CN102776126B - Method for preparing live bacteria biomaterial by multi-stage vacuum drying - Google Patents
Method for preparing live bacteria biomaterial by multi-stage vacuum drying Download PDFInfo
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- CN102776126B CN102776126B CN201110125008.7A CN201110125008A CN102776126B CN 102776126 B CN102776126 B CN 102776126B CN 201110125008 A CN201110125008 A CN 201110125008A CN 102776126 B CN102776126 B CN 102776126B
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 3
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Abstract
The invention discloses a method for preparing a live bacteria biomaterial by multi-stage vacuum drying, comprising the following steps: step A, carrying out vacuum drying on a live bacterial suspension containing a protective solution with dynamic temperature control at -10-20 DEG C under the vacuum degree of lower than 100Pa to reduce the water content of the live bacterial suspension to lower than 12% to let the live bacterial suspension convert into a crystal lamellar solid dry product; step B, crushing the crystal lamellar solid dry product, carrying out vacuum drying for 2-4 h at 10-25 DEG C under the vacuum degree of 10Pa to reduce the water content of the solid dry product to less than 10%; and step C, grinding the solid dry product with the vacuum degree of less than 10% into powder, then carrying out vacuum drying for 4-6 h at 5-25 DEG C under the vacuum degree of less than 10Pa to reduce the water content of the solid dry product to less than 5% to obtain the live bacteria biomaterial. The method has the advantages of short period of the process, time saving and energy saving, and low loss of the biomaterial after vacuum drying.
Description
Technical field
The present invention relates to biomaterial preparing technical field, particularly relate to a kind of multistage segmentation vacuum-drying of bacterium living beings material preparation method that lives.
Background technology
Active biomaterial is as the bacterium living beings material of living, at medicine, in biological products, the field such as food has a wide range of applications, and the bacterium living beings material that will keep for a long time living is in existing state, must make it could realize in low moisture state, therefore different drying meanss is employed wherein, because most bacterium living beings material alive is to thermo-responsive, therefore vacuum-drying and freeze drying process are two technology the most often applying in the dry field of the bacterium living beings material of living conventionally.
Vacuum drying process is to remove one of the best approach of bacterium living beings moisture in materials of living, the feature that the method is compared other drying means is that drying process occurs under different vacuum degree condition, and under low temperature, can farthest keep the activity of bacterium living beings material alive all the time.
But, the existing bacterium living beings material vacuum drying process treatment time alive is longer, be difficult to reach the processing requirement that had both kept the higher survival number of bacterium alive and can remove most of moisture, and existing preparation technology cannot realize large-scale continous way production, preparation method's cost is very high, cannot realize the vacuum-drying preparation of extensive biomaterial, poor practicability.
Summary of the invention
The object of the present invention is to provide a kind of multistage segmentation vacuum-drying of bacterium living beings material preparation method that lives, its process cycle is brief, energy-conservation when joint, and the loss of vacuum-drying artifact material number is little.
The multistage segmentation vacuum-drying of the one bacterium living beings material alive preparation method who provides for realizing the object of the invention, comprises the steps:
A kind of multistage segmentation vacuum-drying of bacterium living beings material preparation method that lives, comprising:
Steps A, the bacterial suspension alive of protection liquid will be added with, the temperature of-10 DEG C to 20 DEG C, under 100Pa vacuum tightness, carry out the vacuum-drying under dynamic temperature control, the moisture of the bacterial suspension that lives was reduced to below 12% in 4-6 hour, and the bacterial suspension that lives is changed the solid-state dry thing of solid-state crystal microchip shape into;
Step B, crushes the solid-state dry thing of described crystal microchip shape, and at 10-25 DEG C, vacuum tightness is less than under 10Pa, and vacuum-drying 2-4 hour is down to solid-state dry thing moisture below 10%;
Step C, the solid-state dry thing that moisture is down to below 10% is ground to powder, and then at 5-25 DEG C, vacuum tightness is less than under 10Pa, carries out the vacuum-drying of 4-6 hour, and the moisture in solid-state dry thing is down to below 5%, prepares bacterium living beings material alive.
More preferably, described bacterium is vibrio cholerae or Shigellae.
More preferably, described steps A comprises the steps:
Steps A 1, to the centrifugal results of bacterium living beings material alive in stable early stage, is mixed with bacterial suspension with vacuum-drying protection liquid by fermentation culture, and described bacterial suspension is evenly tiled;
Steps A 2, the temperature of-10 DEG C to 20 DEG C, under 100Pa vacuum tightness, make bacterial suspension alive vacuum-drying 4-6 hour under dynamic temperature control condition of described tiling, make the moisture of described bacterial suspension alive be evaporated to moisture content and be less than 12%, results obtain the solid-state dry thing of solid-state crystal microchip shape.
More preferably, the composition of described vacuum-drying protection liquid is: 20%-30% trehalose, 2-8% sucrose, 0.1-0.3% polyoxyethylene glycol, 0.05%-0.1% sodium-chlor, 0.01%-0.03% potassium primary phosphate, 0.05%-0.1% Sodium phosphate dibasic;
Described by after the fermentation culture centrifugal results of bacterium living beings material alive in extremely stable early stage, be mixed with bacterial suspension with vacuum-drying protection liquid, specifically comprise the steps:
After above-mentioned substance is weighed in proportion, add in water for injection, adjust pH to 7.0-8.0, then protect liquid with the filter sterile filtration in 0.22um aperture to obtaining vacuum-drying in sterilized container, temperature be at 4 DEG C, preserve stand-by;
Then the bacterium living beings material alive to stable early stage by fermentation culture, pours into after centrifugal results in described vacuum-drying protection liquid, uses overhead stirrer to be stirred to and forms the evenly described bacterial suspension without blocky-shaped particle.
More preferably, in described steps A 1, described bacterial suspension is evenly tiled, comprise the steps:
The fresh feed pump rotating speed of setting Vacuumdrier is that the crawler belt speed of advance of 5rpm, Vacuumdrier is 0.01-0.02m/s, under normal pressure, the crawler belt temperature of Vacuumdrier remains on 4 DEG C, feed time is 5-60min, described bacterial suspension alive is pumped on the crawler belt of Vacuumdrier, make described bacterial suspension alive on crawler belt, evenly tile one deck to 1-2mm.
More preferably, in described steps A 2, described dynamic temperature control condition is as shown in the table:
| Operation stage | Temperature arranges | Time |
| Standby period | -10 DEG C to-5 DEG C | 5-60min |
| In earlier stage | -5 DEG C to 5 DEG C | 30-60min |
| Mid-term | 5 DEG C to 10 DEG C | 30-60min |
| Later stage | 10 DEG C to 20 DEG C | 120min |
More preferably, described step B comprises the steps:
Step B1, by dry dried crystal microchip shape in steps A thing, at normal pressure, temperature 4-15 DEG C, crushes tiling;
Step B2, by dry the crystal microchip shape of tiling thing, controls temperature at 10-25 DEG C, and vacuum tightness is less than under 10Pa, carries out vacuum-drying in 2-4 hour, and the moisture in described dry thing is down to below 10%.
More preferably, in described step B1, dry crystal microchip shape thing is crushed to tiling, comprise the steps:
Use that mortar is manual crushes or use Lowtemperaturepulverizer that dry described crystal microchip shape thing is crushed to material particular diameter and is less than 0.5cm, be then poured onto pallet middle berth even, thickness is 0.3-0.5cm.
More preferably, described step C comprises the steps:
Step C1, after the moisture that completes solid-state dry thing in step B vacuum-drying is down to below 10%, by solid-state dry thing, at normal pressure, temperature 4-15 DEG C, with mortar carry out hand lapping or with shredder grind be screened to fineness more than 60 orders, obtain pulverous solid-state dry thing;
Step C2, by pulverous solid-state dry thing, controls temperature at 5-25 DEG C, and vacuum tightness is less than under 10Pa, carries out vacuum-drying in 4-18 hour, and the moisture of pulverous vacuum-drying thing is reduced to and is less than 5%, prepares the bacterium living beings material alive after vacuum-drying.
More preferably, described step C2 also comprises the steps:
If desired the moisture of controlling final active biomaterial is less than below 3%, controls temperature at 5-15 degree, and vacuum tightness is less than 10Pa, and pulverous vacuum-drying thing is carried out to vacuum-drying in 6-18 hour.
The invention has the beneficial effects as follows: the multistage segmentation vacuum-drying of bacterium living beings material alive of the present invention preparation method, can, at short notice by the moisture removal of the bacterial suspension that lives, reach the moisture below 5%; Can keep the viable count of bacterium in higher level, in other words, in the vacuum-drying preparation method of the embodiment of the present invention, viable bacteria death is less; Can realize large-scale continous way and produce, cost is low.
Brief description of the drawings
Fig. 1 is the multistage segmentation vacuum-drying of embodiment of the present invention bacterium living beings material alive preparation method schematic diagram.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the multistage segmentation vacuum-drying of bacterium living beings material preparation method alive of the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
As shown in Figure 1, as a kind of embodiment, the multistage segmentation vacuum-drying of the biomaterial of embodiment of the present invention preparation method, comprising:
Step S100, the bacterial suspension alive of protection liquid will be added with, the temperature of-10 DEG C to 20 DEG C, under 100Pa vacuum tightness, carry out the vacuum-drying under dynamic temperature control, the moisture of the bacterial suspension that lives was reduced to below 12% in (h) at 4-6 hour, makes bacterial suspension alive change the solid-state dry thing of solid-state crystal microchip shape into;
Step S200, crushes the solid-state dry thing of described crystal microchip shape, and at 10-25 DEG C, vacuum tightness is less than under 10Pa, and vacuum-drying 2-4 hour is down to solid-state dry thing moisture below 10%;
Step S300, processes by step S200 the solid-state dry thing obtaining and is ground to powder, and then at 5-25 DEG C, vacuum tightness is less than under 10Pa, carries out the vacuum-drying of 4-8 hour, and the moisture in solid-state dry thing is down to below 5-3%, prepares active biomaterial.
The multistage segmentation vacuum-drying of the biomaterial preparation method of the embodiment of the present invention, can be at short notice by the moisture removal of the bacterial suspension that lives, and can keep the viable count of bacterium in higher level, and it can realize large-scale continous way and produce, and cost is low.
Below to utilize Vacuumdrier, implement the multistage segmentation vacuum-drying of the biomaterial preparation method's of the embodiment of the present invention detailed process, Vacuumdrier in the embodiment of the present invention, it can be Vacuumdrier of the prior art, also can be that the present patent application people applied in 2009 and the patent No. of authorizing is ZL200920352269.0, the Vacuumdrier that denomination of invention is vacuum low-temperature dryer.
In the embodiment of the present invention, quote that the present patent application people applied in 2009 and the patent No. of authorizing is ZL200920352269.0, denomination of invention be the Vacuumdrier of vacuum low-temperature dryer as a kind of embodiment, and the technical scheme that quotes in full disclosed Vacuumdrier in this ZL200920352269.0 is as Vacuumdrier in the embodiment of the present invention.
Describe step S100 below in detail the bacterial suspension alive that is added with protection liquid is carried out to the vacuum-drying under dynamic temperature control, the moisture of the bacterial suspension that lives was reduced to below 12% in (h) at 4-6 hour, and bacterial suspension is changed the detailed process of the solid-state dry thing of solid-state crystal microchip shape into;
Particularly, as preferably embodiment of one, described step S100 comprises the steps:
Step S110; by after the fermentation culture centrifugal results of bacterium living beings material alive in extremely stable early stage; be mixed with bacterial suspension with vacuum-drying protection liquid; and described bacterial suspension pH value is adjusted to after 7.0-8.0; described bacterial suspension is pumped on the crawler belt of Vacuumdrier, make described bacterial suspension even tiling one deck on crawler belt.
As a kind of embodiment; composition (the weight percent of described vacuum-drying protection liquid; total content≤100%) be: 20%-30% trehalose; 2-8% sucrose; 0.1-0.3% polyoxyethylene glycol, 0.05%-0.1% sodium-chlor, 0.01%-0.03% potassium primary phosphate; 0.05%-0.1% Sodium phosphate dibasic, other are water for injection.
As a kind of embodiment, described by after the fermentation culture centrifugal results of bacterium living beings material alive in extremely stable early stage, be mixed with bacterial suspension with vacuum-drying protection liquid, specifically comprise the steps:
After above-mentioned substance is weighed in proportion, add in water for injection, adjust pH to 7.0-8.0, then protect liquid with the filter sterile filtration in 0.22um aperture to obtaining vacuum-drying in sterilized container, temperature be at 4 DEG C, preserve stand-by; Then the bacterium living beings material alive to the logarithmic phase later stage by fermentation culture, pours into after centrifugal results in described vacuum-drying protection liquid, uses overhead stirrer to be stirred to and forms the evenly described bacterial suspension without blocky-shaped particle.
The extremely stable bacterium living beings material alive in earlier stage of preparation fermentation culture, and are a kind of prior aries by its centrifugal results, therefore, in embodiments of the present invention, be no longer described in detail.
In the embodiment of the present invention, preferably, as a kind of embodiment, described bacterial suspension alive is pumped on the crawler belt of Vacuumdrier, make described bacterial suspension alive even tiling one deck on crawler belt, specifically comprise the steps:
The fresh feed pump rotating speed of setting Vacuumdrier is that the crawler belt speed of advance of 5rpm, Vacuumdrier is 0.01-0.02m/s, under normal pressure (being standard atmospheric pressure), the crawler belt temperature of Vacuumdrier remains on 4 DEG C, feed time is 5-60min, described bacterial suspension alive is pumped on the crawler belt of Vacuumdrier, evenly tiling one deck is to 1-2mm on crawler belt to make described bacterial suspension alive, and the thickness that is laid in the bacterial suspension alive on crawler belt is 1-2mm.
In the embodiment of the present invention, fresh feed pump rotating speed 5rpm, Vacuumdrier crawler belt speed of advance at Vacuumdrier are under 0.01-0.02m/s, the temperature of Vacuumdrier remains on 4 DEG C, feed time is 5-60min, can realize Vacuumdrier leading portion and be continuously pumped into bacterial suspension alive, the continuous drying pattern of the solid-state dry thing of the follow-up crystal microchip shape harvesting drying continuously, this pattern is produced and is significant for extension.
Step S120, the temperature of-10 DEG C to 20 DEG C, under 100Pa vacuum tightness, vacuum-drying 4-6 hour (h) (240-360min) under dynamic temperature control condition to make bacterial suspension described alive on crawler belt, make the moisture of the described bacterial suspension alive on crawler belt be evaporated to moisture content and be less than 12%, results obtain the solid-state dry thing of solid-state crystal microchip shape.
Described dynamic temperature control condition is as shown in table (1).
The bacterial suspension vacuum-drying control table alive of table (1) dynamic temperature control condition
The bacterial suspension that lives is laid in after the crawler belt of Vacuumdrier, live under lower than the 100Pa vacuum tightness boiling point lowering of bacterial suspension, water molecules explosive evaporatoin heat absorption, the outstanding temperature of the bacterium that lives reduces rapidly, control as show the temperature setting of (1), make the bacterial suspension alive of tiling and the shelf chuck medium of crawler belt carry out heat exchange, and make temperature progressively increase, finally reach within the shortest time, and under the prerequisite of the loss of possible minimizing viable count, reach the bacterial suspension preliminarily dried of living, obtain the object of the solid-state dry thing of dry crystal microchip shape.
Experiment shows, carries out the preliminarily dried of step S100 with the method for the embodiment of the present invention, and the loss of viable count is about 0.5-1log left and right, lower than loss number of the prior art.
Describe step S200 below in detail the solid-state dry thing of described crystal microchip shape is crushed, at 10-25 DEG C, vacuum tightness is less than under 10Pa, and vacuum-drying 2-4 hour is down to solid-state vacuum-drying thing moisture the detailed process of 10% step;
First, by the solid-state dry thing of dried crystal microchip shape in step S100, at normal pressure, temperature 4-15 DEG C, crush and tile in pallet, tiling thickness is 0.3-0.5cm;
In the embodiment of the present invention, as a kind of embodiment, use that mortar is manual crushes or use Lowtemperaturepulverizer that dry described crystal microchip shape thing (solid-state dry thing) is crushed to material particular diameter and is less than 0.5cm, be then poured onto pallet middle berth even, thickness is 0.3-0.5cm.
Then, pallet and the solid-state dry thing of crystal microchip shape are wherein placed in Vacuumdrier, control temperature at 10-25 DEG C, vacuum tightness is less than under 10Pa, carry out (h) (120-240min) vacuum-drying in 2-4 hour, the moisture in solid-state dry thing is down to below 10%.
Because the dry thing moisture of dried crystal microchip shape in step S100 is higher, directly grinding can cause viable count to decline, the embodiment of the present invention is in step S200, slight crushing reaches the dry thing surface-area of increase crystal microchip shape, then should be not higher than carrying out vacuum-drying under the maximum growth temperature that can tolerate the bacterium of taking on service jobs to the drying temperature of solid-state dry thing, in the embodiment of the present invention, control temperature at 10-25 DEG C, vacuum tightness is less than under 10Pa, carry out vacuum-drying, moisture is down to below 10%, the level that it can be ground and do not lose viable bacteria, reduce the object of the bacterium loss alive in process of lapping.
Experiment shows, with the method for the embodiment of the present invention carry out the crushing of step S200 dry after, the loss of viable count is about 0.5-1log, lower than loss number of the prior art.
Describe step S300 below in detail the vacuum-drying thing after crushing is ground to powder, then at 10-25 DEG C, vacuum tightness is less than under 10Pa, then carries out vacuum-drying in 4-18 hour, prepares the detailed process of the step of the active biomaterial after vacuum-drying.
First, complete in step S200 vacuum-drying, after the moisture of solid-state dry thing is down to below 10%, in Vacuumdrier, take out the solid-state dry thing crushing, at normal pressure, temperature 4-15 DEG C, with mortar carry out hand lapping or with shredder grind be screened to fineness more than 60 orders, make its complete powdered, obtain pulverous solid-state dry thing;
Then, be 0.3-0.5cm by pulverous solid-state dry thing thickness in pallet that tiles again, pallet is placed in Vacuumdrier, control temperature at 5-25 DEG C, vacuum tightness is less than under 10Pa, carry out (h) vacuum-drying in 4-18 hour, the moisture of pulverous vacuum-drying thing is reduced to and is less than 5%, prepare the bacterium living beings material alive after vacuum-drying.
More preferably, the moisture of if desired controlling final active biomaterial is less than below 3%, controls temperature at 5-15 degree, and vacuum tightness is less than 10Pa, and pulverous vacuum-drying thing is carried out to vacuum-drying in 6-18 hour.
After step S200 vacuum-drying completes, the moisture of bacterial suspension alive is down to below 10%, process of lapping is not too obvious for the impact of viable count, by grinding the complete efflorescence of vacuum-drying thing after crushing, to increase the desiccated surface area of vacuum-drying biomaterial as far as possible, vacuum-drying thing efflorescence order number is larger, granularity is less, the dried moisture of pulverous vacuum-drying thing will be lower, in the embodiment of the present invention, should be not higher than the maximum growth temperature that can tolerate the bacterium of taking on service jobs to the drying temperature of solid-state dry thing, carry out vacuum-drying, in the embodiment of the present invention, control temperature at 5-25 DEG C, vacuum tightness is less than under 10Pa, after vacuum-drying in 4-8 hour in step S300, moisture in vacuum-drying biomaterial is reduced to and is less than 5-3%.Reach biomaterial vacuum-drying and keep its active standard.
Step S200 and step S300 to the processing of solid-state dry thing as table (2) as shown in.
The later stage process of vacuum drying table of the solid-state dry thing of table (2) biomaterial
Experiment shows, carries out after the mill-drying of step S300 with the method for the embodiment of the present invention, and the loss of viable count is about 0.5-1log, lower than loss number of the prior art.
Preferably, biomaterial of the present invention bacterium alive, includes but not limited to vibrio cholerae (Vibrio cholerae), Shigellae (Shigella) etc.
Experiment shows, in the embodiment of the present invention, lives bacterial suspension after step S100-S300 vacuum-drying, and the total losses of the bacterial count of living is about 1.5-2log, lower than loss number of the prior art.
The multistage segmentation vacuum-drying of the biomaterial preparation method of the embodiment of the present invention, its process cycle is brief, energy-conservation when joint, and after vacuum-drying, viable count loss is little; On the other hand, industrialization less investment, cost is low, and apparatus for production line chain is brief, and floor space is little, can realize large-scale continous way and produce.
Using recombinant attenuated vibrio cholerae Peru-15 as sample object, (the recombinant attenuated vibrio cholerae Peru-15 of described sample object preparation method is shown in " Development of Peru-15 (CholeraGarde), a live-attenuated oral cholera vaccine:1991-2009. " Expert Rev Vaccines.2009Dec below; 8 (12): 1643-52.Chowdhury MI, Sheikh A, Qadri F), the multistage segmentation vacuum-drying of the biomaterial preparation method of the experiment embodiment of the present invention, with experimental data explanation preparation method's of the present invention beneficial effect.And, due to the property class of Shigellae (Shigella) and vibrio cholerae (Vibrio cholerae) seemingly, its experimental data is also similar, therefore will not enumerate in embodiments of the present invention the experimental data about Shigellae (Shigella).
1) drying temperature:
A) preparation method of constant drying temperature and the embodiment of the present invention dynamically controls the comparison of drying temperature for biomaterial impact:
Experimental result shows, carries out vacuum-drying remaining under constant temp condition, and the survival rate of sample after being dried will significantly decline.And early stage, sample carried out the evaporation of large quantity of moisture at low temperatures, later stage preparation method's technique of carrying out the embodiment of the present invention of the dry dynamic drying temperature of temperature programming obviously can reach and make bacterium reach very high survival rate.
Experimental result data is as shown in table (3):
Table (3) constant drying temperature and the dynamic drying temperature comparison sheet of controlling
Wherein: the mensuration of * viable count adopts the method for dilution spread flat board to carry out
When sample drying temperature is selected, the vibrio cholerae that this experiment adopts is temperature sensitivity bacterium, and the temperature of selecting after the groping of dry top temperature is the highest 20 to spend.When drying temperature is greater than 20 while spending, although that the moisture of sample can be controlled is better, the viable count survival rate of sample is not high.
Control time of drying:
1) control of step S100 time of drying:
Early stage is-5 DEG C to 5 DEG C left and right low temperature solid stages, within sample should remain on the short period (30-60min), in order to avoid in the time of solid, sample outside surface, owing to forming fast solid-state epitaxial, stops the lasting evaporation of epitaxial inner sample moisture.Through experiment, this stage dry sample treatment rate is that about 1L per hour is advisable.
The temperature-rise period in dry mid-term, in a large amount of evaporations of moisture, sample temperature slowly improves (5-10 DEG C), this stage temperature rise rate should be suitable, be controlled between 0.08-0.2 DEG C/min, being rapidly heated easily causes living that bacterium is too fast because outside temperature changes causes that thereby bacterial film structural damage etc. causes viable count significantly to reduce, and the slow moisture vaporator rate that can affect sample heated up.
Latter temperature is controlled between 10-20 DEG C.
2) control of step S200 time of drying:
The control of step S200 time of drying, depend on dry after sample classification and moisture requirement, the type of the different dry bacteriums that live and moisture requirement have the difference of adaptation.The vibrio cholerae sample of this experiment is sample water being divided and is controlled at below 9% after super-dry 2-4 hour, and under this moisture content, the abrasive flour that carries out vibrio cholerae sample does not have large loss for the activity of sample.
3) control of step S300 time of drying:
Step S200 dry sample is after mesh-of-grind reaches the even higher order number of 60 orders, and sample outer surface area is enough large, and under the vacuum tightness of 10Pa, after vacuum-drying in 4-6 hour, moisture reduction will reach below 5%.If desired it is 3% even lower that the moisture of controlling final sample is less than, drying temperature in step S300 need to be controlled to 5-15 degree left and right, by extend time of drying to the mode of 6-18 hour realize, extend the concrete time per sample required minimum moisture content standard and determine.Three dry sample actual temperatures of vibrio cholerae sample are in 15 about degree, and after extending time of drying and being 18 hours, sample moisture will be lower than 3%.
Below with the multistage segmentation vacuum-drying of the biomaterial preparation method's of one group of vacuum-drying associated sample data declaration embodiment of the present invention beneficial effect:
After vacuum-drying, sample experimental data is as shown in table (4):
Experimental data table after table (4) sample vacuum-drying
Wherein, 20110203 batches and 20110305 batches of vacuum-dryings in table (4); laboratory sample is vibrio cholerae Peru-15 bacterial suspension alive; sample by vibrio cholerae Peru-15 after the centrifugal results of fermenting; be mixed with dry-run protection liquid according to dry liquid proportional, and regulate the pH value of dry-run protection liquid to the scope of 7.0-8.0.
In 20110203 batches, take 4+,3+4 tri-step drying modes, i.e. step S100 vacuum-drying 4 hours; Step S200 vacuum-drying 3 hours; Step S300 vacuum-drying 4 hours, amounts to the time of drying of 11 hours.Wherein after step S100 vacuum-drying, moisture is down to 10.17%; After being crushed to particle, after step S200 vacuum-drying, moisture is down to 8.37%; After grinding efflorescence, after step S300 vacuum-drying, moisture is down to 4.50%, finally obtains survival rate and is greater than 10 again
8and the biomaterial that moisture is less than 5%.
In 20110305 batches, the three step drying modes of 4+4+4 are taked, i.e. step S100 vacuum-drying 4 hours; Step S200 vacuum-drying 4 hours; Step S300 vacuum-drying 4 hours, amounts to the time of drying of 12 hours.Wherein after step S100 vacuum-drying, moisture is down to 11.80%; After crushing macrobead, after step S200 vacuum-drying, moisture is down to 10.67%; After grinding efflorescence, after step S300 vacuum-drying, moisture is down to 4.80%, finally obtains survival rate and is greater than 10
8and the sample that moisture is less than 5%.In 20110305 batches of this batch of samples, the method for also having attempted extending time of drying is in the hope of obtaining more low-moisture sample, and by three dry extending to after 18 hours, obtaining moisture is 3.0%, and survival rate is 8.49*10
7the sample of CFU/ml, experiment shows, obtain more low moisture sample, need to consider that the decline of survival rate is whether within acceptable scope.
Show by above-mentioned experiment, in equal drying area situation, the multistage segmentation vacuum-drying preparation method of the embodiment of the present invention reduces 30%-50% left and right than the investment of freeze-drying process, effectively reduce cost, and use the vacuum drying method of the embodiment of the present invention to complete time that the Peru-15 of same homalographic is dry only for lyophilization method is wanted 1/7th of spended time, improve efficiency, optimized the dry flow process of viable bacteria, improved the quality of viable bacteria drying material.
Finally it should be noted that obviously, those skilled in the art can carry out various changes and modification and not depart from the spirit and scope of the present invention the present invention.Like this, if these amendments of the present invention and within modification belongs to the scope of the claims in the present invention and equivalent technologies thereof, the present invention is also intended to comprise these changes and modification.
Claims (5)
1. the multistage segmentation vacuum-drying of the bacterium living beings material preparation method that lives, is characterized in that, comprising:
Steps A 1, to the centrifugal results of bacterium living beings material alive in stable early stage, survives bacterial suspension with the preparation of vacuum-drying protection liquid by fermentation culture, and described bacterial suspension alive is evenly tiled;
Steps A 2, the temperature of-10 DEG C to 20 DEG C, under 100Pa vacuum tightness, make bacterial suspension alive vacuum-drying 4-6 hour under dynamic temperature control condition of described tiling, make the moisture of described bacterial suspension alive be evaporated to moisture content and be less than 12%, results obtain the solid-state dry thing of solid-state crystal microchip shape;
Step B1, by dry dried crystal microchip shape in steps A 2 thing, at normal pressure, temperature 4-15 DEG C, crushes tiling;
Step B2, by dry the crystal microchip shape of tiling thing, controls temperature at 10-25 DEG C, and vacuum tightness is less than under 10Pa, carries out vacuum-drying in 2-4 hour, and the moisture in described dry thing is down to below 10%;
Step C1, after the moisture that completes solid-state dry thing in step B2 vacuum-drying is down to below 10%, by solid-state dry thing, at normal pressure, temperature 4-15 DEG C, with mortar carry out hand lapping or with shredder grind be screened to fineness more than 60 orders, obtain pulverous solid-state dry thing;
Step C2, by pulverous solid-state dry thing, controls temperature at 5-25 DEG C, and vacuum tightness is less than under 10Pa, carries out vacuum-drying in 4-18 hour, and the moisture of pulverous vacuum-drying thing is reduced to and is less than 5%, prepares the bacterium living beings material alive after vacuum-drying;
The composition of described vacuum-drying protection liquid is: 20%-30% trehalose, 2-8% sucrose, 0.1-0.3% polyoxyethylene glycol, 0.05%-0.1% sodium-chlor, 0.01%-0.03% potassium primary phosphate, 0.05%-0.1% Sodium phosphate dibasic;
Bacterium in described bacterium living beings material alive is vibrio cholerae or Shigellae;
In described steps A 2, described dynamic temperature control condition is as shown in the table:
2. the multistage segmentation vacuum-drying of bacterium living beings material alive according to claim 1 preparation method, is characterized in that,
Described by after the fermentation culture centrifugal results of bacterium living beings material alive in extremely stable early stage, survive bacterial suspension with the preparation of vacuum-drying protection liquid, specifically comprise the steps:
After vacuum-drying being protected above-mentioned substance in the composition of liquid weigh in proportion, add in water for injection, adjust pH to 7.0-8.0, then protect liquid with the filter sterile filtration in 0.22um aperture to obtaining vacuum-drying in sterilized container, temperature be at 4 DEG C, preserve stand-by;
Then the bacterium living beings material alive to stable early stage by fermentation culture, pours into after centrifugal results in described vacuum-drying protection liquid, uses overhead stirrer to be stirred to and forms the evenly bacterial suspension described alive without blocky-shaped particle.
3. the multistage segmentation vacuum-drying of bacterium living beings material alive according to claim 1 preparation method, is characterized in that, in described steps A 1, described bacterial suspension alive is evenly tiled, and comprises the steps:
The fresh feed pump rotating speed of setting Vacuumdrier is that the crawler belt speed of advance of 5rpm, Vacuumdrier is 0.01-0.02m/s, under normal pressure, the crawler belt temperature of Vacuumdrier remains on 4 DEG C, feed time is 5-60min, described bacterial suspension alive is pumped on the crawler belt of Vacuumdrier, make described bacterial suspension alive on crawler belt, evenly tile one deck to 1-2mm.
4. the multistage segmentation vacuum-drying of bacterium living beings material alive according to claim 1 preparation method, is characterized in that, in described step B1, dry crystal microchip shape thing is crushed to tiling, comprises the steps:
Use that mortar is manual crushes or use Lowtemperaturepulverizer that dry described crystal microchip shape thing is crushed to material particular diameter and is less than 0.5cm, be then poured onto pallet middle berth even, thickness is 0.3-0.5cm.
5. the multistage segmentation vacuum-drying of bacterium living beings material alive according to claim 1 preparation method, is characterized in that, described step C2 also comprises the steps:
If desired control final moisture of living bacterium living beings material and be less than 3%, control temperature at 5-15 DEG C, vacuum tightness is less than 10Pa, and pulverous vacuum-drying thing is carried out to vacuum-drying in 6-18 hour.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN86207190U (en) * | 1986-09-23 | 1987-07-22 | 中国人民解放军海军总医院 | Multiple cold-source vacuum freezing and drying unit |
| CN201628450U (en) * | 2009-12-31 | 2010-11-10 | 海南维瑅瑷生物工程技术有限公司 | Vacuum low-temperature dryer |
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| CN86207190U (en) * | 1986-09-23 | 1987-07-22 | 中国人民解放军海军总医院 | Multiple cold-source vacuum freezing and drying unit |
| CN201628450U (en) * | 2009-12-31 | 2010-11-10 | 海南维瑅瑷生物工程技术有限公司 | Vacuum low-temperature dryer |
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