CN106834364A - Pyruvic acid industrial production zymotechnique - Google Patents
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Abstract
The invention discloses a kind of pyruvic acid industrial production zymotechnique, the flat board culture of the technique including torulopsis glabrata, the culture of eggplant bottle, Shaking culture, first class seed pot culture and then carry out 150 cubes of fermentation tanks and carry out industrial volume production zymotechnique and the specific constituent of culture medium and ratio；The technique has filled up the fermentation of pyruvic acid industrial production and has been particularly suited for 150 cubes of large fermentation tank and carried out the technique blank of fermenting and producing, not only fermentation period foreshortens to 56h, yield is obtained compared with high yield, and effectively solve in-fighting and conversion of the strain to zymotic fluid saturation pyruvic acid, it is ensured that product income and quality.
Description
Technical field
The present invention relates to technical field of microbial fermentation, more particularly to a kind of large-scale fermentation for being applied to 150 cubes
The pyruvic acid industrial production zymotechnique of tank.
Background technology
Pyruvic acid, as one of important organic acid, is nothing also known as 2- oxidations propionic acid, α-ketopropionic acid or alpha-Ketopropionic acid
Color, to weak yellow liquid, is one of most important alpha-oxo-carboxyl acid in acetic acid fragrance and happy tart flavour.Pyruvic acid is not only in biology
Had a very important role in energetic supersession, and be the precursor for synthesizing various useful compounds, therefore, it is in pharmacy, day
There is extensive purposes with the industry such as chemical industry, agricultural chemicals and food and scientific research.
At present, the pyruvic acid for being produced relative to chemical synthesis, fermentation method production pyruvic acid has low cost, high-quality
Advantage；In recent years, the research for fermentation method fermentation pyruvic acid technique is carried out therewith, and research is more using torulopsis glabrata fermentation
Production pyruvic acid；Due to the multiple vitamin deficient strain of torulopsis glabrata category be in the production of current fermentation method it is most normal
Most competitive production bacterial strain, therefore, using fermentation method production pyruvic acid in the side such as strain, fermentation condition optimization
Face is used as Research Emphasis.
Currently, following bibliography is provided for the raw pyruvic acid of torulopsis glabrata fermentation：
(1), the 23rd phase in 2003《Finely and specialty chemicals》Fermentative Production of Pyruvic Acid by Torulopsis glabrata Liu Li are bright
Deng；
(2)、《Bioengineering report》The influence of volume 16 2 phase nutritional condition to Fermentative Production of Pyruvic Acid by Torulopsis glabrata
.2000.3. Li Yin etc.；
(3), the 4th phase of volume 31 in 2012《China brewages》Torulopsis glabrata adaptive evolution improves Pyruvate production Zhao
Win；Above with reference to there is certain technological deficiency in document：Firstth, fermentation period is more long, low yield；Secondth, for fermentation
The degraded of the pyruvic acid occurred in reality or transition problem, are not solved effectively；3rd, pilot scale and big production 105
Ten cubes of large fermentation tank is different in the change of fermentation parameter, and the scope of technology controlling and process point is also different, and then realizes work
Industry volume production.
The content of the invention
The purpose of the present invention is to overcome the deficiencies in the prior art, there is provided a kind of to be applied to 150 cubes large-scale
The pyruvic acid industrial production zymotechnique of fermentation tank, the technique fermentation period is short, yield is high, can preferably suppress bacterium in fermentation process
Plant the degraded to the pyruvic acid of saturation or transition problem.
To achieve the above object, the present invention uses following technical proposals：
Pyruvic acid industrial production zymotechnique, concrete technology step is as follows：
(1) flat board culture：
A. plating medium (g/L)：Glucose syrup 3-4, soy peptone 3-4, beef extract-peptone agar 3-4, carbonic acid
Calcium 0.5-1, raw water dissolving, PH natures, 121 DEG C of -124 DEG C of 15min sterilizings are standby；
B. in plating medium, flat board is inverted 28 DEG C of -30 DEG C of cultures, incubation time in culture dish to torulopsis glabrata
After 15h-20h；
(2) eggplant bottle culture：
A. eggplant bottle culture medium (g/L)：Glucose syrup 13-14, soy peptone 11-12, beef extract-peptone agar 10-
12nd, calcium carbonate 2-2.5, ammonium chloride 0.3-0.5, raw water dissolving, PH natures, 121 DEG C of -124 DEG C of 15min sterilizings are standby；
B. sterile working is strictly pressed in desinfection chamber, single bacterium colony is chosen from preservation flat board and is coated on eggplant bottle solid medium
On；
C. condition of culture：28 DEG C -30 DEG C, after incubation time 15h-20h；
D. the refrigerator memory storage for eggplant bottle being placed into 1 DEG C -4 DEG C is standby；
(3) seed flask culture：
I. Shake flask medium (g/L)：Glucose syrup 17-18.5, soy peptone 13.5-15.5, calcium carbonate 2.5-4.5,
Ammonium chloride 0.5-0.8, magnesium chloride 0.32-0.48, dipotassium hydrogen phosphate 0.2-0.4, raw water dissolve and are 1.5mol/L with concentration
HCL regulation culture medium initial ps H is 5-6, and 121 DEG C of -124 DEG C of 15min sterilizings are standby；
The triangular flask or steel cylinder 20 to 30 of II.3000ml or 5000ml, in aseptic indoor sterilized inoculation
Shovel, shovel takes bacterium platform and is connected in the triangular flask of 500ml or steel cylinder in cultured eggplant bottle；Inoculum concentration is 600ml/ bottles of -1000ml/
Bottle；Cultivation temperature is 28 DEG C -30 DEG C, incubation time 22h-24h；
(4) first order seed culture：
1. primary-seed medium (g/L)：Glucose syrup 20-25, soy peptone 16.5-18.5, calcium carbonate 6.5-
8.5th, ammonium chloride 1.5-2.8, magnesium chloride 1.32-3.48, dipotassium hydrogen phosphate 1.2-2.4, raw water dissolve and are 1.5mol/ with concentration
L HCL regulation culture medium initial ps H is 5-6, and 121 DEG C of -124 DEG C of 15min sterilizings are standby；
2. using 800L seeding tanks 3-5；Seed flask is merged to triangle steel cylinder, in an aseptic environment to seeding tank
Inoculation operation is carried out, inoculum concentration is 2000ml/ tank -3000ml/ tanks；
3. condition of culture：28 DEG C -30 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1,6h later to seed maturity throughput
Than：1: 4, tank pressure maintains 0.03MPa-0.05MPa；
4. the standard of seed maturity：OD values are between 40-50；
(5) fermentation tank culture：
First, fermentation mediums (g/L)：Glucose 150-200, dipotassium hydrogen phosphate 5-10, magnesium chloride 0.9-1.5, chlorination
Ammonium 8.5-9.5, calcium carbonate 20-25, sodium acetate 6-8, defoamer 0.01-0.02, liquid microelement 3-5mL, vitamin liquid 3-
5mL, PH concentration are that 40mol/I HCL are adjusted to 5.0-5.5,121 DEG C of -125 DEG C of continuous sterilizations；
2nd, condition of culture：28 DEG C -30 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1, speed of agitator 100r/min-
150min；6h-12h throughputs ratio：1: 2, speed of agitator 200r/min-350min；12h-30h throughputs ratio：1: 3, stirring turns
Fast 400r/min-500min；Throughput is 1: 4, speed of agitator 500r/min-700min after 30h；40h-56h speeds of agitator
800r/min-1000r/min；Tank pressure is maintained：0.03MPa-0.05MPa；
It is with further illustrating, the liquid microelement (g/L)：Zinc chloride 3-6, iron chloride 15-18, copper chloride 0.2-
0.3rd, manganese chloride 15-18, concentration is that 1.5mol/L HCL adjust PH for 5.0-5.5 is settled to 1L-1.5L, 121 DEG C of -125 DEG C of companies
Continuous sterilizing；
It is with further illustrating, the vitamin liquid (g/L)：Vitamin D 0.03-0.04, vitamin E 0.003-
0.005th, vitamin B10.2-0.3, nicotinic acid 6-8, concentration are that 1.5mol/L HCL adjust PH for 5.0-5.5 is settled to 1L-
1.5L, 121 DEG C of -125 DEG C of continuous sterilizations；
It is that the company of the fermentation medium disappears sequentially with further illustrating：Trace element solution, fermentation medium.
It is with further illustrating, in the first order seed culture, the 4. standard of seed maturity：Under the conditions of wavelength 660nm
Spectrophotometer, carries out detection cell concentration, and ripeness standard is：42-45
It is that fermentation tank PH environment during fermentation uses concentration to be controlled for 8-10mol/L NaOH with further illustrating
It is made as 5.0-5.5；
It is that the OD values of the standard of the seed maturity are higher than 50, carry out secondary seed culture with further illustrating；
It is with further illustrating, the secondary seed medium (g/L)：I glucose syrups 10-15, soy peptone 13.5-
15.5th, calcium carbonate 3.5-5.5, ammonium chloride 0.5-1.8, magnesium chloride 0.32-1.48, dipotassium hydrogen phosphate 0.8-1.2, raw water dissolve simultaneously
It is that 1.5mol/L HCL regulations culture medium initial p H is 5-6 with concentration, 121 DEG C of -124 DEG C of 15min sterilizings are standby；
Ii uses 800L seeding tanks；Seed liquor is transferred in two grades of tanks and is further cultured in first class seed pot, inoculum concentration 15L/
Tank -20L/ tanks；
Iii condition of culture：28 DEG C -30 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1,6h later to seed maturity throughput
Than：1: 2, tank pressure maintains 0.03MPa-0.05MPa；
The standard of iv seed maturities：OD values are between 80-120；
Accessed after v secondary seeds maturation and fermented in the fermentation medium；
Needs are with illustrating further, the plating medium (g/L)：Glucose syrup 2-6, soy peptone 1-3, ox
Meat extract peptone agar 3-4, yeast extract 1-3.
Needs be explanatorily again, the fermentation medium (g/L)：Glucose 150-200, dipotassium hydrogen phosphate 5-10, chlorine
Change magnesium 0.9-1.5, ammonium chloride 8.5-9.5, calcium chloride 20-25, sodium acetate 6-8, potassium chloride 2-3.
Needs be explanatorily again：Fermentation tank is mended with the glucose slurries that concentration is 60% in the fermentation process
Sugar operation；The benefit sugar operation is specific as follows：20h mends sugared speed 20L/h, 30min；50h mends sugared speed 20L/h, 10min.
Beneficial effects of the present invention：
(1) this technique realizes and 56h is foreshortened to the fermentation period of pyruvic acid biofermentation, and acetone acid content reaches
89.10g/L；
(2) technique has passed through the fermentation checking of 130 cubes of large fermentation tanks of our factory fermentation plant, the matter of tunning
Amount and process stabilizing, yield are higher, realize to pyruvic acid industrial volume production；
(3) this technique is by adding the drop of vitamin liquid and liquid microelement for the pyruvic acid occurred in fermentation reality
Solution or transition problem, have carried out preferable solution and have overcome in other words, and then ensure that the stabilization and pyruvic acid of product quality are obtained
Improve on rate ground.
Specific embodiment
With reference to experimental data chart and embodiment, the present invention is further described.
Embodiment 1
Pyruvic acid industrial production zymotechnique, concrete technology step is as follows：
(1) flat board culture：
A. plating medium (g/L)：Glucose syrup 3, soy peptone 3, beef extract-peptone agar 3, calcium carbonate 0.5,
Raw water dissolves, PH natures, and 121 DEG C of 15min sterilizings are standby；
B. in plating medium, flat board is inverted 30 DEG C of cultures in culture dish to torulopsis glabrata, after incubation time 20h；
(2) eggplant bottle culture：
A. eggplant bottle culture medium (g/L)：Glucose syrup 13, soy peptone 11, beef extract-peptone agar 10, calcium carbonate 2,
Ammonium chloride 0.3, raw water dissolves, PH natures, and 121 DEG C of 15min sterilizings are standby；
B. sterile working is strictly pressed in desinfection chamber, single bacterium colony is chosen from preservation flat board and is coated on eggplant bottle solid medium
On；
C. condition of culture：30 DEG C, after incubation time 20h；
D. the refrigerator memory storage for eggplant bottle being placed into 1 DEG C is standby；
(3) seed flask culture：
I. Shake flask medium (g/L)：Glucose syrup 17, soy peptone 13.5, calcium carbonate 2.5, ammonium chloride 0.5, chlorination
Magnesium 0.32, dipotassium hydrogen phosphate 0.2, raw water dissolve and are that 1.5mol/L HCL regulation culture medium initial ps H is 5.5,121 with concentration
DEG C 15min sterilizing is standby；
The triangular flask or steel cylinder 20 to 30 of II.3000ml or 5000ml, in aseptic indoor sterilized inoculation
Shovel, shovel takes bacterium platform and is connected in the triangular flask of 500ml or steel cylinder in cultured eggplant bottle；Inoculum concentration be 600ml/ bottles or
1000ml/ bottles；Cultivation temperature is 30 DEG C, incubation time 24h；
(4) first order seed culture：
1. primary-seed medium (g/L)：Glucose syrup 20, soy peptone 16.5, calcium carbonate 6.5, ammonium chloride 1.5,
Magnesium chloride 1.32, dipotassium hydrogen phosphate 1.2, raw water dissolve and are that 1.5mol/L HCL regulation culture medium initial ps H is 5 with concentration,
121 DEG C of 15min sterilizings are standby；
2. 800L seeding tanks 3 are used；Being merged to triangle steel cylinder for seed flask, enters to seeding tank in an aseptic environment
Row inoculation operation, inoculum concentration is 2000ml/ tanks；
3. condition of culture：28 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1,6h later to seed maturity throughput ratio：1: 4,
Tank pressure maintains 0.03MPa；
4. the standard of seed maturity：OD values are in 40-42.5
Illustrated with reference to chart：
| Incubation time | PH | OD | Cell number (individual) | Bud gives birth to % | Sediments microscope inspection |
| 0h | 5.0 | 10.5 | 10 | Strain is neatly healthy and strong | |
| 6h | 4.13 | 26.8 | 23 | Strain is neatly healthy and strong | |
| 12h | 3.35 | 41.3 | 38 | Strain is neatly healthy and strong |
By detect find first class seed pot OD values growth interval more than 14, to strain OD values reach 40-42.5 it
Between when, it is considered to fermented and cultured is carried out in transferred species to fermentation tank.
(5) fermentation tank culture：
First, fermentation mediums (g/L)：Glucose 150, dipotassium hydrogen phosphate 5, magnesium chloride 0.9, ammonium chloride 8.5, calcium carbonate
20th, sodium acetate 6, defoamer 0.01, liquid microelement 3mL, vitamin liquid 3mL, PH concentration are that 40mol/L HCL are adjusted to 5.5,
121 DEG C of continuous sterilizations；
2nd, condition of culture：30 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1, speed of agitator 150min；6h-12h ventilates
Amount ratio：1: 2, speed of agitator 200r/min；12h-30h throughputs ratio：1: 3, speed of agitator 400r/min；Throughput after 30h
It is 1: 4, speed of agitator 500r/min；40h-56h speeds of agitator 800r/min；Tank pressure is maintained：0.03MPa；
Chart is arranged with reference to detection data to illustrate
It should be noted that RD represents residual sugar, when fermentation medium is prepared, the sky that have detected fermentation medium is sampled
White sample PH：5.23、RD：12.2g/100ml.
Embodiment 2
The eggplant bottle of 1 DEG C of preservation carries out shaking flask inoculation, Shake flask medium (g/L) in refrigerator：Glucose syrup 17.5, soybean protein
Peptone 14.5, calcium carbonate 3.0, ammonium chloride 0.55, magnesium chloride 0.38, dipotassium hydrogen phosphate 0.28, raw water dissolve and are with concentration
1.5mol/L HCL regulation culture medium initial ps H is that 5.5,121 DEG C of 15min sterilizings are standby；
The triangular flask or steel cylinder 20 to 30 of II.3000ml or 5000ml, in aseptic indoor sterilized inoculation
Shovel, shovel takes bacterium platform and is connected in the triangular flask of 500ml or steel cylinder in cultured eggplant bottle；Inoculum concentration be 600ml/ bottles or
1000ml/ bottles；Cultivation temperature is 30 DEG C, incubation time 24h；
First order seed culture：
1. primary-seed medium (g/L)：Glucose syrup 21, soy peptone 17.5, calcium carbonate 6.8, ammonium chloride 1.8,
Magnesium chloride 1.50, dipotassium hydrogen phosphate 1.56, raw water dissolve and are that 1.5mol/L HCL regulations culture medium initial p H is with concentration
5.5,121 DEG C of 15min sterilizings are standby；
2. 800L seeding tanks 3 are used；Being merged to triangle steel cylinder for seed flask, enters to seeding tank in an aseptic environment
Row inoculation operation, inoculum concentration is 2000ml/ tanks；
3. condition of culture：28 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1,6h later to seed maturity throughput ratio：1: 4,
Tank pressure maintains 0.03MPa；
The detection data of Binding experiment result is illustrated
| Incubation time | PH | OD | Cell number (individual) | Bud gives birth to % | Sediments microscope inspection |
| 0h | 5.0 | 13.5 | 12 | Strain is neatly healthy and strong | |
| 6h | 4.36 | 28.8 | 25 | Strain is neatly healthy and strong | |
| 12h | 5.02 | 52.3 | 40 | Strain is neatly healthy and strong |
When finding 12h after testing, the OD values in first class seed pot are 52.3, have exceeded 50, it is necessary to carry out second incubation extremely
Strain OD values reach 100 and carry out being transferred to fermentation tank culture medium again.
Secondary seed culture
Glucose syrup 10, soy peptone 13.5, calcium carbonate 3.5, ammonium chloride 0.5, magnesium chloride 0.32, dipotassium hydrogen phosphate
0.8th, raw water dissolves and is that 1.5mol/L HCL adjust culture medium initial p H for 5,121 DEG C of 15min sterilizings are standby with concentration；
Ii uses 800L seeding tanks；Seed liquor is transferred in two grades of tanks and is further cultured in first class seed pot, inoculum concentration 20L/
Tank；
Iii condition of culture：28 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1,6h later to seed maturity throughput ratio：1∶
2, tank pressure maintains 0.03MPa；
The detection data of Binding experiment result is illustrated
| Incubation time | PH | OD | Cell number (individual) | Bud gives birth to % | Sediments microscope inspection |
| 0h | 5.13 | 43.5 | 19.36 | Strain is neatly healthy and strong | |
| 6h | 5.10 | 60.5 | 30.11 | Strain is neatly healthy and strong | |
| 12h | 5.0 | 78.5 | 46.5 | Strain is neatly healthy and strong | |
| 18h | 4.98 | 99.5 | 57.5 | Strain is neatly healthy and strong |
According to secondary seed Testing index, after meeting transferred species requirement, it is transferred in fermentation tank and is cultivated；The fermented and cultured
Base and fermentation are comprised the following steps that：
First, fermentation mediums (g/L)：Glucose 200, dipotassium hydrogen phosphate 10, magnesium chloride 1.5, ammonium chloride 9.5, carbonic acid
Calcium 25, sodium acetate 8, defoamer 0.02, liquid microelement 5mL, vitamin liquid 5mL, PH concentration are that 40mol/L HCL are adjusted to
5.5,121 DEG C of continuous sterilization 30min；
2nd, condition of culture：30 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1, speed of agitator 150min；6h-12h ventilates
Amount ratio：1: 2, speed of agitator 350min；12h-30h throughputs ratio：1: 3, speed of agitator 500min；Throughput is 1 after 30h:
4, speed of agitator 700min；40h-56h speeds of agitator 1000r/min；Tank pressure is maintained：0.03MPa；
Liquid microelement (g/L)：Zinc chloride 6, iron chloride 18, copper chloride 0.3, manganese chloride 18, concentration is 1.5mol/L
HCL adjusts PH for 5.5 are settled to 1.5L, 121 DEG C of continuous sterilization 15min；
Vitamin liquid (g/L)：Vitamin D 0.04, vitamin E 0.005, vitamin B10.3rd, nicotinic acid 8, concentration
For 1.5mol/L HCL adjust PH for 5.5 are settled to 1.5L, 121 DEG C of continuous sterilization 15min；
The company of fermentation medium disappears sequentially：Trace element solution, fermentation medium (miscible vitamin liquid).
Chart is arranged with reference to detection data to illustrate
Further, the zymotic fluid after the 56h that ferments is derived into zymotic fluid 5L to pilot scale 20L by aseptic inoculation sky steel cylinder
Experimental tank in carry out fermentation and be further cultured for, divulge information according to 1: 1, stirring is not opened, and it is as follows to obtain test experience data：
| Incubation time | ph | RD(g/L) | Acetone acid content (g/L) |
| 2h | 4.96 | 0.2 | 89.10 |
| 4h | 4.95 | 0.0 | 89.08 |
| 6h | 4.90 | 1.0 (mending sugar 30s) | 89.07 |
Further, the vitamin liquid and liquid microelement that are formulated by this serve degraded or conversion to pyruvic acid
Problem has carried out effective solution, stabilizes product quality and yield.
Above-mentioned is that specific embodiment of the invention is described, but not limiting the scope of the invention,
One of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art need not pay
Various modifications or deform still within protection scope of the present invention that creative work can be made.
Claims (11)
1. pyruvic acid industrial production zymotechnique, it is characterized in that, the technique is comprised the following steps：
(1) flat board culture：
A. plating medium (g/L)：Glucose syrup 3-4, soy peptone 3-4, beef extract-peptone agar 3-4, calcium carbonate
0.5-1, raw water dissolving, PH natures, 121 DEG C of -124 DEG C of 15min sterilizings are standby；
B. in plating medium, flat board is inverted 28 DEG C of -30 DEG C of cultures, incubation time 15h- in culture dish to torulopsis glabrata
After 20h；
(2) eggplant bottle culture：
A. eggplant bottle culture medium (g/L)：Glucose syrup 13-14, soy peptone 11-12, beef extract-peptone agar 10-12, carbon
Sour calcium 2-2.5, ammonium chloride 0.3-0.5, raw water dissolving, PH natures, 121 DEG C of -124 DEG C of 15min sterilizings are standby：
B. sterile working is strictly pressed in desinfection chamber, single bacterium colony is chosen from preservation flat board and is coated on eggplant bottle solid medium；
C. condition of culture：28 DEG C -30 DEG C, after incubation time 15h-20h；
D. the refrigerator memory storage for eggplant bottle being placed into 1 DEG C -4 DEG C is standby；
(3) seed flask culture：
I. Shake flask medium (g/L)：Glucose syrup 17-18.5, soy peptone 13.5-15.5, calcium carbonate 2.5-4.5, chlorination
Ammonium 0.5-0.8, magnesium chloride 0.32-0.48, dipotassium hydrogen phosphate 0.2-0.4, raw water dissolve and are that 1.5mol/L HCL are adjusted with concentration
Section culture medium initial p H is 5-6, and 121 DEG C of -124 DEG C of 15min sterilizings are standby；
II. the triangular flask of 3000ml or 5000ml or steel cylinder 20 to 30, shovel in aseptic indoor sterilized inoculation,
Shovel takes bacterium platform and is connected in the triangular flask of 500ml or steel cylinder in cultured eggplant bottle；Inoculum concentration is 600ml/ bottles -1000ml/ bottles；
Cultivation temperature is 28 DEG C -30 DEG C, incubation time 22h-24h；
(4) first order seed culture：
1. primary-seed medium (g/L)：Glucose syrup 20-25, soy peptone 16.5-18.5, calcium carbonate 6.5-8.5, chlorine
Change ammonium 1.5-2.8, magnesium chloride 1.32-3.48, dipotassium hydrogen phosphate 1.2-2.4, raw water dissolve and are 1.5mol/L HCL with concentration
Regulation culture medium initial p H is 5-6, and 121 DEG C of -124 DEG C of 15min sterilizings are standby；
2. using 800L seeding tanks 3-5；Being merged to triangle steel cylinder for seed flask, is carried out to seeding tank in an aseptic environment
Inoculation operation, inoculum concentration is 2000ml/ tank -3000ml/ tanks；
3. condition of culture：28 DEG C -30 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1,6h later to seed maturity throughput ratio：1∶
4, tank pressure maintains 0.03MPa-0.05MPa；
4. the standard of seed maturity：OD values are between 40-50；
(5) one hundred five ten cubes of fermentation tank cultures：
First, fermentation mediums (g/L)：Glucose 150-200, dipotassium hydrogen phosphate 5-10, magnesium chloride 0.9-1.5, ammonium chloride
8.5-9.5, calcium carbonate 20-25, sodium acetate 6-8, defoamer 0.01-0.02, liquid microelement 3-5mL, vitamin liquid 3-5mL,
PH concentration is that 40mol/LHCL is adjusted to 5.0-5.5,121 DEG C of -125 DEG C of continuous sterilizations；
2nd, condition of culture：28 DEG C -30 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1, speed of agitator 100r/min-150min；
6h-12h throughputs ratio：1: 2, speed of agitator 200r/min-350min；12h-30h throughputs ratio：1: 3, speed of agitator 400r/
min-500min；Throughput is 1: 4, speed of agitator 500r/min-700min after 30h；40h-56h speeds of agitator 800r/
min-1000r/min；Tank pressure is maintained：0.03MPa-0.05MPa.
2. pyruvic acid industrial production zymotechnique according to claim 1, it is characterized in that, the liquid microelement (g/L)：
Zinc chloride 3-6, iron chloride 15-18, copper chloride 0.2-0.3, manganese chloride 15-18, concentration are that 1.5mol/LHCL regulations PH is 5.0-
5.0 are settled to 1L-1.5L, 121 DEG C of -125 DEG C of continuous sterilizations.
3. pyruvic acid industrial production zymotechnique according to claim 2, it is characterized in that, the vitamin liquid (g/L)：Dimension
Raw element D 0.03-0.04, vitamin E 0.003-0.005, vitamin B10.2-0.3, nicotinic acid 6-8, concentration is
1.5mol/L HCL adjust PH for 5.0-5.5 is settled to 1L-1.5L, 121 DEG C of -125 DEG C of continuous sterilizations.
4. the pyruvic acid industrial production zymotechnique according to claim 1 or 3, it is characterized in that, the fermentation medium
Company disappears sequentially：Trace element solution, fermentation medium.
5. the pyruvic acid industrial production zymotechnique stated according to claim 4, it is characterized in that, in the first order seed culture, 4.
The standard of seed maturity：Spectrophotometer under the conditions of wavelength 660nm, carries out detection cell concentration, and ripeness standard is：42-45
。
6. pyruvic acid industrial production zymotechnique according to claim 1 or 5, it is characterized in that, the fermentation tank is in fermentation
The PH environment of period uses concentration to be controlled to 5.0-5.5 for 8-10mol/L NaOH.
7. pyruvic acid industrial production zymotechnique according to claim 1, it is characterized in that, the standard of the seed maturity
OD values are higher than 50, carry out secondary seed culture.
8. pyruvic acid industrial production zymotechnique according to claim 7, it is characterized in that, the secondary seed medium
(g/L)：I glucose syrups 10-15, soy peptone 13.5-15.5, calcium carbonate 3.5-5.5, ammonium chloride 0.5-1.8, magnesium chloride
0.32-1.48, dipotassium hydrogen phosphate 0.8-1.2, raw water dissolve and are that 1.5mol/L HCL regulations culture medium initial p H is with concentration
5-6,121 DEG C of -124 DEG C of 15min sterilizings are standby；
Ii uses 800L seeding tanks；Seed liquor is transferred in two grades of tanks and is further cultured in first class seed pot, and inoculum concentration 15L/ tanks-
20L/ tanks；
Iii condition of culture：28 DEG C -30 DEG C of cultivation temperature, 1-6h throughputs compare 1: 1,6h later to seed maturity throughput ratio：1∶
2, tank pressure maintains 0.03MPa-0.05MPa；
The standard of iv seed maturities：OD values are between 80-120；
Accessed after v secondary seeds maturation and fermented in the fermentation medium.
9. pyruvic acid industrial production zymotechnique according to claim 1, it is characterized in that, the plating medium (g/L)：
Glucose syrup 2-6, soy peptone 1-3, beef extract-peptone agar 3-4, yeast extract 1-3.
10. pyruvic acid industrial production zymotechnique according to claim 9, it is characterized in that, the fermentation medium (g/
L)：Glucose 150-200, dipotassium hydrogen phosphate 5-10, magnesium chloride 0.9-1.5, ammonium chloride 8.5-9.5, calcium chloride 20-25, acetic acid
Sodium 6-8, potassium chloride 2-3.
The 11. pyruvic acid industrial production zymotechnique according to claim 1 or claim 10, it is characterized in that, the hair
Fermentation tank is carried out to mend sugared operation with the glucose slurries that concentration is 60% during ferment；The benefit sugar operation is specific as follows：20h
Mend sugared speed 20L/h, 30min；50h mends sugared speed 20L/h, 10min.
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Citations (4)
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|---|---|---|---|---|
| CN101157941A (en) * | 2007-09-19 | 2008-04-09 | 江南大学 | Method for controlling dextrose and vitamine concentration and improving fermentation acetonic acid yield |
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| CN102121035A (en) * | 2010-12-10 | 2011-07-13 | 江南大学 | Method for increasing yield of pyruvic acid |
| CN103710274A (en) * | 2013-12-24 | 2014-04-09 | 江南大学 | Genetically engineered bacterium for increasing yield of extracellular pyruvic acid and application thereof |
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2017
- 2017-01-09 CN CN201710012209.3A patent/CN106834364A/en active Pending
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|---|---|---|---|---|
| CN101157941A (en) * | 2007-09-19 | 2008-04-09 | 江南大学 | Method for controlling dextrose and vitamine concentration and improving fermentation acetonic acid yield |
| CN101659925A (en) * | 2009-09-18 | 2010-03-03 | 神舟天辰科技实业有限公司 | Torulopsis glabrata mutant strain and application thereof in fermentation and production of pyruvic acid |
| CN102121035A (en) * | 2010-12-10 | 2011-07-13 | 江南大学 | Method for increasing yield of pyruvic acid |
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