Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1I306A and ScFAS2G1250S significantly improved ethyl hexanoate production and ScFAS1R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

Ethyl hexanoate and ethyl butyrate are indispensable flavor metabolites in strong-flavor Baijiu (SFB), but batch production instability in fermenting grains can reduce the quality of distilled Baijiu. Biofortification of the fermentation process by designing a targeted microbial collaboration pattern is an effective method to stabilize the quality of Baijiu. In this study, we explored the metabolism under co-culture liquid fermentation with Clostridium tyrobutyricum DB041 and Saccharomyces cerevisiae YS219 and investigated the effects of inoculation with two functional microorganisms on physicochemical factors, flavor metabolites, and microbial communities in solid-state simulated fermentation of SFB for the first time. The headspace solid-phase microextraction-gas chromatography-mass spectrometry results showed that ethyl butyrate and ethyl hexanoate significantly increased in fermented grain. High-throughput sequencing analysis showed that Pediococcus, Lactobacillus, Weissella, Clostridium_sensu_stricto_12, and Saccharomyces emerged as the dominant microorganisms at the end of fermentation. Co-occurrence analysis showed that ethyl hexanoate and ethyl butyrate were significantly correlated (|r| > 0.5, P < 0.05) with a cluster of interactions dominated by lactic acid bacteria (Pediococcus, Lactobacillus, Weissella, and Lactococcus), which was driven by the functional C. tyrobutyricum and S. cerevisiae. Mantel test showed that moisture and reducing sugars were the main physicochemical factor affecting microbial collaboration (|r| > 0.7, P < 0.05). Taken together, the collaborative microbial pattern of inoculation with C. tyrobutyricum and S. cerevisiae showed positive results in enhancing typical flavor metabolites and the synergistic effects of microorganisms in SFB.

OBJECTIVE: Ethyl hexanoate, one of the key flavor compounds in strong-flavor Baijiu. To improve the content of ethyl hexanoate in strong-flavor Baijiu, a functional strain with high yield of ethyl hexanoate was screened and its ester-producing performance was studied.
RESULTS: Upon identification, the strain was classified as Candida sp. and designated as ZY002. Under optimal fermentation conditions, the content of ethyl hexanoate synthesized by ZY002 can be as high as 170.56 mg L-1. A fermentation test was carried out using the ZY002 strain bioaugmented Daqu to verify the role of the strain applied to Baijiu brewing. It was found that strain ZY002 could not only improve the moisture and alcohol contents of fermented grains but also diminish the presence of reducing sugar and crude starch. Furthermore, it notably amplified the abundance of flavor compounds.
CONCLUSIONS: In this study, Candida sp. ZY002 with a high yield of ethyl hexanoate provided high-quality strain resources for the actual industrial production of Baijiu.

The popularity of texiang aroma type baijiu (liquor), known for its unique production technology and multilayered flavor, has been steadily increasing among consumers. So far, no research has determined its key aroma compounds for characterizing the unique flavor, which is the purpose of this paper. Using gas chromatography-olfactometry (GC-O) coupled with the GC-mass spectrometry (GC-MS) method, 87 aroma-active compounds were identified and screened out with intensity values ≥2.0. 1,1,6-Trimethyl-1,2-dihydronaphthalene (TDN) was first confirmed by GC-O in Chinese liquor. According to a quantitative study and odor activity values (OAVs), 42 odorants were determined as important aroma compounds. These odorants were recombined based on quantitative concentrations, successfully simulating the overall aroma profile. Omission experiments verified ethyl hexanoate, β-damascenone, and 2-furfuryl ethyl ether as the key aroma compounds and revealed that ethyl acetate, furfural, and ethyl 2-phenylacetate were important aroma compounds to the overall flavor of texiang aroma and flavor type liquor.

The autonomic nervous system (ANS) is implicated in maintaining homeostasis of the internal environment in mammals. Therefore, changes occurring in the ANS can cause alterations of physiological phenomena. Ethyl hexanoate (EH) is known as the aroma component of apples. To study the action of ethyl hexanoate on physiological phenomena, we examined the effect of an intragastric (IG) injection of 1 mL/kg body weight of 0.1 ppm EH solution on sympathetic nerve activity innervating the brown adipose tissue (BAT) and white adipose tissue (WAT) in anesthetized rats. Consequently, IG administration of EH increased activity of the sympathetic nerves innervating both the BAT and WAT. In addition, the effects of the IG injection on body temperature above the interscapular BAT and plasma free fatty acid (FFA) concentration were also examined in conscious rats. In this attempt IG injection of EH elevated both the body temperature and plasma FFA levels. Furthermore, subdiaphragmatic vagotomy eliminated the effects of EH on sympathetic nerves innervating BAT and WAT. These findings suggest that EH causes excitations of sympathetic nerves innervating BAT and WAT, and enhances thermogenesis and lipolysis via the afferent vagus nerve. Thus, these present findings also suggest the possibility that EH might have anti-obesity effects.

Carob liqueur is an alcoholic drink (minimum 15% v/v of ethanol and 100 g/L of sugar) typical for the Mediterranean countries. In the current work, carob macerate produced by maceration of carob pods in hydroalcoholic base at different maceration conditions was characterized for the first time based on its aroma compounds/profile, physicochemical parameters, and chromatic characteristics. The results confirm the migration process of bioactive compounds, aroma compounds, and sugars flowing from the carob pod to the hydroalcoholic base. Changes in ethanol concentration modify the physical properties of the solvent and influence the phenolic and aroma compounds extraction, color, and acidity of the obtained samples. The higher content of phenolic compounds was determinate in the samples obtained in the darkness. The amounts of phenols were in the range of some red fruit liqueurs or walnut liqueurs, and sugars (mostly sucrose) ranging between 96 and 107 g/L. Twenty-six (out of total 94) aroma compounds were detected in all samples, of which 17 esters, 3 alcohols, 4 ketones, and 2 acids. Low molecular weight ethyl esters, ethyl hexanoate, ethyl 2-methyl propanoate, ethyl octanoate, ethyl benzoate, ethyl butanoate, and ethyl cinnamate, were the most abundant. Carob pod maceration in 50% v/v hydroalcoholic base (1:5 solid to liquid ratio) in darkness at room temperature during 8 weeks can be recommended as optimal maceration conditions for production of the aromatic carob macerate with functional properties.

Strong flavor baijiu (SFB), also called Luzhou-flavor liquor, is the most popular Chinese baijiu. It is manufactured via solid fermentation, with daqu as the starter. Microbial diversity of the SFB ecosystem and the synergistic effects of the enzymes and compounds produced by them are responsible for the special flavor and mouthfeel of SFB. The present review covers research studies focused on microbial community analysis of the SFB ecosystem, including the culturable microorganisms, their metabolic functions, microbial community diversity and their interactions. The review specifically emphasizes on the most recently conducted culture-independent analysis of SFB microbial community diversity. Furthermore, the possible application of systems biology approaches for elucidating the molecular mechanisms of SFB production were also reviewed and prospected.

Yeast cells (Saccharomyces cerevisiae), from which β-glucans have been partially extracted, were used to encapsulate flavor inside the lipid bilayer membrane as natural encapsulant. The focus of this study was to investigate the release and stability of flavors (d-limonene and ethyl hexanoate) encapsulated in yeast cells and maltodextrin (MD) (DE = 19) by spray drying. The release behavior of encapsulated flavors from yeast cells was measured at 40, 60, 80, and 105 °C with different moisture content (0, 50, 100, and 200% of powder). Water affected flavor release from the yeast cells. The release rate constants were correlated using Gaussian distribution of the activation energy of the release rate constants. The release of d-limonene from the spray-dried MD powder showed a different trend than that of yeast cells at various temperatures. The activation energies of the release rate constant for ethyl hexanoate and d-limonene from yeast were 55 and 49 kJ/mol, respectively, under a wet condition. The formation rates of limonene oxide and carvone were slower in yeast than that of MD powder at 30 °C after 2 months.

Zhima aroma-type Baijiu with typical sesame aroma is particularly popular in northern China. To our knowledge, it is still uncertain which components are important to make contributions to its unique aroma, although a few pieces of research have reported many volatile compounds in this Baijiu. The aroma-active compounds from the Baijiu were researched in this paper. A total of 56 odorants were identified in Chinese Zhima aroma-type Baijiu by aroma extract dilution analysis (AEDA). Their odor activity values (OAVs) were determined by different quantitative measurements, and then 26 aroma compounds were further confirmed as important odorants due to their OAVs ≥ 1, and these had higher values, such as ethyl hexanoate (OAV 2691), 3-methylbutanal (2403), ethyl pentanoate (1019), and so on. The overall aroma of Zhima aroma-type Baijiu could be simulated by mixing of the 26 key odorants in their measured concentrations. The similarity of the overall aroma profiles between the recombination model and the commercial sample was judged to be 2.7 out of 3.0 points. Omission experiments further corroborated the importance of methional and ethyl hexanoate for the overall aroma of Chinese Zhima aroma-type Baijiu.

Medium-chain fatty acids (MCFAs) and ethyl esters are considered to contribute to some organoleptic properties, such as fatty odor and bitterness in Japanese sake. However, the relationships between these compounds and the organoleptic properties of sake remain unclear. Here, we quantified MCFAs and ethyl hexanoate in ginjo sake using gas chromatography with a flame ionization detector (GC-FID). The hexanoic acid concentration strongly correlated with fatty odor (p < 0.0001). The octanoic acid/hexanoic acid ratio correlated with butanoic acid concentration, which is likely correlated with inharmonious bitter taste. Multiple comparison analysis revealed that the ethyl hexanoate level was negatively correlated with bitterness. We then identified other chemical compounds correlating with fatty odor and bitterness using comprehensive two-dimensional GC coupled with time-of-flight mass spectrometry. By performing correlation analysis between certain compounds and sensory values following statistical selection for chemical compounds, we identified several candidate compounds correlating with fatty odor and bitterness in sake.