CN103205368B - Domestication method for high temperature-resistant ethanol-tolerant aroma-producing yeast - Google Patents
Domestication method for high temperature-resistant ethanol-tolerant aroma-producing yeast Download PDFInfo
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- CN103205368B CN103205368B CN201310004856.1A CN201310004856A CN103205368B CN 103205368 B CN103205368 B CN 103205368B CN 201310004856 A CN201310004856 A CN 201310004856A CN 103205368 B CN103205368 B CN 103205368B
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Abstract
The invention relates to the field of bioengineering technology and provides a domestication method for a high temperature-resistant ethanol-tolerant aroma-producing yeast. The method comprises the following steps: (1) screening a yeast strain with an excellent aroma-producing property from commercially available aroma-producing yeasts; (2) uniformly mixing an aroma-producing yeast bacterial liquid in a logarithmic phase with an aqueous sodium alginate solution at a room temperature under a germ-free condition, adding a mixed liquor into an aqueous calcium chloride solution to prepare a gel ball particle and immobilizing the gel ball particle at a temperature of 2 to 4 DEG C for 4 to 5 h so as to obtain an immobilized gel particle; and (3) subjecting an obtained immobilized aroma-producing yeast to high temperature-resistant ethanol-tolerant cyclic gradient domestication in an ethanol domestication medium and carrying out cyclic domestication a plurality of times so as to obtain the high temperature-resistant ethanol-tolerant aroma-producing yeast. According to the invention, a cyclic gradient domestication process is employed, so the domestication method is easy and convenient to operate and capable of screening a high temperature-resistant ethanol-tolerant aroma-producing yeast strain, the defect of poor high temperature and ethanol resistance of an aroma-producing yeast used in production of sesame-flavor liquor is overcome, and fermentation capacity is improved.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly a kind of acclimation method of high temperature resistant resistance to ethanol SHENGXIANG yeast.
Background technology
SHENGXIANG yeast is called again ester-producing yeast, can, taking ethanol as carbon source, not only possess zymamsis ability but also possess acetic fermentation ability, can produce taking ester perfume (or spice) as main multiple fragrance material.It can be taking sugar, aldehyde, organic acid, salt as nutriment, synthesizing ester under esterase effect.Irises and orchids aromatic white spirit has sauce fragrance and burning concurrently, thereby selects the powerful SHENGXIANG yeast of aroma-producing function, will be conducive to irises and orchids aromatic white spirit and produce ester flavouring, removal of impurities taste, wine body is coordinated, and improves irises and orchids aromatic white spirit quality.
Irises and orchids aromatic white spirit is produced and is adopted high temperature stack-up, and middle gentle low temperature yeast cell growth is suppressed; And carry out ethanol and accumulate gradually along with fermentation, in the time that its concentration reaches certain value, just can suppress saccharomycetic Growth of Cells and metabolism.Therefore good high temperature resistant, the resistance to ethanol SHENGXIANG yeast bacterial strain of seed selection, can improve irises and orchids aromatic white spirit and produce productive rate, can improve again irises and orchids flavor white spirit quality.
The disclosure of the invention of CN101831386A a kind of method for screening salt-tolerant aroma-producing yeasts and technique thereof, its method is: adopt the ultraviolet lamp 30cm place of 15W to irradiate bacteria suspension and mutagenesis bacterial classification, use transparent circle method, just be sieved to the bacterial classification better than the bacterial classification that sets out, produce bent room and be produced into bent synchronized culture and carry out multiple sieve by being placed on again, finally by yeast through repeatedly taming cultivation, plate isolation, make strain screening purifying, obtain high-quality salt-tolerant aroma-producing yeasts.But this yeast can not high temperature resistant, resistance to ethanol, be not suitable for irises and orchids aromatic white spirit and produce.
Summary of the invention
The acclimation method that the object of the present invention is to provide a kind of high temperature resistant resistance to ethanol SHENGXIANG yeast, the method is simple, convenient, can filter out the yeast strain of high temperature resistant resistance to ethanol, is applicable to irises and orchids aromatic white spirit and produces.
The technical solution adopted for the present invention to solve the technical problems is:
An acclimation method for high temperature resistant resistance to ethanol SHENGXIANG yeast, described acclimation method step is as follows:
(1), taking total ester content and Wen Xiang as index, take into account alcoholic strength simultaneously, from commercially available SHENGXIANG yeast, screening obtains raw fragrant good yeast strain;
(2), step (1) screening is obtained in raw fragrant good yeast strain access malt extract medium, be cultured to logarithmic phase, obtain logarithmic phase SHENGXIANG yeast bacterium liquid, logarithmic phase SHENGXIANG yeast bacterium liquid is mixed under room temperature sterile state with sodium alginate aqueous solution, again mixed solution is joined and in calcium chloride water, be prepared into gel ball particle, then at 2-4 DEG C, solidify 4-5 hour to obtain immobilization gel particle, after with sterilized water, immobilization gel particle being washed, obtain immobilization SHENGXIANG yeast;
(3), immobilization SHENGXIANG yeast is carried out in ethanol domestication substratum the circulation gradient domestication of high temperature resistant resistance to ethanol, concrete parameters is: in the ethanol domestication substratum of circulation gradient domestication, ethanol concentration of volume percent scope is: minimum concentration is 1%-5%, and maximum concentration is 10%-15%; The temperature range of circulation gradient domestication is: minimum temperature is 28-32 DEG C, and top temperature is 40-45 DEG C; Each gradient adopts the gradient amplification 1%-3% of ethanol concentration of volume percent and adopts 0.5 DEG C-3 DEG C of thermograde amplification; Circulation domestication repeatedly, finally obtains the SHENGXIANG yeast of high temperature resistant resistance to ethanol.
Domestication is a kind of directive breeding method of microorganism, and microorganism wherein, owing to progressively adapting to certain specified conditions, finally obtains higher tolerance and metabolic activity.The present invention is taking commercially available SHENGXIANG yeast as object, by developing suitable acclimation method, to obtain the SHENGXIANG yeast of the high temperature resistant resistance to ethanol that is applicable to the production of irises and orchids aromatic white spirit.
As preferably, step (1) is specially: after commercially available SHENGXIANG yeast purifying is cultivated, at 30 DEG C, cultivate 7 days to obtain fermented liquid with malt extract medium, taking GB GB/T 10345-2007 as standard, hear fragrant fermented liquid alcoholic strength, the total ester also measured, the yeast strain up to standard fragrant good yeast strain of making a living.
As preferably, in step (2), the concrete preparation method of gel ball particle is: adopt 1.0-3.0 g sodium alginate to add in the 250 mL triangular flasks that have 50 mL distilled water, cooling after heating for dissolving, getting 3-5 mL logarithmic phase SHENGXIANG yeast bacterium liquid adds in triangular flask and mixes, mixed solution injects the calcium chloride water of 1-3% weight percent concentration by syringe, be prepared into gel ball particle.
As preferably, the diameter of described gel ball particle is 2-3mm.
As preferably, in the ethanol domestication substratum of circulation gradient domestication in step (3), ethanol concentration of volume percent scope is: minimum concentration is 1%-3%, and maximum concentration is 10%-12%; The temperature range of circulation gradient domestication is: minimum temperature is 28-30 DEG C, and top temperature is 40-42 DEG C.
As preferably, in the ethanol domestication substratum of circulation gradient domestication, ethanol concentration of volume percent gradient is specially: 2%, 4%, 6%, 8%, 10%, 12%, and the thermograde of circulation gradient domestication is specially: 30 DEG C, 32 DEG C, 34 DEG C, 36 DEG C, 38 DEG C, 40 DEG C, 42 DEG C.
As preferably, in step (3), circulation domestication number of times is 2-5 time.
As preferably, in step (3), the domestication time of each gradient is 24-72 hour.
As preferably, the domestication time of each gradient is 40-48 hour.
As preferably, ethanol domestication substratum is mixed and forms with YPD liquid nutrient medium by ethanol.
The invention has the beneficial effects as follows:
1, select circulation gradient method for domesticating, simple, convenient, can filter out the yeast strain of high temperature resistant resistance to ethanol, solve the poor defect of high temperature resistant, resistance to alcohol performance of SHENGXIANG yeast during irises and orchids aromatic white spirit is produced, having improved fermentation capacity, is also simultaneously other aromatic white spirit correlative study meaning of offering reference.
2, the present invention selects immobilized yeast form to tame, and makes it can under extreme conditions carry out Reusability, has operation fast, simply.
Brief description of the drawings
Fig. 1 is the circulation gradient domestication schematic flow sheet of the embodiment of the present invention 1.
Embodiment
Below by specific embodiment, and by reference to the accompanying drawings, technical scheme of the present invention is described in further detail.
In the present invention, if not refer in particular to, raw material and the equipment etc. adopting all can be buied from market or this area is conventional.Method in following embodiment, if no special instructions, is the ordinary method of this area.
Malt extract medium, YPD solid medium and YPD liquid nutrient medium are existing conventional medium.
embodiment 1
(1), SHENGXIANG yeast song is purchased from Liangshan, Shandong beneficial friend's biotechnology company limited, gets 2 g dry mediums and is placed in 100 mL sterilized waters the triangular flask with granulated glass sphere are housed, 200 r/min vibrate after 30 min and carry out gradient dilution, get l0
-5, 10
-6, 10
-7dilution sample 200 μ L evenly coat on the flat board of YPD solid medium, 4 of the parallel coatings of each sample.After growing bacterium colony, single bacterium colony of selecting to have a products of typical yeast bacterium colony characteristics is further rule and is separated 2 ~ 3 times, after microscopy is purebred, proceeds to respectively YPD culture medium slant, and cryopreservation is for subsequent use.
The purebred yeast strain of depositing of going bail for, at 30 DEG C, cultivate 7 days to obtain fermented liquid with malt extract medium, taking GB GB/T 10345-2007 as standard, hear perfume and measure the alcoholic strength of fermented liquid, total ester, the yeast strain up to standard fragrant good yeast strain (if there is yeast strains more than 5 strains up to standard, choosing three strains that total ester content is the highest as raw fragrant good yeast strain) of making a living.
(2), step (1) screening is obtained in raw fragrant good yeast strain access malt extract medium, be cultured to logarithmic phase, obtain logarithmic phase SHENGXIANG yeast bacterium liquid, logarithmic phase SHENGXIANG yeast bacterium liquid is mixed under room temperature sterile state with sodium alginate aqueous solution, again mixed solution is joined and in calcium chloride water, be prepared into gel ball particle, then at 2-4 DEG C, solidify 4-5 hour to obtain immobilization gel particle, after with sterilized water, immobilization gel particle being washed, obtain immobilization SHENGXIANG yeast;
Adopting 1.5 g sodium alginates to add has in 250 mL triangular flasks of 50 mL distilled water, cooling after heating for dissolving, getting 4 mL logarithmic phase SHENGXIANG yeast bacterium liquid adds in triangular flask and mixes, mixed solution injects the calcium chloride water of 2% weight percent concentration by syringe, be prepared into gel ball particle and then put into curing 4 h of 4 DEG C of refrigerators, with sterilized water, immobilization gel particle is washed to three times and obtain immobilization SHENGXIANG yeast.
(3) (ethanol domestication substratum is mixed and forms with YPD liquid nutrient medium by ethanol, immobilization SHENGXIANG yeast to be tamed to substratum at ethanol, the per-cent of following ethanol is volume percent) in carry out the circulation gradient domestication (as shown in Figure 1) of high temperature resistant resistance to ethanol, concrete steps are as follows:
Preculture: immobilization SHENGXIANG yeast is placed in to YPD liquid nutrient medium (i.e. 0% ethanol), cultivates 48 hours, then carry out according to the following steps for 30 DEG C:
1, under sterile state, immobilization SHENGXIANG yeast after preculture is transferred in 2% ethanol domestication substratum (being ethanol content 2%), cultivate 48 hours for 30 DEG C:
2, immobilization SHENGXIANG yeast is transferred in 2% ethanol domestication substratum, cultivated 48 hours for 32 DEG C;
3, immobilization SHENGXIANG yeast is transferred in 4% ethanol domestication substratum, cultivated 48 hours for 32 DEG C;
4, immobilization SHENGXIANG yeast is transferred in 4% ethanol domestication substratum, cultivated 48 hours for 34 DEG C;
5, immobilization SHENGXIANG yeast is transferred in 6% ethanol domestication substratum, cultivated 48 hours for 34 DEG C;
6, immobilization SHENGXIANG yeast is transferred in 6% ethanol domestication substratum, cultivated 48 hours for 36 DEG C;
7, immobilization SHENGXIANG yeast is transferred in 8% ethanol domestication substratum, cultivated 48 hours for 36 DEG C;
8, immobilization SHENGXIANG yeast is transferred in 8% ethanol domestication substratum, cultivated 48 hours for 38 DEG C;
9, immobilization SHENGXIANG yeast is transferred in 10% ethanol domestication substratum, cultivated 48 hours for 38 DEG C;
10, immobilization SHENGXIANG yeast is transferred in 10% ethanol domestication substratum, cultivated 48 hours for 40 DEG C;
11, immobilization SHENGXIANG yeast is transferred in 12% ethanol domestication substratum, cultivated 48 hours for 40 DEG C;
12, immobilization SHENGXIANG yeast is transferred in 12% ethanol domestication substratum, cultivated 48 hours for 42 DEG C;
Be circulated back to step 1, repeatedly cultivate successively 3 circulations, finally obtain the SHENGXIANG yeast of high temperature resistant resistance to ethanol.
Obtain the SHENGXIANG yeast of high temperature resistant resistance to ethanol by method of the present invention: resistance to temperature range is 36 DEG C-40 DEG C, and Ethanol-torlerance scope is 5%-9%; And total ester content and Wen Xiang result keep stable before with domestication.
embodiment 2
SHENGXIANG yeast song is purchased from Liangshan, Shandong Xu Jia koji factory, and the difference of the present embodiment and embodiment 1 is immobilization SHENGXIANG yeast to carry out in ethanol domestication substratum the circulation gradient domestication parameter of high temperature resistant resistance to ethanol:
Preculture: immobilization SHENGXIANG yeast is placed in to YPD liquid nutrient medium (i.e. 0% ethanol), cultivates 42 hours, then carry out according to the following steps for 30 DEG C:
1, under sterile state, immobilization SHENGXIANG yeast after preculture is transferred in 3% ethanol domestication substratum (being ethanol content 2%), cultivate 42 hours for 30 DEG C:
2, immobilization SHENGXIANG yeast is transferred in 3% ethanol domestication substratum, cultivated 42 hours for 33 DEG C;
3, immobilization SHENGXIANG yeast is transferred in 6% ethanol domestication substratum, cultivated 42 hours for 33 DEG C;
4, immobilization SHENGXIANG yeast is transferred in 6% ethanol domestication substratum, cultivated 42 hours for 36 DEG C;
5, immobilization SHENGXIANG yeast is transferred in 9% ethanol domestication substratum, cultivated 42 hours for 36 DEG C;
6, immobilization SHENGXIANG yeast is transferred in 9% ethanol domestication substratum, cultivated 42 hours for 39 DEG C;
7, immobilization SHENGXIANG yeast is transferred in 12% ethanol domestication substratum, cultivated 42 hours for 39 DEG C;
8, immobilization SHENGXIANG yeast is transferred in 12% ethanol domestication substratum, cultivated 42 hours for 42 DEG C;
Be circulated back to step 1, repeatedly cultivate successively 5 circulations, finally obtain the SHENGXIANG yeast of high temperature resistant resistance to ethanol.
Obtain the SHENGXIANG yeast of high temperature resistant resistance to ethanol by method of the present invention: resistance to temperature range is 36 DEG C-40 DEG C, and Ethanol-torlerance scope is 5%-9%; And total ester content and Wen Xiang result keep stable before with domestication.
embodiment 3
SHENGXIANG yeast song is purchased from Jinan prebiotic thing of a specified duration Engineering Co., Ltd, and method is identical with embodiment 1.Finally obtain the SHENGXIANG yeast of high temperature resistant resistance to ethanol: resistance to temperature range is 36 DEG C-40 DEG C, and Ethanol-torlerance scope is 5%-9%; And total ester content and Wen Xiang result keep stable before with domestication.
The given scope of the claims in the present invention all can be implemented, and its result and embodiment 1-3 are similar, and therefore not to repeat here.
Above-described embodiment is preferably scheme of one of the present invention, not the present invention is done to any pro forma restriction, also has other variant and remodeling under the prerequisite that does not exceed the technical scheme that claim records.
Claims (5)
1. an acclimation method for high temperature resistant resistance to ethanol SHENGXIANG yeast, is characterized in that: described acclimation method step is as follows:
(1), taking total ester content and Wen Xiang as index, take into account alcoholic strength simultaneously, from commercially available SHENGXIANG yeast, screening obtains raw fragrant good yeast strain, be specially: after commercially available SHENGXIANG yeast purifying is cultivated, at 30 DEG C, cultivate 7 days to obtain fermented liquid with malt extract medium, taking GB GB/T 10345-2007 as standard, hear fragrant fermented liquid alcoholic strength, the total ester also measured, the yeast strain up to standard fragrant good yeast strain of making a living;
(2), step (1) screening is obtained in raw fragrant good yeast strain access malt extract medium, be cultured to logarithmic phase, obtain logarithmic phase SHENGXIANG yeast bacterium liquid, logarithmic phase SHENGXIANG yeast bacterium liquid is mixed under room temperature sterile state with sodium alginate aqueous solution, again mixed solution is joined and in calcium chloride water, be prepared into gel ball particle, then at 2-4 DEG C, solidify 4-5 hour to obtain immobilization gel particle, after with sterilized water, immobilization gel particle being washed, obtain immobilization SHENGXIANG yeast;
(3), immobilization SHENGXIANG yeast is carried out in ethanol domestication substratum the circulation gradient domestication of high temperature resistant resistance to ethanol, concrete parameters is: in the ethanol domestication substratum of circulation gradient domestication, ethanol concentration of volume percent gradient is specially: 2%, 4%, 6%, 8%, 10%, 12%, and the thermograde of circulation gradient domestication is specially: 30 DEG C, 32 DEG C, 34 DEG C, 36 DEG C, 38 DEG C, 40 DEG C, 42 DEG C; Circulation domestication 2-5 time, the domestication time of each gradient is 24-72 hour, finally obtains the SHENGXIANG yeast of high temperature resistant resistance to ethanol.
2. acclimation method according to claim 1, it is characterized in that: in step (2), the concrete preparation method of gel ball particle is: adopt 1.0-3.0 g sodium alginate to add in the 250 mL triangular flasks that have 50 mL distilled water, cooling after heating for dissolving, getting 3-5 mL logarithmic phase SHENGXIANG yeast bacterium liquid adds in triangular flask and mixes, mixed solution injects the calcium chloride water of 1-3% weight percent concentration by syringe, be prepared into gel ball particle.
3. acclimation method according to claim 2, is characterized in that: the diameter of described gel ball particle is 2-3mm.
4. acclimation method according to claim 1, is characterized in that: the domestication time of each gradient is 40-48 hour.
5. acclimation method according to claim 1, is characterized in that: ethanol domestication substratum is mixed and forms with YPD liquid nutrient medium by ethanol.
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| CN105400728A (en) * | 2015-12-15 | 2016-03-16 | 重庆大学 | Bacterial strain producing high-temperature-resistant beta-galactosidase and screening method thereof |
| CN109401989B (en) * | 2018-11-30 | 2022-06-28 | 吉林中粮生化有限公司 | Domestication method of industrial saccharomyces cerevisiae |
| CN111607552A (en) * | 2020-06-29 | 2020-09-01 | 重庆工商大学 | Domestication and breeding method of tetrahydrofuran efficient degradation strain |
| CN113957032A (en) * | 2021-11-02 | 2022-01-21 | 宁夏首朗吉元新能源科技有限公司 | Breeding method of high-ethanol-tolerance strain |
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