CN105505807B - Regulate and control the synthetic method of saccharomyces cerevisiae flavor substance by acetate metabolism - Google Patents
Regulate and control the synthetic method of saccharomyces cerevisiae flavor substance by acetate metabolism Download PDFInfo
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Abstract
The invention belongs to technical field of bioengineering, and in particular to regulate and control the synthetic method of saccharomyces cerevisiae flavor substance by acetate metabolism.So that cell is accumulated acetic acid in right amount by strengthening acetic acid route of synthesis, weakening acetic acid and decompose the method that approach or external source add acetic acid, can a step reduce the production quantity of saccharomyces cerevisiae higher alcohol, acetaldehyde and ethyl acetate, the appropriate production quantity for improving acetic acid.This method provides new approach for flavor substance in regulation alcohol product, under the premise of not influencing saccharomyces cerevisiae basic fermenting property, reduces the generation of the micro flavor substance such as all higher alcohols, ester and acetaldehyde on the whole, improves the degree of purity of wine product.
Description
Technical field:
The invention belongs to technical field of bioengineering, are related to industrial micro breeding and drinks brewing, and in particular to pass through
Acetate metabolism regulates and controls the synthesis of the flavor substances such as saccharomyces cerevisiae higher alcohol, acetic acid, ester, aldehyde.
Background technique:
The flavor substances such as alcohol, acid, ester, aldehyde decide the quality of wine, all indispensable for all wine product, but it is specific
Content in wine product must be suitable for, excessive or very few can all have a negative impact to the quality of wine.
Higher alcohol is the important alcohols aroma compound except ethyl alcohol, mainly (about including normal propyl alcohol, isobutanol, isoamyl alcohol
Account for 50% or so of higher alcohol total amount), active-amyl alcohol and bata-phenethyl alcohol etc., every kind of higher alcohol has oneself unique flavor special
Sign, different alcohol ester ratios impart every kind of distinguished fragrance of wine kind.However, if advanced alcohol content is more than certain standard, no
So that wine body is generated different miscellaneous taste, influence the quality of wine, and consumer can be made to generate symptoms such as " dizzy, headaches ", be commonly called as " on
Head ", is detrimental to health.Currently, advanced alcohol content is higher in all drinks, it is therefore desirable to reduce containing for higher alcohol
Amount.
The esters such as ethyl acetate are most important for the fragrance of various wine, for China white wine, the type of ester
It is the main foundation of different flavor white wine classification with content.But ester content is not that the higher the better, and ester content is excessively high in wine body, meeting
So that fragrance is excessively prominent, it is uncoordinated.High concentration beer fermentation process can generate the esters such as excessive ethyl acetate, influence beer
Flavor.
Organic acid is important taste compound, and acid is also the important substance for forming " the rear taste " of wine.The very little wine of acid content,
Vinosity is boring, and rear taste is short, and bitterness has different miscellaneous taste；The excessive wine of acid content, then vinosity is coarse.Suitable acid can play slow in wine
Punching and seasoning effect, can eliminate top and the uncoordinated phenomenon of mouthfeel after drink.Acetic acid is main seasoning acid, acetic acid during wine brewing
It is mainly generated by the miscellaneous bacteria except saccharomyces cerevisiae, saccharomyces cerevisiae itself produces acetic acid ability wretched insufficiency, for passing through wine brewing
The single pure-blood ferment of yeast, the wine brewing process for living contaminants of keeping under strict control need to improve the production acetic acid ability of saccharomyces cerevisiae in right amount.
Acetaldehyde is the volatile flavor compounds in wine, and the acetaldehyde of low concentration has pleasant fruit aroma.But acetaldehyde concentration
When higher, the unhappy smell such as similar herbaceous taste can be generated.Acetaldehyde is the principal element of easy top of drinking, and is a kind of latent
Carcinogen, the wine of high acetaldehyde is pernicious, acetaldehyde beneficiating ingredient absolutely not in wine to alcohol user's body.Second
Aldehyde is usually the generation of Ethanol in Saccharomyces cerevisiae fermentation process, and the miscellaneous bacterias such as some lactic acid bacterias and acetic acid bacteria can also generate acetaldehyde, right
In the purebred saccharomyces cerevisiae drinks brewing process for avoiding living contaminants, the acetaldehyde generative capacity of saccharomyces cerevisiae itself is reduced, is helped
In the drinking safety for improving wine product.
The control of the harmful substances such as wine body Higher Alcohols, acetaldehyde and ester and acid, be mostly by from fermentation work
Set about in skill, for example, application No. is 2012101252759 a kind of low acetaldehyde beers and preparation method thereof, by adjusting beer
Wine fermentation temperature extends fermentation time, and controls other fermentation parameters, so that the acetaldehyde of the semi-finished product beer after fermentation is low
In≤8mg/L.For this predetermined substance of higher alcohol, it can start with from the higher alcohol metabolic pathway of saccharomyces cerevisiae, pass through gene
The engineered reduction for realizing advanced alcohol content.Application No. is 201010227788.1 low-yield higher-alcohol saccharomyces cerevisiae engineering bacteriums
And its construction method, transformant bacterial strain is realized than parent strain by the missing of amino acid transaminase encoding gene (BAT2)
Isobutanol, isoamyl alcohol content reduce 55.19%, 34.43% respectively, and total advanced alcohol content reduces 35.01%.It is overall next
It sees, lacks the method to the disposable global regulation of the flavor substances such as alcohol, aldehyde, acid, ester.
From the angle analysis of metabolism, acetaldehyde generates acetic acid via acetaldehyde dehydrogenase, when the enhancing of acetic acid route of synthesis, second intracellular
When acid content improves, the acetaldehyde as substrate will decrease；Precursor substance of the acetic acid as synthesizing ethyl acetate, works as born of the same parents
When interior acetic acid content improves, the synthesis of ethyl acetate can be enhanced.And the present invention is in experimentation, by improving acetic acid intracellular in right amount
After content, other than reducing acetaldehyde, also it is found surprisingly that the flavor substances content such as wine Higher Alcohols and ester decreases.
Also, not yet occur reaching disposable to flavor substances such as alcohol, aldehyde, acid, esters by adjusting acetic acid in the prior art
The method of global regulation.Therefore, the present invention adds the side of acetic acid by strengthening the yeast level intracellular for generating acetic acid or external source
Method regulates and controls the generation of the flavor substance of alcohol, aldehyde, acid, ester etc. from the overall situation.
Summary of the invention:
To achieve the goals above, the present invention is based on the acetate metabolisms of saccharomyces cerevisiae, provide one kind by making Cellular Accumulation
Acetic acid, to reach the method for carrying out disposable global regulation to flavor substances such as alcohol, aldehyde, acid, esters.It, can one by this method
Step reduces the production quantity of saccharomyces cerevisiae higher alcohol, acetaldehyde and ethyl acetate, the appropriate production quantity for improving acetic acid.
The method for making Cellular Accumulation acetic acid, including but not limited to following approach:
(1) reinforcing acetic acid route of synthesis, including pyruvate decarboxylase (PDC1 gene), acetaldehyde dehydrogenase (ALD1 gene,
ALD2 gene, ALD3 gene, ALD6 gene), alcohol dehydrogenase (ADH2 gene), acetyl coenzyme A hydrolase (ACH1 gene) etc.
The raising of enzyme activity involved by acetic acid biosynthesis.Specific method includes the upper of mutagenesis, directed enzyme evolution or gene expression dose
It adjusts.
(2) weaken acetic acid and decompose approach, the weakening of acetyl-CoA-synthetase (ACS1 gene, ACS2 gene) vigor.Specifically
Method includes the downward of mutagenesis, directed enzyme evolution or gene expression dose.
(3) external source adds acetate pathway, i.e., suitable acetic acid is added in fermentation liquid；
The additive amount of the acetic acid is 2-20mg/L fermentation liquid；
The addition time of the acetic acid is when fermenting for 24 hours.
The present invention also provides a strain gene engineering bacterium, the genetic engineering bacterium is de- by being overexpressed acetaldehyde in saccharomyces cerevisiae
The expressing gene ALD6 gene of hydrogen enzyme, reaching reduces wine product Higher Alcohols, ethyl acetate and acetaldehyde and increases acetic acid generation
The purpose of amount；
The nucleotide sequence of the ALD6 gene is as shown in SEQ ID No.1；
Preferably, the saccharomyces cerevisiae is specially saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC32315.
The utility model has the advantages that
Technology contents of the present invention provide new approach for flavor substance in regulation alcohol product, pass through acetic acid intracellular
Horizontal appropriateness increases and can not influence the basic fermenting property of saccharomyces cerevisiae from the physiological metabolism of global regulation's saccharomyces cerevisiae
Under the premise of, the generation of the micro flavor substances such as higher alcohol, ester and acetaldehyde is reduced on the whole, improves the degree of purity of wine product.Together
When, acetic acid production quantity can fit increase, and wine product flavor richness and harmony can be improved, help to improve vinosity, specific as follows:
1, the bacterial strain provided by the present invention for being overexpressed acetaldehyde dehydrogenase ALD6 gene, compared with parent strain, corn is dense
After wine with dregs liquor fermentation, acetic acid content is 5.8 times of opportunistic pathogen, and the content of acetaldehyde reduces 49.97%, the content drop of normal propyl alcohol
Low 49.58%, the content of isobutanol reduces 22.8%, and the content of isoamyl alcohol reduces 43.1%, the content of ethyl acetate
Reduce 54.95%.
2, the bacterial strain provided by the present invention for being overexpressed acetaldehyde dehydrogenase ALD6 gene, simulation sorghum material solid spirit hair
When ferment is tested, the content of acetic acid improves 1.40 times, and the content of acetaldehyde reduces 81.44%, and isobutyl alcohol content reduces
34.66%, isoamyl alcohol reduces 49.52%, and benzyl carbinol reduces 64.89%, and ethyl acetate content reduces 27.20%.
3, it is provided by the present invention be overexpressed acetaldehyde dehydrogenase ALD6 gene bacterial strain, high concentration beer after fermentation, second
The content of acid is 1.9 times of opportunistic pathogen, and the content of acetaldehyde reduces 9.33%, and the content of normal propyl alcohol reduces 60.8%, isobutanol
Content reduce 21.7%, the content of isoamyl alcohol reduces 25.9%, and ethyl acetate reduces 37.3%.
4, make the effect of appropriate accumulation acetic acid intracellular in such a way that external source acetic acid adds: in corn thick mash fermentation culture item
Under part, in the acetic acid for adding 6.2mg/L afterwards for 24 hours of fermentation, the content for measuring the normal propyl alcohol in wine sample after fermentation is reduced
18.61%, the content of isobutanol is compared with the control without too big difference, and the content of isoamyl alcohol reduces 41.71%, ethyl acetate
12.36% is reduced, the content of acetic acid improves 90.70%.
Detailed description of the invention:
Fig. 1 is the metabolic map of acetic acid in saccharomyces cerevisiae；
The building flow diagram of Fig. 2 recombinant plasmid Yep-PAK；
The PCR of Fig. 3 construction recombination plasmid Yep-PAK verifies electrophoretogram
Wherein, swimming lane M is 5000bp DNA Ladder Marker；It is template PCR amplification that swimming lane 1, which is with saccharomyces cerevisiae α 5,
The 1503bp ALD6 arrived；Swimming lane 2 is the 1503bp ALD6 expanded using Yep-PAK as template；Swimming lane 3 is to be with Yep-PAK
The 3203bp PGK1 that template expandsP-ALD6；Swimming lane 4 is the KANMX of the 1613bp expanded using Yeast genome as template
Segment；Swimming lane 5 is the KANMX segment of the 1613bp expanded using Yep-PAK as template；Swimming lane 6 is to expand by template of Yep-PAK
The A-PGK1 of the 5020bp increasedP-ALD6-PGK1T- KANMX-B segment.
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, swimming lane 1 is the negative control for the upstream fixed point verifying that original strain α 5 is template PCR；Swimming lane 2 is with recombination
The upstream fixed point verifying segment that transformant is the 581bp of template PCR；Swimming lane 3 is the downstream fixed point that original strain α 5 is template PCR
The negative control of verifying；Swimming lane 4 is the downstream fixed point verifying segment using recombinant conversion as the 1077bp of template PCR.
Specific embodiment:
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
Saccharomyces cerevisiae monoploid thallus used in the present invention is the saccharomyces cerevisiae monoploid bacterium that can use any source
Strain.
Embodiment 1: the building of the bacterial strain α 5-A6 of acetic acid content intracellular is accumulated in right amount
(1) building of engineering strain
1) building of Yep-PAK plasmid
It is basic plasmid construction recombinant plasmid Yep-PAK with Yep-PGK1, constructs process as shown in Fig. 2, with saccharomyces cerevisiae
5 genome of (Saccharomyces cerevisiae) CICC32315 monoploid α is template, and ALD6-U, ALD6-D are primer,
PCR amplification obtains the acetaldehyde dehydrogenase gene ALD6 of 1503bp, is inserted into opening on Yep-PGK1 plasmid by XhoI single endonuclease digestion
Mover PGK1p and terminator PGK1TBetween, obtain plasmid Yep-PA；Using pUG6 as template, KAN-U, KAN-D are primer, PCR
Amplification obtains the KanMX gene of 1613bp, and SphI distinguishes digestion KanMX gene and plasmid Yep-PA, with Solution I connection
Enzyme connection, constitutes recombinant plasmid Yep-PAK；The sequence of whole process the primer such as table 1.
1 PCR primer of table
The verifying electrophoretogram that Fig. 3 is recombinant plasmid Yep-PAK: wherein swimming lane M is 5000bp DNA Ladder Marker；
It is 1503bp ALD6 that template PCR amplification arrives that swimming lane 1, which is with saccharomyces cerevisiae α 5,；Swimming lane 2 is to expand to arrive by template of Yep-PAK
1503bp ALD6；Swimming lane 3 is the 3203bp PGK1 expanded using Yep-PAK as templateP-ALD6；Swimming lane 4 is with yeast base
Because organizing the KANMX segment for being the 1613bp that template expands；Swimming lane 5 is the 1613bp's expanded using Yep-PAK as template
KANMX segment；Swimming lane 6 is the A-PGK1 of the 5020bp expanded using Yep-PAK as templateP-ALD6-PGK1T- KANMX-B piece
Section.
2) building of recombinant Saccharomyces cerevisiae bacterial strain
With upper and lower homologous section A, B of citrate synthase CIT2 gene be respectively added to PGK1 upstream primer ALD6-U and
5 ends ' of KANMX downstream primer KAN-D constitute new long primer P-U and K-D；Using recombinant plasmid Yep-PAK as template, length is drawn
Object PCR amplification obtains recombinant fragment A-PGK1P-ALD6-PGK1T- KANMX-B is transformed into wine brewing ferment with lithium acetate transformation method
Genes of brewing yeast engineering haploid strains α 5-A6 in female CICC32315 monoploid α 5, after obtaining homologous recombination.
The verifying of recombinant Saccharomyces cerevisiae bacterial strain:
According to the homologous recombination sequence of the gene order at saccharomyces cerevisiae recombination site both ends and insertion, separately designs two groups and determine
Point verifying upstream and downstream primer carries out PCR amplification respectively using well-grown conversion subgenom as template, verifies recon.
Upstream and downstream fixed point PCR verifying is carried out with primer A-U/A-D and B-U/B-D respectively, wherein upstream primer A-U/A-D
PCR product through 0.8% agarose gel electrophoresis, it can be seen that the specific band of size about 581bp or so, size
It is suitable with expection；The PCR product of downstream primer B-U/B-D through 0.8% agarose gel electrophoresis, it can be seen that a size is about
The specific band of 1077bp or so, size is suitable with expection, illustrates to recombinate box A-PGK1P-ALD6-PGK1T- KANMX-B piece
Section successfully recombinates in saccharomyces cerevisiae genome, and recombinable site is also correct.Electrophoresis result is as shown in Figure 5.
M is 5000bp DNA Ladder Marker in Fig. 5, and swimming lane 1 is the upstream fixed point that original strain α 5 is template PCR
The negative control of verifying；Swimming lane 2 is the upstream fixed point verifying segment using recombinant conversion as the 581bp of template PCR；Swimming lane 3 is
The negative control for the downstream fixed point verifying that original strain α 5 is template PCR；Swimming lane 4 is using recombinant conversion as template PCR's
The downstream fixed point verifying segment of 1077bp.
2: α 5-A6 of embodiment simulates the experiment of maize raw material liquid liquor fermentation
1) zymotechnique route:
Corn flour → immersion → liquefaction → saccharification → cooling → connects bacterium → fermentation → steaming wine → testing index
2) process conditions: soaking conditions: 60~70 DEG C, 20min is impregnated；Liquefaction condition: 85~90 DEG C, high temperature resistant α-is added
Amylase, liquefy 90min；Saccharification condition:, being added carbohydrase and nutritive salt by 55~60 DEG C, and be saccharified 20min；Acidulated condition: 45
DEG C, acid protease, 20min is added；Fermentation condition: 30 DEG C, 4 days.100mL mash is taken when steaming wine, adds 100mL water, steams
100mL wine sample.
3) ingredient: corn flour: 60g；Add water 130mL；Thermostable α-Amylase: 30 μ L；Carbohydrase: 90 μ L；Acidic protein
Enzyme: 1.2mL；Nutritive salt: 1mL；Inoculum concentration: 7.5%；
Carry out corn thick mash white wine hair respectively to saccharomyces cerevisiae engineered yeast α 5-A6 and starting strain α 5 by above-mentioned simulation process
Ferment experiment；It vibrates and weighs every 12h during fermentation, record is weightless；After fermentation, stop cultivating and weighing；Measurement fermentation
Remaining sugar concentration, alcohol by volume score and the main aromatic components content of liquid.With fermentability, remaining sugar concentration and product formation
Its comprehensive performance is characterized, the results are shown in Table 2,3.
The fermenting property of 2 maize raw material liquid liquor fermentation of table
Table 2 shows that present invention saccharomyces cerevisiae engineered yeast obtained compared with initial opportunistic pathogen, simulates maize raw material liquid
When liquor fermentation is tested, alcoholic strength and CO2Compared with the control almost without difference, residual sugar content slightly increases weightlessness.
The flavor substance of 3 maize raw material liquid liquor fermentation of table
Table 3 shows the saccharomyces cerevisiae engineered yeast of present invention yield of higher alcohol obtained and ethyl acetate
(Saccharomyces cerevisiae) α 5-A6 is compared with initial S. cervisiae α 5: corn thick mash liquor fermentation terminates
Afterwards, for transformant bacterial strain compared with parent strain, the content of acetic acid is 5.8 times of opportunistic pathogen, and the content of acetaldehyde is reduced than control strain
49.97%, the content of normal propyl alcohol reduces 49.58% than control strain, the content of isobutanol is reduced than control strain
22.8%, the content of isoamyl alcohol reduces 43.1% than control strain, and the content of ethyl acetate reduces 54.95% than control.
3: α 5-A6 of embodiment simulates sorghum material solid spirit fermenting experiment
1) zymotechnique route:
Sorghum → material moistening → plus rice husk → boiling → airing → connect bacterium → fermentation → distillation
2) process conditions: soaking conditions: 80 DEG C of mixings, sufficiently water suction is without hard-core；Conditions of cooking: rice husk normal pressure is added and steams
60min or so, particle is uniformly, heart is without white.Fermentation condition: 30 DEG C, 5 days.Steam wine condition: 100g raw material adds 200mL water, steams
100mL wine sample out.
3) ingredient: sorghum 100g；Rice husk 20g；Inoculum concentration: 0.6 hundred million/g raw material；
Sorghum material solid-state is carried out respectively to saccharomyces cerevisiae recombinant bacterial strain α 5-A6 and starting strain α 5 by above-mentioned simulation process
Liquor fermentation experiment；It vibrates and weighs every 12h during fermentation, record is weightless；After fermentation, stop cultivating and weighing；It surveys
Determine the remaining sugar concentration, alcohol by volume score and main aromatic components content of fermentation liquid.With CO2Weightless, residual sugar amount and alcoholic strength
Its comprehensive performance is characterized, the results are shown in Table 4,5.
The fermenting property and fragrance matter of 4 solid-state " Daqu " white spirit of table fermentation
Table 4 shows: present invention saccharomyces cerevisiae recombinant bacterium obtained simulates sorghum material solid-state compared with initial opportunistic pathogen
When liquor fermentation is tested, alcoholic strength, weightlessness and residual sugar content are not changed much.
The flavor substance of 5 maize raw material liquid liquor fermentation of table
Table 5 shows: present invention saccharomyces cerevisiae engineered yeast obtained simulates sorghum material solid-state compared with initial opportunistic pathogen
When liquor fermentation is tested, isobutyl alcohol content reduces 34.66% than control, and isoamyl alcohol reduces 49.52%, and benzyl carbinol 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%.
The experiment of 4: α 5-A6 beer fermentation of embodiment
(1) preparation of brewer's wort
Using whole-malt technique, the fructus hordei germinatus of crushing is mixed according to the material-water ratio of 1:4 (w:w) with 52 DEG C of hot water
40min, be then saccharified under the conditions of 65 DEG C about 1h, is warming up to 72 DEG C, keeps 15min, then be warming up to 78 DEG C, keeps 10min.Saccharification
Then filtrate is boiled about 1h by liquid filtered through gauze, during which add 0.4 ‰ hops, filter after boiling, control wheat juice is dense
Degree is 18 ° of Brix.
(2) seed culture
1. actication of culture: saving strain transfer to 28 DEG C of activation culture 2d of YEPD slant tube.
2. first order seed culture: taking one ring of slant strains, be inoculated in the test tube of 18 ° of Brix wheat juice culture mediums equipped with 5mL
In, it is cultivated for 24 hours under the conditions of 28 DEG C, 180rpm.
3. secondary seed culture: primary seed solution is accessed 18 ° of Brix wheat juice for filling 50mL by 10% inoculum concentration
In 150mL triangular flask, 16 DEG C of stationary culture 72h.
(3) high concentration beer ferments
Secondary seed solution obtains yeast paste by centrifugation, and yeast paste is filled 18 ° of 300mL by 0.5% inoculum concentration access
In the 500mL triangular flask of Brix wheat juice, 10 DEG C of standing for fermentation.It vibrates and weighs every 12h during fermentation, record is weightless；Fermentation
After, stop cultivating and weighing；Measure remaining sugar concentration, alcohol by volume score and the main aromatic components content of fermentation liquid.
Its comprehensive performance is characterized with fermentability, remaining sugar concentration and product formation, the results are shown in Table 6,7.
The fermenting property of 6 high concentration beer of table fermentation
As shown in Table 6, present invention saccharomyces cerevisiae engineered yeast α 5-A6 obtained is compared with initial opportunistic pathogen α 5, highly concentrated beer
When wine fermentation is tested, alcoholic strength is in a slight decrease, and residual sugar content has increased slightly.
The flavor substance of 7 high concentration beer of table fermentation
Table 7 it is found that present invention yield of higher alcohol obtained and ethyl acetate saccharomyces cerevisiae engineered yeast
(Saccharomyces cerevisiae) α 5-A6 is compared with initial S. cervisiae α 5: high concentration beer after fermentation, turns
For beggar's bacterial strain compared with parent strain, the content of acetic acid is 1.9 times of opportunistic pathogen, and the content of acetaldehyde reduces 9.33% than control,
The content of normal propyl alcohol reduces 60.8% than control strain, and the content of isobutanol reduces 21.7% than control strain, isoamyl alcohol
Content reduce 25.9% than control strain, ethyl acetate reduces 37.3%.
External source addition acetic acid experiment during 5: α 5 thick mash fermentation of embodiment
1) zymotechnique route:
Corn flour → immersion → liquefaction → saccharification → cooling → connects bacterium → fermentation → steaming wine → testing index
2) process conditions: soaking conditions: 60~70 DEG C, 20min is impregnated；Liquefaction condition: 85~90 DEG C, high temperature resistant α-is added
Amylase, liquefy 90min；Saccharification condition:, being added carbohydrase and nutritive salt by 55~60 DEG C, and be saccharified 20min；Acidulated condition: 45
DEG C, acid protease, 20min is added；Fermentation condition: 30 DEG C, 4 days.100mL mash is taken when steaming wine, adds 100mL water, steams
100mL wine sample.
3) ingredient: corn flour: 60g；Add water 210mL；Thermostable α-Amylase: 30 μ L；Carbohydrase: 90 μ L；Acidic protein
Enzyme: 1.2mL；Nutritive salt: 1mL；Inoculum concentration: 7.5%；
By above-mentioned simulation process starting strain α 5 carry out corn thick mash liquor fermentation experiment, fermentation for 24 hours when fermenting
The acetic acid of 6.2mg/L is added in liquid；It vibrates and weighs every 12h during fermentation, record is weightless；After fermentation, stop culture
And it weighs；Measure remaining sugar concentration, alcohol by volume score and the main aromatic components content of fermentation liquid.With fermentability, residual sugar
Concentration and product formation characterize its comprehensive performance, the results are shown in Table 8,9.
The fermenting property of 8 external source of table addition acetic acid
As shown in Table 8, after the acetic acid of appropriate content is added in fermentation liquid, CO2Weightlessness, alcoholic strength and residual sugar content are almost
There is no difference.
9 external source of table adds the flavor substance that acetic fermentation generates
As shown in Table 9, after acetic acid being added in fermentation liquid, the content of normal propyl alcohol reduces 18.61%, the content of isobutanol
Compared with the control without too big difference, the content of isoamyl alcohol reduces 41.71%, and ethyl acetate reduces 12.36%, acetic acid
Content improves 90.70%.
External source addition 2mg acetic acid experiment during 6: α 5 thick mash fermentation of embodiment
1) zymotechnique route:
Corn flour → immersion → liquefaction → saccharification → cooling → connects bacterium → fermentation → steaming wine → testing index
2) process conditions: soaking conditions: 60~70 DEG C, 20min is impregnated；Liquefaction condition: 85~90 DEG C, high temperature resistant α-is added
Amylase, liquefy 90min；Saccharification condition:, being added carbohydrase and nutritive salt by 55~60 DEG C, and be saccharified 20min；Acidulated condition: 45
DEG C, acid protease, 20min is added；Fermentation condition: 30 DEG C, 4 days.100mL mash is taken when steaming wine, adds 100mL water, steams
100mL wine sample.
3) ingredient: corn flour: 60g；Add water 210mL；Thermostable α-Amylase: 30 μ L；Carbohydrase: 90 μ L；Acidic protein
Enzyme: 1.2mL；Nutritive salt: 1mL；Inoculum concentration: 7.5%；
By above-mentioned simulation process starting strain α 5 carry out corn thick mash liquor fermentation experiment, fermentation for 24 hours when fermenting
The acetic acid of 2mg/L is added in liquid；It vibrates and weighs every 12h during fermentation, record is weightless；After fermentation, stop culture simultaneously
Weighing；Measure remaining sugar concentration, alcohol by volume score and the main aromatic components content of fermentation liquid.It is dense with fermentability, residual sugar
Degree and product formation characterize its comprehensive performance, the results are shown in Table 10,11.
The fermenting property of 10 external source of table addition acetic acid
As shown in Table 10, after the acetic acid of appropriate content is added in fermentation liquid, CO2Weightlessness, alcoholic strength and residual sugar content are several
There is no difference.
11 external source of table adds the flavor substance that acetic fermentation generates
As shown in Table 11, after acetic acid being added in fermentation liquid, the content of normal propyl alcohol reduces 6.65%, the content of isobutanol
Compared with the control without too big difference, the content of isoamyl alcohol reduces 13.23%, and ethyl acetate does not have the area Tai great compared with the control
Not, the content of acetic acid improves 18.68%.
External source addition 20mg acetic acid experiment during 7: α 5 thick mash fermentation of embodiment
1) zymotechnique route:
Corn flour → immersion → liquefaction → saccharification → cooling → connects bacterium → fermentation → steaming wine → testing index
2) process conditions: soaking conditions: 60~70 DEG C, 20min is impregnated；Liquefaction condition: 85~90 DEG C, high temperature resistant α-is added
Amylase, liquefy 90min；Saccharification condition:, being added carbohydrase and nutritive salt by 55~60 DEG C, and be saccharified 20min；Acidulated condition: 45
DEG C, acid protease, 20min is added；Fermentation condition: 30 DEG C, 4 days.100mL mash is taken when steaming wine, adds 100mL water, steams
100mL wine sample.
3) ingredient: corn flour: 60g；Add water 210mL；Thermostable α-Amylase: 30 μ L；Carbohydrase: 90 μ L；Acidic protein
Enzyme: 1.2mL；Nutritive salt: 1mL；Inoculum concentration: 7.5%；
By above-mentioned simulation process starting strain α 5 carry out corn thick mash liquor fermentation experiment, fermentation for 24 hours when fermenting
The acetic acid of 20mg/L is added in liquid；It vibrates and weighs every 12h during fermentation, record is weightless；After fermentation, stop culture simultaneously
Weighing；Measure remaining sugar concentration, alcohol by volume score and the main aromatic components content of fermentation liquid.It is dense with fermentability, residual sugar
Degree and product formation characterize its comprehensive performance, the results are shown in Table 12,13.
The fermenting property of 12 external source of table addition acetic acid
As shown in Table 12, after the acetic acid of appropriate content is added in fermentation liquid, CO2Weightlessness, alcoholic strength and residual sugar content are several
There is no difference.
13 external source of table adds the flavor substance that acetic fermentation generates
As shown in Table 13, after acetic acid being added in fermentation liquid, the content of normal propyl alcohol reduces 48.88%, and isobutanol contains
Amount reduces 66.46%, and the content of isoamyl alcohol reduces 66.20%, and ethyl acetate reduces 57.12%, and the content of acetic acid mentions
It is 3.10 times high.
Claims (7)
1. a kind of regulation saccharomyces cerevisiae flavor substance synthetic method, which is characterized in that by making Cellular Accumulation acetic acid, thus one
Step reduces the production quantity of saccharomyces cerevisiae higher alcohol, acetaldehyde and ethyl acetate.
2. a kind of regulation saccharomyces cerevisiae flavor substance synthetic method as described in claim 1, which is characterized in that described to make carefully
The method that born of the same parents accumulate acetic acid is to strengthen acetic acid route of synthesis, including pyruvate decarboxylase, acetaldehyde dehydrogenase, alcohol dehydrogenase, second
The raising of acyl coenzyme A hydrolysis activity.
3. a kind of regulation saccharomyces cerevisiae flavor substance synthetic method as described in claim 1, which is characterized in that described to make carefully
The method that born of the same parents accumulate acetic acid is to weaken acetic acid to decompose approach, the specially weakening of acetyl-CoA-synthetase vigor.
4. a kind of regulation saccharomyces cerevisiae flavor substance synthetic method as described in claim 1, which is characterized in that described to make carefully
The method that born of the same parents accumulate acetic acid is that external source adds acetic acid in fermentation process.
5. a kind of regulation saccharomyces cerevisiae flavor substance synthetic method as claimed in claim 2, which is characterized in that acetaldehyde-dehydrogenase
The specific method that enzyme activity improves is one strain gene engineering bacterium of building, and the genetic engineering bacterium in saccharomyces cerevisiae by being overexpressed
The expressing gene ALD6 gene of acetaldehyde dehydrogenase, reaching reduces wine product Higher Alcohols, ethyl acetate and acetaldehyde and increases second
The purpose of sour production quantity；The saccharomyces cerevisiae is specially saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC32315；
The nucleotide sequence of the ALD6 gene is as shown in SEQ ID No.1.
6. a kind of regulation saccharomyces cerevisiae flavor substance synthetic method as claimed in claim 5, which is characterized in that the gene
Application of engineering bacteria during wine brewing.
7. a kind of regulation saccharomyces cerevisiae flavor substance synthetic method as claimed in claim 4, which is characterized in that the acetic acid
Additive amount be 2-20mg/L fermentation liquid, addition the time be fermentation for 24 hours when.
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Non-Patent Citations (3)
| Title |
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| Engineering of the Pyruvate Dehydrogenase Bypass in Saccharomyces cerevisiae: Role of the Cytosolic Mg21 and Mitochondrial K1 Acetaldehyde Dehydrogenases Ald6p and Ald4p in Acetate Formation during Alcoholic Fermentation;FABIENNE REMIZE等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20000831;第66卷(第8期);第3151-3159页，尤其是第3153页右栏第2段和第3154页 |
| 酿酒酵母PDC1基因过表达菌株的构建;柯崇榕等;《应用与环境生物学报》;20130825;第19卷(第4期);第704-708页，尤其是摘要 |
| 酿酒酵母乙醇脱氢酶Ⅰ基因的超表达;秦丽娜等;《微生物学通报》;20080220;第35卷(第2期);第261-266页，尤其是摘要 |
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