CN103333811B - Kluyveromyces marxianus as well as composition and application thereof - Google Patents
Kluyveromyces marxianus as well as composition and application thereof Download PDFInfo
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Abstract
The invention provides novel kluyveromyces marxianus, a wine making microbial agent composition containing the kluyveromyces marxianus and applications of the kluyveromyces marxianus and the wine making microbial agent composition in alcoholic fermentation and white spirit making. The invention also provides a method for preparing the wine making microbial agent composition. The kluyveromyces marxianus provided by the invention has the advantages of high temperature resistance, high alcoholic strength resistance, high ester productivity and high alcohol yield. The kluyveromyces marxianus can be used for alcoholic fermentation at high temperature and formation of peculiar white spirit flavor.
Description
Field that the present invention belongs to:
The invention belongs to zymamsis and field is made in wine brewing, specifically, the present invention relates to kluyveromyces marxianus bacterium, containing the wine brewing antimicrobial composition of this bacterium, and this yeast and the application of its composition in zymamsis and brewed spirit.
Background technology:
Thermotolerant yeast has widespread use and is worth in fields such as zymamsis and brewed spirits.Compare with the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) of traditional non-refractory, the zymamsis that thermotolerant yeast carries out under comparatively high temps can reduce the consumption of enzyme, reduce the cooling cost in fermenting process, simultaneously, can realize enzymatic saccharification synchronizes with zymamsis and carries out (Pessani N K, Deng .Bioresource Technology, 2011,102:10618-10624).
Kluyveromyces marxianus (Kluyveromyces marxianus) be a kind of thermotolerant yeast that can produce inulinase (entangle quick etc., Food science, 2010,31(3): 198-202).This yeast strain is identified a kind of generally regarded as safe bacterial strain, can be used for zymin production, the single cell proteins such as beta-galactosidase enzymes and inulinase produces, ferments and produce flavour substances, high temperature alcohol fermentation etc., potentiality (Fonseca is widely used in fields such as zymamsis, food fermentation and zymins, G G, Deng .Appl Microbiol Biotechnol, 2008,79:339 – 354; Lane M M, waits .Antonie van Leeuwenhoek, 2011).
(the industrial microorganism such as Dai Zhenpeng, 2012,42(6): the best ethanol fermentation temperature range of 42-46) having reported heat-resisting kluyveromyces marxianus (K.marxiaus) IFO1777 is 34 ℃～40 ℃, in the time of 45 ℃, can ferment and produce 30.5g/L ethanol.(the modern food science and technology such as Qin Xiaojuan, 2009,25(8): 869-871) three strain wild-types are produced to the physiology patience of the high temperature resistant kluyveromyces marxianus of alcohol under differing temps, pH, ethanol concn, sugared concentration and be studied, result shows: growth under hot conditions of three strain wild-type thermotolerant yeast K.marxianus GX-17, HN-79 and HN-138 bacterial strain and produce alcohol ability apparently higher than Angel Yeast, but the alcohol-tolerant ability of Angel Yeast is apparently higher than this three strains wild-type thermotolerant yeast.(biotechnology journal, 2008,24 (11): 1931-1936) utilize kluyveromyces marxianus YX01 to have inulinase throughput and the well behaved feature of ethanol fermentation, direct fermentation inulin generates ethanol such as Yuan Wenjie.Experimental result shows, the suitableeest leavening temperature of this bacterial strain is 35 ℃.Ma Xiaoyan etc. (brewing science and technology, 2006,146(8): 37-40) with ultraviolet mutagenesis protoplasma selection-breeding method screening fermented whey High-alcohol-yield bacterial strain kluyveromyces marxianus bacterial strain ZR-20.Result shows, this bacterial strain reaches 4.2% at 28 ℃ of bottom fermentation 96h ethanol productions.(the Biotechnol Lett such as Pang Z W, 2010,32:1847-1851) screen the wild-type kluyveromyces marxianus bacterial strain GX-15 of a strain High-alcohol-yield, 40 ℃ of bottom fermentation alcoholic strengths reach 5.8g/100mL, and the mutant strain GX-UN120 fermented wine precision after selection by mutation reaches 6.9g/100mL.(Appl Biochem Biotechnol, 2004,112 (1): 25-35) research shows that K.marxianus IMB3 energy fermenting must under hot conditions produces the alcohol of 3-4% such as Kourkoutas Y.
(the World J Microbiol Biotechnol such as Amaya-Delgado L, 2013,29 (5): 875-881) utilize K.marxianus GRO6 to produce isopropylcarbinol and ethyl acetate ability higher than the feature of yeast saccharomyces cerevisiae (S.cerevisiae), this bacterial strain is produced to improve the content of the volatile flavor substance of wine for industrialized tequila.Urit T etc. (Appl Microbiol Biotechnol, 2012,96 (5): 1313-23.) research shows, in the substratum of limiting the quantity of at iron ion, K.marxianus can utilize whey to produce ethyl acetate.Medeiros AB etc. (Biochem Eng J.2000,6 (1): 33-39) research shows that kluyveromyces marxianus can utilize manioc waste the like waste through flavour substancess such as Produced by Solid-state Fermentation ethyl acetate.
Chinese traditional liquor fermenting process is to utilize song and environment to comprise the solid-state mixed fermentation result of the multiple-microorganisms such as bacterium, yeast, mould, is a kind of spontaneous fermentation, and wherein yeast plays a leading role in the fermenting process of wine unstrained spirits.The yeast kind existing in liquor fermentation process is various, and function also has notable difference, except alcohol produces capacity variance, has substantial connection with the generation of the flavour substances of wine.In alcoholic fermentation process, the generation of ester and special yeast kind have substantial connection.Compare the content of some composition when multiple saccharomycetic mixed fermentation can reduce (Mendoza et al., 2007) or improve the fermentation of (Ciani et al., 2006) single culture with single saccharomycetic pure-blood ferment.The mixed fermentation of kluyveromyces and torula shows low acetate yield, and the existence of Non-Saccharomyces can improve the content of ethyl acetate in white wine, and (Zhao Jun, China brewages, 2011(12): 145-147).Wang Xuliang etc. (wine brewing, 2012,7) be take scent type fermented wine unstrained spirits as screening source, filter out two strains and are applicable to high yield ethyl acetate and acetic acid bacterial strain Ac-47 and the Ac-19 that fen-flavor type white spirit is brewageed, and through identifying, are Picha anomala.Dong Shiwei etc. (China brewages, 2012,2) have carried out the screening and application of ester-producing yeast in soybean-flavor liquor.In soybean-flavor liquor, ethyl acetate content is on the low side.This research is screened functional microorganism in soybean-flavor liquor brewing process, obtains the yeast strain that a plant height produces ethyl acetate, and has carried out aborning Preliminary Applications, has obtained good effect.(the brewing science and technology such as Xu Ling, 2010,12) carried out separation screening and the application of a few strain SHENGXIANG yeast, from sesame cordiale unstrained spirits, separation screening obtains 8 strain SHENGXIANG yeast bacterial strains, therefrom choose 4 strain strain excellents and be applied to the production of sesame cordiale class, can effectively improve the quality of the fragrant former wine of sesame.
In Chinese traditional liquor making method, have the process of a high temperature stack-up, require to pile up temperature and reach more than 45 ℃, therefore, the fermentation of high quality liquor, require wine brewing microbial inoculum there is high temperature resistant, resistance to high ethanol concn, produce ester rate high, produce alcohol rate high.Although China's tradition yeast wine produces, ester rate is high, and liquor output rate is lower, and particularly in Daqu, the composition of microorganism is complicated, and quality of white spirit is unstable; And single yeast or wine brewing antimicrobial composition non-refractory, although liquor output rate is high, product ester rate is low, causes liquor flavor composition too single, local flavor is not true to type, and has reduced the quality of white wine.Therefore, it is very significant obtaining a good wine brewing microbial inoculum, and it is applied to actual production, can improve alcohol output, put forward high ester yield rate, makes white wine have more local flavor.
A kind of manufacture method of kluyveromyces marxianus transformant is disclosed in Chinese patent CN201180021394.3; Chinese patent CN201210005579.1 discloses a kind of preparation method of serum cholesterol reduction Kluyveromyces marxianus freeze-dried powder; Chinese patent CN200710159198.8 discloses a kind of eukaryon expression of kluyveromyces marxianus exoinulinase; Chinese patent CN201180008591.1 provides the high expression method of the transformant goal gene in kluyveromyces marxianus source.The weak point that high temperature resistant kluyveromyces marxianus (K.marxianus) bacterial strain of having reported at present exists shows as: kluyveromyces marxianus alcohol-tolerant ability is poor, not as yeast saccharomyces cerevisiae (Rosa and Sa-Correia.Enzyme Microb Technol1992,14:23-27.); Kluyveromyces marxianus at high temperature produce alcohol ability a little less than; Kluyveromyces marxianus and other yeast compositions are produced to the research of local flavor ability shortage in alcoholic fermentation process.Also do not there is at present kluyveromyces marxianus bacterial strain and the application report of combination microbial inoculum in white wine thereof high temperature resistant, resistance to high alcohol concn.
Invention technology contents:
An object of the present invention is to provide a kind of new kluyveromyces marxianus bacterial strain, this bacterial strain is high temperature resistant, ethanol-tolerant, use this bacterial strain can at high temperature carry out zymamsis, the white wine of fermentative production has high ester yield rate and high ethanol production, can form white wine peculiar flavour.
Another object of the present invention is to provide a kind of wine brewing antimicrobial composition that contains kluyveromyces marxianus.
A further object of the present invention is to provide the application of kluyveromyces marxianus in high temperature alcohol fermentation.
A further object of the present invention is to provide the method for preparing the antimicrobial composition of making wine.
A further object of the present invention is to provide the application of wine brewing antimicrobial composition in liquor fermentation.
The invention discloses the new kluyveromyces marxianus bacterium of a strain, it is characterized in that, this bacterial strain is kluyveromyces marxianus HY32 strain (Kluyveromyces marxianus HY32), be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC M2013258, and preservation date is on June 13rd, 2013.CCTCC is called for short at Chinese Typical Representative culture collection center, is positioned at Wuhan City, Hubei Province Wuhan University in the school, postcode 430072, phone: 027-68752319, Email:cctccwhu.edu.cn.Kluyveromyces marxianus HY32 strain obtains through screening from soil in Yunnan separation.This bacterial strain has following feature:
Kluyveromyces marxianus HY32 strain morphological specificity is: 1～2d grows on YPD solid medium, bacterium colony is oyster white, cheese shape, smooth surface, more moistening, in YPD liquid nutrient medium, at 37 ℃, cultivate 12～14h, cell is spherical in shape, avette or ellipse, and size is (2.2～5.0) μ m * (2.5～5.5) μ m.
Kluyveromyces marxianus HY32 strain biochemical character is: can ferment and utilize sucrose, semi-lactosi, trehalose, maltose, wood sugar, lactose, cellobiose.
Kluyveromyces marxianus HY32 strain is high temperature resistant and growth characteristics is: streak inoculation on YPD flat board, at 42 ℃ and 45 ℃, cultivate equal well-grown, and at 48 ℃, still can grow; And that Marx's Crewe dimension reference culture AS2.1549 grows at 42 ℃ and 45 ℃ is faint, at 48 ℃, cannot grow.
Kluyveromyces marxianus HY32 strain alcohol-tolerant ability feature is: at 37 ℃, and in containing 4% and 6% ethanol substratum, the OD of HY32
600value is respectively 1.508 and 0.580, is 1.6 times and 1.9 times of Angel yeast saccharomyces cerevisiae TRADY.The alcohol-tolerant ability that shows yeast HY32 is good.
Kluyveromyces marxianus HY32 strain thermophilic fermentation produces alcohol ability characteristics: at 42 ℃, HY32 is in liquid zymamsis test, and alcoholic strength reaches 6.61%(v/v), than reference culture AS2.1549 ethanol production, exceed 70.36%.
To kluyveromyces marxianus HY32, the pcr amplification and order-checking experiment of 26S rDNA carried out in strain, analyze and show: the homology of the 26S rDNA of bacterial strain HY32 and Kluyveromyces marxianus GX-17 has reached 99%, determines that bacterial strain HY32 is a kind of kluyveromyces marxianus.
The invention also discloses a kind of wine brewing antimicrobial composition containing kluyveromyces marxianus HY32 strain, this wine brewing antimicrobial composition major ingredient is kluyveromyces marxianus HY32 strain and extracellular products.
Preferentially, the invention also discloses a kind ofly containing kluyveromyces marxianus HY32 strain wine brewing antimicrobial composition, it is characterized in that it contains following component (viable count ratio):
Kluyveromyces marxianus 50%
Distillery yeast 50%
Wherein kluyveromyces marxianus is CCTCC M2013258, and distillery yeast is CCTCC AY92003.
Distillery yeast CCTCC AY92003, for conventional common micro-organisms bacterial classification, publishes in Chinese Typical Representative culture collection center catalogue, and in open state, scientific worker can ask for to Chinese Typical Representative culture collection center.
Wine brewing antimicrobial composition disclosed by the invention is liquid or solid-state.Preferential, wine brewing antimicrobial composition disclosed by the invention is solid-state.
The invention also discloses the method for preparing solid brewing antimicrobial composition, the method comprises the steps:
(1) actication of culture: inoculate respectively kluyveromyces marxianus HY32 strain and distillery yeast CCTCC AY92003 in the wort of the pol Wei12 of sterilizing Bahrain or the bent juice triangular flask substratum of rice, 28～30 ℃ of shaking tables are cultivated 18-24 hour;
(2) solid culture: the batching of solid culture is: wheat bran 80%, whole meal flour 20%, water material moistening, boiling cold loose after, 2 Yeasts liquid of activation culture press to mounted box after the combined inoculation of 1:1 volume ratio, maintenance 30 ℃ of left and right of room temperature.35 ℃ of left and right of product temperature, cultivate and ferment complete in 2 days;
(3) vacuum-drying: the solid sample after fermentation is carried out to vacuum drying treatment at 45 ℃, and sample contains
Water rate is packed lower than 10% final vacuum.
The invention also discloses the application of new kluyveromyces marxianus HY32 strain in zymamsis.
The invention also discloses the application of wine brewing antimicrobial composition in brewed spirit.
Advantage of the present invention:
1, kluyveromyces marxianus HY32 provided by the invention strain is the new bacterial strain of a strain, this bacterial strain can resistance to 48 ℃ of high temperature, and at 45 ℃ well-grown, also higher 3 ℃ than kluyveromyces marxianus reference culture AS2.1549, the latter only can be 45 ℃ of faint growths, cannot be 48 ℃ of growths.
2, kluyveromyces marxianus HY32 strain ethyl acetate productive rate is high.In liquid alcohol fermentation, the ethyl acetate force of labor of HY32 exceeds 31.49% than reference culture AS2.1549, than Angel yeast saccharomyces cerevisiae TRADY, exceeds nearly 10 times.
3, kluyveromyces marxianus HY32 strain ethanol production under thermophilic fermentation condition is high.37 ℃ of fermentations, HY32 ethanol production is suitable with reference culture AS2.1549, but 42 ℃ of fermentations, HY32 exceeds 70.36% than reference culture AS2.1549.
4, HY32 alcohol tolerance is better than Angel yeast saccharomyces cerevisiae TRADY.At 37 ℃, in containing 4% and 6% ethanol substratum, the OD of HY32
600value is respectively 1.6 times and 1.9 times of Angel yeast saccharomyces cerevisiae TRADY.At 42 ℃ and 45 ℃, HY32 alcohol tolerance is all significantly higher than Angel yeast saccharomyces cerevisiae TRADY.
To be that kluyveromyces marxianus HY32 strain has high temperature resistant, ethanol-tolerant, high ethyl acetate force of labor and stronger product alcohol ability.
Accompanying drawing explanation
Fig. 1. kluyveromyces marxianus HY32 strain 26S rDNA D1/D2 region sequence phylogenetic tree
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these embodiment are only for the present invention is described, and can not limit the scope of the invention.
Embodiment
Separation and the evaluation of embodiment 1 kluyveromyces marxianus HY32
The separation of 1.1 kluyveromyces marxianus HY32 strains
Separated acquisition from soil in Yunnan.To after sample activation, join in the YPD liquid nutrient medium of pH4.5-5.0, rotating speed 200rpm, during 30 ℃ of temperature, enrichment culture is 48 hours, then getting the nutrient solution of 100 μ l after certain gradient dilution is applied on YPD substratum solid plate, cultivate after 48 hours for 30 ℃, single bacterium colony of picking products of typical yeast form, then after the purifying three times of repeating to rule on same medium solid plate, 4 ℃ of preservations.By high-temperature cultivation and ethanol-tolerant Preliminary Experiment, screen, finally according to liquid zymamsis test-results and the screening of cassava fermentation test result, produce alcohol ability and produce all higher bacterial strains of ester rate.The inventor is preserved in Chinese Typical Representative culture collection center by the new kluyveromyces marxianus HY32 strain (Kluyveromyces marxianus HY32) of a strain of separation, and deposit number is CCTCC M2013258.Preservation day is on June 13rd, 2013.CCTCC is called for short at Chinese Typical Representative culture collection center, is positioned at Wuhan City, Hubei Province Wuhan University in the school, postcode 430072, phone: 027-68752319, Email:cctccwhu.edu.cn.
The evaluation of 1.2 kluyveromyces marxianus HY32 strains
By morphological specificity observe, Physiology and biochemistry is measured and 26S rDNA D1/D2 regional gene the sequencing results is identified HY32.In authentication, the high temperature resistant yeast saccharomyces cerevisiae TRADY in this experimental selection Angel, kluyveromyces marxianus reference culture AS2.1549 bacterial strain in contrast.Angel is high temperature resistant, and yeast saccharomyces cerevisiae TRADY is market circulation commodity; Kluyveromyces marxianus reference culture AS2.1549 is conventional common micro-organisms bacterial classification, and from DSMZ of the Chinese Academy of Sciences, scientific worker can ask for.
1.2.1 kluyveromyces marxianus HY32 strain morphological specificity is observed
1～the 2d that grows on YPD solid medium, bacterium colony is oyster white, cheese shape, smooth surface, more moistening is cultivated 12～14h at 37 ℃ in YPD liquid nutrient medium, and cell is spherical in shape, avette or ellipse, size is (2.2～5.0) μ m * (2.5～5.5) μ m.
1.2.2 by the analysis of 26S rDNA D1/D2 regional sequence, carry out Phylogenetic evaluation
The genomic dna of yeast strain of take is done pcr amplification reaction as template, amplification yeast strain 26S rDNA D1/D2 region, amplimer adopts universal primer NS1(5'-GTAGTCATATGCTTGTCTC-3') and NS4(5'-CTTCCGTCAATTCCTTTAAG-3').
PCR reaction system (25 μ L): 10 * PCR damping fluid is (containing the Mg of 20mmol/L
2+) 2.5 μ L, primer NS1/NS4(10 μ mol/L) each 1 μ L, template DNA (about 50ng/mL) 1.8/2.8 μ L, dNTP(10mmol/L each) 0.5 μ L, Taq enzyme (5U/ μ L) 0.2 μ L, distilled water 18/17 μ L.
Pcr amplification condition: 94 ℃ of denaturation 5min, 94 ℃ of sex change 40s, 50 ℃ of annealing 40s, 72 ℃ are extended 60s, and after 30 circulations, 72 ℃ are extended 5min.Gained PCR product is served sea raw work biotechnology purifying order-checking, above-mentioned NL1, NL4 for sequencing primer.
After checking order by molecule, the homology of finding the 26SrDNA of bacterial strain HY32 and Kluyveromyces marxianus GX-17 has reached 99%, meet difference between the fixed of the same race interior different strains of Kuttzman & Robnett and be no more than 1% standard, determine that bacterial strain HY32 is a kind of kluyveromyces marxianus.Fig. 1 is the phylogenetic tree of doing according to 26S rDNA D1/D2 regional sequence.
1.2.3 the Physiology and biochemistry measuring result of kluyveromyces marxianus HY32 strain
(1) biochemical character fermentation test
HY32 bacterial strain the results are shown in Table 1 to sugared utilization.
The fermentation test result of the different carbon sources of table 1 kluyveromyces marxianus HY32
(2) kluyveromyces marxianus HY32 heat-resisting ability is measured
Each yeast strain streak inoculation, on YPD flat board, is cultivated respectively at 37 ℃, 42 ℃, 45 ℃ and 48 ℃, observe Yeast Growth situation, the results are shown in Table 2.Result shows that HY32 bacterial strain is at 45 ℃ of well-growns, and reference culture AS2.1549 and yeast saccharomyces cerevisiae TRADY do not grow completely this temperature is next.
The temperature tolerance test-results of table 2HY32 and reference culture
Note: ++ represent well-grown; + represent that growth is general; +-represent, growth was faint;-represent not grow.
Embodiment 2. kluyveromyces marxianus HY32 strains and the comparison of yeast saccharomyces cerevisiae TRADY alcohol-tolerant ability
The seed liquor of bacterial strain TRADY and HY32 is transferred to respectively and contains alcohol concn 0%, 4%(v/v), 6%(v/v) YPD liquid nutrient medium in, be placed at 37 ℃, 42 ℃, 45 ℃ after 170rpm shake-flask culture 6h, survey OD
600value.As shown in table 3, at 37 ℃, added in the substratum of 4% ethanol, TRADY and HY32 all can grow, but the tolerance of HY32 is better than TRADY, the OD of bacterial strain HY32
600value is 1.6 times of TRADY; At 37 ℃, added in the substratum of 6% ethanol, the energy for growth of TRADY and HY32 all a little less than, but the tolerance of HY32 is still better than TRADY, the OD of bacterial strain HY32
600value 1.9 times of TRADY.This shows that the alcohol concn adding is higher, and the relative alcohol-tolerant ability that bacterial strain HY32 shows is more remarkable.
Table 3 yeast strain HY32 and the TRADY alcohol-tolerant ability comparison under differing temps
Ester capability analysis is produced in the fermentation of embodiment 3 kluyveromyces marxianus HY32 strains
In Cassava alcohol fermentation test, no matter be 37 ℃ or 40 ℃ of fermentations, as table 4, the ethyl acetate output of kluyveromyces marxianus HY32 strain is ten times of left and right of Angel yeast saccharomyces cerevisiae TRADY, and n-propyl alcohol and content of ethyl lactate are all lower, there is good fermentation and be worth.
In liquid zymamsis test, as shown in table 5 at 40 ℃ of bottom fermentations, the ethyl acetate output of kluyveromyces marxianus HY32 strain exceeds 31.49% than reference culture AS2.1549, and ethyl lactate is lower by 50.67% than reference culture, is conducive to form the good local flavor of fermented wine.
Table 4 yeast strain HY32 and TRADY Cassava alcohol fermentation sample gas analysis of hplc result
The liquid zymamsis sample gas analysis of hplc result of table 5HY32 and reference culture 2.1549
Embodiment 4.HY32 bacterial strain thermophilic fermentation produces alcohol capability analysis
Kluyveromyces marxianus HY32 strain has higher ethanol production.As shown in table 6 at 37 ℃, the liquid zymamsis test of HY32 shows that its liquor-producing ability is 71.15% of Angel yeast saccharomyces cerevisiae TRADY, and with the liquor-producing ability of kluyveromyces marxianus reference culture 2.1549 quite (table 7); As shown in table 7 at 42 ℃, HY32 is in liquid zymamsis test, and ethanol production exceeds 70.36% than reference culture 2.1549, and TRADY bacterial strain can not be grown completely and be carried out zymamsis effect at 42 ℃.
The glucose zymamsis experiment of table 6 yeast strain
The glucose zymamsis experimental result of table 7 bacterial strain 2.1549 and HY32
The preparation method of embodiment 5HY32 wine brewing antimicrobial composition
5.1 actication of culture: inoculate respectively kluyveromyces marxianus HY32 strain and distillery yeast CCTCC AY92003 in the wort of the pol Wei12 of sterilizing Bahrain or the bent juice triangular flask substratum of rice, 28～30 ℃ of shaking tables are cultivated 18-24 hour;
5.2 solid culture: the batching of solid culture is: wheat bran 80%, whole meal flour 20%, water material moistening, boiling cold loose after, 5.1 activation culture 2 Yeasts liquid press to mounted box after the combined inoculation of 1:1 volume ratio, maintenance 30 ℃ of left and right of room temperature.35 ℃ of left and right of product temperature, cultivate and ferment complete in 2 days;
5.3 vacuum-dryings: the solid sample after fermentation is carried out to vacuum drying treatment at 45 ℃, and sample water ratio is packed lower than 10% final vacuum;
5.4 yeast preparation compositions measurement results: in wine brewing antimicrobial composition prepared by employing dilution plate determination experiment chamber, yeast viable count reaches 2,000,000,000/g left and right.Kluyveromyces marxianus HY32 and CCTCC AY92003 ratio are 1:1
Embodiment 6 kluyveromyces marxianus HY32 and the comparison of yeast saccharomyces cerevisiae combination microbial inoculum high temperature alcohol fermentation ability
Kluyveromyces marxianus HY32 strain and distillery yeast CCTCC AY92003 are made to starter with Different Weight than mixing, and compare with Angel high temperature resistant Active Dry Yeast (TRADY), the zymamsis that to carry out take at 40 ℃ cassava be raw material, the results are shown in Table 8.
40 ℃ of Cassava alcohol fermentation results of table 8 yeast hybrid bacterial strain
As can be seen from Table 8, in the yeast zymamsis sample that HY32 strain mixes by 3 kinds of different ratioss, ethanol content is all high than Angel TRADY Active Dry Yeast fermented sample.The fermented sample ethanol content of wherein, mixing by 1:1 by bacterial strain CCTCC AY92003 and HY32 has improved 12.5% than Angel TRADY active dry yeast product fermented sample.
The application of embodiment 7HY32 wine brewing antimicrobial composition in liquor fermentation
7.1 test method
Steaming after 900kg Chinese sorghum, steamed rice husk is allocated in the cruel steaming of wine after drinking into, and after spreading for cooling, control group adds Daqu, stirs, and Daqu consumption is charging capacity 20%, then carries out high temperature stacking fermentation 3 days, and heap temperature reaches after 45 ℃, and pit entry fermentation goes out pond distillation after 30 days.Experimental group is the HY32 wine brewing antimicrobial composition that adds the Daqu of same amount simultaneously to add to prepare by embodiment 5 methods, and HY32 wine brewing antimicrobial composition consumption is charging capacity 0.1%.
The detection of relevant wine unstrained spirits, wine sample is referring to pertinent literature; The physical and chemical index of wine detects the white wine test method detection of pressing GB10345-89; Key instrument: gas chromatographicanalyzer.
7.2 fermented wine unstrained spirits analytical resultss
From table 9 and table 10, can find out: the starch that goes out pond wine unstrained spirits contrast is content 7.6%.The processing starch content that adds HY32 wine brewing antimicrobial composition is 7.1%, and lower than the content of contrast wine unstrained spirits, and alcoholic strength is 4.9, higher than the alcoholic strength of contrast.Illustrated and in raw material, added HY32 wine brewing antimicrobial composition can utilize more fully starch, fermented more thorough.
Table 9 goes out, enters to store pond wine unstrained spirits physico-chemical analysis result
7.3 vinosity physics and chemistry stratographic analysis result test-results and analyses
As can be seen from Table 10: experimental group wine sample total acid content is more suitable than control group, total ester and ethyl acetate are all high than contrast.The yield of liquor situation statistics is shown, the alcoholic strength 4.5% of control group, the experimental group alcoholic strength that contains HY32 wine brewing antimicrobial composition active dry yeast is 4.9%, the alcoholic strength in wine unstrained spirits has improved 8.9%.In former wine, ethyl acetate content has increased by 11.8% simultaneously, and total ester content has increased by 5.2%, obvious to improving mouthfeel effect.
The main physical and chemical index of table 10 and trace ingredients statistics (unit: g/L)
Sensory evaluation contrast is: control group wine sample mark is at 90-95, and comment is that sweet mellow, fragrance harmony, wine body are plentiful; Experimental group wine sample mark is at 95-100, and comment is that taste alcohol grace, quiet and tastefully laid out exquisiteness, wine body are plentiful.Due to the Main Fragrance that ethyl acetate is white wine, showing to be used in conjunction with HY32 dry yeast can obviously improve vinosity, and the fragrance in wine is coordinated more, and mouthfeel is better.
Claims (10)
1. a strain kluyveromyces marxianus, is characterized in that, this bacterial strain is kluyveromyces marxianus HY32 strain (Kluyveromyces marxianus HY32), is preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC M2013258.
2. containing a microbiobacterial agent for the kluyveromyces marxianus HY32 strain described in claim 1, it is characterized in that its activeconstituents is kluyveromyces marxianus HY32 strain thalline and meta-bolites thereof.
3. the wine brewing antimicrobial composition containing the kluyveromyces marxianus HY32 strain described in claim 1.
4. according to the wine brewing antimicrobial composition described in claim 3, it is characterized in that it contains following component, in viable count ratio:
Kluyveromyces marxianus 50%
Distillery yeast 50%
Wherein kluyveromyces marxianus is CCTCC M2013258, and distillery yeast is CCTCC AY92003.
5. according to the wine brewing antimicrobial composition described in claim 3, it is characterized in that described wine brewing antimicrobial composition is solid-state microbial inoculum.
6. the method for the wine brewing antimicrobial composition described in preparation claim 4, comprises the steps:
(1) actication of culture: inoculate respectively kluyveromyces marxianus HY32 strain and distillery yeast CCTCC AY92003 in the wort of the pol Wei12 of sterilizing Bahrain or the bent juice triangular flask substratum of rice, 28～30 ℃ of shaking tables are cultivated 18-24 hour;
(2) solid culture: the batching of solid culture is: wheat bran 80%, whole meal flour 20%, water material moistening, boiling cold loose after, 2 Yeasts liquid of activation culture press to mounted box after the combined inoculation of 1:1 volume ratio, maintenance 30 ℃ of left and right of room temperature; 35 ℃ of left and right of product temperature, cultivate and ferment complete in 2 days;
(3) vacuum-drying: the solid sample after fermentation is carried out to vacuum drying treatment at 45 ℃, and sample water ratio is packed lower than 10% final vacuum.
7. the application of the kluyveromyces marxianus HY32 strain described in claim 1 in zymamsis.
8. the application of the kluyveromyces marxianus HY32 strain described in claim 1 in brewed spirit.
9. the application of the wine brewing antimicrobial composition described in claim 3 in zymamsis.
10. the application of the wine brewing antimicrobial composition described in claim 3 in brewed spirit.
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| CN107177519A (en) * | 2017-06-20 | 2017-09-19 | 湖北白云边酒业股份有限公司 | Schizosaccharomyces pombe bacterium, its composition and application |
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| CN110628653B (en) * | 2019-09-03 | 2023-04-07 | 江苏大学 | Proliferation medium of kluyveromyces marxianus and preparation method thereof |
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| CN112322509B (en) * | 2020-11-04 | 2022-11-01 | 湖北白云边酒业股份有限公司 | Candida parapsilosis with low temperature resistance and high alcohol yield, and composition and application thereof |
| CN113278536B (en) * | 2021-05-14 | 2022-05-17 | 劲牌有限公司 | Low-yield n-propanol Kluyveromyces marxianus and application thereof in fen-flavor Xiaoqu liquor |
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