CN103614318A - High-temperature-resistant acetic acid bacteria and application thereof in production of acetic acid through fermentation - Google Patents
High-temperature-resistant acetic acid bacteria and application thereof in production of acetic acid through fermentation Download PDFInfo
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Abstract
The invention discloses a high-temperature-resistant Acetobacter pasteurianus strain and an application thereof in production of acetic acid through fermentation. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) and has a preservation number of CGMCC No.8189. The acetic acid bacteria disclosed by the invention can grow and generate acid at 25-45 DEG C; the strain and ethanol serving as a main raw material are used to produce acetic acid through liquid submerged fermentation, and the temperature and ventilation volume during fermentation are controlled in three stages; the concentration of acetic acid obtained after fermentation can reach 60-180g/L, and the acid conversion rate of ethanol reaches 85-101%. The production of acetic acid through fermentation is performed by using the high-temperature-resistant acetic acid bacteria, and a relatively high fermentation temperature is adopted, so that the consumption of cooling electricity and cooling water is reduced, relatively high production efficiency is achieved at the same time, and the production cost is reduced. The high-temperature-resistant acetic acid bacteria and a fermentation process thereof are suitable for production of a variety of table vinegar including food vinegar, fruit vinegar and the like through fermentation.
Description
Technical field
The present invention relates to the high temperature resistant acetic bacteria of a strain, also relate to and utilize this bacterial strain to take ethanol to be that fermenting substrate produces the application of acetic acid, to belong to biological technical field.
Background technology
Acetic acid is a kind of important industrial raw material, in fields such as food, medicine, chemical industry, has a wide range of applications.Alimentary acetic acid general using microbe fermentation method is produced.
Acetic bacteria (Acetic acid bacteria) is that bacterium is used in the major industry of acetic fermentation, oxidation of ethanol can be become to acetic acid.Common are genus acetobacter (
acetobacter) Gluconobacter (
gluconobacter) and gluconobacter suboxydans genus (
gluconacetobacter) etc., wherein the acetic bacteria of genus acetobacter and Gluconobacter is owing to having higher oxidation of ethanol ability, and the liquid pure-blood ferment that is usually used in acetic acid is produced.
Acetic fermentation need to carry out obtaining higher production efficiency under suitable condition.Conventionally, acetic fermentation optimum temperuture is 30-32 ℃, and leavening temperature is too high, and acetic bacteria is easily aging, even causes acetic bacteria dead, causes acetic fermentation rate reduction.Because acetic fermentation is accompanied by the growth metabolism of thalline, fermenting process produces a large amount of heat, and fermented liquid temperature can raise gradually, need to utilize refrigerating unit to fermentor tank lower the temperature, temperature control, to guarantee normally carrying out of acetic fermentation.Majority state and Area during Summer temperature are higher, and numerous vinegar manufacturing enterprise all can produce more cooling cost at high temperature season, if leavening temperature is controlled and improperly can be affected fermentation efficiency, even cause the underproduction, stopping production etc.According to measuring and calculating, if leavening temperature can be brought up to 37 ℃ by 30 ℃, fermentation cooling cost can reduce approximately 50%.The control of acetic fermentation temperature will be applicable to thalli growth on the one hand, to control on the other hand best leavening temperature to obtain higher acetic acid generating rate, therefore, seed selection is high temperature resistant acetic bacteria, industry has important practical significance to vinegar brewing at higher temperature, to carry out acetic fermentation production.
From occurring in nature screening and mutagenic and breeding, it is the most frequently used strain breeding method.Sun Wenying etc. screen the acetic bacteria that a strain has high temperature resistant (36 ℃) high ethanol (11%), after optimizing, its acetate yield reaches 48.6 g/L, produce the lower (Sun Wenying of acid amount, Chen Xiong, Wang Zhi, etc. the screening of high temperature resistant high alcohol acetic bacteria and the initial optimization of zymotechnique thereof, China brewages, 2011, Isosorbide-5-Nitrae 4-47).Shao Jianning etc. by ultraviolet ray, ethyl sulfate step by step mutagenic and breeding obtained two strain acetic bacterias, 40 ℃ of culture temperature, substratum alcohol concn 12% (v/v) condition bottom fermentation, alcohol transformation efficiency reaches respectively 70% and 75%, but rate of producing acid and raw material availability are lower, can not meet demand of industrial production (Shao Jianning, Wang Bingfeng, such as road, etc. high temperature resistant high wine degree acetic bacteria seed selection research. Gansu science journal, 2005,17 (4): 12-14).The Acetobacter pasteurianus CGMCC No. 8189 with high temperature resistant feature obtaining is screened in utilization of the present invention from Shanxi mature vinegar vinegar unstrained spirits, according to its growth and fermentation character, temperature in acetic fermentation process and air flow control are divided into early stage, mid-term, later stage three phases, early stage, controlled fermentation temperature was thalline optimum growth temperature, can obtain higher cell concentration within a short period of time; Mid-term, controlled fermentation temperature was the sour temperature of the suitableeest product of thalline, can guarantee higher acetic acid generating rate; Later stage is suitably reduced the consumption that leavening temperature guarantees residual alcohol, prevents acetic acid peroxidation.Utilize this high temperature resistant acetic bacteria and zymotechnique fermentation gained acetate concentration thereof can reach 60-180 g/L, alcohol transformation efficiency reaches more than 85%, has high industrial application value.
Summary of the invention
One of technical problem to be solved by this invention is to provide the resistant to elevated temperatures acetic fermentation bacterial strain of a strain, in order to realize object of the present invention, intends adopting following technical scheme:
One aspect of the present invention relates to acetic bacteria, and described acetic bacteria is that deposit number is Acetobacter pasteurianus or its thing that goes down to posterity of CGMCC No. 8189.
In one aspect of the invention, described acetic bacteria principal character is as follows: on solid medium, this bacterium bacterium colony is less, and color is creamy white, smooth surface, moistening, and thickness, edge is more neat; Examine under a microscope, this bacterial strain is Gram-negative bacteria, is shaft-like, easily series connection growth; Acid can be grown and produce to this bacterial strain at 25-45 ℃, can take ethanol as fermenting substrate production acetic acid.
The present invention also relates on the other hand above-mentioned acetic bacteria and prepares the application in acetic acid in fermentation.
In a preferred embodiment of the present invention, described fermentation substrate is ethanol or the wine that contains ethanol.
In a preferred embodiment of the present invention, described fermenting process is divided into three phases, and first stage leavening temperature is 30-37 ℃, is preferably 32-35 ℃, and per minute air flow is the volume ratio of 1:0.05-0.1 v/v(liquid and gas); Subordinate phase leavening temperature is 30-45 ℃, be preferably 35 ℃ or more than, 35-38 ℃ more preferably, per minute air flow is the volume ratio of 1:0.08-0.2 v/v(liquid and gas); Phase III leavening temperature is 25-35 ℃, is preferably 28-30 ℃, and per minute air flow is the volume ratio of 1:0.06-0.12 v/v(liquid and gas).
In a preferred embodiment of the present invention, described fermentation comprises the steps:
(1) first order seed is cultivated: the shaking flask of utilizing 250-1000 mL, seed culture medium 30-100 mL is housed, and access Acetobacter pasteurianus CGMCC No. 8189 carries out shake-flask culture, and shaking speed is 100-300 rev/min, temperature is 33-42 ℃, and incubation time is 20-30 hour.
First order seed substratum consists of: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 4-9%(v/v), acetic acid 5-10 g/L, MgSO
40.2 g/L, CaCl
20.3 g/L.
(2) secondary seed is cultivated: utilize 50-1000 L fermentor tank to carry out secondary seed cultivation, inoculum size is 5%-15%(volume ratio).Temperature is 33-40 ℃, and incubation time is 20-30 hour.
Secondary seed medium consists of: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 5-9%(v/v), acetic acid 0-15 g/L, MgSO
40.1 g/L, CaCl
20.2 g/L.
(3) acetic fermentation: carry out in fermentor tank, fermentor tank inoculum size is 5%-30%(v/v), fermenting process is divided into three phases, and first stage (in earlier stage) leavening temperature is 30-37 ℃, and per minute air flow is the volume ratio of 1:0.05-0.1 v/v(liquid and gas); Subordinate phase (mid-term) leavening temperature is 30-45 ℃, and per minute air flow is the volume ratio of 1:0.08-0.2 v/v(liquid and gas); Phase III (later stage) leavening temperature is 25-35 ℃, and per minute air flow is the volume ratio of 1:0.06-0.12 v/v(liquid and gas).Fermentation mode can be batch fermentation, fed-batch fermentation or stuck fermentation.
Fermention medium consists of: glucose 20-35 g/L, yeast extract paste 15-25 g/L, ethanol 7-14%(v/v), acetic acid 0-10 g/L, MgSO
40.1 g/L, CaCl
20.2 g/L.Can also directly utilize and take fruit and carry out fruit vinegar fermentation as the fruit wine that main raw material obtains through zymamsis.
Batch fermentation time 48-120 hour, fermentation ends acetate concentration can reach 60-150 g/L, and ethanol turns sour rate and reaches more than 85%.
In fed-batch fermentation process, add continuously ethanol, in controlled fermentation liquid, alcohol concn is 10-40 g/L, and fermentation ends acetate concentration can reach 60-180 g/L, fermentation time 50-150 hours, and ethanol turns sour rate and reaches more than 85%.
Stuck fermentation takes out fermented liquid 20%-50%(volume ratio at every turn), supplement the fresh fermention medium of same volume, fermentation ends acetate concentration can reach 60-150 g/L, fermentation time 24-100 hour, ethanol turns sour rate and reaches more than 85%.
Feature of the present invention and advantage are:
(1) utilize this bacterial strain under comparatively high temps, to carry out acetic fermentation production, compare with 30 ℃ of condition bottom fermentations, can reduce fermentation refrigeration costs, promoting enterprise energy-saving and emission-reduction have a greater advantage on reducing production costs.
(2) higher leavening temperature can improve the generating rate of acetic acid.Utilize this bacterial strain to carry out acetic fermentation and have that fermentation rate is fast, ethanol conversion advantages of higher, can significantly improve production efficiency and raw material availability, improve the performance of enterprises.
accompanying drawing explanation:
Fig. 1 is the transparent circle that under 45 ℃ of conditions, high temperature resistant acetic bacteria produces on solid medium.
Fig. 2 is that Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 carry out the comparison of acetic fermentation under 37 ℃ of conditions.
Fig. 3 is that Acetobacter pasteurianus CGMCC No. 8189 be take alcohol and produced the conditional curve of acetic acid as raw material batch fermentation.
Fig. 4 is that Acetobacter pasteurianus CGMCC No. 8189 be take hard cider and produced the conditional curve of cider vinegar as raw material batch fermentation.
Fig. 5 is that Acetobacter pasteurianus CGMCC No. 8189 be take hard cider and produced the conditional curve of cider vinegar as raw material stuck fermentation.
microorganism information
Acetobacter pasteurianus (
acetobacter pasteurium) CP-A13, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No. 8189, preservation address: No. 3, No. 1, BeiChen West Road, Chaoyang District, BeiJing City institute, Institute of Microorganism, Academia Sinica, postcode 100101.Preservation date on 09 16th, 2013.
Embodiment
embodiment 1the acquisition of high temperature resistant acetic fermentation bacterial strain
(1) microorganism enrichment is cultivated
Get the vinegar unstrained spirits sample 1-3 g that Shanxi mature vinegar acetic fermentation proceeds to 3-6 days, 15 cm places, Ju Cupei top layer, sample position, join in the shaking flask that 100 mL enrichment mediums are housed, and 180 revs/min of shaking table revolutions, under 42 ℃ of conditions, enrichment culture is 36 hours.
Enrichment medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 4%(v/v), acetic acid 5 g/L.
(2) domestication of continuous high temperature high concentration ethanol is cultivated
The fermented liquid that above-mentioned steps (1) is obtained turns by 10%(v/v) inoculum size transfer and be equipped with in the shaking flask of 100 mL domestication substratum, 180 revs/min of shaking table revolutions, under 42 ℃ of conditions, domestication is cultivated, in the domestication substratum of transferring fresh after 36 hours.
Domestication substratum consists of: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 7%-12%(v/v), acetic acid 15-30 g/L, MgSO
40.2 g/L, CaCl
20.3 g/L.
(3) repeating step (2) is 5-10 time.
(4) get 100 μ L(3) gained fermented liquid, evenly coat Selective solid culture medium surface, under 45 ℃ of conditions, cultivate the acetic fermentation bacterial strain that acquisition can withstand high temperatures.
Selective solid culture medium consists of: glucose 30 g/L, yeast extract paste 10 g/L, peptone 10 g/L, ethanol 12%(v/v), CaCO
35 g/L, agar 25 g/L.
Fig. 1 is the transparent circle that under 45 ℃ of conditions, high temperature resistant acetic bacteria produces on solid medium.
embodiment 2the comparison of Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 acetic fermentation performance
(1) prepare seed liquor
From inclined-plane, getting respectively Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 is inoculated in seed culture medium, at 37 ℃, under 160 revs/min of conditions, shaking table is cultivated 24 hours, by 10%(v/v) inoculum size transfer in fresh seed culture medium and carry out amplification culture.
Seed culture medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 3.5%(v/v).
(2) acetic fermentation
Press 10%(v/v) inoculum size, by obtaining Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 seed liquor in (1), be seeded to respectively and in fermentor tank, carry out acetic fermentation.In fermenting process, controlled fermentation temperature is 37 ℃, and ventilation is 1: 0.1(v/v)/min.
Fermention medium consists of: glucose 30 g/L, yeast extract paste 20 g/L, ethanol 7%(v/v), acetic acid 10 g/L, MgSO
40.1 g/L, CaCl
20.2 g/L.
Utilize Acetobacter pasteurianus CGMCC No. 8189 fermentation 62 h, acetic acid final concentration is 72 g/L, initial acetate concentration 10 g/L, and to turn sour rate be 88.6 % to ethanol, average rate of producing acid is about 1 g/ (Lh).
Utilize acetic bacteria AS1.41 100 h that ferment, acetic acid final concentration is 52 g/L, initial acetate concentration 10 g/L, and to turn sour rate be 60 % to ethanol, average rate of producing acid is about 0.42 g/ (Lh).
Fig. 2 is that Acetobacter pasteurianus CGMCC No. 8189 and acetic bacteria AS1.41 carry out the comparison of acetic fermentation under 37 ℃ of conditions.
embodiment 3utilize Acetobacter pasteurianus CGMCC No. 8189 batch fermentations to produce acetic acid
(1) prepare seed liquor
From inclined-plane, get Acetobacter pasteurianus CGMCC No. 8189 and be inoculated in seed culture medium, at 37 ℃, under 160 revs/min of conditions, shaking table is cultivated 25 hours.By 10%(v/v) inoculum size transfer in fresh seed culture medium and carry out amplification culture.
Seed culture medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 3.5%(v/v).
(2) acetic fermentation
Press 12%(v/v) inoculum size, by obtaining seed liquor in (1), be seeded to and in fermentor tank, carry out acetic fermentation.
Fermention medium consists of: glucose 30 g/L, yeast extract paste 20 g/L, ethanol 8%(v/v), acetic acid 3 g/L, MgSO
40.1 g/L, CaCl
20.2 g/L.
In fermenting process, controlled fermentation temperature is: in earlier stage (continue 20 hours) 33 ℃, 37 ℃ of mid-terms (continuing 38 hours), 30 ℃ of later stages (continuing 4 hours); Ventilation: early stage 1: 0.07(v/v)/min, mid-term 1: 0.11(v/v)/min, later stage 1: 0.08(v/v)/min.
62 h that ferment finish, and acetic acid final concentration is at 74 g/L, initial acetate concentration 3 g/L, and to turn sour rate be 88.8 % to ethanol, average rate of producing acid is about 1.15 g/ (Lh).
Fig. 3 is that Acetobacter pasteurianus CGMCC No. 8189 be take alcohol and produced the conditional curve of acetic acid as raw material batch fermentation.
embodiment 4utilize Acetobacter pasteurianus CGMCC No. 8189 batch fermentations to produce cider vinegar
(1) prepare seed liquor
From inclined-plane, get Acetobacter pasteurianus CGMCC No. 8189 and be inoculated in seed culture medium, at 35 ℃, under 180 revs/min of conditions, shaking table is cultivated 24 hours.By 10%(v/v) inoculum size transfer in fresh seed culture medium and carry out amplification culture.
Seed culture medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 3.5%(v/v).
(2) cider vinegar fermentation
In hard cider, ethanol content is 8%(v/v).
In fermenting process, controlled fermentation temperature is: in earlier stage (continue 24 hours) 32 ℃, 35 ℃ of mid-terms (continuing 36 hours), 28 ℃ of later stages (continuing 4 hours); Ventilation: early stage 1: 0.08(v/v)/min, mid-term 1: 0.12(v/v)/min, later stage 1: 0.1(v/v)/min.
64 h that ferment finish, and acetic acid final concentration is 75 g/L, average rate of producing acid 1.17 g/ (Lh), and ethanol turns sour rate and is about 93 %.
Fig. 4 is that Acetobacter pasteurianus CGMCC No. 8189 be take hard cider and produced the conditional curve of cider vinegar as raw material batch fermentation.
embodiment 5utilize Acetobacter pasteurianus CGMCC No. 8189 stuck fermentations to produce cider vinegar
(1) prepare seed liquor
From inclined-plane, get Acetobacter pasteurianus CGMCC No. 8189 and be inoculated in seed culture medium, at 35 ℃, under 160 revs/min of conditions, shaking table is cultivated 28 hours.By 10%(v/v) inoculum size transfer in fresh seed culture medium and carry out amplification culture.
Seed culture medium forms: glucose 20 g/L, yeast extract paste 15 g/L, ethanol 3.5%(v/v).
(2) initial cider vinegar fermentation
Press 15%(v/v) inoculum size, by obtaining seed liquor in (1), be seeded in fermentor tank, the hard cider of take under 35 ℃ of conditions ferments as raw material.Hard cider ethanol content is 8%(v/v).In fermenting process, controlled fermentation temperature is 35 ℃.
(3) cider vinegar stuck fermentation
Step (2) is fermented after 60 h and is taken out 30%(volume ratio) fermented liquid, supplement the fresh apple wine of same volume, carry out stuck fermentation, fermenting process controlled fermentation temperature is: in earlier stage (continue 6 hours) 35 ℃; 37 ℃ of mid-terms (continuing 12 hours), 35 ℃ of the later stages (continuing 4 hours); Ventilation: early stage 1: 0.1(v/v)/min, mid-term 1: 0.13(v/v)/min, later stage 1: 0.1(v/v)/min.
The repeating step (3) after 24 hours that ferments carries out stuck fermentation next time, obtains acetate concentration in cider vinegar at every turn and can reach 75 g/L, and ethanol turns sour rate and reaches more than 93%.
Fig. 5 is that Acetobacter pasteurianus CGMCC No. 8189 be take hard cider and produced the conditional curve of cider vinegar as raw material stuck fermentation.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited to this, and any variation of expecting without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (5)
1. acetic bacteria, described acetic bacteria is that deposit number is Acetobacter pasteurianus or its thing that goes down to posterity of CGMCC No. 8189.
2. acetic bacteria according to claim 1, described acetic bacteria principal character is as follows: on solid medium, this bacterium bacterium colony is less, and color is creamy white, smooth surface, moistening, thickness, edge is more neat; Examine under a microscope, this bacterial strain is Gram-negative bacteria, is shaft-like, easily series connection growth; Acid can be grown and produce to this bacterial strain at 25-45 ℃, can take ethanol as fermenting substrate production acetic acid.
3. the acetic bacteria described in claim 1 or 2 is prepared the application in acetic acid in fermentation.
4. application according to claim 3, described fermentation substrate is ethanol or the wine that contains ethanol, preferably, described fermentation substrate ethanol can be that raw material carries out zymamsis acquisition with grain or fruit, as corn, rice, glutinous rice, Chinese sorghum, hawthorn, strawberry, lichee, watermelon, apple, pears or date.
5. application according to claim 4, described fermenting process temperature and air flow are controlled and were divided into for three stages, first stage leavening temperature is 30-37 ℃, is preferably 32-35 ℃, and per minute air flow is the volume ratio of 1:0.05-0.1 v/v(liquid and gas); Subordinate phase leavening temperature is 30-45 ℃, be preferably 35 ℃ or more than, 35-38 ℃ more preferably, per minute air flow is the volume ratio of 1:0.08-0.2 v/v(liquid and gas); Phase III leavening temperature is 25-35 ℃, is preferably 28-30 ℃, and per minute air flow is the volume ratio of 1:0.06-0.12 v/v(liquid and gas).
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| CN105368736A (en) * | 2015-08-21 | 2016-03-02 | 云南农业大学 | Bacillus aceticus and application of bacillus aceticus in coffee cherry peel and pulp fermentation vinegar |
| CN106167781A (en) * | 2016-08-25 | 2016-11-30 | 湖北工业大学 | Acetobacter pasteurianus that a kind of temperature capacity is excellent and purposes |
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| CN105018384A (en) * | 2015-07-30 | 2015-11-04 | 江南大学 | Acetobacter strain and application thereof in fermenting apple vinegar |
| CN105018384B (en) * | 2015-07-30 | 2018-07-06 | 江南大学 | One plant of acetic acid bacteria and its application in the apple vinegar that ferments |
| CN105368736A (en) * | 2015-08-21 | 2016-03-02 | 云南农业大学 | Bacillus aceticus and application of bacillus aceticus in coffee cherry peel and pulp fermentation vinegar |
| CN106167781A (en) * | 2016-08-25 | 2016-11-30 | 湖北工业大学 | Acetobacter pasteurianus that a kind of temperature capacity is excellent and purposes |
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| CN110923270A (en) * | 2019-12-06 | 2020-03-27 | 鹤山市东古调味食品有限公司 | Method for producing acetic acid by utilizing chemical ethanol fermentation |
| CN111748491A (en) * | 2020-06-15 | 2020-10-09 | 江苏大学 | Method for promoting fermentation and acid production of acetobacter pasteurianus by using low-frequency alternating magnetic field |
| CN111748491B (en) * | 2020-06-15 | 2022-12-16 | 江苏大学 | Method for promoting fermentation and acid production of acetobacter pasteurianus by using low-frequency alternating magnetic field |
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| CN116286559B (en) * | 2023-05-10 | 2023-09-05 | 阳西美味鲜食品有限公司 | High-yield high-temperature-resistant acetobacter pasteurii M112 and application thereof |
| CN116515704A (en) * | 2023-05-16 | 2023-08-01 | 河北兴台酒业集团有限责任公司 | Making method and process of black bean sauce wine |
| CN116515704B (en) * | 2023-05-16 | 2023-09-26 | 河北兴台酒业集团有限责任公司 | Making method and process of black bean sauce wine |
| CN116814509A (en) * | 2023-08-23 | 2023-09-29 | 仙乐健康科技股份有限公司 | Ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii |
| CN116814509B (en) * | 2023-08-23 | 2023-12-22 | 仙乐健康科技股份有限公司 | Ethanol-resistant, acid-resistant and high-acid-yield acetobacter pasteurii |
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