CN106518962A - Method for preparing reduced glutathione from yeast cells - Google Patents
Method for preparing reduced glutathione from yeast cells Download PDFInfo
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- CN106518962A CN106518962A CN201610901699.8A CN201610901699A CN106518962A CN 106518962 A CN106518962 A CN 106518962A CN 201610901699 A CN201610901699 A CN 201610901699A CN 106518962 A CN106518962 A CN 106518962A
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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 109
- 210000005253 yeast cell Anatomy 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 21
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 20
- 229960003180 glutathione Drugs 0.000 claims abstract description 73
- 238000003756 stirring Methods 0.000 claims abstract description 33
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000013078 crystal Substances 0.000 claims abstract description 9
- 239000007789 gas Substances 0.000 claims abstract description 9
- 239000000047 product Substances 0.000 claims abstract description 8
- 239000000706 filtrate Substances 0.000 claims abstract description 6
- 235000013305 food Nutrition 0.000 claims abstract description 5
- 235000003969 glutathione Nutrition 0.000 claims description 56
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 238000010668 complexation reaction Methods 0.000 claims description 12
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 9
- 239000007900 aqueous suspension Substances 0.000 claims description 8
- 238000001953 recrystallisation Methods 0.000 claims description 8
- 238000005516 engineering process Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 239000002253 acid Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 235000009508 confectionery Nutrition 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 230000000717 retained effect Effects 0.000 claims description 5
- 239000002537 cosmetic Substances 0.000 claims description 4
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 239000002002 slurry Substances 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 3
- 230000000996 additive effect Effects 0.000 claims description 3
- 229910052927 chalcanthite Inorganic materials 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 229910052979 sodium sulfide Inorganic materials 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 3
- 238000002425 crystallisation Methods 0.000 claims 2
- 230000008025 crystallization Effects 0.000 claims 2
- 150000001335 aliphatic alkanes Chemical class 0.000 claims 1
- 239000008367 deionised water Substances 0.000 claims 1
- 229910021641 deionized water Inorganic materials 0.000 claims 1
- 150000002500 ions Chemical class 0.000 claims 1
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 claims 1
- 239000002912 waste gas Substances 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 3
- 239000002244 precipitate Substances 0.000 abstract description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 abstract 1
- 239000000490 cosmetic additive Substances 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 239000002778 food additive Substances 0.000 abstract 1
- 229910000037 hydrogen sulfide Inorganic materials 0.000 abstract 1
- 239000000546 pharmaceutical excipient Substances 0.000 abstract 1
- 238000003809 water extraction Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 10
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 9
- 239000003814 drug Substances 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000008213 purified water Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 238000006087 Brown hydroboration reaction Methods 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- RWSXRVCMGQZWBV-PHDIDXHHSA-N L-Glutathione Natural products OC(=O)[C@H](N)CCC(=O)N[C@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-PHDIDXHHSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 230000003026 anti-oxygenic effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- VDQVEACBQKUUSU-UHFFFAOYSA-M disodium;sulfanide Chemical compound [Na+].[Na+].[SH-] VDQVEACBQKUUSU-UHFFFAOYSA-M 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 230000002443 hepatoprotective effect Effects 0.000 description 1
- 208000018875 hypoxemia Diseases 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 150000002828 nitro derivatives Chemical class 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
一种从酵母细胞中制备还原型谷胱甘肽的方法，利用富含还原型谷胱甘肽的酵母细胞，采取热水浸提得谷胱甘肽溶液；添加谷胱甘肽识别离子Cu2+，搅拌；离心后的沉淀悬浮于水中，通入硫化氢气体，搅拌；离心后的溶液经活性炭脱色，滤液经过浓缩和重结晶处理，得到高纯度还原型谷胱甘肽结晶。本方法操作工艺简单，原材料易得，可进行产业化实施。本方法生产的还原型谷胱甘肽产品可作为医药原料药、食品和化妆品添加剂加以应用。
A method for preparing reduced glutathione from yeast cells, using yeast cells rich in reduced glutathione, taking hot water extraction to obtain glutathione solution; adding glutathione recognition ion Cu 2 + , stirring; the centrifuged precipitate is suspended in water, hydrogen sulfide gas is introduced, and stirred; the centrifuged solution is decolorized by activated carbon, and the filtrate is concentrated and recrystallized to obtain high-purity reduced glutathione crystals. The method has simple operation process, easy-to-obtain raw materials, and can be implemented industrially. The reduced glutathione product produced by the method can be used as a pharmaceutical raw material drug, food and cosmetic additive.
Description
Technical field
The present invention relates to high-purity reduced glutathion purification process and the preparation high-purity reduction from yeast cells
The technique of type glutathion.Belong to biological engineering and medical compoundss field.
Background technology
Reduced glutathion (Glutathione, GSH) is as a kind of important pharmaceutical agent, clinically main to use
In treatment and the auxiliary treatment of the poisoning of chemotherapy, hypoxemia, hepatic disease, organophosphors, amido or nitro compound；While paddy Guang
The antioxygenic property of sweet peptide causes it to be favored as additive in foods and cosmetics processing industry again.It is used on market
The product of sale mainly includes：Hepatoprotective medicine glutathione tabletses (Redued glutathin), the double benefits for the treatment of toxic liver injury medicine are strong
Medical product and the cosmetics such as (glutathione for injection), glutathion whole body whitening breast, glutathion speckle dispelling elite.
2015, GSH domestic and international markets were every year with 10% speed increase.Japanese Kyowa (Kyowa Hakkokogyo Co., Ltd) and
The companies such as Ajinomoto (Ajincomoto Co., Inc) are achieved that commercialization yeast fermenting and producing paddy Guang early in the eighties in 20th century
The industrialization of sweet peptide, has become the main production and supply business of whole world glutathion, while defining technology barriers.It is domestic
Shandong Jincheng Pharmaceutical ＆ Chemicals Co., Ltd.'s Introduced from Japan glutathion purification technology, first is produced from yeast to become the country
The company of glutathion.
Glutathion derives from animal, plant, microorganism and chemosynthesis.Wherein with the content in yeast, Fructus Tritici aestivi
Enrich the most.The purification process of glutathion mainly includes ion exchange, electroosmose process and aqueous two-phase method etc., such side
Fado is limited to the structural instability of glutathion, causes that purification is low, technological process is numerous and diverse, production cost is high.Therefore,
Developing low-cost, reduced glutathion purifying process capable of being industrialized, grind in food medicine field Related product to glutathion
System is particularly important.It is formulation simultaneously《Eutrophy element yeast》National standard establishment provides theory and practice reference.
The content of the invention
It is an object of the present invention to provide a kind of method for preparing high-purity reduced glutathion from yeast cells,
The method can efficiently, low cost apply the industrialization for producing glutathion in yeast cells, provide high-purity for eating medicine field
Degree reduced glutathion raw material.It is formulation simultaneously《Eutrophy element yeast》National standard establishment provides theory and practice reference.
The object of the present invention is achieved like this：A kind of side for preparing high-purity reduced glutathion from yeast cells
Method, comprises the following steps：
(1) preparation rich in reduced glutathion yeast cells：With yeast cells fermentative Production reduced form gluathione
Peptide, is calculated with yeast cells dry weight, and GSH contents are 1-5%, rich in reduced glutathion yeast cells, including：Yeast milk,
The forms such as yeast dry powder, yeast extract.
(2) yeast cells heat is extracted：Take the yeast cells of certain mass, with 5-15 times, preferably 10 times of volumes go from
Sub- aqueous suspension.It is 2.0 to adjust pH value, at 80-100 DEG C, at preferably 85-90 DEG C, is stirred 0-30 minutes, preferably 5-15 point
Clock.After the completion of be immediately placed in frozen water in be cooled to less than 20 DEG C, under 3000-10000rpm/min, preferably 7500rpm/min from
Heart 10-30 minutes, preferably 15 minutes.Disgorging, retains light phase.
(3) preparation of GSH-Cu complex：Using HPLC technology for detection steps 2) content of light phase middle GSH.In 10-
Under 100rpm/min, preferably 50rpm/min stirrings, it is 5.0-7.0 to adjust light phase pH value with NaOH, preferably 6.0.Root
According to the molal weight of light phase middle GSH, according to 1:1-1:20, preferably 1:2-1:15 proportioning, adds CuSO4.Maintain stirring 15-
60 minutes, preferably 30 minutes.20-40 minutes are centrifuged under 3000-10000rpm/min, preferably 7500rpm/min, preferably
30 minutes.Retain heavy phase.
(4) wash：The GSH-Cu complex of certain mass is taken, with 100-200 times, the preferably purified water of 150 times of volumes
Suspend, maintain to stir 20-40 minutes, preferably 30 minutes.It is centrifuged under 3000-10000rpm/min, preferably 7500rpm/min
20-40 minutes, preferably 30 minutes.Retain heavy phase.
(5) solve complexation：The GSH-Cu complex of certain mass is taken, with 2-10 times, the purified water of preferably 4 times of volumes is hanged
It is floating, maintain stirring.It is passed through H2S gases are maintained 5 minutes.10- is centrifuged under 3000-10000rpm/min, preferably 7500rpm/min
30 minutes, preferably 15 minutes.Disgorging, retains light phase.The H being passed through2S gases include：Pure H2S gases, or rich in height
Concentration H2The industrial methane waste product of S compositions, or H is also easy to produce in acid condition2The inorganic and organic salt of S, such as (NH4)2S, Na2S
Deng.
(6) decolourize：In step (5), light phase use salt acid for adjusting pH value is 2.0, adds activated carbon according to 5 ‰ ratio, maintains
20-60 minutes stirring reaction time, preferably 30 minutes.After the completion of reaction, activated carbon is removed with 0.25 μm of membrane filtration, retain
Filtrate.
(7) concentrate：Filtrate in step (6), by concentrating under reduced pressure, obtains containing 500-1000g/L glutathione concentrations
Glutathione solution, preferred concentration are 800g/L.
(8) recrystallization：Stirring is maintained, containing in glutathione solution in step (7), the 50-80% of equivalent volumes is added
(v/v) ethanol water of concentration, preferably 70% ethanol water, at -4-20 DEG C, preferably 4 DEG C, addition glutathion knot
Crystalline substance is used as crystal seed.The addition of crystal seed is 20-200mg/L, preferably 50mg/L.After continuing stirring 30 minutes, stop stirring, dimension
Hold temperature 6-20 hour, preferably 12 hours.Slurry is obtained, high-purity reduced glutathion material, reduced form is filtered to obtain
Glutathion purity>98%.
The reduced glutathion for preparing is used for pharmaceutical reagent, foods and cosmetics additive by the present invention.
Description of the drawings
Fig. 1 is the selection result of GSH- metal complexs.
Fig. 2 be at 90 DEG C of yeast cells hot water (pH 2.0) extract 15 minutes after water phase HPLC collection of illustrative plates.
Fig. 3 is yeast lixiviating solution Jing Cu2+Light phase HPLC collection of illustrative plates after complexation.
Fig. 4 is light phase HPLC collection of illustrative plates after the washing of GSH-Cu complex.
Fig. 5 is light phase HPLC collection of illustrative plates after complex solution complexation.
Fig. 6 is light phase freeze-dried rear gained composition after complex solution complexation1H NMR spectra (400MHz, D2O)。
Fig. 7 is light phase freeze-dried rear gained composition after complex solution complexation13C NMR spectra (400MHz, D2O)。
Fig. 8 is gained composition after recrystallization1H NMR spectra (400MHz, D2O)。
Fig. 9 is gained composition after recrystallization13C NMR spectra (400MHz, D2O)。
Figure 10 is reduced form GSH crude products and recrystallization product appearance, and wherein, the upper left corner is purity>98% recrystallization
Product, right are purity>90% crude product.
Specific embodiment
Embodiment 1
(1) in yeast cells GSH extraction
Yeast milk 100.0g, plus pure water 900mL is weighed, is that 2.0,90 DEG C of water-baths extract 15min with salt acid for adjusting pH value, then
Stirring 5min, rapidly as being cooled to room temperature in ice bath.15min is centrifuged with 7500r/min, supernatant is retained.
(2) preparation of GSH-Cu complex
Yeast extract 400mL in above-mentioned steps, it is 6.0 to adjust pH value with NaOH, by GSH and CuSO4·5H2O mole
Mass ratio is 1:15 add CuSO4·5H2O carries out complexation, maintains stirring, 30min is centrifuged with 7500r/min, must precipitate about
500mg。
(3) wash
Precipitation in above-mentioned steps is washed 3 times, with the purification aqueous suspension of 150 times of volumes, after maintaining stirring 30 minutes,
It is centrifuged 30 minutes under 7500rpm/min.Retain heavy phase.
(4) solve complexation
By the precipitation after washing in above-mentioned steps, 2L aqueous suspensions are added, H is passed through2S gases, maintain stirring 5 minutes,
It is centrifuged 15 minutes under 7500rpm/min.Disgorging, retains light phase.It is 0.022 using spectrophotometer detection OD600 values.
Qualitative and quantitative analysis are carried out to water mutually using HPLC technologies, reduced glutathion purity is 90.5%.
(5) decolourize
Be 2.0 by light phase use salt acid for adjusting pH value in above-mentioned steps, activated carbon is added according to 5 ‰ ratio, maintain stirring
30 minutes response time.After the completion of reaction, activated carbon is removed with 0.25 μm of membrane filtration, retain filtrate.Using spectrophotometer
Detection OD600 values are 0.002, and decolorizing effect is notable.There is no significant changes, reduced form paddy in reduced glutathion absolute content
The sweet peptide purity of Guang is 89.4%.
(6) concentrate
Water in above-mentioned steps was communicated into concentrating under reduced pressure, the glutathione solution that concentration is 800g/L was obtained.
(7) recrystallization
Stirring is maintained, containing in glutathione solution in above-mentioned steps, the second of 70% (v/v) concentration of equivalent volumes is added
Alcohol-water solution, at 4 DEG C, addition glutathion is crystallized as crystal seed.The addition of crystal seed is 50mg/L.Continue stirring 30 minutes
Afterwards, stop stirring, maintain temperature 12 hours.Slurry is obtained, high-purity reduced glutathion material is filtered to obtain.Reduced form paddy
The sweet peptide purity of Guang is 98.5%.
Fig. 1 is the selection result of GSH- metal complexs.Cu is shown in figure2+White complex compound sediment is formed with GSH, and
KMnO4Oxidation is there occurs to GSH, generates brown oxidation product.
Fig. 4 is light phase HPLC collection of illustrative plates after the washing of GSH-Cu complex.Show in figure that the overwhelming majority is water-soluble after 2 washings
Property impurity is washed away, and does not detect reduced form GSH in eluent.
Fig. 5 is light phase HPLC collection of illustrative plates (reduced glutathion purity after complex solution complexation>90%, ebb line is reduction
Type glutathion and oxidized form of glutathione standard substance aggregate sample).
Claims (8)
1. a kind of method for preparing reduced glutathion from yeast cells, it is characterised in that comprise the following steps：
(1) extraction of yeast cells GSH-PX activity：The yeast cells that glutathione content is 1-5% are taken, with 5-15 times of volume
Deionized water suspend, it is 2.0 to adjust pH value, is stirred 0-30 minutes at 80-100 DEG C, after the completion of be immediately placed in it is cold in frozen water
But to less than 20 DEG C, 10-30 minutes under 3000-10000rpm/min, are centrifuged, disgorging is retained light phase；
(2) preparation of GSH-Cu complex：Using the content of the light phase middle GSH of HPLC technology for detection steps (2), in 10-100rpm/
Under min stirrings, it is 5.0-7.0 to adjust light phase pH value with NaOH, according to the molal weight of light phase middle GSH, according to 1：1-1：
20 proportioning, adds CuSO4, maintain stirring 15-60 minutes, 3000-10000rpm/min lower centrifugation 10-40 minutes, to retain weight
Phase, obtains GSH-Cu complex；
(3) wash：GSH-Cu complex is taken, with the purification aqueous suspension of 100-200 times of volume, stirring 20-40 minutes is maintained,
20-40 minutes are centrifuged under 3000-10000rpm/min, retain heavy phase；
(4) solve complexation：The GSH-Cu complex after step (3) washing is taken, with the purification aqueous suspension of 2-10 times of volume, maintains to stir
Mix, be passed through H2S gases are maintained 5 minutes, and 10-30 minutes are centrifuged under 3000-10000rpm/min, and disgorging is retained light phase；
(5) decolourize：In step (4), light phase use salt acid for adjusting pH value is 2.0, adds activated carbon according to the ratio of mass fraction 5 ‰,
20-60 minutes stirring reaction time are maintained, and after the completion of reaction, activated carbon are removed with 0.25 μm of membrane filtration, are retained filtrate；
(6) concentrate：Filtrate in step (5), by concentrating under reduced pressure, obtains the glutathione solution containing glutathion；
(7) recrystallization：Stirring is maintained, containing in glutathione solution in step (6), the 50-80% (v/v) of equivalent volumes is added
The ethanol water of concentration, at -4～20 DEG C, used as crystal seed, the addition of crystal seed is 20- to the crystallization of addition glutathion
200mg/L, after continuing stirring 30 minutes, stops stirring, maintains temperature 6-20 hour, obtain slurry, filter to obtain reduced form paddy
The sweet peptide material of Guang.
2. the method for preparing reduced glutathion from yeast cells according to claim 1, it is characterised in that：Step
(1) in, the extraction of yeast cells GSH-PX activity：The yeast cells that glutathione content is 1-5% are taken, with going for 10 times of volumes
Ion aqueous suspension, adjust pH value be 2.0, at 90 DEG C stir 5 minutes, after the completion of be immediately placed in frozen water in be cooled to 20 DEG C with
Under, it is centrifuged 15 minutes under 7500rpm/min, disgorging, retains light phase.
3. the method for preparing reduced glutathion from yeast cells according to claim 1, it is characterised in that：Step
(2) in, the preparation of GSH-Cu complex：Using the content of the light phase middle GSH of HPLC technology for detection steps (2), stir in 50rpm/min
Mix under state, it is 6.0 to adjust light phase pH value with NaOH, according to the molal weight of light phase middle GSH, according to 1：15 proportioning, addition
CuSO4·5H2O, maintains stirring 15-60 minutes, 7500rpm/min, lower centrifugation 15 minutes to retain heavy phase, obtain GSH-Cu complexations
Thing.
4. the method for preparing reduced glutathion from yeast cells according to claim 1, it is characterised in that：Step
(3) in, washing：GSH-Cu complex is taken, with the purification aqueous suspension of 150 times of volumes, stirring 30 minutes, 7500rpm/min is maintained
Lower centrifugation 30 minutes, retains heavy phase.
5. the method for preparing reduced glutathion from yeast cells according to claim 1, it is characterised in that：Step
(4), in, solve complexation：The GSH-Cu complex after step (3) washing is taken, with the purification aqueous suspension of 4 times of volumes, stirring is maintained, is led to
Enter H2S gases are maintained 5 minutes, are centrifuged 30 minutes, disgorging under 7500rpm/min, are retained light phase.
6. the method for preparing reduced glutathion from yeast cells according to claim 1, it is characterised in that：Pass through
During step (5) carries out complex solution complexation, H is passed through2S gases include：Pure H2S gases, or contain H2The industrial first of S compositions
Alkane waste gas, or H is also easy to produce in acid condition2The inorganic and organic salt of S, including (NH4)2S, Na2S。
7. the method for preparing reduced glutathion from yeast cells according to claim 1, it is characterised in that：Step
(7) in, recrystallization：Stirring is maintained, containing in glutathione solution in step (6), 70% (v/v) concentration of equivalent volumes is added
Ethanol water, at 4 DEG C, used as crystal seed, the addition of crystal seed is 50mg/L, continues stirring 30 for addition glutathion crystallization
After minute, stop stirring, maintain temperature 12 hours, obtain slurry, filter to obtain reduced glutathion material, described reduction
Type glutathion purity>98%.
8. claim 1-7 any one prepares reduced glutathion from yeast cells in pharmaceutical reagent, food and cosmetic
Application on product additive.
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| CN201610901699.8A CN106518962B (en) | 2016-10-14 | 2016-10-14 | A method for preparing reduced glutathione from yeast cells |
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| CN106518962B CN106518962B (en) | 2021-04-02 |
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| CN201610901699.8A Active CN106518962B (en) | 2016-10-14 | 2016-10-14 | A method for preparing reduced glutathione from yeast cells |
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| CN107686503A (en) * | 2016-08-05 | 2018-02-13 | 北大方正集团有限公司 | A kind of method for purifying glutathione |
| CN109021066A (en) * | 2018-08-23 | 2018-12-18 | 上海青平药业有限公司 | A kind of method of broth extraction glutathione |
| CN112321675A (en) * | 2020-10-23 | 2021-02-05 | 内蒙古拜克生物有限公司 | A kind of purification method of glutathione |
| CN116549317A (en) * | 2023-05-30 | 2023-08-08 | 陕西慧康生物科技有限责任公司 | Composition for stabilizing glutathione taste and application thereof |
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| CN109021066A (en) * | 2018-08-23 | 2018-12-18 | 上海青平药业有限公司 | A kind of method of broth extraction glutathione |
| CN112321675A (en) * | 2020-10-23 | 2021-02-05 | 内蒙古拜克生物有限公司 | A kind of purification method of glutathione |
| CN116549317A (en) * | 2023-05-30 | 2023-08-08 | 陕西慧康生物科技有限责任公司 | Composition for stabilizing glutathione taste and application thereof |
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