CN107022583A - A kind of method that fed-batch fermentation produces L alanine - Google Patents
A kind of method that fed-batch fermentation produces L alanine Download PDFInfo
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- CN107022583A CN107022583A CN201710295723.2A CN201710295723A CN107022583A CN 107022583 A CN107022583 A CN 107022583A CN 201710295723 A CN201710295723 A CN 201710295723A CN 107022583 A CN107022583 A CN 107022583A
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- C—CHEMISTRY; METALLURGY
- 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
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The present invention relates to a kind of method that fed-batch fermentation produces L alanine, its be be using concentration of glucose 95 105g/L dextrose culture-medium Lactobacillus delbrueckii mutant strain Lds.0108 is cultivated during, when residual sugar content is less than 45g/L in zymotic fluid, it is disposable into zymotic fluid to add the glucose solution that concentration is 50 75%, the total concentration of the glucose added into zymotic fluid is 125~170g/L, continues to cultivate to fermentation ends.Method of the present invention, L alanine yield is improved by adding glucose solution on the basis of basal fermentation medium, simple and easy to apply, effect is notable, L alanine concentration brings up to 110 120g/L by 80 original 100g/L in fermentation tank, and fermentation conversion rate reaches more than 90%.
Description
Technical field
The present invention relates to technical field of microbial fermentation, and in particular to a kind of method that fed-batch fermentation produces ALANINE.
Background technology
ALANINE is colourless to white crystalline powder, water, ethanol is dissolved in, insoluble in ether and acetone.ALANINE
It is a kind of medicine intermediate, is the primary raw material for producing vitamin B6 and L- aminopropanols.In field of food, ALANINE has
Unique improvement local flavor effect, can significantly improve the utilization rate of protein in food and beverage, improve the tart flavour of organic acid.
At present, it is industrial main using L-Aspartic acid-β-decarboxylation enzymatic conversion L-Aspartic acid production ALANINE, production
Equipment investment is few, and reaction is simple, but primary raw material L-Aspartic acid price fluctuation is too big, it is difficult to ensure long-term steady production.Separately
Outside, company produces L- propionic acid using anaerobism direct fermentation transforming glucose in the recent period.Unareobic fermentation is main based on glucose
Raw material is wanted, it is cheap, but it is converted into that ALANINE concentration is low, conversion ratio is low, causes fermentation method ALANINE production cost
It is higher.
The content of the invention
It is an object of the invention to provide a kind of method that fed-batch fermentation produces ALANINE, it is, at the beginning of using glucose
During beginning concentration is cultivated Lactobacillus delbrueckii mutant strain Lds.0108 for 95-105g/L dextrose culture-medium,
It is disposable into zymotic fluid to add the glucose solution that concentration is 50-75% when residual sugar content is less than 45g/L in zymotic fluid,
The total concentration of the glucose added into zymotic fluid is 125~170g/L, continues to cultivate to fermentation ends.
Method of the present invention, on the one hand, do not add all glucose when initially preparing culture medium, will not be because of one
The glucose of secondary property addition high concentration produces hyperosmosis and thalline is damaged.On the other hand, add during culture
Plus glucose, it can further increase the yield of ALANINE.Lactobacillus delbrueckii mutant strain Lds.0108 during fermentation,
Metabolite ALANINE can be produced, it can cause certain osmotic pressure to thalline, thalline can also be increased by continuing to add glucose
Osmotic pressure, it is 50~75% to control concentration of glucose in the medium to add concentration when being less than 45g/L into culture medium
Grape grain solution, due to the diluting effect of the water in the grape solution added, ALANINE and glucose in nutrient solution are produced
Osmotic pressure bad influence will not be brought to thalli growth.Moreover, the glucose of above-mentioned concentration will not during culture
Because concentration is excessive and chromogenesis class material, do not interfere with the extraction of active principle, influence will not be produced on the growth of microorganism.
It is preferred that, when residual sugar content is 30-41g/L in zymotic fluid, glucose solution is added into zymotic fluid.Work as residual sugar
During excessive concentration, add after high concentration glucose sugar that sugared concentration is too high, form hyperosmosis, big is injured to thalline；Residual sugar is too low, bacterium
Body aging, enzyme system vigor is small, and transformation in planta speed after glucose is added in influence.
As it is a kind of it is preferred add scheme, when residual sugar content is less than 33~35g/L in zymotic fluid, into zymotic fluid one
Secondary property adds the glucose solution that concentration is 68~72%, and the total concentration of the glucose added into zymotic fluid is 160~168g/
L, continues to cultivate to fermentation ends.
It is preferred that, in every liter of dextrose culture-medium, including：Glucose 95-105g, Na2HPO4·12H2O 20g,
KH2PO42.0g, NaCl 0.5g, MgSO4·7H2O 0.2g, small-scale inorganic salting liquid 10mL/L, surplus is purified water；
In the small-scale inorganic salting liquid：FeCl3·6H2O 2.5g/L, CoCl2·6H2O 0.2g/L, CuCl2·2H2O
0.1g/L, ZnCl20.2g/L, Na2MoO4·2H2O 0.3g/L, H3BO30.1g/L, MnCl2·4H2O 0.5g/L, purified water
Surplus.
It is preferred that, it is to the Lactobacillus delbrueckii mutant strain Lds.0108 conditions cultivated using dextrose culture-medium：
30 ± 2 DEG C of temperature；Ventilation 0m3/ h, pressure 0.04MPa-0.06MPa；pH：6.5±0.1.
It is preferred that, method of the present invention adjusts the pH in zymotic fluid by liquefied ammonia.Amino is provided using ammonia simultaneously
Glucose products of sugar decomposition is given, ALANINE is synthesized.
It is preferred that, method of the present invention comprises the following steps:
1) the Lactobacillus delbrueckii mutant strain LDS.0108 seed liquors that OD values are 3.5-7.0 are prepared；
2) into the dextrose culture-medium by kind of volume fraction for 5-10% the seed liquor by planting to being fermented
Culture；
3) when the concentration of glucose in zymotic fluid is less than 45g/L, it is 50-75%'s that concentration is added into the zymotic fluid
Concentration of glucose, into zymotic fluid, the concentration of glucose is higher than the concentration of glucose in the dextrose culture-medium, continues to cultivate
To fermentation ends.
It is preferred that, the OD values of the Lactobacillus delbrueckii mutant strain LDS.0108 seed liquors are 3.5-7.0.
It is preferred that, the preparation of the Lactobacillus delbrueckii mutant strain LDS.0108 seed liquors comprises the following steps：
(1) aseptically, it is inoculated in solid LB media with oese picking Lactobacillus delbrueckii mutant strain strain
Eggplant-shape bottle inclined-plane, is positioned in 28-30 DEG C of constant incubator, cultivates 16-18h；
(2) aseptically, the thalline on inclined-plane is washed down with sterile saline, obtains bacteria suspension；
(3) the bacteria suspension liquid is inoculated in LB liquid medium, is 30 DEG C in temperature, air mass flow is 2L/min,
Cultivated under conditions of tank pressure 0.04-0.06MPa, the OD values to zymotic fluid are 3.5-7.0.
During large scale fermentation is carried out, need to carry out secondary fermentation, i.e. repeat step based on the above method
(3) twice, OD values 5-7.0 seed liquor is obtained.
The Lactobacillus delbrueckii mutant strain Lds.0108 that the present invention is used is preserved in China typical culture collection center, protects
Tibetan number is：CCTCC NO：M2013361.
It is further preferred that method of the present invention comprises the following steps：
1) seed liquor that OD values are 3.5-7.0 is prepared；
2) it is that 5-10% presses kind of the seed liquor by kind of volume fraction into dextrose culture-medium, and carries out fermented and cultured；
The formula of the dextrose culture-medium is in every liter of culture medium, to contain glucose 95-105g, Na2HPO4·
12H2O 20g, KH2PO42.0g, NaCl 0.5g, MgSO4·7H2O0.2g, small-scale inorganic salting liquid 10mL/L, surplus are purifying
Water；
In the small-scale inorganic salting liquid：FeCl3·6H2O 2.5g/L, CoCl2·6H2O 0.2g/L, CuCl2·2H2O
0.1g/L, ZnCl20.2g/L, Na2MoO4·2H2O 0.3g/L, H3BO30.1g/L, MnCl2·4H2O 0.5g/L, purified water
Surplus；
Condition of culture is：30 ± 2 DEG C of temperature；Ventilation 0m3/ h, pressure 0.04MPa-0.06MPa；pH：6.5±0.1；
3) feed-batch culture
When the concentration of glucose in zymotic fluid is 30-41g/L, it is 50-75%'s that concentration is added into the zymotic fluid
Concentration of glucose, the total concentration of the glucose added into zymotic fluid is 125~170g/L, continues to cultivate residual into nutrient solution
Sugared concentration terminates fermentation for≤2g/L.
Most preferably, method of the present invention comprises the following steps：
1) seed liquor that OD values are 3.5-7.0 is prepared；
2) it is that 5-10% presses kind of the seed liquor by kind of volume fraction into dextrose culture-medium, and carries out fermented and cultured；
The formula of the dextrose culture-medium is glucose 95-105g, Na2HPO4·12H2O20g, KH2PO42.0g,
NaCl 0.5g, MgSO4·7H2O 0.2g, small-scale inorganic salting liquid 10mL/L, surplus is purified water；
In the small-scale inorganic salting liquid：FeCl3·6H2O 2.5g/L, CoCl2·6H2O 0.2g/L, CuCl2·2H2O
0.1g/L, ZnCl20.2g/L, Na2MoO4·2H2O 0.3g/L, H3BO30.1g/L, MnCl2·4H2O 0.5g/L, purified water
Surplus；
Condition of culture is：30 ± 2 DEG C of temperature；Ventilation 0m3/ h, pressure 0.04MPa-0.06MPa；pH：6.5±0.1；
3) feed-batch culture
When residual sugar content is less than 33~35g/L in zymotic fluid, into zymotic fluid, disposable concentration of adding is 68~72%
Glucose solution, the total concentration of the glucose added into zymotic fluid is 160~168g/L.
Method of the present invention has the advantages that：
The present invention is starting strain using Lactobacillus delbrueckii mutant strain LDS.0108, by basal fermentation medium base
Add glucose solution on plinth to improve ALANINE yield, method is simple and easy to apply, ALANINE concentration is by original in fermentation tank
80-100g/L, bring up to 110-123g/L, fermentation conversion rate reaches more than 90%, and fermentation remaining sugar concentration keeps constant, although
Though fermentation period increased, unit cost is greatly reduced.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Involved dextrose culture-medium is prepared by following methods in embodiment：
Fermentation medium is prepared by following component content：Glucose 100g/L, Na2HPO4·12H2O 20g/L, KH2PO4
2.0g/L, NaCl 0.5g/L, MgSO4·7H2O0.2g/L, small-scale inorganic salt 10ml/L, surplus is purified water；
Wherein small-scale inorganic salt includes：FeCl3·6H2O 2.5g/L, CoCl2·6H2O 0.2g/L, CuCl2·2H2O
0.1g/L, ZnCl20.2g/L, Na2MoO4·2H2O 0.3g/L, H3BO30.1g/L, MnCl2·4H2O 0.5g/L, surplus is pure
Change water.
By above-mentioned culture medium in 121 DEG C of sterilizings, pressurize 30min cools to 30 DEG C, standby；
The preparation method of glucose solution is in following examples：Volume prepares 50-75% glucose solution on demand,
After constant volume, 115 DEG C of sterilizings, pressurize 20min is cooled down standby.
In following embodiments, embodiment 1~4 is carried out in 10L fermentation tank, and embodiment 5 is entered in 10T fermentation tank
OK, therefore during seed liquor is prepared secondary seed solution is prepared, its OD value is significantly larger than embodiment 1~4.
Embodiment 1
The present embodiment is related to a kind of preparation method of ALANINE, specifically includes following steps：
1st, Lactobacillus delbrueckii mutant strain Lds.0108 seed liquors are prepared
1) aseptically, it is inoculated in solid LB media eggplant with oese picking Lactobacillus delbrueckii mutant strain strain
Shape bottle inclined-plane, is positioned in 28-30 DEG C of constant incubator, cultivates 17h.
2) thalline aseptically, is eluted with the 100ml physiological saline after sterilization, bacteria suspension is obtained.
3) it is 30 DEG C in temperature by bacterial suspension inoculation in LB liquid medium；Air mass flow is 2L/min, tank pressure
0.04-0.06MPa；Cultivate 5.5h to OD (600nm)：4.05, obtain seed liquor.
2nd, the seed liquor is inoculated into above-mentioned dextrose culture-medium by volume fraction 7%, is 30 DEG C in temperature；It is empty
Throughput is 2m3In fermented and cultured under conditions of/h, incubation, tank is kept to press 0.04-0.06MPa by stream plus air；Adopt
PH is controlled to be 6.5 with liquefied ammonia.
3rd, when the residual concentration of glucose of zymotic fluid is 32.8g/L, 50% glucose solution is disposably added into zymotic fluid
The addition concentration of total glucose is 158.4g/L, when the concentration of glucose proceeded in zymotic fluid is 1.85g/L, stops hair
Ferment.
After fermentation ends, it is 110g/L, residual concentration of glucose that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
For 1.85g/L, fermentation conversion rate 90%.
Embodiment 2
The present embodiment is related to a kind of preparation method of ALANINE, specifically includes following steps：
1st, seed liquor is prepared according to method same as Example 1, obtains the seed liquor that OD values are 3.75.
2nd, the seed liquor is inoculated into above-mentioned dextrose culture-medium by volume fraction 7%, is 30 DEG C in temperature；It is empty
Throughput is 2m3In fermented and cultured under conditions of/h, incubation, tank is kept to press 0.04-0.06MPa by stream plus air；Adopt
PH is controlled to be 6.50 with liquefied ammonia.
3rd, when the residual concentration of glucose of zymotic fluid is 40.6g/L, 60% glucose solution is disposably added, into zymotic fluid
The total concentration of glucose is 159.2g/L, when the concentration of glucose proceeded in zymotic fluid is 1.77g/L, stops fermentation.
After fermentation ends, it is 115g/L, residual concentration of glucose that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
To be changed to 1.77g/L, fermentation conversion rate 90.5%.
Embodiment 3
The present embodiment is related to a kind of preparation method of ALANINE, specifically includes following steps：
1st, seed liquor is prepared according to method same as Example 1, obtains the seed liquor that OD values are 3.86.
2nd, the seed liquor is inoculated into above-mentioned dextrose culture-medium by volume fraction 7%, is 30 DEG C in temperature；It is empty
Throughput is 2m3In fermented and cultured under conditions of/h, incubation, tank is kept to press 0.04-0.06MPa by stream plus air；Adopt
PH is controlled to be 6.50 with liquefied ammonia.
3rd, when the residual concentration of glucose of zymotic fluid is 33.5g/L, 70% glucose solution is disposably added, into zymotic fluid
The total concentration of glucose is 166.5g/L, when the concentration of glucose proceeded in zymotic fluid is 1.80g/L, stops fermentation.
After fermentation ends, it is that 123g/L is that residual glucose is dense that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
Spend for 1.80g/L, fermentation conversion rate 91%.
Embodiment 4
The present embodiment is related to a kind of preparation method of ALANINE, specifically includes following steps：
1st, seed liquor is prepared according to method same as Example 1, obtains the seed liquor that OD values are 3.9.
2nd, the seed liquor is inoculated into above-mentioned dextrose culture-medium by volume fraction 7%, is 30 DEG C in temperature；Air
Flow is 2m3In fermented and cultured under conditions of/h, incubation, tank is kept to press 0.04-0.06MPa by stream plus air；Using
Liquefied ammonia controls pH to be 6.50.
3rd, when the residual concentration of glucose of zymotic fluid is 31.0g/L, 75% glucose solution is disposably added, into zymotic fluid
The total concentration of glucose is 127.2g/L, when the concentration of glucose proceeded in zymotic fluid is 1.62g/L, stops fermentation.
After fermentation ends, it is 113g/L, residual concentration of glucose that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
For 1.62g/L, fermentation conversion rate 90.2%.
Embodiment 5
The present embodiment is related to a kind of preparation method of ALANINE, specifically includes following steps：
1st, according to method same as Example 1, seed liquor is prepared by two-stage seed, the seed that OD values are 6.85 is obtained
Liquid.
2nd, the seed liquor is inoculated into above-mentioned dextrose culture-medium by volume fraction 7%, is 30 DEG C in temperature；It is empty
Throughput is 2m3In fermented and cultured under conditions of/h, incubation, tank is kept to press 0.04-0.06MPa by stream plus air；Adopt
PH is controlled to be 6.50 with liquefied ammonia.
3rd, when the residual concentration of glucose of zymotic fluid is 31.0g/L, grape of 75% glucose into zymotic fluid is disposably added
Total addition concentration of sugar is 152.54g/L, when the concentration of glucose proceeded in zymotic fluid is 1.70g/L, stops fermentation.
After fermentation ends, it is 125g/L, residual concentration of glucose that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
For 1.70g/L, fermentation conversion rate 90.2%.
Comparative example 1
Compared with Example 3, difference in this case is that, omit step 3, i.e., glucose is not added additionally, only by most
First medium culture is to fermentation ends.
After fermentation ends, it is 82g/L, residual concentration of glucose that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
For 1.86g/L, fermentation conversion rate 90%.
Comparative example 2
Compared with Example 3, difference in this case is that, in step 3, when glucose in zymotic fluid concentration be 60g/
During L, glucose is added.
After fermentation ends, it is 115g/L, residual concentration of glucose that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
For 2.86g/L, fermentation conversion rate 85%.It can be seen that, glucose is added in the excessive concentration of residual glucose, substantially reduction can be sent out
The conversion ratio of fermentation, is unfavorable for the trans-utilization of glucose in culture medium, and because osmotic pressure is too high, be in concentration of glucose
2.86 microorganisms just stop stopping fermentation.
Comparative example 3
Compared with Example 3, difference in this case is that, in step 3, add concentration be 80% glucose to ferment
Concentration in liquid is 166.5g/L.
After fermentation ends, it is 120g/L, residual concentration of glucose that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
For 1.89g/L%, fermentation conversion rate 88%%.
Comparative example 4
Compared with Example 3, difference in this case is that, in step 3, add after glucose, the grape in zymotic fluid
The concentration of sugar is 140g/L.
After fermentation ends, it is 110g/L, residual concentration of glucose that sampling, which carries out ALANINE content in HPLC measure, zymotic fluid,
For 1.58g, fermentation conversion rate 90.1%.
Although above having made to retouch in detail to the present invention with general explanation, embodiment and experiment
State, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, are belonged to claimed
Scope.
Claims (9)
1. a kind of method that fed-batch fermentation produces ALANINE, it is characterised in that be 95- using glucose initial concentration
During 105g/L dextrose culture-medium is cultivated Lactobacillus delbrueckii mutant strain Lds.0108, when residual in zymotic fluid
It is disposable into zymotic fluid to add the glucose solution that concentration is 50-75% when sugared content is less than 45g/L, add into zymotic fluid
Plus the total concentration of glucose be 125~170g/L, continue to cultivate to fermentation ends.
2. according to the method described in claim 1, it is characterised in that when residual sugar content is 30-41g/L in zymotic fluid, Xiang Fa
Glucose solution is added in zymotic fluid.
3. method according to claim 1 or 2, it is characterised in that when residual sugar content is less than 33~35g/L in zymotic fluid
When, it is disposable into zymotic fluid to add the glucose solution that concentration is 68~72%, the glucose added into zymotic fluid it is total
Concentration is 160~168g/L.
4. the method according to claim 1 or 3, it is characterised in that in every liter of dextrose culture-medium, including：Grape
Sugared 95-105g, Na2HPO4·12H2O 20g, KH2PO42.0g, NaCl 0.5g, MgSO4·7H2O 0.2g, small-scale inorganic salt
Solution 10mL/L, surplus is purified water；
In the small-scale inorganic salting liquid：FeCl3·6H2O 2.5g/L, CoCl2·6H2O 0.2g/L, CuCl2·2H2O
0.1g/L, ZnCl20.2g/L, Na2MoO4·2H2O 0.3g/L, H3BO30.1g/L, MnCl2·4H2O 0.5g/L, purified water
Surplus.
5. the method according to claim 1 or 4, it is characterised in that be mutated using dextrose culture-medium to Lactobacillus delbrueckii
The condition that bacterial strain Lds.0108 is cultivated is：30 ± 2 DEG C of temperature；Ventilation 0m3/ h, pressure 0.04MPa-0.06MPa；
pH6.5±0.1。
6. the method according to any one of claim 1 to 5, it is characterised in that comprise the following steps:
1) Lactobacillus delbrueckii mutant strain Lds.0108 seed liquors are prepared；
2) into the dextrose culture-medium by kind of volume fraction for 5-10% the seed liquor by kind to carrying out fermented and cultured；
3) when the concentration of glucose in zymotic fluid is less than 45g/L, the grape that concentration is 50-75% is added into the zymotic fluid
Sugared concentration, the total concentration of the glucose added into zymotic fluid is 125~170g/L, continue to cultivate to the concentration of glucose for≤
2g/L, terminates fermentation.
7. method according to claim 6, it is characterised in that comprise the following steps：
(1) the Lactobacillus delbrueckii mutant strain Lds.0108 seed liquor seed liquors that OD values are 3.5-7.0 are prepared；
(2) it is that 5-10% is inoculated with the seed liquor by kind of volume fraction into dextrose culture-medium, and carries out fermented and cultured；
During the formula of the dextrose culture-medium is every liter of dextrose culture-medium, including glucose 95-105g, Na2HPO4·
12H2O 20g, KH2PO42.0g, NaCl 0.5g, MgSO4·7H2O 0.2g, small-scale inorganic salting liquid 10mL/L, surplus are pure
Change water；
In the small-scale inorganic salting liquid：FeCl3·6H2O 2.5g/L, CoCl2·6H2O 0.2g/L, CuCl2·2H2O
0.1g/L, ZnCl20.2g/L, Na2MoO4·2H2O 0.3g/L, H3BO30.1g/L, MnCl2·4H2O 0.5g/L, purified water
Surplus；
Condition of culture is：30 ± 2 DEG C of temperature；Ventilation 0m3/ h, pressure 0.04MPa-0.06MPa；pH：6.5±0.1；
(3) feed-batch culture
When the concentration of glucose in zymotic fluid is 30-41g/L, the grape that concentration is 50-75% is added into the zymotic fluid
Sugared concentration, the total concentration of the glucose added into zymotic fluid is 125~170g/L, and the residual sugar for continuing to cultivate into nutrient solution is dense
Spend for≤2g/L, end fermentation.
8. method according to claim 7, it is characterised in that the step 3) it is when residual sugar content is less than 33 in zymotic fluid
It is disposable into zymotic fluid to add the glucose solution that concentration is 68~72%, the grape added into zymotic fluid during~35g/L
The total concentration of sugar is 160~168g/L.
9. the method according to claim 7 or 8, it is characterised in that the Lactobacillus delbrueckii mutant strain Lds.0108 kinds
The preparation of sub- liquid comprises the following steps：
(1) aseptically, the training of solid LB solids is inoculated in oese picking Lactobacillus delbrueckii mutant strain Lds.0108
Primary surface is supported, is placed under 28-30 DEG C of constant temperature and cultivates 16-18h；
(2) aseptically, the thalline on inclined-plane is washed down with sterile saline, obtains bacteria suspension；
(3) the bacteria suspension liquid is inoculated in LB liquid medium, is 30 DEG C in temperature, air mass flow is 2L/min, tank pressure
Cultivated under conditions of 0.04-0.06MPa, the OD values to zymotic fluid are 3.5-7.0, are produced.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109055451A (en) * | 2018-09-13 | 2018-12-21 | 安徽华恒生物科技股份有限公司 | A kind of biofermentation method of l-Alanine |
| CN110982857A (en) * | 2019-09-23 | 2020-04-10 | 安徽丰原生物化学股份有限公司 | Fermentation production method of L-alanine |
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| CN101671706B (en) * | 2009-09-05 | 2013-09-18 | 山东新时代药业有限公司 | Carbohydrate supplementing method in fermentation process of mycophenolic acid |
| CN103602609A (en) * | 2013-09-05 | 2014-02-26 | 淮北新旗氨基酸有限公司 | High-yield strain for producing L-alanine by fermentation and preparation method thereof |
| CN106222309A (en) * | 2016-07-28 | 2016-12-14 | 山东金朗生物科技有限公司 | A kind of fermentable produces the control of additive raw material method improving L alanine yield |
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2017
- 2017-04-28 CN CN201710295723.2A patent/CN107022583B/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055451A (en) * | 2018-09-13 | 2018-12-21 | 安徽华恒生物科技股份有限公司 | A kind of biofermentation method of l-Alanine |
| CN110982857A (en) * | 2019-09-23 | 2020-04-10 | 安徽丰原生物化学股份有限公司 | Fermentation production method of L-alanine |
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