阅读说明：本技术 低产乙醛啤酒工业酵母的高通量筛选方法 (High-throughput screening method for low-acetaldehyde-yield beer industrial yeast ) 是由 董建军 赵鑫锐 尹花 李江华 陈坚 堵国成 孙可澄 陈璐 于 2021-01-08 设计创作，主要内容包括：本发明提出一种低产乙醛啤酒工业酵母的高通量筛选方法,属于发酵工程和微生物育种的技术领域。本发明通过对啤酒工业酵母进行～(60)Coγ诱变育种,获得庞大的突变菌株库；随后通过高浓度乙醛及乙醇-双硫仑平板及其驯养液对突变菌株进行平板筛选和长期驯化,将驯养菌液涂布于筛选平板,获得初筛菌株；再通过低乙醛合成及强乙醛代谢的高通量复筛手段,获得低产乙醛啤酒工业酵母。经摇瓶水平啤酒模拟酿造体系的验证,突变菌株的乙醛产量降低了60％以上,且发酵液主体风味与出发菌株无异。本发明解决了目前对低产乙醛的啤酒工业酵母筛选难度大、效率低的问题。(The invention provides a high-throughput screening method of beer industrial yeast with low acetaldehyde yield, belonging to the technical field of fermentation engineering and microbial breeding. The invention is carried out by carrying out the steps on the yeast in the beer industry 60 Performing Co gamma mutation breeding to obtain a huge mutant strain library; then, carrying out plate screening and long-term domestication on the mutant strain through a high-concentration acetaldehyde and ethanol-disulfiram plate and a domestication solution thereof, and coating the domestication bacterial solution on a screening plate to obtain a primary screening strain; and then the beer industrial yeast with low acetaldehyde yield is obtained by a high-flux re-screening means of low acetaldehyde synthesis and strong acetaldehyde metabolism. Proved by a shake flask horizontal beer simulated brewing system, the acetaldehyde yield of the mutant strain is reduced by more than 60 percent, and the main body wind of the fermentation liquor isThe smell is the same as that of the original strain. The invention solves the problems of high screening difficulty and low efficiency of the beer industrial yeast with low acetaldehyde yield at present.)

1. The high-throughput screening method of the beer industrial yeast with low acetaldehyde yield is characterized by comprising the following steps:

carrying out on yeast in the beer industry⁶⁰Performing Co gamma mutagenesis treatment to construct a mutant strain library;

screening mutant strains by using a high-concentration acetaldehyde plate and an ethanol-disulfiram plate, performing long-term domestication on the mutant strains by using liquid domestication culture media corresponding to the plates, and respectively coating domestication bacteria liquid on the plates to obtain primary-screened strains;

and (3) carrying out high-flux re-screening on the primary screened strain by utilizing the low acetaldehyde synthesis capacity and the strong acetaldehyde metabolic capacity to obtain a re-screened strain, namely the beer industrial yeast with low acetaldehyde yield.

2. High throughput screening method according to claim 1, wherein obtaining the primary screened strain comprises in particular the steps of:

1) carrying out streak culture on beer industrial yeast strains in a 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) subjecting a cell suspension to⁶⁰Co gamma irradiation, coating the irradiated bacterial liquid on a high-concentration acetaldehyde plate and an ethanol-disulfiram plate immediately, and culturing at constant temperature;

3) selecting the growth dominant strains on the flat plates to be respectively and continuously domesticated in domestication culture media corresponding to the flat plates, and gradually increasing the screening concentration of acetaldehyde and disulfiram in each flat plate in the domestication process;

4) and respectively sucking a proper amount of domesticated bacteria liquid and coating the domesticated bacteria liquid on the flat plates to obtain primary screening strains.

3. The high throughput screening method according to claim 2, wherein in the step 1), the growth temperature of the colony is 25-35 ℃ and the culture time is 6-20 h;

the cells were centrifuged, washed, resuspended in sterile physiological saline containing 1-20% glycerol, and diluted to 1X 10⁴-1×10⁸one/mL.

4. The high throughput screening method according to claim 2, wherein in the step 2), an irradiation dose of 0-2.0kGy is selected, and 5-200 μ L of irradiated bacterial liquid is absorbed and coated on the screening plate;

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 temperature culture temperature is 20-40 deg.C, and the culture time is 12-120 h.

5. The high-throughput screening method according to claim 2, wherein the high-concentration acetaldehyde acclimation medium is a YPD medium containing 0.1-10g/L of 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 5-70 d.

6. The high throughput screening method according to claim 1, wherein the high throughput screening method for low acetaldehyde synthesis capacity comprises:

washing and re-suspending the primary screening strain cultured to the middle logarithmic phase with normal saline, and diluting to OD₆₀₀At time 5, a 96 deep well cell culture plate containing a basal carbon source medium containing 5g/L ethanol was inoculated at an inoculum size of 1%, sealed with a 6mm rubber seal cap, cultured at 30 ℃ and 200rpm, and periodically sampled every 24 hours to detect acetaldehyde production.

7. The high-throughput screening method according to claim 1, wherein the high-throughput screening method for strong acetaldehyde metabolic capacity is specifically:

washing the primary-screened strain cultured to the middle logarithmic phase with normal saline, centrifuging, discarding supernatant, adding 100mg/L acetaldehyde standard solution to ensure OD of 60 μ L solution₆₀₀Sampling every 30min at 0.15, and detecting the residual quantity of acetaldehyde.

8. The high throughput screening method of claim 1, further comprising the step of determining the flavor of the re-screened strain obtained by horizontal fermentation in a laboratory shake flask after obtaining the re-screened strain.

9. The efficient selective breeding method according to claim 8, wherein the acetaldehyde yield of the obtained low-acetaldehyde beer industry yeast is reduced by more than 60%, and the main flavor of the yeast is not significantly different from that of the beer industry yeast strain originally screened.

Technical Field

The invention belongs to the technical field of fermentation engineering and microbial breeding, and particularly relates to a high-throughput screening method of beer industrial yeast with low acetaldehyde yield.

Background

Beer is one of three ancient wines in the world, originates in the middle east and ancient Egypt areas more than nine thousand years ago, and is transferred to China in the early twentieth century. At present, China has become a world-wide beer producing and selling country. Along with the development of the beer industry in China becoming mature and the gradual expansion of the beer market, the flavor and the stability are always hot spots for the research of the beer industry.

Acetaldehyde (CH)₃CHO) is a highly volatile, highly reactive, low molecular weight carbonyl compound in beer, directly affecting the flavor and flavor stability of beer. The boiling point is 21 ℃ at normal temperature and normal pressure, the water-soluble organic silicon dioxide is easy to mix with water, and reversible equilibrium exists. In addition, acetaldehyde can react with a large number of nucleophiles and is easy to undergo condensation reaction with other aldehydes, ketones and ester compounds in beer. The proper amount of acetaldehyde can endow beer with unique quality characteristics, and if the content of acetaldehyde is too high, unpleasant tastes of green grass, rotten apples and the like are easily generated; if the storage time is too long, even aging flavor appears.

Factors influencing the acetaldehyde content in the finished beer include raw and auxiliary materials, beer industrial yeast, brewing process, storage and logistics conditions and the like. In long-term industrial practice, researchers have proposed measures to control the acetaldehyde content of beer. However, the control production process has no general applicability, the constructed yeast engineering bacteria do not meet the food safety standard, and the problem of beer flavor is difficult to completely solve.

Beer industryIndustrial yeasts fundamentally determine the formation and accumulation of acetaldehyde, a flavor substance, in beer products. During the fermentation process, the yeast can make the fermentable sugar pass through EMP pathway to generate pyruvate, and acetaldehyde and CO are formed under the action of pyruvate decarboxylase₂. Most of acetaldehyde is reduced into ethanol by the action of yeast alcohol dehydrogenase 1, and the ethanol can be oxidized into acetaldehyde by the action of alcohol dehydrogenase 2; a small amount of acetaldehyde is oxidized to acetic acid by acetaldehyde dehydrogenase. Therefore, screening of excellent low acetaldehyde-producing industrial yeast strains for beer will become one of the ways to effectively solve the problems of beer flavor stability and beer food safety.

The screening indexes of the mutant strain mainly comprise key enzyme activity change and free acetaldehyde content. The breeding of the low acetaldehyde beer industry yeast strain is the beer industry yeast strain with reduced acetaldehyde yield of the screened metabolite. Therefore, accurate determination of acetaldehyde production from starting and improved strains is a necessary means for successful seed selection.

At present, beer production enterprises generally perform fermentation culture in test tubes, shake flasks and EBC tubes by using a multi-purpose screening culture medium, and detect the content of free acetaldehyde by using Gas Chromatography (GC), High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR). The strain culture workload is large and the efficiency is low; the GC method and the HPLC method need to carry out solid phase micro-extraction or derivatization treatment on a sample; the nuclear magnetic resonance apparatus relied on by the NMR technique is expensive, which directly leads to the rise of the detection cost. Therefore, a set of simple, low-cost and high-throughput screening models belonging to the low-acetaldehyde-production beer industry yeast strains needs to be established.

Disclosure of Invention

The invention provides a high-throughput screening method of beer industrial yeast with low acetaldehyde yield, which solves the problems of high screening difficulty and low efficiency of the existing beer industrial yeast with low acetaldehyde yield.

In order to achieve the aim, the invention provides a high-throughput screening method of beer industrial yeast with low acetaldehyde yield, which comprises the following steps:

carrying out on yeast in the beer industry⁶⁰Performing Co gamma mutagenesis treatment to construct a mutant strain library;

screening mutant strains by using a high-concentration acetaldehyde plate and an ethanol-disulfiram plate, performing long-term domestication on the mutant strains by using liquid domestication culture media corresponding to the plates, and respectively coating domestication bacteria liquid on the plates to obtain primary-screened strains;

and (3) carrying out high-flux re-screening on the primary screened strain by utilizing the low acetaldehyde synthesis capacity and the strong acetaldehyde metabolic capacity to obtain a re-screened strain, namely the beer industrial yeast with low acetaldehyde yield.

Preferably, the obtaining of the primary screening strain specifically comprises the following steps:

1) carrying out streak culture on beer industrial yeast strains in a 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) subjecting a cell suspension to⁶⁰Co gamma irradiation, coating the irradiated bacterial liquid on a high-concentration acetaldehyde plate and an ethanol-disulfiram plate immediately, and culturing at constant temperature;

3) selecting the growth dominant strains on the flat plates to be respectively and continuously domesticated in domestication culture media corresponding to the flat plates, and gradually increasing the screening concentration of acetaldehyde and disulfiram in each flat plate in the domestication process;

4) and respectively sucking a proper amount of domesticated bacteria liquid and coating the domesticated bacteria liquid on the flat plates to obtain primary screening strains.

Preferably, in the step 1), the growth temperature of the colony is 25-35 ℃, and the culture time is 6-20 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 irradiation dose of 0-2.0kGy is selected, 5-200 mu L of irradiation bacterium liquid is absorbed and coated on a screening plate;

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 temperature culture temperature is 20-40 deg.C, and the culture time is 12-120 h.

Preferably, the high-concentration acetaldehyde acclimation medium is YPD 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 5-70 d.

Preferably, the high-throughput screening method with low acetaldehyde synthesis capacity specifically comprises the following steps:

washing and re-suspending the primary screening strain cultured to the middle logarithmic phase with normal saline, and diluting to OD₆₀₀At time 5, a 96 deep well cell culture plate containing a basal carbon source medium containing 5g/L ethanol was inoculated at an inoculum size of 1%, sealed with a 6mm rubber seal cap, cultured at 30 ℃ and 200rpm, and periodically sampled every 24 hours to detect acetaldehyde production.

Preferably, the high-throughput screening method for the strong acetaldehyde metabolic capacity specifically comprises the following steps:

washing the primary-screened strain cultured to the middle logarithmic phase with normal saline, centrifuging, discarding supernatant, adding 100mg/L acetaldehyde standard solution to ensure OD of 60 μ L solution₆₀₀Sampling every 30min at 0.15, and detecting the residual quantity of acetaldehyde.

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-production beer industry yeast is reduced by more than 60%, and the main flavor of the yeast is not obviously different from that of the beer industry yeast strain which is originally screened.

Compared with the prior art, the invention has the advantages and positive effects that:

the invention is carried out by carrying out the steps on the yeast in the beer industry⁶⁰Performing mutation breeding on Co gamma to obtain a mutant strain library; then, carrying out plate screening and long-term domestication on the mutant strain through a high-concentration acetaldehyde and ethanol-disulfiram plate and a domestication solution thereof, and coating the domestication bacterial solution on a screening plate to obtain a primary screening strain; and then the beer industrial yeast with low acetaldehyde yield is obtained by a high-flux re-screening means of low acetaldehyde synthesis and strong acetaldehyde metabolism. Through the horizontal of the flaskThe beer simulated brewing system verifies that the acetaldehyde yield of the mutant strain is reduced by more than 60 percent, and the main flavor of the fermentation liquor is not different from that of the original strain, so that the beer simulated brewing method has higher industrial application value.

Drawings

FIG. 1 is a schematic diagram of an ethanol-disulfiram domesticating solution optimization process provided by an embodiment of the invention;

FIG. 2 is a schematic diagram of a process for optimizing the sealing performance of a re-screening experiment provided by an embodiment of the invention;

FIG. 3 is a schematic diagram of the interference of the color reaction of different media provided by the embodiment of the present invention;

FIG. 4 is a schematic diagram of the process for optimizing the conditions of the low acetaldehyde synthesis method according to the embodiment 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-throughput screening method of low-acetaldehyde-yield beer industrial yeast, which comprises the following steps:

carrying out on yeast in the beer industry⁶⁰Performing Co gamma mutagenesis treatment to construct a mutant strain library;

screening mutant strains by using a high-concentration acetaldehyde plate and an ethanol-disulfiram plate, performing long-term domestication on the mutant strains by using liquid domestication culture media corresponding to the plates, and respectively coating domestication bacteria liquid on the plates to obtain primary-screened strains;

and (3) carrying out high-flux re-screening on the primary screened strain by utilizing the low acetaldehyde synthesis capacity and the strong acetaldehyde metabolic capacity to obtain a re-screened strain, namely the beer industrial yeast with low acetaldehyde yield.

In the above-mentioned scheme, the first step of the method,⁶⁰the Co gamma mutagenesis treatment specifically comprises the following steps: single colonies of beer industrial yeast strains are picked, inoculated in YPD medium, and cultured at 30 ℃ and 220rpm to the middle logarithmic phase. The mixture is centrifuged and then is processed by centrifugal separation,discarding supernatant, washing with sterile physiological saline for 2 times and resuspending, the diluted bacterial liquid concentration is 1 × 10⁶one/mL. 2mL of the bacterial suspension is placed in a cell culture dish, sealed by a sealing film, irradiated in an irradiation dose of 0.8kGy, and subjected to gamma ray treatment.

In a preferred embodiment, obtaining the primary screening strain specifically comprises the following steps:

1) carrying out streak culture on beer industrial yeast strains in a 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) subjecting a cell suspension to⁶⁰Co gamma irradiation, coating the irradiated bacterial liquid on a high-concentration acetaldehyde plate and an ethanol-disulfiram plate immediately, and culturing at constant temperature;

3) selecting the growth dominant strains on the flat plates to be respectively and continuously domesticated in domestication culture media corresponding to the flat plates, and gradually increasing the screening concentration of acetaldehyde and disulfiram in each flat plate in the domestication process;

4) and respectively sucking a proper amount of domesticated bacteria liquid and coating the domesticated bacteria liquid on the flat plates to obtain primary screening strains.

In a preferred embodiment, in the step 1), the growth temperature of the colonies is 25-35 ℃, and the culture time is 6-20 h; the bacterial cells were centrifuged, washed, resuspended in sterile physiological saline, and diluted to 1X 10⁴-1×10⁸one/mL.

In a preferred embodiment, in the step 2), an irradiation dose of 0-2.0kGy is selected, 5-200 μ L of irradiation bacterium liquid is absorbed and coated on a screening plate;

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 temperature culture temperature is 20-40 deg.C, and the culture time is 12-120 h.

In a preferred embodiment, high concentration acetaldehyde plates are selected for an initial screening concentration of 2.8g/L and ethanol-disulfiram plates are selected for an initial screening condition in which a basal carbon source containing 10g/L ethanol and 0.3mg/L disulfiram is cultured.

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 3), the high-concentration acetaldehyde acclimation medium is a YPD medium containing 0.1-10g/L of 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 5-70 d.

In a preferred embodiment, a basal carbon source liquid medium having an ethanol concentration of 10g/L and a disulfiram concentration of 2.5mg/L is selected as the initial condition for ethanol-disulfiram acclimatization solution, and a YPD liquid medium having an acetaldehyde concentration of 3.7g/L is selected as the initial condition for high-concentration acetaldehyde acclimatization solution.

In a preferred embodiment, the high throughput screening method for low acetaldehyde synthesis capacity is specifically:

washing and re-suspending the primary screening strain cultured to the middle logarithmic phase with normal saline, and diluting to OD₆₀₀At time 5, a 96 deep well cell culture plate containing a basal carbon source medium containing 5g/L ethanol was inoculated at an inoculum size of 1%, sealed with a 6mm rubber seal cap, cultured at 30 ℃ and 200rpm, and periodically sampled every 24 hours to detect acetaldehyde production.

In a preferred embodiment, the high-throughput screening method for strong acetaldehyde metabolic capacity specifically comprises the following steps:

washing the primary-screened strain cultured to the middle logarithmic phase with normal saline, centrifuging, discarding supernatant, adding 100mg/L acetaldehyde standard solution to ensure OD of 60 μ L solution₆₀₀Sampling every 30min at 0.15, and detecting the residual quantity of acetaldehyde.

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 fermented mud; inoculating to the wort, and sealing;

q) standing and culturing for a period of time, and finishing the primary fermentation when the sugar degree is reduced;

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 beer industry yeast is reduced by more than 60%, and the main flavor has no significant difference with the beer industry yeast strain which is originally screened.

EXAMPLE 1 construction and optimization of screening plates

High-concentration acetaldehyde plates and ethanol-disulfiram plates were selected as primary screening plates for the mutant strains.

Ethanol-disulfiram plate: ethanol is selected as the only carbon source, and 5, 10, 20 and 40g/L ethanol and 0, 0.1, 0.3, 0.5, 1.0, 2.0 and 3.0mg/L disulfiram are respectively added into YNB and a basic carbon source culture medium. After washing, the cells were spread on an ethanol-disulfiram screen plate (20mL) and incubated at 30 ℃ for 60 hours. Observing whether the surface of the plate has the strain with growth dominance.

High-concentration acetaldehyde plate: the acetaldehyde concentrations in the plate were set to 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0g/L, respectively. After washing the bacterial liquid, 50 μ L of the liquid was pipetted and applied to the corresponding plate, and the plate was sealed with a sealing film. And (3) standing and culturing at 30 ℃ for 60 hours, and observing the growth condition of colonies. The initial screening concentration of the high concentration acetaldehyde plate was selected to be 2.8 g/L.

TABLE 1 colony counts in YNB-ethanol-disulfiram plates

Note: the colony numbers are represented by the following symbols: ≧ 50 ≧ 15-50, 5-15, 0-5, or none

TABLE 2 number of colonies in the basic carbon source-ethanol-disulfiram plates

Note: the colony numbers are represented by the following symbols: ≧ 50 ≧ 15-50, 5-15, 0-5, or none

TABLE 3 colony counts in high concentration acetaldehyde plates

Note: the colony numbers are represented by the following symbols: ≧ 50, + + + (15-50), + + (5-15), + (0-5), - (none).

Finally, the initial screening condition of using a basic carbon source culture medium containing 10g/L of ethanol and 0.3mg/L of disulfiram as a primary screening plate is determined, and the initial screening concentration of selecting a high-concentration acetaldehyde plate is 2.8 g/L.

EXAMPLE 2 optimization of domestication conditions

Ethanol-disulfiram domestication solution: ethanol is selected as the only carbon source, and 5, 10, 20 and 40g/L ethanol and 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mg/L disulfiram are respectively added into YNB and a basic carbon source liquid culture medium. The bacterial liquid is diluted to OD after being washed by normal saline₆₀₀3mL of ethanol-disulfiram domestication liquid is inoculated according to the inoculation amount of 1 percent to observe the growth condition of the thalli.

As shown in FIG. 1, the growth of the cells in the liquid medium as the basic carbon source was superior to that in the liquid medium YNB. In order to ensure sufficient carbon source supply and prevent the growth inhibition of the thalli by excessive ethanol, a basal carbon source liquid culture medium with the ethanol concentration of 10g/L and the disulfiram concentration of 2.5mg/L is selected as the initial domestication condition of the ethanol-disulfiram domestication liquid.

High-concentration acetaldehyde domestication liquid: in order to obtain the accurate critical concentration of the acetaldehyde culture solution, the concentration of the acetaldehyde standard solution is set to be 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4 and 4.6g/L respectively, and the activated bacteria solution is inoculated to the initial OD₆₀₀About 0.2. After 20h incubation at 30 ℃ and 220rpm, the growth of the strain was recorded at different concentrations.

TABLE 4 establishment of high concentration acetaldehyde domestication liquid

The result shows that when the concentration of the acetaldehyde standard solution is between 3.6 and 3.8g/L, the growth of the thalli begins to be greatly inhibited. YPD liquid medium with acetaldehyde concentration of 3.7g/L was selected as the initial condition for high concentration acetaldehyde domestication liquid.

And finally: taking 50 mu L of irradiation bacterium liquid, respectively coating the irradiation bacterium liquid on a high-concentration acetaldehyde plate with the acetaldehyde content of 2.8g/L and a basic carbon source plate containing 10g/L of ethanol and 0.3mg/L of disulfiram, and culturing at the constant temperature of 30 ℃ for 60 h. Selecting a strain with growth dominance, respectively inoculating a high-concentration acetaldehyde domestication solution with acetaldehyde content of 3.7g/L and a basic carbon source domestication solution containing 10g/L ethanol and 2.5mg/L disulfiram, continuously domesticating at constant temperature of 30 ℃ for 15 days, and continuously increasing the concentration of acetaldehyde and disulfiram in the domestication solution (keeping the concentration of ethanol in the ethanol-disulfiram domestication solution unchanged, increasing the concentration of disulfiram from 2.5mg/L to 2.7mg/L by taking 0.1mg/L as a gradient, and increasing the concentration of acetaldehyde from 3.7g/L to 3.9g/L in the high-concentration acetaldehyde domestication solution by taking 0.1g/L as a gradient).

Example 3 optimization of the tightness of the rescreening experiment

In order to reduce the volatilization of acetaldehyde in the experiment to the maximum extent, the sealing mode, the specification of the pore plate and the number of gun heads in the experiment process are optimized. The results show that, as shown in FIG. 2, the sealability under the same culture conditions: 6mm rubber sealing cap > BioTss sterile transparent sealing film > 96 shallow-hole plate matching sealing cap; the acetaldehyde allowance of the deep hole plate is larger than that of the shallow hole plate, and the sealing performance is better; the gun head has no obvious influence on the volatilization of acetaldehyde in the acetaldehyde standard solution, but effectively prevents the bacterial sedimentation.

EXAMPLE 4 selection of chromogenic reagents

Preparing acetaldehyde standard solutions with different concentrations, and reacting with Schiff reagent, potassium nitrosoferricyanide and 3-methyl-2-benzothiazolone hydrazone respectively to develop color.

Schiff's reagent method: preparing 0-2000mg/L acetaldehyde standard solution, sucking 5mL solution into clean dry centrifuge tube, adding 1mL Schiff reagent, adding plug, shaking, placing in 20 deg.C water bath, keeping in dark place for 20min, and measuring absorbance OD₅₂₀。

Sodium nitrosoferricyanide: preparing 0-2000mg/L acetaldehyde standard solution, sucking 5mL solution into a clean and dry colorimetric tube, adding 0.5mL, 1% sodium nitroferricyanide solution and 0.5mL, 1mol/L sodium hydroxide solution, keeping in the dark in a water bath at 20 ℃ for 20min, and measuring the light absorption value at 520 nm.

3-methyl-2-benzothiazolone hydrazone method: preparing an acetaldehyde standard solution with the concentration of 0-1000mg/L, sucking 0.5mL of the acetaldehyde standard solution into a clean and dry colorimetric tube, adding 1mL of 0.04g/L MBTR solution, mixing and standing for 20 min; adding 1mL of 0.1g/L ferric chloride solution, mixing and standing for 10 min; 2.5mL of deionized water was added, mixed well, and absorbance was measured at 610 nm.

The high-throughput detection method for the reaction and color development of the 3-methyl-2-benzothiazolone hydrazone and the acetaldehyde comprises the following steps: sucking 25 mu L of sample into a 96-well cell culture plate, adding 50 mu L of 0.04g/L MBTR solution, mixing and standing for 20 min; adding 50 μ L of 0.1g/L ferric chloride solution, mixing and standing for 10 min; adding 125. mu.L deionized water, mixing well, and measuring absorbance at 610 nm.

TABLE 5 establishment of color reagent

The result shows that the precision of the 3-methyl-2-benzothiazolone hydrazone method is relatively good, the color change is relatively obvious under the condition of detection limit, and the detection limit is relatively lowest among the three spectrophotometric methods. When the fermentation is finished, the acetaldehyde content in the beer is basically maintained at 2-20mg/L, the normal linear range of the 3-methyl-2-benzothiazolone hydrazone is wide, and the acetaldehyde content in the beer can be accurately measured. Therefore, 3-methyl-2-benzothiazolone hydrazone was finally determined as a coloring agent.

Example 5 optimization of Low acetaldehyde Synthesis-high throughput screening method

1) Optimization of the culture medium:

in order to prevent the influence of each component in the culture medium on the color development of the final reaction, the color development reaction of 3-methyl-2-benzothiazolone hydrazone was performed on each of the 8 solutions. As shown in FIG. 3, a basal carbon source medium containing 5% ethanol with little basal interference was selected as a medium for the 3-methyl-2-benzothiazolone hydrazone-low acetaldehyde synthesis method.

2) Optimization of sampling time and inoculation amount: the basal carbon source medium containing 5g/L ethanol was aspirated and added to a 96-well plate. Washing, resuspending and diluting the bacterial liquid to OD₆₀₀And (5), inoculating the strain into a 96-deep-well plate according to the inoculum sizes of 1%, 2% and 3% respectively. Sampling is carried out periodically, and the acetaldehyde generation is detected. As shown in FIG. 4, 1% OD was added to the culture medium at 30 ℃ and 200rpm₆₀₀The sampling time points were determined to be 24, 36, 48, 72, 96, 120h for 5 bacteria solutions.

High throughput screening of low acetaldehyde synthesis capacity beer industry yeast:

washing and re-suspending the primary screening strain cultured to the middle logarithmic phase with normal saline, and diluting to OD₆₀₀At 5, a 96 deep well cell culture plate containing a basal carbon source medium of 5g/L ethanol was inoculated at an inoculum size of 1%, a tip was added, and sealed with a 6mm rubber seal cap. Sampling is carried out periodically every 24h, and the acetaldehyde generation is detected.

The detection method comprises the following steps: sucking 25 μ L of sample into 96-well cell culture plate, adding 50 μ L of 0.04 g/L3-methyl-2-benzothiazolone hydrazone solution, mixing and standing for 20 min; adding 50 μ L of 0.1g/L ferric chloride solution, mixing and standing for 10 min; adding 125. mu.L deionized water, mixing well, and measuring absorbance at 610 nm.

The final experimental result shows that the acetaldehyde synthesis amount of the mutant strains is reduced to a certain extent, wherein the mutant strain with the largest reduction amplitude reaches 54.17%, and the mutant strain with the most obvious reduction amplitude effect is subjected to shake flask verification.

Example 6 optimization of Strong acetaldehyde metabolism-high throughput screening method

In order to effectively prevent dispersion of acetaldehyde, the concentration of the acetaldehyde standard solution is optimized. 20, 40, 60, 80, 100, 120, 140, 160, 180 and 200mg/L of acetaldehyde standard solution are added respectively. Sampling is carried out periodically, the volatilization of acetaldehyde is observed, and finally the initial concentration of the acetaldehyde solution is determined to be 100 mg/L.

TABLE 6 Strong acetaldehyde Metabolic method Condition optimization

High-throughput screening of beer industry yeast with strong acetaldehyde metabolic capacity:

and (4) washing the primary screening strain cultured to the middle logarithmic phase with normal saline, centrifuging and removing the supernatant. Adding 100mg/L acetaldehyde standard solution to ensure OD of 60 μ L solution₆₀₀Is 0.15. Sampling every 30min to detect the residual acetaldehyde.

The detection method comprises the following steps: sucking 25 μ L of sample into 96-well cell culture plate, adding 50 μ L of 0.04 g/L3-methyl-2-benzothiazolone hydrazone solution, mixing and standing for 20 min; adding 50 μ L of 0.1g/L ferric chloride solution, mixing and standing for 10 min; adding 125. mu.L deionized water, mixing well, and measuring absorbance at 610 nm.

The final experimental result shows that the acetaldehyde metabolism amount of the mutant strain is increased to a certain extent, wherein the mutant strain with the largest amplification degree reaches 24.42%, and the mutant strain with the most obvious amplification effect is subjected to shake flask verification.

EXAMPLE 7 preparation of wort Medium

At 45 ℃, the ratio of material to water is 1: 4 adding water and feeding. 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 8 laboratory Shake flask horizontal fermentation validation

Single colonies of the 2 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 9 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 7 Main flavor index/(mg/L) in the wine sample at the end of fermentation

The main flavor substances (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 over 60 percent, and the main flavor has no obvious difference with the original strain.