CN107164283A - A kind of L alanine high density bacterium solution culture medium and its cultural method - Google Patents
A kind of L alanine high density bacterium solution culture medium and its cultural method Download PDFInfo
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- CN107164283A CN107164283A CN201710551548.9A CN201710551548A CN107164283A CN 107164283 A CN107164283 A CN 107164283A CN 201710551548 A CN201710551548 A CN 201710551548A CN 107164283 A CN107164283 A CN 107164283A
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- 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
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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
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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Abstract
The present invention relates to fermentation engineering field, a kind of L alanine high density bacterium solution culture medium and its cultural method are specifically disclosed.The culture medium includes the first raw material and the second raw material, and first raw material includes the following component of following parts by weight：1 10 parts of glycerine, 10 30 parts of dusty yeast, 5 15 parts of soy peptone, 1 10 parts of brewer's wort, 15 parts of ammonium phosphate, 15 parts of potassium dihydrogen phosphate, three 3 15 parts of water dipotassium hydrogen phosphates, 15 parts of sodium chloride；Second raw material includes 20 40ppm CoCL2 6H2O, 2 4ppm manganese sulfate monohydrate, 20 40ppm Sodium Molybdate Dihydrate.Through Shaking culture, after culture is finished, seed liquor is obtained, amplified fermentation process obtains L alanine high density bacterium solutions.Shake-flask seed OD600 values of the present invention increase by 84%, and fermentation period shortens 20h, and L alanine output increased 52% reduces production cost.
Description
Technical field
The present invention relates to fermentation engineering field, more particularly to a kind of ALANINE high density bacterium solution culture medium and its training
The method of supporting.
Background technology
ALANINE has extensive purposes in food and medicine field.In field of food, the additive of ALANINE can
Protein utilization in food and beverage is significantly improved, can rapidly set up, inspire enthusiasm after eating.ALANINE can be improved
Pickling effect and improving local flavor for cure foods, improves quality containing alcoholic beverage etc..ALANINE has unique improvement local flavor
Effect, it coordinates with other amino acid can strengthen flavour of food and beverage.It can also improve energy after the Yoghourt of organic acid, addition
Make organic acid closer to the tart flavour of natural tartaric acid.In medical field, ALANINE is synthetic vitamin B6 important source material, simultaneously
It is also the main component that nutritional agents mends glycoprotein amino acid nutrition infusion.ALANINE or a kind of good diuretics.
At present, ALANINE can be using chemical synthesis, enzyme process and fermentation method production.Chemical synthesis is chemical industry
Produce the common solution of various amino acid, but the poor product quality of synthesis, the rate of recovery, cost is high and environmental pollution etc., because
This chemistry and long hair are substantially at superseded edge.Enzymatic conversion method, that is, pass through micro- life rich in L-Aspartic acid-β-decarboxylase activity
Thing cell catalysis L-Aspartic acid and obtain, this method for transformation is expensive due to its raw material L-Aspartic acid, makes the party
The production cost of method is higher.
The content of the invention
For the synthetically produced cost of existing ALANINE it is high the problems such as, the present invention provides a kind of ALANINE high density bacterium solution
Culture medium and its cultural method.
To achieve the above object of the invention, the embodiment of the present invention employs following technical scheme：
A kind of ALANINE high density bacterium solution culture medium, the culture medium includes the first raw material and the second raw material, and described first is former
Material includes the following component of following parts by weight：1-10 parts of glycerine, 10-30 parts of dusty yeast, 5-15 parts of soy peptone, brewer's wort
1-10 parts, 1-5 parts of ammonium phosphate, 1-5 parts of potassium dihydrogen phosphate, three 3-15 parts of water dipotassium hydrogen phosphates, 1-5 parts of sodium chloride；Described second
Raw material includes 20-40ppm CoCL2 6H2O, 2-4ppm manganese sulfate monohydrate, 20-40ppm Sodium Molybdate Dihydrate.
Escherichia coli ability well-grown when nutrient concentrations are suitable in culture medium, can not when nutrient concentrations are too low
Meet needed for Escherichia coli normal growth, may then inhibitory action, such as high concentration be played to Escherichia coli Growth during excessive concentration
Glucide, inorganic salts, heavy metal ion etc. can not only be maintained and promote the growth of microorganism, and antibacterial or sterilization is played on the contrary
Effect.In addition, the concentration proportioning in culture medium between each nutriment also directly affects growth and breeding and (or) the generation of microorganism
Thank to the formation and accumulation of product, the influence of wherein carbon-nitrogen ratio (C/N) is larger.ALANINE high density bacterium solution training proposed by the present invention
Base is supported, with suitable nutrient concentrations, suitable carbon-nitrogen ratio contributes to the amount reproduction of Escherichia coli.
A kind of ALANINE high density bacterium solution cultural method, the cultural method at least comprises the following steps：
Step 1, according to the composition of raw materials of ALANINE high density bacterium solution culture medium as claimed in claim 1 or 2 each component is weighed,
Each component is dissolved in the water, regulation pH is 7.0-7.2, obtains culture medium；
Step 2, culture medium is dispensed after carry out sterilization；
Step 3, access strain in the culture medium, carry out Shaking culture, wherein, the access amount of the strain is the culture
The 0.1% of base gross weight；
After step 4, culture are finished, seed liquor is obtained, amplified fermentation process obtains ALANINE high density bacterium solution, the amplification
The time of fermentation is 24-28h.
Relative to prior art, the ALANINE high density bacterium solution culture medium and its cultural method provided using the present invention,
Shake-flask seed OD600 values increase by 84%, and fermentation period shortens 20h, and ALANINE output increased 52% reduces production cost.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The embodiment of the present invention provides a kind of ALANINE high density bacterium solution culture medium, the culture medium include the first raw material and
Second raw material, first raw material includes the following component of following parts by weight：1-10 parts of glycerine, 10-30 parts of dusty yeast, soybean
5-15 parts of peptone, 1-10 parts of brewer's wort, 1-5 parts of ammonium phosphate, 1-5 parts of potassium dihydrogen phosphate, three 3-15 parts of water dipotassium hydrogen phosphates, chlorine
Change 1-5 parts of sodium；Second raw material includes 20-40ppm CoCL2 6H2O, 2-4ppm manganese sulfate monohydrate, 20-40ppm's
Sodium Molybdate Dihydrate.
Preferably, the mass concentration of the brewer's wort is 12-17%.
A kind of ALANINE high density bacterium solution cultural method, the cultural method at least comprises the following steps：
Step 1, according to the composition of raw materials of ALANINE high density bacterium solution culture medium as claimed in claim 1 or 2 each component is weighed,
Each component is dissolved in the water, regulation pH is 7.0-7.2, obtains culture medium；
Step 2, culture medium is dispensed after carry out sterilization；
Step 3, access strain in the culture medium, carry out Shaking culture, wherein, the access amount of the strain is the culture
The 0.1% of base gross weight；
After step 4, culture are finished, seed liquor is obtained, amplified fermentation process obtains ALANINE high density bacterium solution, the amplification
The time of fermentation is 24-28h.
Preferably, Spawn incubation condition is in the step 3：Cultivation temperature is 35-38 DEG C, and shaking flask rotating speed is 200-
220rpm。
Preferably, before the amplification fermentation, in addition to the seed liquor is detected, examination criteria is met：PH value is
6.8-7.4, OD600 value are 18-30, and microscopy is sterile.
OD600 refers to light absorption value of certain solution at 600nm wavelength, and light absorption value is proportional to the extinction material in solution
Concentration, i.e. OD600 numerical value is higher, shows that the concentration of shake-flask seed is higher.
Preferably, the strain is Escherichia coli.
Preferably, in the step 2, sterilization condition is：120-122 DEG C of temperature, time 20-30min.
Preferably, the pH of the culture medium is adjusted using 20wt% sodium hydroxide.
It is provided in an embodiment of the present invention in order to better illustrate, illustrated below by embodiment is further.
Embodiment 1
A kind of ALANINE high density bacterium solution culture medium that the present embodiment is provided, the culture medium includes the first raw material and the second original
Material, first raw material includes the following component of following parts by weight：Glycerine：1 part, dusty yeast：10 parts, soy peptone：8
Part, 15wt% brewer's worts：1 part, ammonium phosphate：5 parts, potassium dihydrogen phosphate：5 parts, three water dipotassium hydrogen phosphates：5 parts, sodium chloride：4 parts；Institute
Stating the second raw material includes 40ppm CoCL2 6H2O, 2ppm manganese sulfate monohydrate, 32ppm Sodium Molybdate Dihydrate.
The ALANINE high density bacterium solution cultural method that the present embodiment is provided, comprises the following steps：
Step 1, according to the composition of raw materials of above-mentioned ALANINE high density bacterium solution culture medium each component is weighed, each component is added to
In 1L container, with water constant volume to 1L, regulation pH is 7.0-7.2, obtains culture medium；
Step 2, culture medium is divided into 10 parts be respectively placed in the shaking flask that volume is 1L, every part of 100mL, with 8 layers of yarn and brown paper
Shaking flask is sealed, carry out disinfection sterilizing 30min at 121 DEG C；
Step 3, access strain in the culture medium, carry out Shaking culture, condition of culture is shaking flask rotating speed 220rpm, temperature 36
DEG C, time 8h；Wherein, the access amount of the strain is the 0.1% of the culture medium gross weight；
After step 4, culture are finished, seed liquor is obtained, amplifying fermentation process through 5 liters of fermentation tanks obtains ALANINE high density bacterium
Liquid, the time of the amplification fermentation is 28h.
Before the amplification fermentation, in addition to the seed liquor is detected, examination criteria is met：PH value is 7.0,
OD600 values are 21.24, and microscopy is sterile, and the ALANINE yield that amplification fermentation is obtained is 85.8g/L.
Embodiment 2
A kind of ALANINE high density bacterium solution culture medium that the present embodiment is provided, the culture medium includes the first raw material and the second original
Material, first raw material includes the following component of following parts by weight：Glycerine：2 parts, dusty yeast：5 parts, soy peptone：13
Part, 15wt% brewer's worts：10 parts, ammonium phosphate：2 parts, potassium dihydrogen phosphate：5 parts, three water dipotassium hydrogen phosphates：7 parts, sodium chloride：3 parts；
Second raw material includes CoCL2 6H2O：20ppm, manganese sulfate monohydrate：4ppm, Sodium Molybdate Dihydrate 40ppm.
The ALANINE high density bacterium solution cultural method that the present embodiment is provided, comprises the following steps：
Step 1, according to the composition of raw materials of above-mentioned ALANINE high density bacterium solution culture medium each component is weighed, each component is added to
In 1L container, with water constant volume to 1L, regulation pH is 7.0-7.2, obtains culture medium；
Step 2, culture medium is divided into 10 parts be respectively placed in the shaking flask that volume is 1L, every part of 100mL, with 8 layers of yarn and brown paper
Shaking flask is sealed, carry out disinfection sterilizing 30min at 122 DEG C；
Step 3, access strain in the culture medium, carry out Shaking culture, condition of culture is shaking flask rotating speed 220rpm, temperature 37
DEG C, time 8h；Wherein, the access amount of the strain is the 0.1% of the culture medium gross weight；
After step 4, culture are finished, seed liquor is obtained, amplifying fermentation process through 5 liters of fermentation tanks obtains ALANINE high density bacterium
Liquid, the time of the amplification fermentation is 28h.
Before the amplification fermentation, in addition to the seed liquor is detected, examination criteria is met：PH value is 7.2,
OD600 values are 22.86, and microscopy is sterile, and the ALANINE yield that amplification fermentation is obtained is 98g/L.
Embodiment 3
A kind of ALANINE high density bacterium solution culture medium that the present embodiment is provided, the culture medium includes the first raw material and the second original
Material, first raw material includes the following component of following parts by weight：Glycerine：4 parts, dusty yeast：10 parts, soy peptone：6
Part, 15wt% brewer's worts：5 parts, ammonium phosphate：4 parts, potassium dihydrogen phosphate：2 parts, three water dipotassium hydrogen phosphates：4 parts, sodium chloride：5 parts；Institute
Stating the second raw material includes CoCL2 6H2O：30ppm, manganese sulfate monohydrate：3ppm, Sodium Molybdate Dihydrate 30ppm.
The ALANINE high density bacterium solution cultural method that the present embodiment is provided, comprises the following steps：
Step 1, according to the composition of raw materials of above-mentioned ALANINE high density bacterium solution culture medium each component is weighed, each component is added to
In 1L container, with water constant volume to 1L, regulation pH is 7.0-7.2, obtains culture medium；
Step 2, culture medium is divided into 10 parts be respectively placed in the shaking flask that volume is 1L, every part of 100mL, with 8 layers of yarn and brown paper
Shaking flask is sealed, carry out disinfection sterilizing 30min at 120 DEG C；
Step 3, access strain in the culture medium, carry out Shaking culture, condition of culture is shaking flask rotating speed 220rpm, temperature 35
DEG C, time 8h；Wherein, the access amount of the strain is the 0.1% of the culture medium gross weight；
After step 4, culture are finished, seed liquor is obtained, amplifying fermentation process through 5 liters of fermentation tanks obtains ALANINE high density bacterium
Liquid, the time of the amplification fermentation is 28h.
Before the amplification fermentation, in addition to the seed liquor is detected, examination criteria is met：PH value is 7.2,
OD600 values are 27.56, and microscopy is sterile, and the ALANINE yield that amplification fermentation is obtained is 122.89g/L.
In order to better illustrate technical scheme, done further below by comparative example and embodiments of the invention into one
The contrast of step.
Comparative example 1
This comparative example 1, which is provided, uses primordial seed culture medium, and cultural method is consistent with the method that the present embodiment is provided.
Cultivation results are that shake-flask seed culture 8h OD600 is 4.36, through 5 liters of fermentation tank amplification fermentation 48h, ALANINE
Yield is 57.8g/L.
Above-described embodiment 1-3 and the cultivation results of comparative example 1 are listed as follows.
The embodiment 1-3 of table 1 and the cultivation results of comparative example 1
| OD600 | Fermentation period | Yield（g/L） | |
| Embodiment 1 | 21.24 | 28 | 85.8 |
| Embodiment 2 | 22.86 | 28 | 98.84 |
| Embodiment 3 | 27.56 | 28 | 122.89 |
| Comparative example 1 | 4.36 | 48 | 57.8 |
Using the culture medium of this programme, by Shaking culture, amplified fermentation process obtains ALANINE high density bacterium solution, by table
1 can draw, shake-flask seed OD600 values increase by 84%, and fermentation period shortens 20h, ALANINE output increased 52%, reduction production
Cost.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modification, equivalent substitution or improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (8)
1. a kind of ALANINE high density bacterium solution culture medium, it is characterised in that：The culture medium includes the first raw material and the second original
Material, first raw material includes the following component of following parts by weight：1-10 parts of glycerine, 10-30 parts of dusty yeast, soy peptone
5-15 parts, 1-10 parts of brewer's wort, 1-5 parts of ammonium phosphate, 1-5 parts of potassium dihydrogen phosphate, three 3-15 parts of water dipotassium hydrogen phosphates, sodium chloride 1-
5 parts；Second raw material includes 20-40ppm CoCL2 6H2O, 2-4ppm manganese sulfate monohydrate, 20-40ppm molybdate dihydrate
Sour sodium.
2. ALANINE high density bacterium solution culture medium as claimed in claim 1, it is characterised in that：The quality of the brewer's wort is dense
Spend for 12-17%.
3. a kind of ALANINE high density bacterium solution cultural method, it is characterised in that：The cultural method at least comprises the following steps：
Step 1, according to the composition of raw materials of ALANINE high density bacterium solution culture medium as claimed in claim 1 or 2 each component is weighed,
Each component is dissolved in the water, regulation pH is 7.0-7.2, obtains culture medium；
Step 2, culture medium is dispensed after carry out sterilization；
Step 3, access strain in the culture medium, carry out Shaking culture, wherein, the access amount of the strain is the culture
The 0.1% of base gross weight；
After step 4, culture are finished, seed liquor is obtained, amplified fermentation process obtains ALANINE high density bacterium solution, the amplification
The time of fermentation is 24-28h.
4. ALANINE high density bacterium solution cultural method as claimed in claim 3, it is characterised in that：Strain in the step 3
Condition of culture is：Cultivation temperature is 35-38 DEG C, and shaking flask rotating speed is 200-220rpm.
5. ALANINE high density bacterium solution cultural method as claimed in claim 3, it is characterised in that：Before the amplification fermentation,
Also include detecting the seed liquor, examination criteria is met：PH value is 6.8-7.4, and OD600 values are 18-30, microscopy without
Bacterium.
6. ALANINE high density bacterium solution cultural method as claimed in claim 3, it is characterised in that：The strain is large intestine bar
Bacterium.
7. ALANINE high density bacterium solution cultural method as claimed in claim 3, it is characterised in that：In the step 2, sterilization
Sterilising conditions are：120-122 DEG C of temperature, time 20-30min.
8. ALANINE high density bacterium solution cultural method as claimed in claim 3, it is characterised in that：Using 20wt% hydrogen-oxygen
Change the pH that sodium adjusts the culture medium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884565A (en) * | 2006-05-29 | 2006-12-27 | 安徽华恒生物工程有限公司 | Process for producing D-alanine using microbe |
| CN103184172A (en) * | 2011-12-30 | 2013-07-03 | 广州拜迪生物医药有限公司 | Culture medium used in Escherichia coli high-density culturing |
-
2017
- 2017-07-07 CN CN201710551548.9A patent/CN107164283A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884565A (en) * | 2006-05-29 | 2006-12-27 | 安徽华恒生物工程有限公司 | Process for producing D-alanine using microbe |
| CN103184172A (en) * | 2011-12-30 | 2013-07-03 | 广州拜迪生物医药有限公司 | Culture medium used in Escherichia coli high-density culturing |
Non-Patent Citations (2)
| Title |
|---|
| GEOFFREY,ET AL: "Fed-batch two-phase production of alanine by a metabolically engineered Escherichia coli", 《BIOTECHNOL LETT》 * |
| 刘元东，ET AL: "微生物高密度培养的研究概况", 《有色金属科学与工程》 * |
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