CN107699595A - A kind of method of Production by Microorganism Fermentation L hydroxyprolines - Google Patents
A kind of method of Production by Microorganism Fermentation L hydroxyprolines Download PDFInfo
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- CN107699595A CN107699595A CN201711214652.5A CN201711214652A CN107699595A CN 107699595 A CN107699595 A CN 107699595A CN 201711214652 A CN201711214652 A CN 201711214652A CN 107699595 A CN107699595 A CN 107699595A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/24—Proline; Hydroxyproline; Histidine
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Abstract
The invention discloses a kind of method of Production by Microorganism Fermentation L hydroxyprolines, belong to Optimization of fermentation process field.By microorganism in fermentation tank growth metabolism by culture medium raw material（Predominantly glycerine）It is converted into the fermentation production technology of L hydroxyprolines.By using the culture medium prescription of optimization in fermentation process, the control of optimal dissolved oxygen, temperature control, reach the optimal product accumulation state of microorganism.Fermentation manufacturing technique provided by the present invention is simple, high yield, no waste water,waste gas and industrial residue pollution.
Description
Technical field
A kind of method of Production by Microorganism Fermentation L- hydroxyprolines, belongs to Optimization of fermentation process field.
Background technology
L- hydroxyprolines are imino acid, are the chief components of collagen, are not belonging to 20 kinds of common amino acid,
It is the product after proline hydroxylating.In the last few years, the research and development of L- hydroxyprolines had caused medicine, biochemical, food
The extensive concern of the industry such as product and beauty.It can be used as cosmetic additive agent, have anti-oxidant, radiation-resistant effect；It has
There is antiobesity action, it is expected to as more satisfactory slimming medicine；It has different physiological roles and unique bioactivity, both may be used
Using the medicine as various soft tissue diseases, such as Jie Ti tissue damageds, rheumatic arthritis, wound healing can be accelerated again,
And the various skin diseases for the treatment of；It can be as the important component of amino acid injection, and it is to low caused by acute and chronic hepatitis
Proteinemia has the effect of certain；It participates in the emulsification of fat and the formation of red blood cell ferroheme and globulin, has regulation fat
The effects such as fat emulsification；The synthesis of it or multi-medicament, such as third generation antibiotic, antitumor, anti-hypertension and new stomach medicine
Raw material.
The content of the invention
It is an object of the invention to provide a kind of technique that L- hydroxyprolines are produced using microbial fermentation, this method can incite somebody to action
Fermentation raw material（Predominantly glucose）L- hydroxyprolines efficiently are converted into, there is higher turn relative to current fermentation technique
Rate, relatively low cost and environment friendly and pollution-free.
According to experimental result, it can be deduced that during microbial fermentation produces L- hydroxyprolines, different nutritional ingredients
Formula, the control of different dissolved oxygens, different temperatures control, have a great influence to ultimate output.In order to which the maximum amount of raising raw material is with setting
Standby utilization rate, the time required to shortening fermentation, increase the yield of L- hydroxyprolines, the present invention proposes a kind of stable through overtesting
High yield tech method, make microorganism in the nutrient formulation of more conducively product accumulation, dissolved oxygen, fermented under temperature conditionss.Tool
Body operating method is as follows.
Technical scheme：Microorganism fungus kind is seeded to Shaking culture by glycerol tube and activated, and then moves to seed
Tank culture expands, and seed tank culture terminates to move to fermentation tank, control fermentation tank parameters to fermentation ends.Specific steps are such as
Under：
Step 1, Shaking culture based formulas：Yeast extract 5g/L, the g/L of peptone 10, the g/L of sodium chloride 10, configure 150mL
In 1000mL conical flasks, sterilized in high pressure steam sterilization cabinet, 121 DEG C, 0.1MPa sterilizing 20min, after being cooled to room temperature, inoculation
Glycerol tube strain, shaking table culture 12h, shaking speed 200rpm, 36 DEG C of temperature；
Step 2, seed tank culture base are：Yeast extract 6g/L, the g/L of peptone 12, the g/L of sodium chloride 10, defoamer 0.1
G/L, configuration 15L 121 DEG C of high temperature and high pressure steam sterilizing pressurize 0.1MPa, insulation maintenance 15min, are cooled in 50L fermentation tanks
36℃；Shaking culture terminates the seeding tank that rear nutrient solution is moved to after sterilizing cooling, inoculum concentration 150ml, accounts for seeding tank feed liquid body
Product 1%, regulation seeding tank rotating speed 250rpm, ventilation ratio 3vvm, 36 DEG C, pressure 0.05MPa, incubation time 4h of temperature after inoculation；
Step 3, fermentation tank culture based formulas are：The g/L of yeast extract 12, the g/L of glycerine 80, the g/L of ammonium sulfate 10, di(2-ethylhexyl)phosphate
The g/L of hydrogen potassium 4, the g/L of magnesium sulfate 2, the g/L of sodium chloride 2, the g/L of calcium chloride 0.2, the g/L of zinc chloride 0.02, manganese sulfate 0.0002
G/L, the g/L of ferrous sulfate 6, the g/L of defoamer 0.6,20L is in 50L fermentation tanks for configuration, high temperature and high pressure steam sterilizing pressurize
121 DEG C of 0.1MPa, insulation maintenance 15min, are cooled to 36 DEG C.
Step 4, seed tank culture terminate in the fermentation tank after rear culture transferring to the cooling that sterilizes, and fermentation tank culture transferring amount is 5L, is accounted for
The 25% of the initial material liquid volume of fermentation tank, adjusts fermentation tank rotating speed 250rpm after culture transferring, ventilation ratio 2vvm, 36 DEG C of temperature initial value,
Pressure 0.05MPa, correction dissolved oxygen initial value are 100%；
Step 5, speed of agitator is associated with dissolved oxygen level DO values during fermentation tank culture, speed of agitator and dissolved oxygen level DO
Value association is realized by fermentation tank automation control system, when dissolved oxygen level drops to 35% by initial DO values 100%, speed of agitator
Automatically adjust and maintain DO35%, it is 25% that dissolved oxygen DO values are set after fermentation period 20h, and rotating speed automatically adjusts DO25% to fermentation ends；
Step 6, it is 36 DEG C to control temperature in cycle 0h~20h during fermentation tank culture, and fermentation temperature control after 20h as 33
DEG C, until fermentation ends, when L- hydroxyprolines amount is not further added by zymotic fluid, terminate fermentation.
According to experimental result, using the method can significantly improve the yield of L- hydroxyprolines.L- hydroxyls provided by the present invention
Proline zymotechnique is simple, efficient, has important industrial application value, while the fermentation method of other amino acid etc. is produced
With certain guidance and edify meaning.
Embodiment
Microbiological Culture Collection used in the present invention uses glycerol tube ultralow temperature preservation.Fermentation process uses second order fermentation tank
Fermentation.Present invention experiment fermentation tank used is 50L fermentation tanks.
Nutritional ingredient used in 50L tanks of the present invention contained by culture medium and the nutrition contained by industrial production used medium
Composition is identical, so the achievement reached can be easily transformed into commercial scale level very much.
The present invention is in the technique of existing microbial fermentation production L- hydroxyprolines, by improving fermented and cultured basigamy
Side, carbon source nitrogen source vitamin and microelement content ratio is optimal, the control different from temperature two benches of process control dissolved oxygen level DO values
System is horizontal.Experiment shows, fermentation process early stage is mainly the thalli growth stage, and middle and later periods product accumulation is very fast.In many experiments
On the basis of present invention optimizes fermentative medium formula, and it is horizontal, no to propose earlier fermentation and middle and later periods difference dissolved oxygen DO value
Synthermal horizontal control mode, concrete scheme citing：0~20h control DO values 35%, 36 DEG C of temperature, 20h controls DO values afterwards
25%, 33 DEG C of temperature.
As a result L- hydroxyprolines zymotechnique stable yield proposed by the present invention is shown, feed liquid L- hydroxyl dried meat ammonia during fermentation ends
Sour average content reaches 55g/L.38% is improved compared with traditional zymotic technique.And fermentation broth contents are more simple, be advantageous to after simplifying
Continuous extraction and purification process.With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is simultaneously
It is not limited only to this：
Embodiment 1：Microbiological Culture Collection used in the present invention uses glycerol tube ultralow temperature preservation.Fermentation process is sent out using two level
Fermenting pot.Present invention experiment fermentation tank used is 50L fermentation tanks.
Configure the culture of 150mL medicine bottles to be based in 1000mL conical flasks, Shaking culture based formulas：Yeast extract 5g/L, egg
White peptone 10 g/L, the g/L of sodium chloride 10.Sterilized in high pressure steam sterilization cabinet, 121 DEG C, 0.1MPa sterilizings 20min.It is cooled to room temperature
Afterwards, glycerol tube strain, shaking table culture 12h, shaking speed 200rpm, 36 DEG C of temperature are inoculated with.
Seed tank culture base 15L is configured in 50L fermentation tanks, seed tank culture base is：Yeast extract 6g/L, peptone
12 g/L, the g/L of sodium chloride 10, the g/L of defoamer 0.1.121 DEG C of high temperature and high pressure steam sterilizing pressurize 0.1MPa, insulation maintenances
15min.It is cooled to 36 DEG C.Shaking culture terminates the seeding tank that rear nutrient solution is moved to after sterilizing cooling, inoculum concentration 150ml, accounts for
Seeding tank material liquid volume 1%.Regulation seeding tank rotating speed 250rpm, ventilation ratio 3vvm, 36 DEG C of temperature, pressure 0.05MPa after inoculation.
Incubation time 4h.
Fermentation tank culture medium 20L is configured in 50L fermentation tanks, fermentation tank culture based formulas is：The g/L of yeast extract 12,
The g/L of glycerine 80, the g/L of ammonium sulfate 10, the g/L of potassium dihydrogen phosphate 4, the g/L of magnesium sulfate 2, the g/L of sodium chloride 2, the g/ of calcium chloride 0.2
L, the g/L of zinc chloride 0.02, the g/L of manganese sulfate 0.0002, the g/L of ferrous sulfate 6, the g/L of defoamer 0.6.High temperature and high pressure steam goes out
121 DEG C of bacterium pressurize 0.1MPa, insulation maintenance 15min.It is cooled to 36 DEG C.
Seed tank culture terminates in the fermentation tank after rear culture transferring to the cooling that sterilizes, and fermentation tank culture transferring amount is 5L, accounts for fermentation tank
The 25% of initial material liquid volume.Regulation fermentation tank rotating speed 250rpm, ventilation ratio 2vvm, 36 DEG C of temperature initial value, pressure after culture transferring
0.05MPa, the initial DO values of correction dissolved oxygen level are 100%.Speed of agitator is related to dissolved oxygen level DO values during fermented and cultured
Connection, speed of agitator is associated with dissolved oxygen level DO values and realized by fermentation tank automation control system, when dissolved oxygen level is by initial value
100% drops to 35%, and speed of agitator, which automatically adjusts, maintains DO35%.It is 25% that dissolved oxygen DO values are set after fermentation period 20h, and rotating speed is certainly
The dynamic DO25% that adjusts is to fermentation ends.It is 36 DEG C to control temperature in cycle 0h~20h during fermentation tank culture, and hair is controlled after 20h
Ferment temperature is 33 DEG C, until fermentation ends.When L- hydroxyprolines amount is not further added by zymotic fluid, terminate fermentation.Final fermentation
Liquid L- Hydroxyproline concentrations are 55.9g/L.
Embodiment 2：Microbiological Culture Collection used in the present invention uses glycerol tube ultralow temperature preservation.Fermentation process uses two
Level ferment tank.Present invention experiment fermentation tank used is 50L fermentation tanks.
Configure the culture of 150mL medicine bottles to be based in 1000mL conical flasks, Shaking culture based formulas：Yeast extract 5g/L, egg
White peptone 10 g/L, the g/L of sodium chloride 10.Sterilized in high pressure steam sterilization cabinet, 121 DEG C, 0.1MPa sterilizings 20min.It is cooled to room temperature
Afterwards, glycerol tube strain, shaking table culture 12h, shaking speed 200rpm, 36 DEG C of temperature are inoculated with.
Seed tank culture base 15L is configured in 50L fermentation tanks, seed tank culture base is：Yeast extract 6g/L, peptone
12 g/L, the g/L of sodium chloride 10, the g/L of defoamer 0.1.121 DEG C of high temperature and high pressure steam sterilizing pressurize 0.1MPa, insulation maintenances
15min.It is cooled to 36 DEG C.Shaking culture terminates the seeding tank that rear nutrient solution is moved to after sterilizing cooling, inoculum concentration 150ml, accounts for
Seeding tank material liquid volume 1%.Regulation seeding tank rotating speed 250rpm, ventilation ratio 3vvm, 36 DEG C of temperature, pressure 0.05MPa after inoculation.
Incubation time 4h.
Fermentation tank culture medium 20L is configured in 50L fermentation tanks, fermentation tank culture based formulas is：The g/L of yeast extract 12,
The g/L of glycerine 80, the g/L of ammonium sulfate 10, the g/L of potassium dihydrogen phosphate 4, the g/L of magnesium sulfate 2, the g/L of sodium chloride 2, the g/ of calcium chloride 0.2
L, the g/L of zinc chloride 0.02, the g/L of manganese sulfate 0.0002, the g/L of ferrous sulfate 6, the g/L of defoamer 0.6.High temperature and high pressure steam goes out
121 DEG C of bacterium pressurize 0.1MPa, insulation maintenance 15min.It is cooled to 36 DEG C.
Seed tank culture terminates in the fermentation tank after rear culture transferring to the cooling that sterilizes, and fermentation tank culture transferring amount is 5L, accounts for fermentation tank
The 25% of initial material liquid volume.Regulation fermentation tank rotating speed 250rpm, ventilation ratio 2vvm, 36 DEG C of temperature initial value, pressure after culture transferring
0.05MPa, the initial DO values of correction dissolved oxygen level are 100%.Speed of agitator is related to dissolved oxygen level DO values during fermented and cultured
Connection, speed of agitator is associated with dissolved oxygen level DO values and realized by fermentation tank automation control system, when dissolved oxygen level is by initial value
100% drops to 35%, and speed of agitator, which automatically adjusts, maintains DO35%.It is 25% that dissolved oxygen DO values are set after fermentation period 20h, and rotating speed is certainly
The dynamic DO25% that adjusts is to fermentation ends.It is 36 DEG C to control temperature in cycle 0h~20h during fermentation tank culture, and hair is controlled after 20h
Ferment temperature is 33 DEG C, until fermentation ends.When L- hydroxyprolines amount is not further added by zymotic fluid, terminate fermentation.Final fermentation
Liquid L- Hydroxyproline concentrations are 55.1g/L.
Embodiment 3：Microbiological Culture Collection used in the present invention uses glycerol tube ultralow temperature preservation.Fermentation process uses two
Level ferment tank.Present invention experiment fermentation tank used is 50L fermentation tanks.
Configure the culture of 150mL medicine bottles to be based in 1000mL conical flasks, Shaking culture based formulas：Yeast extract 5g/L, egg
White peptone 10 g/L, the g/L of sodium chloride 10.Sterilized in high pressure steam sterilization cabinet, 121 DEG C, 0.1MPa sterilizings 20min.It is cooled to room temperature
Afterwards, glycerol tube strain, shaking table culture 12h, shaking speed 200rpm, 36 DEG C of temperature are inoculated with.
Seed tank culture base 15L is configured in 50L fermentation tanks, seed tank culture base is：Yeast extract 6g/L, peptone
12 g/L, the g/L of sodium chloride 10, the g/L of defoamer 0.1.121 DEG C of high temperature and high pressure steam sterilizing pressurize 0.1MPa, insulation maintenances
15min.It is cooled to 36 DEG C.Shaking culture terminates the seeding tank that rear nutrient solution is moved to after sterilizing cooling, inoculum concentration 150ml, accounts for
Seeding tank material liquid volume 1%.Regulation seeding tank rotating speed 250rpm, ventilation ratio 3vvm, 36 DEG C of temperature, pressure 0.05MPa after inoculation.
Incubation time 4h.
Fermentation tank culture medium 20L is configured in 50L fermentation tanks, fermentation tank culture based formulas is：The g/L of yeast extract 12,
The g/L of glycerine 80, the g/L of ammonium sulfate 10, the g/L of potassium dihydrogen phosphate 4, the g/L of magnesium sulfate 2, the g/L of sodium chloride 2, the g/ of calcium chloride 0.2
L, the g/L of zinc chloride 0.02, the g/L of manganese sulfate 0.0002, the g/L of ferrous sulfate 6, the g/L of defoamer 0.6.High temperature and high pressure steam goes out
121 DEG C of bacterium pressurize 0.1MPa, insulation maintenance 15min.It is cooled to 36 DEG C.
Seed tank culture terminates in the fermentation tank after rear culture transferring to the cooling that sterilizes, and fermentation tank culture transferring amount is 5L, accounts for fermentation tank
The 25% of initial material liquid volume.Regulation fermentation tank rotating speed 250rpm, ventilation ratio 2vvm, 36 DEG C of temperature initial value, pressure after culture transferring
0.05MPa, the initial DO values of correction dissolved oxygen level are 100%.Speed of agitator is related to dissolved oxygen level DO values during fermented and cultured
Connection, speed of agitator is associated with dissolved oxygen level DO values and realized by fermentation tank automation control system, when dissolved oxygen level is by initial value
100% drops to 35%, and speed of agitator, which automatically adjusts, maintains DO35%.It is 25% that dissolved oxygen DO values are set after fermentation period 20h, and rotating speed is certainly
The dynamic DO25% that adjusts is to fermentation ends.It is 36 DEG C to control temperature in cycle 0h~20h during fermentation tank culture, and hair is controlled after 20h
Ferment temperature is 33 DEG C, until fermentation ends.When L- hydroxyprolines amount is not further added by zymotic fluid, terminate fermentation.Final fermentation
Liquid L- Hydroxyproline concentrations are 55.3g/L.
Described above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
Those of ordinary skill for, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (7)
- A kind of 1. method of Production by Microorganism Fermentation L- hydroxyprolines, it is characterised in that including following operating procedure：Step 1：Configure 150mL Shaking cultures to be based in 1000mL conical flasks, Shaking culture based formulas：Yeast extract 5g/L, The g/L of peptone 10, the g/L of sodium chloride 10, high pressure steam sterilization cabinet is interior to sterilize, and 121 DEG C, 0.1MPa sterilizing 20min, is cooled to room Wen Hou, it is inoculated with glycerol tube strain, shaking table culture；Step 2：Seed tank culture base 15L is configured in 50L fermentation tanks, seed tank culture base is：Yeast extract 6g/L, egg White peptone 12 g/L, the g/L of sodium chloride 10, the g/L of defoamer 0.1,121 DEG C of high temperature and high pressure steam sterilizing pressurize 0.1MPa, insulation dimensions 15min is held, 36 DEG C is cooled to, then connects shake-flask seed；Step 3：Configure 20L fermented and cultureds to be based in 50L fermentation tanks, high temperature and high pressure steam sterilizing pressurize 0.1MPa, insulation 121 DEG C maintain 15min, be cooled to 36 DEG C；Step 4：Seed tank culture terminates in the fermentation tank after rear culture transferring to the cooling that sterilizes；Step 5：Speed of agitator is associated with dissolved oxygen level DO values during fermented and cultured, speed of agitator and dissolved oxygen level DO values Association is realized by fermentation tank automation control system, and the control of two benches difference dissolved oxygen is carried out in fermentation process；Step 6：Two benches different temperatures control is carried out during fermentation tank culture, up to L- hydroxyl dried meat in fermentation ends, zymotic fluid When propylhomoserin amount is not further added by, terminate fermentation.
- 2. the method for Production by Microorganism Fermentation L- hydroxyprolines as claimed in claim 1, it is characterised in that configuration 150mL Shaking culture is based in 1000mL conical flasks, disinfection inoculation, shaking table culture 12h, shaking speed 200rpm, 36 DEG C of temperature.
- 3. the method for Production by Microorganism Fermentation L- hydroxyprolines as claimed in claim 1, it is characterised in that Shaking culture knot The seeding tank that nutrient solution is moved to after sterilizing cooling after beam, inoculum concentration 150ml, accounts for seeding tank material liquid volume 1%, is adjusted after inoculation Seeding tank rotating speed 250rpm, ventilation ratio 3vvm, 36 DEG C, pressure 0.05MPa, incubation time 4h of temperature.
- 4. the method for Production by Microorganism Fermentation L- hydroxyprolines as claimed in claim 1, it is characterised in that fermentation tank culture Based formulas is：The g/L of yeast extract 12, the g/L of glycerine 80, the g/L of ammonium sulfate 10, the g/L of potassium dihydrogen phosphate 4, the g/ of magnesium sulfate 2 L, the g/L of sodium chloride 2, the g/L of calcium chloride 0.2, the g/L of zinc chloride 0.02, manganese sulfate .0002 g/L, the g/L of ferrous sulfate 6, defoaming The g/L of agent 0.6.
- 5. the method for Production by Microorganism Fermentation L- hydroxyprolines as claimed in claim 1, it is characterised in that move to fermentation tank Kind of amount be 5L, accounts for the 25% of the initial material liquid volume of fermentation tank, regulation fermentation tank rotating speed 250rpm, ventilation ratio 2vvm, temperature after culture transferring 36 DEG C, pressure 0.05MPa of initial value, the initial DO values of correction dissolved oxygen level are 100%.
- 6. the method for Production by Microorganism Fermentation L- hydroxyprolines as claimed in claim 1, it is characterised in that fermentation tank culture During cycle 0h~20h controls dissolved oxygen level DO values be 35%, 20h after control the dissolved oxygen level DO values to be 25%, until fermentation Terminate.
- 7. the method for Production by Microorganism Fermentation L- hydroxyprolines as claimed in claim 1, it is characterised in that fermentation tank culture During to control fermentation temperature in cycle 0h~20h be 36 DEG C, it is 33 DEG C that fermentation temperature control after 20h, up to fermentation ends.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108949550A (en) * | 2018-10-16 | 2018-12-07 | 无锡荣丰生物工程有限公司 | A kind of seed can system and its working method expanding culture for amino acids production strain |
| CN109355327A (en) * | 2018-12-18 | 2019-02-19 | 山东金洋药业有限公司 | A kind of L- hydroxyproline bacteria suspension is into tank inoculation method |
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| CN101962663A (en) * | 2010-11-02 | 2011-02-02 | 天津科技大学 | High-efficiency fermenting method for producing L-isoleucine |
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| JPH07233147A (en) * | 1994-02-23 | 1995-09-05 | Konika Zerachin Kk | Production of purified l-hydroxyproline |
| CN101962663A (en) * | 2010-11-02 | 2011-02-02 | 天津科技大学 | High-efficiency fermenting method for producing L-isoleucine |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949550A (en) * | 2018-10-16 | 2018-12-07 | 无锡荣丰生物工程有限公司 | A kind of seed can system and its working method expanding culture for amino acids production strain |
| CN109355327A (en) * | 2018-12-18 | 2019-02-19 | 山东金洋药业有限公司 | A kind of L- hydroxyproline bacteria suspension is into tank inoculation method |
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