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Biotechnology as a source of natural volatile flavours
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ScienceDirect Biotechnology as a source of natural volatile flavours Ralf G Berger It has been a long way from the endogenous flavour-forming microflora of traditional fermented food to genetically engineered prokaryotic and eukaryotic flavour producer strains in fully controlled bioreactors. More than 100 flavours from advanced bioprocesses are commercially available. Apart from its inherent principal advantages, biocatalysis matches the increasing re-orientation to sustainable industrial processes. To create valuable and pure food flavours in high yields from low-valued industrial side-streams using metabolically streamlined, safe microorganisms in specifically engineered bioreactors has been a vision which turns more and more into reality. Addresses Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Hannover, Germany Corresponding author: Berger, Ralf G ([email protected])
Current Opinion in Food Science 2015, 1:38–43 This review comes from a themed issue on Food chemistry and biochemistry Edited by Delia B. Rodriguez Amaya
doi:10.1016/j.cofs.2014.09.003 S2214-7993/# 2014 Elsevier Ltd. All rights reserved.
Introduction The human sense of smell is triggered by small, non-polar to medium polar molecules which dock onto receptor proteins of the olfactory epithelium. They signal freshness, quality and authenticity of a food, hence guiding our choice of food. Flavours from plant sources occur as complex mixtures with very different, generally low to ultra-trace concentrations, and this is not their only disadvantage. Traditional agriculture suffers from much imponderability. Chemical synthesis seems to be an immediate alternative, but the resulting products must not carry the label ‘natural’ which, although scientifically unfounded, is preferred by the consumer. According to effective European law (EG 1334/2008) a ‘natural flavouring substance’ shall mean a compound ‘obtained by appropriate physical, enzymatic or microbiological processes from material of vegetable, animal or microbiological origin . . ..’. In the US, the Code of Federal Regulation (CFR — Title 21) of the FDA contains a Current Opinion in Food Science 2015, 1:38–43
similar definition including the terms ‘enzymolysis’ and ‘fermentation’. Apart from the legal preference, biotechnology offers advantages especially for the generation of volatile flavours. Like other agonists, volatile flavours often carry stereo-centres, and both odour intensity and quality are usually affected by the stereochemistry [1]. To execute their physiological functions, volatile flavours possess an internal clock. Not only the physical volatility, but also the chemical instability of the structures (thiol groups and aldehyde functions, tertiary alcohols, Z-double bonds, among others) limits their activity. As most bioprocesses run under ambient conditions without highly reactive chemicals in the system, volatile flavours should be among the preferred targets. More recent driving forces for a biotechnology of flavours are the world-wide focus on a ‘greener chemistry’ and the association of certain flavours with beneficial health effects [2].
Fermentation flavours are the historical roots The obvious change of aroma from a fruit must to a fermented beverage are easily recognized without knowing about the (micro)biological reasons. In fact, the food industry has rediscovered fermentation as a gentle, versatile and natural means to create new products [3]. Most of the more than 100 commercial volatile flavours from biotechnology were inspired by the empirical prototypes. Lactic acid bacteria, such as Lactococcus, Lactobacillus, Lecuconostoc, or also Enterococcus faecium (!) were grown in milk with supplements of amino acids. The headspace volatiles identified were carbonyls and esters with simple structures, but great impact on the flavour of fermented dairy products [4]. Work of such kind is still required to find facultative flavour precursors, here leucine, phenylalanine and methionine, and to derive theoretical pathways of formation. Due to their hydrophobic nature, unsaturated fatty acids are popular flavour precursors. Lactobacillus helveticus converted oleic, linoleic and linolenic acids to hexanal, octanal, nonanal, 2-octenal, 2octanal and the corresponding alcohols [5]. Isotopes labelled precursors, such as carbon labelled linoleic acid, build the metabolic bridge from substrate to flavour product. Monoxygenase P 450 or dioxygenase genes code for the enzymes typically responsible for the formation of these volatile oxylipins. However, no such genes were ever detected in L. helveticus. It appears high time to analyze the enzymology of flavour formation in more depth in flavour formers. Prototypic work on heterofermentative Leuconostoc and Lactobacillus strains dealt with esterase and aminotransferase activities [6]. A genomewide model of carbon and nitrogen flow in L. lactis www.sciencedirect.com
Flavour biotechnology Berger 39
coupled with the pathways resulting in flavour formation showed that a more systematic, pathway based approach is now possible, at least with fully sequenced prokaryotics [7]. Two applications of the newly gained metabolic knowledge can be envisaged: Deliberately shifted flavour profiles of the classical products of the dairy industry, or a concerted over-production of a sought-after flavour chemicals. Similar work is going on with the more complex yeasts, particularly Saccharomyces. However, even with state-of-the-art molecular biology tools for strain differentiation, micro-vinification experiments were required to correlate genetics with oenological traits [8].
resulting flavour mixtures showed pleasant fruity, flowery and cinnamon-like sensorial attributes suitable to flavour a new non-alcoholic fermented beverage [11]. The food industry is currently re-considering the traditional routes of flavour formation to create new opportunities using clean technologies [12,13]. Particularly higher fungi possess large genomes and suggest themselves as suitable catalysts to generate a multitude of plant-like flavour compounds, as they are appreciated by the consumers (Figure 1).
Another de´ja` vu: from trash to treasure
Among the well amenable biotech-derived flavours are phenylpropanoids, esters and lactones, and terpenoids. Not only phenylpropanoids, but also some of their catabolic derivatives, such as anethole, isoeugenol, and isosafrole were found [14]. Isosafrole is itself precursor to piperonal, a constituent of composed vanilla flavours.
The food industry inevitably produces huge volumes of side-streams, such as pomace, peels and husks which still contain flavour precursors. To consume a portion of a side-stream as a fermentation substrate and to produce a high-value flavour at the same time means to kill two birds with one stone. Along this current trend, a Brazilian patent application described the conversion of cassava and malt bagasse to the fruity smelling volatile ethyl hexanoate by Neurospora sitophila [9]. Cassava wastewater served as the substrate to evaluate the production of 2phenylethanol by Geotrichum fragrans, Kluyveromyces marxianus and Saccharomyces cerevisiae through the Ehrlich pathway [10]. Likewise, higher fungi, such as Tyromyces chioneus, were grown on apple pomace, and potent odorants, such as 3-phenylpropanal, 3-phenyl-1-propanol, cinnamaldehyde and methyl cinnamate were identified. The
Wild-type strains cultivated in defined nutrient media
Esters, such as 2-phenylethyl acetate, impart fruity notes to yeast cultures. The reaction using lipophilic Yarrowia yeast was optimized, and the cell wall specifically permeabilized [15]. Compared to a reverse hydrolysis using an isolated esterase, the whole cell approach may show advantages in terms of enzyme regeneration or stability and result in a more economic process. Fruit esters and lactones with fruit, milk, cream and nutty attributes are now the best researched and economically most important
Figure 1
L-Phenylalanine
(1) is a versatile precursor of volatile microbial flavours, such as vinylbenzenes (2), benzylalcohols, benzaldehydes, acetophenones, benzoic and salicylic acids, anthranilic acids and their esters (3), phenylethanals/ols, phenylethanoic acids, and their esters (4), cinnamyl alcohols, cinnamaldehydes, cinnamates, and their esters (5), phenylpropanals/ols, phenylpropanoic acids, and their esters (6), eugenol, safrol and derivatives (7), phenylbutenoids (8), and phenylbutanoids (9), such as frambinone (‘raspberry ketone’).
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Current Opinion in Food Science 2015, 1:38–43
40 Food chemistry and biochemistry
microbial flavour compounds. Metabolic engineering strategies for the various pathways and bioreactor operation were examined [16]. Hydroxylation and b-oxidation of a fatty acid precursor leads to 4- and 5-alkanolides; cytochrome catalysis presents another route to lactones through Baeyer-Villiger-type oxidation. Comprising more than 30,000 representatives, oligoisoprenoids derived from the acetate-mevalonate or from the triose-pyruvate pathway are the most diverse class of substances in nature. The primary products of isoprene addition, the terpene hydrocarbons, predominate in plant essential oils. The oxygenated terpenoids are secondary products. Starting in the early 1960s, microorganisms, such as Pseudomonas, were used for the biotransformation of the hydrocarbons [17]. Cytochrome and other oxidoreductase activities yielded high-valued flavour compounds [18]. Current work is searching for new species, such as fungal endophytes growing inter- or intracellularly in plants [19]. Common biotransformation substrates were the abundant monoterpenes limonene, citronellol, a- and b-pinene. The strains were distinguished by a high tolerance towards the generally cytotoxic hydrocarbons and were identified as Penicillia and Aspergilli [20]. Further transformations of the resulting carbonyls were achieved using the high reduction power of yeasts, such as Candida, Debaryomyces, or Kluyveromyces [21]. (4R)-( )-carvone and (1R)-( )-myrtenal gave (1R,4R)-dihydrocarvone and (1R)-myrtenol as the main products. As many of these transformation reactions could as well be achieved by chemical means, analytical tools are needed to differentiate between the various origins. Chiral gaschromatography or, if stereocentres are missing, stable isotope analysis on the levels of natural abundance are the techniques of choice [22]. Using intact cells as biocatalysts means to entertain many metabolic routes not required for the formation of the target flavour. As the isolation of an enzyme may turn out complicated, lyophilisates retaining the catalytic activity are a viable compromise. DyP-type peroxidases of the basidiomycete Marasmius scorodonius (garlic mushroom) capable of the asymmetric cleavage of tetraterpenes yielded C13-orisoprenoid flavour compounds, such as b-ionone [23], and a lipoxygenase-like enzyme from Pleurotus species converted b-myrcene and related monoterpenes to furanoterpenoids [24]. The initial incorporation of dioxygen was similar to a 2 + 4 cycloaddition of 1,3dienes and was followed by a spontaneous decay to furans. The cyclic peroxides 3,6-dihydro-4-(2-(3,3dimethyloxiran-2-yl)ethyl)-1,2-dioxine and 5-(3,6-dihydro-1,2-dioxin-4-yl)-2-methylpentan-2-ol were identified as key intermediates. This biotransformation not only allowed to present a substantiated biogenetic scheme for the formation of monoterpenoid furans, but also opened access to potent flavours, such as perillene and rosefurane (Figure 2). Some orthologous lipoxygenases from other Current Opinion in Food Science 2015, 1:38–43
Figure 2
b-Myrcene (1), a common starter in chemical flavour synthesis, is transformed to hydroxy derivatives, such as dihydrolinallol (2) or aterpineol (3) by Cytochromes of procaryotic organisms [17], while eukaryotes may also introduce dioxygen to result in myrcene hydroperoxides (4, 6) which decay to monoterpene ethers, such as rose furane or perillene (5) or the melonal-like nonadienal (7) [24].
Pleurotus species were characterized for their specificity in converting the uncommon terpenic substrates [25].
Enzymes, the most straightforward approach The use of lipases for lipolysis, reverse hydrolysis and resolution of racemic esters, glycosidases to release flavours from glycosidic precursors, peptidases, and a number of oxidoreductases and synthases is established [26]. The observation that some lipases maintained their activity in organic solvents was a breakthrough. Since then, numerous papers showed the capacity of the concept. Recently, a carboxylesterase from Bacillus licheniformis was reported to synthesize isoamyl acetate from isoamyl alcohol and p-nitrophenyl acetate in n-hexane [27]. Although the choice of the acyl donor facilitated the analytics, another (natural) source, such as vinegar, will be required to produce a natural flavour. Following the principles of sustainability, Lipozyme was used for the transesterification of coconut oil and fusel alcohols, both renewable and low-cost natural materials [28]. Octanoic acid ethyl-, butyl-, isobutyl-, propyl- and (iso)amyl esters were formed. The enzyme was re-used several times without significant loss of activity after a washing step was introduced. www.sciencedirect.com
Flavour biotechnology Berger 41
Biocatalysts from transgenic sources Regardless of the controversial public discussion, the tremendous advances in genetic engineering currently stimulate scientific progress in flavour biotechnology. Full genomes of food microorganisms, such as Saccharomyces and Propionibacterium are electronically available, and many tools can help expressing a metabolic trait in a cellular host. Exotic sources of genes, such as sediment from the Chinese Sea were explored [29]. An esterase gene was found there, and the enzyme with specificity towards short chain fatty acids was expressed in Escherichia coli. Enzymes from extremophiles are supposed to feature high tolerance against chemical and physical inactivation resulting in the requested improved operational stability. Recently, the production of flavour precursors is gaining attention. Ferulic acid, the precursor of biotech-vanillin, was generated in recombinant Pseudomonas fluorescens by targeted mutation of the vanillin dehydrogenase gene and concurrent expression of structural genes for feruloyl-CoA synthetase and hydratase/ aldolase [30]. A strain of Saccharomyces cerevisiae was engineered to convert eugenol to the same precursor by chromosomal integration of a vanillyl-alcohol oxidase gene [31]. The expression of stress or insect-induced genes of terpene synthases from higher plants in E. coli presents a remarkable progress looking at the large metabolic distance between donor and host. The formation of monoterpenes [32], and of sesquiterpenes [33,34], sometimes amplified by a genetically improved provision of endogenous diphosphate precursors, was reported. The recombinant production of key compounds of sandalwood oil, such as santalol in yeast, and of patchouli oil, such as patchoulol in E. coli, has proven the principle. The expression of a sesquiterpene synthase gene in the edible mushroom Schizophyllum commune may contribute to divert public concerns on the safety of recombinant food ingredients [35]. At the same time, biotechnology helps to overcome the destructive exploitation of tropical sources of highly appreciated flavours opening ways to a more bioeconomic production. Among the obstacles of heterologous production are low expression rates, labile and non-natural character of the chemo-synthetic precursor diphosphates, and the emotional objections of the public. Thus, the expression of flavour forming activities in plant hosts is worth being considered. When a melon hydroperoxide lyase gene, a tomato peroxygenase gene and a potato epoxide hydrolase gene were incorporated into tobacco leaves, unsaturated fatty acids were transformed to C9-aldehydes [36]. Advantages include easy handling, and savings of time and costs. However, to establish or reinforce aroma formation in a fruit may affect other metabolic pathways, for example by competition for the same precursors. Although it is obvious that rational metabolic engineering www.sciencedirect.com
has to rely on knowledge of the metabolic pathways, still not enough efforts have been made [37].
Scaling up and streaming down The scale-up of laboratory experiments to pilot or larger scale involves a number of problems owing to the chemistry of the volatile targets. Both substrate and product are often not well water soluble, may be sensitive towards acid or oxygen and cytotoxic towards their producers. Various procedural solutions were developed. The loss of volatile product through gas stripping by the exhaust gas stream may be turned into a down-stream step using adsorbent traps for the recovery; co-cultivation of an adsorbent is another option. Fed-batch protocols avoid high substrate concentrations, in situ recovery is mandatory to prevent further conversion of the product. Ionic liquids replaced water as the reaction medium, for example in reverse hydrolytic reactions. High cell density cultivations counteract the problem of insufficient yield. Two-phase systems harbour the biocatalyst (cell or enzyme) in an aqueous environment, while substrate and product are dissolved in a lipophilic compartment. A recent example is a solid–liquid two-phase partitioning bioreactor used for vanillin production [38]. A thermoplastic polymer was used as the sequestering phase, and a final vanillin concentration of 19.5 g per litre was reached. Vanillin was recovered from the polymer by extraction into an organic solvent, simultaneously regenerating the polymer beads for reuse.
Conclusions The industrial feasibility of a bioprocess mainly depends on its productivity. Two digit yields per litre and day have been achieved for volatile flavours [39]. Kluyveromyces marxianus produced >26 g per litre of 2-phenylethanol, and Candida or Mucor produced up to 40 g per litre of gdecalactone. Still unclear is the legal status of ‘natural’ flavours obtained from recombinant hosts. However, the foreseeable depletion of petrochemicals exerts a strong pressure on the flavour industry. Advances in molecular methods will detect new enzymes associated with flavour formation. Tailored enzymes [40], over-producers selected by transcription analysis or created by gene knock-out (CRISPR), and genetically altered cells [41,42] will become the silver bullets for producing structurally complicated volatile flavours in economic yields.
Acknowledgements Own work related to this review was supported by the ‘Biokatalyse2021’ cluster of the BMBF and by the German Ministry of Economics and Technology (via AiF), and the FEI (Forschungskreis der Erna¨hrungsindustrie e.V., Bonn) (Project AiF ZN 299).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest Current Opinion in Food Science 2015, 1:38–43
42 Food chemistry and biochemistry
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10. Menegatti de Oliveira SM, Gome SD, Sene L, Machado Coelho SR, Barana AC, Cereda MP, Christ D, Piechontcoski J: Production of 2-phenylethanol by Geotrichum fragrans, Saccharomyces cerevisiae and Kluyveromyces marxianus in cassava wastewater. J Food Agric Environ 2013, 11:158-163. 11. Bosse AK, Fraatz MA, Zorn H: Formation of complex natural flavours by biotransformation of apple pomace with basidiomycetes. Food Chem 2013, 141:2952-2959. Basidiomycetes not only produce fruiting bodies with a mushroom flavour; they can be cultivated as mycelia or as pellets in a classical fermentation for the production of a non-alcoholic, cider-like beverage. 12. Marriott RJ: Greener chemistry preparation of traditional flavour extracts and molecules. Agro Food Ind Hi-Tech 2010, 21:46-48. 13. Berger RG: White biotechnology: sustainable options for the generation of natural volatile flavors, Expression of Multidisciplinary Flavour Science. In Proceedings of the Weurman Symposium, 12th, Interlaken, Switzerland, July 1–4, 2008. Edited by Blank I, Wuest M, Yeretzian C. 2010: 319–327. 14. Han D, Ryu J-Y, Lee H, Hur HG: Bacterial biotransformation of phenylpropanoid compounds for producing flavor and fragrance compounds. J Korean Soc Appl Biol Chem 2013, 56:125-133. 15. Bialecka-Florjanczyk E, Krzyczkowska J, Stolarzewicz I, Kapturowska A: Synthesis of 2-phenylethyl acetate in the presence of Yarrowia lipolytica KKP 379 biomass. J Mol Catal B: Enzym 2012, 74:241-245. 16. Romero-Guido C, Belo I. Ta TMN, Lan CH, Alchihab M, Gomes N, Thonart P, Teixeira JA, Destain J, Wache Y: Biochemistry of lactone formation in yeast and fungi and its utilisation for the production of flavour and fragrance compounds. Appl Microbiol Biotechnol 2011, 89:535-547. A review of the leading group on the state-of-the-art of lactone production using bioprocesses including strategies for optimization based on metabolic knowledge. Current Opinion in Food Science 2015, 1:38–43
17. Molina G, Pimentel MR, Pastore GM: Pseudomonas: a promising biocatalyst for the bioconversion of terpenes. Appl Microbiol Biotechnol 2013, 97:1851-1864. A comprehensive review on the enormous metabolic versatility of this genus, especially for the biotransformation of terpenes. Addressed are statistical tools for process optimization, genetic engineering for improvement of biocatalysts, and downstream process improvement. 18. Krings U, Berger RG:: Terpene bioconversion — how does its future look? Nat Prod Commun 2010, 5:1507-1522. 19. Molina G, Pastore GM:: Biotransformation of monoterpenes by endophytes isolated from Brazilian fruits. Int Proc Chem Biol Environ Eng 2013, 50(Food Engineering and Biotechnology):59-63. 20. Molina G, Pinheiro DM, Pimentel MR, dos Santos R, Pastore GM:: Monoterpene bioconversion for the production of aroma compounds by fungi isolated from Brazilian fruits. Food Sci Biotechnol 2013, 22:999-1006. 21. Goretti M, Turchetti B, Cramarossa MR, Forti L, Buzzini P:: Production of flavours and fragrances via bioreduction of (4R)(S)-carvone and (1R)-(S)-myrtenal by non-conventional yeast whole-cells. Molecules 2013, 18:5736-5748. 22. Brenna E, Fronza G, Fuganti C, Gatti FG, Serra S: Biotechnological tools to produce natural flavors and methods to authenticate their origin. In Innovation in Food Engineering. Edited by Passos ML, Ribeiro CP. 2010:81-106. How natural is a flavour compound claimed ‘natural’ on the data sheet? Mere calculations of demand and natural abundance may not be sufficient. Advanced analytical tools are required to distinguish between original and copy. 23. Zelena K, Hardebusch B, Huelsdau B, Berger RG, Zorn H: Generation of norisoprenoid flavors from carotenoids by fungal peroxidases. J Agric Food Chem 2009, 57:9951-9955. 24. Kruegener S, Schaper C, Krings U, Berger RG: Pleurotus species convert monoterpenes to furanoterpenoids through 1,4endoperoxides. Bioresour Technol 2009, 100:2855-2860. 25. Leonhardt R-H, Plagemann I, Linke D, Zelena K, Berger RG: Orthologous lipoxygenases of Pleurotus spp. — a comparison of substrate specificity and sequence homology. J Mol Catal B: Enzym 2013, 97:189-195. 26. Cheetham PSJ: Enzymes for flavor production. In Encyclopedia Industrial Biotechnology, vol. 4. Edited by Flickinger, Michael C. 2010:2149-2175. 27. Torres S, Baigori MD, Swathy SL, Pandey A, Castro GR: Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase. Food Res Int 2009, 42:454460. 28. Sun J, Yu B, Curran P, Liu S-Q: Lipase-catalysed transesterification of coconut oil with fusel alcohols in a solvent-free system. Food Chem 2012, 134:89-94. 29. Peng Q, Zhang X, Shang M, Wang X, Wang G, Li B, Guan G, Li Y, Wang Y: A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea. Microbial Cell Factories 2011, 10:95. The metagenome approach also worked for the detection of flavour enzymes. 30. Di Gioia D, Luziatelli F, Negroni A, Ficca AG, Fava F, Ruzzi M: Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid. J Biotechnol 2011, 156:309-316. 31. Lambert F, Zucca J, Ness F, Aigle M:: Production of ferulic acid and coniferyl alcohol by conversion of eugenol using a recombinant strain of Saccharomyces cerevisiae. Flavour Fragrance J 2014, 29:14-21. 32. Navia-Gine WG, Yuan JS, Mauromoustakos A, Murphy JB, Chen F, Korth KL: Medicago truncatula (E)-b-ocimene synthase is induced by insect herbivory with corresponding increases in emission of volatile ocimene. Plant Physiol Biochem 2009, 47:416-425. 33. Diaz-Chavez ML, Moniodis J, Madilao LL, Jancsik S, Keeling CI, Barbour EL, Ghisalberti EL, Plummer JA, Jones CG, Bohlmann J: Biosynthesis of sandalwood oil: santalum album CYP76F www.sciencedirect.com
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cytochromes P450 produce santalols and bergamotol. PLoS One 2013, 8:e75053. Work showing the promise and the problems of using monooxygenases for the generation of volatile terpenoids.
High product yields by using a novel bioreactor concept based on polymer beads as the partitioning phase preventing the catabolism of the product as a carbon source by the producer cells; possibly transferable to other bioflavour processes.
34. Hartwig S, Frister T, Alemdar S, Li Z, Krings U, Berger RG, Scheper T, Beutel S: Expression, purification and activity assay of a mutant patchoulol synthase cDNA fused to thioredoxin in Escherichia coli. Protein Exp Purif 2014, 97:61-71.
39. Gounaris Y: Biotechnology for the production of essential oils, flavours and volatile isolates. Flavour Fragrance J 2010, 25: 367-386.
35. Scholtmeijer K, Cankar K, Beekwilder J, Woesten HAB, Lugones LG, Bosch D: Production of (+)-valencene in the mushroom-forming fungus S. commune. Appl Microbiol Biotechnol 2014 http://dx.doi.org/10.1007/s00253-014-5581-2. 36. Huang F-C, Schwab W: Overexpression of hydroperoxide lyase, peroxygenase and epoxide hydrolase in tobacco for the biotechnological production of flavours and polymer precursors. Plant Biotechnol J 2012, 10:1099-1109. A different approach: heterologous expression of plant genes in a plant cell using the well-growing tobacco cells in vitro. 37. Aragueez I, Valpuesta Fernandez V: Metabolic engineering of aroma components in fruits. Biotechnol J 2013, 8:1144-1158. 38. Ma X-K, Daugulis AJ: Transformation of ferulic acid to vanillin using a fed-batch solid-liquid two-phase partitioning bioreactor. Biotechnol Progr 2014, 30:207-214.
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40. Lu X, Liu S, Feng Y, Rao S, Zhou X, Wang M, Du G, Chen J:: Enhanced thermal stability of Pseudomonas aeruginosa lipoxygenase through modification of two highly flexible regions. Appl Microbiol Biotechnol 2014, 98:1663-1669. 41. Berger RG (Ed): Flavour and Fragrances — Chemistry Bioprocessing and Sustainability. Berlin: Springer; 2007. 42. Beekwilder J, van Rossum HM, Koopman F, Sonntag F, Buchhaupt M, Schrader J, Halla RD, Boscha D, Pronk JT, van Maris AJA, Daran J-M: Polycistronic expression of a b-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to b-ionone production. J Biotechnol 2014:10. doi: 1016/ j.jbiotec.2013.12.016. Ground-breaking genetic engineering work in which a several fungal open reading frames of the carotenoid pathway were co-expressed with a plant dioxygenase. Possibly the approach of the future, once genetically modified strains will be accepted for food applications.
Current Opinion in Food Science 2015, 1:38–43
ScienceDirect Biotechnology as a source of natural volatile flavours Ralf G Berger It has been a long way from the endogenous flavour-forming microflora of traditional fermented food to genetically engineered prokaryotic and eukaryotic flavour producer strains in fully controlled bioreactors. More than 100 flavours from advanced bioprocesses are commercially available. Apart from its inherent principal advantages, biocatalysis matches the increasing re-orientation to sustainable industrial processes. To create valuable and pure food flavours in high yields from low-valued industrial side-streams using metabolically streamlined, safe microorganisms in specifically engineered bioreactors has been a vision which turns more and more into reality. Addresses Gottfried Wilhelm Leibniz University Hannover, Institute of Food Chemistry, Hannover, Germany Corresponding author: Berger, Ralf G ([email protected])
Current Opinion in Food Science 2015, 1:38–43 This review comes from a themed issue on Food chemistry and biochemistry Edited by Delia B. Rodriguez Amaya
doi:10.1016/j.cofs.2014.09.003 S2214-7993/# 2014 Elsevier Ltd. All rights reserved.
Introduction The human sense of smell is triggered by small, non-polar to medium polar molecules which dock onto receptor proteins of the olfactory epithelium. They signal freshness, quality and authenticity of a food, hence guiding our choice of food. Flavours from plant sources occur as complex mixtures with very different, generally low to ultra-trace concentrations, and this is not their only disadvantage. Traditional agriculture suffers from much imponderability. Chemical synthesis seems to be an immediate alternative, but the resulting products must not carry the label ‘natural’ which, although scientifically unfounded, is preferred by the consumer. According to effective European law (EG 1334/2008) a ‘natural flavouring substance’ shall mean a compound ‘obtained by appropriate physical, enzymatic or microbiological processes from material of vegetable, animal or microbiological origin . . ..’. In the US, the Code of Federal Regulation (CFR — Title 21) of the FDA contains a Current Opinion in Food Science 2015, 1:38–43
similar definition including the terms ‘enzymolysis’ and ‘fermentation’. Apart from the legal preference, biotechnology offers advantages especially for the generation of volatile flavours. Like other agonists, volatile flavours often carry stereo-centres, and both odour intensity and quality are usually affected by the stereochemistry [1]. To execute their physiological functions, volatile flavours possess an internal clock. Not only the physical volatility, but also the chemical instability of the structures (thiol groups and aldehyde functions, tertiary alcohols, Z-double bonds, among others) limits their activity. As most bioprocesses run under ambient conditions without highly reactive chemicals in the system, volatile flavours should be among the preferred targets. More recent driving forces for a biotechnology of flavours are the world-wide focus on a ‘greener chemistry’ and the association of certain flavours with beneficial health effects [2].
Fermentation flavours are the historical roots The obvious change of aroma from a fruit must to a fermented beverage are easily recognized without knowing about the (micro)biological reasons. In fact, the food industry has rediscovered fermentation as a gentle, versatile and natural means to create new products [3]. Most of the more than 100 commercial volatile flavours from biotechnology were inspired by the empirical prototypes. Lactic acid bacteria, such as Lactococcus, Lactobacillus, Lecuconostoc, or also Enterococcus faecium (!) were grown in milk with supplements of amino acids. The headspace volatiles identified were carbonyls and esters with simple structures, but great impact on the flavour of fermented dairy products [4]. Work of such kind is still required to find facultative flavour precursors, here leucine, phenylalanine and methionine, and to derive theoretical pathways of formation. Due to their hydrophobic nature, unsaturated fatty acids are popular flavour precursors. Lactobacillus helveticus converted oleic, linoleic and linolenic acids to hexanal, octanal, nonanal, 2-octenal, 2octanal and the corresponding alcohols [5]. Isotopes labelled precursors, such as carbon labelled linoleic acid, build the metabolic bridge from substrate to flavour product. Monoxygenase P 450 or dioxygenase genes code for the enzymes typically responsible for the formation of these volatile oxylipins. However, no such genes were ever detected in L. helveticus. It appears high time to analyze the enzymology of flavour formation in more depth in flavour formers. Prototypic work on heterofermentative Leuconostoc and Lactobacillus strains dealt with esterase and aminotransferase activities [6]. A genomewide model of carbon and nitrogen flow in L. lactis www.sciencedirect.com
Flavour biotechnology Berger 39
coupled with the pathways resulting in flavour formation showed that a more systematic, pathway based approach is now possible, at least with fully sequenced prokaryotics [7]. Two applications of the newly gained metabolic knowledge can be envisaged: Deliberately shifted flavour profiles of the classical products of the dairy industry, or a concerted over-production of a sought-after flavour chemicals. Similar work is going on with the more complex yeasts, particularly Saccharomyces. However, even with state-of-the-art molecular biology tools for strain differentiation, micro-vinification experiments were required to correlate genetics with oenological traits [8].
resulting flavour mixtures showed pleasant fruity, flowery and cinnamon-like sensorial attributes suitable to flavour a new non-alcoholic fermented beverage [11]. The food industry is currently re-considering the traditional routes of flavour formation to create new opportunities using clean technologies [12,13]. Particularly higher fungi possess large genomes and suggest themselves as suitable catalysts to generate a multitude of plant-like flavour compounds, as they are appreciated by the consumers (Figure 1).
Another de´ja` vu: from trash to treasure
Among the well amenable biotech-derived flavours are phenylpropanoids, esters and lactones, and terpenoids. Not only phenylpropanoids, but also some of their catabolic derivatives, such as anethole, isoeugenol, and isosafrole were found [14]. Isosafrole is itself precursor to piperonal, a constituent of composed vanilla flavours.
The food industry inevitably produces huge volumes of side-streams, such as pomace, peels and husks which still contain flavour precursors. To consume a portion of a side-stream as a fermentation substrate and to produce a high-value flavour at the same time means to kill two birds with one stone. Along this current trend, a Brazilian patent application described the conversion of cassava and malt bagasse to the fruity smelling volatile ethyl hexanoate by Neurospora sitophila [9]. Cassava wastewater served as the substrate to evaluate the production of 2phenylethanol by Geotrichum fragrans, Kluyveromyces marxianus and Saccharomyces cerevisiae through the Ehrlich pathway [10]. Likewise, higher fungi, such as Tyromyces chioneus, were grown on apple pomace, and potent odorants, such as 3-phenylpropanal, 3-phenyl-1-propanol, cinnamaldehyde and methyl cinnamate were identified. The
Wild-type strains cultivated in defined nutrient media
Esters, such as 2-phenylethyl acetate, impart fruity notes to yeast cultures. The reaction using lipophilic Yarrowia yeast was optimized, and the cell wall specifically permeabilized [15]. Compared to a reverse hydrolysis using an isolated esterase, the whole cell approach may show advantages in terms of enzyme regeneration or stability and result in a more economic process. Fruit esters and lactones with fruit, milk, cream and nutty attributes are now the best researched and economically most important
Figure 1
L-Phenylalanine
(1) is a versatile precursor of volatile microbial flavours, such as vinylbenzenes (2), benzylalcohols, benzaldehydes, acetophenones, benzoic and salicylic acids, anthranilic acids and their esters (3), phenylethanals/ols, phenylethanoic acids, and their esters (4), cinnamyl alcohols, cinnamaldehydes, cinnamates, and their esters (5), phenylpropanals/ols, phenylpropanoic acids, and their esters (6), eugenol, safrol and derivatives (7), phenylbutenoids (8), and phenylbutanoids (9), such as frambinone (‘raspberry ketone’).
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40 Food chemistry and biochemistry
microbial flavour compounds. Metabolic engineering strategies for the various pathways and bioreactor operation were examined [16]. Hydroxylation and b-oxidation of a fatty acid precursor leads to 4- and 5-alkanolides; cytochrome catalysis presents another route to lactones through Baeyer-Villiger-type oxidation. Comprising more than 30,000 representatives, oligoisoprenoids derived from the acetate-mevalonate or from the triose-pyruvate pathway are the most diverse class of substances in nature. The primary products of isoprene addition, the terpene hydrocarbons, predominate in plant essential oils. The oxygenated terpenoids are secondary products. Starting in the early 1960s, microorganisms, such as Pseudomonas, were used for the biotransformation of the hydrocarbons [17]. Cytochrome and other oxidoreductase activities yielded high-valued flavour compounds [18]. Current work is searching for new species, such as fungal endophytes growing inter- or intracellularly in plants [19]. Common biotransformation substrates were the abundant monoterpenes limonene, citronellol, a- and b-pinene. The strains were distinguished by a high tolerance towards the generally cytotoxic hydrocarbons and were identified as Penicillia and Aspergilli [20]. Further transformations of the resulting carbonyls were achieved using the high reduction power of yeasts, such as Candida, Debaryomyces, or Kluyveromyces [21]. (4R)-( )-carvone and (1R)-( )-myrtenal gave (1R,4R)-dihydrocarvone and (1R)-myrtenol as the main products. As many of these transformation reactions could as well be achieved by chemical means, analytical tools are needed to differentiate between the various origins. Chiral gaschromatography or, if stereocentres are missing, stable isotope analysis on the levels of natural abundance are the techniques of choice [22]. Using intact cells as biocatalysts means to entertain many metabolic routes not required for the formation of the target flavour. As the isolation of an enzyme may turn out complicated, lyophilisates retaining the catalytic activity are a viable compromise. DyP-type peroxidases of the basidiomycete Marasmius scorodonius (garlic mushroom) capable of the asymmetric cleavage of tetraterpenes yielded C13-orisoprenoid flavour compounds, such as b-ionone [23], and a lipoxygenase-like enzyme from Pleurotus species converted b-myrcene and related monoterpenes to furanoterpenoids [24]. The initial incorporation of dioxygen was similar to a 2 + 4 cycloaddition of 1,3dienes and was followed by a spontaneous decay to furans. The cyclic peroxides 3,6-dihydro-4-(2-(3,3dimethyloxiran-2-yl)ethyl)-1,2-dioxine and 5-(3,6-dihydro-1,2-dioxin-4-yl)-2-methylpentan-2-ol were identified as key intermediates. This biotransformation not only allowed to present a substantiated biogenetic scheme for the formation of monoterpenoid furans, but also opened access to potent flavours, such as perillene and rosefurane (Figure 2). Some orthologous lipoxygenases from other Current Opinion in Food Science 2015, 1:38–43
Figure 2
b-Myrcene (1), a common starter in chemical flavour synthesis, is transformed to hydroxy derivatives, such as dihydrolinallol (2) or aterpineol (3) by Cytochromes of procaryotic organisms [17], while eukaryotes may also introduce dioxygen to result in myrcene hydroperoxides (4, 6) which decay to monoterpene ethers, such as rose furane or perillene (5) or the melonal-like nonadienal (7) [24].
Pleurotus species were characterized for their specificity in converting the uncommon terpenic substrates [25].
Enzymes, the most straightforward approach The use of lipases for lipolysis, reverse hydrolysis and resolution of racemic esters, glycosidases to release flavours from glycosidic precursors, peptidases, and a number of oxidoreductases and synthases is established [26]. The observation that some lipases maintained their activity in organic solvents was a breakthrough. Since then, numerous papers showed the capacity of the concept. Recently, a carboxylesterase from Bacillus licheniformis was reported to synthesize isoamyl acetate from isoamyl alcohol and p-nitrophenyl acetate in n-hexane [27]. Although the choice of the acyl donor facilitated the analytics, another (natural) source, such as vinegar, will be required to produce a natural flavour. Following the principles of sustainability, Lipozyme was used for the transesterification of coconut oil and fusel alcohols, both renewable and low-cost natural materials [28]. Octanoic acid ethyl-, butyl-, isobutyl-, propyl- and (iso)amyl esters were formed. The enzyme was re-used several times without significant loss of activity after a washing step was introduced. www.sciencedirect.com
Flavour biotechnology Berger 41
Biocatalysts from transgenic sources Regardless of the controversial public discussion, the tremendous advances in genetic engineering currently stimulate scientific progress in flavour biotechnology. Full genomes of food microorganisms, such as Saccharomyces and Propionibacterium are electronically available, and many tools can help expressing a metabolic trait in a cellular host. Exotic sources of genes, such as sediment from the Chinese Sea were explored [29]. An esterase gene was found there, and the enzyme with specificity towards short chain fatty acids was expressed in Escherichia coli. Enzymes from extremophiles are supposed to feature high tolerance against chemical and physical inactivation resulting in the requested improved operational stability. Recently, the production of flavour precursors is gaining attention. Ferulic acid, the precursor of biotech-vanillin, was generated in recombinant Pseudomonas fluorescens by targeted mutation of the vanillin dehydrogenase gene and concurrent expression of structural genes for feruloyl-CoA synthetase and hydratase/ aldolase [30]. A strain of Saccharomyces cerevisiae was engineered to convert eugenol to the same precursor by chromosomal integration of a vanillyl-alcohol oxidase gene [31]. The expression of stress or insect-induced genes of terpene synthases from higher plants in E. coli presents a remarkable progress looking at the large metabolic distance between donor and host. The formation of monoterpenes [32], and of sesquiterpenes [33,34], sometimes amplified by a genetically improved provision of endogenous diphosphate precursors, was reported. The recombinant production of key compounds of sandalwood oil, such as santalol in yeast, and of patchouli oil, such as patchoulol in E. coli, has proven the principle. The expression of a sesquiterpene synthase gene in the edible mushroom Schizophyllum commune may contribute to divert public concerns on the safety of recombinant food ingredients [35]. At the same time, biotechnology helps to overcome the destructive exploitation of tropical sources of highly appreciated flavours opening ways to a more bioeconomic production. Among the obstacles of heterologous production are low expression rates, labile and non-natural character of the chemo-synthetic precursor diphosphates, and the emotional objections of the public. Thus, the expression of flavour forming activities in plant hosts is worth being considered. When a melon hydroperoxide lyase gene, a tomato peroxygenase gene and a potato epoxide hydrolase gene were incorporated into tobacco leaves, unsaturated fatty acids were transformed to C9-aldehydes [36]. Advantages include easy handling, and savings of time and costs. However, to establish or reinforce aroma formation in a fruit may affect other metabolic pathways, for example by competition for the same precursors. Although it is obvious that rational metabolic engineering www.sciencedirect.com
has to rely on knowledge of the metabolic pathways, still not enough efforts have been made [37].
Scaling up and streaming down The scale-up of laboratory experiments to pilot or larger scale involves a number of problems owing to the chemistry of the volatile targets. Both substrate and product are often not well water soluble, may be sensitive towards acid or oxygen and cytotoxic towards their producers. Various procedural solutions were developed. The loss of volatile product through gas stripping by the exhaust gas stream may be turned into a down-stream step using adsorbent traps for the recovery; co-cultivation of an adsorbent is another option. Fed-batch protocols avoid high substrate concentrations, in situ recovery is mandatory to prevent further conversion of the product. Ionic liquids replaced water as the reaction medium, for example in reverse hydrolytic reactions. High cell density cultivations counteract the problem of insufficient yield. Two-phase systems harbour the biocatalyst (cell or enzyme) in an aqueous environment, while substrate and product are dissolved in a lipophilic compartment. A recent example is a solid–liquid two-phase partitioning bioreactor used for vanillin production [38]. A thermoplastic polymer was used as the sequestering phase, and a final vanillin concentration of 19.5 g per litre was reached. Vanillin was recovered from the polymer by extraction into an organic solvent, simultaneously regenerating the polymer beads for reuse.
Conclusions The industrial feasibility of a bioprocess mainly depends on its productivity. Two digit yields per litre and day have been achieved for volatile flavours [39]. Kluyveromyces marxianus produced >26 g per litre of 2-phenylethanol, and Candida or Mucor produced up to 40 g per litre of gdecalactone. Still unclear is the legal status of ‘natural’ flavours obtained from recombinant hosts. However, the foreseeable depletion of petrochemicals exerts a strong pressure on the flavour industry. Advances in molecular methods will detect new enzymes associated with flavour formation. Tailored enzymes [40], over-producers selected by transcription analysis or created by gene knock-out (CRISPR), and genetically altered cells [41,42] will become the silver bullets for producing structurally complicated volatile flavours in economic yields.
Acknowledgements Own work related to this review was supported by the ‘Biokatalyse2021’ cluster of the BMBF and by the German Ministry of Economics and Technology (via AiF), and the FEI (Forschungskreis der Erna¨hrungsindustrie e.V., Bonn) (Project AiF ZN 299).
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17. Molina G, Pimentel MR, Pastore GM: Pseudomonas: a promising biocatalyst for the bioconversion of terpenes. Appl Microbiol Biotechnol 2013, 97:1851-1864. A comprehensive review on the enormous metabolic versatility of this genus, especially for the biotransformation of terpenes. Addressed are statistical tools for process optimization, genetic engineering for improvement of biocatalysts, and downstream process improvement. 18. Krings U, Berger RG:: Terpene bioconversion — how does its future look? Nat Prod Commun 2010, 5:1507-1522. 19. Molina G, Pastore GM:: Biotransformation of monoterpenes by endophytes isolated from Brazilian fruits. Int Proc Chem Biol Environ Eng 2013, 50(Food Engineering and Biotechnology):59-63. 20. Molina G, Pinheiro DM, Pimentel MR, dos Santos R, Pastore GM:: Monoterpene bioconversion for the production of aroma compounds by fungi isolated from Brazilian fruits. Food Sci Biotechnol 2013, 22:999-1006. 21. Goretti M, Turchetti B, Cramarossa MR, Forti L, Buzzini P:: Production of flavours and fragrances via bioreduction of (4R)(S)-carvone and (1R)-(S)-myrtenal by non-conventional yeast whole-cells. Molecules 2013, 18:5736-5748. 22. Brenna E, Fronza G, Fuganti C, Gatti FG, Serra S: Biotechnological tools to produce natural flavors and methods to authenticate their origin. In Innovation in Food Engineering. Edited by Passos ML, Ribeiro CP. 2010:81-106. How natural is a flavour compound claimed ‘natural’ on the data sheet? Mere calculations of demand and natural abundance may not be sufficient. Advanced analytical tools are required to distinguish between original and copy. 23. Zelena K, Hardebusch B, Huelsdau B, Berger RG, Zorn H: Generation of norisoprenoid flavors from carotenoids by fungal peroxidases. J Agric Food Chem 2009, 57:9951-9955. 24. Kruegener S, Schaper C, Krings U, Berger RG: Pleurotus species convert monoterpenes to furanoterpenoids through 1,4endoperoxides. Bioresour Technol 2009, 100:2855-2860. 25. Leonhardt R-H, Plagemann I, Linke D, Zelena K, Berger RG: Orthologous lipoxygenases of Pleurotus spp. — a comparison of substrate specificity and sequence homology. J Mol Catal B: Enzym 2013, 97:189-195. 26. Cheetham PSJ: Enzymes for flavor production. In Encyclopedia Industrial Biotechnology, vol. 4. Edited by Flickinger, Michael C. 2010:2149-2175. 27. Torres S, Baigori MD, Swathy SL, Pandey A, Castro GR: Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase. Food Res Int 2009, 42:454460. 28. Sun J, Yu B, Curran P, Liu S-Q: Lipase-catalysed transesterification of coconut oil with fusel alcohols in a solvent-free system. Food Chem 2012, 134:89-94. 29. Peng Q, Zhang X, Shang M, Wang X, Wang G, Li B, Guan G, Li Y, Wang Y: A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea. Microbial Cell Factories 2011, 10:95. The metagenome approach also worked for the detection of flavour enzymes. 30. Di Gioia D, Luziatelli F, Negroni A, Ficca AG, Fava F, Ruzzi M: Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid. J Biotechnol 2011, 156:309-316. 31. Lambert F, Zucca J, Ness F, Aigle M:: Production of ferulic acid and coniferyl alcohol by conversion of eugenol using a recombinant strain of Saccharomyces cerevisiae. Flavour Fragrance J 2014, 29:14-21. 32. Navia-Gine WG, Yuan JS, Mauromoustakos A, Murphy JB, Chen F, Korth KL: Medicago truncatula (E)-b-ocimene synthase is induced by insect herbivory with corresponding increases in emission of volatile ocimene. Plant Physiol Biochem 2009, 47:416-425. 33. Diaz-Chavez ML, Moniodis J, Madilao LL, Jancsik S, Keeling CI, Barbour EL, Ghisalberti EL, Plummer JA, Jones CG, Bohlmann J: Biosynthesis of sandalwood oil: santalum album CYP76F www.sciencedirect.com
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cytochromes P450 produce santalols and bergamotol. PLoS One 2013, 8:e75053. Work showing the promise and the problems of using monooxygenases for the generation of volatile terpenoids.
High product yields by using a novel bioreactor concept based on polymer beads as the partitioning phase preventing the catabolism of the product as a carbon source by the producer cells; possibly transferable to other bioflavour processes.
34. Hartwig S, Frister T, Alemdar S, Li Z, Krings U, Berger RG, Scheper T, Beutel S: Expression, purification and activity assay of a mutant patchoulol synthase cDNA fused to thioredoxin in Escherichia coli. Protein Exp Purif 2014, 97:61-71.
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35. Scholtmeijer K, Cankar K, Beekwilder J, Woesten HAB, Lugones LG, Bosch D: Production of (+)-valencene in the mushroom-forming fungus S. commune. Appl Microbiol Biotechnol 2014 http://dx.doi.org/10.1007/s00253-014-5581-2. 36. Huang F-C, Schwab W: Overexpression of hydroperoxide lyase, peroxygenase and epoxide hydrolase in tobacco for the biotechnological production of flavours and polymer precursors. Plant Biotechnol J 2012, 10:1099-1109. A different approach: heterologous expression of plant genes in a plant cell using the well-growing tobacco cells in vitro. 37. Aragueez I, Valpuesta Fernandez V: Metabolic engineering of aroma components in fruits. Biotechnol J 2013, 8:1144-1158. 38. Ma X-K, Daugulis AJ: Transformation of ferulic acid to vanillin using a fed-batch solid-liquid two-phase partitioning bioreactor. Biotechnol Progr 2014, 30:207-214.
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40. Lu X, Liu S, Feng Y, Rao S, Zhou X, Wang M, Du G, Chen J:: Enhanced thermal stability of Pseudomonas aeruginosa lipoxygenase through modification of two highly flexible regions. Appl Microbiol Biotechnol 2014, 98:1663-1669. 41. Berger RG (Ed): Flavour and Fragrances — Chemistry Bioprocessing and Sustainability. Berlin: Springer; 2007. 42. Beekwilder J, van Rossum HM, Koopman F, Sonntag F, Buchhaupt M, Schrader J, Halla RD, Boscha D, Pronk JT, van Maris AJA, Daran J-M: Polycistronic expression of a b-carotene biosynthetic pathway in Saccharomyces cerevisiae coupled to b-ionone production. J Biotechnol 2014:10. doi: 1016/ j.jbiotec.2013.12.016. Ground-breaking genetic engineering work in which a several fungal open reading frames of the carotenoid pathway were co-expressed with a plant dioxygenase. Possibly the approach of the future, once genetically modified strains will be accepted for food applications.
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