The purpose of this invention is to provide a strain and can produce the saccharomyces cerevisiae engineered yeast of alcohol and ergosterol simultaneously.
Saccharomyces cerevisiae engineered yeast ZHJM-1 CGMCC № 0578 of the present invention was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (it abbreviates CGMCC as) in 05 month 16 days calendar year 2001, and deposit number is CGMCC № 0578.
The building process of saccharomyces cerevisiae engineered yeast ZHJM-1 is: according to the physio-biochemical characteristics of different barmses, distillery yeast bacterial classification 2.558 (the Saccharomyces cerevisiae 2.558 of alcohol is produced in screening respectively, can buy in Chinese common micro-organisms culture presevation administrative center) and high bread yeast bacterial classification 2.403 (the Saccharomyces cerevisiae 2.403 of cell ergosterol content, can buy in Chinese common micro-organisms culture presevation administrative center), above-mentioned bacterial classification is given birth to spore to be cultivated, monoploid separates, through the ultraviolet ray monoploid mutant strain (2.558-12 a his and 2.403-8 α trp) that screening has different mating types and different genetic markers with nitrosoguanidine mutagenesis;
The yeast monoploid mutant strain that the yeast monoploid mutant strain of the high yield alcohol that is separated to and cell ergosterol content is high hybridizes;
Screening obtains the Yeast engineering bacteria ZHJM-1 of high yield alcohol and ergosterol on the YNB minimum medium.
Another object of the present invention provides a kind of method of utilizing above-mentioned saccharomyces cerevisiae engineered yeast ZHJM-1 while high yield alcohol and ergosterol.
For realizing this purpose, the present invention takes following design: a kind of method of producing alcohol and ergosterol simultaneously may further comprise the steps:
1) above-mentioned saccharomyces cerevisiae engineered yeast ZHJM-1 being inoculated in total reducing sugars concentration is 30%-60%, contains 0.1-0.5% ammonium sulfate, and 0.1% phosphoric acid, pH are in the fermention medium of 4.5-7.0, cultivates 35-60 hour under 28-40 ℃ of condition;
2) fermented liquid that obtains is filtered, collect supernatant liquor and filter residue respectively;
3) distillation obtains alcohol in supernatant liquor, extracts ergosterol in filter residue.
In said process, the inoculum size of described bacterial classification is 10%-40%, 20%-30% preferably wherein, most preferably 25%.
The reducing sugar of described substratum is W-Gum saccharification liquid, Starch rice saccharification liquid, sweet potato starch saccharification liquid or molasses.
The described cultivation under 28-40 ℃ of condition 35-60 hour is meant at first stir culture 15-30 hour, static then cultivation 20-30 hour.
In order to make the better effects if of fermentation, the saccharomyces cerevisiae engineered yeast ZHJM-1 of described inoculation should be with the actication of culture of slant culture before inoculation, cultivates to be liquid spawn, and liquid spawn is inoculated in the fermention medium after cultivating through at least three grades of liquid seeds.Described slant strains is to be on the wort solid inclined-plane or YEPD solid inclined-plane of 2-35% in total sugar content with saccharomyces cerevisiae engineered yeast ZHJM-1 bacterial classification inoculation, after cultivating 20-72 hour under the 28-40 ℃ of condition, puts into 4 ℃ of refrigerators and preserves.After described liquid spawn is the saccharomyces cerevisiae engineered yeast ZHJM-1 actication of culture that will preserve, connect a garland cells in the triangular flask of wort that 150 milliliters of total sugar contents are housed is 8-35% or YEPD liquid nutrient medium, in 28-40 ℃ of stir culture 14-20 hour, be liquid spawn.Described at least three grades of liquid seeds are cultivated and are meant: the level liquid seed culture, be that liquid spawn is equipped with 250 milliliters by the inoculum size access of 5-30%, in the triangular flask of the saccharification liquid of total sugar content 8-35%,, be the level liquid inoculum in 28-40 ℃ of stir culture 14-20 hour; Described secondary liquid seeds is cultivated, be that the first order seed nutrient solution is equipped with 300 liters by the inoculum size access of 5-30%, total sugar content is the seed culture jar of the saccharification liquid of 10-35%, under 28-40 ℃ of condition stir culture 14-20 hour, is secondary liquid seeds culture; Described three grades of liquid seeds are cultivated: be that the secondary seed nutrient solution is equipped with 3000 liters by the inoculum size access of 10-30%, total sugar content is the seed culture jar of the saccharification liquid of 10-35%, under 28-40 ℃ of condition stir culture 14-20 hour, be three grades of liquid seeds cultures.
Saccharomyces cerevisiae engineered yeast ZHJM-1 of the present invention has the characteristics such as ergosterol content height of high temperature resistant, osmophilic strain, resisting high-concentration alcohol, yeast cell.The ethanol concn of fermenting-ripening wine with dregs reaches more than 13% (v/v), and the ergosterol content of yeast cell reaches more than 2% of dry cell weight.Like this, can produce alcohol and two products of ergosterol by one time fermentation.
The fermentation substrate that the present invention uses is common wort, starch saccharificating liquid or molasses etc., only needs to regulate its reducing sugar total content, suitably adds ammonium sulfate and phosphoric acid, can carry out ordinary production, and method is simple, and is with low cost.
Fermentation period of the present invention is short, the wider range of normal fermentation, and this method does not have particular requirement to fermentation equipment and condition, and the equipment and the condition of general zymamsis factory all can be produced, and the production investment is less.
There is not any difference qualitatively with the alcohol of method production of the present invention and these two kinds of products of ergosterol and independent fermentative production.Method of the present invention is practical, and is easy and simple to handle, is easy to promote.Because one time fermentation obtains two products simultaneously, so less investment, instant effect, high efficiency have the very strong market competitiveness.
The invention will be further described below by specific embodiment.
Embodiment 1,
Utilize saccharomyces cerevisiae engineered yeast ZHJM-1 fermentative production alcohol and ergosterol, step is as follows:
1, slant strains: is on 3% the wort solid inclined-plane with saccharomyces cerevisiae engineered yeast ZHJM-1 bacterial classification inoculation in containing total sugar content, after cultivating 70 hours under 30 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves;
2, liquid spawn: after the saccharomyces cerevisiae engineered yeast ZHJM-1 slant strains activation of preserving, connect a garland cells in being equipped with 150 milliliters, total sugar content is in the triangular flask of 10% malt juice liquid medium, cultivates 20 hours down in 30 ℃ of agitation conditions, is liquid spawn;
3, level liquid seed culture: liquid spawn is equipped with 250 milliliters by 8% inoculum size access, in the W-Gum saccharification liquid triangular flask of total sugar content 10%, cultivated 19 hours down, obtain the level liquid inoculum in 30 ℃ of agitation conditions;
4, the secondary liquid seeds is cultivated: the first order seed nutrient solution is inserted by 8% inoculum size 300 liters of total sugar contents are housed is 15% W-Gum saccharification liquid seed culture jar, under 30 ℃ of agitation conditions, cultivated 19 hours, obtain secondary liquid seeds culture;
5, three grades of liquid seeds are cultivated: the secondary seed nutrient solution is inserted by 15% inoculum size 3000 liters of total sugar contents are housed is 15% W-Gum saccharification liquid seed culture jar, under 30 ℃ of agitation conditions, cultivated 19 hours, obtain three grades of liquid seeds cultures.
6, the preparation of fermention medium
Substratum is that total reducing sugars concentration is 35% W-Gum saccharification liquid, adds 0.15% ammonium sulfate, and 0.1% phosphoric acid, the pH of substratum are 5;
7, ferment tank: it is that stir culture is 28 hours under 30 ℃ of conditions, static then cultivation 28 hours in 35% the W-Gum saccharification liquid culture medium that three grades of seed culture fluids are inserted the total sugar content that 2000 liters of previous steps preparations are housed by 15% inoculum size;
8, according to a conventional method the fermented liquid that obtains is filtered, collect supernatant liquor and filter residue (being yeast cell in the filter residue more than 80%) respectively;
9, in supernatant liquor, distill acquisition alcohol by the ordinary method in the industrial production, in yeast cell, extract ergosterol.
Embodiment 2
The step of present embodiment is identical with embodiment 1, just culture medium prescription, culture condition has been done suitable adjustment, and its step is as follows:
1, slant strains: is on 10% the YEPD solid inclined-plane with saccharomyces cerevisiae engineered yeast ZHJM-1 bacterial classification inoculation in containing total sugar content, after cultivating 50 hours under 35 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves;
2, liquid spawn: after the saccharomyces cerevisiae engineered yeast ZHJM-1 slant strains activation of preserving, connect a garland cells in being equipped with 150 milliliters, total sugar content is in the triangular flask of 20% YEPD liquid nutrient medium, cultivates 18 hours down in 35 ℃ of agitation conditions, is liquid spawn;
3, level liquid seed culture: liquid spawn is equipped with 250 milliliters by 18% inoculum size access, in the Starch rice saccharification liquid triangular flask of total sugar content 18%, cultivated 18 hours down, obtain the level liquid inoculum in 35 ℃ of agitation conditions;
4, the secondary liquid seeds is cultivated: the first order seed nutrient solution is inserted by 8% inoculum size 300 liters of total sugar contents are housed is 20% Starch rice saccharification liquid seed culture jar, under 35 ℃ of agitation conditions, cultivated 18 hours, obtain secondary liquid seeds culture;
5, three grades of liquid seeds are cultivated: the secondary seed nutrient solution is inserted by 15% inoculum size 3000 liters of total sugar contents are housed is 20% Starch rice saccharification liquid seed culture jar, under 35 ℃ of agitation conditions, cultivated 18 hours, obtain three grades of liquid seeds cultures.
6, fermention medium:
Substratum is that total reducing sugars concentration is 45% Starch rice saccharification liquid, adds 0.25% ammonium sulfate, and 0.1% phosphoric acid, the pH of substratum are 6;
7, ferment tank: it is that stir culture is 22 hours under 30 ℃ of conditions, static then cultivation 25 hours in 45% the W-Gum saccharification liquid culture medium that three grades of seed culture fluids are inserted the total sugar content that 2000 liters of previous steps preparations are housed by 20% inoculum size;
8, according to a conventional method the fermented liquid that obtains is filtered, collect supernatant liquor and filter residue (being yeast cell in the filter residue more than 80%) respectively;
9, in supernatant liquor, distill acquisition alcohol by the ordinary method in the industrial production, in yeast cell, extract ergosterol.
Embodiment 3
Utilize yeast saccharomyces cerevisiae ZHJM-1 fermentative production alcohol and ergosterol, step is as follows:
1, slant strains: is on 20% the YEPD solid inclined-plane with saccharomyces cerevisiae engineered yeast ZHJM-1 bacterial classification inoculation in containing total sugar content, after cultivating 22 hours under 40 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves;
2, liquid spawn: after the saccharomyces cerevisiae engineered yeast ZHJM-1 slant strains activation of preserving, connect a garland cells in being equipped with 150 milliliters, total sugar content is in the triangular flask of 33% YEPD liquid nutrient medium, cultivates 14 hours down in 30 ℃ of agitation conditions, is liquid spawn;
3, level liquid seed culture: liquid spawn is equipped with 250 milliliters by 28% inoculum size access, in the Starch rice saccharification liquid triangular flask of total sugar content 32%, cultivated 14 hours down, obtain the level liquid inoculum in 38 ℃ of agitation conditions;
4, the secondary liquid seeds is cultivated: the first order seed nutrient solution is inserted by 8% inoculum size 300 liters of total sugar contents are housed is 20% Starch rice saccharification liquid seed culture jar, under 38 ℃ of agitation conditions, cultivated 14 hours, obtain secondary liquid seeds culture;
5, three grades of liquid seeds are cultivated: the secondary seed nutrient solution is inserted by 15% inoculum size 3000 liters of total sugar contents are housed is 20% Starch rice saccharification liquid seed culture jar, under 38 ℃ of agitation conditions, cultivated 14 hours, obtain three grades of liquid seeds cultures.
6, the level Four liquid seeds is cultivated: three grades of seed culture fluids are inserted by 15% inoculum size 30000 liters of total sugar contents are housed is 20% Starch rice saccharification liquid seed culture jar, under 38 ℃ of agitation conditions, cultivated 14 hours, obtain level Four liquid seeds culture.
7, fermention medium:
Substratum is that total reducing sugars concentration is 52% sweet potato starch saccharification liquid, adds 0.35% ammonium sulfate, and 0.1% phosphoric acid, the pH of substratum are 4.5;
8, ferment tank: it is that stir culture is 15 hours under 35 ℃ of conditions, static then cultivation 20 hours in 52% the sweet potato starch saccharification liquid culture medium that the level Four seed culture fluid is inserted the total sugar content that 2000 liters of previous steps preparations are housed by 25% inoculum size;
9, according to a conventional method the fermented liquid that obtains is filtered, collect supernatant liquor and filter residue (being yeast cell in the filter residue more than 80%) respectively;
10, in supernatant liquor, distill acquisition alcohol by the ordinary method in the industrial production, in yeast cell, extract ergosterol.
Embodiment 4
Utilize saccharomyces cerevisiae engineered yeast ZHJM-1 fermentative production alcohol and ergosterol, step is as follows:
1, slant strains: is on 35% the wort solid inclined-plane with saccharomyces cerevisiae engineered yeast ZHJM-1 bacterial classification inoculation in containing total sugar content, after cultivating 30 hours under 32 ℃ of conditions, puts into 4 ℃ of refrigerators and preserves;
2, liquid spawn: after the saccharomyces cerevisiae engineered yeast ZHJM-1 slant strains activation of preserving, connect a garland cells in being equipped with 150 milliliters, total sugar content is in the triangular flask of 35% malt juice liquid medium, cultivates 20 hours down in 35 ℃ of agitation conditions, is liquid spawn;
3, level liquid seed culture: liquid spawn is equipped with 250 milliliters by 20% inoculum size access, in the W-Gum saccharification liquid triangular flask of total sugar content 20%, cultivated 19 hours down, obtain the level liquid inoculum in 35 ℃ of agitation conditions;
4, the secondary liquid seeds is cultivated: the first order seed nutrient solution is inserted by 20% inoculum size 300 liters of total sugar contents are housed is 20% W-Gum saccharification liquid seed culture jar, under 35 ℃ of agitation conditions, cultivated 19 hours, obtain secondary liquid seeds culture;
5, three grades of liquid seeds are cultivated: the secondary seed nutrient solution is inserted by 20% inoculum size 3000 liters of total sugar contents are housed is 20% W-Gum saccharification liquid seed culture jar, under 35 ℃ of agitation conditions, cultivated 19 hours, obtain three grades of liquid seeds cultures.
6, the level Four liquid seeds is cultivated: three grades of seed culture fluids are inserted by 20% inoculum size 30000 liters of total sugar contents are housed is 20% W-Gum saccharification liquid seed culture jar, under 35 ℃ of agitation conditions, cultivated 19 hours, obtain level Four liquid seeds culture.
7, the Pyatyi liquid seeds is cultivated: the level Four seed culture fluid is inserted by 20% inoculum size 300000 liters of total sugar contents are housed is 20% W-Gum saccharification liquid seed culture jar, under 35 ℃ of agitation conditions, cultivated 19 hours, and obtained Pyatyi liquid seeds culture.
8, the preparation of fermention medium
Substratum is that total reducing sugars concentration is 56% beet sirup, and 1.0% corn hydrolyzed solution adds 0.1% ammonium sulfate, and 0.1% phosphoric acid, the pH of substratum are 6;
9, ferment tank: the Pyatyi seed culture fluid is equipped with in the substratum of 2000 liters of previous steps preparations by 26% inoculum size access, and stir culture is 22 hours under 30 ℃ of conditions, static then cultivation 25 hours;
10, according to a conventional method the fermented liquid that obtains is filtered, collect supernatant liquor and filter residue (being yeast cell in the filter residue more than 80%) respectively;
11, in supernatant liquor, distill acquisition alcohol by the ordinary method in the industrial production, in yeast cell, extract ergosterol.