CN103131750A - Method for detecting aging of yeast - Google Patents
Method for detecting aging of yeast Download PDFInfo
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- CN103131750A CN103131750A CN2013100465803A CN201310046580A CN103131750A CN 103131750 A CN103131750 A CN 103131750A CN 2013100465803 A CN2013100465803 A CN 2013100465803A CN 201310046580 A CN201310046580 A CN 201310046580A CN 103131750 A CN103131750 A CN 103131750A
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Abstract
The invention provides a method for detecting aging of yeast. The method includes steps of firstly carrying out subculture on yeast bodies so as to obtain yeast cell fermentation broth in different algebra, centrifugally separating the yeast fermentation nutrient solution to obtain yeast cells, freeze-drying the yeast cells, then weighing a certain number of freeze-dried cells and dissolving the freeze-dried cells with wall-breaking buffering solution, breaking the freeze-dried cells through ultrasonic, freeze-centrifuging the broken yeast cells, testing superroxide dismutase (SOD) activity of supernatant, and generation speed and content of superoxide anion free radical O2-, analyzing mutual relationship among the three factors, setting up a standard relationship curve of contents and generation speeds of the SOD activity and the superoxide anion free radical, and finding algebra of the yeast according to the standard curve and the known SOD activity.
Description
Technical field
The present invention relates to a kind of aging method of yeast that detects, with the bacterial classification cultivation of going down to posterity, set up the active mutual relationship standard control figure with ultra-oxygen anion free radical of SOD in different algebraically yeast cell, and then detect the method for yeast degree of aging.
Background technology
Yeast is the most widely used microorganisms of the mankind, is used for traditionally foodstuffs industry and makes the wheaten food such as bread and steamed bun, is mainly used in wine brewing in fermentation industry.Brewageing on the process nature of drinks is the metabolic process of yeast, and the metabolic rate of yeast is aging closely-related with yeast, can directly have influence on enterprise's production output and economic benefit.On the other hand, the meta-bolites of yeast consists of wine body and the distinctive local flavor of wine.The good local flavor of wine is mainly comprehensively to be gone into by the various flavour substancess that the fermentation by saccharomyces cerevisiae metabolism forms, and that the shortcoming in wine and assorted flavor also come from yeast saccharomyces cerevisiae greatly is aging, and self-dissolving is especially even more important for beer and cereuisiae fermentum.Therefore to obtain high-quality wine, just must have energetic, the yeast that quality is good.This shows, the problem of aging of yeast saccharomyces cerevisiae directly affects the quality of fermenting speed and wine.Yeast is aging usually from its hypoglycemic speed, the indexs such as alcohol growing amount are characterized, but be a progressive formation because yeast is aging, influence factor is more, at present without comparison accurately method identify the degree of aging of yeast, cause and only use the less yeast of algebraically in Process of Beer Brewing, cause yeast even to be had influence on production by large quantities of wastes.Therefore, finding a kind of feasible aging method of evaluation yeast to produce for modernization industry has great significance.
The SOD(superoxide-dismutase) be the antioxidase of ultra-oxygen anion free radical in single-minded scavenger cell, stability is strong, is prevalent in yeast, animals and plants and all eukaryotic biologies, plays a very important role at the aspect such as pre-anti-aging, under the yeast growth condition, O
2 -Can in time be removed by SOD, can not produce injury to cell, but in the situations such as aging, active oxygen will exert an influence to yeast physiology, the accumulation of ultra-oxygen anion free radical is the one of the main reasons of yeast aging.
This research obtains the yeast of different algebraically by cultivation that yeast saccharomyces cerevisiae 2144 and 2146 is gone down to posterity, and measures its SOD activity and O
2 -.Content with produce speed, set up SOD activity and O
2 -.Between mutual relationship standard control figure, SOD activity and the O of the yeast by measuring production and application
2 -.Content with produce speed, comparison standard control figure just can judge saccharomycetic degree of aging, thereby provides reliable foundation for production decision.Experimental results show that this method is a kind of accurate, reliably detection method.
Summary of the invention
The invention provides a kind of aging method of yeast that detects, at first to the cultivation of going down to posterity of yeast thalline, obtain the yeast cell fermented liquid of different algebraically, the yeast fermentation nutrient solution is obtained yeast cell by centrifugation, yeast cell is after lyophilize, and the cell that takes a certain amount of freeze-drying dissolves with the broken wall damping fluid, ultrasonication, yeast cell frozen centrifugation after fragmentation is got supernatant liquor and is measured SOD activity and ultra-oxygen anion free radical O
2 -.Generating rate and content;
Concrete operation steps is as follows:
The first step, culturing yeast cell, wherein:
1) substratum: dipotassium hydrogen phosphate 10.5g, potassium primary phosphate 4.5g, ammonium sulfate 1g, trisodium citrate 0.5g, glucose 60g is without amino yeast nitrogen substratum 6.7g, Histidine 10mg, methionine(Met) 20mg, tryptophane 20mg, 1000ml deionized water;
2) culture condition: according to 3～8 * 10
6The inoculum size inoculation of cfu/mL at 28～30 ℃, is cultivated fermentation termination, fermentation ends under 150～180r/min condition;
3) be defined as first-generation yeast from the strain transfer on the solid slant culture base to liquid nutrient medium, after arriving fermentation termination, with 5.05 * 10
6The inoculum size switching of cfu/mL is once gone down to posterity;
4) collecting cell: fermented liquid under the 4000r/min condition centrifugal 20 minutes, with sterile water wash 3～5 times, each 4000r/min centrifugal 10 minutes, obtains clean yeast cell.By lyophilize (48 ℃, 4Pa), obtain the yeast of freeze-drying, standby.
Wherein said fermentation termination is that residual sugar content in substratum is when 1～3mg/mL.
Second step, yeast cell is adopted the multigelation ultrasonication, take the yeast of 2g freeze-drying, with the broken wall damping fluid dissolving of 30ml; freezing 4h under-20 ℃ dissolves 0.5h under 30 ℃, twice repeatedly; carry out ultrasonication, work 2s, intermittently 3s during ultrasonication; omnidistance time 25min, power 400w, 4 ℃ of protection temperature; mixed solution frozen centrifugation after fragmentation, centrifugation rate 10000r/min, centrifugation time 10min; 4 ℃ of protection temperature are got supernatant liquor, measure immediately.
Wherein, the broken wall buffer formulation is to contain in every 1000ml: the Tris-HCl of 50mmol/L, 1.491gKCl, 0.146gEDTA.
The 3rd step, the method that adopts the pyrogallol autotrophy and azanol oxidation style are measured SOD activity and ultra-oxygen anion free radical generating rate and content in cell extract, analyze the mutual relationship between the three, set up the standard relationship curve between SOD activity and ultra-oxygen anion free radical content and generation speed, and then according to typical curve, known SOD is active, finds the algebraically of yeast.
The present invention is by analyzing SOD activity and O in different algebraically yeast born of the same parents
2 -.Between mutual relationship, set up SOD activity and O
2 -.Between mutual relationship standard control figure, as SOD activity and O
2 -.When generation speed presents different variation tendency from content, illustrate that yeast cell has occurred aging.This can be proved from hypoglycemic speed.Judge the degree of aging of yeast, only need to set up SOD activity and the O of different algebraically yeast
2 -.Between mutual relationship standard control figure, then it is active to measure the SOD of yeast of production and application, with the standard map contrast, namely can find out the degree of aging of yeast cell.Because SOD is the high intracellular anti-oxidation enzyme of stability, this method is accurate, and is reliable, can provide reliable foundation for production decision.
Table 1, different algebraically yeast saccharomyces cerevisiae 2144SOD activity, ultra-oxygen anion free radical produces speed and content:
Table 2, different algebraically yeast saccharomyces cerevisiae 2146SOD activity, ultra-oxygen anion free radical produces speed and content:
Description of drawings
Fig. 1, bovine serum albumin (BSA) typical curve
Fig. 2, yeast saccharomyces cerevisiae 2144SOD activity and free-radical generating speed correlogram, wherein--be that SOD is active ,-●-be free-radical generating speed.
Fig. 3, yeast saccharomyces cerevisiae 2144SOD activity and free-radical contents correlogram, wherein--be that SOD is active ,-●-be free-radical contents.
Fig. 4, yeast saccharomyces cerevisiae 2146SOD activity and free-radical generating speed correlogram, wherein--be that SOD is active ,-●-be free-radical generating speed.
Fig. 5, yeast saccharomyces cerevisiae 2146SOD activity and free-radical generating speed correlogram, wherein--be that SOD is active ,-●-be free-radical generating speed.
Embodiment
In conjunction with the embodiments, technical solution of the present invention is further elaborated, but institute of the present invention protection domain is not limited only to this.In embodiment, bacterial classification used and substratum are:
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 2144,2146, bacterial classification is provided by Dalian Polytechnic University's culture presevation.
Substratum: K
2HPO
410.5g, KH
2PO
44.5g, (NH
4)
2SO
41g, trisodium citrate 0.5g, glucose 60g, YNB substratum 6.7g, L-His10mg, L-Met20mg, L-try20mg, 1000ml deionized water.
Yeast saccharomyces cerevisiae 2144
The first step, different algebraically yeast cell obtain:
1) seed culture: the bacterial classification on the solid slant culture base, be transferred in seed culture medium, 28 ℃, 170r/min cultivates 15h, arrives logarithmic phase, and this is as seed liquor.
2) fermentation culture: according to 5.05 * 10
6The inoculum size inoculation of cfu/mL, 28 ℃, 170r/min cultivates 65h, arrives fermentation termination.Be defined as first-generation yeast from the strain transfer on the solid slant culture base to liquid nutrient medium, after fermentation ends, with identical inoculum size switching, go down to posterity.
3) collecting cell: fermented liquid under the 4000r/min condition centrifugal 20 minutes, with sterile water wash 3～5 times, each 4000r/min centrifugal 10 minutes, obtains clean yeast cell.By lyophilize (48 ℃, 4Pa), obtain the yeast of freeze-drying, standby.
Second step, the preparation of sample liquid:
Take the method smudge cells of multigelation ultrasonication; step is as follows: the yeast that takes the 2g freeze-drying; with the broken wall damping fluid dissolving of 30ml, freezing 4h under-20 ℃ dissolves 0.5h under 30 ℃; twice repeatedly; carry out ultrasonication (work 2s, 3s intermittently, omnidistance time 25min; power 400w, 4 ℃ of protection temperature).Mixed solution frozen centrifugation after fragmentation (4 ℃, 10000r/min, 10min) is got supernatant liquor, measures immediately.
The 3rd step, index determining:
Adopt pyrogallol autotrophy method to measure SOD active, the azanol oxidation style is measured O
2 -.Produce speed and content, set up SOD activity and O
2 -.Between mutual relationship standard control figure.Data such as the table 1 measured, the SOD activity reaches maximum when the 9th generation, and after this SOD activity begins to descend, and O
2 -.Generation speed accelerate, content continues to rise, and aging node just occurred this moment, and yeast after this begins aging, can be after cultivation 65h, in substratum, residual sugar content is proved, and yeast saccharomyces cerevisiae 2144 the 1st, 3,6,9,12 generations, after cultivation 65h, residual sugar content (mg/ml) respectively is: 2.46,1.79,1.51,1.38,1.49.
The 4th step, practical application:
Cultivate according to above-mentioned cultural method 2nd generation, the 5th generation and the 10th generation of yeast saccharomyces cerevisiae 2144 in experiment, obtain yeast cell, according to the said method of this paper, the SOD of mensuration 2,5,10 generation cell is active, be respectively 24.08,26.98,29.03U/mgprotein, and according to the standard map contrast, 2,5,10 generation the cell SOD activity theoretical value be 23.89,27.41,29.83U/mgprotein, basically identical with the data that experiment obtains, provable this method is feasible thus.
Yeast saccharomyces cerevisiae 2146
The first step, different algebraically yeast cell obtain:
1) seed culture: the bacterial classification on the solid slant culture base, be transferred in seed culture medium, 28 ℃, 170r/min cultivates 17h, arrives logarithmic phase, and this is as seed liquor.
2) fermentation culture: according to 5.05 * 10
6The inoculum size inoculation of cfu/mL, 28 ℃, 170r/min cultivates 72h, arrives fermentation termination.Be defined as first-generation yeast from the strain transfer on the solid slant culture base to liquid nutrient medium, after fermentation ends, with identical inoculum size switching, go down to posterity.
3) collecting cell: fermented liquid under the 4000r/min condition centrifugal 20 minutes, with sterile water wash 3～5 times, each 4000r/min centrifugal 10 minutes, obtains clean yeast cell.By lyophilize (48 ℃, 4Pa), obtain the yeast of freeze-drying, standby.
Second step, the preparation of sample liquid:
Take the method smudge cells of multigelation ultrasonication; step is as follows: the yeast that takes the 2g freeze-drying; with the broken wall damping fluid dissolving of 30ml, freezing 4h under-20 ℃ dissolves 0.5h under 30 ℃; twice repeatedly; carry out ultrasonication (work 2s, 3s intermittently, omnidistance time 25min; power 400w, 4 ℃ of protection temperature).Mixed solution frozen centrifugation after fragmentation (4 ℃, 10000r/min, 10min) is got supernatant liquor, measures immediately.
The 3rd step, index determining:
Adopt pyrogallol autotrophy method to measure SOD active, the azanol oxidation style is measured O
2 -.Produce speed and content, set up SOD activity and O
2 -.Between mutual relationship standard control figure.Data such as the table 2 measured, the SOD activity reaches maximum when the 7th generation, and after this SOD activity begins to descend, and O
2 -.Generation speed accelerate, content continues to rise, and aging node just occurred this moment, yeast after this begins aging, can be after cultivation 72h, in substratum, residual sugar content is proved, yeast saccharomyces cerevisiae 2146 the 1st, 3,5,7,9,10 generations, after cultivating 72h, residual sugar content (mg/ml) respectively is: 2.06,1.87,1.48,1.22,1.28,1.31.
The 4th step, practical application:
Cultivate according to above-mentioned cultural method the 4th generation and the 8th generation of yeast saccharomyces cerevisiae 2146 in experiment, obtain yeast cell, according to the said method of this paper, the SOD of mensuration 4,8 generation cell is active, be respectively 39.08,60.53U/mgprotein, and according to the standard map contrast, 4,8 generation the cell SOD activity theoretical value be 38.19,60.07U/mgprotein, basically identical with the data that experiment obtains, provable this method is feasible thus.
In sum, the present invention can provide reliable foundation for production decision can be used as a kind of easy, reliable means aspect evaluation yeast degree of aging.
Claims (1)
1. one kind is detected the aging method of yeast, it is characterized in that the cultivation of going down to posterity of yeast thalline, obtain the yeast cell fermented liquid of different algebraically, the yeast fermentation nutrient solution is obtained yeast cell by centrifugation, yeast cell is after lyophilize, and the cell that takes a certain amount of freeze-drying dissolves with the broken wall damping fluid, ultrasonication, yeast cell frozen centrifugation after fragmentation is got supernatant liquor and is measured SOD activity and ultra-oxygen anion free radical generating rate and content;
Concrete operation steps is as follows:
The first step, culturing yeast cell, wherein:
1) substratum: dipotassium hydrogen phosphate 10.5g, potassium primary phosphate 4.5g, ammonium sulfate 1g, trisodium citrate 0.5g, glucose 60g is without amino yeast nitrogen substratum 6.7g, Histidine 10mg, methionine(Met) 20mg, tryptophane 20mg, 1000ml deionized water;
2) culture condition: according to 3～8 * 10
6The inoculum size inoculation of cfu/mL at 28～30 ℃, is cultivated fermentation termination, fermentation ends under 150～180r/min condition;
3) be defined as first-generation yeast from the strain transfer on the solid slant culture base to liquid nutrient medium, after arriving fermentation termination, with 5.05 * 10
6The inoculum size switching of cfu/mL is once gone down to posterity;
4) collecting cell: fermented liquid under the 4000r/min condition centrifugal 20 minutes, with sterile water wash 3～5 times, each 4000r/min, centrifugal 10 minutes, obtain clean yeast cell, by lyophilize (48 ℃, 4Pa), obtain the yeast of freeze-drying, standby;
Wherein said fermentation termination is that residual sugar content in substratum is when 1～3mg/mL;
Second step, yeast cell is adopted the multigelation ultrasonication, take the yeast of 2g freeze-drying, with the broken wall damping fluid dissolving of 30ml, freezing 4h under-20 ℃ dissolves 0.5h under 30 ℃, twice repeatedly, carry out ultrasonication, work 2s, intermittently 3s during ultrasonication, omnidistance time 25min, power 400w, 4 ℃ of protection temperature, mixed solution frozen centrifugation after fragmentation, centrifugation rate 10000r/min, centrifugation time 10min, 4 ℃ of protection temperature are got supernatant liquor, measure immediately;
Wherein, the broken wall buffer formulation is to contain in every 1000ml: the Tris-HCl of 50mmol/L, 1.491gKCl, 0.146gEDTA;
The 3rd step, the method that adopts the pyrogallol autotrophy and azanol oxidation style are measured SOD activity and ultra-oxygen anion free radical generating rate and content in cell extract, analyze the mutual relationship between the three, set up the standard relationship curve between SOD activity and ultra-oxygen anion free radical content and generation speed, and then according to typical curve, known SOD is active, finds the algebraically of yeast.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104705764A (en) * | 2015-03-06 | 2015-06-17 | 广州甘蔗糖业研究所 | Solid-liquid separation method for fermentation product |
| CN114507601A (en) * | 2021-11-05 | 2022-05-17 | 南京郢天健康管理有限公司 | Wall breaking method for preparing nanoscale selenium-enriched yeast |
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| CN101255436A (en) * | 2008-01-24 | 2008-09-03 | 中山大学 | Food-grade expression method for superoxide dismutase gene and special vectors thereof |
| CN101368163A (en) * | 2008-09-25 | 2009-02-18 | 山东农业大学 | Pichia engineering bacterial strain for expression of thermosacus aurantiancus miehe light spore variation gene Mn-sod |
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2013
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| CN101168747A (en) * | 2007-11-01 | 2008-04-30 | 上海交通大学 | Method for preparing ethanol and yeast superoxide dismutase from sweet sorghum stem liquor |
| CN101255436A (en) * | 2008-01-24 | 2008-09-03 | 中山大学 | Food-grade expression method for superoxide dismutase gene and special vectors thereof |
| CN101368163A (en) * | 2008-09-25 | 2009-02-18 | 山东农业大学 | Pichia engineering bacterial strain for expression of thermosacus aurantiancus miehe light spore variation gene Mn-sod |
Non-Patent Citations (3)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104705764A (en) * | 2015-03-06 | 2015-06-17 | 广州甘蔗糖业研究所 | Solid-liquid separation method for fermentation product |
| CN104705764B (en) * | 2015-03-06 | 2016-08-24 | 广州甘蔗糖业研究所 | A kind of solid-liquid separating method of tunning |
| CN114507601A (en) * | 2021-11-05 | 2022-05-17 | 南京郢天健康管理有限公司 | Wall breaking method for preparing nanoscale selenium-enriched yeast |
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Application publication date: 20130605 |