CN102296056A - Mycobacteriophage D29 particles and preparation method and use thereof - Google Patents
Mycobacteriophage D29 particles and preparation method and use thereof Download PDFInfo
- Publication number
- CN102296056A CN102296056A CN2011102527468A CN201110252746A CN102296056A CN 102296056 A CN102296056 A CN 102296056A CN 2011102527468 A CN2011102527468 A CN 2011102527468A CN 201110252746 A CN201110252746 A CN 201110252746A CN 102296056 A CN102296056 A CN 102296056A
- Authority
- CN
- China
- Prior art keywords
- mycobacteriophage
- pfu
- particle
- lactose
- protective material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses mycobacteriophage D29 particles and a preparation method and use thereof. The active ingredients of the mycobacteriophage D29 particles include mycobacteriophage D29 and a protective agent; and the protective agent is sugar and/or protein and/or amino acid and/or alcohol. In the invention, inhalable particles are prepared by using a nano spray drying technique, a unique stabilizing technique is adopted in spray drying, and thus, the technical problems in the preparation process of the mycobacteriophage D29 dry powder inhalant are solved; and the inhalation administration can give better play to the treatment effect of the mycobacteriophage D29 on tuberculosis in clinic, solves the technical problem in the use of the mycobacteriophage D29 and makes the development of the mycobacteriophage D29 into a medicine possible.
Description
Technical field
The present invention relates to a kind of mycobacteriophage D29 particle and its production and application.
Background technology
Tuberculosis is that human health is threatened very big infectious diseases common to human beings and animals always, owing to movement of population, extensive resistance and factor affecting such as multiple-drug resistance tuberculosis bacterium (MDR-TB) propagation, acquired immune deficiency syndrome (AIDS), the tuberculosis epidemic situation is gone up in the world over particularly past two, 30 years.The World Health Organization (WHO) once announced in 1993: the global tuberculosis emergency state; Reaffirmed again in 1998: the action of containment tuberculosis is very urgent, and being decided to be the World Tuberculosis Prevention and Cure Day annual March 24.The WHO latest data shows: newly-increased tuberculosis patient 9,400,000 people in 2009, and the patient who dies from tuberculosis reaches 1,700,000 people; Newly-increased patient MDR-TB 440,000 people in 2008, that dies from MDR-TB then reaches 150,000 people.China is one of country of the high burden of 22 tuberculosis in the whole world, and belongs to high resistance country, and the tuberculosis patient numerical digit occupies the whole world the second, and the multiple-drug resistance tuberculosis disease accounts for 13.4%.But up to the present, also there is not the medicine of ideal novel anti multiple-drug resistance tuberculosis to can be used for clinical.
(Bacteriophage phage) is a bacterioid virus to phage, and some bacteriums are had the specificity of height, when phage is infected these bacteriums, can breed in bacterium and killing bacteria, and it does not have toxicity to animals and plants.Therefore, phage is expected to become antibacterials preferably with its distinctive physical feature.20th century frontal lobe, once as the effective tool of treatment, prevention infectation of bacteria, but along with antibiotic invention and widespread use, many countries furtherd investigate this phage.Yet in the face of the continuous growth of drug-fast bacteria infection ratio, the someone foretells that phage will become a big research focus of nearly ten years field of biological pharmacy.
Phage successfully has been applied to the treatment of multiple animal bacterial infection, as bird, fish, calf, lamb etc.Also there are a lot of clinical studyes bacterial infection aspect to prevention and treatment people.Weber-Dabrowska etc. use the phage treatment to 20 routine tumour patients and 27 routine infectation of bacteria persons, find all patients' all very fast disappearances of concurrent phenomenon such as suppuration, wound, pneumonia.Bretscher etc. propose to use the infection once again of phage treatment acquired immune deficiency syndrome (AIDS), can slow down the speed that the anti-multiple medicines of hiv virus is evolved.
Mycobacteriophage D29 is a kind of dna virus that parasitizes mycobacterium, form capsid by protein enclosure, interior is DNA, there is not independently metabolism system, must combine with the host bacterium and enter in the born of the same parents, utilize the metabolic enzyme system of host bacterium to duplicate, final cracking bacterium discharges progeny phage.The virion of mycobacteriophage D29 is similar round, and size has the wide spectrum host between 75～150nm, comprise M. smegmatics and mycobacterium tuberculosis etc.The host is 90～180min the proliferating cycle in M. smegmatics, and be more than 3～6h the proliferating cycle in mycobacterium tuberculosis.
Mycobacteriophage D29 has been widely used in the rapid detection and the chemical sproof detection of mycobacterium tuberculosis of mycobacterium tuberculosis.Mycobacteriophage D29 is used for the research of antituberculosis therapy, experiment in vitro finds that mycobacteriophage D29 can reduce the M. smegmatics in the scavenger cell of growth in vitro and the number of viable of mycobacterium tuberculosis, have the ability of killing host bacterium in the scavenger cell, have treatment possibility lungy; Animal experiment proves that also mycobacteriophage D29 can alleviate organ disease degree and the internal organs lotus bacterium amount of tuberculosis cavy, have with Rifampin approaching, even better therapeutic, and cavy is free from side effects, be hopeful development object as novel antitubercular agent.
Mycobacteriophage D29 is used for tuberculotherapy its potential advantages.Phage has the host specificity of height, only at pathogenic bacterium, can not have influence on other flora, therefore has the advantage that has no side effect, and has reduced the resistance problem of thereupon being brought simultaneously; Phage can breed with the propagation of host bacterium, and plays a role in the whole process of infectation of bacteria, and unlike microbiotic As time goes on curative effect reduce gradually.Be used for the application of tuberculotherapy at mycobacteriophage D29, the problem that emphasis will solve is how to arrive site of action and which kind of form to arrive site of action effectively with.
Mycobacteriophage D29 is as a kind of microorganism, and cultivating the product that obtains generally all is the liquid that comprises developing medium.For solid, liquid store, transportation and have problems such as inconvenience and stability when using, so microniological proudcts desire to make medicine, solidification has its advantage.Microbial preparation adopts lyophilize favourable for keeping its stability in the solidification process, so all adopt freeze-drying usually in the preservation of microbial preparation.
Summary of the invention
The purpose of this invention is to provide a kind of mycobacteriophage D29 particle and its production and application.
Mycobacteriophage D29 particle provided by the invention, its activeconstituents is made up of mycobacteriophage D29 and protective material (double as thinner); Described protective material is sugar and/or albumen and/or amino acid and/or alcohol.
Described sugar can be at least a in sucrose, lactose and the trehalose.Described albumen can be at least a in milk powder and the bovine serum albumin.Described amino acid can be leucine.Described alcohol can be N.F,USP MANNITOL.
Described protective material can be a kind of sugar, also can be the mixture of multiple sugar.Described sugar can be at least a in sucrose, lactose and the trehalose.Described protective material also can be albumen, as milk powder (as skim-milk) or bovine serum albumin.Described protective material also can be sugar and proteic mixture, as the mixture of lactose and milk powder, or the mixture of lactose and bovine serum albumin.Described protective material also can be sugar and amino acid whose mixture, as lactose and leucic mixture.Described protective material also can be the mixture of sugar and alcohol, as the mixture of lactose and N.F,USP MANNITOL.
Described protective material is preferably any one in following (1) to (9): (1) sucrose; (2) lactose; (3) trehalose; (4) lactose and trehalose; (5) lactose and N.F,USP MANNITOL; (6) milk powder; (7) lactose and milk powder; (8) lactose and bovine serum albumin; (9) lactose and leucine.
Described mycobacteriophage D29 particle specifically can be made up of described mycobacteriophage D29, described protective material and water.
In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival can be 1 * 10
2PFU/g to 1 * 10
9PFU/g, the quality percentage composition of moisture can be below 10%; Described mycobacteriophage D29 particulate granularity median D50 can be below the 20 μ m.
In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is preferably 1 * 10
4PFU/g to 7 * 10
7PFU/g, the quality percentage composition of moisture most preferably is 1% to 9%; Described mycobacteriophage D29 particulate granularity median D50 is preferably 5.00 μ m to 18.00 μ m.
In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival most preferably is 1.99 * 10
4PFU/g to 6.66 * 10
7PFU/g, the quality percentage composition of moisture most preferably is 1.23% to 8.30%; Described mycobacteriophage D29 particulate granularity median D50 most preferably is 5.60 μ m to 17.31 μ m.
In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival can be (2.72 ± 0.73) * 10
4PFU/g is to (3.10 ± 1.12) * 10
4PFU/g, (3.10 ± 1.12) * 10
4PFU/g is to (1.29 ± 0.16) * 10
6PFU/g, (1.29 ± 0.16) * 10
6PFU/g is to (1.81 ± 0.19) * 10
6PFU/g, (1.81 ± 0.19) * 10
6PFU/g is to (1.86 ± 0.10) * 10
6PFU/g, (1.86 ± 0.10) * 10
6PFU/g is to (2.06 ± 0.23) * 10
6PFU/g, (2.06 ± 0.23) * 10
6PFU/g is to (2.41 ± 0.12) * 10
6PFU/g, (2.41 ± 0.12) * 10
6PFU/g is to (4.49 ± 0.99) * 10
6PFU/g, (4.49 ± 0.99) * 10
6PFU/g is to (6.53 ± 0.13) * 10
6PFU/g, or (6.53 ± 0.13) * 10
6PFU/g is to (5.14 ± 1.52) * 10
7PFU/g.
In the described mycobacteriophage D29 particle, the quality percentage composition of moisture can be (1.34 ± 0.11) % to (2.11 ± 0.08) %, (2.11 ± 0.08) % is to (2.30 ± 0.57) %, (2.30 ± 0.57) % is to (2.50 ± 0.20) %, (2.50 ± 0.20) % is to (2.74 ± 0.04) %, (2.74 ± 0.04) % is to (2.84 ± 0.57) %, (2.84 ± 0.57) % is to (2.88 ± 0.04) %, (2.88 ± 0.04) % is to (3.27 ± 0.35) %, (3.27 ± 0.35) % is to (5.08 ± 0.22) %, or (5.08 ± 0.22) % is to (8.08 ± 0.27) %.
Described mycobacteriophage D29 particulate granularity median D50 can be (5.65 ± 0.05) μ m to (7.49 ± 0.59) μ m, (7.49 ± 0.59) μ m is to (7.91 ± 0.39) μ m, (7.91 ± 0.39) μ m is to (8.14 ± 0.17) μ m, (8.14 ± 0.17) μ m is to (8.69 ± 0.94) μ m, (8.69 ± 0.94) μ m is to (8.94 ± 0.29) μ m, (8.94 ± 0.29) μ m is to (9.34 ± 0.40) μ m, (9.34 ± 0.40) μ m is to (10.94 ± 0.45) μ m, (10.94 ± 0.45) μ m is to (10.56 ± 0.65) μ m, or complete (15.66 ± 1.65) μ m of (10.56 ± 0.65) μ m.
The present invention also protects described mycobacteriophage D29 particulate preparation method, is that described mycobacteriophage D29 is carried out solidification (curing) with described protective material as raw material, obtains mycobacteriophage D29 particle.
When described protective material was lactose and trehalose, described lactose and described trehalose were preferably 1: 1 as the mass ratio of raw material.When described protective material was lactose and milk powder, described lactose and described milk powder were preferably 1: 5 as the mass ratio of raw material.When described protective material was lactose and bovine serum albumin, described lactose and described bovine serum albumin were preferably 1: 1 as the mass ratio of raw material.When described protective material was lactose and leucine, described lactose and described leucine were preferably 5: 1 as the mass ratio of raw material.When described protective material was lactose and N.F,USP MANNITOL, described lactose and described N.F,USP MANNITOL were preferably 1: 1 as the mass ratio of raw material.
Described protective material and described mycobacteriophage D29 can be 1g as proportion of raw materials: (1 * 10
4PFU to 1 * 10
12PFU).Described protective material and described mycobacteriophage D29 specifically can be 1g as proportion of raw materials: (1 * 10
6PFU to 1 * 10
10PFU).Described protective material and described mycobacteriophage D29 specifically are preferably 1g as proportion of raw materials: (1.88 * 10
6PFU to 5.16 * 10
9PFU).
Described protective material and described mycobacteriophage D29 can be 1g as proportion of raw materials: (2.68 ± 0.80) * 10
6PFU is to (0.74 ± 0.40) * 10
8PFU, 1g: (0.74 ± 0.40) * 10
8PFU is to (0.96 ± 0.09) * 10
8PFU, 1g: (0.96 ± 0.09) * 10
8PFU is to (1.08 ± 0.27) * 10
8PFU, 1g: (1.08 ± 0.27) * 10
8PFU is to (1.30 ± 0.33) * 10
8PFU, 1g: (1.30 ± 0.33) * 10
8PFU is to (1.86 ± 0.78) * 10
8PFU, 1g: (1.86 ± 0.78) * 10
8PFU is to (2.78 ± 0.15) * 10
8PFU or 1g: (2.78 ± 0.15) * 10
8PFU is to (4.4 ± 0.76) * 10
9PFU.
Described solidified method specifically can be spray-drying process.When using spray-drying process and carrying out described solidification, the parameter of described spray-drying process specifically can be: temperature in 80 ℃ to 120 ℃ (as 80 ℃ to 100 ℃, 100 ℃ to 120 ℃), gas velocity 90L/min to 150L/min (as 90L/min to 120L/min, 120L/min to 150L/min), spray rate 40% to 100% (as 40% to 60%, 60% to 80%, 80% to 100%), nozzle cap apertures are 4.0 μ m to 7.0 μ m (as 4.0 μ m to 5.5 μ m, 5.5 μ m to 7.0 μ m).The used instrument of described spraying drying specifically can be Switzerland BUCHI B-90 nanometer spray-drier.
Be used for described solidified raw material, described protective material specifically can add with the form of the aqueous solution.Described solidified raw material is preferably aseptic raw material.
The present invention is by after adopting suitable protective material, suitable plant and instrument, optimized parameters, and the mycobacteriophage D29 particulate matter (Foradil Aerolizer formoterol fumarate) and the active retention value of mycobacteriophage D29 that can obtain to be suitable for inhalation are very high.If do not use protective material, or protective material select for use improper, mycobacteriophage D29 in spray-drying process, the overwhelming majority (＞95%) with inactivation, lost its solidified meaning.The present invention finds that in carbohydrate, protein, amino acids and the alcohols one or more can be used as the solidified protective material of mycobacteriophage D29; adopt carbohydrate admixture the active reservation of mycobacteriophage D29 to be reached more than 70% and (see embodiment 5) as protective material; the mixture that adopts carbohydrate and aminoacid protein class is as protective material; then can make the active reservation of mycobacteriophage D29 reach about 80% (seeing embodiment 9,10), even 90% above (seeing embodiment 8).Conventional spraying drying, it obtains the particulate size usually at tens of microns even more than 100 microns, and Da Xiao particle can't suck and enter lower respiratory tract like this, therefore is not suitable for lung's inhalation.The present invention has explored the influence of drying process with atomizing to acquisition particulate matter size, is suitable for the particle of inhalation.
Mycobacteriophage D29 particle provided by the invention, the content of the mycobacteriophage D29 of survival can be 1 * 10
2PFU/g to 1 * 10
9PFU/g, the quality percentage composition of moisture can be below 10%, and granularity median D50 can be below the 20 μ m.
The present invention adopts the nanometer spray drying technology to prepare pellet, and has adopted unique stabilization technology in spraying drying, thereby has solved the technological difficulties in the mycobacteriophage D29 Foradil Aerolizer formoterol fumarate preparation process; Adopt inhalation can better bring into play the result of treatment of mycobacteriophage D29 in clinical lung tuberculosis, solved the technological difficulties during mycobacteriophage D29 uses.Finally, mycobacteriophage D29 provides possibility for developing into a medicine.
Description of drawings
Fig. 1 be embodiment 1 with sucrose the electromicroscopic photograph during as protective material
Fig. 2 be embodiment 3 with trehalose the electromicroscopic photograph during as protective material
Fig. 3 be embodiment 4 with lactose the electromicroscopic photograph during as protective material
Electromicroscopic photograph when Fig. 4 is embodiment 5 with lactose and trehalose as protective material
Electromicroscopic photograph when Fig. 5 is embodiment 6 with lactose and N.F,USP MANNITOL as protective material
Fig. 6 be embodiment 7 with skim-milk the electromicroscopic photograph during as protective material
Electromicroscopic photograph when Fig. 7 is embodiment 8 with lactose and skim-milk as protective material
Electromicroscopic photograph when Fig. 8 is embodiment 9 with lactose and bovine serum albumin as protective material
Electromicroscopic photograph when Fig. 9 is embodiment 10 with lactose and leucine as protective material
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.CFU is a colony-forming unit, and PFU is a plaque forming unit.
7H9 substratum: U.S. company BD (the former factory of product numbering 271310).
Mycobacteriophage D29: reference is: Peng Li, Luo Yongai, Chen Baowen etc. phage D29 is to infecting the curative effect of sensitive strain mycobacterium tuberculosis cavy. and Chinese Amphixenosis's journal .2009,25 (8): 733-736.
Used M. smegmatics is M. smegmatics (Mycobacterium smegmatis) ATCC607 among the embodiment: reference: Liu Keyang, Du Qian, Wen Zhanbo etc. the preliminary study of aerocolloidal spraying of mycobacteriophage D29 and sampling media. institute of Military Medical Science Institute periodical, 2010,34 (4): 347-350.
The method of calculation of yield are as follows: (the mycobacteriophage D29 particulate quality of results/add as raw material protectant quality) * 100%.
The method of calculation of water content are as follows: get and clean air dried weighing bottle (the about 45mm of diameter), dry 2h in 100 ℃ ± 2 ℃, put to the room temperature the precision (W that weighs
0); About 1g mycobacteriophage D29 particle is tiled in the weighing bottle precision (W that weighs
1); Weighing bottle is transferred in the baking oven, opens bottle cap and dry processing and (under 115 ℃ ± 2 ℃ conditions, dry 20h when protective material is carbohydrate; Under 80 ℃ ± 2 ℃ conditions, dry 20h when protective material contains protein), build bottle cap then, take out under room temperature (relative humidity is lower than 60%) condition and place 10min, the precision (W that weighs
2); Water content (%)=[(W
1-W
2)/(W
1-W
0)] * 100%.
Mycobacteriophage D29 particulate particle size determination is as follows: adopt LS908 (A) laser particle size analyzer (the American-European gram in Zhuhai Instr Ltd.) to measure among the embodiment of this patent, concrete grammar is: (1) adds 7mL medium (ethyl oleate) in the static sample pond, put into instrument measuring system, after treating instrument self-centering, zero clearing, instrument enters state to be measured; (2) after employing dispersion medium (ethyl oleate) is made the even suspension of about 5mg/mL with mycobacteriophage D29 particle, getting 3 suspensions (0.1mL-0.2mL) joins in the medium in static sample pond, dispel evenly, put into instrument measuring system, after treating that light beam is stable, can carry out particle size measurement, D50 represents the particulate size with the granularity median.Also can adopt other commercial obtainable particle size analyzers, concrete steps need to be adjusted accordingly according to the working specification of institute's use instrument.
Mycobacteriophage D29 particulate Electronic Speculum measuring method: with mycobacteriophage D29 uniform particles be layered in the sample holder, behind the E-1010 of Hitachi ion sputtering instrument metal spraying, observe under the S-3400N of the Hitachi scanning electron microscope, take a picture.Adopt other commercial obtainable scanning electron microscope also can obtain electromicroscopic photograph, concrete steps need to be adjusted accordingly according to the working specification of institute's use instrument.
The method of calculation of the phage survival rate in the mycobacteriophage D29 particle are as follows: after mycobacteriophage D29 particle is dissolved with SM liquid, carry out 10 times of gradient dilutions with SM liquid again; Get the 0.1mL diluent respectively to different 7H9 bottom culture medium flat plates (Ф 90mm), evenly with the coating of " L " rod; Adding concentration according to the ratio of 3mL bacterium liquid/50mL substratum in being heated to about 60 ℃ 7H9 upper strata substratum is 10
7The M. smegmatics bacterium liquid of CFU/mL, shake up gently the back to above-mentioned each be coated with in the 7H9 bottom substratum plate of phage D29 and poured the 7H9 upper strata substratum that 5mL contains M. smegmatics into; After treating that the upper strata substratum solidifies fully, plate is positioned in the constant incubator 37 ℃ is just putting and cultivated 2 days, observe, the PFU (10～300 result is the credibility interval) on the statistics flat board; According to the survival phage number in the extent of dilution calculating mycobacteriophage D29 particle; 3 plates of each dilution sample coating, results averaged; Survival rate=(the phage number/yield that adds during the survival phage number in the mycobacteriophage D29 particle that finally obtains/spraying drying) * 100%.
SM liquid: take by weighing 2.9g NaCl and 1.0g MgSO
4.7H
2O adds 25ml 1mol/L Tris-Cl (pH 7.5) and 2.5ml 2% (g/100ml) aqueous gelatin solution, adds water to 500ml, in 121 ℃ of 30min that sterilize, puts to room temperature and promptly gets SM liquid behind the mixing.
7H9 bottom substratum: take by weighing 1.6g 7H9 substratum, 5.6g agar powder, add water 315mL, 121 ℃ of sterilization 30min behind the mixing, aseptic technique adds calf serum complex solution (OADC solution) 35ml 45～50 ℃ the time then, makes flat board (12ml/ plate) behind the mixing; Put solidify to room temperature after, 4 ℃ of refrigerators are preserved.
7H9 upper strata substratum: take by weighing 1.6g 7H9 substratum, 2.8g agar powder, add water 315mL, 121 ℃ of sterilization 30min place 4 ℃ of preservations behind the mixing under the sterile seal condition.
OADC solution: 8.5g NaCl, 50g BSA (the bSA V factor), 20g glucose are fully dissolved with 900mL water, get reagent A; With 1.28g oleic acid, 6mL 0.1M NaOH solution and 54mL water, fully dissolving mixes, and gets reagent B; With 37.5mg vitamin H and 12.5mg vitamin B6 10mL water dissolution, get jolting behind 1mL and 25mg catalase and the 59mL water, make abundant dissolving, reagent C; 800mL reagent A, 50mL reagent B and 50mL reagent C are mixed, obtain OADC stoste; OADC stoste with aseptic membrane filtration after (filtration unit lower floor is 0.22 μ m filter membrane, and the upper strata is 0.45 μ m filter membrane) be OADC solution (add a cover back-20 ℃ of preservations standby).
Embodiment 1, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing 20g sucrose (promptly protectant quality of adding as raw material is 20g), join stirring and dissolving in the 930g water, in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, [concentration is (1.07 ± 0.32) * 10 to add 50g mycobacteriophage D29 bacterium liquid in filtrate
6PFU/g], mix the back and adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 100 ℃ of temperature ins, gas velocity 120L/min, spray rate 40%, nozzle cap 7.0 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (83.56 ± 2.15) %, and water content is (2.84 ± 0.57) %; Spherical (see figure 1), size is between several microns to tens microns; The grain spectrum is measured and is shown that D50 is (10.56 ± 0.65) μ m; The phage survival rate is (42.47 ± 11.43) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (2.72 ± 0.73) * 10
4PFU/g.
Embodiment 2, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
Use the lactose place of sucrose, other is fully with the step 1 of embodiment 1.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (78.72 ± 1.41) %, and water content is (5.08 ± 0.22) %; The class sphere, size is between several microns to tens of microns; The grain spectrum is measured and is shown that D50 is (15.66 ± 1.65) μ m; The phage survival rate is (45.58 ± 16.50) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (3.10 ± 1.12) * 10
4PFU/g.
Embodiment 3, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing 50g trehalose (promptly protectant quality of adding as raw material is 50g), join stirring and dissolving in the 150g water, in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, in filtrate, add 50g mycobacteriophage D29 bacterium liquid [(1.30 ± 0.33) * 10
8PFU/g], mix the back and adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 80 ℃ of temperature ins, gas velocity 90L/min, spray rate 60%, nozzle cap 5.5 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (72.57 ± 2.49) %; Water content is (8.08 ± 0.27) %; Spherical (see figure 2), size is at several microns to tens microns; The grain spectrum is measured and is shown that D50 is (8.69 ± 0.94) μ m; The phage survival rate is (50.40 ± 5.22) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (1.81 ± 0.19) * 10
6PFU/g.
Embodiment 4, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing 50g lactose (promptly protectant quality of adding as raw material is 50g), join stirring and dissolving in the 400g water, in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, in filtrate, add 50g mycobacteriophage D29 bacterium liquid [(4.4 ± 0.76) * 10
9PFU/g], mix the back and adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 120 ℃ of temperature ins, gas velocity 150L/min, spray rate 100%, nozzle cap 4.0 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (71.05 ± 0.22) %; Water content is (3.27 ± 0.35) %; Spherical, part particle surface has the depression (see figure 3), and granular size is at several microns; The grain spectrum is measured and is shown that D50 is (5.65 ± 0.05) μ m; The phage survival rate is (41.50 ± 12.30) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (5.14 ± 1.52) * 10
7PFU/g.
Embodiment 5, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing 25g lactose and 25g trehalose (promptly protectant quality of adding as raw material is 50g), join stirring and dissolving in the 400g water, in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, in filtrate, add 50g mycobacteriophage D29 solution [(0.74 ± 0.40) * 10
8PFU/g], mix the back and adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 120 ℃ of temperature ins, gas velocity 120L/min, spray rate 80%, nozzle cap 5.5 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (84.99 ± 0.62) %; Water content is (2.88 ± 0.04) %; Class sphere, part particle surface have fold, depression (see figure 4), and granular size is between several microns to tens microns; The grain spectrum is measured and is shown that D50 is (10.94 ± 0.45) μ m; The phage survival rate is (73.80 ± 9.05) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (1.29 ± 0.16) * 10
6PFU/g.
Embodiment 6, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing 25g lactose and 25g N.F,USP MANNITOL (promptly protectant quality of adding as raw material is 50g), join stirring and dissolving in the 400g water, in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, in filtrate, add 50g mycobacteriophage D29 solution [(0.96 ± 0.62) * 10
8PFU/g], after mixing, adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 120 ℃ of temperature ins, gas velocity 120L/min, spray rate 80%, nozzle cap 5.5 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (57.16 ± 3.57) %; Water content is (1.34 ± 0.11) %; Sphere also has certain adhesion (see figure 5), and the microscopic examination granular size is at several microns to tens microns; The grain spectrum is measured and is shown that D50 is (8.94 ± 0.29) μ m; The phage survival rate is (71.60 ± 3.68) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (2.41 ± 0.12) * 10
6PFU/g.
Embodiment 7, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing the commercially available skim-milk of 50g (Inner Mongolia Yili Industry Group Co., Ltd) (promptly protectant quality of adding as raw material is 50g); join stirring and dissolving in the 400g water; in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, in filtrate, add 50g mycobacteriophage D29 solution [(0.96 ± 0.09) * 10
8PFU/g], after mixing, adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 120 ℃ of temperature ins, gas velocity 120L/min, spray rate 80%, nozzle cap 5.5 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (83.40 ± 2.90) %; Water content is (2.50 ± 0.20) %; Sphere, part particle surface depression (see figure 6), the microscopic examination granular size is at several microns; The grain spectrum is measured and is shown that D50 is (7.91 ± 0.39) μ m; The phage survival rate is (80.71 ± 4.34) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (1.86 ± 0.10) * 10
6PFU/g.
Embodiment 8, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing the commercially available skim-milk of 5g lactose and 25g (Inner Mongolia Yili Industry Group Co., Ltd) (promptly protectant quality of adding as raw material is 30g); join in the 400g water after the stirring and dissolving; in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, in filtrate, add 50g mycobacteriophage D29 solution [(1.67 ± 0.09) * 10
8PFU/g], after mixing, adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 120 ℃ of temperature ins, gas velocity 120L/min, spray rate 80%, nozzle cap 5.5 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (81.10 ± 3.76) %; Water content is (2.11 ± 0.08) %; The class sphere, part particle surface indent even be irregular shape (see figure 7), the microscopic examination granular size is between several microns to 20 microns; The grain spectrum is measured and is shown that D50 is (7.49 ± 0.59) μ m; The phage survival rate is (95.10 ± 1.88) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (6.53 ± 0.13) * 10
6PFU/g.
Embodiment 9, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing 25g lactose and 25g bovine serum albumin (promptly protectant quality of adding as raw material is 50g), join stirring and dissolving in the 400g water, in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, in filtrate, add 50g mycobacteriophage D29 solution [(1.86 ± 0.78) * 10
8PFU/g], after mixing, adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 120 ℃ of temperature ins, gas velocity 120L/min, spray rate 80%, nozzle cap 5.5 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (70.58 ± 1.09) %; Water content is (2.74 ± 0.04) %; Sphere, there is the fold (see figure 8) on the small quantities of particles surface, and the microscopic examination granular size is between several microns to tens microns; The grain spectrum is measured and is shown that D50 is (8.14 ± 0.17) μ m; The phage survival rate is (85.10 ± 18.95) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (4.49 ± 0.99) * 10
6PFU/g.
Embodiment 10, spray drying method for preparation mycobacteriophage D29 particle
One, mycobacteriophage D29 particulate preparation
1, takes by weighing 25g lactose and 5g leucine (promptly protectant quality of adding as raw material is 30g), join stirring and dissolving in the 400g water, in Biohazard Safety Equipment,, obtain filtrate with 0.22 μ m filtering with microporous membrane degerming.
2, in filtrate, add 50g mycobacteriophage D29 solution [(0.65 ± 0.16) * 10
8PFU/g], after mixing, adopt Switzerland BUCHI B-90 type nanometer spray-drier spraying drying, obtain mycobacteriophage D29 particle.Instrument parameter is set at: 120 ℃ of temperature ins, gas velocity 120L/min, spray rate 80%, nozzle cap 5.5 μ m; Phegma is collected in the sterile flask.
Two, mycobacteriophage D29 particulate characterizes
Mycobacteriophage D29 particulate characterization data is as follows: yield is (82.85 ± 0.16) %; Water content is (2.30 ± 0.57) %; Class sphere, or the spherical (see figure 9) of irregular class, the microscopic examination granular size is between several microns to 20 microns; The grain spectrum is measured and is shown that D50 is (9.34 ± 0.40) μ m; The phage survival rate is (78.70 ± 8.91) %; In the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is (2.06 ± 0.23) * 10
6PFU/g.
Claims (10)
1. mycobacteriophage D29 particle, its activeconstituents is made up of mycobacteriophage D29 and protective material; Described protective material is sugar and/or albumen and/or amino acid and/or alcohol.
2. mycobacteriophage D29 particle as claimed in claim 1 is characterized in that: described sugar is at least a in sucrose, lactose and the trehalose; Described albumen is at least a in milk powder and the bovine serum albumin; Described amino acid is leucine; Described alcohol is N.F,USP MANNITOL.
3. particle as claimed in claim 1 or 2 is characterized in that: in the described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of survival is 1 * 10
2PFU/g to 1 * 10
9PFU/g, the quality percentage composition of moisture is below 10%; Described mycobacteriophage D29 particulate granularity median D50 is below the 20 μ m.
4. claim 1 or 2 or 3 described mycobacteriophage D29 particulate preparation methods are that described mycobacteriophage D29 is carried out solidification with described protective material as raw material, obtain mycobacteriophage D29 particle.
5. method as claimed in claim 4 is characterized in that: described protective material is any one in following (1) to (9): (1) sucrose; (2) lactose; (3) trehalose; (4) lactose and trehalose; (5) lactose and N.F,USP MANNITOL; (6) milk powder; (7) lactose and milk powder; (8) lactose and bovine serum albumin; (9) lactose and leucine.
6. as claim 4 or 5 described methods, it is characterized in that: described protective material and described mycobacteriophage D29 are 1g as proportion of raw materials: 1 * 10
4PFU to 1 * 10
12PFU.
7. method as claimed in claim 6 is characterized in that: described protective material and described mycobacteriophage D29 are 1g as proportion of raw materials: 1.88 * 10
6PFU to 5.16 * 10
9PFU.
8. as arbitrary described method in the claim 4 to 7, it is characterized in that: described solidified method is a spray-drying process.
9. method as claimed in claim 8 is characterized in that: the parameter of described spray-drying process is: 80 ℃ to 120 ℃ of temperature ins, gas velocity 90L/min to 150L/min, spray rate 40% to 100%, nozzle cap aperture are 4.0 μ m to 7.0 μ m.
10. as arbitrary described method in the claim 4 to 9, it is characterized in that: described solidified raw material is aseptic.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102527468A CN102296056B (en) | 2011-08-30 | 2011-08-30 | Mycobacteriophage D29 particles and preparation method and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011102527468A CN102296056B (en) | 2011-08-30 | 2011-08-30 | Mycobacteriophage D29 particles and preparation method and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102296056A true CN102296056A (en) | 2011-12-28 |
| CN102296056B CN102296056B (en) | 2013-06-05 |
Family
ID=45356732
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011102527468A Expired - Fee Related CN102296056B (en) | 2011-08-30 | 2011-08-30 | Mycobacteriophage D29 particles and preparation method and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102296056B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108651522A (en) * | 2018-04-09 | 2018-10-16 | 广州诺晶生物技术有限公司 | A kind of vibrio alginolyticus phage preparation, preparation method and applications |
| CN112063594A (en) * | 2020-09-24 | 2020-12-11 | 瑞科盟(青岛)生物工程有限公司 | High-temperature-resistant salmonella bacteriophage RDP-SA-18056 and preparation process of microcapsules thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5773267A (en) * | 1992-02-07 | 1998-06-30 | Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University | D29 shuttle phasmids and uses thereof |
| CN1209172A (en) * | 1995-12-15 | 1999-02-24 | 生物技术实验室有限公司 | Method to detect bacteria |
| RU2214829C2 (en) * | 2001-11-05 | 2003-10-27 | Новосибирский научно-исследовательский институт туберкулеза | Method for tuberculosis phagotherapy |
| CN101302498A (en) * | 2007-05-09 | 2008-11-12 | 中国药品生物制品检定所 | Preservation method of mycobacteriophage |
| CN101570777A (en) * | 2009-06-12 | 2009-11-04 | 郑州安图绿科生物工程有限公司 | Method for quickly detecting mycobacterium tuberculosis |
| CN101912048A (en) * | 2010-08-13 | 2010-12-15 | 中牧实业股份有限公司 | T4 lysozyme premix for animals and preparation method thereof |
-
2011
- 2011-08-30 CN CN2011102527468A patent/CN102296056B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5773267A (en) * | 1992-02-07 | 1998-06-30 | Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University | D29 shuttle phasmids and uses thereof |
| CN1209172A (en) * | 1995-12-15 | 1999-02-24 | 生物技术实验室有限公司 | Method to detect bacteria |
| RU2214829C2 (en) * | 2001-11-05 | 2003-10-27 | Новосибирский научно-исследовательский институт туберкулеза | Method for tuberculosis phagotherapy |
| CN101302498A (en) * | 2007-05-09 | 2008-11-12 | 中国药品生物制品检定所 | Preservation method of mycobacteriophage |
| CN101570777A (en) * | 2009-06-12 | 2009-11-04 | 郑州安图绿科生物工程有限公司 | Method for quickly detecting mycobacterium tuberculosis |
| CN101912048A (en) * | 2010-08-13 | 2010-12-15 | 中牧实业股份有限公司 | T4 lysozyme premix for animals and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| 《中国博士论文全文数据库 医药卫生科技辑2008年第11期》 20081115 温占波 分枝杆菌噬菌体D29气溶胶特性和动物气溶胶暴露***的初步研究 第10-13页 1-10 , 第11期 * |
| 刘克洋等: "分枝杆菌噬菌体D29气溶胶的喷雾和采样介质的初步研究", 《军事医学科学院院刊》 * |
| 彭丽: "分枝杆菌噬菌体生物学特性探讨", 《微生物学免疫学进展》 * |
| 温占波: "分枝杆菌噬菌体D29气溶胶特性和动物气溶胶暴露***的初步研究", 《中国博士论文全文数据库 医药卫生科技辑2008年第11期》 * |
| 温占波等: "分枝杆菌噬菌体D29气溶胶特性的研究", 《中国颗粒学会第六届学术年会暨海峡两岸颗粒技术研讨会》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108651522A (en) * | 2018-04-09 | 2018-10-16 | 广州诺晶生物技术有限公司 | A kind of vibrio alginolyticus phage preparation, preparation method and applications |
| CN112063594A (en) * | 2020-09-24 | 2020-12-11 | 瑞科盟(青岛)生物工程有限公司 | High-temperature-resistant salmonella bacteriophage RDP-SA-18056 and preparation process of microcapsules thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102296056B (en) | 2013-06-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021174738A1 (en) | Bionic nanoparticle coated with mesenchymal stem cell membrane having surface overexpressing pd-l1 molecule, and preparation therefor and application thereof | |
| JP2019071890A (en) | Method of making agglomerated microbiological media, and compositions thereof | |
| US20220142933A1 (en) | Method for preparing probiotic-loaded microcapsule, product obtained from the same, and use of the same | |
| CN111662880B (en) | Clostridium perfringens bacteriophage, bacteriostatic agent containing bacteriophage, preparation method and application | |
| CN100571715C (en) | The compositions and preparation and the processing method that contain bioactive substance | |
| CN102296056B (en) | Mycobacteriophage D29 particles and preparation method and use thereof | |
| CN113367130B (en) | Pesticide compound drug-loaded microsphere for honeysuckle and preparation method thereof | |
| CN112841222B (en) | Antibacterial and antivirus material and application thereof | |
| Sheng et al. | Infection microenvironment-responsive multifunctional peptide coated gold nanorods for bimodal antibacterial applications | |
| Hittinger et al. | Autologous co-culture of primary human alveolar macrophages and epithelial cells for investigating aerosol medicines. Part II: Evaluation of IL-10-loaded microparticles for the treatment of lung inflammation | |
| Iwasaki et al. | An immunofluorescent study of generalized Coxsackie virus B3 infection in a newborn infant | |
| CN114504586A (en) | Antiallergic composition and application thereof in preparing antiallergic preparation | |
| CN102834101A (en) | Biodegradable material containing silicon, for pro-angiogenetic therapy | |
| CN111346222A (en) | Preparation method of plague attenuated live vaccine dry powder | |
| CN107873731B (en) | Fe for resisting influenza virus3O4Nano material and activity evaluation method and application thereof | |
| EP3395829B1 (en) | Gelatin particles, method for producing gelatin particles, gelatin particle-containing cell, and method for producing gelatin particle-containing cell | |
| CN108721248A (en) | A kind of preparation method, product and the application of pH response type nanos silver assembly | |
| CN111440195A (en) | Cefuroxime magnesium compound, composition, preparation method and application | |
| CN112587563A (en) | Antibacterial Chinese and western medicine powder for amoxicillin and scutellaria baicalensis for livestock and preparation method thereof | |
| RU2448730C2 (en) | Preparation, containing biologically active ingredients | |
| CN109644988A (en) | A kind of machine perfusion system mitigating organ transplant postoperative infection | |
| RU2440098C2 (en) | Preparation containing biologically active substances | |
| CN109731148A (en) | A kind of antibacterial Heat Conduction Material | |
| CN107261106A (en) | Thymopeptide-5, chitosan inclusion compound of rifamycin isoniazid and preparation method thereof | |
| CN1110299C (en) | Triazazole nucleoside aerosol and its preparing method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130605 Termination date: 20210830 |