CN109182399B - Immobilized acetic acid fermentation method for improving acetic acid producing capability of acetic acid bacteria - Google Patents
Immobilized acetic acid fermentation method for improving acetic acid producing capability of acetic acid bacteria Download PDFInfo
- Publication number
- CN109182399B CN109182399B CN201810971578.XA CN201810971578A CN109182399B CN 109182399 B CN109182399 B CN 109182399B CN 201810971578 A CN201810971578 A CN 201810971578A CN 109182399 B CN109182399 B CN 109182399B
- Authority
- CN
- China
- Prior art keywords
- acetic acid
- fermentation
- acid bacteria
- immobilized
- minutes
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/54—Acetic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an immobilized acetic acid fermentation method for improving the acetic acid producing capability of acetic acid bacteria, and belongs to the field of food processing. The invention adopts a reconstructed wall membrane compound (yeast cell wall and acetic acid bacteria cell membrane at the fermentation end) to cover corncob microparticles as an acetic acid bacteria carrier, thereby achieving the purpose of improving the acetic acid resistance of the acetic acid bacteria and finally improving the acetic acid production.
Description
Technical Field
The invention belongs to the field of food brewing, and particularly relates to an immobilized acetic acid fermentation method for improving the acetic acid producing capability of acetic acid bacteria.
Background
The preparation of acetic acid can be realized by two methods of artificial synthesis and biological fermentation, and the fermentation method for producing the acetic acid or the vinegar is a process of oxidizing ethanol into the acetic acid under the action of acetic acid bacteria. Biofermentation represents only 10% of the world's production. However, food safety regulations in many countries stipulate that vinegar in foods must be prepared by biological methods, and thus biological fermentation methods are still the most important methods for producing acetic acid, especially vinegar (acetic acid is the main component of vinegar).
Acetic acid, as low as 0.5%, can inhibit the growth of most microorganisms. Microbial inhibition makes acetic acid a common preservative. The acetic acid bacteria are important microorganisms for producing acetic acid by a fermentation method, and have excellent acetic acid tolerance compared with common microorganisms. However, in the fermentation production, acetic acid still has a toxic effect on acetic acid bacteria and is the biggest limiting factor faced by the acetic acid bacteria, so that the acetic acid bacteria have a limited ability to produce acetic acid. The acid resistance of different types of acetic acid bacteria is very different, and it has been found that Ga. europaeus can tolerate the environment with the highest acetic acid concentration, 10% of acetic acid in the environment can be tolerated, and the two acetic acid bacteria, A. aceti and A. pateuricenus, which are most commonly used in acetic acid fermentation can only tolerate no more than 6% of acetic acid. The common vinegar brewing microorganism in China is A. pateurianus AS1.41, the acid resistance of the microorganism is gradually induced by acetic acid and temporarily improved in the fermentation process, and the acetic acid can be produced to 9 percent; but the tolerance capacity can not be maintained genetically, and the initial tolerance capacity of acetic acid bacteria producing 9 percent of acetic acid is still lower than 6 percent after the acetic acid bacteria are cultured in a culture medium without ethanol and acetic acid for 3 generations.
The toxicity mechanism of acetic acid: high concentration acetic acid easily permeates into cytoplasm through cell membrane to adversely affect the growth and metabolism of thallus, and its toxicity mechanism includes H+And CH3COO-Resulting in intracellular environment disorder. At present, the acid resistance mechanism of acetic acid bacteria is not completely clarified. Studies have been reported to discover some of the acid-tolerant molecular mechanisms in Acetobacter and gluconacetobacter: (1) ethanol oxidation-related mechanisms including membrane-bound ethanol dehydrogenase (Adh), acetaldehyde dehydrogenase (Aldh) mechanisms; (2) an acetic acid assimilation mechanism; (3) transport mechanisms that may pump acetic acid out; (4) general stress resistance mechanism.
The fermentation of vinegar is divided into solid fermentation and liquid fermentation. The liquid submerged acetic acid fermentation adopts pure fermentation, has short period, high acid production and strong production capacity, and is gradually adopted by manufacturers due to obvious advantages.
A method (CN 201510492742.5) for brewing banana full fruit vinegar by using corncob immobilized acetic acid bacteria provides a method for utilizing corncob immobilized acetic acid bacteria; a method (CN 201610895642.1) for preparing cigarette laurel extract by immobilized acetic acid bacteria comprises adding wet thallus into mixed solution of polyvinyl alcohol and sodium alginate, and adding CaCl containing 1-3%2And saturated Na2SO4The immobilized acetic acid bacteria pellets are obtained by crosslinking in the solution, but the method does not relate to the improvement of the acetic acid resistance of the acetic acid bacteria.
Based on the acetic acid fermentation process, it is essential to design a better strategy to improve the acid stability of industrial microorganisms. The acetic acid bacteria immobilized fermentation method improves the acetic acid resistance of the acetic acid bacteria, and finally achieves the aim of improving the acetic acid production.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an immobilized acetic acid fermentation method for improving the acetic acid producing capability of acetic acid bacteria.
In order to achieve the purpose of the present invention, the present inventors finally obtained the following technical solutions through a great deal of experimental research and diligent research:
an immobilized acetic acid fermentation method for improving the acetic acid producing capability of acetic acid bacteria, which comprises the following steps:
(1) preparing corncobs: soaking corn cob in hydrochloric acid solution of pH5.0 for 10-12 hr, cleaning with clear water, and standing at 80 deg.C overnight;
(2) preparing acetic acid bacteria cell membranes at the fermentation end: A. taking acetic acid fermentation liquor fermented in batches, centrifuging at 3000-;
B. re-suspending the collected thallus in distilled water containing lysozyme in 0.8-1.0mg/mL for 1 hr at 30 deg.c;
C. centrifuging at 10000rpm for 10-15 minutes at 8000-;
(3) preparing a carrier: resuspending yeast cell walls by water, uniformly spraying the yeast cell walls on the corncobs prepared in the step (1), and rotationally drying the corncobs for 10-15 minutes by using sterile wind at the temperature of 30-35 ℃ to prepare a carrier;
(4) spraying the acetic acid bacteria cell membrane prepared in the step (2) on the carrier in the step (3), and rotationally drying for 10-15 minutes by using sterile wind at the temperature of 30-35 ℃;
(5) preparing immobilized acetic acid bacteria: concentrating acetic acid bacteria seed liquid in the middle logarithmic phase, spraying the acetic acid bacteria seed liquid on the carrier prepared in the step (4), standing at normal temperature for 40-60 minutes, and horizontally rolling in an air box at the temperature of 32-35 ℃ for 40-60 minutes;
(6) fixing the immobilized acetic acid prepared in the step (5) on a stirring paddle of a fermentation tank for liquid acetic acid fermentation, and immersing the corn cob carrier in mash of 6% ethanol in the fermentation tank to start acetic acid fermentation; when the acetic acid concentration reaches 35g/L, the wine mash of pure alcohol begins to flow, the total acid of the fermentation liquid is measured not to rise, and the acetic acid fermentation is stopped.
Compared with the prior art, the invention has the following advantages compared with the prior vinegar processing technology:
(1) the invention adopts a reconstructed wall membrane compound (yeast cell wall and acetic acid bacteria cell membrane at the fermentation end) to cover corncob microparticles as an acetic acid bacteria carrier, thereby achieving the purposes of improving the acetic acid resistance of the acetic acid bacteria, greatly improving the acid resistance of the acetic acid and obviously improving the yield of the acetic acid.
(2) The immobilized acetic acid bacteria can be repeatedly used, and the production cost is reduced.
The specific implementation mode is as follows:
the following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.
Example 1
(1) Preparing corncobs: 3 corncobs were soaked in a hydrochloric acid solution of pH5.0 for 12 hours, washed with clean water, and left overnight at 80 ℃.
(2) Preparing acetic acid bacteria cell membranes at the fermentation end: A. 30L of acetic acid fermentation liquor fermented in batches (the sampling point is the fermentation end point, the acetic acid concentration is 6 percent) is taken, and thalli are collected by centrifugation at 4000 rpm. B. Resuspended in distilled water (containing 1.0mg/mL lysozyme) for 1 hour at 30 ℃. C. Centrifugation is carried out at 10000rpm for 10-15 minutes, and the pellet is resuspended with 200-300mL of PEG800 (6%).
(3) Preparing a carrier: resuspending 30g of commercial yeast cell walls in 300mL of water, spraying the cell walls onto the corncobs prepared in step (1), and rotary blowing the corncobs for 15 minutes at 30 ℃ by sterile wind.
(4) And (3) spraying the acetic acid bacteria cell membranes prepared in the step (2) on the corncobs in the step (3), and rotationally drying for 15 minutes by sterile wind at the temperature of 30 ℃.
(5) Preparing immobilized acetic acid bacteria: and (3) concentrating 500mL of acetic acid bacteria seed liquid in the middle logarithmic phase to 200mL, spraying the acetic acid bacteria seed liquid on the carrier prepared in the step (4), standing at normal temperature for 60 minutes, rotationally drying at 30 ℃ for 15 minutes by using sterile wind, and fixing the acetic acid bacteria seed liquid on a 60L fermentation tank stirring paddle for liquid acetic acid fermentation.
(6) Acetic acid fermentation: immersing corn cob (liquid loading amount is about 45L) with 6% ethanol wine mash in a 60L fermentation tank, and starting acetic fermentation; when the acetic acid concentration is 35g/L, the beer mash added with pure alcohol (0.05L/h) is started to carry out acetic fermentation, and the acetic fermentation is stopped when the total acid of the fermentation liquor is not rising.
Comparative example 1 (without using the immobilization technique of the invention):
(1) A60L fermenter was filled with 45L of 6% ethanol mash.
(2) Adding 500mL of acetic acid bacteria seed liquid in the middle logarithmic phase to start acetic acid fermentation; when the acetic acid concentration is 35g/L, pure alcohol (0.05L/h) is fed in, and the acetic acid fermentation is stopped when the total acid of the fermentation liquid is not increased.
Comparative example 2 (without using acetate cell membrane as immobilization material):
the specific operation is to delete steps (2) and (4), and the rest is the same as that of embodiment 1.
Comparative example 3 (without addition of yeast cell wall as immobilization material):
the procedure of example 1 was otherwise the same as except for the step (3).
Comparative example 4 (PEG 800 was not used in the preparation of cell membranes):
c in the step (2) is removed: centrifugation was carried out at 10000rpm for 10-15 minutes, and the pellet was resuspended in 200-300mL of the medium, which was otherwise the same as in example 1.
Comparative example 5 (comparative example for examining the efficiency of reuse of the immobilized cells in the present method)
After completion of example 1, acetic acid fermentation in example 1 (6) was carried out.
Comparative example 6 (comparative example for examining the efficiency of reuse of the immobilized cells in the present method)
Upon completion of comparative example 5, acetic acid fermentation in example 1 (6) was carried out.
The samples prepared in examples and comparative examples 1-6 were compared in acetic acid content, and the test results are statistics of three repeated tests, and the specific results are shown in the following table.
TABLE 1 comparison of acetic acid content in examples
aDetermination by titration
From the above test results, it is clear that the content of acetic acid in the sample prepared in example 1 is significantly higher than that in comparative examples 1 to 4, and is as high as 9.3 ± 0.1%, and it is clear from comparative examples 5 and 6 that the immobilized acetic acid bacteria of the present invention can be repeatedly used.
Claims (1)
1. An immobilized acetic acid fermentation method for improving the acetic acid producing capability of acetic acid bacteria is characterized in that: the method comprises the following steps:
(1) preparing corncobs: soaking corn cob in hydrochloric acid solution of pH5.0 for 10-12 hr, cleaning with clear water, and standing at 80 deg.C overnight;
(2) preparing acetic acid bacteria cell membranes at the fermentation end: A. taking acetic acid fermentation liquor fermented in batches, centrifuging at 3000-;
B. re-suspending the collected thallus in distilled water containing lysozyme in 0.8-1.0mg/mL for 1 hr at 30 deg.c;
C. centrifuging at 10000rpm for 10-15 minutes at 8000-;
(3) preparing a carrier: resuspending yeast cell walls by water, uniformly spraying the yeast cell walls on the corncobs prepared in the step (1), and rotationally drying the corncobs for 10-15 minutes by using sterile wind at the temperature of 30-35 ℃ to prepare a carrier;
(4) spraying the acetic acid bacteria cell membrane prepared in the step (2) on the carrier in the step (3), and rotationally drying for 10-15 minutes by using sterile wind at the temperature of 30-35 ℃;
(5) preparing immobilized acetic acid bacteria: concentrating acetic acid bacteria seed liquid in the middle logarithmic phase, spraying the acetic acid bacteria seed liquid on the carrier prepared in the step (4), standing at normal temperature for 40-60 minutes, and horizontally rolling in an air box at the temperature of 32-35 ℃ for 40-60 minutes;
(6) fixing the immobilized acetic acid bacteria prepared in the step (5) on a stirring paddle of a fermentation tank for liquid acetic acid fermentation, and immersing the corncob carrier in mash of 6% ethanol in the fermentation tank to start acetic acid fermentation; when the acetic acid concentration reaches 35g/L, the wine mash of pure alcohol begins to flow, the total acid of the fermentation liquid is measured not to rise any more, and the acetic acid fermentation is stopped.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810971578.XA CN109182399B (en) | 2018-08-24 | 2018-08-24 | Immobilized acetic acid fermentation method for improving acetic acid producing capability of acetic acid bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810971578.XA CN109182399B (en) | 2018-08-24 | 2018-08-24 | Immobilized acetic acid fermentation method for improving acetic acid producing capability of acetic acid bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109182399A CN109182399A (en) | 2019-01-11 |
| CN109182399B true CN109182399B (en) | 2021-09-03 |
Family
ID=64919445
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810971578.XA Active CN109182399B (en) | 2018-08-24 | 2018-08-24 | Immobilized acetic acid fermentation method for improving acetic acid producing capability of acetic acid bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109182399B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734841A (en) * | 2019-10-28 | 2020-01-31 | 广西农业职业技术学院 | Preparation method of banana composite fruit vinegar |
| CN113151245B (en) * | 2021-05-28 | 2022-06-03 | 湖北工业大学 | Flavor compound microbial inoculum for reducing rice wine fusel oil and application thereof |
Citations (5)
| 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 |
-
2018
- 2018-08-24 CN CN201810971578.XA patent/CN109182399B/en active Active
Patent Citations (5)
| 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)
| Title |
|---|
| Adaptation and tolerance of bacteria against acetic acid;Janja Trček等;《Appl Microbiol Biotechnol》;20150705;第99卷;第6215-6229页 * |
| 醋醅中醋酸菌的分离及产酸耐酸能力分析;张琳等;《中国调味品》;20151130;第40卷(第11期);第19-27页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109182399A (en) | 2019-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109182399B (en) | Immobilized acetic acid fermentation method for improving acetic acid producing capability of acetic acid bacteria | |
| CN111961603B (en) | Saccharomyces cerevisiae and bacterial agents and their use in the preparation of fermented products, in particular in the brewing of Huai drop of water basin wines | |
| CN103436403B (en) | Highland barley za wine and preparation method thereof | |
| EP2641962A1 (en) | Pichia kluyveri strain and uses thereof | |
| CN108384701A (en) | A kind of preparation method of the fermented type fruit vinegar rich in anthocyanin | |
| CN111961601B (en) | Yeast for high yield of isoamyl acetate by solid state fermentation and application thereof | |
| CN112175845B (en) | Saccharomyces cerevisiae suitable for brewing tea wine and application thereof | |
| CN108165501B (en) | Saccharomyces cerevisiae strain and culture method and application thereof | |
| CN109486693A (en) | A kind of S. cervisiae and its purposes in alcohol fermentation | |
| CN111621430B (en) | Saccharomyces cerevisiae suitable for brewing yellow peach fruit wine and application thereof | |
| CN106754526A (en) | Preparation method of lactobacillus direct vat set powder applied to yellow wine brewing | |
| TW200819532A (en) | Method of manufacturing vinegar with high extract content | |
| CN113337499B (en) | Method for increasing total acid content in gluconobacter oxydans fermented vinegar | |
| CN111690504B (en) | Method for increasing content of non-volatile organic acid in acetification stage of apple vinegar | |
| CN112442453B (en) | Ormokodak yeast for degrading biogenic amine and application of same in white spirit brewing | |
| CN109666594B (en) | Method for intensively producing Shanxi mature vinegar by utilizing excellent native high-alcohol-yield saccharomyces cerevisiae and Artemisia annua Candida | |
| CN111961599B (en) | Solid-state fermentation yeast with flower and fruit flavor and application thereof | |
| CN103320300A (en) | Double-strain immobilization method for brewing grape vinegar | |
| CN111909875A (en) | Persimmon gluconacetobacter and application thereof | |
| CN111820340A (en) | Biological feed leavening agent for aquaculture and preparation process thereof | |
| JPH1118750A (en) | Production of citrus wine and citrus fermented vinegar | |
| CN116948847B (en) | Zygosaccharomyces bailii and application thereof in white spirit production | |
| CN112625929B (en) | Tannin-tolerant yeast and application thereof in fruit vinegar fermentation | |
| CN115141762B (en) | Lepidii yeast, acquisition method, preparation method of composite freeze-drying starter and application of composite freeze-drying starter in vinegar preparation | |
| CN116103171B (en) | Saccharomyces cerevisiae resistant to environmental stress and capable of producing ethanol through rapid fermentation and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |