Biocatalytic Production of Odor-Active Fatty Aldehydes from Fungal Lipids.

Odor-active fatty aldehydes are important compounds for the flavor and fragrance industry. By a coupled enzymatic reaction using an α-dioxygenase (α-DOX) and an aldehyde dehydrogenase (FALDH), scarcely available aldehydes from the biotransformation of margaroleic acid [17:1(9Z)] were characterized and have shown highly interesting odor profiles, including citrus-like, soapy, herbaceous, and savory notes. In particular, (Z)-8-hexadecenal and (Z)-7-pentadecenal exhibited notable meaty odor characteristics. Submerged cultivation of Mortierella hyalina revealed the accumulation of the above-mentioned, naturally uncommon fatty acid 17:1(9Z). Its production was significantly increased by the modulation of culture conditions, whereas the highest accumulation was observed after 4 days at 24 °C and l-isoleucine supplementation. The lipase-, α-DOX-, and FALDH-mediated biotransformation of M. hyalina lipid extract resulted in a complex aldehyde mixture with a high aldehyde yield of ∼50%. The odor qualities of the formed aldehydes were assessed by means of gas chromatography-olfactometry, and several of the obtained fatty aldehydes have been sensorially described for the first time. To assess the aldehyde mixture's potential as a flavor ingredient, a sensory evaluation was conducted. The obtained product exhibited intense citrus-like, green, and soapy odor impressions.

Upcycling of black currant pomace for the production of a fermented beverage with Wolfiporia cocos.

Pomace as a side stream from black currant juice production is mostly discarded, even though it is rich in nutrients like protein, fiber, sugars, anthocyanins, polyphenols, and other secondary metabolites. Fungi from the division of Basidiomycota have a great enzymatic toolbox to recycle these complex mixtures of nutrients. In particular, the edible medicinal fungus Wolfiporia cocos has been described as a suitable biocatalyst to form pleasant aroma compounds in fermentation processes. Therefore, medium optimization, upscaling, and filtration were performed to produce a beverage based on black currant pomace fermented with W. cocos. A trained panel described the beverage as highly pleasant, reminiscent of honey, flowers and berries with a well-balanced sour and sweet taste. The flavor compounds linalool (citrus), geraniol (flowery), phenylacetic acid (honey), methyl phenylacetate (honey), eugenol (clove), and 2-phenylethanol (rose) were produced during fermentation and the concentrations exceeded their respective odor thresholds. The produced beverage was evaluated with 8.0 ± 1.4 from 10 for the question of whether panelists would buy the product. Fungal fermentation with the edible fungus W. cocos enabled the production of a highly pleasant beverage and additionally may reduce waste by using pomace and table sugar as sole ingredients. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05677-4.

An enzymatic tandem reaction to produce odor-active fatty aldehydes.

Aldehydes represent a versatile and favored class of flavoring substances. A biocatalytic access to odor-active aldehydes was developed by conversion of fatty acids with two enzymes of the α-dioxygenase pathway. The recombinant enzymes α-dioxygenase (α-DOX) originating from Crocosphaera subtropica and fatty aldehyde dehydrogenase (FALDH) from Vibrio harveyi were heterologously expressed in E. coli, purified, and applied in a coupled (tandem) repetitive reaction. The concept was optimized in terms of number of reaction cycles and production yields. Up to five cycles and aldehyde yields of up to 26% were achieved. Afterward, the approach was applied to sea buckthorn pulp oil as raw material for the enzyme catalyzed production of flavoring/fragrance ingredients based on complex aldehyde mixtures. The most abundant fatty acids in sea buckthorn pulp oil, namely palmitic, palmitoleic, oleic, and linoleic acid, were used as substrates for further biotransformation experiments. Various aldehydes were identified, semi-quantified, and sensorially characterized by means of headspace-solid phase microextraction-gas chromatography-mass spectrometry-olfactometry (HS-SPME-GC-MS-O). Structural validation of unsaturated aldehydes in terms of double-bond positions was performed by multidimensional high-resolution mass spectrometry experiments of their Paternò-Büchi (PB) photoproducts. Retention indices and odor impressions of inter alia (Z,Z)-5,8-tetradecadienal (Z,Z)-6,9-pentadecadienal, (Z)-8-pentadecenal, (Z)-4-tridecenal, (Z)-6-pentadecenal, and (Z)-8-heptadecenal were determined for the first time. KEY POINTS: • Coupled reaction of Csα-DOX and VhFALDH yields chain-shortened fatty aldehydes. • Odors of several Z-unsaturated fatty aldehydes are described for the first time. • Potential for industrial production of aldehyde-based odorants from natural sources.

Aroma characterization of a wild plant (Sanguisorba albanica) from Kosovo using multiple headspace solid phase microextraction combined with gas chromatography-mass spectrometry-olfactometry.

A wild plant (Sanguisorba albanica) from Kosovo exhibits an appealing aroma, which was perceived as intensely fresh, green, and slightly sweetish with hints of cucumber, melon, and green tea. The characteristic aroma of S. albanica was analyzed for the first time by employing advanced molecular sensory techniques coupled with multiple headspace solid phase microextraction (MHS-SPME). Thirty-six different odor impressions were perceived, and 34 corresponding compounds were identified by a gas chromatography system equipped with a mass spectrometric detector and an olfactory detection port. By aroma dilution analysis, sixteen key odorants with flavor dilution factors higher than 16 were identified. Their respective concentrations were quantified by means of MHS-SPME according to the determined ß values (0.43-0.83), and odor activity values (OAVs) were calculated. (E,Z)-2,6-Nonadienal (green, melon-like, and sweetish odor; OAV 20,046), followed by linalool (floral and citrus-like odor; OAV 10,563), (Z)-6-nonenal (cucumber-like, green, and melon-like odor; OAV 4200), ethyl isobutyrate (floral and fruity odor; OAV 1315), and (E)-2-nonenal (green and fatty odor; OAV 1062) were representative odorants contributing to the overall aroma of S. albanica. Differences between stems, leaves, and flowers of S. albanica with regard to the key odorants were also presented. Intensely green and floral aromas of the leaves and flowers were greatly attributed to (E,Z)-2,6-nonadienal and linalool, respectively. Apart from these compounds, (Z)-6-nonenal contributed to the overall aroma of the stems as well.

Aroma Investigation of Chios Mastic Gum (Pistacia lentiscus Variety Chia) Using Headspace Gas Chromatography Combined with Olfactory Detection and Chiral Analysis.

Chios mastic gum (Pistacia lentiscus var. Chia) exhibits intensely sourish, green, resinous, and woody odor notes with hints of citrus and pine. Despite its attractive flavor, no description of its aroma properties by molecular sensory techniques has been published thus far. A total of 25 odor-active compounds with flavor dilution (FD) factors of 1-512 were identified by gas chromatography-mass spectrometry-olfactometry (GC-MS-O) combined with headspace solid-phase microextraction (HS-SPME) and stir bar sorptive extraction (HS-SBSE). Quantitative analyses performed by multiple HS-SPME and calculation of odor activity values of 10 odorants with high FD factors revealed an essential role of several minor components (e.g., ß-myrcene, limonene, ß-linalool, and perillene) for the overall aroma of mastic gum, besides the dominating compound α-pinene. The indispensable contribution of the minor odorants to mastic gum was further confirmed by aroma recombination and omission tests. Varying enantiomeric excess values of the key odorants were observed by multidimensional GC-MS.

Biosynthesis of Stereoisomers of Dill Ether and Wine Lactone by Pleurotus sapidus.

The white-rot fungus Pleurotus sapidus (PSA) biosynthesizes the bicyclic monoterpenoids 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (dill ether) (1) and 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one (wine lactone) (2). Submerged cultures grown in different media were analyzed by gas chromatography-mass spectrometry. The stereochemistry of the formed isomers was elucidated by comparing their retention indices to those of reference compounds by enantioselective multidimensional gas chromatography. The basidiomycete produced the rare (3R,3aR,7aS) and (3S,3aR,7aS) stereoisomers of dill ether and wine lactone. Kinetic analyses of the volatilome and bioprocess parameters revealed that the biosynthesis of the bicyclic monoterpenoids correlated with the availability of the primary carbon source glucose. Spiking the media with 13C-labeled glucose demonstrated that the compounds were produced de novo. Supplementation studies i.a. with isotopically labeled substrates further identified limonene and p-menth-1-en-9-ol as intermediate compounds in the fungal pathways. PSA was able to biotransform all enantiomeric forms of the latter compounds to the respective isomers of dill ether and wine lactone.

Enantiomeric ratios of 2-methylbutanoic acid and its methyl ester: Elucidation of novel biogenetic pathways towards (R)-methyl 2-methylbutanoate in a beverage fermented with shiitake.

Up to 35% of (R)-methyl 2-methylbutanoate (M2MB) was observed in a beverage fermented with shiitake. As M2MB naturally occurs typically in high excesses of the (S)-enantiomer, the origin of the (R)-ester was elucidated by stable isotope labeled precursor-feeding studies. (R)-2-Methylbutanoic acid was identified as the main precursor in the substrate wort. Trace amounts of (R)-M2MB were produced by transformation of unsaturated secondary metabolites (tiglic aldehyde and tiglic acid) derived from l-isoleucine. Surprisingly, shiitake esterified (R)-2-methylbutanoic acid faster to (R)-M2MB than the corresponding (S)-enantiomer. Concurrently, spontaneous non-enantioselective degradation of M2MB occurred in shiitake. This explains diverse enantiomeric ratios of M2MB and different enantiomeric ratios of 2-methylbutanoic acid and M2MB in the beverage. As the odor threshold values of (R)-and (S)-M2MB differ significantly, these findings are of high relevance for the overall flavor of the fermented beverage and elucidate the discrepancy of enantiomeric ratios of 2-alkyl-branched acids and esters reported in nature.

Biotechnological Production of Methyl-Branched Aldehydes.

A number of methyl-branched aldehydes impart interesting flavor impressions, and especially 12-methyltridecanal is a highly sought after flavoring compound for savory foods. Its smell is reminiscent of cooked meat and tallow. For the biotechnological production of 12-methyltridecanal, the literature was screened for fungi forming iso-fatty acids. Suitable organisms were identified and successfully grown in submerged cultures. The culture medium was optimized to increase the yields of branched fatty acids. A recombinant carboxylic acid reductase was used to reduce 12-methyltridecanoic acid to 12-methyltridecanal. The efficiency of whole-cell catalysis was compared to that of the purified enzyme preparation. After lipase-catalyzed hydrolysis of the fungal lipid extracts, the released fatty acids were converted to the corresponding aldehydes, including 12-methyltridecanal and 12-methyltetradecanal.

Submerged Cultivation of Pleurotus sapidus with Molasses: Aroma Dilution Analyses by Means of Solid Phase Microextraction and Stir Bar Sorptive Extraction.

The basidiomycete Pleurotus sapidus (PSA) was grown in submerged cultures with molasses as substrate for the production of mycelium as a protein source for food applications. The volatilomes of the substrate, the submerged culture, and the mycelia were analyzed by gas chromatography-tandem mass spectrometry-olfactometry. For compound identification, aroma dilution analyses by means of headspace solid phase microextraction and stir bar sorptive extraction were performed via variation of the split vent flow rate. Among the most potent odorants formed by PSA were arylic compounds (e.g., p-anisaldehyde), unsaturated carbonyls (e.g., 1-octen-3-one, ( E)-2-octenal, ( E, E)-2,4-decadienal), and cyclic monoterpenoids (e.g., 3,9-epoxy- p-menth-1-ene, 3,6-dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3 H)-one). Several compounds from the latter group were described for the first time in Pleurotus spp. After separation of the mycelia from the medium, the aroma compounds were mainly enriched in the culture supernatant. The sensory analysis of the mycelium correlated well with the instrumental results.

Aroma Characterization and Safety Assessment of a Beverage Fermented by Trametes versicolor.

A cereal-based beverage was developed by fermentation of wort with the basidiomycete Trametes versicolor. The beverage possessed a fruity, fresh, and slightly floral aroma. The volatiles of the beverage were isolated by liquid-liquid extraction (LLE) and additionally by headspace solid phase microextraction (HS-SPME). The aroma compounds were analyzed by a gas chromatography system equipped with a tandem mass spectrometer and an olfactory detection port (GC-MS/MS-O) followed by aroma (extract) dilution analysis. Thirty-four different odor impressions were perceived, and 27 corresponding compounds were identified. Fifteen key odorants with flavor dilution (FD) factors ranging from 8 to 128 were quantitated, and their respective odor activity values (OAVs) were calculated. Six key odorants were synthesized de novo by T. versicolor. Furthermore, quantitative changes during the fermentation process were analyzed. To prepare for the market introduction of the beverage, a comprehensive safety assessment was performed.

Identification of potent odorants in a novel nonalcoholic beverage produced by fermentation of wort with shiitake (Lentinula edodes).

Novel refreshments with pleasant flavors were developed by fermentation of wort with basidiomycetes. Among 31 screened fungi, shiitake (Lentinula edodes) produced the most pleasant flavor. It was perceived as fruity, slightly sour, and plum-like. Flavor compounds were isolated by liquid-liquid extraction (LLE) and by headspace solid phase microextraction (HS-SPME). The key odor-active compounds were analyzed by a gas chromatography system equipped with a tandem mass spectrometry detector and an olfactory detection port (GC-MS/MS-O) and aroma extract dilution analysis (AEDA). For HS-SPME, a revised method of increasing the GC inlet split ratio was used. Most of the key odor-active compounds (e.g., 2-acetylpyrrole, ß-damascenone, (E)-2-nonenal, and 2-phenylethanol) were detected with both extraction techniques. However, distinct differences between these two methods were observed.

Food and feed enzymes.

Humans have benefited from the unique catalytic properties of enzymes, in particular for food production, for thousands of years. Prominent examples include the production of fermented alcoholic beverages, such as beer and wine, as well as bakery and dairy products. The chapter reviews the historic background of the development of modern enzyme technology and provides an overview of the industrial food and feed enzymes currently available on the world market. The chapter highlights enzyme applications for the improvement of resource efficiency, the biopreservation of food, and the treatment of food intolerances. Further topics address the improvement of food safety and food quality.