CN105670971A - Clostridium kluyveri JZZ and application thereof - Google Patents
Clostridium kluyveri JZZ and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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Abstract
The invention discloses clostridium kluyveri JZZ and application thereof.The clostridium kluyveri JZZ is separated out of pit mud.It is proved by experiments that the strain is a hexanoic acid high-producing strain, has both temperature tolerance and ethanol tolerance, and has the advantages of adapting to changes of the temperature and ethanol concentration of a traditional pit fermentation system, being high in adaptability and capable of meeting the production requirements in the traditional baijiu brewing technique, improving the quality of baijiu and increasing the brewing production benefit.
Description
Technical field
The present invention relates to strain clostridium kluyveri JZZ and an application thereof, belong to biological technical field.
Background technology
China's liquor-making history is long, and spirits culture inside information is deep. In very long wine brewing practice, define differ from one another, the Chinese liquor of the multiple odor type of different style, wherein, the feature such as aromatic Chinese spirit has fragrance soft sweet cold, smell coordination strong, continuous, enters sweet taste in the mouth, the mouth silk floss that falls, tail remainder length, become the main product of liquor industry. The bulk composition constituting rich fragrance wine typical style is ethyl hexanoate, and this component content is high and fragrance is prominent. One key character of conventional solid-state brewed spirit is to utilize cellar mud microorganisms to participate in sweat. In Luzhou-flavor pit mud, key function bacterium caproic acid bacteria serves vital effect in fragrance is formed. The metabolite caproic acid of caproic acid bacteria, Chinese liquor not only act as in effect fragrant, that help perfume (or spice), minimizing wine body to stimulate, but also the ethanol generation esterification that can produce with the fermentation of grain unstrained spirits, generate the fragrance matters such as ethyl hexanoate, thus improving local flavor and the mouthfeel of aromatic Chinese spirit.
Caproic acid bacteria is not the species name in biological classification meaning, is an appellation sanctified by usage in liquor industry, comprises multiple microorganism kind, and the common trait of this quasi-microorganism is that metabolite has caproic acid, can as the precursor of ethyl hexanoate synthesis. Searching document is known, existing substantial amounts of caproic acid bacteria separation, purification, qualification and applied research, wherein, in strain identification, report that except patent 201010238106 except 1 example caproic acid bacteria (ClostridiumbutyriCum) GK13 bacterial strain, the caproic acid bacteria of all the other reports is kirschner clostridium (Clostridiumkluyveri).
Kirschner clostridium (Clostridiumkluyveri) was just found as far back as the thirties in last century, is one and typically has the strain producing caproic acid ability. But in actual production, due to the natural environment of various places, weather conditions, pit mud physicochemical property, wine-making technology difference, each wine enterprise separating hexanoic acid bacterium habit different. The habit of caproic acid bacteria industrial strain is different, and the performance in actual production just has very big-difference, and this is directly connected to the product wine effect brewageed.
In brewed spirit process, notable change can be there is along with inside grain unstrained spirits course of fermentation fermentation system.One of them factor is fermentation system variations in temperature, and during blanking, initial temperature is generally about 20 DEG C, along with starch degradation heat release, gradually rise up to 35 DEG C-40 DEG C, this level can be maintained afterwards or slightly decline, certainly, at the diverse location of pit, local temperature also can be different, have even more than more than 40 DEG C, further, since the open production of pit, by season, Changes in weather sooner or later, rain or shine, generation is also changed by fermentation system temperature; Another factor is concentration of alcohol change in fermentation system, and along with course of fermentation, concentration of alcohol accumulates gradually, and the fermentation later stage reaches 3-4%, and local is likely to higher. Fermentation system temperature and concentration of alcohol change, it is desirable to pit mud functional bacteria has the toleration of a degree of temperature, ethanol. At present, the high yield caproic acid bacteria not yet having higher temperature toleration and alcohol resistance is reported.
The high caproic acid yield of caproic acid bacteria is always pursuing a goal of brewageing of aromatic Chinese spirit. Although some caproic acid bacteria bacterial strain high yields, but it is not strong that variations in temperature and concentration of alcohol are changed adaptability, causes that production effect is unstable. Owing to fermentation system is among dynamically change, temperature, alcohol resistance also should be listed in examination index by caproic acid bacteria, and this is significant for the stability in caproic acid bacteria bacterial strain commercial production and adaptation traditional fermentation technique.
Summary of the invention
It is an object of the present invention to provide strain kirschner clostridium (Clostridiumkluyveri) JZZ.
The deposit number of kirschner clostridium (Clostridiumkluyveri) JZZ provided by the invention is CGMCCNo.11948.
The Classification And Nomenclature of the bacterial strain JZZ of the present invention is kirschner clostridium (Clostridiumkluyveri), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 29th in December in 2015 and (is called for short CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCCNo.11948.
It is a further object to provide above-mentioned kirschner clostridium or its bacteria suspension or its culture fluid or its tunning or the new application of the microbial inoculum containing it.
The invention provides above-mentioned kirschner clostridium or its bacteria suspension or its culture fluid or its tunning or containing its microbial inoculum following 1)-4) in application in any one:
1) caproic acid and/or ethyl hexanoate are produced;
2) preparation produces the product of caproic acid and/or ethyl hexanoate;
3) wine brewing;
4) product of preparation wine brewing; Described product is specially pit mud.
It is a still further object of the present invention to provide a kind of method producing caproic acid.
The method of production caproic acid provided by the invention comprises the steps: kirschner clostridium (Clostridiumkluyveri) JZZ in the system containing ethanol described in fermentation culture claim 1, collects tunning, obtains caproic acid.
In said method, the temperature of described fermentation culture is 36-40 DEG C.
In said method, the temperature of described fermentation culture is 37 DEG C.
In said method, the ethanol volumn concentration in the described system containing ethanol is 2-10%; The described system containing ethanol is sodium acetate fluid medium; The solvent of sodium acetate fluid medium is water, solute and concentration thereof is: sodium acetate 0.5% (mass fraction), yeast extract 0.1% (mass fraction), magnesium sulfate 0.02% (mass fraction), dipotassium hydrogen phosphate 0.04% (mass fraction), ammonium sulfate 0.05% (mass fraction), calcium carbonate 1% (mass fraction), ethanol 2% (mass fraction).
In said method, the ethanol volumn concentration in the described system containing ethanol is 2%.
In said method,
The time of described fermentation culture is 6-8 days;
The time of described fermentation culture is specially 7 days.
In said method, after collecting tunning, also comprise the steps: tunning described in organic solvent extraction, collect organic facies and obtain caproic acid.
Said method application in producing caproic acid and/or producing ethyl hexanoate and/or make wine falls within protection scope of the present invention.
The present invention separates from pit mud and obtains a strain clostridium kluyveri JZZ. It is experimentally confirmed: this bacterial strain is caproic acid superior strain, with temperature, alcohol resistance, it provides the benefit that the variations in temperature having adapted to traditional pit fermentation system and concentration of alcohol change, and strong adaptability, disclosure satisfy that production requirement in traditional liquor brewage process, not only increase the quality of Chinese liquor, also improve wine brewing productivity effect.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Culture medium of sodium acetate compound method in following embodiment:
1) solvent of sodium acetate fluid medium is water, solute and concentration thereof is: sodium acetate 0.5% (mass fraction), yeast extract 0.1% (mass fraction), magnesium sulfate 0.02% (mass fraction), dipotassium hydrogen phosphate 0.04% (mass fraction), ammonium sulfate 0.05% (mass fraction), calcium carbonate 1% (mass fraction), ethanol 2% (mass fraction), adding after sterilizing, pH is 6.5. Weigh after dissolving, 121 DEG C of sterilizing 30min.
2) sodium acetate solid medium is the culture medium that add 2% in the sodium acetate fluid medium agar of (mass fraction) obtains.
Embodiment 1, clostridium kluyveri JZZ acquisition
One, the separation of bacterial strain
1, the fermentation culture of pit mud
Sodium acetate fluid medium is loaded in test tube (liquid amount 90%), in test tube, add 1g aged pit mud (Anhui Golden Seed Brewery Co., Ltd.), 80 DEG C of water-bath heat treatment 10min, seal, 37 DEG C of fermentation culture 7d, obtain fermentation liquid.
2, the purification of fermentation liquid is cultivated
The fermentation liquid obtained by ether extraction step 1, and with copper sulfate development process primary dcreening operation, blueness is the positive; Take the bacterium solution 1mL that primary dcreening operation is the positive, add in the test tube equipped with sodium acetate fluid medium, 80 DEG C of water-bath heat treatment 10min, seal, cultivate 7d for 37 DEG C; Repeat purification 2-3 time, obtain purification after fermentation liquid.
3, purification after fermentation liquid
Purification after fermentation liquid step 2 obtained carries out gradient dilution, and is coated with flat board in sodium acetate solid medium, 37 DEG C cultivate 7d after, the single bacterium colony of picking ferments, and in bacterium solution fermentation culture obtained by gas chromatogram, acetic acid content detects. Finishing screen is chosen a plant height and is produced the bacterial strain of caproic acid, by its called after JZZ.
Two, the qualification of JZZ
1, identification of morphology
Through the morphologic observation to JZZ bacterial strain, the form of JZZ bacterial strain is as follows: direct rod shape, and bud embraces end life or near-end is raw, after solid medium cultivates 7d, forms circular colonies, and surface is smooth, milky, glossy, and neat in edge is opaque.
2, Molecular Identification
Extract the STb gene of bacterial strain JZZ as template, adopt universal primer 27f and 1492r to carry out pcr amplification, obtain the fragment containing bacterial strain JZZ16SrDNA conserved region and it is checked order. Primer sequence is as follows:
27f (forward primer): 5 '-AGAGTTTGATCCTGGCTCAG-3 ';
1492r (downstream primer): 5 '-TACGGTTACCTTGTTACGACTT-3 '.
The 16SrDNA sequencing of JZZ bacterial strain is completed by Shanghai Major Biological Medical Technology Co., Ltd. (building, No. 3399 epoch Yi Chuan gardens the 3rd of Pudong New Area, Shanghai Kang Xin highway, 021-51875086). Bacterial strain JZZ16SrDNA is sequence 1. And it is compared in GeneBank.
Comparison result at GeneBank shows, the similarity of the 16SrDNA gene of JZZ and kirschner clostridium (JN592512.1) reaches 100%.
3, the preservation of bacterial strain
According to morphological characteristic and Molecular Identification result and analysis, bacterial strain JZZ is defined as kirschner clostridium. The Classification And Nomenclature of bacterial strain JZZ is kirschner clostridium (Clostridiumkluyveri), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on the 29th in December in 2015 and (is called for short CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCCNo.11948.
Embodiment 2, clostridium kluyveri JZZ application in high yield caproic acid
One, JZZ fermentation culture and produce caproic acid amount detection
1, the fermentation culture of JZZ
(1) the JZZ bacterial strain implementing 1 acquisition is carried out activation culture in the test tube equipped with sodium acetate fluid medium, 37 DEG C of activation culture 7d, obtain seed liquor;
(2) seed liquor 5% inoculum concentration is inoculated in the triangular flask equipped with sodium acetate fluid medium and carries out fermentation culture, 37 DEG C of fermentation culture 7d, obtain fermentation liquid.
2, caproic acid vapor detection condition
1) caproic acid extracts
0.22 μm of filtering with microporous membrane of fermentation liquor step 1 obtained, obtains filtrate; Taking filtrate 500 μ L in 5mL volumetric flask, add diethyl ether and be settled to 5mL, fully vibrate 2min, stands 1min, draws upper strata ether extraction liquid, is caproic acid, standby survey. Setup Experiments repeatedly repeats, and repeats laboratory sample and is numbered 1,2,3,4,5,6 respectively.
2) detection
By gas chromatogram, the caproic acid content in ether extraction liquid is detected. Chromatographic condition is: injector temperature 240 DEG C; Post case initial temperature: 100 DEG C (1min); Heating rate: 10 DEG C/min; Column temperature finishing temperature: 220 DEG C (2min); Detector temperature 250 DEG C; Gas flow: hydrogen 40mL/min, air 400mL/min, make-up gas 30mL/min; Input mode: split sampling, split ratio 20:1; Sampling volume: 1 μ L.
Measurement result is in Table 1, and as can be seen from the table, caproic acid yield is up to 4.55g/L, and meansigma methods is 4.36g/L, it was shown that JZZ bacterial strain is stable caproic acid superior strain.
Table 1, clostridium kluyveri JZZ produce the detection of caproic acid amount
Two, the temperature of JZZ fermentation product caproic acid is groped
The seed liquor obtained (1) in step one is inoculated in the triangular flask (sample-loading amount 90%) equipped with sodium acetate fluid medium according to the inoculum concentration of 5%, the Anaerobic culturel 7d when 36 DEG C, 37 DEG C, 38 DEG C, 39 DEG C and 40 DEG C respectively, respectively obtain fermentation liquid, extract caproic acid and use gas chromatography to detect the caproic acid yield in each fermentation liquid, the repetition of three, each sample in above-mentioned one.
Testing result is as shown in table 2, from Table 2, it can be seen that under 37 DEG C of conditions, caproic acid yield is the highest, for 4.36g/L ± 0.09g/L, for producing the optimum temperature of acetic acid; Caproic acid yield when 39 DEG C, 40 DEG C be respectively 94% when 37 DEG C, 88%, but remain on and show high yield characteristics, illustrate that JZZ bacterial strain has higher temperature resistance characteristics.
The caproic acid yield of clostridium kluyveri JZZ under table 2, condition of different temperatures
Three, the reaction system ethanol content of JZZ fermentation product caproic acid is groped
The seed liquor obtained (1) in step one is inoculated into the triangular flask (sample-loading amount 90%) equipped with sodium acetate fluid medium according to the inoculum concentration of 5%, and in triangular flask, add ethanol, make ethanol volume fraction in the medium respectively 2%, 4%, 6%, 8% and 10%, after 37 DEG C of Anaerobic culturel 7d, respectively obtain fermentation liquid. Extract caproic acid and use gas chromatography to detect the caproic acid yield in each fermentation liquid, the repetition of three, each sample in above-mentioned one.
Testing result shows, when concentration of alcohol (volume fraction) is 2%, caproic acid yield is 4.36 ± 0.09g/L, along with gradually rising of ethanol mass fraction, caproic acid yield is gradually reduced, concentration of alcohol (volume fraction) is caproic acid yield when 4%, 6% to be concentration of alcohol (volume fraction) respectively be 95% when 2%, 90%. JZZ bacterial strain is when concentration of alcohol (volume fraction) is 6%, and JZZ bacterial strain still shows the characteristic of high yield acetic acid, illustrates that JZZ bacterial strain has higher ethanol resistance characteristics.
The caproic acid yield of clostridium kluyveri JZZ under table 3, different concentration ethanol
Claims (10)
1. strain kirschner clostridium (Clostridiumkluyveri) JZZ, its deposit number is CGMCCNo.11948.
2. kirschner clostridium described in claim 1 or its bacteria suspension or its culture fluid or its tunning or containing its microbial inoculum following 1)-4) and in application in any one:
1) caproic acid and/or ethyl hexanoate are produced;
2) preparation produces the product of caproic acid and/or ethyl hexanoate;
3) wine brewing;
4) product of preparation wine brewing.
3. the method producing caproic acid, comprises the steps: kirschner clostridium (Clostridiumkluyveri) JZZ described in fermentation culture claim 1 in the system containing ethanol, collects tunning, obtains caproic acid.
4. method according to claim 3, it is characterised in that: the temperature of described fermentation culture is 36-40 DEG C.
5. the method according to claim 3 or 4, it is characterised in that: the temperature of described fermentation culture is 37 DEG C.
6. method according to claim 3, it is characterised in that: the ethanol volumn concentration in the described system containing ethanol is 2-10%.
7. the method according to claim 3 or 6, it is characterised in that: the ethanol volumn concentration in the described system containing ethanol is 2%.
8. according to described method arbitrary in claim 3-7, it is characterised in that:
The time of described fermentation culture is 6-8 days;
The time of described fermentation culture is specially 7 days.
9. according to described method arbitrary in claim 3-8, it is characterised in that: after collecting tunning, also comprise the steps: tunning described in organic solvent extraction, collect organic facies and obtain caproic acid.
10. in claim 3-9, arbitrary described method is producing caproic acid and/or is producing the application in ethyl hexanoate and/or wine brewing.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834177A (en) * | 2017-02-21 | 2017-06-13 | 中国科学院成都生物研究所 | One plant of cud bacterium and its application |
| CN107177639A (en) * | 2017-06-02 | 2017-09-19 | 安徽迎驾贡酒股份有限公司 | A kind of method that utilization bean product processing byproduct bean dregs improve clostridium klebsi fermenting and producing caproic acid yield |
| CN113186055A (en) * | 2021-03-25 | 2021-07-30 | 宜宾五粮液股份有限公司 | Method for improving quality of Luzhou-flavor liquor distiller's grains by using clostridium |
| CN114276948A (en) * | 2021-11-08 | 2022-04-05 | 泸州老窖股份有限公司 | Lysine bacillus for high-yield caproic acid and application thereof |
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| CN103255085A (en) * | 2013-04-24 | 2013-08-21 | 济南瑞丰生物工程有限公司 | Preparation method of concentrated caproic acid bacterial liquid |
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2016
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| CN103255085A (en) * | 2013-04-24 | 2013-08-21 | 济南瑞丰生物工程有限公司 | Preparation method of concentrated caproic acid bacterial liquid |
Non-Patent Citations (2)
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106834177A (en) * | 2017-02-21 | 2017-06-13 | 中国科学院成都生物研究所 | One plant of cud bacterium and its application |
| CN106834177B (en) * | 2017-02-21 | 2019-11-08 | 中国科学院成都生物研究所 | One plant of cud bacterium and its application |
| CN107177639A (en) * | 2017-06-02 | 2017-09-19 | 安徽迎驾贡酒股份有限公司 | A kind of method that utilization bean product processing byproduct bean dregs improve clostridium klebsi fermenting and producing caproic acid yield |
| CN107177639B (en) * | 2017-06-02 | 2021-02-12 | 安徽迎驾贡酒股份有限公司 | Method for improving caproic acid yield in fermentation production of clostridium kluyveri by using bean dregs which are byproducts of bean product processing |
| CN113186055A (en) * | 2021-03-25 | 2021-07-30 | 宜宾五粮液股份有限公司 | Method for improving quality of Luzhou-flavor liquor distiller's grains by using clostridium |
| CN113186055B (en) * | 2021-03-25 | 2023-08-01 | 宜宾五粮液股份有限公司 | Method for improving quality of strong aromatic Chinese liquor waste distillers' grains by using clostridium |
| CN114276948A (en) * | 2021-11-08 | 2022-04-05 | 泸州老窖股份有限公司 | Lysine bacillus for high-yield caproic acid and application thereof |
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Application publication date: 20160615 |