CN107213171A - A kind of preparation method of bacteriophagic Bdellovibrio freeze-dried powder - Google Patents

A kind of preparation method of bacteriophagic Bdellovibrio freeze-dried powder Download PDF

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CN107213171A
CN107213171A CN201710450413.3A CN201710450413A CN107213171A CN 107213171 A CN107213171 A CN 107213171A CN 201710450413 A CN201710450413 A CN 201710450413A CN 107213171 A CN107213171 A CN 107213171A
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China
Prior art keywords
freeze
bacteriophagic bdellovibrio
dried powder
bdellovibrio
bacteriophagic
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CN201710450413.3A
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Chinese (zh)
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郭立
敬科举
林茂
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厦门昶科生物工程有限公司
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Priority to CN201710450413.3A priority Critical patent/CN107213171A/en
Publication of CN107213171A publication Critical patent/CN107213171A/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET 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

Abstract

The invention provides a kind of production method of high, the powdered freeze dried preparation of bacteriophagic Bdellovibrio of survival rate, including according to bacteriophagic Bdellovibrio conventional culture methods obtain bacterium solution, or by harmful host separate after bacteriophagic Bdellovibrio bacteria suspension；Prepare protection agent solution；By after after the freeze-drying of lyophilized mixed liquor, vacuumize repeatedly, rush inert gas so that preparation ultimately becomes powdered；Above-mentioned bacteriophagic Bdellovibrio freeze-dried powder preparation is packed under vacuum or under conditions of filling inert gas, bacteriophagic Bdellovibrio freeze-dried powder preparation finished product is made.By the compounding of a variety of freeze drying protectants, the lyophilized survival rate of Bdellovibrio is improved, and the character of bacteriophagic Bdellovibrio freeze-dried powder preparation is powdered, is conducive to the scattered and compounding of freeze-dried powder preparation.

Description

A kind of preparation method of bacteriophagic Bdellovibrio freeze-dried powder
Technical field
The invention belongs to field of biological, and in particular to a kind of preparation method of bacteriophagic Bdellovibrio freeze-dried powder.
Background technology
Animal bacteria disease is prevented and treated using biotechnology to be widely used by people.Particularly leech arc The application of bacterium is more and more paid close attention to by people.Due to bacteriophagic Bdellovibrio major cleavage Gram-negative bacteria, such as Escherichia coli, The gram negative pathogenic bacterias such as salmonella, Aeromonas hydrophila, vibrios, and it is harmless to humans and animals, compared to traditional thing Physicochemical antibiotic, which eliminates pathogenic bacteria, obvious advantage.
The drawbacks of traditional physical chemistry antibiotic has obvious on harmful levels of pathogens is eliminated, physical method is caused to various The elimination effect of germ can be only applied to a certain link of association area, it is impossible to realize the covering in whole field.It is chemical and anti- Rear pathogenic bacteria easily form resistance to raw element method by long-term use, using effect are reduced, while also destruction inhibits beneficial bacterium , there is potential environmental hazard, can not finally efficiently control, eliminate pathogenic bacteria in group.So, bacteriophagic Bdellovibrio is gradually wide It is general to be applied to water body treating in aquaculture, the prevention and treatment of vibrios in fish shrimp crab, and chicken and duck class intestines problem control.
Harsh to environmental requirement because bacteriophagic Bdellovibrio does not generate gemma, its environment temperature survived is 4~45 DEG C, existing It is liquid bio preparation in the biological agent of the bacteriophagic Bdellovibrio used.It is continuous with itself bacterium due in liquid formulation Growth and breeding, can cause the metabolism product in liquid mechanism constantly to accumulate, pH value is gradually changed, and miscellaneous bacteria also can increasingly increase Value.Meanwhile, when environment temperature is higher than 25 DEG C, the viable count of the bacteriophagic Bdellovibrio in liquid formulation is decayed rapidly, and its curative effect is also fast Speed declines, therefore, and the shelf-life of liquid preparation at normal temperatures only has three, four months or so, has had a strong impact on the biological products Popularization and application.Therefore, people start by Bdellovibrio freeze to be prepared into freeze-dried powder in order to long-term preservation.
Existing patent (application number 99112389.1) mixes bacteriophagic Bdellovibrio (viable bacteria body) with the viable bacteria body of streptococcus fecalis Bacteria suspension is prepared into, is mixed by a certain percentage with protective agent by the bacteria suspension prepared, its protective agent is sucrose and gelatin group Conjunction or the combination of sucrose and skimmed milk power.Freeze-dried powder is obtained after mixed liquor is carried out into pre-freeze, distillation, parsing again, its Bdellovibrio Lose nearly an order of magnitude after freeze drying, survival rate is between 50~70%.The protective agent composition of the patent is simple, and freeze-dried powder is bitten Bacterium Bdellovibrio survival rate is not high, is 50~70%.Meanwhile, specific lyophilized technique is not disclosed, lacks specific Operating Guideline.Most Host is streptococcus fecalis afterwards, and host type is single, general for the preservation effect of Bdellovibrio.
Existing patent (application number 00112230.4) elaborates the freeze-drying process that a kind of host is dead host.Phagocytosis leech Bacterium solution is made according to inactivation host's cultural method in vibrios, obtains bacteriophagic Bdellovibrio aqua.By bacteriophagic Bdellovibrio aqua and stabilizer Mixing, obtains lyophilized mixed liquor, is freeze-dried.Wherein its freeze drying protectant is skimmed milk power sucrose stabilizer, and freezing is dry Dry temperature is -50 DEG C~37 DEG C.Pre-freezing temperature is -20~-50 DEG C.But, bacteriophagic Bdellovibrio host is lethal Fu Shi will Hayes dysentery bacillus either Escherichia coli.Using the Bdellovibrio of host's culture of inactivation, its infection ability is weaker, while using chlorine There is potential environmental hazard in imitative progress inactivation, while host, which is Fu Shi Hayes dysentery bacillus or Escherichia coli, is respectively provided with stronger cause Characteristic of disease, if inactivation does not have powerful destructiveness thoroughly to environment.Meanwhile, freeze drying protectant component is simple, is not carried in patent And lyophilized survival rate is how many, effect is unknown, there is obvious uncertainty, lacks referential.
The content of the invention
In view of this, it is an object of the invention to provide a kind of high, the powdered freeze dried preparation of bacteriophagic Bdellovibrio of survival rate Production method, comprise the following steps：
A. the acquisition of bacteriophagic Bdellovibrio bacteria suspension：Bacterium solution, Su Zhuwei are obtained according to the conventional culture methods of bacteriophagic Bdellovibrio The Gram-negative bacteria of no pathogenicity, or by harmful host separate after bacteriophagic Bdellovibrio bacteria suspension；
B. the preparation of agent solution is protected：The bacteriophagic Bdellovibrio bacteria suspension of agent solution and the step a will be protected according to 1：1 body Product ratio mixing, obtained bacteriophagic Bdellovibrio mixed liquor to be freezed；
C. the freeze-drying of mixed liquor to be freezed：
Bacteriophagic Bdellovibrio mixed liquor to be freezed is positioned in vacuum freeze dryer and carries out vacuum freeze drying, is obtained Obtain freeze dried preparation of bacteriophagic Bdellovibrio；
D. block lyophilized formulations are vacuumized repeatedly, rushes inert gas so that preparation ultimately becomes powdered；
E. above-mentioned bacteriophagic Bdellovibrio freeze-dried powder preparation is carried out under vacuum or under conditions of filling inert gas Pack, bacteriophagic Bdellovibrio freeze-dried powder preparation finished product is made.
Preferably, in the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation of the present invention, the step a no pathogenicities Host is：Bacillus subtilis, bafillus natto, bifidobacterium bifidum, enterococcus faecalis, lactobacillus bulgaricus, lactic acid sheet Coccus, streptococcus lactis, VREF, bacillus licheniformis, lactobacillus acidophilus, Lactobacillus casei, lactobacillus lactis, plant breast Bacillus or the one or more of Pediococcus pentosaceus.
Preferably, in the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation of the present invention, the phagocytosis leech of the step a Vibrios bacteria suspension is the bacteriophagic Bdellovibrio bacteria suspension of the Gram-negative bacteria containing no pathogenicity.
Preferably, in the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation of the present invention, the no pathogenicity leather is blue Family name's negative bacterium source is host in itself or by manually adding acquisition, the Gram-negative of final bacteriophagic Bdellovibrio and no pathogenicity Bacterium quantitative proportion is：1:10~1：100.
Preferably, in the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation of the present invention, described no pathogenicity Gram-negative bacteria be bacillus subtilis, bafillus natto, bifidobacterium bifidum, enterococcus faecalis, lactobacillus bulgaricus, Pediococcus acidilactici, streptococcus lactis, VREF, bacillus licheniformis, lactobacillus acidophilus, Lactobacillus casei, lactobacillus lactis, Lactobacillus plantarum or the one or more of Pediococcus pentosaceus.
Preferably, in the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation of the present invention, the protective agent of the step b Solution compound method is to dissolve skimmed milk power, beta-schardinger dextrin, trehalose, mannitol, lucid asparagus respectively using the purified water of sterilizing Propylhomoserin so that its final mass concentration is respectively 2%~10%, 2%~10%, 2%~4%, 2%~6%, 2%~8%.
Preferably, in the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation of the present invention, phagocytosis leech in the step c The mixed volume ratio of vibrios bacteria suspension and protection agent solution is 1:1~1:2.
Preferably, in the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation of the present invention, the step d freezes to described The parameter of dry preparation processing is that pre-freezing temperature is -30~-20 DEG C, and the pre-freeze time is 2~5 hours, freezing dry process temperature model Enclose for -15~37 DEG C, vacuum range is 0.05 millibar~0.2 millibar, freeze-drying time is 24~48 hours.
Preferably, in the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation of the present invention, to described in the step d The character of block lyophilized formulations is：Loose block structure, water content is 1~5%, and survival rate is more than 85%.
Another object of the present invention is to provide the bacteriophagic Bdellovibrio freeze-dried powder preparation of above method acquisition.
Compared with prior art, the present invention has advantages below to the present invention：
1), the present invention improves the lyophilized survival rate of Bdellovibrio by the compounding of a variety of freeze drying protectants.
2), secondly, the character of bacteriophagic Bdellovibrio freeze-dried powder preparation of the invention is powdered, is conducive to freeze-dried powder preparation It is scattered, i.e.,：According to actual needs, bacteriophagic Bdellovibrio powder preparation dilution is carried out by other powder, having reached preferably makes Use effect.
3) finally, by the host added with beneficial ecological environment, bacteriophagic Bdellovibrio Bdellovibrio in freeze-dried powder is not only increased The speed and stability of recovery.Meanwhile, positive effect is also played in actual production application, not only without potential Environmental hazard, and also help the protection of environment.
Embodiment
In one embodiment of the invention there is provided the production method of freeze dried preparation of bacteriophagic Bdellovibrio, including following step Suddenly：
A. the acquisition of bacteriophagic Bdellovibrio bacteria suspension：Bacterium solution, Su Zhuwei are obtained according to the conventional culture methods of bacteriophagic Bdellovibrio The Gram-negative bacteria of no pathogenicity, or by harmful host separate after bacteriophagic Bdellovibrio bacteria suspension；
B. the preparation of agent solution is protected：The bacteriophagic Bdellovibrio bacteria suspension of agent solution and the step a will be protected according to 1：1 body Product ratio mixing, obtained bacteriophagic Bdellovibrio mixed liquor to be freezed；
C. the freeze-drying of mixed liquor to be freezed：
Bacteriophagic Bdellovibrio mixed liquor to be freezed is positioned in vacuum freeze dryer and carries out vacuum freeze drying, is obtained Obtain freeze dried preparation of bacteriophagic Bdellovibrio；
D. block lyophilized formulations are vacuumized repeatedly, rushes inert gas so that preparation ultimately becomes powdered；
E. above-mentioned bacteriophagic Bdellovibrio freeze-dried powder preparation is carried out under vacuum or under conditions of filling inert gas Pack, bacteriophagic Bdellovibrio freeze-dried powder preparation finished product is made.
Preferably, in another embodiment of the present invention, the no pathogenicity host in the step a is：Withered grass gemma Bacillus, bafillus natto, bifidobacterium bifidum, enterococcus faecalis, lactobacillus bulgaricus, Pediococcus acidilactici, streptococcus lactis, VREF, bacillus licheniformis, lactobacillus acidophilus, Lactobacillus casei, lactobacillus lactis, Lactobacillus plantarum or Pediococcus pentosaceus One or more.
Preferably, in another embodiment of the present invention, the bacteriophagic Bdellovibrio bacteria suspension of the step a is containing whetheing there is cause The bacteriophagic Bdellovibrio bacteria suspension of the Gram-negative bacteria of characteristic of disease.
Preferably, in another embodiment of the present invention, the no pathogenicity derived from gram-negative bacteria is host's sheet Body or by manually adding acquisition, the Gram-negative bacteria quantitative proportion of final bacteriophagic Bdellovibrio and no pathogenicity is：1:10~ 1：100.
Preferably, in another embodiment of the present invention, the Gram-negative bacteria of described no pathogenicity is withered grass bud Spore bacillus, bafillus natto, bifidobacterium bifidum, enterococcus faecalis, lactobacillus bulgaricus, Pediococcus acidilactici, nisin Bacterium, VREF, bacillus licheniformis, lactobacillus acidophilus, Lactobacillus casei, lactobacillus lactis, Lactobacillus plantarum or pentose piece The one or more of coccus.
Preferably, in another embodiment of the present invention, the protection agent solution compound method of the step b is to use The purified water of sterilizing dissolves skimmed milk power, beta-schardinger dextrin, trehalose, mannitol, asparatate respectively so that its final mass Concentration is respectively 2%~10%, 2%~10%, 2%~4%, 2%~6%, 2%~8%.
Preferably, in another embodiment of the present invention, bacteriophagic Bdellovibrio bacteria suspension and protective agent are molten in the step c The mixed volume ratio of liquid is 1:1~1:2.
Preferably, in yet another embodiment of the present invention, the parameter that the step d is handled the lyophilized formulations is pre- It is -30~-20 DEG C to freeze temperature, and the pre-freeze time is 2~5 hours, and freezing dry process temperature range is -15~37 DEG C, vacuum model Enclose for 0.05 millibar~0.2 millibar, freeze-drying time is 24~48 hours.
Preferably, in another embodiment of the present invention, to the characters of the block lyophilized formulations in the step d For：Loose block structure, water content is 1~5%, and survival rate is more than 85%.
Below in conjunction with the embodiment in the present invention, the technical scheme in the present invention is clearly and completely described.It is aobvious So, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based on the reality in the present invention Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made all belongs to In the scope of protection of the invention.
The production technology 1 of the bacteriophagic Bdellovibrio freeze-dried powder preparation of embodiment 1
A. the acquisition of bacteriophagic Bdellovibrio bacteria suspension.
Conventional culture methods：Take 50ml Bdellovibrio liquid (107Pfu/ml) with 50ml bacillus licheniformis liquid (108cfu/ Ml) mix, be inoculated in 20 liters of fermentation tanks, liquid amount is 10 liters, and temperature is 28 DEG C, and rotating speed is 150rpm, and throughput is 10L/L/ Min, ferments 3 days, obtains Bdellovibrio zymotic fluid
The culture of Bdellovibrio zymotic fluid is formulated：1% peptone, 0.3% beef extract, 0.5% sodium chloride, 0.3% dusty yeast.
After fermentation ends, 1 liter of bacillus subtilis bacterium solution (10 is added to zymotic fluid13Cfu/ml), it is ensured that bacteriophagic Bdellovibrio It is with Bacillus subtilis number ratio：1:10.
B. the preparation and the preparation of lyophilized liquid of protection agent solution
It is to dissolve skimmed milk power, beta-schardinger dextrin, sea respectively using 10 liters of purified waters of sterilizing to protect agent solution process for preparation Algae sugar, mannitol, asparatate so that its final mass concentration is respectively 2%, 2%, 2%, 2%, 2%.Protective agent is molten Liquid and the bacteriophagic Bdellovibrio bacteria suspension of a technical process are according to 1：1 volume ratio is mixed, and 20 liters of bacteriophagic Bdellovibrios are made and treat lyophilized mixed Close liquid；
C. the preparation of freeze dried preparation of bacteriophagic Bdellovibrio
Bacteriophagic Bdellovibrio mixed liquor to be freezed in technique b is put into Dong Fulong vacuum freeze dryers (LYO-2m2) in Freezed, its pre-freezing temperature is -30 DEG C, the pre-freeze time is 2 hours, and vacuum is 0.2 millibar, wherein the temperature once distilled For -15 DEG C, the time is 24 hours, and secondary sublimation temperature is 10 DEG C, and the time is 24 hours, and it is thin finally to obtain 800g lyophilized formulations The block structure of pine, its water content is 5%, and Bdellovibrio survival rate is 95%
D. block lyophilized formulations are vacuumized repeatedly, gas flow is controlled by flowmeter using nitrogen cylinder, with 0.5L/min speed rushes nitrogen to the freeze-dried powder that vacuum packet is installed, untill packaging bag sticks out, and so inflation is vacuumized, So that lyophilized formulations ultimately become powdered, final Bdellovibrio survival rate is 95%.
The production technology 2 of the bacteriophagic Bdellovibrio freeze-dried powder preparation of embodiment 2
A. the acquisition of bacteriophagic Bdellovibrio bacteria suspension.
Bacterium solution is obtained according to the following conventional culture methods of bacteriophagic Bdellovibrio
Conventional culture methods：Take 100ml Bdellovibrio liquid (107Pfu/ml) with 100ml bacillus licheniformis liquid (108cfu/ Ml) mix, be inoculated in 30 liters of fermentation tanks, liquid amount is 20 liters, and temperature is 28 DEG C, and rotating speed is 150rpm, and throughput is 20L/L/ Min, ferments 3 days, obtains Bdellovibrio zymotic fluid
The culture of Bdellovibrio zymotic fluid is formulated：1% peptone, 0.3% beef extract, 0.5% sodium chloride, 0.3% dusty yeast.
After fermentation ends, 1 liter of Bacillus licheniformis liquid (10 is added to zymotic fluid13Cfu/ml), it is ensured that bacteriophagic Bdellovibrio It is with bacillus licheniformis quantitative proportion：1:100.
B. the preparation and the preparation of lyophilized liquid of protection agent solution
Protection agent solution process for preparation is to dissolve skimmed milk power, beta-schardinger dextrin, sea respectively using the purified water of 20 liters of sterilizings Algae sugar, mannitol, asparatate so that its final mass concentration is respectively 5%, 5%, 5%, 5%, 5%.Protective agent is molten Liquid and the bacteriophagic Bdellovibrio bacteria suspension of a technical process are according to 1：1 volume ratio is mixed, and 40 liters of bacteriophagic Bdellovibrios are made and treat lyophilized mixed Close liquid；
C. the preparation of freeze dried preparation of bacteriophagic Bdellovibrio
Bacteriophagic Bdellovibrio mixed liquor to be freezed in technique b is put into Dong Fulong vacuum freeze dryers (LYO-2m2) in Freezed, its pre-freezing temperature is -25 DEG C, the pre-freeze time is 4 hours, and vacuum is 0.2 millibar, wherein the temperature once distilled For -5 DEG C, the time is 18 hours, and secondary sublimation temperature is 25 DEG C, and the time is 18 hours, and it is thin finally to obtain 1600g lyophilized formulations The block structure of pine, its water content is 5%, and Bdellovibrio survival rate is 95%
D. block lyophilized formulations are vacuumized repeatedly, gas flow is controlled by flowmeter using nitrogen cylinder, with 0.5L/min speed rushes nitrogen to the freeze-dried powder that vacuum packet is installed, untill packaging bag sticks out, and so inflation is vacuumized, So that lyophilized formulations ultimately become powdered；
E. block lyophilized formulations are vacuumized repeatedly, rushes inert gas so that lyophilized formulations ultimately become powder Shape.
The production technology 3 of the bacteriophagic Bdellovibrio freeze-dried powder preparation of embodiment 3
A. the acquisition of bacteriophagic Bdellovibrio bacteria suspension.
Bacterium solution is obtained according to the conventional culture methods of bacteriophagic Bdellovibrio
Conventional culture methods：Take 50ml Bdellovibrio liquid (107Pfu/ml) with 50ml enterococcus faecalis bacterium solution (108cfu/ml) Mixing, is inoculated in 10 liters of fermentation tanks, and liquid amount is 5 liters, and temperature is 28 DEG C, and rotating speed is 100rpm, and throughput is 5L/L/min, hair Ferment 3 days, obtains Bdellovibrio zymotic fluid
The culture of Bdellovibrio zymotic fluid is formulated：1% peptone, 0.3% beef extract, 0.5% sodium chloride, 0.3% dusty yeast.
B. the preparation and the preparation of lyophilized liquid of protection agent solution
Protection agent solution process for preparation is to dissolve skimmed milk power, beta-schardinger dextrin, sea respectively using the purified water of 10 liters of sterilizings Algae sugar, mannitol, asparatate so that its final mass concentration is respectively 10%, 10%, 10%, 10%, 10%.It will protect Agent solution is protected with the bacteriophagic Bdellovibrio bacteria suspension of a technical process according to 1：2 volume ratios are mixed, and 15 liters of bacteriophagic Bdellovibrios are made and treat Lyophilized mixed liquor；
C. the preparation of freeze dried preparation of bacteriophagic Bdellovibrio
Bacteriophagic Bdellovibrio mixed liquor to be freezed in technique b is put into Dong Fulong vacuum freeze dryers (LYO-2m2) in Freezed, its pre-freezing temperature is -20 DEG C, the pre-freeze time is 5 hours, and vacuum is 0.1 millibar, wherein the temperature once distilled For 0 DEG C, the time is 15 hours, and secondary sublimation temperature is 37 DEG C, and the time is 15 hours, and it is thin finally to obtain 1200g lyophilized formulations The block structure of pine, its water content is 5%, and Bdellovibrio survival rate is 93%
D. block lyophilized formulations are vacuumized repeatedly, gas flow is controlled by flowmeter using nitrogen cylinder, with 0.5L/min speed rushes nitrogen to the freeze-dried powder that vacuum packet is installed, untill packaging bag sticks out, and so inflation is vacuumized, So that lyophilized formulations ultimately become powdered；
E. block lyophilized formulations are vacuumized repeatedly, rushes inert gas so that lyophilized formulations ultimately become powder Shape.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (10)

1. a kind of production method of freeze dried preparation of bacteriophagic Bdellovibrio, comprises the following steps：
A. the acquisition of bacteriophagic Bdellovibrio bacteria suspension：Conventional culture methods according to bacteriophagic Bdellovibrio obtain bacterium solution, and host is without cause The Gram-negative bacteria of characteristic of disease, or by harmful host separate after bacteriophagic Bdellovibrio bacteria suspension；
B. the preparation of agent solution is protected：The bacteriophagic Bdellovibrio bacteria suspension of agent solution and the step a will be protected according to 1：1 volume ratio Example mixing, obtained bacteriophagic Bdellovibrio mixed liquor to be freezed；
C. the freeze-drying of mixed liquor to be freezed：
Bacteriophagic Bdellovibrio mixed liquor to be freezed is positioned in vacuum freeze dryer and carries out vacuum freeze drying, is bitten Bacterium Bdellovibrio lyophilized formulations；
D. block lyophilized formulations are vacuumized repeatedly, rushes inert gas so that preparation ultimately becomes powdered；
E. above-mentioned bacteriophagic Bdellovibrio freeze-dried powder preparation is sealed under vacuum or under conditions of filling inert gas Packaging, is made bacteriophagic Bdellovibrio freeze-dried powder preparation finished product.
2. the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation according to claim 1, it is characterised in that the step a No pathogenicity host is：Bacillus subtilis, bafillus natto, bifidobacterium bifidum, enterococcus faecalis, bulgarian milk bar The newborn bar of bacterium, Pediococcus acidilactici, streptococcus lactis, VREF, bacillus licheniformis, lactobacillus acidophilus, Lactobacillus casei, lactic acid The one or more of bacterium, Lactobacillus plantarum or Pediococcus pentosaceus.
3. the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation according to claim 1, it is characterised in that the step a Bacteriophagic Bdellovibrio bacteria suspension be the Gram-negative bacteria containing no pathogenicity bacteriophagic Bdellovibrio bacteria suspension.
4. the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation according to claim 3, it is characterised in that the nothing is caused a disease Property derived from gram-negative bacteria be host in itself or by manually adding acquisitions, the leather orchid of final bacteriophagic Bdellovibrio and no pathogenicity Family name's negative bacterium quantitative proportion is：1:10~1：100.
5. the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation according to claim 3, it is characterised in that described without cause The Gram-negative bacteria of characteristic of disease is bacillus subtilis, bafillus natto, bifidobacterium bifidum, enterococcus faecalis, Bulgaria Lactobacillus, Pediococcus acidilactici, streptococcus lactis, VREF, bacillus licheniformis, lactobacillus acidophilus, Lactobacillus casei, lactic acid Lactobacillus, Lactobacillus plantarum or the one or more of Pediococcus pentosaceus.
6. the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation according to claim 1, it is characterised in that the step b Protection agent solution compound method be to dissolve skimmed milk power, beta-schardinger dextrin, trehalose, sweet dew respectively using the purified water of sterilizing Alcohol, asparatate so that its final mass concentration is respectively 2%~10%, 2%~10%, 2%~4%, 2%~6%, 2%~8%.
7. the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation according to claim 1, it is characterised in that the step c The mixed volume ratio of middle bacteriophagic Bdellovibrio bacteria suspension and protection agent solution is 1:1~1:2.
8. bacteriophagic Bdellovibrio freeze-dried powder preparation production method according to claim 1, it is characterised in that d pairs of the step The parameter of the lyophilized formulations processing is that pre-freezing temperature is -30~-20 DEG C, and the pre-freeze time is 2~5 hours, freezing dry process Temperature range is -15~37 DEG C, and vacuum range is 0.05 millibar~0.2 millibar, and freeze-drying time is 24~48 hours.
9. the production method of bacteriophagic Bdellovibrio freeze-dried powder preparation according to claim 1, it is characterised in that the step d In be to the characters of the block lyophilized formulations：Loose block structure, water content is 1~5%, and survival rate is more than 85%.
10. the bacteriophagic Bdellovibrio freeze-dried powder preparation that the method according to claim 1~9 any one is obtained.
CN201710450413.3A 2017-06-15 2017-06-15 A kind of preparation method of bacteriophagic Bdellovibrio freeze-dried powder CN107213171A (en)

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