Cell factor powder and preparation method thereof

Abstract

The invention relates to the technical field of biology, in particular to cytokine powder and a preparation method thereof. The preparation method provided by the invention comprises the following steps: adding a composite protective agent into a cytokine solution, dissolving and uniformly mixing, and carrying out spray drying at the temperature of 60-100 DEG C to obtain powder, namely cytokine powder, wherein the composite protective agent is a mixture of L-leucine and trehalose, and the mass ratio of the L-leucine to the trehalose is (6-8): (15-20). The composite protective agent is added into the cytokine powder, and spray drying is performed under a specific temperature condition, so that high antiviral activity is ensured, the powder yield of the cytokine powder and the production efficiency of the cytokine powder can be improved, the cytokine can be stored at normal temperature, the storage life of the cytokine is prolonged, and the cytokine powder has a good application prospect. The transportation is convenient.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a cytokine powder and a preparation method thereof. Background Art

[0002] One of the major challenges facing the poultry industry is production losses caused by pathogenic microorganisms. With the ban on the addition of antibiotics to animal feed, cytokine preparations have become a research hotspot to prevent adverse effects on the livestock industry. Although cytokines have excellent therapeutic and preventive effects against viruses, there are currently no products on the market that have received a positive market response. One of the most pressing issues that needs to be addressed is the transportation and storage of cytokines. Cytokines are protein-based biological products that are conventionally transported and stored in liquid form under refrigeration to ensure their effectiveness. However, when cytokines are transported and stored in liquid form, the cost is high, the protein degrades rapidly, and the activity loss is high.

[0003] Currently, some researchers use freeze-drying technology (referred to as lyophilization technology) to mix cytokines with protective agents and first freeze them in -80°C or liquid nitrogen, and then sublime the frozen water by reducing the air pressure to achieve the purpose of drying. The whole process takes 24-48 hours. Although freeze-drying technology can keep cytokines active for a long time and facilitate transportation, the production process is complex, the production cycle is long, the equipment is expensive, the energy consumption is high, and a low-temperature vacuum environment is required, which is not suitable for continuous large-scale production. Therefore, it is very important to find a suitable cytokine production process to reduce storage and transportation costs and facilitate product preservation. This will provide technical support for the promotion and application of this product in the market. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a cytokine powder and a preparation method thereof. The present invention improves the powder extraction rate and production efficiency of the cytokine powder by adding a composite protective agent to the cytokine powder and spray-drying the powder under specific temperature conditions, so that the cytokine can be stored at room temperature, thereby extending the shelf life of the cytokine and facilitating transportation.

[0005] To this end, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides, in an optional embodiment, a method for preparing a cytokine powder, comprising the following steps:

[0007] After adding the composite protective agent to the cytokine solution and dissolving and mixing, the solution is spray-dried at a temperature of 60-100°C to obtain a powder which is the cytokine powder;

[0008] Wherein, the composite protective agent is a mixture of L-leucine and trehalose, and the mass ratio of the L-leucine to the trehalose is 6-8:15-20.

[0009] Preferably, the volume-to-mass ratio of the cytokine and the composite protective agent is 1 L:23-26 g. Furthermore, the mass ratio of L-leucine to trehalose is 6.55:17.

[0010] Preferably, the spray drying temperature is 60°C. During spray drying, the feed rate of the mixed solution containing the composite protective agent and cytokine is 8-12 rpm / min; and / or the needle passage time is 3-10 seconds; and / or the fan frequency is 30-50 Hz. The atomization pressure is 0.1-0.4 MPa; and / or the needle passage pressure is 0.2-0.4 MPa. The cytokine is selected from chicken bicytokine or bovine interferon lambda.

[0011] In the present invention, the chicken dual cytokine is a product in which a flexible linker connects the gene sequences of ChIL-2 and ChIFN-α to express the fusion protein ChIL-2-IFN-α.

[0012] In a second aspect, the present invention provides a cytokine powder in an optional embodiment, which is prepared using the above-mentioned preparation method.

[0013] Preferably, the storage time of the cytokine powder at 4°C is at least 9 months. The storage time of the cytokine powder at room temperature is 6 months. The antiviral activity of the cytokine powder is 10 5 u / 0.1mL or more, and the powder extraction rate exceeds 240%.

[0014] Compared with the prior art, the present invention has one of the following beneficial effects:

[0015] 1. The present invention adds a composite protective agent to the cytokine powder and spray-dries it under specific temperature conditions. While ensuring high antiviral activity, it can improve the powder yield and production efficiency of the cytokine powder, enable the cytokines to be stored at room temperature, extend the shelf life of the cytokines, and facilitate transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1These are the SDS-PAGE test results of the chicken double cytokine powder and the chicken double cytokine solution prepared in Example 1 and Comparative Example 14 of the present invention, wherein M is a protein marker, 1 is a chicken double cytokine solution, 2 is the chicken double cytokine powder of Example 1, and 3 is the chicken double cytokine powder of Comparative Example 14.

[0018] Figure 2 The SDS-PAGE test results of the chicken double cytokine powder prepared in Example 1 of the present invention at different temperatures and time samples are shown, wherein: Figure 2 A is the SDS-PAGE test result of the powder stored at 4℃. Figure 2 B is the SDS-PAGE test result of the powder stored at room temperature, M is the protein marker, Figure 2 1-5 in A are the protein contents of the powder in the 0th month, 3rd month, 4th month, 6th month and 9th month respectively. Figure 2 1-5 in B are the protein contents of the powder in month 0, 3, 4, 6 and 9 respectively.

[0019] Figure 3 These are the SDS-PAGE test results of the chicken double cytokine powder, product A, and product B prepared in Example 1 of the present invention, wherein M is a protein marker, and 1-3 are chicken double cytokine powder, product A, and product B, respectively. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0021] The technical solution of the present invention will be described below with reference to embodiments.

[0022] Example 1

[0023] This embodiment provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0024] Take 2L of chicken double cytokine solution, add 47.1g of composite protective agent (13.1g L-leucine and 34g trehalose) to the solution, dissolve and mix, and then install the machine components according to the manual of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.). Turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then introduce the above solution. After the spray drying is completed, rinse the pipes and nozzle with clean water for 5 minutes. Then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0025] Example 2

[0026] This embodiment provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0027] Take 2L of chicken double cytokine solution, add 47.1g of composite protective agent (13.1g L-leucine and 34g trehalose) to the solution, dissolve and mix, and then install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.). Turn on the power, click "Enter System" on the screen, set the inlet air temperature to 80°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then add the above solution. After the spray drying is completed, rinse the pipes and nozzle with clean water for 5 minutes. Then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0028] Example 3

[0029] This embodiment provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0030] Take 2L of chicken double cytokine solution, add 47.1g of composite protective agent (13.1g L-leucine and 34g trehalose) to the solution, dissolve and mix, and then install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.). Turn on the power, click "Enter System" on the screen, set the inlet air temperature to 100°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then introduce the above solution. After the spray drying is completed, rinse the pipes and nozzle with clean water for 5 minutes. Then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0031] Example 4

[0032] This embodiment provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0033] Take 2L of chicken double cytokine solution, add 46g of composite protective agent (16g L-leucine and 30g trehalose) to the solution and dissolve and mix. Then install the machine components according to the manual of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60℃, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait for 5-10 minutes for the temperature to stabilize, observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, first add clean water for 3 minutes, and then add the above solution. After the spray drying is completed, first add clean water for 5 minutes to clean the pipes and nozzles, then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60℃, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0034] Example 5

[0035] This embodiment provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0036] Take 2L of chicken double cytokine solution, add 52g of composite protective agent (12g L-leucine and 40g trehalose) to the solution and dissolve and mix. Then install the machine components according to the manual of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then introduce the above solution. After the spray drying is completed, rinse the pipes and nozzle with clean water for 5 minutes. Then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0037] Example 6

[0038] This embodiment provides a method for preparing bovine interferon lambda powder, comprising the following steps:

[0039] To 2L of bovine interferon λ solution, add 47.1g of a composite protective agent (13.1g L-leucine and 34g trehalose) to the solution and dissolve and mix thoroughly. Install the machine components according to the instructions for the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.). Turn on the power and click "Enter System" on the screen. Set the inlet air temperature to 60°C, the feed rate to 10 rpm / min, the needle-passing time to 10 seconds, and the fan frequency to 50 Hz. Turn on the air compressor, fan, and heater in sequence. Wait 5-10 minutes for the temperature to stabilize. Observe the atomization pressure and needle-passing pressure, maintaining a range of 0.1 MPa ≤ 0.4 MPa and 0.2 MPa ≤ 0.4 MPa. Turn on the peristaltic pump and introduce clean water for 3 minutes before adding the solution. After spray drying is complete, rinse the pipes and nozzle with clean water for 5 minutes. Then, turn off the peristaltic pump, air compressor, needle-passing time, and heater in sequence. Once the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain bovine interferon λ powder.

[0040] Comparative Example 1

[0041] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0042] Take 2L of chicken double cytokine solution, add 50mL of water to the solution, and install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.). Turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then introduce the above solution. After the spray drying is completed, introduce clean water for 5 minutes to clean the pipes and nozzle, then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and remove the solid powder inside to obtain chicken double cytokine powder.

[0043] Comparative Example 2

[0044] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0045] Take 2L of chicken double cytokine solution, add 34g of trehalose to the solution and dissolve and mix. Then install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60℃, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait for 5-10 minutes for the temperature to stabilize, observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, first add clean water for 3 minutes, and then add the above solution. After the spray drying is completed, first clean water for 5 minutes to clean the pipes and nozzle, then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60℃, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0046] Comparative Example 3

[0047] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0048] Take 2L of chicken double cytokine solution, add 13.1g of L-leucine to dissolve and mix, then install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, and 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then add the above solution. After the spray drying is completed, rinse the pipes and nozzle with clean water for 5 minutes. Then, turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0049] Comparative Example 4

[0050] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0051] Take 2L of chicken double cytokine solution, add 34g of lactose to the solution and dissolve and mix. Then, install the machine components according to the manual of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait for 5-10 minutes for the temperature to stabilize, observe the atomization pressure and needle pressure, where 0.1Mpa≤atomization pressure≤0.4Mpa, 0.2Mpa≤needle pressure≤0.4Mpa. Turn on the peristaltic pump, advance clean water for 3min and then enter the above solution. After the spray drying is completed, advance clean water for 5min to clean the pipes and nozzle, then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0052] Comparative Example 5

[0053] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0054] Take 2L of chicken double cytokine solution, add 18g of whey protein to the solution and dissolve and mix. Then install the machine components according to the manual of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait for 5-10 minutes for the temperature to stabilize, observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, advance clean water for 3 minutes, and then enter the above solution. After the spray drying is completed, advance clean water for 5 minutes to clean the pipes and nozzles. Then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0055] Comparative Example 6

[0056] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0057] Take 2L of chicken double cytokine solution, add 100g of maltodextrin to the solution and dissolve and mix. Then install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, first add clean water for 3 minutes, and then add the above solution. After the spray drying is completed, first clean water for 5 minutes to rinse the pipes and nozzle, then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0058] Comparative Example 7

[0059] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0060] Take 2L of chicken double cytokine solution, add 15g of β-dextrin to the solution and dissolve and mix. Then install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, first add clean water for 3 minutes, and then add the above solution. After the spray drying is completed, first clean water for 5 minutes to rinse the pipes and nozzle, then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0061] Comparative Example 8

[0062] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0063] Take 2L of chicken double cytokine solution, add 100g of soy peptone to dissolve and mix, then install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, and 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, first add clean water for 3 minutes, and then add the above solution. After the spray drying is completed, first rinse the pipes and nozzle with clean water for 5 minutes, then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0064] Comparative Example 9

[0065] This embodiment provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0066] Take 2L of chicken double cytokine solution, add 47.1g of composite protective agent (13.1g L-leucine and 34g lactose) to the solution and dissolve and mix. Then, install the machine components according to the manual of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60℃, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait for 5-10 minutes for the temperature to stabilize, observe the atomization pressure and needle pressure, where 0.1Mpa≤atomization pressure≤0.4Mpa, 0.2Mpa≤needle pressure≤0.4Mpa. Turn on the peristaltic pump, advance clean water for 3 minutes, and then enter the above solution. After spray drying is completed, advance clean water for 5 minutes to clean the pipes and nozzles. Then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60℃, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0067] Comparative Example 10

[0068] This embodiment provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0069] Take 2L of chicken double cytokine solution, add 31.1g of composite protective agent (13.1g L-leucine and 18g whey protein) to the solution and dissolve and mix. Then, install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 60°C, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, and 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then add the above solution. After the spray drying is completed, rinse the pipes and nozzle with clean water for 5 minutes. Then, turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0070] Comparative Example 11

[0071] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0072] Take 2L of chicken double cytokine solution, add 47.1g of composite protective agent (13.1g L-leucine and 34g trehalose) to the solution, dissolve and mix, and then install the machine components according to the instructions of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.). Turn on the power, click "Enter System" on the screen, set the inlet air temperature to 120℃, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, and observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then introduce the above solution. After the spray drying is completed, rinse the pipes and nozzle with clean water for 5 minutes. Then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60℃, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0073] Comparative Example 12

[0074] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0075] Take 2L of chicken double cytokine solution, add 47.1g of composite protective agent (13.1g L-leucine and 34g trehalose) to the solution and dissolve and mix. Then install the machine components according to the manual of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 140℃, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, first add clean water for 3 minutes, and then add the above solution. After the spray drying is completed, first add clean water for 5 minutes to clean the pipes and nozzles, then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60℃, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0076] Comparative Example 13

[0077] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0078] Take 2L of chicken double cytokine solution, add 47.1g of composite protective agent (13.1g L-leucine and 34g trehalose) to the solution and dissolve and mix. Then install the machine components according to the manual of the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.), turn on the power, click "Enter System" on the screen, set the inlet air temperature to 160℃, the feed rate to 10rpm / min, the needle time to 10s, and the fan frequency to 50Hz. Turn on the air compressor, fan, and heater in sequence, wait 5-10 minutes for the temperature to stabilize, observe the atomization pressure and needle pressure, where 0.1Mpa ≤ atomization pressure ≤ 0.4Mpa, 0.2Mpa ≤ needle pressure ≤ 0.4Mpa. Turn on the peristaltic pump, introduce clean water for 3 minutes, and then introduce the above solution. After the spray drying is completed, introduce clean water for 5 minutes to clean the pipes and nozzles. Then turn off the peristaltic pump, air compressor, needle, and heater in sequence. When the inlet air temperature drops to 60℃, turn off the fan. Remove the receiving bottle and take out the solid powder inside to obtain chicken double cytokine powder.

[0079] Comparative Example 14

[0080] This comparative example provides a method for preparing chicken double cytokine powder, comprising the following steps:

[0081] Take 2L of chicken double cytokine solution, add 50g of lactose to the solution, stir evenly to thoroughly mix, and then dispense into vials, 10mL per bottle. Loosely place the rubber stopper on the vial and freeze at -80°C for 12 hours. Place the pre-frozen material on a capping drying rack, then on a cold trap. After checking that the seal is intact and free of debris, cover with a plexiglass cover. Turn on the switch, adjust the vacuum value to <20Pa, and dry for 24-48 hours to obtain freeze-dried chicken double cytokine powder.

[0082] Comparative Example 15

[0083] This comparative example provides a method for preparing bovine interferon lambda powder, comprising the following steps:

[0084] Take 2L of bovine interferon λ solution and add 47% water to the solution. Install the machine components according to the instructions for the experimental spray dryer (model QFN-8000S, Shanghai Qiaofeng Industrial Co., Ltd.). Turn on the power and click "Enter System" on the screen. Set the inlet air temperature to 60°C, the feed rate to 10 rpm / min, the needle-passing time to 10 seconds, and the fan frequency to 50 Hz. Turn on the air compressor, fan, and heater in sequence. Wait 5-10 minutes for the temperature to stabilize. Observe the atomization pressure and needle-passing pressure, maintaining a constant temperature of 0.1 MPa ≤ atomization pressure ≤ 0.4 MPa and a constant temperature of 0.2 MPa ≤ needle-passing pressure ≤ 0.4 MPa. Turn on the peristaltic pump and introduce clean water for 3 minutes before adding the solution. After spray drying is complete, rinse the pipes and nozzle with clean water for 5 minutes. Then, turn off the peristaltic pump, air compressor, needle-passing time, and heater in sequence. When the inlet air temperature drops to 60°C, turn off the fan. Remove the receiving bottle and remove the solid powder inside to obtain bovine interferon λ powder.

[0085] Experimental example

[0086] 1. The chicken double cytokine powders prepared in Examples 1-5 and Comparative Examples 1-13 were weighed. Based on the sample powder yield of Comparative Example 1, the powder yield was recorded as 100%. The powder yield of the preparation methods of Examples 1-5 and Comparative Examples 2-13 was calculated according to the formula (powder yield - amount of protective agent added) / 102 = powder yield%. The results are shown in Table 1.

[0087] The chicken double cytokine powders prepared in Examples 1-5 and Comparative Examples 1-13 were added to 2 L of DMEM culture medium (containing 2% FBS) and reduced in a ratio of 1:1 for 4 -1 -4 -10 After the DF1 cells grew to 80-90%, the culture medium in the wells was discarded, and the plate was washed twice with PBS. Then 100 μL of the 16 samples that had been diluted in multiples were added, and each dilution was repeated in 6 wells. The plate was placed in a cell culture incubator at a temperature of 37°C and a carbon dioxide concentration of 5% for 24 hours. According to the VSV test on DF1 cells, 10 6.5 TCID 50 / 100μL results, use DMEM medium (containing 2% FBS) to dilute VSV to a concentration of 100TCID 50 / 100μL. Add 100μL of 100TCID to each well. 50VSV was added to the 11th column as a VSV positive control well. The plate was incubated at 37°C for 1 hour, the virus solution was discarded, the plate was washed three times, and DMEM medium (containing 2% FBS) was added. The plate was cultured in a 37°C, 5% CO2 cell culture incubator and observed every 12 hours. When the VSV positive control wells completely developed pathological changes, the highest dilution that inhibited the development of pathological changes in 50% of the cells in each well was considered one unit of sample activity. The antiviral activity of the chicken dual cytokine powders prepared in Examples 1-5 and Comparative Examples 1-13 was tested. The results are shown in Table 1.

[0088] Table 1 Powder yield, powder yield and activity of Examples 1-5 and Comparative Examples 1-13

[0089]

[0090]

[0091] Table 1 shows that the chicken dual cytokine powder exhibited the best powder yield and antiviral activity when a mixture of L-leucine and trehalose was added as the composite protective agent. A drying temperature of 60-100°C improved the powder yield of the chicken dual cytokine powder while maintaining high antiviral activity. 2. The chicken dual cytokine powders prepared in Example 1 and Comparative Example 14 were each randomly divided into two equal portions.

[0092] One of the two parts was added to DMEM medium (containing 2% FBS) and reduced in a 1:1 ratio. -1 -4 -10 After the DF1 cells grew to 80-90%, the culture medium in the wells was discarded, the plate was washed twice with PBS, and then 100 μL of the two diluted samples were added respectively. Each dilution was repeated for 6 wells and cultured in a cell culture incubator at 37°C and 5% carbon dioxide for 24 hours. 6.5 TCID 50 / 100μL results, use DMEM medium (containing 2% FBS) to dilute VSV to a concentration of 100TCID 50 / 100μL. Add 100μL of 100TCID to each well. 50 The VSV was added to the 11th column as a VSV positive control well, incubated at 37°C for 1 hour, the virus solution was discarded, the plate was washed 3 times, and DMEM medium (containing 2% FBS) was added. The plate was cultured in a 37°C, 5% carbon dioxide cell culture incubator and observed every 12 hours. When the VSV positive control wells were completely lesioned, the highest dilution that could inhibit 50% of the cells in each cell well from developing lesions was considered one unit of sample activity. The antiviral activity of the chicken double cytokine powder prepared in Example 1 was 3.67×10 5u / 0.1mL, the antiviral activity of the chicken double cytokine powder prepared in Comparative Example 14 was 3.69×10 5 It can be seen that the chicken double cytokine powders prepared in Example 1 and Comparative Example 14 have little difference in antiviral activity.

[0093] Another spray-dried sample and freeze-dried sample were respectively reduced and dissolved in PBS buffer at a ratio of 1:1 according to 1L of solution. 1mL of the dissolved solution was taken and concentrated with TCA. The protein level was detected by SDS-PAGE, and chicken double cytokine solution was used as a positive control.

[0094] The results are as follows Figure 1 As shown, after the chicken dual cytokine solution was prepared into a powder by the preparation method of Example 1 or Comparative Example 14, there was no protein loss compared to the chicken dual cytokine solution. 1 mL of the dissolved solution was then taken, and the protein content of the chicken dual cytokine supernatant, Example 1, and Comparative Example 14 was 8.4 mg / mL, 8.3 mg / mL, and 8.2 mg / mL, respectively.

[0095] 3. The chicken dual cytokine powder prepared in Example 1 was divided equally into 40 portions and stored at 4°C and room temperature, respectively. Four portions each of 4°C and room temperature were collected at the 0th, 3rd, 4th, 6th, and 9th month of storage. Cytokine antiviral activity was tested on DF1 cells using the method of Experimental Example 1 for three of these portions, and the average value was calculated.

[0096] The results are shown in Table 2. When the samples were stored at 4°C, the antiviral activity at 0, 3, 4, 6 and 9 months was 10 5 u / 0.1mL or more; when the sample is stored at room temperature, the activity at 0, 3, 4, and 6 months is within 10 5 u / 0.1mL or more, the activity after storage for 9 months is 8.27×10 4 u / 0.1mL.

[0097] Table 2 Antiviral activity of chicken double cytokine powder at different holding temperatures and times

[0098]

[0099] The remaining sample was added to 1 L of PBS buffer and dissolved in a 1:1 ratio. 1 mL of the dissolved solution was taken and concentrated with TCA, and the protein level was detected by SDS-PAGE.

[0100] like Figure 2As shown, samples stored at 4°C showed no protein degradation at 0, 3, 4, 6, and 9 months; samples stored at room temperature showed no protein degradation at 0, 3, 4, and 6 months, but protein degradation occurred at 9 months. Therefore, the chicken double cytokine powder can be stored at 4°C for more than 9 months and at room temperature for 6 months.

[0101] 4. Two chicken cytokine products were purchased from a biological product company and named Product A and Product B. These two products need to be refrigerated and have a shelf life of 3 months. 2L of DMEM medium (containing 2% FBS) was added to the chicken double cytokine powder prepared in Example 1 and reduced in a 1:1 ratio; Product A and Product B were diluted according to the product instructions, and the cytokine antiviral activity was tested (see the method of Experimental Example 1). The antiviral activities of the chicken double cytokine powder, Product A, and Product B in Example 1 were 3.38×10 5 u / 0.1mL、1.20×10 4 u / 0.1mL、9.35×10 3 u / 0.1mL.

[0102] Take 1 mL of the liquid, concentrate it with TCA, and then detect the protein level by SDS-PAGE. Figure 3 As shown, the protein bands of Chicken Dual Cytokine Powder are thicker than those of Products A and B. BCA protein quantification revealed protein contents of Chicken Dual Cytokine Powder, Product A, and Product B at 8.1 mg / mL, 3.2 mg / mL, and 1.6 mg / mL, respectively. Therefore, Chicken Dual Cytokine Powder demonstrates stronger antiviral activity and a higher protein concentration than similar commercially available products.

[0103] 5. The bovine interferon λ powders prepared in Example 6 and Comparative Example 15 were weighed. The powder weight of Example 6 was 339 g, and the powder weight of Comparative Example 15 was 101 g. Based on the powder yield of the sample in Comparative Example 15, the powder yield was recorded as 100%. The powder yield of the preparation method of Example 6 was calculated according to the formula (powder yield - amount of protective agent added) / 102 = powder yield%, and the powder yield of the preparation method of Example 6 was 289.1%.

[0104] The bovine interferon λ powder prepared in Example 6 and Comparative Example 15 was added to 2 L of RPMI 1640 culture medium (containing 10% FBS) and reduced in a ratio of 1:1. -1 -4 -10 After the MDBK cells grew to 80-90%, the culture medium in the wells was discarded, and the plate was washed twice with PBS. Then 100 μL of the two samples that had been diluted in a doubling ratio were added, and 6 wells were repeated for each dilution. The plates were placed in a cell culture incubator at a temperature of 37°C and a carbon dioxide concentration of 5% for 24 hours. 6.5 TCID50 / 100μL results, use RPMI 1640 medium (containing 10% FBS) to dilute VSV to a concentration of 100TCID 50 / 100μL. Add 100μL of 100TCID to each well. 50 The VSV was transferred to the 11th column as a VSV positive control well and incubated at 37°C for 1 hour. The virus solution was discarded, the plate was washed three times, and RPMI 1640 medium (containing 10% FBS) was added. The plate was cultured in a 37°C, 5% CO2 cell culture incubator and observed every 12 hours. When the VSV positive control wells were completely lesioned, the highest dilution that could inhibit lesion formation in 50% of the cells in each well was considered one unit of sample activity. The results showed that the antiviral activity of the bovine interferon λ powder of Example 6 was 3.54×10 5 u / 0.1mL, the antiviral activity of the bovine interferon λ powder of Comparative Example 15 was 1.03×10 2 u / 0.1mL.

[0105] Although the principles of the present invention have been described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Without departing from the spirit and scope of the present invention, any obvious changes such as equivalent transformations and simple substitutions based on the technical solution of the present invention fall within the scope of protection of the present invention.

Claims

1. A method for preparing cytokine powder, characterized in that: The following steps are involved: After adding the composite protective agent to the cytokine solution and dissolving and mixing, spray drying is performed at a temperature of 60-100° C., and the obtained powder is the cytokine powder; Wherein, the composite protective agent is a mixture of L-leucine and trehalose, and the mass ratio of L-leucine to trehalose is 6-8:15-20.

2. The method for preparing the cytokine powder according to claim 1, characterized in that: The volume-to-mass ratio of the cytokine and the composite protective agent is 1L:23-26g.

3. The method for preparing the cytokine powder according to claim 1, characterized in that: The mass ratio of L-leucine to trehalose is 6.55:

17.

4. The method for preparing the cytokine powder according to claim 1, characterized in that: The spray drying temperature is 60°C; During spray drying, the feeding rate of the solution containing the composite protective agent and cytokine is 8-12 rpm / min; and / or, Needle retention time is 3-10 seconds; and / or, The fan frequency is 30-50Hz.

5. The method for preparing the cytokine powder according to claim 1, characterized in that: During spray drying, the atomization pressure is 0.1-0.4Mpa; and / or, The needle pressure is 0.2-0.4Mpa.

6. The method for preparing the cytokine powder according to claim 1, characterized in that: The cytokine is selected from one of chicken bicytokine or bovine interferon lambda.

7. A cytokine powder, characterized in that: The preparation method is described in any one of claims 1 to 6.

8. The cytokine powder according to claim 7, characterized in that The cytokine powder can be stored at 4° C. for at least 9 months.

9. The cytokine powder according to claim 7, characterized in that The storage time of the cytokine powder at room temperature is 6 months.

10. The cytokine powder according to claim 7, characterized in that: The antiviral activity of the cytokine powder is 10 5 u / 0.1mL or more, and the powder extraction rate exceeds 240%.

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