Isoamyl Acetate Production Research Articles

Investigation of novel metabolites generated by Torulaspora delbrueckii to promote the aroma biosynthesis in Saccharomyces cerevisiae during wine fermentation

The co-fermentation of Saccharomyces cerevisiae and non-Saccharomyces has been recognized as an effective strategy for improving the quality of wine aroma. One of the most critical factors for the aroma enhancement is the yeast-produced metabolites, but the underlying mechanisms remain poorly understood. In this study, we used a cell/medium separation strategy to investigate the effects of metabolites generated by Torulaspora delbrueckii on the aroma biosynthesis in S. cerevisiae. The results demonstrated that T. delbrueckii metabolites enhanced the production of ethyl octanoate, ethyl decanoate, isoamyl acetate, and isopentanol within S. cerevisiae. Upregulation of aroma-related genes, including EEB1, SLI1, BAP3, BAT1, PDC1, and PDC5 in S. cerevisiae, was responsible for the aroma enhancement. The untargeted metabolomic analysis determined a total of 869 T. delbrueckii metabolites, in which three novel metabolites were identified to promote the aroma formation in S. cerevisiae. Specifically, 3-amino-4-methylpentanoic acid stimulated the levels of ethyl octanoate (up to 42.55%) and ethyl decanoate (up to 31.92%), while 4-methylaminobutyric acid and citraconic acid increased the production of ethyl hexanoate (up to 59.19%) and isoamyl acetate (up to 32.26%). A comprehensive understanding of the specific yeast metabolites provides a robust foundation for winemakers to purposefully modulate the aromatic characteristics of wines.

Enhanced production of isobutyl and isoamyl acetate using Yarrowia lipolytica.

Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters.

Recycled Brewer's Spent Grain (BSG) and Grape Juice: A New Tool for Non-Alcoholic (NAB) or Low-Alcoholic (LAB) Craft Beer Using Non-Conventional Yeasts.

Non-alcoholic beer (NAB) and low-alcoholic beer (LAB) are taking over the market with growing sales. Sustainable recycling and valorization of exhausted brewer's spent grain (BSG) coming from craft beer is a relevant issue in the brewing process. In this work, recycled BSG and BSG + GJ (supplemented with 10% grape juice) were used as a wort substrate to inoculate Lachancea thermotolerans, Wickeramhomyces anomalus, Torulaspora delbruecki and Pichia kluyveri non-conventional yeasts to produce NABLAB craft beer. Results showed that wort composed of only recycled BSG produced appreciated NAB beers (ethanol concentration from 0.12% to 0.54% v/v), while the addition of 10% grape juice produced LAB beers (ethanol concentration from 0.82 to 1.66% v/v). As expected, volatile compound production was highest with the addition of grape juice. L. thermotolerans showed lactic acid production, characterizing both worts with the production of ethyl butyrate and isoamyl acetate. T. delbrueckii exhibited relevant amounts of hexanol, phenyl ethyl acetate and β-phenyl ethanol (BSG + GJ). W. anomalus and P. kluyveri showed consistent volatile production, but only in BSG + GJ where fermentation activity was exhibited. The overall results indicated that reused BSGs, non-conventional yeasts and grape juice are suitable bioprocesses for specialty NABLAB beer.

Pervaporation Membrane-Catalytic Reactors for Isoamyl Acetate Production

This study reports the analysis and design of a liquid phase esterification process to convert acetic acid with isoamyl alcohol into isoamyl acetate via reactive pervaporation, in the presence of an Amberlite IR-120 ion exchange resin catalyst. To accomplish this, a catalytic reactor is coupled with a separation membrane unit (Pervaporation Membrane Reactor (PVMR)). In the proposed unit, the chemical reaction equilibrium is favorably shifted towards isoamyl acetate formation by removing water with the help of a separation membrane. The study is developed by using relevant thermodynamics, kinetics, and membrane transport models, and by considering different catalytic reactor-pervaporator membrane configurations such as: (a) a two-step continuous fixed bed-pervaporator process (FBR+PVMU), (b) a two-step continuous slurry reactor-pervaporator process (SR+PVMU), (c) a single-step integrated fixed bed-pervaporator reactor (IFBPVMR), and d) a single step integrated slurry-pervaporator reactor (ISPVMR). The performance of the PVMRs is evaluated by using a R recycle ratio, a Ω membrane area to reactor volume ratio, and Da Damköhler dimensionless parameters. From the various proposed configurations, it is shown that the integrated plug flow reactor-pervaporation reactor (IFBPVMR) provides the best performance. On the basis of various simulations and design charts developed in the present study, operational conditions leading to optimum ester yields as high as 0.94 are predicted. These results provide a valuable prospect for the industrial scale-up and implementation of isoamyl acetate production units.

Aroma Profiles of Vitis vinifera L. cv. Gewürztraminer Must Fermented with Co-Cultures of Saccharomyces cerevisiae and Seven Hanseniaspora spp.

In this study, the aroma-production profiles of seven different Hanseniaspora strains, namely H. guilliermondii, H. meyeri, H. nectarophila, H. occidentalis, H. opuntiae, H. osmophila and H. uvarum were determined in a simultaneous co-inoculation with the wine yeast Saccharomyces cerevisiae Champagne Epernay Geisenheim (Uvaferm CEG). All co-inoculated fermentations with Hanseniaspora showed a dramatic increase in ethyl acetate levels except the two (H. occidentalis and H. osmophila) that belong to the so-called slow-evolving clade, which had no meaningful difference, compared to the S. cerevisiae control. Other striking observations were the almost complete depletion of lactic acid in mixed-culture fermentations with H. osmophila, the more than 3.7 mg/L production of isoamyl acetate with H. guilliermondii, the significantly lower levels of glycerol with H. occidentalis and the increase in certain terpenols, such as citronellol with H. opuntiae. This work allows for the direct comparison of wines made with different Hanseniapora spp. showcasing their oenological potential, including two (H. meyeri and H. nectarophila) previously unexplored in winemaking experiments.

Gradually supply of isoamyl alcohol increases the isoamyl acetate production in solid-state fermentation.

Nowadays, isoamyl acetate production is carried out by chemical synthesis with a recent interest in developing biological producing processes, mainly based on microorganisms in submerged fermentation. This work assayed producing isoamyl acetate through solid-state fermentation (SSF), feeding the precursor in the gas phase. Polyurethane foam functioned as the inert support to contain 20ml of a solution of molasses (10%w/v, pH 5.0). The yeast Pichia fermentans was inoculated at 3×107 cells per gram of initial dry weight. The airstream to supply oxygen also served to supply the precursor. Slow supply was obtained using an isoamyl alcohol solution of 5g l-1 in the bubbling columns and an air stream of 50ml min-1. For fast supply, fermentations were aerated using 10g l-1 and 100ml min-1 for isoamyl alcohol solution and air stream, respectively. It demonstrated the feasibility of isoamyl acetate production in SSF. Moreover, the slow supply of the precursor increased isoamyl acetate production up to 390mg l-1, which is 12.5 times higher than that obtained without precursor (32mg l-1). On the other hand, fast supply caused an evident inhibition of the growth and production capacity of the yeast.

Screening of Lactiplantibacillus plantarum with High Stress Tolerance and High Esterase Activity and Their Effect on Promoting Protein Metabolism and Flavor Formation in Suanzhayu, a Chinese Fermented Fish.

In this study, three Lactiplantibacillus plantarum, namely 3-14-LJ, M22, and MB1, with high acetate esterase activity, acid, salt, and high-temperature tolerance were selected from 708 strains isolated from fermented food. Then, L. plantarum strains MB1, M22, and 3-14-LJ were inoculated at 107 CFU/mL in the model and 107 CFU/g in actual Suanzhayu systems, and the effects during fermentation on the physicochemical properties, amino acid, and volatile substance were investigated. The results showed that the inoculated group had a faster pH decrease, lower protein content, higher TCA-soluble peptides, and total amino acid contents than the control group in both systems (p < 0.05). Inoculation was also found to increase the production of volatile compounds, particularly esters, improve the sour taste, and decrease the bitterness of the product (p < 0.05). L. plantarum M22 was more effective than the other two strains in stimulating the production of isoamyl acetate, ethyl hexanoate, and ethyl octanoate. However, differences were discovered between the strains as well as between the model and the actual systems. Overall, the isolated strains, particularly L. plantarum M22, have good fermentation characteristics and have the potential to become excellent Suanzhayu fermenters in the future.

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, ketoacids, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including ketoisovalerate submodule for converting pyruvate to ketoisovalerate, ketoacid elongation submodule for producing longer carbon-chain ketoacids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23–31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (>0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

Incorporation of metallic particles in activated carbon used in lipase immobilization for production of isoamyl acetate

AbstractBACKGROUNDBiotechnology has revolutionized the use of enzymes for application in industrial processes. The present work evaluated the effect of functionalizing activated carbon with metallic nanoparticles obtained from corn cob residue on lipase immobilization. The aim was to improve the enzymatic activity of the immobilized enzyme and subsequent application of derivatives in the synthesis of isoamyl acetate ester (banana flavor).RESULTSThe activated carbon (CCAC) had a mesoporous structure and specific surface area of 1135 m2 g−1, characteristic of supports from precursor material with a high cellulose content, which was reduced in metalized carbon (CCACM) due to the insertion of metallic particles (760 m2 g−1). CCACM showed a slightly higher lipase absorptive capacity than CCAC (280.72 ± 0.27 mg g−1 and 276.46 ± 0.03 mg g−1, respectively). Furthermore, for derivatives the optimal reaction medium conditions were pH 7.0 and 30 °C, confirming the advantage associated with lipase immobilization, obtaining activity values of 84.0 ± 2.12 U, 72.3 ± 1.7 U and 31.4 ± 1.9 U, for lipase immobilized on CCACM, CCAC and native form, respectively. The derivatives showed potential application in the synthesis of isoamyl acetate ester, with conversions above 90% and stability during five cycles of use.CONCLUSIONThe activated carbon metallization process showed an increase in lipase immobilization efficiency and proved to be a promising support, since there was an increase in its enzymatic activity and the derivatives obtained showed potential application in aroma synthesis, reaching conversion above 90%. © 2022 Society of Chemical Industry (SCI).

Producción de acetato de isoamilo durante el cultivo continuo de Pichia fermentans

Pichia fermentans ITD-00165 is a yeast with a high potential to produce isoamyl acetate, but no previous works report its growth in continuous culture. A bioreactor containing a chemically defined medium allowed continuous cultures to develop three dilution rates (0.25, 0.30 y 0.35 h-1), measuring the isoamyl acetate production. Steady-state isoamyl acetate concentration was measured using an entrapping system based on a bubbling column with n-decane. Results demonstrate that the entrapping system showed a linear correlation between the isoamyl acetate concentration into the culture medium and the absorption rate into the column. Airstream for oxygen supply was used to continuously feed isoamyl alcohol to the culture to diminish the inhibitory effect of the precursor. Finally, it was demonstrated that the yeast used the precursor to produce isoamyl acetate (0.144 mg L-1).

Development of a green catalyst for production of synthetic banana flavoring based on low transition temperature mixture through numerical optimization, LCA assessment and sustainability metrics

The transition to cleaner production of chemicals to abate the prolonged environmental impairment has been an emerging concern worldwide, calling for alternative cost effective and greener process routes. The present study witnesses the ecofriendly preparation of synthetic banana flavoring or Isoamyl acetate (IAA) catalyzed by a low transition temperature mixture (LTTM) comprised of abundantly available natural resources. The chosen LTTM exhibited bifunctional properties of highly active acidic catalyst as well as an excellent solvent media which could phase out the product as an independent layer after the reaction assuring simpler downstream processing and direct recycle of the catalytic media for subsequent runs. Numerical optimization, executed to obtain the optimal process parameters using the Box-Behnken design yielded a maximum conversion of 99.81%. The LTTM catalyzed reaction kinetics is deciphered through the pseudo-homogeneous model inclusive of the system non-ideality estimated through the UNIFAC model and the thermodynamic functions of the reaction were also evaluated to aid the realistic process design. A comparison of the proposed route and that using sulfuric acid as catalyst has been performed through a cradle to gate life cycle assessment (LCA) using the Simapro 9.1 software for the laboratory production of 1 kg IAA which revealed that the adopted green approach decreased the damage to ecosystem and resource availability by 48.8% and 30.2% respectively. Furthermore, the environment profiling of the conventional and intensified reaction pathways using green metrics also indicated the LTTM mediated production of IAA to be extremely promising that paves the path for sustainable chemical industries.

Continuous production of isoamyl acetate from fusel oil under supercritical CO2: A mass transfer approach

Isoamyl acetate esters were synthesized under supercritical CO2 (SC-CO2) in a continuous packed-bed reactor (PBR) using immobilized lipase (Novozym 435) as the catalyst. A fractional factorial design (24−1) was applied to evaluate the effects of pressure (10–20 MPa), temperature (40–60 °C), substrates (fusel oil + acetic anhydride) flow rate (QS1/S2: 0.2–0.6 mL/min), and CO2 flow rate (QCO2: 0.4–1.2 L/min) on the isoamyl acetate conversion (X, %). Results showed that only the effects of QS1/S2 were statistically significant at the level of 5%. Mathematical modeling of the PBR was applied using the law of conservation of mass that resulted in two partial differential equations for fluid and enzymes. The model was solved using finite difference methods and successfully validated with the experimental data in terms of isoamyl alcohol concentration. Finally, the model was employed to examine the impact of the input molar ratio of fusel oil per acetic anhydride (IMFA) and the length per diameter ratio (L/D) of the PBR on its performance.

Bioprospecting of indigenous yeasts involved in cocoa fermentation using sensory and chemical strategies for selecting a starter inoculum

Cocoa fermentation is the key and most relevant process in the synthesis of aroma and flavor precursor molecules in dry beans or raw material for producing chocolate. Because this process occurs in an uncontrolled manner, the chemical and sensory quality of beans can vary and be negatively affected. One of the strategies for the standardization and improvement of the sensory quality of chocolate is the introduction of microbial starter cultures. Among these, yeasts involved in fermentation have been studied because of their pectinolytic and metabolic potential in the production of volatile compounds. This study was aimed at isolating and characterizing, both sensory and chemically, yeasts involved in cocoa fermentation that could be used as starter cultures from two agro-ecological regions for the cultivation of cocoa in Colombia. The microbiological analyses identified 22 species represented mostly by Saccharomyces cerevisiae, Wickerhamomyces anomalus and Pichia sp. The preliminary sensory analysis of eight of these species showed that Hanseniaspora thailandica and Pichia kluyveri presented sensory profiles characterized by high intensity levels of fruity notes, which could be ascribed to the production of ethyl acetate, isoamyl acetate, and 2-phenylethyl acetate.

Mejorando la biosíntesis de acetato de isoamilo por Pichia fermentans

Microbial production of aroma compounds is a promising alternative to extracting plants or chemical synthesis. The yeast Pichia fermentans ITD-00165 has demonstrated a high potential to produce isoamyl acetate. The yeast was cultured in a bioreactor on a chemically defined medium to evaluate the influence of airflow (0.5, 0.75, and 1.0 vvm) and agitation speed (150, 175, and 200 rpm) on the isoamyl acetate production. Isoamyl acetate was retained using an in situ entrapping system, which was obtained, adding a cap of n-decane in a ratio of 80:20 (medium:decane). This work demonstrated the relevance of well-controlled aeration to enhance isoamyl acetate production using isoamyl alcohol as a precursor. Culture operating near to 0.71 vvm and 168 rpm (kLa = 0.16 min-1) achieved production of 2.14 g L-1, which means an almost 2.5-fold increase in respect to the higher value previously reported. On the other hand, the minimum production occurred at the best aeration conditions (kLa = 0.22 min-1), meaning that excessive aeration could negatively affect the aroma production. Nevertheless, it appears that the concomitant ethanol production negatively affects aroma production.

Evaluation of the Effect of Auxiliary Starter Yeasts With Enzyme Activities on Kazak Cheese Quality and Flavor.

To investigate the effect of yeasts on Kazak cheese quality and flavor, three isolated yeasts (Kluyveromyces marxianus A2, Pichia kudriavzevii A11, and Pichia fermentans A19) were used to ferment cheeses and designated as StC, LhC, and WcC, respectively. The cheese fermented with a commercial lactic acid starter without adding yeast was used as control named LrC. The results showed that the texture of cheese added with yeasts were more brittle. K. marxianus A2 contributed to the formation of free amino acids and organic acids, especially glutamate and lactic acid. Moreover, K. marxianus A2 provides cheese with onion, oily, and floral aromas. Furthermore, P. kudriavzevii A11 promotes a strong brandy, herbaceous, and onion flavor. Although no significant aroma change was observed in PfC, it promoted the production of acetic acid, isoamyl acetate, and phenethyl acetate. These results indicate that yeasts are important auxiliary starters for cheese production.

Coffee flavour modification through controlled fermentation of green coffee beans by Saccharomyces cerevisiae and Pichia kluyveri: Part II. Mixed cultures with or without lactic acid bacteria

This study attempted to achieve coffee flavour biotransformation through controlled fermentation of sterilsed green coffee beans with a coculture of Saccharomyces cerevisiae and Pichia kluyveri (FYco) and a sequential inoculation of Lc. lactis subsp. cremoris and the yeast coculture (FLYco). Isoamyl acetate, 2-phenylethyl acetate, and ethyl octanoate were produced by 5.76, 1.35 and 0.54 mg/kg, respectively, in FYco fermented green coffee beans. Compared to the green coffee bean fermented by the yeast monocultures in previous study, FYco led to a 1.2- and 4.1-times elevation in production of isoamyl acetate and 2-phenylethyl acetate, respectively. FLYco further increased acetate ester production by more than 2 times relative to FYco. The esters produced in FYco and FLYco partially survived the roasting process and imparted the roasted coffees with considerable fruity and winey aromas. The lactic acid fermentation in FLYco increased the acidity in green coffee beans, which promoted the formation of caramel-smelling furfurals and preservation of acidity and sweetness in the roasted coffees. Apart from the mere additions of flavour modification from individual strains, the proper combination of multiple strains can result in synergistic effects that enhanced the modulating activities of individual strains and further enhance flavour complexity of the resulted coffee.

Biocatalytic production of isoamyl acetate from fusel oil in supercritical CO2

Isoamyl acetate esters were enzymatically synthesized from fusel oil, a by-product generated in bioethanol industrial plants, in supercritical carbon dioxide (SC-CO2) using immobilized lipase (Lipozyme 435). Effects of enzyme load (2.5–10.0 %), substrates’ molar ratio (acetic anhydride to fusel oil; 1:1–4:1), acyl donor (acetic anhydride, ethyl acetate, and acetic acid) and solvent type (SC-CO2, n-hexane, and solvent-free system) on the biocatalytic isoamyl acetate conversion and the specific productivity were investigated. Higher volumetric expansions of the reaction medium were observed at higher temperature (60 °C) between 10 and 20 MPa. A monophasic system was observed at 15 MPa and 60 °C. Acetic anhydride and SC-CO2 were the best acyl donor and solvent, respectively. An elementary mathematical model was adjusted to the experimental data. 15 MPa, 60 °C, 2:1 substrates’ molar ratio and 2.5 % of enzyme concentration yielded the highest conversion (88.4 ± 0.5 %) and specific productivity (36.9 ± 0.2 %).

Fluid phase equilibria for the isoamyl acetate production by reactive distillation

A new set of revised parameters for NRTL activity coefficient model was correlated for the quaternary mixture of acetic acid + isoamyl alcohol + isoamyl acetate + water based on available scientific experimental vapor – liquid, liquid – liquid and vapor – liquid – liquid equilibrium data. The structure of residue curve maps for the non-reactive quaternary mixture presents four binary azeotropes and two ternary azeotropes. No reactive azeotropes were found for the reactive system. The new set of parameters for NRTL activity coefficient model is more consistent with experimental data than current models and their predictions are used to calculate a complete thermodynamic characterization for the reactive and nonreactive fluid phase equilibria. The results can be used in reactive distillation calculations such as process synthesis and conceptual design.

Parameters study on the production of isoamyl acetate via milli-reactor in a solvent-free system

Isoamyl acetate is an aromatic ester that has a similar smell to banana. This ester is high in demand for various industries application, especially in the flavour and fragrance sectors. This research aims to investigate the effects of synthesis parameters on the production of isoamyl acetate in a milli-reactor. Non-catalyzed reaction was performed by reacting isoamyl alcohol with acetic anhydride without further dilution. The flow rate of 40 to 80µL/min, reaction temperature of 20 to 50°C, and acid-alcohol molar ratio of 0.5 to 2.0 were chosen as the reaction parameters. All samples were analyzed through gas chromatography with flame-ionization detection (GC-FID). The results revealed that the highest production of isoamyl acetate was obtained at the flow rate of 60µL/min and temperature of 50°C with the acid-molar ratio of 0.5. In conclusion, these synthesis parameters significantly influences the non-catalyzed esterification reaction of isoamyl acetate production in a milli-reactor.

Effects of Isoamyl Acetate Production in Makgeolli According to Fermentation Conditions

Effects of Isoamyl Acetate Production in Makgeolli According to Fermentation Conditions