CN109182399A - A kind of immobilization acetic fermentation method for improving acetic acid bacteria and producing acetic acid ability - Google Patents

A kind of immobilization acetic fermentation method for improving acetic acid bacteria and producing acetic acid ability Download PDF

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Publication number
CN109182399A
CN109182399A CN201810971578.XA CN201810971578A CN109182399A CN 109182399 A CN109182399 A CN 109182399A CN 201810971578 A CN201810971578 A CN 201810971578A CN 109182399 A CN109182399 A CN 109182399A
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China
Prior art keywords
acetic acid
fermentation
acid bacteria
acetic
prepared
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Pending
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CN201810971578.XA
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Chinese (zh)
Inventor
胡勇
唐斌
王安
李冬生
徐宁
柳志杰
周梦舟
祁勇刚
吴茜
汪超
石勇
龚元元
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Hubei University of Technology
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Hubei University of Technology
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Priority to CN201810971578.XA priority Critical patent/CN109182399A/en
Publication of CN109182399A publication Critical patent/CN109182399A/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/40Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/54Acetic acid
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier

Abstract

The invention discloses a kind of immobilization acetic fermentation methods that raising acetic acid bacteria produces acetic acid ability, belong to food processing field.The invention, as acetic acid bacteria carrier, is reached using wall membrane complex (the acetic acid bacteria cell membrane of yeast cell wall, fermentation termination) the covering corncob microparticle of reconstruct and improves the resistance to acetic acid ability of acetic acid bacteria, be finally reached the purpose for improving and producing acetic acid.

Description

A kind of immobilization acetic fermentation method for improving acetic acid bacteria and producing acetic acid ability
Technical field
The invention belongs to food brewing fields, and in particular to a kind of immobilization acetic acid hair for improving acetic acid bacteria and producing acetic acid ability Fermenting process.
Background technique
The preparation of acetic acid can be by two methods of artificial synthesized and biofermentation, and fermentation method produces acetic acid or vinegar is second Alcohol is oxidized to the process of acetic acid under the action of acetic acid bacteria.Biological fermentation process only accounts for the 10% of All Around The World yield.But many states The food safety regulation of family provides that the vinegar in food must be prepared by bioanalysis, therefore biological fermentation process is still production second The most important method of acid, especially vinegar (acetic acid is vinegar main component).
Acetic acid down to 0.5% can grow generation inhibiting effect to most of microbe.Antimicrobial effect make acetic acid at For common preservative.Acetic acid bacteria is the important microbe of fermentation method production acetic acid, relative to common microorganism, acetic acid bacteria tool There is excellent acetic acid tolerance.But in fermenting and producing, acetic acid still Dichlorodiphenyl Acetate bacterium forms toxic action, and is vinegar The maximum limiting factor that sour bacterium is faced causes acetic acid bacteria that there is production acetic acid ability to be restricted.Different types of acetic acid bacteria Acid resistance is widely different, it has been found that be resistant to highest acetate concentration environment is Ga. europaeus, is resistant in environment 10% acetic acid, two kinds of acetic acid bacterias of most common A. aceti and A. pasteurianus, which can only be resistant to, in acetic fermentation does not surpass Cross 6% acetic acid.It is A. pasteurianus AS1.41, during the fermentation, acidproof energy that vinegar microorganism is commonly made in China Power is gradually induced by acetic acid, is temporarily improved, and can produce up to 9% acetic acid;But its tolerance cannot obtain hereditary guarantor It stays, after production acetic acid cultivated for 3 generations in the culture medium of no ethyl alcohol and acetic acid up to 9% acetic acid bacteria, initial resistance to acetic acid ability is still low In 6%.
The toxic mechanism of acetic acid: the acetic acid of high concentration is easy to enter cytoplasm by membrane permeability, to thalli growth And metabolism has an adverse effect, toxic mechanism includes H+And CH3COO-Caused environment disturbance intracellular.At present, acetic acid bacteria is resistance to Sour mechanism is not verified thoroughly also.Report that research has found one in Acetobacter and Gluconoacetobacter A little acidproof molecular mechanisms: (1) the relevant mechanism of oxidation of ethanol, including film combination alcohol dehydrogenase (Adh), acetaldehyde dehydrogenase (Aldh) mechanism;(2) acetic acid assimilates mechanism;(3) transporting mechanism that acetic acid may be pumped out;(4) general degeneration-resistant mechanism.
The fermentation of vinegar is divided into solid state fermentation and liquid state fermentation.Liquid deep layer acetic fermentation uses pure-blood ferment, and the period is short, produces Sour high, production capacity is strong, is gradually used by manufacturer because advantage is significant.
A kind of method (CN201510492742.5) offer using corn core immobilized acetic acid bacteria brewing full banana fruits vinegar It is a kind of to utilize corn core immobilized acetic acid bacteria method;A kind of immobilization acetic acid bacteria prepares the cigarette method of laurel extract (CN201610895642.1) wet thallus is added in the mixed solution of polyvinyl alcohol and sodium alginate, and with contain 1~3% CaCl2Boric acid saturated solution and saturation Na2SO4Immobilization acetic acid bacteria bead is crosslinked to obtain in solution, but above method is not related to Improve the resistance to acetic acid ability of acetic acid bacteria.
Based on acetic fermentation process, it is essential for designing the absolute acid stability that preferably strategy improves industrial microorganism. The present invention improves the resistance to acetic acid ability of acetic acid bacteria by a kind of acetic acid bacteria immobilization fermentation method, is finally reached the mesh for improving and producing acetic acid 's.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides the immobilization acetic acid hairs that a kind of raising acetic acid bacteria produces acetic acid ability Fermenting process, the invention cover corn using the wall membrane complex (the acetic acid bacteria cell membrane of yeast cell wall, fermentation termination) of reconstruct Core microparticle reaches as acetic acid bacteria carrier and improves the resistance to acetic acid ability of acetic acid bacteria, is finally reached the purpose for improving and producing acetic acid.
In order to achieve the object of the present invention, the present inventor is finally obtained by a large number of experiments research and unremitting exploration Following technical solution:
A kind of immobilization acetic fermentation method for improving acetic acid bacteria and producing acetic acid ability, this method comprises the following steps:
(1) prepared by corncob: corncob is impregnated 10-12 hours with pH5.0 hydrochloric acid solution, then is cleaned with clear water, overnight in 80 DEG C;
(2) the acetic acid bacteria cell film preparation of fermentation termination: A, taking the acetic acid fermentation liquid of batch fermentation, and 3000-4000rpm centrifugation is received Collect thallus, wherein the sample point of acetic acid fermentation liquid is fermentation termination, acetate concentration 6%;
B, the thallus of collection is resuspended in distilled water 1 hour, 30 DEG C, wherein distilled water lysozyme containing 0.8-1.0mg/mL;
C, 8000-10000rpm is centrifuged 10-15 minutes, precipitating 200-300mL PEG800(4-6%) it is resuspended;
(3) prepared by carrier: yeast cell wall is resuspended with water, on corncob prepared by its even spraying to step (1), and 30-35 The rotation of DEG C sterile wind drying 10-15 minute, obtained carrier;
(4) acetic acid bacteria cell membrane prepared by step (2) is sprayed on the carrier of step (3), 30-35 DEG C of sterile wind rotation drying 10-15 minutes;
(5) prepared by immobilization acetic acid bacteria: the carrier of step (4) preparation is sprayed to after the acetic acid bacteria seed liquor of mid-log phase is concentrated On, room temperature stands 40-60 minutes, horizontal rolling 40-60 minutes in 32-35 DEG C of bellows;
(6) immobilization acetic acid prepared by step (5) is fixed on fermentor agitating paddle, is used for liquid acetic acid fermentation, fermentor The middle distiller's wort with 6% ethyl alcohol submerges corn core carrier, starts acetic fermentation;When acetate concentration reaches 35g/L, start stream plus pure wine The distiller's wort of essence, measures fermentation liquid total acid and is not rising, and terminates acetic fermentation.
Compared with prior art, the present invention has the advantages that compared with existing vinegar processing technology
(1) invention covers corn using the wall membrane complex (the acetic acid bacteria cell membrane of yeast cell wall, fermentation termination) of reconstruct Core microparticle reaches as acetic acid bacteria carrier and improves the resistance to acetic acid ability of acetic acid bacteria, greatly improve the acid-fast ability of acetic acid, shows The yield for improving acetic acid write.
(2) immobilization acetic acid bacteria of the present invention can Reusability, reduce production cost.
Specific embodiment:
The following is specific embodiments of the present invention, and technical scheme of the present invention is further described, but guarantor of the invention Shield range is not limited to these examples.It is all be included in without departing substantially from the change of present inventive concept or equivalent substitute it is of the invention Within protection scope.
Embodiment 1
(1) prepared by corncob: 3 corncobs are impregnated 12 hours with pH5.0 hydrochloric acid solution, then are cleaned with clear water, overnight in 80 DEG C.
(2) the acetic acid bacteria cell film preparation of fermentation termination: A, take 30L batch fermentation acetic acid fermentation liquid (sample point for hair Ferment terminal, acetate concentration 6%), thalline were collected by centrifugation by 4000rpm.B, it is small to be resuspended in distilled water (lysozyme containing 1.0mg/mL) 1 When, 30 DEG C.C, 10000rpm is centrifuged 10-15 minutes, precipitating 200-300mL PEG800(6%) it is resuspended.
(3) prepared by carrier: 30g is resuspended with 300mL water, yeast cell wall is commercialized, even spraying to step (1) preparation On corncob, 30 DEG C of sterile wind rotations are dried up 15 minutes.
(4) acetic acid bacteria cell membrane prepared by step (2) is sprayed on the corncob of step (3), 30 DEG C of sterile wind rotations Drying 15 minutes.
(5) prepared by immobilization acetic acid bacteria: taking the acetic acid bacteria seed liquor of 500mL mid-log phase to be concentrated into 200mL and is sprayed to step Suddenly on the carrier of (4) preparation, room temperature stands 60 minutes, and 30 DEG C of sterile wind rotations dry up 15 minutes, is fixed on the stirring of 60L fermentor On paddle, it to be used for liquid acetic acid fermentation.
(6) acetic fermentation: submerging corncob (liquid amount is about 45L) with the distiller's wort of 6% ethyl alcohol in 60L fermentor, starts vinegar Acid fermentation;When acetate concentration 35g/L, the distiller's wort for starting stream plus absolute alcohol (0.05L/h) carries out acetic fermentation, and it is total to measure fermentation liquid Acid is not rising, and terminates acetic fermentation.
Comparative example 1(is without using the immobilization technology in the present invention):
(1) distiller's wort of 60L fermentor filling 6% ethyl alcohol of 45L.
(2) acetic acid bacteria seed liquor of 500mL mid-log phase is added, starts acetic fermentation;When acetate concentration 35g/L, start Stream plus absolute alcohol (0.05L/h), measure fermentation liquid total acid and are not rising, and terminate acetic fermentation.
Comparative example 2(does not have to fixing of acetic acid bacteria cell membrane raw material):
Concrete operations are left out except step (2) and (4), other same as Example 1.
It is immobilization raw material that comparative example 3(, which does not add yeast cell wall):
Concrete operations remove step (3), other same as Example 1.
Without using PEG800) when comparative example 4(prepares cell membrane:
Except the C of step (2) are as follows: 10000rpm be centrifuged 10-15 minute, precipitating with 200-300mL culture medium resuspension, it is other with it is real It is identical to apply example 1.
Comparative example 5 (comparative example is that detection this method immobilized cell reuses efficiency)
After the completion of embodiment 1, at once carry out embodiment 1(6) in acetic fermentation.
The comparative example 6(comparative example is that detection this method immobilized cell reuses efficiency)
After the completion of comparative example 5, at once carry out embodiment 1(6) in acetic fermentation.
Acetic acid content comparison is carried out to sample prepared by embodiment and comparative example 1-6, test result is to weigh three times Multiple test statistics, concrete outcome see the table below.
Acetic acid content compares in each embodiment of table 1
aTitration measuring
By the above test result it is found that embodiment 1, which prepares acetic acid content in sample, is significantly higher than comparative example 1-4, highest Reach 9.3 ± 0.1%, by comparative example 5,6 it is found that the immobilization acetic acid bacteria in the present invention can Reusability.

Claims (1)

1. a kind of immobilization acetic fermentation method for improving acetic acid bacteria and producing acetic acid ability, it is characterised in that: this method includes as follows Step:
(1) prepared by corncob: corncob is impregnated 10-12 hours with pH5.0 hydrochloric acid solution, then is cleaned with clear water, overnight in 80 DEG C;
(2) the acetic acid bacteria cell film preparation of fermentation termination: A, taking the acetic acid fermentation liquid of batch fermentation, and 3000-4000rpm centrifugation is received Collect thallus, wherein the sample point of acetic acid fermentation liquid is fermentation termination, acetate concentration 6%;
B, the thallus of collection is resuspended in distilled water 1 hour, 30 DEG C, wherein distilled water lysozyme containing 0.8-1.0mg/mL;
C, 8000-10000rpm is centrifuged 10-15 minutes, precipitating 200-300mL PEG800(4-6%) it is resuspended;
(3) prepared by carrier: yeast cell wall is resuspended with water, on corncob prepared by its even spraying to step (1), and 30-35 The rotation of DEG C sterile wind drying 10-15 minute, obtained carrier;
(4) acetic acid bacteria cell membrane prepared by step (2) is sprayed on the carrier of step (3), 30-35 DEG C of sterile wind rotation drying 10-15 minutes;
(5) prepared by immobilization acetic acid bacteria: the carrier of step (4) preparation is sprayed to after the acetic acid bacteria seed liquor of mid-log phase is concentrated On, room temperature stands 40-60 minutes, horizontal rolling 40-60 minutes in 32-35 DEG C of bellows;
(6) immobilization acetic acid bacteria prepared by step (5) is fixed on fermentor agitating paddle, is used for liquid acetic acid fermentation, fermentation Corn core carrier is submerged with the distiller's wort of 6% ethyl alcohol in tank, starts acetic fermentation;When acetate concentration reaches 35g/L, start stream plus it is pure The distiller's wort of alcohol measures fermentation liquid total acid and is not rising, and terminates acetic fermentation.
CN201810971578.XA 2018-08-24 2018-08-24 A kind of immobilization acetic fermentation method for improving acetic acid bacteria and producing acetic acid ability Pending CN109182399A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101319188A (en) * 2008-07-15 2008-12-10 山东农业大学 Normal juice fruit vinegar immobilization preparation method
CN103131688A (en) * 2013-01-05 2013-06-05 武汉工业学院 Preparation method for corncob and/or straw bearing photosynthetic bacteria and application thereof
CN105018328A (en) * 2015-08-12 2015-11-04 广西壮族自治区农业科学院农产品加工研究所 Method for making banana fruit vinegar through corncob immobilized acetobacter
CN105851470A (en) * 2015-01-20 2016-08-17 陈留成 Producing process and use thereof of straw stalks/grass meal feed
CN106167780A (en) * 2016-08-25 2016-11-30 湖北工业大学 A kind of ethanol heat-resisting, resistance to, the acetic acid bacteria of resistance to acetic acid

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101319188A (en) * 2008-07-15 2008-12-10 山东农业大学 Normal juice fruit vinegar immobilization preparation method
CN103131688A (en) * 2013-01-05 2013-06-05 武汉工业学院 Preparation method for corncob and/or straw bearing photosynthetic bacteria and application thereof
CN105851470A (en) * 2015-01-20 2016-08-17 陈留成 Producing process and use thereof of straw stalks/grass meal feed
CN105018328A (en) * 2015-08-12 2015-11-04 广西壮族自治区农业科学院农产品加工研究所 Method for making banana fruit vinegar through corncob immobilized acetobacter
CN106167780A (en) * 2016-08-25 2016-11-30 湖北工业大学 A kind of ethanol heat-resisting, resistance to, the acetic acid bacteria of resistance to acetic acid

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JANJA TRČEK等: "Adaptation and tolerance of bacteria against acetic acid", 《APPL MICROBIOL BIOTECHNOL》 *
张琳等: "醋醅中醋酸菌的分离及产酸耐酸能力分析", 《中国调味品》 *

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