CN103757076B - A kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire - Google Patents
A kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire Download PDFInfo
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- CN103757076B CN103757076B CN201410012170.1A CN201410012170A CN103757076B CN 103757076 B CN103757076 B CN 103757076B CN 201410012170 A CN201410012170 A CN 201410012170A CN 103757076 B CN103757076 B CN 103757076B
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Abstract
The invention discloses a kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire, comprise the following steps: (1) Spawn preparation: yeast saccharomyces cerevisiae is inoculated into cultivation in seed culture medium and obtains seed culture fluid; (2) fermentation culture: be inoculated in fermention medium by the seed culture fluid in step (1), then add aseptic oxygen carrier, the add-on of oxygen carrier is 0.5 ~ 10% of fermention medium volume, and fermentation obtains gsh.The present invention, by adding suitable oxygen carrier in the medium, is not adding other material, under the condition not having additional energy source to consume, is significantly improve the dissolved oxygen concentration in substratum and membrane passage; Carrier of the present invention not only plays the effect of oxygen carrier in process, improves membrane passage simultaneously, successfully can realize the object significantly improving expression product output, shorten fermentation period, improve production efficiency.
Description
Technical field
What the present invention relates to is a kind of microbial fermentation processes, in particular a kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire.
Background technology
Gsh (GSH) is a kind of tripeptide compound be made up of Pidolidone, Cys and glycine, is a kind of important physiologically active substance in organism, plays very important effect especially to maintenance organism internal oxidition reducing environment.Gsh belongs to containing sulfydryl, small-molecule peptide material, has two kinds of important antioxygenations and integrate detoxification.Sulfydryl in glutathione molecules structure on halfcystine is its active group, therefore gsh is often abbreviated as GSH, the easy and heavy metallic salt complexing such as iodoacetic acid, yperite (a kind of poison gas), lead, mercury, arsenic, and is provided with integration detoxification.Gsh (gsh especially in liver cell) has very important physiological action and integrates detoxification exactly, can with the combinations such as some drugs (as Paracetamol), toxin (as free radical, heavy metal), participate in biotransformation, thus poisonous substance harmful in body is converted into harmless material, excrete external.Its application on clinical medicine, foodstuffs industry, sports nutrition constantly increases, and demand constantly increases.Gsh is used for the treatment of and prophylaxis of tumours, hepatitis and liver injury, removing toxic substances, Radiation sickness and radio-protective, antianaphylaxis, the course of disease improving some disease and symptom, skin maintenance skin care, increase 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 improvement function, Ginseng Extract and suppress the effect of hiv virus.Widely use in modern food processing industry as antioxidant, and can food value be strengthened, improve flavour of food products.
Another major physiological effect of gsh is that it can dispose the free radical in human body as important antioxidant a kind of in body, and clean and purification human internal environment pollutes, thus enhances the physical and mental health of people.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 by objectionable impuritiess such as such as free radicals in vivo, thus protein and enzyme equimolecular is allowed to play its physiological function.The content of human erythrocyte GSH-PX activity is a lot; this is in reduced state to the sulfydryl of protein on protection erythrocyte membrane; prevent haemolysis from protecting red corpuscle to exempt from oxidative damage significant, but also oxyphorase can be protected not by the oxidation such as hydrogen peroxide oxidation, free radical thus make it continue normally in the ability playing transport oxygen.In red corpuscle, part oxyphorase is under the effect of the oxygenants such as hydrogen peroxide, and wherein ferrous oxidising is ferric iron, makes oxyphorase change methemoglobin into, thus loses band oxygen ability.Reduced glutathion can directly be 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 vitamins C reversibly hydrogenation or dehydrogenation, therefore vitamins C plays an important role in many redox reactions in vivo.Such as, the active group of many enzymes is sulfydryl (-SH), and vitamins C can maintain-SH and be in reduced state and the activity keeping enzyme; 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 from oxidation.Therefore, when using gsh, with vitamins C and use, its effect can be improved.
GSH can play a protective role for symptoms such as radioactive rays, radiopharmaceuticals or the oligoleukocythemia that causes due to antitumor drug.GSH can combine with entering the toxic compounds of body, heavy metal ion or carcinogenic substance etc., and short its excretes, in playing and detoxification.SH also can Cell protection film, makes it to exempt from oxidative damage, prevents erythrocyte hemolysis and promotes the reduction of methemoglobin, to anoxenia, to 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 also has fine effect in treatment cornea disease and improvement function aspects.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 constantly finds, plays a greater and greater role to the healthy cause of human body.
First gsh prepared Patent Publication 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 extracts from 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 obtained is DL body, separation difficulty, and product purity is not high.Enzyme transforming process is also in the laboratory study stage at present.In current 4 kinds of methods, microbe fermentation method most is potential.
Production by Microorganism Fermentation GSH studies mainly from the nineties in last century in China, achieve some achievements, but domestic fermentation level rests on 2 ~ 3g/L always, as: the method for method for synthesizing glutathione by fermentation disclosed in the special CN200710036925.1 of China.Although the zymotechnique of a Chinese patent CN200810019761.6 gsh, also the method utilized through genetic engineering modified methanol using type glutathione production of Saccharomyces cerevisiae is disclosed, gsh output can reach 6.6g/L, but ferments with Japanese consonance of world level 7 ~ 10g/L() also have larger distance.In recent years, utilize the report of Glutathione Production by Microbial Fermentation more, fermentation level also improves gradually.Such as Tan Tian is big waits people at Process Biochemistry(2006) utilize cereuisiae fermentum high density fermentation in 41:2424 ~ 2428, by adding precusor amino acids during the fermentation, in fermented liquid, dry cell weight reaches 140g/L, and the concentration of gsh reaches 2190mg/L.The gsh of fermentative Production is present in the born of the same parents of microorganism more; so while raising glutathione content; the feedback inhibition of product reductive glutathione to γ-L-glutamyl-Cys synthetic enzyme is also increasing the weight of, and limits the further raising of gsh output.In addition, the somatic cells permeability in fermenting process is bad, makes the substrate amino acid added be difficult to enter in born of the same parents for the synthesis of gsh, cause gsh not high relative to added amino acid whose yield, and the fermentative production cycle is long, productivity is low, is 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 method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire, 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) Spawn preparation: yeast saccharomyces cerevisiae is inoculated into cultivation in seed culture medium and obtains seed culture fluid;
(2) fermentation culture: be inoculated in fermention medium by the seed culture fluid in step (1), then add aseptic oxygen carrier, the add-on of oxygen carrier is 0.5 ~ 10% of fermention medium volume, and fermentation obtains gsh.
In described step (1), in seed culture medium, add oxygen carrier, the add-on of described oxygen carrier is 1 ~ 8% of seed culture medium volume.In seed culture medium, add oxygen carrier, improve membrane passage on the one hand, improve donor energy on the other hand, increase the oxygen delivery capacity of entire system.
In described step (1), yeast saccharomyces cerevisiae in seed culture medium, 30 ~ 40 DEG C, 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, pH=7, and pH value adopts ammoniacal liquor to regulate.
In described step (2), the seed culture fluid that step (1) obtains is inoculated in fermention medium, 30 ~ 40 DEG C, 200 ~ 300rpm, aseptic oxygen carrier is added in fermentor tank, 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, treat that cell density reaches more than 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, yeast extract 20g, calcium chloride 0.2g, potassium primary phosphate 5g, magnesium sulfate 10g, inorganic ionic solution 1ml, water 1000ml, pH7, and 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, and it is formulated to add following material: 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 is by adding suitable oxygen carrier in the medium, do not adding other material, under the condition not having additional energy source to consume, significantly improve the dissolved oxygen concentration in substratum and membrane passage, and the catalytic capability of bacterial classification can not be affected, change the process that cell and environment carry out exchange of substance, thus promote 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 process, improves membrane passage simultaneously, successfully can realize the object significantly improving expression product output, shorten fermentation period, improve production efficiency.
Embodiment
Elaborate to embodiments of the invention below, the present embodiment is implemented under premised on technical solution of the present invention, give 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) Spawn preparation: get in yeast saccharomyces cerevisiae access seed culture medium, at 30 DEG C, cultivate 8h under stirring velocity 150 ~ 200rpm condition, then proceed in two generation seed culture mediums, condition is pH6.5 ~ 7, and temperature 30 DEG C is cultivated 10 ~ 15 hours, prepares secondary seed for subsequent use;
Seed culture medium is: yeast extract 5 ~ 10g/L, peptone 10 ~ 20g/L, glucose 5 ~ 20g/L, pH=7, and 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 Culture Collection, and it is numbered CICC1898;
(2) fermentation culture: the seed culture fluid obtained is inoculated in fermention medium, 30 DEG C, 200 ~ 300rpm, add aseptic oxygen carrier normal hexane in the fermentation medium, 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 ammoniacal liquor is adjusted to 7, 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, and 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) Spawn preparation: get in yeast saccharomyces cerevisiae access seed culture medium, at 35 DEG C, 8h is cultivated under stirring velocity 150 ~ 200rpm condition, then proceed in two generation seed culture mediums, add aseptic oxygen carrier n-dodecane, the add-on of oxygen carrier is 8%, pH=7 of seed culture medium volume simultaneously, temperature 35 DEG C is cultivated 10 ~ 15 hours, prepares secondary seed for subsequent use;
(2) fermentation culture: the seed culture fluid obtained is inoculated in fermention medium, 35 DEG C, 200 ~ 300rpm, aseptic oxygen carrier n-dodecane is added in fermentor tank, 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 ammoniacal liquor is adjusted to 7, 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 of obtained 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 of obtained 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 of obtained 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 obtained glutathione concentrations is 1.5g/L.
As can be seen from embodiment 1 ~ 7, after adding oxygen carrier, obtained glutathione concentrations significantly improves.
Claims (7)
1. the method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire, is characterized in that, comprise the following steps:
(1) Spawn preparation: yeast saccharomyces cerevisiae is inoculated into cultivation in seed culture medium and obtains 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, and fermentation obtains gsh,
In described step (1), seed culture medium is: yeast extract 5 ~ 10g/L, peptone 10 ~ 20g/L, glucose 5 ~ 20g/L, pH=7, and pH value adopts ammoniacal liquor to regulate.
2. a kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire according to claim 1, it is characterized in that, in described step (1), in seed culture medium, add oxygen carrier, the add-on of described oxygen carrier is 1 ~ 8% of seed culture medium volume.
3. a kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire according to claim 1, it is characterized in that, in described step (1), yeast saccharomyces cerevisiae is in seed culture medium, 30 ~ 40 DEG C, 150 ~ 200rpm constant temperature culture 10 ~ 15 hours.
4. a kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire according to claim 1, it is characterized in that, in described step (2), the seed culture fluid that step (1) obtains is inoculated in fermention medium, 30 ~ 40 DEG C, 200 ~ 300rpm, aseptic oxygen carrier is added in fermentor tank, 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, treat that cell density reaches more than OD600nm=50, add precusor amino acids Pidolidone 5 ~ 30mmol/L, Cys 5 ~ 20mmol/L and glycine 5 ~ 30mmol/L, final fermentation obtains gsh.
5. a kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire according to claim 4, 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.
6. a kind of method utilizing oxygen carrier to improve fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire according to claim 5, it is characterized in that, the preparation method of described inorganic ionic solution is: in 1000ml distilled water, and it is formulated to add following material: 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.
7. a kind of method utilizing oxygen carrier raising fermentation by saccharomyces cerevisiae liquid GSH-PX activity to tire according to claim 1 and 2, 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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