CN1203185C

CN1203185C - Process for raising glutathion yield by fermentation of tornla yeast

Info

Publication number: CN1203185C
Application number: CN 03113418
Authority: CN
Inventors: 陈坚; 李寅; 卫功元; 堵国成; 伦世仪
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2003-05-09
Filing date: 2003-05-09
Publication date: 2005-05-25
Anticipated expiration: 2023-05-09
Also published as: CN1450168A

Abstract

The present invention discloses a method for enhancing glutathion yield by fermentation of tornla yeast, which relates to the method for preparing glutathione by a fermentation method. The method adopts tornla yeast as a fermentation bacterial strain; after slant culture and seed culture, the fermentation bacterial strain is inoculated in a fermentation medium for shaking culture or fermenting tank culture. L-cysteine is added to the fermentation medium to adding the supply of the L-cysteine in fermentation liquor, so the synthesized speed and the yield of the glutathione of the product are enhanced. The method has the advantages that under the condition that other culture conditions are the same, the yield of the glutathione and the intracellular content can be enhanced by one time after the L-cysteine is added to the fermentation liquor. Thus, the method is favorable for realizing the industrial production for preparing glutathione by the fermentation method.

Description

A kind of method that improves Candida utilis glutathion production by fermentation output

Technical field

A kind of method that improves Candida utilis glutathion production by fermentation output relates to the method that fermentation method prepares gsh.

Technical background

Gsh (GSH) is a kind of tripeptide compound of being made up of L-glutamic acid, halfcystine and glycine.GSH mainly is present in yeast, animal livers, muscle and the blood at occurring in nature, and some plants such as vegetables, beans, cereal, potato class and mushroom class also contain GSH.As a kind of peptide material that contains sulfydryl, GSH is a kind of important biochemical drug, and scientists is being studied it in the intravital physiological action of various biologies always, and the relation of the content in the various histocytes of human body and various diseases and tissue injury.In addition, GSH also gets more and more people's extensive concerning in fundamental research fields such as biological chemistry always.

Gsh is as a kind of important physical active substance, and with growing, and demand constantly increases people to its interest on clinical medicine, foodstuff additive and nutrition in sport.Clinically in radioprotective, tumour, cancer, oxygen intoxication, aging with coordinate in the endocrine treatment effect obviously and have no side effect.In addition, GSH also has the effect of sexual function improving and Ginseng Extract, finds also that in recent years GSH has the effect that suppresses hiv virus.At food processing field, as a kind of antioxidant, gsh has functions such as the food value of enhancing and nutrient fortified food local flavor.In the sports field, GSH can improve the content of oxyphorase in the body, and the protection red corpuscle exempts from oxidisability destruction.Therefore, no matter be discussion in exercise induced anemia mechanism, the discussion of the prevention of still overtraining, sports fatigue mechanism, and the additional aspect GSH of sports nutrition people's attention extremely all.

Since the patent the earliest of the GSH preparation of delivering in 1938, Chinese scholars has been carried out a large amount of research around the production of GSH.In short, the production method of gsh mainly contains solvent extration, chemical synthesis, enzyme transforming process and fermentation method at present.Extraction process mainly is the separation and Extraction of carrying out GSH by extraction and sedimentary method from the animal vegetable tissue that contains GSH, because raw material is difficult for obtaining and the content of GSH is extremely low, so the actual application value of this method is little.Chemical synthesis is produced GSH, is about to L-L-glutamic acid, L-halfcystine and glycine and is condensed into GSH.This method early is used for GSH production, but complicated operating process, consuming time and mixture that GSH that obtain is levo form and dextrorotatory form, separates very difficultly, cause product purity not high, and biological value is difficult to be consistent.The enzyme process of GSH is synthetic to be to utilize the GSH synthetase series, forms amino acid catalytic with three kinds and form GSH in the presence of ATP.This method at first needs to obtain GSH synthetic related enzyme systems, secondly needs to add expensive precursor amino acid and ATP, also is in the laboratory study stage at present.Relatively comprehensive, fermentative Production GSH has competitive power most, and owing to microorganism is cultivated easily, so this method will have great application potential.

Fermentation method is exactly to adopt cheap saccharide raw material, utilizes the pathways metabolism of bacterium or yeast substance in vivo to carry out the biosynthetic method of GSH.Generally speaking, the content of GSH not high (＜1%) in the microorganism cells, the GSH of too high amount destroy in the body equilibrated redox environment already easily.Biosynthetic GSH is an intracellular product, needs in the actual production process to extract, and lower content can improve production cost undoubtedly greatly.Therefore, the most critical issue of fermentative Production GSH is to improve the intravital GSH content of microorganism, normally realize, adopt conventional selection by mutation breeding high-yield bacterial strain or utilize gene engineering to make up genetic engineering bacterium with GSH composite reactive by strain improvement.In addition, fermenting process being controlled and optimized also is the effective way that improves GSH output.

Japan begins fermentative Production GSH is studied very early, wherein utilizes yeast-leavened method to be successfully used to the suitability for industrialized production of GSH by Japan.And at home, since late 1980s, units such as Zhongshan University and Zhejiang University successively carried out the research of this respect, but the result is not really desirable.From the mid-90, Southern Yangtze University has carried out systematic research to the biosynthesizing of GSH, and wherein the research of relevant " fermentative Production gsh " has obtained gratifying achievement.

L-halfcystine (L-cysteine) is one of precursor amino acid of synthesizing glutathion, in utilizing yeast fermentation method synthesizing glutathion process, and the under-supply resultant velocity and the output that has had a strong impact on gsh of L-halfcystine in the born of the same parents.Therefore it will be feasible adopting the method that interpolation L-halfcystine is produced gsh output with the raising yeast fermentation in fermented liquid, but not see the relevant report of adding L-halfcystine and obvious raising gsh output in utilizing the Candida utilis synthesizing glutathion so far.

Summary of the invention

The purpose of this invention is to provide a kind of method that improves Candida utilis glutathion production by fermentation output.

Technical solution of the present invention is to adopt to add the L-halfcystine in fermented liquid, and the interpolation time is 0-20 hour, and interpolation concentration is 6-10mmol/L.

(1) bacterial strain Candida utilis (Candida utilis) WSH 02-08, biotechnology institute of Southern Yangtze University Environmental Biotechnology research department preservation and providing, at " biotechnology journal " 2003,19 (3), 358-363 is open for this bacterial strain.

(2) substratum

Slant medium: 10% malt extract medium;

Seed culture medium (g/L): glucose 20.0, peptone 20.0, yeast extract paste 10.0, pH6.0; Fermention medium (g/L): glucose 30.0, ammonium sulfate 8.0, potassium primary phosphate 3.0, sal epsom 0.25, pH5.5.

(3) shake-flask culture

The inclined-plane seed activation inserted seed culture medium after 4 hours, cultivate after 20 hours again and to insert the 500mL that the 50mL fermention medium is housed by 10% inoculum size and shake and carry out fermentation culture in the bottle, fermentation time 24 hours, leavening temperature 28-32 ℃, the speed governing of HYG-II type rotary type constant temperature is shaken a bottle cabinet, rotating speed 200r/min.Add the L-halfcystine in fermented liquid, the interpolation time is 0-20 hour, and interpolation concentration is 6-10mmol/L.

(4) fermentor cultivation

The inclined-plane seed activation inserted seed culture medium after 4 hours, cultivate automatic fermenter KFT-7L (the KoBio Techco. that the 4L fermention medium is housed by 10% inoculum size access after 20 hours again, Ltd, Korea S) in, fermentation time 24 hours, leavening temperature 28-32 ℃, mixing speed 300r/min, air flow 5.0L/min, pH5.5.Add the L-halfcystine in fermented liquid, the interpolation time is 0-20 hour, and interpolation concentration is 6-10mmol/L.

The best interpolation time is 16 hours, and best interpolation concentration is 8mmol/L.

(5) extraction of born of the same parents' glutathion inside

Behind the fresh yeast usefulness distilled water wash that fermentation culture obtains 3 times, handled 2 hours down for 30 ℃ in 40% ethanol, the centrifuging and taking supernatant liquor carries out gsh as sample and measures.

(6) mensuration of dry cell weight

Centrifugal back distilled water wash 3 times of a certain amount of fermented liquid, the new fresh cell that obtains dries by the fire 48 hours down to constant weight at 60 ℃.

(7) definition of gsh born of the same parents intensive amount

(8) mensuration of glucose quality concentration: 3,5-dinitrosalicylic acid method.

(9) mensuration of gsh

Adopt DTNB[5,5 '-two sulphur two (2-nitrobenzoic acid)]-the glutathione reductase circulation method.

The beneficial effect of this patent: GSH is a kind of important medicine, and purposes clinically is extremely wide.The antioxidant property of GSH makes it gain great popularity in food-processing industry as foodstuff additive again.The art of this patent changes the situation of China's gsh bulk drug dependence on import for the production domesticization that realizes GSH, lays the foundation for GSH obtains large-scale application as foodstuff additive simultaneously.No matter from theory still from angle of practice, realize the industrialization of fermentative Production GSH, can not only fill up the blank of China in this field, China's medicine industry, clinical medicine and foodstuffs industry are all had important social benefit and economic benefit.

By shake in bottle and the fermentor tank the result more as can be seen, interpolation L-halfcystine all is very effective to the raising of Candida utilis glutathion production by fermentation output.Under the identical situation of other culture condition, the interpolation of L-halfcystine is the highest can to double gsh output and gsh born of the same parents intensive amount.

Embodiment

Embodiment 1 to specifications described in fermentation condition prepare gsh, shake the experimental result of not adding the L-halfcystine in the bottle

Dry cell weight: 9-10g/L; GSH output: 180-200mg/L; GSH content: 1.8-2.1%.

The fermentation time that produces optimum is 24 hours.

Embodiment 2 to specifications described in fermentation condition prepare gsh, shake in the bottle experimental result of adding the L-halfcystine

The interpolation time: 0 one 20 hours; Add concentration: 6-10mmol/L;

Dry cell weight: 9-10g/L; GSH output: 300-410mg/L; GSH content: 2.0-4.2%.

The wherein best interpolation time is 16 hours; Best interpolation concentration is 8mmol/L.

Embodiment 3 to specifications described in fermentation condition prepare gsh, do not add the experimental result of L-halfcystine in the 7L fermentor tank

Dry cell weight: 14-16g/L; GSH output: 220-300 mg/L; GSH content: 2.0-2.2%.

Embodiment 4 to specifications described in fermentation condition prepare gsh, add the experimental result of L-halfcystine in the 7L fermentor tank

Interpolation time: 0-20 hour; Add concentration: 6-10mmol/L;

Dry cell weight: 14-16g/L; GSH output: 400-520mg/L; GSH content: 3.5-4.0%.

Claims

1. A method for preparing glutathione by fermentation is characterized in that it adopts Candida utilis (Candidautilis) WSH 02-08 as starting bacterial strain, after slant culture and seed culture, it is inoculated in fermentation medium and shakes the flask Cultivation or fermenter cultivation, adding L-cysteine to the fermentation broth, the addition time is 0-20 hours, and the addition concentration is 6-10mmol/L.

2. The method according to claim 1, characterized in that the fermentation conditions are:

Strain: Candida utilis WSH 02-08;

Incline medium: 10% wort medium;

Seed medium (g/L): glucose 20.0, peptone 20.0, yeast extract 10.0, pH 6.0;

Fermentation medium (g/L): glucose 30.0, ammonium sulfate 8.0, potassium dihydrogen phosphate 3.0, magnesium sulfate 0.25, pH 5.5;

Shake flask culture: after 4 hours of activation, the slant seeds were inserted into the seed medium, and after 20 hours of cultivation, they were inserted into the fermentation medium according to the inoculum size of 10%. -32°C, fermentation time 24 hours, shaker speed 200r/min, add L-cysteine to the fermentation broth, the addition time is 0-20 hours, and the concentration is 6-10mmol/L;

Or fermenter culture: After 4 hours of activation, the slant seeds are inserted into the seed medium, and after 20 hours of cultivation, they are inserted into the fermentation medium according to the inoculation amount of 10%. The liquid volume is 4L/7L automatic fermenter, the fermentation temperature is 28 -32°C, fermentation time 24 hours, pH 5.5, stirring speed 300r/min, air flow 5.0L/min, add L-cysteine to the fermentation broth, the addition time is 0-20 hours, and the concentration is 6 -10mmol/L.

3. The preparation method according to claim 1, characterized in that L-cysteine is added to the fermentation broth, the optimum addition time is 16 hours, and the optimum addition concentration is 8mmol/L.