Natural Food Additives, Ingredients and Flavourings || Natural aroma chemicals for use in foods and beverages

© Woodhead Publishing Limited, 2012

10

Natural aroma chemicals for use in foods and beverages D. J. Rowe, Riverside Aromatics Ltd, UK

Abstract: The origins of organic chemistry are contiguous with the isolation of natural materials, including what we now term aroma chemicals. The development of isolation techniques has enabled materials to be isolated from sources in which they are at low concentrations and to obtain them at purities that match those of synthetic origin. Methodologies include isolation from essential oils, isolation as by-products of other processing, ‘soft chemistry’ and biotechnology (including fermentation). The commercial importance of natural materials has led to the advances in the techniques used to investigate the authenticity of such materials.

Key words: natural aroma chemicals, isolation, by-products, ‘soft chemistry’, biotechnology, isotope ratios.

10.1 Natural aroma chemicals and the origins of organic chemistry

To the general public, this title is a contradiction in terms! Some of the most important natural aroma chemicals were among the fi rst organic chemicals to be purifi ed and characterised, beginning with menthol in 1771 ( Table 10.1 ). The fundamental difference, of course, is that in the early days of organic chemistry, it was believed that life was essential to the creation of complex molecules, the so-called ‘vital force’ or ‘vitalism’. We understand now that the reverse is true, i.e. it’s the complexity of organic chemistry that is the mechanism of life (Byrne and Rowe 2009).

The nineteenth and twentieth centuries saw the development of chemistry to the level that most organic molecules can be made in vitro ; despite this, recent years have seen continually growing interest in natural materials.

Natural aroma chemicals for use in foods and beverages 213


Table 10.1 The development of organic chemistry

Date Event

Fourth century BC Hippocrates, Aristotle and others posit that the spark of life creates what we now know as the complexity of organic chemistry.

1771 Menthol isolated.1828 Dr Freidrich Wöhler and the beginning of the end of vitalism –

preparation of urea from ammonium isocyanate, described by him in a letter to Jöns Jacob Berzelius as ‘the slaying of a beautiful hypothesis by an ugly fact’.

1834 Cinnamaldehdye identifi ed.1837 Benzaldehyde isolated.1845 Dr Adolph Wilhelm Hermann Kolbe, the supposed inventor of the

modern term ‘synthesis’: Synthesis of acetic acid from carbon – THE COMPLEXITY OF ORGANIC CHEMISTRY CREATES LIFE.

1858 Vanillin identifi ed.2006 Doctor Who (A British science fi ction TV series produced by the

BBC): ‘What’s life? Nothing. A quirk of matter. Nature’s way of keeping meat fresh’ (BBC 2006).

Source: Byrne and Rowe (2009). Adapted and reprinted with permission from Perfumer & Flavorist magazine, Allured Business Media, Carol Stream, IL, USA.

10.2 Naturals in the twenty-fi rst century 10.2.1 Driving forces In the case of aroma chemicals, there is rarely, if ever, a technical advantage in using a natural material over a synthetic one. This is refl ected in the fact that a ‘natural’ and ‘synthetic’ material have the same ‘identifi ers’ – Flavour and Extracts Manufacturers Association (FEMA) number, Chemical Abstracts Service (CAS) number, Flavis, Joint FAO/WHO Expert Committee on Food Additives (JECFA), etc. The sole purpose of using naturals is to enable the fi nal product to be sold with a natural label – the so-called ‘clean label’ concept – and this is not the place to discuss the pros and cons of this.

10.2.2 Sources This is an area of continuous expansion. As well as isolation from the ‘traditional’ sources such as essential oils, we can now isolate material from fermentation mixes of many kinds – both as the primary product and as by-products, or co-products. In the case of isolation from effl uent gases, it can claim a positive environmental advantage. This, and the isolation from waste materials, is discussed later.

214 Natural food additives, ingredients and fl avourings


10.2.3 Techniques While the basic techniques of distillation and crystallisation remain the cornerstones of isolation techniques, the increased demand for natural aroma chemicals means that greater effort is expended in isolating components at low concentrations – materials that might be said to be ‘down in the noise’ on chromatographic traces!

10.3 Quality control and natural aroma chemicals There is an irony here. In general, the aim of the supplier of natural aroma chemicals, refl ecting the general desire in the fl avour industry, is for the natural materials to be as close in quality to that of the already established ‘synthetic’ material; this enables the fl avourist to replace a nature-identical fl avour with a natural one with minimal effort. Yet at the same time this emphasises that the lack of technical advantage to using natural aroma chemicals, that ‘natural’ is purely a marketing conceit. It has the added impact that as natural materials increasingly approximate the quality of their synthetic cousins, it becomes harder to tell them apart!

Originally, natural aroma chemicals were often best described as fractions or cuts from distillations; one still comes across things like ‘decanal 50% in orange oil’. Higher purity materials always have an advantage that the potential variations in quality are less (a similar phenomenon has taken place in synthetics, especially where materials are a mixture of isomers, e.g. the ‘industry standard’ trans -2-hexenal moving from 95% to 98%). Quality requirements have also risen in organoleptic terms. Dimethyl sulfi de [10.1], formerly isolated from mint oils (in the form of ‘peppermint heads’, the most volatile fraction from mint oils) was generally ca. 95% pure, with the remainder consisting of a range of materials with volatilities ranging from acetaldehyde to furfural, and whose odour standard was ‘if it doesn’t smell of mint, it’s OK’. This is now produced as a fermentation by-product (see below), assays in the region of 99% and approximates to the clean, sweetcorn–asparagus note of good quality, washed and redistilled synthetic material.

[10.1]

10.4 Natural aroma chemicals by direct isolation 10.4.1 Natural aroma chemicals from essential oils and extracts This is still the single most important approach, especially in terms of sheer volume. It is also potentially the most turgidly dull to write or read about, so the author will try to avoid the worst ‘book of lists’ approach and NOT do a table of citrus oils and their terpenes! However, it is equally true that the citrus oils, produced in enormous



quantities both in their own right and as the by-products of the juice industry, are of great commercial importance as sources of natural aroma chemicals (Margetts 2004), some of which are used to prepare other natural aroma chemicals. One useful differentiator might be that between materials that are directly isolated from the oil (often mono-terpenes and their derivatives) and those that require additional processing (usually sesqui- and di-terpenes and their derivatives).

Citrus oils are often ‘folded’, i.e. the more volatile terpenes (frequently hydrocarbons that provide little organoleptic impact and have a negative effect on water solubility) are distilled off, the remaining pot fraction being the ‘folded oil’. For example, grapefruit oil can be folded to give a terpene fraction, which along with the ubiquitous d-limonene [10.2], contains the grapefruit mercaptan, p -menthen-8-thiol [10.3]; the less volatile material, the ‘folded oil’, can be further processed to give the oxygenated sesquiterpene nootkatone [10.4].

[10.2] [10.3] [10.4]

Orange oil, the cheapest and highest volume of all the oils, can be processed to give d-limonene, aliphatic aldehydes such as octanal [10.5] and decanal [10.6] from the terpene fraction, and the sesquiterpene valencene [10.7] from the folded oil.

[10.5] [10.6] [10.7]

Other examples of ‘volatile derivatives’ are the pinenes, alpha - [10.8] and beta - [10.9] from pine oils; these also yield alpha -terpineol [10.10]. Mandarin oil is unusual in yielding a nitrogen derivative, methyl N -methylanthranilate [10.11].

[10.8] [10.9] [10.10] [10.11]



This approach is not unique to the citrus oils; for example, mint oils ( Mentha arvensis and variations thereof) yield menthol [10.12] very easily, simply by cooling the oil and fi ltering off the resulting crystalline menthol. Distillation of the resulting ‘dementholised oil’ gives a terpene fraction, which can be fractionally distilled to give natural cis -3-hexenol [10.13].

[10.12] [10.13]

Other important natural aroma chemicals from essential oils include linalool [10.14] from ho wood oil, cinnamaldehyde [10.15] from cinnamon bark oil and (mostly) cassia oil, eugenol [10.16] from clove oil and citral [10.17] from Litsea cubeba oil. In the latter case, production is de facto solely for the production of natural citral.

[10.14] [10.15] [10.16] [10.17]

10.4.2 Natural aroma products from food by-products and ‘waste streams’ This potentially is the most important approach for the future. There are ethical issues arising from the growth of non-food crops, most recently around palm oil and its contribution to deforestation in Indonesia. The growth of crops solely for ‘chemicals’ is certainly questionable, to say the least, and unlikely to win favour with the wider public. This is especially a concern in the production of natural aroma chemicals, when marketing departments are at the same time trying to associate the word ‘natural’ with a green, unspoiled, picture, a veritable Garden of Eden. . . . In this context, green credentials deriving from the use of material which would otherwise be disposed of can offer great advantage.

The stones, or pits, of peach and apricot can be processed to give natural benzaldehyde [10.18]. The leaves and stems of tomato vine can be processed to yield 2-isobutylthiazole [10.19] and 2-isopropyl-4-methylthiazole [10.20].



[10.18] [10.19] [10.20]

Less obvious, but also of great importance, is the isolation from ‘off-gases’. Since aroma chemicals are, by their nature, volatile components of food, it should be no surprise that, when food and related natural materials are processed, useful materials are lost and that unless trapped or in some way ‘scrubbed’ from the effl uent gasses, they would fi ll the surrounding area with an unwanted ‘miasma’! Those of us who have lived, or worked, near to a brewery know the strength of aromas given off during fermentation. Since this process is also used in the production of fuel alcohol, a lot of such effl uent gas is produced.

Trapping the volatiles, either cryogenically or by means of activated charcoal, both reduces the nuisance value of such odours and provides a feedstock for natural aroma chemicals, most especially dimethyl sulfi de [10.1], which was mentioned earlier. Other examples are 2-methyltetrahydrofuranone [10.21], commonly known as coffee furanone from, most appropriately, the effl uent gases from coffee roasting, and trans –2- trans -4-decadienal [10.22], which is produced during the deodorisation of vegetable oils.

[10.1] [10.21] [10.22]

There is, perhaps, a limit to this approach. Certain materials, which are both food by-products and a waste stream, could in theory be processed to yield natural skatole [10.23], a material of interest for fl avours of the camembert cheese type, with their goaty-indolic notes. It must be admitted, however, that the marketing of such a material would perhaps provide a challenge above and beyond the call of duty . . . .

[10.23]

10.5 Natural aroma chemicals by traditional food preparation processes: cooking chemistry

One of the classic insults thrown at chemistry students, usually by physics undergraduates, is that ‘chemistry is just cooking’. The correct riposte to this is,



of course ‘No, Iain: it’s the other way round – cooking is chemistry’ (O’Hare and Grigor 2004).

10.5.1 Natural esters While these are perhaps of limited interest chemically, they are probably the largest volume of all the ‘synthesised’ natural aroma chemicals. Natural alcohols and natural acids are readily available from fermentation, especially ethanol itself and isoamyl alcohol derived from fusel oils (see below), i.e. the higher boiling residues from distillation of ‘liquors’ such as whisky and brandy.

The simplest approach to esters is direct reaction between an alcohol and an acid, with the water produced being removed by azeotropic distillation. With the more volatile esters, the ester itself can be used to form an azeotrope; with esters of water-soluble alcohols such as ethyl esters, the presence of too much alcohol in the distillate can stop the water from separating out and a volatile hydrocarbon such as toluene may be added to help overcome this. From n -butyl esters onwards this is not a problem as the alcohol is immiscible with water and separation is not an issue.

All of this applies to naturals as much as synthetics. However, the reaction is slow and the ‘chemical’ approach of adding a strong acid catalyst is not possible. For esters of higher boiling alcohols such as isoamyl alcohol [10.24] and cis -3-hexenol [10.13], this is not much of a problem as the reaction mix can be heated to a suffi ciently high temperature to achieve a reaction in a matter of a few hours, for example the formation of the familiar isoamylacetate [10.25]. With the formation of ethyl esters, however, the low boiling point of ethanol (78°C) means the reaction is very slow, leading to reaction times running into many days, with each passing day increasing the temptation to add a catalyst! This can be done naturally, of course, by using esterases (lipases) to accelerate the reaction, especially in transesterifi cation reactions.

[10.24] [10.25]



Many aldol-type reactions take place spontaneously, or with gentle heating; for example, 5-methyl-2-phenyl-2-hexenal [10.26] is readily formed from isovaleraldehyde [10.27] (obtained from eucalyptus oil) and phenylacetaldehyde [10.28] (from the fermentation of phenylalanine – see below).

[10.26] [10.27] [10.28]

10.6 Natural aroma chemicals by biotechnology 10.6.1 Fermentation products Language is a tricky beast, and never more so than here. ‘Biotechnology’ invokes scientists in white coats in laboratories and scientists are, as we are informed by the press, and know from the movies, at best, naive and bit barmy (‘the nutty professor’), and even more often deranged sociopaths (Dr Strangelove, Dr Frankenstein). The term ‘fermentation’ is more acceptable and ‘brewing’ is positively cuddly. So this is the cuddly fl uffy section on brewing derivatives, or more generally by the use of different varieties of yeast ( Saccharomyces spp.)

The single biggest use of yeast (in chemical terms) is in the production of ethanol, both for consumption in alcoholic liquors and for use in fuel. Ethanol [10.29] is also of great importance in fl avours, where it can contribute to the fl avour, act as a solvent or be considered an additive (as E1510). However, while it is the primary product of fermentation, other highly valuable materials are produced as well. The isolation of dimethyl sulfi de [10.1] from the effl uent gases has already been mentioned; the residues after the ethanol has been distilled off, the fusel oils (from the German ‘fusel’, referring to ‘bad spirits’) are the main source of ‘isoamyl alcohol’, a mixture of 2-methyl- and 3-methyl-butan-1-ol [10.30], together with smaller amounts of other alcohols such as butan-1-ol and hexan-1-ol. The residues after the removal of the isoamyl alcohols can be further processed to give natural pyrazines. These are the simple alkyl pyrazines, predominantly the isomers of dimethylpyrazine, e.g. 2,3-dimethylpyrazine [10.31], trimethylpyrazine and ethyldimethylpyrazines (such as [10.32]); there is little tetramethylpyrazine and the cyclic pyrazines such as 5,6,7,8-tetrahydroquinoxaline are not present. Finally, from the residues of the residues of the residues, the carotenoid derivative β -damascenone [10.33] can be isolated.

Table 10.2 shows the structures of these materials and their boiling points. The commercial feasibility of isolating these materials derives solely from their origins



as de facto waste from brewing/fermentation. If the process was carried out in order to produce (say) the natural pyrazines, the cost would add several zeros to the current market price.

10.6.2 Microbial methods Again, as with brewing, the origins of this lie in prehistory, for example, the use of ‘moulds’ ( Penicillium spp.) to modify the fl avour of cheese is the technology behind all ‘blue’ cheeses such as Stilton, Roquefort and Gorgonzola. This indicates that there are three uses of microbial systems: (i) an in situ modifi cation of the fl avours of foods; (ii) the more recent development of the use of microorganisms to produce aroma chemicals for isolation; and (iii) their use as ‘natural reagents’ to carry out specifi c chemical reactions. This is an area of great activity. There are a number of excellent reviews (Longo and Sanroman 2006; Schrader 2007; Brenna et al. 2010) and hence only a few examples, those concerning the production of natural aroma chemicals, are given here.

Amino acids are interesting precursors, in some ways, of course, paralleling their roles in secondary metabolism. Fermentation of phenylalanine [10.34]

Table 10.2 Structure of materials derived from yeast fermentation and their boiling points

Name Boiling point Structure

DMS 37 [10.1]

Ethanol 78 [10.29]

Isoamyl alcohol 130

[10.30]

2,3-Dimethylpyrazine 156

[10.31]

2-Ethyl-3,5-dimethylpyrazine 181

[10.32]

beta-Damascenone 274

[10.33]



leads to the rose-like phenyl ethyl alcohol [10.35] and phenylacetaldehyde [10.26], which are in turn precursors to the cheesy phenylacetic acid [10.36].

[10.34] [10.26] [10.35] [10.36]

The ‘C6 wound gases’, i.e. cis -3-hexenol [10.13], trans -2-hexenal [10.37] and trans -2-hexenol [10.38], are produced by plants as a by-product of cellular damage, as molecular oxygen, a deadly cytotoxin, is ‘trapped’ by reaction with the highly unsaturated carboxylic linolenic acid. We can use the same chemistry in the manufacture of these aroma chemicals. Linoleic acid [10.39], readily available from vegetable oils, is treated with lipoxygenase and the resulting hydroperoxide [10.40] cleaved with (unsurprisingly) hydroperoxide lyase to give cis -3-hexenal [10.41], which rearranges to trans -2-hexenal [10.37]. This can then be reduced by bakers’ yeast (Seebach et al. 1985) to the alcohol [10.38], which in turn can be converted to esters such as the acetate [10.42]. Cis -3-hexenal can also be reduced to give cis -3-hexenol [10.13], but at the moment this is not economic compared with its isolation from mint oil – though, of course, the production of this by the mint plant utilises exactly the same chemistry!

[10.39]

[10.40]

[10.13] [10.41] [10.37]



[10.42] [10.38]

10.7 Assessing the natural status of aroma chemicals 10.7.1 Hi-tech methods

Measurement of 14 C level The measurement of the 14 C level of an aroma chemical of plant origin gives the date that the carbon in the molecule was last in the atmosphere. This is de facto radiocarbon dating, making use of the fact that the upper atmosphere has a constant ‘steady state’ concentration of 14 C, a balance between its formation from 14 N by impact of cosmic rays and its subsequent decay back to 14 N over a half-life of 5700 years. The measurement is usually carried out by traditional scintillation counting methods, and indicates whether the material is ‘of recent biological origin’ or whether it contains carbon of fossil origin, in which essentially all the 14 C has decayed. While this only shows the source of the carbon and says nothing about the methods used to prepare the material, it remains a good test for materials which are usually isolates; for example, cinnamaldehyde [10.16] is usually isolated from cassia oil, and the 14 C test would generally confi rm its natural status. In theory, it could be prepared from natural benzaldehyde [10.18] by ‘chemical’ means, but this would not be economic – a greater concern would be chemical conversion of cinnamaldehyde to benzaldehyde!

[10.16] [10.18]

In some cases, however, 14 C would give little or no information about natural status. For example, furfural [10.43] is produced from cereal (carbohydrate) waste and hence would pass as natural by the 14 C test, as would any of the materials into which it is converted without the addition of other carbon atoms. It is hydrogenated to yield furfuryl alcohol [10.44] and 2-methylfuran [10.45], with the latter a precursor to 2-methyl-3-furanthiol [10.46]; all of these would appear ‘natural’ by radiocarbon testing!



[10.43] [10.44] [10.45] [10.46]

13 C isotopic ratio mass spectrometry (IRMS) All chemical reactions differentiate to some extent between isotopes of the same element. This phenomenon is related to the zero-point energy of the chemical bond, which in turn refl ects the mass of the atoms thus combined. Hence the greatest difference occurs when hydrogen is replaced by deuterium, as the change in mass is the greatest. But it is also observable with other light elements, especially carbon, where 13 C is ca. 1% of all terrestrial carbon. This can enable us to determine the nature of transformations that have taken place in the production of a specifi c material.

In the context of natural aroma chemicals this can, in certain cases, enable us to differentiate between chemical and enzymatic conversions, and in the latter case, between enzymatic and microbial conversions, and between different types of organism. One of the most useful makes use of the different routes plants use in photosynthesis; the three routes, C3 and C4, so-called as the carbon dioxide is fi rst incorporated into a C3 and a C4 chain respectively, and the less common CAM pathway (named after the type of plant where it was fi rst recognised and the storage of CO 2 as an acid). All three pathways ‘disfavour’ incorporation of 13 C, but to different degrees. This difference, delta 13 C (or d13C) is the ratio of stable isotopes 13 C: 12 C, expressed in parts per thousand (per mil, ‰) when compared to the ratio in the mineral PeeDee belemnite. Typical values for bulk 13 C ratios are −10 to −16 for C4 plants, −23 to −32 for C3 plants, with CAM plants having a wider range, ca. −12 to −30. It is important to note that this test does not of itself indicate natural status, as ‘fossil fuels’ are of organic origin and themselves have a range of ca. −15 to −33 (Asche et al. 2003).

One of the most clearest differentiated material is vanillin [10.47]. The vanilla plant ( Vanilla planifolia or Vanilla tahitensis ) is in fact a tropical orchid, which makes use of the CAM pathway, and the 13 C signature of ‘vanillin ex vanilla’ is typically in the –12 region, making it clearly distinguishable from material of petrochemical origin (ca. −24) and that derived from ferulic acid [10.48]. The latter is usually obtained from rice, a C3 plant, and has a delta 13 C value more in the region of −31.

[10.47] [10.48]



The above work has focused on the so-called ‘bulk’ 13 C analysis. By looking at fragmentation pathways, it has been possible to identify the delta 13 C of individual sites within a molecule; for example, the different 13 C ‘signatures’ within the vanillin molecule have been elucidated (Dennis et al. 1998).

Other isotopes have been studied as markers. Hydrogen/deuterium (H/D) ratios can be used, for example with the ‘green’ molecules trans -2-hexenol and trans -2-hexenal (Hor et al. 2001), though they can be affected by other ‘non-biological’ reactions such as ‘acid’ protons, including proton exchange at active sites such as those alpha- to a carbonyl group. The 16 O/ 18 O ratio has also been studied and found to be of interest.

Site-specifi c natural isotope fractionation nuclear magnetic resonance (SNIF-NMR) This wonderfully appropriately named technique, sites-specifi c natural isotope fractionation nuclear magnetic resonance, makes use of the tendency towards differential use of isotopes in biological systems (Mosandl 2007). Theoretically a number of nuclei can be investigated, but a combination of natural abundance and nuclear properties make the δ H/D values the most practical. This method can be used to make detailed investigation of δ H/D values at many (theoretically all) sites even in a complex molecule; but several factors combine to make this method more of a research tool than a day-to-day quality instrument:

1 A pure sample is generally needed, precluding the analysis of fl avours and foodstuffs.

2 The technique requires specialised understanding to both carry out and interpret the results.

3 The instrumentation (and running costs) is extremely expensive; at the time of writing (spring 2011) the world’s largest NMR spectrometer is the CNRS instrument at Lyon in France costing in the region of EUR 11 million.

10.7.2 False fl ags: chirality and isomer ratios This is one of the most misunderstood, yet potentially most valuable, areas concerning natural aroma chemicals. The fi rst thing we must eliminate is the idea that geometric isomers, cis–trans (or E–Z) ratios, provide any information about the naturalness, or otherwise, of a material. The different molecular geometry of such isomers means their intermolecular forces are signifi cantly different; in lay terms, they’re different shapes and stick together differently, Hence they have different boiling points, and since the vast majority of aroma chemicals are isolated and/or purifi ed by distillation, the cis and trans isomers can be separated, at least to some extent. Hence such ratios tell us about how the distillation has been carried out, not whether a material is natural or not. Examples of natural aroma chemicals with geometric isomers include citral [10.17], 5-methyl-2-phenyl-2-hexenal [10.28] and trans -2- trans -4-decadienal [10.22]; in each case



the structures below are shown in the trans form for the sole reason that the author is too lazy to draw the cis -isomer as well and fi nds the trans -isomers to be more aesthetically pleasing. 2-Methyl-4-propyloxathiane (tropathiane) [10.49] is an example of cis–trans geometry across a ring.

[10.17] [10.28] [10.22] [10.49]

‘Chirality’, or optical isomerism, is a feature of many molecules used as aroma chemicals. As our sensory system is based on biomolecules that are themselves chiral, we can often differentiate enantiomers by their odour. The best known, and perhaps most extreme, case is the enantiomers of carvone; the R -isomer [10.50] is spearmint and the S -isomer [10.51] caraway. However, these extremes are rare and more commonly both enantiomers have some shared character, with one of the enantiomers being more powerful and more ‘typical’ than the other.

In addition, and most importantly, most aroma chemicals are found as mixtures of both isomers, i.e. even if one enantiomer dominates, the other is usually found at a lower level. Very few, if any, aroma chemicals are found in nature in ‘homochiral’ form (corresponding to an enantiomeric excess of 100%). Similarly, they are rarely found to be truly ‘racemic’, i.e. an enantiomeric excess of zero. We can illustrate this with the enantiomers of hazeltone (5-methyl-2-hepten-4-one) [10.52] and [10.53]. Both enantiomers have the nutty, buttery, metallic character implied by the name, with the d-isomer [10.53] (which has the S -confi guration) having the lower odour threshold, and both are found in nature: ca. 80:20 ( S:R ) in raw hazelnuts and 70:30 in roasted hazelnuts.

[10.50] [10.51] [10.52] [10.53]

With this in mind, we strongly wish to dispel the concept that ‘only chiral (or more strictly, homochiral) natural aroma chemicals are truly natural’. The reality is both more complex and more interesting! An area where this has become something of a cause celebre is alpha -ionone [10.54] and [10.55].



[10.54] [10.55]

It has been known for many years that the alpha-ionone found in raspberry extract does approach homochirality, with an enantiomeric excess of at least 99.9% R -isomer [10.54] (by headspace analysis). However, in a simplifi cation which rivals Orwell’s ‘two legs good, four legs bad’, this has been converted into ‘100% R -isomer = natural, <100% R -isomer = synthetic’. This wretched dictum is even being used to promote chiral gas chromatography (GC) systems, to enable people to ‘prove’ whether their raspberry fl avours are natural or not. Yet it is not based on scientifi c fact. Both isomers are found in nature; work by Haarman and Reimer (Werkhoff et al. 1991) on a variety of foodstuffs found that while the R -isomer predominates, the S -isomer [10.55] is found at the level of 5% in Darjeeling tea (by solvent extraction), 3% in vanilla pods and 5% in carrots (both by headspace analysis). Boronia absolute was found to have 9% S -isomer. Even in raspberry there was some S -isomer, if only 0.1%! In addition, work by Firmenich and the University of Hamburg found that the S -isomer, not the R -isomer, was the main isomer in black tea (Konig et al. 1989). This is a particular problem, as the ionones can be obtained by air oxidation of carotenes; the elevated temperature and purifi cation by distillation leads to loss of chirality as the chiral centre has an active proton alpha - to two double bonds, one of which is also conjugated to a carbonyl group. This loss of chirality is shown below in this case via a carbanion mechanism; as a result, the alpha -ionone thus obtained has little enantiomeric excess, typically R:S = 60:40.

We have seen above that both enantiomers are found in nature. The techniques used fulfi l all the criteria of the European Union’s Regulation (EC) No. 1334/2008 and the US 21 CFR 101.22, yet due to the ‘two legs good, four legs bad’, the present paradigm of naturalness in the ionones, there is resistance to use what is potentially a very important natural aroma chemical. This absurdity has even spilled over into issues with beta -ionone [10.56], which itself is not and cannot be chiral! Distillation of beta -ionone leads to formation of alpha -ionone, an energetically unfavourable transformation as this takes a double bond out of conjugation, but since the alpha -form has the lower boiling point, the equilibrium is always shifted in its favour as the distillation proceeds. Hence, distilled beta -ionone always contain traces of alpha-ionone, and since beta -ionone is achiral, the resulting alpha -ionone has zero enantiomeric excess leading, once again, into accusations of ‘un-naturalness’ when the material is analysed on a chiral GC column!



[10.56]

Issues around chirality can be illustrated well with the lactones, especially the wide-spread gamma -lactones. All the gamma-lactones [10.57] (except the simplest, gamma -butyrolactone, R = H) have a chiral centre alpha - to the ring oxygen.

[10.57]

Another piece of ‘received wisdom’ is that all naturally occurring gamma -lactones are found exclusively in the R -confi guration. Table 10.3 shows that while the R -form predominates in most though not all sources, with the possible exception of gamma -dodecalactone, both are found in nature. In the case of the wonderfully

Table 10.3 Sources of naturally occurring γ -lactones

Source C8 C9 C10 C12 Reference

Apricot 89/11 84/16 94/6 100/0 Bernreuther et al. (1989)85/15 100/0 100/0 Greger and Schieberle (2007)

Mango 53/47 73/27 66/34 100/0 Bernreuther et al. (1989)Peach 87/13 85/15 87/13 96/4 Bernreuther et al. (1989)Passionfruit 55/45- 50/50- 87/13- 99/1- Nitz et al. (1991)

78/22 56/44 93/7 100/0Raspberry 44/56 28/72 49/51 50/50 Nitz et al. (1991)Lulo del chocó 23/77 20/80 Morales et al. (2000)



named lulo del Chocó (‘chocolate banana’ in Portuguese?), things are the reverse with the S forms dominating (Brown 2007).

By using different culture media, both high- R and low enantiomeric excess gamma -lactones can be obtained by fermentation of natural long-chain carboxylic acids; essentially we have enzymatic oxidation at position 3 followed by cyclisation. The organoleptic properties of the high- R forms are subtly different to that of the R / S mixture, and this gives the fl avourist an extra opportunity to infl uence the fl avour.

We have a similar situation with the six-membered delta -lactones. For example, the delta-decalactone in raspberries is 98% S -isomer [10.58], whereas in peach the situation is reversed at 97% R -isomer [10.59], and in cheddar cheese we have a 72:28 mixture!

[10.58] [10.59]

10.8 Future trends For those of us not possessed of crystal balls, this is an impossible question to answer. The marketing departments of fl avour and food companies, the supermarkets and other major retailers are unlikely to reverse their policies of promoting their subliminal (and sometimes not so subliminal) formula of ‘natural = healthy’. This also chimes with the current general ‘fl ight from reason’ in which scientifi c thought takes second place to our ‘gut feelings’ and general vague spirituality – whatever that means. All too often, ‘science’ is presented as some sort of conspiracy theory, something that THEY want you to think (whoever THEY are is never specifi ed). A brief session on the blogs and message boards of the internet will demonstrate all too clearly that we are in a ‘golden age’ of conspiracy theories and general irrationality. In this climate, there’s unlikely to be any opposition to the naturals concept.

In terms of new aroma chemicals, continuing developments in isolations and (especially) microbial transformations means that more materials become available. However, the industry is on a knife edge. Growing populations, especially in the developing countries, may lead to pressure to stop the use of valuable agricultural land to, in effect, ‘grow chemicals’. At the moment it’s a rhetorical question (to which the author does not know the answer), but is the best use of land in India to grow mint in order to make menthol, when the same material is available from non-agricultural sources? Will a time come when ‘petrochemical’, currently a term of abuse, becomes fashionable – perhaps in the form ‘this fl avour contains no materials derived from food crops’? There is added irony in that the yields of natural aroma chemicals could be increased by genetic modifi cation of



the producing organism – except that the term ‘genetically modifi ed’ beats even ‘petrochemical’ in the pantheon of horrors! To a chemist, the situation is bizarre – vitalism lives on, and a mere 150 years on, the work of Wöhler and Kolbe is still not accepted by the general public!

10.9 References ASCHE , S. , MICHAUD , A.L. and BRENNA , J.T. ( 2003 ), ‘Sourcing organic compounds based on

natural isotopic variations measured by high precision isotope ratio mass spectrometry’ , Current Organic Chemistry , 7 , 1527 – 1543 .

BBC ( 2005 ), ‘The Doctor Dances’ , Doctor Who , BBC , London . BERNREUTHER , A. , CHRISTOPH , N. and SCHREIER , P. ( 1989 ), ‘ Determination of the enantiomeric

composition of γ -lactones in complex natural matrices using multidimensional capillary gas chromatography ’, Journal of Chromatography A , 481 , 363 – 367 .

BRENNA E. , FRONZA , G. , FUGANTI , C. , GATTI , F.G. and SERRA , S. ( 2010 ), ‘ Biotechnological tools to produce, natural fl avors and methods to authenticate their origin ’, in Passos , M. L. and Ribeiro , C.P. ( eds ,), Innovation in Food Engineering: New Techniques and Products , CRC Press , Boca Raton, FL , pp. 81 – 106 .

BROWN , R.C. ( 2007 ), γ-Lactones in wine: Synthesis, quantifi cation and sensory studies , PhD thesis, Flinders University of South Australia .

BYRNE , B. and ROWE , D.J. ( 2009 ), ‘ Natural aroma chemicals: Nothing new under the Sun? ’, Perfumer & Flavorist , ( 34 ) 9 , 36 – 46 .

DENNIS , M.J. , WILSON , P. , KELLY , S. and PARKER , I. ( 1998 ), ‘ The use of pyrolytic techniques to estimate site specifi c isotope data of vanillin ’, Journal of Analytical & Applied Pyrolysis , 47 , 95 – 103 .

GREGER , P. and SCHIEBERLE , P. ( 2007 ), ‘ Characterization of the key aroma compounds in apricots ( Prunus armeniaca ) by application of the molecular sensory science concept ’, Journal of Agricultural and Food Chemistry , 55 , 5221 – 5228 .

HOR , K. , RUFF , C. , WECKERLE , B. , KONIG , T. and SCHREIER , P. ( 2001 ), ‘ Flavor authenticity studies by 2H/1H ratio determination using on-line gas chromatography pyrolysis isotope ratio mass spectrometry ’, Journal of Agricultural and Food Chemistry , 49 , 21 – 25 .

KÖNIG , W.A. , EVERS , P. , KREBBER , R. , SCHULZ , S. , FEHR , C. et al. ( 1989 ), ‘ Determination of the absolute confi guration of α -damascone and α -ionone from black tea by enantioselective capillary gas chromatography ’, Tetrahedron , 45 , 7003 – 7006 .

LONGO , M.A. and SANROMAN , M.A. ( 2006 ), ‘ Production of food aroma compounds: microbial and enzymatic methodologies ’, Food Technology and Biotechnology , 44 , 335 – 353 .

MARGETTS , J. ( 2004 ), ‘ Aroma chemicals V: Natural aroma chemicals ’, in Rowe , D.J. (ed.), Chemistry and Technology of Flavours and Fragrances , Wiley-Blackwell , Oxford , pp. 169 – 198 .

MORALES , A.L. , DUQUE , C. and BAUTISTA , E.J. ( 2000 ), ‘ Identifi cation of free and glycosidically bound volatiles and glycosides by capillary GC and capillary GC-MS in Lulo del Chocó ( Solanum topiro )’ , Journal of High Resolution Chromatography , 23 , 379 – 385 .

MOSANDL , A. ( 2007 ), ‘ Enantioselective and isotope analysis: Key steps to fl avour authentication ’, in Berger R.G. (ed.), Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability , Springer-Verlag , Berlin , pp. 379 – 407 .

NITZ , S. , KOLLMANNSBERGER , H. , WEINREICH , B. and DRAWERT , F.J. ( 1991 ), ‘ Enantiomeric distribution and 13C/12C isotope ratio determination of gamma-lactones: Appropriate methods for the differentiation between natural and non-natural fl avours? ’, Journal of Chromatography , 557 , 187 – 197 .

O’HARE , L. and GRIGOR , J. ( 2004 ), ‘ Flavour generation in food ’, in Rowe , D.J. (ed.), Chemistry and Technology of Flavours and Fragrances , Wiley-Blackwell , Oxford , pp. 35 – 55 .



SCHRADER , J. ( 2007 ), ‘ Microbial fl avour production ’, in Berger R.G. (ed.), Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability , Springer-Verlag , Berlin , pp. 507 – 574 .

SEEBACH , D. , SUTTER , M.A. , WEBER , R.H. and ZÜGER , M.F. ( 1985 ), ‘Yeast reduction of ethyl acetoacetate: ( S )-(+)-Ethyl 3-hydroxybutanoate’ , Organic Syntheses, 63 , 1 and Collected Volume VII , 215 – 220 .

WERKHOFF , P. , BRETSCHNEIDER , W. , GÜNTERT , M. , HOPP , R. and SURBURG , H. ( 1991 ), ‘ Chirospecifi c analysis in fl avor and essential oil chemistry Part B. Direct enantiomer resolution of trans-a-ionone and trans-a-damascone by inclusion gas chromatography ’, Zeitschrift für Lebensmitteluntersuchung-und-Forschung A, 192 , 1431 – 4630 .

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Natural Food Additives, Ingredients and Flavourings || Natural aroma chemicals for use in foods and beverages