CN112592918A - Efficient breeding method of low-yield acetaldehyde beer yeast strain - Google Patents
Efficient breeding method of low-yield acetaldehyde beer yeast strain Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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Abstract
The invention discloses a high-efficiency breeding method of low-yield acetaldehyde beer yeast, belonging to the technical field of strain breeding. According to the invention, an ARTP mutagenesis treatment is carried out on a beer yeast strain to construct a mutant strain library; screening the mutant strain library by using an ethanol-disulfiram resistance plate and a high-concentration acetaldehyde screening plate to obtain a growth dominant strain, domesticating the growth dominant strain by using a liquid domestication culture medium, and establishing a mutagenic-screening-domesticated primary screening strain; and carrying out fermentation culture on the obtained primary screening strain, and carrying out secondary screening by using the enzyme activity changes of the ethanol dehydrogenases I and II and the acetaldehyde dehydrogenase as indexes to obtain the low-yield acetaldehyde beer yeast with the acetaldehyde yield reduced by more than 50%. The invention solves the problems of high screening difficulty and low efficiency of the beer industrial yeast with low acetaldehyde yield at present.
Description
Technical Field
The invention belongs to the technical field of beer brewing, and particularly relates to a high-efficiency breeding method of a low-acetaldehyde-yield beer yeast strain.
Background
Beer has a shorter shelf life than other alcoholic beverages due to the accumulation of aged flavor substances during its storage. These aged flavor compounds are composed mainly of aldehydes with a low flavor threshold. When beer is filled, the aldehyde compounds mainly exist in a combined state, and the content is below a flavor threshold; along with the prolonging of the wine storage time, a large amount of aldehyde compounds are released, the aging flavor in the beer is prominent, and the flavor stability is reduced. Acetaldehyde (CH)3CHO) is a main aging aldehyde compound in beer, has higher volatility and reactivity, and directly influences the flavor and the flavor stability of the beer.
With the development of the beverage industry, the desire of beer production enterprises to maintain stable flavor of wine and prolong the shelf life of products is more urgent. Therefore, researchers have proposed many process steps for controlling acetaldehyde content in succession, however, most process optimizations are not generally applicable, and the same steps may have different or even opposite results when applied to different species and process conditions.
Beer yeast fundamentally determines the formation and accumulation of acetaldehyde, a flavor substance, in beer products. On one hand, the yeast metabolizes to generate aging substances such as acetaldehyde and the like; on the other hand, the beer yeast has good reduction activity, and can reduce acetaldehyde in fermentation liquor. Therefore, the breeding of the low-yield acetaldehyde beer yeast strain with excellent performance becomes one of the ways of effectively improving the flavor stability of beer and solving the safety problem of beer food.
To date, researchers have adopted many technical approaches, mainly including traditional breeding approaches, homologous recombination approaches, and yeast "genetic self-cloning techniques", in order to obtain experiments and production strains with low acetaldehyde production. In view of the specificity of the application field of the beer yeast, beer researchers and brewers are more inclined to adopt the traditional breeding technology to breed the low-yield acetaldehyde beer yeast strain. However, the probability of obtaining the required phenotype by adopting the traditional breeding technology is low, and the character reversion rate is high. Therefore, the research of efficient, stable and safe breeding technology has important practical application value in constructing beer industrial yeast with low acetaldehyde yield.
Disclosure of Invention
The invention provides a high-efficiency breeding method of a beer yeast strain with low acetaldehyde yield, which solves the problems of high difficulty and low efficiency in screening beer industrial yeast with low acetaldehyde yield at present.
In order to achieve the aim, the invention provides a high-efficiency breeding method of a low-acetaldehyde-production beer yeast strain, which is characterized by comprising the following steps of:
carrying out ARTP mutagenesis treatment on the beer yeast strain to construct a mutant strain library;
screening the mutant strain library by using an ethanol-disulfiram resistance plate and a high-concentration acetaldehyde screening plate to obtain a growth dominant strain, domesticating the growth dominant strain by using a liquid domestication culture medium, and establishing a mutagenic-screening-domesticated primary screening strain;
and carrying out fermentation culture on the obtained primary screening strain, and carrying out secondary screening by using the enzyme activity changes of the ethanol dehydrogenases I and II and the acetaldehyde dehydrogenase as indexes to obtain a secondary screening strain, namely the low-acetaldehyde-production beer yeast.
Preferably, the ARTP mutagenesis treatment of the Saccharomyces cerevisiae strain is specifically as follows:
the cells cultured to mid-log phase were diluted to 1X 106Per mL, yeast cells were subjected to ARTP mutagenesis treatment using a helium ion beam.
Preferably, the conditions for the ARTP mutagenesis treatment are:
helium gas flow rate 15.0L/min, input power 100W, treatment distance 2mm, treatment temperature < 40 ℃ and mutagenesis time 90 s.
Preferably, the establishing of the mutagenesis-screening-domestication primary screening strain specifically comprises the following steps:
1) carrying out streak culture on beer yeast strains in the mutant strain library, selecting a single colony to grow in a YPD culture medium, centrifuging the thallus, washing and resuspending to obtain a cell suspension;
2) performing ARTP mutagenesis treatment on the cell suspension to obtain a mutagenesis bacterial liquid;
3) respectively coating the mutagenic bacterium liquid on a high-concentration acetaldehyde flat plate and an ethanol-disulfiram flat plate, carrying out constant-temperature culture, and selecting the growth dominant strains on the flat plates to be respectively subjected to continuous domestication in domestication culture media corresponding to the flat plates;
4) respectively sucking proper amounts of domesticated bacteria liquid, repeating the steps 2) -3) for at least 2-6 rounds, increasing the screening concentration and the domesticated culture medium concentration of each round of plates, and respectively coating the final domesticated bacteria liquid on two plates to obtain a primary screening strain.
Preferably, in the step 1), the growth temperature of the colony is 25-35 ℃, and the culture time is 6-48 h;
the cells were centrifuged, washed, resuspended in sterile physiological saline containing 1-20% glycerol, and diluted to 1X 104-1×108one/mL.
Preferably, in the step 2), the dosage of the cell suspension is 1-20 μ L;
the ARTP mutagenesis treatment conditions are as follows: helium gas flow rate of 5-45L/min, input power of 70-120W, treatment distance of 1-4mm, treatment temperature below 60 deg.C and mutagenesis time of 30-180 s.
Preferably, in the step 3), the suction amount of the mutagenic bacterium liquid is 5-200 μ L;
the high-concentration acetaldehyde plate is a YPD culture medium containing 0.1-10g/L acetaldehyde, and the ethanol-disulfiram plate is a basic carbon source culture medium containing 0-50g/L ethanol and 0-5mg/L disulfiram;
the constant culture temperature is 20-40 deg.C, and the culture time is 1-5 days;
the high-concentration acetaldehyde acclimation culture medium is an YPD culture medium containing 0.1-10g/L acetaldehyde;
the ethanol-disulfiram domestication culture medium is a basic carbon source culture medium containing 0-50g/L of ethanol and 0-10mg/L of disulfiram;
the domestication temperature is 20-40 deg.C, and the domestication time is 1-5 days.
Preferably, the enzyme activity changes of the alcohol dehydrogenase I, II and the acetaldehyde dehydrogenase are used as indexes, and three primary screening strains with the sum of the positive mutation amplitude of the enzyme activity of more than 65 percent are selected as secondary screening strains.
Preferably, after the rescreened strain is obtained, the method further comprises the step of measuring the flavor substances of the rescreened strain obtained by horizontal fermentation in a laboratory shake flask.
Preferably, the acetaldehyde yield of the obtained low-acetaldehyde producing Saccharomyces cerevisiae is reduced by more than 50%, and the main flavor of the low-acetaldehyde producing Saccharomyces cerevisiae is not obviously different from that of the originally screened Saccharomyces cerevisiae strain.
Compared with the prior art, the invention has the advantages and positive effects that:
the method adopts beer industrial yeast as an initial strain, constructs a mutant strain library by a multi-round ARTP mutation breeding technology, and completes the plate screening and domestication of the strain by using an ethanol-disulfiram resistant plate, a high-concentration acetaldehyde screening plate and a corresponding domestication liquid thereof to form a multi-round primary screening system of mutation-screening-domestication; meanwhile, key enzyme activity is changed into a low-yield acetaldehyde re-screening standard, a beer simulated brewing system at the laboratory shake flask level is used, and finally the mutant strain with acetaldehyde reduction amplitude of more than 50% is obtained, and the main flavor of mutant strain fermentation liquor is not obviously different from that of the original strain. The invention is further optimization and improvement of production strains, and has the potential of being applied to the industrial production of beer.
Drawings
FIG. 1 is a graph of lethality of strains as a function of mutagenesis time when mutagenesis conditions were established as provided in the examples of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides a high-efficiency breeding method of a low-acetaldehyde-production beer yeast strain, which comprises the following steps:
carrying out ARTP mutagenesis treatment on the beer yeast strain to construct a mutant strain library;
screening the mutant strain library by using an ethanol-disulfiram resistance plate and a high-concentration acetaldehyde screening plate to obtain a growth dominant strain, domesticating the growth dominant strain by using a liquid domestication culture medium, and establishing a mutagenic-screening-domesticated primary screening strain;
and carrying out fermentation culture on the obtained primary screening strain, and carrying out secondary screening by using the enzyme activity changes of the ethanol dehydrogenases I and II and the acetaldehyde dehydrogenase as indexes to obtain a secondary screening strain, namely the low-acetaldehyde-production beer yeast.
In the above scheme, the ARTP mutagenesis treatment of the Saccharomyces cerevisiae strain is specifically:
the cells cultured to the middle logarithmic phase were centrifuged and washed 3 times with a sterile physiological saline containing 5% glycerol, and the amount of the diluted cells was 1X 106one/mL. 10 μ L of the suspension was applied to a sterile metal slide (8mm) and the yeast cells were mutagenized with a helium plasma beam.
ARTP mutagenesis treatment conditions: the helium flow rate QHe was maintained at 15.0L/min, the power input was 100W, the treatment distance was 2mm, the treatment temperature was < 40 ℃ and the mutagenesis time was 90 s. The metal slide was placed in 1mL of sterile physiological saline and washed with shaking.
It is understood that, in the above mutagenesis treatment, under the conditions of keeping the helium gas flow rate QHe at 15.0L/min, the input power at 100W, the treatment distance at 2mm, and the treatment temperature at < 40 ℃, in order to obtain the optimum mutagenesis conditions, the treatment time is set to 0, 50, 70, 90, 110, and 130s, and the metal slide is placed in 1mL of sterile physiological saline and washed with shaking. And (3) coating the diluted bacterium liquid on a YPD culture medium, culturing at 30 ℃ for 48h, drawing a lethality curve, and determining the optimal irradiation time. If the fatality rate is low, the saccharomyces cerevisiae cannot be effectively improved; if the fatality rate is too high, the performance of the strain is easily changed greatly, so that the main flavor of the finished beer is influenced. Thus, the strain lethality was maintained between 80-90%, and the optimal mutagenesis time was selected to be 90s, as shown in FIG. 1.
In a preferred embodiment, the establishment of the mutagenesis-screening-domesticated prescreened strain is specifically as follows:
1) carrying out streak culture on beer yeast strains in the mutant strain library, selecting a single colony to grow in a YPD culture medium, centrifuging the thallus, washing and resuspending to obtain a cell suspension;
2) performing ARTP mutagenesis treatment on the cell suspension to obtain a mutagenesis bacterial liquid;
3) respectively coating the mutagenic bacterium liquid on a high-concentration acetaldehyde flat plate and an ethanol-disulfiram flat plate, carrying out constant-temperature culture, and selecting the growth dominant strains on the flat plates to be respectively subjected to continuous domestication in domestication culture media corresponding to the flat plates;
4) respectively sucking proper amounts of domesticated bacteria liquid, repeating the steps 2) -3) for at least 2-6 rounds, increasing the screening concentration and the domesticated culture medium concentration of each round of plates, and respectively coating the final domesticated bacteria liquid on two plates to obtain a primary screening strain.
In a preferred embodiment, in the step 1), the growth temperature of the colony is 25-35 ℃, and the culture time is 6-48 h; the cells were centrifuged, washed, resuspended in sterile physiological saline containing 1-20% glycerol, and diluted to 1X 104-1×108one/mL.
In a preferred embodiment, in the step 2), the amount of the cell suspension is 1 to 20 μ L;
the ARTP mutagenesis treatment conditions are as follows: helium gas flow rate of 5-45L/min, input power of 70-120W, treatment distance of 1-4mm, treatment temperature below 60 deg.C and mutagenesis time of 30-180 s.
In a preferred embodiment, in the step 3), the aspiration amount of the mutagenic bacteria liquid is 5-200 μ L;
the high-concentration acetaldehyde plate is a YPD culture medium containing 0.1-10g/L acetaldehyde, and the ethanol-disulfiram plate is a basic carbon source culture medium containing 0-50g/L ethanol and 0-5mg/L disulfiram;
the constant culture temperature is 20-40 deg.C, and the culture time is 1-5 days;
the high-concentration acetaldehyde acclimation culture medium is an YPD culture medium containing 0.1-10g/L acetaldehyde;
the ethanol-disulfiram domestication culture medium is a basic carbon source culture medium containing 0-50g/L of ethanol and 0-10mg/L of disulfiram;
the domestication temperature is 20-40 deg.C, and the domestication time is 1-5 days.
In a preferred embodiment, in the step 3), the strain with the growth dominance is a mutant strain with high growth speed, regular shape of single colony and large volume.
In a preferred embodiment, in the step 4), the usage amount of the acclimatized bacterial liquid is 10 to 200 μ L;
the screening concentration and the acclimatization culture medium concentration of each round of plates are gradually increased, and are specifically shown in table 1-2, wherein the acetaldehyde concentrations in the high-concentration acetaldehyde plates are respectively 2.0, 2.2, 2.4, 2.6 and 2.8g/L, and the disulfiram concentrations in the ethanol-disulfiram plates are respectively 0.2, 0.3, 0.4, 0.5 and 0.6 mg/L; the acetaldehyde concentrations in the high-concentration acetaldehyde acclimation culture medium are respectively 3.0, 3.2, 3.4, 3.6 and 3.8g/L, and the disulfiram concentrations in the ethanol-disulfiram acclimation culture medium are respectively 2.0, 2.1, 2.2, 2.3 and 2.4 mg/L.
In a preferred embodiment, the primary screening strain is subjected to fermentation culture, and secondary screening is carried out by taking the enzyme activity changes of the alcohol dehydrogenases I and II and the aldehyde dehydrogenase as indexes, wherein the screening process comprises the following steps:
a) inoculating the mutant strain obtained by primary screening to a microporous cell culture plate, and performing fermentation culture;
b) centrifuging to collect yeast cells, and washing and suspending; breaking cells, centrifuging and taking supernatant for later use;
c) determination of alcohol dehydrogenase I: adding appropriate amount of cell leaching solution and buffer solution into microporous cell culture plate, and measuring light absorption value.
d) Determination of alcohol dehydrogenase II: adding cell leaching solution and buffer solution into the microporous cell culture plate, and measuring the light absorption value.
e) Determination of acetaldehyde dehydrogenase: adding cell leaching solution and buffer solution into the microporous cell culture plate, and measuring the light absorption value.
f) And synthesizing the positive mutation amplitudes of the three key enzyme activities to obtain the low-yield acetaldehyde re-screening bacterial strain.
In a preferred embodiment, in step a), the microporous cell culture plate is a 96-well cell culture plate; the growth temperature of the thalli is 25-35 ℃, and the culture time is 6-20 h.
In a preferred embodiment, in step b), the bacterial cells are washed and resuspended in a phosphate buffer solution with pH of 6.0-10.0 and a concentration of 0.01-0.5 mol/L;
breaking the cells with an ultrasonic breaker for 2-8s, stopping for 1-10s, and taking 2-10min in total;
the cell extract is stored at 0-10 deg.C for further use.
In a preferred embodiment, in step c), the alcohol dehydrogenase I is determined by:
the adding amount of the cell leaching solution is 10-100 mu L, and the adding amount of the buffer solution is 80-300 mu L;
the buffer solution contains 0.01-0.2mol/L glycine, 0.01-0.2mol/L potassium hydroxide and 0.0001-0.01mol/L NAD+、1×10-5-1×10-3mol/L acetaldehyde, and the pH value of the buffer solution is 7.0-11.0;
the absorbance at 200-500nm was recorded by single cell time scanning for 2-8 min.
In a preferred embodiment, in step d), the alcohol dehydrogenase II is determined by:
the adding amount of the cell leaching solution is 10-100 mu L, and the adding amount of the buffer solution is 80-300 mu L;
the buffer solution contains 0.01-0.2mol/L glycine, 0.01-0.2mol/L potassium hydroxide, 0.0001-0.01mol/L NADH and 0.01-1mol/L ethanol, and the pH value of the buffer solution is 7.0-11.0;
the absorbance at 200-500nm was recorded by single cell time scanning for 2-8 min.
In a preferred embodiment, in step e), the acetaldehyde dehydrogenase is determined by:
the adding amount of the cell leaching solution is 1-20 mu L, and the adding amount of the buffer solution is 80-350 mu L;
the buffer solution contains 0.05-0.25mol/L potassium phosphate and 0.0001-0.01mol/L NAD+、1×10-6-1×10-2mol/L dithiothreitol solution, 1X 10-5-1×10-3mol/L acetaldehyde, and the pH value of the buffer solution is 6.0-10.0;
the absorbance at 200-500nm was recorded by single cell time scanning for 2-8 min.
In a preferred embodiment, in the step f), the primary screening strains with the sum of the positive mutation amplitudes of the enzyme activities of three primary screening strains with the enzyme activities of more than 65 percent are selected as the secondary screening strains by taking the enzyme activity changes of the alcohol dehydrogenases I and II and the aldehyde dehydrogenase as indexes.
In a preferred embodiment, after obtaining the rescreened strain, the method further comprises the step of measuring the flavor substances of the rescreened strain obtained by horizontal fermentation in a laboratory shake flask. The specific method comprises the following steps:
p) carrying out variable-temperature expansion culture on the strains, and standing to obtain the fermented mud. Inoculating to the wort, and sealing.
And q) standing and culturing for a period of time until the sugar degree is reduced and the primary fermentation is finished.
r) standing and fermenting for a period of time, and finishing post-fermentation.
s) detecting the content of main flavor substances such as acetaldehyde in the fermentation liquor by using headspace gas chromatography, wherein the internal standard is 3-heptanone.
In a preferred embodiment, in step p), the inoculum size is 1X 104-1×108CFU/mL；
The sealing mode is fermentation plug sealing and water liquid sealing.
In a preferred embodiment, in step q), the static culture conditions are 5-20 ℃ for 5-15 d;
the end of the pre-fermentation is marked by a reduction in sugar to 3-8 ° Bx.
In a preferred embodiment, in step r), the static culture conditions are 0-8 ℃ and 5-15d of static culture.
In a preferred embodiment, in step s), the host flavor comprises acetaldehyde, ethyl acetate, isoamyl acetate, n-propanol, isobutanol, and isoamyl alcohol;
the concentration of the 3-heptanone is 10-50 mg/L.
In a preferred embodiment, the acetaldehyde yield of the obtained low acetaldehyde producing Saccharomyces cerevisiae is reduced by more than 50%, and the main flavor has no significant difference from the originally screened Saccharomyces cerevisiae strain.
EXAMPLE 1 establishment of mutagenesis conditions
The cells in log phase were washed by centrifugation and diluted. 10 μ L of the suspension was applied to a sterile metal slide (8mm) and the yeast cells were mutagenized with a helium plasma beam. Maintaining helium flow rate QHe15.0L/min, input power of 100W, processing distance of 2mm and processing temperature of less than 40 ℃. To obtain optimal mutagenesis conditions, treatment times were set at 0, 50, 70, 90, 110 and 130s, and the metal slides were placed in 1mL of sterile saline and washed with shaking. And (3) coating the diluted bacterium liquid on a YPD culture medium, culturing at 30 ℃ for 48h, drawing a lethality curve, and determining the optimal irradiation time. If the fatality rate is low, the saccharomyces cerevisiae cannot be effectively improved; if the fatality rate is too high, the performance of the strain is easily changed greatly, so that the main flavor of the finished beer is influenced. Thus, the strain lethality is maintained between 80-90%, and the optimal mutagenesis time is selected to be 90 s.
Example 2 Primary screening Breeding Process and Effect
Absorbing a proper amount of mutant strain suspension, coating the mutant strain suspension on a resistance plate with acetaldehyde concentration of 2.0g/L, and culturing at 30 ℃ for 72 h. Selecting the strain with the growth dominance, placing the strain in a liquid culture medium containing 3.0g/L acetaldehyde for domestication, and culturing for 48h at 30 ℃. The domesticated strain was washed for the next round of mutagenesis, and the procedure of strain mutagenesis-plate screening-liquid domestication was repeated three times.
Absorbing a proper amount of mutant strain bacterial suspension, coating the mutant strain bacterial suspension on a resistance plate which takes ethanol as a unique carbon source and has the disulfiram concentration of 0.2mg/L, and culturing for 96h at 30 ℃. Selecting the strain with the growth dominance, putting the strain into a liquid culture medium containing 10g/L ethanol and 2.0mg/L disulfiram for domestication, and culturing for 72 hours at 30 ℃. The domesticated strain was washed for the next round of mutagenesis, and the procedure of strain mutagenesis-plate screening-liquid domestication was repeated three times.
TABLE 1 number of mutant strains in different rounds of screening of plates
Note: the colony numbers are represented by the following symbols: + ++ (≧ 50), + ++ (15.50), + + (5-15), + (0-5), - (none).
TABLE 2 OD of different rounds of domestication liquid600
Note: light absorption OD of bacterial liquid600Expressed in the following notation: + + + + + + (≧ 6.0), + + + + (2.0-6.0), + (0.5-2.0), + (0-0.5), (-none).
The results show that after three rounds of selection, the mutant strain can grow in a resistant plate with an acetaldehyde concentration of 2.6g/L and an acclimatized solution with an acetaldehyde concentration of 3.6 g/L. The mutant strain can be grown in a resistant plate with a disulfiram concentration of 0.4mg/L and an acclimatization solution of 2.2 mg/L. And coating the third round of domesticated bacteria liquid on a screening plate to obtain 20 primary screening strains with low acetaldehyde yield.
EXAMPLE 3 screening of Key enzyme Activity in acetaldehyde metabolism
Determination of key enzyme activity: three key enzyme activities of the alcohol dehydrogenase I, II and the acetaldehyde dehydrogenase of the original strain and the primary screening strain are respectively measured, and the sum of positive mutation amplitudes of the three enzyme activities is used as a re-screening index. Centrifuging to collect yeast cells, washing with phosphate buffer (pH 8.0, concentration of 0.05mol/L), resuspending, ultrasonically breaking cells (breaking for 4s and stopping for 5s, totally 5min), centrifuging to obtain leaching solution, and storing at 4 deg.C for use.
Alcohol dehydrogenase I: selecting 250 mu L enzyme activity reaction system (0.05mol/L glycine-potassium hydroxide buffer solution (pH 9.0), 0.001mol/L NAD)+，1×10-4mol/L acetaldehyde solution) into a 96-well plate. Acetaldehyde was used as a reaction substrate, and 50. mu.L of the cell extract was added. Ultraviolet spectrophotometry adopts single cell time scanning, and records the time within 5minRecord the absorbance value at 340 nm.
Alcohol dehydrogenase II: 250 mu L of enzyme activity reaction system (0.05mol/L glycine-potassium hydroxide buffer solution (pH 9.0) buffer solution, 0.001mol/L NADH, 0.1mol/L ethanol solution) is added into a 96-well plate. Ethanol was used as a reaction substrate, and 50. mu.L of the cell extract was added. And (3) scanning the ultraviolet spectrophotometry by adopting a single cell for 5min continuously, and recording the absorbance value at 340 nm.
Acetaldehyde dehydrogenase: selecting 300 mu L enzyme activity reaction system (0.15mol/L potassium phosphate buffer solution (pH7.5), 0.001mol/L NAD)+，5×10-4mol/L dithiothreitol solution, 1X 10-4mol/L acetaldehyde solution) was added to the cuvette. Acetaldehyde was used as a reaction substrate, and 10. mu.L of the cell extract was added. And (3) scanning the ultraviolet spectrophotometry by adopting a single cell for 5min continuously, and recording the absorbance value at 340 nm.
The absorbance value of NADH increased by 0.001 activity units (U) at 340 nm. In this example, the enzyme activity was expressed as the total activity value divided by the mass of the corresponding yeast paste (unit U/mg), and the key enzyme activity was measured for 20 yeasts obtained from the preliminary screening, using the rate of change of the enzyme activity as the screening index.
TABLE 3 measurement results of key enzyme activity
Primary screening of key enzyme activity and change rate of strain
The results show that the positive mutation rate of various key enzyme activities of the primary screened strains all reaches more than 70%, and the primary screened strains with the sum of the positive mutation amplitudes of three enzyme activities of more than 65% are taken as secondary screened strains to obtain 3 secondary screened strains in total.
EXAMPLE 4 preparation of wort Medium
At 45 ℃, water is added for feeding according to the material-water ratio of 1: 4. Heating to 48 deg.C, and maintaining the temperature for 30 min; heating to 65 deg.C, and maintaining for 40 min; heating to 72 deg.C, and maintaining for 10 min; heating to 78 deg.C, maintaining for 10min, and saccharifying. Filtering while hot, boiling for 1 hr, and adding flos Lupuli (0.25 ‰). Adjusting the concentration to 12 ° P to obtain clarified wort.
Example 5 laboratory Shake flask horizontal fermentation validation
Single colonies of the 3 rescreened strains were picked from the plates and inoculated into 1mL of wheat juice medium for seed culture at 25 ℃ and 220rpm for 12 h. The 1mL of the above-mentioned bacterial suspension was transferred to 9mL of wort medium and cultured at 20 ℃ and 220rpm for 12 hours. Then 10mL of the bacterial solution was transferred to 90mL of the corresponding wort medium and cultured at 15 ℃ and 220rpm for 12 hours. Standing to obtain fermented mud, inoculating to wheat juice (1 × 10)6CFU/mL). And (4) buckling a fermentation plug and sealing in liquid. Standing at 12 deg.C for 8 days, and finishing primary fermentation when sugar degree is about 4 ° Bx. Standing at 4 deg.C for 7d, fermenting to maturity, and finishing post-fermentation.
Example 6 measurement of Main flavor substances such as acetaldehyde
Taking 4.5mL of beer fermentation liquor, adding 0.5mL of 300 mg/L3-heptanone, and measuring by adopting a headspace gas chromatograph. Headspace injector equilibrium temperature: 70 ℃, equilibration time: and (3) 30 min. Transmission line temperature: 130 ℃, sample injection time: 0.04min, injection port temperature: 200 ℃, detector temperature: at 250 ℃ to obtain a mixture. Initial temperature of chromatographic column: the temperature is programmed to 180 ℃ at 40 ℃ and 10 ℃/min. Column flow rate: 1.2mL/min, N2Flow rate: 30mL/min, H2Flow rate: 47mL/min, air flow: 400 mL/min.
TABLE 4 Main flavor index/(mg/L) in the wine sample at the end of fermentation
The results show that: the acetaldehyde content in the mutant strain fermentation liquor is reduced by more than 50%, and the main flavor has no obvious difference from the original strain.
Claims (10)
1. The efficient breeding method of the low acetaldehyde producing beer yeast strain is characterized by comprising the following steps:
carrying out ARTP mutagenesis treatment on the beer yeast strain to construct a mutant strain library;
screening the mutant strain library by using an ethanol-disulfiram resistance plate and a high-concentration acetaldehyde screening plate to obtain a growth dominant strain, domesticating the growth dominant strain by using a liquid domestication culture medium, and establishing a mutagenic-screening-domesticated primary screening strain;
and carrying out fermentation culture on the obtained primary screening strain, and carrying out secondary screening by using the enzyme activity changes of the ethanol dehydrogenases I and II and the acetaldehyde dehydrogenase as indexes to obtain a secondary screening strain, namely the low-acetaldehyde-production beer yeast.
2. The efficient breeding method according to claim 1, wherein the ARTP mutagenesis treatment on the Saccharomyces cerevisiae strain is specifically:
the cells cultured to mid-log phase were diluted to 1X 106Per mL, yeast cells were subjected to ARTP mutagenesis treatment using a helium ion beam.
3. The efficient breeding method according to claim 2, wherein the ARTP mutagenesis treatment conditions are as follows:
helium gas flow rate 15.0L/min, input power 100W, treatment distance 2mm, treatment temperature < 40 ℃ and mutagenesis time 90 s.
4. The efficient breeding method according to claim 1, wherein the establishment of the mutagenic-screening-domesticated primary screening strain specifically comprises:
1) carrying out streak culture on beer yeast strains in the mutant strain library, selecting a single colony to grow in a YPD culture medium, centrifuging the thallus, washing and resuspending to obtain a cell suspension;
2) performing ARTP mutagenesis treatment on the cell suspension to obtain a mutagenesis bacterial liquid;
3) respectively coating the mutagenic bacterium liquid on a high-concentration acetaldehyde flat plate and an ethanol-disulfiram flat plate, carrying out constant-temperature culture, and selecting the growth dominant strains on the flat plates to be respectively subjected to continuous domestication in domestication culture media corresponding to the flat plates;
4) respectively sucking proper amounts of domesticated bacteria liquid, repeating the steps 2) -3) for at least 2-6 rounds, increasing the screening concentration and the domesticated culture medium concentration of each round of plates, and respectively coating the final domesticated bacteria liquid on two plates to obtain a primary screening strain.
5. The efficient breeding method according to claim 4, wherein in the step 1), the growth temperature of the colony is 25-35 ℃, and the culture time is 6-48 h;
the cells were centrifuged, washed, resuspended in sterile physiological saline containing 1-20% glycerol, and diluted to 1X 104-1×108one/mL.
6. The efficient breeding method according to claim 4, wherein in the step 2), the cell suspension is taken in an amount of 1-20 μ L;
the ARTP mutagenesis treatment conditions are as follows: helium gas flow rate of 5-45L/min, input power of 70-120W, treatment distance of 1-4mm, treatment temperature below 60 deg.C and mutagenesis time of 30-180 s.
7. The efficient breeding method according to claim 4, wherein in the step 3), the amount of the mutagenized bacterium liquid absorbed is 5-200 μ L;
the high-concentration acetaldehyde plate is a YPD culture medium containing 0.1-10g/L acetaldehyde, and the ethanol-disulfiram plate is a basic carbon source culture medium containing 0-50g/L ethanol and 0-5mg/L disulfiram;
the constant culture temperature is 20-40 deg.C, and the culture time is 1-5 days;
the high-concentration acetaldehyde acclimation culture medium is an YPD culture medium containing 0.1-10g/L acetaldehyde;
the ethanol-disulfiram domestication culture medium is a basic carbon source culture medium containing 0-50g/L of ethanol and 0-10mg/L of disulfiram;
the domestication temperature is 20-40 deg.C, and the domestication time is 1-5 days.
8. The efficient selective breeding method according to claim 1, wherein the primary screening strains with the sum of positive mutation amplitudes of enzyme activities of three primary screening strains with the sum of positive mutation amplitudes of enzyme activities of more than 65% are selected as secondary screening strains by taking the enzyme activity changes of the alcohol dehydrogenases I and II and the aldehyde dehydrogenase as indexes.
9. The efficient breeding method according to claim 1, further comprising the step of measuring the flavor substances of the obtained re-screened strain by laboratory shake flask horizontal fermentation after the re-screened strain is obtained.
10. The efficient selective breeding method according to claim 9, wherein the acetaldehyde yield of the obtained low acetaldehyde producing Saccharomyces cerevisiae is reduced by more than 50%, and the main flavor of the obtained low acetaldehyde producing Saccharomyces cerevisiae has no significant difference from that of the originally screened Saccharomyces cerevisiae strain.
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