CN107287254A - The zymotechnique of γ polyglutamic acids - Google Patents
The zymotechnique of γ polyglutamic acids Download PDFInfo
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- CN107287254A CN107287254A CN201710576817.7A CN201710576817A CN107287254A CN 107287254 A CN107287254 A CN 107287254A CN 201710576817 A CN201710576817 A CN 201710576817A CN 107287254 A CN107287254 A CN 107287254A
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Abstract
The invention discloses a kind of zymotechnique of γ polyglutamic acids, including：S1：Prepare bacillus subtilis seed liquor；S2：Bacillus subtilis seed liquor is inoculated in liquid state fermentation culture medium, in after 37 DEG C, the 16h of 120r/min Shaking cultures 12, zymotic fluid is obtained；After frequency is used for the 5min of ultrasonication zymotic fluid 3 that 10 20Khz and power are 5W/mL, wherein, the ultrasonication is concretely comprised the following steps：After ultrasound 30s, interval 30s is ultrasonic again；Zymotic fluid Cord blood that ultrasonication is obtained simultaneously adds peptide glycan freezing capsule, and feed supplement is further continued for 37 DEG C, 120r/min Shaking cultures 96h；S3：Zymotic fluid is extracted, purified, γ polyglutamic acids are obtained.The present invention has the advantages that saving cost, yield are high, can be widely applied to technical field of microbial fermentation.
Description
Technical field
The present invention relates to technical field of microbial fermentation, it is more particularly related to a kind of hair of gamma-polyglutamic acid
Ferment technique.
Background technology
Gamma-polyglutamic acid is widely used in fields such as medicine, food, agriculturals, and its economic value is self-evident.
In the prior art, the main stream approach of production gamma-polyglutamic acid is microbe fermentation method.Gamma-polyglutamic acid generating bacterium mainly has four
Kind：Withered grass (natto) bacillus, bacillus licheniformis, high temperature resistant bacillus, Bacillus anthracis.Although sending out microorganism
Ferment method production gamma-polyglutamic acid has carried out numerous studies, but the yield of gamma-polyglutamic acid is still pessimistic.Low yield cause γ-
The production cost of polyglutamic acid is high, limits the production and application of gamma-polyglutamic acid.Due to gamma-polyglutamic acid biosynthesis
Mechanism is still not clear so far, and metabolic pathway is complicated, and gamma-polyglutamic acid yield is improved at present and relies primarily on seed selection excellent species and excellent
Change zymotechnique.Therefore, the yield for improving gamma-polyglutamic acid is large-scale production with applying important topic urgently to be resolved hurrily.
The content of the invention
It is an object of the invention to solve at least the above, and provide the advantage that at least will be described later.
It is a still further object of the present invention to provide a kind of zymotechnique for saving the high gamma-polyglutamic acid of cost, yield.
In order to realize that there is provided a kind of fermentation work of gamma-polyglutamic acid according to object of the present invention and further advantage
Skill, including：S1：Prepare bacillus subtilis seed liquor；S2：Bacillus subtilis seed liquor is inoculated in liquid state fermentation culture medium
In, in after 37 DEG C, 120r/min Shaking cultures 12-16h, obtain zymotic fluid；Frequency is used for 10-20Khz and power is 5W/mL's
After ultrasonication zymotic fluid 3-5min, wherein, the ultrasonication is concretely comprised the following steps：After ultrasound 30s, 30s is spaced
It is ultrasonic again；Zymotic fluid Cord blood that ultrasonication is obtained simultaneously adds peptide glycan freezing capsule, and feed supplement is further continued for 37
DEG C, 120r/min Shaking cultures 96h；S3：Zymotic fluid is extracted, purified, gamma-polyglutamic acid is obtained；Wherein, peptide is prepared to gather
Sugar freezing capsule specific step be：S4：In mass ratio 3:1 mixes peptide glycan and konjaku flour, and quality is put into thereto and is
The magnetized water stirring that 2 times of peptide glycan, 4 DEG C are down to 3 DEG C/min of cooling velocity, are protected in magnetic field intensity for 0.45T magnetic field environment
Temperature keeps standing 24h, obtains peptide glycan mixed liquor；S5：Then peptide glycan mixed liquor is dispensed into multiple starch capsule shells, wherein
Each starch capsule shells contain the peptide glycan mixed liquor that 3g steps s4 is obtained；S6：Gathered again with 5 DEG C/min of cooling velocity by peptide is contained
The starch capsule shells of sugared mixed liquor are cooled to -12 DEG C, are incubated 6h, obtain peptide glycan freezing capsule.
Preferably, the carbon source concentration of the liquid state fermentation culture medium is 10-15g/L.
Preferably, the zymotechnique of described gamma-polyglutamic acid also includes supplying technicses, the specific step of the supplying technicses
Suddenly it is：By after zymotic fluid Cord blood in S2, addition feed supplement maintains carbon source concentration in zymotic fluid to be 40-60g/L.
Preferably, the feed supplement is the mixture of glycerine and maltose.
Preferably, the feed supplement number of times of the supplying technicses is twice；Feed supplement time first time is：It is in S2 that zymotic fluid is low
After temperature is preserved；The interval time of second of feed supplement and first time feed supplement is 36-40h.
Preferably, zymotic fluid Cord blood and add peptide glycan freezing capsule and concretely comprise the following steps：Step one:Prepare precooling
To -12 DEG C of peptide glycan freezing capsule；Step 2:By zymotic fluid 4 DEG C stand 20 minutes after, in super-clean bench by 1/
15min is gradually added step one into zymotic fluid and obtains peptide glycan freezing capsule 4, is gently shaken when adding；Step 3：
The incubator that zymotic fluid is placed into 20 DEG C stands 20 minutes；Step 4:20 minutes will be stood in 30 ° of incubators of zymotic fluid.
Preferably, ultrasonic 30s is concretely comprised the following steps：S7：The ultrasonic wave that frequency is 5W/mL for 10Khz and power is used to send out
Zymotic fluid 2s；S8：Use the ultrasonic fermentation liquid 3s that frequency is 5W/mL for 15Khz and power；S9：Use frequency for 20Khz and
Power is 5W/mL ultrasonic fermentation liquid 15s；S10：Use the ultrasonic fermentation liquid that frequency is 5W/mL for 15Khz and power
5s；S11：Use the ultrasonic fermentation liquid 5s that frequency is 5W/mL for 10Khz and power.
Preferably, the process for preparing the mixture of glycerine and maltose is：S12：Mass fraction is preheated to for 10 water
60 DEG C, mass fraction is slowly added into water for 1 maltose, it is stirring while adding be completely dissolved to maltose after be cooled to room
Suction filtration after temperature, obtains the first mixed solution；S13：The first mixed solution obtained in S12 is complete for 1 glycerine with mass fraction
The second mixed solution is mixed to get entirely, it is standby that the second mixed solution is placed on into 37 ° of incubators.
The present invention at least includes following beneficial effect：
First, compared with prior art, the yield of gamma-polyglutamic acid of the invention is high, and the equipment and reagent that use are equal
It is that laboratory is commonly used, therefore invention is a kind of zymotechnique of cost-effective gamma-polyglutamic acid.
2nd, compared with prior art, the yield of gamma-polyglutamic acid of the invention is up to 45g/L, therefore invention is a kind of section
The zymotechnique of the gamma-polyglutamic acid of cost-saving.
Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Embodiment
With reference to embodiment, the present invention is described in further detail, to make those skilled in the art with reference to specification
Word can be implemented according to this.
It should be noted that experimental technique described in following embodiments, is common process unless otherwise specified, institute
Reagent and material are stated, unless otherwise specified, is commercially obtained.
The present invention provides a kind of zymotechnique of gamma-polyglutamic acid, including：S1：Prepare bacillus subtilis seed liquor；
S2：Bacillus subtilis seed liquor is inoculated in liquid state fermentation culture medium, in 37 DEG C, 120r/min Shaking cultures 12-16h
Afterwards, zymotic fluid is obtained；After frequency is used for the ultrasonication zymotic fluid 3-5min that 10-20Khz and power are 5W/mL, wherein, institute
State concretely comprising the following steps for ultrasonication：After ultrasound 30s, interval 30s is ultrasonic again；The zymotic fluid that ultrasonication is obtained
Cord blood simultaneously adds peptide glycan freezing capsule, and feed supplement is further continued for 37 DEG C, 120r/min Shaking cultures 96h；S3：To zymotic fluid
Extracted, purified, obtain gamma-polyglutamic acid；Wherein, prepare peptide glycan freezing capsule specific step be：S4：By quality
Than 3:1 mixes peptide glycan and konjaku flour, and quality is put into thereto and is stirred for the magnetized water of 2 times of peptide glycan, with cooling velocity 3
DEG C/min is down to 4 DEG C, keep standing 24h for 0.45T magnetic field environment insulation in magnetic field intensity, obtain peptide glycan mixed liquor；S5：So
Peptide glycan mixed liquor is dispensed into multiple starch capsule shells afterwards, wherein each starch capsule shells contain the peptide that 3g steps s4 is obtained
Glycan mixed liquor；S6：The starch capsule shells for containing peptide glycan mixed liquor are cooled to by -12 DEG C, guarantor with 5 DEG C/min of cooling velocity again
Warm 6h, obtains peptide glycan freezing capsule.The present invention during the fermentation, uses ultrasonication zymotic fluid, can extend withered grass gemma
Bacillus is in logarithmic phase and the time of stationary phase；And after ultrasonication zymotic fluid, with the method for Cord blood, delay withered
The multiplication rate and metabolic rate of careless bacillus, make its of short duration in mastery state, and are added during Cord blood
Peptide glycan stimulates the reparation of cell membrane, and adds after low-temperature treatment feed supplement, the carbon source of afterfermentation liquid, in order to avoid fermentation time
Extension causes carbon source not enough, causes cell to enter decline phase in advance.Peptide glycan is prepared into peptide glycan freezing capsule by the present invention, and
And allow it to be stored in -12 DEG C of low-temperature conditions, and be mixed with konjaku flour during peptide glycan freezing capsule is prepared, be all for
Peptide glycan is allowed after zymotic fluid is added to, can slowly be discharged, in order to avoid destruction of the complicated fermentation broth contents to peptide glycan.Together
When, the process of cooling is an interim temperature-fall period, is first cooled to 4 DEG C, is in order to which the temperature difference that prevents from disposably cooling causes greatly
The precipitation of peptide glycan.By temperature from normal temperature drop to 4 DEG C of quick coolings for using 3 DEG C/min be in order to accelerate to test into
Degree, -12 DEG C of quick coolings for using 5 DEG C/min are cooled to from 4 DEG C, partly in order to accelerating experiment progress, the opposing party
Face is to quickly through maximum ice crystal area, prevent ice crystal from destroying the gel state of peptide glycan and konjaku flour formation.In peptide glycan
With konjaku flour formation gel state after, be placed on magnetic field intensity be 0.45T magnetic field environment insulation keep stand 24h, be for
In the case where the structure of gel will not be damaged, make peptide glycan and the konjaku flour adhesion bigger.Prepare peptide glycan freezing capsule
The reagent used is sterile, and it is also sterile to prepare environment, and the peptide glycan freezing capsule prepared is also sterile.
The carbon source concentration of the liquid state fermentation culture medium is 10-15g/L.In earlier fermentation, bacillus subtilis is in slow
Postpone a deadline, be metabolized it is not vigorous, it is necessary to carbon source it is less, therefore in cost consideration, the carbon source concentration of liquid state fermentation culture medium is set
It is scheduled on 10-15g/L.
The zymotechnique of described gamma-polyglutamic acid also includes supplying technicses, and the supplying technicses are concretely comprised the following steps：
By after zymotic fluid Cord blood in S2, addition feed supplement maintains carbon source concentration in zymotic fluid to be 40-60g/L.Liquid state fermentation culture medium
Carbon source concentration be set in 10-15g/L, and be depleted substantially by lag phase carbon source, after cell enters logarithmic phase, carbon source
Concentration is needed in 40-60g/L, could maintain its steady growth.The yield of the feed supplement influence fermentation of addition, adds the volume of feed supplement
It is the smaller the better.
The feed supplement is the mixture of glycerine and maltose.Carbon source selects glycerine and maltose to be that of the invention one is preferred
Scheme, but this is not limited only to, every carbon source that can reach supplementary carbon source purpose is within the scope of the present invention.
The feed supplement number of times of the supplying technicses is twice；Feed supplement time first time is：By zymotic fluid Cord blood in S2
Afterwards；The interval time of second of feed supplement and first time feed supplement is 36-40h.After zymotic fluid Cord blood, bacillus subtilis by
Stepping enters exponential phase, in order to maintain its growth, it is necessary to feed supplement at this moment.And bacillus subtilis is passing through exponential phase
Afterwards, the consuming to carbon source is also larger, if supplementary carbon source can cause it to rapidly enter decline phase not in time, therefore is mended in first time
After material, interval 36-40h carries out second of feed supplement, and second of feed supplement also maintains the carbon source concentration in zymotic fluid to be 40-60g/L.
Zymotic fluid Cord blood simultaneously adds concretely comprising the following steps for peptide glycan freezing capsule：Step one:Preparation is cooled to -12 DEG C in advance
Peptide glycan freezing capsule；Step 2:By zymotic fluid after 4 DEG C stand 20 minutes, 1/15min is pressed in super-clean bench to fermentation
Step one is gradually added in liquid and obtains peptide glycan freezing capsule 4, is gently shaken when adding；Step 3：Zymotic fluid is placed
The incubator for entering 20 DEG C stands 20 minutes；Step 4:20 minutes will be stood in 30 ° of incubators of zymotic fluid.Adding, peptide glycan is cold
Zymotic fluid is cooled to 4 DEG C before frozen capsule, delays the multiplication rate and metabolic rate of bacillus subtilis, allows its of short duration is in repair
The state of supporting, meanwhile, it is easy to it to adapt to the temperature that peptide glycan freezes capsule, adds peptide glycan freezing capsule and progressively heated up by it, allowed
It progressively adapts to fermentation temperature.
Ultrasonic 30s's concretely comprises the following steps：S7：Use the ultrasonic fermentation liquid 2s that frequency is 5W/mL for 10Khz and power；
S8：Use the ultrasonic fermentation liquid 3s that frequency is 5W/mL for 15Khz and power；S9：Frequency is used for 20Khz and power is 5W/
ML ultrasonic fermentation liquid 15s；S10：Use the ultrasonic fermentation liquid 5s that frequency is 5W/mL for 15Khz and power；S11：Using
The ultrasonic fermentation liquid 5s that frequency is 10Khz and power is 5W/mL.Step up ultrasonic frequency, allow bacillus subtilis by
Step adapts to the stimulation of ultrasonic wave, after ultrasonic wave reaches peak frequency processing, then gradually reduces ultrasonic frequency, allows it progressively to fit
Should the environment without ultrasonic stimulation, this gradient, which is set, to be provided to protect bacillus subtilis.
The process for preparing the mixture of glycerine and maltose is：S12：Mass fraction is preheated to 60 DEG C for 10 water, will
Mass fraction is slowly added into water for 1 maltose, it is stirring while adding be completely dissolved to maltose after be cooled to after room temperature and take out
Filter, obtains the first mixed solution；S13：The the first mixed solution mass fraction obtained in S12 is thoroughly mixed for 1 glycerine
To the second mixed solution, the second mixed solution is placed on 37 DEG C of incubators standby.It will be preheating to for the water for dissolving maltose
60 DEG C, accelerate maltose dissolving speed, this kind of mode than maltose is put into the method progressively dissolved by heating again in water by
It is hot uniform, it is to avoid the situation of the too high destruction maltose of local temperature occur.Maltose, water, the quality parts ratio of glycerine are 1:
10:1, it is only the preferred scheme of the present invention.In the environment that second mixed solution is put into 37 DEG C, it is to avoid the addition of feed supplement is to hair
The influence of zymotic fluid temperature.
The embodiment of the present invention one：
(1) preparation of seed liquor
Produce the bacillus subtilis that gamma-polyglutamic acid is used.Seed culture medium is constituted：Peptone 10g/L, yeast extract
5g/L, NaCl 10g/L, pH7.0, remaining is distilled water or running water, liquid amount 50mL/250mL triangular flasks, in 121 DEG C of sterilizings
30min, after cooling, the bacillus subtilis for taking a ring to pre-save in slant medium is inoculated in seed culture medium, 37
DEG C, 120r/min Shaking culture 24h, bacillus subtilis seed liquor.
(2) shake flask fermentation culture
The composition of the liquid state fermentation culture medium：Peptone 3g/L, citric acid 1g/L, glucose 6g/L, glutamic acid 15g/
L, NH4Cl 7g/L, K2HPO4 0.5g/L,MgSO4·7H2O 0.5g/L, FeCl3·6H2O 0.04g/L, CaCl2·2H2O
0.15g/L, MnSO4·H2O 0.104g/L, initial pH value is 7.0.Bacillus subtilis seed liquor presses 1%~6% inoculum concentration
Be inoculated in liquid state fermentation culture medium, liquid amount 1L/3L plastics shaking flasks, 37 DEG C, 120r/min Shaking culture 12h, use frequency for
After 10-20Khz and power is 5W/mL ultrasonication zymotic fluid 3min, wherein, the specific steps of the ultrasonication
For：After ultrasound 30s, interval 30s is ultrasonic again；Zymotic fluid Cord blood that ultrasonication is obtained and to add peptide glycan cold
Frozen capsule, feed supplement is further continued for 37 DEG C, second of feed supplement after 120r/min Shaking cultures 36h, is further continued for 37 DEG C, 120r/min shakes
Bottle culture 60h is terminated.
Wherein, the feed supplement is the mixture of glycerine and maltose, mends zymotic fluid Cord blood and second in S2
After material, addition feed supplement maintains carbon source concentration in zymotic fluid to be 40g/L.
(3) extraction of gamma-polyglutamic acid
By zymotic fluid in centrifuging 30min, supernatant 1mol/L HCl tune pH to 3.0 under 3000r/min, 4 times of bodies are added
95% long-pending ethanol, is put in fridge overnight at 4 DEG C.Then in centrifuging 30min under 3000r/min, ethanol washing precipitate is used,
Collect viscous precipitate thing, dry gamma-polyglutamic acid product, the yield of gained gamma-polyglutamic acid is 38g/L.
The embodiment of the present invention two：
(1) preparation of seed liquor, ibid.
(2) Shaking culture
The composition of the liquid state fermentation culture medium：Peptone 4g/L, citric acid 1.5g/L, glucose 7g/L, glutamic acid
15g/L, NH4Cl 7g/L, K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, FeCl3·6H2O 0.04g/ L, CaCl2·
2H2O 0.15g/L, MnSO4·H2O 0.104g/L, initial pH value is 7.0.Bacillus subtilis seed liquor connects by 1%~6%
The amount of kind is inoculated in liquid state fermentation culture medium, liquid amount 1L/3L plastics shaking flasks, 37 DEG C, 120r/min Shaking culture 14h, using frequency
After the ultrasonication zymotic fluid 4min that rate is 10-20Khz and power is 5W/mL, wherein, the specific step of the ultrasonication
Suddenly it is：After ultrasound 30s, interval 30s is ultrasonic again；Zymotic fluid Cord blood that ultrasonication is obtained simultaneously adds peptide glycan
Freeze capsule, feed supplement is further continued for 37 DEG C, second of feed supplement after 120r/min Shaking cultures 38h, is further continued for 37 DEG C, 120r/min
Shaking culture 58h is terminated.
Wherein, the feed supplement is the mixture of glycerine and maltose, mends zymotic fluid Cord blood and second in S2
After material, addition feed supplement maintains carbon source concentration in zymotic fluid to be 50g/L.
(3) extraction of gamma-polyglutamic acid, ibid, the yield of gained gamma-polyglutamic acid is 41g/L.
The embodiment of the present invention three：
(1) preparation of seed liquor, ibid.
(2) Shaking culture
The composition of the liquid state fermentation culture medium：Peptone 5g/L, citric acid 2g/L, glucose 8g/L, glutamic acid 15g/
L, NH4Cl 7g/L, K2HPO40.5g/L, MgSO4·7H2O 0.5g/L, FeCl3·6H2O 0.04g/L, CaCl2·2H2O
0.15g/L, MnSO4·H2O 0.104g/L, initial pH value is 7.0.Bacillus subtilis seed liquor is connect by 1%~6% inoculum concentration
Plant in liquid state fermentation culture medium, liquid amount 1L/3L plastics shaking flasks, 37 DEG C, 120r/min Shaking culture 16h use frequency for 10-
After 20Khz and power is 5W/mL ultrasonication zymotic fluid 5min, wherein, the ultrasonication is concretely comprised the following steps：Often
After ultrasonic 30s, interval 30s is ultrasonic again；Zymotic fluid Cord blood that ultrasonication is obtained simultaneously adds peptide glycan frozen rubber
Capsule, feed supplement is further continued for 37 DEG C, second of feed supplement after 120r/min Shaking cultures 40h, is further continued for 37 DEG C, the training of 120r/min shaking flasks
56h is supported to terminate.
Wherein, the feed supplement is the mixture of glycerine and maltose, mends zymotic fluid Cord blood and second in S2
After material, addition feed supplement maintains carbon source concentration in zymotic fluid to be 60g/L.
(3) extraction of gamma-polyglutamic acid, ibid, the yield of gained gamma-polyglutamic acid is 45g/L.
Comparative example：
In the zymotechnique of existing gamma-polyglutamic acid, the average yield of gamma-polyglutamic acid is 33g/L.
| The yield (g/L) of gamma-polyglutamic acid | |
| Embodiment one | 38 |
| Embodiment two | 41 |
| Embodiment three | 45 |
| Comparative example | 33 |
From form, the yield of three embodiments of the invention is higher than comparative example.
Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed
With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art
Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited
In specific details and shown here as the embodiment with description.
Claims (8)
1. a kind of zymotechnique of gamma-polyglutamic acid, it is characterised in that including：
S1：Prepare bacillus subtilis seed liquor；
S2：Bacillus subtilis seed liquor is inoculated in liquid state fermentation culture medium, in 37 DEG C, 120r/min Shaking cultures 12-
After 16h, zymotic fluid is obtained；
After frequency is used for the ultrasonication zymotic fluid 3-5min that 10-20Khz and power are 5W/mL, wherein, the ultrasonic wave
What is handled concretely comprises the following steps：After ultrasound 30s, interval 30s is ultrasonic again；
Zymotic fluid Cord blood that ultrasonication is obtained simultaneously adds peptide glycan freezing capsule, feed supplement, be further continued for 37 DEG C,
120r/min Shaking cultures 96h；
S3：Zymotic fluid is extracted, purified, gamma-polyglutamic acid is obtained；
Wherein, prepare peptide glycan freezing capsule specific step be：
S4：In mass ratio 3:1 mixes peptide glycan and konjaku flour, and quality is put into thereto and is stirred for the magnetized water of 2 times of peptide glycan,
4 DEG C are down to 3 DEG C/min of cooling velocity, keeps standing 24h for 0.45T magnetic field environment insulation in magnetic field intensity, obtains peptide glycan
Mixed liquor；
S5：Then peptide glycan mixed liquor is dispensed into multiple starch capsule shells, wherein each starch capsule shells contain 3g steps
The peptide glycan mixed liquor that s4 is obtained；
S6：The starch capsule shells for containing peptide glycan mixed liquor are cooled to -12 DEG C with 5 DEG C/min of cooling velocity again, 6h is incubated, obtains
Peptide glycan freezes capsule.
2. the zymotechnique of gamma-polyglutamic acid as claimed in claim 1, it is characterised in that the liquid state fermentation culture medium
Carbon source concentration is 10-15g/L.
3. the zymotechnique of gamma-polyglutamic acid as claimed in claim 2, it is characterised in that also including supplying technicses, the benefit
Expect concretely comprising the following steps for technique：By after zymotic fluid Cord blood in S2, addition feed supplement maintains carbon source concentration in zymotic fluid to be 40-
60g/L。
4. the zymotechnique of gamma-polyglutamic acid as claimed in claim 3, it is characterised in that the feed supplement is glycerine and malt
The mixture of sugar.
5. the zymotechnique of gamma-polyglutamic acid as claimed in claim 3, it is characterised in that the feed supplement time of the supplying technicses
Number is twice；Feed supplement time first time is：By after zymotic fluid Cord blood in S2；Between second of feed supplement and first time feed supplement
It is 36-40h every the time.
6. the zymotechnique of gamma-polyglutamic acid as claimed in claim 1, it is characterised in that zymotic fluid Cord blood is simultaneously added
Peptide glycan freezes concretely comprising the following steps for capsule：
Step one:Prepare the pre- peptide glycan freezing capsule for being cooled to -12 DEG C；
Step 2:By zymotic fluid after 4 DEG C stand 20 minutes, it is gradually added in super-clean bench by 1/15min into zymotic fluid
Step one obtains peptide glycan freezing capsule 4, is gently shaken when adding；
Step 3：The incubator that zymotic fluid is placed into 20 DEG C stands 20 minutes；
Step 4:20 minutes will be stood in 30 ° of incubators of zymotic fluid.
7. the zymotechnique of gamma-polyglutamic acid as claimed in claim 1, it is characterised in that ultrasonic 30s's concretely comprises the following steps：
S7：Use the ultrasonic fermentation liquid 2s that frequency is 5W/mL for 10Khz and power；
S8：Use the ultrasonic fermentation liquid 3s that frequency is 5W/mL for 15Khz and power；
S9：Use the ultrasonic fermentation liquid 15s that frequency is 5W/mL for 20Khz and power；
S10：Use the ultrasonic fermentation liquid 5s that frequency is 5W/mL for 15Khz and power；
S11：Use the ultrasonic fermentation liquid 5s that frequency is 5W/mL for 10Khz and power.
8. the zymotechnique of gamma-polyglutamic acid as claimed in claim 4, it is characterised in that prepare the mixed of glycerine and maltose
The process of compound is：
S12：Mass fraction is preheated to 60 DEG C for 10 water, mass fraction is slowly added into water for 1 maltose, Bian Jia
While stir to maltose be completely dissolved after be cooled to suction filtration after room temperature, obtain the first mixed solution；
S13：The first mixed solution obtained in S12 and mass fraction are thoroughly mixed for 1 glycerine and obtain the second mixed solution,
Second mixed solution is placed on 37 DEG C of incubators standby.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113528404A (en) * | 2021-08-31 | 2021-10-22 | 淮阴师范学院 | Method for improving yield of bacillomycin D based on segmented fermentation of corn straw enzymatic hydrolysate base material |
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| CN113528404A (en) * | 2021-08-31 | 2021-10-22 | 淮阴师范学院 | Method for improving yield of bacillomycin D based on segmented fermentation of corn straw enzymatic hydrolysate base material |
| CN113698244A (en) * | 2021-08-31 | 2021-11-26 | 武汉光华时代生物科技有限公司 | Application of poly-gamma-glutamic acid as fertilizer synergist in crops |
| CN115820758A (en) * | 2023-02-07 | 2023-03-21 | 山东嘉禾海洋生物科技有限公司 | Polyglutamic acid fermentation liquor and preparation method and application thereof |
| CN115820758B (en) * | 2023-02-07 | 2023-04-14 | 山东嘉禾海洋生物科技有限公司 | Polyglutamic acid fermentation liquor and preparation method and application thereof |
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