CN102154135B - Novel strain JP2 for biological fermentation of fruit wine and application thereof - Google Patents
Novel strain JP2 for biological fermentation of fruit wine and application thereof Download PDFInfo
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- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 22
- 230000001580 bacterial effect Effects 0.000 claims description 19
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- 238000000034 method Methods 0.000 claims description 7
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a novel strain JP2 for biological fermentation of fruit wine and application thereof. The novel strain JP2 is named as saccharomyces cerevisiae JP2 and preserved in China Center for Type CultureCollection in Wuhan city on Sep. 1st, 2010, and has the preservation number of CCTCC NO:M2010214. The invention also relates to the application of the strain in deacidification of the fruit wine. The saccharomyces cerevisiae JP2 provided by the invention has favorable alcoholic fermentation capacity and very strong acid metabolic capability, can degrade organic acid components with stimulating sourness, such as citric acid, malic acid and the like in original juice and improve the mellowness of the fruit wine, and has great advancing effect on brewing of high-quality fruit wine and increasing of market share of the fruit wine.
Description
Technical field
The present invention relates to a kind of yeast saccharomyces cerevisiae (Saccharomyces cerevisiae); Be particularly related to a kind of fruit wine biological fermentation bacterial strain JP2 and application thereof; Called after yeast saccharomyces cerevisiae JP2; This bacterial classification is deposited in the Chinese typical culture collection center that is positioned at Lopa Nationality an ancient woman's ornament mountain, wuchang, wuhan Wuhan University on September 1st, 2010, and preserving number is CCTCC NO:M 2010214.
Background technology
Fruit is that the ideal of nutritive substances such as human body meals VITAMINs, inorganic salt, Mierocrystalline cellulose, organic acid supplies the source, and protein and lipid content are low, and the fruit wine of brew is nutritious, and unique flavor is often drunk in right amount and had certain function of health care, and market outlook are wide.But fruit wine often because organic acid content is higher, causes that the wine body is sour and astringent, harsh feeling is stronger, and this has become the 'bottleneck' restrictions that the fruit wine industry is further widened market and large-scale development.The yeast kind is one of key factor of brewed fruit wine, and the quality of its leavening property directly has influence on the mouthfeel and the local flavor of institute's fermented fruit wine, the quality of decision fruit wine quality.Therefore; The yeast that employing has acid metabolic ability and high comprehensive performance carries out the total acid decline that zymamsis can make fruit wine; Sour and astringent sense and harsh feeling reduce, and mouthfeel is soft, mellow and full, and the share of market that improves fruit wine quality, expansion fruit wine is had important pushing effect.
The fruit wine acid reduction method mainly adopts chemistry to fall acid and inserts milk-acid bacteria Secondary Fermentation MLF biological acid reduction at present.Acid produces effect though chemistry falls, fireballing characteristics, introduces too much Na when falling acid
+And Ca
2+, wine body local flavor and quality are obviously descended, and the unsettled calcium precipitation of generation causes wine body stability decreases in the seasoning process.Milk-acid bacteria can utilize oxysuccinic acid to carry out malolactic fermentation for substrate, under the catalysis of malolactic acid enzyme, is transformed into the soft lactic acid of sour, but that sour amplitude falls in milk-acid bacteria is limited, and harsh to nutritional requirement, and it is grown in the wine body and is vulnerable to alcohol, SO again
2Suppress Deng comprehensive, still be difficult to apply.Therefore; The yeast that employing has the acid metabolic ability carries out zymamsis, is used for the higher brewing fruit wine of tartaric acid content, falls the integrated application of technic acid in conjunction with lactic acid bacteria biological; Can effectively improve the pungency sour of wine body; Brewage soft, the mellow and full fruit wine of mouthfeel, the fruit wine quality is significantly improved, the share of market that enlarges fruit wine is had important pushing effect.
Summary of the invention
To problem that exists in the above-mentioned brewing fruit wine and defective, the objective of the invention is to select a kind of fruit wine biological fermentation bacterial strain JP2 and application thereof with acid metabolic ability and high comprehensive performance.
Fruit wine biological fermentation bacterial strain JP2 of the present invention; That from spontaneous fermentation No. six, clock " early " loquat wine, separates, filters out has very strong acid metabolic ability and a comprehensive good saccharomycete strain of leavening property; Called after Saccharomyces cerevisiae JP2; Be called for short JP2, deliver the Chinese typical culture collection center preservation that is positioned at Wuhan on September 1st, 2010, preserving number is CCTCC NO:M 2010214.
Yeast saccharomyces cerevisiae JP2 of the present invention (being fruit wine biological fermentation bacterial strain JP2) is except that having the zymamsis ability; Also has the acid metabolic ability; Can effectively reduce oxysuccinic acid, citric acid content in the pulp, thereby make soft, the coordination of the fruit wine mouthfeel of brewageing, lay the foundation for improving the fruit wine quality.
The application method of yeast saccharomyces cerevisiae JP2 of the present invention in brewing fruit wine is characterized in that: with yeast saccharomyces cerevisiae JP2 in the seed fermentation substratum after the activation, with 1.03 * 10
6～9.63 * 10
7The cfu/mL inoculum size inserted total sulphur concentration 40.22～123.85mg/L, pH value in 2.85～6.0 pulp, 20～25 ℃ of bottom fermentations 5～20 days.
Description of drawings:
Fruit wine biological fermentation bacterial strain JP2 of the present invention, called after Saccharomyces cerevisiae JP2 is called for short JP2, delivers the Chinese typical culture collection center preservation that is positioned at Wuhan on September 1st, 2010, and preserving number is CCTCC NO:M2010214.
Figure 118 S rDNA sequential system is grown evolutionary tree.
Fig. 2 flat-plate bacterial colony form.
Fig. 3 liquid culture microscopic morphology
Fig. 4 cell shape figure (ESEM).
Fig. 5 cell profile structure iron (transmission electron microscope).
Embodiment:
Embodiment 1:
Below will combine accompanying drawing, introduce the microbial characteristic of fruit wine biological fermentation bacterial strain JP2 yeast saccharomyces cerevisiae JP2 of the present invention, such as morphology, Physiology and biochemistry, 18S rDNA sequencing and homology analysis.
The isolation and selection of yeast saccharomyces cerevisiae JP2
1, the separation of yeast strain
With No. six, clock " early " loquat is raw material, and technologies such as selected, cleaning, fragmentation are processed loquat juice, regulate the total SO of pulp
2Concentration is 80mg/L, is divided in the 5L fermentor tank, 20-25 ℃ of following spontaneous fermentation, as yeast seed selection bacterium source.
When treating that sample has great amount of bubbles in fermentor tank, get fermented liquid 10% and insert in the yeast enrichment medium, cultivate 1d for 28 ℃.Under aseptic condition, fermented liquid is carried out 10 times of gradient dilutions, choose 3 suitable extent of dilution 10
-1, 10
-2With 10
-3, make flat-plate bacterial colony to be separated from each other, each draws 0.1mL, coat on the known malt extract medium flat board, each extent of dilution do 2 parallel, place 28 ℃ of incubator constant temperature culture 2d.Picking has single bacterium colony of products of typical yeast colonial morphology, on the malt extract medium flat board, carries out 4～5 line and goes down to posterity, and through sediments microscope inspection, from the sample of gathering, is divided into from obtaining 56 saccharomycete strains.At first 56 strain bacterial strains are carried out the TTC test, pick out primary dcreening operation bacterial strain 23 strains with products of typical yeast bacterium colony characteristics.The primary dcreening operation bacterium is tested through Du Shi tubule aerogenesis; Obtain the bacterial strain that 11 strains have aroma; Have wherein that 5 strains produce great amount of bubbles in 12h, aroma is strong, it is numbered JP1, JP2, JP3, JP4, JP5 and is transferred on the wort agar slant medium preservation, subsequent use under 4 ℃ of conditions respectively.
2, the seed selection of yeast saccharomyces cerevisiae JP2
The JP1 that separation is obtained, JP2, JP3, JP4, JP5 yeast are respectively with 1 * 10
7～9 * 10
7The cfu/mL inoculum size is inoculated in and contains total SO
2In the Vitis davidi pulp of 60mg/L, titratable acid 8.7g/L (oxysuccinic acid meter); In 20～25 ℃ of spontaneous fermentations 10 days; Carry out fuzzy comprehensive evoluation with fall sour ability, fermentation rate, product wine ability, produce that perfume is flat, sensory evaluation etc., filter out the yeast strain that is suitable for wine fermentation, has acid metabolic ability and high comprehensive performance.Result such as table 1, yeast saccharomyces cerevisiae JP2 liquor output rate is the highest, and it is the strongest to fall sour ability, the fruit wine total acid of the brewageing 1.4g/L that descended; Carry out comprehensive sensory evaluation from color and luster, clarity, fragrance, flavour and the typicalness of fruit wine; Also the fruit wine score value with the JP2 brew is the highest, is 95 minutes, and the fruit wine local flavor is pure, coordination; Mouthfeel is soft, mellow and full, explains that yeast saccharomyces cerevisiae JP2 is the quality yeast bacterial strain that 1 strain has the acid metabolic ability.Yeast saccharomyces cerevisiae JP2 has been deposited in the Chinese typical culture collection center that is positioned at Wuhan, preserving number CCTCC NO:M 2010214.The component of the seed fermentation substratum that CCTCC NO:M 2010214 JP2 are suitable is: wort agar is cultivated: 10 ° of ripple woods wort 1L, add 18～20g agar, and cultivate 3d for 25～30 ℃.
Table 1 different strain fermentation comparative test result
Annotate: total acid is 8.7g/L (an oxysuccinic acid meter) before brewageing.
3, the evaluation of yeast saccharomyces cerevisiae JP2
(1) morphology of JP2 and physiologic character
(2) the sugar-fermenting ability of JP2
| Project | The result | Project | The result |
| D-glucose | + | Sucrose | + |
| The D-semi-lactosi | + | SANMALT-S | + |
| Alpha-lactose | - | The D-cellobiose | - |
| Zulkovsky starch | - |
Annotate: "-" negative reaction; "+" positive reaction, the positive weak reaction of " W " expression
(3) the carbon assimilation ability of JP2
| Project | The result | Project | The result |
| Alpha-lactose | - | The L-sorbose | - |
| The D-cellobiose | - | The D-trehalose | - |
| Zulkovsky starch | + | The D-wood sugar | - |
| The D-pectinose | - | The L-rhamnosyl | - |
| D-ribose | - | Erythritol | - |
| D-N.F,USP MANNITOL | - | The D-sorbyl alcohol | - |
| The D-saligenin | - | Succsinic acid | - |
| Hydrocerol A | - | Inositol | - |
| Ethanol | - | Glycerine | - |
Annotate: "-" negative reaction; "+" positive reaction, the positive weak reaction of " W " expression
(4) the nitrogenous source assimilative capacity of JP2
| Project | The result | Project | The result |
| Ammonium sulfate | + | Saltpetre | - |
| Urea | + |
Annotate: "-" negative reaction; "+" positive reaction, the positive weak reaction of " W " expression
(5) 18S rDNA sequencing and homology analysis
The yeast genome DNA extracting reagent kit of employing TIANGEN company extracts the genomic dna of the aimed strain of pure culture.With primer Au2-F and its 18S rDNA gene conserved regions of Au4-R amplification.Increase with 50 μ LPCR reaction systems, reaction system is: 10*buffer 5.0 μ L, dNTP 4.0 μ L, Au2-F 2 μ L, Au4-R 2 μ L, DNA 4 μ L, rTaq 1.0 μ L, dd H
2O 32 μ L.94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 1min then, 54 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 35 circulations, last 72 ℃ are fully extended 10min, 4 ℃ of preservations.The PCR product detects: get the PCR product of 5 μ L, and the sample-loading buffer of 2 μ L, gel electrophoresis separates in 1.0% agarose, and the 10min that on the EB dyestuff, dyes analyzes with gel imaging system, and the product that will contain target fragment checks order.The 18S rDNA sequence input Genebank of JP2 is carried out sequence homology relatively with Blast software, utilize MEGA 4.0 software building phylogenetic evolution tree, carry out sibship and Phylogenetic Analysis, its 18S rDNA sequential system is grown evolutionary tree, like Fig. 1.The result shows; Being mainly yeast saccharomyces cerevisiae with the higher sequence of JP2 strain gene sequence similarity belongs to; With its similarity the highest be that yeast saccharomyces cerevisiae (Saccharomyces sp) in this genus is planted; Reach more than 98% with the homology of the known yeast saccharomyces cerevisiae of many strains, explain that this bacterium belongs to a kind in the yeast saccharomyces cerevisiae genus on the Molecular Phylogeny taxonomy.Through 18S rDNA sequence homology analysis; According to principle of classification and combining form, the physiological and biochemical property of homology in phylogeny; With reference to " saccharomycetic characteristic and identification handbook ", confirm that finally the JP2 bacterial strain is a yeast saccharomyces cerevisiae, called after Saccharomyces cerevisiae JP2.
In order fully to disclose a kind of fruit wine biological fermentation bacterial strain JP2 of the present invention and application thereof, combine accompanying drawing and contriver to provide the application of yeast saccharomyces cerevisiae JP2 in brewing fruit wine at present, further specify beneficial effect of the present invention.
Embodiment 2: the application of yeast saccharomyces cerevisiae JP2 in " early No. six, clock " loquat fermented glutinous rice is made
With yeast saccharomyces cerevisiae JP2 in the seed fermentation substratum after the activation; Press total sulphur concentration and pH value and inoculation that table 2 orthogonal test scheme is regulated loquat juice; 23 ± 1 ℃ of bottom fermentations 5～20 days, after the fermentation ends fermentation capacity of yeast saccharomyces cerevisiae JP2 is investigated, the result sees table 3.The result shows, " early No. six, clock " the loquat fruit wine alcoholic strength average out to 11.3% that uses yeast saccharomyces cerevisiae JP2 to brewage, and total acid has reduced by 0.8～1.4g/L than loquat magma, and residual sugar content all is lower than 0.4%, and wine body local flavor is pure, mouthfeel is soft, aroma is mellow.It is thus clear that yeast saccharomyces cerevisiae JP2 is the yeast strain that 1 strain has acid metabolic ability and high comprehensive performance, be applicable to the production of " early No. six, clock " loquat wine.
Table 2 orthogonal test L
9(3
4) the level of factor table
The influence that table 3 different fermentations condition changes main physical and chemical index before and after " early No. six, clock " loquat wine fermentation
Embodiment 3: the application of yeast saccharomyces cerevisiae JP2 in " No. one, Portugal, osmanthus " wine
With yeast saccharomyces cerevisiae JP2 in the seed fermentation substratum after the activation, with 5.0 * 10
7Inoculum size insert 80mg/L, pH value 3.85, initial total acid is in " No. one, Portugal, osmanthus " grape pulp of 8.6g/L, 25 ± 1 ℃ of bottom fermentations 15 days, after the fermentation ends fermentation capacity of yeast saccharomyces cerevisiae JP2 investigated, the result sees table 4,5.Yeast saccharomyces cerevisiae JP2 can degrade sour than the oxysuccinic acid and the Hydrocerol A that stimulate, produces the softer lactic acid of sour; " No. one, Portugal, osmanthus " dregs of grape wine precision of using yeast saccharomyces cerevisiae JP2 to brewage is 11.6%, and total acid has reduced 1.1g/L than grape magma, and wine body local flavor is pure, mouthfeel is soft, aroma is mellow.It is thus clear that yeast saccharomyces cerevisiae JP2 is the yeast strain that 1 strain has acid metabolic ability and high comprehensive performance, be applicable to " No. one, Portugal, osmanthus " production vinous.
The variation of " No. one, Portugal, osmanthus " main physical and chemical index of wine before and after table 4 fermentation
The variation of " No. one, Portugal, osmanthus " main organic acid index of wine before and after table 5 fermentation
Test Example 4: the application of yeast saccharomyces cerevisiae JP2 in " China " Yangtao wine is brewageed
With yeast saccharomyces cerevisiae JP2 in the seed fermentation substratum after the activation, with 1.25 * 10
7～9.53 * 10
7The cfu/mL inoculum size inserts 41.35～119.66mg/L, pH value in " China " mashed fruit of kiwi fruit of 2.85～5.77,20 ± 1 ℃ of bottom fermentations 12 days, after the fermentation ends fermentation capacity of yeast saccharomyces cerevisiae JP2 is investigated.Can know by table 6, " China " the Yangtao wine alcoholic strength average out to 11.3% that uses yeast saccharomyces cerevisiae JP2 to brewage, total acid has reduced by 0.7～1.1g/L than grape magma, and wine body local flavor is pure, mouthfeel is soft, aroma is mellow.It is thus clear that yeast saccharomyces cerevisiae JP2 is the yeast strain that 1 strain has acid metabolic ability and high comprehensive performance, be applicable to the production of " China " Yangtao wine.
The variation of the main physical and chemical index of table 6 " China " Yangtao wine
It is following more than to test involved TP:
1, total acid: indicating meter method (GB/T15038-2006), in oxysuccinic acid.
2, total sulfur, free sulphur: direct iodimetry,iodometry (GB/T15038-2006).
3, alcoholic strength: Ebullioscope method (GB/T15038-2006).
4, the mensuration of total reducing sugar: film reagent method (GB/T15038-2006).
5, the mensuration of volatile acid: undertaken by GB/T15038-2006.
6, the mensuration of wine colourity: wine is through 0.4 μ m aperture membrane filtration; Survey its pH value; After 10 times of Sodium phosphate, dibasic one citrate buffer solution of identical pH dilutions, in the 1cm cuvette at wavelength 420,520; Measure its light absorption value under the 620nm respectively, colourity vinous is the product of three's sum and extension rate.
7, sensory evaluation: with reference to GB/T15038-2006, and somewhat modified: investigate from color and luster, clarity, fragrance, flavour and four angles of typicalness respectively, gave respectively 5 fens, 5 minutes, 30 minutes, 40 minutes, 20 minutes weight was calculated with the summation of each item at last.
8, oxysuccinic acid, Hydrocerol A, determination of lactic acid: adopt HPLC, carry out with reference to GB GB/T 5009.157-2003
Claims (2)
1. fruit wine biological fermentation bacterial strain JP2; This bacterium is the yeast saccharomyces cerevisiae that yeast saccharomyces cerevisiae belongs to; Called after yeast saccharomyces cerevisiae JP2 (Saccharomyces cerevisiae JP2); This bacterial classification has been deposited in the Chinese typical culture collection center that is positioned at Lopa Nationality an ancient woman's ornament mountain, wuchang, wuhan Wuhan University, preserving number CCTCC NO:M 2010214.
2. utilize the described fruit wine biological fermentation of claim 1 bacterial strain JP2 to carry out the method for brewing fruit wine, it is characterized in that: with said yeast saccharomyces cerevisiae JP2 in the seed fermentation substratum after the activation, with 1.03 * 10
6～9.63 * 10
7The cfu/mL inoculum size inserted total sulphur concentration 40.22～123.85mg/L, pH value in 2.85～6.0 pulp, 20～25 ℃ of bottom fermentations 5～20 days.
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