CN102296056A - Mycobacteriophage D29 particles and preparation method and use thereof - Google Patents

Abstract

The invention discloses a mycobacteriophage D29 particle, a preparation method and application thereof. The active ingredient of the mycobacteriophage D29 particles provided by the invention consists of mycobacteriophage D29 and a protective agent; the protective agent is sugar and/or protein and/or amino acid and/or alcohol. The present invention adopts the nano-spray drying technology to prepare inhalable particles, and adopts a unique stabilization technology in the spray-drying, thus solving the technical difficulties in the preparation process of the mycobacteriophage D29 dry powder inhaler; Better play the therapeutic effect of mycobacteriophage D29 in clinical tuberculosis, solve the technical difficulties in the application of mycobacteriophage D29, and finally provide the possibility for the development of mycobacteriophage D29 into a drug.

Description

Mycobacteriophage D29 particle and its preparation method and application

technical field

The invention relates to a mycobacteriophage D29 particle, a preparation method and application thereof.

Background technique

Tuberculosis has always been a zoonotic infectious disease that poses a great threat to human health. The epidemic is picking up around the world. The World Health Organization (WHO) declared in 1993: a global tuberculosis emergency; in 1998, it reiterated that action to curb tuberculosis is urgent, and every year March 24 was designated as World Tuberculosis Day. The latest WHO data shows that in 2009, there were 9.4 million new tuberculosis patients, and 1.7 million people died of tuberculosis; in 2008, 440,000 new MDR-TB patients were added, and 150,000 people died of MDR-TB. my country is one of the 22 countries with a high burden of tuberculosis in the world, and is a country with high drug resistance. The number of tuberculosis patients ranks second in the world, and multi-drug resistant tuberculosis accounts for 13.4%. But so far, there is no ideal new anti-multidrug-resistant tuberculosis drug available for clinical use.

Phage (Bacteriophage, phage) is a type of bacterial virus, which is highly specific to some bacteria. When the phage infects these bacteria, it can reproduce in the bacteria and kill the bacteria, but it is not toxic to animals and plants. Therefore, phages are expected to become better antibacterial drugs due to their unique natural characteristics. In the first half of the 20th century, phages were once used as an effective tool to treat and prevent bacterial infections, but with the invention and wide application of antibiotics, many countries have not conducted in-depth research on this. However, in the face of the increasing proportion of drug-resistant bacterial infections, some people predict that phages will become a major research hotspot in the field of biopharmaceuticals in the past decade.

Phages have been successfully applied to the treatment of bacterial infections in various animals, such as poultry, fish, calves, lambs, etc. There are also many clinical studies on the prevention and treatment of bacterial infections in humans. Weber-Dabrowska et al. used phage therapy on 20 tumor patients and 27 bacterial infections, and found that all patients' concomitant phenomena such as suppuration, trauma, and pneumonia disappeared quickly. Bretscher et al proposed that the use of phages to treat AIDS reinfection can slow down the evolution of HIV multidrug resistance.

Mycobacteriophage D29 is a DNA virus parasitic on mycobacteria. It consists of a protein shell and a DNA inside. It has no independent metabolic system and must be combined with the host bacteria to enter the cell. It uses the metabolic enzyme system of the host bacteria Replication occurs and the bacteria are eventually lysed, releasing progeny phages. The virus particles of mycobacteriophage D29 are round, with a size between 75 and 150 nm, and have a broad spectrum of hosts, including Mycobacterium smegmatis and Mycobacterium tuberculosis. The multiplication period of the host in Mycobacterium smegmatis is 90-180 minutes, and the multiplication period in Mycobacterium tuberculosis is more than 3-6 hours.

Mycobacteriophage D29 has been widely used in the rapid detection of Mycobacterium tuberculosis and the detection of drug resistance of Mycobacterium tuberculosis. In the study of mycobacteriophage D29 used in anti-tuberculosis treatment, in vitro experiments found that mycobacteriophage D29 can reduce the number of viable bacteria of Mycobacterium smegmatis and Mycobacterium tuberculosis in macrophages grown in vitro, and has the ability to kill The ability to host bacteria in macrophages has the potential to treat tuberculosis; animal experiments have also proved that mycobacteriophage D29 can reduce the degree of organ lesions and the amount of bacteria in the organs of tuberculosis guinea pigs, which is close to or even better than rifampicin Curative effect, and no side effects on guinea pigs, hopeful as the development of new anti-tuberculosis drugs.

Mycobacteriophage D29 has potential advantages in the treatment of tuberculosis. Phage has a high degree of host specificity, only targeting pathogenic bacteria, and will not affect other flora, so it has the advantage of no side effects, and at the same time reduces the resulting problem of drug resistance; phage can follow the host bacteria Proliferates and proliferates, and works throughout the course of a bacterial infection, unlike antibiotics, which gradually lose efficacy over time. In the application of mycobacteriophage D29 in the treatment of tuberculosis, the key issues to be solved are how to reach the action site and in what form to effectively reach the action site.

Mycobacteriophage D29 is a kind of microorganism, and the product obtained by culture is generally a liquid containing a culture medium. Compared with solids, liquids have problems such as inconvenient storage, transportation and use, and stability. Therefore, if microbial products are to be made into drugs, solidification has its advantages. The use of freeze-drying of microbial preparations is beneficial to maintaining its stability during the solidification process, so freeze-drying is usually used in the preservation of microbial preparations.

Contents of the invention

The object of the present invention is to provide a mycobacteriophage D29 particle and its preparation method and application.

The mycobacteriophage D29 particle provided by the present invention has an active ingredient consisting of mycobacteriophage D29 and a protective agent (also serving as a diluent); the protective agent is sugar and/or protein and/or amino acid and/or alcohol.

The sugar may be at least one of sucrose, lactose and trehalose. The protein can be at least one of milk powder and bovine serum albumin. The amino acid may be leucine. The alcohol may be mannitol.

The protective agent can be a kind of sugar or a mixture of multiple sugars. The sugar may be at least one of sucrose, lactose and trehalose. The protective agent can also be protein, such as milk powder (such as skimmed milk powder) or bovine serum albumin. The protective agent can also be a mixture of sugar and protein, such as a mixture of lactose and milk powder, or a mixture of lactose and bovine serum albumin. The protecting agent may also be a mixture of sugars and amino acids, such as a mixture of lactose and leucine. The protectant may also be a mixture of sugar and alcohol, such as a mixture of lactose and mannitol.

The protective agent is preferably any one of the following (1) to (9): (1) sucrose; (2) lactose; (3) trehalose; (4) lactose and trehalose; (5) lactose and mannose (6) milk powder; (7) lactose and milk powder; (8) lactose and bovine serum albumin; (9) lactose and leucine.

The mycobacteriophage D29 particle may specifically consist of the mycobacteriophage D29, the protecting agent and water.

In the mycobacteriophage D29 particles, the content of the surviving mycobacteriophage D29 may be 1×10 ² PFU/g to 1×10 ⁹ PFU/g, and the mass percentage of water may be less than 10%; The median particle size D50 of the mycobacteriophage D29 particles may be below 20 μm.

In the mycobacteriophage D29 particles, the content of surviving mycobacteriophage D29 is preferably 1×10 ⁴ PFU/g to 7×10 ⁷ PFU/g, and the mass percentage of water is most preferably 1% to 9% %; The median particle size D50 of the mycobacteriophage D29 particles is preferably 5.00 μm to 18.00 μm.

In the mycobacteriophage D29 particles, the content of surviving mycobacteriophage D29 is most preferably 1.99×10 ⁴ PFU/g to 6.66×10 ⁷ PFU/g, and the mass percentage of water is most preferably 1.23% to 8.30%; the median particle size D50 of said mycobacteriophage D29 particles is most preferably 5.60 μm to 17.31 μm.

In the mycobacteriophage D29 particles, the content of the surviving mycobacteriophage D29 may be (2.72±0.73)×10 ⁴ PFU/g to (3.10±1.12)×10 ⁴ PFU/g, (3.10±1.12) ×10 ⁴ PFU/g to (1.29±0.16)×10 ⁶ PFU/g, (1.29±0.16)×10 ⁶ PFU/g to (1.81±0.19)×10 ⁶ PFU/g, (1.81±0.19)×10 ⁶ PFU/g to (1.86±0.10)×10 ⁶ PFU/g, (1.86±0.10)×10 ⁶ PFU/g to (2.06±0.23)×10 ⁶ PFU/g, (2.06±0.23)×10 ⁶ PFU /g to (2.41±0.12)×10 ⁶ PFU/g, (2.41±0.12)×10 ⁶ PFU/g to (4.49±0.99)×10 ⁶ PFU/g, (4.49±0.99)×10 ⁶ PFU/g to (6.53±0.13)×10 ⁶ PFU/g, or (6.53±0.13)×10 ⁶ PFU/g to (5.14±1.52)×10 ⁷ PFU/g.

In the mycobacteriophage D29 particles, the mass percentage of water can be from (1.34±0.11)% to (2.11±0.08)%, (2.11±0.08)% to (2.30±0.57)%, (2.30±0.57)% )% to (2.50±0.20)%, (2.50±0.20)% to (2.74±0.04)%, (2.74±0.04)% to (2.84±0.57)%, (2.84±0.57)% to (2.88±0.04) %, (2.88±0.04)% to (3.27±0.35)%, (3.27±0.35)% to (5.08±0.22)%, or (5.08±0.22)% to (8.08±0.27)%.

The median particle size D50 of the mycobacteriophage D29 particles can be (5.65±0.05) μm to (7.49±0.59) μm, (7.49±0.59) μm to (7.91±0.39) μm, (7.91±0.39) μm to (8.14±0.17) μm, (8.14±0.17) μm to (8.69±0.94) μm, (8.69±0.94) μm to (8.94±0.29) μm, (8.94±0.29) μm to (9.34±0.40) μm, (9.34±0.40) μm to (10.94±0.45) μm, (10.94±0.45) μm to (10.56±0.65) μm, or (10.56±0.65) μm to (15.66±1.65) μm.

The invention also protects the preparation method of the mycobacteriophage D29 particle, which is to solidify (solidify) the mycobacteriophage D29 and the protective agent together as raw materials to obtain the mycobacteriophage D29 particle.

When the protective agent is lactose and trehalose, the mass ratio of the lactose and the trehalose as raw materials is preferably 1:1. When the protective agent is lactose and milk powder, the mass ratio of the lactose and the milk powder as raw materials is preferably 1:5. When the protective agent is lactose and bovine serum albumin, the mass ratio of the lactose and the bovine serum albumin as raw materials is preferably 1:1. When the protecting agent is lactose and leucine, the mass ratio of lactose and leucine as raw materials is preferably 5:1. When the protective agent is lactose and mannitol, the mass ratio of the lactose and the mannitol as raw materials is preferably 1:1.

The ratio of the protective agent to the mycobacteriophage D29 as a raw material may be 1 g: (1×10 ⁴ PFU to 1×10 ¹² PFU). The proportion of the protective agent and the mycobacteriophage D29 as a raw material may specifically be 1 g: (1×10 ⁶ PFU to 1×10 ¹⁰ PFU). The proportion of the protective agent and the mycobacteriophage D29 as raw materials is preferably 1 g: (1.88×10 ⁶ PFU to 5.16×10 ⁹ PFU).

The proportion of the protective agent and the mycobacteriophage D29 as raw materials can be 1g: (2.68±0.80)×10 ⁶ PFU to (0.74±0.40)×10 ⁸ PFU, 1g: (0.74±0.40)×10 ⁸ PFU to (0.96±0.09)×10 ⁸ PFU, 1g: (0.96±0.09)×10 ⁸ PFU to (1.08±0.27)×10 8 PFU, 1g: (1.08± ^0.27 )×10 ⁸ PFU to (1.30± 0.33)×10 ⁸ PFU, 1g: (1.30±0.33)×10 ⁸ PFU to (1.86±0.78)×10 ⁸ PFU, 1g: (1.86±0.78)×10 ⁸ PFU to (2.78±0.15)×10 ⁸ PFU , or 1 g: (2.78±0.15)×10 ⁸ PFU to (4.4±0.76)×10 ⁹ PFU.

The solidification method can specifically be a spray drying method. When the spray drying method is used for the solidification, the parameters of the spray drying method can specifically be: inlet temperature 80°C to 120°C (such as 80°C to 100°C, 100°C to 120°C), air velocity 90L/min to 150L/min (such as 90L/min to 120L/min, 120L/min to 150L/min), spray rate 40% to 100% (such as 40% to 60%, 60% to 80%, 80% to 100%), The pore diameter of the nozzle cover is 4.0 μm to 7.0 μm (such as 4.0 μm to 5.5 μm, 5.5 μm to 7.0 μm). The used instrument of described spray drying can be Swiss BUCHI B-90 nanometer spray dryer specifically.

In the raw material for solidification, the protective agent may be added in the form of an aqueous solution. The solidified raw material is preferably a sterile raw material.

The present invention can obtain mycobacteriophage D29 particles (dry powder inhalation) suitable for inhalation administration after adopting suitable protective agent, suitable equipment and optimized process parameters, and the activity retention value of mycobacteriophage D29 is very high . If no protective agent is used, or the protective agent is improperly selected, most of the mycobacteriophage D29 (>95%) will be inactivated during the spray drying process, and the meaning of solidification will be lost. The present invention finds that one or more of carbohydrates, proteins, amino acids and alcohols can be used as a protective agent for the solidification of mycobacteriophage D29, and a mixture of carbohydrates is used as a protective agent to retain the activity of mycobacteriophage D29 to reach More than 70% (see embodiment 5), adopt the mixture of carbohydrate and amino acid protein as protective agent, then can make mycobacteriophage D29 active retention reach about 80% (see embodiment 9,10), even more than 90% (See Example 8). The size of particles obtained by conventional spray drying is usually tens of microns or even more than 100 microns. Particles of this size cannot be inhaled into the lower respiratory tract, so they are not suitable for pulmonary inhalation administration. The present invention explores the effect of the spray drying process on the size of the obtained particles, which are suitable for inhalation and administration of particles.

In the mycobacteriophage D29 particle provided by the present invention, the content of the surviving mycobacteriophage D29 can be 1×10 ² PFU/g to 1×10 ⁹ PFU/g, and the mass percentage of water can be less than 10%. The median particle size D50 may be below 20 μm.

The present invention adopts the nano-spray drying technology to prepare inhalable particles, and adopts a unique stabilization technology in the spray-drying, thus solving the technical difficulties in the preparation process of the mycobacteriophage D29 dry powder inhaler; Better exert the therapeutic effect of mycobacteriophage D29 in clinical pulmonary tuberculosis, and solve the technical difficulties in the application of mycobacteriophage D29. Finally, it is possible to develop mycobacteriophage D29 into a drug.

Description of drawings

Fig. 1 is the electron micrograph when embodiment 1 uses sucrose as protective agent

Fig. 2 is the electron micrograph when embodiment 3 uses trehalose as protective agent

Fig. 3 is the electron micrograph when embodiment 4 uses lactose as protective agent

Fig. 4 is the electron micrograph when embodiment 5 uses lactose and trehalose as protective agent

Fig. 5 is the electron micrograph when embodiment 6 uses lactose and mannitol as protective agent

Fig. 6 is the electron micrograph when using skimmed milk powder as protective agent in embodiment 7

Fig. 7 is the electron micrograph when embodiment 8 uses lactose and skimmed milk powder as protective agent

Fig. 8 is the electron micrograph when embodiment 9 uses lactose and bovine serum albumin as protective agent

Fig. 9 is the electron micrograph when embodiment 10 uses lactose and leucine as protective agent

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged. CFU stands for colony forming unit and PFU stands for plaque forming unit.

7H9 medium: BD Company of the United States (original product number 271310).

Mycobacteriophage D29: References: Peng Li, Luo Yongai, Chen Baowen, etc. Efficacy of phage D29 on guinea pigs infected with sensitive strains of Mycobacterium tuberculosis. Chinese Journal of Zoonoses. 2009, 25(8): 733 -736.

Mycobacterium smegmatis used in the examples is Mycobacterium smegmatis ATCC607: References: Liu Keyang, Du Qian, Wen Zhanbo, etc. Preliminary study on the spray and sampling medium of mycobacteriophage D29 aerosol .Proceedings of Academy of Military Medical Sciences, 2010, 34(4): 347-350.

The calculation method of the yield is as follows: (mass of harvested mycobacteriophage D29 particles/mass of protective agent added as raw material)×100%.

The calculation method of water content is as follows: take a washed and dried weighing bottle (about 45mm in diameter), dry it at 100°C±2°C for 2 hours, and accurately weigh it after it is placed at room temperature (W ₀ ); add about 1g of mycobacteria Spread the phage D29 particles in a weighing bottle, and weigh them precisely (W ₁ ); transfer the weighing bottle to an oven, open the bottle cap for drying treatment (when the protective agent is sugar, at 115°C±2°C Dry for 20 hours; if the protective agent contains protein, dry for 20 hours at 80°C±2°C), then cover the bottle cap, take it out and place it at room temperature (relative humidity below 60%) for 10 minutes, and weigh it accurately (W ₂ ); water content (%)=[(W ₁ -W ₂ )/(W ₁ -W ₀ )]×100%.

The particle size determination method of mycobacteriophage D29 particle is as follows: adopt LS908 (A) laser particle size analyzer (Zhuhai European and American Ke Instrument Co., Ltd.) to measure in the embodiment of this patent, concrete method is: (1) add in the static sample pool 7mL medium (ethyl oleate), put it into the measuring system of the instrument, and after the instrument is automatically centered and cleared, the instrument enters the state to be tested; (2) use the dispersion medium (ethyl oleate) to dissolve the After the particles are made into a uniform suspension of about 5mg/mL, take 3 drops of the suspension (0.1mL-0.2mL) and add it to the medium of the static sample pool, blow it evenly, put it into the instrument measurement system, and wait for the light column to stabilize. The particle size measurement can be carried out, and the particle size is represented by the particle size median value D50. Other commercially available particle size analyzers can also be used, and the specific steps need to be adjusted accordingly according to the operating procedures of the instrument used.

Electron microscope determination method of mycobacteriophage D29 particles: spread mycobacteriophage D29 particles evenly on the sample holder, spray gold with Hitachi E-1010 ion sputtering instrument, observe and take pictures under Hitachi S-3400N scanning electron microscope. Electron micrographs can also be obtained by using other commercially available scanning electron microscopes, and the specific steps need to be adjusted accordingly according to the operating procedures of the instruments used.

The calculation method of the phage survival rate in the mycobacteriophage D29 particles is as follows: after dissolving the mycobacteriophage D29 particles with SM solution, then carry out 10-fold gradient dilution with SM solution; respectively take 0.1mL dilution solution to different 7H9 bottom layers On the medium plate (Ф90mm), spread it evenly with an "L"rod; add smegma at a concentration of 10 ⁷ CFU/mL to the 7H9 upper layer medium heated to about 60°C according to the ratio of 3mL bacterial solution/50mL medium Mycobacterium bacterium liquid, after gently shaking, pour 5mL of 7H9 upper layer medium containing Mycobacterium smegmatis into each above-mentioned 7H9 bottom medium plate coated with bacteriophage D29; after the upper layer medium is completely solidified, Place the plate in a constant temperature incubator at 37°C for upright culture for 2 days, observe and count the PFU on the plate (the result of 10-300 is the confidence interval); calculate the number of surviving phages in mycobacteriophage D29 particles according to the dilution The sample of each dilution is coated with 3 plates, and the results are averaged; Survival rate=(the phage number/yield/yield added during the surviving phage number/spray-drying in the mycobacteriophage D29 particle that finally obtains )×100%.

SM solution: Weigh 2.9g NaCl and 1.0g MgSO ₄ .7H ₂ O, add 25ml 1mol/L Tris-Cl (pH 7.5) and 2.5ml 2% (g/100ml) gelatin aqueous solution, add water to 500ml, mix well Sterilize at 121°C for 30 minutes, and let it cool to room temperature to obtain SM solution.

7H9 bottom medium: Weigh 1.6g 7H9 medium, 5.6g agar powder, add 315mL water, mix well, sterilize at 121°C for 30min, then add calf serum complex solution (OADC solution) aseptically at 45-50°C 35ml, mix well and make a flat plate (12ml/plate); let it solidify at room temperature, and store it in a refrigerator at 4°C.

7H9 upper layer medium: Weigh 1.6g 7H9 medium, 2.8g agar powder, add 315mL water, mix well, sterilize at 121°C for 30min, store at 4°C under sterile airtight condition.

OADC solution: 8.5g NaCl, 50g BSA (bovine serum albumin factor V), 20g glucose were fully dissolved in 900mL water to obtain reagent A; 1.28g oleic acid, 6mL 0.1M NaOH solution and 54mL water were fully dissolved and mixed , to obtain reagent B; dissolve 37.5mg biotin and 12.5mg vitamin B6 with 10mL water, take 1mL, 25mg catalase and 59mL water and shake to fully dissolve to obtain reagent C; mix 800mL reagent A, 50mL reagent Mix B and 50mL of reagent C to obtain the OADC stock solution; the OADC stock solution is filtered through a sterile filter (the lower layer of the filter device is a 0.22 μm filter membrane, and the upper layer is a 0.45 μm filter membrane) is the OADC solution (covered and stored at -20°C for later use) ).

Embodiment 1, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

1. Weigh 20g of sucrose (that is, the quality of the protective agent added as raw material is 20g), add it to 930g of water, stir and dissolve, filter and sterilize with a 0.22 μm microporous membrane in a biological safety cabinet to obtain a filtrate.

2. Add 50 g of mycobacteriophage D29 bacterial liquid [concentration is (1.07 ± 0.32) × 10 ⁶ PFU/g] to the filtrate, and after mixing evenly, adopt Swiss BUCHI B-90 nanometer spray dryer to spray dry to obtain branches Bacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 100°C, air velocity 120L/min, spray rate 40%, nozzle cap 7.0μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (83.56 ± 2.15)%, and the water content is (2.84 ± 0.57)%; spherical (see Figure 1), the size is between several microns and more than ten microns; particle spectrum Determination shows that D50 is (10.56±0.65) μm; phage survival rate is (42.47±11.43)%; in the mycobacteriophage D29 particles, the content of surviving mycobacteriophage D29 is (2.72±0.73)×10 ⁴ PFU/g.

Embodiment 2, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

Lactose is used to replace sucrose, and the others are completely the same as Step 1 of Example 1.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (78.72 ± 1.41)%, and the water content is (5.08 ± 0.22)%; spherical shape, the size is between several microns and tens of microns; particle spectrometry shows that D50 is (15.66±1.65) μm; phage survival rate (45.58±16.50)%; in the mycobacteriophage D29 particles, the content of surviving mycobacteriophage D29 is (3.10±1.12)×10 ⁴ PFU/g.

Embodiment 3, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

1. Weigh 50g of trehalose (that is, the quality of the protective agent added as a raw material is 50g), add it to 150g of water and stir to dissolve, filter and sterilize with a 0.22 μm microporous membrane in a biological safety cabinet to obtain a filtrate.

2. Add 50 g of mycobacteriophage D29 bacterial liquid [(1.30±0.33)×10 ⁸ PFU/g] to the filtrate, mix well and spray dry with Swiss BUCHI B-90 nanometer spray dryer to obtain mycobacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 80°C, airflow velocity 90L/min, spray rate 60%, nozzle cap 5.5μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (72.57 ± 2.49)%; the water content is (8.08 ± 0.27)%; spherical (see Figure 2), the size is from several microns to more than ten microns; particle spectrometry It shows that D50 is (8.69±0.94) μm; the phage survival rate is (50.40±5.22)%; in the mycobacteriophage D29 particles, the content of surviving mycobacteriophage D29 is (1.81±0.19)×10 ⁶ PFU /g.

Embodiment 4, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

1. Weigh 50g of lactose (that is, the quality of the protective agent added as raw material is 50g), add it to 400g of water, stir and dissolve, filter and sterilize with a 0.22 μm microporous membrane in a biological safety cabinet to obtain a filtrate.

2. Add 50 g of mycobacteriophage D29 bacterial liquid [(4.4±0.76)×10 ⁹ PFU/g] to the filtrate, mix well and then spray dry with Swiss BUCHI B-90 nanometer spray dryer to obtain mycobacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 120°C, air velocity 150L/min, spray rate 100%, nozzle cap 4.0μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of the mycobacteriophage D29 particles are as follows: the yield is (71.05 ± 0.22)%; the water content is (3.27 ± 0.35)%; spherical, with depressions on the surface of some particles (see Figure 3), and the particle size is several microns; Particle spectrum measurement shows that D50 is (5.65 ± 0.05) μm; Phage survival rate is (41.50 ± 12.30)%; In described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of surviving is (5.14 ± 1.52) × 10 ⁷ PFU/g.

Embodiment 5, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

1. Weigh 25g of lactose and 25g of trehalose (that is, the quality of the protective agent added as raw materials is 50g), add to 400g of water and stir to dissolve, filter and sterilize with a 0.22 μm microporous membrane in a biological safety cabinet to obtain a filtrate.

2. Add 50 g of mycobacteriophage D29 solution [(0.74±0.40)×10 ⁸ PFU/g] to the filtrate, mix well and then spray dry it with a Swiss BUCHI B-90 nano spray dryer to obtain mycobacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 120°C, air velocity 120L/min, spray rate 80%, nozzle cap 5.5μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (84.99 ± 0.62)%; the water content is (2.88 ± 0.04)%; spherical, with wrinkles and depressions on the surface of some particles (see Figure 4), and the particle size Between a few microns and more than a dozen microns; particle spectrometry shows that D50 is (10.94±0.45) μm; the phage survival rate is (73.80±9.05)%; The content is (1.29±0.16)×10 ⁶ PFU/g.

Embodiment 6, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

1. Take by weighing 25g lactose and 25g mannitol (that is, the quality of the protective agent added as raw materials is 50g), add to 400g water and stir to dissolve, filter and sterilize with a 0.22 μm microporous membrane in a biological safety cabinet to obtain a filtrate.

2. Add 50 g of mycobacteriophage D29 solution [(0.96±0.62)×10 ⁸ PFU/g] to the filtrate, mix evenly, and spray dry with Swiss BUCHI B-90 nano spray dryer to obtain mycobacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 120°C, air velocity 120L/min, spray rate 80%, nozzle cap 5.5μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (57.16 ± 3.57)%; the water content is (1.34 ± 0.11)%; the spherical shape has certain adhesion (see Figure 5), and the microscopic observation particle size is several microns to more than ten microns; particle spectrometry shows that D50 is (8.94 ± 0.29) μm; the phage survival rate is (71.60 ± 3.68)%; in the mycobacteriophage D29 particles, the content of surviving mycobacteriophage D29 is ( 2.41±0.12)×10 ⁶ PFU/g.

Embodiment 7, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

1. Weigh 50g of commercially available skimmed milk powder (Inner Mongolia Yili Industrial Group Co., Ltd.) (that is, the quality of the protective agent added as a raw material is 50g), add it to 400g of water and stir to dissolve it, and filter it through a 0.22μm microporous filter in a biological safety cabinet. Sterilize by membrane filtration to obtain a filtrate.

2. Add 50 g of mycobacteriophage D29 solution [(0.96±0.09)×10 ⁸ PFU/g] to the filtrate, mix well, and spray dry with Swiss BUCHI B-90 nano spray dryer to obtain mycobacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 120°C, air velocity 120L/min, spray rate 80%, nozzle cap 5.5μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (83.40±2.90)%; the water content is (2.50±0.20)%; micron; Particle spectrum measurement shows that D50 is (7.91 ± 0.39) μm; Phage survival rate is (80.71 ± 4.34)%; In described mycobacteriophage D29 particle, the content of the mycobacteriophage D29 of surviving is (1.86 ± 0.10 )×10 ⁶ PFU/g.

Embodiment 8, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

1. Weigh 5g of lactose and 25g of commercially available skimmed milk powder (Inner Mongolia Yili Industrial Group Co., Ltd.) (that is, the quality of the protective agent added as raw materials is 30g), add it to 400g of water and stir to dissolve, then use 0.22 Filter and sterilize with a μm microporous membrane to obtain a filtrate.

2. Add 50 g of mycobacteriophage D29 solution [(1.67±0.09)×10 ⁸ PFU/g] to the filtrate, mix evenly, and spray dry with Swiss BUCHI B-90 nano spray dryer to obtain mycobacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 120°C, air velocity 120L/min, spray rate 80%, nozzle cap 5.5μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (81.10 ± 3.76)%; the water content is (2.11 ± 0.08)%; spherical, and some of the particle surfaces are concave or even irregular (see Figure 7), Microscopic observation of particle size between several microns to twenty microns; particle spectrometry shows that D50 is (7.49 ± 0.59) μm; phage survival rate is (95.10 ± 1.88)%; in the mycobacteriophage D29 particles, survival The content of mycobacteriophage D29 was (6.53±0.13)×10 ⁶ PFU/g.

Embodiment 9, spray-drying method prepares mycobacteriophage D29 particle

1. Preparation of mycobacteriophage D29 particles

1. Take by weighing 25g lactose and 25g bovine serum albumin (that is, the quality of the protective agent added as raw material is 50g), add to 400g water and stir to dissolve, filter and sterilize with a 0.22 μm microporous membrane in a biological safety cabinet to obtain filtrate.

2. Add 50 g of mycobacteriophage D29 solution [(1.86±0.78)×10 ⁸ PFU/g] to the filtrate, mix well, and spray dry with a Swiss BUCHI B-90 nano spray dryer to obtain mycobacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 120°C, air velocity 120L/min, spray rate 80%, nozzle cap 5.5μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (70.58 ± 1.09)%; the water content is (2.74 ± 0.04)%; spherical, a small amount of particles have wrinkles on the surface (see Figure 8), and the particle size is observed under a microscope Between a few microns and more than a dozen microns; particle spectrometry shows that D50 is (8.14±0.17) μm; the phage survival rate is (85.10±18.95)%; in the mycobacteriophage D29 particles, the survival mycobacteriophage D29 The content of is (4.49±0.99)×10 ⁶ PFU/g.

Example 10, preparation of mycobacteriophage D29 particles by spray drying method

1. Preparation of mycobacteriophage D29 particles

1. Weigh 25g of lactose and 5g of leucine (that is, the quality of the protective agent added as raw materials is 30g), add to 400g of water and stir to dissolve, filter and sterilize with a 0.22μm microporous membrane in a biological safety cabinet to obtain the filtrate .

2. Add 50 g of mycobacteriophage D29 solution [(0.65±0.16)×10 ⁸ PFU/g] to the filtrate, mix well, and spray dry with a Swiss BUCHI B-90 nano spray dryer to obtain mycobacteriophage D29 particles. The instrument parameters are set as follows: inlet temperature 120°C, air velocity 120L/min, spray rate 80%, nozzle cap 5.5μm; reflux liquid is collected into a sterile flask.

2. Characterization of Mycobacteriophage D29 Particles

The characterization data of mycobacteriophage D29 particles are as follows: the yield is (82.85 ± 0.16)%; the water content is (2.30 ± 0.57)%; spherical, or irregular spherical (see Figure 9), microscopically observe the particle size Between a few microns and twenty microns; particle spectrometry shows that D50 is (9.34±0.40) μm; the phage survival rate is (78.70±8.91)%; The content is (2.06±0.23)×10 ⁶ PFU/g.

Claims (10)

1. A mycobacteriophage D29 particle, whose active ingredient is made up of mycobacteriophage D29 and a protective agent; the protective agent is sugar and/or protein and/or amino acid and/or alcohol. 2. mycobacteriophage D29 particles as claimed in claim 1, is characterized in that: described sugar is at least one in sucrose, lactose and trehalose; Described albumen is at least one in milk powder and bovine serum albumin ; The amino acid is leucine; The alcohol is mannitol. 3. The particle according to claim 1 or 2, characterized in that: in the mycobacteriophage D29 particle, the content of surviving mycobacteriophage D29 is 1×10 ² PFU/g to 1×10 ⁹ PFU /g, the mass percentage of moisture is below 10%; the median particle size D50 of the mycobacteriophage D29 particles is below 20 μm. 4. The method for preparing the mycobacteriophage D29 particles according to claim 1, 2 or 3, comprising solidifying the mycobacteriophage D29 and the protective agent as raw materials to obtain the mycobacteriophage D29 particles. 5. The method according to claim 4, characterized in that: the protective agent is any one of the following (1) to (9): (1) sucrose; (2) lactose; (3) trehalose; (4) lactose and trehalose; (5) lactose and mannitol; (6) milk powder; (7) lactose and milk powder; (8) lactose and bovine serum albumin; (9) lactose and leucine. 6. The method according to claim 4 or 5, characterized in that: the proportion of the protective agent and the mycobacteriophage D29 as raw materials is 1 g: 1×10 ⁴ PFU to 1×10 ¹² PFU. 7 . The method according to claim 6 , wherein the proportion of the protective agent and the mycobacteriophage D29 as raw materials is 1 g: 1.88×10 ⁶ PFU to 5.16×10 ⁹ PFU. 8. The method according to any one of claims 4 to 7, characterized in that: the solidification method is a spray drying method. 9. The method according to claim 8, characterized in that: the parameters of the spray drying method are: inlet temperature 80°C to 120°C, air velocity 90L/min to 150L/min, spray rate 40% to 100%, The nozzle cap has a pore size of 4.0 μm to 7.0 μm. 10. The method according to any one of claims 4 to 9, characterized in that the solidified raw material is sterile.

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