CN101012465A - Aminoglutaric acid fermentation production method for secondary inoculation superimposing bioepiderm - Google Patents
Aminoglutaric acid fermentation production method for secondary inoculation superimposing bioepiderm Download PDFInfo
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Abstract
The invention discloses a manufacturing method of fermented glutamic acid of secondary seeded superimposed biotin, which comprises the following steps: allocating ferment culture medium; setting the sum of biotin quantity and the ferment liquid of first batch of seed at 5.0-12.0mug/L corresponding to initial bulk; sterilizing; cooling; seeding the first batch of mature seed liquid; stirring; fermenting 9-18h; seeding the second batch of mature seed liquid; carrying biotin through the second batch of seed liquid into ferment tank; setting the biotin quantity of second batch of seed liquid at 2.0-5.0mug/L corresponding to ferment liquid; aerating gas continuously to ferment 28-36h.
Description
Technical field
The invention belongs to biological technical field, be meant that specifically a kind of glucose that utilizes is raw material, serves as to produce bacterial strain with the strain of vitamin H defective type glutamate-producing strain, by the aminoglutaric acid fermentation production method of secondary inoculation superimposing bioepiderm.
Background technology
At present, the vitamin H deficient strain is adopted in domestic glutamic acid fermentation production mostly, and adopts the acid yield height of vitamin H " super suboptimal dose " zymotechnique than traditional biological element " suboptimal dose " zymotechnique.But, the cell concentration of vitamin H " super suboptimal dose " zymotechnique is higher, it requires oxyty also higher, owing to be subjected to the restriction of fermentor tank dissolved oxygen efficiency, many factories are when using vitamin H " super suboptimal dose " zymotechnique, often cause acid yield and glucose acid invert ratio inharmonious, promptly acid yield is higher and glucose acid invert ratio is on the low side, and perhaps acid yield and glucose acid invert ratio are all on the low side.We are by transforming fermenter stirrer to improve dissolved oxygen efficiency, adopt vitamin H " super suboptimal dose " zymotechnique can obtain satisfied acid yield and glucose acid invert ratio, but often occur the raw material batch difference aborning and cause the fluctuation of vitamin H content, when vitamin H total amount in the substratum surmounts " super suboptimal dose " scope a little, it is quite difficult that fermentation control can become, above-mentioned phenomenon can appear in L-glutamic acid acid yield and glucose acid invert ratio equally, and (acid yield and glucose acid invert ratio are inharmonious, be that acid yield is higher and glucose acid invert ratio is on the low side, perhaps acid yield and glucose acid invert ratio are all on the low side).Moreover although the improved dissolved oxygen efficiency of fermenter stirrer increases, its dissolved oxygen efficiency is always limited, and this is the restriction bottleneck that further improves acid yield and glucose acid invert ratio all the time.
Summary of the invention
The objective of the invention is at existing problems in the existing glutamic acid fermentation production, provide a kind of glucose that utilizes to be raw material, with the strain of vitamin H defective type glutamate-producing strain serves as to produce bacterial strain, aminoglutaric acid fermentation production method by secondary inoculation superimposing bioepiderm, improve single jar of output of glutamic acid fermentation, reduce production costs, obtain remarkable economic efficiency.
The objective of the invention is to be realized by following technical proposals: a kind of aminoglutaric acid fermentation production method of secondary inoculation superimposing bioepiderm comprises the steps:
(1) cultivation of first seed:
Prepare first seed culture medium, making the vitamin H amount of first seed culture medium is excessive with respect to seed growth demand in the culture cycle, after sterilization and being cooled to production bacterium suitable growth temperature scope, kind amount by 0.005～0.03% (with seed liquor original volume ratio) inserts shake-flask seed, and 8～20h is cultivated in aeration-agitation.
The cultivation of (2) second batches of seeds:
Prepare second batch of seed culture medium, making the vitamin H amount of second batch of seed culture medium is 2.0～5.0 μ g/L with respect to the fermented liquid original volume, after sterilization and being cooled to production bacterium suitable growth temperature scope, kind amount by 0.005～0.03% (with seed liquor original volume ratio) inserts shake-flask seed, and 8～16h is cultivated in aeration-agitation.
(3) glutamic acid fermentation of secondary inoculation superimposing bioepiderm:
The preparation fermention medium, making the vitamin H amount of first seed culture medium and the vitamin H amount sum of fermention medium is 5.0～12.0 μ g/L with respect to the fermented liquid original volume, after sterilization and being cooled to production bacterium suitable growth temperature scope, kind amount by 1～20% (with fermented liquid original volume ratio) inserts first seed liquor that step (1) is cultivated gained, aeration-agitation fermentation 9～18h, promptly the bacterium cell that inserts first seed liquor by growth form after type of production transforms fully, kind amount by 0.08～10% (with fermented liquid original volume ratio) inserts second batch of seed liquor that step (2) is cultivated gained, and carry vitamin H remaining in the seed culture medium by second batch of seed liquor and enter fermentor tank, and continue aeration-agitation fermentation 28～36h and can finish fermentation.
In order to realize the present invention better, the vitamin H source material of described first seed culture medium, the second batch of seed culture medium or fermention medium adopts one or more mixtures in molasses (vitamin H content is generally 1200～2000 μ g/kg), corn steep liquor (vitamin H content is generally 300～600 μ g/kg) and the vitamin H (reagent).
The vitamin H consumption of seed culture medium must be greater than the seed growth demand in the described step (1), and the best vitamin H total amount of substratum must be decided according to the dissolved oxygen efficiency of concrete fermentor tank in step (1) and the step (3), the best vitamin H total amount of the fermentor tank that dissolved oxygen efficiency is higher is also higher, but requires that the highest oxygen consumption rate must be less than the highest dissolved oxygen efficiency in the fermenting process.
Substratum vitamin H consumption also is excessive with respect to seed growth demand in the culture cycle in the described step (2), but its optimum amount also will be decided according to the dissolved oxygen efficiency and the fermentation period of concrete fermentor tank, in the fermentation period of restriction, best vitamin H amount is also higher in the step of the fermentor tank that dissolved oxygen efficiency is higher (2).
Sterilization method be real jar of sterilization or continuous sterilization in described step (1), step (2) and the step (3), and sterilising temp and sterilization time design meet the requirement of the principle of sterilize (N after the sterilization is got in the residual law calculating of employing logarithm
s=10
-3).
Culture temperature is 30～34 ℃ in described step (1) and the step (2), cultivates pH and is controlled to be 6.6～8.0, and air flow is controlled to be 0.10～0.50vvm (with respect to the seed liquor volume), and mixing speed is 100～300r/min.
In the described step (3), the leavening temperature span of control is 30～40 ℃, adopt multistage stage by stage temperature control (30～37 ℃ of the general controls of growth phase temperature according to somatic cells growth, transition, metabolic optimum temperature range difference, 34～38 ℃ of the general controls of phase temperature transition, 36～40 ℃ of general controls of metabolism L-glutamic acid stage), leavening temperature control curve is ascendant trend gradually, is increased to higher metabolic temperature gradually by lower growth temperature; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; Adopt intermittent injecting Glucose Liquid or Continuous Flow to add Glucose Liquid mode supplementary carbon source, the concentration of Glucose Liquid is 300～650g/L, additional amount, adds number of times or stream dosage and specifically consumes sugared situation according to thalline and decide; According to thalline oxygen requirement control air flow is 0.10～0.50vvm (with respect to the fermented liquid original volume), and the control mixing speed is 100～200r/min.
Described production bacterial strain is the strain of vitamin H defective type glutamate-producing strain, comprises Brevibacteriumtianjinese S9114, Brevibacterium tianjinese FM
84-415, Corynebacterium PekineseAs1.299, Brevibacterium lactofermentum L
B8801, Corynebacterium PekineseAs1.524, Brevibacterium tianjinese T
6-13, or Brevibacterium tianjinese T
G866Deng.
The present invention adds vitamin H technology with existing once inoculation and compares, and has following advantage and beneficial effect:
1, suitably improving the vitamin H consumption helps improving the cell concentration of glutamic acid fermentation and promotes the sugared speed of consumption.In the fermentor tank of same jar of type condition, the present invention determines that according to the dissolved oxygen speed of concrete fermentor tank best vitamin H consumption carries out adding the first time vitamin H, the an amount of seed liquor that when vitamin H is consumed to a certain degree, superposes again and an amount of vitamin H, its vitamin H consumption can be more than the vitamin H consumption that once adds vitamin H technology, final cell concentration and consumption sugar amount can be bigger, put tank volume and acid yield and therefore be significantly improved, single jar of output can improve about 15%.
2, in once inoculating interpolation vitamin H technology, if vitamin H is excessive, cell concentration can be excessive, and the highest oxygen consumption rate can be greater than the dissolved oxygen speed of fermentor tank in the fermenting process, and the eubolism of thalline can be had a strong impact on.But, the present invention has utilized vitamin H stack, somatic cells growth and the synergetic principle of metabolism, though final cell concentration increases, but the highest oxygen consumption rate does not increase, the existing dissolved oxygen efficiency of fermentor tank can satisfy the demand of thalline to dissolved oxygen fully, somatic cells in time produces the synergetic effect of fermentation result transition in the fermenting process.If once the used vitamin H amount of inoculation interpolation vitamin H technology is the same with vitamin H total amount of the present invention, single jar of glutamic acid yield of the present invention and glucose acid invert ratio have improved about 20% and about 15% than once inoculating interpolation vitamin H technology respectively.Coordinate because glucose acid invert ratio and acid yield increase, production cost significantly reduces.
3, in temperature higher season, because cooling difficulty in the glutamic acid fermentation process needs to reduce an addition of vitamin H, so that reduce the peak value of heat of fermentation, so once the inoculation fermentation level that adds vitamin H technology can so and reduce.Adopt method of the present invention, though reduce the addition first time of vitamin H, by the secondary superimposing bioepiderm, its fermentation level adds vitamin H technology far above once inoculating.Therefore, the present invention is applied to high temperature season fermentative production L-glutamic acid, helps reducing the peak value of heat of fermentation, and obtains ideal fermentation result.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited thereto.
Embodiment 1---at 80m
3Implement on the fermentor tank (biotin concentration 5.0 μ g/L superpose 2.0 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese S9114, South China Science ﹠ Engineering University's cloud meet continuous heavy rain and Zhou Wanbing seed selection, have been widely used in producing)
Seeding tank: 1.0m
3The general form fermentor tank
Fermentor tank: 80m
3The general form fermentor tank
The cultivation of first seed: glucose 18kg, urea 3.5kg, KH
2PO
41.1kg, MgSO
47H
2O 0.5kg, molasses 20kg, corn steep liquor 30kg, FeSO
4And MnSO
4Each 1.4g, defoamer 0.15kg regulates pH to 7.0 with alkali lye, constant volume 700L, real jar of sterilization inserts the 1000mL shake-flask seed to 121～122 ℃ of insulation 10min when being cooled to 31～32 ℃.In the culturing process, mixing speed is 250～300r/min, and temperature is controlled to be 30～33 ℃, is 0.20～0.28vvm (with respect to the seed liquor volume) according to thalline oxygen requirement control air flow, cultivates 8h.
The cultivation of second batch of seed: glucose 12kg, urea 2kg, KH
2PO
40.62kg, MgSO
47H
2O0.28kg, corn steep liquor 30kg, vitamin H (reagent is pure) 81mg, FeSO
4And MnSO
4Each 1.4g, defoamer 0.10kg regulates pH to 7.0 with alkali lye, constant volume 400L, real jar of sterilization inserts the 600mL shake-flask seed to 121～122 ℃ of insulation 10min when being cooled to 31～32 ℃.In the culturing process, mixing speed is 250～300r/min, and temperature is controlled to be 30～33 ℃, is 0.20～0.28vvm (with respect to the seed liquor volume) according to thalline oxygen requirement control air flow, cultivates 8h.
The glutamic acid fermentation of secondary inoculation superimposing bioepiderm: glucose 7200kg, KH
2PO
456kg, KCL48kg, MgSO
47H
2O 55kg, corn steep liquor 90kg, vitamin H (reagent is pure) 150mg, FeSO
4, MnSO
4Each 96g/m
3, it is 48m that defoamer 3kg, constant volume make the fermented liquid original volume
3, continuous sterilization, sterilising temp control is also kept 8～10min for 121～122 ℃, is cooled to 32～33 ℃ of first mature seed liquid of access and ferments.Fermentation 16h, by microscopic examination, first seed cell is transformed to type of production by growth form fully, inserts second batch of mature seed liquid, carries remaining vitamin H by second batch of seed liquor and enters in the fermentor tank, proceeds fermentation.In the fermenting process, temperature controlling range is 32～40 ℃, adopt multistage stage by stage temperature control (30～37 ℃ of the general controls of growth phase temperature according to somatic cells growth, transition, metabolic optimum temperature range difference, 34～38 ℃ of the general controls of phase temperature transition, 36～40 ℃ of general controls of metabolism L-glutamic acid stage), leavening temperature is increased to higher metabolic temperature gradually by lower growth temperature, is ascendant trend gradually; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; The Glucose Liquid supplementary carbon source of intermittent injecting 320g/L was added once in per 2 hours, and each additional amount specifically consumes sugared situation on thalline to be decided; According to thalline oxygen requirement control air flow is 0.12～0.32vvm (fermented liquid original volume relatively), and the control mixing speed is 160～200r/min.Fermentation 36h finishes, and the fermentation and acid level is 118.3g/L, and putting tank volume is 62.5m
3, glutamic acid yield is 7.46t, glucose acid invert ratio is 62.97%.
Embodiment 2---at 120m
3Implement on the fermentor tank (biotin concentration 12 μ g/L superpose 5.0 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese FM
84-415, the kindhearted seed selection of Zheng of Fudan University has been widely used in producing)
Seeding tank: 20m
3General form fermentor tank (being used for first seed culture), 10m
3General form fermentor tank (being used for second batch of seed culture)
Fermentor tank: 120m
3The general form fermentor tank
The cultivation of first seed: glucose 700kg, KH
2PO
426kg, MgSO
47H
2O 9.6kg, molasses 150kg, corn steep liquor 400kg, vitamin H (reagent is pure) 200mg, FeSO
4And MnSO
4Each 24g, defoamer 3.0kg, constant volume 12000L, real jar of sterilization regulated pH to 7.0 with liquefied ammonia to 121～122 ℃ of insulation 10min when being cooled to 30～32 ℃, inserts the 1200mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 160r/min, according to thalline oxygen requirement control air flow is 0.20～0.50vvm (with respect to the seed liquor volume), cultivates 18h.
The cultivation of second batch of seed: glucose 330kg, KH
2PO
412kg, MgSO
47H
2O 4.8kg, corn steep liquor 200kg, vitamin H (reagent is pure) 200mg, FeSO
4And MnSO
4Each 10g, defoamer 1.5kg, constant volume 6000L, real jar of sterilization regulated pH to 7.0 with liquefied ammonia to 121～122 ℃ of insulation 10min when being cooled to 30～32 ℃, inserts the 1200mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 160r/min, according to thalline oxygen requirement control air flow is 0.22～0.48vvm (with respect to the seed liquor volume), cultivates 16h.
The glutamic acid fermentation of secondary inoculation superimposing bioepiderm: glucose 7200kg, vitamin H (reagent is pure) 95mg, 85% H
3PO
465kg, KCL100kg, MgSO
47H
2O 70kg, FeSO
4, MnSO
4Each 120g/m
3, it is 60m that defoamer 4kg, constant volume make the fermented liquid original volume
3, continuous sterilization, sterilising temp control is also kept 8～10min for 121～122 ℃, is cooled to 33～34 ℃ of first mature seed liquid of access and ferments.Fermentation 9h, by microscopic examination, first seed cell is transformed to type of production by growth form fully, inserts second batch of mature seed liquid, carries remaining vitamin H by second batch of seed liquor and enters in the fermentor tank, proceeds fermentation.In the fermenting process, temperature controlling range is 33～40 ℃, adopt multistage stage by stage temperature control (30～37 ℃ of the general controls of growth phase temperature according to somatic cells growth, transition, metabolic optimum temperature range difference, 34～38 ℃ of the general controls of phase temperature transition, 36～40 ℃ of general controls of metabolism L-glutamic acid stage), leavening temperature is increased to higher metabolic temperature gradually by lower growth temperature, is ascendant trend gradually; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; Continuous Flow adds the Glucose Liquid supplementary carbon source of 450g/L, and the stream dosage specifically consumes sugared situation on thalline to be decided; According to thalline oxygen requirement control air flow is 0.24～0.50vvm (fermented liquid original volume relatively), and the control mixing speed is 150r/min.Fermentation 28h finishes, and the fermentation and acid level is 139.5g/L, and putting tank volume is 94m
3, glutamic acid yield is 13.11t, glucose acid invert ratio is 62.94%.
Embodiment 3---at 150m
3Implement on the fermentor tank (biotin concentration 7.0 μ g/L superpose 2.5 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese S9114, South China Science ﹠ Engineering University's cloud meet continuous heavy rain and Zhou Wanbing seed selection, have been widely used in producing)
Seeding tank: 10m
3The general form fermentor tank
Fermentor tank: 150m
3The general form fermentor tank
The cultivation of first seed: glucose 190kg, urea 38kg, KH
2PO
412kg, MgSO
47H
2O 6kg, molasses 60kg, corn steep liquor 250kg, vitamin H (reagent is pure) 150mg, FeSO
4And MnSO
4Each 15g, defoamer 1.2kg regulates pH to 7.0 with alkali lye, constant volume 7500L, real jar of sterilization inserts the 400mL shake-flask seed to 121～122 ℃ of insulation 10min when being cooled to 30～32 ℃.In the culturing process, mixing speed is 160r/min, and temperature is controlled to be 31～33 ℃, is 0.10～0.28vvm (with respect to the seed liquor volume) according to thalline oxygen requirement control air flow, cultivates 9h.
The cultivation of second batch of seed: glucose 190kg, urea 38kg, KH
2PO
412kg, MgSO
47H
2O 6kg, molasses 50kg, corn steep liquor 200kg, vitamin H (reagent is pure) 50mg, FeSO
4And MnSO
4Each 15g, defoamer 1.2kg regulates pH to 7.0 with alkali lye, constant volume 400L, real jar of sterilization inserts the 400mL shake-flask seed to 121～122 ℃ of insulation 10min when being cooled to 31～32 ℃.In the culturing process, mixing speed is 160r/min, and temperature is controlled to be 31～33 ℃, is 0.10～0.28vvm (with respect to the seed liquor volume) according to thalline oxygen requirement control air flow, cultivates 9h.
The glutamic acid fermentation of secondary inoculation superimposing bioepiderm: glucose 12600kg, vitamin H (reagent is pure) 115mg, molasses 100kg, 85% H
3PO
495kg, KCL 150kg, MgSO
47H
2O 100kg, FeSO
4, MnSO
4It is 90m that each 180g, defoamer 5.5kg, constant volume make the fermented liquid original volume
3, continuous sterilization, sterilising temp control is also kept 8～10min for 121～122 ℃, is cooled to 32～34 ℃ of first mature seed liquid of access and ferments.Fermentation 18h, by microscopic examination, first seed cell is transformed to type of production by growth form fully, inserts second batch of mature seed liquid, carries remaining vitamin H by second batch of seed liquor and enters in the fermentor tank, proceeds fermentation.In the fermenting process, temperature controlling range is 32～40 ℃, adopt multistage stage by stage temperature control (30～37 ℃ of the general controls of growth phase temperature according to somatic cells growth, transition, metabolic optimum temperature range difference, 34～38 ℃ of the general controls of phase temperature transition, 36～40 ℃ of general controls of metabolism L-glutamic acid stage), leavening temperature is increased to higher metabolic temperature gradually by lower growth temperature, is ascendant trend gradually; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; Continuous Flow adds the Glucose Liquid supplementary carbon source of 650g/L, and the stream dosage specifically consumes sugared situation on thalline to be decided; According to thalline oxygen requirement control air flow is 0.12～0.42vvm (fermented liquid original volume relatively), and the control mixing speed is 140r/min.Fermentation 34h finishes, and the fermentation and acid level is 139.0g/L, and putting tank volume is 117.5m
3, glutamic acid yield is 16.33t, glucose acid invert ratio is 62.87%.
Embodiment 4---at 200m
3Implement on the fermentor tank (biotin concentration 8.0 μ g/L superpose 3.0 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese FM
84-415, the kindhearted seed selection of Zheng of Fudan University has been widely used in producing)
Seeding tank: 10m
3The general form fermentor tank
Fermentor tank: 200m
3The general form fermentor tank
The cultivation of first seed: glucose 300kg, KH
2PO
412kg, MgSO
47H
2O 6kg, molasses 100kg, corn steep liquor 200kg, vitamin H (reagent is pure) 150mg, FeSO
4And MnSO
4Each 15g, defoamer 1.5kg, constant volume 7500L, real jar of sterilization regulated pH to 7.0 with liquefied ammonia to 121～122 ℃ of insulation 10min when being cooled to 31～32 ℃, inserts the 4000mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 160r/min, according to thalline oxygen requirement control air flow is 0.22～0.44vvm (with respect to the seed liquor volume), cultivates 12h.
The cultivation of second batch of seed: glucose 160kg, KH
2PO
46.4kg, MgSO
47H
2O 3.2kg, corn steep liquor 100kg, vitamin H (reagent is pure) 310mg, FeSO
4And MnSO
4Each 8g, defoamer 0.50kg, constant volume 4000L, real jar of sterilization regulated pH to 7.0 with liquefied ammonia to 121～122 ℃ of insulation 10min when being cooled to 31～32 ℃, inserts the 4000mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 160r/min, according to thalline oxygen requirement control air flow is 0.25～0.44vvm (with respect to the seed liquor volume), cultivates 12h.
The glutamic acid fermentation of secondary inoculation superimposing bioepiderm: glucose 18500kg, 85% H
3PO
4120kg, KCL 200kg, MgSO
47H
2O 140kg, molasses 100kg, corn steep liquor 200kg, vitamin H (reagent is pure) 310mg, FeSO
4, MnSO
4It is 120m that each 240g, defoamer 10kg, constant volume make the fermented liquid original volume
3, continuous sterilization, sterilising temp control is also kept 8～10min for 121～122 ℃, is cooled to 33～34 ℃ of first mature seed liquid of access and ferments.Fermentation 14h, by microscopic examination, first seed cell is transformed to type of production by growth form fully, inserts second batch of mature seed liquid, carries remaining vitamin H by second batch of seed liquor and enters in the fermentor tank, proceeds fermentation.In the fermenting process, temperature controlling range is 33～40 ℃, adopt multistage stage by stage temperature control (30～37 ℃ of the general controls of growth phase temperature according to somatic cells growth, transition, metabolic optimum temperature range difference, 34～38 ℃ of the general controls of phase temperature transition, 36～40 ℃ of general controls of metabolism L-glutamic acid stage), leavening temperature is increased to higher metabolic temperature gradually by lower growth temperature, is ascendant trend gradually; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; Continuous Flow adds the Glucose Liquid supplementary carbon source of 500g/L, and the stream dosage specifically consumes sugared situation on thalline to be decided; According to thalline oxygen requirement control air flow is 0.22～0.48vvm (fermented liquid original volume relatively), and the control mixing speed is 120r/min.Fermentation 32h finishes, and the fermentation and acid level is 139.6g/L, and putting tank volume is 155m
3, glutamic acid yield is 21.64t, glucose acid invert ratio is 62.80%.
Embodiment 5---at 350m
3Implement on the fermentor tank (biotin concentration 10.0 μ g/L superpose 4.0 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese S9114, South China Science ﹠ Engineering University's cloud meet continuous heavy rain and Zhou Wanbing seed selection, have been widely used in producing)
Seeding tank: 30m
3The general form fermentor tank
Fermentor tank: 350m
3The general form fermentor tank
The cultivation of first seed: glucose 1600kg, KH
2PO
450kg, MgSO
47H
2O 18kg, molasses 300kg, corn steep liquor 600kg, vitamin H (reagent is pure) 500mg, FeSO
4And MnSO
4Each 45g, defoamer 4kg, constant volume 22500L, continuous sterilization to 121～122 ℃ insulation 10min regulates pH to 7.0 with liquefied ammonia when being cooled to 31～32 ℃, inserts the 2000mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 150r/min, according to thalline oxygen requirement control air flow is 0.10～0.50vvm (with respect to the seed liquor volume), cultivates 20h.
The cultivation of second batch of seed: glucose 500kg, KH
2PO
417kg, MgSO
47H
2O 8.5kg, molasses 150kg, corn steep liquor 200kg, vitamin H (reagent is pure) 555mg, FeSO
4And MnSO
4Each 20g, defoamer 2.0kg, constant volume 10000L, real jar of sterilization regulated pH to 7.0 with liquefied ammonia to 121～122 ℃ of insulation 10min when being cooled to 31～32 ℃, inserts the 2000mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 150r/min, according to thalline oxygen requirement control air flow is 0.20～0.44vvm (with respect to the seed liquor volume), cultivates 15h.
The glutamic acid fermentation of secondary inoculation superimposing bioepiderm: glucose 35200kg, 85% H
3PO
4220kg, KCL 360kg, MgSO
47H
2O 250kg, molasses 180kg, corn steep liquor 300kg, vitamin H (reagent is pure) 530mg, FeSO
4, MnSO
4It is 220m that each 440g, defoamer 18kg, constant volume make the fermented liquid original volume
3, continuous sterilization, sterilising temp control is also kept 8～10min for 121～122 ℃, is cooled to 33～34 ℃ of first mature seed liquid of access and ferments.Fermentation 16h, by microscopic examination, first seed cell is transformed to type of production by growth form fully, inserts second batch of mature seed liquid, carries remaining vitamin H by second batch of seed liquor and enters in the fermentor tank, proceeds fermentation.In the fermenting process, temperature controlling range is 33～40 ℃, adopt multistage stage by stage temperature control (30～37 ℃ of the general controls of growth phase temperature according to somatic cells growth, transition, metabolic optimum temperature range difference, 34～38 ℃ of the general controls of phase temperature transition, 36～40 ℃ of general controls of metabolism L-glutamic acid stage), leavening temperature is increased to higher metabolic temperature gradually by lower growth temperature, is ascendant trend gradually; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; Continuous Flow adds the Glucose Liquid supplementary carbon source of 520g/L, and the stream dosage specifically consumes sugared situation on thalline to be decided; According to thalline oxygen requirement control air flow is 0.24～0.50vvm (fermented liquid original volume relatively), and the control mixing speed is 120r/min.Fermentation 34h finishes, and the fermentation and acid level is 141.4g/L, and putting tank volume is 275m
3, glutamic acid yield is 38.89t, glucose acid invert ratio is 64.07%.
Embodiment 6---at 350m
3Implement on the fermentor tank (biotin concentration 8.5 μ g/L superpose 3.0 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese S9114, South China Science ﹠ Engineering University's cloud meet continuous heavy rain and Zhou Wanbing seed selection, have been widely used in producing)
Seeding tank: 30m
3The general form fermentor tank
Fermentor tank: 350m
3The general form fermentor tank
The cultivation of first seed: glucose 1100kg, KH
2PO
436kg, MgSO
47H
2O 18kg, molasses 300kg, corn steep liquor 500kg, vitamin H (reagent is pure) 500mg, FeSO
4And MnSO
4Each 45g, defoamer 4kg, constant volume 22500L, continuous sterilization to 121～122 ℃ insulation 10min regulates pH to 7.0 with liquefied ammonia when being cooled to 31～32 ℃, inserts the 3000mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 150r/min, according to thalline oxygen requirement control air flow is 0.16～0.48vvm (with respect to the seed liquor volume), cultivates 16h.
The cultivation of second batch of seed: glucose 480kg, KH
2PO
415kg, MgSO
47H
2O 8.0kg, molasses 150kg, corn steep liquor 250kg, vitamin H (reagent is pure) 310mg, FeSO
4And MnSO
4Each 20g, defoamer 2.0kg, constant volume 10000L, real jar of sterilization regulated pH to 7.0 with liquefied ammonia to 121～122 ℃ of insulation 10min when being cooled to 31～32 ℃, inserts the 3000mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 150r/min, according to thalline oxygen requirement control air flow is 0.22～0.44vvm (with respect to the seed liquor volume), cultivates 14h.
The glutamic acid fermentation of secondary inoculation superimposing bioepiderm: glucose 35200kg, 85% H
3PO
4220kg, KCL 360kg, MgSO
47H
2O 250kg, molasses 200kg, corn steep liquor 300kg, vitamin H (reagent is pure) 220mg, FeSO
4, MnSO
4It is 220m that each 440g, defoamer 18kg, constant volume make the fermented liquid original volume
3, continuous sterilization, sterilising temp control is also kept 8～10min for 121～122 ℃, is cooled to 33～34 ℃ of first mature seed liquid of access and ferments.Fermentation 14h, by microscopic examination, first seed cell is transformed to type of production by growth form fully, inserts second batch of mature seed liquid, carries remaining vitamin H by second batch of seed liquor and enters in the fermentor tank, proceeds fermentation.In the fermenting process, temperature controlling range is 33～40 ℃, adopt multistage stage by stage temperature control (30～37 ℃ of the general controls of growth phase temperature according to somatic cells growth, transition, metabolic optimum temperature range difference, 34～38 ℃ of the general controls of phase temperature transition, 36～40 ℃ of general controls of metabolism L-glutamic acid stage), leavening temperature is increased to higher metabolic temperature gradually by lower growth temperature, is ascendant trend gradually; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; Continuous Flow adds the Glucose Liquid supplementary carbon source of 500g/L, and the stream dosage specifically consumes sugared situation on thalline to be decided; According to thalline oxygen requirement control air flow is 0.24～0.50vvm (fermented liquid original volume relatively), and the control mixing speed is 120r/min.Fermentation 34h finishes, and the fermentation and acid level is 137.7g/L, and putting tank volume is 273.5m
3, glutamic acid yield is 37.66t, glucose acid invert ratio is 64.34%.
Comparative Examples 1---at 80m
3Fermentor tank top fermentation (once adding biotin concentration 5.0 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese S9114, South China Science ﹠ Engineering University's cloud meet continuous heavy rain and Zhou Wanbing seed selection, have been widely used in producing)
Seeding tank: 1.0m
3The general form fermentor tank
Fermentor tank: 80m
3The general form fermentor tank
The cultivation of seed: identical with first seed culture among the embodiment 1.
Once the glutamic acid fermentation of vitamin H is added in inoculation: fermention medium, sterilising method are identical with embodiment's 1, and mature seed liquid is inserted in sterilization cooling back, and fermenting process is controlled identical with the control among the embodiment 1.Fermentation 36h finishes, and the fermentation and acid level is 109.5g/L, and putting tank volume is 58.5m
3, glutamic acid yield is 6.41t, glucose acid invert ratio is 60.56%.
Comparative Examples 2---at 200m
3Fermentor tank top fermentation (once adding biotin concentration 8.0 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese FM
84-415, the kindhearted seed selection of Zheng of Fudan University has been widely used in producing)
Seeding tank: 10m
3The general form fermentor tank
Fermentor tank: 200m
3The general form fermentor tank
The cultivation of seed: identical with first seed culture among the embodiment 4.
Once the glutamic acid fermentation of vitamin H is added in inoculation: fermention medium, sterilising method are identical with embodiment's 4, and mature seed liquid is inserted in sterilization cooling back, and fermenting process is controlled identical with the control among the embodiment 4.Fermentation 32h finishes, and the fermentation and acid level is 131.2g/L, and putting tank volume is 142m
3, glutamic acid yield is 18.66t, glucose acid invert ratio is 62.52%.
Comparative Examples 3---at 200m
3Fermentor tank top fermentation (once adding biotin concentration 11.0 μ g/L)
Produce bacterial classification: (Brevibacterium tianjinese FM
84-415, the kindhearted seed selection of Zheng of Fudan University has been widely used in producing)
Seeding tank: 10m
3The general form fermentor tank
Fermentor tank: 200m
3The general form fermentor tank
The cultivation of seed: identical with first seed culture among the embodiment 4.
The cultivation of first seed: glucose 300kg, KH
2PO
412kg, MgSO
47H
2O 6kg, molasses 100kg, corn steep liquor 200kg, vitamin H (reagent is pure) 150mg, FeSO
4And MnSO
4Each 15g, defoamer 1.5kg, constant volume 7500L, real jar of sterilization regulated pH to 7.0 with liquefied ammonia to 121～122 ℃ of insulation 10min when being cooled to 31～32 ℃, inserts the 4000mL shake-flask seed.In the culturing process, temperature is controlled to be 31～32 ℃, and Continuous Flow adds the additional nitrogenous source of liquefied ammonia and controls pH is 7.0～7.2, and mixing speed is controlled to be 160r/min, according to thalline oxygen requirement control air flow is 0.22～0.44vvm (with respect to the seed liquor volume), cultivates 12h.
Once the glutamic acid fermentation of vitamin H is added in inoculation: glucose 18500kg, 85% H
3PO
4120kg, KCL 200kg, MgSO
47H
2O 140kg, molasses 300kg, corn steep liquor 400kg, vitamin H (reagent is pure) 270mg, FeSO
4, MnSO
4It is 120m that each 240g, defoamer 10kg, constant volume make the fermented liquid original volume
3, continuous sterilization, sterilising temp control is also kept 8～10min for 121～122 ℃, is cooled to 33～34 ℃ of access mature seed liquid and ferments.In the fermenting process, temperature controlling range is 33～40 ℃, adopt multistage stage by stage temperature control (30～37 ℃ of the general controls of growth phase temperature according to somatic cells growth, transition, metabolic optimum temperature range difference, 34～38 ℃ of the general controls of phase temperature transition, 36～40 ℃ of general controls of metabolism L-glutamic acid stage), leavening temperature is increased to higher metabolic temperature gradually by lower growth temperature, is ascendant trend gradually; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; Continuous Flow adds the Glucose Liquid supplementary carbon source of 500g/L, and the stream dosage specifically consumes sugared situation on thalline to be decided; According to thalline oxygen requirement control air flow is 0.22～0.50vvm (fermented liquid original volume relatively), and the control mixing speed is 120r/min.Fermentation 32h finishes, and the fermentation and acid level is 118.9g/L, and putting tank volume is 146m
3, glutamic acid yield is 17.36t, glucose acid invert ratio is 54.59%.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (6)
1, a kind of aminoglutaric acid fermentation production method of secondary inoculation superimposing bioepiderm is characterized in that comprising the steps:
(1) cultivation of first seed:
Prepare first seed culture medium, making the vitamin H amount of first seed culture medium is excessive with respect to seed growth demand in the culture cycle, after sterilization and being cooled to production bacterium suitable growth temperature scope, kind amount by 0.005～0.03% inserts shake-flask seed, and 8～20h is cultivated in aeration-agitation;
The cultivation of (2) second batches of seeds:
Prepare second batch of seed culture medium, making the vitamin H amount of second batch of seed culture medium is 2.0～5.0 μ g/L with respect to the fermented liquid original volume, after sterilization and being cooled to production bacterium suitable growth temperature scope, the kind amount by 0.005～0.03% inserts shake-flask seed, and 8～16h is cultivated in aeration-agitation;
(3) glutamic acid fermentation of secondary inoculation superimposing bioepiderm:
The preparation fermention medium, making the vitamin H amount of first seed culture medium and the vitamin H amount sum of fermention medium is 5.0～12.0 μ g/L with respect to the fermented liquid original volume, after sterilization and being cooled to production bacterium suitable growth temperature scope, kind amount by 1～20% inserts first seed liquor that step (1) is cultivated gained, aeration-agitation fermentation 9～18h, promptly the bacterium cell that inserts first seed liquor by growth form after type of production transforms fully, kind amount by 0.08～10% inserts second batch of seed liquor that step (2) is cultivated gained, and continues aeration-agitation fermentation 28～36h and promptly finish fermentation.
2, the aminoglutaric acid fermentation production method of a kind of secondary inoculation superimposing bioepiderm according to claim 1 is characterized in that: the vitamin H source material of described first seed culture medium, the second batch of seed culture medium or fermention medium adopts one or more mixtures in molasses, corn steep liquor and the vitamin H.
3, the aminoglutaric acid fermentation production method of a kind of secondary inoculation superimposing bioepiderm according to claim 1 is characterized in that: described sterilization method is real jar of sterilization or continuous sterilization.
4, the aminoglutaric acid fermentation production method of a kind of secondary inoculation superimposing bioepiderm according to claim 1, it is characterized in that: culture temperature is 30～34 ℃ in described step (1) and the step (2), cultivate pH and be controlled to be 6.6～8.0, air flow is controlled to be 0.10～0.50vvm, and mixing speed is 100～300r/min.
5, the aminoglutaric acid fermentation production method of a kind of secondary inoculation superimposing bioepiderm according to claim 1 is characterized in that: in the described step (3), the leavening temperature span of control is 30～40 ℃; The mode that adopts Continuous Flow to add liquefied ammonia is replenished nitrogenous source and controlled pH is 6.5～7.7; Adopt intermittent injecting Glucose Liquid or Continuous Flow to add Glucose Liquid mode supplementary carbon source, the concentration of Glucose Liquid is 300～650g/L; According to thalline oxygen requirement control air flow is 0.10～0.50vvm, and the control mixing speed is 100～200r/min.
6, the aminoglutaric acid fermentation production method of a kind of secondary inoculation superimposing bioepiderm according to claim 1, it is characterized in that: described production bacterial strain is the strain of vitamin H defective type glutamate-producing strain, comprises Brevibacteriumtianjinese S9114, Brevibacterium tianjinese FM
84-415, Corynebacterium PekineseAs1.299, Brevibacterium lactofermentum L
B8801, Corynebacterium PekineseAs1.524, Brevibacterium tianjinese T
6-13, or Bevibacterium tianjinese T
G866
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703537A (en) * | 2012-06-26 | 2012-10-03 | 呼伦贝尔东北阜丰生物科技有限公司 | Novel production method for glutamic acid |
| CN107099563A (en) * | 2017-06-02 | 2017-08-29 | 卢松 | It is a kind of the method that power technology prepares monosodium glutamate such as to utilize |
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2007
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703537A (en) * | 2012-06-26 | 2012-10-03 | 呼伦贝尔东北阜丰生物科技有限公司 | Novel production method for glutamic acid |
| CN107099563A (en) * | 2017-06-02 | 2017-08-29 | 卢松 | It is a kind of the method that power technology prepares monosodium glutamate such as to utilize |
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