CN108120833A - A kind of method of quick detection yeast activity - Google Patents
A kind of method of quick detection yeast activity Download PDFInfo
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- CN108120833A CN108120833A CN201810157204.4A CN201810157204A CN108120833A CN 108120833 A CN108120833 A CN 108120833A CN 201810157204 A CN201810157204 A CN 201810157204A CN 108120833 A CN108120833 A CN 108120833A
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Abstract
The invention discloses a kind of methods of quick detection yeast activity, belong to microbiological analysis detection field.The present invention is realized by detecting yeast intracellular ATP contents, the quick, vigor of Sensitive Detection brewer's yeast.The result that this method is evaluated is used to deposit the extension between brewer's yeast storage, the failure of intracellular nutriment, yeast intracellular ATP contents decline.When stress in storage condition can cause yeast intracellular pH variations, there are significant linear regression relations with intracellular pH value for ATP relative amounts.When the brewer's yeast intracellular ATP contents during storage, which are reduced to, has just enter into storage stage intracellular ATP contents 60% when, it is not recommended that continue to produce using brewer's yeast under this state.This method is easy to operate, time saving, can Fast Evaluation yeast activity variation.
Description
Technical field
The present invention relates to a kind of methods of quick detection yeast activity, belong to microbiological analysis detection field.
Background technology
Brewer's yeast is microorganism used in beer brewing, is divided into two class of top yeast and bottom yeast.Brewer's yeast
Physiological status affect beer fermentation generation the substances such as organic acid, ester, higher alcohol, aldehydes content, so as to finished product beer
The quality of wine impacts.There is various abiotic stress effect in Process of Beer Brewing, such as tank pressure, the failure of nutriment, fermentation
Continuous accumulation of the substances such as ethyl alcohol etc. in liquid.In addition, the yeast that brewing uses has the feature recycled, i.e. fermentation is tied
The yeast paste recycled after beam can generally can be used for 5 to 10 generations as the yeast of next round fermentation inoculation, Domestic Beer yeast.Beer
The recycling of coercion and yeast during liquor brewing causes the vigor of yeast constantly to decline.The vigor of brewer's yeast
Directly affect the quality of finished beer.Therefore, the vigor of brewer's yeast in time, is quickly and accurately detected for Beer Brewage
Process is highly important.
In Brewery, usually using the death rate as judge brewer's yeast whether the parameter that can be continuing with, so
And the method for the death rate is more coarse, can not react the nuance of yeast activity.When dead state has not yet been reached in yeast,
It just has occurred that the decline of different physiological roles, such as the change of cellular morphology and the failure of mitochondrial function, and finally leads
Cause the decline of fermentability.Therefore, the physiological status for judging brewer's yeast with the death rate can not accurately reflect yeast cells
The variation of vigor.At present, have the research much measured on cell viability, but for the research of brewer's yeast vitality test
It is relatively fewer.In addition, although existing many vigor measuring methods, due to lacking sensitivity, operating comfort etc., this
A little method not extensive uses in brewery.
The content of the invention
Atriphos (ATP) is direct energy source in organism, is played the part of in the growth of cell and metabolic process
The vital role of person.When yeast has higher vigor, yeast maintains the shape of generation and consumption in balance of intracellular ATP
State, when yeast cells impaired mitochondrial function or intracellular lack the energy, and vigor is caused to decline, ATP contents decline.Therefore, ATP
Content become and judge the good index of yeast activity.
The object of the present invention is to provide a kind of methods of quick detection yeast activity, and the method is by detecting yeast born of the same parents
The variation of the reacting condition yeast activity of interior ATP contents.
In one embodiment of the invention, when intracellular ATP contents are less than 60%, cell survival rate is less than 95%.
In one embodiment of the invention, the method for the detection yeast intracellular ATP contents is to wash yeast cells
It is crushed after washing dilution, after centrifugation removal cell fragment, ATP contents in supernatant is detected using ATP detection kits.
In one embodiment of the invention, the dilution refers to yeast cells being diluted to 103~106A cell/mL
Yeast cells suspension.
In one embodiment of the invention, described crush refers to that carrying out concussion using bead crushes.
In one embodiment of the invention, the broken treatment conditions of the concussion are：Middle addition is hanged according to 1ml bacterium
200~400 μ l beades, take 0.4~1mm beades add in dilution after bacteria suspension in, in concussion 4 on the broken instrument of concussion~
8min, concussion speed are 4~8m/s, and sample often is placed in 20~40s of ice bath after 1~2min of concussion.
In one embodiment of the invention, the method for the detection yeast intracellular ATP contents is specially：
(1) yeast bacteria suspension is taken, washs 2~3 times, is diluted to 103~106The yeast cells suspension of a cell/mL；
(2) 0.4~1mm beades are added in broken wall pipe, is placed on the broken instrument of concussion and shakes 4~8min, shake speed
For 4~8m/s, sample is often placed in 20~40s of ice bath after 1~2min of concussion；
(3) centrifugation cell fragment after shaking, collects supernatant, is preserved on ice；
(4) above-mentioned supernatant is taken in light tight 96 orifice plate, and ATP contents are detected using ATP kits.
Second object of the present invention is to provide application of the method in beer fermentation.
In one embodiment of the invention, the application is lived using the cell of above method detection beer distiller's yeast
Power.
In one embodiment of the invention, the application be specifically when detect intracellular ATP contents be less than 60% when,
Cell survival rate is less than 95%, and beer yeast cells cannot be continuing with.
Beneficial effects of the present invention：Compared with other yeast activity measuring methods, ATP methods detection yeast activity is easy to operate
And sensitive, the nuance of energy effecting reaction brewer's yeast vigour changes during storage.In the ATP detection methods of the present invention
Decline 20% or so when intracellular pH only declines 0.1 or so, ATP contents, it being capable of sensitive reacting cells vigor condition.The present invention's
ATP luminous values have high correlation (10 with the dense number of saccharomyces cerevisiae3-106), when using the dense number of bacterium 103-106Between beer
When yeast carries out ATP content detections, relative standard deviation<1.68%.There is the method for the present invention relatively low detection to limit, and use cell
Number is 103Beer yeast cells when can carry out ATP content detections.The present invention it is easy to operate, time saving, method is sensitive can
It leans on.
Description of the drawings
The variation of intracellular ATP contents, intracellular pH and survival rate during the 25 DEG C of storages of Fig. 1 brewer's yeasts；
The variation of intracellular ATP contents, intracellular pH and survival rate during the 4 DEG C of storages of Fig. 2 brewer's yeasts；
The variation of intracellular ATP contents and intracellular trehalose content during the brewer's yeast storages of Fig. 3 after fermentation.
Specific embodiment
Embodiment 1：
By taking brewer's yeast G-03 as an example, impregnated after brewer's yeast G-03 when culture 24 is small in YPD is collected by centrifugation, is washed
In sterile water, 25 DEG C are positioned over, measures intracellular ATP, survival rate and intracellular pH variations.Per 1ml bacteria suspensions are taken for 24 hours, precooling is used
Sterile water washing cell, centrifugation goes supernatant to collect thalline, washes repeatedly 1 time, and the thalline after washing is resuspended to 1ml in sterile water,
Bacteria suspension is diluted to concentration as 106The yeast cells suspension of a cell/ml.In 2ml broken wall pipes, 1ml bacterium solutions and 300 are added in
The a diameter of 0.5mm beades of μ l.Broken wall pipe containing bacterium solution and bead is placed on the broken instrument of concussion and shakes 5min, extends shake
Detected ATP contents are not further added by when swinging the time.Centrifugation cell fragment after concussion collects supernatant, after crushing
Sample in being preserved on ice bath, be ready to use in ATP content detections.100 μ l of clasmatosis liquid are taken in light tight 96 orifice plate, are used
ATP contents kit detects yeast intracellular ATP contents.Yeast intracellular ATP contents are defined as 100% when will just store, remaining
Sample intracellular ATP contents are opposite ATP contents compared with the control group.
Implementation result：
Experimental result is as shown in Figure 1, the results showed that, under 25 DEG C of conditions of storage, with the extension of yeast storage time, yeast
Intracellular ATP contents decline.The yeast cells for storing first day is set to control sample, intracellular ATP contents are defined as 100%,
Other samples intracellular ATP contents are the relative amount compared with control sample.Measurement show that first day intracellular pH is 5.93.Storage
Second day yeast intracellular ATP content becomes 82.68%, and intracellular pH is 5.81.The 3rd day yeast intracellular ATP content is stored to become
58.68%, intracellular pH are 5.66.It stores the 4th day yeast intracellular ATP content and becomes 52.47%, intracellular pH is 5.61.Storage the
Five days yeast intracellular ATP contents become 38.52%, and intracellular pH is 5.54.The decline of intracellular pH shows prolonging with storage time
Long yeast activity declines.Intracellular ATP contents have significant correlation, correlation coefficient r with yeast intracellular pH contents2=0.997.
The detecting step of ATP is simple, time saving.Therefore, detection ATP contents are a kind of quick, methods of Sensitive Detection brewer's yeast vigor.
In the actual use of brewer's yeast, there are one section of storage process, usually depositing yeast before the use of a yeast input new round
Motility rate is controlled more than 95%, and corresponding intracellular ATP is containing being about 60% of intracellular ATP contents when just storing at this time, therefore, when
When yeast intracellular ATP contents drop to less than 60%, being continuing with the yeast at this time will be unfavorable for producing.
Embodiment 2：
By taking brewer's yeast G-03 as an example, impregnated after brewer's yeast G-03 when culture 24 is small in YPD is collected by centrifugation, is washed
In sterile water, 4 DEG C are positioned over, measures intracellular ATP, survival rate and intracellular pH variations.Per 1ml bacteria suspensions are taken for 24 hours, with precooling
Sterile water washing cell, centrifugation go supernatant to collect thalline, wash repeatedly 1 time, and the thalline after washing is resuspended to 1ml in sterile water, will
Bacteria suspension is diluted to concentration as 106The yeast cells suspension of a cell/ml.In 2ml broken wall pipes, 1ml bacterium solutions and 300 μ l are added in
A diameter of 0.5mm beades.Broken wall pipe containing bacterium solution and bead is placed on the broken instrument of concussion and shakes 5min, extends concussion
Detected ATP contents are not further added by during the time.Centrifugation cell fragment after concussion collects supernatant, broken
Sample is ready to use in ATP content detections in being preserved on ice bath.100 μ l of clasmatosis liquid is taken to use ATP in light tight 96 orifice plate
Content kit detects yeast intracellular ATP contents.Yeast intracellular ATP contents are defined as 100% when will just store, remaining sample
Intracellular ATP contents are opposite ATP contents compared with the control group.
Implementation result：
Experimental result is as shown in Figure 2, the results showed that, under 4 DEG C of conditions of storage, with the extension of yeast storage time, yeast
Intracellular ATP contents decline.The yeast cells for storing first day is set to control sample, intracellular ATP contents are defined as 100%,
Other samples intracellular ATP contents are the relative amount compared with control sample.At 4 DEG C, metabolic activity in cells is faint, and vigor is subtle
The detection of difference becomes increasingly difficult, and from result, when being stored at 4 DEG C, yeast intracellular pH and survival rate be not apparent
Variation, and the variation of intracellular ATP contents is apparent, convenient for the judgement of vigor difference.After storage 5 days, survival rate declines yeast
To about 95%, 60% when corresponding intracellular ATP contents are about just deposited at this time, therefore it is proposed that when cell intracellular ATP contains
It just no longer comes into operation when measuring so far content.When the brewer's yeast death rate changes very little, significant change does not occur for intracellular pH, born of the same parents
Interior ATP methods still can delicately react the change of yeast state.The sensitivity of ATP methods detection yeast activity is demonstrated again.Therefore,
Detection ATP contents are a kind of quick, methods of Sensitive Detection brewer's yeast vigor.
Embodiment 3：
With brewer's yeast e43, brewer's yeast 11#, exemplified by brewer's yeast e77, the yeast after beer fermentation is collected,
Storage is in sterile water, being positioned over 20 DEG C.Per 1ml bacteria suspensions are taken for 24 hours, with the sterile water washing cell of precooling, supernatant is removed in centrifugation
Thalline is collected, is washed repeatedly 1 time, after the thalline after sterile water resuspension washing to 1ml, surveys intracellular ATP and intracellular trehalose content.
Bacteria suspension is diluted to concentration as 106The yeast cells suspension of a cell/ml.In 2ml broken wall pipes, 1ml bacterium solutions and 300 are added in
The a diameter of 0.5mm beades of μ l.Broken wall pipe containing bacterium solution and bead is placed on the broken instrument of concussion and is shaken, when extending concussion
Between when detected ATP contents be not further added by.Centrifugation cell fragment after concussion takes supernatant, broken sample
In being preserved on ice bath, ATP content detections are ready to use in.100 μ l of clasmatosis liquid is taken to use ATP contents in light tight 96 orifice plate
Kit detects yeast intracellular ATP contents.Yeast intracellular ATP contents are defined as 100% when will just store, remaining sample intracellular
ATP contents are opposite ATP contents compared with the control group.
Implementation result：
Experimental result is as shown in Figure 3, the results showed that, yeast intracellular ATP contents during storage decline after fermentation.It will be each
Intracellular ATP contents are defined as 100% when yeast stores first day, other storage times intracellular ATP contents are and first day intracellular
The relative amount that ATP contents are compared.When just storing compared with the ATP contents of yeast intracellular, during storage the 5th day, brewer's yeast
E43, brewer's yeast 11#, brewer's yeast e77 intracellular ATP content distributions become 84.8%, 78%, 96%, have different degrees of
Decline.At the same time, three plants of brewer's yeast intracellular trehalose contents constantly decline with the extension of storage time, illustrate that intracellular supplies
The content of energy substance is constantly reduced, and cell viability is declining.Therefore, detection ATP contents are a kind of quick, Sensitive Detections
The method of brewer's yeast vigor.
Embodiment 4：
By taking brewer's yeast G-03 as an example, yeast liquid is diluted to 103、104、105、106The bacteria suspension of various concentration, according to
Secondary its ATP content of detection.With the sterile water washing cell of precooling, centrifugation goes supernatant to collect thalline, washes repeatedly 1 time, sterile water
The thalline after washing is resuspended to 1ml, bacteria suspension is diluted to concentration as 106The yeast cells suspension of a cell/ml.It is broken in 2ml
In wall pipe, 1ml bacterium solutions and a diameter of 0.5mm beades of 300 μ l are added in.Broken wall pipe containing bacterium solution and bead is placed in concussion
5min is shaken on broken instrument, detected ATP contents are not further added by when extending the concussion time.Centrifugation is thin after concussion
Born of the same parents' fragment, collects supernatant, and broken sample is ready to use in ATP content detections in being preserved on ice bath.Take 100 μ l of clasmatosis liquid
In light tight 96 orifice plate, yeast intracellular ATP luminous values are detected using ATP contents kit, and calculate standard deviation and opposite
Standard deviation implementation result：
The accuracy of 1 brewer's yeast intracellular ATP content measuring methods of table
The result shows that ATP luminous values have high correlation (10 with the dense number of saccharomyces cerevisiae3-106), when using the dense number of bacterium
103-106Between brewer's yeast carry out ATP content detections when, relative standard deviation<1.68%.The method has relatively low inspection
Limit is surveyed, using cell number 103Beer yeast cells when can carry out ATP content detections.Therefore, it is one to detect ATP contents
Kind is quick, the method for Sensitive Detection brewer's yeast vigor.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art without departing from the spirit and scope of the present invention, can do various change and modification, therefore the protection model of the present invention
Enclosing be subject to what claims were defined.
Claims (10)
- A kind of 1. method of quick detection yeast activity, which is characterized in that the method is by detecting yeast intracellular ATP contents Reacting condition yeast activity variation.
- 2. according to the method described in claim 1, it is characterized in that, when intracellular ATP contents are less than 60%, cell survival rate is low In 95%.
- 3. according to the method described in claim 1, it is characterized in that, the method for the detection yeast intracellular ATP contents is by ferment It is crushed after mother cell washing dilution, after centrifugation removal cell fragment, ATP contents in supernatant is detected using ATP detection kits.
- 4. according to the method described in claim 3, it is characterized in that, the dilution refers to yeast cells being diluted to 103~106 The yeast cells suspension of a cell/mL.
- 5. according to the method described in claim 3, it is characterized in that, described crush refers to that carrying out concussion using bead crushes.
- 6. according to the method described in claim 5, it is characterized in that, the treatment conditions that the concussion crushes are：It is hanged according to 1ml bacterium 200~400 μ l beades of middle addition, take 0.4~1mm beades to add in the bacteria suspension after diluting, and are shaken on the broken instrument of concussion 4~8min is swung, concussion speed is 4~8m/s, and sample often is placed in 20~40s of ice bath after 1~2min of concussion.
- 7. according to the method described in claim 1, it is characterized in that, the method for the detection yeast intracellular ATP contents is specially：(1) yeast bacteria suspension is taken, washs 2~3 times, is diluted to 103~106The yeast cells suspension of a cell/mL；(2) 0.4~1mm beades are added in broken wall pipe, is placed on the broken instrument of concussion and shakes 4~8min, concussion speed for 4~ Sample is often placed in 20~40s of ice bath by 8m/s after 1~2min of concussion；(3) centrifugation cell fragment after shaking, collects supernatant, is preserved on ice；(4) above-mentioned supernatant is taken in light tight 96 orifice plate, and ATP contents are detected using ATP kits.
- 8. application of the method described in claim 1 in beer fermentation.
- 9. application according to claim 8, which is characterized in that the application detects beer distiller's yeast using the above method Cell viability.
- 10. application according to claim 9, which is characterized in that the application is specifically to work as to detect that intracellular ATP contents are low When 60%, cell survival rate is less than 95%, and beer yeast cells cannot be continuing with.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444766A (en) * | 2021-06-04 | 2021-09-28 | 佛山市海天（江苏）调味食品有限公司 | Enrichment medium for spoilage bacteria in fermented wine aging process and detection method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1072957A (en) * | 1992-12-18 | 1993-06-09 | 华东化工学院 | A kind of fixed yeast cell and the application on adenosine triphosphate is produced thereof |
| CN103614308A (en) * | 2013-08-16 | 2014-03-05 | 新乡拓新生化股份有限公司 | Saccharomyces cerevisiae for ATP synthesis and applications thereof |
| CN104789600A (en) * | 2015-04-24 | 2015-07-22 | 江南大学 | Method for improving acid stress resistance of candida glabrata |
-
2018
- 2018-02-24 CN CN201810157204.4A patent/CN108120833A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1072957A (en) * | 1992-12-18 | 1993-06-09 | 华东化工学院 | A kind of fixed yeast cell and the application on adenosine triphosphate is produced thereof |
| CN103614308A (en) * | 2013-08-16 | 2014-03-05 | 新乡拓新生化股份有限公司 | Saccharomyces cerevisiae for ATP synthesis and applications thereof |
| CN104789600A (en) * | 2015-04-24 | 2015-07-22 | 江南大学 | Method for improving acid stress resistance of candida glabrata |
Non-Patent Citations (1)
| Title |
|---|
| MAGDALENA KWOLEK-MIREK ET AL: "Comparison of methods used for assessing the viability and vitality of yeast cells", 《FEDERATION OF EUROPEAN MICROBIOLOGICAL SOCIETIES》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113444766A (en) * | 2021-06-04 | 2021-09-28 | 佛山市海天（江苏）调味食品有限公司 | Enrichment medium for spoilage bacteria in fermented wine aging process and detection method |
| CN113444766B (en) * | 2021-06-04 | 2024-05-24 | 海天醋业集团有限公司 | Enrichment medium for spoilage bacteria in fermentation wine ageing process and detection method |
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Application publication date: 20180605 |