CN105505801A - Saccharomyces cerevisiae for high yield of glutathione and application of saccharomyces cerevisiae - Google Patents

Saccharomyces cerevisiae for high yield of glutathione and application of saccharomyces cerevisiae Download PDF

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CN105505801A
CN105505801A CN201511003961.9A CN201511003961A CN105505801A CN 105505801 A CN105505801 A CN 105505801A CN 201511003961 A CN201511003961 A CN 201511003961A CN 105505801 A CN105505801 A CN 105505801A
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saccharomyces cerevisiae
yeast
glutathione
gsh
fermentation
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张卫元
蔡俊
戴军
高经纬
郝康
代兵
李硕
张靖
杨永荣
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HUBEI YANGSHENG BIOTECHNOLOGY CO Ltd
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Abstract

The invention discloses saccharomyces cerevisiae for a high yield of glutathione and application of the saccharomyces cerevisiae. The preservation date of the saccharomyces cerevisiae GSH-HS-001 is December 4, 2015, and the preservation number is CCTCC NO: M 2015724. After the saccharomyces cerevisiae is subjected to fermentation culture, the yield of glutathione is high, and the fermentation period is short. By the adoption of a fed-batch mode, a carbon and nitrogen source, vitamins and precursor amino acid are added respectively in a fed-batch mode to promote synthesis of glutathione, so that the content of glutathione in yeast cells is increased, and the problems of poor glutathione synthesis ability of yeast and low glutathione content in dry yeast are effectively solved. The application range of glutathione-rich dry yeast is expanded, and the added value of the product can be effectively increased.

Description

Yeast saccharomyces cerevisiae of a kind of high-yield glutathione and uses thereof
Technical field
The invention belongs to microbial technology field, relate to a kind of yeast saccharomyces cerevisiae and uses thereof, be specifically related to yeast saccharomyces cerevisiae (Saccharomycescerevisiae) GSH-HS-001 of the efficient synthesizing glutathion of a strain energy and uses thereof.
Background technology
Gsh (Glutathione, GSH) is formed through peptide bond condensation by L-glutamic acid, Cys and glycine, is non-protein class sulfhydryl compound the abundantest in organism, is extensively present in animal vegetable tissue and microorganism cells.Organism glutathion inside exists in two forms: reduced glutathion (GSH) and Sleep-promoting factor B (GSSG), and exists in a large number in body and that work is GSH.Due to the existence of sulfydryl (-SH) on halfcystine in GSH molecule, make GSH have multiple important physiological function in vivo, be mainly three aspects, as anti-oxidant, removing toxic substances and immunomodulatory.First, GSH plays a part to maintain appropriate redox environment in organism, and as a kind of important antioxidant, GSH can oxyradical (reactiveoxygenspecies in purged body, ROS), protect DNA, protein and other biomolecules not oxidized; The second, as toxinicide, the active group sulfydryl on halfcystine easily and the combination such as some drugs, toxin, heavy metal, and has integration detoxification; 3rd, can enhancing body immunological competence, in higher eucaryotic cells, GSH can the immunological competence of enhancing body rapidly.Therefore, gsh has market outlook widely in fields such as food, medicine, makeup, fermentation, fodder additivess.
The production method of GSH mainly contains chemical synthesis, solvent extration, enzyme transforming process and fermentation method.Wherein, fermentation method utilizes cheap carbohydrate, proteinaceous materials, carried out the method for GSH synthesis by the pathways metabolism of material in microorganism cells.This method has the advantages such as bacterial classification is easy to cultivation, the convenient cheapness of raw material sources, cost is low, transformation efficiency is high, throughput rate is fast, has become the main method of current production GSH.Abroad just achieved the suitability for industrialized production of GSH as far back as the eighties in 20th century, China obtains substantive breakthroughs not yet due to the suitability for industrialized production of the current GSH that starts late.
In the animal and plant cells and microorganism of all synthesis GSH, yeast is the microorganism of synthesis GSH most potentiality.Meanwhile, yeast contains rich in protein and VITAMIN, widely uses already in food, medicine, makeup, fermentation, fodder industry.Utilize yeast that fermentative Production is rich in gsh all to can be used as the raw material of above-mentioned industry, abundant functionality active component GSH is provided simultaneously, to improving HUMAN HEALTH, improve the aspects such as animal function and play a significant role.
Summary of the invention
Technical problem to be solved by this invention is the yeast of the efficient synthesizing glutathion of screening one strain energy and utilizes this yeast fermentation to prepare the dry yeast being rich in gsh, effectively solves the problem that yeast synthesizing glutathion ability is low and dry yeast Glutathione peptide content is low.Expansion is rich in the range of application of gsh dry yeast by the present invention, effectively improves value-added content of product.
To achieve these goals, the present invention adopts following technical scheme:
A kind of yeast saccharomyces cerevisiae, its Classification And Nomenclature is yeast saccharomyces cerevisiae (Saccharomycescerevisiae) GSH-HS-001, be preserved in China typical culture collection center on December 4th, 2015, deposit number is CCTCCNO:M2015724, address: China. Wuhan. and Wuhan University.
As preferably, yeast saccharomyces cerevisiae of the present invention is the yeast saccharomyces cerevisiae being rich in gsh for fermentation culture.
Yeast saccharomyces cerevisiae of the present invention is rich in the application in the fodder additives of gsh, food, medicine or makeup in preparation.
Yeast saccharomyces cerevisiae of the present invention is preparing the application of being rich in the dry yeast of gsh.
The deposit number that the present invention relates to is that yeast saccharomyces cerevisiae (Saccharomycescerevisiae) the GSH-HS-001 tool of CCTCCNO:M2015724 has the following advantages and progress significantly:
(1) ability of yeast saccharomyces cerevisiae (Saccharomycescerevisiae) the GSH-HS-001 synthesizing glutathion screened is strong, and fermentation period is short, clear through qualification genetic background, lifeless matter safety risks.
(2) in the dry yeast born of the same parents of fed-batch fermentation and spraying dry gained, reduced glutathion content, up to 200 ~ 220mg/g, is significantly higher than yeast saccharomyces cerevisiae (Saccharomycescerevisiae) CCCG reported.Efficiently solve the problem that yeast synthesizing glutathion ability is low and dry yeast Glutathione peptide content is low.Expansion is rich in the range of application of gsh dry yeast by this, effectively improves added value of product.
(3) be 60 ~ 80mg/g by reduced glutathion content in shake flask fermentation gained yeast born of the same parents, adopt reduced glutathion content in speed change fed-batch cultivation gained yeast born of the same parents to be 200 ~ 220mg/g by 10L fermentor tank, adopt reduced glutathion content in speed change fed-batch cultivation gained yeast born of the same parents to be 200 ~ 220mg/g by 20 cubes of fermentor tanks.
(4) yeast cell film and cell walls play natural cover for defense effect and reduce the oxidation of gsh, thus without the need to purifying to gsh, can use together with yeast cell, and this is by the cost of the loss and product that reduce gsh greatly.
Accompanying drawing explanation
Fig. 1 is that photo observed by GSH-HS-001 microscope (400X);
Fig. 2 is that GSH-HS-001 flat-plate bacterial colony observes photo.
Embodiment
The present invention repeatedly carries out bacterial screening by lot of experiments from the different samples that national different areas gather, finally obtain yeast saccharomyces cerevisiae (Saccharomycescerevisiae) GSH-HS-001 of the efficient synthesizing glutathion of a strain energy, be preserved in China typical culture collection center on December 4th, 2015, Patent Deposit Designation is CCTCCNO:M2015724.
Strain morphology feature: bacterium colony is creamy white, matt, flat, large and thick, opaque, smooth surface, neat in edge, moistening sticky, easily provoke, pros and cons, edge and central part solid colour.
Bacterial strain physiological property: the common carbon sources such as glucose, sucrose, maltose can be utilized.
Bacterial strain metabolic characteristic: metabolic process utilizes the efficient synthesizing glutathion of precursor substance.
Embodiment one: high-yield glutathione bacteria selection
(1) Screening Samples
Soil, fresh water, fruit, plant, animal excrement etc.
(2) substratum
Enrichment medium (g/L): glucose 50, (NH 4) 2sO 42, KH 2pO 42.5, MgSO 4﹒ 7H 2o1, yeast extract paste 0.5, pH4.5,115 DEG C of sterilizing 30min;
Primary dcreening operation plate culture medium (g/L): glucose 50, urea 1, (NH 4) 2sO 41, KH 2pO 42.5, Na 2hPO 40.5, MgSO 4﹒ 7H 2o1, FeSO 4﹒ 7H 2o0.1, yeast extract paste 0.5, rose-bengal 0.03, pH4.5, agar 20,115 DEG C of sterilizing 30min;
Slant medium (g/L): glucose 20, peptone 20, yeast extract paste 10, pH4.5, agar 20,115 DEG C of sterilizing 30min;
Seed culture medium (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
Fermention medium (g/L): glucose 50, yeast extract paste 10, urea 4, (NH 4) 2sO 44, (NH 4) 2hPO 410, KH 2pO 42.5, Na 2hPO 40.5, MgSO 4﹒ 7H 2o1, CaCl 21, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
Mixed vitamin solution (g/L): vitamins B 11, vitamins B 21, vitamins B 31, vitamins B 41, vitamins B 63, calcium pantothenate 1;
Precusor amino acids solution (g/L): Sodium Glutamate 40, L-cysteine hydrochloride 40, glycine 10.
(3) enrichment culture
Get 10g Screening Samples in 100ml sterile saline, get 10ml bacterium liquid after 200 ~ 220rpm shaking table vibration, 1 ~ 2h at 28 ~ 32 DEG C and be inoculated in enrichment medium, 28 ~ 32 DEG C, 200 ~ 220rpm shaking table shaking culture, 36 ~ 42h.Enrichment culture 3 times.
(4) dilution spread
Get in the small test tube of enrichment culture liquid 0.5mL after sterilizing, add 4.5ml sterilized water, dilute successively, obtain 10 respectively -1to 10 -7deng 7 dilution gradients.Get 10 respectively -5to 10 -7deng the dilution bacterium liquid 100 μ l of 3 gradients on primary dcreening operation plate culture medium, coating evenly, each gradient do respectively 3 parallel.28 ~ 32 DEG C of constant temperature culture 36 ~ 48h.
(5) plate streaking divides pure
Get colony diameter on dilution spread flat board comparatively large, the good bacterium colony of growing way is numbered respectively and line divides pure, in 28 ~ 32 DEG C of constant temperature culture 36 ~ 48h on agar plate.Line point pure rear choosing colony diameter is comparatively large, grows bacterial strain faster and to transfer 4 DEG C of Storage in refrigerator after slant medium 28 ~ 32 DEG C of constant temperature culture 36 ~ 48h.Primary dcreening operation screens 904 bacterial strains altogether.
(6) primary dcreening operation bacterial strain activation
Slant medium sterilizing is for subsequent use, is transferred by primary dcreening operation bacterial strain in test tube slant activation, cultivates 36 ~ 48h for 28 ~ 32 DEG C.
(7) seed liquor preparation
Seed culture medium packing (100ml/500ml shaking flask) sterilizing is for subsequent use, gets the strain transfer after activation in seed culture medium.28 ~ 32 DEG C, 200 ~ 220rpm shaking table cultivation, 18 ~ 20h.
(8) inoculation fermentation substratum fermentation
Fermention medium packing (100ml/500ml shaking flask) sterilizing is for subsequent use, gets 5mL mixed vitamin solution and 10ml seed liquor (inoculum size 8 ~ 10%) adds in fermention medium, 28 ~ 32 DEG C, 200 ~ 220rpm shaking table cultivates.Add 30% glucose solution 15mL respectively when shaking table cultivates 12h, 24h, add 15mL precusor amino acids solution when 24h cultivated by shaking table, shaking table terminates fermentation after cultivating 36 ~ 42h.
(9) breaking yeast cellule membrane release gsh
Get the bacterium liquid of certain volume in centrifuge tube, centrifugal 5 ~ the 10min of 5000 ~ 8000rpm collects thalline, after brine somatic cells 2 ~ 3 times, suspend with distilled water,-80 DEG C of refrigerators are put into freezing rapidly after shaking up, freezing thalline is melted rapidly under boiling water bath condition, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm after multigelation 2 times, get supernatant liquor as test sample.
(10) mensuration of dry cell weight
Get the bacterium liquid of certain volume in centrifuge tube, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm collects thalline, and after brine somatic cells 2 ~ 3 times, the wet thallus obtained is dried to constant weight under 105 DEG C of conditions.
(11) mensuration of born of the same parents' glutathion inside content
Adopt iodometric determination supernatant liquor Glutathione peptide content, then calculate the content of born of the same parents' glutathion inside in conjunction with sporoderm-broken rate and dry cell weight.
See Fig. 1 and Fig. 2, through primary dcreening operation and multiple sieve, the ability being numbered the bacterial strain synthesizing glutathion of GSH-HS-001 is the strongest, it is 60 ~ 80mg/g by reduced glutathion content in shake flask fermentation gained yeast born of the same parents, identified by 18SrRNA, ITS and 26SrRNA, finally determine that this bacterial strain is yeast saccharomyces cerevisiae (Saccharomycescerevisiae), be preserved in China typical culture collection center on December 4th, 2015, deposit number is CCTCCNO:M2015724.Using the test strain of this bacterial strain as subsequent embodiment.Wherein, the sequence of 18SrRNA is the SEQIDNO.1 in sequence table, the ITS sequence SEQIDNO.3 that to be the SEQIDNO.2 in sequence table, 26SrRNA sequence be in sequence table.
Embodiment two: cultivate in the horizontal top fermentation of shaking flask
(1) bacterial classification
Yeast saccharomyces cerevisiae (Saccharomycescerevisiae) GSH-HS-001, deposit number CCTCCNO:M2015724.
(2) substratum
Slant medium (g/L): glucose 20, peptone 20, yeast extract paste 10, pH4.5, agar 20,115 DEG C of sterilizing 30min;
Seed culture medium (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
Fermention medium (g/L): glucose 50, yeast extract paste 10, urea 4, (NH 4) 2sO 44, (NH 4) 2hPO 410, KH 2pO 42.5, Na 2hPO 40.5, MgSO 4﹒ 7H 2o1, CaCl 21, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
Mixed vitamin solution (g/L): vitamins B 11, vitamins B 21, vitamins B 31, vitamins B 41, vitamins B 63, calcium pantothenate 1;
Precusor amino acids solution (g/L): Sodium Glutamate 40, L-cysteine hydrochloride 40, glycine 10.
(3) actication of culture
Slant medium sterilizing is for subsequent use, is activated by strain transfer in test tube slant, cultivates 36 ~ 48h for 28 ~ 32 DEG C.
(4) seed liquor preparation
Seed culture medium packing (100ml/500ml shaking flask) sterilizing is for subsequent use, gets the strain transfer after activation in seed culture medium.28 ~ 32 DEG C, 200 ~ 220rpm shaking table cultivation, 18 ~ 20h.
(5) inoculation fermentation substratum fermentation
Fermention medium packing (100ml/500ml shaking flask) sterilizing is for subsequent use, gets 5mL mixed vitamin solution and 10ml seed liquor (inoculum size 8 ~ 10%) adds in fermention medium, 28 ~ 32 DEG C, 200 ~ 220rpm shaking table cultivates.Add 30% glucose solution 15mL respectively when shaking table cultivates 12h, 24h, add 15mL precusor amino acids solution when 24h cultivated by shaking table, shaking table terminates fermentation after cultivating 36 ~ 42h.
(6) breaking yeast cellule membrane release gsh
Get the bacterium liquid of certain volume in centrifuge tube, centrifugal 5 ~ the 10min of 5000 ~ 8000rpm collects thalline, after brine somatic cells 2 ~ 3 times, suspend with distilled water,-80 DEG C of refrigerators are put into freezing rapidly after shaking up, freezing thalline is melted rapidly under boiling water bath condition, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm after multigelation 2 times, get supernatant liquor as test sample.
(7) mensuration of dry cell weight
Get the bacterium liquid of certain volume in centrifuge tube, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm collects thalline, and after brine somatic cells 2 ~ 3 times, the wet thallus obtained is dried to constant weight under 105 DEG C of conditions.
(8) mensuration of born of the same parents' glutathion inside content
Adopt iodometric determination supernatant liquor Glutathione peptide content, then calculate the content of born of the same parents' glutathion inside in conjunction with sporoderm-broken rate and dry cell weight.
(9) result
Fermentation time: 36-42 hour; Dry cell weight: 10-15g/L; Born of the same parents' glutathion inside content: 60-80mg/g.
Embodiment three: cultivate in the top fermentation of 10L fermentation tank level
(1) bacterial classification
Yeast saccharomyces cerevisiae (Saccharomycescerevisiae) GSH-HS-001, deposit number CCTCCNO:M2015724.
(2) substratum
Slant medium (g/L): glucose 20, peptone 20, yeast extract paste 10, pH4.5, agar 20,115 DEG C of sterilizing 30min;
Seed culture medium (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
(end water 2L) fermention medium (g/L) on 10L fermentation tank level: yeast extract paste 30, CaCl 22.5, MgSO 4﹒ 5H 2o7.5, ZnSO 40.008, KCl5, pH4.5 ~ 6.0, defoamer 4mL;
Fed-batch cultivation substratum: 300g/L glucose sugar (4L), 80g/L urea, 35g/L ammonium sulfate (600mL), 57g/L primary ammonium phosphate (300mL), mixed vitamin solution 100mL is (containing vitamins B 10.1%, vitamins B 20.1%, vitamins B 30.1%, vitamins B 40.1%, vitamins B 60.3%, calcium pantothenate 0.1%), precusor amino acids solution 600ml (containing Sodium Glutamate 4.33%, L-cysteine hydrochloride 4.67%, glycine 1.17%).
(3) actication of culture
Slant medium sterilizing is for subsequent use, is activated by strain transfer in test tube slant, cultivates 36 ~ 48h for 28 ~ 32 DEG C.
(4) fermentation seed liquid preparation
Getting inclined-plane seed one to two articulating activated, to enter liquid amount be in the seed culture medium of 50mL/250mL, 28 ~ 32 DEG C, 200 ~ 220rpm shaking table cultivates 18 ~ 20h and namely obtains first order seed, first order seed to be accessed in identical seed culture medium 28 ~ 32 DEG C according to the ratio of inoculum size 8% ~ 10%, 200 ~ 220rpm shaking table cultivates 14 ~ 18h and namely obtain secondary seed.
(5) inoculation fermentation tank fermentation
The secondary seed getting above-mentioned preparation is inoculated in fermentor tank according to the inoculum size of 8% ~ 10%, utilize glucose for carbon source, urea, ammonium sulfate, Secondary ammonium phosphate are nitrogen phosphorus source control by stages feed-batch process, control leavening temperature 28 ~ 32 DEG C, mixing speed 200 ~ 600rmp, air flow 5 ~ 15L/min, tank pressure 0.03MPa ~ 0.04MPa, pH4.5 ~ 6.0.Disposablely during inoculation add mixed vitamin solution 100mL, add precusor amino acids solution 600ml nitrogen, phosphorus stream are disposable after adding 1 ~ 2h.Fermenting process adjusting rotary speed and air flow, control by stages dissolved oxygen levels.Glucose continuous flow adduction controls glucose content 1%, and 36 ~ 42h terminates fermentation.
(6) breaking yeast cellule membrane release gsh
Get the bacterium liquid of certain volume in centrifuge tube, centrifugal 5 ~ the 10min of 5000 ~ 8000rpm collects thalline, after brine somatic cells 2 ~ 3 times, suspend with distilled water,-80 DEG C of refrigerators are put into freezing rapidly after shaking up, freezing thalline is melted rapidly under boiling water bath condition, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm after multigelation 2 times, get supernatant liquor as test sample.
(7) mensuration of dry cell weight
Get the bacterium liquid of certain volume in centrifuge tube, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm collects thalline, and after brine somatic cells 2 ~ 3 times, the wet thallus obtained is dried to constant weight under 105 DEG C of conditions.
(8) mensuration of born of the same parents' glutathion inside content
Adopt iodometric determination supernatant liquor Glutathione peptide content, then calculate the content of born of the same parents' glutathion inside in conjunction with sporoderm-broken rate and dry cell weight.
(9) result
Fermentation time: 36-42 hour; Dry cell weight: 45-50g/L; Born of the same parents' glutathion inside content: 200-220mg/g.
Embodiment four: cultivate 20 cubes of fermentation tank level top fermentations
(1) bacterial classification
Yeast saccharomyces cerevisiae (Saccharomycescerevisiae) GSH-HS-001, deposit number CCTCCNO:M2015724.
(2) substratum
Slant medium (g/L): glucose 20, peptone 20, yeast extract paste 10, pH4.5, agar 20,115 DEG C of sterilizing 30min;
Primary-seed medium (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
Secondary seed medium (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
Three grades of seed culture mediums (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
20m 3(end water 4000L) fermention medium (g/L) on fermentation tank level: yeast extract paste 30, CaCl 22.5, MgSO 4﹒ 5H 2o7.5, ZnSO 40.008, KCl5, pH4.5 ~ 6.0;
Fed-batch cultivation substratum: 300g/L glucose sugar (8000L), 80g/L urea, 35g/L ammonium sulfate (1300L), 57g/L primary ammonium phosphate (600L), mixed vitamin solution 200L is (containing vitamins B 10.1%, vitamins B 20.1%, vitamins B 30.1%, vitamins B 40.1%, vitamins B 60.3%, calcium pantothenate 0.1%), precusor amino acids solution 1200L (containing Sodium Glutamate 4.33%, L-cysteine hydrochloride 4.67%, glycine 1.17%).
(3) actication of culture
Slant medium sterilizing is for subsequent use, is activated by strain transfer in test tube slant, cultivates 36 ~ 48h for 28 ~ 32 DEG C.
(4) fermentation seed liquid preparation
Getting inclined-plane seed one to two articulating activated, to enter liquid amount be in the seed culture medium of 1000mL/5000mL, 28 ~ 32 DEG C, 200 ~ 220rpm shaking table cultivate 18 ~ 20h namely obtain first order seed, according to inoculum size, first order seed accesses in the 800L fermentor tank of identical seed culture medium by the ratio of 1% ~ 3% (V/V) to be cultivated 20 ~ 24h and namely obtains secondary seed, by the inoculum size of 8% ~ 10% (V/V), secondary seed is accessed the 2m of identical seed culture medium afterwards again 3preparation in 12-16 hour three grades of seeds are cultivated in fermentor tank.
(5) inoculation fermentation tank fermentation
The three grades of seeds getting above-mentioned preparation are inoculated in fermentor tank according to the inoculum size of 10% ~ 15%, utilize glucose for carbon source, urea, ammonium sulfate, Secondary ammonium phosphate are nitrogen phosphorus source control by stages feed-batch process, control leavening temperature 28 ~ 32 DEG C, air flow 300 ~ 700m 3/ h, tank pressure 0.03MPa ~ 0.04MPa, pH4.5 ~ 6.0.Disposablely during inoculation add mixed vitamin solution 200L, add precusor amino acids solution 1200L nitrogen, phosphorus stream are disposable after adding 1 ~ 2h.Fermenting process adjusting rotary speed and air flow, control by stages dissolved oxygen levels, controls the content of alcohol in fermented liquid at 0.1%-0.5% (W/V) simultaneously.Glucose continuous flow adduction controls glucose content 1%, and 36 ~ 42h terminates fermentation.
(6) breaking yeast cellule membrane release gsh
Get the bacterium liquid of certain volume in centrifuge tube, centrifugal 5 ~ the 10min of 5000 ~ 8000rpm collects thalline, after brine somatic cells 2 ~ 3 times, suspend with distilled water,-80 DEG C of refrigerators are put into freezing rapidly after shaking up, freezing thalline is melted rapidly under boiling water bath condition, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm after multigelation 2 times, get supernatant liquor as test sample.
(7) mensuration of dry cell weight
Get the bacterium liquid of certain volume in centrifuge tube, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm collects thalline, and after brine somatic cells 2 ~ 3 times, the wet thallus obtained is dried to constant weight under 105 DEG C of conditions.
(8) mensuration of born of the same parents' glutathion inside content
Adopt iodometric determination supernatant liquor Glutathione peptide content, then calculate the content of born of the same parents' glutathion inside in conjunction with sporoderm-broken rate and dry cell weight.
(9) result
Fermentation time: 36-42 hour; Dry cell weight: 45-50g/L; Born of the same parents' glutathion inside content: 200-220mg/g.
Embodiment five: the preparation of being rich in gsh dry yeast
(1) bacterial classification
Yeast saccharomyces cerevisiae (Saccharomycescerevisiae) GSH-HS-001, deposit number CCTCCNO:M2015724.
(2) substratum
Slant medium (g/L): glucose 20, peptone 20, yeast extract paste 10, pH4.5, agar 20,115 DEG C of sterilizing 30min;
Primary-seed medium (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
Secondary seed medium (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
Three grades of seed culture mediums (g/L): glucose 20, peptone 20, yeast extract paste 10, NaCl5, pH4.5 ~ 6.0,115 DEG C of sterilizing 30min;
20m 3(end water 4000L) fermention medium (g/L) on fermentation tank level: yeast extract paste 30, CaCl 22.5, MgSO 4﹒ 5H 2o7.5, ZnSO 40.008, KCl5, pH4.5 ~ 6.0;
Fed-batch cultivation substratum: 300g/L glucose sugar (8000L), 80g/L urea, 35g/L ammonium sulfate (1300L), 57g/L primary ammonium phosphate (600L), mixed vitamin solution 200L is (containing vitamins B 10.1%, vitamins B 20.1%, vitamins B 30.1%, vitamins B 40.1%, vitamins B 60.3%, calcium pantothenate 0.1%), precusor amino acids solution 1200L (containing Sodium Glutamate 4.33%, L-cysteine hydrochloride 4.67%, glycine 1.17%).
(3) actication of culture
Slant medium sterilizing is for subsequent use, is activated by strain transfer in test tube slant, cultivates 36 ~ 48h for 28 ~ 32 DEG C.
(4) fermentation seed liquid preparation
Getting inclined-plane seed one to two articulating activated, to enter liquid amount be in the seed culture medium of 1000mL/5000mL, 28 ~ 32 DEG C, 200 ~ 220rpm shaking table cultivate 18 ~ 20h namely obtain first order seed, according to inoculum size, first order seed accesses in the 800L fermentor tank of identical seed culture medium by the ratio of 1% ~ 3% (V/V) to be cultivated 20 ~ 24h and namely obtains secondary seed, by the inoculum size of 8% ~ 10% (V/V), secondary seed is accessed the 2m of identical seed culture medium afterwards again 3preparation in 12-16 hour three grades of seeds are cultivated in fermentor tank.
(5) inoculation fermentation tank fermentation
The three grades of seeds getting above-mentioned preparation are inoculated in fermentor tank according to the inoculum size of 10% ~ 15%, utilize glucose for carbon source, urea, ammonium sulfate, Secondary ammonium phosphate are nitrogen phosphorus source control by stages feed-batch process, control leavening temperature 28 ~ 32 DEG C, air flow 300 ~ 700m 3/ h, tank pressure 0.03MPa ~ 0.04MPa, pH4.5 ~ 6.0.Disposablely during inoculation add mixed vitamin solution 200L, add precusor amino acids solution 1200L nitrogen, phosphorus stream are disposable after adding 1 ~ 2h.Fermenting process adjusting rotary speed and air flow, control by stages dissolved oxygen levels, controls the content of alcohol in fermented liquid at 0.1%-0.5% (W/V) simultaneously.Glucose continuous flow adduction controls glucose content 1%, and 36 ~ 42h terminates fermentation.
(6) preparation of gsh dry yeast is rich in
The fermented liquid obtained in (5) centrifugal 5 ~ 10min under 5000 ~ 8000rpm condition is collected thalline, with after brine somatic cells 2 ~ 3 times clean thalline, with the physiological saline having dissolved 0.1% ~ 0.3% gum arabic, clean thalline is configured to the yeast juice of 10% ~ 15% again, drying machine with centrifugal spray is utilized to control inlet temperature 140 ~ 160 DEG C, air outlet temperature 80 ~ 90 DEG C, finally obtain dry matter content 92% ~ 96% dry yeast.
(7) breaking yeast cellule membrane release gsh
Get 1g dry yeast 99g distilled water to suspend, put into-80 DEG C of refrigerators after shaking up freezing rapidly, freezing thalline is melted rapidly under boiling water bath condition, the centrifugal 5 ~ 10min of 5000 ~ 8000rpm after multigelation 2 times, get supernatant liquor as test sample.
(8) mensuration of dry yeast moisture content
The dry yeast getting certain mass is dried to constant weight under 105 DEG C of conditions, measures moisture content.
(9) mensuration of born of the same parents' glutathion inside content
Adopt iodometric determination supernatant liquor Glutathione peptide content, then calculate the content of born of the same parents' glutathion inside in conjunction with sporoderm-broken rate and dry cell weight.
(10) result
Dry yeast dry matter content: 92% ~ 96%; Born of the same parents' glutathion inside content: 200-220mg/g.

Claims (4)

1. a yeast saccharomyces cerevisiae, is characterized in that: described yeast saccharomyces cerevisiae Classification And Nomenclature be yeast saccharomyces cerevisiae ( saccharomycescerevisiae) GSH-HS-001, deposit number is CCTCCNO:M2015724.
2. yeast saccharomyces cerevisiae according to claim 1, is characterized in that: described yeast saccharomyces cerevisiae is the yeast saccharomyces cerevisiae being rich in gsh for fermentation culture.
3. the yeast saccharomyces cerevisiae described in claim 1 or 2 is rich in the application in the fodder additives of gsh, food, medicine or makeup in preparation.
4. the yeast saccharomyces cerevisiae described in claim 1 or 2 is preparing the application of being rich in the dry yeast of gsh.
CN201511003961.9A 2015-12-25 2015-12-25 Saccharomyces cerevisiae for high yield of glutathione and application of saccharomyces cerevisiae Pending CN105505801A (en)

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CN106119139A (en) * 2016-06-21 2016-11-16 南京农业大学 A kind of selenium-rich produces glutathion compound bacteria and its preparation method and application
US10053744B2 (en) 2016-10-11 2018-08-21 Ningxia RisingMark Intellectual Property Consulting Co., Ltd. Yeast strain with high yield of glutathione
CN106617109A (en) * 2016-11-09 2017-05-10 北京东方兴企食品工业技术有限公司 Functional nutrition composition for improving and treating liver disease and preparation method of functional nutrition composition
CN107090483A (en) * 2017-04-24 2017-08-25 吉林大学 Application of the brewer's yeast in glutathione biosynthesis
CN107090483B (en) * 2017-04-24 2021-03-05 吉林大学 Application of saccharomyces cerevisiae in glutathione biosynthesis
CN114761539A (en) * 2019-10-29 2022-07-15 Cj第一制糖株式会社 Yeast strain for producing glutathione and method for producing glutathione using same
CN114761539B (en) * 2019-10-29 2023-11-14 Cj第一制糖株式会社 Glutathione producing yeast strain and method for producing glutathione using the same
CN114847467A (en) * 2021-02-03 2022-08-05 安琪酵母股份有限公司 Yeast extract rich in cysteine and preparation method thereof
CN114276942A (en) * 2021-12-30 2022-04-05 安琪酵母股份有限公司 Glutathione yeast, preparation method and application of glutathione yeast product
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