CN109750073A - A method of extracting glutathione from candida utili fermentation liquid - Google Patents
A method of extracting glutathione from candida utili fermentation liquid Download PDFInfo
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- candida utili
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 96
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 54
- 241000235646 Cyberlindnera jadinii Species 0.000 title claims abstract description 53
- 238000000855 fermentation Methods 0.000 title claims abstract description 40
- 230000004151 fermentation Effects 0.000 title claims abstract description 40
- 229960003180 glutathione Drugs 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 33
- 239000007788 liquid Substances 0.000 title claims abstract description 32
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 32
- 230000003834 intracellular effect Effects 0.000 claims abstract description 20
- 238000000605 extraction Methods 0.000 claims abstract description 14
- 239000006228 supernatant Substances 0.000 claims abstract description 13
- 238000011218 seed culture Methods 0.000 claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- 238000005119 centrifugation Methods 0.000 claims abstract description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 9
- 235000018102 proteins Nutrition 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 238000012366 Fed-batch cultivation Methods 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 235000001014 amino acid Nutrition 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000013028 medium composition Substances 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 3
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 230000010261 cell growth Effects 0.000 claims description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 3
- 235000018417 cysteine Nutrition 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000004615 ingredient Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 2
- 235000013922 glutamic acid Nutrition 0.000 claims description 2
- 239000004220 glutamic acid Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- 239000011591 potassium Substances 0.000 claims 1
- 229910052700 potassium Inorganic materials 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 19
- 239000002253 acid Substances 0.000 abstract description 10
- 238000007710 freezing Methods 0.000 abstract description 7
- 230000008014 freezing Effects 0.000 abstract description 7
- 150000001720 carbohydrates Chemical class 0.000 abstract description 4
- 210000002421 cell wall Anatomy 0.000 abstract description 4
- 150000007523 nucleic acids Chemical class 0.000 abstract description 4
- 108020004707 nucleic acids Proteins 0.000 abstract description 4
- 102000039446 nucleic acids Human genes 0.000 abstract description 4
- 238000012545 processing Methods 0.000 abstract description 4
- 238000000746 purification Methods 0.000 abstract description 4
- 230000035699 permeability Effects 0.000 abstract description 3
- 229920002521 macromolecule Polymers 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 238000003809 water extraction Methods 0.000 description 5
- 210000005253 yeast cell Anatomy 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000001728 nano-filtration Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000638 solvent extraction Methods 0.000 description 3
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000003729 cation exchange resin Substances 0.000 description 2
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- -1 sulfhydryl compound Chemical class 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 206010027439 Metal poisoning Diseases 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
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- 239000003480 eluent Substances 0.000 description 1
- 230000006571 energy metabolism pathway Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 125000002642 gamma-glutamyl group Chemical group 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 208000010501 heavy metal poisoning Diseases 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
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- 210000004185 liver Anatomy 0.000 description 1
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- 150000002772 monosaccharides Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
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- 239000012879 subculture medium Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The method that the invention discloses a kind of to extract glutathione from candida utili fermentation liquid, comprising the following steps: candida utili seed culture and fermentation；The fermentation liquid that fermented and cultured finishes is collected, centrifugation, the cell freezing of collection；Candida utili cell after freezing is added in sulfuric acid solution, is centrifugated after placing 15~30 minutes, supernatant is collected；Reduced glutathione intracellular at this time is dissolved in sulfuric acid solution from intracellular release, and completes to extract.The present invention handles the cell freezing of collection to increase cell wall permeability；Cell after freezing processing is added in strong acid solution, reduced glutathione intracellular releases completely in strong acid solution, and it is substantially remained in if other macromolecular substances such as protein, carbohydrate, nucleic acid etc. intracellular intracellular, simplify subsequent purification process, reduce purifying cost, realize efficiently, clean extraction；Reduced glutathione is not destroyed under extraction conditions of the invention simultaneously.
Description
The application is application No. is 201510720495.X, and the applying date is on October 30th, 2015, and invention and created name is
The divisional application of the application for a patent for invention of " method of glutathione is extracted from candida utili fermentation liquid ".
Technical field
The present invention relates to a kind of preparation methods of glutathione, and in particular to one kind is mentioned from candida utili fermentation liquid
The method for taking glutathione.
Background technique
Glutathione, i.e. γ-L- glutamy-L- cysteinyl glycine are by Pidolidone, L-cysteine and sweet ammonia
One kind made of acid is condensed through peptide bond while the bioactivity tripeptide compound with γ-glutamyl and sulfydryl.As biology
Intracorporal non-protein sulfhydryl compound, glutathione mainly have two kinds of forms of reduced form (G-SH) and oxidized form (G-S-S-G),
What is largely existed and play a major role in body is reduced glutathione.Reduced glutathione has unique physiology function
Energy, referred to as longevity factor and the anti-aging factor.Reduced glutathione can be used for treating liver as amino acids biochemical drug
The diseases such as disease, heavy metal poisoning, it has recently been found that reduced glutathione also has effects that AIDS resisting poison.
The main method of production reduced glutathione is fermentation method at present, i.e., using raw materials such as cheap carbohydrates as nutrients
Matter, the method for carrying out reduced glutathione biosynthesis using matter and energy metabolic pathway in microbial body.General feelings
The content of reduced glutathione is not high in microbial cell under condition, only the 0.5%~1.0% of dry weight, because too high amount
Reduced glutathione is easily destroyed internal Balanced redox environment.Since reduced glutathione is a kind of production intracellular
Object needs to extract reduced glutathione purifying after fermentation.
There are solvent extraction, hot-water extraction method and ultrasonic wave broken from extraction reduced glutathione main method intracellular at present
Broken extraction process.
Wherein solvent extraction is by increase cell wall permeability to complete to extract, type, temperature and the extraction of solvent
Time is affected to extraction yield, and recovery rate is low when making solvent extraction with formic acid, liquefied ammonia, sulfuric acid or trichloroacetic acid；It is mentioned with ethyl alcohol
Cell wall can not be destroyed when taking, impurity content is few, and low energy consumption, but ethanolic extraction finishes and needs to be recycled, and increases and mentions
Take cost.Ultrasonic disruption main problem is that the impurity such as intracellular protein, nucleic acid all dissolve out after broken wall, is easy after centrifugation muddy
It is turbid, it is unfavorable for subsequent processing；Consuming time is long for ultrasonication, needs effective cooling system, and energy consumption is high, is unfavorable for industrial life
It produces.
Hot-water extraction method is to extract glutathione from fermentation liquid to apply method, such as Chinese patent literature CN earlier
100455592 C(application numbers 200610040606.3) it discloses and a kind of extracts glutathione from glutathione fermented broth
Method adjusts yeast cells by the centrifuged yeast cells liquid of glutathione fermented broth, with sulfuric acid using boiling water cooking method
Liquid pH value is 1 to 2, the yeast cells liquid for regulating pH value is poured into after progress boiling water broken wall, cation exchange in boiling water and is collected
Concentrate rich in glutathione, be concentrated in vacuo glutathione hyper-concentration liquid, glutathione hyper-concentration liquid is freeze-dried
To white powder glutathione, finished product packing and obtain finished product glutathione.
104130310 A(application number 201410231642.2 of Chinese patent literature CN) disclose a kind of glutathione
Yeast fermentation broth is centrifuged by isolation and purification method, collects yeast, and 50 DEG C~90 DEG C 5~30min of hot water extraction are added, and is adjusted and is taken out
The pH value for taking liquid is 1.5~3, and extracting solution is filtered or is centrifuged, collects extracting solution；Extracting solution is using big point of ultrafiltration removal
Then the impurity such as protein, the polysaccharide of son remove most small molecule amino acid, monosaccharide using the method for nanofiltration membrane
The impurity such as class, inorganic salts；Using nanofiltration concentrate as raw material, glutathione, absorption are adsorbed using storng-acid cation exchange resin
It is eluted after reaching breakthrough point；Eluent is concentrated under reduced pressure, the ethyl alcohol that three times volume is added is precipitated, sediment freezing drying,
Obtain glutathione product, purity is up to 95% or more.
Hot-water extraction method method is simple, and cost is relatively low, but is easy that reduced form G-SH is made to be transformed into G-S-S-G, and intracellular
Other substances such as macromoleculars such as protein, carbohydrate and nucleic acid can also release therewith, increase subsequent purification difficulty.
Summary of the invention
Technical problem to be solved by the invention is to provide it is a kind of extract it is complete, at low cost from candida utili ferment
The method of glutathione is extracted in liquid.
The technical solution for realizing the object of the invention is a kind of side that glutathione is extracted from candida utili fermentation liquid
Method, comprising the following steps:
1. candida utili seed culture and fermentation.
2. the collection step candida utili fermentation liquid that 1. fermented and cultured finishes, removes supernatant after centrifugation, collection
Cell freezes 20~30 minutes at -15 DEG C~-25 DEG C.
3. candida utili cell 2. step is freezed after is added in sulfuric acid solution, the pH value of sulfuric acid solution is 1~
After placing 15~30 minutes at 2,20 DEG C~35 DEG C, the intracellular reduced glutathione of candida utili is from release intracellular
It is dissolved in sulfuric acid solution out, completes that the extraction of glutathione is centrifugated, is collected from candida utili fermentation liquid
Supernatant.
Above-mentioned steps 3. in, in sulfuric acid solution the concentration of candida utili cell be 0.08g/mL~0.12g/mL.
Above-mentioned steps 1. candida utili seed culture when, by candida utili strain inoculated in seed culture medium
In, it is cultivated 18~28 hours, 200~250rpm of shaking speed at 28 DEG C~32 DEG C, stops culture when culture is to logarithmic phase, obtain
Seed liquor wait for fermented and cultured.
Seed liquor is first seeded in fermentation basic training by 8%~10% inoculum concentration when 1. candida utili ferments by step
8~10h of batch culture in base is supported, according still further to specific cell growth rate 0.15h-1~0.25h-1Exponential fed-batch 11~13h of culture,
Then with 5.0~6.0 g(h ﹒ L)-1After 23~25h of permanent number fed-batch cultivation；Three kinds of amino acid, paddy in tank after addition are added into tank
5.5~6.5 mmol/L of propylhomoserin, 5.0~6.0mmol/L of glycine, 6.0~7.0 mmol/L of cysteine, continue culture 25
The fermented and cultured of~35h completion candida utili.
Fermentation minimal medium composition when above-mentioned batch culture are as follows: 15 g/L of glucose, 5 g/L of ammonium sulfate, yeast
2 g/L of cream, 1.5 g/L of potassium dihydrogen phosphate, 0.25 g/L of magnesium sulfate, the pH value for the minimal medium that ferments are 5.5；
It is fed-batch medium used in above-mentioned exponential fed-batch culture and permanent number fed-batch cultivation, fed-batch medium composition is as follows: Portugal
Grape sugar 500 g/L, 36 g/L of ammonium sulfate, 5 g/L of potassium dihydrogen phosphate, 0.5 g/L of magnesium sulfate, after wherein glucose individually sterilizes with
Other ingredient mixing.
The present invention has the effect of positive: (1) extracting method of the invention is first by candida utili (Candida
Utilis is abbreviated as C.utilis) it cultivates and ferments, obtained fermentation liquid centrifuge separation removes supernatant, the cell of collection
The freezing processing at -20 DEG C~-30 DEG C, to increase C.utilis cell wall permeability；C.utilis after freezing processing is thin
Born of the same parents are added in the strong acid solution of low ph value, and reduced glutathione intracellular releases completely in the strong acid solution of low ph value,
And substantially remain in intracellular if other macromolecular substances such as protein, carbohydrate, nucleic acid etc. intracellular, subsequent purification process is simplified,
Reduce purifying cost, realize efficiently, clean extract C.utilis cell in G-SH；Simultaneously in extraction conditions of the invention
Lower reduced glutathione is not destroyed.
(2) when extracting GSH with the strong acid solution of low ph value, after need to only a small amount of strong acid solution, G-SH being used to extract,
Strong acid solution can also be recycled further, have environment friendly.
(3) extracting method extraction time of the invention is short, and intracellular glutathione can discharge completely within 20 minutes or so
Into strong acid solution, therefore this method extraction efficiency is high.
(4) extracting method simple process of the invention, economy, environmental protection, meet the needs of recycling economy development, have good
Prospects for commercial application.
Specific embodiment
(embodiment 1)
In the slave candida utili fermentation liquid of the present embodiment extract glutathione method the following steps are included:
1. candida utili seed culture and fermentation.Using candida utili (Candida utilis WSH 02-08) as
Bacterial strain is produced, first progress seed culture, stops culture when culture is to late log phase, obtained seed liquor waits for fermented and cultured.
Seed culture medium forms following (g/L): glucose 20, peptone 20, yeast extract 10, solvent are water, pH value 5.5
~6.5.
When specific seed culture, by candida utili (Candida utilis WSH 02-08) strain inoculated in kind
It in sub- culture medium, is cultivated 18~28 hours, 200~250rpm of shaking speed at 28 DEG C~32 DEG C, when late log phase is arrived in culture
Stop culture, obtained seed liquor waits for fermented and cultured.
It is packed into 3L fermentation minimal medium in 5L fermentor, above-mentioned seed liquor is pressed into 8%~10%(v/v) inoculum concentration inoculation
It is 9h in batch culture 8~10h(the present embodiment in fermentation minimal medium), according still further to specific cell growth rate 0.15h-1~
0.25h-1It is 12h in exponential fed-batch culture 11~13h(the present embodiment), then with 5.0~6.0 g(h ﹒ L)-1Permanent number stream adds training
Support in 23~25h(the present embodiment as 24 hours) after, three kinds of amino are added up to 102 g/L in fermentor inner cell concentration into tank at this time
Acid, 6 mmol/L of glutamic acid, 5.5 mmol/L of glycine, 6.5 mmol/L of cysteine in tank after addition；Continue culture 25
In~35h(the present embodiment h) for 30, the fermented and cultured of candida utili is completed.
Above-mentioned fermentation minimal medium forms following (g/L): glucose 15, ammonium sulfate 5, yeast extract 2, potassium dihydrogen phosphate
1.5, magnesium sulfate 0.25, solvent is water, pH value 5.5.
It is fed-batch medium used in exponential fed-batch culture and permanent number fed-batch cultivation, fed-batch medium forms following (g/
L): glucose 500, ammonium sulfate 36, potassium dihydrogen phosphate 5, magnesium sulfate 0.5, solvent are water,；After wherein glucose individually sterilizes with
Other ingredient mixing.
The fermentation liquid 25mL that fermented and cultured finishes in above-mentioned reactor tank is taken, 10min is centrifuged at revolving speed 3000rpm, is removed
Supernatant in centrifuge tube collects candida utili cell.The candida utili cell of collection is added in ethyl alcohol, it is false to produce protein
The volume ratio of silk yeast cells and ethyl alcohol is 0.4:1, and after extracting 30 minutes in ethanol, centrifuge separation, supernatant injects efficient
G-SH content is measured in liquid chromatogram, being computed step, 1. G-SH content is in the candida utili cell of fermented and cultured
1.81%, further, in the fermentation liquid that fermented and cultured finishes in reactor tank, the yield of reduced glutathione is 1847mg/L.
In addition, according to 104130310 A(application number 201410231642.2 of Chinese patent literature CN) method extract
Glutathione intracellular takes in nanofiltration concentrate injection high performance liquid chromatography and measures GSH content, calculates step 1. fermented and cultured
GSH content is 1.81% in candida utili cell.
2. the candida utili fermentation liquid that the fermentation cylinder for fermentation culture of collection step 1. finishes is collected in the present embodiment
To fermentation liquid 5000g, fermentation liquid is centrifuged 10min under 3000rpm revolving speed, centrifugation removal supernatant, the cell of collection is -15
DEG C~-25 DEG C (they being -20 DEG C in the present embodiment) under freeze 20~30 minutes (being 30 minutes in the present embodiment).
3. the candida utili cell 2.5g 2. step is freezed after is added in 25mL sulfuric acid solution, wherein sulfuric acid solution
PH value to be in 1~2(the present embodiment be 1.2), the concentration of candida utili cell is 0.1g/mL in sulfuric acid solution.
It is placed at 20 DEG C~35 DEG C of room temperature 15~30 minutes (being 20 minutes in the present embodiment), candida utili is thin at this time
Reduced glutathione intracellular is dissolved in sulfuric acid solution from intracellular release, and completes the present embodiment from candida utili
To the extraction of glutathione in fermentation liquid；Then it is centrifugated 10min under 3000rpm revolving speed, collects supernatant.
Taking step, 3. the 0.2 μ L of supernatant containing reduced glutathione injects measurement GSH content in high performance liquid chromatography,
The concentration for measuring reduced glutathione in supernatant is 0.16wt%, and the production protein that step 1. fermented and cultured is then calculated is false
GSH content is 1.80wt% in silk yeast cells, further, in the fermentation liquid that fermented and cultured finishes in reactor tank, reduced form paddy
The yield of the sweet peptide of Guang is 1843mg/L.
By above-mentioned data it is found that the present invention extracted from candida utili cell the effect of the method for glutathione with it is molten
Agent extraction and hot-water extraction method are suitable.
The purifying of supernatant GSH-PX activity is carried out according to the prior art, and cation exchange resin can be used for example.
Claims (3)
1. a kind of method for extracting glutathione from candida utili fermentation liquid, it is characterised in that the following steps are included:
1. candida utili seed culture and fermentation；By candida utili strain inoculated in seed culture medium, 28 DEG C~
It is cultivated 18~28 hours, 200~250rpm of shaking speed at 32 DEG C, stops culture, obtained seed liquor when culture is to logarithmic phase
To fermented and cultured；
2. the collection step candida utili fermentation liquid that 1. fermented and cultured finishes, removes supernatant, the cell of collection after centrifugation
It is freezed 20~30 minutes at -15 DEG C~-25 DEG C；
3. the candida utili cell 2. step is freezed after is added in sulfuric acid solution, candida utili is thin in sulfuric acid solution
The concentration of born of the same parents is 0.08g/mL~0.12g/mL, and the pH value of sulfuric acid solution is to place 15~30 minutes at 1~2,20 DEG C~35 DEG C
Afterwards, the intracellular reduced glutathione of candida utili is dissolved in sulfuric acid solution from intracellular release, and is completed from production
Supernatant is collected in extraction in protein Candida fermentation liquid to glutathione, centrifuge separation.
2. the method according to claim 1 for extracting glutathione from candida utili fermentation liquid, it is characterised in that:
Step 1. candida utili ferment when, first by seed liquor by 8%~10% inoculum concentration be seeded in fermentation minimal medium in point
8~10h of culture is criticized, according still further to specific cell growth rate 0.15h-1~0.25h-1Exponential fed-batch 11~13h of culture, then with 5.0
~6.0g(h ﹒ L)-1After 23~25h of permanent number fed-batch cultivation；Three kinds of amino acid are added into tank, glutamic acid 5.5 in tank after addition~
6.5 mmol/L, 5.0~6.0mmol/L of glycine, 6.0~7.0 mmol/L of cysteine continue 25~35h of culture completion
The fermented and cultured of candida utili.
3. the method according to claim 2 for extracting glutathione from candida utili fermentation liquid, it is characterised in that:
Fermentation minimal medium composition when batch culture are as follows: 15 g/L of glucose, 5 g/L of ammonium sulfate, 2 g/L of yeast extract, phosphoric acid
1.5 g/L of potassium dihydrogen, 0.25 g/L of magnesium sulfate, the pH value for the minimal medium that ferments are 5.5；
It is fed-batch medium used in exponential fed-batch culture and permanent number fed-batch cultivation, fed-batch medium composition is as follows: glucose
500 g/L, 36 g/L of ammonium sulfate, 5 g/L of potassium dihydrogen phosphate, 0.5 g/L of magnesium sulfate, after wherein glucose individually sterilizes with it is other
Ingredient mixing.
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| KR20110118492A (en) * | 2010-04-23 | 2011-10-31 | 샘표식품 주식회사 | Novel yeast mutant containing increased glutathione content, method for producing the same and use thereof |
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