CN113230215A - Phage freeze-dried powder preparation and preparation method, preservation method and application thereof - Google Patents
Phage freeze-dried powder preparation and preparation method, preservation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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Abstract
The invention discloses a phage freeze-dried powder preparation, a preparation method, a preservation method and application thereof. The phage freeze-dried powder preparation is a freeze-dried mixture formed by freeze-drying a mixed solution containing phage with biological activity and a freeze-drying protective agent; wherein, in the lyophilization mixture, the titer of the bacteriophage is 109～1011PFU/g, the concentration of the freeze-drying protective agent is 0.3-1 mol/L. The phage freeze-dried powder preparation can be stored for a long time and has low requirement on the storage condition, thereby improving the convenience of transportation and storage of the phage freeze-dried powder preparationThe activity of the phage after long-term storage and redissolution of the phage freeze-dried powder preparation is high, and the practical applicability of the phage freeze-dried powder preparation is improved. The preparation method of the phage freeze-dried powder preparation has the advantages that the process conditions are easy to control, the activity of the prepared phage freeze-dried powder preparation is stable, and the preparation cost is effectively reduced.
Description
Technical Field
The invention belongs to the technical field of biological medicine preparations, and particularly relates to a phage freeze-dried powder preparation, and a preparation method, a preservation method and application thereof.
Background
China is a big country for aquaculture. In recent years, the aquaculture industry in China is rapidly developed. With the increasing expansion of the scale of mariculture and the continuous popularization of intensive culture modes, the frequent occurrence of diseases poses serious threats to the healthy development of large-scale aquaculture. The pathogens of aquaculture mainly include bacteria, viruses, parasites, etc., wherein bacterial diseases are a group of diseases with serious harm degree.
Vibrio is a bacillus brevis capable of moving, widely distributed in seawater and organisms in coastal areas and estuary areas, is one of the most common bacterial groups in the ocean, and is also the most important pathogenic bacteria causing bacterial diseases of marine culture fishes and shrimps. The pathogenic intensity of vibrio is related to the physiological state of host, water quality, environment condition and other comprehensive factors, and belongs to conditional pathogenic bacteria, which are mainly infected through mouth or wound to generate toxin, so that wound muscle ulceration and suppuration and severe pathological changes of internal organs cause death of fishes and shrimps.
The disease caused by vibrio is also called vibriosis, has the characteristics of wide epidemic area, high morbidity, strong lethality and the like, is a main disease of aquaculture animals, is determined to be an important limiting factor for hindering the development of aquaculture industry, and causes great harm to the aquaculture industry. Among them, vibriosis caused by Vibrio anguillarum (Vibrio anguillarum), Vibrio alginolyticus (Vibrio algirybacteria), Vibrio harveyi (Vibrio harveyi), Vibrio parahaemolyticus (Vibrio parahaemolyticus) and Vibrio salmonicida (Vibrio salmonicida) is considered to be one of the most serious diseases in the cultivation of fish and shrimp. Early Mortality Syndrome (EMS) or hepatopancreas acute necrosis syndrome is a rather new disease affecting shrimp, which first appeared in china in 2009, spreads to vietnam in 2010, to northern malaysia and borea in 2011, and to thailand in 2012. In 2013 EMS was first reported outside asia to occur in mexico-due to infected live shrimp imported from asia. Studies at the university of Arizona have established that the disease is caused by the bacterial agent Vibrio parahaemolyticus, which is transmitted orally and colonizes the gastrointestinal tract of shrimp and then produces toxins, resulting in tissue destruction and dysfunction of the digestive organs of shrimp.
The existing methods for preventing and controlling animal diseases related to aquaculture mainly adopt a chemical antibiotic method and an immune vaccine method, wherein the chemical antibiotic is mainly used. As the main means for controlling the diseases of the aquatic animals at present, the chemical antibiotic method has the advantages of convenient use, quick response, good curative effect and the like. However, the frequent use of antibiotics in large quantities greatly promotes the drug resistance of pathogenic bacteria and accelerates the generation of broad-spectrum drug-resistant bacteria while controlling pathogenic bacteria. This makes the problem of drug resistance of pathogenic bacteria increasingly serious. In addition, chemical drug residues are also one of the important causes for the generation of human food-borne diseases, and cause serious harm to human health.
Bacteriophage, also called bacterial virus, is a virus for specifically cracking bacteria, and has wide distribution, various types, simple structure and strong host specificity in the environment. Most importantly, the mechanism of phage lysis of bacteria is not affected by bacterial drug resistance, and thus it passes through receptor recognition and adsorbs on specific host bacterial surface, injects self genetic material into the host body for self-replication constitution, and finally releases progeny through lysis of host bacteria so as to realize self-growth and propagation.
Due to its specificity and its rapid proliferation, bacteriophages have been a powerful tool for the treatment of bacterial diseases. Firstly, the treatment of the bacteriophage has specificity, and can selectively kill a certain specific harmful bacterial strain, so that the normal flora balance in the organism or the living environment of the organism is influenced to the minimum extent, compared with antibiotics, the bacteriophage has small toxic and side effects on the environment, and the food safety is also ensured; secondly, the phage can proliferate as the host bacteria proliferate and play a role in the whole process of bacterial infection, unlike antibiotics, which gradually decrease in efficacy over time; thirdly, the phage freeze-dried powder preparation is convenient to obtain and low in price.
Phage is used for controlling pathogenic bacteria before antibiotics are discovered, but because phage is insufficient in level and antibiotics have broad-spectrum bacteriostatic effects at that time, the curative effect is good and the effect is quick, the phage is not widely used. In recent years, due to a series of drug-resistant bacteria problems caused by abuse of antibiotics, phages regress to the visual field of people, and research on application of the phages to control pathogenic bacteria also has attracted wide interest in the scientific community. The clinical application potential of bacteriophage as an important weapon for antibiotic resistance is huge, and phage preparations such as liquid preparations and freeze-dried preparations have been reported in public at present, but in practical use and research, the bacteriophage preparations still have some potential disadvantages in the application process: the phage mainly contains protein and nucleic acid, so that the activity of the phage in the transportation and storage processes after preparation is particularly critical, but the phage containing the phage in the phage preparation which is reported in the prior publication has unsatisfactory survivability and short preservation time, wherein the freeze-dried preparation also has the problem of unsatisfactory phage activity after reconstitution, and the application of the phage preparation is limited. Meanwhile, the vibrio phage has particularity when being mainly used for aquaculture, and the product can be diffused to the whole water body in the using process, so that no component harmful to the environment exists, and the balance between the cost and the process needs to be made.
Disclosure of Invention
The invention aims to provide a phage freeze-dried powder preparation, a preparation method, a preservation method and application thereof, and aims to solve the technical problems of unsatisfactory survival, short preservation time of phage contained in the conventional phage freeze-dried powder preparation, unsatisfactory activity after further reconstitution and the like.
In order to achieve the above object, according to one aspect of the present invention, there is provided a lyophilized powder preparation of bacteriophage. The phage freeze-dried powder preparation of the invention comprises phage and jelly with biological activityThe mixed solution of the dry protective agent is subjected to freeze-drying treatment to form a freeze-dried mixture; wherein, in the lyophilization mixture, the titer of the bacteriophage is 109～1011PFU/g, wherein the mass ratio of the freeze-drying protective agent is 0.3-1 mol/L.
Preferably, the freeze-drying protective agent comprises at least one of polyhydroxy compound and polymer composite protective agent.
Further preferably, the polyol comprises at least one of sucrose and sorbitol, and the polymer composite protective agent comprises skimmed milk powder.
Preferably, the phage is a phage whose host is an aquatic pathogen.
Further preferably, the aquatic pathogenic bacteria comprise vibrio.
Further preferably, the bacteriophage comprises at least one of a vibrio alginolyticus bacteriophage, a vibrio parahaemolyticus bacteriophage, and an escherichia coli bacteriophage.
In another aspect of the present invention, a method for preparing a lyophilized powder preparation of a bacteriophage of the present invention comprises the following steps:
mixing a solution containing phage and the freeze-drying protective agent in proportion to prepare a mixed solution;
and carrying out freeze-drying treatment on the mixed solution to form a freeze-dried mixture, thus obtaining the phage freeze-dried powder preparation.
Preferably, the conditions of the lyophilization process are:
the freeze-drying temperature is-30 ℃ to-40 ℃, the time is 18-36 h, and the vacuum degree is 0.05-0.10 mbar.
Preferably, the phage-containing solution is a phage stock solution, and the method for preparing the phage stock solution comprises the following steps:
carrying out amplification culture on the host bacteria to obtain host bacteria liquid;
inoculating the phage into the host bacterium liquid for phage culture to obtain a phage culture solution;
and (3) carrying out separation treatment on the phage culture solution to remove impurities including host bacteria which are not lysed, so as to obtain the phage stock solution.
Further preferably, the step of inoculating the phage into the host bacterial liquid is to inoculate the phage into the host bacterial liquid according to the proportion that the MOI value is 1-10.
Further preferably, the concentration of the host bacterium in the host bacterium liquid is 109～1010CFU/ml。
Further preferably, the separation treatment includes the steps of centrifuging the phage culture solution to obtain a supernatant, and filtering the supernatant with a micron filter to collect the filtrate.
In still another aspect of the present invention, a method for preserving the phage lyophilized powder preparation of the present invention is provided. The preservation method of the phage freeze-dried powder preparation comprises the following steps:
and (3) preserving the phage freeze-dried powder preparation at the temperature of 4-27 ℃.
Preferably, the temperature of the preservation treatment is 23-27 ℃.
In another aspect of the invention, the invention provides the application of the phage freeze-dried powder preparation in aquaculture or preparation of aquaculture drugs.
Compared with the prior art, the phage freeze-dried powder preparation, the preparation method, the preservation method and the application thereof have the following technical effects:
the phage freeze-dried powder preparation is subjected to freeze-drying treatment together with the phage with biological activity by the freeze-drying protective agent, and the content ratio of the freeze-drying protective agent to the phage is controlled, so that the freeze-drying protective agent can protect the phage, the phage freeze-dried powder preparation can be stored for a long time, and the requirement on the storage condition is low, thereby improving the convenience for transportation and storage of the phage freeze-dried powder preparation, reducing the economic cost for transportation and storage of the phage freeze-dried powder preparation, ensuring high activity of the phage after long-term storage and re-dissolution of the phage freeze-dried powder preparation, and improving the practical applicability of the phage freeze-dried powder preparation. In addition, according to the specificity of the bacteriophage, the type of the bacteriophage can be flexibly controlled according to the type of actual pathogenic bacteria, targeted bacterial disease control is realized, other flora in the environment is not damaged, and no influence is generated on cultured animals and consumers. Compared with antibiotics, the antibiotic does not cause the drug resistance of pathogenic bacteria. And secondly, the phage freeze-dried powder preparation does not contain any toxic or harmful substances, has no drug residue and is harmless to the environment.
The preparation method of the phage freeze-dried powder preparation only needs to freeze-dry the mixed solution formed by the freeze-drying protective agent and the phage, the process conditions are easy to control, the activity of the prepared phage freeze-dried powder preparation is stable, and the preparation cost is effectively reduced.
The phage freeze-dried powder preparation can be stably stored for a long time, has low requirement on storage conditions, and has high activity after redissolution, so that the transportation and storage convenience of the phage freeze-dried powder preparation is improved, the economic cost of transportation and storage of the phage freeze-dried powder preparation is reduced, the practical applicability of the phage freeze-dried powder preparation is effectively improved, and particularly, the phage freeze-dried powder preparation is applied to aquaculture or preparation of aquaculture drugs, has high specificity, strong pertinence on prevention and treatment of aquatic pathogenic bacteria, is harmless to water, has no drug residue, improves the health safety and environmental safety of aquatic products, and reduces the economic cost.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise.
FIG. 1 is a schematic flow chart of a method for preparing a lyophilized phage powder preparation according to an embodiment of the present invention;
FIG. 2 is a histogram of the titer of Vibrio alginolyticus phage provided in example 1 of the present invention after being stored at 4 deg.C, 25 + -2 deg.C for 6 months;
FIG. 3 is a graph showing the bacteriostatic effect of Vibrio alginolyticus phage and a control group on inhibiting Vibrio alginolyticus provided in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and the embodiments described below are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention shall fall within the scope of protection of the present invention. Those whose specific conditions are not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the description of the present invention, the term "and/or" describing an association relationship of associated objects means that there may be three relationships, for example, a and/or B, may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In the description of the present invention, "at least one" means one or more, "a plurality" means two or more. "at least one of the following" or similar expressions refer to any combination of these items, including any combination of the singular or plural items. For example, "at least one (a), b, or c", or "at least one (a), b, and c", may each represent: a. b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple respectively.
It should be understood that the weight of the related components mentioned in the embodiments of the present invention may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, it is within the scope of the disclosure that the content of the related components is scaled up or down according to the embodiments of the present invention. Specifically, the weight described in the embodiments of the present invention may be a unit of weight known in the chemical field such as μ g, mg, g, kg, etc.
In addition, unless the context clearly uses otherwise, an expression of a word in the singular is to be understood as including the plural of the word. The terms "comprises" or "comprising" are intended to specify the presence of stated features, quantities, steps, operations, elements, portions, or combinations thereof, but are not intended to preclude the presence or addition of one or more other features, quantities, steps, operations, elements, portions, or combinations thereof.
In one aspect, embodiments of the present invention provide a lyophilized phage powder formulation. The phage freeze-dried powder preparation is a freeze-dried mixture formed by freeze-drying a mixed solution containing phage with biological activity and a freeze-drying protective agent.
The phage contained in the phage freeze-dried powder preparation of the embodiment of the invention is a phage with biological activity. The bacteriophage with biological activity is a biological functional active component of a bacteriophage freeze-dried powder preparation. Therefore, the types of the bacteriophage can be flexibly controlled according to the types of actual pathogenic bacteria according to the specificity of the bacteriophage, targeted bacterial disease control is realized, other flora in the environment is not damaged, and any influence on cultured animals and consumers is avoided. Compared with antibiotics, the antibiotic does not cause the drug resistance of pathogenic bacteria. And the phage freeze-dried powder preparation does not contain any toxic or harmful substances, has no drug residue and is harmless to the environment.
In an embodiment, the biologically active host is a bacteriophage of an aquatic pathogen. Further, the aquatic pathogenic bacteria include, but are not limited to, Vibrio. At this time, the corresponding bacteriophage with biological activity can be selected according to the type of vibrio, so that the bacteriophage freeze-dried powder preparation provided by the embodiment of the invention can be used for performing targeted prevention and treatment on specific aquatic pathogenic bacteria such as vibrio types.
In an embodiment, the bacteriophage with biological activity comprises at least one of a vibrio alginolyticus bacteriophage, a vibrio parahaemolyticus bacteriophage, and a coliphage. In a specific example, when the bacteriophage is a Vibrio alginolyticus bacteriophage, the Vibrio alginolyticus bacteriophage is Vibrio alginolyticus bacteriophage ValSw 4-1. According to the specific characteristics of the bacteriophage, when the bacteriophage with biological activity contained in the bacteriophage freeze-dried powder preparation comprises vibrio alginolyticus bacteriophage such as ValSw4-1, the bacteriophage freeze-dried powder preparation can be used for treating or further preventing and treating aquatic organisms infected with vibrio alginolyticus. And has no harm to other fungi and aquatic organisms and no drug residue.
The freeze-drying protective agent contained in the phage freeze-drying powder preparation and the phage with biological activity are subjected to freeze-drying treatment to form a freeze-drying mixture, and the freeze-drying protective agent and the content ratio of the phage are controlled, so that the freeze-drying protective agent can protect the phage, the phage freeze-drying powder preparation can be stored for a long time, and the requirement on the storage condition is low, thereby improving the transportation and storage convenience of the phage freeze-drying powder preparation, reducing the economic cost of transportation and storage of the phage freeze-drying powder preparation, ensuring high activity of the phage after long-term storage and redissolution of the phage freeze-drying powder preparation, and improving the practical applicability of the phage freeze-drying powder preparation.
In an embodiment, the freeze-drying protective agent comprises a polyhydroxy compound and a polymer composite protective agent, in a specific embodiment, the polyhydroxy compound comprises at least one of sucrose and sorbitol, and the polymer composite protective agent comprises skimmed milk powder. The research of the inventor finds that the polyhydroxy compounds such as sucrose and sorbitol have higher glass transition temperature, play an obvious role in preventing the change of the secondary structure of protein and the extension and aggregation of protein polypeptide chains in the freeze-drying treatment process and the storage period, and can obviously improve the biological activity of the phage in the phage freeze-dried powder preparation. When the high-molecular composite protective agent such as the skimmed milk powder is subjected to freeze drying, the whey protein can form a protein film outside the bacteria to play a role in protection, and other components in the skimmed milk powder can also improve the freeze drying survival rate of the phage and provide a light and porous structure for a freeze-dried product, so that the high-molecular composite protective agent such as the skimmed milk powder can obviously improve the biological activity of the phage in the phage freeze-dried powder preparation.
In an embodiment, the bacteriophage having biological activity and the solution for supporting the bacteriophage having biological activity may be a filtrate collected by a filtration process after culturing the bacteriophage. Therefore, the filtrate used as a solvent carrier is more beneficial to the stability of the activity of the phage with biological activity, and has a synergistic effect with the components of the freeze-drying protective agent, so that the stability of the biological activity of the phage with biological activity is improved, the stability of the biological activity of the phage in the transportation and storage of the phage freeze-dried powder preparation is improved, and the biological activity of the phage is prolonged, thereby prolonging the storage time of the phage freeze-dried powder preparation and ensuring that the high biological activity is still maintained after long-time storage. Of course, the solution of the mixed solution may also be used with other reagents that are beneficial to the biological activity of the phage, such as but not limited to buffers.
Therefore, the freeze-dried powder preparation of the bacteriophage in each embodiment utilizes the freeze-drying protective agent contained in the freeze-dried powder preparation to play a role in protecting the biological activity of the bacteriophage so as to ensure the biological activity of the bacteriophage, particularly prolong the biological activity of the bacteriophage, endow the biological activity of the bacteriophage in the processes of transportation, storage and application of the freeze-dried powder preparation of the bacteriophage in the embodiment of the invention, and realize the effectiveness and effect of treatment and control of corresponding pathogenic bacteria. And the specific characteristic of the bacteriophage can realize the specific prevention, treatment and treatment of bacterial diseases in the environment, does not destroy other flora in the environment, does not generate toxic and harmful components, does not have drug residue, is harmless to the environment, and does not have any influence on cultured animals and consumers. And the type and the content of the contained freeze-drying protective agent can be adjusted to improve the biological activity stability of the phage freeze-dried powder preparation, prolong the storage time of the phage, and reduce the cost and the application cost of the phage freeze-dried powder preparation.
In another aspect, the embodiment of the invention also provides a preparation method of the phage freeze-dried powder preparation. The preparation method of the phage freeze-dried powder preparation of the embodiment of the invention has the flow shown in figure 1, and comprises the following steps:
s01: mixing the solution containing the phage and a freeze-drying protective agent in proportion to prepare a mixed solution;
s02: and carrying out freeze-drying treatment on the mixed solution to form a freeze-dried mixture, thus obtaining the phage freeze-dried powder preparation.
Wherein, the phage in step S01 is the phage contained in the above phage lyophilized powder preparation, and in the embodiment, the solution containing phage may be a phage stock solution. The phage should be a biologically active phage. Therefore, the kinds of the phage are as those contained in the above phage lyophilized powder preparation, and the lyoprotectant is also as that contained in the above phage lyophilized powder preparation, and for the sake of brevity, the kinds of the phage and the lyoprotectant in step S01 will not be described in detail herein.
In the examples, the mixing ratio of the phage-containing solution to the lyoprotectant during the mixing process in step S01 ensures that the titer of phage in the prepared phage lyophilized powder preparation is 109～1011PFU/g, the concentration of the freeze-drying protective agent is 0.3-1 mol/L, and further 0.3-0.5 mol/L. By controlling the mixing proportion of the freeze-drying protective agent and the bacteriophage, the freeze-drying protective agent can protect the bacteriophage, and the prepared bacteriophage freeze-drying powder preparation can be stored for a long time and has high biological activity.
The mixing process in step S01 may be a mixing process that is conventional in the field of biology, as long as the phage-containing solution and the lyoprotectant can be uniformly mixed. Such as including stirring, shaking, etc.
In the examples, when the phage-containing solution in step S01 is a phage stock solution, the phage stock solution is prepared by a method comprising the steps of:
s011: carrying out amplification culture on the host bacteria to obtain host bacteria liquid;
s012: inoculating the phage into host bacterium liquid for phage culture to obtain a phage culture solution;
s013: separating the phage culture solution to remove impurities including host bacteria which are not lysed, and obtaining phage stock solution.
In step S011, the host bacteria are expanded by a corresponding expanding culture method according to the species or biological characteristics of the host bacteria, so as to obtain a large amount of phage host bacteria. As an example, the culture can be carried out by the following scale-up culture method:
transferring the host strain into a culture bottle for culture according to the proportion of 1-5 percent, wherein the culture conditions are as follows: the temperature is 30-37 ℃, the rotating speed is 220-250 rpm, and the culture time is 3-5 hours, so that the concentration of the host bacterial liquid reaches 109～1010CFU/ml。
In step S012, the phage is inoculated into the host bacteria obtained through mass culture in step S011, and the phage infects the host bacteria, and self-replication assembly is performed in the host bacteria, so as to obtain a large amount of phage after phage culture. In this process, the host bacteria are lysed along with the self-replication of the phage, and thus, the content of the host bacteria is gradually reduced and the content of the phage is gradually increased during the culture of the phage. Due to the specificity of the phage, the host bacterium should be the host bacterium to which the phage corresponds.
In the embodiment, the step of inoculating the phage into the host bacterial liquid is to inoculate the phage into the host bacterial liquid according to the proportion that the MOI value is 1-10. Wherein, the bacteriophage used for inoculation can be titer of 1010PFU/ml phage.
The phage culture in step S012 should be sufficient, for example, the culture time is 6-8 hours according to the above-mentioned host bacterium concentration and the inoculation amount of phage inoculation, the culture solution gradually becomes clear, and the culture is finished.
In step S013, the phage culture solution is subjected to a separation process to remove the culture medium and the host bacteria or possibly other bacteria that have not been lysed, thereby obtaining a pure phage solution, i.e. a phage stock solution. In an example, the separation process in step S013 includes the steps of centrifuging the phage culture solution to obtain a supernatant, and collecting the filtrate by filtering the supernatant with a microfiltration membrane. The microfiltration membrane should be a microfiltration membrane capable of retaining host bacteria, and may be a 0.22 micron filtration membrane.
In step S02, the solvent such as water contained in the mixed solution obtained through the lyophilization process step S01 is sublimation-dried, thereby obtaining a lyophilized mixture. In the examples, the conditions of the lyophilization process were: the freeze-drying temperature is-30 ℃ to-40 ℃, the time is 18-36 h, and the vacuum degree is 0.05-0.10 mbar (5-10 Pa). Specifically, the temperature is adjusted to a preset freeze-drying temperature, such as-30 ℃ to-40 ℃, and then the solvent to be freeze-dried, specifically the mixed solution prepared in step S01, is placed at the preset freeze-drying temperature for freeze-drying treatment. Through the condition control optimization of the freeze-drying treatment, the activity of the phage in the mixed solution can be effectively ensured, and the freeze-drying treatment efficiency can be improved.
The preparation method of the phage freeze-dried powder preparation provided by the embodiment of the invention only needs to carry out mixing treatment and freeze-drying treatment on the freeze-drying protective agent and the phage, the process conditions are easy to control, the activity of the prepared phage freeze-dried powder preparation is stable, and the preparation cost is effectively reduced. And the treatment and prevention of the bacteriophage freeze-dried powder preparation on bacterial diseases can be improved, the biological activity stability of the bacteriophage freeze-dried powder preparation can be improved, and the storage time can be prolonged by the types of the bacteriophage, the types of the freeze-drying protective agents and the mixing ratio of the bacteriophage freeze-dried protective agents.
In another aspect, the embodiments of the present invention further provide a method for preserving the phage lyophilized powder preparation according to the above embodiments of the present invention. The preservation method of the phage freeze-dried powder preparation comprises the following steps:
and (3) preserving the phage freeze-dried powder preparation at the temperature of 4-27 ℃.
Based on the embodiment of the invention, the phage freeze-dried powder preparation can be stably stored for a long time, has low requirement on storage conditions, can be stored at a wider temperature such as 4-27 ℃, can effectively ensure the biological activity of the phage, and prolongs the storage time, so that the transportation and storage convenience of the phage freeze-dried powder preparation is improved, the economic cost of transportation and storage of the phage freeze-dried powder preparation is reduced, the practical applicability of the phage freeze-dried powder preparation is effectively improved, and the application cost of the phage freeze-dried powder preparation is reduced. According to detection, after the phage freeze-dried powder preparation is stored at the temperature of 4-27 ℃ for 6 months, the survival rate of the phage can still be kept above 91%. Further, the temperature of the preservation treatment is 23 to 27 ℃. Through detection, after the phage freeze-dried powder preparation disclosed by the embodiment of the invention is stored at the temperature of 23-27 ℃ for 6 months, the survival rate of the phage still has activity of more than 92%. Therefore, the phage freeze-dried powder preparation in the embodiment of the invention has low requirement on storage conditions, can keep high biological activity at normal temperature, effectively reduces the economic cost of storage, also reduces the requirement on transportation and the economic cost, enhances the applicability of the phage freeze-dried powder preparation in the embodiment of the invention and reduces the economic cost of application.
In still another aspect, the embodiment of the present invention provides an application of the phage lyophilized powder preparation of the present invention, and an application of the phage lyophilized powder preparation of the present invention in aquaculture or in preparation of aquaculture drugs. As described above, the phage freeze-dried powder preparation in the embodiment of the present invention can be stably stored for a long time, and has low requirement on storage conditions, so that the transportation and storage convenience of the phage freeze-dried powder preparation in the embodiment of the present invention is improved, the economic cost for transportation and storage of the phage freeze-dried powder preparation in the embodiment of the present invention is reduced, the practical applicability of the phage freeze-dried powder preparation in the embodiment of the present invention is effectively improved, particularly, the phage freeze-dried powder preparation is applied to aquaculture or preparation of aquaculture drugs, the specificity is high, the pertinence for preventing and controlling aquatic pathogenic bacteria is strong, and the phage freeze-dried powder preparation is harmless to water and has no drug residue, the health safety and the environmental safety of aquatic products are improved, and the economic cost is reduced.
In order to make the details and operation of the above-mentioned embodiments of the present invention clearly understood by those skilled in the art and to make the progress of the phage lyophilized powder preparation and the preparation method thereof obvious, the above-mentioned technical solution is illustrated by the following examples.
Example 1
The embodiment provides a phage freeze-dried powder preparation and a preparation method thereof, and the phage freeze-dried powder preparation comprises Vibrio alginolyticus phage ValSw4 and a sucrose freeze-drying protective agent. Wherein, in the phage freeze-dried powder preparation, the titer of the Vibrio alginolyticus phage ValSw4 is 10.91 multiplied by 1010PFU/g, sucrose concentration 0.3 mol/L.
The preparation method of the phage freeze-dried powder preparation comprises the following steps:
s1: preparing a vibrio alginolyticus phage stock solution:
s11: transferring the vibrio alginolyticus strain into a culture bottle according to the proportion of 1 percent for cultureCulturing under the following conditions: 2216E culture medium (2216E liquid culture medium is autoclaved and cooled), temperature is 30 deg.C, rotation speed is 220rpm, culture time is 4 hr, and bacterial liquid concentration is 109CFU/ml；
S12: adding titer 10 to the culture solution of step S11 according to MOI 110PFU/ml Vibrio alginolyticus phage ValSw4-1, starting phage culture;
s13: the culture time is 6 hours, the culture solution begins to become clear, the culture is finished, and the culture solution of the vibrio alginolyticus phage ValSw4-1 is obtained;
s14: centrifuging the phage culture solution of the step S13 for 10 minutes at 9000g, filtering the supernatant with 0.22 micron filter membrane to obtain sterile Vibrio alginolyticus phage ValSw4-1 stock solution with phage concentration of 1010PFU/ml；
S2: the stock solution containing the vibrio alginolyticus phage ValSw4 and a sucrose freeze-drying preservative are subjected to freeze-drying treatment according to the following method to obtain a phage freeze-dried powder preparation:
s21: mixing stock solution of Vibrio alginolyticus phage ValSw4 in the step S1 with 0.3M sucrose in a ratio of 1:9, and pre-freezing for 3 hours in a refrigerator at-20 ℃ to form a solid sample to be frozen;
s22: starting a refrigeration function of a Labconco freeze dryer, and cooling the temperature of a cold trap to be lower than-40 ℃;
s23: placing the solidified sample to be frozen into a freeze dryer, starting a vacuum pumping mode of the freeze dryer, enabling the air pressure in a cold trap to reach 0.07mbar, and freeze-drying for 36 hours to obtain the freeze-dried powder of the bacteriophage of Vibrio alginolyticus, thus obtaining the titer of the phage of the freeze-dried powder of 10.91 multiplied by 1010PFU/g。
Example 2
The embodiment provides a phage freeze-dried powder preparation and a preparation method thereof, and the phage freeze-dried powder preparation comprises Vibrio alginolyticus phage ValSw4 and a sucrose freeze-drying protective agent. Wherein, in the phage freeze-dried powder preparation, the titer of the Vibrio alginolyticus phage ValSw4 is 10.91 multiplied by 1010PFU/g, sucrose concentration 1.0 mol/L.
The preparation method of the phage freeze-dried powder preparation comprises the following steps:
s1: preparing a vibrio alginolyticus phage stock solution:
s11: transferring the vibrio alginolyticus strains into a culture bottle for culture according to the proportion of 1 percent, wherein the culture conditions are as follows: 2216E culture medium (2216E liquid culture medium is autoclaved and cooled), temperature is 30 deg.C, rotation speed is 220rpm, culture time is 4 hr, and bacterial liquid concentration is 109CFU/ml；
S12: adding titer 10 to the culture solution of step S11 according to MOI 110PFU/ml Vibrio alginolyticus phage ValSw4-1, starting phage culture;
s13: the culture time is 6 hours, the culture solution begins to become clear, the culture is finished, and the culture solution of the vibrio alginolyticus phage ValSw4-1 is obtained;
s14: centrifuging the phage culture solution of the step S13 for 10 minutes at 9000g, filtering the supernatant with 0.22 micron filter membrane to obtain sterile Vibrio alginolyticus phage ValSw4-1 stock solution with phage concentration of 1010PFU/ml；
S2: the stock solution containing the vibrio alginolyticus phage ValSw4 and a sucrose freeze-drying preservative are subjected to freeze-drying treatment according to the following method to obtain a phage freeze-dried powder preparation:
s21: mixing stock solution of Vibrio alginolyticus phage ValSw4 in the step S1 with 1.0M sucrose in a ratio of 1:9, and pre-freezing the mixture in a refrigerator at the temperature of-20 ℃ for 3 hours to form a solid sample to be frozen;
s22: starting a refrigeration function of a Labconco freeze dryer, and cooling the temperature of a cold trap to be lower than-40 ℃;
s23: placing the solidified sample to be frozen into a freeze dryer, starting a vacuum pumping mode of the freeze dryer, enabling the air pressure in a cold trap to reach 0.07mbar, and freeze-drying for 36 hours to obtain lyophilized powder of Vibrio alginolyticus bacteriophage, wherein the titer of the lyophilized powder of the bacteriophage is 10.35 × 1010PFU/g。
Example 3
Compared with the preparation of example 1, the freeze-drying protective agent of the phage freeze-drying powder preparation is sorbitol, and the rest is unchanged.
The preparation method of the phage freeze-dried powder preparation in the comparative example comprises the following steps:
s1: a stock solution of Vibrio alginolyticus phage ValSw4 was prepared according to step S1 of the example:
s2: and (4) carrying out freeze-drying treatment on the stock solution of the vibrio alginolyticus phage ValSw4 and sorbitol according to the mixing ratio and the freeze-drying treatment method in the step S2 to obtain a phage freeze-dried powder preparation.
Example 4
Compared with the preparation of example 1, the freeze-drying protective agent of the phage freeze-drying powder preparation is skimmed milk powder, and the rest is unchanged.
The preparation method of the phage freeze-dried powder preparation in the comparative example comprises the following steps:
s1: stock solutions of Vibrio alginolyticus phage ValSw4 were prepared according to step S1 of the examples;
s2: and (4) freeze-drying the stock solution of the vibrio alginolyticus phage ValSw4 and the skimmed milk powder according to the mixing ratio and the freeze-drying method in the step S2 to obtain a phage freeze-dried powder preparation.
Example 5
This comparative example provides a lyophilized phage powder formulation, which is, compared to example 1, phage that is E.coli phage, the others being unchanged.
Example 6
This comparative example provides a phage lyophilized powder formulation, compared to example 1, the lyoprotectant was Vibrio parahaemolyticus phage, and the others were unchanged.
Comparative example 1
This comparative example provides a lyophilized powder preparation of bacteriophage which does not contain components of a lyoprotectant such as sucrose, as compared with examples.
The preparation method of the phage freeze-dried powder preparation in the comparative example comprises the following steps:
s1: a stock solution of Vibrio alginolyticus phage ValSw4 was prepared according to step S1 of the example:
s2: and (4) carrying out freeze-drying treatment on the stock solution of the vibrio alginolyticus phage ValSw4 according to the freeze-drying treatment method in the step S2 to obtain a phage freeze-dried powder preparation.
Comparative example 2
The embodiment provides a phage freeze-dried powder preparation and a preparation method thereof, wherein the phage freeze-dried powder preparation comprises vibrio alginolyticus phageValSw4 and sucrose lyoprotectants. Wherein, in the phage freeze-dried powder preparation, the titer of the Vibrio alginolyticus phage ValSw4 is 10.91 multiplied by 1010PFU/g, sucrose concentration 0.1 mol/L.
The preparation method of the phage freeze-dried powder preparation comprises the following steps:
s1: preparing a vibrio alginolyticus phage stock solution:
s11: transferring the vibrio alginolyticus strains into a culture bottle for culture according to the proportion of 1 percent, wherein the culture conditions are as follows: 2216E culture medium (2216E liquid culture medium is autoclaved and cooled), temperature is 30 deg.C, rotation speed is 220rpm, culture time is 4 hr, and bacterial liquid concentration is 109CFU/ml；
S12: adding titer 10 to the culture solution of step S11 according to MOI 110PFU/ml Vibrio alginolyticus phage ValSw4-1, starting phage culture;
s13: the culture time is 6 hours, the culture solution begins to become clear, the culture is finished, and the culture solution of the vibrio alginolyticus phage ValSw4-1 is obtained;
s14: centrifuging the phage culture solution of the step S13 for 10 minutes at 9000g, filtering the supernatant with 0.22 micron filter membrane to obtain sterile Vibrio alginolyticus phage ValSw4-1 stock solution with phage concentration of 1010PFU/ml；
S2: the stock solution containing the vibrio alginolyticus phage ValSw4 and a sucrose freeze-drying preservative are subjected to freeze-drying treatment according to the following method to obtain a phage freeze-dried powder preparation:
s21: mixing stock solution of Vibrio alginolyticus phage ValSw4 in the step S1 with 0.1M sucrose in a ratio of 1:9, and pre-freezing the mixture in a refrigerator at the temperature of 20 ℃ below zero for 3 hours to form a solid sample to be frozen;
s22: starting a refrigeration function of a Labconco freeze dryer, and cooling the temperature of a cold trap to be lower than-40 ℃;
s23: placing the solidified sample to be frozen into a freeze dryer, starting a vacuum pumping mode of the freeze dryer, enabling the air pressure in a cold trap to reach 0.07mbar, and freeze-drying for 36 hours to obtain the freeze-dried powder of the bacteriophage of Vibrio alginolyticus, so as to obtain the freeze-dried powder with the bacteriophage titer of 9.95 multiplied by 1010PFU/g。
Relevant performance experiment of phage freeze-dried powder preparation
1. Comparative experiment of the effect of freeze-drying protective agent contained in the phage freeze-dried powder preparation:
the phage freeze-dried powder preparations of the examples 1 to 6 and the comparative examples 1 to 2 are subpackaged and stored in an environment of 25 +/-2 ℃ for 6 months. And detecting the titer and survival rate of the vibrio alginolyticus phage according to the following method (7 samples are parallelly made in each group and respectively numbered 0, 1, 2, 3, 4, 5 and 6, and the average value is taken):
the phage titer is measured by a double-layer plate method at an interval of 30 days, and the specific method comprises the following steps: 2216E medium 10, 10 for taking proper amount of product from tank2、……108Double dilution, 10. mu.L of each dilution was mixed with the host bacteria and counted by pouring the double-layered plate. After culturing at 30 ℃ for 12 hours, the number of plaques in the plate was counted and the phage content in the original sample was calculated. Among them, the results of measuring the phage lyophilized powder formulations of examples 1 to 6 and comparative examples 1 to 2 after being stored in an environment of 25 ℃ ± 2 ℃ for 6 months are shown in the following table 1:
TABLE 1
As can be seen from Table 1 and FIG. 2, the activity of the lyophilized phage powder preparations of examples 1 to 6 containing sucrose, sorbitol and skimmed milk powder as the lyophilized protectant still has high survival rate after 6 months storage, and as in examples 1 and 2, the activity is as high as 92.59% and 93.83%, respectively, and is maintained above 90%, which are all higher than that of the phage in comparative example 1. Embodiment 1 is relatively preferred to embodiments 1 and 2 in view of cost. In addition, compared with ValSw4, coliphage and Vibrio parahaemolyticus phage, the freeze-drying protection effect on the Vibrio alginolyticus phage is relatively good, but the freeze-drying protection effect on the phage can be effectively achieved, and the high biological activity of the phage freeze-dried powder preparation can be still ensured after the phage freeze-dried powder preparation is stored in an environment of 25 +/-2 ℃ for 6 months.
2. Phage freeze-dried powder preparation preservation temperature experiment:
the phage freeze-dried powder preparations of the embodiments 1 to 6 are subpackaged and stored in the environment of 4 ℃ and 25 +/-2 ℃ for 6 months respectively. The titer and survival rate of the vibrio alginolyticus phage were respectively detected according to the method in the comparative experiment of the protective agent action contained in section 1 phage freeze-dried powder preparation (7 samples were made in parallel in each group, numbered 0, 1, 2, 3, 4, 5, 6, respectively, and the average value was taken). The results of example 1, among others, are shown in figure 2 and table 2 below:
TABLE 2
As can be seen from Table 2 and FIG. 2, the freeze-dried powder preparation of Vibrio alginolyticus phage of the embodiment of the invention has high stability at a wide storage temperature of 4-25 + -2 ℃, wherein the survival rate of the Vibrio alginolyticus phage is still maintained to be more than 91% after 6 months of storage. Wherein, the Vibrio alginolyticus bacteriophage ValSw4-1 in the Vibrio alginolyticus bacteriophage freeze-dried powder preparation of the embodiment of the invention has the highest activity after being stored for 6 months in an environment with 25 +/-2 ℃, and still has 92.59 percent of activity. Based on the survival rate of the preservation and the cost of preservation and transportation of the phage lyophilized powder preparation according to the embodiment of the present invention, the preservation at room temperature, e.g., 25. + -. 2 ℃ is preferable. Other examples provide phage lyophilized powder preparations which have high survival rates at 4 ℃ and normal temperature, wherein the ideal selection is at 25 + -2 ℃ for storage at normal temperature, as the storage temperature experiment is similar to that of example 1.
3. Bacteriostatic effect or bioactivity experiment of the phage freeze-dried powder preparation:
the phage lyophilized powder preparations provided in the above examples 1 to 6 were subjected to bacteriostatic effect experiments according to the following methods, wherein SM buffer was used as a control group:
1) adding 180 mu L of 2216E culture medium into each hole of a 96-hole enzyme label plate;
2) dissolving 0.5g of the phage freeze-dried powder preparation in 1ml of SM buffer solution, adding 10 mu L of the dissolved phage into each well, adding 10 mu L of Vibrio alginolyticus cultured to logarithmic phase and 10 mu L of SM buffer solution into each well without adding the product;
3) placing the ELISA plate into an EPOCH 2 ELISA reader, linearly shaking the plate at 30 ℃, continuously shaking and culturing for 24 hours, and measuring the OD600 value of the culture system every 60 minutes.
Wherein, the measurement results of the Vibrio alginolyticus phage lyophilized powder preparation and the control group in the example 1 are shown in FIG. 3, and it can be known from FIG. 3 that the Vibrio alginolyticus phage lyophilized powder preparation of the embodiment of the invention can inhibit the growth of Vibrio alginolyticus for 12 hours. The phage freeze-dried powder preparation provided by other embodiments has stability based on a storage temperature experiment, and a bacteriostatic effect experiment of the phage freeze-dried powder preparation also has high biological activity, and can realize bacteriostatic action on corresponding specific bacteria for a long time.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A bacteriophage freeze-dried powder preparation is a freeze-dried mixture formed by freeze-drying treatment of a mixed solution containing bacteriophage with biological activity and a freeze-drying protective agent; wherein, in the lyophilization mixture, the titer of the bacteriophage is 109～1011PFU/g, the concentration of the freeze-drying protective agent is 0.3-1 mol/L.
2. The lyophilized powder preparation of bacteriophage according to claim 1, wherein: the freeze-drying protective agent comprises at least one of polyhydroxy compound and polymer composite protective agent; and/or
The phage is the phage with host being aquatic pathogenic bacteria.
3. The lyophilized powder preparation of bacteriophage according to claim 2, wherein: the aquatic pathogenic bacteria comprise vibrio;
the polyhydroxy compound comprises at least one of sucrose and sorbitol;
the polymer composite protective agent comprises skimmed milk powder.
4. The lyophilized powder preparation of bacteriophage according to claim 3, wherein: the bacteriophage includes at least one of a vibrio alginolyticus bacteriophage, a vibrio parahaemolyticus bacteriophage, and an escherichia coli bacteriophage.
5. The method for preparing a lyophilized phage powder preparation according to any one of claims 1 to 4, comprising the steps of:
mixing a solution containing phage and the freeze-drying protective agent in proportion to prepare a mixed solution;
and carrying out freeze-drying treatment on the mixed solution to form a freeze-dried mixture, thus obtaining the phage freeze-dried powder preparation.
6. The method for preparing a drug according to claim 5, wherein the conditions of the lyophilization process are as follows:
the freeze-drying temperature is-30 ℃ to-40 ℃, the time is 18-36 h, and the vacuum degree is 0.05-0.10 mbar;
and/or
The solution containing the phage is phage stock solution, and the preparation method of the phage stock solution comprises the following steps:
carrying out amplification culture on the host bacteria to obtain host bacteria liquid;
inoculating the phage into the host bacterium liquid for phage culture to obtain a phage culture solution;
and (3) carrying out separation treatment on the phage culture solution to remove impurities including host bacteria which are not lysed, so as to obtain the phage stock solution.
7. The method of claim 6, wherein: inoculating the phage into the host bacterial liquid according to the proportion that the MOI value is 1-10; and/or
In the host bacterium liquid, the concentration of the host bacterium is 109～1010CFU/ml; and/or
The separation treatment comprises the steps of centrifuging the phage culture solution to obtain a supernatant, and filtering the supernatant by adopting a micron filter membrane to collect filtrate.
8. A method for preserving a lyophilized phage powder preparation according to any one of claims 1 to 4, comprising the steps of:
and (3) preserving the phage freeze-dried powder preparation at the temperature of 4-27 ℃.
9. The saving method according to claim 8, wherein: the temperature of the preservation treatment is 23-27 ℃.
10. Use of a lyophilized phage powder formulation according to any one of claims 1-4 in aquaculture or in the manufacture of a medicament for aquaculture.
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