CN108004180A - The method that a kind of control pH and feed supplement double fluid increase density fermentation acetobacter - Google Patents
The method that a kind of control pH and feed supplement double fluid increase density fermentation acetobacter Download PDFInfo
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Abstract
The invention belongs to technical field of food biotechnology, discloses the method that a kind of control pH and feed supplement double fluid increase density fermentation acetobacter.Method is：(1) thalline activates；(2) Shaking culture；(3) seeding tank spreads cultivation；(4) ferment tank：The seed liquor that step (3) obtains is accessed in fermentation tank by 1%~20% inoculum concentration and is cultivated on culture medium, condition of culture：The sodium hydroxide of 100~300rpm of speed of agitator, 0.1~1.0vvm of throughput, 28~30 DEG C of temperature, stream plus 5~10M, control pH5.5~6.0 of zymotic fluid, while flow and add yeast extract and corn pulp, 8~12h of incubation time.The present invention greatly shortens the production cycle so that thalline comparatively fast can be more enriched with, and realize 1.56~3.34 × 10 by way of controlling pH and taking yeast extract and the double flow feedings of corn pulp9The high density fermentation of cfu/mL.
Description
Technical field
The present invention relates to a kind of method that control pH and feed supplement double fluid increase density fermentation acetobacter, belong to biological food
Technical field.
Background technology
Acetobacter, Gram-negative, does not produce gemma, can be used to vinegar processed, aerobic respiration, aerobic, neutral and sour
Ethanol (alcohol) can be oxidized to acetic acid under the conditions of property.Thalline in alcohol and the fermentation of bottom acid content Dichlorodiphenyl Acetate in fermentation medium
Growth and production acid amount have a significant impact.Although acetobacter Dichlorodiphenyl Acetate has certain tolerance, with fermentation time
Extend, produce the gradually increase of acid amount, acetobacter may be killed by the acid of oneself production.
Compared to the high speed development of dairy industry, the research of China's acetobacter microbial inoculum falls behind relatively, traditional zymotic technique
Frequently with Batch fermentation and single batch fed-batch fermentation technique, and analysis and optimization is carried out to preliminary fermentation culture medium.According to text
Offer《It is prepared by direct putting type high activity fruits and vegetables acetic acid fermenting agent》The shaking flask Batch fermentation of report, the viable count under optimal conditions of fermentation
Only 2.74~5.56 × 108Cfu/mL, its fermentation level cannot still meet industrialized production.According to document《Pasteur's acetic acid bar
The optimization of bacterium acetic fermentation process》Report, using the zymotechnique of single batch feed supplement, dry cell weight up to 0.2~0.5g/L,
Fermentation period is up to 80~85h.The Chinese patent of publication number CN103820368A discloses that " acetic acid bacteria freezes the preparation side of bacterium powder
Method and purposes ", the sodium hydrate aqueous solution for employing 5~25wt% maintains the pH of fermentation medium, but cultivation cycle is longer,
Need 20~30h.Therefore, traditional zymotic technique is optimized, improves fermentation level and shorten fermentation time, be to produce at present
The major issue that acetobacter microbial inoculum is faced.
The content of the invention
The object of the present invention is to provide a kind of method that control pH and feed supplement double fluid increase density fermentation acetobacter, improves
Zymotechnique, improves biomass growth rate, shortens the production cycle, increases the quantity of thalline in unit volume zymotic fluid, makes
Obtain cell density and reach 1.56~3.34 × 109cfu/mL。
Technical scheme is as follows：
A kind of control pH and the fermentation process in high density of feed supplement double fluid plus acetobacter, include the following steps：
(1) thalline activates：Acetobacter strain is seeded on culture medium and is cultivated, realizes that thalline activates；Step (1)
Described in the condition cultivated be that 24~48h is cultivated at 28~30 DEG C；The culture medium is agar medium, the group of agar medium
Become：Yeast extract 1~2%, glucose 1~2%, agar 1.5~2%, ethanol 2~4%, pH5.5~6.0, percentage are matter
Percentage is measured, surplus is water；
(2) Shaking culture：Strain through overactivation in step (1) is seeded in shaking flask on culture medium and carries out vibration training
Support, obtain seed liquor；Condition of culture：Shaking flask 100~200rpm of rotating speed, 28~30 DEG C of temperature, 10~24h of shaken cultivation；Step
(2) culture medium prescription of shaking flask described in：Yeast extract 1~2%, glucose 1~2%, KH2PO40.15~1%, ethanol 2~
4%, pH5.5~6.0；250mL shaking flasks liquid amount is 20~100mL；
(3) seeding tank spreads cultivation：The seed liquor that step (2) obtains is accessed in seeding tank by 1%~20% inoculum concentration and is trained
Support and cultivated on base；Condition of culture：100~300rpm of speed of agitator, 0.1~1.0vvm of throughput, 28~30 DEG C of temperature, stream
Add the sodium hydroxide of 5~10M, control pH5.5~6.0 of zymotic fluid, 10~12h of incubation time；Seeding tank described in step (3)
Culture medium composition：Yeast extract 1~5%, glucose 1~5%, KH2PO40.15~1%, ethanol 2~4%；Seed tank volume
10L, liquid amount 50~70%；
(4) ferment tank：The seed liquor that step (3) obtains is accessed in fermentation tank by 1%~20% inoculum concentration and is trained
Support and cultivated on base, condition of culture：100~300rpm of speed of agitator, 0.1~1.0vvm of throughput, 28~30 DEG C of temperature, stream
Add the sodium hydroxide of 5~10M, control pH5.5~6.0 of zymotic fluid, while flow and add yeast extract and corn pulp, incubation time
8~12h.
Yeast extract described in step (4) and corn pulp add according to the additive amount stream of 1~10g/L, after fermentation starts
5~8h in stream add it is complete.
The culture medium composition of fermentation tank in step (4)：Yeast extract 1~5%, glucose 1~5%, KH2PO40.15~1%,
Ethanol 2~4%；Fermenter volume 100L, liquid amount 50~70%.
Compared with prior art, the invention has the advantages that and beneficial effect：
The present invention provides a kind of method that control pH and feed supplement double fluid increase density fermentation acetobacter, this method passes through
Control pH and the mode for taking yeast extract and the double flow feedings of corn pulp, greatly shorten the production cycle so that thalline energy
Enough very fast more enrichments are got up, and realize 1.56~3.34 × 109The high density fermentation of cfu/mL.
Embodiment
It is that the present invention is described in further detail in conjunction with the embodiments below, but embodiments of the present invention are not limited to
This.
The method Dichlorodiphenyl Acetate bacillus of the present invention has versatility, acetobacter (Acetobacter used in embodiment
Pasteurianus), it is preserved in Guangdong Province's Culture Collection, deposit number GIM1.67.Yeast extract is purchased from
Huankai Microbes Tech Co., Ltd., Guangdong, Dried Corn Steep Liquor Powder are purchased from Angel Yeast Co., Ltd.Activate and use in embodiment
Solid medium refer on the basis of seed culture medium, with the addition of agar powder, the seed culture based formulas be culture acetic acid
The more common formula of bacillus.
Embodiment 1
A kind of control pH and the fermentation process in high density of feed supplement double fluid plus acetobacter, include the following steps：
(1) actication of culture：The acetobacter seed that picking grows fine from storage medium is seeded to agar medium
On, cultivate 24h at 30 DEG C；The composition of agar medium is：Yeast extract 1%, glucose 1%, agar 2%, ethanol 2%, pH5.5
~6.0, percentage is mass percent, and surplus is water；
(2) Shaking culture：The strain activated in step (1) will be seeded in shaking flask, shaken cultivation 18h, is planted
Sub- liquid；Shaking culture based formulas：Yeast extract 1%, glucose 1%, KH2PO40.25%, ethanol 2%, pH5.5~6.0, percentage
Number is mass percent, and surplus is water；250mL shaking flasks liquid amount is 50mL；Condition of culture：Shaking flask rotating speed 150rpm, temperature 30
℃；
(3) seeding tank spreads cultivation：Using the liquid of Shaking culture as seed liquor by 5% inoculum concentration access 10L seeding tanks into
Row culture；10L seeding tanks liquid amount is 60%；Culture medium forms：Yeast extract 2%, glucose 2%, KH2PO40.25%, ethanol
2%, pH5.5~6.0, percentage are mass percent, and surplus is water；Condition of culture：Speed of agitator 100rpm, throughput
The sodium hydroxide of 0.1vvm, 30 DEG C of temperature, stream plus 5M, control pH5.5~6.0 of zymotic fluid, incubation time 10h；
(4) ferment tank：By pressure difference culture transferring, the seed liquor in the seeding tank of step (3) is connect by 5% inoculum concentration
Enter in fermentation tank and ferment；100L fermentation tanks liquid amount 70%；Culture medium forms：Yeast extract 4%, glucose 5%, KH2PO4
0.75%, ethanol 2%, pH5.5~6.0, percentage is mass percent, and surplus is water；Condition of culture：Speed of agitator
The sodium hydroxide of 300rpm, throughput 0.1vvm, 30 DEG C of temperature, stream plus 5M, control pH5.5~6.0 of zymotic fluid, while flow and add
Yeast extract and corn pulp, incubation time 8h, the yeast extract and corn pulp add according to the additive amount stream of 1g/L, are sending out
Ferment start after 5h in stream add it is complete.In zymotic fluid, viable count 1.56 × 109cfu/mL。
Embodiment 2
A kind of control pH and the fermentation process in high density of feed supplement double fluid plus acetobacter, include the following steps：
(1) actication of culture and Shaking culture are the same as embodiment 1；
(2) seeding tank spreads cultivation：Using the liquid of Shaking culture as seed liquor 10L seeding tanks are accessed by 10% inoculum concentration；
10L seeding tanks liquid amount is 60%；Culture medium forms：Yeast extract 4%, glucose 5%, KH2PO40.75%, ethanol 2%,
PH5.5~6.0；Condition of culture：The sodium hydroxide of speed of agitator 300rpm, throughput 1vvm, 30 DEG C of temperature, stream plus 5M, control
PH5.5~6.0 of zymotic fluid, incubation time 10h；
(3) ferment tank：By pressure difference culture transferring, the seed liquor in seeding tank is accessed into fermentation tank by 10% inoculum concentration
In ferment；100L fermentation tanks liquid amount 70%；Culture medium forms：Yeast extract 4%, glucose 5%, KH2PO40.75%,
Ethanol 2%, pH5.5~6.0；Condition of culture：The hydroxide of speed of agitator 300rpm, throughput 1vvm, 30 DEG C of temperature, stream plus 5M
Sodium, controls pH5.5~6.0 of zymotic fluid, while flows and add yeast extract and corn pulp, incubation time 12h, the yeast extraction
Thing and corn pulp add according to the additive amount stream of 10g/L, and stream adds complete in the 8h after fermentation starts.In zymotic fluid, viable count
3.34×109cfu/mL。
Embodiment 3
A kind of control pH and the fermentation process in high density of feed supplement double fluid plus acetobacter, include the following steps：
(1) actication of culture and Shaking culture be with embodiment 1, and seeding tank spreads cultivation same embodiment 2
(2) ferment tank：By pressure difference culture transferring, the seed liquor in seeding tank is accessed into fermentation tank by 10% inoculum concentration
In ferment；100L fermentation tanks liquid amount 70%；Culture medium forms：Yeast extract 4%, glucose 5%, KH2PO40.75%,
Ethanol 2%, pH5.5~6.0；Condition of culture：The hydroxide of speed of agitator 300rpm, throughput 1vvm, 30 DEG C of temperature, stream plus 5M
Sodium, controls pH5.5~6.0 of zymotic fluid, while flows and add yeast extract and corn pulp, incubation time 12h, the yeast extraction
Thing and corn pulp add according to the additive amount stream of 5g/L, and stream adds complete in the 5h after fermentation starts.In zymotic fluid, viable count 2.78
×109cfu/mL。
The acetobacter fermentation process of embodiment 1~3 is contrasted with traditional zymotic technique, the results are shown in Table 1.
The acetobacter fermentation process of 1 embodiment 1~3 of table and the contrast of traditional zymotic technique
By table 1 as it can be seen that high degree is shortened fermentation time by this method, and the fermentation density of acetobacter is improved, had
Good industrial application value.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (6)
1. a kind of control pH and the fermentation process in high density of feed supplement double fluid plus acetobacter, it is characterised in that：Include the following steps：
(1) thalline activates：Acetobacter strain is seeded on culture medium and is cultivated, realizes that thalline activates；
(2) Shaking culture：Strain through overactivation in step (1) is seeded in shaking flask on culture medium and carries out shaken cultivation, is obtained
Obtain seed liquor；Condition of culture：Shaking flask 100~200rpm of rotating speed, 28~30 DEG C of temperature, 10~24h of shaken cultivation；
(3) seeding tank spreads cultivation：Culture medium in the seed liquor access seeding tank for obtaining step (2) by 1%~20% inoculum concentration
On cultivated；Condition of culture：100~300rpm of speed of agitator, 0.1~1.0vvm of throughput, 28~30 DEG C of temperature, stream plus 5
The sodium hydroxide of~10M, controls pH5.5~6.0 of zymotic fluid, 10~12h of incubation time；
(4) ferment tank：Culture medium in the seed liquor access fermentation tank for obtaining step (3) by 1%~20% inoculum concentration
On cultivated, condition of culture：100~300rpm of speed of agitator, 0.1~1.0vvm of throughput, 28~30 DEG C of temperature, stream plus 5
The sodium hydroxide of~10M, controls pH5.5~6.0 of zymotic fluid, while flows and add yeast extract and corn pulp, and incubation time 8~
12h。
2. pH and the fermentation process in high density of feed supplement double fluid plus acetobacter are controlled according to claim 1, it is characterised in that：
Yeast extract described in step (4) and corn pulp add according to the additive amount stream of 1~10g/L, in 5~8h after fermentation starts
Stream adds complete.
3. pH and the fermentation process in high density of feed supplement double fluid plus acetobacter are controlled according to claim 1, it is characterised in that：
The condition cultivated described in step (1) is that 24~48h is cultivated at 28~30 DEG C；Culture medium described in step (1) is agar culture
Base, the composition of agar medium are：Yeast extract 1~2%, glucose 1~2%, agar 1.5~2%, ethanol 2~4%, pH5.5
~6.0, percentage is mass percent, and surplus is water.
4. pH and the fermentation process in high density of feed supplement double fluid plus acetobacter are controlled according to claim 1, it is characterised in that：
The culture medium prescription of shaking flask described in step (2)：Yeast extract 1~2%, glucose 1~2%, KH2PO40.15~1%, ethanol 2
~4%, pH5.5~6.0；Flask volume 250mL, liquid amount are 20~100mL.
5. pH and the fermentation process in high density of feed supplement double fluid plus acetobacter are controlled according to claim 1, it is characterised in that：
The culture medium composition of seeding tank described in step (3)：Yeast extract 1~5%, glucose 1~5%, KH2PO40.15~1%, second
Alcohol 2~4%；Seed tank volume 10L, liquid amount 50~70%.
6. pH and the fermentation process in high density of feed supplement double fluid plus acetobacter are controlled according to claim 1, it is characterised in that：
The culture medium composition of fermentation tank in step (4)：Yeast extract 1~5%, glucose 1~5%, KH2PO40.15~1%, ethanol 2~
4%；Fermenter volume 100L, liquid amount 50~70%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109329916A (en) * | 2018-10-31 | 2019-02-15 | 华南协同创新研究院 | It is a kind of to flow the method and hill gooseberry's ferment for adding formula quickly to produce hill gooseberry's ferment |
| CN113637596A (en) * | 2021-08-24 | 2021-11-12 | 广东海天创新技术有限公司 | Saccharomyces cerevisiae ZB421 and application thereof |
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| CN103243132A (en) * | 2013-05-28 | 2013-08-14 | 山东祥维斯生物科技有限公司 | Method for producing glutamic acid through double-feeding fermentation optimization of corn steep liquor and glucose |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109329916A (en) * | 2018-10-31 | 2019-02-15 | 华南协同创新研究院 | It is a kind of to flow the method and hill gooseberry's ferment for adding formula quickly to produce hill gooseberry's ferment |
| CN113637596A (en) * | 2021-08-24 | 2021-11-12 | 广东海天创新技术有限公司 | Saccharomyces cerevisiae ZB421 and application thereof |
| CN113637596B (en) * | 2021-08-24 | 2023-03-21 | 广东海天创新技术有限公司 | Saccharomyces cerevisiae ZB421 and application thereof |
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