CN1141377C - High-content mycose saccharomycetes and its preparing process - Google Patents
High-content mycose saccharomycetes and its preparing process Download PDFInfo
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- CN1141377C CN1141377C CNB011018585A CN01101858A CN1141377C CN 1141377 C CN1141377 C CN 1141377C CN B011018585 A CNB011018585 A CN B011018585A CN 01101858 A CN01101858 A CN 01101858A CN 1141377 C CN1141377 C CN 1141377C
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Abstract
The present invention relates to yeast with a high content of trehalose and a production method thereof. In the method, after yeast strains are treated by haploid separation and mutagenesis under the condition of a radiation space, haploids with relatively high content of trehalose are selected; then diploids are obtained by protoplast fusion; a strain with high content is selected from the diploids; on the basis that a fermentative condition of a shake flask is determined, a technology of reasonably feeding a carbon source in small can fermentation and a technology of raising temperature in the later period of the fermentation are established; yeast with high biomass and high trehalose content is obtained after large can production. The yeast is classified and named as Saccharomyces cerevisia which is a microbe strain of DZTA8, the preservation number is China General Microbiological Culture Collection Center (CGMCC) No 0535, and the preservation date is Feb. 7th, 2001.
Description
Technical field
The present invention relates to the production technology of microbial strains, the particularly cultivation of the Wine brewing yeast strain of a plant height content of trehalose and add and the later stage of fermenting is improved temperature by rational carbon source stream obtains the processing method of the Wine brewing yeast strain of high-biomass and high content of trehalose.
Background technology
As everyone knows, trehalose (trehalose) be by two glucose molecules through α, the irreducibility disaccharide of α-1 → 1 keyed jointing because there is the axial symmetrical structure of two-fold in its molecular conformation, thereby trehalose has extremely strong stability and low colour generation.Many organisms are suffering poor environment, when coercing as drying, high temperature, freezing, high osmotic pressure etc., by regulating the injury that trehalose synthesis is resisted extraneous adverse circumstance in the body.And the major cause that trehalose is had a sudden rise in social status is ectogenic trehalose organism and biomacromolecule there is good non-specific provide protection equally.As the stablizer and the protective material of biologically active substance, trehalose has wide application prospect in fields such as food, healthcare products, makeup, medicine, molecular biology and agriculturals.Yet the price limit of trehalose costliness its widespread use.
To the later stage nineties 20th century, the various countries scientist begins one's study and develops the production technology of trehalose.Because trehalose can't be synthetic with chemical method, biological process is produced trehalose to be had following several substantially:
1, extraction method: directly extract from natural phant or algae, its major defect is: content is low, the restriction of resource-constrained does not re-use because this method is subjected to;
2. Enzymatic transformation method: be to be medium, change into trehalose by the Starch phosphorylase reaction with glucose, sucrose or maltose; With starch is that substrate changes into trehalose by several enzyme synergies, and its major defect is: the trehalose that the Enzymatic transformation method obtains contains other assorted sugar, and product purity does not reach edible or medical requirement;
3. fermentation method: separate the preparation trehalose by microbial fermentation, the microorganism of utilization comprises bacterium (Arthrobacter, coccus, excellent bacillus), yeast, fungi etc.Its major defect is: it is too low and extract difficulty because of content to separate trehalose from inoculum, can't suitability for industrialized production.Comparatively speaking, yeast is the more satisfactory material of Production by Microorganism Fermentation trehalose, but the content of trehalose of common bread yeast is lower, generally only is 8-10%, and the trehalose commodity of selling use fermentation method to cultivate the bread yeast preparation mostly at present.Its major defect is: because can be lower for the content of trehalose that extracts in the cell, make the products obtained therefrom manufacturing cost too high, cost an arm and a leg, be not suitable for applying widely.
Summary of the invention
The object of the present invention is to provide a kind of yeast saccharomyces cerevisiae and production method thereof of high content of trehalose, low at fermentative Production trehalose used yeast bacterial classification content of trehalose, the defective that lacks the zymotechnique of effectively synthetic and accumulation trehalose, Wine brewing yeast strain and the suitable zymotechnique that produces high-biomass and high trehalose semi-invariant by the high content of trehalose of seed selection, overcome the drawback of prior art, reach and produce high-biomass and high-load trehalose yeast cell, from these cells, separate the purpose of the production cost of preparation trehalose and reduction mycose saccharomycetes.
The object of the present invention is achieved like this: a kind of yeast saccharomyces cerevisiae of high content of trehalose, it is characterized in that: it is that yeast strain separates through monoploid, after the mutagenic treatment of space radiation condition, select the monoploid of relative high-content trehalose, merge the acquisition diploid through protoplastis, therefrom filter out a plant height content of trehalose bacterial strain, on the basis of determining conditions of flask fermentation, set up in the canister fermentation that rational carbon source stream adds and the technology of the later stage raising temperature of ferment, produce the yeast that obtains high-biomass and high content of trehalose through big jar, classification name: Saccharomyces Cerevisiae in S accharomyces cerevisiae, microbial strain: DZTA8, preserving number is the common micro-organisms center C GMCC No of China Committee for Culture Collection of Microorganisms 0535,7 days February calendar year 2001 of preservation date.
The present invention also provides a kind of production method of yeast saccharomyces cerevisiae of high content of trehalose, it is characterized in that: it comprises the steps:
(1) seed selection of the Wine brewing yeast strain of high content of trehalose:
(1). monoploid is cultivated: the yeast strain that preserve in the laboratory is cultivated on the YPD inclined-plane, under 25-30 ℃ condition, be transferred to pre-living spore substratum after the cultivation, under 25-30 ℃ condition, cultivated 2-3 days; Be forwarded to Kleyn again and give birth to the spore substratum, under 25-30 ℃ condition, cultivate and generate spore after 5-7 days; When examining under a microscope most of cell and the ascus of four spores being arranged in forming, phosphoric acid buffer with 0.2M washes ascus, transferring to the sterilization centrifuge tube carries out centrifugal, be suspended from same damping fluid, add beta-mercaptoethanol 1-3% to enlarge the cell permeability, add the broken ascus wall of helicase of 1-3% concentration again, question response liquid is extremely thick, the thecasporous release of microscopy, centrifugal reaction solution, wash 1-3 time with damping fluid, suitably be coated with the YPD plate after the dilution, under 25-30 ℃ condition, cultivate, single bacterium colony is chosen, with the monoploid mating of known mating type,, be stored in the refrigerator standby to determine that isolating monoploid is a or α mating type;
(2). satellite carries: before satellites transmits, a that above-mentioned separation is obtained and α monoploid are carried the bacterial classification atom cabin of packing into; Temperature is 17-26 ℃ in the cabin, and average ionizing radiation dose is 0.177GY/d, and the time is 15 days;
(3) mating is merged and formed diploid: the sample bacterial classification returns the laboratory, the beginning ground test, carry bacterial strain and ground control strain, after cultivating, measure the variation of cell intracellular trehalose semi-invariant, the monoploid that has different mating types is merged the formation diploid cell by mating, in order to be beneficial to The Fusants Screen, makes monoploid be with the auxotrophy selective marker by chemomorphosis;
(4). mutagenic treatment: a and α haploid strains are carried out mutagenesis with methylsulphonic acid ester, bacterium liquid after the processing is through suitably being coated on the YPD plate after the dilution, choose YPD and YNB basic medium respectively to detect auxotroph with growing bacterium colony on the YPD plate, selected content of trehalose higher H is in a mating type
-Have Met in defective strain and the α mating type
-, Thr
-The double defect strain is carried out protoplastis and is merged; Merging strain selects not adding on any amino acid whose YNB plate, from the diploid of strain more than 200, filter out the 10-20 strain, after three culturing cells are measured trehalose output, determine the trehalose bacterial classification of 1 plant height content again, be used to shake the test of bottle and canister optimization for fermentation technology;
(2) shake flask fermentation is cultivated: the substratum moiety: it is 2-10% that treated molasses are amounted to into glucose content; Yeast extract 1.5-5.0%; Corn steep liquor 3.0-5.0%; (NH
4)
2SO
4, (NH
4)
2HPO
4And micro-0-10%;
(1), culture temperature: after normal temperature is cultivated certain hour 1-20 hour, improve temperature again to 33-45 ℃;
(2), incubation time: the later stage that is accumulated in yeast growth of trehalose, the incubation time harvested cell at 16-36 hour can obtain higher trehalose;
(3) canister fermentation culture:
(1). strain inclined plane is cultivated: bacterial classification inoculation places 25-30 ℃ of incubator to cultivate 12-48 hour on the YPD inclined-plane;
(2). Boiling tube is cultivated: the bacterial classification on the above-mentioned YPD inclined-plane is inserted 5-10ml sterile malt juice body Boiling tube is housed, placed 20-40 ℃ of shaking table shaking culture 12-48 hour;
(3). triangular flask one-level enlarged culturing: above-mentioned tube culture is transferred and is handled the back waste molasses in 1/2 times of volume wort and 1/2 times of volume that 50-200ml is housed, amount in the triangular flask that glucose content is the 2-10% nutrient solution, put 20-40 ℃ of shaking table shaking culture 12-48 hour;
(4). triangular flask secondary enlarged culturing: the inoculum size of above-mentioned culture being pressed 5-20% inserts with the waste molasses after handling, and to amount to glucose content be 2%-10%, yeast extract 0.5-5.0% and comprise corn steep liquor, (NH
4)
2SO
4, (NH
4)
2HPO
4, vitamin H and the trace element in the liquid nutrient medium that interior at least three kinds of nutrition compositions of each 0.1-5% are made into, under 20-40 ℃ condition, shaking culture 12-48 hour;
(5). three grades of enlarged culturing of triangular flask: the inoculum size of above-mentioned culture being pressed 5-20% inserts with the molasses sugar after handling, and amounting to glucose content is 2%-10%, (NH
4)
2SO
4Or (NH
4)
2HPO
4In the liquid nutrient medium that 0.5-10%, vitamin H and micro-0-2% are made into, under 20-40 ℃ condition, cultivated 12-48 hour;
(6). the canister fermentation culture:
1). the fermention medium component: the waste molasses after the processing, ammonium salt, microcosmic salt and calcium, magnesium salts and trace element, each component concentration is at 0-10%;
2) the reasonable stream of carbon source and nutritive substance adds: instantaneous than growth rate and change to set up and to add by the index upwelling earlier according to zymic in the fermenting process, the stream that adds by index decreased stream adds curve again, makes stream add carbon source and nutritive substance is converted into thalline to greatest extent;
3) by improving temperature during the fermentation, obtain higher trehalose output to 30-45 ℃;
4) improve the trehalose accumulation by prolonging fermentation time: on the basis of conventional fermentation time, prolong 1-15 hour again, cell intracellular trehalose content is improved;
(4) produce for big jar:
Canister fermentation parameter and processing condition according to above-mentioned foundation, carry out factory's bulk fermentation of 2-10 batch, the molasses that stream adds are amounted to into glucose calculating, average every gram conversion of glucose produces 0.3-0.5 gram thalline, per 100 gram dry weight cells on average contain 20-40 gram trehalose, the yeast saccharomyces cerevisiae of the high content of trehalose of producing, classification name: Saccharomyces Cerevisiae in S accharomyces cerevisiae, microbial strain: DZTA8, preserving number is the common micro-organisms center C GMCC No of China Committee for Culture Collection of Microorganisms 0535,7 days February calendar year 2001 of preservation date.
This satellite carries and changes the radiation cultivation into, and a and α monoploid bacterial classification that above-mentioned separation obtains are put into the radiation case, and 136/cm of high energy particle density is provided and the identical condition of satellite lift-launch cardinal principle
2, temperature is 17-26 ℃, and average ionizing radiation dose is 0.177GY/ days, and the time is 15-30 days.
Major advantage of the present invention is:
1, because the present invention for the fermentative Production mycose saccharomycetes provides strain excellent, adopts this bacterial strain to add and earlier through the zymotechnique that conventional temperature is cultivated, the later stage is improved temperature, obtain high-biomass in conjunction with rational carbon source stream;
2, simultaneously, the yeast cell of high content of trehalose is in multiple batches of big jar is produced, the molasses that stream adds are amounted to into glucose calculating, average every gram conversion of glucose produces 0.3-0.6 gram thalline, per 100 gram dry weight cells on average contain 20-40 gram trehalose, and its content of trehalose is common zymic 2.4-3.0 times;
3, use same raw material and fermentation equipment with common method, can increase trehalose output 1.4-2.0 doubly, the 1.4-2.0 that just reduces cost doubly.
Further specify below in conjunction with preferred embodiment and accompanying drawing.
Description of drawings
Fig. 1 is a technological process of production synoptic diagram of the present invention.
Embodiment
Embodiment 1
Consult Fig. 1, the yeast saccharomyces cerevisiae of high content of trehalose of the present invention, it is characterized in that: it is that yeast strain separates through monoploid, after the mutagenic treatment of radiation (space) condition, select the monoploid of relative high-content trehalose, merge the acquisition diploid through protoplastis, therefrom filter out a plant height content bacterial strain, on the basis of determining conditions of flask fermentation, set up in the canister fermentation that rational carbon source stream adds and the technology of the later stage raising temperature of ferment, produce acquisition high-biomass and high content of trehalose yeast through big jar, classification name: Saccharomyces Cerevisiae in S accharomyces cerevisiae, microbial strain: DZTA8, preserving number is the common micro-organisms center C GMCC No of China Committee for Culture Collection of Microorganisms 0535,7 days February calendar year 2001 of preservation date.
Its production technique comprises the steps:
(1) seed selection of the Wine brewing yeast strain of high content of trehalose:
1, strain selection:
The yeast strain that preserve in the laboratory is cultivated at YPD (peptone 2%, yeast powder 1%, glucose 2%, agar 1.5%) on the inclined-plane, under 25-30 ℃ condition, be transferred to pre-living spore substratum (peptone 0.8%, yeast powder 0.3%, glucose 1% after the cultivation, agar 1.5%), under 25-30 ℃ condition, cultivated 2-3 days, be forwarded to Kleyn again and give birth to spore substratum (KH
2PO
40.012%, K
2HPO
40.02%, NaAc0.5%, glucose 0.062%, NaCl 0.062%, vitamin H 0.2mg/100ml, micro-1ml/100ml.(wherein micro-component: MgSO
47H
2O 0.3-0.5%, CuSO
45H
2O0.001-0.003%, MnSO
44H
2O 0.1-0.3%, FeSO
44H
2O 0.1-0.3%), under 25-30 ℃ condition, cultivate and generate spore after 5-7 days.When examining under a microscope most of cell and the ascus of four spores being arranged in forming, phosphoric acid buffer with 0.2M washes ascus, transferring to the sterilization centrifuge tube carries out centrifugal, be suspended from same damping fluid, add beta-mercaptoethanol 1-3% to enlarge the cell permeability, add the broken ascus wall of helicase of 1-3% concentration again, question response liquid is extremely thick, the thecasporous release of microscopy, centrifugal reaction solution, wash 1-3 time with damping fluid, suitably be coated with the YPD plate after the dilution, under 25-30 ℃ condition, cultivate, single bacterium colony is chosen, with the monoploid mating of known mating type,, deposit in the refrigerator standby to determine that isolating monoploid is a or α mating type.
2. satellite carries: before satellites transmits, a that above-mentioned separation is obtained and α monoploid are carried the bacterial classification atom cabin of packing into, and general, the satellite flight orbital inclination is 63 °, far point 354km, near point 175km, microgravity level 5 * 10
-5G, 136/cm of high energy particle density
2(35.6 ± 6/cm of ground contrast
2).Record shows that during 15 days flight, temperature is 17-26 ℃ in the cabin, and average ionizing radiation dose is 0.177GY/ days.
3. mating is merged and formed diploid: the radiation sample bacterial classification that above-mentioned satellite carries returns the laboratory, the beginning ground test, carry bacterial strain and ground control strain, after cultivating, measure the variation of cell intracellular trehalose semi-invariant, the content of trehalose of a and α monoploid ground control strain is about 8%, and it is respectively 1.26 times to 1.72 times that contrast that a and α monoploid are carried bacterial strain.The monoploid that has different mating types is merged the formation diploid cell by mating.In order to be beneficial to The Fusants Screen, make monoploid be with the auxotrophy selective marker by chemomorphosis.
4. mutagenic treatment:
With the own ester of methylsulphonic acid a and α haploid strains are carried out mutagenesis, the bacterium liquid after the processing is through suitably being coated on the YPD plate after the dilution, and bacterium colony is chosen YPD respectively and the YNB basic medium (contains glucose 10-30g, (NH in every liter with growing on the YPD plate
4)
2SO
42-8g, KH
2PO
40.5-0.9g, K
2HPO
40.1-0.2g, MgSO
47H
2O 0.2-0.8g, NaCl 0.05-0.2g, CaCl
22H
2O 0.05-0.2g, trace element: H
3BO
4400-600 μ g, CuSO
45H
2O 20-60 μ g, KI 50-200 μ g, FeCl
26H
2O100-300 μ g, MnSO
4H
2O 300-500 μ g, Na
2MoO
42H
2O 100-300 μ g, ZnSO
47H
2O300-500 μ g, VITAMIN: growth hormone 1-3 μ g, calcium pantothenate 300-500 μ g, inositol 1000-3000 μ g, nicotinic acid 300-500 μ g, para-amino benzoic acid 100-300 μ g, VitB1 300-500 μ g, riboflavin 100-300 μ g, pyridoxol 300-500 μ g) to detect auxotroph.Select content of trehalose higher H is in a mating type
-Have Met in defective strain and the α mating type
-, Thr
-The double defect strain is carried out protoplastis and is merged.Merging strain selects not adding on any amino acid whose YNB plate.From the diploid of strain more than 200, filter out the 10-20 strain, after three culturing cells are measured trehalose output, determine that 1 strain is used for research and shakes bottle and canister optimization for fermentation technology test sample again.
(2) shake flask fermentation is cultivated: the substratum moiety: it is 2-10% that treated molasses are amounted to into glucose content; Yeast extract 1.5-5.0%; Corn steep liquor 3.0-5.0%; (NH
4)
2SO
4, (NH
4)
2HPO
4And micro-0-8%.
1, culture temperature: normal temperature was cultivated 1-20 hour, improved temperature again to 33-45 ℃.
2, incubation time: the later stage that is accumulated in yeast growth of trehalose, the incubation time harvested cell at 16-36 hour can obtain higher trehalose.
(3) canister fermentation culture:
1. strain inclined plane is cultivated: bacterial classification inoculation is put 25-30 ℃ of incubator and was cultivated 12-48 hour on the YPD inclined-plane.
2. Boiling tube is cultivated: the bacterial classification on the above-mentioned YPD inclined-plane is inserted 5-10ml sterile malt juice body Boiling tube is housed, place 20-40 ℃ shaking table shaking culture 12-48 hour.
3. triangular flask one-level enlarged culturing: above-mentioned tube culture is transferred and is handled the back waste molasses in 1/2 times of volume wort and 1/2 times of volume that 50-200ml is housed, amount in the triangular flask that glucose content is the 2-10% nutrient solution, place 20-40 ℃ shaking table shaking culture to carry out enlarged culturing in 12-48 hour.
4. triangular flask secondary enlarged culturing: the inoculum size of above-mentioned culture being pressed 5-20% inserts with the waste molasses after handling, and to amount to glucose content be 2%-10%, yeast extract 0.5-5.0% and comprise corn steep liquor, (NH
4)
2SO
4, (NH
4)
2HPO
4, vitamin H and the trace element in the liquid nutrient medium that interior at least three kinds of nutrition compositions of each 0.1-5.0% are made into, under 20-40 ℃ condition, shaking culture 12-48 hour.
5. three grades of enlarged culturing of triangular flask: the inoculum size of above-mentioned culture being pressed 5-20% inserts with the molasses sugar after handling, and amounting to glucose content is 2%-10%, (NH
4)
2SO
4Or (NH
4)
2HPO
4In the liquid nutrient medium that 0.5-10%, biomass and micro-0-2% are made into, under 20-40 ℃ condition, cultivated 12-48 hour.
6. canister fermentation culture:
(1) fermention medium component: the waste molasses after the processing, ammonium salt, microcosmic salt and calcium, magnesium salts and trace element, each component concentration is at 0-10%.
(2) the reasonable stream of carbon source and nutritive substance adds: instantaneous according to zymic in the fermenting process than growth rate and variation thereof, and set up an elder generation and added by the index upwelling, the stream that adds by index decreased stream adds curve again, and the carbon source that stream is added is converted into thalline to greatest extent.
(3) improve temperature by the fermentation later stage and increase the trehalose accumulation: trehalose is through adverse circumstance, induces the synthetic metabolite as arid, high temperature, high osmotic pressure etc., suitably improves temperature during the fermentation to 30-45 ℃, obtains higher trehalose output.
(4) improve the trehalose accumulation by prolonging fermentation time: on the basis of conventional fermentation time, prolong 1-15 hour again, cell intracellular trehalose semi-invariant is improved greatly.
(4) produce for big jar:
According to the canister fermentation parameter and the processing condition of above-mentioned foundation, carry out factory's bulk fermentation test of 2-10 batch.The molasses that stream adds are amounted to into glucose calculating, and average every gram conversion of glucose produces 0.3-0.5 gram thalline, and per 100 gram dry weight cells on average contain 20-40 gram trehalose, and the content of its trehalose is common zymic 2.4-4.0 times.Use same raw material and fermentation equipment, can increase trehalose output 1.4-3.0 doubly, the 1.4-3.0 that just reduces cost doubly.The yeast saccharomyces cerevisiae of high content of trehalose of the present invention, classification name: Saccharomyces Cerevisiae in S accharomyces cerevisiae, microbial strain: DZTA8, preserving number are the common micro-organisms center C GMCC No of China Committee for Culture Collection of Microorganisms 0535,7 days February calendar year 2001 of preservation date.
Embodiment 2
In the present embodiment, its satellite carries and changes radiation into and cultivate, with on reach and separate a and the α monoploid bacterial classification that obtain and put into the radiation case, provide and satellite carries identical substantially condition, 136/cm of high energy particle density
2, temperature is 17-26 ℃, and average ionizing radiation dose is 0.177GY/ days, and the time is 15-30 days.Other conditions obtain the yeast saccharomyces cerevisiae strain of high content of trehalose equally with embodiment 1, and content of trehalose reaches 〉=and 20%.
In sum, the present invention has following characteristics:
1, utilizing the means of space flight condition radioinduction, make the yeast saccharomyces cerevisiae strain of high content of trehalose in conjunction with conventional breeding, content of trehalose reaches 〉=and 20%.
2. according to the bacterial classification characteristics, the carbon source index stream of having set up suitable acquisition high-biomass yeast thalline adds discharge curve.
3. replace the carbon source of glucose with waste molasses, both utilized the dead meal of sugar industry, reduced production cost again as fermention medium.
4. according to the generation principle of trehalose, by leavening temperature being brought up to 30-45 ℃, set up the accumulation of leavening temperature and cell intracellular trehalose and increased between the output and concerned, improved output.
5. according to the accumulation principle of trehalose,, set up the prolongation fermentation time and improved relation between the accumulation of cell intracellular trehalose by on conventional fermentation time basis, prolonging 1-15 hour.
6. supply and the stream that has increased some trace elements according to different condition adds, and comprises corn steep liquor, yeast extract, vitamin H, urea, ammonia, calcium and magnesium etc., has improved the output of trehalose.
Claims (8)
1, a kind of yeast saccharomyces cerevisiae of high content of trehalose, it is characterized in that: it is that yeast strain separates through monoploid, after the mutagenic treatment of radiation space condition, select the monoploid of relative high-content trehalose, merge the acquisition diploid through protoplastis, therefrom filter out a plant height content bacterial strain, on the basis of determining conditions of flask fermentation, set up in the canister fermentation that rational carbon source stream adds and the technology of the later stage raising temperature of ferment, produce acquisition high-biomass and high content of trehalose yeast through big jar, classification name: the brilliant SaccharomycesCerevisiae of yeast saccharomyces cerevisiae, microbial strain: DZTA8, preserving number are the common micro-organisms center C GMCC No of China Committee for Culture Collection of Microorganisms 0535.
2, the yeast saccharomyces cerevisiae of high content of trehalose as claimed in claim 1 is characterized in that: content of trehalose is that per 100 gram dry weight cells on average contain 20-40 gram trehalose.
3, a kind of production method of yeast saccharomyces cerevisiae of high content of trehalose, it is characterized in that: it comprises the steps:
(1) seed selection of the Wine brewing yeast strain of high content of trehalose:
(1). monoploid is cultivated: the yeast strain that preserve in the laboratory is cultivated on the YPD inclined-plane, under 25-30 ℃ condition, be transferred to pre-living spore substratum after the cultivation, under 25-30 ℃ condition, cultivated 2-3 days; Be forwarded to Kleyn again and give birth to the spore substratum, under 25-30 ℃ condition, cultivate and generate spore after 5-7 days; When examining under a microscope most of cell and the ascus of four spores being arranged in forming, phosphoric acid buffer with 0.2M washes ascus, transferring to the sterilization centrifuge tube carries out centrifugal, be suspended from same damping fluid, add beta-mercaptoethanol 1-3% to enlarge the cell permeability, add the broken ascus wall of helicase of 1-3% concentration again, question response liquid is extremely thick, the thecasporous release of microscopy, centrifugal reaction solution, wash 1-3 time with damping fluid, suitably be coated with the YPD plate after the dilution, under 25-30 ℃ condition, cultivate, single bacterium colony is chosen, with the monoploid mating of known mating type,, be stored in the refrigerator standby to determine that isolating monoploid is a or α mating type;
(2). satellite carries: before satellites transmits, a that above-mentioned separation is obtained and α monoploid are carried the bacterial classification atom cabin of packing into; Temperature is 17-26 ℃ in the cabin, and average ionizing radiation dose is 0.177GY/d, and the time is 15 days;
(3). mating is merged and formed diploid: the sample bacterial classification returns the laboratory, the beginning ground test, carry bacterial strain and ground control strain, after cultivating, measure the variation of cell intracellular trehalose semi-invariant, the monoploid that has different mating types is merged the formation diploid cell by mating, in order to be beneficial to The Fusants Screen, makes monoploid be with the auxotrophy selective marker by chemomorphosis;
(4). mutagenic treatment: a and α haploid strains are carried out mutagenesis with methylsulphonic acid ester, bacterium liquid after the processing is through suitably being coated on the YPD plate after the dilution, choose YPD and YNB basic medium respectively to detect auxotroph with growing bacterium colony on the YPD plate, selected content of trehalose higher H is in a mating type
-Have Met in defective strain and the α mating type
-, Thr
-The double defect strain is carried out protoplastis and is merged; Merging strain selects not adding on any amino acid whose YNB plate, from the diploid of strain more than 200, filter out the 10-20 strain, after three culturing cells are measured trehalose output, determine the trehalose bacterial classification of 1 plant height content again, be used to shake the test of bottle and canister optimization for fermentation technology;
(2) shake flask fermentation is cultivated: the substratum moiety: it is 2-10% that treated molasses are amounted to into glucose content; Yeast extract 1.5-5.0%; Corn steep liquor 3.0-5.0%; (NH
4)
2SO
4, (NH
4)
2HPO
4And micro-0-10%;
(1), culture temperature: after normal temperature is cultivated certain hour 1-20 hour, improve temperature again to 33-45 ℃;
(2), incubation time: the later stage that is accumulated in yeast growth of trehalose, the incubation time harvested cell at 16-36 hour can obtain higher trehalose;
(3) canister fermentation culture:
(1). strain inclined plane is cultivated: bacterial classification inoculation places 25-30 ℃ of incubator to cultivate 12-48 hour on the YPD inclined-plane;
(2). Boiling tube is cultivated: the bacterial classification on the above-mentioned YPD inclined-plane is inserted 5-10ml sterile malt juice body Boiling tube is housed, placed 20-40 ℃ of shaking table shaking culture 12-48 hour;
(3). triangular flask one-level enlarged culturing: above-mentioned tube culture is transferred and is handled the back waste molasses in 1/2 times of volume wort and 1/2 times of volume that 50-200ml is housed, amount in the triangular flask that glucose content is the 2-10% nutrient solution, put 20-40 ℃ of shaking table shaking culture 12-48 hour;
(4). triangular flask secondary enlarged culturing: the inoculum size of above-mentioned culture being pressed 5-20% inserts with the waste molasses after handling, and to amount to glucose content be 2%-10%, yeast extract 0.5-5.0% and comprise corn steep liquor, (NH
4)
2SO
4, (NH
4)
2HPO
4, vitamin H and the trace element in the liquid nutrient medium that interior at least three kinds of nutrition compositions of each 0.1-5% are made into, under 20-40 ℃ condition, shaking culture 12-48 hour;
(5). three grades of enlarged culturing of triangular flask: the inoculum size of above-mentioned culture being pressed 5-20% inserts with the molasses sugar after handling, and amounting to glucose content is 2%-10%, (NH
4)
2SO
4Or (NH
4)
2HPO
4In the liquid nutrient medium that 0.5-10%, vitamin H and micro-0-2% are made into, under 20-40 ℃ condition, cultivated 12-48 hour;
(6). the canister fermentation culture:
1). the fermention medium component: the waste molasses after the processing, ammonium salt, microcosmic salt and calcium, magnesium salts and trace element, each component concentration is at 0-10%;
2) the reasonable stream of carbon source and nutritive substance adds: add by the index upwelling earlier than growth rate and variation foundation thereof according to zymic in the fermenting process is instantaneous, add by index decreased stream, carbon source and nutritive substance that stream is added are converted into thalline to greatest extent;
3) by improving temperature during the fermentation, obtain higher trehalose output to 30-45 ℃;
4) improve the trehalose accumulation by prolonging fermentation time: on the basis of conventional fermentation time, prolong 1-15 hour again, cell intracellular trehalose content is improved;
(4) produce for big jar:
Canister fermentation parameter and processing condition according to above-mentioned foundation, carry out factory's bulk fermentation of 2-10 batch, the waste molasses that stream adds is amounted to into glucose calculating, average every gram conversion of glucose produces 0.3-0.5 gram thalline, per 100 gram dry weight cells on average contain 20-40 gram trehalose, produce the yeast saccharomyces cerevisiae of high content of trehalose, classification name: Saccharomyces Cerevisiae in S accharomyces cerevisiae, microbial strain: DZTA8, preserving number are the common micro-organisms center C GMCC No of China Committee for Culture Collection of Microorganisms 0535.
4, the production method of the yeast saccharomyces cerevisiae of high content of trehalose as claimed in claim 3, it is characterized in that: this satellite carries by radiation cultivates replacement, a and α monoploid bacterial classification that above-mentioned separation obtains are put into the radiation case, provide with satellite and carry identical substantially condition, 136/cm of high energy particle density
2, temperature is 17-26 ℃, and average ionizing radiation dose is 0.177GY/ days, and the time is 15-30 days.
5, the production method of the yeast saccharomyces cerevisiae of high content of trehalose as claimed in claim 3 is characterized in that: the cultivation YPD inclined-plane of this yeast strain is a peptone 2%, yeast powder 1%, glucose 2%, agar 1.5%.
6, the production method of the yeast saccharomyces cerevisiae of high content of trehalose as claimed in claim 3 is characterized in that: this gives birth to the spore substratum in advance is peptone 0.8%, yeast powder 0.3%, glucose 1%, agar 1.5%.
7, the production method of the yeast saccharomyces cerevisiae of high content of trehalose as claimed in claim 3 is characterized in that: it is KH that this Kleyn gives birth to the spore substratum
2PO
40.012%, K
2HPO
40.02%, NaAc 0.5%, glucose 0.062%, and NaCl 0.062%, vitamin H 0.2mg/100ml, micro-1ml/100ml; Wherein micro-component: MgSO
47H
2O 0.3-0.5%, CuSO
45H
2O 0.001-0.003%, MnSO
44H
2O 0.1-0.3%, FeSO
44H
2O 0.1-0.3%.
8, the production method of the yeast saccharomyces cerevisiae of high content of trehalose as claimed in claim 3 is characterized in that: this YNB basic medium contains glucose 10-30g, (NH in being every liter
4)
2SO
42-8g, KH
2PO
40.5-0.9g, K
2HPO
40.1-0.2g, MgSO
47H
2O 0.2-0.8g, NaCl 0.05-0.2g, CaCl
22H
2O0.05-0.2g; Trace element: H
3BO
4400-600 μ g, CuSO
45H
2O 20-60 μ g, KI 50-200 μ g, FeCl
26H
2O 100-300 μ g, MnSO
4H
2O 300-500 μ g, Na
2MoO
42H
2O 100-300 μ g, ZnSO
47H
2O 300-500 μ g; VITAMIN: growth hormone 1-3 μ g, calcium pantothenate 300-500 μ g, inositol 1000-3000 μ g, nicotinic acid 300-500 μ g, para-amino benzoic acid 100-300 μ g, VitB1 300-500 μ g, riboflavin 100-300 μ g, pyridoxol 300-500 μ g.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011018585A CN1141377C (en) | 2001-02-09 | 2001-02-09 | High-content mycose saccharomycetes and its preparing process |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011018585A CN1141377C (en) | 2001-02-09 | 2001-02-09 | High-content mycose saccharomycetes and its preparing process |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1302864A CN1302864A (en) | 2001-07-11 |
| CN1141377C true CN1141377C (en) | 2004-03-10 |
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| Application Number | Title | Priority Date | Filing Date |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101665772B (en) * | 2009-08-14 | 2012-06-20 | 中国食品发酵工业研究院 | Yeast strain of beer and application thereof |
| CN105861344A (en) * | 2016-05-30 | 2016-08-17 | 湖北工业大学 | Synchronous culture method for improving yeast biomass and intracellular trehalose content |
| CN107245458B (en) * | 2017-06-19 | 2019-12-24 | 湖北大学 | Screening and application of high-resistance trehalose-producing saccharomyces cerevisiae strain |
| CN108841897A (en) * | 2018-07-16 | 2018-11-20 | 安徽民祯生物工程有限公司 | It is a kind of for producing the rhodozyma culture method of trehalose |
| CN113151367A (en) * | 2021-05-10 | 2021-07-23 | 湖北工业大学 | Fermentation method for synthesizing xylitol de novo by self-protected zygosaccharomyces rouxii |
| CN114958707B (en) * | 2022-04-22 | 2024-04-09 | 南京同凯兆业生物技术有限责任公司 | Method for maintaining activity of yeast in fermentation process |
| CN116210886A (en) * | 2022-12-30 | 2023-06-06 | 安琪酵母股份有限公司 | Trehalose-enriched extract and process for its preparation |
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2001
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