Flavor is the main quality characteristic for beer and requires the flavor-active components presented as raw materials or developed by yeast metabolism to be controlled within a certain range in order to maintain flavor balance.
During the shelf life of beer, numerous reactions take place, resulting in a decrease of fresh flavor notes and the appearance of typical aged flavors . Hence, improving the flavor stability of beer during its shelf life is of great concern for brewers as it is important for a commercial beer to have a consistent sensory experience and satisfy the expectations of consumers at all times.
Aldehydes such as (E)-2-nonenal, 5-hydroxymethylfurfural, hexanal, and acetaldehyde are characterized as the aged flavor components in beer, and higher concentrations of these aldehydes greatly impair flavor stability . Thus, many strategies have been applied to reduce the production of these aldehydes during industrial manufacturing, for example, reducing the aeration levels of wort, limiting the time of boiling and cooling, and increasing the inoculation volume of cells . Notably, the natural reducing activity of yeast can also facilitate the reduction of these components .
There are many powerful methods to create microbial mutations, of which atmospheric and room temperature plasma (ARTP) mutagenesis is the new developed technology with the advantages of high efficiency, consistency, nontoxicity, environmental-friendliness, and low cost . Therefore, generating non-genetically modified mutant yeast cells with this method would be a good choice. In addition, prior findings suggested that the uncoupler 2,4-dinitrophenol (DNP) could block nicotinamide adenine dinucleotide hydride (NADH) oxidation and affect cell growth ; therefore, it might be a promising selective marker for NADH perturbation varients.
2. DNP Serves as a Selective Marker for the NADH Perturbation Variants
To explore the optimal ARTP treatment time for YJ-002, the death rates of YJ-002 at different treatment times were assessed. The death rate of YJ-002 reached 87.2% after treatment for 45 s, but reached 95.4% when treated for 60 s (Figure 1). Thus, 45 s was chosen as the optimal treatment time and used in subsequent ARTP mutagenesis studies.
Figure 1. The death rate of YJ-002 at different times of ARTP treatment.
Then, three runs of ARTP mutagenesis were performed on strain YJ-002 and 172 colonies were obtained from the YPD agar plate containing 0.10 mM DNP. To investigate whether DNP could serve as a selective marker for NADH perturbation variants, the mutants were cultured in YPD media for 2 days and the cells were collected to measure the cellular NADH/NAD+ ratio. Of these 172 mutants, 142 strains exhibited higher NADH/NAD+ ratios than the parental strain YJ-002 with the values from 0.180 to 0.329, while 30 mutants exhibited lower NADH/NAD+ ratios with the values from 0.150 to 0.180 (Figure 2). This result suggested that DNP could effectively serve as a selective marker for NADH perturbation variants, especially for mutants with higher NADH levels, with the positive rate approximately at 82.6%.
Figure 2. Heatmap for the NADH/NAD+ ratios of the mutants selected from the DNP plate. The NADH/NAD+ ratio of YJ-002 was 0.180. The strains with an increased NADH level are marked in red, and those with a decreased value are marked in green.
3. Screening the Optimal Strain with Industrial Potential
To screen an optimal strain with improved flavor stability as well as industrial potential, several indices were considered (Figure 3
a). Lab-scale fermentation was performed using the 142 mutants with an increased NADH/NAD+
ratio, and the TBA method was first employed to assess the flavor stability of the fermentation liquid. A total of 126 strains exhibited improved flavor stability (Figure 3
b). Of these 126 strains, 108 strains showed great improvements in flavor stability, with the reduction rates of TBA values exceeding 10% (Figure 3
c). To continue the screening process, 62 strains with TBA reduction rates of over 40% were chosen for next selection (Figure 3
a). Ethanol is the main product obtained from yeast during alcoholic fermentation, and lower ethanol production generally indicates incomplete carbohydrate utilization resulting in greater economic losses to breweries 
Figure 3. Screening an optimal strain with improved flavor stability. (a) Schematic representation of the entire selection. (b) Heat map for the TAB value of the mutants; strains with reduced TBA value than YJ-002 are marked in pink, strains with increased TBA value are marked in blue, strains with reduced NADH levels are marked in grey, and the TBA values have not been detected. (c) The reduction rates of the TBA value in those strains with decreased TBA value (126 of 142) were calculated to reflect the improvement in the flavor stability. (d) Fermentation stability is an important criterion for the industrial producing strain; therefore, five runs of fermentation were carried out and the fluctuations in acetaldehyde production were compared to judge this characteristic.
For industrial scale-producing strains, the fermentation stability was important as the yeast cells were collected and reused for fermentation for more than four runs. Acetaldehyde was the most abundant aldehyde in beer and was identified as a key contributor to beer staling 
. Therefore, the fermentation stability of these seven mutants was evaluated by analyzing fluctuations in acetaldehyde production over five runs of fermentation. The acetaldehyde concentration of these seven strains was lower than that of YJ-002 during five runs of continuous fermentation (Figure 3
d). However, large fluctuations in acetaldehyde production were identified in the different generations of strains YDR-1, YDR-57, YDR-95, YDR-106, and YDR-144. In contrast, acetaldehyde content was almost constant among the different generations of YDR-46 and YDR-63.
4. Screening Strains with Hgher NADH Levels
Beer staling is of great concern to brewers as it leads to an irreversible change in flavor. Therefore, to improve the flavor stability of beer during shelf life, it is imperative to reduce the production of aldehydes in the final product. In previous studies, strains with lower acetaldehyde production and improved flavor stability have been developed, based on ARTP mutagenesis coupled with 4-methylpyrazole (inhibitor of alcohol dehydrogenase 2) selection or disulfiram (inhibitor of aldehyde dehydrogenase) selection 
. However, other aldehydes, such as (E
)-2-nonena and 5-Hydroxymethylfurfura, have also been identified as key factors contributing toward beer staling, despite their extremely low concentrations 
. Thus, reducing the production of these aldehydes is also of great interest to the beer production industry.
DNP is already used as a dye, as well as in wood preserver, herbicides, munitions, and photographic developer, and was also initially popularized as a weight loss drug, as the consumption of DNP led to significant weight loss 
. The mechanism underlying this weight loss property was that it increases the basal metabolic rate by uncoupling oxidative phosphorylation and stimulating the glycolysis rate 
. However, this chemical was soon labeled “not fit for human consumption” owing to many adverse effects