CN102776258A - Fermentation method for coproduction of S-adenosyl-L-methionine and glutathione - Google Patents
Fermentation method for coproduction of S-adenosyl-L-methionine and glutathione Download PDFInfo
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- CN102776258A CN102776258A CN2012102859006A CN201210285900A CN102776258A CN 102776258 A CN102776258 A CN 102776258A CN 2012102859006 A CN2012102859006 A CN 2012102859006A CN 201210285900 A CN201210285900 A CN 201210285900A CN 102776258 A CN102776258 A CN 102776258A
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Abstract
The invention discloses a fermentation method for coproduction of S-adenosyl-L-methionine and glutathione. The fermentation method comprises the following steps of: adopting a Candidautilis strain with the collection number of CCTCC M209298, performing seed culture, then performing fermentation culture for 18-30h, and further extracting from a fermentation solution to get S-adenosyl-L-methionine and glutathione, and the fermentation method is characterized in that sodium citrate is added into a culture solution within 0-15h from the beginning of the fermentation culture, and 2-10g of the sodium citrate is added in each liter of the culture solution. According to the fermentation method disclosed by the invention, the way of exogenous adding of the low-price sodium citrate is adopted, the coproduction yield of SAM (S-adenosyl-L-methionine) and GSH (glutathione) can be further improved under the situation of not additionally adding other precursor amino acids, and the indirect improvement of the level of intracellular ATP (adenosine triphosphate) during the aerobic fermentation process becomes possible.
Description
Technical field
The present invention relates to a kind of microbial fermentation processes, be specifically related to a kind of
S-adenosine-L-methionine(Met) and gsh coproduction fermentation process.
Background technology
S-adenosine-L-methionine(Met) (SAM) and gsh (GSH) are two kinds of important biological molecules in the organism.
SAM has another name called adenosylmethionine, is the activity form of methionine(Met), in animal and plant body, extensively exists, by substrate L-methionine(Met) and Triphosaden (ATP) warp
S-adenosine methilanin synthase (EC 2.5.1.6) enzymatic synthetic.Many metabolic processes are closely related in SAM and the human body; It is a kind of biochemical drug that improves cellular metabolism; Can increase GSH, sulfate radical and taurine level in the liver through changeing sulfenyl; Prevent hepatitis, fatty liver, hepatic fibrosis, liver cirrhosis and liver cancer, also can prevent the damage of alcohol, medicine and cytokine liver.SAM also has good result of treatment for diseases such as sacroiliitis, fibering muscle, migraine, and spinoff is little.As far back as the seventies in last century, use SAM in Europe as the prescription drugs of treatment of arthritis.1999, drugs approved by FDA SAM went on the market as healthcare products, and oneself becomes one of best-selling nutritious prod in the U.S..
GSH is distributed widely in main and low molecule tripeptides that content is the abundantest in Mammals, plant and the microorganism cells; It also is one of most important sulfhydryl-group activity compound in the organism; Be widely used as toxinicide, inhibitor, immunostimulant, flavour agent and sanitas, all have a wide range of applications in a lot of fields such as clinical medicine, sports health, food-processing.Clinicing aspect; The adjuvant therapy medicaments of GSH Chang Zuowei hepatitis, ABO-HD and keratitis, cataract and retinal diseases; Sulfydryl in can protective in numerous protein and the enzyme equimolecular is not by like objectionable impurities oxidations such as radicals; Thereby let its physiological function of protein and enzyme equimolecular performance, proteinic sulfydryl on the protection red cell membrane is in reduced state, prevent that haemolysis is significant.GSH for radioactive rays, radiopharmaceuticals or since the symptoms such as oligoleukocythemia that antitumor drug causes also can play a protective role.Simultaneously GSH can combine with toxic compounds, heavy metal ion or the carcinogenic substance etc. that get into body, and short its excrete, in playing and detoxification.Current research also shows; GSH can correct the imbalance of vagusstoff, Pseudocholinesterase in the body, plays anti-allergic effects, also can prevent skin aging and pigmentation; Reduce melanic formation; Improve the skin resistance of oxidation and make skin produce gloss, in addition, GSH also has fine effect aspect sexual function improving.In recent years, the western sciences man finds that gsh also has the function that suppresses hiv virus.The food-processing aspect joins GSH in the flour products, can play reductive action, and it is original 1/2nd or 1/3rd that the time of making bread is foreshortened to, and labor condition is improved significantly, and plays the effect of nutrient fortified food nutrition; It is joined in sour milk and the infant or baby food, be equivalent to vitamins C, can play function of stabilizer; It is mixed in the breaded fish stick, can prevent the color and luster intensification; Be added in the food such as meat product and cheese, have the effect of strong taste.
Because SAM and GSH be in many-sided effect, the outstanding role of medical aspect particularly, people also rapidly increase the demand of the two, but the present main dependence on import of domestic needs, and hold at high price.
The working method of SAM and GSH has chemical synthesis, enzyme process and microbe fermentation method.Wherein chemical synthesis has limited its industrial applications owing to have process complicacy, defective consuming time.The synthetic SAM of enzyme process has the product purity height with GSH, is prone to advantages such as extraction, but raw material A TP costs an arm and a leg and becomes the restrictive factor that enzyme process synthesizes SAM and GSH.At present yeast fermentation method is considered to SAM and the most potential method of GSH biosynthesizing, yet mostly concentrates in the production of a kind of product wherein for the existing research of fermentative Production SAM and GSH.In fact, because SAM and GSH all are the important node materials in the yeast cell S-contained substance metabolism network, the two exists complicated association in building-up process.More noticeablely be: can be in the catabolic process of SAM through the synthetic L-halfcystine of transsulfuration, thus for providing important as precursors, GSH synthetic promoted that GSH's is synthetic.In recent years, utilize a kind of microorganism strains fermentation coproduction SAM and GSH also to cause investigator's extensive concern gradually.
"
SThe envrionment conditions of-adenosine methyllanthionine and gsh coproduction fermentation " in (the 9th the 2nd phase of volume of " biological processing " March in 2011, the 23rd～28 page) literary composition, disclose the employing Candida utilis (
Candida utilisCCTCC M 209298) carries out fermentation culture coproduction SAM and GSH, and culture condition is studied.
In the biosynthetic process of SAM and GSH, ATP is as restricted substrate and energy matter, and its effect is irreplaceable, and can the transformation efficiency that how much is determining substrate of ATP content and SAM and GSH efficiently synthesize in the born of the same parents.There are some researches show that the external source ATP that in good time in fermented liquid, adds proper concn can promote the raising of SAM and GSH output effectively.But because ATP costs an arm and a leg, exogenous ATP addition manner obviously is difficult to be applied to the suitability for industrialized production of SAM and GSH.Therefore search out the substituting cheap material of a kind of ATP, for the raising of SAM and GSH output, and even the suitability for industrialized production of SAM and GSH is significant undoubtedly.
Summary of the invention
Goal of the invention of the present invention provides a kind of
S-adenosine-L-methionine(Met) and gsh coproduction fermentation process through the improvement to method, improve the coproduction amount of SAM and GSH with lower cost.
For reaching the foregoing invention purpose, the technical scheme that the present invention adopts is: a kind of
S-adenosine-L-methionine(Met) and gsh coproduction fermentation process, the employing Candida utilis (
Candida utilis, preserving number CCTCC M 209298) and bacterial strain, after seed culture, fermentation culture 18 to 30 hours is extracted acquisition again from fermented liquid
S-adenosine-L-methionine(Met) and gsh in 0～15 hour of beginning in fermentation culture, adds Trisodium Citrate in nutrient solution, add 2～10 gram Trisodium Citrates in every liter of nutrient solution.
Optimized technical scheme, Trisodium Citrate is added in 15 hours of beginning in fermentation culture in nutrient solution, add 2 gram Trisodium Citrates in every liter of nutrient solution.
In the technique scheme, the inoculum size of fermentation culture is 5～10%, 28～32 ℃ of leavening temperatures, and mixing speed 300～400 rpm, air flow 3.0～4.0 L/min, pH are 5.0.
Optimized technical scheme, fermented incubation time are 24～30 hours.
Because the utilization of technique scheme, the present invention compared with prior art has advantage:
1. the present invention takes exogenous mode of adding cheap Trisodium Citrate, is not having further to have improved SAM and GSH coproduction output under the amino acid whose situation of other precursors of extra interpolation, makes the level of ATP in aerobic fermentation process indirect improves born of the same parents become possibility.
2. the present invention has increased new approach for the optimization of SAM and GSH coproduction fermenting process, for the industrialization production of SAM and GSH provides technical basis, also is that excessive the synthesizing of similar power consumption synthetic compound provides new thinking.
Description of drawings
Fig. 1 is the result of variations figure of SAM and GSH in the coproduction fermenting process among the embodiment five.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Embodiment one: a kind of
S-adenosine-L-methionine(Met) and gsh coproduction fermentation process adopt the Candida utilis bacterial strain, through seed culture and fermentation culture, from fermented liquid, extract acquisition
S-adenosine-L-methionine(Met) and gsh.Its concrete grammar is following:
(1) bacterial strain
Candida utilis (
Candida utilis) CCTCC M 209298, University Of Suzhou's industrial microorganism laboratory screening is also preserved.
(2) substratum
Inclined-plane and seed culture medium (g/L): glucose 20, peptone 10, yeast extract paste 10, pH 6.0;
Fermention medium (g/L): sucrose 35, ammonium sulfate 10, potassium primary phosphate 12.3, L-methionine(Met) 4.6, sal epsom 0.05, pH 5.0.
(3) seed culture
The inclined-plane seed activation inserts after 4 hours in the 500 mL triangular flasks that 50 mL seed culture mediums are housed and carries out shaking culture, 30 ℃ of culture temperature, shaking speed 200 rpm, incubation time 24 hours.
(4) batch fermentation is cultivated
The fresh seeds nutrient solution is seeded in the 5 L fermentor tanks (BIOTECH-5BGZ, Shanghai Baoxing Biology Equipment Engineering Co., Ltd), liquid amount 3 L; Inoculum size 10%, 30 ℃ of leavening temperatures, mixing speed 350 rpm; Air flow 3.0 L/min, pH 5.0 is (through auto-feeding 3 mol/L H
2SO
4Regulate with 3 mol/L NaOH solution), incubation time 30 hours.At 0 hour, 6 hours or 15 hours of fermentation culture, in nutrient solution, add ATP (Comparative Examples) or Trisodium Citrate, ATP adds concentration 0.2 mmol/L, and Trisodium Citrate adds concentration 2 g/L or 10 g/L.
(5) mensuration of yeast bio amount
With dried cell weight (DCW) expression yeast bio amount.Get 25 mL fermented liquids, centrifugal 10 min of 3500 rpm, zero(ppm) water centrifuge washing 3 times is collected thalline, dries to constant weight for 70 ℃.
(6) extraction and the mensuration of GSH in the born of the same parents
The fresh yeast that fermentation culture obtains with distilled water wash 3 times after, in 40% ethanolic soln, handled 2 hours under 30 ℃, the centrifuging and taking supernatant is as testing sample.Adopt DTNB [5,5'-two sulphur two (2-nitrobenzoic acid)]-NADPH-GSSG reductase circulation method to measure.
(7) extraction and the mensuration of SAM in the born of the same parents
The fresh yeast that fermentation culture obtains with distilled water wash 3 times after, handled 2 hours down for 4 ℃ with 0.35 mol/L dilute sulphuric acid, centrifugal, supernatant warp 0.22 μ m membrane filtration is as testing sample.Adopt the HPLC method to measure: moving phase is 0.5 mol/L ammonium formate solution (pH 4.0), flow velocity 0.8 mL/min, 25 ℃ of column temperatures.
(8) extraction and the mensuration of ATP in the born of the same parents
Get 5 mL fermented liquids, the centrifugal yeast cell that wets that obtains is suspended in wet cell in 5 mL, the 0.2 mol/L phosphate buffered saline buffers (pH 7.0) again; Ultrasonic disruption 10 min, the supernatant that obtains after centrifugal adopt the HPLC method to detect ATP content as testing sample; Moving phase is PBS solution; Flow velocity 1 mL/min detects wavelength 254 nm, 35 ℃ of column temperatures.
Comparative Examples one: according to fermentation condition described in the embodiment one, batch fermentation added the experimental result of 0～0.2 mmol/L ATP in 0 hour:
Dried cell weight: 14.47～14.86 g/L; SAM output: 270.1～296.2 mg/L; GSH output: 257.1～281.1 mg/L; SAM and GSH coproduction output: 528.8～562.9 mg/L; SAM content in the born of the same parents: 1.96～1.98%; GSH content in the born of the same parents: 1.78～1.81%.
Comparative Examples two: according to fermentation condition described in the embodiment one, batch fermentation added the experimental result of 0～0.2 mmol/L ATP in 15 hours:
Dried cell weight: 14.47～14.77 g/L; SAM output: 270.1～360.3 mg/L; GSH output: 257.1～350.9 mg/L; SAM and GSH coproduction output: 528.8～701.3 mg/L; SAM content in the born of the same parents: 1.96～2.39%; GSH content in the born of the same parents: 1.78～2.35%.
Embodiment two: according to fermentation condition described in the embodiment one, batch fermentation added the experimental result of 2～10 g/L Trisodium Citrates in 0 hour:
Dried cell weight: 14.01～14.27 g/L; SAM output: 329.7～344.0 mg/L; GSH output: 269.4～310.3 mg/L; SAM and GSH coproduction output: 582.5～636.3 mg/L; SAM content in the born of the same parents: 2.23～2.41%; GSH content in the born of the same parents: 1.91～2.33%.
Embodiment three: according to fermentation condition described in the embodiment one, batch fermentation added the experimental result of 2～10 g/L Trisodium Citrates in 6 hours:
Dried cell weight: 13.93～14.20 g/L; SAM output: 310.7～328.6 mg/L; GSH output: 270.0～301.5 mg/L; SAM and GSH coproduction output: 588.6～607.3 mg/L; SAM content in the born of the same parents: 2.01～2.42%; GSH content in the born of the same parents: 2.05～2.32%.
Embodiment four: according to fermentation condition described in the embodiment one, batch fermentation added the experimental result of 2～10 g/L Trisodium Citrates in 15 hours:
Dried cell weight: 14.02～14.33 g/L; SAM output: 308.4～331.5 mg/L; GSH output: 281.8～344.6 mg/L; SAM and GSH coproduction output: 571.4～663.9 mg/L; SAM content in the born of the same parents: 2.44～2.55%; GSH content in the born of the same parents: 2.06～2.47%.
Embodiment five: according to embodiment one said fermentation condition, batch fermentation added 2 g/L Trisodium Citrates in 15 hours, the variation of SAM and GSH in the detection culturing process, and the result is as shown in Figure 1.
From the foregoing description and Comparative Examples, can find out: the present invention has realized the further raising of SAM and GSH coproduction amount through in the coproduction fermenting process, adding allogenic material ATP and Trisodium Citrate; Compare with adding ATP, add the raising that Trisodium Citrate helps interior SAM of born of the same parents and GSH content more.
Claims (4)
1. one kind
S-adenosine-L-methionine(Met) and gsh coproduction fermentation process, the employing Candida utilis (
Candida utilis, preserving number CCTCC M 209298) and bacterial strain, after seed culture, fermentation culture 18 to 30 hours is extracted acquisition again from fermented liquid
S-adenosine-L-methionine(Met) and gsh is characterized in that: in begin in fermentation culture 0～15 hour, in nutrient solution, add Trisodium Citrate, add 2～10 gram Trisodium Citrates in every liter of nutrient solution.
2. according to claim 1
S-adenosine-L-methionine(Met) and gsh coproduction fermentation process is characterized in that: begin in fermentation culture 15 hours, in nutrient solution, add Trisodium Citrate, and add 2 gram Trisodium Citrates in every liter of nutrient solution.
3. according to claim 1
S-adenosine-L-methionine(Met) and gsh coproduction fermentation process is characterized in that: the inoculum size of fermentation culture is 5～10%, 28～32 ℃ of leavening temperatures, and mixing speed 300～400 rpm, air flow 3.0～4.0 L/min, pH are 5.0.
4. according to claim 1
S-adenosine-L-methionine(Met) and gsh coproduction fermentation process is characterized in that: fermented incubation time is 24～30 hours.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878059A (en) * | 2015-03-31 | 2015-09-02 | 浙江大学宁波理工学院 | Method for preparing S-adenosylmethionine |
| CN106119318A (en) * | 2016-07-05 | 2016-11-16 | 苏州大学 | Method for improving co-production fermentation yield of S-adenosylmethionine and glutathione |
| CN106349311A (en) * | 2016-08-23 | 2017-01-25 | 北京金阳利康医药有限公司 | Method for extracting S-adenosylmethionine from yeast fermentation liquor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781625A (en) * | 2010-01-04 | 2010-07-21 | 苏州大学 | Ethionine resistance Candida utilis and application thereof |
| WO2011118807A1 (en) * | 2010-03-26 | 2011-09-29 | アサヒビール株式会社 | Method for culturing yeast |
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- 2012-08-13 CN CN2012102859006A patent/CN102776258A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781625A (en) * | 2010-01-04 | 2010-07-21 | 苏州大学 | Ethionine resistance Candida utilis and application thereof |
| WO2011118807A1 (en) * | 2010-03-26 | 2011-09-29 | アサヒビール株式会社 | Method for culturing yeast |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104878059A (en) * | 2015-03-31 | 2015-09-02 | 浙江大学宁波理工学院 | Method for preparing S-adenosylmethionine |
| CN104878059B (en) * | 2015-03-31 | 2018-06-26 | 浙江大学宁波理工学院 | A kind of method for preparing s-adenosylmethionine |
| CN106119318A (en) * | 2016-07-05 | 2016-11-16 | 苏州大学 | Method for improving co-production fermentation yield of S-adenosylmethionine and glutathione |
| CN106119318B (en) * | 2016-07-05 | 2019-05-24 | 苏州大学 | Method for improving co-production fermentation yield of S-adenosylmethionine and glutathione |
| CN106349311A (en) * | 2016-08-23 | 2017-01-25 | 北京金阳利康医药有限公司 | Method for extracting S-adenosylmethionine from yeast fermentation liquor |
| CN106349311B (en) * | 2016-08-23 | 2018-04-13 | 北京金阳利康医药有限公司 | The method that S Ademetionines are extracted from yeast fermentation broth |
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Application publication date: 20121114 |