CN103757076A - Method for increasing valence of glutathione in saccharomyces cerevisiae fermentation liquor by utilizing oxygen carrier - Google Patents
Method for increasing valence of glutathione in saccharomyces cerevisiae fermentation liquor by utilizing oxygen carrier Download PDFInfo
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- CN103757076A CN103757076A CN201410012170.1A CN201410012170A CN103757076A CN 103757076 A CN103757076 A CN 103757076A CN 201410012170 A CN201410012170 A CN 201410012170A CN 103757076 A CN103757076 A CN 103757076A
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Abstract
The invention discloses a method for increasing the valence of glutathione in saccharomyces cerevisiae fermentation liquor by utilizing an oxygen carrier. The method comprises the following steps: (1) preparation of seeds, namely inoculating saccharomyces cerevisiae in a seed medium for culturing so as to obtain a seed culture solution; and (2) fermentation culture, namely inoculating the seed culture solution obtained in step (1) in a fermentation medium, then adding an aseptic oxygen carrier of which the added amount is 0.5-10 percent of the volume of the fermentation medium, and fermenting to obtain the glutathione. According to the method disclosed by the invention, by adding the proper oxygen carrier into the medium without addition of other substances and extra energy consumption, the concentration of dissolved oxygen and the permeability of cell membranes in the medium are greatly increased; in the process, the carrier disclosed by the invention acts as the oxygen carrier, and the permeability of the cell membranes is improved as well, so the aim of obviously increasing the yield of an expression product can be successfully achieved, the fermentation period is shortened and the production efficiency is improved.
Description
Technical field
What the present invention relates to is a kind of microbial fermentation processes, and in particular a kind of oxygen carrier that utilizes improves the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired.
Background technology
Gsh (GSH) is a kind of tripeptide compound being comprised of Pidolidone, Cys and glycine, is a kind of important physiologically active substance in organism, plays very important effect especially to maintaining redox environment in organism.Gsh be belong to contain sulfydryl, small-molecule peptide material, there are two kinds of important antioxygenations and integrate detoxification.Sulfydryl in gsh molecular structure on halfcystine is its active group, therefore gsh is often abbreviated as GSH, easily and the heavy metallic salt complexing such as iodoacetic acid, yperite (a kind of poison gas), lead, mercury, arsenic, and has had integration detoxification.Gsh (the especially gsh in liver cell) has very important physiological action and integrates exactly detoxification, can with the combinations such as some drugs (as Paracetamol), toxin (as free radical, heavy metal), participate in biotransformation, thereby harmful poisonous substance in body is converted into harmless material, excretes external.Its application on clinical medicine, foodstuffs industry, sports nutrition constantly increases, and demand constantly increases.Gsh is used for the treatment of with prophylaxis of tumours, hepatitis and liver injury, removing toxic substances, Radiation sickness and radio-protective, antianaphylaxis, the course of disease of improving some disease and symptom, skin maintenance skin care, increases eyesight and ophthalmic diseases and the anti-ageing effect of waiting for a long time clinically.Successful in the treatment of endocrine regulation, and there is the effect that improves sexual function, Ginseng Extract and suppress hiv virus.As antioxidant, in modern food processing industry, be widely used, and can strengthen food value, improve flavour of food products.
Another major physiological effect of gsh is that it can dispose the free radical in human body as a kind of important antioxidant in body, and clean and purification human internal environment pollutes, thereby has promoted people's physical and mental health.Because reduced glutathion itself is subject to Cucumber oxidation, so it can protect the sulfydryl in numerous protein and enzyme equimolecular not to be oxidized as objectionable impuritiess such as free radicals in vivo, thereby allow protein and enzyme equimolecular bring into play its physiological function.The content of human erythrocyte GSH-PX activity is a lot; this is to protecting the sulfydryl of protein on erythrocyte membrane in reduced state; prevent that haemolysis protection red corpuscle from exempting from oxidisability destruction significant, thereby but also can protect oxyphorase not to be subject to the oxidations such as hydrogen peroxide oxidation, free radical that its is continued normally in ability of bringing into play transportation oxygen.In red corpuscle, part oxyphorase is under the effect of the oxygenants such as hydrogen peroxide, and wherein ferrous iron is oxidized to ferric iron, makes oxyphorase change methemoglobin into, thereby has lost band oxygen ability.Reduced glutathion can be directly combined with the oxygenant such as hydrogen peroxide, generates water and Sleep-promoting factor B, also methemoglobin can be reduced to oxyphorase, improves the content of oxyphorase in body.
In addition, vitamins C is also a kind of important antioxidant in body.Due to reversibly hydrogenation or dehydrogenation of vitamins C, therefore vitamins C plays an important role in many redox reactions in vivo.For example, the active group of many enzymes is sulfydryl (SH), and can maintain-SH of vitamins C keeps the activity of enzyme in reduced state; Vitamins C can make Sleep-promoting factor B change reduced glutathion (GSH) into, the hydrogen peroxide (H that organism metabolism is produced
2o
2) reduction; Vitamins C also can protect vitamin A, E and some vitamin B group to avoid oxidation.Therefore,, while using gsh, with vitamins C use, can improve its effect.
GSH can play a protective role for radioactive rays, radiopharmaceuticals or the symptoms such as oligoleukocythemia that cause due to antitumor drug.GSH can combine with toxic compounds, heavy metal ion or the carcinogenic substance etc. that enter body, and short its excrete, in playing and detoxification.SH also can Cell protection film, makes it to exempt from oxidisability and destroys, and prevents erythrocyte hemolysis and promotes the reduction of methemoglobin, to anoxenia, feel sick and discomfort that hepatic diseases causes has mitigation.Current research also shows, GSH can correct the imbalance of vagusstoff, Pseudocholinesterase, play anti-allergic effects, also can prevent skin aging and pigmentation, reduce melanic formation, improve skin resistance of oxidation and make skin produce gloss, in addition, GSH is sick and improve aspect sexual function and also have fine effect at treatment cornea.The wide spectrum detoxification that gsh has, not only can be used for medicine, more can be used as the base-material of functional foodstuff, delaying senility, strengthening immunity, the functional foodstuff widespread use such as antitumor, along with deepening continuously of peptide matters research, the magical function that peptide matters has is constantly found, the healthy cause of human body is played a greater and greater role.
First gsh is prepared patent and is published in 1938, and various countries scientist has carried out large quantity research to its production development subsequently.The method of generally speaking producing gsh has extraction process, chemical synthesis, enzyme transforming process and microbe fermentation method.Extraction process is from extracting containing the animals and plants of GSH.Because the too low this method of content does not have actual application value.Chemical synthesis was once produced for early stage GSH, but complex process, the product obtaining is DL body, separation difficulty, and product purity is not high.Enzyme transforming process is at present also in the laboratory study stage.In 4 kinds of current methods, microbe fermentation method has potentiality most.
The research of Production by Microorganism Fermentation GSH China is mainly since the nineties in last century, some achievements have been obtained, but domestic fermentation level rests on 2～3g/L always, as: China is the method for the disclosed method for synthesizing glutathione by fermentation of CN200710036925.1 specially.Although the zymotechnique of a Chinese patent CN200810019761.6 gsh, the method through genetic engineering modified methanol using type glutathione production of Saccharomyces cerevisiae of utilizing is also disclosed, gsh output can reach 6.6g/L, but with world level 7～10g/L(Japan consonance fermentation) also have larger distance.In recent years, utilize the report of Glutathione Production by Microbial Fermentation more, fermentation level also improves gradually.Such as people such as Tan Tianwei at Process Biochemistry(2006) utilize cereuisiae fermentum high density fermentation in 41:2424～2428, by adding during the fermentation precusor amino acids, in fermented liquid, dry cell weight has reached 140g/L, and the concentration of gsh has reached 2190mg/L.The gsh of fermentative Production is present in the born of the same parents of microorganism more; so when improving glutathione content; product reductive glutathione is also increasing the weight of the feedback inhibition of γ-L-glutamyl-Cys synthetic enzyme, has limited the further raising of gsh output.In addition, the somatic cells permeability in fermenting process is bad, makes the substrate amino acid adding be difficult to enter in born of the same parents for the synthesis of gsh, cause gsh not high with respect to added amino acid whose yield, and the fermentative production cycle is long, and productivity is low, be also unfavorable for the separation and Extraction of product.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of oxygen carrier that utilizes to improve the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, utilize oxygen carrier to promote yeast saccharomyces cerevisiae high density fermentation.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
(1) seed preparation: yeast saccharomyces cerevisiae is inoculated in seed culture medium and cultivates and obtain seed culture fluid;
(2) fermentation culture: the seed culture fluid in step (1) is inoculated in fermention medium, then adds aseptic oxygen carrier, the add-on of oxygen carrier is 0.5～10% of fermention medium volume, fermentation obtains gsh.
In described step (1), add oxygen carrier in seed culture medium, the add-on of described oxygen carrier is 1～8% of seed culture medium volume.In seed culture medium, add oxygen carrier, improve on the one hand the permeability of cytolemma, improve on the other hand donor energy, increase the oxygen delivery capacity of entire system.
In described step (1), yeast saccharomyces cerevisiae in seed culture medium, 30～40 ℃, 150～200rpm constant temperature culture 10～15 hours.
In described step (1), seed culture medium is: yeast extract 5～10g/L, peptone 10～20g/L, glucose 5～20g/L, and pH=7, pH value adopts ammoniacal liquor to regulate.
In described step (2), the seed culture fluid that step (1) is obtained is inoculated in fermention medium, 30～40 ℃, 200～300rpm, in fermentor tank, add aseptic oxygen carrier, the add-on of oxygen carrier is 0.5～10% of fermention medium volume, constant temperature culture, control dissolved oxygen 30～50%, stream adds glucose 100～500g/L and maintains thalli growth, more than treating that cell density reaches OD600nm=50, add precusor amino acids Pidolidone 5～30mmol/L, Cys 5～20mmol/L and glycine 5～30mmol/L, final fermentation obtains gsh.
In described step (2), fermention medium is: glucose 70g, and yeast extract 20g, calcium chloride 0.2g, potassium primary phosphate 5g, magnesium sulfate 10g, inorganic ionic solution 1ml, water 1000ml, pH7, stream adds with glucose 100～500g/L, and pH value adopts ammoniacal liquor to regulate.
The preparation method of described inorganic ionic solution is: in 1000ml distilled water, add following material formulated: ZnCl
20.65～0.70g, MnCl
210.10～10.20g, H
3bO
30.05～0.07g, CuCl
20.45～0.50g, FeCl
35.2～5.6g, CoCl
211～15g, (NH
4)
6mo
7o
244H
2o0.23～0.27g, 3mol/LH
2sO
418～22ml.
In described step (2), oxygen carrier is one or more the mixture in n-dodecane, normal hexane or soybean oil.
The present invention has the following advantages compared to existing technology: the present invention by adding suitable oxygen carrier in substratum, do not adding other material, do not have under the condition of additional energy source consumption, dissolved oxygen concentration in substratum and the permeability of cytolemma have significantly been improved, and can not affect the catalytic capability of bacterial classification, changed the process that cell and environment carry out exchange of substance, thereby promoted yeast saccharomyces cerevisiae high density fermentation, during fermentation ends, stratification reclaims oxygen carrier cycling and reutilization; Carrier of the present invention not only plays the effect of oxygen carrier in technique, improves the permeability of cytolemma simultaneously, can successfully realize the object of obvious raising expression product output, has shortened fermentation period, has improved production efficiency.
Embodiment
Below embodiments of the invention are elaborated, the present embodiment is implemented take technical solution of the present invention under prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
The present embodiment comprises the following steps:
(1) seed preparation: get in yeast saccharomyces cerevisiae access seed culture medium, at 30 ℃, cultivate 8h under stirring velocity 150～200rpm condition, then proceed in two generation seed culture mediums, condition is pH6.5～7, and 30 ℃ of cultivations of temperature 10～15 hours, prepare secondary seed standby;
Seed culture medium is: yeast extract 5～10g/L, peptone 10～20g/L, glucose 5～20g/L, and pH=7, pH value adopts ammoniacal liquor to regulate;
In the present embodiment, yeast saccharomyces cerevisiae is that market purchase obtains, and described yeast saccharomyces cerevisiae is preserved by Chinese industrial microbial strains preservation center, and it is numbered CICC1898;
(2) fermentation culture: the seed culture fluid obtaining is inoculated in fermention medium, 30 ℃, 200～300rpm, in fermention medium, add aseptic oxygen carrier normal hexane, the add-on of oxygen carrier is 5% constant temperature culture of fermention medium volume, control dissolved oxygen 30～50%, stream adds glucose 100g/L and maintains thalli growth, pH value is adjusted to 7 with ammoniacal liquor, cell density OD600nm=90, add precusor amino acids Pidolidone 20mmol/L, Cys 20mmol/L, glycine 30mmol/L, final fermented liquid GSH-PX activity 3g/L.
Embodiment 2
In the present embodiment, the mixture of oxygen carrier n-dodecane, normal hexane and soybean oil, add-on is 0.5% of fermention medium volume, final fermented liquid GSH-PX activity 2.1g/L, other embodiments are identical with embodiment 1.Embodiment 3
The present embodiment comprises the following steps:
(1) seed preparation: get in yeast saccharomyces cerevisiae access seed culture medium, at 35 ℃, under stirring velocity 150～200rpm condition, cultivate 8h, then proceed in two generation seed culture mediums, add aseptic oxygen carrier n-dodecane, the add-on of oxygen carrier is 8% of seed culture medium volume, pH=7 simultaneously, 35 ℃ of cultivations of temperature 10～15 hours, prepare secondary seed standby;
(2) fermentation culture: the seed culture fluid obtaining is inoculated in fermention medium, 35 ℃, 200～300rpm, in fermentor tank, add aseptic oxygen carrier n-dodecane, the add-on of oxygen carrier is 8% constant temperature culture of fermention medium volume, control dissolved oxygen 30～50%, stream adds glucose 500g/L and maintains thalli growth, pH value is adjusted to 7 with ammoniacal liquor, cell density OD600nm=120, add precusor amino acids Pidolidone 20mmol/L, Cys 20mmol/L, glycine 20mmol/L, final fermented liquid GSH-PX activity 6.5g/L.Other embodiments are identical with embodiment 1.
Embodiment 4
In the present embodiment, oxygen carrier is the mixture of n-dodecane and soybean oil, 3% of the seed culture medium volume of oxygen carrier add-on in the seed culture medium of step (1); In the fermention medium of step (2), add-on is 10% of fermention medium volume, and the concentration that makes gsh is 7.2g/L.Other embodiments are identical with embodiment 3.
Embodiment 5
In the present embodiment, oxygen carrier is the mixture of normal hexane and soybean oil, 1% of the seed culture medium volume of oxygen carrier add-on in the seed culture medium of step (1); In the fermention medium of step (2), add-on is 10% of fermention medium volume, and the concentration that makes gsh is 5.4g/L.Other embodiments are identical with embodiment 3.
Embodiment 6
In the present embodiment, oxygen carrier is soybean oil, 5% of the seed culture medium volume of oxygen carrier add-on in the seed culture medium of step (1); In the fermention medium of step (2), add-on is 8% of fermention medium volume, and the concentration that makes gsh is 4.5g/L.Other embodiments are identical with embodiment 3.
Embodiment 7
In the present embodiment, do not add oxygen carrier, other embodiments are identical with embodiment 1, and the glutathione concentrations making is 1.5g/L.
By embodiment 1～7, can be found out, add after oxygen carrier, the glutathione concentrations making significantly improves.
Claims (8)
1. utilize oxygen carrier to improve the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, it is characterized in that, comprise the following steps:
(1) seed preparation: yeast saccharomyces cerevisiae is inoculated in seed culture medium and cultivates and obtain seed culture fluid;
(2) fermentation culture: the seed culture fluid in step (1) is inoculated in fermention medium, then adds aseptic oxygen carrier, the add-on of oxygen carrier is 0.5～10% of fermention medium volume, fermentation obtains gsh.
2. a kind of oxygen carrier that utilizes according to claim 1 improves the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, it is characterized in that, in described step (1), add oxygen carrier in seed culture medium, the add-on of described oxygen carrier is 1～8% of seed culture medium volume.
3. a kind of oxygen carrier that utilizes according to claim 1 improves the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, it is characterized in that, in described step (1), yeast saccharomyces cerevisiae is in seed culture medium, 30～40 ℃, 150～200rpm constant temperature culture 10～15 hours.
4. a kind of oxygen carrier that utilizes according to claim 1 improves the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, it is characterized in that, in described step (1), seed culture medium is: yeast extract 5～10g/L, peptone 10～20g/L, glucose 5～20g/L, pH=7, pH value adopts ammoniacal liquor to regulate.
5. a kind of oxygen carrier that utilizes according to claim 1 improves the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, it is characterized in that, in described step (2), the seed culture fluid that step (1) is obtained is inoculated in fermention medium, 30～40 ℃, 200～300rpm, in fermentor tank, add aseptic oxygen carrier, the add-on of oxygen carrier is 0.5～10% of fermention medium volume, constant temperature culture, control dissolved oxygen 30～50%, stream adds glucose 100～500g/L and maintains thalli growth, more than treating that cell density reaches OD600nm=50, add precusor amino acids Pidolidone 5～30mmol/L, Cys 5～20mmol/L and glycine 5～30mmol/L, final fermentation obtains gsh.
6. a kind of oxygen carrier that utilizes according to claim 5 improves the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, and it is characterized in that, in described step (2), fermention medium is: glucose 70g, yeast extract 20g, calcium chloride 0.2g, potassium primary phosphate 5g, magnesium sulfate 10g, inorganic ionic solution 1ml, water 1000ml, pH7, stream adds with glucose 100～500g/L, and pH value adopts ammoniacal liquor to regulate.
7. a kind of oxygen carrier that utilizes according to claim 6 improves the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, and it is characterized in that, the preparation method of described inorganic ionic solution is: in 1000ml distilled water, add following material formulated: ZnCl
20.65～0.70g, MnCl
210.10～10.20g, H
3bO
30.05～0.07g, CuCl
20.45～0.50g, FeCl
35.2～5.6g, CoCl
211～15g, (NH
4)
6mo
7o
244H
2o 0.23～0.27g, 3mol/LH
2sO
418～22ml.
8. a kind of oxygen carrier that utilizes according to claim 1 and 2 improves the method that fermentation by saccharomyces cerevisiae liquid GSH-PX activity is tired, and it is characterized in that, described oxygen carrier is one or more the mixture in n-dodecane, normal hexane or soybean oil.
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| CN114350731A (en) * | 2021-12-15 | 2022-04-15 | 华南理工大学 | Preparation method of glutathione |
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| CN114350731A (en) * | 2021-12-15 | 2022-04-15 | 华南理工大学 | Preparation method of glutathione |
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