Aroma Compounds 2-phenylethanol Research Articles

Styrene Oxide Isomerase Catalyzed Meinwald Rearrangement Reaction: Discovery and Application in Single-Step and One-Pot Cascade Reactions

Styrene oxide isomerase (SOI) is a unique enzyme that catalyzes the Meinwald rearrangement of aryl epoxides into carbonyl compounds. In this review, we first summarize the scientific literature on the discovery and characterization of SOI’s catalytic properties such as activity, substrate scope, and enantioselectivity. We then discuss its potential use for biocatalytic synthesis of aldehydes via single-step reactions using cell-free extracts and whole cells. Emerging technologies, such as the use of a stability tag, could enhance SOI biotransformation to reach product concentrations as high as 3.37 M. SOI has also been utilized in the engineering of biocatalytic cascades to produce valuable compounds such as chiral acids, alcohols, and amines from styrene. The best systems developed to date could reach product concentrations up to 100 mM. Additionally, recent efforts are focused on the metabolic engineering of the “styrene-derived” pathway involving SOI for microbial production of the aroma compound 2-phenylethanol (2-PE) from renewable sources such as l-phenylalanine, glucose, or glycerol, achieving concentrations up to 85 mM. The application of two-phase aqueous–organic systems in practical biotransformation reactions involving SOI is also notable. Finally, some perspectives and recommendations for future work are given to set the stage for development of SOI research toward applications in sustainable industrial biocatalysis.

Novel wine yeast with ARO4 and TYR1 mutations that overproduce 'floral' aroma compounds 2-phenylethanol and 2-phenylethyl acetate.

It is well established that the choice of yeast used to perform wine fermentation significantly influences the sensory attributes of wines; different yeast species and strains impart different profiles of esters, volatile fatty acids, higher alcohols, and volatile sulphur compounds. Indeed, choice of yeast remains one of the simplest means by which winemakers can modulate the sensory characteristics of wine. Consequently, there are more than 100 commercially available Saccharomyces cerevisiae wine yeast strains available, mostly derived by isolation from vineyards and successful fermentations. Nevertheless, some desirable characteristics such as 'rose' and 'floral' aromas in wine are not present amongst existing strains. Such aromas can be conferred from the higher alcohol 2-phenylethanol (2-PE) and its acetate ester, 2-phenylethyl acetate (2-PEA). These metabolites of the aromatic amino acid phenylalanine are present at concentrations below their aroma detection thresholds in many wines, so their contribution to wine style is often minimal. To increase the concentration of phenylalanine metabolites, natural and chemically mutagenised populations of a S. cerevisiae wine strain, AWRI796, were exposed to toxic analogues of phenylalanine. Resistant colonies were found to overproduce 2-PE and 2-PEA by up to 20-fold, which resulted in a significant increase in 'floral' aroma in pilot-scale white wines. Genome sequencing of these newly developed strains revealed mutations in two genes of the biosynthetic pathway of aromatic amino acids, ARO4 and TYR1, which were demonstrated to be responsible for the 2-PE overproduction phenotype.

Sodium Chloride and Its Influence on the Aroma Profile of Yeasted Bread.

The impact of sodium chloride (NaCl) concentration on the yeast activity in bread dough and its influence on the aroma profile of the baked bread was investigated. Key aroma compounds in the bread samples were analysed by two-dimensional high-resolution gas chromatography-mass spectrometry in combination with solvent-assisted flavour evaporation distillation. High-sensitivity proton-transfer-reaction mass spectrometry was used to detect and quantify 2-phenylethanol in the headspace of the bread dough during fermentation. The analyses revealed significant (p < 0.05) changes in the aroma compounds 2-phenylethanol, (E)-2-nonenal, and 2,4-(E,E)-decadienal. Descriptive sensory analysis and discriminating triangle tests revealed that significant differences were only determinable in samples with different yeast levels but not samples with different NaCl concentrations. This indicates that a reduction in NaCl does not significantly influence the aroma profile of yeasted bread at levels above the odour thresholds of the relevant compounds, thus consumers in general cannot detect an altered odour profile of low‑salt bread crumb.

Microtiter plate-based cultivation to investigate the growth of filamentous fungi.

Microscale bioprocessing techniques are rapidly emerging as a means to increase the speed of bioprocess design and to reduce material consumption. However, there is still a lack of suitable parallelized techniques to investigate the industrially important group of filamentous bacteria and fungi. Cultivation of filamentous organisms in shake flasks is still the favored technique for comparing and optimizing cultivation conditions of production strains at mL-scale. In this paper, the application of a microtiter plate-based cultivation system in combination with the filamentous fungus Aspergillus niger was investigated. A protocol for reproducible cultivation was developed and evaluated. Productivity of A. niger concerning the rose-like aroma compound 2-phenylethanol showed low standard deviations while regular and consistent morphologies appeared in the parallelized system. Furthermore, the effect of addition of microparticles on the morphology was investigated. The results can be used to accelerate the process development with A. niger and other filamentous organisms.

Phase equilibrium and bioproduction of the aroma compound 2-phenylethanol in a biphasic aqueous system

2-Phenylethanol (PEA) is a very important natural aroma compound with a roselike odor. The solid–liquid phase equilibrium (SLE) with higher alkanols (decan-1-ol, hexadecan-1-ol, octadecan-1-ol and eicosan-1-ol), alkanes (eicosane) and oleic acid has been determined at ambient pressure in order to explore their potential application for continuous product extraction during fermentation. A dynamic method was used over a broad range of mole fractions and temperatures from (246 to 338) K. Complete liquid miscibility and simple liquidus curves were observed for all binary (alkan-1-ol + PEA) systems. Liquid–liquid equilibrium (LLE) with the upper critical solution temperature was observed in case of (eicosane, or oleic acid + PEA) mixtures. Decan-1-ol was chosen as the best solvent for PEA. The influence of pressure on SLE was determined up to 700 MPa and at three different temperatures (303.15, 313.15 and 323.15) K. The experimental data were correlated with the non-random two-liquid equation, and both SLE and LLE data were described with average low root-mean-square deviation σ T = 1.25 K for SLE and in mole fraction σ x = 0.004 for LLE. The experimental data were in a very good agreement with the predictions of the group contribution model Mod. UNIFAC(Do) using published parameters. The main aim of this study was to determine the best solvent for a biphasic in situ product removal at temperature T = 303.15 K. Two solvents, decan-1-ol and oleic acid, were further tested for their influence on yeast cell growth, and both were found to be biocompatible. The well-known oleic acid was found to give better results than decan-1-ol in terms of extraction capability, biocompatibility and overall performance in the bioprocess.

Adsorption of volatile aroma compound 2-phenylethanol from synthetic solution onto granular activated carbon in batch and continuous modes

This paper reports a study on the adsorption of the aroma component 2-phenylethanol from an aqueous solution onto granular activated carbon, derived from coconut husks, using batch and continuous (packed-bed column) systems. In the batch mode, the contact time required to attain the adsorption equilibrium was 90min. The adsorption isotherms were evaluated at 20.0°C, 30.0°C and 40.0°C. In the continuous mode, the effects of flow rate and bed length were studied. Experimental data showed that the breakthrough and exhaustion times decreased with higher flow rates and increased with higher packed beds. The adsorption in the fixed-bed column was more efficient under the following conditions: flow rate of 3.5mLmin−1 and bed length of 11cm. Based on the data acquired, adsorption appears to represent a promising method for the recovery of 2-phenylethanol lost during the instant coffee production process.

Modeling of an integrated fermentation/membrane extraction process for the production of 2-phenylethanol and 2-phenylethylacetate

An unstructured model for an integrated fermentation/membrane extraction process for the production of the aroma compounds 2-phenylethanol and 2-phenylethylacetate by Kluyveromyces marxianus CBS 600 was developed. The extent to which this model, based only on data from the conventional fermentation and separation processes, provided an estimation of the integrated process was evaluated. The effect of product inhibition on specific growth rate and on biomass yield by both aroma compounds was approximated by multivariate regression. Simulations of the respective submodels for fermentation and the separation process matched well with experimental results. With respect to the in situ product removal (ISPR) process, the effect of reduced product inhibition due to product removal on specific growth rate and biomass yield was predicted adequately by the model simulations. Overall product yields were increased considerably in this process (4.0g/L 2-PE+2-PEA vs. 1.4g/L in conventional fermentation) and were even higher than predicted by the model. To describe the effect of product concentration on product formation itself, the model was extended using results from the conventional and the ISPR process, thus agreement between model and experimental data improved notably. Therefore, this model can be a useful tool for the development and optimization of an efficient integrated bioprocess.

Bioproduction of the aroma compound 2‐Phenylethanol in a solid–liquid two‐phase partitioning bioreactor system by Kluyveromyces marxianus

The rose-like aroma compound 2-phenylethanol (2-PE) is an important fragrance and flavor ingredient. Several yeast strains are able to convert l-phenylalanine (l-phe) to 2-PE among which Kluyveromyces marxianus has shown promising results. The limitation of this process is the low product concentration and productivity primarily due to end product inhibition. This study explored the possibility and benefits of using a solid-liquid Two-Phase Partition Bioreactor (TPPB) system as an in situ product removal technique. The system applies polymer beads as the sequestering immiscible phase to partition 2-PE and reduce the aqueous 2-PE concentration to non-inhibitory levels. Among six polymers screened for extracting 2-PE, Hytrel 8206 performed best with a partition coefficient of 79. The desired product stored in the polymer was ultimately extracted using methanol. A 3 L working volume solid-liquid batch mode TPPB using 500 g Hytrel as the sequestering phase generated a final overall 2-PE concentration of 13.7 g/L, the highest reported in the current literature. This was based on a polymer phase concentration of 88.74 g/L and aqueous phase concentration of 1.2 g/L. Even better results were achieved via contact with more polymers (approximately 900 g) with the aqueous phase applying a semi-continuous reactor configuration. In this system, a final 2-PE concentration (overall) of 20.4 g/L was achieved with 1.4 g/L in the aqueous and 97 g/L in the polymer phase. The overall productivities of these two reactor systems were 0.38 and 0.43 g/L h, respectively. This is the first report in the literature of the use of a polymer sequestering phase to enhance the bioproduction of 2-PE, and exceeds the performance of two-liquid phase systems in terms of productivity as well as ease of operation (no emulsions) and ultimate product recovery.

Medium optimization for the production of the aroma compound 2-phenylethanol using a genetic algorithm

Using a genetic algorithm, 13 medium constituents and the temperature were varied to improve the bioconversion of l-phenylalanine (l-phe) to 2-phenylethanol (2-PE) with Kluyveromyces marxianus CBS 600. Within four generations plus an additional temperature screening, corresponding to 98 parallel experiments altogether, a maximum 2-PE concentration of 5.6g/l, equivalent to an increase of 87% compared to the non-optimized medium was obtained. The vitamin content of the medium had to be raised significantly.

Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium.

Fourteen yeast strains were screened for production of 2-phenylethanol from L-phenylalanine with molasses as carbon source. Up to 1 g 2-phenylethanol l-1 was obtained. Using oleyl alcohol as a second phase for in situ product removal to enhance the production of 2-phenylethanol increased the yield to about 3 g 2-phenylethanol l-1 at 35 degrees C. The most productive strains were Kluyveromyces marxianus CBS 600 and CBS 397.

Metabolic physiology of aroma-producing Kluyveromyces marxianus.

Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2-phenylethanol, which is derived in a bioconversion from L-phenylalanine. In the present work the product yield of K. marxianus in batch cultivation was estimated as 0.65 mol 2-phenylethanol/mol L-phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol. By a comprehensive approach of stoichiometric balancing and GC-MS analysis of various substrates and products of K. marxianus a detailed insight into its metabolism was gained. For this purpose ring-labelled ((13)C(6)) L-phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture. The produced aroma compounds 2-phenylethanol and 2-phenylethylacetate stem exclusively from the supplied L-phenylalanine, whereas glucose was not converted into these products because of efficient feed-back inhibition of prephenate dehydratase in the L-phenylalanine biosynthetic pathway. It could be further shown that the supplied L-phenylalanine completely covers the anabolic cellular demand for this amino acid. Quantification of (13)CO(2) in the exhaust gas provided clear evidence for catabolic breakdown of L-phenylalanine during cultivation. Metabolic balancing around the pool of free intracellular L-phenylalanine revealed a significant loss of L-phenylalanine into catabolic and anabolic pathways. While 73.3% of L-phenylalanine was converted into 2-phenylethanol or 2-phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis. Catabolic breakdown of L-phenylalanine via hydroxylation to L-tyrosine could be excluded. In addition to an insight into metabolic functioning and regulation of 2-phenylethanol-producing K. marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2-phenylethanol-producing yeasts.