CN105505807A - Method for regulating synthesis of saccharomyces cerevisiae flavor substances through acetic acid metabolism - Google Patents

Method for regulating synthesis of saccharomyces cerevisiae flavor substances through acetic acid metabolism Download PDF

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CN105505807A
CN105505807A CN201610056297.2A CN201610056297A CN105505807A CN 105505807 A CN105505807 A CN 105505807A CN 201610056297 A CN201610056297 A CN 201610056297A CN 105505807 A CN105505807 A CN 105505807A
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acetic acid
saccharomyces cerevisiae
yeast saccharomyces
acetaldehyde
regulate
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陈叶福
肖冬光
钱泓
罗伟伟
薛星祥
郭学武
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Tianjin University of Science and Technology
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Abstract

The invention belongs to the technical field of bioengineering and particularly relates to a method for regulating synthesis of saccharomyces cerevisiae flavor substances through acetic acid metabolism. Cells are made to accumulate an appropriate amount of acetic acid by reinforcing acetic acid synthesis or weakening acetic acid decomposition or adding acetic acid through an external source, the generation amount of saccharomyces cerevisiae higher alcohols, acetaldehyde and ethyl acetate can be further lowered, and the generation amount of acetic acid is raised properly. By means of the method, a new path is provided for regulating the flavor substances in liquor products, on the premise that basic fermentation performance of saccharomyces cerevisiae is not influenced, generation of such trace flavor substances as the higher alcohols, esters and acetaldehyde is lowered on the whole, and the purity degree of the liquor products is raised.

Description

By the method for the synthesis of acetate metabolism regulation and control yeast saccharomyces cerevisiae flavour substances
Technical field:
The invention belongs to technical field of bioengineering, relate to industrial micro breeding and drinks is brewageed, be specifically related to the synthesis by flavour substancess such as acetate metabolism regulation and control yeast saccharomyces cerevisiae higher alcohols, acetic acid, ester, aldehyde.
Background technology:
The flavour substancess such as alcohol, acid, ester, aldehyde decide the quality of wine, all indispensable for all wine product, but its content in specific wine product must be suitable for, and too much or very fewly all can have a negative impact to the quality of wine.
Higher alcohols is the important alcohols flavour cpds outside ethanol, mainly comprise n-propyl alcohol, isopropylcarbinol, primary isoamyl alcohol (accounting for about 50% of higher alcohols total amount), active-amyl alcohol and bata-phenethyl alcohol etc., often kind of higher alcohols has the flavor feature of oneself uniqueness, and different alcohol ester ratios imparts often kind of distinguished fragrance of wine kind.But, if higher alcohols content exceedes certain standard, not only make wine body produce different assorted taste, affect the quality of wine, and human consumer can be made to produce symptoms such as " dizzy, headaches ", be commonly called as " top ", be detrimental to health.Current, higher alcohols content is all higher in the middle of all drinks, therefore needs the content reducing higher alcohols.
The ester classes such as ethyl acetate are most important for the fragrance of various wine, and especially for China white wine, the kind of ester and content are the Main Basiss of different flavor white wine classification.But ester content is not more high better, and in wine body, ester content is too high, and fragrance can be made too outstanding, inharmonious.High concentration beer fermenting process, can produce the ester classes such as too much ethyl acetate, affect the local flavor of beer.
Organic acid is important taste compound, and acid is also the important substance forming wine " aftertaste ".Acid content wine very little, vinosity is boring, and aftertaste is short, and bitterness has different assorted taste; The wine that acid content is excessive, then vinosity is coarse.Appropriate acid can play buffering and seasoning effect in wine, can eliminate the rear top of drink and the inharmonious phenomenon of mouthfeel.Acetic acid is main seasoning acid, in wine brewing process, acetic acid is mainly produced by the miscellaneous bacteria outside yeast saccharomyces cerevisiae, yeast saccharomyces cerevisiae itself produces acetic acid ability wretched insufficiency, for passing through the single pure-blood ferment of yeast saccharomyces cerevisiae, to keep under strict control the wine brewing process of living contaminants, need the appropriate product acetic acid ability improving yeast saccharomyces cerevisiae.
Acetaldehyde is the volatile flavor compounds in wine, and the acetaldehyde of lower concentration has happy fruit aroma.But when acetaldehyde concentration is higher, can the unhappy smells such as similar Herbaceous Taste be produced.Acetaldehyde is the principal element of easy top of drinking, and is a kind of potential carcinogenic substance, and the wine of high acetaldehyde is pernicious to alcohol user's health, acetaldehyde beneficiating ingredient absolutely not in wine.Acetaldehyde is generally the generation of Ethanol in Saccharomyces cerevisiae fermenting process, some miscellaneous bacterias such as milk-acid bacteria and acetic bacteria also can produce acetaldehyde, for the purebred yeast saccharomyces cerevisiae drinks brewing process avoiding living contaminants, reduce the acetaldehyde generative capacity of yeast saccharomyces cerevisiae self, contribute to the drinking safety improving wine product.
The objectionable impuritiess such as wine body Higher Alcohols, acetaldehyde, and the control of ester and acid, mostly by setting about from zymotechnique, such as, application number is low acetaldehyde beer of one of 2012101252759 and preparation method thereof, by adjustment beer fermentation temperature, extends fermentation time, and control other fermentation parameters, make the acetaldehyde of the work in-process beer after fermenting lower than≤8mg/L.For this predetermined substance of higher alcohols, can start with from the higher alcohols pathways metabolism of yeast saccharomyces cerevisiae, by the genetic engineering modified reduction realizing higher alcohols content.Application number is low-yield higher-alcohol saccharomyces cerevisiae engineering bacterium and the construction process thereof of 201010227788.1, achieve transformant bacterial strain by the disappearance of amino acid transaminase encoding gene (BAT2) and reduce 55.19%, 34.43% respectively than the isopropylcarbinol of parent strain, primary isoamyl alcohol content, total higher alcohols content reduces 35.01%.All in all, the method to disposable global regulations of flavour substances such as alcohol, aldehyde, acid, esters is lacked.
From the angle analysis of metabolism, acetaldehyde generates acetic acid via acetaldehyde dehydrogenase, and when acetic acid route of synthesis strengthens, when in born of the same parents, acetic acid content improves, the acetaldehyde as substrate will decrease; Acetic acid, as the precursor substance of synthesizing ethyl acetate, when acetic acid content in born of the same parents improves, can strengthen the synthesis of ethyl acetate.And the present invention is at experimentation, by improving in born of the same parents after acetic acid content in right amount, except reducing acetaldehyde, also unexpected flavour substances content such as discovery wine Higher Alcohols and ester etc. all decreases.
Further, not yet occur in prior art by regulating acetic acid to reach method to disposable global regulations of flavour substances such as alcohol, aldehyde, acid, esters.Therefore, the present invention is by generating the level of acetic acid in strengthening yeast born of the same parents, or external source adds the method for acetic acid, regulates and controls the generation of the flavour substances of alcohol, aldehyde, acid, ester etc. from the overall situation.
Summary of the invention:
To achieve these goals, the present invention is based on the acetate metabolism of yeast saccharomyces cerevisiae, provide a kind of by making Cellular Accumulation acetic acid, thus reach the method for the flavour substancess such as alcohol, aldehyde, acid, ester being carried out to disposable global regulation.By present method, can the growing amount of the low yeast saccharomyces cerevisiae higher alcohols of a step-down, acetaldehyde and ethyl acetate, the appropriate growing amount improving acetic acid.
The described method making Cellular Accumulation acetic acid, includes but not limited to following approach:
(1) acetic acid route of synthesis is strengthened, comprise pyruvic carboxylase (PDC1 gene), acetaldehyde dehydrogenase (ALD1 gene, ALD2 gene, ALD3 gene, ALD6 gene), ethanol dehydrogenase (ADH2 gene), the raising of enzyme activity involved by the acetic acid biosynthesizing such as acetyl coenzyme A hydrolase (ACH1 gene).Concrete grammar comprises the rise of mutagenesis, directed enzyme evolution or gene expression dose.
(2) weaken acetic acid and decompose approach, the weakening of acetyl-CoA-synthetase (ACS1 gene, ACS2 gene) vigor.Concrete grammar comprises the downward of mutagenesis, directed enzyme evolution or gene expression dose.
(3) external source adds acetate pathway, namely in fermented liquid, adds appropriate acetic acid;
The addition of described acetic acid is 2-20mg/L fermented liquid;
When the interpolation time of described acetic acid is for fermentation 24h.
The present invention also provides a strain gene engineering bacterium, and described genetic engineering bacterium, by the expressing gene ALD6 gene of process LAN acetaldehyde dehydrogenase in yeast saccharomyces cerevisiae, reaches and reduces wine product Higher Alcohols, ethyl acetate and acetaldehyde and the object increasing acetic acid growing amount;
The nucleotide sequence of described ALD6 gene is as shown in SEQIDNo.1;
Preferably, described yeast saccharomyces cerevisiae is specially yeast saccharomyces cerevisiae (Saccharomycescerevisiae) CICC32315.
Beneficial effect:
Technology contents of the present invention is that regulation and control alcohol product flavor substances provides new approach, being raised by the appropriateness of acetic acid level in born of the same parents can from the physiological metabolism of global regulation's yeast saccharomyces cerevisiae, under the prerequisite not affecting the basic leavening property of yeast saccharomyces cerevisiae, reduce the generation of the micro-flavour substancess such as higher alcohols, ester and acetaldehyde on the whole, improve the purity of wine product.Meanwhile, acetic acid growing amount can fit increase, can improve wine product local flavor richness and Harmony, contributes to improving vinosity, specific as follows:
1, the bacterial strain of process LAN acetaldehyde dehydrogenase ALD6 gene provided by the present invention, compared with parent strain, after corn thick mash liquor fermentation terminates, acetic acid content is 5.8 times of former bacterium, the content of acetaldehyde reduces 49.97%, and the content of n-propyl alcohol reduces 49.58%, and the content of isopropylcarbinol reduces 22.8%, the content of primary isoamyl alcohol reduces 43.1%, and the content of ethyl acetate reduces 54.95%.
2, the bacterial strain of process LAN acetaldehyde dehydrogenase ALD6 gene provided by the present invention, during simulation sorghum material solid spirit fermenting experiment, the content of acetic acid improves 1.40 times, the content of acetaldehyde reduces 81.44%, isopropylcarbinol content reduces 34.66%, primary isoamyl alcohol reduces 49.52%, and phenylethyl alcohol reduces 64.89%, and ethyl acetate content reduces 27.20%.
3, the bacterial strain of process LAN acetaldehyde dehydrogenase ALD6 gene provided by the present invention, after high concentration beer fermentation ends, the content of acetic acid is 1.9 times of former bacterium, the content of acetaldehyde reduces 9.33%, the content of n-propyl alcohol reduces 60.8%, the content of isopropylcarbinol reduces 21.7%, and the content of primary isoamyl alcohol reduces 25.9%, and ethyl acetate reduces 37.3%.
The mode of 4, being added by external source acetic acid makes the effect of appropriate accumulation acetic acid in born of the same parents: under corn thick mash fermentation culture condition, the acetic acid of 6.2mg/L is added after the 24h of fermentation, the content recording the n-propyl alcohol in wine sample after fermentation ends reduces 18.61%, the content of isopropylcarbinol does not have too big difference compared with the control, the content of primary isoamyl alcohol reduces 41.71%, ethyl acetate reduces 12.36%, and the content of acetic acid improves 90.70%.
Accompanying drawing illustrates:
Fig. 1 is the metabolic map of acetic acid in yeast saccharomyces cerevisiae;
The structure schematic flow sheet of Fig. 2 recombinant plasmid Yep-PAK;
The PCR of Fig. 3 construction recombination plasmid Yep-PAK verifies electrophorogram
Wherein, swimming lane M is 5000bpDNALadderMarker; Swimming lane 1 is the 1503bpALD6 arrived for masterplate pcr amplification with yeast saccharomyces cerevisiae α 5; Swimming lane 2 is take Yep-PAK as the 1503bpALD6 that masterplate increases; Swimming lane 3 is take Yep-PAK as the 3203bpPGK1 that masterplate increases p-ALD6; Swimming lane 4 is take Yeast genome as the KANMX fragment of the 1613bp that masterplate increases; Swimming lane 5 is take Yep-PAK as the KANMX fragment of the 1613bp that masterplate increases; Swimming lane 6 is take Yep-PAK as the A-PGK1 of the 5020bp that masterplate increases p-ALD6-PGK1 t-KANMX-B fragment.
The homologous recombination schematic diagram of Fig. 4 recombinant fragment and Yeast genome;
The PCR of Fig. 5 recombinant Saccharomyces cerevisiae bacterial strain verifies gel electrophoresis figure
Wherein, the negative control of the upstream fixed point checking of swimming lane 1 to be original strain α 5 be masterplate PCR; The upstream fixed point checking fragment that swimming lane 2 is is the 581bp of masterplate PCR with recombinant conversion; The negative control of the downstream fixed point checking of swimming lane 3 to be original strain α 5 be masterplate PCR; The downstream fixed point checking fragment that swimming lane 4 is is the 1077bp of masterplate PCR with recombinant conversion.
Embodiment:
The present invention is described below by specific embodiment.Unless stated otherwise, technique means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Yeast saccharomyces cerevisiae monoploid thalline used in the present invention is the yeast saccharomyces cerevisiae haploid strains that can adopt any source.
Embodiment 1: the structure of the bacterial strain α 5-A6 of acetic acid content in appropriate accumulation born of the same parents
(1) structure of engineering strain
1) structure of Yep-PAK plasmid
Plasmid construction recombinant plasmid Yep-PAK based on Yep-PGK1, build flow process as shown in Figure 2, with yeast saccharomyces cerevisiae (Saccharomycescerevisiae) CICC32315 monoploid α 5 genome for template, ALD6-U, ALD6-D are primer, pcr amplification obtains the acetaldehyde dehydrogenase gene ALD6 of 1503bp, is inserted into promotor PGK1p on Yep-PGK1 plasmid and terminator PGK1 by XhoI single endonuclease digestion tbetween, obtain plasmid Yep-PA; Take pUG6 as template, KAN-U, KAN-D are primer, and pcr amplification obtains the KanMX gene of 1613bp, and SphI respectively enzyme cuts KanMX gene and plasmid Yep-PA, connects with SolutionI ligase enzyme, form recombinant plasmid Yep-PAK; The sequence of whole process the primer is as table 1.
Table 1PCR primer
Fig. 3 is the checking electrophorogram of recombinant plasmid Yep-PAK: wherein swimming lane M is 5000bpDNALadderMarker; Swimming lane 1 is the 1503bpALD6 arrived for masterplate pcr amplification with yeast saccharomyces cerevisiae α 5; Swimming lane 2 is take Yep-PAK as the 1503bpALD6 that masterplate increases; Swimming lane 3 is take Yep-PAK as the 3203bpPGK1 that masterplate increases p-ALD6; Swimming lane 4 is take Yeast genome as the KANMX fragment of the 1613bp that masterplate increases; Swimming lane 5 is take Yep-PAK as the KANMX fragment of the 1613bp that masterplate increases; Swimming lane 6 is take Yep-PAK as the A-PGK1 of the 5020bp that masterplate increases p-ALD6-PGK1 t-KANMX-B fragment.
2) structure of recombinant Saccharomyces cerevisiae bacterial strain
With Oxalacetic transacetase CIT2 gene up and down between homologous region A, B add to respectively PGK1 upstream primer ALD6-U and KANMX downstream primer KAN-D 5 ＇ end, form new long primer P-U and K-D; With recombinant plasmid Yep-PAK for template, long primer pcr amplification obtains recombinant fragment A-PGK1 p-ALD6-PGK1 t-KANMX-B, is transformed in yeast saccharomyces cerevisiae CICC32315 monoploid α 5 by lithium acetate transformation method, obtains the genes of brewing yeast engineering haploid strains α 5-A6 after homologous recombination.
The checking of recombinant Saccharomyces cerevisiae bacterial strain:
According to the gene order at yeast saccharomyces cerevisiae recombination site two ends and the homologous recombination sequence of insertion, design two groups of fixed point checking upstream and downstream primers respectively, respectively to grow good transformant genome for template, carry out pcr amplification, checking recon.
Carry out upstream and downstream fixed point PCR checking with primer A-U/A-D and B-U/B-D respectively, wherein the PCR primer of upstream primer A-U/A-D is through the agarose gel electrophoresis of 0.8%, can see that a size is about the specific band of about 581bp, and its size and expection are quite; The PCR primer of downstream primer B-U/B-D, through the agarose gel electrophoresis of 0.8%, can see that a size is about the specific band of about 1077bp, and its size and expection quite, illustrate restructuring box A-PGK1 p-ALD6-PGK1 t-KANMX-B fragment is successfully recombinated in genes of brewing yeast group, and recombinable site is also correct.Electrophoresis result as shown in Figure 5.
In Fig. 5, M is 5000bpDNALadderMarker, the negative control of the upstream fixed point checking of swimming lane 1 to be original strain α 5 be masterplate PCR; The upstream fixed point checking fragment that swimming lane 2 is is the 581bp of masterplate PCR with recombinant conversion; The negative control of the downstream fixed point checking of swimming lane 3 to be original strain α 5 be masterplate PCR; The downstream fixed point checking fragment that swimming lane 4 is is the 1077bp of masterplate PCR with recombinant conversion.
Embodiment 2: α 5-A6 simulates the liquid liquor fermentation experiment of maize raw material
1) zymotechnique route:
Semen Maydis powder → immersion → liquefaction → saccharification → cool → connect bacterium → fermentation → steaming wine → testing index
2) processing condition: soaking conditions: 60670 DEG C, dipping 20min; Liquefaction condition: 85690 DEG C, add Thermostable α-Amylase, liquefaction 90min; Saccharification condition: 55660 DEG C, adds saccharifying enzyme and nutritive salt, saccharification 20min; Acidulated condition: 45 DEG C, add aspartic protease, 20min; Fermentation condition: 30 DEG C, 4 days.Get 100mL mash when steaming wine, add 100mL water, steam 100mL wine sample.
3) prepare burden: Semen Maydis powder: 60g; Add water 130mL; Thermostable α-Amylase: 30 μ L; Saccharifying enzyme: 90 μ L; Aspartic protease: 1.2mL; Nutritive salt: 1mL; Inoculum size: 7.5%;
By above-mentioned simulation process, the experiment of corn thick mash liquor fermentation is carried out respectively to saccharomyces cerevisiae engineered yeast α 5-A6 and starting strain α 5; Vibrate every 12h between yeast phase and weigh, record is weightless; After fermentation ends, stop cultivating and weighing; Measure the remaining sugar concentration of fermented liquid, alcohol by volume mark and main aromatic components content.Characterize its over-all properties with fermentation capacity, remaining sugar concentration and product formation, the results are shown in Table 2,3.
The leavening property of the liquid liquor fermentation of table 2 maize raw material
Table 2 shows, the saccharomyces cerevisiae engineered yeast that the present invention obtains compared with initial former bacterium, during the liquid liquor fermentation experiment of simulation maize raw material, alcoholic strength and CO 2weightlessness does not almost have difference compared with the control, and residual sugar content slightly raises.
The flavour substances of the liquid liquor fermentation of table 3 maize raw material
Table 3 shows, saccharomyces cerevisiae engineered yeast (Saccharomycescerevisiae) the α 5-A6 of the yield of higher alcohol that the present invention obtains and ethyl acetate is compared with initial S. cervisiae α 5: after corn thick mash liquor fermentation terminates, transformant bacterial strain is compared with parent strain, the content of acetic acid is 5.8 times of former bacterium, the content of acetaldehyde reduces 49.97% than control strain, the content of n-propyl alcohol reduces 49.58% than control strain, the content of isopropylcarbinol reduces 22.8% than control strain, the content of primary isoamyl alcohol reduces 43.1% than control strain, the content of ethyl acetate reduces 54.95% than contrast.
Embodiment 3: α 5-A6 simulates sorghum material solid spirit fermenting experiment
1) zymotechnique route:
Chinese sorghum → material moistening → add rice husk → boiling → airing → connect bacterium → fermentation → distillation
2) processing condition: soaking conditions: 80 DEG C of mixings, fully water suction is without hard-core; Conditions of cooking: add rice husk normal pressure and steam about 60min, uniform particles, heart are without in vain.Fermentation condition: 30 DEG C, 5 days.Steam wine condition: 100g raw material, adds 200mL water, steams 100mL wine sample.
3) prepare burden: Chinese sorghum 100g; Rice husk 20g; Inoculum size: 0.6 hundred million/g raw material;
By above-mentioned simulation process, sorghum material solid spirit fermenting experiment is carried out respectively to yeast saccharomyces cerevisiae recombinant bacterial strain α 5-A6 and starting strain α 5; Vibrate every 12h between yeast phase and weigh, record is weightless; After fermentation ends, stop cultivating and weighing; Measure the remaining sugar concentration of fermented liquid, alcohol by volume mark and main aromatic components content.With CO 2weightlessness, residual sugar amount and alcoholic strength characterize its over-all properties, the results are shown in Table 4,5.
The leavening property of the solid-state " Daqu " white spirit fermentation of table 4 and fragrance matter
Table 4 shows: the yeast saccharomyces cerevisiae recombinant bacterium that the present invention obtains is compared with initial former bacterium, and during simulation sorghum material solid spirit fermenting experiment, alcoholic strength, weightlessness and residual sugar content are all less than too large change.
The flavour substances of the liquid liquor fermentation of table 5 maize raw material
Table 5 shows: the saccharomyces cerevisiae engineered yeast that the present invention obtains is compared with initial former bacterium, during simulation sorghum material solid spirit fermenting experiment, isopropylcarbinol content reduces 34.66% than contrast, primary isoamyl alcohol reduces 49.52%, phenylethyl alcohol reduces 64.89%, ethyl acetate content reduces 27.20%, and the content of acetic acid improves 1.40 times, and the content of acetaldehyde reduces 81.44%.
Embodiment 4: α 5-A6 beer fermentation is tested
(1) preparation of wort
Adopt whole-malt technique, by the Fructus Hordei Germinatus of pulverizing according to the material-water ratio of 1:4 (w:w) and the hot water mixing 40min of 52 DEG C, then under 65 DEG C of conditions, saccharification is about 1h, is warming up to 72 DEG C, keeps 15min, then is warming up to 78 DEG C, keeps 10min.Saccharified liquid filtered through gauze, then boils about 1h by filtrate, the hops of period interpolation 0.4 ‰, and boil after terminating and filter, controlling wort concentration is 18 ° of Brix.
(2) seed culture
1. actication of culture: preserve strain transfer to YEPD slant tube 28 DEG C of activation culture 2d.
2. first order seed is cultivated: get slant strains one ring, be inoculated in the test tube of 18 ° of Brix wheat juice substratum that 5mL is housed, 28 DEG C, cultivate 24h under 180rpm condition.
3. secondary seed is cultivated: primary seed solution fills in the 150mL triangular flask of 18 ° of Brix wheat juice of 50mL by the inoculum size access of 10%, 16 DEG C of quiescent culture 72h.
(3) high concentration beer fermentation
Secondary seed solution obtains yeast slurry through centrifugal, and yeast slurry fills in the 500mL triangular flask of 18 ° of Brix wheat juice of 300mL by the inoculum size access of 0.5%, 10 DEG C of standing for fermentation.Vibrate every 12h between yeast phase and weigh, record is weightless; After fermentation ends, stop cultivating and weighing; Measure the remaining sugar concentration of fermented liquid, alcohol by volume mark and main aromatic components content.Characterize its over-all properties with fermentation capacity, remaining sugar concentration and product formation, the results are shown in Table 6,7.
The leavening property of table 6 high concentration beer fermentation
As shown in Table 6, the saccharomyces cerevisiae engineered yeast α 5-A6 that the present invention obtains is compared with initial former bacterium α 5, and during high concentration beer fermenting experiment, alcoholic strength is in a slight decrease, and residual sugar content has increased slightly.
The flavour substances of table 7 high concentration beer fermentation
Table 7 is known, saccharomyces cerevisiae engineered yeast (Saccharomycescerevisiae) the α 5-A6 of the yield of higher alcohol that the present invention obtains and ethyl acetate is compared with initial S. cervisiae α 5: after high concentration beer fermentation ends, transformant bacterial strain is compared with parent strain, the content of acetic acid is 1.9 times of former bacterium, the content of acetaldehyde reduces 9.33% than contrast, the content of n-propyl alcohol reduces 60.8% than control strain, the content of isopropylcarbinol reduces 21.7% than control strain, the content of primary isoamyl alcohol reduces 25.9% than control strain, ethyl acetate reduces 37.3%.
In embodiment 5: α 5 thick mash fermentation process, external source adds acetic acid experiment
1) zymotechnique route:
Semen Maydis powder → immersion → liquefaction → saccharification → cool → connect bacterium → fermentation → steaming wine → testing index
2) processing condition: soaking conditions: 60670 DEG C, dipping 20min; Liquefaction condition: 85690 DEG C, add Thermostable α-Amylase, liquefaction 90min; Saccharification condition: 55660 DEG C, adds saccharifying enzyme and nutritive salt, saccharification 20min; Acidulated condition: 45 DEG C, add aspartic protease, 20min; Fermentation condition: 30 DEG C, 4 days.Get 100mL mash when steaming wine, add 100mL water, steam 100mL wine sample.
3) prepare burden: Semen Maydis powder: 60g; Add water 210mL; Thermostable α-Amylase: 30 μ L; Saccharifying enzyme: 90 μ L; Aspartic protease: 1.2mL; Nutritive salt: 1mL; Inoculum size: 7.5%;
Carry out the experiment of corn thick mash liquor fermentation by above-mentioned simulation process starting strain α 5, in fermented liquid, add the acetic acid of 6.2mg/L when the 24h fermented; Vibrate every 12h between yeast phase and weigh, record is weightless; After fermentation ends, stop cultivating and weighing; Measure the remaining sugar concentration of fermented liquid, alcohol by volume mark and main aromatic components content.Characterize its over-all properties with fermentation capacity, remaining sugar concentration and product formation, the results are shown in Table 8,9.
Table 8 external source adds the leavening property of acetic acid
As shown in Table 8, add the acetic acid of appropriate content in fermented liquid after, CO 2weightlessness, alcoholic strength and residual sugar content almost do not have difference.
Table 9 external source adds the flavour substances that acetic acid fermentation produces
As shown in Table 9, add acetic acid in fermented liquid after, the content of n-propyl alcohol reduces 18.61%, the content of isopropylcarbinol does not have too big difference compared with the control, the content of primary isoamyl alcohol reduces 41.71%, and ethyl acetate reduces 12.36%, and the content of acetic acid improves 90.70%.
In embodiment 6: α 5 thick mash fermentation process, external source adds the experiment of 2mg acetic acid
1) zymotechnique route:
Semen Maydis powder → immersion → liquefaction → saccharification → cool → connect bacterium → fermentation → steaming wine → testing index
2) processing condition: soaking conditions: 60670 DEG C, dipping 20min; Liquefaction condition: 85690 DEG C, add Thermostable α-Amylase, liquefaction 90min; Saccharification condition: 55660 DEG C, adds saccharifying enzyme and nutritive salt, saccharification 20min; Acidulated condition: 45 DEG C, add aspartic protease, 20min; Fermentation condition: 30 DEG C, 4 days.Get 100mL mash when steaming wine, add 100mL water, steam 100mL wine sample.
3) prepare burden: Semen Maydis powder: 60g; Add water 210mL; Thermostable α-Amylase: 30 μ L; Saccharifying enzyme: 90 μ L; Aspartic protease: 1.2mL; Nutritive salt: 1mL; Inoculum size: 7.5%;
Carry out the experiment of corn thick mash liquor fermentation by above-mentioned simulation process starting strain α 5, in fermented liquid, add the acetic acid of 2mg/L when the 24h fermented; Vibrate every 12h between yeast phase and weigh, record is weightless; After fermentation ends, stop cultivating and weighing; Measure the remaining sugar concentration of fermented liquid, alcohol by volume mark and main aromatic components content.Characterize its over-all properties with fermentation capacity, remaining sugar concentration and product formation, the results are shown in Table 10,11.
Table 10 external source adds the leavening property of acetic acid
As shown in Table 10, add the acetic acid of appropriate content in fermented liquid after, CO 2weightlessness, alcoholic strength and residual sugar content almost do not have difference.
Table 11 external source adds the flavour substances that acetic acid fermentation produces
As shown in Table 11, add acetic acid in fermented liquid after, the content of n-propyl alcohol reduces 6.65%, the content of isopropylcarbinol does not have too big difference compared with the control, the content of primary isoamyl alcohol reduces 13.23%, and ethyl acetate is not too large difference compared with the control, and the content of acetic acid improves 18.68%.
In embodiment 7: α 5 thick mash fermentation process, external source adds the experiment of 20mg acetic acid
1) zymotechnique route:
Semen Maydis powder → immersion → liquefaction → saccharification → cool → connect bacterium → fermentation → steaming wine → testing index
2) processing condition: soaking conditions: 60670 DEG C, dipping 20min; Liquefaction condition: 85690 DEG C, add Thermostable α-Amylase, liquefaction 90min; Saccharification condition: 55660 DEG C, adds saccharifying enzyme and nutritive salt, saccharification 20min; Acidulated condition: 45 DEG C, add aspartic protease, 20min; Fermentation condition: 30 DEG C, 4 days.Get 100mL mash when steaming wine, add 100mL water, steam 100mL wine sample.
3) prepare burden: Semen Maydis powder: 60g; Add water 210mL; Thermostable α-Amylase: 30 μ L; Saccharifying enzyme: 90 μ L; Aspartic protease: 1.2mL; Nutritive salt: 1mL; Inoculum size: 7.5%;
Carry out the experiment of corn thick mash liquor fermentation by above-mentioned simulation process starting strain α 5, in fermented liquid, add the acetic acid of 20mg/L when the 24h fermented; Vibrate every 12h between yeast phase and weigh, record is weightless; After fermentation ends, stop cultivating and weighing; Measure the remaining sugar concentration of fermented liquid, alcohol by volume mark and main aromatic components content.Characterize its over-all properties with fermentation capacity, remaining sugar concentration and product formation, the results are shown in Table 12,13.
Table 12 external source adds the leavening property of acetic acid
As shown in Table 12, add the acetic acid of appropriate content in fermented liquid after, CO 2weightlessness, alcoholic strength and residual sugar content almost do not have difference.
Table 13 external source adds the flavour substances that acetic acid fermentation produces
As shown in Table 13, add acetic acid in fermented liquid after, the content of n-propyl alcohol reduces 48.88%, and the content of isopropylcarbinol reduces 66.46%, and the content of primary isoamyl alcohol reduces 66.20%, and ethyl acetate reduces 57.12%, and the content of acetic acid improves 3.10 times.

Claims (9)

1. regulate and control a method for yeast saccharomyces cerevisiae flavour substances synthesis, it is characterized in that, by making Cellular Accumulation acetic acid, thus the growing amount of the low yeast saccharomyces cerevisiae higher alcohols of a step-down, acetaldehyde and ethyl acetate.
2. as claimed in claim 1 a kind of regulate and control yeast saccharomyces cerevisiae flavour substances synthesis method, it is characterized in that, the described method of Cellular Accumulation acetic acid that makes, for strengthening acetic acid route of synthesis, comprises the raising of pyruvic carboxylase, acetaldehyde dehydrogenase, ethanol dehydrogenase, acetyl coenzyme A hydrolase vigor.
3. as claimed in claim 1 a kind of regulate and control the method for yeast saccharomyces cerevisiae flavour substances synthesis, it is characterized in that, described in make the method for Cellular Accumulation acetic acid decompose approach for weakening acetic acid, be specially the weakening of acetyl-CoA-synthetase vigor.
4. as claimed in claim 1 a kind of regulate and control yeast saccharomyces cerevisiae flavour substances synthesis method, it is characterized in that, described in make the method for Cellular Accumulation acetic acid be in fermenting process external source add acetic acid.
5. as claimed in claim 2 a kind of regulate and control the method for yeast saccharomyces cerevisiae flavour substances synthesis, it is characterized in that, the concrete grammar that described enzyme activity improves comprises: the rise of mutagenesis, directed enzyme evolution or gene expression dose.
6. as claimed in claim 3 a kind of regulate and control the method for yeast saccharomyces cerevisiae flavour substances synthesis, it is characterized in that, the concrete grammar that described enzyme activity weakens comprises the downward of mutagenesis, directed enzyme evolution or gene expression dose.
7. as claimed in claim 2 a kind of regulate and control yeast saccharomyces cerevisiae flavour substances synthesis method, it is characterized in that, the concrete grammar that acetaldehyde-dehydrogenase enzyme activity improves is structure one strain gene engineering bacterium, described genetic engineering bacterium, by the expressing gene ALD6 gene of process LAN acetaldehyde dehydrogenase in yeast saccharomyces cerevisiae, reaches and reduces wine product Higher Alcohols, ethyl acetate and acetaldehyde and the object increasing acetic acid growing amount; Described yeast saccharomyces cerevisiae is specially yeast saccharomyces cerevisiae (Saccharomycescerevisiae) CICC32315; The nucleotide sequence of described ALD6 gene is as shown in SEQIDNo.1.
8. as claimed in claim 7 a kind of regulate and control yeast saccharomyces cerevisiae flavour substances synthesis method, it is characterized in that, the application of described genetic engineering bacterium in wine brewing process.
9. as claimed in claim 4 a kind of regulate and control yeast saccharomyces cerevisiae flavour substances synthesis method, it is characterized in that, the addition of described acetic acid is 2-20mg/L fermented liquid, add the time for fermentation 24h time.
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CN113416664A (en) * 2021-02-02 2021-09-21 天津科技大学 Saccharomyces cerevisiae genetic engineering strain, construction method thereof and application thereof in high-yield ethyl acetate

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CN110804561A (en) * 2019-11-01 2020-02-18 天津科技大学 Saccharomyces cerevisiae with high yield of C6-C10 ethyl ester and construction method and application thereof
CN110804561B (en) * 2019-11-01 2021-04-09 天津科技大学 Saccharomyces cerevisiae with high yield of C6-C10 ethyl ester and construction method and application thereof
CN113416664A (en) * 2021-02-02 2021-09-21 天津科技大学 Saccharomyces cerevisiae genetic engineering strain, construction method thereof and application thereof in high-yield ethyl acetate
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CN113278537A (en) * 2021-06-15 2021-08-20 天津科技大学 Series of low-yield higher alcohol saccharomyces cerevisiae and construction method and application thereof

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