CN108486019A - The method for preserving of nitrococcus - Google Patents

The method for preserving of nitrococcus Download PDF

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CN108486019A
CN108486019A CN201810436739.5A CN201810436739A CN108486019A CN 108486019 A CN108486019 A CN 108486019A CN 201810436739 A CN201810436739 A CN 201810436739A CN 108486019 A CN108486019 A CN 108486019A
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freeze
nitrococcus
thalline
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drying
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CN108486019B (en
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汤江武
孙宏
李园成
吴逸飞
姚晓红
王新
沈琦
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Zhejiang Academy of Agricultural Sciences
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Abstract

The invention discloses a kind of method for preserving of nitrococcus, include the following steps：1) protective agent is configured：2~4 parts of sodium glutamates, 18~25 parts of tripotassium phosphates and 1~3 part of cysteine are added in 90~120 parts of sterile waters, is uniformly mixed and obtains protective agent；2) thalline is added：0.8~1.2 times of volume nitrococcus thalline is added in the protective agent that step 1) obtains, concussion is resuspended, and obtains mixed liquor；3) freezing：The mixed liquor freeze-drying that step 2) is obtained, the freezing at 70~90 DEG C carried out activation switching every 2~3 months.Advantageous effect：Damage of the freeze-drying to nitrococcus thalline can substantially be reduced, improve the activity of nitrococcus survival rate and ATP enzyme, ammona monooxygenase (AMO) in preserving process.

Description

The method for preserving of nitrococcus
Technical field
The present invention relates to cell preservation technique field, the method for preserving of specifically a kind of nitrococcus.
Background technology
Ammonia nitrogen is converted into nitrite under nitrococcus effect first, then in the case where nitrifier acts on nitrite into one Step is converted into nitrate.The net reaction of nitration reaction is：NH4 ++1.815O2+0.1304CO2→0.0261C5H7O2+ 0.973NO3 --N+0.921H2O+1.973H+.The means of oxidative metabolism of nitrococcus nitrogen cycle includes mainly ammonia (NH3) pass through ammonia Monooxygenase (AMO) is oxidized to azanol (NH20H), azanol continues to be oxidized to nitrous under hydroxylamine reductase (HAO) catalytic action Acid group.Distribution of the nitrite bacteria in nature is universal, there is the presence of nitrite bacteria in soil, ocean and fresh water, and Only account for the minimum ratio of wherein bacteria total amount.The environment that the nitrite bacteria of different genera is distributed is also different.Such as European Asia Nitromonas is distributed widely in soil, sewage, fresh water and ocean；Nitrous acid spirillum is distributed mainly in soil in white；And Ocean nitrous acid coccus only exists in ocean.The growth of nitrite bacteria is extremely slow, for 24 hours could need under appropriate conditions Complete a division cycle.The several months is generally needed during carrying out solid culture just can see bacterium colony growth.
Freeze Drying Technique is in numerous culture collection process in one of ideal method, but is lyophilized thin to nitrococcus Born of the same parents can cause certain physiological damage, reduce cell survival rate, be mainly manifested in the mechanical damage of ice crystal in freezing process, freeze And caused solute damage, the front and back structure of cell membrane of freeze-drying, the variation of function and ingredient, key enzyme lose in drying process Living, DNA structure variation etc..
(1) mechanical damage:Before it is dried, thalline is freezed, during freezing, intracellular a large amount of water meeting Ice crystal is formed, ice crystal causes the rupture of cell membrane, and the mechanical damage caused by cell membrane system leads to the destruction of membrane structure, from And influence the normal physiological metabolism of cell；
(2) solute damages:Its volume very little, specific surface area be very big for cell, the water penetration rate of cell membrane is very high, Intracellular water content declines when temperature reduces, and extracellular and intracellular electrolyte concentrates, cell transition dehydration, catastrophic collapse；This A little damages for resulting in cell are so that dead；This damage is referred to as " solution damage " or " solute damage "；
(3) variation of the structure of cell membrane and ingredient:Freeze-drying is a strong stress procedure, in this process The physical state of cell membrane lipid substance is necessarily caused to change.Cell membrane phospholipid is bilayer, and the both ends of film are polar end, It is existing for hydrated form, to be separated by water between the polar end of each phosphatide to a certain extent.When freeze-drying and dehydrating, Phosphatide polar end removes hydration hydrogen bond, and acyl group is forced to be clustered together, and polar end density becomes larger, and intermolecular Van der Waals force increases, Liquid crystalline phase adipose membrane changes gel phase, and this structure change results in cell membrane permeability increase so that cell is unable to control intracellular The two-way exchange of outer substance, to cause the metabolic impairment of cell.
Invention content
The purpose of the present invention is to provide a kind of damages that can substantially reduce freeze-drying to nitrococcus thalline, improve sub- Nitrifier survival rate and ATP enzyme, ammona monooxygenase (AMO) active nitrococcus method for preserving.
To achieve the above object, the technical scheme is that：The method for preserving of nitrococcus, includes the following steps：
1) protective agent is configured：In 90~120 parts of sterile waters be added 2~4 parts of sodium glutamates, 18~25 parts of tripotassium phosphates and 1~3 part of cysteine is uniformly mixed and obtains protective agent；
2) thalline is added：0.8~1.2 times of volume nitrococcus thalline, concussion are added in the protective agent that step 1) obtains It is resuspended, obtains mixed liquor；
3) freezing：The mixed liquor freeze-drying that step 2) is obtained, the freezing at -70~-90 DEG C, every 2 Carry out activation switching within~3 months.
Cold and hot Stress treatment is carried out before freeze-drying：By obtained mixed liquor in 30~40 DEG C of constant temperature heat treatment 10~ Then 20min handles 1~2h in 4~8 DEG C of constant temperature.
Freeze drying process is：Pre-freeze：- 40~-30 DEG C of 3~5h of pre-freeze；Freeze-drying：- 70~-60 DEG C, 30~50Pa It is freeze-dried 5~7h, -20~-10 DEG C, 60~100Pa is freeze-dried 2~3h；Lyophilization：5~15 DEG C, 100~200Pa liters Dry 3~the 4h of China；Parsing-desiccation:10~20 DEG C, 3~5Pa parsing-desiccations, 2~4h.
To optimize said program, the measure that the present invention takes is：
1) protective agent is configured：In 90~120 parts of sterile waters be added 2~4 parts of sodium glutamates, 18~25 parts of tripotassium phosphates and 1~3 part of cysteine is uniformly mixed and obtains protective agent.Sodium glutamate, tripotassium phosphate and cysteine three have synergistic effect, During thalline freezes, the formation of ice crystal can be significantly prevented, stablizes phospholipid bilayer and Membrane protein conformation, and The activity of cell membrane-ATP enzyme can be protected, is reduced because freezing or moisture constantly distil to the damage caused by nitrococcus cell；
2) absorption carrier is prepared：According to the cationic of chitosan, gelatin obtains both sexes feature, using the two as raw material, penta Dialdehyde is crosslinking agent, and chitosan/gelatin-compounded microballoon is prepared by inverse suspension crosslinking method；
3) thalline is added：The shell that 30~50 parts of steps 2) obtain is added in the protective agent that 100~120 parts of steps 1) obtain Glycan/gelatin-compounded microballoon and 100~180 parts of nitrococcus thalline, concussion are resuspended, and obtain mixed liquor.Nitrococcus thalline is inhaled It is attached to chitosan/gelatin-compounded microsphere surface, increases cell density, when survival of the nitrococcus under hungry environment can be improved Between.And above-mentioned system can induce the synthesis of unsaturated fatty acid in nitrococcus thalline, increase unsaturated fat in membrane lipid The ratio of acid significantly reduces ice crystal and is formed and the injury to cell membrane of intracellular osmotic pressure, maintains the mobility, complete of cell membrane Whole property, ATP enzyme (Mg2+ATPase, Ca2+ATPase, Na+-K+ATPase the activity of activity and ammona monooxygenase (AMO)), from And improve the freeze-drying resistance of thalline；
4) cold and hot Stress treatment：Obtained mixed liquor is heat-treated 10~20min in 30~40 DEG C of constant temperature, then in 4~8 DEG C constant temperature handles 1~2h.Nitrococcus will produce the stress protein of some low molecular weights under conditions of cold stress and heat stress, The stress protein generated under cold stress conditions is referred to as Cold-inducible protein gene, cold shock protein；It is generated under heat stress conditions Stress protein is referred to as heat shock protein.Cold shock protein and heat shock protein play the role of molecular chaperones, can improve cell membrane Mobility, effectively improve the correct folding of albumen, improve resistance of the nitrococcus to freeze-drying, reduce freeze-drying to thalline Damage.And translation and the transcription that DNA can also be effectively adjusted under -70~-90 DEG C of cryogenic conditions, are greatly reduced nitrous The mutation probability for changing bacterium, retains original excellent performance；
5) freezing：The mixed liquor freeze-drying that step 4) is obtained, the freezing at -70~-90 DEG C, every 8 Carry out activation switching within~12 months.Scheme improves the mobility, integrality, ATP of nitrococcus cell membrane after present invention optimization Enzyme (Mg2+ATPase, Ca2+ATPase, Na+-K+ATPase the activity of activity and ammona monooxygenase (AMO)), and reduce Actication of culture switching number, preferably remains the excellent performance of nitrococcus.
Preferably, the specific preparation process of absorption carrier is：1~3 part of chitosan is dissolved in 80~100 part 2~4% In acetum, 40~50 parts of 18~25g/mL gelatin solutions are added after stirring and dissolving, are mixed evenly to obtain water phase；By 1 ~4 parts of atoleines and 1~4 part of surfactant span-80 mixing, 50~65 DEG C are heated under stirring well, is obtained To oil phase；Oil phase is added to water phase, is cooled down after emulsifying 15~30min, when temperature drop to 15 DEG C hereinafter, be added 1~2 part 40~ 55% glutaraldehyde carries out crosslinking curing, and reaction was completed after 0.8~1.5h.Product centrifuges 5 under the conditions of 800~1500r/min~ 10min, after, distinguish rinse with propyl alcohol, isopropanol, until atoleine is cleaned, it is multiple to be finally dried to obtain chitosan/gelatin Close microballoon.
Preferably, freeze drying process is：Pre-freeze：- 40~-30 DEG C of 3~5h of pre-freeze；Freeze-drying：- 70~-60 DEG C, 30~50Pa is freeze-dried 5~7h, and -20~-10 DEG C, 60~100Pa is freeze-dried 2~3h；Lyophilization：5~15 DEG C, 100 3~4h of~200Pa lyophilizations；Parsing-desiccation:10~20 DEG C, 3~5Pa parsing-desiccations, 2~4h.Above-mentioned freeze-drying temperature Under, freezing speed balances each other to the permeability of moisture with nitrococcus cell.If chilling rate is too fast, intracellular moisture comes Not as good as exudation, it is trapped in cell interior, a large amount of ice crystal can be formed, mechanical damage is caused to cell membrane；If freezing speed mistake Slowly, intracellular moisture largely oozes out, and intracellular ion concentration rises too high, and causes solute effect, causes intracellular quick The inactivation for feeling albumen, influences the vigor of cell.
Compared with the prior art, the advantages of the present invention are as follows：
1. the correct folding for improving the mobility of cell membrane by cold and hot Stress treatment, effectively improving albumen improves sub- Nitrifier reduces damage of the freeze-drying to thalline to the resistance of freeze-drying.And it can also be under -70~-90 DEG C of cryogenic conditions Translation and the transcription for effectively adjusting DNA, are greatly reduced the mutation probability of nitrococcus, retain original excellent performance；
2. by the synergistic effect of protective agent and carrier, during thalline freezes, ice crystal can be significantly prevented It is formed, stablizes phospholipid bilayer and Membrane protein conformation, and induce the synthesis of unsaturated fatty acid in nitrococcus thalline, increased The ratio of unsaturated fatty acid in blooming lipid significantly reduces the injury of ice crystal formation and intracellular osmotic pressure to cell membrane, Maintain the mobility, integrality, ATP enzyme (Mg of cell membrane2+ATPase, Ca2+ATPase, Na+-K+ATPase activity) and ammonia list The activity of oxygenase (AMO) is reduced because freezing or moisture constantly distil to the damage caused by nitrococcus cell；
3. control freezing speed balances each other to the permeability of moisture with nitrococcus cell, by freeze-drying to the damage of thalline Wound is preferably minimized.Thalline is in dry, anoxic and low-temperature condition, vital movement are in suspend mode, can reach the mesh of long term storage 's.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
The method for preserving of nitrococcus：
1) protective agent is configured：3 parts of sodium glutamates, 20 parts of tripotassium phosphates and 2 part of half Guang ammonia are added in 100 parts of sterile waters Acid is uniformly mixed and obtains protective agent；
2) thalline is added：160 parts of nitrococcus thalline are added in the protective agent that 110 parts of steps 1) obtain, concussion is resuspended, Obtain mixed liquor；
3) cold and hot Stress treatment：Obtained mixed liquor is heat-treated 12min in 35 DEG C of constant temperature, is then handled in 6 DEG C of constant temperature 1.7h；
4) freezing：The mixed liquor freeze-drying that step 3) is obtained, the freezing at -80 DEG C, every 8~12 The moon carries out activation switching.Freeze drying process is：Pre-freeze：- 30 DEG C of pre-freeze 4h；Freeze-drying：- 60 DEG C, 40Pa freeze-dryings 6h, -20 DEG C, 70Pa is freeze-dried 2.2h；Lyophilization：10 DEG C, 150Pa lyophilizations 3.5h；Parsing-desiccation:16 DEG C, 4Pa solutions The dry 3h of analysis.The correct folding that the present invention is improved the mobility of cell membrane by cold and hot Stress treatment, effectively improves albumen, changes Kind nitrococcus reduces damage of the freeze-drying to thalline to the resistance of freeze-drying.And can also have under -80 DEG C of cryogenic conditions Effect adjusts translation and the transcription of DNA, and the mutation probability of nitrococcus is greatly reduced, retains original excellent performance.Paddy ammonia Sour sodium, tripotassium phosphate and cysteine three have synergistic effect, during thalline freezes, can significantly prevent ice crystal It is formed, stablizes phospholipid bilayer and Membrane protein conformation, and the activity of cell membrane-ATP enzyme can be protected, reduced because of freezing or water Divide constantly distillation to the damage caused by nitrococcus cell.Moisture is oozed by control freezing speed and nitrococcus cell Saturating rate balances each other, and freeze-drying is preferably minimized the damage of thalline.Thalline is in dry, anoxic and low-temperature condition, and life is lived It is dynamic to be in suspend mode, it can achieve the purpose that long term storage.
Embodiment 2：
The method for preserving of nitrococcus：
1) protective agent is configured：3.5 parts of sodium glutamates, 22 parts of tripotassium phosphates and 2 part of half Guang ammonia are added in 110 parts of sterile waters Acid is uniformly mixed and obtains protective agent；
2) thalline is added：140 parts of nitrococcus thalline are added in the protective agent that 100 parts of steps 1) obtain, concussion is resuspended, Obtain mixed liquor；
3) cold and hot Stress treatment：Obtained mixed liquor is heat-treated 10min in 37 DEG C of constant temperature, is then handled in 7 DEG C of constant temperature 1.2h；
4) freezing：The mixed liquor freeze-drying that step 3) is obtained, the freezing at -85 DEG C, every 8~12 The moon carries out activation switching.Freeze drying process is：Pre-freeze：- 30 DEG C of pre-freeze 4h；Freeze-drying：- 58 DEG C, 40Pa freeze-dryings 6h, -20 DEG C, 70Pa is freeze-dried 2.2h；Lyophilization：10 DEG C, 150Pa lyophilizations 3.5h；Parsing-desiccation:16 DEG C, 4Pa solutions The dry 3h of analysis.The correct folding that the present invention is improved the mobility of cell membrane by cold and hot Stress treatment, effectively improves albumen, changes Kind nitrococcus reduces damage of the freeze-drying to thalline to the resistance of freeze-drying.And can also have under -85 DEG C of cryogenic conditions Effect adjusts translation and the transcription of DNA, and the mutation probability of nitrococcus is greatly reduced, retains original excellent performance.Paddy ammonia Sour sodium, tripotassium phosphate and cysteine three have synergistic effect, during thalline freezes, can significantly prevent ice crystal It is formed, stablizes phospholipid bilayer and Membrane protein conformation, and the activity of cell membrane-ATP enzyme can be protected, reduced because of freezing or water Divide constantly distillation to the damage caused by nitrococcus cell.Moisture is oozed by control freezing speed and nitrococcus cell Saturating rate balances each other, and freeze-drying is preferably minimized the damage of thalline.Thalline is in dry, anoxic and low-temperature condition, and life is lived It is dynamic to be in suspend mode, it can achieve the purpose that long term storage.
Embodiment 3：
The method for preserving of nitrococcus：
1) protective agent is configured：2.8 parts of sodium glutamates, 20 parts of tripotassium phosphates and 2 part of half Guang ammonia are added in 100 parts of sterile waters Acid is uniformly mixed and obtains protective agent；
2) thalline is added：150 parts of nitrococcus thalline are added in the protective agent that 100 parts of steps 1) obtain, concussion is resuspended, Obtain mixed liquor；
3) cold and hot Stress treatment：Obtained mixed liquor is heat-treated 10min in 37 DEG C of constant temperature, is then handled in 7 DEG C of constant temperature 1.2h；
4) freezing：The mixed liquor freeze-drying that step 3) is obtained, the freezing at -80 DEG C, every 8~12 The moon carries out activation switching.Freeze drying process is：Pre-freeze：- 35 DEG C of pre-freeze 4h；Freeze-drying：- 60 DEG C, 40Pa freeze-dryings 6h, -20 DEG C, 70Pa is freeze-dried 2.2h；Lyophilization：10 DEG C, 140Pa lyophilizations 3.5h；Parsing-desiccation:16 DEG C, 4Pa solutions The dry 3h of analysis.The correct folding that the present invention is improved the mobility of cell membrane by cold and hot Stress treatment, effectively improves albumen, changes Kind nitrococcus reduces damage of the freeze-drying to thalline to the resistance of freeze-drying.And can also have under -80 DEG C of cryogenic conditions Effect adjusts translation and the transcription of DNA, and the mutation probability of nitrococcus is greatly reduced, retains original excellent performance.Paddy ammonia Sour sodium, tripotassium phosphate and cysteine three have synergistic effect, during thalline freezes, can significantly prevent ice crystal It is formed, stablizes phospholipid bilayer and Membrane protein conformation, and the activity of cell membrane-ATP enzyme can be protected, reduced because of freezing or water Divide constantly distillation to the damage caused by nitrococcus cell.Moisture is oozed by control freezing speed and nitrococcus cell Saturating rate balances each other, and freeze-drying is preferably minimized the damage of thalline.Thalline is in dry, anoxic and low-temperature condition, and life is lived It is dynamic to be in suspend mode, it can achieve the purpose that long term storage.
Embodiment 4：
The method for preserving of nitrococcus：
1) protective agent is configured：3 parts of sodium glutamates, 20 parts of tripotassium phosphates and 2 part of half Guang ammonia are added in 100 parts of sterile waters Acid is uniformly mixed and obtains protective agent；
2) absorption carrier is prepared：2 parts of chitosans are dissolved in 100 part 3% of acetum, 45 parts are added after stirring and dissolving 20g/mL gelatin solutions are mixed evenly to obtain water phase；2 parts of atoleines and 2 parts of surfactant span-80 are mixed, It is heated to 60 DEG C under stirring well, obtains oil phase；Oil phase is added to water phase, is cooled down after emulsifying 20min, when temperature drops To 15 DEG C hereinafter, 1.3 part of 50% glutaraldehyde progress crosslinking curing is added, reaction was completed after 1.0h.Product is in 1000r/min conditions After lower centrifugation 8min, distinguishes rinse with propyl alcohol, isopropanol, until atoleine is cleaned, be finally dried to obtain chitosan/bright Glue complex microsphere；
3) thalline is added：Chitosan/gelatin that 40 parts of steps 2) obtain is added in the protective agent that 110 parts of steps 1) obtain Complex microsphere and 160 parts of nitrococcus thalline, concussion are resuspended, and obtain mixed liquor；
4) cold and hot Stress treatment：Obtained mixed liquor is heat-treated 12min in 35 DEG C of constant temperature, is then handled in 6 DEG C of constant temperature 1.7h；
5) freezing：The mixed liquor freeze-drying that step 4) is obtained, the freezing at -80 DEG C, every 8~12 The moon carries out activation switching.Freeze drying process is：Pre-freeze：- 30 DEG C of pre-freeze 4h；Freeze-drying：- 60 DEG C, 40Pa freeze-dryings 6h, -20 DEG C, 70Pa is freeze-dried 2.2h；Lyophilization：10 DEG C, 150Pa lyophilizations 3.5h；Parsing-desiccation:16 DEG C, 4Pa solutions The dry 3h of analysis.The correct folding that the present invention is improved the mobility of cell membrane by cold and hot Stress treatment, effectively improves albumen, changes Kind nitrococcus reduces damage of the freeze-drying to thalline to the resistance of freeze-drying.And can also have under -80 DEG C of cryogenic conditions Effect adjusts translation and the transcription of DNA, and the mutation probability of nitrococcus is greatly reduced, retains original excellent performance.Pass through The synergistic effect of protective agent and carrier can significantly prevent the formation of ice crystal during thalline freezes, and it is double to stablize phosphatide Molecular layer and Membrane protein conformation, and the synthesis of unsaturated fatty acid in nitrococcus thalline is induced, increase insatiable hunger in membrane lipid With the ratio of aliphatic acid, the injury of ice crystal formation and intracellular osmotic pressure to cell membrane is significantly reduced, the stream of cell membrane is maintained Dynamic property, integrality, ATP enzyme (Mg2+ATPase, Ca2+ATPase, Na+-K+ATPase activity) and ammona monooxygenase (AMO) Activity is reduced because freezing or moisture constantly distil to the damage caused by nitrococcus cell.Pass through control freezing speed and Asia Nitrifier cell balances each other to the permeability of moisture, and freeze-drying is preferably minimized the damage of thalline.Thalline is in dry, scarce Oxygen and low-temperature condition, vital movement are in suspend mode, can achieve the purpose that long term storage.
Embodiment 5：
The method for preserving of nitrococcus：
1) protective agent is configured：3.5 parts of sodium glutamates, 22 parts of tripotassium phosphates and 2 part of half Guang ammonia are added in 110 parts of sterile waters Acid is uniformly mixed and obtains protective agent；
2) absorption carrier is prepared：2 parts of chitosans are dissolved in 100 part 3% of acetum, 42 parts are added after stirring and dissolving 20g/mL gelatin solutions are mixed evenly to obtain water phase；3 parts of atoleines and 2 parts of surfactant span-80 are mixed, It is heated to 60 DEG C under stirring well, obtains oil phase；Oil phase is added to water phase, is cooled down after emulsifying 20min, when temperature drops To 15 DEG C hereinafter, 1.3 part of 50% glutaraldehyde progress crosslinking curing is added, reaction was completed after 1.0h.Product is in 1000r/min conditions After lower centrifugation 8min, distinguishes rinse with propyl alcohol, isopropanol, until atoleine is cleaned, be finally dried to obtain chitosan/bright Glue complex microsphere；
3) thalline is added：Chitosan/gelatin that 40 parts of steps 2) obtain is added in the protective agent that 100 parts of steps 1) obtain Complex microsphere and 140 parts of nitrococcus thalline, concussion are resuspended, and obtain mixed liquor；
4) cold and hot Stress treatment：Obtained mixed liquor is heat-treated 10min in 37 DEG C of constant temperature, is then handled in 7 DEG C of constant temperature 1.2h；
5) freezing：The mixed liquor freeze-drying that step 4) is obtained, the freezing at -85 DEG C, every 8~12 The moon carries out activation switching.Freeze drying process is：Pre-freeze：- 30 DEG C of pre-freeze 4h；Freeze-drying：- 58 DEG C, 40Pa freeze-dryings 6h, -20 DEG C, 70Pa is freeze-dried 2.2h；Lyophilization：10 DEG C, 150Pa lyophilizations 3.5h；Parsing-desiccation:16 DEG C, 4Pa solutions The dry 3h of analysis.The correct folding that the present invention is improved the mobility of cell membrane by cold and hot Stress treatment, effectively improves albumen, changes Kind nitrococcus reduces damage of the freeze-drying to thalline to the resistance of freeze-drying.And can also have under -85 DEG C of cryogenic conditions Effect adjusts translation and the transcription of DNA, and the mutation probability of nitrococcus is greatly reduced, retains original excellent performance.Pass through The synergistic effect of protective agent and carrier can significantly prevent the formation of ice crystal during thalline freezes, and it is double to stablize phosphatide Molecular layer and Membrane protein conformation, and the synthesis of unsaturated fatty acid in nitrococcus thalline is induced, increase insatiable hunger in membrane lipid With the ratio of aliphatic acid, the injury of ice crystal formation and intracellular osmotic pressure to cell membrane is significantly reduced, the stream of cell membrane is maintained Dynamic property, integrality, ATP enzyme (Mg2+ATPase, Ca2+ATPase, Na+-K+ATPase activity) and ammona monooxygenase (AMO) Activity is reduced because freezing or moisture constantly distil to the damage caused by nitrococcus cell.Pass through control freezing speed and Asia Nitrifier cell balances each other to the permeability of moisture, and freeze-drying is preferably minimized the damage of thalline.Thalline is in dry, scarce Oxygen and low-temperature condition, vital movement are in suspend mode, can achieve the purpose that long term storage.
Embodiment 6：
The method for preserving of nitrococcus：
1) protective agent is configured：2.8 parts of sodium glutamates, 20 parts of tripotassium phosphates and 2 part of half Guang ammonia are added in 100 parts of sterile waters Acid is uniformly mixed and obtains protective agent；
2) absorption carrier is prepared：2 parts of chitosans are dissolved in 120 part 3% of acetum, 40 parts are added after stirring and dissolving 20g/mL gelatin solutions are mixed evenly to obtain water phase；3 parts of atoleines and 2 parts of surfactant span-80 are mixed, It is heated to 60 DEG C under stirring well, obtains oil phase；Oil phase is added to water phase, is cooled down after emulsifying 20min, when temperature drops To 15 DEG C hereinafter, 1.5 part of 50% glutaraldehyde progress crosslinking curing is added, reaction was completed after 1.0h.Product is in 800r/min conditions After lower centrifugation 10min, with propyl alcohol, isopropanol distinguish rinse, until atoleine clean until, be finally dried to obtain chitosan/ Gelatin-compounded microballoon；
3) thalline is added：Chitosan/gelatin that 45 parts of steps 2) obtain is added in the protective agent that 100 parts of steps 1) obtain Complex microsphere and 150 parts of nitrococcus thalline, concussion are resuspended, and obtain mixed liquor；
4) cold and hot Stress treatment：Obtained mixed liquor is heat-treated 10min in 37 DEG C of constant temperature, is then handled in 7 DEG C of constant temperature 1.2h；
5) freezing：The mixed liquor freeze-drying that step 4) is obtained, the freezing at -80 DEG C, every 8~12 The moon carries out activation switching.Freeze drying process is：Pre-freeze：- 35 DEG C of pre-freeze 4h；Freeze-drying：- 60 DEG C, 40Pa freeze-dryings 6h, -20 DEG C, 70Pa is freeze-dried 2.2h；Lyophilization：10 DEG C, 140Pa lyophilizations 3.5h；Parsing-desiccation:16 DEG C, 4Pa solutions The dry 3h of analysis.The correct folding that the present invention is improved the mobility of cell membrane by cold and hot Stress treatment, effectively improves albumen, changes Kind nitrococcus reduces damage of the freeze-drying to thalline to the resistance of freeze-drying.And can also have under -80 DEG C of cryogenic conditions Effect adjusts translation and the transcription of DNA, and the mutation probability of nitrococcus is greatly reduced, retains original excellent performance.Pass through The synergistic effect of protective agent and carrier can significantly prevent the formation of ice crystal during thalline freezes, and it is double to stablize phosphatide Molecular layer and Membrane protein conformation, and the synthesis of unsaturated fatty acid in nitrococcus thalline is induced, increase insatiable hunger in membrane lipid With the ratio of aliphatic acid, the injury of ice crystal formation and intracellular osmotic pressure to cell membrane is significantly reduced, the stream of cell membrane is maintained Dynamic property, integrality, ATP enzyme (Mg2+ATPase, Ca2+ATPase, Na+-K+ATPase activity) and ammona monooxygenase (AMO) Activity is reduced because freezing or moisture constantly distil to the damage caused by nitrococcus cell.Pass through control freezing speed and Asia Nitrifier cell balances each other to the permeability of moisture, and freeze-drying is preferably minimized the damage of thalline.Thalline is in dry, scarce Oxygen and low-temperature condition, vital movement are in suspend mode, can achieve the purpose that long term storage.
Embodiment 7：Damaged membrane situation
Damaged membrane situation is indicated with the ratio of the undamaged cells on total cells of cell membrane, selects LIVE/ BacLightTWBacterialViability fluorescent dyes to after NPs stress tests sample dyeing hatching after in fluorescence microscopy The undamaged cell of cell membrane and total cell are counted respectively under mirror, then calculates and obtains.Respectively prepared by Example 1~6 Obtained freeze-dried powder 0.1g is used as test group after 10mL sterile water rehydrations are added, and 10mL sterile waters are as blank control group, respectively It is measured using said determination method.Each test group carries out parallel repetition verification experimental verification with blank control group 3 times, is averaged Value, measurement result are as shown in Figure 1.
As seen from Figure 1 using the present invention be not optimised before technical solution to nitrococcus carry out preservation, cell membrane it is complete Whole property is 90% or so, and the technical solution after being optimized using the present invention carries out preservation to nitrococcus, and the integrality of cell membrane is 99% or so.It can thus be seen that the present invention program is excellent to the protecting effect of nitrococcus cell integrity.
Embodiment 8：AMO enzymatic activitys
With unit interval N02 -The yield of-N characterizes the activity of AMO.Concrete operation step is as follows：
(1) bacterioprotein extracts：Sample after NPs stress tests inclines after 10000rpm centrifuges 10min in major part Clear liquid carries out protein extraction to sediment, and operating procedure refers to one-step method bacterial activity Protein Extraction Reagent specification；
(2) determination of protein concentration：According to BCA determination of protein concentration kit specification operating procedures, to extracted egg It is white to carry out concentration mensuration；
(3) hatch：In the bacterioprotein supernatant to the serum bottle of the sealing of 10mL for taking 200 μ L extractions, 0.01M phosphorus is added Phthalate buffer 1.8mL (wherein contains 2mM (NH4)2S04, pH7.4).Then it in 21 DEG C of shaking tables after shaken cultivation 30min, surveys Determine nitrite nitrogen content；
(4) it calculates:Formula is：
The freeze-dried powder 0.1g that Example 1~6 is prepared respectively is used as test group after 10mL sterile water rehydrations are added, 10mL sterile waters are respectively adopted said determination method and are measured as blank control group.Each test group and blank control group are equal It carries out parallel repetition verification experimental verification 3 times, is averaged.
Technical solution before being not optimised as seen from Figure 2 using the present invention carries out preservation to nitrococcus, and AMO enzymatic activitys are 0.82((μgNO2 -- N)/(mg albumen min)) left and right, nitrococcus is protected using the technical solution after present invention optimization It hides, AMO enzymatic activitys are 0.95 ((μ gNO2 -- N)/(mg albumen min)) left and right.It can thus be seen that the present invention program can be effectively Protect the activity of AMO enzymes.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention, Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.

Claims (3)

1. the method for preserving of nitrococcus, it is characterised in that include the following steps：
1) protective agent is configured：2~4 parts of sodium glutamates, 18~25 parts of tripotassium phosphates and 1~3 are added in 90~120 parts of sterile waters Part cysteine, is uniformly mixed and obtains protective agent；
2) thalline is added：0.8~1.2 times of volume nitrococcus thalline is added in the protective agent that step 1) obtains, concussion is resuspended, Obtain mixed liquor；
3) freezing：The mixed liquor freeze-drying that step 2) is obtained, the freezing at -70~-90 DEG C, every 2~3 The moon carries out activation switching.
2. the method for preserving of nitrococcus according to claim 1, it is characterised in that：It is carried out before the freeze-drying cold Heat stress processing：Obtained mixed liquor is heat-treated 10~20min in 30~40 DEG C of constant temperature, then in 4~8 DEG C of constant temperature processing 1 ~2h.
3. the method for preserving of nitrococcus according to claim 1, it is characterised in that：The freeze drying process is： Pre-freeze：- 40~-30 DEG C of 3~5h of pre-freeze；Freeze-drying：- 70~-60 DEG C, 30~50Pa be freeze-dried 5~7h, -20~-10 DEG C, 60~100Pa is freeze-dried 2~3h；Lyophilization：5~15 DEG C, 100~200Pa lyophilizations, 3~4h；Parsing-desiccation: 10~20 DEG C, 3~5Pa parsing-desiccations, 2~4h.
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