CN104962485A - Preparation method of Saccharomyces cerevisiae with high content of glutathione - Google Patents
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Abstract
The invention relates to a preparation method of Saccharomyces cerevisiae with high content of glutathione. The method comprises the steps of activated culture, seed culture, fermentation, separation, washing and drying of Saccharomyces cerevisiae CICC1532. A Saccharomyces cerevisiae product with high content of glutathione can be obtained by optimizing culture conditions and feeding strategies and adopting relevant process means. The method provided by the invention can enable the intracellular content of glutathione in the Saccharomyces cerevisiae to be improved by more than five times, and thereby solves the problem that the content of glutathione in microbial glutathione dry yeast is low.
Description
Technical field
The present invention relates to a kind of preparation method of homoglutathion content yeast saccharomyces cerevisiae, particularly a kind of method utilizing agricultural and sideline resource to prepare homoglutathion content yeast saccharomyces cerevisiae.
Technical background
Gsh (GSH) has the important physiological function such as scavenging free radicals, removing toxic substances, suppression aging, prevent diabetes and cancer, inhibiting HIV, raising body immunity; Nearest discovery gsh, improving the immunological competence of animal, reduces antibiotic consumption aspect and has important left and right.Therefore, the yeast saccharomyces cerevisiae of homoglutathion content has market outlook widely in fields such as medical science, food, makeup and protection of animal.
The production method of gsh mainly contains extraction process, chemical synthesis, enzyme process and fermentation method.Wherein fermentation method is the method for most potentiality.But there is the problem of the following aspects in the fermentative production of state's glutathion inside at present: the microorganism glutathione synthesis productive rate 1, producing gsh is low; 2, the metabolic regulation imperfection in glutathione synthesis process; 3, gsh is very easily oxidized in aqueous, causes loss in gsh leaching process large.Due to the restriction of these factors, the production of gsh is greatly affected.
Gsh is very easily oxidized in aqueous, and the gsh of therefore microorganism cells synthesis is generally present in cell.Yeast is the microorganism of glutathione synthesis most potentiality, and yeast rich in proteins, in food and medicine industry, tool has been widely used, the yeast producing glutathione content high can as the raw material of food and medicine industry, also can providing abundant functional ingredient gsh simultaneously, to improving function of human body, there is very important effect.
There are reports for the fermentative production of gsh, as: patent CN1203185C, with Candida utilis WSH02-08 for bacterial strain, disposable interpolation Cys during the fermentation, 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 all lower.Patent CN101024818A is with yeast saccharomyces cerevisiae YA03083 for bacterial strain, and adopt fed-batch fermentation, though biomass can reach 60g/L, the content of gsh only has 15mg/g.The patent CN102653722A that the applicant once declared disclose yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) CICC1532 fermentative production is rich in the yeast of gsh, it is in shake flask fermentation, and add L-glutamic acid, halfcystine, glycine, ATP, fermentation period is 56 hours, dry cell weight: 12.5g/L, stem cell Glutathione peptide content: 40mg/g; In 30L fermentor tank, adopt flow feeding batch fermentation, disposable interpolation precusor amino acids and ATP, ferment 47 hours, dry cell weight: 46g/L, stem cell Glutathione peptide content: 35-40mg/g; Fermented liquid GSH-PX activity output: 1.814g/L.But because the technical parameter of zymotechnique is not optimized, therefore its gsh productive rate is lower.
Summary of the invention
The present inventor on the basis of early-stage Study, Optimizing Process Parameters again, thus the preparation method that a kind of homoglutathion content yeast saccharomyces cerevisiae is provided.The method can make the gsh born of the same parents intensive amount of yeast saccharomyces cerevisiae improve more than 5 times, thus solves the low problem of microorganism gsh dry yeast Glutathione peptide content further.
In order to realize object of the present invention, contriver studies technique and technical parameter repeatedly by lot of experiments, and finally obtains following production method:
A preparation method for homoglutathion content yeast saccharomyces cerevisiae, the method comprise by yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) the activated cultivation of CICC1532, seed culture, fermentation, separation, washing and drying step, described fermentation step is shake flask fermentation, does not all add L-glutamic acid, halfcystine, glycine and ATP before fermentation and in fermenting process.
The method being prepared by above-mentioned shake flask fermentation to homoglutathion content yeast saccharomyces cerevisiae is done further preferably, and fermentation step is wherein shake flask fermentation, and substratum consists of glucose 60g/L, yeast powder 13g/L, ammonium sulfate 6g/L, potassium primary phosphate 2.5g/L, magnesium sulfate 0.3g/L, pH5.5-6.0; Technological condition for fermentation is: shaking speed 180-200rmp/min, leavening temperature 28-32 DEG C, fermentation time 32-48 hour.
A preparation method for homoglutathion content yeast saccharomyces cerevisiae, the method comprise by yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) the activated cultivation of CICC1532, seed culture, fermentation, separation, washing and drying step, described fermentation step is ferment tank, bacterial classification access after seed culture is equipped with in the fermentor tank of end water, adopt the mode of multi-speed change fed-batch cultivation, at 30L fermentation cylinder for fermentation 32-48h, leavening temperature 28-32 DEG C;
It is 350g/L glucose solution that stream adds carbon source; Fermentation 0-4 hour, the flow acceleration of carbon source is 80mL/h ~ 120mL/h, and dissolved oxygen amount controls at 2-4%; Fermentation 4-20 hour, the flow acceleration of carbon source is 120mL/h ~ 180mL/h, and dissolved oxygen amount controls at 10-20%;
Stream adds nitrogen, the formula in phosphorus source is urea 80g/L, ammonium sulfate 35g/L and potassium primary phosphate 60g/L; Fermentation 0-20 hour, the flow acceleration in nitrogen, phosphorus source is 60mL/h ~ 90mL/h;
Fermentation is to 18-20 hour, biomass dry weight reaches 30-50g/L, start stream and add L-glutamic acid 8-12mmol/L, halfcystine 8-12mmol/L and glycine 5-7mol/L, control flow check acceleration, in 8-12 hour, stream adds complete, start stream add amino acid whose while, the flow acceleration controlling carbon source is that 50mL/h ~ 90mL/h continues fermentation 6-16 hour;
In whole fermenting process, in fermented liquid, the content of alcohol controls at 0.1% ~ 0.3%(W/V).
The method being prepared by above-mentioned ferment tank to homoglutathion content yeast saccharomyces cerevisiae is done further preferably, and end water wherein contains: water 9L, yeast powder 270g, 22.5g calcium chloride, 67.5g five water magnesium sulfate, 0.072g zinc sulfate, 45g Repone K, 0.45g VITMAIN B1,0.45g Lin Suanna Vitamin B2 Sodium Phosphate, 13.5g vitamin B6,0.45g nicotinic acid, 0.45g calcium pantothenate, natural pH.
The method being prepared by above-mentioned ferment tank to homoglutathion content yeast saccharomyces cerevisiae remakes further preferably, and separating step wherein adopts vacuum drum to be separated, vacuum tightness 0.08-0.1Mpa, rotary drum rotating speed per minute 50-80rpm.
Compared with prior art, the method that the present invention relates to can prepare the yeast saccharomyces cerevisiae of homoglutathion content, embodies as follows:
(1) in shake flask fermentation, contriver be surprised to find that add amino acid and ATP can suppress yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) the gsh conversion yield of CICC1532, therefore select not add L-glutamic acid, halfcystine, glycine and ATP, reach 16-24g/L in fermentation 32-48 little yeast cell constantly dry weight, the content of dry yeast cell GSH-PX activity reaches 80-100 mg/g;
(2) in 30L fermentor tank, variable flow is adopted to add carbon source, nitrogenous source and phosphorus source, L-glutamic acid, halfcystine, the glycine of certain proportioning is added at different time internal speed-changing stream, do not add ATP, reach 40-60g/L in fermentation 32-48 little yeast cell constantly dry weight, dry yeast cell Glutathione peptide content is up to 180-220mg/g.
Embodiment
Form is described in further detail foregoing of the present invention again by the following examples, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment, all technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment one: in shaking flask, fermentation culture prepares homoglutathion yeast saccharomyces cerevisiae
(1) bacterial strain:
Yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) CICC1532, Hubei University Of Technology's biotechnology 209 Laboratories Accession.
(2) substratum:
Described in every 1L, slant medium contains: glucose 20g, peptone 20g, yeast extract paste 5g, agar 20g, described slant medium pH5.8-6.2;
Described in every 1L, seed culture medium contains: glucose 20g, peptone 20g, yeast extract paste 5g, described seed culture medium pH5.8-6.2;
The horizontal top fermentation substratum of shaking flask described in every 1L contains: glucose 60g, yeast powder 13g, ammonium sulfate 6g, potassium primary phosphate 2.5g, magnesium sulfate 0.3g, pH5.5-6.0;
(3) seed enlarged culturing:
Access in the triangular flask loading seed culture medium after inclined-plane seed activation 12-16 hour and cultivate, namely liquid amount 25-30mL, 12-18 hour obtain first order seed;
(4) medicine bottle fermentation culture:
Seed is accessed 25mL/250mL substratum to consist of: glucose 60g/L, yeast powder 13g/L, ammonium sulfate 6g/L, potassium primary phosphate 2.5g/L, in the shaking flask of magnesium sulfate 0.3g/L, pH5.5-6.0, at 28-32 DEG C, shaking speed is 180-200rmp/min, does not add L-glutamic acid, halfcystine, glycine, ATP, fermentation 32-48 hour.
(5) mensuration of dry cell weight
The centrifugal rear distilled water wash of certain fermented liquid 2 times, the fresh cells obtained is dried to constant weight at 105 DEG C.
(6) mensuration of glutathione content
Adopt iodimetry,iodometry.
(7) result:
Fermentation time: 48 hours; Dry cell weight: 20g/L; Dry yeast cell Glutathione peptide content: 90mg/g.
Embodiment two 30L fermentor tank adopts fed-batch cultivation cultivate and prepare homoglutathion content yeast saccharomyces cerevisiae
(1) bacterial strain
Yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) CICC1532, Hubei University Of Technology's biotechnology 209 Laboratories Accession.
(2) substratum
Described in every 1L, slant medium contains: glucose 20g, peptone 20g, yeast extract paste 5g, agar 20g, described slant medium pH5.8-6.2;
Described in every 1L, seed culture medium contains: glucose 20g, peptone 20g, yeast extract paste 5g, described seed culture medium pH5.8-6.2;
In described fermentation tank level top fermentation, end water contains: water 9L, yeast powder 270g, 22.5g calcium chloride, 67.5g five water magnesium sulfate, 0.072g zinc sulfate, 45g Repone K.Nature pH.Add the defoamer of 4ml.In addition: add 0.45g VITMAIN B1,0.45g Lin Suanna Vitamin B2 Sodium Phosphate, 13.5g vitamin B6,0.45g nicotinic acid, 0.45g calcium pantothenate, is dissolved in 500ml water together, and 115 DEG C of sterilizings 20 minutes, join in the end water after sterilizing.
Stream adds carbon source: the volume that stream adds carbon source is 9.0L, glucose concn 350g/L, tap water lysate, 108 DEG C of sterilizings 20 minutes.
Stream adds nitrogenous source, phosphorus source: stream adds nitrogen, the volume in phosphorus source is 3L, and filling a prescription is: urea 80g/L, ammonium sulfate 35g/L, potassium primary phosphate 60 g/L.
Stream adds precusor amino acids: the volume that stream adds precusor amino acids is 1.8L, and filling a prescription is: L-glutamic acid 8-12mmol/L, halfcystine 8-12mmol/L, glycine 5-7mol/L.
(3) seed culture
Inclined-plane seed activation, after 14 hours, is cultivated in the 250mL triangular flask of access loading 25mL seed culture medium and is namely obtained first order seed in 12-18 hour; Afterwards again by 10%(V/V) inoculum size first order seed is accessed identical fermention medium cultivate within 12-18 hour, prepare secondary seed, culture temperature 28-32 DEG C, constant temperature culture oscillator, rotating speed 200rpm;
(4) fermentation culture
Multistage fed-batch cultivation: by ready secondary seed by 10% inoculum size access be equipped with in the automatic fermenter of water at the bottom of 9L, leavening temperature 28-32 DEG C, fermentation period 32-48h.Earlier fermentation 0-4 hour, the flow acceleration of carbon source is: 80mL/h ~ 120mL/h, and dissolved oxygen amount controls at 2-4%, ferment middle 4-20 hour, and the flow acceleration of carbon source is: 120mL/h ~ 180mL/h, dissolved oxygen amount controls at 10-20%.Fermentation 0-20 hour, the flow acceleration in nitrogen, phosphorus source is 60mL/h ~ 90mL/h.Fermentation is to 18-20 hour, biomass dry weight reaches 30-50g/L, start stream and add L-glutamic acid 8-12mmol/L, halfcystine 8-12mmol/L, glycine 5-7mol/L, control flow check acceleration, in 8-12 hour, stream adds complete, start stream add amino acid whose while, the flow acceleration controlling carbon source is that 50mL/h ~ 90mL/h continues fermentation 6-16 hour.In whole fermenting process, in fermented liquid, the content of alcohol controls at 0.1% ~ 0.3%(W/V).
(5) mensuration of dry cell weight
The centrifugal rear distilled water wash of certain fermented liquid 2 times, the fresh cells obtained is dried to constant weight at 105 DEG C.
(6) mensuration of glutathione content
Adopt iodimetry,iodometry.
(7) result:
Fermentation time: 48 hours; Dry cell weight: 50g/L; Born of the same parents' glutathion inside content: 180-220mg/g; The output of fermented liquid GSH-PX activity is: 9-11g/L.
Embodiment three: the separation of homoglutathion content yeast saccharomyces cerevisiae, washing and drying
(1) separation of homoglutathion content yeast saccharomyces cerevisiae
Fermented liquid being pumped into vacuum tightness is that 0.08-0.1Mpa vacuum drum is separated, and rotary drum rotating speed per minute 50-80rpm, is continuously separated yeast cell, is formed after filter cake until rotary drum, filtrate second time is pumped into rotary drum, reclaims the cell run off in filtrate;
(2) washing of homoglutathion content yeast saccharomyces cerevisiae
Wherein said washing is in storage tank, add the tap water Disseminating yeast cell that the doubly separating obtained yeast cell bulk temperature of 2-4 is normal temperature, then under aforementioned identical condition, adopt vacuum drum to be separated, repeated washing like this 2-3 time.
(3) drying of homoglutathion content yeast saccharomyces cerevisiae
The yeast cell collected after washing being separated is scattered in the tap water of normal temperature, makes cell concn reach 220-260g/L, and the yeast dispersion liquid of gained adopts spray-drying tower dry, and the inlet temperature controlling spray-drying tower is 130-150 DEG C and carries out drying.
(4) result
The yield 95-98% of yeast cell;
The moisture of dry yeast cell is 10-12%(W/W);
The rate of loss of yeast cell GSH-PX activity is 0.4-0.6%.
Claims (5)
1. a preparation method for homoglutathion content yeast saccharomyces cerevisiae, the method comprise by yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) the activated cultivation of CICC1532, seed culture, fermentation, separation, washing and drying step, it is characterized in that: described fermentation step is shake flask fermentation, before fermentation and in fermenting process, all do not add L-glutamic acid, halfcystine, glycine and ATP.
2. the preparation method of homoglutathion content yeast saccharomyces cerevisiae according to claim 1, is characterized in that: described fermentation step is shake flask fermentation, and substratum consists of glucose 60g/L, yeast powder 13g/L, ammonium sulfate 6g/L, potassium primary phosphate 2.5g/L, magnesium sulfate 0.3g/L, pH5.5-6.0; Technological condition for fermentation is: shaking speed 180-200rmp/min, leavening temperature 28-32 DEG C, fermentation time 32-48 hour.
3. a preparation method for homoglutathion content yeast saccharomyces cerevisiae, the method comprise by yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) the activated cultivation of CICC1532, seed culture, fermentation, separation, washing and drying step, it is characterized in that: described fermentation step is ferment tank, bacterial classification access after seed culture is equipped with in the fermentor tank of end water, adopt the mode of multi-speed change fed-batch cultivation, at 30L fermentation cylinder for fermentation 32-48h, leavening temperature 28-32 DEG C;
It is 350g/L glucose solution that stream adds carbon source; Fermentation 0-4 hour, the flow acceleration of carbon source is 80mL/h ~ 120mL/h, and dissolved oxygen amount controls at 2-4%; Fermentation 4-20 hour, the flow acceleration of carbon source is 120mL/h ~ 180mL/h, and dissolved oxygen amount controls at 10-20%;
Stream adds nitrogen, the formula in phosphorus source is urea 80g/L, ammonium sulfate 35g/L and potassium primary phosphate 60g/L; Fermentation 0-20 hour, the flow acceleration in nitrogen, phosphorus source is 60mL/h ~ 90mL/h;
Fermentation is to 18-20 hour, biomass dry weight reaches 30-50g/L, start stream and add L-glutamic acid 8-12mmol/L, halfcystine 8-12mmol/L and glycine 5-7mol/L, control flow check acceleration, in 8-12 hour, stream adds complete, start stream add amino acid whose while, the flow acceleration controlling carbon source is that 50mL/h ~ 90mL/h continues fermentation 6-16 hour;
In whole fermenting process, in fermented liquid, the content of alcohol controls at 0.1% ~ 0.3%(W/V).
4. the preparation method of homoglutathion content yeast saccharomyces cerevisiae according to claim 3, is characterized in that: described end water contains: water 9L, yeast powder 270g, 22.5g calcium chloride, 67.5g five water magnesium sulfate, 0.072g zinc sulfate, 45g Repone K, 0.45g VITMAIN B1,0.45g Lin Suanna Vitamin B2 Sodium Phosphate, 13.5g vitamin B6,0.45g nicotinic acid, 0.45g calcium pantothenate, natural pH.
5. the preparation method of homoglutathion content yeast saccharomyces cerevisiae according to claim 3, is characterized in that: described separating step adopts vacuum drum to be separated, vacuum tightness 0.08-0.1Mpa, rotary drum rotating speed per minute 50-80rpm.
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| CN112118741A (en) * | 2018-03-26 | 2020-12-22 | 丹斯塔发酵股份公司 | Method for increasing the content of thiol precursors in plants |
| CN112118741B (en) * | 2018-03-26 | 2022-07-19 | 丹斯塔发酵股份公司 | Method for increasing the content of thiol precursors in plants |
| CN114350731A (en) * | 2021-12-15 | 2022-04-15 | 华南理工大学 | Preparation method of glutathione |
| CN114276942A (en) * | 2021-12-30 | 2022-04-05 | 安琪酵母股份有限公司 | Glutathione yeast, preparation method and application of glutathione yeast product |
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