CN101029300A - Basylous yeast and its use in brewing myrica rubra - Google Patents
Basylous yeast and its use in brewing myrica rubra Download PDFInfo
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Abstract
An acid-decreasing yeast (Issatchenkia orientalis)xlc6 is prepared by separating it from red bayberry pulp, citric acid-alcohol fermenting and degrading alcohol from citric acid. The acid-decreasing total is above 90% and can be used to ferment fruit wine.
Description
Technical field
The present invention relates to microorganism field, be specifically related to the Issatchenkia orientalis of a primary yeast, particularly Issatchenkia, also relate to this yeast and fall application in the acid at fruit wine.
Background technology
In the production process of red bayberry fruit wine, the problem that acidity is too high is usually perplexing manufacturer and consumers in general.Conventional acid reduction method is that acid falls in chemistry and acid falls in physics.To fall sour effect remarkable though acid falls in chemical method, very big to local flavor and color and luster influence.Ion exchange resin and electrodialysis are the main method that acid falls in domestic and international physics.Studies show that the sour effect of falling of ion exchange resin is remarkable, but resin adsorbs a large amount of polyphenols simultaneously, cause wine body colour pool seriously to be faded and the loss of health-care components; Electrodialysis fall sour effect chemistry acid is fallen and ion-exchange-resin process will be got well, but facility investment is big.
Biological acid reduction is the method that adopts some milk-acid bacteria or yeast that the organic acid in fruit wine or the fruit juice is decomposed or transforms, this method not only can be fallen acid, very little to the influence of fruit wine local flavor and color and luster simultaneously, be considered at present than chemistry and the more feasible method of physics acid lowering method.Wherein utilize milk-acid bacteria the oxysuccinic acid in the grape wine to be converted into technology existing very long applicating history in Production of Wine of lactic acid.In addition, find that also fission yeast also can degrade the oxysuccinic acid in the fruit wine.Main organic acid is tartrate and oxysuccinic acid in the grape wine, and wherein oxysuccinic acid is a diprotic acid, and has pungency, and sour is stronger, after lactic acid bacteria tranformation is monoprotic acid lactic acid, grape wine acidity is reduced, and it is more soft that mouthfeel becomes.
State Intellectual Property Office has announced the application for a patent for invention of " a kind of acidity reducing yeast of grape wine and cultural method thereof " on January 18th, 2006, the disclosed acidity reducing yeast of grape wine of this patent application is to be parental plant with wine yeast 1450 and drinks wine coccus SD-2a, uses that Protoplast Fusion Technique builds.Its form, size and colony characteristics are all similar to wine yeast 1450, can carry out malo-lactic fermentation under the environment of high acidity, reduce the malic acid content of fruit wine.The ability of its fermentation alcohol is fallen sour ability near Oenococcus Oeni SD-2a near wine yeast 1450, falls sour amount and can arrive 77.6%.Therefore yet the disclosed yeast of this patent application does not but transform the effect of citric acid, and inapplicable organic acid is the acid of falling of the cranberry juice of citric acid or red bayberry fruit wine more than 80%.
Disclose the pichia yeast Pichia ohmer Y19 that a strain separates in the Ph D dissertation that the Zhao Yu of University Of Science and Technology Of Tianjin in 2003 is flat " hawthorn comprehensive utilization and development research " and can carry out citric acid-ethanol fermentation from hawthorn fruit, and the application of this yeast in brewing hawthorn wine.Y19 has in haw juice and well falls sour ability, falls sour total amount up to 80%, and very little to the flavones content influence, but this article does not relate to the bacterial strain of red bayberry fruit wine biological acid reduction.
Summary of the invention
The object of the present invention is to provide a kind of new acid leaven that falls.
Another object of the present invention is to provide the application of this yeast in the red bayberry brewing fruit wine.
The present invention realizes that the above-mentioned purpose technical scheme is: Issatchenkia orientalis (Issatchenkia orientalis) xlc6, CCTCC NO.M 207003.
Issatchenkia orientalis (Issatchenkia orientalis) xlc6 separates from waxberry flesh, is preserved in Chinese typical culture collection center (being called for short CCTCC) on January 15th, 2007, and preserving number is M 207003.
Issatchenkia orientalis (Issatchenkia orientalis) xlc6 25 ℃ of cultivations in falling sour substratum were observed after 2 days, and cell is a long oval or cylindrical, single-ended gemmation, the citric acid fermented generation ethanol of energy; Bacterium colony is flat in malt extract medium fold, tarnish, and the edge is irregular, can form about 1～6 thecaspore, belongs to syngenesis.Wherein, describedly fall sour substratum and be dissolved in the 1L deionized water by ammonium sulfate 5g, potassium primary phosphate 1g, sal epsom 0.5g, Calcium Chloride Powder Anhydrous 0.1g, sodium-chlor 0.1g, yeast extract paste 0.2g and citric acid 20g and make; Described malt extract medium is that pol is that 10 degree Beaume, pH are 5.4 wort, contains 2% agar.
Issatchenkia orientalis (Issatchenkia orientalis) xlc6 can cultivate with common yeast culture base, and as the YEPD substratum, culture temperature is 20～30 ℃, and is aerobic; Can adopt the preservation of whiteruss preserving process, preservation is made up of 2% glucose, 1% peptone, 0.5% yeast extract paste, 2% agar and distilled water with substratum.
The citric acid fermented generation ethanol of Issatchenkia orientalis (Issatchenkia orientalis) xlc6 energy, can in the process of fermentation, reduce the content of citric acid, it falls sour amount and can reach more than 90%, can be used for mainly containing acid and is the fruit brewing fruit wine of citric acid, as the red bayberry brewing fruit wine.
Issatchenkia orientalis (Issatchenkia orientalis) xlc6 is applied to the method for red bayberry brewing fruit wine, and this method may further comprise the steps:
(1) with yeast saccharomyces cerevisiae with 37 ℃ of activation of 1～6% sucrose solution, 20～40min, after the sodium alginate of sterilization cooled 0.5～6% mixes, inject 1～10% calcium chloride or liquor alumini chloridi, form gelled pill, wash with sterilized water after leaving standstill 20～60min;
(2) serve as the fermentation base-material with the clarification original juice of red bayberry, adjusting soluble solid content with sugar is 18～28%, and inoculation is through the immobilized yeast saccharomyces cerevisiae 0.1～2% of alginate calcium/aluminium, ferment the total reducing sugar 0～12g/L to the mash that ferments, membrane filtration in 10～30 ℃, get filtrate, get wine base A;
(3) with xlc6 after the domestication of anti-2～12% alcohol, be inoculated in the clarification original juice of red bayberry, inoculum size is 10
6～10
9Cfu/mL, 15～25 ℃ of total acid 0～6g/L to the original juice of red bayberry that ferment, sour Normal juice must be fallen, serve as the fermentation base-material to fall sour Normal juice then, adjusting soluble solid content with sugar is 18～28%, and inoculation is fermented through the immobilized yeast saccharomyces cerevisiae 0.1～2% of alginate calcium/aluminium, total reducing sugar 0～12g/L to the mash ferments, membrane filtration is got filtrate, gets wine base B;
(4) wine base A and wine base B are blent by 1: 1～5, get final product.
Aforesaid method is brewageed red bayberry fruit wine and is needed 15～25 days, the red bayberry fruit wine alcoholic strength 9～16% of gained, total reducing sugar≤12g/L, total acid≤8g/L, be red-purple to red-brown, clear is glossy, have refreshing happy, soft taste and fresh please into former fruital flavor, the wine body is complete, sour is suitable; The red bayberry fruit wine of blending gained can also carry out ageing more than 1 year or 1 year under 5～20 ℃, its color and luster, local flavor and mouthfeel are further improved.
The evaluation of Issatchenkia orientalis (Issatchenkia orientalis) xlc6:
1, molecular biology identification:
Extract the DNA (extracting method is seen 84 pages of A Adam Si works " yeast genetics method experiment guide ") of micro-xlc6, according to Kurtzman﹠amp; The method of Robnett, adopt primer NL1 (5 '-GCA TAT CAA TAA GCG GAG GAA AAG-3 ') and NL4 (5 '-GGT CCG TGTTTC AAG ACG G-3 ') pcr amplification xlc6 bacterial strain 26S rDNA nearly 5 ' the D1/D2 zone of holding, and carry out 36 circulations by following reaction conditions: 94 ℃, 1min; 53 ℃, 1min; 72 ℃, 1min 20s.Gained PCR product is delivered to the handsome Bioisystech Co., Ltd in Shanghai and is checked order.
Xlc6 bacterial strain PCR product is 561 bases altogether, and is as follows.
GCGGCGAGTGAaGCGGCAAGAGCTCAGATTTGAAATCGTGCTTTGCGGCACGAGTTGTAGATTG
CAGGTTGGAGTCTGTGTGGAAGGCGGTGTCCAAGTCCCTTGGAACAGGGCGCCCAGGAGGGTGAGA
GCCCCGTGGGATGCCGGCGGAAGCAGTGAGGCCCTTCTGACGAGTCGAGTTGTTTGGGAATGCAGCT
CCAAGCGGGTGGTAAATTCCATCTAAGGCTAAATACTGGCGAGAGACCGATAGCGAACAAGTACTGT
GAAGGAAAGATGAAAAGCACTTTGAAAAGAGAGTGAAACAGCACGTGAAATTGTTGAAAGGGAAG
GGTATTGCGCCCGACATGGGGATTGCGCACCGCTGCCTCTCGTGGGCGGCGCTCTGGGCTTTCCCTGG
GCCAGCATCGGTTCTTGCTGCAGGAGAAGGGGTTCTGGAACGTGGCTCTTCGGAGTGTTATAGCCAG
GGCCAGATGCTGCGTGCGGGGACCGAGGACTGCGGCCGTGTAGGTCACGGATGCTGGCAGAACGGC
GCAACACCGCCCGTCTTGAAACACCGGACCA
Choose kluyveromyces, candiyeast, her Sa yeast and pichia yeast and show bacterial strain and the part representative species that homology is higher with xlc6 blast result in belonging to, with S.pombe is outgroup, xlc6 is done evolutionary tree with 49 bacterial classifications choosing, and the bacterial classification that is used to compare is as shown in table 1.From the cluster result of Fig. 1 as can be seen, the bacterial strain of the several genus of selected this mainly is divided into 4 classes.Wherein kluyveromyces is separately poly-is a class, Issatchenkia orientalis (I.orientalis) with the 554bp of xlc6 comparison in, matching rate 100%, rDNA difference base number is 0; In evolutionary tree, I.orientalis and xlc6 demonstrate higher homology (98%), and with the 559bp of nearest C.pseudolambica of I.orientalis and I.orientalis comparison in, matching rate only 92.84%, rDNA difference base number is 40.According to Kurtzman ﹠amp; The theory of Robnett " belong to a kind of bacterial strain; its D1/D2 district base difference is no more than 1%; and prediction can think to belong to not of the same race if this difference surpasses 1%, and if have only 0-3 base alternative, just can think to belong to a kind of or sibling species (Kurtzman; Robnett, 1998) ", can think that xlc6 belongs to I.orientalis, or its subspecies.
Table 1 and xlc6 do 49 strain bacterium of 26S rDNA sequence alignment
| Bacterial classification | GenBank database numbering | Be called for short |
| Candida boleticola | U45777 | C.boleticola |
| Candida cantarellii | U45814 | C.cantarellii |
| Candida dendrica | U62301 | C.dendrica |
| Candida entomophila | U62302 | C.entomophila |
| Candida ethanolica | U71073 | C.ethanolica |
| Candida friedrichii | U45781 | C.friedrichii |
| Candida gropengiesseri | U45721 | C.gropengiesseri |
| Candida inconspicua | U71062 | C.inconspicua |
| Candida intermedia | U44809 | C.intermedia |
| Candida lyxosophila | U76204 | C.lyxosophila |
| Candida methanosorbosa | U70186 | C.methanosorbosa |
| Candida pseudolambica | U71063 | C.pseudolambica |
| Candida rugopelliculosa | U71069 | C.rugopelliculosa |
| Candida rugosa | U45727 | C.rugosa |
| Candida sake | U45728 | C.sake |
| Candida thaimueangensis | AB264014 | C.thaimueangensis |
| Candida valida | AJ508586 | C.valida |
| Issatchenkia hanoiensis | DQ466541 | I.hanoiensis |
| Issatchenkia occidentalis | U76348 | I.occidentalis |
| Issatchenkia orientalis | U76347 | I.orientalis |
| Issatchenkia scutulata var.scutulata | U76529 | I.scutulata |
| Issatchenkia terricola | U76345 | I.terricola |
| Kluyveromyces aestuarii | U69579 | K.aestuarii |
| Kluyveromyces africanus | U68550 | K.africanus |
| Kluyveromyces bacillisporus | U69583 | K.bacillisporus |
| Kluyveromyces blattae | U69580 | K.blattae |
| Kluyveromyces delphensis | U69576 | K.delphensis |
| Kluyveromyces dobzhanskii | U69575 | K.dobzhanskii |
| Kluyveromyces lactis var.lactis | U94922 | K.lactis |
| Kluyveromyces lodderae | U6855l | K.lodderae |
| Kluyveromyces marxianus | U94924 | K.marxianus |
| Kluyveromyces phaffii | U69578 | K.phaffii |
| Kluyveromyces piceae | U84346 | K.piceae |
| Kluyveromyces polysporus | U68548 | K.polysporus |
| Kluyveromyces thermotolerans | U69581 | K.thermotolerans |
| Kluyveromyces waltii | U69582 | K.waltii |
| Kluyveromyces wickerhamii | U69577 | K.wickerhamii |
| Kluyveromyces yarrowii | U68559 | K.yarrowii |
| Pichia(Komagataella)pastoris | U75963 | Pichia.pastoris |
| Pichia(Yamadazyma)stipitis | U45741 | Pichia.stipitis |
| Pichia angophorae | U75521 | P.angophorae |
| Pichia bispora | U74589 | p.bispora |
| Pichia canadensis | U75415 | P.canadensis |
| Pichia dryadoides | U75422 | P.dryadoides |
| Pichia galeiformis | U75738 | P.galeiformis |
| Pichia membranifaciens | U75725 | P.membranifaciens |
| Pichia methanolica | U75523 | P.methanolica |
| Pichia scaptomyzae | DQ409150 | P.scaptomyzae |
| Schizosaccharomyces pombe | U40085 | S.pombe |
2, Physiology and biochemistry is identified:
1) fermenting carbohydrate test:
Identify that required fermentation carbon source is: raffinose, D-semi-lactosi, lactose, sucrose, maltose, glucose.Getting 12.5% bean sprouts juice is basal liquid, and packing Du Shi pipe was sterilized 15 minutes for 115 ℃; The sugar of test is not made into 10% solution with sterilized water, boiled 15 minutes, and is cold slightly, is sub-packed in the Du Shi pipe with a certain amount of liquid glucose of aseptic pipette, extract, makes sugared concentration reach 2%, and raffinose is 4%.Yeast with fresh culture is inoculated in fermentation tube respectively, 25 ℃ of cultivations down, and the colour-change of observing interior gas volume burden of Du Shi pipe and substratum observed for two weeks altogether, and write down once first every day in week, checked altogether in second week 2～3 times.The result explains according to following principle:
+ positive 7 days in the Du Shi pipe all be full of by gas
The slow positive of d all is full of by gas more than 7 days Du Shi pipes
-feminine gender does not have gas build
2) carbon assimilation culture experiment (seeing Table 2)
The experiment of table 2 carbon assimilation
| The carbon source type | Carbon source kind |
| Hexose disaccharide trisaccharide five-carbon sugar polysaccharide alcohols organic acid | The D-semi-lactosi, the D-seminose, glucose saccharose, maltose, lactose raffinose D-wood sugar, L-arabinose, D-ribose, L-rhamnosyl Zulkovsky starch glycerine, sorbyl alcohol, D-N.F,USP MANNITOL succsinic acid, citric acid, lactic acid |
1. the preparation of substratum: join yeast carbon assimilation effect substratum YNB1000ml, add the carbon-source cpd suitable with 5g glucose, raffinose doubles, if the carbon source of assimilation is an organic acid, then earlier acidity is transferred to pH5.7, filtration sterilization is drawn the above solution of 5ml and is injected 16 * 160mm test tube.
2. assimilate result's observation and recording method: get a white card and draw the wide straight line of about 3/4cm with prepared Chinese ink or black ink on it, the assimilation pipe of cultivation after 1 month fully shaken up, be affixed on this card, see through nutrient solution and observe black line on the card, and according to following principle record:
+++see through test tube be cannot see black line fully;
++ visible black line but present the fuzzy bar of dispersing;
+ black line can be seen clearly but edge blurry;
-black line is high-visible and the edge is not fuzzy.
Be recorded as " +++" and the test tube of " ++ " and show that then anabolic reaction is positive, be recorded as "+" and show that the weak test tube positive and "-" of assimilate into is then negative.Form film, mould etc. in this way and also be recorded as the positive.For the ease of observations, make positive and negative control and total blank, promptly be the positive contrast of substratum of sole carbon source with glucose, be designated as +++; And the negative contrast of the substratum that does not contain any carbon source.
3) assimilation nitrogenous source test:
The general auxanography that adopts, concrete grammar is as follows:
1. bacterium liquid preparation: yeast one articulating of picking fresh culture is gone in the 1ml stroke-physiological saline solution, behind the mixing, makes cell suspending liquid.
2. cast the flat board that carries disease germs: every pipe is equipped with the basic medium that 20ml does not have nitrogenous source, be chilled to after the fusing about 45 ℃, the bacterium liquid of 1ml is all poured in the basic medium test tube of no nitrogenous source, fully shake up, dish inclines, solidify back 28 ℃ and be inverted several hours down, make the surface dried slightly, put on the mark of nitrogenous source title in the bottom of plate.For fear of the interference of contiguous different nitrogen sources, between the junction region of different nitrogen sources, open a ditch (not ditching in dull and stereotyped nearly center) with aseptic small steel spade.Every ware is advisable with six kinds of nitrogenous sources.
3. point sample: add nitrogenous source a little (about grain of rice size) with aseptic stainless steel spoon (or using aseptic toothpick) by mark, just putting 2～4 hours earlier, put 25 ℃ then and be inverted down and cultivated observationss 1～2 day.
4) generate the kind of starch compound test:
Generate at yeast on the slant medium of kind of starch compound, line inserts the yeast that has activated, after 25 ℃ of 1～2 weeks of cultivation, 1～2 of dropping road, surface Ge Shi iodine liquid.The all yeast that can give birth to the kind of starch compound are blue in periphery of bacterial colonies.
5) no VITAMIN growth test
Get the no vitamin(e)-producing yeast bacterium basic medium of 16.7 grams, be dissolved in the 1000ml distilled water, filtration sterilization is placed in 4 ℃ of refrigerators standby.
Cultivate for the first time: with transfering loop picking one ring activatory bacterial classification, insert the test tube that 5ml does not have the VITAMIN basic medium, cultivated 3～7 days for 25 ℃.
Cultivate for the second time: get 0.1ml nutrient solution access for the first time and contain in the test tube of fresh culture, cultivated 3～7 days for 25 ℃.Observe and the record result.
6) growth test in 50% (w/v) and 60% (w/v) glucose-yeast extract paste:
50 gram glucose are dissolved in the water that 50ml contains 0.1% yeast extract paste, add 3% (w/w) agar dissolving back packing test tube then, every pipe 5ml sterilized 10 minutes for 115 ℃, taking-up do the inclined-plane (note: sterilization excessively will cause cultivating brown stain, after the sterilization the substratum of brown stain should not use again).The yeast that has activated is rule on above-mentioned medium slant, cultivate to observe after 30 days through 25 ℃ and have or not the growth phenomenon.For preventing that the osmotic pressure that causes cultivating owing to moisture evaporation between incubation period from further strengthening, the most handy rubber stopper seal of test tube.
7) decomposing urea test
Get the yeast of new cultivation, be inoculated on the agar slant of doing hydrolyze urea test, 25 ℃ of cultivations, every day observations.After 5～7 days, as presenting incarnadine on the agar slant, then this yeast can decomposing urea.
8) litmus milk test
Mainly contain compositions such as lactose and casein in the milk.Add reindeer moss as soda acid and oxidation-reduction indicator at milk, get the yeast of new cultivation, be inoculated in this substratum, cultivate observation situation after 30 days.Reindeer moss is lavender when neutrality, pinkiness when acid is blue during alkalescence, then fade from bottom to top during reduction.Can not add reindeer moss in the milk when generally identifying yeast.
9) 37 ℃ of growth tests
Yeast-inoculated was cultivated 2～4 days in 37 ℃ on YEPD agar, if weak growth, then renewed vaccination is again cultivated the result who observed in 2～4 days with same temperature and is as the criterion.
10) producing ester measures
Get 10% bean sprouts juice and lead to 5% glucose solution, insert the new yeast of cultivating, cultivated 3～5 days down, judged whether esters fragrance for 25 ℃ with sense of smell.
11) thecasporous microscopic examination
Yeast is inserted on the malt extract medium inclined-plane, cultivated 24 hours in 25 ℃～28 ℃; Cultured thalline is changed in the Fructus Hordei Germinatus liquid line, cultivated 24 hours in 28 ℃; Cultured bacterium liquid is all poured in the centrifuge tube into centrifugal 10 minutes of 3000r/min, abandoning supernatant; Repetitive operation three times; To precipitate yeast slurry and be applied to sporogenic substratum, cultivate 3～5 days in 25 ℃.
The used substratum of above-mentioned experiment is as follows:
A.YEPD substratum (37 ℃)
YEPD liquid nutrient medium: yeast extract 1%, peptone 2%, glucose 2%, natural pH malt juice liquid medium
Malt juice liquid medium transfers to the pol of 10 degree Beaume with wort, transfers pH to 5.4; Be sub-packed in test tube after the filtration, every pipe 4ml; 112 ℃, sterilization in 20 minutes.Add 2% agar and be solid medium.
B.McClary substratum (thecaspore)
Glucose 0.1%, Repone K 0.18%, yeast water 0.25%, sodium-acetate 0.82%, agar 2% is joined with distilled water, the test tube of packing into.112 ℃, sterilization in 15 minutes.
C.Gorodkowa substratum (thecaspore)
Glucose 0.1%, peptone 1%, 12% sodium-chlor, washing agar 2%, distilled water is joined, the test tube of packing into; 112 ℃, sterilization in 20 minutes.
D.PDA (false silk is observed)
Peeling potatoes 200g, glucose 20g, agar 20g, tap water 1L.Potato is cleaned peeling, and water is put on slitting inclined-plane, in order to avoid oxidized.Boiled 30 minutes, and filtered through gauze, filtrate adds water to 1L, adds glucose and agar, fusion back packing triangular flask or test tube, 112 ℃, sterilization in 20 minutes.
E. sugar-fermenting basic medium
12.5% bean sprouts juice: soybean sprout 125 grams add water 1L, boil half an hour, supply water after the filtration to 1L.112 ℃, sterilization in 30 minutes.
F. produce kind of starch compound substratum
Ammonium sulfate 0.1%, potassium primary phosphate 0.1%, 7 water magnesium sulfate 0.05%, glucose 1%, washing agar 2.5% is joined with distilled water, and pH4.5 is sub-packed in small test tube, and 112 ℃, sterilization in 20 minutes.Shelve the inclined-plane.
G. produce the ester substratum
Glucose 5%, is sub-packed in the 50ml triangular flask every bottle of 20ml, 112 ℃, sterilization in 20 minutes with 10% bean sprouts juice preparation.
H. there is not the VITAMIN basic medium
Ammonium sulfate 0.5%, potassium primary phosphate 0.1%, 7 water magnesium sulfate 0.05%, glucose 2%, sodium-chlor 0.01%, Calcium dichloride dihydrate 0.01%, adding distil water 100ml, packing test tube, every pipe 5ml, 112 ℃, sterilization in 20 minutes.
I. urea decomposition is measured substratum
Peptone 0.1g, sodium-chlor 0.5g, potassium primary phosphate 0.2g, it is phenol red in 100ml distilled water to add 0.0012g, regulate pH6.8, add agar 2g, packing test tube, every pipe dress 2.7ml, after the sterilization, every again pipe adds 20% urea soln that 0.3ml sterilizes after filtration, shelves the inclined-plane after the mixing.
J. high osmotic pressure substratum
Take by weighing 50g and 60g glucose, add 50ml and 40ml respectively, in 1% yeast extract paste solution, add the fusing of 3% agar after, the packing test tube, every pipe 3～5ml shelves the inclined-plane after the sterilization.
K. assimilate the nitrogenous source basic medium
Glucose 2%, potassium primary phosphate 0.1%, 7 water magnesium sulfate 0.05%, yeast extract paste 0.02%, washing agar 2%, adding distil water 100ml filters back packing test tube, every pipe 20ml, 112 ℃, sterilization in 15 minutes.
L. litmus milk enrichment
Get fresh milk powder 1L and add 2.5% reindeer moss aqueous solution 4ml.The packing test tube, tyndallization 3 times, or 112 ℃ the sterilization 15 minutes.
M. yeast carbon assimilation effect substratum YNB (0.67% yeast nitrogen base, 2% glucose, 3% agar, pH6.2.Another prescription is: glucose 10g (NH
4)
2SO
41g, K
2HPO
40.125g, KHPO
40.875g, KI 0.0001g, MgSO
47H
2O 0.5g, CaCl
22H
2O 0.1g, NaCl 0.1g, dimension secondary element mother liquor 1mL, VITAMIN mother liquor 1mL (mother liquor is preparation routinely all), water 1000mL, pH5.8～6.0.)
The physio-biochemical characteristics of Xlc6 are as shown in table 3, according to morphological specificity and physiological characteristic, along dendriform key (saccharomycetic feature and identification handbook, the special ﹠amp in [English] J.A. Barney; R.W. Penn is outstanding) identify that xlc6 is an Issatchenkia orientalis, binding molecule biological assay result, determine that xlc6 is an Issatchenkia Issatchenkia orientalis kind, but can not carry out the bacterial strain of citric acid-ethanol fermentation in the known Issatchenkia orientalis, therefore, determine that xlc6 is the subspecies or the mutation of Issatchenkia orientalis.
The physio-biochemical characteristics of table 3 xlc6
| Experiment | Project | The result | Project | The result |
| The fermenting carbohydrate test | Glucose | + | Lactose | - |
| Maltose | - | The D-semi-lactosi | - | |
| Sucrose | - | Raffinose | - | |
| The assimilation experiment | Saltpetre | - | The D-seminose | + |
| Ammonium sulfate | + | The L-rhamnosyl | - | |
| Lactose | L-arabinose | - | ||
| The D-wood sugar | + | Lactic acid | + | |
| The D-semi-lactosi | +/- | Glycerine | + | |
| Sucrose | +/- | Citric acid | +/- | |
| Maltose | +/- | Succsinic acid | - | |
| Raffinose | +/- | Zulkovsky starch | +/- | |
| Sorbyl alcohol | +/- | D-ribose | - | |
| Glucose | +/- | N.F,USP MANNITOL | - | |
| Other experiments | VITAMIN is cultivated | + | Kind of starch | - |
| The product ester is measured | + | High osmotic pressure (50%) | + | |
| The urease test | - | High osmotic pressure (60%) | + | |
| 37 ℃ of growths | + | Litmus milk | - |
Annotate :+the positive ,-feminine gender ,+/-performance is uncertain
To prove by experiment that below xlc6 has the ability of degraded citric acid and the advantage that is used for the red bayberry brewing fruit wine.
Xlc6 is inoculated into to be equipped with to fall in the sour substratum cultivates that (inoculum size is 10
9Cfu/mL, 25 ℃), measure the variation (with citrometer) of total acid in the substratum, the results are shown in Table 4, xlc6 was degraded to 1.20g/L to the total acid content that falls in the sour substratum from 18.6g/L in 7 days, fall sour total amount up to 93%.Xlc6 after the domestication of anti-6% alcohol, is inoculated in and cultivates in the red bayberry juice that (inoculum size is 10
9Cfu/mL, 25 ℃), measure the variation (with citrometer) of total acid in the fruit juice, the results are shown in Table 5, xlc6 in 11 days with fruit juice in total acid content drop to 4.8g/L by 9.5g/L, fall sour total amount and reach 50%, as seen, xlc6 has the ability of very strong degraded citric acid.
Table 4 contains the variation that sour substratum total acid content falls in xlc6
| Time/d | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Total acid content/(g/L) | 18.6 | 14.3 | 11.1 | 5.20 | 2.20 | 1.40 | 1.30 | 1.20 | 1.20 |
The situation of acid falls in the anti-6% alcohol xlc6 yeast of table 5 in a collection of cranberry juice
| Time/d | The pH value | Total acid (with citrometer)/(g/L) |
| 1 | 3.11 | 9.5 |
| 3 | 3.15 | 8.9 |
| 5 | 3.18 | 7.8 |
| 7 | 3.19 | 7.5 |
| 9 | 3.22 | 6.5 |
| 10 | 3.26 | 5.7 |
| 11 | 3.30 | 4.8 |
Description of drawings
Fig. 1 is to be outgroup with fission yeast Schizosaccharomyces pombe (S.pombe), the evolutionary tree that xlc6 and 49 bacterial classifications choosing are done.
The biological material specimens preservation
Issatchenkia orientalis, preservation date: on January 15th, 2007, depositary institution: Chinese typical culture collection center, CCTCC NO.M207003.
Embodiment
To further set forth the present invention and effect thereof by embodiment below.
Example 1
1, the selection of raw material:
The clarification original juice of red bayberry: with Ningbo of Zhejiang water chestnut kind red bayberry really is raw material, through squeeze the juice, enzymolysis and purifying treatment are obtained the clarification original juice of red bayberry (soluble solid 96g/L, total acid 9.5g/L, pH2.9).
Yeast saccharomyces cerevisiae is the Angel active dry yeast.
2, brewageing of the inventive method red bayberry fruit wine:
(1) with yeast saccharomyces cerevisiae with 37 ℃ of activation of 3% sucrose solution 30min, mixes with cooled 3% sodium alginate of sterilization, inject 3% calcium chloride solution, the formation gelled pill washs with sterilized water after when leaving standstill 30min;
(2) serve as the fermentation base-material with the clarification original juice of red bayberry, adjusting soluble solid content with sugar is 25%, and inoculation is through the immobilized yeast saccharomyces cerevisiae 0.5% of alginate calcium/aluminium, ferments in 25 ℃, the total reducing sugar 3.5g/L to the mash that ferments, and membrane filtration is got filtrate, gets wine base A;
(3) with xlc6 after the domestication of anti-8% alcohol, be inoculated in the clarification original juice of red bayberry, inoculum size is 10
9Cfu/mL, 25 ℃, fermenting, total acid 5g/L finishes to the original juice of red bayberry, must fall sour Normal juice, serves as the fermentation base-material to fall sour Normal juice then, adjusting soluble solid content with sugar is 25%, inoculation is fermented through the immobilized yeast saccharomyces cerevisiae of alginate calcium/aluminium 0.5%, the total reducing sugar 3.5g/L to the mash that ferments, cellulose acetate membrane filtration, get filtrate, get wine base B;
(4) wine base A and wine base B were blent by 1: 2, get final product.
3, the physical and chemical index of a collection of red bayberry fruit wine of gained sees Table 4.
</entry></row></tbody></tgroup></table></tables>
4, the red bayberry fruit wine brewageed of example 1 described method and traditional method are brewageed red bayberry fruit wine and are compared as follows:
Traditional method is brewageed red bayberry fruit wine
1) raw material: the clarification original juice of red bayberry: with Ningbo of Zhejiang water chestnut kind red bayberry really is raw material, through squeeze the juice, enzymolysis and purifying treatment are obtained the clarification original juice of red bayberry (soluble solid 96g/L, total acid 9.5g/L, pH2.9).
Yeast saccharomyces cerevisiae is the Angel active dry yeast.
2) method:
With yeast saccharomyces cerevisiae with 37 ℃ of activation of 3% sucrose solution 30min; With the clarification original juice of red bayberry is the fermentation base-material, and adjusting soluble solid content with sugar is 25%, and the good yeast saccharomyces cerevisiae 0.5% of inoculation activation ferments in 25 ℃, and the total reducing sugar 4g/L to the mash that ferments gets new wine, membrane filtration; The CaCO that adds 1.0g/L filtrate
3, stir, leave standstill 2d, treat total acid 6g/L in the wine juice, then use the cellulose acetate membrane filtration, promptly get and fall tart fruit wine.
3) result:
The a collection of red bayberry fruit wine index such as the table 5 that adopt traditional free cell fermentation, acid-base neutralisation acid lowering method to produce.
</entry></row></tbody></tgroup></table></tables>
From table 4 and 5 as can be seen, the inventive method utilizes xlc6 that the citric acid fermentation in the red bayberry juice is ethanol, do not need to add any chemical acid lowering agent and just can reach the purpose of falling acid, the total phenol loss of red bayberry fruit wine flavour substances of adopting immobilization technology to brewage in conjunction with biological acid reduction method is few, and the color everyway is better than the red bayberry fruit wine that traditional method is brewageed.
Example 2
1, the selection of raw material:
The clarification original juice of red bayberry: with Ningbo of Zhejiang water chestnut kind red bayberry really is raw material, through squeeze the juice, enzymolysis and purifying treatment are obtained the clarification original juice of red bayberry (soluble solid 90g/L, total acid 13.5g/L, pH2.7).
Yeast saccharomyces cerevisiae is the Angel active dry yeast.
2, brewageing of the inventive method red bayberry fruit wine:
(1) with yeast saccharomyces cerevisiae with 37 ℃ of activation of 1% sucrose solution 40min, mixes with cooled 0.5% sodium alginate of sterilization, inject 1% calcium chloride solution, the formation gelled pill washs with sterilized water after when leaving standstill 60min;
(2) serve as the fermentation base-material with the clarification original juice of red bayberry, adjusting soluble solid content with sugar is 25%, and inoculation is through the immobilized yeast saccharomyces cerevisiae 2% of alginate calcium/aluminium, ferment the total reducing sugar 2g/L to the mash that ferments, cellulose acetate membrane filtration in 10 ℃, get filtrate, get wine base A;
(3) with xlc6 after the domestication of anti-2% alcohol, be inoculated in the clarification original juice of red bayberry, inoculum size is 10
9Cfu/mL, 25 ℃, sour Normal juice must fall in the total acid 6g/L to the original juice of red bayberry that ferments, and serves as the fermentation base-material to fall sour Normal juice then, adjusting soluble solid content with sugar is 28%, inoculation is fermented through the immobilized yeast saccharomyces cerevisiae of alginate calcium/aluminium 2%, and fermenting, total reducing sugar 2g/L finishes the cellulose acetate membrane filtration to the mash, get filtrate, get wine base B;
(4) wine base A and wine base B were blent by 1: 1.
Example 3
1, the selection of raw material:
The clarification original juice of red bayberry: with Ningbo of Zhejiang water chestnut kind red bayberry really is raw material, through squeeze the juice, enzymolysis and purifying treatment are obtained the clarification original juice of red bayberry (soluble solid 110g/L, total acid 8.5g/L, pH3.2).
Yeast saccharomyces cerevisiae is the Angel active dry yeast.
2, brewageing of the inventive method red bayberry fruit wine:
(1) with yeast saccharomyces cerevisiae with 37 ℃ of activation of 6% sucrose solution 30min, mixes with cooled 6% sodium alginate of sterilization, inject 10% calcium chloride solution, the formation gelled pill washs with sterilized water after when leaving standstill 30min;
(2) serve as the fermentation base-material with the clarification original juice of red bayberry, adjusting soluble solid content with sugar is 28%, and inoculation is through the immobilized yeast saccharomyces cerevisiae 0.1% of alginate calcium/aluminium, ferment the total reducing sugar 5g/L to the mash that ferments, cellulose acetate membrane filtration in 10 ℃, get filtrate, get wine base A;
(3) with xlc6 after the domestication of anti-12% alcohol, be inoculated in the clarification original juice of red bayberry, inoculum size is 10
6Cfu/mL, 15 ℃, fermenting, total acid 1g/L finishes to the original juice of red bayberry, must fall sour Normal juice, serves as the fermentation base-material to fall sour Normal juice then, adjusting soluble solid content with sugar is 18%, inoculation is fermented through the immobilized yeast saccharomyces cerevisiae of alginate calcium/aluminium 2%, the total reducing sugar 12g/L to the mash that ferments, cellulose acetate membrane filtration, get filtrate, get wine base B;
(4) wine base A and wine base B were blent by 1: 5, get final product.
Example 4
1, the selection of raw material:
The clarification original juice of red bayberry: with Ningbo of Zhejiang water chestnut kind red bayberry really is raw material, through squeeze the juice, enzymolysis and purifying treatment are obtained the clarification original juice of red bayberry (soluble solid 100g/L, total acid 10g/L, pH3).
Yeast saccharomyces cerevisiae is the Angel active dry yeast.
2, brewageing of the inventive method red bayberry fruit wine:
(1) with yeast saccharomyces cerevisiae with 37 ℃ of activation of 3% sucrose solution 30min, mixes with cooled 4% sodium alginate of sterilization, inject 7% calcium chloride solution, the formation gelled pill washs with sterilized water after when leaving standstill 30min;
(2) serve as the fermentation base-material with the clarification original juice of red bayberry, adjusting soluble solid content with sugar is 20%, and inoculation is through the immobilized yeast saccharomyces cerevisiae 1% of alginate calcium/aluminium, ferment the total reducing sugar 11g/L to the mash that ferments, cellulose acetate membrane filtration in 20 ℃, get filtrate, get wine base A;
(3) with xlc6 after the domestication of anti-6% alcohol, be inoculated in the clarification original juice of red bayberry, inoculum size is 10
8Cfu/mL, 20 ℃, sour Normal juice must fall in the total acid 4g/L to the original juice of red bayberry that ferments, and serves as the fermentation base-material to fall sour Normal juice then, adjusting soluble solid content with sugar is 25%, inoculation is fermented through the immobilized yeast saccharomyces cerevisiae of alginate calcium/aluminium 1%, the total reducing sugar 10g/L to the mash that ferments, cellulose acetate membrane filtration, get filtrate, get wine base B;
(4) wine base A and wine base B were blent by 1: 3, get final product.
The red bayberry fruit wine of blending gained can also carry out ageing 1 year under 20 ℃.
Example 5
1, the selection of raw material:
The clarification original juice of red bayberry: with Ningbo of Zhejiang water chestnut kind red bayberry really is raw material, through squeeze the juice, enzymolysis and purifying treatment are obtained the clarification original juice of red bayberry (solubility skeuomorph 95g/L, total acid 11g/L, pH3).
Yeast saccharomyces cerevisiae is the Angel active dry yeast.
2, brewageing of the inventive method red bayberry fruit wine:
(1) with yeast saccharomyces cerevisiae with 37 ℃ of activation of 5% sucrose solution 30min, mixes with cooled 3% sodium alginate of sterilization, inject 10% calcium chloride solution, the formation gelled pill washs with sterilized water after when leaving standstill 30min;
(2) serve as the fermentation base-material with the clarification original juice of red bayberry, adjusting soluble solid content with sugar is 26%, and inoculation is through the immobilized yeast saccharomyces cerevisiae 2% of alginate calcium/aluminium, ferment in 25 ℃, total reducing sugar 8g/L to the mash ferments, the cellulose acetate membrane filtration is got filtrate, gets wine base A;
(3) with xlc6 after the domestication of anti-8% alcohol, be inoculated in the clarification original juice of red bayberry, inoculum size is 10
9Cfu/mL, 18 ℃, fermenting, total acid 0.5g/L finishes to the original juice of red bayberry, must fall sour Normal juice, serves as the fermentation base-material to fall sour Normal juice then, adjusting soluble solid content with sugar is 20%, inoculation is fermented through the immobilized yeast saccharomyces cerevisiae of alginate calcium/aluminium 2%, the total reducing sugar 8g/L to the mash that ferments, cellulose acetate membrane filtration, get filtrate, get wine base B;
(4) wine base A and wine base B were blent by 1: 4, get final product.
The red bayberry fruit wine of blending gained can also carry out ageing 1 year half under 5 ℃.
Example 6
1, the selection of raw material:
The clarification original juice of red bayberry: with Ningbo of Zhejiang water chestnut kind red bayberry really is raw material, through squeeze the juice, enzymolysis and purifying treatment are obtained the clarification original juice of red bayberry (soluble solid 95g/L, total acid 11g/L, pH3).
Yeast saccharomyces cerevisiae is the Angel active dry yeast.
2, brewageing of the inventive method red bayberry fruit wine:
(1) with yeast saccharomyces cerevisiae with 37 ℃ of activation of 5% sucrose solution 30min, mixes with cooled 3% sodium alginate of sterilization, inject 10% calcium chloride solution, the formation gelled pill washs with sterilized water after when leaving standstill 30min;
(2) serve as the fermentation base-material with the clarification original juice of red bayberry, adjusting soluble solid content with sugar is 26%, and inoculation is through the immobilized yeast saccharomyces cerevisiae 2% of alginate calcium/aluminium, ferment in 25 ℃, total reducing sugar 0g/L to the mash ferments, the cellulose acetate membrane filtration is got filtrate, gets wine base A;
(3) with xlc6 after the domestication of anti-8% alcohol, be inoculated in the clarification original juice of red bayberry, inoculum size is 10
9Cfu/mL, 18 ℃, fermenting, total acid 0.5g/L finishes to the original juice of red bayberry, must fall sour Normal juice, serves as the fermentation base-material to fall sour Normal juice then, adjusting soluble solid content with sugar is 20%, inoculation is fermented with the yeast saccharomyces cerevisiae 2% of fixedization through alginate calcium/aluminium, the total reducing sugar 8g/L to the mash that ferments, cellulose acetate membrane filtration, get filtrate, get wine base B;
(4) wine base A and wine base B were blent by 1: 4, get final product.
The red bayberry fruit wine of blending gained can also carry out ageing 1 year half under 5 ℃.
＜110〉Shaoguan College
＜120〉a kind of acid leaven and the application in the red bayberry brewing fruit wine thereof fall
<160>1
<170>PatentIn version 3.3
<210>1
<211>561
<212>DNA
<213>Issatchenkia orientalis
<220>
<221>CDS
<222>(1)..(561)
<400>1
gcg gcg agt gaa gcg gca aga gct cag att tga aat cgt gct ttg cgg 48
cac gag ttg tag att gca ggt tgg agt ctg tgt gga agg cgg tgt cca 96
agt ccc ttg gaa cag ggc gcc cag gag ggt gag agc ccc gtg gga tgc 144
cgg cgg aag cag tga ggc cct tct gac gag tcg agt tgt ttg gga atg 192
cag ctc caa gcg ggt ggt aaa ttc cat cta agg cta aat act ggc gag 240
aga ccg ata gcg aac aag tac tgt gaa gga aag atg aaa agc act ttg 288
aaa aga gag tga aac agc acg tga aat tgt tga aag gga agg gta ttg 336
cgc ccg aca tgg gga ttg cgc acc gct gcc tct cgt ggg cgg cgc tct 384
ggg ctt tcc ctg ggc cag cat cgg ttc ttg ctg cag gag aag ggg ttc 432
tgg aac gtg gct ctt cgg agt gtt ata gcc agg gcc aga tgc tgc gtg 480
cgg gga ccg agg act gcg gcc gtg tag gtc acg gat gct ggc aga acg 528
gcg caa cac cgc ccg tct tga aac acc gga cca 561
Claims (4)
1, Issatchenkia orientalis (Issatchenkia orientalis) xlc6, CCTCC NO.M 207003.
2, the application of the described xlc6 of claim 1 in the brewageing of red bayberry fruit wine.
3, xlc6 according to claim 2 is applied to brewage the method for red bayberry fruit wine, and this method may further comprise the steps:
(1) with yeast saccharomyces cerevisiae with 37 ℃ of activation of 1～6% sucrose solution, 20～40min, after the sodium alginate of sterilization cooled 0.5～6% mixes, inject 1～10% calcium chloride or liquor alumini chloridi, form gelled pill, wash with sterilized water after leaving standstill 20～60min;
(2) serve as the fermentation base-material with the clarification original juice of red bayberry, adjusting soluble solid content with sugar is 18～28%, and inoculation is through the immobilized yeast saccharomyces cerevisiae 0.1～2% of alginate calcium/aluminium, ferment the total reducing sugar 0～12g/L to the mash that ferments, membrane filtration in 10～30 ℃, get filtrate, get wine base A;
(3) with xlc6 after the domestication of anti-2～12% alcohol, be inoculated in the clarification original juice of red bayberry, inoculum size is 10
6～10
9Cfu/mL, 15～25 ℃ of total acid 0～6g/L to the original juice of red bayberry that ferment, sour Normal juice must be fallen, serve as the fermentation base-material to fall sour Normal juice then, adjusting soluble solid content with sugar is 18～28%, and inoculation is fermented through the immobilized yeast saccharomyces cerevisiae 0.1～2% of alginate calcium/aluminium, total reducing sugar 0～12g/L to the mash ferments, membrane filtration is got filtrate, gets wine base B;
(4) wine base A and wine base B are blent by 1: 1～5, get final product.
4, xlc6 according to claim 3 is applied to brewage the method for red bayberry fruit wine, and this method is further comprising the steps of: the red bayberry fruit of blending gained can also carry out ageing more than 1 year or 1 year under 5～20 ℃.
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| CN1721524A (en) * | 2005-04-11 | 2006-01-18 | 西北农林科技大学 | Acidity reducing yeast of grape wine and its culturing method |
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