CN110724639A - Marine luminous bacteria freeze-dried preparation and preparation method thereof - Google Patents
Marine luminous bacteria freeze-dried preparation and preparation method thereof Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 33
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- 238000004108 freeze drying Methods 0.000 claims abstract description 36
- 239000003223 protective agent Substances 0.000 claims abstract description 26
- 238000002156 mixing Methods 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 238000007710 freezing Methods 0.000 claims abstract description 8
- 230000008014 freezing Effects 0.000 claims abstract description 8
- 239000012452 mother liquor Substances 0.000 claims description 24
- 229920001661 Chitosan Polymers 0.000 claims description 17
- 229930182555 Penicillin Natural products 0.000 claims description 14
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 14
- 229940049954 penicillin Drugs 0.000 claims description 14
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- 235000020183 skimmed milk Nutrition 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- OTPDWCMLUKMQNO-UHFFFAOYSA-N 1,2,3,4-tetrahydropyrimidine Chemical compound C1NCC=CN1 OTPDWCMLUKMQNO-UHFFFAOYSA-N 0.000 claims description 11
- 241000607620 Aliivibrio fischeri Species 0.000 claims description 11
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 11
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 11
- 229940073490 sodium glutamate Drugs 0.000 claims description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 229930006000 Sucrose Natural products 0.000 claims description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 10
- 229960004793 sucrose Drugs 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 9
- 238000001816 cooling Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 5
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- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 229940045110 chitosan Drugs 0.000 claims description 3
- 230000006196 deacetylation Effects 0.000 claims description 3
- 238000003381 deacetylation reaction Methods 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000009630 liquid culture Methods 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000012137 tryptone Substances 0.000 claims description 3
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- 230000005855 radiation Effects 0.000 description 4
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- 150000003839 salts Chemical class 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
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- 230000008569 process Effects 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
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- 230000007774 longterm Effects 0.000 description 2
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- 241000193830 Bacillus <bacterium> Species 0.000 description 1
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- 241001602685 Vibrio qinghaiensis Species 0.000 description 1
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- 238000009529 body temperature measurement Methods 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
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- 231100000160 chronic toxicity Toxicity 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
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- 238000009472 formulation Methods 0.000 description 1
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- 230000036541 health Effects 0.000 description 1
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- 230000035790 physiological processes and functions Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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- 239000013535 sea water Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention discloses a marine luminous bacteria freeze-dried preparation and a preparation method thereof, belonging to the field of microbial freeze-dried preparations, wherein the freeze-dried preparation comprises bacterial mud and a freeze-drying protective agent, and is prepared by the steps of bacterial liquid preparation, bacterial mud preparation, mixing, pre-freezing, freeze-drying and the like, in the marine luminous bacteria freeze-dried powder prepared by using the protective agent, after the marine luminous bacteria freeze-dried powder is stored for 18 months at the temperature of 2 ~ 8 ℃, the luminescence rate of a strain recovered and the luminescence performance of the strain are maintained stable, the storage effect and the quality of the marine luminous bacteria strain can be greatly improved, and the use of the strain in environmental toxicity detection is facilitated.
Description
Technical Field
The invention designs a freeze-dried preparation specially for marine luminous bacteria and a preparation method thereof, belonging to the field of microbial freeze-dried preparations.
Background
With the rapid development of industrial and agricultural in China, various ecological environment pollution problems are severe, and great threats are brought to the ecological environment and human health. At present, the biotoxicity detection experiments mainly comprise fish acute toxicity, fish chronic toxicity, flea toxicity, algae toxicity, microorganism toxicity experiments (such as luminous inhibition of luminous bacteria on pollutant toxicity) and the like. The biotoxicity detection method based on the luminous bacteria as the indicator is widely applied to detection of pesticide residues, heavy metal toxicity, biotoxins and the like. The luminous physiological process of luminous bacteria is interfered under the interference of external toxicants, so that the luminous intensity is changed, the reduction degree of the luminous value is in a proportional relation with the external toxicants to a certain degree, and the environmental toxicity is reflected by the change of the luminous intensity. The luminescent bacteria method is of great interest because of its characteristics of fast detection speed, simplicity, reliable sensitivity, low cost, etc.
When the luminous bacteria are used in the environmental toxicity detection process, the strains are important influencing factors. Currently, the commonly used bacteria species in the market include two types of seawater bacteria (Photobacterium brightens and Vibrio fischeri) and one type of freshwater bacteria (Vibrio qinghaiensis). Wherein the photobacterium lucidum is used in the luminous bacteria method for determining acute toxicity of water (GB/T15441-1995); vibrio fischeri is used in the European Union standards.
At present, most of luminous bacteria used for toxicity detection are fresh and alive bacteria and cannot be stored for a long time, and the strains can be stored for a long time after being prepared into freeze-dried products, so that earlier work of using a luminous bacteria method is simplified. The freeze-dried product is directly prepared by sublimating ice crystals, has a porous structure, can keep the original activity of bacteria, and cannot damage the shape and the characteristics of cells. Freeze-drying is a drying method in which a bacterial suspension is frozen below the freezing point to be converted into a solid crystalline state, and then the solid is sublimated at a low temperature and a low pressure (vacuum) to be dehydrated. The drying process can destroy the cell membrane of the microorganism to cause massive cell death, and the cryoprotectant is added to suspend the bacteria and reduce the death of live bacteria in the freeze-drying process. Therefore, the formulation of the protective agent added before lyophilization becomes a crucial determinant, which affects both the activity and stability of the cells during storage and the survival rate of the cells upon reconstitution.
Therefore, in order to improve the survival rate of the strain after freeze drying, the recovery luminous rate and the long-term storage stability, the luminous bacteria freeze-dried preparation which has simple formula, good safety and good effect has positive practical value.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a formula and a preparation method of a marine luminous bacterium freeze-dried preparation. The invention surprisingly discovers that the preparation prepared by using the freeze-drying protective agent and adding chitosan with specific molecular weight and mixing the mixture into a freeze-dried product not only has excellent properties such as appearance, re-solubility, structural strength and the like, but also obviously improves the survival rate, the recovered luminous rate and the long-term storage stability of the strain after freeze drying. The present invention has been completed based on the above findings.
In order to achieve the above purpose, the invention adopts the technical scheme that:
the preparation method of the freeze-dried preparation comprises the following steps:
(1) preparing bacterial liquid, inoculating strain on slant, culturing at 20 deg.C for 24 ~ 48h, repeatedly activating for 2 ~ 3 times, inoculating activated pure strain in 100mL liquid culture medium at 1% inoculum size, culturing at 20 deg.C under constant temperature, shaking at 140 ~ 160rpm/min for 10 ~ 17 hr, and passing OD600nmJudging the concentration of the bacterial liquid, controlling the initial OD600 of the concentration of the bacterial liquid to be 0.8 ~ 1.0.0, and obtaining fresh bacterial liquid for later use;
(2) preparing bacterial mud: subpackaging the bacterial liquid, centrifuging, and removing supernatant to obtain bacterial sludge;
(3) preparation of the freeze-drying protective agent: the freeze-drying protective agent consists of the following raw materials: 15wt% of skim milk, 6wt% of sucrose, 0.6wt% of chitosan, 1wt% of sodium glutamate, 0.4wt% of tetrahydropyrimidine and the balance of distilled water. The preparation method comprises the following steps:
a, preparing liquid: dissolving skim milk, cane sugar, chitosan, sodium glutamate and tetrahydropyrimidine respectively by using distilled water to prepare mother liquor of each raw material, wherein the concentration of the mother liquor is 5 times of the concentration of the formula;
b, sterilization: sterilizing skim milk mother liquor and sucrose mother liquor at 115 deg.C under high temperature and high pressure for 15min, rapidly cooling, and refrigerating at 4 deg.C; sterilizing the chitosan mother liquor and the tetrahydropyrimidine mother liquor by using a 0.22 mu m sterile microporous filter; sterilizing the sodium glutamate mother liquor at 121 deg.C for 21min under high temperature and high pressure, rapidly cooling, and refrigerating at 4 deg.C;
c, mixing: after each mother liquor was sterilized, the ratio of 1: 1: 1: 1: 1, uniformly mixing to obtain a protective agent;
(4) mixing the bacterial sludge with a freeze-drying protective agent: adding the bacterial sludge into a freeze-drying protective agent, mixing, fully shaking and uniformly mixing;
(5) pre-freezing, namely transferring the mixed bacterial suspension into a penicillin bottle, rapidly cooling to ~ -70 ℃ below zero 40 ℃ within 1 ~ 2h, changing the feed liquid into a blocky structure, and pre-freezing for 24 ~ 48 h;
(6) freeze-drying: vacuum freeze-drying the pre-frozen penicillin bottle in the step (5) to obtain the pre-frozen penicillin bottle;
the formula of the culture medium in the step (1) is as follows: 0.3% of glycerol, 3% of sodium chloride, 0.5% of disodium hydrogen phosphate, 0.5% of tryptone, 0.1% of potassium dihydrogen phosphate and 0.5% of yeast extract.
The strain in the step (1) is one or more of luminous bacillus and vibrio fischeri;
in the step (2), the centrifugal speed is 6000 ~ 10000g/min, and the centrifugal time is 5-10 minutes;
and (3) mixing the bacterial sludge and the protective agent in the step (4) according to the volume of the bacterial sludge before centrifugation and the volume of the protective agent of 1: 1, mixing;
in the step (6), the vacuum degree is 0 ~.0 Pa, the temperature is-40 ℃, and the freeze-drying is continuously carried out for 60 ~ h.
The fat content of the skim milk is less than or equal to 2.0 percent, and the protein content is more than or equal to 30 percent.
The molecular weight of the chitosan is 100KDa, and the deacetylation degree is 80%.
Compared with the prior art, the invention has the following outstanding advantages and beneficial effects:
on the basis of the conventional skimmed milk powder and sucrose protective agent, chitosan, sodium glutamate and tetrahydropyrimidine are added. Wherein the tetrahydropyrimidine is derived from high halophilic bacteria, and can effectively protect marine bacteria from being damaged under extreme conditions. Under severe conditions of severe cold, drying and dehydration, the chitosan with specific molecular weight and other components can generate good compatibility, and can also form a special protective film on the cell surface to fully and effectively protect the biomolecular structure from being damaged, thereby maintaining the biological characteristics of the strain and effectively improving the freeze-drying resistance and anti-drying capability of the strain in the freeze-drying process. Sodium glutamate can raise the collapse temperature of the lyophilized product, preventing the disruption of the protein structure due to collapse. After the protective agent and the bacterial mud are mixed according to a specific proportion to prepare a freeze-dried product, the obtained freeze-dried preparation has the advantages of excellent appearance, good re-solubility and good preparation strength, and the bacterial can stably survive for a long time.
The freeze-dried preparation of the marine luminous bacteria has the advantages of simple preparation process and simple and convenient operation, and is suitable for large-scale industrial production and application.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1
Preparing freeze-dried powder:
(1) the preparation method comprises inoculating luminous bacillus strain on slant, culturing at 20 deg.C for 24 ~ 48h, repeatedly activating for 3 times, inoculating the activated pure strain in 100mL liquid culture medium at 1% inoculum size, culturing at 20 deg.C under constant temperature, shaking at 150rpm/min for 15 hr, wherein the culture medium comprises glycerol 0.3%, sodium chloride 3%, disodium hydrogen phosphate 0.5%, tryptone 0.5%, potassium dihydrogen phosphate 0.1%, and yeast extract 0.5%, and passing through OD600nmJudging the concentration of the bacterial liquid, controlling the initial OD600 of the concentration of the bacterial liquid to be 0.8 ~ 1.0.0, and obtaining fresh bacterial liquid for later use;
(2) preparing bacterial mud: subpackaging the bacteria liquid, centrifuging at the rotating speed of 6000g/min for 10 minutes, and removing the supernatant to obtain bacteria mud;
(3) preparation of the freeze-drying protective agent: the freeze-drying protective agent consists of the following raw materials: 15wt% of skim milk (fat content is less than or equal to 2.0%, and protein content is more than or equal to 30%), 6wt% of sucrose, 0.6wt% of chitosan (molecular weight is 100KDa, degree of deacetylation is 80%), 1wt% of sodium glutamate, 0.4wt% of tetrahydropyrimidine, and the balance of distilled water. The preparation method comprises the following steps:
a, preparing liquid: dissolving skim milk, cane sugar, chitosan, sodium glutamate and tetrahydropyrimidine respectively by using distilled water to prepare mother liquor of each raw material, wherein the concentration of the mother liquor is 5 times of the concentration of the formula;
b, sterilization: sterilizing skim milk mother liquor and sucrose mother liquor at 115 deg.C under high temperature and high pressure for 15min, rapidly cooling, and refrigerating at 4 deg.C; sterilizing the chitosan mother liquor and the tetrahydropyrimidine mother liquor by using a 0.22 mu m sterile microporous filter; sterilizing the sodium glutamate mother liquor at 121 deg.C for 21min under high temperature and high pressure, rapidly cooling, and refrigerating at 4 deg.C;
c, mixing: after each mother liquor was sterilized, the ratio of 1: 1: 1: 1: 1, uniformly mixing to obtain a protective agent;
(4) mixing the bacterial sludge with a freeze-drying protective agent: mixing the bacterial sludge and the protective agent according to the volume of the bacterial sludge before centrifugation and the volume of the protective agent 1: 1, mixing, fully shaking and uniformly mixing;
(5) pre-freezing: transferring the mixed bacterial suspension into a penicillin bottle, rapidly cooling to-40 ℃ within 1h, changing the feed liquid into a blocky structure, and pre-freezing for 48 h;
(6) freeze-drying, namely performing vacuum freeze-drying on the pre-frozen penicillin bottle in the step (5), keeping the vacuum degree of 0 ~ 1.0.0 Pa, keeping the temperature at-40 ℃, continuously performing freeze-drying for 60 hours, pressing a plug and covering after the freeze-drying is finished, marking, and storing in a refrigerator at 4 ℃ for later use.
Example 2
And (3) freeze-drying, namely, the step (1) to the step (4) are pre-frozen in the same way as in the example 1 (5), namely, the mixed bacterial suspension is transferred into a penicillin bottle, the temperature is rapidly reduced to-70 ℃ within 2 hours, the feed liquid is changed into a blocky structure, the pre-frozen is frozen for 24 hours, (6) freeze-drying is carried out, namely, the pre-frozen penicillin bottle in the step (5) is subjected to vacuum freeze-drying, the vacuum degree is kept at 0 ~ 1.0.0 Pa, the temperature is kept at-40 ℃, freeze-drying is continuously carried out for 72 hours, after the freeze-drying is finished, the cover is pressed.
Example 3
And (3) freeze-drying, namely, the step (1) to the step (4) are pre-frozen in the same way as the step (5), namely, the mixed bacterial suspension is transferred into a penicillin bottle, the temperature is rapidly reduced to-55 ℃ within 1.5h, the feed liquid is changed into a blocky structure, the pre-frozen suspension is freeze-dried for 36h, (6) the pre-frozen penicillin bottle in the step (5) is subjected to vacuum freeze drying, the vacuum degree is kept at 0 ~ 1.0.0 Pa, the temperature is kept at-40 ℃, freeze drying is continuously carried out for 66h, after the freeze drying is finished, the penicillin bottle is plugged and covered, and the penicillin bottle is marked and.
Example 4
Preparing freeze-dried powder: the strain in step (1) is Vibrio fischeri, and the preparation method is the same as that in example 1.
Example 5
Preparing freeze-dried powder: the strain of step (1) is Vibrio fischeri and the preparation method is the same as example 2.
Example 6
Preparing freeze-dried powder: the strain of step (1) is Vibrio fischeri and the preparation method is the same as example 3.
Comparative example 1
Preparing freeze-dried powder: the molecular weight of the chitosan in the step (3) is 50KDa, and other steps are the same as the example 1.
Comparative example 2
Preparing freeze-dried powder: the molecular weight of the chitosan of the step (3) is 150KDa, and other steps are the same as the example 1.
Comparative example 3
Preparing freeze-dried powder: the molecular weight of the chitosan in the step (3) is 200KDa, and other steps are the same as the example 1.
Test example 1
The collapse temperatures of the lyophilized powders of example 1 ~ 6 and comparative example 1 ~ 3 were measured (the measurement method was analyzed with reference to the influence of the freeze-drying parameters on the collapse temperature, journal of agricultural machinery, 2011, 42 (2)), and the appearance and solubility (the solubility is measured as the time for dissolving the lyophilized powder completely in an equal amount of distilled water), and the results are shown in table 1.
TABLE 1 Freeze-dried powder appearance, redissolution and collapse temperature measurements
As can be seen from Table 1, compared with the lyophilized powder of comparative example 1 ~ 3, the lyophilized powder of example 1 ~ 6 maintained good appearance and re-dissolution, and the lyophilized powder of example had higher collapse temperature and better strength.
Test example 2
The lyophilized powders of example 1 ~ 6 and comparative example 1 ~ 3 were subjected to high salt (reconstituted with 15% NaCl), high temperature (at 37 ℃), high ultraviolet radiation (continuous irradiation with UVA (340) lamp, irradiance of 0.89W/m), respectively2) ) for 24 hours and the bacterial activity was measured by the MTT (0.5% tetramethylazozolium) method, the results of which are shown in table 2.
TABLE 2 results of the determination of bacterial activity under high salt, high temperature, high UV radiation conditions
As shown in Table 2, compared with comparative example 1 ~ 3, the lyophilized product of example 1 ~ 3 containing Leuconostoc brilliant bacteria still maintains higher level of bacterial activity in the lyophilized product of the invention of example 1 ~ 3 after being lyophilized, and is significantly higher than the level of bacterial activity in comparative example 1 ~ 3. the lyophilized product of example 1 ~ 3 maintains higher level of bacterial activity under high salt, high temperature and high ultraviolet radiation, and is significantly higher than the level of bacterial activity in comparative example 1 ~ 3. the lyophilized product of example 4 ~ 6 containing Vibrio fischeri maintains higher level of bacterial activity after being lyophilized, and maintains higher level of bacterial activity under high salt, high temperature and high ultraviolet radiation.
Test example 3
The lyophilized powder of example 1 ~ 6 and comparative example 1 ~ 3 were stored at 4 ℃ for 1 st, 3 rd, 6 th, 12 th, and 18 th months, respectively, 2% NaCl was added to the lyophilized powder to restore the original volume, and after mixing, the luminescence value was measured, and the luminescence rate for recovery = (luminescence value before resuscitation/luminescence value after resuscitation) × 100%, the results are shown in table 3.
TABLE 3 test results of luminescence recovery of lyophilized powder stored at 4 deg.C
As can be seen from Table 3, the lyophilized Vibrio shigella powder prepared in example 1 ~ 3 maintained a high level of restored luminescence after 18 months of storage at 4 ℃ and was significantly higher than that of comparative example 1 ~ 3, and the lyophilized Vibrio fischeri powder prepared in example 4 ~ 6 maintained a high level of restored luminescence after 18 months of storage at 4 ℃.
Compared with the lyophilized powder of the photobacterium lucidum in the comparative example 1 ~ 3, the lyophilized powder of the example 1 ~ 3 has higher recovery luminous efficiency and better effect.
In conclusion, the marine luminous bacteria freeze-dried powder prepared by the marine luminous bacteria freeze-dried preparation has the advantages of excellent appearance, good re-solubility, good preparation strength, high recovered luminous efficiency and stable bacterial activity, and can be stored for a long time.
Claims (3)
1. A freeze-dried preparation of marine luminous bacteria is characterized in that: the method comprises the following steps:
(1) the preparation method comprises inoculating strain on slant, culturing at 20 deg.C for 24 ~ 48h, repeatedly activating for 2 ~ 3 times, inoculating activated pure strain in 100mL liquid culture medium at 1% inoculum size, culturing at 20 deg.C under constant temperature of 140 ~ 160rpm/min under shaking for 10 ~ 17 hr, and passing through OD600nmJudging the concentration of the bacterial liquid, controlling the initial OD600 of the concentration of the bacterial liquid to be 0.8 ~ 1.0.0, and obtaining fresh bacterial liquid for later use;
(2) preparing bacterial mud: subpackaging the bacterial liquid, centrifuging, and removing supernatant to obtain bacterial sludge;
(3) preparation of the freeze-drying protective agent: the freeze-drying protective agent consists of the following raw materials: 15wt% of skim milk, 6wt% of sucrose, 0.6wt% of chitosan, 1wt% of sodium glutamate, 0.4wt% of tetrahydropyrimidine and the balance of distilled water;
the preparation method comprises the following steps:
a, preparing liquid: dissolving skim milk, cane sugar, chitosan, sodium glutamate and tetrahydropyrimidine respectively by using distilled water to prepare mother liquor of each raw material, wherein the concentration of the mother liquor is 5 times of the concentration of the formula;
b, sterilization: sterilizing skim milk mother liquor and sucrose mother liquor at 115 deg.C under high temperature and high pressure for 15min, rapidly cooling, and refrigerating at 4 deg.C; sterilizing the chitosan mother liquor and the tetrahydropyrimidine mother liquor by using a 0.22 mu m sterile microporous filter; sterilizing the sodium glutamate mother liquor at 121 deg.C for 21min under high temperature and high pressure, rapidly cooling, and refrigerating at 4 deg.C;
c, mixing: after each mother liquor was sterilized, the ratio of 1: 1: 1: 1: 1, uniformly mixing to obtain a protective agent;
(4) mixing the bacterial sludge with a freeze-drying protective agent: adding the bacterial sludge into a freeze-drying protective agent, mixing, fully shaking and uniformly mixing;
(5) pre-freezing, namely transferring the mixed bacterial suspension into a penicillin bottle, rapidly cooling to ~ -70 ℃ below zero 40 ℃ within 1 ~ 2h, changing the feed liquid into a blocky structure, and pre-freezing for 24 ~ 48 h;
(6) freeze-drying: vacuum freeze-drying the pre-frozen penicillin bottle in the step (5) to obtain the pre-frozen penicillin bottle;
the formula of the culture medium in the step (1) is as follows: 0.3% of glycerol, 3% of sodium chloride, 0.5% of disodium hydrogen phosphate, 0.5% of tryptone, 0.1% of potassium dihydrogen phosphate and 0.5% of yeast extract;
the strain in the step (1) is one or more of luminous bacillus and vibrio fischeri;
in the step (2), the centrifugal speed is 6000 ~ 10000g/min, and the centrifugal time is 5-10 minutes;
and (3) mixing the bacterial sludge and the protective agent in the step (4) according to the volume of the bacterial sludge before centrifugation and the volume of the protective agent of 1: 1, mixing;
in the step (6), the vacuum degree is 0 ~.0 Pa, the temperature is-40 ℃, and the freeze-drying is continuously carried out for 60 ~ h.
2. The lyoprotectant according to claim 1, wherein the fat content of said skim milk is not more than 2.0% and the protein content is not less than 30%.
3. The lyoprotectant of claim 1, wherein said chitosan has a molecular weight of 100KDa and a degree of deacetylation of 80%.
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