CN109401986A - A method of improving yeast oxidation resistance - Google Patents
A method of improving yeast oxidation resistance Download PDFInfo
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- CN109401986A CN109401986A CN201811231503.4A CN201811231503A CN109401986A CN 109401986 A CN109401986 A CN 109401986A CN 201811231503 A CN201811231503 A CN 201811231503A CN 109401986 A CN109401986 A CN 109401986A
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Abstract
The invention discloses a kind of methods for improving yeast oxidation resistance, belong to bioengineering field.The H of present invention debita spissitudo2O2Stimulate yeast cells, with the bacterial strain that the 96 orifice plate minisize liquid cultural methods screening speed of growth is fast, activity is high, obtained bacterial strain ROS accumulating level intracellular compared with starting strain reduces 30% or so, and in addition to one plant of fruit wine yeast, ATP content ascensional range intracellular is more than 50%.Yeast ROS cumulant intracellular can be effectively reduced in the method for the present invention, increases ATP content intracellular, to achieve the purpose that improve yeast strain oxidation resistance.
Description
Technical field
The present invention relates to a kind of methods for improving yeast oxidation resistance, belong to bioengineering field.
Background technique
Yeast is a kind of important industrial producing strain, it has, and fermenting speed is fast, growth cycle is short, it is extensive to carry out
The advantages that culture.Yeast will receive in the industrial production various stress pressure (such as oxidative stress, osmotic pressure stress, low ph value,
High alcohol content stress, cold or thermostimulation, starvation, high concentration SO2Deng) influence, these stress are to the survival ability and hair of yeast
Ferment effect has a great impact.Wherein, oxidative stress is to influence maximum stress to Yeast fermentation process, it is mainly by activity
Caused by a large amount of accumulation of oxygen cluster (ROS).
ROS is the Main By product of cell metabolism, it includes H2O2, singlet oxygen, hydroxy radical and superoxides yin from
Son etc..Since ROS and ATP participates in oxidative phosphorylation process, the presence of the two in the cell is necessary.But when ROS with
Time increases accumulation in the cell excessively, and the mechanism that cell itself removes endogenous ROS cannot balance out excessive ROS,
A large amount of cumulative damages macromolecular substances intracellular of ROS, so that the miopragias such as cell membrane transmission, cell metabolism, brewer's yeast
Bioactivity and fermentability all can be by serious influences.The damage that ROS induces is complicated and is not under normal conditions
It is reversible.ROS especially intracellular can directly damage mitochondrial function, so that mitochondria is towards the direction for more easily producing ROS
Variation.Vicious circle more accelerates the rate of cell senescence between mitochondria and ROS.The aggregation of ROS and cellular oxidation pressure
Increase the important feature for being identified as cell ageing, therefore, the oxidation resistance for improving yeast has to the Fermented for improving yeast
There can be important meaning.
Summary of the invention
The present invention provides a kind of method for improving yeast oxidation resistance, is the logarithmic phase in the yeast growth period, uses
H2O21~2h of yeast cells is stimulated, then, yeast cells is inoculated in stand in 96 orifice plates containing fresh YPD medium and is trained
It supports, separates single colonie, obtain the yeast of oxidation resistance raising.
The yeast includes brewer's yeast, saccharomyces cerevisiae, fruit wine yeast, Lu Shi yeast.
The raising oxidation resistance, which refers to, reduces ROS cumulant intracellular, increases ATP content intracellular.
Specifically using 10mmol/L H in the logarithmic phase in yeast growth period2O21~2h of yeast cells is stimulated, and
It is inoculated in 96 orifice plates containing fresh YPD medium afterwards, dilutes step by step, separates single colonie, obtain oxidation resistance raising
Yeast.
Specifically, yeast is cultivated to logarithmic growth phase in YPD culture medium, collects yeast thallus, it is dilute with PBS buffer solution
It releases to (1~2) × 107Cfu/mL adds H2O2, make H2O2Final concentration reach 10mmol/L, in 28 DEG C, 180r/min cultivate
1h takes bacterium solution to be inoculated in 96 orifice plates containing fresh YPD medium, in 28 DEG C of stationary cultures, separates single colonie.
In one embodiment of the invention, using ROS content intracellular, ATP content intracellular as Testing index, screening is passed through
The yeast that oxidation resistance after hydrogen peroxide treatment improves.
The H of present invention various concentration2O2Yeast cells is stimulated, with the training of 96 orifice plate minisize liquids under the conditions of lethasl concentration
The bacterial strain that the method for the supporting screening speed of growth is fast, activity is high, obtained bacterial strain ROS accumulating level intracellular compared with starting strain reduce 30%
Left and right, and in addition to one plant of fruit wine yeast, ATP content ascensional range intracellular is more than 50%.Therefore, the method for the present invention can be effective
Yeast ROS cumulant intracellular is reduced, ATP content intracellular is increased, to achieve the purpose that improve yeast strain oxidation resistance.
Detailed description of the invention
Fig. 1 be in the growth brewer's yeast pilsner of logarithmic phase, saccharomyces cerevisiae 12, saccharomyces cerevisiae 14, fruit wine yeast JNB,
Fruit wine yeast D254, Lu Shi yeast WLL186, by 10mmol/L H2O2Before and after the processing, the content of ROS intracellular.
Fig. 2 be in the growth brewer's yeast pilsner of logarithmic phase, saccharomyces cerevisiae 12, saccharomyces cerevisiae 14, fruit wine yeast JNB,
Fruit wine yeast D254, Lu Shi yeast WLL186, by 10mmol/L H2O2Before and after the processing, the content of ATP intracellular.
12 plants of bacterium of Fig. 3 are containing H2O2Plating medium in growing state；(A)H2O2Concentration is 4mmol/L, (B)
H2O2Concentration is 6mmol/L.
Specific embodiment
The measuring method of ROS intracellular: yeast starter liquid is accessed in YPD culture medium with 1% inoculum concentration, 28 DEG C, 180r/
Min cultivate for 24 hours, take 1mL bacterium solution, thalline were collected by centrifugation, after cell washing, with phosphate buffer by cell concentration adjust to
OD600=0.8, take 1mL OD600=0.8 cell suspension 5 μ L 0.02mmol/L DCFH-DA solution of addition, 180r/min, 28
DEG C culture 30min, with microplate reader detect DCF fluorescence.λ ex=485nm, λ em=530nm.
The detection method of ATP vigor: yeast starter liquid is accessed in YPD culture medium with 1% inoculum concentration, 28 DEG C, 180r/
Min is cultivated for 24 hours, takes 1mL bacterium solution, and thalline were collected by centrifugation, and somatic cells are suspended in phosphate buffer, adjusts cell concentration
To OD600=0.8, take 1mL OD600=0.8 bacterium solution uses in the breaking-wall cell pipe containing bead- 24 homogeneous
Broken instrument smudge cells 5min (concussion 1min, ice bath 30s), 12000r/min is centrifuged 5min, collects supernatant.Take 100 μ L
ATP detects box reagent in 96 orifice plates, and the supernatant of 100 μ L clasmatosis liquid is added, and detects its chemistry with microplate reader after mixing
Luminous value.
Embodiment 1
1. the screening of anti-oxidant yeast
(1) the brewer's yeast pilsner for saving laboratory, saccharomyces cerevisiae 12,14, fruit wine yeast JNB, D254, Lu Shi
Yeast WLL186 is inoculated into fresh YPD culture solution with 1% inoculum concentration, and 28 DEG C of 180r/min cultivate 12h, takes appropriate culture solution,
10 are diluted respectively-1、10-2、10-3、10-4、10-5、10-6、10-7, the dilution of 20 each gradients of μ L is taken respectively, is added and is contained 180 μ L
In the hole of 96 orifice plates of YPD fluid nutrient medium, in 28 DEG C of stationary cultures.28 DEG C after stationary culture 2~3 days, in extension rate foot
In the case where reaching greatly, the bottom hole of 96 orifice plates will appear single colonie.
(2) the single colonie yeast in step (1) is taken, culture grows to logarithm early period, 4000r/min centrifugation in YPD
5min collects thallus, and thallus, which is suspended in PBS buffer solution, makes cell concentration reach (1~2) × 107Cfu/mL adds H2O2,
Make H2O2Final concentration reach 10mmol/L, 28 DEG C, 180r/min cultivates 1h, takes 20 μ L bacterium solutions to be inoculated in fresh containing 180 μ L
In 96 orifice plates of YPD culture medium, in 28 DEG C of stationary cultures, separates single colonie and cultivate 2~3 days.Picking grows faster single bacterium
It falls and is inoculated in inclined-plane preservation.
(3) single colonie bacterial strain progress ROS intracellular, the ATP intracellular of acquisition are detected, and is carried out anti-oxidant plate verifying.
(I) by starting strain brewer's yeast pilsner, saccharomyces cerevisiae 12, saccharomyces cerevisiae 14, fruit wine yeast JNB, fruit wine ferment
Female D254, Lu Shi yeast WLL186 and step (2) collect that treated strain S yeast pilsner-1, saccharomyces cerevisiae 12-
1, saccharomyces cerevisiae 14-1, fruit wine yeast JNB-1, fruit wine yeast D254-1, Lu Shi yeast WLL186-1 are inoculated into 1% inoculum concentration
In YPD fluid nutrient medium, 28 DEG C of 180r/min cultures for 24 hours, and carry out ROS detection intracellular.
As a result as shown in Figure 1, by H2O2Bacterial strain after processed, ROS level intracellular reduce obvious.Compared to bacterium germination out
Strain, by H2O2The ROS content intracellular of six plants of bacterium after processed reduces by 42%, 32%, 24%, 42%, 12%, 21% respectively,
For reduction amplitude 30% or so, amplitude of variation may be related to bacterial strain itself antioxidant system.In short, the treatment process for
Yeast ROS accumulation intracellular is effectively reduced, improving oxidation resistance has more significant effect.
(II) by above-mentioned 12 plants of bacterium (brewer's yeast pilsner, saccharomyces cerevisiae 12, saccharomyces cerevisiae 14, fruit wine yeast JNB, fruit
Brewer yeast D254, Lu Shi yeast WLL186, brewer's yeast pilsner-1, saccharomyces cerevisiae 12-1, saccharomyces cerevisiae 14-1, fruit wine ferment
Female JNB-1, fruit wine yeast D254-1, Lu Shi yeast WLL186-1) 1% inoculum concentration is inoculated into YPD fluid nutrient medium, and 28 DEG C
180r/min cultivates 12h, and carries out ATP detection intracellular.
ATP is the important indicator for embodying cell viability.From figure 2 it can be seen that compared to starting strain, by H2O2Place
Bacterial strain ATP content intracellular after reason has been respectively increased 78%, 110%, 87%, 52%, 21%, 78%.Except one plant of fruit wine yeast
Outside, more than 50%, ATP content intracellular rises significant other five plants of bacterium ascensional ranges.This result becomes with ROS changes of contents intracellular
Gesture is consistent, and ATP content increases, and ROS content accumulation intracellular is effectively reduced, so that the antioxygenic property of yeast be promoted to be promoted.
(III) by above-mentioned 12 plants of bacterium (brewer's yeast pilsner, saccharomyces cerevisiae 12, saccharomyces cerevisiae 14, fruit wine yeast JNB, fruit
Brewer yeast D254, Lu Shi yeast WLL186, brewer's yeast pilsner-1, saccharomyces cerevisiae 12-1, saccharomyces cerevisiae 14-1, fruit wine ferment
Female JNB-1, fruit wine yeast D254-1, Lu Shi yeast WLL186-1) 1% inoculum concentration is inoculated into YPD fluid nutrient medium, and 28 DEG C
180r/min cultivates 12h, and carries out H2O2Flat-plate spotting verifies (bacterial concentration 105、104、103、102CFU/mL)。
As shown in figure 3, from the H of various concentration2O2It can be found that working as H in flat-plate spotting result2O2Concentration is 4mmol/L
When, 6 plants of bacterium do not show notable difference；Work as H2O2When concentration is increased to 6mmol/L, by this method handle bacterial strain compared with
Opportunistic pathogen strain, significant difference show stronger activity.Its result explanation, the H of 4mmol/L2O2For 6 plants of bacterium stress compared with
It is small, the difference of the oxidation resistance between bacterial strain cannot be disclosed；The H of 6mmol/L2O2Concentration selection is more appropriate, and treated
Bacterial strain has stronger oxidation resistance compared with opportunistic pathogen strain, which can effectively improve the oxidation resistance of yeast strain.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method for improving yeast oxidation resistance, which is characterized in that be the logarithmic phase in the yeast growth period, use
H2O2Yeast cells is then inoculated in stationary culture in 96 orifice plates containing fresh YPD medium by stimulation yeast cells, is separated
Single colonie obtains the yeast of oxidation resistance raising.
2. a kind of method for improving yeast oxidation resistance according to claim 1, which is characterized in that the yeast includes
Brewer's yeast, saccharomyces cerevisiae, fruit wine yeast, Lu Shi yeast.
3. a kind of method for improving yeast oxidation resistance according to claim 1 or 2, which is characterized in that the raising
Oxidation resistance, which refers to, reduces ROS cumulant intracellular, increases ATP content intracellular.
4. the method for any a kind of raising yeast oxidation resistance according to claim 1~3, which is characterized in that be
The logarithmic phase in yeast growth period uses 10~12mmol/L H2O2Stimulation 1~2h of yeast cells then connects yeast cells
Kind stationary culture in 96 orifice plates containing fresh YPD medium, separates single colonie, obtains the yeast of oxidation resistance raising.
5. a kind of method for improving yeast oxidation resistance according to claim 4, which is characterized in that be in yeast growth
The logarithmic phase in period uses 10mmol/L H2O21~2h of yeast cells is stimulated, is then inoculated in containing fresh YPD medium
Stationary culture in 96 orifice plates separates single colonie, obtains the yeast of oxidation resistance raising.
6. a kind of method for improving yeast oxidation resistance according to claim 4 or 5, which is characterized in that yeast exists
Culture collects yeast thallus, is diluted to (1~2) × 10 with PBS buffer solution to logarithmic growth phase in YPD culture medium7Cfu/mL,
Add H2O2, make H2O2Final concentration reach 10mmol/L, in 28 DEG C, 180r/min cultivate 1h, take bacterium solution be inoculated in containing newly
In 96 orifice plates of fresh YPD culture medium, in 28 DEG C of stationary cultures, single colonie is separated.
7. the method for any a kind of raising yeast oxidation resistance according to claim 1~5, which is characterized in that with born of the same parents
Interior ROS content, ATP content intracellular are Testing index, screen the yeast that the oxidation resistance after hydrogen peroxide treatment improves.
8. a kind of method for improving yeast oxidation resistance according to claim 1, which is characterized in that with 10mmol/L
H2O2Stimulate yeast cells.
9. the yeast that the oxidation resistance that method culture according to claims 1 to 9 obtains improves.
10. application of the yeast described in claim 9 in beer brewing.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283771A (en) * | 2019-07-24 | 2019-09-27 | 四川大学 | The method for improving survival rate of the methamidophos under stress conditions |
| CN114437952A (en) * | 2020-11-04 | 2022-05-06 | 伽蓝（集团）股份有限公司 | Soil-derived saccharomyces cerevisiae and fermentation product and application thereof |
| CN116042422A (en) * | 2023-02-07 | 2023-05-02 | 广东丸美生物技术股份有限公司 | Yeast-derived exosome and preparation method and application thereof |
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| CN106916759A (en) * | 2015-12-24 | 2017-07-04 | 新疆康美瑞农业发展有限公司 | A kind of preparation method of high anti-oxidation lactic acid bacteria powder |
| CN107446984A (en) * | 2017-07-12 | 2017-12-08 | 西南大学 | A kind of evaluation method of polyphenoils antioxidation activity |
| CN108559714A (en) * | 2018-06-01 | 2018-09-21 | 江南大学 | A kind of Yeast strain of beer with high anti-oxidation ability |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106916759A (en) * | 2015-12-24 | 2017-07-04 | 新疆康美瑞农业发展有限公司 | A kind of preparation method of high anti-oxidation lactic acid bacteria powder |
| CN107446984A (en) * | 2017-07-12 | 2017-12-08 | 西南大学 | A kind of evaluation method of polyphenoils antioxidation activity |
| CN108559714A (en) * | 2018-06-01 | 2018-09-21 | 江南大学 | A kind of Yeast strain of beer with high anti-oxidation ability |
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| MING J. WU等: "Anantioxidant screeningassay based on oxidant-induced growth arrest inSaccharomyces cerevisiae", 《FEMS YEAST RES》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110283771A (en) * | 2019-07-24 | 2019-09-27 | 四川大学 | The method for improving survival rate of the methamidophos under stress conditions |
| CN114437952A (en) * | 2020-11-04 | 2022-05-06 | 伽蓝（集团）股份有限公司 | Soil-derived saccharomyces cerevisiae and fermentation product and application thereof |
| CN114437952B (en) * | 2020-11-04 | 2023-09-12 | 伽蓝（集团）股份有限公司 | Saccharomyces cerevisiae derived from soil, fermentation product and application thereof |
| CN116042422A (en) * | 2023-02-07 | 2023-05-02 | 广东丸美生物技术股份有限公司 | Yeast-derived exosome and preparation method and application thereof |
| CN116042422B (en) * | 2023-02-07 | 2024-03-26 | 广东丸美生物技术股份有限公司 | Yeast-derived exosome and preparation method and application thereof |
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