CN102899212A - Method for increasing content of 4-vinyl guaiacol in top fermentation wheat beer - Google Patents
Method for increasing content of 4-vinyl guaiacol in top fermentation wheat beer Download PDFInfo
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- CN102899212A CN102899212A CN2012104448624A CN201210444862A CN102899212A CN 102899212 A CN102899212 A CN 102899212A CN 2012104448624 A CN2012104448624 A CN 2012104448624A CN 201210444862 A CN201210444862 A CN 201210444862A CN 102899212 A CN102899212 A CN 102899212A
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Abstract
The invention discloses a method for increasing the content of 4-vinyl guaiacol in top fermentation wheat beer. The method comprises the following steps of: cloning the PADC (Poly Ethylene Glycol Ester Alkyl Carbonate) gene of Bacillus subtilis, reserving the hereditary capacity of the Bacillus subtilis, expressing the Bacillus subtilis in escherichia coli and transforming the PADC gene into a top fermentation yeast strain W303-1A so as to establish the top fermentation beer yeast W303+padc. By utilizing the method, the content of the 4-vinyl guaiacol in the top fermentation wheat beer is remarkably increased, and the typical lilac flavor in the wheat beer is highlighted.
Description
Technical field
The present invention relates to a kind of method of utilizing genetic engineering technique to improve 4-vinyl guaiacol content in the beer, belong to gene engineering technology field.
Background technology
4-vinyl guaiacol sense organ threshold value is very low, only be 0.3mg/L, even trace, also can make beer present pharmacy flavor, phenol flavor, cloves flavor or tangy flavour, thereby in most of bottom fermentation beer, its local flavor also is out of favour, and b referred to as " phenol peculiar smell (phenolic off-flavour) ".Although be smell substance for bottom fermentation beer, but well-known, the 4-vinyl guaiacol but is the requisite fragrance matter that forms the top fermented beers such as Belgian white beer (brewageing with the wheat that does not germinate), spelt beer (using the wheat malt brew) and sootiness beer, also is the indispensable characteristic composition of the comprehensive flavor evaluation of this type beer.
In Process of Beer Brewing, by hydrolysis and enzymolysis, many phenolic acid can dissociate out, in the above in the fermentation beer yeast under the effect of phenolic acid decarboxylase, can be with phenolic acid decarboxylation wherein, form the characteristic flavour substancess such as 4-vinyl guaiacol (being the Flos Caryophylli flavor), can improve significantly the fragrance of top fermentation wheat beer.
The 4-vinyl guaiacol mainly is to synthesize by chemistry or biological process at present.As: Chinese patent literature CN101885669A(application number 201010229531.X) a kind of preparation method of 4-vinyl guaiacol is disclosed, step is: (1) drops into quinoline, forulic acid in the reactor and successively as the Resorcinol of stopper, open heating unit, connect nitrogen protection device,, continue to stir 40-60 minute without the gas overflowing stopped heating to system; (2) will be in the above-mentioned reaction add in the solvent benzol and stir, and add hydrochloric acid in this solution, stir, standing demix then detects aqueous pH values and is till the neutrality; Add the siccative Calcium Chloride Powder Anhydrous in the solution after the most backward deionized water is washed; (3) dried solution is put into distiller, under normal pressure, will steam as the benzene of solvent, then connect vacuum pump and under the gas phase temperature below 100 ℃, product steamed and get final product.Because the method generating portion by product and raw material are residual, can't directly add in the Process of Beer Brewing.
Chinese patent literature CN101397568A(application number 200810233517.X) disclose a kind of method by gene clone obtain to decompose among the ENTEROBACTER SP.PX6-4 coding region sequence that forulic acid generates the key enzyme ferulic acid decarboxylase of 4-vinyl guaiacol, and by the heterogenous expression technology in intestinal bacteria (E.COLI BL21) great expression this enzyme; Obtained the sterling of ferulic acid decarboxylase by the method for purifying; Use the hanging drop crystallization process to obtain the crystallization of protein of ferulic acid decarboxylase, and grasped structure and the active centre of this enzyme.Because forulic acid is the precursor substance of 4-vinyl guaiacol, adds ferulic acid decarboxylase in Process of Beer Brewing, although can improve the 4-vinyl guaiacol content in the beer, effect can't practical requirement.
In fact, part bacterium and fungi contain the PADC gene, coding phenolic acid decarboxylase (phenolic acid decarboxylase), in the catabolic pathway of metabolism of biology, play very important effect, such as acinetobacter calcoaceticus Acinetobacter, Pseudomonas fluorescens Pseudomonasfluorescens and subtilis Bacillus subtilis etc., but the PADC gene is applied to there is not yet in the beer brewing technique report.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of method of utilizing genetic engineering technique to improve 4-vinyl guaiacol content in the top fermentation wheat beer is provided.
A kind of method that improves 4-vinyl guaiacol content in the top fermentation wheat beer, step is as follows:
(1) pass through pcr amplification PADC gene from subtilis, the PADC gene nucleotide series is shown in SEQ ID NO.1, and pcr amplification primer sequence is as follows:
Upstream primer: F-padc5 '-GCG
TCTAGAATGGAAAACTTTATCG-3 '
Downstream primer: R-padc5 '-GCG
AAGCTTTTATAATCTTCCCGC-3 ';
(2) after the PADC gene that step (1) is made is purified, be connected with restriction endonuclease HindIII double digestion and equally be connected plasmid YEp352 with restriction endonuclease HindIII double digestion through restriction endonuclease XbaI and be connected through restriction endonuclease XbaI, make recombinant plasmid YPADC, recombinant plasmid YPADC is converted into top fermenting yeast W303-1A, obtains top fermentation restructuring yeast strains W303+padc;
(3) utilize 11 ° of P wheat juice that the top fermentation restructuring yeast strains W303+padc that step (2) makes is carried out enlarged culturing, culture condition is: 25～28 ℃ of shaking tables, and 220～240r/min obtains top fermenting yeast W303+padc;
(4) the top fermenting yeast W303+padc that step (3) is made is inoculated in 11 ° of P wheat juice, be 16～18 ℃ in temperature and carry out fermentation culture to pol when being 4 ° of P, sealing and fermenting container pressurize to diacetyl content is lower than 0.1mg/L, be cooled to 0～2 ℃, preserved 5～8 days, making 4-vinyl guaiacol content is the beer of 1.6～1.8mg/L.
Preferred according to the present invention, in the described step (1), the PCR response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of template sex change 30s, 52.6 ℃ of primer annealing 30s, 72 ℃ of primer extension 45s, 30 circulations; 72 ℃ are extended 10min.
Preferred according to the present invention, in the described step (2), recombinant plasmid YPADC transforms top fermenting yeast W303-1A and adopts the Lithium Acetate conversion method.
Preferred according to the present invention, the preparation method of 11 ° of P wheat juice in described step (3) and the step (4) is as follows: Fructus Hordei Germinatus and wheat malt are pulverized with EBC standard mill after mixing by weight 3:2, adopt infusion mashing after saccharification, after filtration, boil after, make 11 ° of P wheat juice.
Further preferred according to the present invention, the concrete steps of above-mentioned saccharification are: feed temperature is 37 ℃, keeps 20min; Be warming up to 45 ℃ and keep 30min, then be warming up to 52 ℃ and keep 40min; Be warming up to again 65 ℃, keep 70min; Being warming up at last 78 ℃ of leachings gets final product.
Beneficial effect
First passage of the present invention carries out the cereuisiae fermentum that genetic engineering modified acquisition has specific function to top fermenting yeast, and this yeast is used for brewage, thereby significantly improved the 4-vinyl guaiacol content in the top fermentation wheat beer, given prominence to the typical Flos Caryophylli flavor of top fermentation wheat.The method provides new method for improving beer flavor and fragrance, lays a good foundation for genetically engineered is applied to the brewage field.
Description of drawings
Fig. 1 is the electrophorogram of the PADC gene of PCR acquisition;
Wherein: M is D2000DNA Marker, and 1,2,3,4,5,6,7 is the PADC gene;
Fig. 2 is the double digestion electrophorogram of plasmid YEp352 and PADC gene PCR purified product;
Wherein: A is 1kb DNA Maker, and B is the YEp352 plasmid, and C is PADC PCR purified product, and D is D2000DNA Maker;
Fig. 3 is the building process schematic diagram of recombinant plasmid YPADC;
Fig. 4 is that the enzyme of recombinant plasmid YPADC is cut the evaluation electrophorogram;
Wherein: M is 1kb DNAMarker, and 1 is XbaI and HindIII double digestion recombinant plasmid YPADC, and 2 is recombinant plasmid YPADC, and 3 is XbaI and HindIII double digestion plasmid YEp352, and 4 is XbaI and HindIII double digestion PADC gene, and 5 is D2000DNAMarker;
Fig. 5 is the bacterium colony PCR checking electrophorogram of clone strain W303+padc;
M-D2000DNAMarker wherein, 1 is starting strain W303-1A, 2～21 is clone strain W303+padc;
Fig. 6 is the SDS-PAGE electrophorogram of starting strain W303-1A and clone strain W303+padc;
Wherein M is marker, and 1 is starting strain W303-1A, and 2 is clone strain W303+padc;
Fig. 7 is the column contrast schematic diagram of starting strain W303-1A and clone strain W303+padc enzymic activity;
Fig. 8 is outward appearance concentration and the diacetyl content change curve of the beer brewageed of starting strain W303-1A and clone strain W303+padc;
Fig. 9 is the 4-vinyl guaiacol content column contrast schematic diagram of the beer brewageed of starting strain W303-1A and clone strain W303+padc;
Figure 10 is the sensory evaluation schematic diagram of starting strain W303-1A and clone strain W303+padc beer brewing.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is described further, but institute of the present invention protection domain is not limited to this.
Biological material source:
Subtilis strangles arraying biomolecule Science and Technology Ltd. available from Shanghai, and goods number is: ACCC10627;
Top fermented beer yeast W303-1A is available from German Doemens beer institute;
Restriction endonuclease XbaI, restriction endonuclease HindIII, plasmid YEp352 is available from Fermentas company;
Substratum:
The bacillus subtilis bacterium culture medium:
Then sucrose 30.0g, potassium primary phosphate 4.5g, ammonium sulfate 2.5g, peptone 10g, calcium carbonate 2g, yeast extract paste 5g and agar 20g fully dissolve in 1000mL water, 121 ℃ of autoclavings again.
Intestinal bacteria E.coli DH5 α substratum LB:
Yeast extract 5g, Tryptones 10g, NaCL10g is dissolved in the 950mL distilled water, transfers pH to 7.0 with 5M NaOH, if adding distil water is settled to 1000mL(preparation solid medium, also need add the agar of 1.5wt%), packing, 121 ℃ of autoclaving 20min.
Microzyme culture medium YPD(1L):
Yeast extract 10g, Tryptones 20g, be dissolved in (if preparation solid medium in the 950mL distilled water, the agar that also need add 1.5wt%), the pH nature is behind 121 ℃ of autoclaving 20min, be cooled to 55 ℃, then add 115 ℃ of separately glucose solutions of sterilization 30min, making the glucose final concentration is 2wt%, and distilled water is settled to 1L.
SC solid plate substratum (1L): without amino acid whose yeast nitrogen (YNB) 6.7g, VITAMIN B4 50mg, uridylic 50mg, Histidine 100mg, tryptophane 100mg, leucine 100mg, arginase 12 0mg, aspartic acid 100mg, L-glutamic acid 100mg, Isoleucine 30mg, Methionin 30mg, methionine(Met) 20mg, phenylalanine 50mg, Serine 150mg, Threonine 150mg, tyrosine 30mg, α-amino-isovaleric acid 150mg, regulating the pH value is 6.5, the agar of adding 1.5%, adding 40wt% glucose, to make it final concentration be 2wt% again, and distilled water is settled to 1L, at 121 ℃ of lower sterilization 15min.
SC
-uraSolid plate substratum: for omitting the SC solid plate substratum of uridylic.
Equipment
EBC standard mill is available from German B ü hler company;
The full-automatic saccharification instrument of LB8 is available from Lochner company;
Embodiment
1. the extraction of subtilis Bacillus subtilis DNA
The picking subtilis is inoculated in the bacillus subtilis bacterium culture medium, 36 ℃ of concussion incubated overnight.Get 100mL bacterium liquid, the centrifugal 10min of 5000r/min abandons supernatant liquor; Bacterial sediment suspends with 10mL TE damping fluid (Tris-edta buffer liquid), adds the 0.5mL10wt% sodium lauryl sulphate, 50 μ L Proteinase Ks, mixing, 37 ℃ of insulation 1h.Add 1.5mL5mol/L NaCl, mixing.Add 1.5mL CTAB/NaCl solution (NaCl1.4M, CTAB2%, TrisCl100mM, EDTA20mM, mercaptoethanol 0.2%), mixing, 65 ℃ of insulation 20min.With equal-volume extract I extracting, extract I is by phenol: chloroform: the volume ratio of primary isoamyl alcohol is that 25:24:1 mixes, the centrifugal 10min of 5000r/min, pipette supernatant liquor to clean centrifuge tube, with equal-volume extract II extracting, extract II is by chloroform: the volume ratio of primary isoamyl alcohol is that 24:1 mixes, and gets supernatant liquor and moves in the clean pipe.Add 1 times of volume Virahol, put upside down mixing, leave standstill 10min under the room temperature, the centrifugal 15min of 10000r/min obtains the DNA precipitation.Add 700 μ l volumetric concentrations and be 70% ethanol, 70 μ l3mol/LNaAc rinsing DNA precipitation is dissolved in 1mL TE damping fluid ,-20 ℃ of preservations.
Remove RNA: add 5 μ LRNase, add 1/10 volume 3mol/LNaAc, add 2 times of volume dehydrated alcohol 10min after, the DNA precipitation is dissolved in 1mL TE ,-20 ℃ of preservations make subtilis DNA.
2. the clone of design of primers and PADC gene fragment
Upstream primer: F-padc5 '-GCG
TCTAGAATGGAAAACTTTATCG-3 ' SEQIDNO2
Downstream primer: R-padc5 '-GCG
AAGCTTTTATAATCTTCCCGC-3 ' SEQIDNO.3
Upstream primer 5 ' end is introduced XbaI enzyme cutting site (underscore part), and downstream primer 5 ' end is introduced HindIII restriction enzyme site (underscore part), and is synthetic by Invitrogen company.
Take F-padc and R-padc as primer, subtilis DNA is template, carries out polymerase chain reaction (PCR), and reaction system is 50 μ L, and is specific as follows:
The PCR response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 52.6 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations; 72 ℃ are extended 10min.
Detect through agarose gel electrophoresis, the result as seen from Figure 1, has amplified the specific fragment of 1 treaty 504bp as shown in Figure 1, meets the big or small 504bp of expection PADC gene fragment.
3.PCR the purifying of product
Get PCR product 200 μ L and 6xLoading Buffer40 μ L, and 5 μ L D2000DNA Marker loadings, run 1.5% agarose gel electrophoresis.Then, reclaim the purifying that test kit carries out the PCR product with the GenClean of Shanghai Jierui Biology Engineering Co., Ltd pillar sepharose DNA.
4.PCR the enzyme of product is cut and is connected
Respectively the PADCPCR product of purifying and plasmid YEp352 are carried out enzyme with restriction enzyme XbaI and HindIII and cut, as shown in Figure 2, reaction system is respectively 30 μ L and 20 μ L.Reaction mixture will add successively, and attack mixes gently, whizzer centrifugal 5 seconds.Put 37 ℃ of water-baths.
According to T4DNA Ligase specification sheets enzyme is cut product and connect, 16 ℃ of water-baths are spent the night, and namely are built into recombinant plasmid YPADC, and building process is seen Fig. 3.Reaction system is:
5. recombinant plasmid YPADC is converted into intestinal bacteria E.coli DH5 α
In the aseptic centrifuge tube of 1.5mL that 100 μ L competent cell E.coli DH5 α are housed, add to connect product 5 μ L, pipe is flicked behind the mixing place 30min on ice; Centrifuge tube put into 42 ℃ water bath with thermostatic control 90s; Fast centrifuge tube is transferred in the ice bath, made cell in ice bath, cool off 2min; Add 500 μ L in the centrifuge tube and be preheated to 37 ℃ intestinal bacteria E.coli DH5 α substratum LB, shaking culture 60min(rotating speed surpasses 200r/min on 37 ℃ of shaking tables); The centrifugal 20s of 13000r/min discards most of clear liquid, and cell precipitation suspends with residue clear liquid 100 μ L, is coated on the intestinal bacteria E.coli DH5 α substratum LB solid plate (when melting the LB solid medium, to add penbritin 1 μ L/mL); First just put culture dish 30min, after bacterium liquid absorbs fully, be inverted culture dish, 37 ℃ of lower overnight incubation.Choose positive monoclonal in 4mL LB liquid nutrient medium, add penbritin 4 μ L, shake bacterium in 37 ℃.Approximately 8-10h left and right sides bacterium liquid can be muddy, notes detecting OD with SmartSpec plus nucleic acid-protein determinator
600Value is treated OD
600During=0.8-1.0, can carry out the extraction of recombinant plasmid.
6. the evaluation of recombinant plasmid YPADC
Adopt the method for XbaI and HindIII double digestion that recombinant plasmid YPADC is identified, reaction system is as follows:
Then put 37 ℃ of water-baths, leave standstill 1～16h, through the electrophoresis checking, the result as shown in Figure 4.
7. recombinant plasmid YPADC is converted into top fermented beer yeast W303-1A
Adopt the Lithium Acetate conversion method that recombinant plasmid YPAD1 is converted among the top fermented beer yeast W303-1A, concrete grammar is as follows:
(1) top fermented beer yeast W303-1A is inoculated among the 5mL microzyme culture medium YPD, 230r/min, 30 ℃ of lower shaking culture are spent the night.
(2) counting overnight culture cell density is with final 5 * 10
6The cell density of individual/mL (approximately 24-28h) is inoculated in 50mL microzyme culture medium YPD.
(3) 30 ℃, 200r/min shaking table shaking culture, approximately behind the 3-5h, the OD value is 0.8-1.0(approximately 10
8Cells/mL); There is the thalline of drift sand sample flowing in the microzyme culture medium YPD.
(4) pour 48mL microzyme culture medium YPD into aseptic 50mL centrifuge tube, the centrifugal 5min harvested cell of 5000r/min (cell of this culture enough transforms for 10 times).All the other 2mL microzyme culture medium YPD, 1mL are used for frozen thalline, in addition 1mL coated plate contrast.
(5) abandon nutrient solution, resuspended with the 50mL sterilized water, the centrifugal 5min harvested cell of 5000r/min.
(6) abandon water, cell suspension in the LiAC of the 0.1mol/L of 1mL solution, is turned the centrifuge tube of suspended substance to an aseptic 1.5mL.
(7) whizzer is with the centrifugal 15s precipitation of maximum speed of revolution (14000r/min) thalline, with the careful sucking-off sucking-off of micropipet supernatant liquor (LiAC solution).
(8) resuspended thalline is in the LiAC of the 0.1mol/L of 400 μ L solution.
(9) by the packing of 50 μ L/ pipe, transform immediately.
(10) boil 1mL salmon sperm dna 5min, ice bath is with preparation strand carrier DNA rapidly.Salmon sperm dna mainly is the degraded that prevents goal gene, assists the conversion of goal gene.
(11) vibration step (9) cell suspending liquid, centrifugation cell is with micropipet sucking-off LiAC solution.Add transformation mixture (totally 360 μ L)." transformation mixture " is comprised of following ingredients, adds in the following order:
240 μ L polyoxyethylene glycol (PEG) 3350(50%, W/V)
The LiAC of 36 μ L1.0mol/L
34 μ L strand salmons essence (Salmon sperm) carrier DNA(2.0mg/mL)
50 μ L water and plasmid DNA (0.1-10 μ g)
(12) each reaction tubes of thermal agitation is until the complete mixing of cell needs about 1min usually.
(13) 30 ℃ of water-baths, insulation 30min.
(14) 42 ℃ of water-baths, heat shock 20-25min.Attention: the suitableeest heat shock time is different with different yeast.If need to obtain high transformation frequency, need in advance test Best Times.
(15) with the centrifugal 1-2min of 8000r/min, remove the conversion mixed solution with micropipet.
(16) inhale 0.2-1.0mL sterilized water (can consider 0.5mL) and be added in each reaction tubes, with pipettor up and down gently extracting with the precipitation that suspends.
(17) 200 μ L with equal portions transform mixed solution, are applied to the SC of nutritious defective type
-UraSubstratum is selected dull and stereotyped.Cultivated 3 days for 28-30 ℃, observe and the record result, and compare with unconverted top fermented beer yeast W303-1A.
8. the bacterium colony PCR of clone strain W303+padc
In order to verify that whether recombinant plasmid YPADC has changed among the top fermented beer yeast W303-1A, gets SC
-uraThe bacterium colony that forms on the solid plate substratum carries out PCR.
(1) chooses SC
-ura20 of the bacterium colonies that forms on the solid plate substratum are chosen into 20 10 μ L ddH are housed
2In the EP pipe of O, corresponding one by one.
(2) get top fermented beer yeast W303-1A bacterium liquid 1 μ L, add 1 10 μ L ddH are housed
2In the EP pipe of O, compare.
(3) again topple over SC
-UraSelect 2 of plate culture mediums, and setting-out at the bottom of ware.
(4) dip in (1) the bacterium liquid in EP pipe a little, corresponding setting-out is in SC one by one
-uraThe solid plate substratum.
(5) the bacterium liquid of step (1) and the bacterium liquid of step (2) are boiled 5min, as the bacterium colony pcr template.
The PCR reaction system is 50 μ L, adds successively in order: F-padc1 μ L, R-padc1 μ L, dNTPs4 μ L, Easy Taq10xbuffer5 μ L, bacterium colony pcr template 1 μ L, ddH
2O37 μ L, Easy Taq DNA Polymerase1 μ L.
The PCR response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of sex change 30s, 52.6 ℃ of annealing 30s, 72 ℃ are extended 45s, 30 circulations; Last 72 ℃ are extended 10min.
Agarose gel electrophoresis the results are shown in Figure 5.As seen from Figure 5, clone strain W303+padc(swimming lane 4-21) about 500bp, there is band to occur, starting strain W303-1A(swimming lane 1) then do not have band to occur about 500bp, this further specifies, and goal gene PADC has changed starting strain W303-1A over to.
9.PADC the protein expression of gene
Sheet glass is fixed on the encapsulating support, and the preparation separation gel adds rapidly the sheet glass interlayer, and control is 6cm highly approximately, lives liquid level with the 1mL water seal, leaves standstill to gel polymerisation complete.Pour out water, with filter paper remaining moisture is blotted.Add concentrated glue, insert comb, leave standstill to gel polymerisation complete.Extract comb, add the groove damping fluid.Get an amount of sample, add 10 μ L sample-loading buffers, mixing heats 5min in boiling water.Add electrophoretic buffer, carry out electrophoresis.The beginning current constant changes 20mA at 10mA into after the tetrabromophenol sulfonphthalein line enters separation gel, electrophoresis finishes when tetrabromophenol sulfonphthalein moves to from 5mm place, gel edge.Dyeing a few hours or spend the night, to decolour with destainer clear to protein band, the result is as shown in Figure 6.
10. the mensuration of clone strain W303+padc phenolic acid decarboxylase
Cut the protein in the glue extraction electrophoretic band, add the forulic acid of 25mmol/L phosphate buffered saline buffer (pH6) and 0.3mmol/L.Behind the 24h, add the Tris-HCl of 20mmol/L and the SDS(pH6 of 3g/L), make 16 times of mixed solution dilutions, with termination reaction.The enzyme work of phenolic acid decarboxylase is defined as every milligram of protein at degrade in the time μ mol number of substrate of per minute.
Because decarboxylation and the reduction of phenolic acid can cause UV spectrum to be offset, thereby the activity of phenolic acid decarboxylase can detect by ultraviolet spectrophotometer.Forulic acid peak value occurs at the 300nm place, and the 4-vinyl guaiacol peak value occurs at the 258nm place.The formation of the disappearance of 300nm peak value and 258nm peak value has shown the disappearance of forulic acid and the generation of 4-vinyl guaiacol.
After inducing with forulic acid, clone strain W303+padc phenolic acid decarboxylase greatly improves, and approximately is 2.1 times of (see figure 7)s of control strain W303-1A activity.
11. the Stability Determination of clone strain W303+padc
Clone strain W303+padc is accessed in the 10mLYPD liquid nutrient medium incubated overnight with 1% inoculum size.After 10 generations, be applied to respectively SC and SC
-uraThe solid plate substratum, calculate with following formula:
Through calculating, the stability of clone strain W303+padc is 53.23%, illustrates that the ability that goes down to posterity of clone strain W303+padc is stronger.
12. beer saccharification fermenting experiment
Wort preparation: Fructus Hordei Germinatus and wheat malt in mass ratio 3:2 mix, and pulverize with EBC standard mill, carry out saccharification with the full-automatic saccharification instrument of LB8, adopt infusion mashing.Feed temperature is 37 ℃, keeps 20min; Protein stops 44 ℃ and 52 ℃, keeps respectively 30min and 40min; 65 ℃ of amylolysis keep 70min; Be warming up to 78 ℃, filter with gauze.Boiling time 90min boils rear concentration and is controlled at 11 ° of P.After the cooling of wheat juice, inject the PET bottle at Bechtop.
The cultivation of clone strain W303+padc: get the clone strain W303+padc an amount of (W303-1A compares with starting strain) that preserves on the inclined-plane, access is equipped with in the test tube of 10mL wheat juice, place 25 ℃ of shaking tables, 230r/min, after the cultivation overnight, changing in the little triangular flask that 100mL wheat juice is housed, is 0.8～1.0 o'clock (approximately behind 5～6h) until the OD value, and the PET bottle of 1000mL wheat juice is housed in the Bechtop access.
Beer fermentation: the PET bottle that 1000mL wheat juice will be housed rocks evenly, unclamps bottle cap, is semi-closed state, places constant incubator, and main fermentation temperature is controlled at 20 ℃.When the outward appearance pol is down to 4 ° of P, the PET bottle cap to be tightened, pressurize 4 days detects diacetyl content, if content is lower than 0.1mg/L, this PET bottle is placed 4 ℃ of refrigerators, is cooled to 0-2 ℃.During the fermentation, the variation of outward appearance concentration and diacetyl content as shown in Figure 8.As seen from Figure 8, the fermentation character of starting strain W303-1A and clone strain W303+padc is very close, and just clone strain W303+padc hypoglycemic speed and reduction of diacetyl speed are slower.
13. mensuration and the sensory evaluation thereof of 4-vinyl guaiacol content in the beer
After preserving for 1 week, measure the content of the flavour substancess such as alcohols, ester class and 4-vinyl guaiacol in the beer, and carry out sensory evaluation.
The measuring method of 4-vinyl guaiacol content: adopt high performance liquid chromatography, chromatographic condition is C
18Pillar 250mmx4mm, moving phase is H
2O/CH
3OH/H
3PO4, ratio is 540:450:10(V/V/V), flow l mL/min, temperature is room temperature.W303-1A compares with starting strain, and the 4-vinyl guaiacol content of clone strain W303+padc beer brewing has been brought up to 1.71mg/L from 1.13mg/L, and amplification reaches 51.3%(and sees Fig. 9).
Sensory evaluation: will be stored in 5 ℃ with the finished beer that starting strain W303-1A and clone strain W303+padc brewage, and judge with preparation.The personnel that one group rich abundant judges experience to brew beer and carry out the sense organ taste, the organoleptics propertys such as " Flos Caryophylli " flavor, " dizziness " sense, ester perfume, taste, bitter taste and aftertaste that beer embodies have been carried out the marking judge, assign to 5 minutes from 1, represent respectively from being poor to fine.Sample has two groups, adopts the blind method of commenting.Through sensory evaluation, very close with taste, bitter taste, the aftertaste characteristic of starting strain W303-1A and clone strain W303+padc beer brewing.As the result of expectation, W303-1A compares with starting strain, beer " Flos Caryophylli " flavor and the ester perfume (or spice) brewageed with clone strain W303+padc are more outstanding, contain more 4-vinyl guaiacol content in this and the beer and ester class closely bound up (see Table 1, Figure 10).In addition, " dizzy sense " of clone strain W303+padc beer brewing is more obvious, and this is relevant with higher higher alcohols content in the beer.
Table 1
14. when brewageing with starting strain W303-1A and clone strain W303+padc, the contrast of alcohols, ester class flavour substances content in the beer
Through gas chromatographic analysis, the beer that clone strain W303+padc is brewageed, except ethyl acetate, Isoamyl Acetate FCC, n-propyl alcohol, primary isoamyl alcohol content have obviously increase, all the other flavour substances content are little, concrete outcome is referring to table 1.
Reference examples
Being CN101397568A(application number 200810233517.X according to publication number) method put down in writing in the specification sheets embodiment prepares ferulic acid decarboxylase, and in the saccharification operation of Process of Beer Brewing, under 44 ℃ of (the forulic acid burst size is maximum) conditions, add, method by embodiment step 12 and step 13 is carried out, and detects the content of the 4-vinyl guaiacol in the beer.
After testing, the content of the 4-vinyl guaiacol in the method institute beer brewing that adds ferulic acid decarboxylase is as 1.53mg/L, 4-vinyl guaiacol content 1.13mg/L than starting strain W303-1A beer brewing is high, and lower than the 4-vinyl guaiacol content 1.71mg/L of the inventive method clone strain W303+padc beer brewing.
By to above data analysis, can find out that the beer that is made by technical solution of the present invention contains higher 4-vinyl guaiacol content, thereby can significantly improve the fragrance of top fermentation wheat beer, outstanding its typical Flos Caryophylli flavor.And compare with reference examples, do not need extra preparation or buy ferulic acid decarboxylase, saved brewing time, reduced production cost.
Claims (5)
1. a method that improves 4-vinyl guaiacol content in the top fermentation wheat beer is characterized in that, step is as follows:
(1) pass through pcr amplification PADC gene from subtilis, the PADC gene nucleotide series is shown in SEQ IDNO.1, and pcr amplification primer sequence is as follows:
Upstream primer: F-padc5 '-GCG
TCTAGAATGGAAAACTTTATCG-3 '
Downstream primer: R-padc5 '-GCG
AAGCTTTTATAATCTTCCCGC-3 ';
(2) after the PADC gene that step (1) is made is purified, be connected with restriction endonuclease HindIII double digestion and equally be connected plasmid YEp352 with restriction endonuclease HindIII double digestion through restriction endonuclease XbaI and be connected through restriction endonuclease XbaI, make recombinant plasmid YPADC, recombinant plasmid YPADC is converted into top fermenting yeast W303-1A, obtains top fermentation restructuring yeast strains W303+padc;
(3) utilize 11 ° of P wheat juice that the top fermentation restructuring yeast strains W303+padc that step (2) makes is carried out enlarged culturing, culture condition is: 25～28 ℃ of shaking tables, and 220～240r/min obtains top fermenting yeast W303+padc;
(4) the top fermenting yeast W303+padc that step (3) is made is inoculated in 11 ° of P wheat juice, be 16～18 ℃ in temperature and carry out fermentation culture to pol when being 4 ° of P, sealing and fermenting container pressurize to diacetyl content is lower than 0.1mg/L, be cooled to 0～2 ℃, preserved 5～8 days, making 4-vinyl guaiacol content is the beer of 1.6～1.8mg/L.
2. the method for claim 1 is characterized in that, in the described step (1), the PCR response procedures is: 94 ℃ of denaturation 3min; 94 ℃ of template sex change 30s, 52.6 ℃ of primer annealing 30s, 72 ℃ of primer extension 45s, 30 circulations; 72 ℃ are extended 10min.
3. the method for claim 1 is characterized in that, in the described step (2), recombinant plasmid YPADC transforms top fermenting yeast W303-1A and adopts the Lithium Acetate conversion method.
4. the method for claim 1, it is characterized in that, the preparation method of 11 ° of P wheat juice in described step (3) and the step (4) is as follows: after Fructus Hordei Germinatus and wheat malt mix by weight 3:2, pulverize with EBC standard mill, adopt infusion mashing after saccharification, after filtration, boil after, make 11 ° of P wheat juice.
5. method as claimed in claim 4 is characterized in that, the concrete steps of above-mentioned saccharification are: feed temperature is 37 ℃, keeps 20min; Be warming up to 45 ℃ and keep 30min, then be warming up to 52 ℃ and keep 40min; Be warming up to again 65 ℃, keep 70min; Being warming up at last 78 ℃ of leachings gets final product.
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| CN106010847A (en) * | 2016-08-02 | 2016-10-12 | 青岛啤酒股份有限公司 | Method for brewing muddy whole-malt white beer |
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| CN106753945A (en) * | 2016-12-23 | 2017-05-31 | 江南大学 | A kind of method for improving wheat beer characteristic flavor on basis |
| CN108315271A (en) * | 2018-04-09 | 2018-07-24 | 佛山市海天(高明)调味食品有限公司 | One primary yeast and its application |
| CN108315315A (en) * | 2018-04-23 | 2018-07-24 | 齐鲁工业大学 | A method of preparing ferulic acid decarboxylase |
| CN108315315B (en) * | 2018-04-23 | 2021-12-10 | 齐鲁工业大学 | Method for preparing ferulic acid decarboxylase |
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