CN106377543B - A kind of preparation method of spleen source transfer factor - Google Patents
A kind of preparation method of spleen source transfer factor Download PDFInfo
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- CN106377543B CN106377543B CN201610760863.8A CN201610760863A CN106377543B CN 106377543 B CN106377543 B CN 106377543B CN 201610760863 A CN201610760863 A CN 201610760863A CN 106377543 B CN106377543 B CN 106377543B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- Immunology (AREA)
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- Developmental Biology & Embryology (AREA)
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- Biotechnology (AREA)
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- Hematology (AREA)
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- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
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- Animal Behavior & Ethology (AREA)
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Abstract
The invention belongs to field of veterinary, disclose a kind of preparation method of spleen source transfer factor.This method step includes spleen pre-treatment, Minced Steak homogenate, acidification, liquid nitrogen frozen, broken microwave thawing, centrifugal purification and drying, wherein the aqueous citric acid solution of diamond dust, TritonX-100 and concentration 70 ~ 80% are added when homogenate.The present invention can significantly shorten the preparation extraction time of spleen source transfer factor TF and improve the recovery rate of transfer factor, it is higher with simple process, mechanization degree, easily operates with convenient for scale process standard, continuous operation, significantly improves spleen transfer factor filter efficiency, reduce labor intensity and Purification by filtration easily reverse cleaning the characteristics of, the preparation method of the spleen source transfer factor of the invention is suitable for the technique production application of industrialization spleen transfer factor, has promotion prospect outstanding.
Description
Technical field
The invention belongs to field of veterinary, and in particular to a kind of preparation method of spleen source transfer factor.
Background technique
Transfer factor (Transfer factor, TF) is used as a kind of novel immune exciting agent, is T lymphocyte release
One kind can shift the substance of sensitization information, the cellular immunity information of donor special, efficiently can be transferred to receptor by it,
To enhance the immune function of receptor.From immunology, TF can be divided into specific transfer factor and non-single transfer factor two
Major class.
It has now been found that 1. the function of TF transfer factor and the mechanism of action, which are respectively as follows:, enhances cellular immune function；2. increasing
Strong humoral immunity；3. enhancing nospecific immunity；4. anti-immunity inhibits function；5. into the growth and development of body.
Splenic T F is rich in a variety of amino acid with it, and no antigen has no toxic side effect, and becomes a kind of safe novel disease-resistant
Malicious biological agent is widely used in viral disease, bacteriosis, fungal disease, parasitic diseases of people and animal etc.
Disease prevention and cure.Especially specificity splenic T F is to the disease that can not be effectively controlled with antibacterials, antiviral drugs, vaccine etc. at present
Viral disease has unique curative effect, has bright prospects in the application of veterinary clinic.Transfer factor is present in the leaching of spleen
In bar cell, the transfer factor preparation method of the prior art is generally to rub animal spleen, destroy eucaryotic cell structure and make born of the same parents
Interior transfer factor is discharged into slurries, then the transfer factor in slurries is carried out separation and purification and finally obtains transfer factor product.
Multigelation method and dialysis are to use more universal method during producing TF at present, and multigelation method refers to animal spleen
The dirty process for carrying out -30 DEG C of cryogenic freezings and thawing again.It is several that dialysis refers to that the centrifuged supernatant of animal spleen homogenate carries out
Secondary dialysis, realization isolate and purify transfer factor, and above-mentioned two classes method is easy to operate, but the production cycle is long, and operating cost is high,
Large labor intensity, mechanical degrees are weaker, and TF recovery rate is not high, it is difficult to carry out industrially scalable and produce in bulk, therefore having must
Develop a kind of novel preparation method of spleen source transfer factor (TF).
Summary of the invention
To solve the problems, such as not high, the of the invention mesh of the length of production cycle existing for existing transfer factor preparation method, recovery rate
The preparation method for being intended to provide a kind of spleen source transfer factor, this method can significantly shorten preparation time and improve TF extract
Rate.
To achieve the above object, the technical solution adopted by the present invention is as follows:
A kind of preparation method of spleen source transfer factor, steps are as follows:
S1, pre-treatment: the qualified animal spleen of screening quarantine, then impurity elimination, cleaning, disinfection, drained and standby；
S2, Minced Steak homogenate:
(a) rubbing processing Minced Steak: is carried out to the spleen after pre-treatment, it is desirable that the granularity of the spleen particulate matter after rubbing reaches
0.5~1.0cm；
(b) it is homogenized: into the spleen particulate matter after rubbing, the lemon of diamond dust, TritonX-100 and concentration 70 ~ 80% is added
Lemon aqueous acid is homogenized, and obtains Spleen homogenate, and every 100g spleen particulate matter adds the diamond dust of 3 ~ 5 g, 0.1 ~ 0.5
The aqueous citric acid solution of the TritonX-100 of mL and 4 ~ 6 mL；Wherein, " % " concentration of aqueous citric acid solution refer to citric acid with
The mass/volume percent concentration that water is measured with g/mL；
S3, acidification: it adjusts Spleen homogenate pH value to 3.0 ~ 3.5, obtains spleen homogenate acidifying solution；
S4, freezing, broken defrosting:
(a), acidifying solution is homogenized using liquid nitrogen frozen processing spleen, obtains spleen homogenate acidifying solution ice cube；
(b), spleen homogenate acidifying solution ice cube is smashed, then carries out defrosting operation again, thaw point is 30 ~ 37 DEG C, is made
The industry time is 0.5 ~ 1h；
(c), defrosting spleen liquid is collected, then presses S4(a) ~ (b) repetition 1 ~ 2 time；
S5, centrifugal purification: carrying out centrifugal purification using self-control centrifugal purification system, the centrifugal purification system include tubular type from
Scheming, centrifugation liquid storage tank, power transmitting assembly I, power transmitting assembly II, power transmitting assembly III, grade one filter, second level
Filter, tertiary filter and its extraneous subsystem；Power transmitting assembly I is connected in series by kinetic pump I and check-valves I；It is dynamic
Power transmission assembly II is connected in series by kinetic pump II and check-valves II；Power transmitting assembly III is by kinetic pump III and check-valves
III is connected in series；Grade one filter and secondary filter are conventional cartridge filter；Tertiary filter is self-control filter,
Structure includes cylinder, doughnut tread assembly, pedestal；Cylinder body bottom and top are respectively equipped with inlet and liquid outlet, outside cylinder
Portion is equipped with interlayer, and the upper and lower ends of interlayer are respectively equipped with interlayer import and interlayer outlet；Doughnut tread assembly includes being evenly equipped with
Porous package shell, several doughnuts, these doughnuts merge bunchy and are placed in inside package shell；Pedestal is cylinder
Body, cylindrical body upper section be it is hollow be cylindrical in shape, middle and lower sections it is solid, the middle and lower sections of pedestal it is solid equipped with doughnut quantity and its
The compatible through-hole of size；Pedestal is located at below liquid outlet, is close to cylinder roof and is firmly connected with it；The upper end of doughnut
Opening is simultaneously threaded through in the through-hole of pedestal, and lower end closed is simultaneously fixedly connected with package shell bottom wall；The external world of tertiary filter is attached
Category system includes kinetic pump IV, check-valves IV, constant temperature refrigeration water tank, and successively contact check-valves IV, constant temperature chilled water for interlayer outlet
Case and kinetic pump IV form a closed cycle system to interlayer import；Tube centrifuge by pipeline successively contact centrifugate storage
Tank, kinetic pump I, check-valves I, grade one filter, kinetic pump II, check-valves II, secondary filter, kinetic pump III, check-valves
III to tertiary filter inlet；Grade one filter and be respectively 4.5 ~ 5 μm with the filter core aperture of secondary filter, 0.2 ~
0.22μm；The molecular cut off of doughnut is 5000 ~ 6000 dalton；
Steps by centrifugation are as follows:
(a) first will S4 obtain spleen homogenate thawing solution input tube centrifuge inner cavity in, start the equipment carry out from
Heart work, centrifugal rotational speed are centrifuged 20 ~ 30min up to 4000 ~ 6000rpm/min；The centrifugal clear liquid that will acquire in time after centrifugation
It is transferred to centrifugation liquid storage tank, and temperature controlling range is 5 ~ 10 DEG C in the tank；
(b) the cooling centrifugate that upper process obtains is crossed check-valves I by the road and is delivered to grade one filter by starting kinetic pump I
Interior, 0.2 ~ 0.6 Mpa of input pressure realizes first time coarse filtration；
(c) the by-pass filtration liquid obtained after grade one filter is crossed check-valves II by the road and is delivered to by starting kinetic pump II
In secondary filter, 0.2 ~ 0.6 Mpa of input pressure realizes second of coarse filtration；
(d) start kinetic pump IV, by temperature obtained in constant temperature refrigeration water tank be 5 ~ 15 DEG C cold water by the road from folder
Layer import enters in interlayer, and crosses check-valves IV by the road by interlayer outlet and be recycled in constant temperature refrigeration water tank；To in cylinder
Chamber temperature restarts kinetic pump III and the secondary filtration liquid obtained after secondary filter is crossed non-return by the road when reaching 5 ~ 15 DEG C
Valve III is delivered in tertiary filter by inlet, 0.2 ~ 0.6 MPa of input pressure, and secondary filtration liquid is first from package shell
It is porous enter package shell inside, due to the crown_interception of doughnut, the impurity in secondary filtration liquid is blocked in hollow fibre
The outside of dimension, and the secondary filtration liquid containing transfer factor then passes through doughnut and enters pedestal from doughnut top opening, then
It comes out from liquid outlet to get the stoste containing transfer factor；
S6, dry finished product: by the stoste containing transfer factor obtained by S5 through vacuum freeze drying or spray drying to get at
Product.
Specifically, the process of S1 are as follows: the qualified animal spleen of screening quarantine, it is desirable that spleen is fresh, without extravasated blood, without lesion and
Without bad smell；Then the fat and connective tissue of spleen are removed under conditions of 100,000 grades of working environment, while guaranteeing spleen
Integrality；The watery blood for cleaning spleen exudation again, drains away the water；Spleen is finally immersed to the first of concentration of volume percent 0.3 ~ 0.6%
Carry out disinfection processing in phenol aqueous solution, and the time is 20 ~ 30min, drained and standby.
Specifically, S2 is that the spleen for draining S1 disinfection carries out Minced Steak and homogenized respectively, and operating environment is in cleaning
It is carried out in the clean room of ten thousand grades of degree, process are as follows:
(a) Minced Steak: the spleen that disinfection is drained is packed into meat grinder machine cavity, and starting working power carries out spleen rubbing and makees
Industry, operating rate are 100 ~ 150kg/h, spleen particle of the granularity of spleen particulate matter up to 0.5 ~ 1.0cm, after rubbing after rubbing
Object is temporarily stored in sterilized plastic containers for use together；
(b) it is homogenized: the spleen particulate matter that upper step obtains is put into the cavity of the high-speed homogenization machine of chummery, then to height
The aqueous citric acid solution of diamond dust, TritonX-100 and concentration 70 ~ 80%, every 100g spleen are added in the cavity of fast refiner
Grain object add the diamond dust of 3 ~ 5 g, 0.1 ~ 0.5 mL TritonX-100 and 4 ~ 6 mL aqueous citric acid solution, start work
Power supply carry out spleen be homogenized operation, homogenate rotating speed of flail be 9000 ~ 15000rpm/min, homogenized temperature control at 32 DEG C hereinafter,
Time is 20 ~ 30min；Wherein, " % " concentration of aqueous citric acid solution refers to the matter that citric acid and water are measured with g/mL in the process
Amount/concentration of volume percent.
Specifically, the process of S3 are as follows: Spleen homogenate pH value is adjusted to 3.0 ~ 3.5, acidulant used in the step
For the mixed liquor of aqueous citric acid solution 2:1 ~ 4:1 mixing by volume of the aqueous hydrochloric acid solution and concentration 10% of concentration 15%；Acidification
Middle acidulant is added in Spleen homogenate using constant flow pump, while at the uniform velocity being stirred, acidulant flow rate and stirring rate point
It Wei not 10 ~ 20 mL/min, 600 ~ 800 rpm/min；Wherein, " % " concentration of aqueous hydrochloric acid solution refers to quality percentage in the process
Specific concentration refers to the mass/volume percent concentration that citric acid and water are measured with g/mL with " % " concentration of aqueous citric acid solution.
Specifically, the process of S4 are as follows:
(a), spleen homogenate acidifying solution is dispensed it is intracavitary to liquid nitrogen tank is gone in container again, the processing time 10 ~
20min；
(b), the spleen formed after liquid nitrogen frozen homogenate acidifying solution ice cube is dosed in ice crusher and smashes ice cube to straight
Diameter is the particle of 0.8 ~ 1.5cm；Then the pellet frozen object that will acquire again be dosed in the machine cavity of continuous-type microwave unfreezing machine into
Row defrosting operation, thaw point are 30 ~ 37 DEG C, and the activity duration is 0.5 ~ 1h；
(c), defrosting spleen liquid is collected, then presses S4(a) ~ (b) repetition 1 ~ 2 time.
Further, S5 further includes that step (e) is instead cleaned: at the end of work to be filtered, can carry out the reverse cleaning of the system
Operation, manual 180 ° of integrated regulation power transmitting assemblies I, power transmitting assembly II, power transmitting assembly III direction, that is, it is original
Import become exporting, reconnect corresponding pipeline, while starting above-mentioned kinetic pump I, kinetic pump II, kinetic pump III, will clean
Purified water enter in cylinder through barrel liquid discharge mouth, then ejected wash water is finally from centrifugation liquid storage tank discharge, each filter pressure in operation
Power list index must not be higher than 0.3 MPa, repeat 2 ~ 3 times.
Preferably, be centrifuged between liquid storage tank and kinetic pump I, between check-valves I and grade one filter, grade one filter and dynamic
Power pumps between II, between check-valves II and secondary filter, between secondary filter and kinetic pump III, check-valves III and three-level
Pipeline between filter takes movable connection method.
Further, the movable connection method is clamp connection.
Preferably, tube centrifuge is GF- divergence type.
Spleen source transfer factor (TF) is obtained from the animal spleen separation and Extraction of normal health, and the animal is related to
Pig, ox, chicken and duck etc..
The utility model has the advantages that the present invention takes the multigelation method of liquid nitrogen and microwave thawing, (- 30 DEG C cold for more traditional multigelation
Freeze, 37 DEG C of thawings) it can significantly shorten preparation extraction time of spleen source transfer factor TF, diamond dust, the TritonX-100 of addition
The recovery rate of transfer factor (TF) can be significantly improved with aqueous citric acid solution, have simple process, mechanization degree higher, easily just
It operated in scale process standard, continuous operation, significantly improve spleen transfer factor (TF) filter efficiency, reduce labor
Fatigue resistance and Purification by filtration easily reverse cleaning the characteristics of, the preparation method of the spleen source transfer factor (TF) of the invention is suitable for producing
The technique production application of industry spleen transfer factor (TF) has promotion prospect outstanding.
Detailed description of the invention
Fig. 1: the structural schematic diagram of centrifugal purification system is made in the present invention by oneself；
Fig. 2: structural schematic diagram when centrifugal purification system is in anti-cleaning state is made in the present invention by oneself；
Description of symbols: 1- tube centrifuge, 2- are centrifuged liquid storage tank, 31- kinetic pump I, 41- check-valves I, 32- power
Pump II, 42- check-valves II, 33- kinetic pump III, 43- check-valves III, 34- kinetic pump IV, 44- check-valves IV, 5- by-pass filtration
Device, 6- secondary filter, 7- cylinder, 8- inlet, 9- liquid outlet, 10- interlayer, 11- interlayer import, the outlet of 12- interlayer, 13-
Pedestal, 14- package shell, 15- doughnut, 16- constant temperature refrigeration water tank.
Specific embodiment
The contents of the present invention are described in further detail with reference to the accompanying drawings and detailed description.
Embodiment 1
A kind of preparation method of pig spleen source transfer factor, steps are as follows:
S1, pre-treatment: the qualified pig spleen of screening quarantine, it is desirable that spleen is fresh, without extravasated blood, without lesion and without bad gas
Taste；Then the fat and connective tissue of spleen are removed under conditions of 100,000 grades of working environment, while guaranteeing spleen integrality；Again
The watery blood for cleaning spleen exudation, drains away the water；Finally by spleen immerse concentration of volume percent 0.5% cresols aqueous solution in into
Row disinfection treatment, time 25min are drained and standby；
S2, Minced Steak homogenate: S2 are that the spleen for draining S1 disinfection carries out Minced Steak and homogenized respectively, and operating environment exists
It is carried out in ten thousand grades of cleanliness of clean room, process are as follows:
(a) Minced Steak: the spleen that disinfection is drained is packed into meat grinder machine cavity, and starting working power carries out spleen rubbing and makees
Industry, operating rate 120kg/h, the granularity of spleen particulate matter reaches 0.8cm after rubbing, and the spleen particulate matter after rubbing is temporary together
There are stand-by in sterilized hard plastic vessels；
(b) it is homogenized: the spleen particulate matter that upper step obtains is put into the cavity of the high-speed homogenization machine of chummery, then to height
The aqueous citric acid solution of diamond dust, TritonX-100 and concentration 75%, every 100g spleen particle are added in the cavity of fast refiner
Object add the diamond dust of 4 g, 0.3 mL TritonX-100 and 5 mL aqueous citric acid solution, starting working power carries out spleen
Dirty homogenate operation, homogenate rotating speed of flail are 13000rpm/min, and homogenized temperature control is at 32 DEG C hereinafter, the time is 25min；Its
In, the mass/volume percentage that " % " concentration of aqueous citric acid solution refers to that citric acid and water are measured with g/mL in the process is dense
Degree；
S3, acidification: being adjusted to 3.0 for Spleen homogenate pH value, and acidulant used in the step is the hydrochloric acid of concentration 15%
The mixed liquor of aqueous solution and the 3:1 mixing by volume of the aqueous citric acid solution of concentration 10%；Acidulant is added using constant flow pump in acidification
It adds in Spleen homogenate, while at the uniform velocity stirring, acidulant flow rate and stirring rate are respectively 15 mL/min, 700
rpm/min；Wherein, " % " concentration of aqueous hydrochloric acid solution refers to mass percent concentration in the process, with aqueous citric acid solution
" % " concentration refers to the mass/volume percent concentration that citric acid and water are measured with g/mL；
S4, freezing, broken defrosting:
(a), spleen homogenate acidifying solution is dispensed intracavitary to liquid nitrogen tank is gone in container again, handles time 20min；
(b), the spleen formed after liquid nitrogen frozen homogenate acidifying solution ice cube is dosed in ice crusher and smashes ice cube to straight
Diameter is the particle of 1.0cm；Then the pellet frozen object that will acquire again is dosed in the machine cavity of continuous-type microwave unfreezing machine and is solved
Freeze operation, thaw point is 35 DEG C, activity duration 0.5h；
(c), defrosting spleen liquid is collected, then by S4(a) ~ (b) be repeated 1 times；
S5, centrifugal purification: centrifugal purification is carried out using self-control centrifugal purification system, as shown in Figure 1, the centrifugal purification system
Including GF- divergence type tube centrifuge 1, centrifugation liquid storage tank 2(tank in temperature controlling range be 5~10 DEG C), power transmitting assembly
I, power transmitting assembly II, power transmitting assembly III, grade one filter 5, secondary filter 6, tertiary filter and its external world are attached
Category system；
Power transmitting assembly I is connected in series by kinetic pump I 31 and check-valves I 41；Power transmitting assembly II is by kinetic pump
II 32 and check-valves II 42 are connected in series；Power transmitting assembly III by kinetic pump III 33 and check-valves III 43 series winding and
At；
Grade one filter 5 and secondary filter 6 are conventional cartridge filter；Tertiary filter is self-control filter, knot
Structure includes cylinder 7, doughnut tread assembly, pedestal 13；7 bottom and top of cylinder are respectively equipped with inlet 8 and liquid outlet 9, cylinder
Interlayer 10 is equipped with outside body 7, the upper and lower ends of interlayer 10 are respectively equipped with interlayer import 11 and interlayer outlet 12；Hollow fiber bundle group
Part includes being evenly equipped with porous package shell 14, quantity 600, molecular cut off for the doughnut 15 of 5000 dalton, this
A little doughnuts 15 merge bunchy and are placed in inside package shell 14；Pedestal 13 be cylindrical body, cylindrical body upper section be it is hollow be cylindrical in shape,
Middle and lower sections are solid, and the middle and lower sections of pedestal 13 are solid to be equipped with through-hole compatible with 15 quantity of doughnut and its size；Pedestal
13 are located at 9 lower section of liquid outlet, are close to 7 roof of cylinder and are firmly connected with it；The upper end of doughnut 15 is open and is threaded through base
In the through-hole of seat 13, lower end closed is simultaneously fixedly connected with 14 bottom wall of package shell；
The extraneous subsystem of tertiary filter includes kinetic pump IV 34, check-valves IV 44, constant temperature refrigeration water tank 16, folder
Successively contact check-valves IV 44, constant temperature refrigeration water tank 16 (controllable temperature range are as follows: 5~15 DEG C) and kinetic pump IV 34 for layer outlet 12
A closed cycle system is formed to interlayer import 11；
Tube centrifuge 1 is successively contacted by pipeline and is centrifuged liquid storage tank 2, kinetic pump I 31, check-valves I 41, by-pass filtration
Device 5, kinetic pump II 32, check-valves II 42, secondary filter 6, kinetic pump III 33, check-valves III 43 to tertiary filter
Inlet 8；Wherein, be centrifuged between liquid storage tank 2 and kinetic pump I 31, between check-valves I 41 and grade one filter 5, level-one mistake
Between filter 5 and kinetic pump II 32, between check-valves II 42 and secondary filter 6, secondary filter 6 and kinetic pump III 33
Between, the pipeline between check-valves III 43 and tertiary filter take clamp connection；
Aforementioned grade one filter 5 and secondary filter 6 pass through commercially available acquisition, the filter of grade one filter 5 and secondary filter 6
Core is folding type filter element, grade one filter 5 and be respectively 5 and 7, by-pass filtration with the filter core number of secondary filter 6
The filter core aperture of device 5 and secondary filter 6 is respectively 5 μm, 0.22 μm；The filter core material of grade one filter 5 and secondary filter 6
For polypropylene, frame of filter element (support frame) is rigid plastics, filter core can compression resistance be 0.4 MPa；
Steps by centrifugation are as follows:
(a) first will S4 obtain spleen homogenate thawing solution input 1 inner cavity of tube centrifuge in, start the equipment carry out from
Heart work, centrifugal rotational speed reach 5000rpm/min, are centrifuged 30min；The centrifugal clear liquid that will acquire in time after centrifugation be transferred to from
In heart liquid storage tank 2, and temperature controlling range is 10 DEG C in the tank；
(b) the cooling centrifugate that upper process obtains is crossed check-valves I 41 through pipeline and is delivered to level-one by starting kinetic pump I 31
In filter 5,0.2 Mpa of input pressure realizes first time coarse filtration；
(c) the by-pass filtration liquid obtained after grade one filter 5 is crossed check-valves II through pipeline by starting kinetic pump II 32
42 are delivered in secondary filter 6,0.2 Mpa of input pressure, realize second of coarse filtration；
(d) start kinetic pump IV 34, by temperature obtained in constant temperature refrigeration water tank 16 be 5 DEG C cold water through pipeline from
Interlayer import 11 enters in interlayer 10, and crosses check-valves IV 44 through pipeline by interlayer outlet 12 and be recycled to constant temperature refrigeration water tank
In 16；Restart the second level mistake that kinetic pump III 33 will be obtained after secondary filter 6 when 7 inner cavity temperature of cylinder reaches 5 DEG C
Filtrate is crossed check-valves III 43 through pipeline and is delivered in tertiary filter by inlet 8,0.2 Mpa of input pressure, secondary filtration
Liquid elder generation is inside the porous entrance package shell 14 on package shell 14, due to the crown_interception of doughnut 15, secondary filtration
Impurity in liquid is blocked in the outside of doughnut 15, and the secondary filtration liquid containing transfer factor then pass through doughnut 15 and from
15 top opening of doughnut enters pedestal 13, then comes out from liquid outlet 9, at this time up to the stoste containing transfer factor；
(e) device is counter cleans: at the end of work to be filtered, can carry out the reverse cleaning operation of the equipment series, i.e., first
Between disassembly centrifugation liquid storage tank 2 and kinetic pump I 31, between check-valves I 41 and grade one filter 5, grade one filter 5 and power
Pump between II 32, between check-valves II 42 and secondary filter 6, between secondary filter 6 and kinetic pump III 33, check-valves
The clip on pipeline between III 43 and tertiary filter, manual 180 ° of integrated regulation power transmitting assemblies I, power transmission group
The direction (original import becomes exporting) of part II, power transmitting assembly III, then pass through the corresponding pipeline of clamp connection, adjustment
Rear state, will be clean pure as shown in Fig. 2, start above-mentioned kinetic pump I 31, kinetic pump II 32, kinetic pump III 33 simultaneously
Change water to enter in cylinder 7 through 7 liquid outlet 9 of cylinder, then ejected wash water is finally discharged from centrifugation liquid storage tank 2, each filter pressure in operation
Power list index must not be higher than 0.3 MPa, be repeated 2 times；
S6, dry finished product: the stoste containing transfer factor obtained by S5 is spray-dried to get finished product -- transfer factor is dry
Powder.
The S1-S6 entire production cycle is 12h.The produced in conventional processes period generally but needs 2 ~ 3d, in comparison, this hair
Bright method significantly shortens manufacturing cycle.
Gained transfer factor (TF) dry powder is detected, TF dry powder detection method is specific as follows:
[character]
This product is light yellow loose block or powder.
[identification]
(1) this product 20mg is taken, adds 1ml water to dissolve, adds few drops of ninhydrin solution, is heated, solution should show bluish violet.
(2) this product 10-15mg is taken, 1ml water is added to dissolve, is measured according to Ultraviolet spectrophotometry.In 261 ± 3nm(ox spleen
It is dirty) or 251 ± 3nm(pig spleen) wavelength at have absorption maximum.
[inspection] pH value takes this product, adds suitable quantity of water that the solution in every 1ml containing 1mg is made, and checks (Chinese Pharmacopoeia in accordance with the law
Two VI H of annex of version in 2010), pH value should be 5.0-7.5.
Clarity this product of solution, adds suitable quantity of water that the solution in every 1ml containing 2mg is made, and solution should be clarified；It is such as aobvious mixed
It is turbid, it, must not be denseer compared with No. 1 turbidity standard (two Ⅸ B of annex of Chinese Pharmacopoeia version in 2010).
Protein takes this product 4mg, adds 2ml water to dissolve, adds 20 %(g/ml) sulfosalisylic acid solution 1ml, it mixes, solution
It should clarify, muddy or precipitating must not occur.
Moisture takes this product appropriate, measures according to aquametry (two annex of Chinese Pharmacopoeia version in 2010, VIII the first method of M),
Moisture content must not exceed 6.0%
Heavy metal content of beary metal must not cross 20ppm.
Bacterium escherichia coli and salmonella must not detect, and total bacteria must not cross 300CFU/g.
Determination of activity takes this product appropriate, takes off the measurement of E receptor method, the E rose of test sample pipe according to T cell activation measurement-
The difference for tying the E rosette percentage of percentage and control tube should must not be lower than 10.0%.
[assay]
Polypeptide takes this product 10mg, adds 2ml water that mixed solution is made, and as test solution, precision measures 1.0ml, shines
Forint phenol measuring method measures content of peptides.
Ribose
It is appropriate that the preparation precision of reference substance solution weighs D-ribose reference substance, with 5%(g/ml, similarly hereinafter) trichloroacetic acid solution
The solution in every 1ml containing 20ug is dissolved and be made, is shaken up.
The preparation of test solution takes this product appropriate, is dissolved and is made in every 1ml containing ribose with 5% trichloroacetic acid solution
The solution of 5ug, as test solution.
The preparation precision of standard curve measures reference substance solution 0.0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml
It sets in tool plug test tube respectively, each 5% trichloroacetic acid solution to be added in right amount to 2.0ml, each to be added 1% 3,5- dihydroxymethyl benzole soln
[3,5- xylenediol 1g is taken, 0.1% ferric trichloride-hydrochloric acid solution is added (to take ferric trichloride 0.5g, enriching dissolving with hydrochloric acid makes into
500ml can be used for a long time) make to dissolve in 100ml, face with newly matching] 2.0ml, it shakes up, sets and accurately heat 30min in boiling water bath, it is fast
Speed is cooled to room temperature, using No. 0 pipe as blank, according to spectrophotometry (two annex IV A of Chinese Pharmacopoeia version in 2010).In 650nm
Wavelength at measure absorbance, using concentration as abscissa, trap is that ordinate draws standard curve and carries out linear regression,
Related coefficient should be greater than 0.995.
Measuring method precision measures test solution 2.0ml, the preparation method of sighting target directrix curve, from " each addition 1% 3,5-
Dihydroxymethyl benzole soln 2.0ml " rises, and measures in accordance with the law.Ribose concentration is found out from regression equation.
Testing result such as table 1.
Diamond dust, TritonX-100 and the aqueous citric acid solution of reference examples 1-- Different adding amount extract TF transfer factor
The influence of rate
Difference from example 1 is that: the aqueous citric acid solution of adjustment diamond dust, TritonX-100 and concentration 75%
Additive amount it is as shown in table 2；Other steps and technological parameter are the same as embodiment 1.
The TF recovery rate results of comparison of the diamond dust of Different adding amount, TritonX-100 and aqueous citric acid solution is shown in Table 2,
Known to: embodiment 1, reference examples 1 2# ~ 5# be above reference examples 1 1# TF recovery rate, i.e., " addition diamond dust, TritonX-
100 and aqueous citric acid solution " be above " not adding the TF recovery rate of diamond dust, TritonX-100 and aqueous citric acid solution ", together
When embodiment 1, reference examples 1 2#, 3# TF recovery rate be higher than reference examples 1 4#, 5# TF recovery rate, show diamond dust,
TritonX-100, aqueous citric acid solution can be played within the scope of additive amount of the invention and preferably be acted synergistically, and promote cell membrane
It preferably cracks, improves the recovery rate of TF.
Finally, it should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although reference
Preferred embodiment describes the invention in detail, those skilled in the art should understand that, it can be to of the invention
Technical solution is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered
In scope of the presently claimed invention.
Claims (9)
1. a kind of preparation method of spleen source transfer factor, which is characterized in that steps are as follows:
S1, pre-treatment: the qualified animal spleen of screening quarantine, then impurity elimination, cleaning, disinfection, drained and standby；
S2, Minced Steak homogenate:
(a) rubbing processing Minced Steak: is carried out to the spleen after pre-treatment, it is desirable that the granularity of the spleen particulate matter after rubbing up to 0.5 ~
1.0cm；
(b) it is homogenized: into the spleen particulate matter after rubbing, the citric acid of diamond dust, TritonX-100 and concentration 70 ~ 80% is added
Aqueous solution is homogenized, and obtains Spleen homogenate, and every 100g spleen particulate matter adds the diamond dust of 3 ~ 5 g, 0.1 ~ 0.5 mL
The aqueous citric acid solution of TritonX-100 and 4 ~ 6 mL；Wherein, " % " concentration of aqueous citric acid solution refer to citric acid and water with
The mass/volume percent concentration of g/mL metering；
S3, acidification: it adjusts Spleen homogenate pH value to 3.0 ~ 3.5, obtains spleen homogenate acidifying solution；
S4, freezing, broken defrosting:
(a), acidifying solution is homogenized using liquid nitrogen frozen processing spleen, obtains spleen homogenate acidifying solution ice cube；
(b), spleen homogenate acidifying solution ice cube is smashed, then carries out defrosting operation again, thaw point is 30 ~ 37 DEG C, when operation
Between be 0.5 ~ 1h；
(c), defrosting spleen liquid is collected, then presses S4(a) ~ (b) repetition 1 ~ 2 time；
S5, centrifugal purification: centrifugal purification is carried out using self-control centrifugal purification system, which includes tubular type centrifugation
Machine, centrifugation liquid storage tank, power transmitting assembly I, power transmitting assembly II, power transmitting assembly III, grade one filter, second level mistake
Filter, tertiary filter and its extraneous subsystem；Power transmitting assembly I is connected in series by kinetic pump I and check-valves I；Power
Transmission assembly II is connected in series by kinetic pump II and check-valves II；Power transmitting assembly III is by kinetic pump III and check-valves III
It is connected in series；Grade one filter and secondary filter are conventional cartridge filter；Tertiary filter is self-control filter, structure
Including cylinder, doughnut tread assembly, pedestal；Cylinder body bottom and top are respectively equipped with inlet and liquid outlet, set outside cylinder
There is interlayer, the upper end of interlayer is equipped with interlayer outlet, lower end is equipped with interlayer import；Doughnut tread assembly is porous including being evenly equipped with
Package shell, several doughnuts, these doughnuts merge bunchy and are placed in inside package shell；Pedestal is cylindrical body, circle
Cylinder upper section be it is hollow be cylindrical in shape, middle and lower sections it is solid, the middle and lower sections of pedestal it is solid equipped with and doughnut quantity and its size
Compatible through-hole；Pedestal is located at below liquid outlet, is close to cylinder roof and is firmly connected with it；The upper end of doughnut is open
And be threaded through in the through-hole of pedestal, lower end closed is simultaneously fixedly connected with package shell bottom wall；The attached system, the external world of tertiary filter
System include kinetic pump IV, check-valves IV, constant temperature refrigeration water tank, interlayer outlet successively contact check-valves IV, constant temperature refrigeration water tank and
Kinetic pump IV to interlayer import formed a closed cycle system；Tube centrifuge is successively contacted by pipeline to be centrifuged liquid storage tank, moves
Power pumps I, check-valves I, grade one filter, kinetic pump II, check-valves II, secondary filter, kinetic pump III, check-valves III to three
The inlet of grade filter；Grade one filter and be respectively 4.5 ~ 5 μm, 0.2 ~ 0.22 μm with the filter core aperture of secondary filter；
The molecular cut off of doughnut is 5000 ~ 6000 dalton；
Steps by centrifugation are as follows:
(a) in the spleen homogenate thawing solution input tube centrifuge inner cavity first obtained S4, start the equipment and carry out centrifugation work
Make, centrifugal rotational speed is centrifuged 20 ~ 30min up to 4000 ~ 6000rpm/min；The centrifugal clear liquid that will acquire in time after centrifugation is transferred to
Temperature controlling range is 5 ~ 10 DEG C to centrifugation liquid storage tank, and in the tank；
(b) the cooling centrifugate that upper process obtains is crossed check-valves I by the road and is delivered in grade one filter by starting kinetic pump I,
0.2 ~ 0.6 Mpa of input pressure realizes first time coarse filtration；
(c) the by-pass filtration liquid obtained after grade one filter is crossed check-valves II by the road and is delivered to second level by starting kinetic pump II
In filter, 0.2 ~ 0.6 Mpa of input pressure realizes second of coarse filtration；
(d) start kinetic pump IV, by temperature obtained in constant temperature refrigeration water tank be 5 ~ 15 DEG C cold water by the road from interlayer into
Mouth enters in interlayer, and crosses check-valves IV by the road by interlayer outlet and be recycled in constant temperature refrigeration water tank；To cylinder lumen temperature
Degree restarts kinetic pump III and the secondary filtration liquid obtained after secondary filter is crossed check-valves by the road when reaching 5 ~ 15 DEG C
III is delivered in tertiary filter by inlet, 0.2 ~ 0.6 MPa of input pressure, and secondary filtration liquid is first from package shell
Porous to enter inside package shell, due to the crown_interception of doughnut, the impurity in secondary filtration liquid is blocked in doughnut
Outside, and the secondary filtration liquid containing transfer factor then passes through doughnut and enters pedestal from doughnut top opening, then from
Liquid outlet comes out to get the stoste containing transfer factor；
S6, dry finished product: by the stoste containing transfer factor obtained by S5 through vacuum freeze drying or spray drying to get finished product.
2. preparation method as described in claim 1, which is characterized in that the detailed process of S1 are as follows: the qualified animal of screening quarantine
Spleen, it is desirable that spleen is fresh, without extravasated blood, without lesion and without bad smell；Then it is removed under conditions of 100,000 grades of working environment
The fat and connective tissue of spleen, while guaranteeing spleen integrality；The watery blood for cleaning spleen exudation again, drains away the water；Finally will
Carry out disinfection processing in the cresols aqueous solution of spleen immersion concentration of volume percent 0.3 ~ 0.6%, and the time is 20 ~ 30min, is drained
It is spare.
3. preparation method as described in claim 1, which is characterized in that S2 is that the spleen for draining S1 disinfection carries out Minced Steak respectively
And homogenized, operating environment carry out in ten thousand grades of cleanliness of clean room, detailed process are as follows:
(a) Minced Steak: the spleen that disinfection is drained is packed into meat grinder machine cavity, and starting working power carries out spleen and rubs operation, work
Making rate is 100 ~ 150kg/h, spleen particulate matter one of the granularity of spleen particulate matter up to 0.5 ~ 1.0cm, after rubbing after rubbing
And it is temporarily stored in sterilized plastic containers stand-by；
(b) it is homogenized: the spleen particulate matter that upper step obtains being put into the cavity of the high-speed homogenization machine of chummery, then even to high speed
The aqueous citric acid solution of diamond dust, TritonX-100 and concentration 70 ~ 80%, every 100g spleen particulate matter are added in the cavity of pulp grinder
The aqueous citric acid solution of the diamond dust of 3 ~ 5 g, the TritonX-100 and 4 ~ 6 mL of 0.1 ~ 0.5 mL is added, working power is started
It carries out spleen and is homogenized operation, homogenate rotating speed of flail is 9000 ~ 15000rpm/min, and homogenized temperature control is at 32 DEG C hereinafter, the time
For 20 ~ 30min；Wherein, in the process " % " concentration of aqueous citric acid solution refer to quality that citric acid and water measures with g/mL/
Concentration of volume percent.
4. preparation method as described in claim 1, which is characterized in that the detailed process of S3 are as follows: by Spleen homogenate pH value tune
To 3.0 ~ 3.5, acidulant used in the step is the aqueous hydrochloric acid solution of concentration 15% and the aqueous citric acid solution of concentration 10%
The mixed liquor of 2:1 ~ 4:1 mixing by volume；Acidulant is added in Spleen homogenate using constant flow pump in acidification, while at the uniform velocity
Stirring, acidulant flow rate and stirring rate are respectively 10 ~ 20 mL/min, 600 ~ 800 rpm/min；Wherein, in the process
" % " concentration of aqueous hydrochloric acid solution refers to mass percent concentration, with " % " concentration of aqueous citric acid solution refer to citric acid and water with
The mass/volume percent concentration of g/mL metering.
5. preparation method as described in claim 1, which is characterized in that the detailed process of S4 are as follows:
(a), spleen homogenate acidifying solution is dispensed intracavitary to liquid nitrogen tank is gone in container again, handles 10 ~ 20min of time；
(b), the spleen formed after liquid nitrogen frozen homogenate acidifying solution ice cube is dosed to smash ice cube to diameter in ice crusher and is
The particle of 0.8 ~ 1.5cm；Then the pellet frozen object that will acquire again is dosed in the machine cavity of continuous-type microwave unfreezing machine and is solved
Freeze operation, thaw point is 30 ~ 37 DEG C, and the activity duration is 0.5 ~ 1h；
(c), defrosting spleen liquid is collected, then presses S4(a) ~ (b) repetition 1 ~ 2 time.
6. preparation method as described in claim 1, which is characterized in that S5 further includes that step (e) is instead cleaned: work knot to be filtered
Shu Shi, can carry out the reverse cleaning operation of the system, manual 180 ° of integrated regulation power transmitting assemblies I, power transmitting assembly II,
The direction of power transmitting assembly III, that is, original import becomes exporting, and reconnects corresponding pipeline, while starting above-mentioned kinetic pump
I, kinetic pump II, kinetic pump III enter clean purified water in cylinder through barrel liquid discharge mouth, then ejected wash water is finally from centrifugation
Liquid storage tank is discharged, and each filter pressure list index must not be higher than 0.3 MPa in operation, repeats 2 ~ 3 times.
7. preparation method as described in claim 1, it is characterised in that: centrifugation liquid storage tank and kinetic pump I between, check-valves I with
Between grade one filter, between grade one filter and kinetic pump II, between check-valves II and secondary filter, secondary filter with
Pipeline between kinetic pump III, between check-valves III and tertiary filter takes movable connection method.
8. preparation method as claimed in claim 7, it is characterised in that: the movable connection method is clamp connection.
9. preparation method as described in claim 1, it is characterised in that: tube centrifuge is GF- divergence type.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102397293A (en) * | 2010-09-19 | 2012-04-04 | 曹吉祥 | Spleen immune factor and its preparation method |
| CN103054902A (en) * | 2013-01-24 | 2013-04-24 | 重庆永健生物技术有限责任公司 | Method for producing transfer factor in scale |
| CN105030827A (en) * | 2015-07-14 | 2015-11-11 | 天津瑞普生物技术股份有限公司 | Transfer factor and application thereof |
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2016
- 2016-08-30 CN CN201610760863.8A patent/CN106377543B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102397293A (en) * | 2010-09-19 | 2012-04-04 | 曹吉祥 | Spleen immune factor and its preparation method |
| CN103054902A (en) * | 2013-01-24 | 2013-04-24 | 重庆永健生物技术有限责任公司 | Method for producing transfer factor in scale |
| CN105030827A (en) * | 2015-07-14 | 2015-11-11 | 天津瑞普生物技术股份有限公司 | Transfer factor and application thereof |
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