CN104458543A - Detection method of flocculation proteins on yeast cell surface based on flow cytometry - Google Patents
Detection method of flocculation proteins on yeast cell surface based on flow cytometry Download PDFInfo
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- CN104458543A CN104458543A CN201410816248.5A CN201410816248A CN104458543A CN 104458543 A CN104458543 A CN 104458543A CN 201410816248 A CN201410816248 A CN 201410816248A CN 104458543 A CN104458543 A CN 104458543A
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Abstract
The invention belongs to the technical field of beer, relates to a detection method of flocculability of beer yeast, and particularly relates to a detection method of flocculation proteins on yeast cell surface based on flow cytometry. The detection method comprises the following steps of firstly washing yeast, secondly, carrying out heavy suspension and dilution on the yeast, then adding an FITC-avidin solution into yeast bacterium suspension for dying the yeast bacterium suspension, and finally determining the flocculation performance of yeast by a flow cytometer. The detection method provided by the invention is low in detection error, is good in parallism, can be further used for researching the distribution of flocculation proteins of the yeast bacterium group, can act as a performance evaluation means for the yeast culture to differentiate the yeast culture with different flocculation capacities, and can act as a yeast flocculation performance index for providing powerful technical support for yeast breeding and bacterial strain management.
Description
Technical field
The invention belongs to the detection method of beer technical field for brewer's yeast flocculability, particularly a kind of detection method of the flocculation of the yeast cell surface based on Flow Cytometry albumen.
Background technology
The flocculability of yeast has very important significance for the management of Process of Beer Brewing.In Process of Beer Brewing, not only require that yeast is quick, fully wheat juice Middle nutrition composition fermented, and require that yeast forms dense precipitation when fermentation ends.The too strong yeast of flocculation ability is due to premature flocculation, and the mature indicator such as wine body biacetyl, acetaldehyde do not reduce, thus affect degree of ripeness and the local flavor of beer; On the contrary, the yeast of flocculability difference can cause filtration difficulty, affects the clarity of fermentation liquor.Therefore, the flocculating property of yeast is the factor that winemaker carries out yeast strain seed selection and Technical innova-tion and must consider.
Mostly the primary evaluation approach of current yeast flocculence is to be based upon the modification method on Helm method basis, its Cleaning Principle be by contrast Yeast Flocculation before and after free cell number or the change of optical density, determine Yeast Flocculation level.But, therefore there is the common problem that the large and different batches of error detects data redundancy difference in the status and appearance of used yeast when this kind of method by the quantitative Assessment of Changes flocculating property of yeast is owing to relying on detection to a great extent.
The visualize of the flocculability of yeast is the quantitative change of yeast, and the essence that flocculation phenomenon occurs between the yeast cells mannose residue selectivity of albumen on adjacent yeast cell wall of then flocculating secreted by yeast cell surface is combined and causes.The people such as R.Alex Speers utilize fluorospectrophotometer to study the distribution of yeast cell surface flocculation protein density, but detecting what obtain that fluorescence signal shows owing to utilizing fluorospectrophotometer is yeast samples average to be measured, cannot obtain the details such as the classification of the flora of yeast or distribution, the method therefore utilizing fluorospectrophotometer to detect yeast cell surface flocculation albumen cannot realize the analysis to yeast flora flocculation albumen distribution in yeast life cycle.
Summary of the invention
The error that the present invention is directed to Yeast Flocculation performance evaluation in prior art is large, poor reproducibility, the deficiency that cannot analyze yeast flora, provides a kind of Flow Cytometry that utilizes to flocculate the method that albumen detects to yeast cell surface.
Technical scheme of the present invention is:
A kind of detection method of the flocculation of the yeast cell surface based on Flow Cytometry albumen, comprise the steps: first yeast washing, secondly the resuspended and dilution of yeast, in saccharomycete suspension, again add FITC-avidin solution hatch and carry out the dyeing of saccharomycete suspension, finally utilize cells were tested by flow cytometry Yeast Flocculation performance.
Concrete steps are as follows:
Step 1: yeast wash, to the nutrient solution of yeast carry out centrifugal after, with 5mL 100mM EDTA wash twice, then spend deionized water twice;
Step 2: yeast resuspended, carries out resuspended with flocculation damping fluid to the yeast after washing;
Step 3: the dilution of the bacteria suspension of yeast, according to the yeast concentration after resuspended, using described flocculation damping fluid saccharomycete suspension to be diluted to yeast concentration is 4 ~ 5 × 10
6cell/mL;
Step 4: the dyeing of saccharomycete suspension, adds FITC-avidin solution in saccharomycete suspension, makes FITC-avidin concentration in saccharomycete suspension reach 2 ~ 2.5mg FITC-avidin/10
9cell, hatches 1hr under dark reaction condition on ice;
Step 5: flow cytomery, the saccharomycete suspension got after 1ml dyeing carries out flow cytomery.
Preferably, the centrifugal condition described in step 1 is 5000 × g, 5min.
Preferably, the flocculation damping fluid described in step 2 is for comprising 0.2mM CaCl
220mM acetate delay solution.
Preferably, flow cytomery: the saccharomycete suspension got after 1ml dyeing carries out flow cytometer (BD Accuri C6) and detects, not add the saccharomycete suspension of FITC-avidin as negative control (determining the autofluorescence of yeast scope and elimination sample itself).
Flow cytomery condition: the FL1 passage of selective excitation optical wavelength 488nm, receives sample speed 66uL/min, each sample collection 2 × 10
4individual yeast cells, utilizes CFlow Sampler software to analyze result, the parameter of the protein content that flocculates using the fluorescent value of FL1 passage as yeast surface, the parameter using FSC value as yeast volume.
Above-mentioned FITC-avidin, purchased from invitrogen; Article No. 434411; Lot number 1324041A.
Flow Cytometry owing to can realize the measurement to individual cells unlike signal under hydrokinetic congregation by sheath fluid, be more conducive to the physiological status accurately reflecting yeast flora, and will realize using Flow Cytometry to detect yeast cell surface flocculation albumen, first to determine flocculate Protein Detection specific antibody and can with the fluorescein of the applicable flow cytomery of antibody conjugate, FITC-avidin is utilized to detect yeast cell surface flocculation albumen in the present invention, avidin wherein containing mannose side chain can to flocculate protein combination with yeast cell surface as specific antibody, the fluorescein FITC of conjugation provides the fluorescence signal that can be detected by fluorospectrophotometer, secondly the present invention determines the flocculation buffer solution system being beneficial to flocculation albumen and being combined with specific antibody, and the specific antibody of the best and the reaction ratio of sterilised yeast suspension and dyeing time.
The invention has the beneficial effects as follows:
One, the present invention is relative to the current detection method based on the quantitative Assessment of Changes flocculating property of yeast, and metrical error is low, and collimation is good;
Its two, the present invention relative to the detection method based on fluorescence spectrophotometry, can further to yeast flora flocculation albumen distribution study;
Its three, the present invention can distinguish as the yeast strain of performance evaluation means to different flocculation ability of bacterial classification;
Therefore the present invention as Yeast Flocculation performance evaluation index, can provide strong technical support for yeast sreen and bacterial strain manage.
Accompanying drawing explanation
Accompanying drawing 1 is the streaming figure of the negative control sample (being unstained) of 2 of the specific embodiment of the invention
Wherein, scheming A is FSC-SSC scatter diagram; Figure B is FL1-FSC scatter diagram;
Accompanying drawing 2 is the streaming figure of the sample of the specific embodiment of the invention 2
Wherein, scheming A is FSC-SSC scatter diagram; Figure B is FL1-FSC scatter diagram; Figure C is FSC-A histogram and grouping (M1-M5);
Accompanying drawing 3 is the streaming FL1-A histogram that the M1-M5 of the specific embodiment of the invention 2 respectively organizes yeast surface flocculation albumen
Wherein, the streaming FL1-A histogram that A is the flocculation albumen of M1 group yeast is schemed; Figure B is the streaming FL1-A histogram of the flocculation albumen of M2 group yeast; Figure C is the streaming FL1-A histogram of the flocculation albumen of M3 group yeast; Figure D is the streaming FL1-A histogram of the flocculation albumen of M4 group yeast; Figure E is the streaming FL1-A histogram of the flocculation albumen of M5 group yeast;
Accompanying drawing 4 is yeast (M1-M5) the surface flocculation protein level difference of the different sizes of the specific embodiment of the invention 2;
Accompanying drawing 5 is the growth curve of the specific embodiment of the invention 3 sweat yeast.
Embodiment
The specific embodiment of the present invention is as follows:
Embodiment 1:
When getting certain Beer Brewage factory sweat 20hr, 60hr, 110hr, each 100mL of fermentation broth sample, respectively gets 1mL fermentation broth sample, uses yeast counts instrument (BD Z1 proton analyser) to detect the yeast quantity in sample; Respectively get 5mL fermentation liquor to carry out centrifugal (5000 × g, 5min), use 5mL 100mM EDTA to the yeast paste washing twice after centrifugal, then with 5mL deionized water washing twice; Flocculating with 5mL, (20mM acetate buffer, comprises 0.2mM CaCl to damping fluid
2) carry out resuspended to the yeast after washing; According to the yeast number that each sample detects, using flocculation damping fluid saccharomycete suspension to be diluted to yeast concentration is 4 ~ 5 × 10
6cell/mL; Get 1mL saccharomycete suspension, add 4 μ L FITC-avidin solution (2.5 mg/mL), hatch 1hr on ice, under dark reaction condition; The saccharomycete suspension got after 1mL dyeing carries out flow cytometer (BD Accuri C6) and detects, not add the saccharomycete suspension of FITC-avidin as negative control, each fermentation broth sample Parallel testing 3 times.Flow cytomery condition: the FL1 passage of selective excitation optical wavelength 488nm, receives sample speed 66uL/min, each sample collection 2 × 10
4individual yeast cells, utilizes CFlow Sampler software to analyze result, the parameter of the protein content that flocculates using the fluorescent value of FL1 passage as yeast surface, and testing result is as shown in table 1 below:
The flocculation protein level of yeast in table 1 different fermentations liquid sample
From above testing result and analyze, the detection CV value of the yeast flocculence detection method that the present invention uses is less than 5%, and method collimation is good.
Embodiment 2
Get Beer Brewage factory sweat fermentation broth sample 100mL, after mixing, get 1mL fermentation broth sample, use yeast counts instrument (BD Z1 proton analyser) to detect the yeast quantity in sample; Get 5mL fermentation liquor to carry out centrifugal (5000 × g, 5min), use 5mL 100mM EDTA to the yeast paste washing twice after centrifugal, then with 5mL deionized water washing twice; Flocculating with 5mL, (20mM acetate buffer, comprises 0.2mM CaCl to damping fluid
2) carry out resuspended to the yeast after washing; According to the yeast number that each sample detects, using flocculation damping fluid saccharomycete suspension to be diluted to yeast concentration is 4 ~ 5 × 10
6cell/mL; Get 1mL saccharomycete suspension, add 4 μ L FITC-avidin solution (2.5 mg/mL), hatch 1hr on ice, under dark reaction condition; The saccharomycete suspension got after 1mL dyeing carries out flow cytometer (BD Accuri C6) and detects, not add the saccharomycete suspension of FITC-avidin as negative control (see Fig. 1).Flow cytomery condition: the FL1 passage of selective excitation optical wavelength 488nm, receives sample speed 66uL/min, each sample collection 2 × 10
4individual yeast cells, utilizes CFlowSampler software to analyze result, and to dyeing 2 × 10
4individual yeast is divided into 5 groups from small to large by FSC value (for representing the volume of yeast): M1, M2, M3, M4, M5, and FSC value is less, represents that the volume of yeast is less; FSC value is larger, represents the volume larger (as shown in Figure 2) of yeast.The parameter of flocculation protein content using the fluorescent value of FL1 passage as yeast surface, the flocculation protein content often organizing yeast is shown in shown in Fig. 3 and table 2, (see Fig. 4) is analyzed to the flocculation protein level of 5 groups of yeast of the large small size of difference known, the yeast cell surface flocculation protein content of different size is different, cellule flocculation protein content is low, maxicell flocculation protein content is high, and namely at yeast propagation process, yeast cell surface flocculation protein content increases.
Table 2 M1 ~ M5 respectively organizes Yeast Flocculation protein level
| ％of This Plot | Mean FL1-A (flocculation protein content) | |
| M1 | 1.31％ | 23610 |
| M2 | 28.07％ | 25056 |
| M3 | 55.28％ | 50615 |
| M4 | 4.55％ | 92335 |
| M5 | 3.56％ | 126929 |
Embodiment 3
Use the yeast strain (L830, L820) that 2 strain flocculabilities are different, first 200mL Beer Brewage wheat juice is used to carry out triangular flask activation culture (cultivating 2d for 25 DEG C) to yeast strain, centrifugal (5000 × g is carried out to the fermentation liquor after cultivating, 5min), take 1.1g centrifugal after the yeast paste Beer Brewage wheat juice that adds 220mL carry out EBC pipe fermenting experiment, cultivate 6 days for 12 DEG C, the yeast number of tracing detection inoculation after fermentation liquid and Yeast Flocculation protein content, concrete detecting step is:
(1) sweat yeast number detects: get 1mL fermentation broth sample when fermenting 0hr, 48hr, 52hr, 68hr, 72hr, 76hr, 92hr, 96hr, 100hr, 116hr, 120hr, 144hr respectively, yeast counts instrument (BD Z1 proton analyser) is used to detect the yeast quantity in sample, draw the growth curve of yeast, see Fig. 5;
(2) after fermentation ends, get 5ml fermentation liquor and carry out centrifugal (5000 × g, 5min), use 5mL 100mM EDTA to the yeast paste washing twice after centrifugal, then with 5mL deionized water washing twice; Flocculating with 5mL, (20mMacetate buffer comprises 0.2mM CaCl to damping fluid
2) carry out resuspended to the yeast after washing; According to the yeast number detected, using flocculation damping fluid saccharomycete suspension to be diluted to yeast concentration is 4 ~ 5 × 10
6cell/mL; Get 1mL saccharomycete suspension, add 4uLFITC-avidin solution (2.5 mg/mL), hatch 1hr on ice, under dark reaction condition; The saccharomycete suspension got after 1mL dyeing carries out flow cytometer (BD Accuri C6) and detects, not add the saccharomycete suspension of FITC-avidin as negative control.Flow cytomery condition: the FL1 passage of selective excitation optical wavelength 488nm, receives sample speed 66uL/min, each sample collection 2 × 10
4individual yeast cells, utilize CFlow Sampler software to analyze result, flocculate using the fluorescent value of FL1 passage as yeast surface protein content, analyzes the flocculation protein content of 2 strain yeast:
Table 3 yeast strain L830, L820 flocculation protein level difference
| Yeast strain | Mean FL1-A (flocculation protein level) |
| L830 | 63067 |
| L820 | 80035 |
As can be seen from Fig. 5 and table 3, after yeast strain L820 fermentation ends, the yeast number be suspended in fermentation liquor is less than bacterial strain L830, illustrate that the flocculability of bacterial strain L830 is better than L820, this is consistent with the flocculation expressing quantity of yeast strain L830, therefore utilizes Yeast Flocculation method of protein detection disclosed by the invention to can be used in distinguishing the yeast of different flocculation ability.
Wherein, the Latin name of L830 is called Saccharomyces pastorianus, buys from U.S. White Labs, is numbered WLP830 GERMAN LAGER YEAST.The Latin name of L820 is called Saccharomyces pastorianus, buys from U.S. White Labs, is numbered WLP820
lAGER YEAST.
Above-described embodiment is illustrative instead of determinate, can list several embodiments, therefore in the change do not departed under general plotting of the present invention and amendment, should belong within protection scope of the present invention according to institute's limited range.
Claims (5)
1. the detection method of the flocculation of the yeast cell surface based on a Flow Cytometry albumen, it is characterized in that, comprise the steps: first yeast washing, secondly the resuspended and dilution of yeast, in saccharomycete suspension, again add FITC-avidin solution hatch and carry out the dyeing of saccharomycete suspension, finally utilize cells were tested by flow cytometry Yeast Flocculation performance.
2. the detection method of the flocculation of the yeast cell surface based on Flow Cytometry albumen according to claim 1, it is characterized in that, concrete steps are as follows:
Step 1: yeast wash, to the nutrient solution of yeast carry out centrifugal after, with 100mM EDTA wash twice, then spend deionized water twice;
Step 2: yeast resuspended, carries out resuspended with flocculation damping fluid to the yeast after washing;
Step 3: the dilution of the bacteria suspension of yeast, according to the yeast concentration after resuspended, using described flocculation damping fluid saccharomycete suspension to be diluted to yeast concentration is 4 ~ 5 × 10
6cell/mL;
Step 4: the dyeing of saccharomycete suspension, adds FITC-avidin solution in saccharomycete suspension, makes FITC-avidin concentration in saccharomycete suspension reach 2 ~ 2.5mg FITC-avidin/10
9cell, hatches 1hr under dark reaction condition on ice;
Step 5: flow cytomery, the saccharomycete suspension got after 1ml dyeing carries out flow cytomery.
3. the detection method of the flocculation of the yeast cell surface based on Flow Cytometry albumen according to claim 2, it is characterized in that, the centrifugal condition described in step 1 is 5000 × g, 5min.
4. the detection method of the flocculation of the yeast cell surface based on Flow Cytometry albumen according to claim 2, it is characterized in that, the flocculation damping fluid described in step 2 is for comprising 0.2mM CaCl
220mM acetate delay solution.
5. the detection method of the flocculation of the yeast cell surface based on Flow Cytometry albumen according to claim 2, it is characterized in that, flow cytomery condition described in step 5: the FL1 passage of selective excitation optical wavelength 488nm, receives sample speed 66uL/min, each sample collection 2 × 10
4individual yeast cells, utilizes software to analyze result, the parameter of the protein content that flocculates using the fluorescent value of FL1 passage as yeast surface, the parameter using FSC value as yeast volume.
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