CN103468573B - Vacuum freeze-drying protective agent for Edwardsiella tarda and freeze-drying method thereof - Google Patents
Vacuum freeze-drying protective agent for Edwardsiella tarda and freeze-drying method thereof Download PDFInfo
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Abstract
The invention provides a vacuum freeze-drying protective agent for Edwardsiella tarda and a freeze-drying method thereof, and belongs to the technical field of vacuum freeze-drying preservation of marine bacteria. The vacuum freeze-drying protective agent comprises the following components: dried skim milk, lactose, mycose, vitamin C, NaCl and sterile water. The viable bacterium suspension of the Edwardsiella tarda is sufficiently and evenly mixed with the freeze-drying protective agent in the volume ratio of 1: 4 to 1: 1 under a sterile condition; the mixed solution is subpackaged, and quickly frozen for 5-6 hours under the condition of -80 DEG C and then freeze-dried for 18 hours under the conditions of the vacuum degree of 0.28 mbar and the freeze-drying chamber temperature of -54 DEG C; next, the degree of vacuum is reduced to 0.034 mbar, and the subpackaged solutions are continuously freeze-dried for 2 hours at the temperature of -54 DEG C. After the completion of freeze-drying, 99.99% dry carbon dioxide gas is introduced into the freeze-drying chamber to achieve the standard atmospheric pressure and then the freeze-drying chamber is sealed with a gland. According to the invention, a technical method for long-term vacuum freeze-drying preservation of the Edwardsiella tarda is established, and technical support is provided for carrying out the related research of the Edwardsiella tarda in a deep-going way.
Description
Technical field
The invention belongs to marine bacteria vacuum freezedrying Techniques of preserving field, relate to a kind of the vacuum lyophilization protective material and the freeze drying process thereof that are applicable to Edwardsiella tarda particularly.
Background technology
Edwardsiella tarda (Edwardsiella tarda), have another name called Edwardsiella tarda, worldwide be distributed widely in sea, fresh water environment, it is hydrobiological important pathogenic bacteria, the disease of the multiple aquatic products economic animal such as fish, batrachians can be caused, serious threat (Zheng great Hai etc. 2004) is formed to breeding production.Therefore, research about Edwardsiella tarda becomes a focus in aquaculture Study on etiology field gradually, Chinese scholars has all carried out comparatively deep research (Matt J.Griffin to the classification of Edwardsiella tarda, pathogenesis, the aspect such as antigenic characteristic and vaccine, et al.2013, Jong Earn Yu, et al.2013, Meng Zhang, et al.2012, Seung Hyuk Choi, et al.2011).
Nearly ten years, along with the fast development of China's marine fish culture industry, the disease caused by Edwardsiella tarda is all found in different marine fish culture kinds.Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science has been separated to a strain predominant bacteria from the turbot spleen and kidney of trouble " spleen kidney tubercle disease ", through intensive etiology and pathological research, confirm that the pathogenic former of turbot " spleen kidney tubercle disease " is Edwardsiella tarda (aquatic product journal, 31st volume the 4th phase in 2007), and deeply carried out multiplex PCR detection technique and the fluorescence quantitative PCR detection technique research (Liu Zhichao of Edwardsiella tarda and other common seawater fish pathogenic bacterias, Chinese Marine University master Diplomarbeit, 2012).Meanwhile, a kind of multiple-effect Chinese herb compound (national inventing patent ZL201010100782.8) has been screened for the multiple turbot encountered pathogenic comprising Edwardsiella tarda.These study the aspects such as epidemiology, etiology, pathology and the Prevention Technique mainly concentrating on the morbidity of Edwardsiella tarda infection Isolated from Diseased Scophthalmus maximus, do not relate to the long-term Techniques of preserving research of Edwardsiella tarda.
Because to be fish comparatively common and the pathogenic bacteria that virulence is stronger for Edwardsiella tarda, one of pattern species of aquatic economic animal bacteriosis Study on etiology are acknowledged as.Have extensively studied after in the pathogenic characteristic and pathogenesis etc. of this bacterium, lot of domestic and international scholar's research thinks that developing corresponding vaccine is one of most effective measures of prevention Edwardsiella tarda disease, and how long term storage has the Edwardsiella tarda of biologic activity or its vaccine and becomes key technique wherein.Vacuum Freezing & Drying Technology is current long conserving microorganism bacterial strain and preserves the most effective technological method of its activity, is suitable for very much the permanent preservation of Edwardsiella tarda live strain or vaccine.But; because the biological characteristics, site conditions, nutritional needs, artificial culture method etc. of Edwardsiella tarda self have its specificity; the very difficult store method using for reference other microorganism strains, therefore finds suitable vacuum lyophilization protective material and freeze drying process realizes Edwardsiella tarda freeze-drying to preserve the technical barrier that must solve for a long time.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of lyophilized vaccine for vacuum freezedrying preservation Edwardsiella tarda bacterial strain and concrete freeze drying process; Edwardsiella tarda long term storage at normal temperatures and preservation vigor can be realized, for the correlative study carrying out Edwardsiella tarda in a deep going way provides technical support.
The present invention adopts following technical scheme to be achieved:
A vacuum lyophilization protective material for Edwardsiella tarda, its composition is: skimmed milk powder, lactose, trehalose, vitamins C, NaCl and sterile distilled water.
Further, the vacuum lyophilization protective material of described Edwardsiella tarda, its composition and final concentration thereof are: skimmed milk powder 50 ~ 150g/L; lactose 50 ~ 100g/L, trehalose 100g/L, vitamins C 5 ~ 20g/L; NaCl 15g/L, solvent is sterile distilled water.
A kind of described protectant compound method is: skimmed milk powder, lactose and trehalose are dissolved in aseptic NaCl solution, under 106 DEG C of conditions, moist heat sterilization 30min, is mixed with solution A after naturally cooling; Vitamins C is dissolved in aseptic NaCl solution, and is mixed with solution B after 0.22 μm of cellulose mixture membrane filtration; The solution A configured and solution B are aseptically mixed; and after shaken well, being mixed with frozen-dried protective agent solution, the final concentration of its each composition is skimmed milk powder 50 ~ 150g/L, lactose 50 ~ 150g/L, trehalose 100g/L, vitamins C 5 ~ 20g/L, NaCl 15g/L.
The present invention also provides a kind of freeze drying process of Edwardsiella tarda.
The present invention realizes by following technical scheme:
A freeze drying process for Edwardsiella tarda vacuum freezedrying preservation, concrete steps are that the aseptic 1.5%NaCl solution preparation of the Edwardsiella tarda of purified cultivation is become 10
7~ 10
9the viable bacteria suspension of cfu/mL, and after the ratio of viable bacteria suspension and above-mentioned frozen-dried protective agent solution 1:4 ~ 1:1 by volume is fully mixed, point to be filled in ampere bottle and quick freezing 5 ~ 6 hours under being placed in-80 DEG C of conditions, put into the vacuum freeze drier freeze-drying cabin of precooling again, at vacuum tightness 0.28mbar, under the condition of freeze-drying cabin temperature-54 DEG C, freeze-drying is after 18 hours, vacuum tightness is down to 0.034mbar, and continue freeze-drying 2 hours under maintaining the condition of freeze-drying cabin temperature-54 DEG C, treat freeze-drying complete in backward freeze-drying cabin be filled with 99.99% the dry carbon dioxide of high purity to standard atmospheric pressure and gland seal.Seal the Edwardsiella tarda bacterium powder that namely freeze-drying is housed in complete ampere bottle, preserve for a long time under normal temperature can be placed in.
The beneficial effect that the present invention is compared with the prior art:
1. optimize the formula of lyophilized vaccine, and with the addition of certain salinity wherein, make it be more applicable for the freeze-drying preservation of Edwardsiella tarda.
2. improve the freeze-drying program of vacuum freezedrying, be two stages of different vacuum tightness by freeze-drying programming, make freeze-drying more thorough, preservation effect is better.
3. in freeze-drying cabin, be filled with high-pureness carbon dioxide gas after freeze-drying and directly gland seal; because carbon dioxide gas volume density is larger; effectively completely cut off freeze-drying bacterial strain to contact with the direct of air, under the protection of rare gas element, made the preservation period of freeze-drying bacterial strain longer.
Embodiment
Below by embodiment, technical scheme of the present invention is described in detail:
Embodiment 1
From In Shandong Weihai turbot cultivation factory collect troubles " spleen kidney tubercle disease " turbot disease fish several, oxygenation of being packed is taken back Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science laboratory and is carried out pathogen separation.The spleen of a little sick fish of aseptic technique difference clip and renal tissue, put it in sterile petri dish to drip gently by the NaCl solution of 1.5% of sterilizing and wash for several times, shredded by tissue.With the tissue that transfering loop picking shreds on a small quantity, pancreas peptone soybean broth nutrient agar carries out line separation of bacterial.The complete substratum of line is placed in 28 DEG C of constant incubators and cultivates 48h, from the dominant colony that form is consistent, choose single bacterium colony, be again forwarded to a new pancreas peptone soybean broth nutrient agar carries out streak culture.Repeat above step 2 ~ 3 time, until obtain the pure culture bacterium colony of pathogenic bacteria.The pure growth of acquisition is made the viable bacteria suspension of high density, and the healthy turbot of abdominal injection is to verify that it is pathogenic.The biochemical phenotype experiment of customary physiological and 16S rDNA sequence alignment analysis are carried out to the pathogenic bacteria after checking, is accredited as Edwardsiella tarda (Edwardsiella tarda).
The Edwardsiella tarda list bacterium colony of picking pure culture is connected in pancreas peptone soybean broth liquid nutrient medium, be placed in biochemical cultivation case 28 DEG C, cultivate 20h under 150rpm condition after, supernatant is abandoned in the centrifugal 8min of 4000r/min, collect bacterial sediment, then the concentration adding thalline volume about 15 times is after the abundant shaken well of aseptic NaCl solution of 1.5%, again in the centrifugal 8min of 4000r/min, abandon supernatant, collect bacterial sediment, after repeating same step twice, be that the aseptic NaCl solution of 1.5% is mixed with 10 by resuspended for the thalline of collection by concentration
7~ 10
9the bacteria suspension of cfu/mL is for subsequent use.
First respectively 5g skimmed milk powder, 5g lactose and 10g trehalose being dissolved in 90mL concentration is in the NaCl solution of 1.5%, and under 106 DEG C of conditions, moist heat sterilization 30min, is mixed with solution A after naturally cooling.0.5g vitamins C being dissolved in 10mL concentration is in the aseptic NaCl solution of 1.5%, and is mixed with solution B after 0.22 μm of cellulose mixture membrane filtration.The solution A configured and solution B are aseptically mixed; and after shaken well, being mixed with the frozen-dried protective agent solution of 100mL, the final concentration of its each composition is skimmed milk powder 50g/L, lactose 50g/L, trehalose 100g/L, vitamins C 5g/L, NaCl 15g/L.
Aseptically; by the bacteria suspension that configured and protective material solution by volume 1:1 ratio mixing and after shaken well; dividing is filled in the aseptic ampere bottle of several 2mL; the mixed solution of 1.5mL is housed in each ampere of bottle, by after ampere bottle cap top cement plug in-80 DEG C of Ultralow Temperature Freezers freezing 5h.Then the mixing solutions frozen is put into rapidly the vacuum freeze drier freeze-drying cabin of precooling, freeze-drying 18 hours under vacuum tightness 0.28mbar, freeze-drying cabin temperature are the condition of-54 DEG C, afterwards vacuum tightness is reduced to 0.034mbar, maintains freeze-drying cabin temperature-54 DEG C and continue freeze-drying 2 hours.After freeze-drying program completes, the dry carbon dioxide of high purity being slowly filled with 99.99% in freeze-drying cabin to standard atmospheric pressure and the vacuum freezedrying that namely gland seal completes Edwardsiella tarda preserve.Preserve under ampere bottle after freeze-drying also seals is placed in normal temperature.
The same day after freeze-drying completes, 7th day and the 90th day, random choose 5 ampere bottles respectively, aseptically open and to seal and the sterile purified water adding 1.5mL carries out rehydration, gradient dilution is carried out by the aseptic NaCl solution that concentration is 1.5% after lyophilized powder dissolves completely, and carry out live bacterial count, to measure the survival rate of freeze-drying bacterial strain with the coating of pancreas peptone soybean broth Agar Plating.Each gradient carries out 2 parallel laboratory tests, gets its mean value, the results are shown in Table 1.
This results show, the Edwardsiella tarda freeze-dried vaccine powder normal temperature obtained by present method preserves the average viability after 1 day, 7 days and 90 days all higher than 19%, achieves preferably freeze-drying preservation effect.
Survival rate after table 1 Edwardsiella tarda freeze-dried vaccine powder rehydration
Embodiment 2
2012, Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science obtains Chinese Marine University's interchange and gives Edwardsiella tarda one strain, this bacterium is that Chinese Marine University laboratory is obtained by streak plating separation with Luria-Bertani nutrient agar (LB substratum) from ill Cultured Flounder, Paralichthys Olivaceus internal organ, can cause " Ascites Disease " of lefteye flounder, cause serious death.
In the lab, with pancreas peptone soybean broth nutrient agar, switching is carried out to this strain bacterium and cultivate, obtained the pure growth of this strain Edwardsiella tarda by streak plating.Single colony inoculation of picking pure culture is in pancreas peptone soybean broth liquid nutrient medium, be placed in biochemical cultivation case 28 DEG C, cultivate 18h under 150rpm condition after, in the centrifugal 8min of 4000r/min, abandon supernatant, collect bacterial sediment, the concentration adding thalline volume about 15 times is after the abundant shaken well of aseptic NaCl solution of 1.5%, again in the centrifugal 8min of 4000r/min, abandon supernatant, collect bacterial sediment, after repeating same step twice, be that the aseptic NaCl solution of 1.5% is mixed with 10 by resuspended for the thalline of collection by concentration
7~ 10
9the bacteria suspension of cfu/mL is for subsequent use.
First respectively 15g skimmed milk powder, 10g lactose and 10g trehalose being dissolved in 90mL concentration is in the NaCl solution of 1.5%, and under 106 DEG C of conditions, moist heat sterilization 30min, is mixed with solution A after naturally cooling.2g vitamins C being dissolved in 10mL concentration is in the aseptic NaCl solution of 1.5%, and is mixed with solution B after 0.22 μm of cellulose mixture membrane filtration.The solution A configured and solution B are aseptically mixed; and after shaken well, being mixed with 100mL frozen-dried protective agent solution, the final concentration of its each composition is skimmed milk powder 150g/L, lactose 100g/L, trehalose 100g/L, vitamins C 20g/L, NaCl 15g/L.
Aseptically; by the bacteria suspension that configured and protective material solution by volume 1:4 ratio mixing and after shaken well; dividing is filled in the aseptic ampere bottle of several 2mL; the mixed solution of 1.5mL is housed in each ampere of bottle, by after ampere bottle cap top cement plug in-80 DEG C of Ultralow Temperature Freezers quick freezing 6h.Then the mixing solutions frozen is put into rapidly the vacuum freeze drier freeze-drying cabin of precooling, freeze-drying 18 hours under vacuum tightness 0.28mbar, freeze-drying cabin temperature are the condition of-54 DEG C, again vacuum tightness is reduced to 0.034mbar, maintains freeze-drying cabin temperature-54 DEG C and continue freeze-drying 2 hours.After freeze-drying program completes, the dry carbon dioxide of high purity being slowly filled with 99.99% in freeze-drying cabin to standard atmospheric pressure and the vacuum freezedrying that namely gland seal completes this strain Edwardsiella tarda preserve.Preserve under ampere bottle after freeze-drying also seals is placed in normal temperature.
The same day after freeze-drying completes, 7th day and the 90th day, random choose 5 ampere bottles respectively, aseptically open and to seal and the sterile purified water adding 1.5mL carries out rehydration, gradient dilution is carried out by the aseptic NaCl solution that concentration is 1.5% after lyophilized powder dissolves completely, and carry out live bacterial count, to measure the survival rate of freeze-drying bacterial strain with the coating of pancreas peptone soybean broth Agar Plating.Each gradient carries out 2 parallel laboratory tests, gets its mean value, the results are shown in Table 2.
This results show, the Edwardsiella tarda freeze-dried vaccine powder normal temperature obtained by present method preserves the average viability after 1 day, 7 days and 90 days all higher than 19%, and frozen-dried protective effectiveness comparison is desirable.
Survival rate after table 2 Edwardsiella tarda freeze-dried vaccine powder rehydration
Claims (1)
1., to a freeze drying process for the vacuum freezedrying preservation of Edwardsiella tarda, it is characterized in that its method steps is:
To treat that the Edwardsiella tarda pure culture bacterium colony of freeze-drying is mixed with 10 with aseptic 1.5%NaCl solution
7~ 10
9the viable bacteria suspension of cfu/mL, and after aseptically the ratio of viable bacteria suspension and protective material solution 1:4 ~ 1:1 by volume fully being mixed, point to be filled in ampere bottle and quick freezing 5 ~ 6 hours under being placed in-80 DEG C of conditions, put into the vacuum freeze drier freeze-drying cabin of precooling again, vacuum tightness 0.28mbar is set, under the condition of freeze-drying cabin temperature-54 DEG C, freeze-drying is after 18 hours, vacuum tightness is down to 0.034mbar, maintain freeze-drying cabin temperature-54 DEG C and continue freeze-drying 2 hours, treat freeze-drying complete in backward freeze-drying cabin be filled with 99.99% the dry carbon dioxide of high purity to standard atmospheric pressure and gland seal,
The composition of described protective material solution is: skimmed milk powder; lactose; trehalose; vitamins C, NaCl and sterile distilled water, the final concentration of described various compositions is: skimmed milk powder 50 ~ 150g/L, lactose 50 ~ 100g/L; trehalose 100g/L; vitamins C 5 ~ 20g/L, NaCl15g/L, solvent is sterile distilled water.
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Application publication date: 20131225 Assignee: SHANDONG SINDER TECHNOLOGY Co.,Ltd. Assignor: YELLOW SEA FISHERIES Research Institute CHINESE ACADEMY OF FISHERY SCIENCES Contract record no.: X2020980008902 Denomination of invention: Protective agent and freeze-drying method of Edwardsiella tarda Granted publication date: 20150325 License type: Exclusive License Record date: 20201208 |