number of plaques is counted. Each sample was replicated three times.
The detection results are as follows:
TABLE 1 Effect of different inulin concentrations on the preservation of phages in inventive example 1
The difference of the preservation effect of the phage in 10% and 15% of inulin is not significant, but is significantly better than that in 5% of inulin, and 10% of inulin is selected as the preservation use concentration in consideration of the use cost of the reagent.
10% inulin was selected for preservation of Vibrio parahaemolyticus phage, and the preservation effect at different temperatures is shown in Table 2 below:
TABLE 2 preservation Effect of Vibrio parahaemolyticus phage Using protective agent of example 1 of the present invention
As can be seen from Table 2, the survival rate of the phage added with the protective agent is obviously better than that of the phage not added, and especially at room temperature, the effect is more prominent, and 2-3 orders of magnitude can be improved.
The second embodiment of the invention is as follows: a phage protectant comprises inulin, trehalose, polyvinylpyrrolidone, skimmed milk powder, sodium alginate and sodium carboxymethylcellulose, and is prepared by dissolving the above raw materials and phage in SM buffer (NaCl 5.89g, MgSO 5)4·7H2O29 g, 1M Tris-HCl (pH 7.5)50ml, 2% gelatin 5ml, distilled water 1000ml) and then dropped into 1.5% w/v calcium chloride to be fixed into ballsMicrocapsules of the form of granules. Wherein, inulin accounts for 10% w/v, trehalose accounts for 5% w/v, polyvinylpyrrolidone accounts for 1% w/v, skimmed milk powder accounts for 5% w/v, sodium alginate accounts for 1% w/v, and sodium carboxymethylcellulose accounts for 1% w/v.
The protective agent is applied to the preservation of vibrio parahaemolyticus phage and vibrio alginolyticus phage, wherein the preparation process of the phage suspension refers to example 1, 0.1g of spherical particles are weighed and placed in a 1.5ml EP tube, 1ml of SM buffer solution is added, and the spherical particles are respectively preserved at 4 ℃, room temperature (25 ℃) and 37 ℃.
Similarly, the preservation effect was measured by the double-plate phage assay, which was performed in a manner similar to that of example one, and the results of the measurement of the Vibrio parahaemolyticus phage are shown in Table 3 below:
TABLE 3 protective Effect of the phage protective agent of example 2 of the present invention
As can be seen from the above table, the preparation of microcapsules with protective agents and phages allows the survival of the phages to be further improved.
The results of the Vibrio alginolyticus phage assay are shown in Table 4 below:
TABLE 4 protective Effect of the phage protective agent of example 2 of the present invention
Experiments prove that the protective agent of the component can reduce the inactivation rate of the bacteriophage and improve the survival rate of the bacteriophage, particularly the survival rate of the bacteriophage at room temperature. The phage used in the experiment can resist high temperature, so that the phage can still survive in a large amount after being treated at 37 ℃ for two months. Particularly, the survival rate of the phage at 37 ℃ can be remarkably improved by adopting the technology of wrapping the phage by sodium alginate.
Inulin is a biological polysaccharide widely present in nature, is present in various plants and vegetables in the form of energy, and is present in large amounts in the Asteraceae familyThe root tuber of plants such as Jerusalem artichoke, herba Cichorii, etc. mainly comprises fructose and glucose, wherein fructose molecule is connected via β - (2, 1) glycosidic bond, polymerization degree is from 2-60 (usually 10), and terminal is glucose unit, and molecular formula can be GFnWherein G is a terminal glucose unit, F represents a fructose molecule, and n represents the number of fructose molecules. If the Degree of Polymerization is low (DP 2-9), it may be called Fructo-OligoSaccharide (Fructo OligoSaccharide). The functional oligosaccharide used as the freeze-drying protective agent can enhance the resistance of bacteria to freeze-drying damage and improve the survival rate of the bacteria. In addition, due to the special structure of the inulin, the inulin can selectively promote the growth of beneficial bacteria such as bifidobacteria and lactobacilli in intestinal tracts, inhibit the growth and reproduction of pathogenic microorganisms such as escherichia coli and the like, create an ideal animal intestinal micro-ecological environment and improve the production performance and the immune function of animals. Inulin is not digested and utilized in intestinal tracts, is a prebiotic, is a feed additive, promotes the growth of beneficial bacteria, has no residue in bodies, does not generate drug resistance, and is a resource which is worthy of being developed and applied to the breeding industry.
Sodium Alginate (SA) is a polysaccharide formed by connecting beta-D-mannuronic acid and alpha-L-guluronic acid by a (1 → 4) bond. The method of using sodium alginate as microcapsule wall material is that it can form heat stable gel at room temperature, and SA is commonly used as additive and adjuvant in food, cosmetic and pharmaceutical industries because of its good gelling property, stability and high viscosity in aqueous solution. In addition, SA is widely used for the preparation of biomedical materials due to its low toxicity and excellent biocompatibility. Alginate can be used as a film-forming material for preparing the microbial capsule, the gelation is formed by cross-linking carboxylate anions of guluronic acid and calcium ions, and gel can be formed after sodium ions in the sodium alginate are replaced by calcium ions in calcium chloride.
The phage HS15 in the above examples was isolated from wastewater collected from the aquaculture market of Guangzhou, and the host bacterium Vibrio parahaemolyticus ATCC17802 was purchased from the culture Collection of the microorganism institute in Guangdong province. 2216E liquid medium and 2216E semisolid medium are both commercially available. In the examples, "% w/v" represents "g/100 ml".
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention in the specification or directly or indirectly applied to the related technical field are included in the scope of the present invention.
Claims (8)
1. A phage protective agent characterized by: the phage protective agent consists of the following components: inulin, trehalose, polyvinylpyrrolidone, skim milk powder, sodium alginate and sodium carboxymethylcellulose; the protective agent is dissolved in a buffer solution, the mass concentration of the inulin is 100g/L, the mass concentration of the trehalose is 50g/L, the mass concentration of the polyvinylpyrrolidone is 10g/L, the mass concentration of the skim milk powder is 50g/L, the mass concentration of the sodium alginate is 10g/L, and the mass concentration of the sodium carboxymethylcellulose is 10 g/L; the buffer solution is SM buffer solution.
2. A phage preparation, characterized by: the formulation comprises the protective agent of claim 1 and a bacteriophage.
3. Phage preparation according to claim 2, characterized in that: the volume ratio of the protective agent to the phage is 1 (1.5-2.5).
4. The phage preparation of claim 3, wherein: the volume ratio of the protective agent to the phage is 1: 2.
5. Phage preparation according to claim 2, characterized in that: the phage includes at least one of phage HS15, Vibrio parahaemolyticus phage or Vibrio alginolyticus phage.
6. A method for preparing a phage preparation, wherein the raw materials of the phage preparation comprise phage, a protective agent and a buffer solution, and the method is characterized in that: the protective agent consists of the following components: inulin, trehalose, polyvinylpyrrolidone, skim milk powder, sodium alginate and sodium carboxymethyl cellulose, and the method comprises the following steps: dissolving inulin, trehalose, polyvinylpyrrolidone, skimmed milk powder, sodium alginate and bacteriophage in buffer solution, dropping into calcium chloride solution, mixing, fixing and molding; the inulin is added according to the proportion of 100g per liter of buffer solution, the mass concentration of trehalose is 50g/L, the mass concentration of polyvinylpyrrolidone is 10g/L, the mass concentration of skimmed milk powder is 5g/L, the mass concentration of sodium alginate is 10g/L, and the mass concentration of sodium carboxymethylcellulose is 10 g/L; the buffer solution is SM buffer solution.
7. The method for preparing a phage preparation according to claim 6, wherein: the mass concentration ratio of the calcium chloride to the sodium alginate is 1 (1-2).
8. The method for preparing a phage preparation according to claim 7, wherein: the mass concentration ratio of the calcium chloride to the sodium alginate is 1: 1.5.
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| CN111826324A (en) * | 2020-08-05 | 2020-10-27 | 厦门惠盈动物科技有限公司 | Preparation method of lactobacillus rhamnosus powder |
| CN113230215A (en) * | 2021-05-21 | 2021-08-10 | 中国科学院深圳先进技术研究院 | Phage freeze-dried powder preparation and preparation method, preservation method and application thereof |
| CN115707389B (en) * | 2021-08-20 | 2023-07-21 | 江苏省农业科学院 | Phage gel and preparation method and application thereof |
| CN114403290B (en) * | 2022-01-28 | 2023-07-07 | 青岛诺安百特生物技术有限公司 | Phage microcapsule and preparation method thereof |
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| WO2010138522A2 (en) * | 2009-05-26 | 2010-12-02 | Advanced Bionutrition Corporation | Stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making |
| CN103041399A (en) * | 2013-01-07 | 2013-04-17 | 江苏省农业科学院 | Heat-resisting protective agent and application thereof |
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| CN106065399A (en) * | 2016-06-14 | 2016-11-02 | 苏州埃瑞特生物技术有限公司 | A kind of bacteriophage preserves protective agent and its preparation method and application |
| CN105950567A (en) * | 2016-06-14 | 2016-09-21 | 苏州埃瑞特生物技术有限公司 | Preservation method for freeze-dried powder capsules of bacteriophage |
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| WO2010138522A2 (en) * | 2009-05-26 | 2010-12-02 | Advanced Bionutrition Corporation | Stable dry powder composition comprising biologically active microorganisms and/or bioactive materials and methods of making |
| CN103041399A (en) * | 2013-01-07 | 2013-04-17 | 江苏省农业科学院 | Heat-resisting protective agent and application thereof |
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