CN101955889B - Cadmium salt resistant Candida tropicalis and application thereof - Google Patents
Cadmium salt resistant Candida tropicalis and application thereof Download PDFInfo
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- CN101955889B CN101955889B CN2010105031597A CN201010503159A CN101955889B CN 101955889 B CN101955889 B CN 101955889B CN 2010105031597 A CN2010105031597 A CN 2010105031597A CN 201010503159 A CN201010503159 A CN 201010503159A CN 101955889 B CN101955889 B CN 101955889B
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Abstract
The invention discloses a strain of cadmium salt resistant Candida tropicalis and a method for producing glutathione(GSH)-enriched yeast mycelium by using the strain. The Candida tropicalis CV26 has been preserved in China General Microbiological Culture Collection Center (CGMCC) on September 21, 2010, and the collection number is CGMCC No.4190. The strain can accumulate GSH in cells by taking xylose or hemicellulose hydrolysis sugar liquid as a carbon source, has higher biomass, and is used for producing the glutathione (GSH)-enriched yeast mycelium. The glutathione-enriched yeast can be used for extracting the glutathione to prepare glutathione and mycoprotein, and also can be directly used as functional yeast. The glutathione (GSH)-enriched yeast mycelium is produced by using the xylose in the yeast-fermented lignocellulose hydrolysate, so a novel path for high-efficiency utilization of the xylose can be developed.
Description
[technical field]
The invention belongs to the microbial fermentation technology field, be specifically related to a strain cadmium salt resistance candida tropicalis and be rich in the application in the gsh 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 glutathion molecule, can participate in multiple biological respinse, significant to the standard state of keeping cell.Gsh has detoxifcation, anti-oxidant and anti-aging effect, and direct or indirect participation protein and DNA are synthetic, the important physiological function such as transportation, metabolism and cytoprotective of material, has a extensive future.
The working method of gsh has solvent extration, chemical synthesis, biological process.Biological process comprises enzyme process and fermentation method, and wherein fermentation method is to utilize mikrobe self metabolism to produce gsh, has advantages such as easy, easy control, easy expansion production with data by MoM and MEI, thereby becomes main production methods.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 genetic engineering bacterium with GSH synthase activity.
At home, the bacterial classification that is used for the gsh fermentative prodn is yeast saccharomyces cerevisiae and Candida utilis mostly, and fermenting carbon source is molasses, glucose, sucrose mostly, is that carbon source through fermentation production gsh does not appear in the newspapers as yet with the wood sugar.Lignocellulose is the abundantest on the earth, the most cheap renewable resources; And semicellulose accounts for about 30% of lignocellulosic material; Semicellulose obtains the mixed sugar liquid of five-carbon sugar and hexose after hydrolysis; Wherein wood sugar is the maximum sugar of content in the semicellulose, also is the second largest sugar that content is only second to glucose in the whole lignocellulose material.For example just contain a large amount of semicelluloses in the corn straw, its structural unit has wood sugar, pectinose, glucose etc., and wherein wood sugar accounts for over half.China is large agricultural country, and the agricultural crop straw YO surpasses 600,000,000 tons, but utilization ratio is lower, few part is only arranged as feed or as the raw material of papermaking and production chemical product, a large amount of stalks are used to fuel wood or rot discard, and cause the resource significant wastage.Cellulosic ethanol production is the effective way of a present wooden cellulosic material bio-transformation of generally acknowledging; But because of wood sugar ethanol fermentation difficulty, cause existing cellulosic ethanol production technology to be ignored mostly or helplessly abandoned wood-sugar fermentation, only utilized the Mierocrystalline cellulose part in the fibrous material; Cause raw material availability on the low side; Basically in 30%～35% level, produce 1 ton of alcohol fuel and need 6～7 tons of lignocellulose raw materials, this has not only increased production cost; Cause the wasting of resources, and strengthened intractability and the cost that contains five-carbon sugar waste water.If can the wood sugar in the straw-like materials be converted into the yeast thalline that is rich in gsh, both can be gsh production exploitation new raw material, new road is opened up in the effective utilization that can be wood sugar again.
[summary of the invention]:
The objective of the invention is one strain cadmium salt resistance candida tropicalis is provided, but this bacterial strain xylose-fermenting or hydrolysis of hemicellulose liquid glucose to obtain being rich in the yeast thalline of gsh to above-mentioned existing problems.
Technical scheme of the present invention::
One strain cadmium salt resistance candida tropicalis CV26; Said candida tropicalis CV26 (Candida tropicalis CV26) is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 21st, 2010, and preserving number is CGMCC No.4190.
A kind ofly use the method that above-mentioned candida tropicalis CV26 production is rich in the yeast thalline of gsh, adopt wood sugar and hemicellulose hydrolysate shake flask fermentation, step is:
1) picking one ring CV26 bacterial classification inserts the seed culture medium from activated inclined plane, and 30 ℃, 160r/min shaking table cultivation 24h get seed liquor;
2) with seed liquor with 5～15% inoculum size; Insert in wood-sugar fermentation substratum or the hydrolysis of hemicellulose liquid glucose fermention medium and ferment, fermentation condition is: liquid amount 20～60mL/250mL, shaking speed 140～180r/min, 26～32 ℃ of temperature, pH value 5～8, fermentation time are 28～40h.
A kind ofly use the method that above-mentioned candida tropicalis CV26 production is rich in the yeast thalline of gsh, adopt the wood-sugar fermentation of 5L fermentor tank, step is:
1) picking one ring CV26 bacterial classification inserts the seed culture medium from activated inclined plane, and 30 ℃, 160r/min shaking table cultivation 24h get seed liquor;
2) seed culture fluid inserts in the 5L fermentor tank that contains 3L wood-sugar fermentation substratum and ferments by 5～15% inoculum size, and fermentation condition is: be 25%～35% at dissolved oxygen, pH value 5～8, temperature be to cultivate 14～20h under 26～32 ℃ the condition.
Consisting of of said seed culture medium: wood sugar 20g/L, peptone 20g/L, yeast soak powder 10g/L.
Consisting of of said wood-sugar fermentation substratum: wood sugar (25～35) g/L, urea (2.5～5.5) g/L, steeping water (0.5～3) % (v/v), KH
2PO
4(0.5～1.5) g/L, K
2HPO
43H
2O (1.5～3.5) g/L, MgSO
4(0.05～0.15) g/L.
Consisting of of said hydrolysis of hemicellulose liquid glucose fermention medium: the hemicellulose hydrolysate of xylose concentration (30～35) g/L, glucose concn (2～6) g/L; According to the composition of said wood-sugar fermentation substratum, gained after interpolation other medium components except that wood sugar.
Advantage of the present invention and positively effect:
The present invention combines the cadmium salt resistance screening to obtain the candida tropicalis mutant strain CV26 of cadmium salt resistance, the raising of born of the same parents' glutathion inside growing amount through ultraviolet mutagenesis; And, thalline living weight and born of the same parents' glutathion inside content when this mutant strain is carbon source with the wood sugar have effectively been improved through fermention medium and fermentation condition optimization.Use bacterial classification of the present invention and can the wood sugar in wood sugar or the hydrolysis of hemicellulose liquid glucose efficiently be converted into the yeast thalline that is rich in gsh; GSH content can reach the 13.996mg/g dry mycelium in the born of the same parents of wood sugar shake flask fermentation; Living weight 8.65mg/L; GSH content can reach the 11.615mg/g dry mycelium, living weight 7.8mg/L in the cellulosic hydrolysate shake flask fermentation born of the same parents; The GSH output of 5L fermentor tank wood-sugar fermentation is 135.885mg/L, GSH content 9.991mg/g dry mycelium in the born of the same parents, living weight 14.4g/L.The yeast thalline that is rich in gsh both can be used for the gsh extraction and obtain gsh and two kinds of products of tropina, also can directly use as functional yeast.The present invention has opened up new way for effective utilization of wood sugar, has also developed new raw material for gsh production, can effectively improve the raw material availability of lignocellulose biotransformation such as cellulosic ethanol, and increase business economic benefit reduces environmental pollution.
[description of drawings]:
Accompanying drawing is a 5L fermentor tank wood-sugar fermentation process curve.
[embodiment]:
Embodiment 1: the mutagenesis screening of the torrid zone false silk mutant strain CV26 of cadmium salt resistance, wood sugar capable of using accumulation gsh
Candida tropicalis bacterium (Candida tropicalis) CT1463 can utilize wood sugar is a starting strain; Selecting lethality rate is that 85% ultraviolet mutagenesis dosage (irradiation 90s) carries out mutagenesis to candida tropicalis; Bacterium liquid after the mutagenesis is evenly coated on the cadmium salt resistant panel of 0.7mmol/L; Picking mutant strain behind 30 ℃ of lucifuges cultivation 2～3d obtains the yeast mutant that 80 strain cadmium salt resistances improve, called after CV1～CV80 at last.Mutant strain is carried out the gsh fermenting experiment, measure born of the same parents' glutathion inside growing amount behind the fermentation 28h, do contrast with the strain of setting out, obtain the mutant strain CV26 that strain gsh output and content obviously improve, GSH output improves 37% than starting strain; Born of the same parents' intensive amount improves 49%, and the result sees table 1.
Table 1 mutant strain CV26 compares with starting strain gsh growing amount
Embodiment 2: wood sugar and hemicellulose hydrolysate shake flask fermentation
With mutant strain CV26 is experimental strain, is carbon source with the wood sugar, through substratum and fermentation condition optimization, is changed the wood-sugar fermentation substratum and is: wood sugar 30g/L, urea 4.5g/L, steeping water 1% (v/v), KH
2PO
41g/L, K
2HPO
43H
2O 2.5g/L, MgSO
40.1g/L, the 0.1MPa 15min that sterilizes; Optimization of fermentation conditions is: inoculum size 10%, and liquid amount 30mL/250mL, shaking speed 160r/min, 30 ℃ of temperature, pH value 7, incubation time is 36h.The preparation method of corn cob hydrolyzed solution fermention medium is following: be raw material with the corn cob earlier; Make hemicellulose hydrolysate through dilute acid hydrolysis; Form according to the substratum of wood-sugar fermentation substratum again, in hemicellulose hydrolysate, add other medium components except that wood sugar.The actual conditions of corn cob dilute acid hydrolysis is: sulfuric acid concentration 1.5%, solid-liquid ratio 1: 10,120 ℃ of hydrolysis temperatures, hydrolysis time 3h.Under this condition in the hydrolyzed solution xylose concentration be 33g/L, glucose concn 4g/L.
Picking one ring CV26 thalline is seeded to the 250mL triangular flask that the 50mL seed culture medium is housed and cultivates from activated inclined plane, and 30 ℃ of 160r/min shaking tables are cultivated 24h, get seed liquor.Seed culture medium consists of: wood sugar 20g/L, peptone 20g/L, yeast soak powder 10g/L.Seed liquor inserts in above-mentioned wood-sugar fermentation substratum and the hemicellulose hydrolysate fermention medium with 10% inoculum size, and 30 ℃ of 160r/min shake flask fermentations are cultivated 36h, and the result sees table 2.
Table 2. mutant strain CV26 shake flask fermentation experimental result
Can know that by table 2 mutant strain CV26 utilizes the fermentation of hydrolyzed solution a little less than other wood-sugar fermentation, suppress composition because contain thalli growths such as furfural, hydroxymethylfurfural, acetate and aldehydes matter in the hydrolyzed solution.
The wood-sugar fermentation of embodiment 3:5L fermentor tank
On the basis of shake flask fermentation, carry out the experiment of CV26 xylose fermentation for producing gsh through the 5L fermentor tank, investigated yeast bio amount and gsh generation situation in the fermentor cultivation.Seed culture medium and seed culture method, wood-sugar fermentation substratum form with embodiment 2 in identical.
5L ferment tank condition is specially: the fermentor tank liquid amount is 60% of jar appearance, and inoculum size 10%, original ph are 7, and leavening temperature is 30 ℃, and dissolved oxygen is 30%, fermentation 18h.Gsh output, residual sugar amount, living weight and pH are measured in certain hour sampling at interval, paint to such an extent that course of fermentation figure is as shown in Figure 1.On the jar of 5L, fermenting to the 14h wood sugar is exhausted basically, and living weight no longer increases, and shortens greatly than the 36h of shake flask fermentation.Living weight is greatly improved than shake flask fermentation, is up to 14.4g/L, and gsh output slightly improves, and is up to 135.885mg/L, and GSH content is 9.991mg/g in the born of the same parents, decreases than shake flask fermentation.But born of the same parents' glutathion inside content and GSH output still are increase trend to 18h, explain if prolong fermentation time, still have the possibility of further raising GSH born of the same parents' intensive amount and output.
Claims (5)
1. strain cadmium salt resistance candida tropicalis (Candida tropicalis) CV26; It is characterized in that: said candida tropicalis CV26 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on September 21st, 2010, and preserving number is CGMCC No.4190.
2. an application rights requires 1 said candida tropicalis CV26 production to be rich in the method for the yeast thalline of gsh, and it is characterized in that: adopt wood sugar and hemicellulose hydrolysate shake flask fermentation, step is:
1) picking one ring CV26 bacterial classification inserts the seed culture medium from activated inclined plane, and 30 ℃, 160r/min shaking table cultivation 24h get seed liquor;
2) with seed liquor with 5～15% inoculum size; Insert in wood-sugar fermentation substratum or the hydrolysis of hemicellulose liquid glucose fermention medium and ferment, fermentation condition is: liquid amount 20～60mL/250mL, shaking speed 140～180r/min, 26～32 ℃ of temperature, pH value 5～8, fermentation time are 28～40h.
3. an application rights requires 1 said candida tropicalis CV26 production to be rich in the method for the yeast thalline of gsh, and it is characterized in that: adopt the wood-sugar fermentation of 5L fermentor tank, step is:
1) picking one ring CV26 bacterial classification inserts the seed culture medium from activated inclined plane, and 30 ℃, 160r/min shaking table cultivation 24h get seed liquor;
2) seed culture fluid inserts in the 5L fermentor tank that contains 3L wood-sugar fermentation substratum and ferments by 5～15% inoculum size, and fermentation condition is: be 25%～35% at dissolved oxygen, pH value 5～8, temperature be to cultivate 14～20h under 26～32 ℃ the condition.
4. the method for producing the yeast thalline that is rich in gsh according to claim 2 or 3 said application candida tropicalis CV26 is characterized in that: the consisting of of said seed culture medium: wood sugar 20g/L, peptone 20g/L, yeast soak powder 10g/L.
5. the method for producing the yeast thalline that is rich in gsh according to claim 2 or 3 said application candida tropicalis CV26 is characterized in that: the consisting of of said wood-sugar fermentation substratum: wood sugar 25～35g/L, urea 2.5～5.5g/L, steeping water 0.5～3 volume %, KH
2PO
40.5～1.5g/L, K
2HPO
43H
2O1.5～3.5g/L, MgSO
40.05～0.15g/L.
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| CN2010105031597A CN101955889B (en) | 2010-10-12 | 2010-10-12 | Cadmium salt resistant Candida tropicalis and application thereof |
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| CN101955889B true CN101955889B (en) | 2012-07-04 |
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| CN104531551A (en) * | 2014-11-24 | 2015-04-22 | 苏州嘉禧萝生物科技有限公司 | Cadmium contaminated soil microbe repairing agent and application thereof |
| CN104560746B (en) * | 2014-12-26 | 2017-07-28 | 盐城工学院 | One plant can handle high salinity organic chemical waste water candida tropicalis and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1844407A (en) * | 2005-04-06 | 2006-10-11 | 北京化工大学 | Method for simultaneous production of ergosterol and glutathione by yeast fermentation |
| CN101255454A (en) * | 2008-03-14 | 2008-09-03 | 华东理工大学 | Method for biosynthesis of glutathione by using yeast |
| CN101709318A (en) * | 2009-11-26 | 2010-05-19 | 苏州大学 | Method for preparing glutathione by fed-batch fermentation of Candida utilis |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1844407A (en) * | 2005-04-06 | 2006-10-11 | 北京化工大学 | Method for simultaneous production of ergosterol and glutathione by yeast fermentation |
| CN101255454A (en) * | 2008-03-14 | 2008-09-03 | 华东理工大学 | Method for biosynthesis of glutathione by using yeast |
| CN101709318A (en) * | 2009-11-26 | 2010-05-19 | 苏州大学 | Method for preparing glutathione by fed-batch fermentation of Candida utilis |
Non-Patent Citations (1)
| Title |
|---|
| 王晓琼 等.利用木糖高产谷胱甘肽酵母菌株的诱变选育及培养条件优化.《现代食品科技》.2011,第27卷(第5期),546-549,567. * |
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