CN105062903A - Low-temperature-resistant high-sugar-yield Torulaspora delbrueckii strain and application thereof - Google Patents
Low-temperature-resistant high-sugar-yield Torulaspora delbrueckii strain and application thereof Download PDFInfo
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Abstract
The invention relates to a low-temperature-resistant high-sugar-yield Torulaspora delbrueckii strain and application thereof. The invention is characterized in that the strain is named YT-22, the classification designation is Torulaspora delbrueckii, the strain is collected by China General Microbiological Culture Collection Center on July 3rd, 2014, and the collection number is CGMCC No.9408. The Torulaspora delbrueckii has normal fermenting property at 15-40 DEG C, can generate abundant reducing sugar in the dough fermentation process. When being mixed with other microbes, the Torulaspora delbrueckii can provide abundant nutrient substances for other strains in the fermentation process, promote metabolism of the other strains and obviously enhance the fermentation characteristics of the other microzymes and lactobacillus. When being added into the dough, the Torulaspora delbrueckii can lower the toughness of the dough under the fermentation action and improve the ductility of the dough, so that the finally obtained steamed bread has favorable quality.
Description
Technical field
The invention belongs to microorganism field, relate to a kind of bacterial classification of new screening, especially a kind of low temperature resistant high yield sugar Dare is furnished with spore torula bacterium bacterial classification and application thereof.
Background technology
Dare is furnished with spore torula bacterium (Torulasporadelbrueckii) and belongs to Pezizale, cup fungi section.Data shows, and Dare is furnished with spore torula and is easy to cultivate, and can produce ethanol, be widely used in the fermentation of fruit wine, improves the character of wine, has become a very important non-Saccharomyces.
Applying more in flour products fermentation is yeast saccharomyces cerevisiae, its temperature applicability is at 28 DEG C-35 DEG C, but under the condition that envrionment temperature is lower, its leavening property is suppressed sometimes, ferment effect is bad, the measure heated up is taked so have to when practical application, labor intensive, material resources and financial resources, therefore obtain the focus that the yeast that can ferment at a lower temperature becomes research.During flour products mixed fungus fermentation, microorganism wherein needs a large amount of energy matters, as carbohydrate, but carbon source can be utilized in flour limited, select suitable method to supply the available carbon source of these microorganisms, promote their metabolism, improve leavening property and also there is important Research Significance.
In addition, the local flavor of steamed bun and mouthfeel are the important organoleptic qualities of steamed bun, modern people more wish to consume the steamed bun product with traditional organoleptic quality, except adopting the mixed fungus fermentation agent production traditional flavor steamed buns such as traditional leavening dough or old face, the pure culture yeast bacterial strain that utilization can produce particular flavor has unrivaled advantage, is not only convenient to control product fermentation, and can shorten fermentation time, enhance productivity, meet people in many-sided demands such as consumption and productions.
Summary of the invention
The one that object of the present invention provides based on above-mentioned prior art situation just low temperature resistant high yield sugar Dare is furnished with spore torula bacterium bacterial classification and application thereof, this yeast all has normal leavening property under 15-40 DEG C of condition, and can produce a large amount of reducing sugars in panary fermentation process.This yeast mixes with other microorganism to be applied, and during the fermentation for other bacterial strain provides abundant nutritive substance, can promote the metabolism of other bacterial strain, obviously strengthens the fermentation character of other yeast and milk-acid bacteria.Good preparation characteristic is had with the steamed bun that it makes.
The object of the invention is to be achieved through the following technical solutions: a kind of low temperature resistant high yield sugar Dare is furnished with spore torula bacterium, the title YT-22 of bacterial strain, and Classification And Nomenclature is: Dare be furnished with spore torula bacterium (
torulasporadelbrueckii), this bacterial strain was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 2014 07 year 03 day, and preservation is numbered: CGMCCNo.9408.Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
This yeast all has normal leavening property under 15-40 DEG C of condition, and can produce a large amount of reducing sugars in panary fermentation process and comprise maltose, trisaccharide maltose, Oligomeric maltose.
Low temperature resistant high yield sugar Dare of the present invention is furnished with spore torula bacterium and can carries out mixed fermentation application with other microorganism, as Dare is furnished with spore torula bacterium YT-22 and other yeast as yeast saccharomyces cerevisiae, Bradley yeast and the mixed fermentation of Brunswick debaryomyces hansenii, with milk-acid bacteria as Pediococcus pentosaceus, plant lactobacillus, Enterococcus durans and acetobacter mixed fermentation, yeast YT-22 can provide abundant nutritive substance for other bacterial strain during the fermentation, promote the metabolism of other bacterial strain, obviously strengthen the fermentation character of other yeast and milk-acid bacteria.
Dare of the present invention is furnished with spore torula and adds the toughness that can be reduced dough in dough by its fermentative action to, improve the ductility of dough, make the steamed bun finally obtained have good quality characteristic.
Dare of the present invention is furnished with spore torula bacterium and can be used as steamed bun starter and apply, utilize the steamed bun that it makes, the special volatile flavor substance that can produce has ethyl acetate, ethyl hexanoate, 1,4-caprylolactone, dimixo-octyl phthalate, 2-heptanone, 2,3-acetyl caproyls, trans-3-nonene-2-ketone, 3-methyl-4-amylene-1-ol, (Z)-2-octen-1-ol, 2-butyl-2-octenal and oranges and tangerines ring.
The invention has the advantages that:
1, Dare is furnished with spore torula and can produces a large amount of reducing sugars by the metabolism of self in panary fermentation process, comprises maltose, trisaccharide maltose, Oligomeric maltose.
2, mix with other starter when fermenting, abundant nutritive substance can be provided for other bacterial strain, promote the metabolism of other bacterial strain, improve fermentation rate.
3, Dare is furnished with spore torula and adds the maximum stretching force that can be reduced dough in dough by its fermentative action to, extend the stretching distance of dough, reduce the toughness of dough, increasing the ductility of dough, should be that the small molecular sugar owing to there being spore torula to produce in metabolic process changes the viscosity of dough thus changes dough tensile characteristic.
4, the steamed bun utilizing it to make has special volatile flavor substance.
Accompanying drawing explanation
Fig. 1: Dare is furnished with spore torula heatproof fermentation character and dough volume growth chart in time.
Fig. 2: Dare is furnished with spore torula panary fermentation process sugar content.
Fig. 3: Dare is furnished with spore torula bacterium to the impact of saccharomyces boulardii fermenting power.
Embodiment
Below in conjunction with specific examples, the screening and identification process of yeast strain of the present invention and correlation properties experiment are described in further detail, but are not to limit the present invention.
The experimental technique used in following embodiment if no special instructions, is ordinary method; The material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Rose-bengal solid medium: peptone 5g/L, potassium primary phosphate 1g/L, magnesium sulfate 0.5g/L, glucose 10g/L, paraxin 0.1g/L, rose-bengal 0.033g/L, agar powder 18.5g/L.
YPD liquid nutrient medium: yeast extract paste 10g/L, peptone 20g/L, glucose 20g/L, paraxin 0.1g/L.
Phenol: chloroform (1:1V/V)
TE solution (pH7.5): 10mMTris-HCl (pH7.5), 1mMEDTA (pH8.0)
STES damping fluid: 0.2MTris-HCl (pH7.6), 0.5MNaCl, 0.1% (m/V) SDS, 0.01MEDTA
50 × TAE (100mL): Tris24.2g, Glacial acetic acid 5.71mL, 0.5MEDTA10mL, distilled water is settled to 100mL, 121 DEG C of sterilizing 30min, uses 1 × TAE(50 × TAE to be diluted 50 times and use afterwards during electrophoresis detection).
one, the Isolation and ldentification of bacterial strain
Strains separation sample is commercially available traditional zymotic agent---the leavening dough of Nanyang, henan.
1. sample diluting liquid preparation and strains separation
Get and pulverize rear sample 25g and put into the Erlenmeyer flask (sterile glass beads of the preset proper amt of bottle) that 225mL stroke-physiological saline solution is housed, shake well, makes the suspension of 1:10.
Draw 1:10 sample suspension 1mL, slowly note in the sterile test tube that 9mL physiological saline is housed along tube wall, jolting test tube or use 1 aseptic straw instead and repeatedly blow and beat and make it mix, make the sample suspension of 1:100.Draw 1mL1:100 sample suspension again by aforesaid operations order, do 10 times of dilute sample suspension, by that analogy.
According to the estimation of measuring samples total viable count, select 2 ~ 3 continuous print acceptable diluent degree, each extent of dilution is drawn 0.2mL sample suspension and is coated 2 rose-bengal agar plates, cultivates 48h for 30 DEG C.
2. bacterial strain purifying
Single bacterium colony on inoculating needle picking flat board, new identical flat board carries out streak inoculation, and after dull and stereotyped purifying 2 generation, slant preservation also carries out colony morphological observation and microscopic examination.
3.ITSrDNA identifies
YT-22 bacterium colony on picking rose-bengal flat board, adds in YPD liquid nutrient medium, and 30 DEG C are shaken bacterium overnight incubation, gets in 3mL bacteria suspension to 5mL centrifuge tube centrifugal, 6000r, 3min; Supernatant discarded, with TE washing, centrifugal (6000r, 2min) washs, and is colourless to supernatant, supernatant discarded; Get 0.2mLSTES in centrifuge tube, add quartz sand, add 60 μ l phenol chloroforms, vortex oscillation 5min, interval concussion, cooling; Bacteria suspension is transferred in 1.5mL centrifuge tube, centrifugal (6000r, 2min) centrifugal rear layering; Get supernatant liquor in 1.5mL centrifuge tube, with equal-volume phenol chloroform 3 times; Get and reset and add 2 times of volume dehydrated alcohols ,-20 DEG C of precipitates overnight; 10,000rpm centrifugal 5min at 4 DEG C, collect nucleic acid precipitation, and by 70% washing with alcohol precipitation, 10,000 centrifugal 1min, dry air DNA is precipitated to does not have alcohol smell, and be dissolved in 40 μ lTE, the system of getting is PCR.Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd.:
The sequence of primer I TS4 is: 5 '-TCCTCCGCTGACTAATATGC-3 '
The sequence of primer I TS5 is: 5 '-GGAAGTAAAAGTCGTAACAAGG-3 '
PCR primer being carried out electrophoresis detection, sending Sangon Biotech (Shanghai) Co., Ltd. to check order by there being the product of band.
The ITS sequence recording YT-22 bacterial strain, as shown in SEQIDNO:1, is shown in sequence table.
The fungi ITSrDNA reported in sequencing result blast program and US National Biotechnology Information central database compares, and result shows, the ITS sequence of YT-22 bacterial strain and Dare be furnished with spore justify (
torulasporadelbrueckii) homology be greater than 99%.
From above qualification result, YT-22 bacterial strain be Dare be furnished with spore yeast (
torulasporadelbrueckii).This bacterium was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number: CGMCCNo.9408 on 07 03rd, 2014.
two, Dare is furnished with spore torula bacterium enlarged culturing
Streak culture in the YT-22 bacterial strain transfering loop streak inoculation to rose-bengal flat board that inclined-plane is preserved, again the bacterium on flat board is inoculated in YPD liquid nutrient medium, cultivate 12h for 30 DEG C and obtain bacteria suspension, again by bacteria suspension with 1% inoculum size to be inoculated in the triangular flask that YPD liquid nutrient medium is housed 30 DEG C and to cultivate 24h, centrifugally abandon supernatant, wash 3 times fresh wet thallus is for subsequent use with sterilized water.
three, fermentation character
1, stream becomes fermentation character
Experimental technique: fresh Dare is furnished with spore torula and adds flour neutral plane with flour quality 3.2%, takes 315g dough and carries out stream change fermenting experiment, and instrument is that F3 stream becomes fermentograph (French Chopin).
Experimental result is as follows:
T
1(curve maximum fermentation time): 135min
Hm(dough maximum swelling height): 29.4mm
The maximum height of H ' m(air release curve): 38.2mm
Aerogenesis total amount: 579mL
R(holds gas): 98.6%
2, heatproof fermentation character: graduated cylinder method measures Dough fermentation ability
This measuring Dare is furnished with the heatproof fermentation character of spore torula, with 100g flour for raw material, add the fresh Dare of 3.2g and be furnished with spore torula, 45mL water, the dough of becoming reconciled is placed in graduated cylinder, record original volume, by three groups of samples, it proofs under being placed in 20 DEG C, 30 DEG C and 40 DEG C of conditions respectively, and the change procedure that record surface group is long-pending.See Fig. 1.
four, Dare is furnished with spore torula and produces sugared ability mensuration
Fresh Dare is furnished with spore torula bacterium to add in flour with 3.2% of flour quality and carry out and face, be placed in 35 DEG C, relative humidity is under the condition of 80%, and timing sampling measures sugared content in dough.Adopt Megazyme maltose, sucrose and D-Glucose detection kit to measure the maltose in dough, sucrose and glucose, select 3,5-dinitrosalicylic Acid Colorimetry to measure reducing sugar content, experimental result is see Fig. 2.
As can be seen from Figure 2, Dare is furnished with spore torula bacterium dough maltose content and sharply rises at the fermentation initial stage, keeps very high content during the fermentation, and in dough, maltose content is 0.285% at the content at fermentation initial stage, rises to 0.721% after fermentation 8h.Fermentation initial reduction sugar content is 0.98%, and during fermentation 4h, reducing sugar content rises to 1.45%, slightly declines afterwards.Dare is furnished with spore torula can produce a large amount of reducing sugars by the metabolism of self in panary fermentation process, comprises the Oligomeric maltose such as maltose, trisaccharide maltose.
five, Dare is furnished with spore torula and Bradley yeast for fermentation
Dare is furnished with spore torula to mix with Bradley yeast 1:9 ratio and add flour to and carry out and face, yeast total addition level is 3.2% of flour quality, graduated cylinder method is adopted to measure dough volume change (identical with method described in 2), be placed in 35 DEG C, relative humidity is ferment under 80% condition, the change of dough volume in record fermenting process, and contrast, see Fig. 3 with saccharomyces boulardii fermentation..
As can be seen from Figure 3, when Dare is furnished with spore torula bacterium and saccharomyces boulardii mixed fermentation, the fermenting power of dough significantly improves, and illustrates that Dare is furnished with the metabolism that spore torula facilitates Bradley yeast, thus improves the fermenting power of Bradley yeast.Should be Dare be furnished with spore torula bacterium mix with other starter ferment time, abundant nutritive substance can be provided for other bacterial strain, promote the metabolism of other bacterial strain, improve fermentation rate.
six, Dare is furnished with the impact of spore torula on dough tensile properties
Fresh Dare is furnished with spore torula bacterium to add in flour with 3.2% of flour quality and carry out and face, be placed in 35 DEG C, relative humidity is proof under the condition of 80%, measures Dare respectively and is furnished with spore torula without the tensile properties of proofing of dough and the tensile properties proofing 40min dough.
(1) choose the matter structure tensile properties that TA-XTPlus texture analyser measures dough, result is as table 1:
Table 1: Dare is furnished with spore torula dough matter structure tensile properties table
| Sample | Dough tensile maximum, force (g) | Dough tensile distance (mm) | Dough tensile disruptive force (g) |
| Non-proofing of dough | 69.391 | 24.384 | 69.391 |
| Proof 40 min doughs | 32.291 | 37.281 | 32.291 |
(2) choose the NG type instrument that froths and measure the tensile properties that froths of dough, result is as table 2:
Table 2: Dare is furnished with spore torula dough and froths tensile properties
| Sample | P mm | L mm | P/L |
| Non-proofing of dough | 232 | 27 | 8.59 |
| Proof 40 min doughs | 118 | 67 | 1.76 |
P: represent that the sample ordinate zou in curve that froths holds the peak value of atmospheric pressure, unit is mmH
2o, represents the toughness of dough herein;
L: represent the corresponding vertical range (mm) that curve starting point declines suddenly to papula parting pressure, represents dough ductility;
P/L: be commonly referred to curved configuration ratio, represents the profile ratio frothed, and P/L value shows that greatly dough resistance is strong, and extensibility is poor.
As can be seen from Table 1 and Table 2, Dare is furnished with spore torula and adds the maximum stretching force that can be reduced dough in dough by its fermentative action to, extend the stretching distance of dough, reduce the toughness of dough, increasing the ductility of dough, should be that the small molecular sugar owing to there being spore torula to produce in metabolic process changes the viscosity of dough thus changes dough tensile characteristic.
seven, Dare is furnished with the making of spore torula bacterium steamed bun
Fresh for 9.6g Dare is furnished with spore torula bacterium, 135mL water and 300g flour to put into dough mixing machine and stir 12min, it is shaping then dough to be carried out piecemeal, and is placed in 35 DEG C, and relative humidity is proof 40min under 80% condition, steams 30min afterwards.
eight, Dare is furnished with spore torula steamed bun physical and chemical property determining
(1) choose TA-XTPlus texture analyser and matter structure analysis (TPA) is carried out to steamed bun, adopt P/36R probe, program setting: before surveying, speed is 3.00mm/s; Survey medium velocity is 1.00mm/s; After surveying, speed is 5.00mm/s; Strain 50%; Time 3.00s; Trigger force is 5.0g.
(2) the volume displaced method of millet is adopted to measure steamed bun specific volume.
(3) JBDZ20 whiteness instrument is adopted to measure steamed bun whiteness.
Dare is furnished with the physical and chemical index result of spore torula steamed bun as table 3:
| Physical and chemical index | Hardness | Recovery | Elasticity | Gumminess | Chewiness | Specific volume (mL/g) | Epidermis whiteness | Inner whiteness |
| Steamed bun | 2175 | 47 | 94 | 1775 | 1662 | 2.085 | 50.8 | 48.1 |
Table 3: Dare is furnished with spore torula steamed bun physical and chemical index result
As can be seen from Table 3, Dare is furnished with spore torula steamed bun and has good texture characteristic, and specific volume 2.085mL/g is greater than the 1.7mL/g of national regulations.
nine, Dare is furnished with the flavour substance-measuring that spore torula steamed bun produces
Application headspace solid-phase microextraction-gas chromatography mass spectrometry measures the flavour substances that Dare is furnished with the generation of spore torula steamed bun, its special volatile flavor substance produced has ethyl acetate, ethyl hexanoate, 1,4-caprylolactone, dimixo-octyl phthalate, 2-heptanone, 2,3-acetyl caproyls, trans-3-nonene-2-ketone, 3-methyl-4-amylene-1-ol, (Z)-2-octen-1-ol, 2-butyl-2-octenal, oranges and tangerines ring etc.Wherein ethyl acetate has fruital; Ethyl hexanoate has fruital, is the theme fragrance of strong type wine; Isosorbide-5-Nitrae-caprylolactone has herbal, the beans perfume of coconut sample breath, lasting fragrance.
sequence table
<110> He'nan University of Technology
<120> mono-kind low temperature resistant high yield sugar Dare is furnished with spore torula bacterium and application thereof
<160>1
<210>1
<211>796
<212>DNA
<213>dare is furnished with spore torula bacterium YT-22
<400>1
GGGGCATTCTACTGCATTTGAGGTCAACTTTGAGAGTATTGTTCGCCGGATTGCGTATGC60
GAAAAGCTCAAACAAACGATAAATCGTTTACGACAAAGTCTTTAAAGCGCCCGAGCCAAC120
ACACAGTCTGCCGAAGTTGGTAAAACCTAATACGACAATTCGGTATTAGCTAGACTGGAC180
AAAGTCATCCAAGCCAGCAAAATCACAACCAAATAACAGGCTAGCAATTTCAAGTTAACC240
CTTGACAGAGTATCACTCACTACCAAACATGATTGTTTGAGAAGGAAATGACGCTCAAAC300
AGGCATGCCCCCTGGAATACCAAGGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACG360
GAATTCTGCAATTCACATTACGTATCGCATTTCGCTGCGTTCTTCATCGATGCGAGAACC420
AAGAGATCCGTTGTTGAAAGTTTTAAATATTTTTAATTTCATAACGAAATTGGTTTTGAC480
TGGTTTATAATAATAAAAAGTTGTTTGTGTTTGTCACCTCTGGGAAGCACGCACAAGACG540
TATCTTCCCAAAGAAGAAAAGTTGTGTAGAAAACTCCACAGTGTTGTAGAAGTTGAAGTA600
GAGTTAACAACGAGAAAAGACATGTTCCGCAAAAGGCGAACCAAGCCGCAACCGCGCAAT660
TAAGCGCTGGTCTTGAACCAGATCCTCTCTTACAGTATCTTTAGACGTCCTCTAACTTCA720
TTCATATAGATTTCTCTAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTACATT780
TTTTATCTTCCAGGCC796
Claims (8)
1. low temperature resistant high yield sugar Dare is furnished with a spore torula bacterium, and it is characterized in that: the title YT-22 of bacterial strain, Classification And Nomenclature is: Dare be furnished with spore torula bacterium (
torulasporadelbrueckii), this bacterial strain was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center in 2014 07 year 03 day, and preservation is numbered: CGMCCNo.9408.
2. low temperature resistant high yield sugar Dare according to claim 1 is furnished with spore torula bacterium, it is characterized in that: this yeast all has normal leavening property under 15-40 DEG C of condition.
3. low temperature resistant high yield sugar Dare according to claim 1 is furnished with spore torula bacterium, it is characterized in that: this yeast can produce a large amount of reducing sugars in panary fermentation process.
4. low temperature resistant high yield sugar Dare according to claim 3 is furnished with spore torula bacterium, it is characterized in that: described reducing sugar is maltose, trisaccharide maltose, Oligomeric maltose.
5. a low temperature resistant high yield sugar Dare as claimed in claim 1 is furnished with the application in mixed fermentation of spore torula bacterium and other microorganism, it is characterized in that: described Dare is furnished with spore torula bacterium YT-22 and other yeast as yeast saccharomyces cerevisiae, Bradley yeast and the mixed fermentation of Brunswick debaryomyces hansenii, with milk-acid bacteria as Pediococcus pentosaceus, plant lactobacillus, Enterococcus durans and acetobacter mixed fermentation, Dare is furnished with spore torula bacterium YT-22 can provide abundant nutritive substance for other bacterial strain during the fermentation, promote the metabolism of other bacterial strain, the fermentation character of other yeast of obvious enhancing and milk-acid bacteria.
6. a Dare as claimed in claim 1 is furnished with the application of spore torula bacterium in panary fermentation, it is characterized in that: Dare is furnished with spore torula and adds the toughness that can be reduced dough in dough by its fermentative action to, improve the ductility of dough, make the steamed bun finally obtained have good quality characteristic.
7. a Dare as claimed in claim 1 is furnished with the application of spore torula bacterium as steamed bun starter.
8. the steamed bun utilizing the Dare described in claims 1 to be furnished with the making of spore torula bacterium, it is characterized in that: this steamed bun utilizes Dare to be furnished with spore torula and makes, the special volatile flavor substance that made steamed bun produces has ethyl acetate, ethyl hexanoate, 1,4-caprylolactone, dimixo-octyl phthalate, 2-heptanone, 2,3-acetyl caproyls, trans-3-nonene-2-ketone, 3-methyl-4-amylene-1-ol, (Z)-2-octen-1-ol, 2-butyl-2-octenal and oranges and tangerines ring.
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| CN108251318A (en) * | 2018-04-20 | 2018-07-06 | 中国农业大学 | A kind of Dell is furnished with spore torula and its application in Wine Aroma quality is improved |
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