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.

Odor perception is the impetus for important animal behaviors with two predominate modes of processing: odors pass through the front of the nose (orthonasal) while inhaling and sniffing, or through the rear (retronasal) during exhalation and while eating. Despite the importance of olfaction for an animal\'s well-being and that ortho and retro naturally occur, it is unknown how the modality (ortho vs. retro) is even transmitted to cortical brain regions, which could significantly affect how odors are processed and perceived. Using multielectrode array recordings in tracheotomized anesthetized rats, which decouples ortho-retro modality from breathing, we show that mitral cells in rat olfactory bulb can reliably and directly transmit orthonasal versus retronasal modality with ethyl butyrate, a common food odor. Drug manipulations affecting synaptic inhibition via GABAA lead to worse decoding of ortho versus retro, independent of whether overall inhibition increases or decreases, suggesting that the olfactory bulb circuit may naturally favor encoding this important aspect of odors. Detailed data analysis paired with a firing rate model that captures population trends in spiking statistics shows how this circuit can encode odor modality. We have not only demonstrated that ortho/retro information is encoded to downstream brain regions but also used modeling to demonstrate a plausible mechanism for this encoding; due to synaptic adaptation, it is the slower time course of the retronasal stimulation that causes retronasal responses to be stronger and less sensitive to inhibitory drug manipulations than orthonasal responses.NEW & NOTEWORTHY Whether ortho (sniffing odors) versus retro (exhalation and eating) is encoded from the olfactory bulb to other brain areas is not completely known. Using multielectrode array recordings in anesthetized rats, we show that the olfactory bulb transmits this information downstream via spikes. Altering inhibition degrades ortho/retro information on average. We use theory and computation to explain our results, which should have implications on cortical processing considering that only food odors occur retronasally.

Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. Here, we present a generalizable framework to enable the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Overall, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.

Ethyl butyrate is one of the most important flavor substances in Chinese Baijiu and is also an ingredient in various daily-use chemical essences and food flavorings. In this study, to produce ethyl butyrate, we first introduced a butyryl-CoA synthesis pathway into Saccharomyces cerevisiae. Subsequently, three different alcohol acyltransferases, SAAT, VAAT, and CmAAT, were separately introduced into S. cerevisiae to catalyze the reaction of butyryl-CoA with ethanol to produce ethyl butyrate, and the results showed that strain EBS with SAAT produced the most ethyl butyrate (20.06 ± 2.23 mg/L). Furthermore, as the reaction catalyzed by Bcd to produce butyryl-CoA from crotonyl-CoA is a rate-limiting step, we replaced Bcd with Ter, and the modified strain EST produced 77.33 ± 4.79 mg/L ethyl butyrate. Finally, the copy numbers of Ter and SAAT were further increased, and the resulting modified strain EST-dST produced 99.65 ± 7.32 mg/L ethyl butyrate.

The synthesis of ethyl butyrate catalyzed by lipases A (CALA) or B (CALB) from Candida antarctica immobilized onto magnetic nanoparticles (MNP), CALA-MNP and CALB-MNP, respectively, is hereby reported. MNPs were prepared by co-precipitation, functionalized with 3-aminopropyltriethoxysilane, activated with glutaraldehyde, and then used as support to immobilize either CALA or CALB (immobilization yield: 100 ± 1.2% and 57.6 ± 3.8%; biocatalysts activities: 198.3 ± 2.7 Up-NPB/g and 52.9 ± 1.7 Up-NPB/g for CALA-MNP and CALB-MNP, respectively). X-ray diffraction and Raman spectroscopy analysis indicated the production of a magnetic nanomaterial with a diameter of 13.0 nm, whereas Fourier-transform infrared spectroscopy indicated functionalization, activation and enzyme immobilization. To determine the optimum conditions for the synthesis, a four-variable Central Composite Design (CCD) (biocatalyst content, molar ratio, temperature and time) was performed. Under optimized conditions (1:1, 45 °C and 6 h), it was possible to achieve 99.2 ± 0.3% of conversion for CALA-MNP (10 mg) and 97.5 ± 0.8% for CALB-MNP (12.5 mg), which retained approximately 80% of their activity after 10 consecutive cycles of esterification. Under ultrasonic irradiation, similar conversions were achieved but at 4 h of incubation, demonstrating the efficiency of ultrasound technology in the enzymatic synthesis of esters.

Complexes of ethyl butyrate and hexanal encapsulated by β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) were prepared by coprecipitation, and gas chromatography was used to quantity the flavour compounds in the complexes. The ethyl butyrate-γ-CD complex had the highest inclusion ratio (12.20%) followed by the ethyl butyrate-β-CD, hexanal-β-CD and hexanal-γ-CD complexes (11.29, 4.41 and 3.33%, respectively). Release experiments were performed under different relative humidities (RH 93, 75 and 52%) and temperatures (4 and 25 °C). The flavour release behaviours of the complexes were described by the Avrami equation. The rate of flavour release was enhanced with both increasing temperature and RH, although the effect of RH was stronger. Physicochemical characterisation using FT-IR, XRD, DSC and SEM analyses demonstrated that crystalline complexes were formed. Both β-CD and γ-CD were able to encapsulate ethyl butyrate and hexanal, and lower RH and temperature were more suitable for the storage of these complexes.

Immobilization of biocatalysts onto particulate carriers has been widely explored for recycling of biocatalyst. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work was to elucidate the importance of nanoimmobilization system in organic synthesis. The surface of multiwalled carbon nanotubes (MWCNTs) was functionalized with a mixture of concentrated acids to create an interface for enzyme immobilization. Successful functionalization and enzyme immobilization was structurally evidenced by transmision electron microscopy analysis and Fourier-transform infrared spectroscopy analysis. Furthermore, immobilized enzyme was exploited for the synthesis of flavoured ester ethyl butyrate in the presence of n-heptane. Optimized conditions for enhanced ester synthesis was found to be 8.5 pH, 40 °C, 150 rpm, 0.15:0.2 M substrate molar ratio (ethanol/butyric acid) and n-heptane as reaction medium. Utmost 81% of ester synthesis was obtained using immobilized lipase quite higher in comparison to that of free lipase. The activation energy indicated a lower energy requirement for immobilization of lipase on the surface of functionalized MWCNTs. In summary, immobilization of lipase on functionalized MWCNTs by simple adsorption method displayed excellent properties for enzyme stability and reusability, indicating its potential for application in organic synthesis.

Sensation profiles are observed all around us and are made up of many different molecules, such as esters. These profiles can be mimicked in everyday items for their uses in foods, beverages, cosmetics, perfumes, solvents, and biofuels. Here, we developed a systematic \'natural\' way to derive these products via fermentative biosynthesis. Each ester fermentative pathway was designed as an exchangeable ester production module for generating two precursors- alcohols and acyl-CoAs that were condensed by an alcohol acyltransferase to produce a combinatorial library of unique esters. As a proof-of-principle, we coupled these ester modules with an engineered, modular, Escherichia coli chassis in a plug-and-play fashion to create microbial cell factories for enhanced anaerobic production of a butyrate ester library. We demonstrated tight coupling between the modular chassis and ester modules for enhanced product biosynthesis, an engineered phenotype useful for directed metabolic pathway evolution. Compared to the wildtype, the engineered cell factories yielded up to 48 fold increase in butyrate ester production from glucose.

The effect of sucrose, glucose and fructose on the retention of ethyl butyrate by low acyl gellan gels was investigated by static headspace gas chromatography. The air/biopolymer partition coefficient (K) and percentage of retention (R%) were determined. When 5 g of sample were left to equilibrate at 37 °C for 24 h, the obtained results were explained in terms of gel rigidity, as increased rigidity resulted in increased aroma retention. Glucose showed the greatest aroma release among the sugars and resulted in either the same or increased aroma release with increasing concentration. Increasing concentrations of fructose and sucrose did not alter aroma release significantly. For 15 g of sample mass, sucrose exhibited the lowest partition coefficient values among the sugars. The two higher sucrose concentrations resulted in decreased coefficient values. For fructose and glucose, aroma retention decreased with increasing concentration. The percentage of retention values were positive for all sugars, throughout their concentration range and for both experiments.

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