CN101955890B - Cadmium salt-resistant saccharomyces cerevisiae and application thereof - Google Patents
Cadmium salt-resistant saccharomyces cerevisiae and application thereof Download PDFInfo
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- CN101955890B CN101955890B CN201010503178XA CN201010503178A CN101955890B CN 101955890 B CN101955890 B CN 101955890B CN 201010503178X A CN201010503178X A CN 201010503178XA CN 201010503178 A CN201010503178 A CN 201010503178A CN 101955890 B CN101955890 B CN 101955890B
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- bread yeast
- cadmium salt
- saccharomyces cerevisiae
- gsh
- glutathione
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Abstract
The invention relates to cadmium salt-resistant saccharomyces cerevisiae BV54-11-11, which is collected in China General Microbiological Culture Collection Center on September 21st, 2010 and has the collection number of CGMCC No.4189. A method for producing glutathione-enriched saccharomyces cerevisiae thalli by using the cadmium salt-resistant saccharomyces cerevisiae comprises the following steps of: 1) picking BV54-11-11 thalli from an activated slope and inoculating the thalli to a seed culture medium to prepare seed liquid; and 2) inoculating 5 to 15 percent of seed liquid to an optimized fermentation medium for fermentation. The invention has the advantages that: in the glutathione-enriched saccharomyces cerevisiae thalli obtained by fermentation, 19mg of glutathione exists in each gram of dry thalli; and the glutathione-enriched saccharomyces cerevisiae thalli can be used for extracting the glutathione to obtain two products, namely the glutathione and mycoprotein and can be directly used as functional yeast.
Description
[technical field]
The invention belongs to the microbial fermentation technology field, be specifically related to a strain cadmium salt resistance bread yeast and be rich in the application in the gsh bread yeast thalline in production.
[background technology]:
Gsh (glutathione; GSH) be the biological activity tripeptides that forms by L-glutamic acid, halfcystine and glycocoll condensation; Be divided into reduced form and oxidized form in vivo; Wherein contain γ-Gu Anxianji and sulfhydryl-group activity simultaneously in the reduced form GSH molecule, can participate in multiple biological respinse, significant to the standard state of keeping cell.GSH anti-oxidant, remove radical, detoxifcation, strengthening immunity, delay senility, aspect such as anticancer, anti-radioactive rays plays a significant role, and is the important function factor, therefore, in foodstuffs industry, medicine industry, uses very extensively, have a extensive future.
The working method of gsh has solvent extration, chemical synthesis, biological process; Biological process comprises enzyme process and fermentation method; Wherein fermentation method is to utilize mikrobe self metabolism to produce gsh; Having advantages such as easy, easy control, easy expansion production with data by MoM and MEI, is present main gsh working method.The key issue of fermentative Production gsh is how to improve cell density and intracellular glutathione content, and the combination of the two will help increasing substantially of gsh output.The raising of cell density can obtain through the optimal control (comprising high-density culture) to fermenting process; And the raising of born of the same parents' glutathion inside content is normally realized by strain improvement; Mainly contain two kinds of methods: the one, adopt conventional selection by mutation breeding high-yield bacterial strain, the 2nd, utilize gene engineering to make up and have the active genetic engineering bacterium of glutathione synthetase.
The bacterial classification that can accumulate gsh at present is mainly yeast morely; The strain breeding method that adopts mainly is chemical mutagenesis (nitrosoguanidine, ethyl sulfate etc.) and physical mutagenesis method (ultraviolet ray and X ray); The mutant strain that obtains is main with ethionine resistant strain, methionine(Met) sensitive strain, zine ion resistant strain etc.; The present invention is through ultraviolet and ethyl sulfate complex mutation; With the Cadmium Sulphate resistance is selection markers, and screening obtains the bread yeast bacterial strain that the GSH growing amount improves in a strain cadmium salt resistance, the born of the same parents.
[summary of the invention]:
The object of the invention provides a strain cadmium salt resistance bread yeast mutant strain and utilizes this bacterial strain production to be rich in the method for gsh yeast thalline.
Technical scheme of the present invention:
One strain cadmium salt resistance bread yeast, said bread yeast (Saccharomyces cerevisiae) BV54-11-11 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 21st, 2010, and preserving number is CGMCC No.4189.
A kind ofly use the method that gsh bread yeast thalline is rich in the production of said cadmium salt resistance bread yeast, step is:
1) picking one ring BV54-11-11 thalline inserts the seed culture medium from activated inclined plane, is to leave standstill under 26～30 ℃ to cultivate 20～24h in temperature, promptly gets seed liquor;
2) with seed liquor with 5～15% inoculum sizes, insert to optimize fermention medium and ferment, fermentation condition is: liquid amount (30～80) mL/250mL, rotating speed 150～200r/min, 26～30 ℃ of temperature, pH value 5.5～6.5, incubation time 20～30h.
Consisting of of said seed culture medium: glucose 20g/L, peptone 20g/L, yeast soak powder 10g/L.
Consisting of of said optimization fermention medium: glucose 25～35g/L, steeping water 0.3～1.0% (v/v), (NH
4) H
2PO
4(1～3) g/L, KH
2PO
4(0.5～2) g/L, MgSO
47H
2O (0.05～0.3) g/L.
Advantage of the present invention and positively effect:
The invention provides a strain cadmium salt resistance, can be in born of the same parents the bread yeast bacterial strain of enrichment gsh, use this bread yeast and can ferment and obtain being rich in the bread yeast thalline of gsh, gsh born of the same parents intensive amount can reach the 19mg/g dry mycelium; The bread yeast thalline that gained is rich in gsh both can be used for gsh and extract and obtain gsh and two kinds of products of tropina, also can be directly as functional yeast use.
[embodiment]:
Embodiment 1: but the mutagenesis screening of cadmium salt resistance enrichment gsh bread yeast bacterial strain
1) ultraviolet mutagenesis and screening
BV54 is a starting strain with bread yeast (Saccharomyces cerevisiae), and picking one ring BV54 places 50mL YPD substratum from the inclined-plane, and 30 ℃ leave standstill cultivation 36h, the preparation first order seed.Be inoculated in the 250mL triangular flask that 50mL YPD is housed with 2% inoculum size, 30 ℃, 150r/min cultivates 10h, makes thalli growth be in logarithmic growth latter stage.Get 1mL bacterium liquid after shaking up, the saline water with 0.85% is diluted to cell concentration 10
3, get the 0.1mL diluent respectively and coat on the resistance checking flat board that contains the different concns cadmium salt, cultivate 3～4d for 30 ℃, observe its growing state, as shown in table 1.
The checking of table 1 bread yeast BV54 cadmium salt resistance
" ++ ": expression has stronger growth; "+": expression has more weak growth; "-": expression can not be grown
Can know by table 1; Bread yeast BV54 has more weak growth on 0.90mmol/L cadmium salt resistant panel; And on 1.00mmol/L cadmium salt resistant panel, can not grow, so the YPD flat board of selecting to contain the 1.00mmol/L Cadmium Sulphate is as bread yeast BV54 ultraviolet mutagenesis primary dcreening operation flat board.
With bread yeast BV54 is starting strain, according to the relation of yeast GSH growing amount and its cadmium salt resistance, utilizes the cadmium salt resistant panel to screen the mutant strain that GSH output improves.Selecting lethality rate is that 85% ultraviolet mutagenesis dosage (irradiation 120s) carries out mutagenesis to bread yeast; Bacterium liquid after the mutagenesis is evenly coated on the cadmium salt resistant panel of 1.0mmol/L; Picking mutant strain behind 30 ℃ of lucifuges cultivation 3～4d; Obtain the bread yeast mutant strain that 39 strain GSH obviously improve at last, called after BV54-1～BV54-39 selects glutathione content to improve tangible BV54-11 bacterial strain and carries out chemomorphosis.
2) chemomorphosis and screening
Cultivate BV54-11 by above-mentioned bread yeast BV54 cultural method, confirm that the cadmium salt resistance screening is dull and stereotyped.The result sees table 2.
The checking of table 2 bread yeast BV54-11 cadmium salt resistance
" ++ ": expression has stronger growth; "+": expression has more weak growth; "-": expression can not be grown
Bread yeast BV54-11 has more weak growth on 1.00mmol/L cadmium salt resistant panel; And on 1.10mmol/L cadmium salt resistant panel, can not grow, so the YPD flat board of selecting to contain the 1.10mmol/L Cadmium Sulphate is as bread yeast BV54-11 chemomorphosis primary dcreening operation flat board.
To select lethality rate be 80.5% DES consumption (0.146%) carries out mutagenic treatment 15min to the bread yeast BV54-11 that is in logarithmic growth latter stage, the bacterium liquid after the mutagenesis is evenly coated on the YPD flat board that contains the 1.10mmol/L cadmium salt, cultivates 3～4d for 30 ℃.After treating that bacterium colony grows, select single bacterium colony point that growth is fast, bacterium colony is big and be connected on the YPD flat board, and numbering BV54-11-1～BV54-11-51.The GSH fermenting experiment is carried out in the resistant mutant strain and the strain of setting out respectively, measure born of the same parents' glutathion inside growing amount behind the fermentation 22h, obtain the mutant strain BV54-11-11 that a strain GSH output obviously improves, more former bacterial strain GSH output improves 68%, and the result sees table 3.
Table 3 mutant strain and starting strain GSH growing amount are relatively
Embodiment 2: the shake flask fermentation experiment
With bread yeast BV54-11-11 is fermented bacterium; In conjunction with Plackett-Burman (PB) design and response surface design fermention medium and the fermentation condition that its fermentative prodn is rich in gsh bread yeast thalline is optimized; The fermention medium that is optimized is: glucose 30g/L; Steeping water 0.5% (v/v), (NH
4) H
2PO
42g/L, KH
2PO
41g/L, MgSO
47H
2O 0.1g/L.Optimization of fermentation conditions is: liquid amount 50mL/250mL, rotating speed 180r/min, 28 ℃ of temperature, pH value 6.0, incubation time 22h.
Picking one ring BV54-11-11 thalline inserts the seed culture medium from activated inclined plane, and 30 ℃ leave standstill cultivation 22h, get seed liquor.Seed culture medium consists of: glucose 20g/L, peptone 20g/L, yeast soak powder 10g/L.Seed liquor with 5% inoculum size, is inserted the optimization fermention medium and under optimization of fermentation conditions, ferments.3 batches of shake flask fermentations are tested the result who records and are seen table 8, and GSH output MV is 122.02mg/L, and GSH content MV is 19.07mg/g, and dried cell weight MV is 6.40g/L.
Table 4 bread yeast BV54-11-11 shake flask fermentation result
Claims (3)
1. a strain cadmium salt resistance bread yeast; It is characterized in that: said bread yeast (Saccharomyces cerevisiae) BV54-11-11 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 21st, 2010, and preserving number is CGMCC No.4189.
2. an application rights requires 1 said cadmium salt resistance bread yeast production to be rich in the method for gsh bread yeast thalline, it is characterized in that step is:
1) picking one ring BV54-11-11 thalline inserts the seed culture medium from activated inclined plane, is to leave standstill under 26～30 ℃ to cultivate 20～24h in temperature, promptly gets seed liquor;
2) with seed liquor with 5～15% inoculum sizes, insert to optimize fermention medium and ferment, fermentation condition is: liquid amount 30～80mL/250mL, rotating speed 150～200r/min, 26～30 ℃ of temperature, pH value 5.5～6.5, incubation time 20～30h;
Consisting of of said optimization fermention medium: glucose 25～35g/L, steeping water 0.3～1.0% (v/v), (NH
4) H
2PO
41～3g/L, KH
2PO
40.5～2g/L, MgSO
47H
2O 0.05～0.3g/L.
3. the method for gsh bread yeast thalline is rich in production according to the said application cadmium salt of claim 2 resistance bread yeast, it is characterized in that: the consisting of of said seed culture medium: glucose 20g/L, peptone 20g/L, yeast soak powder 10g/L.
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| JP2012213361A (en) * | 2011-03-31 | 2012-11-08 | Kyoto Univ | Method for creating baker's yeast with enhanced glutathione production |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699552A (en) * | 2005-06-24 | 2005-11-23 | 中国科学院微生物研究所 | Beer yeast engineering strain and its construction method |
| CN1934982A (en) * | 2006-10-23 | 2007-03-28 | 杜冰 | Method for preparing yeast peptide utilizing beer yeast |
| CN1948462A (en) * | 2006-10-13 | 2007-04-18 | 中国科学院微生物研究所 | Beer yeast engineering bacteria, its preparation method and application |
| CN101255454A (en) * | 2008-03-14 | 2008-09-03 | 华东理工大学 | Method for biosynthesis of glutathione by using yeast |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699552A (en) * | 2005-06-24 | 2005-11-23 | 中国科学院微生物研究所 | Beer yeast engineering strain and its construction method |
| CN1948462A (en) * | 2006-10-13 | 2007-04-18 | 中国科学院微生物研究所 | Beer yeast engineering bacteria, its preparation method and application |
| CN1934982A (en) * | 2006-10-23 | 2007-03-28 | 杜冰 | Method for preparing yeast peptide utilizing beer yeast |
| CN101255454A (en) * | 2008-03-14 | 2008-09-03 | 华东理工大学 | Method for biosynthesis of glutathione by using yeast |
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