Hybrid Process Flow Diagram for Separation of Fusel Oil into Valuable Components

This study was performed to analyze the content of 6 different fusel oils in 9 types of liquor distributed in domestic market. GC-FID method was employed for quantifying fusel oil (1-propanol, iso-butanol, 1-butanol, 2-butanol, iso-amyl alcohol, active amyl alcohol) levels in 260 liquor samples of liquor. Relative standard deviations (%) of intraand interday measurements were under 1.56 and 2.44%, respectively, while recovery rates (%) were 98.22-105.26% and 98.53-107.15%, respectively. Pretreatment method (filtering and centrifugation) of Takju did not affect analytic results. The average of total fusel oil contents in Yakju (39 types) and fruit wines (30 types) were 497.6 and 151.9 mg/L, showing Yakju contains more fusel oils than Takju or fruit wines. In fruit wines, iso-amyl alcohol was the major fusel oil component (at 6.8-249.0 mg/L). The highest content of fusel oil was found in foreign brandy, whereas the diluted Soju did not contain fusel oils. However, the average of total fusel oil contents was high at 764.5 mg/L in the three types of distilled Soju and iso-amyl alcohol content ranged from 114.2 to 421.0 mg/L. Domestic and foreign beers were similar in terms of their fusel oil compositions and contents. In conclusion, excluding the diluted Soju, the contents of total fusel oils ranged from 114.8 to 1447.3 mg/L in the monitored liquors.

Fusel oil is a byproduct obtained from bioethanol distilleries, composed of a mixture of higher alcohols such as isoamyl alcohol, isobutyl alcohol, and others. This study aimed to evaluate the industrial distillation process of fusel oil to obtain isoamyl alcohol using the Aspen Plus simulator, considering fusel oil as a mixture of nine components. Fusel oil samples collected in Brazilian industrial mills were analyzed by gas chromatography. An investigation of phase equilibrium (vapor–liquid equilibrium and liquid–liquid equilibrium) was carried out for the components involved in this mixture. Three configurations for the fusel oil separation process were proposed. The best design with minimum total annual cost (TAC) resulted in a recovery of 99.53% of isoamyl alcohol of product containing the isomers isoamyl alcohol (0.818 w/w) and active amyl alcohol (0.178 w/w). Dynamic control of this configuration was investigated, and the results show that reasonable control performance can be achieved.

Application of an immobilized Rhizopus oryzae lipase to batch and continuous ester synthesis with a mixture of a lauric acid and fusel oil

A study was made of the higher alcohols (fusel oils) produced during the Indonesian tapé ketan fermentation using Amylomyces rouxii as the principal mold, alone or in combination with yeasts belonging to genera commonly found in the tapé ketan fermentation (Endomycopsis, Candida, and Hansenula). Total fusel oils increased with length of fermentation. Fusel oils detected in the product distillate included isobutanol and isoamyl and active amyl alcohols. No n-propanol was detected. Isobutanol and isoamyl alcohols were formed in the largest amounts. A. rouxii alone produced nearly the same quantity of fusel oils (total production, 275 mg/liter at 192 h) as it did in combination with Endomycopsis burtonii (total production, 292 mg/liter at 192 h).A. rouxii and Endomycopsis fibuliger produced fusel oils totaling 72 mg/liter at 32 h and 558 mg/liter at 192 h. A. rouxii in combination with Candida yeasts produced somewhat more fusel oils, ranging from 590 to 618 mg/liter at 192 h. A. rouxii in combination with Hansenula yeasts produced the least fusel oils, totaling 143 to 248 mg/liter at 192 h. During the first 36 h, production of fusel oils was higher at 30 and 35 degrees C than at 25 degrees C. At 48 h fusel oil production was slightly higher at 30 degrees C than at 35 degrees C. Beyond 48 h, production of fusel oils was higher at 25 degrees C. A. rouxii in combination with Hansenula anomala and Hansenula subpelliculosa produced considerable ethyl acetate, ranging from 145 to 199 mg/liter at 36 h and 354 to 369 mg/liter at 192 h.

Excessive fusel alcohols in red wine will bring an uncomfortable bitterness and generate an intoxicating effect, which affects the quality and attractivity of the red wine. In order to achieve better regulation of fusel alcohols in red wine, strains with LEU1 and PDC5 deletions were constructed, and seven engineered yeast strains based on THI3 and BAT2 deletions were applied to red wine fermentation to dissect the effects of four critical genes on fusel alcohols during wine fermentation. The fermentation results of these recombinant strains showed that the deletion of THI3 increased the contents of n-propanol, isobutanol, and isoamyl alcohol by 48.46%, 42.01%, and 7.84%, respectively; the deletion of BAT2 decreased isoamyl alcohol and isobutanol by 32.81% and 44.91%; the deletion of PDC5 and LEU1 decreased isoamyl alcohol by 40.21% and 68.28%, while increased isobutanol by 24.31% and 142%, respectively; the deletion of THI3 exerted a negative influence on the reduction of isoamyl alcohol caused by BAT2 or PDC5 deletion; the deletion of THI3 and PDC5 had a synergistic effect on the increase of isobutanol, while BAT2 and PDC5 deletion presented no additive property to the decrease of isoamyl alcohol. Hence, it is concluded that either BAT2, PDC5, or LEU1 deletion can effectively decrease fusel alcohols, especially isoamyl alcohol, which provides an important reference for the control of fusel alcohols in red wine.

Fusel oil, a byproduct of ethanol fermentation, mainly consists of isoamyl alcohol and isobutanol. These compounds negatively impact distillation by causing fouling and corrosion in rectification columns, increasing maintenance costs, and affecting ethanol quality and sensory properties. Despite these drawbacks, fusel oil has extensive applications in the fine chemical industry. Its quality and yield vary depending on raw materials and fermentation conditions. This research investigates the effects of pH, supplementation, and refrigeration on isoamyl alcohol and isobutanol formation during sugarcane molasses fermentation at a microdistillery. Fermentations were performed in fed-batches with molasses must at 25 °Brix and commercial dry yeast (25% v/v) for 10 h. A complete 2³ factorial design analyzed the effects on fermentation efficiency (nf), process efficiency (np), ethanol productivity (P), substrate-to-cell conversion (YX/S), isoamyl alcohol production (A), isobutanol production (B), and the A/B ratio. Statistical analysis included analysis of variance (ANOVA) and Tukey’s test. Results showed significant interactions influencing isoamyl alcohol and isobutanol production and the A/B ratio. Fermentation at pH 3.5, without supplementation, and with refrigeration achieved the highest A/B ratio (2.26). Conversely, fermentation at pH 5.0, with supplementation, and without refrigeration, yielded the highest isoamyl alcohol (0.4372 g L-1) and isobutanol (0.2666 g L-1) concentrations, without selectively enhancing isoamyl alcohol.

I S O M E R I C amyl alcohols, active amyl alcohol [ (-1-2methylbutanol] and isoamyl alcohol (3-methylbutanol) are the principal compounds in fusel oil (25) . A by-product of normal alcoholic fermentation, fusel oil is produced to the extent of 0.1 to 0.7 per cent of the ethyl alcohol yield (16). The mixed amyl alcohols are used as solvents and extractives in large amounts in many industrial processes where a five-carbon branched-chain primary alcohol is needed. The purified active amyl alcohol might be used as a starting material for the synthesis of compounds having optical activity. Le Bel (30) tried to purify the optically active isomer by chlorination, because isoamyl alcohol is more rapidly chlorinated. Pasteur (33) separated the amyl sulfates by their different solubilities. Isom and Hunt (27) found only a very slight separation of the alcohols on passing a mixture through a 50-foot column packed with activated carbon a t 91 C., and Blessin, Kretschmer, and Wiebe (5 ) reported that separation of normal alcohols by thermal diffusion was virtually impossible. Isoamyl and active amyl alcohols are difficult to separate by fractional distillation, as their boiling points are 132.0 and 128.7 C. ( 2 , 6), respectively, a t atmospheric pressure. Whitmore and Olewine (37) reported tha t several fractionations on a 101-plate column were necessary to obtain 95T pure active amyl alcohol. Brauns reported (6) that fractional distillation seemed the only practicable method. Separating the two branched-chain primary amyl alcohols by fractional distillation after addition of a third component which would azeotrope with one or both alcohols has never been investigated. Horsley (23) records several azeotropes involving isoamyl alcohol, but only one study of systems containing active amyl alcohol ( 4 ) . The azeotroping agent should ideally form a low boiling azeotrope with active amyl and none with isoamyl, or a high boiling azeotrope with isoamyl and none with active amyl alcohol. Or, if it azeotropes with both alcohols, the boiling points should differ by considerably more than 3.3O C. To determine whether or not an azeotrope had formed in the distillation fractions of binary mixtures containing either isoamyl or active amyl alcohol and another component, the physical properties of such mixtures had to be evaluated. Possible azeotroping agents, chosen with consideration of the principles developed by Ewell, Harrison, and Berg (II), were mixed with pure isoamyl and active amyl alcohols to make known solutions for determinations of refractive indices, densities and, for active amyl solutions, optical activities. In nearly every case nine mixtures of each binary combination were prepared, successive samples differing by approximately 10 weight % in azeotroping agent content. The amyl alcohols were obtained from grape brandy fusel oil by repeated fractional distillations. The active amyl alcohol was distilled until the optical rotation agreed with the values of Markwald and McKenzie (32), who used fractional crystallization of the 1-amyl-3-nitrophthalate acid esters to separate the last traces of isoamyl from the active amyl derivative. After saponification, distillation, drying, and redistillation, the physical constants of the active amyl alcohol were measured. The possibi.!ity of some (+) isomer of active amyl alcohol being present was not considered in the research reported. Cohen, Marshall, and Woodman (8) attempted resolution of the 3-nitrophthalic acid 2-monoester of active amyl alcohol by conversion to the brucine salts, bu t obtained active amyl alcohol of decreased optical activity. This they attributed to racemization by the hot base during hydrolysis of the ester-salt which seems unlikely, as the center of optical activity is not involved directly in the hydrolysis reaction. I t was desirable, therefore, to attempt resolution of the active amyl nitrophthalate monoester using other optically active bases, to be certain tha t Markwald and McKenzie's value was that of the single pure optical isomer.

Several auxotrophic mutants requiring branched chain amino acids (valine, leucine, or isoleucine) were isolated in a strain of Montrachet wine yeast. They were tested for their ability to produce lowered amounts of higher alcohols (‘fusel oil’: isobutyl, active amyl, and isoamyl alcohols) in grape juice fermentations. One strain which required leucine was especially good in this respect. This mutation is recessive and is the result of a deficiency for the enzyme α-isopropylamate dehydratase. In trial fermentations with this mutant, the resulting wines contained up to 20% less total fusel oil and 50% less isoamyl alcohol compared to the parent Montrachet strain. An experienced taste panel did not discern any gross degradation of taste quality in wine made with the mutant strain compared to that made with the parent strain. The mutant strain could be of commercial importance in preparation of distilling material for alcoholic beverages since the reduced fusel oil content would not require any special distillation procedures which are normally used to avoid the unpleasant flavour associated with concentrated higher alcohols. Reduction of the isoamyl alcohol content is particularly significant since this fusel oil component is usually present in the highest amount.

Many food, cosmetic and pharmaceutical industries have increased their interest in short-chain esters due to their flavor properties. From the industrial standpoint, enzyme reactions are the most economical strategy to reach green products with neither toxicity nor damage to human health. Isoamyl butyrate (pear flavor) was synthesized by isoamyl alcohol (a byproduct of alcohol production) and butyric acid with the use of the immobilized lipase Lipozyme TL IM and hexane as solvents. Reaction variables (temperature, butyric acid concentration, isoamyl alcohol:butyric acid molar ratio and enzyme concentration) were investigated in ester conversion (%), concentration (mol L-1) and productivity (mmol ester g-1 mixture . h), by applying a sequential strategy of the Fractional Factorial Design (FFD) and the Central Composite Rotatable Design (CCRD). High isoamyl butyrate conversion of 95.8% was achieved at 24 hours. At 3 hours, the highest isoamyl butyrate concentration (1.64 mol L-1) and productivity (0.19 mmol ester g-1 mixture . h) were obtained under different reaction conditions. Due to high specificity and selectivity of lipases, process parameters of this study and their interaction with the Lipozyme TL IM are fundamental to understand and optimize the system so as to achieve maximum yield to scale up. Results show that fusel oil may be recycled by the green chemistry process proposed by this study.

Fusel oil, a residue from ethanol production, can be used as a source of alcohol in the synthesis of biodiesel. Babassu oil is a Brazilian native plant that has high oil productivity, and its use in transesterification reactions may be a source of triglycerides. This study aimed to purify fusel oil in order to obtain isoamyl alcohol for use in the alkali-transesterification reaction with babassu oil. Purification of fusel oil was promising since achieving 96% purity in the content of isoamyl alcohol, adding value to the said residue, was possible. The reaction of babassu oil with isoamyl alcohol showed 94% conversion into esters and was higher than that with soybean oil, which showed 86% conversion, under the same reaction conditions at 25 °C, 1 h, and oil: alcohol ratio of 1:10. These temperature conditions are considered mild for obtaining biodiesel, which is obtained at higher temperatures when larger chain alcohols are used. Physicochemical properties were in line with the literature and met the standards.

Fusel oil has been reported to have undesirable side effects such as hangover. However, the relationship between fusel oil and hangover has been investigated insufficiently. In this study, we investigated the effects of fusel oil and their ingredients contained in alcoholic beverages by using animal hangover models. Ethanol and fusel oil were simultaneously administered to Suncus murinus, and emetic responses were observed for 60 min. Ethanol and fusel oil were simultaneously administered to mice immediately after intake of saccharin solution; on the next day, the mouse's saccharin solution intake was measured. The volatile fraction (fusel oil) of whisky had no remarkable effect on ethanol-induced emetic responses in suncus. Whisky had the most suppressive effect on ethanol-induced conditioned taste aversion in mice among the various alcoholic beverages tested. The volatile fraction (fusel oil) of whisky suppressed the ethanol-induced conditioned taste aversion. In contrast, the nonvolatile fraction of whisky had no effect. The administration of isoamyl alcohol (5 mg/kg) and isoamyl acetate (10 and 40 microg/kg), ingredients of fusel oil, significantly suppressed the ethanol-induced conditioned taste aversion. The fusel oil in whisky had no effect on the ethanol-induced emetic response, but it suppressed taste-aversion behavior in animal models of hangover symptoms. These results suggest that the fusel oil in whisky alleviates hangover, contrary to the common belief.

Saccharomyces cerevisiae has been used for at least eight millennia in the production of alcoholic beverages (41). Along with ethanol and carbon dioxide, fermenting cultures of this yeast produce many low-molecular-weight flavor compounds. These alcohols, aldehydes, organic acids, esters, organic sulfides, and carbonyl compounds have a strong impact on product quality. Indeed, the subtle aroma balance of these compounds in fermented foods and beverages is often used as an organoleptic fingerprint for specific products and brands (42). Food fermentation by yeast and lactic acid bacteria is accompanied by the formation of the aliphatic and aromatic alcohols known as fusel alcohols. Fusel oil, which derives its name from the German word fusel (bad liquor), is obtained during the distillation of spirits and is enriched with these higher alcohols. While fusel alcohols at high concentrations impart off-flavors, low concentrations of these compounds and their esters make an essential contribution to the flavors and aromas of fermented foods and beverages. Fusel alcohols are derived from amino acid catabolism via a pathway that was first proposed a century ago by Ehrlich (13). Amino acids represent the major source of the assimilable nitrogen in wort and grape must, and these amino acids are taken up by yeast in a sequential manner (23, 32). Amino acids that are assimilated by the Ehrlich pathway (valine, leucine, isoleucine, methionine, and phenylalanine) are taken up slowly throughout the fermentation time (32). After the initial transamination reaction (Fig. ​(Fig.1),1), the resulting α-keto acid cannot be redirected into central carbon metabolism. Before α-keto acids are excreted into the growth medium, yeast cells convert them into fusel alcohols or acids via the Ehrlich pathway. FIG. 1. The Ehrlich pathway. Catabolism of branched-chain amino acids (leucine, valine, and isoleucine), aromatic amino acids (phenylalanine, tyrosine, and trytophan), and the sulfur-containing amino acid (methionine) leads to the formation of fusel acids and ... Current scientific interest in the Ehrlich pathway is supported by increased demands for natural flavor compounds such as isoamyl alcohol and 2-phenylethanol, which can be produced from amino acids in yeast-based bioconversion processes (14), as well as by the need to control flavor profiles of fermented food products. The goal of this paper is to present a concise centenary overview of the biochemistry, molecular biology, and physiology of this important pathway in S. cerevisiae.

The reduced yields of acetaldehyde and fusel alcohols through fermentation by Saccharomyces cerevisiae is of significance for the improvement of the flavor and health of alcoholic beverages. In this study, the ADH2 (encode alcohol dehydrogenase) and THI3 (encode decarboxylase) genes of the industrial diploid strain S. cerevisiae XF1 were deleted. Results showed that single-gene-deletion mutants by separate gene deletion of ADH2 or THI3 led to a reduced production of the acetaldehyde or fusel alcohols, respectively. In the meantime, the double-gene-deletion mutant S. cerevisiae XF1-AT was constructed by deleting the ADH2 and THI3 simultaneously. An equivalent level of the ethanol production by the S. cerevisiae XF1-AT could be achieved but with the yields of acetaldehyde, isoamyl alcohol and iso-butanol reduced by 42.09%, 15.65% and 20.16%, respectively. In addition, there was no interaction between the ADH2 deletion and THI3 deletion in reducing the production of acetaldehyde and fusel alcohols. The engineered S. cerevisiae XF1-AT provided a new strategy to alcoholic beverages brewing industry for reducing the production of acetaldehyde as well as the fusel alcohols.

Many yeasts can produce filamentous elongated cells identifiable as hyphae, pseudohyphae or invasive filaments. Filament formation has been understood as a foraging response that occurs in nutrient-poor conditions. However, fusel alcohols were observed to induce filament formation in rich nutrient conditions in every yeast species examined. Fusel alcohols, e.g., 3-methyl-1-butanol (3Me-BuOH; 'isoamyl alcohol'), 2-methyl-1-propanol (isobutyl alcohol), (-)-2-methyl-1-butanol ('active amyl alcohol'), 2-phenylethanol and 3-(2-hydroxyethyl)indole (tryptophol) (the end products of leucine, valine, isoleucine, phenylalanine and tryptophan catabolism, respectively) are the end products of amino acid catabolism that accumulate when nutrients become limiting. Thus, yeast responds to its own metabolic by-products. Considerable effort was made to define the cell biological and biochemical changes that take place during 3Me-BuOH-induced filamentation. In Saccharomyces cerevisiae filaments contain significantly greater mitochondrial mass and increased chitin content in comparison with yeast-form cells. The global transcriptional response of S. cerevisiae during the early stages of 3Me-BuOH-induced filament formation has been described. Four ORFs displayed very significant (more than 10-fold) increases in their RNA species, and 12 ORFs displayed increases in transcription of more than 5-fold. The transcription of five genes (all of which encode transporters) decreased by similar amounts. Where examined, the activity of the proteins encoded reflected the transcriptional pattern of their respective mRNAs. To understand this regulation, studies were performed to see whether deletion or overexpression of key genes affects the ability to filament and invade solid YEPD medium. This has led to identification of those proteins that are essential for filament formation, repressors and those which are simply not required. It also leads to the conclusion that 3Me-BuOH-induced filament formation is not a foraging response but a response to reduced growth rate.

Esterification of fusel oil using reactive distillation – Part I: Reaction kinetics