CN104962485B - A kind of preparation method of homoglutathion content saccharomyces cerevisiae - Google Patents
A kind of preparation method of homoglutathion content saccharomyces cerevisiae Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of homoglutathion content saccharomyces cerevisiae, this method is included saccharomyces cerevisiae（Saccharomyces cerevisiae）CICC1532 activated culture, seed culture, fermentation, separation, washing and drying steps, by optimum culture condition and feed-batch process and related process means, a kind of product of homoglutathion content saccharomyces cerevisiae can be obtained.The method of the present invention can make the glutathione intracellular content of the saccharomyces cerevisiae improve more than 5 times, so as to solve the problems, such as that microorganism glutathione dry ferment Glutathione peptide content is low.
Description
Technical field
It is more particularly to a kind of to utilize agricultural and sideline money the present invention relates to a kind of preparation method of homoglutathion content saccharomyces cerevisiae
The method that source prepares homoglutathion content saccharomyces cerevisiae.
Technical background
Glutathione（GSH）With removing free radical, removing toxic substances, suppress aging, prevention diabetes and cancer, suppression AIDS
Poison, improve the important physiological functions such as body immunity；Find that glutathione is improving the immunocompetence of animal recently, reduce antibiosis
There is important left and right in terms of the dosage of element.Therefore, the saccharomyces cerevisiae of homoglutathion content medical science, food, cosmetics and
The fields such as animal protection have extensive market prospects.
The production method of glutathione mainly has extraction, chemical synthesis, enzyme process and fermentation method.Wherein fermentation method is most
Has the method for potentiality.But there is the problem of the following aspects in the fermenting and producing of current state glutathion inside：1st, glutathione is produced
Microorganism glutathione synthesis low yield；2nd, the metabolic regulation imperfection during glutathione synthesis；3rd, glutathione exists
Easily it is oxidized in the aqueous solution, causes to lose greatly in glutathione extraction process.Due to the restriction of these factors, make gluathione
The production of peptide is greatly affected.
Glutathione is easily oxidized in aqueous, therefore the glutathione of microbial cell synthesis is generally present in carefully
In born of the same parents.Yeast is the most potential microorganism of glutathione synthesis, and yeast is rich in protein, has in food and medicine industry
Have been widely used, the raw material that the high yeast of production glutathione content can be industrial as food and medicine, while can also
Abundant functional components glutathione is provided, there is very important effect to improving function of human body.
There are reports for the fermenting and producing of glutathione, such as：Patent CN1203185C, with candida utili WSH02-
08 is bacterial strain, during the fermentation disposable addition Cys, in 7L fermentation cylinder for fermentation, dry cell weight 14-16g/L,
Stem cell glutathione content 3.5-4.0%, its biomass and glutathione content are relatively low.Patent CN101024818A is to make
Brewer yeast YA03083 is bacterial strain, uses fed-batch fermentation, though biomass can reach 60g/L, the content of glutathione only has
15mg/g.The patent CN102653722A that the applicant once declared discloses saccharomyces cerevisiae（Saccharomyces cerevisiae）CICC1532 fermenting and producings are rich in the yeast of glutathione, and it is in shake flask fermentation, addition glutamic acid, half Guang
Propylhomoserin, glycine, ATP, fermentation period are 56 hours, dry cell weight：12.5g/L, stem cell Glutathione peptide content：40mg/
g；It is disposable to add precusor amino acids and ATP using flow feeding batch fermentation in 30L fermentation tanks, ferment 47 hours, carefully
Born of the same parents' dry weight：46g/L, stem cell Glutathione peptide content：35-40mg/g；Zymotic fluid GSH-PX activity yield：1.814g/L.So
And because the technical parameter of zymotechnique is not optimized, therefore its glutathione yield is relatively low.
The content of the invention
The present inventor is on the basis of early-stage Study, and Optimizing Process Parameters, contain so as to provide a kind of homoglutathion again
Measure the preparation method of saccharomyces cerevisiae.This method can make the glutathione intracellular content of saccharomyces cerevisiae improve more than 5 times, so as to enter one
Step solves the problems, such as that microorganism glutathione dry ferment Glutathione peptide content is low.
In order to realize the purpose of the present invention, inventor studies technique and technical parameter by lot of experiments repeatedly, and finally
Obtain the method for being produced by below：
A kind of preparation method of homoglutathion content saccharomyces cerevisiae, this method are included saccharomyces cerevisiae
（Saccharomyces cerevisiae）The activated cultures of CICC1532, seed culture, fermentation, separation, washing and dry step
Suddenly, described fermentation step is shake flask fermentation, and glutamic acid, cysteine, glycine are not added before fermentation and in fermentation process
And ATP.
The method that homoglutathion content saccharomyces cerevisiae is prepared to above-mentioned shake flask fermentation makees further preferred, hair therein
Ferment step is shake flask fermentation, and culture medium composition is glucose 60g/L, dusty yeast 13g/L, ammonium sulfate 6g/L, potassium dihydrogen phosphate
2.5g/L, magnesium sulfate 0.3g/L, pH5.5-6.0；Technological condition for fermentation is：Shaking speed 180-200rmp/min, fermentation temperature
28-32 DEG C, fermentation time 32-48 hours.
A kind of preparation method of homoglutathion content saccharomyces cerevisiae, this method are included saccharomyces cerevisiae
（Saccharomyces cerevisiae）The activated cultures of CICC1532, seed culture, fermentation, separation, washing and dry step
Suddenly, described fermentation step is ferment tank, the strain access after seed culture is equipped with the fermentation tank of bottom water, using more
The mode of level speed change fed-batch cultivation, in 30L fermentation cylinder for fermentation 32-48h, 28-32 DEG C of fermentation temperature；
Stream plus carbon source are 350g/L glucose solutions；Ferment 0-4 hours, the flow acceleration of carbon source is 80mL/h~120mL/
H, dissolved oxygen amount are controlled in 2-4%；Ferment 4-20 hours, the flow acceleration of carbon source is 120mL/h~180mL/h, and dissolved oxygen amount control exists
10-20%；
Stream plus nitrogen, the formula of phosphorus source are urea 80g/L, ammonium sulfate 35g/L and potassium dihydrogen phosphate 60g/L；The 0-20 that ferments is small
When, nitrogen, the flow acceleration of phosphorus source are 60mL/h~90mL/h；
Ferment and reach 30-50g/L to 18-20 hours, biomass dry weight, start stream plus glutamic acid 8-12mmol/L, half Guang
Propylhomoserin 8-12mmol/L and glycine 5-7mol/L, flow acceleration is controlled, stream adds complete within 8-12 hours, starts to flow ammonification base
While sour, the flow acceleration of carbon source is controlled to continue 6-16 hours of fermenting for 50mL/h~90mL/h；
The content of alcohol is controlled 0.1%~0.3% in zymotic fluid in whole fermentation process（W/V）.
It is further preferred that the method work of homoglutathion content saccharomyces cerevisiae is prepared to above-mentioned ferment tank, it is therein
Bottom water contains：Water 9L, dusty yeast 270g, 22.5g calcium chloride, the water magnesium sulfates of 67.5g five, 0.072g zinc sulfate, 45g potassium chloride,
0.45g vitamin B1s, 0.45g vitamin B2s, 13.5g vitamin B6s, 0.45g nicotinic acid, 0.45g calcium pantothenates, natural pH.
The method that homoglutathion content saccharomyces cerevisiae is prepared to above-mentioned ferment tank remakes further preferably, wherein
Separating step using vacuum drum separate, vacuum 0.08-0.1Mpa, rotary drum rotating speed 50-80rpm per minute.
Compared with prior art, method of the present invention can prepare the saccharomyces cerevisiae of homoglutathion content, embody such as
Under：
（1）In shake flask fermentation, inventor is surprised to find that saccharomyces cerevisiae can be suppressed by adding amino acid and ATP
（Saccharomyces cerevisiae）CICC1532 glutathione conversion yield, therefore glutamic acid, half are not added in selection
Cystine, glycine and ATP, when fermenting 32-48 hours, yeast cells dry weight reaches 16-24g/L, paddy Guang in dry yeast cell
The content of sweet peptide reaches 80-100 mg/g；
（2）In 30L fermentation tanks, using non-uniform flow plus carbon source, nitrogen source and phosphorus source, add necessarily in different time internal speed-changing stream
Glutamic acid, cysteine, the glycine of proportioning, do not add ATP, and when fermenting 32-48 hours, yeast cells dry weight reaches 40-
60g/L, dry yeast cell Glutathione peptide content are up to 180-220mg/g.
Embodiment
Form is described in further detail again to the above of the present invention by the following examples, but should not manage this
The scope solved as the above-mentioned theme of the present invention is only limitted to following embodiment, and all technologies for being realized based on the above of the present invention are equal
Belong to the scope of the present invention.
Embodiment one:Fermented and cultured prepares homoglutathion saccharomyces cerevisiae in shaking flask
（1）Bacterial strain：
Saccharomyces cerevisiae（Saccharomyces cerevisiae）CICC1532, Hubei University Of Technology's bioengineering 209 are real
Test room preservation.
（2）Culture medium：
Slant medium described in per 1L contains：Glucose 20g, peptone 20g, yeast extract 5g, agar 20g, it is described tiltedly
Face medium pH 5.8-6.2；
Seed culture medium described in per 1L contains：Glucose 20g, peptone 20g, yeast extract 5g, the seed culture medium
pH5.8-6.2；
The horizontal top fermentation culture medium of shaking flask described in per 1L contains：Glucose 60g, dusty yeast 13g, ammonium sulfate 6g, di(2-ethylhexyl)phosphate
Hydrogen potassium 2.5g, magnesium sulfate 0.3g, pH5.5-6.0；
（3）Seed expands culture：
Access, which is fitted into the triangular flask of seed culture medium, after inclined-plane seed activation 12-16 hours cultivates, liquid amount 25-
30mL, 12-18 hour produce first order seed；
（4）Medicine bottle fermented and cultured：
It is by seed access 25mL/250mL culture medium compositions：Glucose 60g/L, dusty yeast 13g/L, ammonium sulfate 6g/L,
Potassium dihydrogen phosphate 2.5g/L, in magnesium sulfate 0.3g/L, pH5.5-6.0 shaking flask, at 28-32 DEG C, shaking speed 180-
200rmp/min, glutamic acid, cysteine, glycine, ATP are not added, ferment 32-48 hours.
（5）The measure of dry cell weight
With water washing is distilled 2 times after certain zymotic fluid centrifugation, obtained fresh cells are dried to constant weight at 105 DEG C.
（6）The measure of glutathione content
Using iodimetric titration.
（7）As a result：
Fermentation time：48 hours；Dry cell weight：20g/L；Dry yeast cell Glutathione peptide content：90mg/g.
Homoglutathion content saccharomyces cerevisiae is prepared using fed-batch cultivation culture on the 30L fermentation tanks of embodiment two
（1）Bacterial strain
Saccharomyces cerevisiae（Saccharomyces cerevisiae）CICC1532, Hubei University Of Technology's bioengineering 209 are real
Test room preservation.
（2）Culture medium
Slant medium described in per 1L contains：Glucose 20g, peptone 20g, yeast extract 5g, agar 20g, it is described tiltedly
Face medium pH 5.8-6.2；
Seed culture medium described in per 1L contains：Glucose 20g, peptone 20g, yeast extract 5g, the seed culture medium
pH5.8-6.2；
The fermentation tank level top fermentation midsole water contains：Water 9L, dusty yeast 270g, 22.5g calcium chloride, the water of 67.5g five
Magnesium sulfate, 0.072g zinc sulfate, 45g potassium chloride.Natural pH.Add 4ml defoamer.In addition：Add 0.45g vitamin B1s,
0.45g vitamin B2s, 13.5g vitamin B6s, 0.45g nicotinic acid, 0.45g calcium pantothenates, it is dissolved in together in 500ml water, 115 DEG C go out
Bacterium 20 minutes, it is added in the bottom water after sterilizing.
Stream plus carbon source：The volume of stream plus carbon source is 9.0L, concentration of glucose 350g/L, originally water-soluble liquid, and 108 DEG C go out
Bacterium 20 minutes.
Stream plus nitrogen source, phosphorus source：Stream plus nitrogen, the volume of phosphorus source are 3L, are formulated and are：Urea 80g/L, ammonium sulfate 35g/L, phosphoric acid
The g/L of potassium dihydrogen 60.
Stream plus precusor amino acids：Stream plus the volumes of precusor amino acids are 1.8L, are formulated and are：Glutamic acid 8-12mmol/L, half
Cystine 8-12mmol/L, glycine 5-7mol/L.
（3）Seed culture
After inclined-plane seed activation 14 hours, it is small that access is fitted into culture 12-18 in the 250mL triangular flasks of 25mL seed culture mediums
When produce first order seed；Press 10% again afterwards（V/V）Inoculum concentration first order seed is accessed into identical fermentation medium culture 12-18
Hour prepares secondary seed, 28-32 DEG C of cultivation temperature, constant temperature culture oscillator, rotating speed 200rpm；
（4）Fermented and cultured
Multistage fed-batch cultivation：Ready secondary seed is equipped with to the full-automatic hair of 9L bottoms water by 10% inoculum concentration access
In fermentation tank, 28-32 DEG C of fermentation temperature, fermentation period 32-48h.Earlier fermentation 0-4 hours, the flow acceleration of carbon source are：80mL/h
~120mL/h, dissolved oxygen amount are controlled in 2-4%, and ferment middle 4-20 hours, the flow acceleration of carbon source is：120mL/h~180mL/
H, dissolved oxygen amount are controlled in 10-20%.Ferment 0-20 hours, nitrogen, the flow acceleration of phosphorus source are 60mL/h~90mL/h.Ferment to 18-
20 hours, biomass dry weight reached 30-50g/L, started stream plus glutamic acid 8-12mmol/L, cysteine 8-12mmol/L, sweet
Propylhomoserin 5-7mol/L, flow acceleration is controlled, stream adds complete within 8-12 hours, while starting stream plus amino acid, controls carbon source
Flow acceleration for 50mL/h~90mL/h continue ferment 6-16 hours.In whole fermentation process in zymotic fluid alcohol content control
System is 0.1%~0.3%（W/V）.
（5）The measure of dry cell weight
With water washing is distilled 2 times after certain zymotic fluid centrifugation, obtained fresh cells are dried to constant weight at 105 DEG C.
（6）The measure of glutathione content
Using iodimetric titration.
（7）As a result：
Fermentation time：48 hours；Dry cell weight：50g/L；Intracellular glutathione content：180-220mg/g；In zymotic fluid
The yield of glutathione is：9-11g/L.
Embodiment three:Separation, washing and the drying of homoglutathion content saccharomyces cerevisiae
（1）The separation of homoglutathion content saccharomyces cerevisiae
Zymotic fluid is pumped into vacuum to separate for 0.08-0.1Mpa vacuum drums, rotary drum rotating speed 50-80rpm per minute, even
Continuous separation yeast cells, after rotary drum forms filter cake, filter liquor is pumped into rotary drum for the second time, is lost in recovery filter liquor thin
Born of the same parents；
（2）The washing of homoglutathion content saccharomyces cerevisiae
Wherein described washing be added in storage tank 2-4 times of separating obtained yeast cells bulk temperature for normal temperature from
Water Disseminating yeast cell, then using vacuum drum separation separation, such repeated washing 2-3 times under conditions of same as before.
（3）The drying of homoglutathion content saccharomyces cerevisiae
The yeast cells collected after washing is separated is scattered in the running water of normal temperature, cell concentration is reached 220-
260g/L, the yeast dispersion liquid of gained are dried using spray drying tower, and the EAT for controlling spray drying tower is 130-150 DEG C
It is dried.
（4）As a result
The yield 95-98% of yeast cells；
The moisture of dry yeast cell is 10-12%（W/W）；
The loss late of yeast cells GSH-PX activity is 0.4-0.6%.
Claims (3)
1. a kind of preparation method of homoglutathion content saccharomyces cerevisiae, this method is included saccharomyces cerevisiae（Saccharomyces cerevisiae）CICC1532 activated culture, seed culture, fermentation, separation, washing and drying steps, it is characterised in that：Institute
The fermentation step stated is shake flask fermentation, and glutamic acid, cysteine, glycine and ATP are not added before fermentation and in fermentation process；
Described fermentation step is shake flask fermentation, and culture medium composition is glucose 60g/L, dusty yeast 13g/L, ammonium sulfate 6g/L,
Potassium dihydrogen phosphate 2.5g/L, magnesium sulfate 0.3g/L, pH5.5-6.0；Technological condition for fermentation is：Shaking speed 180-200rmp/
Min, 28-32 DEG C of fermentation temperature, fermentation time 32-48 hours.
2. a kind of preparation method of homoglutathion content saccharomyces cerevisiae, this method is included saccharomyces cerevisiae（Saccharomyces cerevisiae）CICC1532 activated culture, seed culture, fermentation, separation, washing and drying steps, it is characterised in that：Institute
The fermentation step stated is ferment tank, by fermentation tank of the strain access equipped with bottom water after seed culture, is become using multistage
The mode of fast fed-batch cultivation, in 30L fermentation cylinder for fermentation 32-48h, 28-32 DEG C of fermentation temperature；
Stream plus carbon source are 350g/L glucose solutions；Ferment 0-4 hours, the flow acceleration of carbon source is 80mL/h~120mL/h, molten
Oxygen content control is in 2-4%；Ferment 4-20 hours, the flow acceleration of carbon source is 120mL/h~180mL/h, and dissolved oxygen amount is controlled in 10-
20%；
Stream plus nitrogen, the formula of phosphorus source are urea 80g/L, ammonium sulfate 35g/L and potassium dihydrogen phosphate 60g/L；Ferment 0-20 hours,
Nitrogen, the flow acceleration of phosphorus source are 60mL/h~90mL/h；
Ferment and reach 30-50g/L to 18-20 hours, biomass dry weight, start stream plus glutamic acid 8-12mmol/L, cysteine
8-12mmol/L and glycine 5-7mol/L, flow acceleration is controlled, stream adds complete within 8-12 hours, starts stream and adds amino acid
Meanwhile the flow acceleration of carbon source is controlled to continue 6-16 hours of fermenting for 50mL/h~90mL/h；
The content of alcohol is controlled 0.1%~0.3% in zymotic fluid in whole fermentation process（W/V）；
Described bottom water contains：Water 9L, dusty yeast 270g, 22.5g calcium chloride, the water magnesium sulfates of 67.5g five, 0.072g zinc sulfate,
45g potassium chloride, 0.45g vitamin B1s, 0.45g vitamin B2s, 13.5g vitamin B6s, 0.45g nicotinic acid, 0.45g calcium pantothenates, from
Right pH.
3. the preparation method of homoglutathion content saccharomyces cerevisiae according to claim 2, it is characterised in that：Described separation
Step is separated using vacuum drum, vacuum 0.08-0.1Mpa, rotary drum rotating speed 50-80rpm per minute.
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| CN105505801A (en) * | 2015-12-25 | 2016-04-20 | 湖北泱盛生物科技有限公司 | Saccharomyces cerevisiae for high yield of glutathione and application of saccharomyces cerevisiae |
| CN106579464B (en) * | 2016-11-03 | 2018-07-17 | 首都医科大学附属北京佑安医院 | A kind of alimentation composition and preparation method thereof for treating hepatic sclerosis |
| CN106722883A (en) * | 2016-12-21 | 2017-05-31 | 天津中天精科科技有限公司 | A kind of mushroom effervescent tablet and its preparation |
| CN106858040A (en) * | 2017-03-15 | 2017-06-20 | 湖北绿科乐华生物科技有限公司 | High content reduced glutathione Cultures of S. cerevisiae and its production method |
| AU2019246299B2 (en) * | 2018-03-26 | 2022-03-24 | Danstar Ferment Ag | Method for increasing the content of thiol precursors in plants |
| CN114350731B (en) * | 2021-12-15 | 2024-06-18 | 华南理工大学 | Preparation method of glutathione |
| CN114276942B (en) * | 2021-12-30 | 2024-05-28 | 安琪酵母股份有限公司 | Glutathione yeast, preparation method and application of product |
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Effective date of registration: 20201021 Address after: 678025 in Lujiang town in Longyang District of Baoshan city of Yunnan Province, xiaopingtian Patentee after: BAOSHAN REXXAM YEAST Co.,Ltd. Address before: 430068, No. 1, Li Jia, Nanhu Lake, Wuchang, Hubei, Wuhan Patentee before: HUBEI University OF TECHNOLOGY |